@article {pmid42283406,
year = {2026},
author = {Zhu, Y and Cao, D and Guo, T and Zhang, X and Luo, Y and Li, X and Yang, H and Zhang, M and Guo, S and Song, F},
title = {RNase III-deficient Bacillus thuringiensis enables stable dsRNA production and enhances HD1 activity against Spodoptera frugiperda.},
journal = {Pest management science},
volume = {82},
number = {10},
pages = {9889-9898},
doi = {10.1002/ps.71023},
pmid = {42283406},
issn = {1526-4998},
support = {CAAS-CSCB-202402//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 024YFD1200203//National Key R&D Program of China/ ; 32372623//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Spodoptera/growth & development/genetics ; *Bacillus thuringiensis/genetics/enzymology ; *RNA, Double-Stranded/metabolism/genetics ; *Ribonuclease III/genetics/metabolism/deficiency ; *Pest Control, Biological/methods ; RNA Interference ; Larva/growth & development/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Spodoptera frugiperda is one of the most important invasive agricultural pests in China. Integrating RNA interference (RNAi) with Bacillus thuringiensis (Bt)-based biocontrol represents a promising strategy for pest control. However, efficient dsRNA production in Bt remains challenging as endogenous RNase activity limits dsRNA stability and accumulation.<br><br>RESULTS: In this study, an RNase III-deficient Bt strain, DB&#x394;rnc, was generated using an optimized CRISPR-Cas9 system. Deficiency of RNase III markedly enhanced intracellular dsRNA accumulation. A hairpin dsRNA construct exhibited higher transcript levels than the dual-promoter construct, especially in the DB&#x394;rnc background. Oral delivery of Bt expressed hairpin dsRNA targeting the endochitinase gene SfCHI of S.&#x2009;frugiperda induced effective gene silencing and caused pupation defects. Moreover, co-application with Bt HD1 strain significantly increased larval mortality in both neonate and 2[nd]-instar larvae.<br><br>CONCLUSION: Collectively, our findings highlight that the RNase III-deficient strain DB&#x394;rnc is an efficient chassis for dsRNA expression. Furthermore, the enhanced insecticidal activity from co-application of DB&#x394;rnc(hpdsCHI) and HD1 demonstrates the feasibility of integrating RNAi with Bt-based pest control. &#xa9; 2026 Society of Chemical Industry.},
}
@article {pmid42537346,
year = {2026},
author = {Yang, G and Qin, H and Wang, Q and Cheng, J and Du, L and Fan, A and Lin, W and Huang, J and Hu, X and Li, Y and Wang, H},
title = {In vivo CRISPR screening identifies LTA4H as a target for potentiating immunotherapy efficacy via the LTB4-neutrophil axis.},
journal = {International immunopharmacology},
volume = {187},
number = {},
pages = {117213},
doi = {10.1016/j.intimp.2026.117213},
pmid = {42537346},
issn = {1878-1705},
mesh = {Animals ; *Epoxide Hydrolases/genetics/metabolism ; *Neutrophils/immunology ; *Leukotriene B4/metabolism ; Mice ; Humans ; Signal Transduction ; *Immunotherapy/methods ; Receptors, Leukotriene B4/antagonists &amp; inhibitors/metabolism ; Mice, Inbred C57BL ; Cell Line, Tumor ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/metabolism ; CRISPR-Cas Systems ; CD8-Positive T-Lymphocytes/immunology ; Killer Cells, Natural/immunology ; },
abstract = {Despite the clinical success of PD-1 blockade, therapeutic resistance remains a major barrier. Using an in vivo CRISPR-Cas9 screen with a metabolism-focused sgRNA library in MC38 and B16 syngeneic tumor models, we identified leukotriene A4 hydrolase (LTA4H) as a candidate regulator of PD-1 resistance. Lta4h ablation was associated with enhanced PD-1 responsiveness, increased CD8+ T and NK-cell infiltration and function, and reduced myeloid accumulation. Single-cell and spatial transcriptomic analyses showed that LTA4H is expressed in both malignant and myeloid compartments, with LTA4H-high tumor regions associated with myeloid-enriched niches and TGF-β related signaling. Mechanistically, hypoxia-induced HIF1α upregulated Lta4h in tumor cells, supporting tumor cells as one relevant source of LTA4H/LTB4. LTA4H-LTB4 signaling was linked to myeloid remodeling and TGF-β1 production in LTB4-conditioned neutrophils. Pharmacological blockade of LTB4 receptor signaling with the BLT1 antagonist CP-105696 improved tumor control in combination with PD-1 blockade. These findings identify intratumoral LTA4H-LTB4 signaling as a metabolic-immune pathway associated with suppressive myeloid remodeling and support further investigation of LTB4 receptor blockade to improve PD-1 blockade responses.},
}
@article {pmid42659797,
year = {2026},
author = {Fu, R and Hong, J and Qu, Q and Hong, Z and Jiang, Q and Xianyu, Y},
title = {CRISPR-Cas12a-based querying of DNA-stored MRI and PET imaging data.},
journal = {Medical image analysis},
volume = {114},
number = {},
pages = {104272},
doi = {10.1016/j.media.2026.104272},
pmid = {42659797},
issn = {1361-8423},
mesh = {*Positron-Emission Tomography/methods ; *Magnetic Resonance Imaging/methods ; *DNA/genetics ; Sensitivity and Specificity ; Reproducibility of Results ; *Information Storage and Retrieval/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Storing magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging data in DNA offers a promising solution for the long-term archival management of rapidly expanding biomedical image volumes. However, querying these data typically requires polymerase chain reaction and sequencing, which increases readout complexity and retrieval latency. Here, we propose a CRISPR-Cas12a-based querying strategy that leverages the programmability of CRISPR RNA (crRNA) for precise matching with identifier (ID) sequences. In this approach, crRNA functions as a query tool that identifies matching IDs and triggers detectable signals. Through a one-to-one mapping between ID and payload sequences established during encoding, the corresponding image data can be retrieved upon query. Computational simulations demonstrate over 99% query accuracy for both MRI and PET data using crRNA. CRISPR-Cas12a cleavage assays and molecular docking further validate the high specificity of crRNA-ID recognition. This work demonstrates the potential of CRISPR-Cas12a for biochemical querying of DNA-encoded biomedical imaging data, enabling targeted access to indexed sequences.},
}
@article {pmid42700392,
year = {2026},
author = {Pittman, CC and Xu, C and Catchpole, RJ and Garrett, S and Fuchs, R and Chu, X and Makarova, KS and Koonin, EV and Zhao, P and Wells, L and Graveley, BR and Ke, A and Terns, MP},
title = {Type IV-C CRISPR-Cas effector complexes recognize double-stranded DNA and switch on collateral cleavage of ssDNA and RNA.},
journal = {Cell reports},
volume = {45},
number = {9},
pages = {117939},
doi = {10.1016/j.celrep.2026.117939},
pmid = {42700392},
issn = {2211-1247},
abstract = {Type IV-C CRISPR-Cas systems remain enigmatic compared to other class 1 systems. Here, we expand the type IV-C catalog, identifying two phylogenetically distinct clades primarily found in archaea (IV-C1) or bacteria (IV-C2), distinguishable by the Cas10IVc subunit architecture. We functionally and structurally characterize type IV-C1 systems from Thermococcus onnurineus (Ton) and Pyrococcus abyssi (Pab). Type IV-C complexes assemble with crRNAs derived from distinct CRISPR arrays and recognize a 5'-GGG-3' protospacer adjacent motif (PAM) to bind double-stranded DNA targets. Target recognition activates the HD domain of Cas10IVc, triggering metal-dependent collateral cleavage of single-stranded DNA and RNA. This behavior is explained by allosteric alignment of the HD active site, triggered by PAM-dependent R-loop formation, as revealed by cryo-EM. Together, our findings suggest that type IV-C systems provide immunity via non-specific cleavage of nucleic acids generated during mobile genetic element replication or transcription.},
}
@article {pmid42700448,
year = {2026},
author = {Allehyani, N and Alissa, M and Alghamdi, A and Alshehri, MA and Abusalim, GS and Alhegaili, AS and Juraybi, TN and Alsuwat, MA},
title = {CRISPR-Engineered CAR-T Cell Therapy for Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma: A Review of Emerging Therapeutic Prospects.},
journal = {Reviews in medical virology},
volume = {36},
number = {5},
pages = {e70199},
pmid = {42700448},
issn = {1099-1654},
support = {PSAU/2026/R/1448//Prince Sattam bin Abdulaziz University/ ; },
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Herpesvirus 4, Human/immunology/physiology ; *Nasopharyngeal Carcinoma/therapy/virology/immunology ; *Epstein-Barr Virus Infections/complications/virology/immunology/therapy ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics/immunology ; Gene Editing ; *Nasopharyngeal Neoplasms/therapy ; Tumor Microenvironment ; },
abstract = {Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) remains a clinically challenging malignancy, particularly in recurrent or metastatic disease where durable responses to chemoradiotherapy and immune checkpoint blockade are limited. The viral aetiology of NPC provides a strong biological rationale for immune-based treatment; however, translation of chimaeric antigen receptor (CAR) T-cell therapy into this solid tumour setting is constrained by poor tumour trafficking, antigen heterogeneity, limited surface accessibility of EBV latent antigens, T-cell exhaustion, and an immunosuppressive tumour microenvironment. This review critically evaluates the emerging therapeutic prospects of CRISPR-engineered CAR-T cell therapy for EBV-associated NPC. It synthesises evidence on EBV latency biology, NPC immune evasion, solid-tumour CAR-T limitations, and genome-engineering strategies including conventional CRISPR-Cas9, base editing, prime editing, and double-strand-break-sparing targeted integration. Particular attention is given to genotoxicity, chromosomal rearrangements, chromosome loss, bystander and off-target editing, manufacturing heterogeneity, and the regulatory and biological barriers that currently separate technical feasibility from NPC-specific clinical implementation. Available clinical evidence from checkpoint blockade, EBV-specific adoptive T-cell therapy, base-edited CAR-T cells in haematologic malignancy, and early CRISPR-edited T-cell trials supports the feasibility of immune and genetic redirection but does not establish efficacy of a clinically validated CRISPR-engineered CAR-T platform for NPC. Future development should prioritise surface-accessible antigen validation, fit-for-purpose selection of editing technology, genomic safety, scalable manufacturing, and biomarker-driven early-phase trials.},
}
@article {pmid42700480,
year = {2026},
author = {He, R and Miao, L and Deng, R and Xia, X},
title = {Advances in cascaded CRISPR for preamplification-free nucleic acid assays.},
journal = {Biosensors &amp; bioelectronics},
volume = {314},
number = {},
pages = {119197},
doi = {10.1016/j.bios.2026.119197},
pmid = {42700480},
issn = {1873-4235},
abstract = {The development of rapid, sensitive, and specific nucleic acid assays is pivotal for advancing molecular detection in clinical diagnosis, food safety, and environmental monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems, renowned for their programmable and signal amplification capacity, have emerged as efficient tools for meeting these challenges. However, the intrinsic sensitivity of CRISPR/Cas assays relying on a single Cas effector is typically confined to the picomolar level, often necessitating complex nucleic acid preamplification. Cascaded CRISPR systems, which integrate sequential enzymatic reactions or multiple CRISPR effectors, can address this limitation by achieving nucleic acid preamplification-free signal enhancement. This review starts with the introduction of the design principles and working mechanisms of cascaded CRISPR strategies, encompassing Enzyme-Coupled cascades, Multi-effector class 2 CRISPR cascades, and Type III CRISPR-mediated cascades. Then, we highlight the deployment of these techniques across diverse bio-sensing scenarios, ranging from disease diagnosis to food and environmental surveillance. Finally, critical challenges and emerging frontiers are discussed, including integration with digital detection platforms and AI-assistant algorithms.},
}
@article {pmid42700916,
year = {2026},
author = {Zhang, X and Zhang, L and Guo, T and Du, W and Wang, S and Li, Y},
title = {Application of emerging technologies in the antiviral field.},
journal = {Antiviral research},
volume = {255},
number = {},
pages = {106529},
doi = {10.1016/j.antiviral.2026.106529},
pmid = {42700916},
issn = {1872-9096},
abstract = {Viral diseases pose a serious threat to global public health, agriculture, and biosecurity. Conventional antiviral strategies are often limited by an incomplete understanding of disease mechanisms, poor targeting precision, and slow response times. Emerging technologies are now reshaping the landscape of antiviral research. This review examines the roles of four key frontiers, including organoid models, gene editing, AI-driven molecular design, and synthetic biology. Organoids provide physiologically relevant platforms that model virus-host interactions and disease progression. Viral infections remain a major challenge to human and animal health, agriculture, and biosecurity. Progress in antiviral research is constrained by the complexity of viral pathogenesis, the diversity and rapid evolution of viruses, and the limited translational relevance of some traditional model systems. Recent advances in organoid technology, gene editing, artificial intelligence, and synthetic biology are expanding the toolkit available for antiviral research and development. In this review, we discuss how these four technological frontiers contribute to disease modeling, target discovery, molecular design, and translational innovation. Organoids, in particular, provide physiologically relevant systems for investigating viral infection, tissue tropism, host responses, and pathogenesis. Gene editing tools, such as CRISPR, enable precise manipulation of host and viral genomes, facilitating the development of resistant organisms and next-generation vaccine platforms. AI technologies, including AlphaFold for structure prediction and platforms for de novo protein design, address long-standing bottlenecks in structural biology and offer powerful means to engineer antiviral proteins, antibodies, and vaccine antigens. Synthetic biology, guided by the Design-Build-Test-Learn cycle, integrates computational design, genetic assembly, and functional validation into a cohesive pipeline. Together, these technologies form a synergistic workflow that spans disease modeling, target discovery, molecular design, construction, testing, and iterative optimization. This integrated approach is shifting antiviral development from traditional empirical methods toward more precise, intelligent strategies. The review also highlights ongoing challenges in integration and scalability, stressing that high-quality biological datasets and stronger interdisciplinary collaboration are essential for realizing translational potential. By presenting a cohesive view of these converging methodologies, this review offers a framework to guide the intelligent evolution of antiviral strategies in both human and animal health.},
}
@article {pmid42702425,
year = {2026},
author = {Li, J and Yuan, M and Cui, X and Ding, Y and Yang, M and Liu, R and Li, L},
title = {A cascaded signal amplification strategy combining Au@Cu2O core-shell nanozymes and CRISPR-Cas12a self-cycling for ultrasensitive colorimetric detection of miRNA-21.},
journal = {Analytica chimica acta},
volume = {1421},
number = {},
pages = {345987},
doi = {10.1016/j.aca.2026.345987},
pmid = {42702425},
issn = {1873-4324},
mesh = {*MicroRNAs/blood/analysis ; *Colorimetry/methods ; *Copper/chemistry ; *Gold/chemistry ; Humans ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {As a critical biomarker for early-stage tumor diagnosis, miRNA-21 poses significant challenges for colorimetric detection due to its ultra-low physiological abundance and high sequence homology. In this study, a multi-stage cascade amplification sensing platform was engineered by integrating an entropy-driven molecular circuit with a heterogeneous nanozyme catalytic network. The platform utilizes Catalytic Hairpin Assembly (CHA) for initial signal transduction, coupled with a CRISPR-Cas12a positive feedback self-cycling system to achieve exponential signal amplification. At the signal output interface, Au@Cu2O core-shell nanocomposites, synthesized via epitaxial growth, exhibit significantly enhanced peroxidase-like activity through interfacial electronic coupling. Experimental results demonstrate a limit of detection (LOD) as low as 1.39 fM, with the capacity to precisely discriminate between serum samples from lung cancer patients and healthy individuals.},
}
@article {pmid42702653,
year = {2026},
author = {Hu, S and Liu, J and Deng, J and Yuan, Y and Peng, S and Le, M and Li, J and Xu, K},
title = {Recent advances in CRISPR/Cas12a-based biosensors for heavy metal analysis: a review of signal readout strategies and analytical performance.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {42702653},
issn = {1618-2650},
support = {GZC20261162//the China Postdoctoral Science Foundation/ ; 82173572//the National Natural Science Foundation of China/ ; 82574159//the National Natural Science Foundation of China/ ; 2023JJ30430//the Science and Technology Program of Hunan Province/ ; 2026JJ80902//the Science and Technology Program of Hunan Province/ ; 2026JK2006//the Science and Technology Program of Hunan Province/ ; 23A0075//the Department of Education of Hunan Province/ ; },
abstract = {Heavy metals are environmental hazards owing to their toxicity, persistence, and bioaccumulation in living organisms. These metals often accumulate or transform into more toxic compounds, and some accumulate in the human body through the food chain, which is extremely harmful to health. Although conventional detection methods are reliable, they are often hampered by high costs and complex operations, motivating the exploration of alternative sensing strategies. CRISPR/Cas (Clustering Regularly Interspaced Short Palindromic Repeats and Related Proteins) systems have shown potential for developing sensitive assays with significant target specificity, programmability, and signal amplification capabilities. This review discusses research progress on using CRISPR/Cas12a to detect and prevent heavy metal contamination in food. This review introduces methodological innovations in various signal readout methods, including fluorescence, colorimetry, electrochemistry, and microfluidics, and critically evaluates their analytical performance, such as sensitivity, selectivity, and practical applicability in complex food matrices. Finally, this review aims to provide valuable guidance for researchers to develop next-generation diagnostic tools that meet the requirements of rapid screening and precision testing for food safety.},
}
@article {pmid42703025,
year = {2026},
author = {Li, B and Liu, L and Jin, K and Huang, Z and Zhang, T and Gao, R and Chen, H and Niu, L and Fan, C and Zhang, H and Huang, P and Wang, H},
title = {One-Pot RPA-CRISPR/Cas12a Assay With Visual Readout for the Ultra-Specific Detection of Monkeypox Virus Clade I.},
journal = {Microbial biotechnology},
volume = {19},
number = {9},
pages = {e70437},
pmid = {42703025},
issn = {1751-7915},
support = {2021YFF0703600//National Key Research and Development Program of China/ ; 20250203114SF//the science and technology development program of Jilin Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Monkeypox virus/genetics/isolation &amp; purification/classification ; *Nucleic Acid Amplification Techniques/methods ; *Mpox, Monkeypox/diagnosis/virology ; Recombinases/metabolism ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; Humans ; Animals ; Limit of Detection ; },
abstract = {In 2024, Monkeypox virus (MPXV) clade I has triggered outbreaks in several countries world-wide. MPXV clade I demonstrates enhanced virulence and transmissibility, with a case fatality rate reaching 10%. In response, we have developed a one-pot detection assay specifically targeting MPXV clade I, combining recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. The assay can be completed within 40 min and achieved a 95% limit of detection (LOD95) of 27.16 copies/μL. No cross-reactivity was observed with MPXV clade II or other tested viral templates, including Vaccinia virus (Tiantan strain). A preliminary room-temperature evaluation showed that the assay retained detectable performance at 25°C, supporting its potential use in equipment-limited settings. The assay also showed good intra-assay and inter-assay repeatability for recombinant plasmid templates, with all coefficient of variation (CV) values below 10%. In simulated clinical samples, the RPA-CRISPR/Cas12a assay detected more low-concentration plasmid-spiked samples than quantitative polymerase chain reaction (qPCR). These results indicate that the established assay is specific, sensitive, repeatable, and easy to perform, providing a practical tool for field-based screening and decentralized detection of MPXV clade I.},
}
@article {pmid41120666,
year = {2026},
author = {Goudy, L and Ha, A and Borah, AA and Umhoefer, JM and Chow, L and Tran, C and Winters, A and Talbot, A and Hernandez, R and Li, Z and Subramanya, S and Arab, A and Kale, N and Lee, JHJ and Muldoon, JJ and Liu, C and Schmidt, R and Santangelo, P and Carnevale, J and Eyquem, J and Shy, BR and Marson, A and Gilbert, LA},
title = {Integrated epigenetic and genetic programming of primary human T cells.},
journal = {Nature biotechnology},
volume = {44},
number = {9},
pages = {1470-1481},
pmid = {41120666},
issn = {1546-1696},
support = {K08 CA273529/CA/NCI NIH HHS/United States ; L30TR002983//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; UM1HG012660//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 2021316455)//National Science Foundation (NSF)/ ; 1K08CA252605-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K08CA273529//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *T-Lymphocytes/metabolism/cytology ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Epigenome Editing ; *Gene Editing/methods ; Animals ; Receptors, Chimeric Antigen/genetics ; },
abstract = {Targeted epigenetic engineering of gene expression in cell therapies would allow programming of desirable phenotypes without many of the challenges and safety risks associated with double-strand break-based genetic editing approaches. Here, we develop an all-RNA platform for efficient, durable and multiplexed epigenetic programming in primary human T cells, stably turning endogenous genes off or on using CRISPRoff and CRISPRon epigenetic editors. We achieve epigenetic programming of diverse targeted genomic elements without the need for sustained expression of CRISPR systems. CRISPRoff-mediated gene silencing is maintained through numerous cell divisions, T cell stimulations and in vivo adoptive transfer, avoiding cytotoxicity or chromosomal abnormalities inherent to multiplexed Cas9-mediated genome editing. Lastly, we successfully combined genetic and epigenetic engineering using orthogonal CRISPR Cas12a-dCas9 systems for targeted chimeric antigen receptor (CAR) knock-in and CRISPRoff silencing of therapeutically relevant genes to improve preclinical CAR-T cell-mediated in vivo tumor control and survival.},
}
@article {pmid42314573,
year = {2026},
author = {Landi, E and van Beusekom, E and Ben-Dor, S and Albert, S and Dickman, MM and LaPointe, VLS and van Bokhoven, H},
title = {Biallelic rescue of CTG18.1 in two Fuchs endothelial corneal dystrophy-derived iPSC lines (SCTCi047-A-2, SCTCi046-A-2) following a two-step gene editing strategy.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104032},
doi = {10.1016/j.scr.2026.104032},
pmid = {42314573},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism ; *Gene Editing/methods ; Cell Line ; CRISPR-Cas Systems/genetics ; *Transcription Factor 4/genetics/metabolism ; *Alleles ; },
abstract = {Fuchs endothelial corneal dystrophy (FECD) is an age-related condition distinguished by the degeneration of the corneal endothelium. An intronic CTG18.1 repeat in the transcription factor 4 (TCF4) gene has been associated with a 78-fold increased risk of developing the disease when at least one copy of the CTG18.1 expands above 50 repeats. Employing patient-derived material, we applied a dual CRISPR/Cas9-mediated editing approach to rescue the expansion. Combining non-homologous end-joining (NHEJ) and homologous direct repair (HDR) events, we generated two FECD-derived [+/+](CTG)8 induced pluripotent stem cell (iPSC) lines, which were then successfully characterized, providing relevant isogenic controls for disease-modelling purposes.},
}
@article {pmid42349105,
year = {2026},
author = {Hamam-Marawi, G and Papadopoulou, M and Ramachandran, H and Dobner, J and Binder, S and Hildebrandt, B and Krutmann, J and Rossi, A},
title = {CRISPR/Cas9-mediated editing of ERCC6 in iPSCs: A disease model for Cockayne Syndrome type B.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104044},
doi = {10.1016/j.scr.2026.104044},
pmid = {42349105},
issn = {1876-7753},
mesh = {Humans ; Poly-ADP-Ribose Binding Proteins/genetics ; *Cockayne Syndrome/genetics/pathology/metabolism ; *DNA Helicases/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *DNA Repair Enzymes/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Cell Line ; Base Sequence ; Cell Differentiation ; Excision Repair ; },
abstract = {Cockayne Syndrome type B (CSB) is caused by mutations in the ERCC6 gene, which encodes a key protein involved in transcription-coupled nucleotide excision repair (TC-NER) and chromatin remodeling. Deficiency in CSB leads to defective transcriptional recovery after DNA damage, oxidative stress accumulation, and progressive neurodegeneration. In this work, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, IUFi004-A-12, carrying a homozygous mutation in ERCC6 causing a premature stop codon in its 10th exon. The modified iPSCs displayed normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This model enables mechanistic studies of CSB dysfunction and facilitates therapeutic development for Cockayne Syndrome.},
}
@article {pmid42361764,
year = {2026},
author = {Liu, NN and Baljinnyam, E and Hu, R and Salemi, SE and Webb, BD and Marro, SG},
title = {Dual CRISPR/Cas9 correction of compound heterozygous MARS2 mutations in the iPSC line ISMMSi060-A from a patient with COXPD25.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104041},
doi = {10.1016/j.scr.2026.104041},
pmid = {42361764},
issn = {1876-7753},
support = {R21 NS130319/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Heterozygote ; Cell Line ; *Mitochondrial Proteins/genetics ; Base Sequence ; Cell Differentiation ; },
abstract = {We previously described the induced pluripotent stem cell (iPSC) line ISMMSi060-A derived from a patient with Combined Oxidative Phosphorylation Deficiency 25 (COXPD25) carrying compound heterozygous pathogenic variants in the mitochondrial methionyl-tRNA synthetase gene, MARS2. Here, we report the generation of the isogenic control line ISMMSi060-A-1 by CRISPR/Cas9-mediated correction of the MARS2 variants c.424C>T (p.Arg142Trp) and c.550C>T (p.Gln184*). The corrected line retained normal morphology, pluripotency, genomic integrity, and differentiation capacity, providing a valuable resource to study MARS2-related mitochondrial dysfunction and therapeutic strategies for COXPD25.},
}
@article {pmid42361765,
year = {2026},
author = {Fan, J and Zhang, S and Sun, H and Sun, M and Gu, X and He, Y},
title = {Generation of a FRMD5 knockout human embryonic stem cell line by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104048},
doi = {10.1016/j.scr.2026.104048},
pmid = {42361765},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Gene Editing/methods ; *Membrane Proteins/genetics/metabolism ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {FRMD5 is a protein-coding gene on human chromosome 15q15.3 encoding a FERM domain-containing protein. Its mutations are linked to NEDEMA, a rare autosomal dominant neurodevelopmental disorder characterized by developmental delay, intellectual disability, ataxia, epilepsy, and eye movement abnormalities (Keller Sarmiento et al., 2024).We established a FRMD5 knockout (FRMD5[-]/[-]) human embryonic stem cell line via CRISPR/Cas9. This line shows normal karyotype, expresses pluripotency markers, and differentiates into three germ layers, serving as a valuable tool for studying FRMD5 in neural development.},
}
@article {pmid42378908,
year = {2026},
author = {Lorthongpanich, C and Srisook, P and Jiamvoraphong, N and Klaihmon, P and Kumsui, S and Laowtammathron, C and Issaragrisil, S},
title = {A human iPSC model with LATS1/2 knockdown (MUSIi012-A-9) for investigating Hippo signaling in stem cell.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104052},
doi = {10.1016/j.scr.2026.104052},
pmid = {42378908},
issn = {1876-7753},
mesh = {Humans ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Signal Transduction ; *Tumor Suppressor Proteins/genetics/metabolism ; Cell Differentiation ; Hippo Signaling Pathway ; Gene Knockdown Techniques ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {This study reports the generation and comprehensive characterization of the LATS1/2 knockdown (KD) induced pluripotent stem cell (iPSC) line, MUSIi012-A-9. Created via CRISPR/Cas9-mediated modification of the LATS2 gene in a parental LATS1-KD line, this resource serves as a crucial human model to investigate the roles of LATS1/2 kinases, key regulators of the Hippo signaling pathway. Comprehensive validation confirmed normal iPSC morphology, pluripotency marker expression (OCT3/4, NANOG, SOX2), genetic stability (46,XX karyotype), and robust multilineage differentiation potential. This well-characterized iPSC line is a vital tool for advancing research into Hippo pathway regulation, lineage specification, and cell fate determination in development and disease.},
}
@article {pmid42385351,
year = {2026},
author = {Buchmann, S and Aldinger, A and Klopocki, E and Gerull, B},
title = {Establishment of CRISPR/Cas9-edited LEMD2 knock-in (UKWCHFi001-B-1) and knock-out (UKWCHFi001-B-2) iPSC lines to investigate the mechanisms of LEMD2-associated cardiomyopathy.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104049},
doi = {10.1016/j.scr.2026.104049},
pmid = {42385351},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Membrane Proteins/genetics/metabolism ; *Cardiomyopathies/genetics/metabolism/pathology ; Cell Line ; Gene Knockout Techniques ; *Nuclear Proteins/genetics/metabolism ; *Gene Editing ; Cell Differentiation ; *Gene Knock-In Techniques ; },
abstract = {LEMD2 is an inner nuclear membrane protein. The pathogenic LEMD2 variant (NM_181336.4: c.38 T > G, p.L13R) has been associated with an inherited cardiomyopathy characterized by left ventricular dysfunction and severe arrhythmias. To gain more insights into this disease and investigate the role of LEMD2 more broadly, LEMD2 p.L13R knock-in (LEMD2-KI; UKWCHFi001-B-1) and knock-out (LEMD2-KO; UKWCHFi001-B-2) iPSC lines were generated in a healthy control iPSC (UKWCHFi001-B) line using CRISPR/Cas9 gene editing. Both new iPSC lines retained pluripotency, normal karyotypes, and differentiation potential, whereby expression of LEMD2 was successfully disrupted in LEMD2-KO cells. Thus, these lines provide a suitable model for analyzing LEMD2-associated diseases.},
}
@article {pmid42391322,
year = {2026},
author = {Wiesbeck, M and Alard, EL and Merino, F and Chowdhury, N and Egert, L and Danese, A and Imhof, S and Iraci Borgia, M and Rajan, A and Fernandez-Novel Marx, N and Kepesidis, E and Köferle, A and Cerron-Alvan, LM and Vierl, F and Truong, TT and Thorwirth, M and Bilalli, L and Santos Dias Mourão, A and Ninkovic, J and Schieweck, R and Diefenbacher, M and Hauck, SM and Trainor, PA and Mardakheh, FK and Götz, M and Stricker, SH},
title = {Manipulation of protein translation and stem cell self-renewal by CRISPR activation of rRNA transcription.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6816},
pages = {eaeh1348},
doi = {10.1126/science.aeh1348},
pmid = {42391322},
issn = {1095-9203},
mesh = {*Protein Biosynthesis/genetics ; *RNA, Ribosomal/genetics ; Animals ; *Transcription, Genetic ; *Neural Stem Cells/cytology/metabolism/physiology ; *Cell Self Renewal/genetics ; Cell Proliferation ; Humans ; Mice ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Nucleolus/metabolism ; },
abstract = {Ribosomal RNA (rRNA) transcription rates vary during development, and their dysregulation is linked to diseases such as cancer and ribosomopathies. Owing to their high abundance and genomic redundancy, the functional significance of rRNA levels remains unclear. We developed TAPIR (Targeted Activation of Protein Translation), a CRISPR-based approach to elevate rRNA levels by inducing 47S ribosomal DNA transcription. TAPIR increased nucleolar size and enhanced protein synthesis, even in rapidly proliferating cells. In neural stem cells, elevated translation promoted self-renewal and proliferation in vitro and in vivo. Furthermore, TAPIR enabled the modeling and partial rescue of associated disease phenotypes. Our findings reveal that rRNA levels directly regulate translational output and that protein synthesis capacity can act as a key determinant of mammalian stem cell behavior.},
}
@article {pmid42447733,
year = {2026},
author = {Ntzani, ER and Ramachandran, H and Papadopoulou, M and Binder, S and Hildebrandt, B and Haarmann-Stemmann, T and Rossi, A},
title = {CRISPR/Cpf1-mediated knockout of FLG in human induced pluripotent stem cells generates a model for studying epidermal barrier dysfunction.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104056},
doi = {10.1016/j.scr.2026.104056},
pmid = {42447733},
issn = {1876-7753},
mesh = {Humans ; Filaggrin Proteins ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Epidermis/metabolism/pathology ; *Gene Knockout Techniques ; Cell Line ; *Intermediate Filament Proteins/genetics/metabolism ; },
abstract = {Loss of filaggrin (FLG) function impairs skin barrier formation and contributes to common inflammatory skin diseases. In this study, we established a FLG knockout human induced pluripotent stem cell (iPSC) line based on KOLF2.1 J using CRISPR/Cas12a (Cpf1)-mediated genome editing. A guide RNA targeting exon 2 introduced a homozygous mutation, which was confirmed by sequencing. The edited cells maintained typical pluripotent stem cell morphology, expressed key undifferentiated markers, and retained the ability to differentiate into all three germ layers. Karyotype and copy number variation (CNV) analyses confirmed genomic stability and parental origin; the cells were free of mycoplasma. This cell line enables studies of FLG-associated skin biology and pathology.},
}
@article {pmid42470827,
year = {2026},
author = {Dong, Q and Luo, S and Sun, J and Liu, H},
title = {Generation of FCGR3A-EGFP knock-in reporter human embryonic stem cell line, WAe001-A-3S, using CRISPR/Cas9n-based gene targeting.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104061},
doi = {10.1016/j.scr.2026.104061},
pmid = {42470827},
issn = {1876-7753},
mesh = {Humans ; *Receptors, IgG/genetics/metabolism ; *Green Fluorescent Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Differentiation ; Cell Line ; *Gene Targeting ; Genes, Reporter ; },
abstract = {Fc gamma receptor IIIA (FCGR3A) encodes CD16a, a key mediator of antibody-dependent cellular cytotoxicity (ADCC) that regulates innate and adaptive immunity, especially in natural killer (NK) cells and monocytes. We generated an FCGR3A-EGFP knock-in human embryonic stem cell (hESC) line via CRISPR/Casn9n. The cell line showed a normal karyotype, maintained expression ofthe pluripotency markers OCT4, SOX2, and NANOG, and retained trilineage differentiation potential. This reporter line enables real-time tracking of FCGR3A expression during immune cell differentiation, serving as a useful tool for studying FCGR3A[+] immune cell development and related immune mechanisms.},
}
@article {pmid42492408,
year = {2026},
author = {Duan, C and Sun, X and Ding, C and Mao, S and Liang, Y and Liang, Y and Zhang, R and Chen, H and Chen, J and Tang, S},
title = {Generation of a human iPSC line (CSUASOi016-A) modeling X-linked retinoschisis by introducing the RS1 c.214G&gt;A mutation using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104060},
doi = {10.1016/j.scr.2026.104060},
pmid = {42492408},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retinoschisis/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Eye Proteins/genetics/metabolism ; Cell Line ; Mutation ; Cell Differentiation ; Base Sequence ; },
abstract = {X-linked retinoschisis (XLRS) is an inherited retinal degenerative disease caused by mutations in the RS1 gene, leading to visual impairment. The RS1 c.214G>A mutation is a clinically relevant variant associated with XLRS. In this study, we generated a human induced pluripotent stem cell (iPSC) line (CSUASOi016-A) carrying the RS1 c.214G>A (p.E72K) mutation using CRISPR/Cas9. The edited iPSC line exhibited typical pluripotent stem cell morphology, expressed pluripotency markers (OCT4, SSEA4, SOX2, and NANOG), and retained the ability to differentiate into all three germ layers. This cell line provides a valuable resource for modeling XLRS pathogenesis and developing therapeutic strategies.},
}
@article {pmid42508498,
year = {2026},
author = {Liu, J and Bian, Z and Chen, J and Kang, H and Li, Q and Lin, J and Gou, H and Li, C},
title = {Rapid and visual detection of Lawsonia intracellularis via an RPA-CRISPR/Cas12a assay.},
journal = {Journal of microbiological methods},
volume = {249},
number = {},
pages = {107645},
doi = {10.1016/j.mimet.2026.107645},
pmid = {42508498},
issn = {1872-8359},
mesh = {*Lawsonia Bacteria/isolation &amp; purification/genetics ; Animals ; *Desulfovibrionaceae Infections/diagnosis/veterinary/microbiology ; Swine ; *Swine Diseases/diagnosis/microbiology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; },
abstract = {The swine industry faces a substantial economic threat from Lawsonia intracellularis (LI), the bacterium responsible for porcine proliferative enteropathy. Infection commonly presents with diarrhea and increased enterocyte proliferation in the ileum and colon, ultimately resulting in impaired growth performance in affected pigs. Effective management of porcine proliferative enteropathy requires timely and accurate diagnosis. The method provides significant advantages over existing detection techniques, offering enhanced sensitivity and high accuracy. The assay achieves detection within one hour, with no observed cross-reactivity against a panel of common swine pathogens. Results can be visually interpreted under blue light. Clinical validation using 123 samples demonstrated 100% concordance with qPCR results (14 positive and 109 negative). Characterized by simplicity, rapidity, and high sensitivity, the RPA-CRISPR/Cas12a method represents a novel solution for Lawsonia intracellularis detection.},
}
@article {pmid42544618,
year = {2026},
author = {Li, X and Li, D and Wang, Y and Xiang, C and Tan, S and Fan, R and Shi, K and Zhou, W},
title = {HCR-assisted CRISPR/Cas12a platform enables logic-gate and multimodal detection of kanamycin and bisphenol A.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {34},
pages = {7385-7395},
doi = {10.1039/d6ay01075j},
pmid = {42544618},
issn = {1759-9679},
mesh = {Bisphenol A Compounds ; *Benzhydryl Compounds/analysis ; *Phenols/analysis ; *Biosensing Techniques/methods ; *Kanamycin/analysis ; *CRISPR-Cas Systems/genetics ; Animals ; Nucleic Acid Hybridization/methods ; Milk/chemistry ; Limit of Detection ; Colorimetry/methods ; },
abstract = {A novel triple-modal biosensor integrating hybridization chain reaction (HCR) with CRISPR/Cas12a was developed for sensitive and selective detection of kanamycin (KANA) and bisphenol A (BPA). The system employs a phosphorothioate-modified G-rich hairpin (SHG4-2) as a dual-functional reporter probe, which resists Cas12a trans-cleavage and enables multimodal signal output via SG-quadruplex (SG4) formation. Upon target recognition by aptamers, an initiator strand is released to trigger HCR amplification, generating long double-stranded DNA products that activate Cas12a trans-cleavage. This cleaves the (SHG4-2) probe, releasing SG-rich sequences that self-assemble into SG4 structures, yielding fluorescence (with Thioflavin T), colorimetric (via SG4/hemin-catalyzed TMB oxidation), and smartphone-readable RGB signals. This platform enables parallel detection of a single target analyte, allowing flexible detection modes. Under optimized conditions, the sensor achieved detection limits as low as 20.1 pM for KANA and 6.8 pM for BPA in fluorescence mode, 32.3 pM for KANA and 17.1 pM for BPA in colorimetric mode, and 74.3 pM for KANA and 35.8 pM for BPA in smartphone mode, with excellent selectivity against interfering analogues. Successful application in spiked milk samples demonstrated high recovery rates and good reproducibility. In addition, the platform supports the logical gate operations of OR (single-target detection) and AND (dual-target detection), which allows flexible detection modes. This work presents a versatile, amplification-enhanced multimodal sensing strategy for environmental and food safety monitoring, highlighting its potential for logic-driven biosensing applications.},
}
@article {pmid42556251,
year = {2026},
author = {Clémençon, M and Brogard, J and Rozen, M and Hourton, C and Meléndez García, R and Bigou, S and Tsang, SH and Thouvenin, O and Grieve, K and Reichman, S},
title = {Generation of retinitis pigmentosa patient-derived hiPSC lines (IDVi007-A, IDVi007-B) carrying the RHO c.68C &gt; A variant (p.P23H) and CRISPR/Cas9-corrected isogenic hiPSC lines (IDVi007-A-1, IDVi007-A-2, IDVi007-A-3).},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104042},
doi = {10.1016/j.scr.2026.104042},
pmid = {42556251},
issn = {1876-7753},
mesh = {Humans ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; Cell Differentiation ; Mutation ; Fibroblasts/metabolism/cytology ; *rho GTP-Binding Proteins/genetics ; },
abstract = {The p.Pro23His (c.68C > A; P23H) mutation leads to autosomal dominant retinitis pigmentosa (adRP). Here, we reprogrammed adRP patient fibroblasts in human induced pluripotent stem cells (hiPSCs) using Sendai virus. We then generated two mutated hiPSC clones and three isogenic controls using CRISPR/Cas9. All five hiPSC lines express pluripotency genes and are able to differentiate into the three germ layers as well as retinal organoids. Altogether, these hiPSCs constitute unique biological tools to elucidate mechanisms of adRP linked to the RHO-P23H mutation.},
}
@article {pmid42566814,
year = {2026},
author = {Wang, S and Feng, X and Shang, B and Wang, H and Hu, X and Wang, Z and Li, Z},
title = {Generation of an NKX2-1-EGFP reporter iPSC line with inducible Cas9 for lung progenitor cell tracing.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104072},
doi = {10.1016/j.scr.2026.104072},
pmid = {42566814},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Thyroid Nuclear Factor 1/metabolism/genetics ; *Lung/cytology/metabolism ; *Green Fluorescent Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Differentiation ; Genes, Reporter ; },
abstract = {NK2 homeobox 1 (NKX2-1), a master regulator robustly expressed in lung, thyroid, and forebrain, is indispensable for specifying lung epithelial fate and serves as a definitive marker of lung progenitors. Here, we generated a human induced Pluripotent Stem Cell (iPSC) line harboring a doxycycline (dox)-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair. This dual-function line combines inducible genome editing with real-time tracing of early lung progenitors, enabling their prospective isolation and screening for stage-specific maturation regulators. Therefore, this engineered iCas9-NKX2-1 EGFP line is a key resource for dissecting human lung development, modeling pulmonary disease, and advancing regenerative therapies.},
}
@article {pmid42566815,
year = {2026},
author = {Zhou, H and Li, Y and Jia, M and Du, J and Zheng, K},
title = {CRISPR/Cas9-mediated generation of a homozygous MT4 knockout mouse embryonic stem cell line.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104070},
doi = {10.1016/j.scr.2026.104070},
pmid = {42566815},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Mouse Embryonic Stem Cells/metabolism/cytology ; Homozygote ; Cell Line ; Mice, Knockout ; Cell Differentiation ; *Metallothionein/genetics/deficiency/metabolism ; Gene Knockout Techniques ; },
abstract = {Metallothionein 4 (MT4) is a low-molecular-weight, cysteine-rich metal-binding protein belonging to the metallothionein family. It exhibits unique skin developmental and differentiation inhibitory activity when functionally impaired and regulates skin cell growth and disease through multiple mechanisms. However, its exact role in cell fate determination remains unknown. Here, we utilized the CRISPR/Cas9 system to generate a homozygous Mt4 knockout (Mt4[-]/[-]) mouse embryonic stem cell (mESC) line. This cell line maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers. It provides a valuable resource for investigating the mechanisms underlying skin diseases caused by MT4 gene mutations.},
}
@article {pmid42615464,
year = {2026},
author = {Kotler, ED and Ashraf, S and Santerre, JP and Kandel, RA},
title = {Optimization of CRISPR dCas9 Lentiviral Transduction in Nucleus Pulposus Cells for Use in Tissue Engineering.},
journal = {Tissue engineering. Part C, Methods},
volume = {32},
number = {8-9},
pages = {310-320},
doi = {10.1177/19373384261477635},
pmid = {42615464},
issn = {1937-3392},
mesh = {Humans ; *Lentivirus/genetics/metabolism ; *Tissue Engineering/methods ; HEK293 Cells ; *Nucleus Pulposus/cytology/metabolism ; *Transduction, Genetic/methods ; *CRISPR-Cas Systems/genetics ; Transfection ; Hepatocyte Nuclear Factor 3-beta/metabolism/genetics ; },
abstract = {Efficient delivery of large gene-editing plasmids, such as the mCherry-CRISPR dCas9 system, into nucleus pulposus (NP) cells is a key step in generating sufficient cells for tissue-engineered intervertebral disc (IVD) constructs and other regenerative therapies for degenerative disc disease (DDD). However, the transfection and transduction of these environmentally sensitive cells remain challenging. This study aimed to identify the best protocol for delivery with minimal cytotoxicity and greatest efficiency. Transfection conditions in HEK293T cells were evaluated using Lipofectamine 3000, Lipofectamine Classic, and ViaFect at different reagent-to-DNA ratios and DNA amounts. Transfection efficiency was quantified by flow cytometry based on mCherry expression. Lentivirus was produced and concentrated by comparing PEG8000, a commercial Lenti-X concentrator, and ultracentrifugation. For NP cell transduction, polybrene and protamine sulfate were tested at multiple concentrations to maximize efficiency and viability. The optimized protocol was validated by delivering a CRISPR/dCas9 Synergistic Activation Mediator (SAM) system to activate endogenous FOXA2, and by seeding the resulting cells onto membranes to assess in vitro NP-like tissue formation. Lipofectamine 3000 at a 2:1 reagent-to-DNA ratio with 0.5 µg DNA per well yielded the highest transfection efficiency in HEK293T cells while minimizing cytotoxicity. Coprecipitation methods for lentiviral concentration, particularly the in-house PEG8000 concentrator, were better than ultracentrifugation. Protamine sulfate at 30 µg/mL yielded efficient NP cell transduction with higher viability than polybrene, as assessed by survival after antibiotic selection. Application of this protocol upregulated endogenous FOXA2 mRNA and protein expression, demonstrating functional efficacy. SAM-FOXA2 cells produced thicker tissue on membrane inserts than SAM controls, confirming compatibility of the protocol with downstream tissue engineering applications. Therefore, an optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD.},
}
@article {pmid42624408,
year = {2026},
author = {Zhang, X and Wang, L and Shi, Y and Wang, J and Liu, L and Sun, X and Liang, B and Wang, J and Xu, X},
title = {Development and preliminary evaluation of real-time fluorescence-based RPA and RPA-CRISPR/Cas12a assays for rapid detection of Coxiella burnetii.},
journal = {Journal of microbiological methods},
volume = {249},
number = {},
pages = {107674},
doi = {10.1016/j.mimet.2026.107674},
pmid = {42624408},
issn = {1872-8359},
mesh = {*Coxiella burnetii/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Q Fever/diagnosis/microbiology ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Animals ; DNA Primers/genetics ; Fluorescence ; DNA, Bacterial/genetics ; },
abstract = {Coxiella burnetii (C. burnetii) is a ubiquitous zoonotic pathogen that exists widely in nature and is an obligate intracellular parasite of eukaryotic cells, causing Q fever in both humans and animals. Therefore, the surveillance and prevention of Q fever are of great importance for public health and the livestock industry. In this study, specific RPA primers, probes, and crRNAs were designed and synthesized targeting the IS1111 gene of C. burnetii, a real-time fluorescence recombinase polymerase amplification (qRPA) assay and a RPA combined with CRISPR/Cas12a (RPA-CRISPR/Cas12a) assay for the rapid detection of C. burnetii were developed. The qRPA assay completed target gene amplification within 20 min, and the amplification curves were monitored in real time on a portable Genie III instrument at 39 °C. The entire single-tube RPA-CRISPR/Cas12a assay, including RPA amplification and CRISPR/Cas12a detection, was completed within 40 min, and the results could be directly visualized under blue-light illumination. Both assays exhibited high specificity for C. burnetii and showed no cross-reactivity with the tested microorganisms. The LOD of the qRPA assay for C. burnetii DNA standard was 54 copies/μL, whereas the LOD of the RPA-CRISPR/Cas12a assay was 1.3 copies/μL. The assays were further evaluated on 456 clinical samples for the detection of C. burnetii. The postive rates of the qRPA and RPA-CRISPR/Cas12a assays were 3.73% (17/456) and 4.17% (19/456), respectively. Compared with qPCR detection results, the concordance rates were 99.34% and 99.78%, respectively. In conclusion, the qRPA and RPA-CRISPR/Cas12a assays developed in this study are rapid, specific and sensitive, making them suitable for the on-site detection of C. burnetii at primary level.},
}
@article {pmid42673729,
year = {2026},
author = {Jones, BM and Xu, M and Zou, J and Liu, C and Zou, Y and Pathak, P and Lomash, RM and Stan, R and Bönnemann, CG and Chen, CZ},
title = {Establishment of induced pluripotent stem cell line TRNDi045-A-38 carrying homozygous DOK7-related Congenital Myasthenia patient-mutation knock-in variant from parental KOLF2.1J.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104090},
doi = {10.1016/j.scr.2026.104090},
pmid = {42673729},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Myasthenic Syndromes, Congenital/genetics/pathology ; *Muscle Proteins/genetics/metabolism ; Cell Line ; Homozygote ; *Mutation ; Gene Knock-In Techniques ; Female ; CRISPR-Cas Systems ; },
abstract = {DOK7-related Congenital Myasthenic Syndrome (CMS) is a rare genetic neuromuscular junction disorder. This is one of the most common of the recessive forms of CMS, often presenting with more static proximal weakness (hence also referred to as limb girdle CMS). Whole-genome sequencing of affected patients implicates frameshift duplication mutations in DOK7 as drivers of impaired neuromuscular-junction signaling. In this study, we generated a human induced pluripotent stem cell (hiPSC) line TRNDi045-A-38 from the KOLF2.1J reference line, engineered to carry homozygous DOK7 c.1124_1127dupTGCC mutation knock-in using CRISPR/Cas9. This iPSC line could be used for in vitro disease modeling to study disease pathophysiology and for therapeutic development.},
}
@article {pmid42696150,
year = {2026},
author = {Sharma, NN and Kumari, A and Vashisht, I and Sharma, MK},
title = {Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.},
journal = {Plant cell reports},
volume = {45},
number = {9},
pages = {},
pmid = {42696150},
issn = {1432-203X},
support = {DST/INSPIRE Fellowship/2021/IF210110//Department of Science and Technology, Ministry of Science and Technology, India/ ; CRG/2023/007038//Anusandhan National Research Foundation,India/ ; ANRF/PAIR/2025/000029/PAIR//JNU-PAIR Network of Anusandhan National Research Foundation, India/ ; },
mesh = {*Gene Editing/methods ; *Transcription Factors/genetics/metabolism ; *Crops, Agricultural/genetics ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics/metabolism ; Genome, Plant/genetics ; },
abstract = {The development of CRISPR multiplex genome-editing (MGE) tools is rapidly transforming plant functional genomics and accelerating crop improvements. By simultaneously targeting two or more DNA loci, it allows scientists to precisely edit multiple genes at the single-nucleotide level, within the target genome. Simultaneous manipulation of multiple targets has revolutionized the functional elucidation studies, particularly the dissection of complex genetic pathways. Due to its superior precision and feasibility, CRISPR-MGE is widely accepted and has largely replaced alternative editing tools such as TALENs and ZFNs. Several CRISPR-MGE strategies, including the use of individual expression cassettes, tRNA-processing enzymes, Csy4 or ribozymes, have been successfully deployed in plants. Recent advancements, such as Cpf1, transgene-free methods, or ultra-multiplexing approaches, have further refined the technology into a powerful, efficient, and robust toolkit. MGE enables complex genome editing, including multiple-gene knockouts, base alterations, transcriptional regulation, metabolic engineering, or their combinations. Consequently, it is ideal for elucidating the function of transcription factors that are key molecular players in regulating diverse plant responses, especially in stress pathways. Several stress-responsive TFs have been functionally characterized via CRISPR-MGE, and more advanced tools are being employed. This review evaluates multiplexing tools, their diverse applications, and the current progress toward developing advanced MGE tools. Ultimately, we provide evidence to encourage the use of advanced MGE tools for functional characterization studies of stress-responsive TFs, thereby highlighting their potential to accelerate crop improvement.},
}
@article {pmid42697642,
year = {2026},
author = {Ye, WN and Xing, Q and Chen, RY and Zhang, YC and Wu, SF and Gao, CF},
title = {Field resistance monitoring and nAChR α6 target validation support spinetoram as a candidate insecticide for rotation in Nilaparvata lugens resistance management.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107264},
doi = {10.1016/j.pestbp.2026.107264},
pmid = {42697642},
issn = {1095-9939},
mesh = {Animals ; *Receptors, Nicotinic/genetics/metabolism ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; *Hemiptera/drug effects/genetics ; *Macrolides/pharmacology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Drug Combinations ; },
abstract = {The brown planthopper (BPH), Nilaparvata lugens (Stål), is a major pest threatening rice production in Asia. Severe resistance to commonly used insecticides has become a major challenge for its effective control, highlighting the need for alternative insecticides. In this study, we evaluated the susceptibility of field populations of N. lugens to spinetoram and functionally validated the nicotinic acetylcholine receptor (nAChR) α6 subunit, Nlα6, as a molecular determinant of spinosyn susceptibility. Resistance monitoring showed that field populations remained highly susceptible to spinetoram despite exhibiting moderate to high resistance to several commonly used insecticides. A homozygous Nlα6 knockout strain generated using CRISPR/Cas9 developed high-level resistance to spinosad and spinetoram, with resistance ratios of 554.1 and 357.1, respectively, but did not show broad cross-resistance to nine other nAChR-targeting insecticides. Co-segregation analysis further confirmed tight linkage between Nlα6 loss of function and spinosyn resistance. Inheritance analysis showed that spinetoram resistance mediated by Nlα6 knockout was autosomal and completely recessive. Transcriptome analysis revealed that Nlα6 deficiency was associated with reduced expression of immune defense genes, enrichment of functions related to axonemes and neural processes, and coordinated activation of energy metabolism pathways, suggesting that Nlα6 disruption may alter neural homeostasis and energy demand. These results demonstrate that Nlα6 is a key subunit mediating spinosyn susceptibility in N. lugens and support spinetoram as a candidate rotation insecticide for resistance management.},
}
@article {pmid42697651,
year = {2026},
author = {Zhou, J and Zang, Z and Gao, H and Sun, J and Xu, L and Gao, Q and He, H and Ding, W and Qiu, L and Li, Y},
title = {CRISPR/Cas9-mediated knockout of CsRDL1 decreases the susceptibility of Chilo suppressalis to cyproflanilide.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107282},
doi = {10.1016/j.pestbp.2026.107282},
pmid = {42697651},
issn = {1095-9939},
mesh = {Animals ; *CRISPR-Cas Systems ; *Insecticides/pharmacology ; Gene Knockout Techniques ; Insecticide Resistance/genetics ; *Insect Proteins/genetics/metabolism ; *Moths/drug effects/genetics ; Ivermectin/analogs &amp; derivatives ; Female ; *Anilides/pharmacology ; *Receptors, GABA/genetics ; Larva/drug effects ; },
abstract = {The novel meta-diamide insecticide cyproflanilide represents a promising tool for pest control, yet its precise molecular target remains uncharacterized. The RDL1 encodes a subunit of insect ionotropic GABA receptors (GABAr), which is targeted by meta-diamide pesticides. This study investigated the functional role of the resistance to dieldrin 1 (RDL1) subunit in Chilo suppressalis, a major rice pest, by generating a homozygous CsRDL1 knockout strain (CsRDL1[-/-], lacking 101 bp) via CRISPR/Cas9-mediated gene editing. Bioassay results showed that the CsRDL1[-/-] strain exhibited a 2.67-fold increase in tolerance to cyproflanilide and a 3.08-fold increase to broflanilide, whereas its susceptibility to abamectin and emamectin benzoate, which also act on the CsRDL1 subunit, remained unchanged. These results indicate that knockout of CsRDL1 decreased susceptibility to cyproflanilide and broflanilide, and that the mode of action of these meta-diamides differs from that of abamectin and emamectin benzoate. Meanwhile, the CsRDL1[-/-] strain exhibited significant changes in key biological traits: 3rd-6th instar larval duration shortened by 9.45%, fecundity (eggs per female) reduced by 29.49%, and both pupal duration and female adult longevity were significantly shortened relative to the wild-type population. These findings provide in vivo evidence that CsRDL1 contributes to cyproflanilide susceptibility and plays important roles in development and reproduction in C. suppressalis. This study lays a foundation for further mechanistic studies on the interaction between cyproflanilide and CsRDL1 subunit, and offers insights for the development of insecticides targeting this subunit.},
}
@article {pmid42697663,
year = {2026},
author = {Pang, X and Zhang, Z and Qin, D and Bachler, A and Yang, Y and Wu, Y},
title = {Knockout of SfVipR2 confers Vip3Aa resistance in Spodoptera frugiperda with evidence of genetic complementation among non-allelic mutations of SfVipR1, SfVipR2, and SfCHS2.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107294},
doi = {10.1016/j.pestbp.2026.107294},
pmid = {42697663},
issn = {1095-9939},
mesh = {Animals ; *Spodoptera/genetics/drug effects ; *Bacterial Proteins/genetics ; Gene Knockout Techniques ; Mutation ; Genetic Complementation Test ; *Insect Proteins/genetics ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; },
abstract = {The Vip3Aa protein from Bacillus thuringiensis is widely deployed in transgenic crops to control lepidopteran pests, including the fall armyworm Spodoptera frugiperda, a highly invasive, polyphagous pest of global importance. Loss-of-function mutations in SfVipR1 (a thyroglobulin-like gene) and SfCHS2 (chitin synthase 2) have previously been confirmed to confer high-level, recessive resistance to Vip3Aa in S. frugiperda. A second thyroglobulin-like gene (HaVipR2) has recently been recognized as an important determinant of Vip3Aa resistance in Helicoverpa armigera. In this study, we first confirmed that knockout of SfVipR2 using CRISPR/Cas9 gene editing also confers high-level, recessive resistance to Vip3Aa. Then, three isogenic knockout strains (SfVipR1-KO established previously; and SfVipR2-KO and SfCHS2-KO, newly generated; all in the YJ-19 genetic background) were used to investigate functional relationships among SfVipR1, SfVipR2, and SfCHS2. Reciprocal crosses between any pair of these resistant strains yielded fully susceptible hybrid progeny, demonstrating complete genetic complementation and no epistasis among the non-allelic mutations. Disruption of any of these genes can result in high-level resistance to Vip3Aa, indicating a substantial resistance risk in S. frugiperda and underscoring the need for resistance management tactics such as establishing an adequate proportion of non-Bt host refuges and deploying pyramided crops expressing Vip proteins and Bt proteins with a different mode of action than Vip3Aa (e.g., Cry proteins).},
}
@article {pmid42697672,
year = {2026},
author = {Kang, R and Qian, Y and Zuo, Y and Li, R and Zhang, L},
title = {Development of an RPA-CRISPR/Cas12a-based rapid visual detection method for two insecticide resistance-associated mutations in Aphis gossypii: A302S in the ace1 gene and M918L in the para gene.},
journal = {Pesticide biochemistry and physiology},
volume = {223},
number = {},
pages = {107303},
doi = {10.1016/j.pestbp.2026.107303},
pmid = {42697672},
issn = {1095-9939},
mesh = {Animals ; *Insecticide Resistance/genetics ; Mutation ; *CRISPR-Cas Systems ; Insecticides/pharmacology ; *Insect Proteins/genetics ; },
abstract = {The resistance of Aphis gossypii to pyrethroids and organophosphate/carbamate insecticides has become increasingly severe. The para M918L and ace1 A302S target-site mutations are the key mechanisms conferring resistance to these two types of insecticides, respectively. However, rapid and field-deployable methods for detecting these mutations are still lacking. In this study, the genotype and allele frequencies of eight field populations collected in 2023 were investigated, and a rapid visual detection method based on RPA-CRISPR/Cas12a was developed. By optimizing the concentration of the reaction components, specific recognition of both mutations was achieved. The optimized assay could be completed within 1 h under a single-temperature condition of 37 °C, with fluorescence signals directly visualized using a portable 440-460 nm blue-light illuminator without PCR thermocyclers, fluorescence readers or sequencing platforms. Validation using the tested field samples showed complete concordance with Sanger sequencing. In addition, Sanger sequencing further showed that no homozygous susceptible genotype was detected at the para M918 locus across all populations, whereas the 302S allele frequency at the ace1 A302S locus exhibited significant geographic variation, ranging from 0% to 100%. The RPA-CRISPR-based visual detection method developed in this study is rapid, accurate, and does not require expensive instrumentation. Together with previously developed neonicotinoid resistance detection techniques, it forms a comprehensive monitoring system for multiple resistance in A. gossypii, providing a practical tool for rapid field diagnosis and more informed insecticide selection.},
}
@article {pmid42699674,
year = {2026},
author = {Mohanty, A and Sahoo, RK and Sanket, AS and Rout, E},
title = {Combating New Delhi metallo-beta-lactamase-1 (NDM-1): an integrated review of inhibitor development and next-generation therapeutic platforms.},
journal = {RSC advances},
volume = {},
number = {},
pages = {},
pmid = {42699674},
issn = {2046-2069},
abstract = {New Delhi metallo-β-lactamase-1 (NDM-1), which has emerged globally, exhibits resistance to almost all β-lactam antibiotics, including carbapenems, posing a major challenge to modern antibiotic therapy. Deep sequencing has revealed the evolution of several new NDM variants (some of the variants are more thermostable than NDM-1) due to mutations that increase their zinc-binding and catalytic efficiency and establish a selective pressure on the enzymes. In addition to enzymatic breakdown, NDM-mediated resistance is exorbitantly exhibited via porin loss, efflux pump activity, biofilm formation, and outer membrane vesicle-mediated dissemination. Despite the urgent need for effective countermeasures, no clinically approved inhibitor of NDM-1 has been developed. This review explores the structural and functional characteristics of NDM-1, together with the epidemiology of bla NDM-1-harbouring bacterial pathogens. It further elaborates the catalytic basis of the broad-spectrum β-lactam hydrolysis mechanism, attributed to the presence of dinuclear Zn(ii); the conserved αβ/βα metallo-β-lactamase fold, and flexible active-site loops that govern substrate recognition are also highlighted. In addition, this review provides details on the known inhibitor classes, including thiol-based compounds, hydroxamates, bicyclic boronates, repurposed drugs, and natural products, and discusses the challenges related to pharmacokinetics, toxicity, and variant-specific effectiveness. Other emerging approaches, such as CRISPR-Cas systems for targeted gene editing, nanoparticle-based delivery platforms, and artificial intelligence-driven drug discovery, are also explored as promising options for future therapy development.},
}
@article {pmid42364585,
year = {2027},
author = {Wang, T and Wen, Y and Ma, H and Wang, Y and Liao, K and Xue, F},
title = {RAA-CRISPR/Cas12a-mediated SERS magnetic biosensor combined with a portable Raman spectrometer for rapid and ultrasensitive detection of Lumpy skin disease virus.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130222},
doi = {10.1016/j.talanta.2026.130222},
pmid = {42364585},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; Animals ; *CRISPR-Cas Systems ; *Lumpy skin disease virus/isolation &amp; purification/genetics ; Cattle ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/genetics ; Rapid Diagnostic Tests ; Recombinases/metabolism ; Gold/chemistry ; },
abstract = {Lumpy skin disease virus (LSDV) poses a severe threat to global cattle farming, causing significant economic losses. Current laboratory-based methods such as qPCR are accurate procedures; however, they are time-consuming and require sophisticated instrumentation, thus hindering onsite outbreak control. We report a novel magnetic biosensor integrating recombinase-aided amplification (RAA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a, and surface-enhanced Raman scattering (SERS) for ultrasensitive and portable detection of LSDV. The biosensor operates through a synergistic cascade: (i) RAA isothermally amplifies the viral DNA target; (ii) the amplified product activates the trans-cleavage activity of CRISPR/Cas12a, thus generating numerous short ssDNA fragments; and (iii) these fragments cleave linker DNA, thereby releasing AuNS@DTNB SERS nanoprobes from the magnetic bead surface and enabling signal readout via a handheld Raman spectrometer. This triple-amplification strategy yielded an ultrahigh sensitivity at 1.2 copies/μL, with a total assay time of only 90 min. The platform exhibited exceptional specificity, discriminating LSDV from genetically homologous Capripoxviruses (up to 97% similarity). In blind tests on 30 beef samples spiked with LSDV nucleic acid, it achieved 100% concordance with qPCR results, thus demonstrating excellent robustness. By integrating a handheld Raman spectrometer and a portable thermostat, this platform delivers laboratory-grade performance in a field-deployable format. This biosensor represents a powerful new tool for onsite LSDV surveillance; given its modular design, it can be readily adapted for detecting other emerging nucleic acid targets.},
}
@article {pmid42379036,
year = {2027},
author = {Chen, Y and Liu, S and Yuan, Z and He, X and Zhu, X and Wang, G and Wang, X},
title = {Dual-engine amplification: Integrating catalytic hairpin assembly with CRISPR-Cas12a for ultrasensitive point-of-care testing of Chikungunya virus RNA.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130191},
doi = {10.1016/j.talanta.2026.130191},
pmid = {42379036},
issn = {1873-3573},
mesh = {*RNA, Viral/genetics/analysis ; *Chikungunya virus/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *Chikungunya Fever/diagnosis/virology ; Rapid Diagnostic Tests ; Limit of Detection ; Sensitivity and Specificity ; },
abstract = {Chikungunya virus (CHIKV) has emerged as a globally important arthropod-borne pathogen with rapid geographical expansion, urging high-performance yet simple diagnostic tools. Herein, we develop an isothermal dual-engine amplification biosensor by integrating catalytic hairpin assembly (CHA) with CRISPR-Cas12a for ultrasensitive and rapid detection of CHIKV RNA. This strategy combines the enzyme-free isothermal amplification of CHA and the high-specificity trans-cleavage activity of CRISPR-Cas12a to realize cascade signal enhancement. By systematically optimizing reaction parameters including probe ratio, temperature, buffer, and incubation time, we achieve superior analytical performance. The biosensor exhibits a low limit of detection of 4 fM, a wide linear range from 10 fM to 1 μM (R[2] > 0.98), and excellent specificity against dengue virus, Zika virus, and other common blood-borne viruses. It also shows strong resistance to interference from hemolysis, icterus, and lipemia. The whole assay can be finished within 60 min under isothermal conditions without sophisticated thermal cyclers. In clinical serum samples, this method achieves 97.5% concordance with RT-qPCR, with a sensitivity of 96.8% and a specificity of 100%. This CHA-CRISPR-Cas12a dual-amplification platform provides a sensitive, specific, rapid, and cost-effective approach for CHIKV detection and holds great promise for point-of-care testing in resource-limited settings.},
}
@article {pmid42402225,
year = {2027},
author = {Jia, X and Wang, J},
title = {Aptamer-CRISPR Glucose Transducer for point-of-care IgE detection.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130221},
doi = {10.1016/j.talanta.2026.130221},
pmid = {42402225},
issn = {1873-3573},
mesh = {*Immunoglobulin E/analysis/blood ; Humans ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Glucose/analysis ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Limit of Detection ; Rapid Diagnostic Tests ; },
abstract = {Immunoglobulin E (IgE) is a critical biomarker for the diagnosis and therapeutic monitoring of allergic diseases. Conventional IgE detection methods (e.g., ELISA, ImmunoCAP) generally offer satisfactory sensitivity but require hours to days, rely on specialized equipment and trained personnel, and are unsuitable for point-of-care or home-based testing. Here, we developed a portable point-of-care testing platform termed Aptamer-CRISPR Glucose Transducer (ACGT), based on aptamer competition and CRISPR-Cas12a trans-cleavage. The platform employs the D17.4 aptamer as the molecular recognition element. In the presence of target IgE, the aptamer specifically binds IgE, triggering a strand displacement reaction that releases the blocker as ssDNA. This released blocker (now serving as the trigger) activates the trans-cleavage of Cas12a, which cleaves ssDNA linkers on magnetic beads, thereby releasing invertase into the supernatant. The released invertase hydrolyzes sucrose into glucose, which is quantitatively measured using a personal glucose meter. The method achieves a detection limit as low as 76.5 kU/L, with a total assay time of approximately 80 min. By integrating the signal amplification capability of CRISPR with the accessibility of glucose-based transduction, the ACGT platform provides a sensitive, cost-effective, and user-friendly solution for allergen diagnosis and biomarker monitoring in decentralized settings, offering great potential for primary care and home-based testing.},
}
@article {pmid42420522,
year = {2026},
author = {Price, D and Zemlyanskiy, G and Trakarnphornsombat, W and Forne, I and Volkova, N and Dubusse, L and Cooper, AJ and Imhof, A and Radzisheuskaya, A},
title = {Identifying critical lysines in mammalian histone H3 with high-throughput CRISPR prime editing.},
journal = {Nature genetics},
volume = {58},
number = {9},
pages = {2303-2319},
pmid = {42420522},
issn = {1546-1718},
support = {UKRI698//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; EP/Y000331/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; RG\R1\241175//Royal Society/ ; CRC1309/325871075 and SPP2191/419067076//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Histones/genetics/metabolism/chemistry ; Animals ; *Lysine/genetics/metabolism ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Protein Processing, Post-Translational/genetics ; Mouse Embryonic Stem Cells/metabolism ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; Genomic Instability ; Saccharomyces cerevisiae/genetics ; },
abstract = {Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.},
}
@article {pmid42664856,
year = {2026},
author = {Huang, J and Zhang, Q and Yu, FW and Liu, MQ and Jing, HK and Wang, SY and Wang, HY and Li, JQ and Zheng, L and Shao, H and He, XL and Shen, RF and Zhu, XF},
title = {Disruption of OsGONST3 reduces grain cadmium accumulation without yield penalty in field-grown rice.},
journal = {Journal of hazardous materials},
volume = {516},
number = {},
pages = {143397},
doi = {10.1016/j.jhazmat.2026.143397},
pmid = {42664856},
issn = {1873-3336},
mesh = {*Oryza/metabolism/genetics/growth &amp; development ; *Cadmium/metabolism ; *Plant Proteins/genetics/metabolism ; *Soil Pollutants/metabolism ; Plants, Genetically Modified ; Plant Roots/metabolism ; CRISPR-Cas Systems ; },
abstract = {Cadmium (Cd) pollution in paddy soils threatens global food safety. Identifying molecular gateways controlling grain Cd accumulation is essential for breeding low-Cd rice. A field-based screen of a large-scale CRISPR/Cas9 mutant library identified OsGONST3, a Golgi nucleotide sugar transporter family member localized to the plasma membrane. After 1 μM Cd treatment for 7 d, OsGONST3 transcript abundance increased approximately 3.4-fold in root and 5.7-fold in shoot, accompanied by increased OsGONST3-GFP protein accumulation. Heterologous OsGONST3 expression increased yeast Cd content by approximately 23% relative to the empty-vector control. Under hydroponic exposure to 1 μM Cd for 7 d, two independent osgonst3 mutants contained 22-25% less Cd in root, approximately 13% less in shoot, and 16-21% less in xylem sap than Nipponbare. Conversely, OsGONST3-overexpressing lines contained 40-42%, 38-40%, and 28-37% more Cd in root, shoot, and xylem sap, respectively. Under tested field condition, OsGONST3 disruption reduced grain Cd by approximately 25-26% without a significant yield penalty and was associated with secondary transcriptional adjustments in Cd-homeostasis genes. These findings identify OsGONST3 as a plasma membrane-localized Cd influx-associated protein and a promising target for breeding low-Cd rice.},
}
@article {pmid42690973,
year = {2026},
author = {Qin, Y and Zhang, G and Hu, Y},
title = {CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01856},
pmid = {42690973},
issn = {2379-3694},
support = {32401261//National Natural Science Foundation of China/ ; },
abstract = {Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.},
}
@article {pmid42692561,
year = {2026},
author = {Zheng, Z and Xu, D},
title = {CRISPR-Cas9 screen to identify genes regulating cell death.},
journal = {Methods in cell biology},
volume = {210},
number = {},
pages = {83-96},
doi = {10.1016/bs.mcb.2026.05.010},
pmid = {42692561},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Cell Death/genetics ; Animals ; *Apoptosis/genetics ; },
abstract = {Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Understanding the mechanisms of cell death is crucial for elucidating the control of cellular homeostasis and developing therapies for related diseases. Despite extensive research efforts spanning decades, many aspects of cell death mechanisms remain elusive, highlighting the need for continued exploration. Here, we describe how to identify novel regulators involved in cell death pathways using a genome-wide screening approach.},
}
@article {pmid42692894,
year = {2026},
author = {Gaizauskaite, U and Songailiene, I and Sasnauskas, G and Siksnys, V},
title = {Building CRISPR immunity: evolution and mechanisms of spacer acquisition.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2026.08.004},
pmid = {42692894},
issn = {0968-0004},
abstract = {CRISPR-Cas systems in prokaryotes serve as adaptive immune systems that neutralize phage infections through RNA-guided nucleases. Immunization is achieved during the adaptation stage through Cas1-Cas2 integrase-mediated insertion of short foreign DNA snippets, termed spacers, into a CRISPR array in the host genome. This review examines the evolutionary origins of Cas1-Cas2 and the mechanisms of spacer acquisition in DNA-targeting CRISPR-Cas systems. Particular emphasis is placed on the recently characterized effector-assisted adaptation pathways, in which CRISPR effector proteins, such as Cascade and Cas9, typically involved in target interference, are repurposed for prespacer capture and integration into a CRISPR array.},
}
@article {pmid42694431,
year = {2026},
author = {Putignani, L and Marsiglia, R and Turco, L and Russo, A and Pane, S and Fusco, A and Lopetuso, L and Trecarichi, EM},
title = {The gut reservoir of carbapenem-resistant Enterobacterales: from dysbiosis and colonization to infection and decolonization, with a focus on patients with hematologic malignancies - a narrative review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1939690},
pmid = {42694431},
issn = {2235-2988},
mesh = {Humans ; *Hematologic Neoplasms/complications/microbiology ; *Dysbiosis/microbiology ; *Enterobacteriaceae Infections/microbiology/therapy ; *Carbapenem-Resistant Enterobacteriaceae/drug effects ; *Gastrointestinal Microbiome ; Fecal Microbiota Transplantation ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Probiotics ; },
abstract = {Carbapenem-resistant Enterobacterales (CRE) remain among the highest-priority antimicrobial-resistant pathogens worldwide, and intestinal colonization is increasingly recognized as the key precursor of invasive infections, particularly in patients with hematological malignancies. Increasing evidence indicates that disruption of the gut microbial ecosystem, reflected in reduced diversity, depletion of beneficial anaerobic taxa, intestinal barrier dysfunction, immune dysregulation, and expansion of Enterobacterales, plays a central role in the transition from colonization to infection. Consequently, restoring colonization resistance through microbiome-targeted interventions has emerged as a promising preventive strategy. This narrative review summarizes the current evidence on the epidemiology and clinical impact of CRE colonization and infection, with particular emphasis on the ecological alterations of the gut microbiome linking gut dysbiosis to epithelial barrier dysfunction, immune dysregulation, and loss of colonization resistance to CRE persistence and invasive infection. We critically discuss both conventional and emerging decolonization approaches, including selective digestive decontamination, probiotics, prebiotics and synbiotics, fecal microbiota transplantation (FMT), bacteriophage therapy, and CRISPR-Cas-based technologies, highlighting their mechanisms of action, available clinical evidence, and current limitations. Particular attention is given to patients with hematological malignancies, in whom the clinical need for effective decolonization strategies is greatest. Although FMT currently represents the most promising microbiome-based intervention, the available evidence remains heterogeneous and largely derived from small studies. Overall, durable and standardized decolonization strategies have yet to be established, underscoring the need for well-designed multicenter randomized clinical trials to define effective microbiome-directed approaches for preventing CRE-related infections in high-risk populations.},
}
@article {pmid42694879,
year = {2026},
author = {Huang, CW and Chen, HH and Jao, TM and Li, CJ and Sung, JM and Tsai, YS and Chen, JS},
title = {CRISPR/Cas9-Driven Fndc5 Knockout Reveals Augmented Mitochondrial Structural and Dynamic Alterations in Diabetic Nephropathy.},
journal = {International journal of medical sciences},
volume = {23},
number = {9},
pages = {2884-2898},
pmid = {42694879},
issn = {1449-1907},
mesh = {Animals ; Mice ; *Diabetic Nephropathies/pathology/genetics ; *Mitochondria/pathology/metabolism/genetics/ultrastructure ; Mice, Knockout ; *Diabetes Mellitus, Experimental/pathology/genetics/chemically induced/complications ; *Fibronectins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Male ; Diet, High-Fat/adverse effects ; Mitochondrial Dynamics/genetics ; Kidney/pathology ; Mice, Inbred C57BL ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism ; Oxidative Stress/genetics ; Humans ; Streptozocin/toxicity ; Lipid Peroxidation/genetics ; },
abstract = {FNDC5 has been implicated in glucose homeostasis and is associated with mitochondrial function. Its role in diabetes and diabetic nephropathy (DN) remains unclear. This study hypothesizes that FNDC5 deficiency predisposes the kidney to accelerated mitochondrial dysfunction in diabetes and DN. Systemic Fndc5 knockout (KO) C57BL/6 mice were generated using CRISPR/Cas9. DN were induced in six-week-old Fndc5 wild-type (WT) and KO mice using high-fat diet combined with streptozotocin injection. Weekly blood and urine analyses assessed glucose, cholesterol, triglycerides, blood urea nitrogen, creatinine, and proteinuria. At 15 weeks, kidneys and metabolic tissues including pancreas, muscle and adipose were collected for histological and molecular analyses. Results showed that while both Fndc5 WT and KO mice were successfully induced with hyperglycemia, the Fndc5 KO DN group exhibited a slightly lower cumulative glycemic burden compared with the WT DN group. Despite this milder metabolic stress, proteinuria remained comparable between the two groups. Furthermore, histological analysis revealed that Fndc5 KO DN mice displayed more severe mesangial expansion, glomerular basement membrane thickening, and podocyte effacement compared with WT DN mice. The elevated lipid peroxidation, reduced PGC-1α expression, and increased DNA fragmentation were also evident in Fndc5 KO DN. More swollen mitochondria with a significantly higher percentage of disrupted cristae were observed in Fndc5 KO DN mice compared with WT DN mice. This was accompanied by the upregulation of mitochondrial fission-related genes (Dnm1l and Fis1), downregulation of the fusion-related gene (Mfn1), and reduced expression of ATP synthase subunits (ATP5A1 and ATP5B). Systemic analysis of other metabolic tissues, including the pancreas and muscle and adipose tissues, revealed increased lipid peroxidation and decreased PGC-1α expression. These findings underscore FNDC5's role in maintaining mitochondrial integrity and cellular health under diabetic conditions, positioning FNDC5 as a potential therapeutic target.},
}
@article {pmid42695329,
year = {2026},
author = {Chowdhury, S and Nayak, SP and Pattanayak, R and Sardar, S and Majhi, A and Yadav, B and Dasari, A and Mandlik, R and Sonah, H and Deshmukh, R and Gupta, I and Bauer, P and Ram, H},
title = {Establishment of a CRISPR-Cas9 Library for Indica Rice and Identification of OsOPR5 (LOC_Os06g11210) as a Regulator of Root Architecture.},
journal = {Physiologia plantarum},
volume = {178},
number = {5},
pages = {e71097},
pmid = {42695329},
issn = {1399-3054},
support = {SRGJ2021/001495//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; BT/PR53626/BSA/33/96/2024//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; BT/PR56697/AMRIT/165/21/2025//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; //BRIC-National Institute of Plant Genome Research/ ; //Alexander von Humboldt Foundation/ ; },
mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Roots/genetics/anatomy &amp; histology/growth &amp; development/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Gene Library ; Gene Expression Regulation, Plant ; Mutation ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Gene Editing ; },
abstract = {Functional characterization of a large number of rice genes remains a major challenge despite the availability of genome sequences and large-scale transcriptomic datasets. CRISPR-Cas9 library is a powerful approach for high-throughput targeted mutagenesis; however, its application in indica rice cultivars remains limited due to low transformation and regeneration efficiencies. In this study, we developed a CRISPR-Cas9 library targeting 12,000 rice genes and evaluated its utility for functional genomics in the indica cultivar MTU-1010. Sanger sequencing and NGS analysis of the plasmid library revealed high sgRNA coverage and more than 80% accuracy. Transformation of the developed library into the indica cultivar MTU-1010 resulted in a high target editing efficiency, with 90% of analyzed transgenic plants carrying mutations at the intended target site. Functional analysis of one homozygous mutant identified a previously uncharacterized role for OsOPR5 (LOC_Os06g11210), a member of the 12-oxophytodienoate reductase family in root architecture. The opr5 mutants exhibited significant reductions in lateral root number, seminal and crown root number, and root length, demonstrating that OsOPR5 positively regulates root system architecture in rice. Notably, endogenous jasmonic acid (JA) and JA-isoleucine levels were not significantly altered in the mutant, suggesting potential functional specialization or redundancy among rice OPR family members for JA accumulation. The root system architecture is a key determinant of water and nutrient acquisition; our results suggest that OsOPR5 may play an important role in adaptation under adverse environmental conditions. Collectively, this study establishes an efficient genome-editing platform for indica rice and identifies OsOPR5 as a novel regulator of root development.},
}
@article {pmid42695976,
year = {2026},
author = {de Kreek, F and Hertzberger, R and van Eeden, F and Illidge, S and Teunis, EJ and Hanemaaijer, M and Lievens, E and Rienstra, F and Wiedhaup, DE and Lisotto, P and Butler, D and Molenaar, D and Kort, R},
title = {Intra-individual genetic diversity of vaginal Lactobacillus crispatus revealed through citizen science-driven isolation and pangenome analysis.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag218},
pmid = {42695976},
issn = {1365-2672},
abstract = {AIMS: A vaginal microbiome dominated by Lactobacillus crispatus is associated with positive reproductive and sexual health outcomes, yet intra-individual genetic diversity within this species remains largely unexplored. This study characterised inter- and intra-individual genomic variation in L. crispatus strains isolated through a citizen science initiative and assessed implications for multi-strain probiotic development.<br><br>METHODS AND RESULTS: Fifty-three women participated in this citizen science project. Self-sampling resulted in 48 shotgun metagenomes. Twenty-two participants isolated their own L. crispatus strains using selective enrichment and LAMP-based species confirmation, resulting in 53 whole-genome-sequenced isolates. L. crispatus dominated 20 of 48 metagenomes (50.1-99.6% relative abundance). Pangenome analysis revealed 3&#x2009;456 gene families, of which 43.7% were core and 56.3% accessory. A 14-kb plasmid harbouring a Fic-domain toxin-antitoxin protein, but devoid of antimicrobial resistance genes, was present in 44 of 53 strains. Strains from the same individual clustered closely together yet harboured 1-123 gene differences. Intra-individual variation was observed in the pullulanase type I gene required for glycogen degradation: 40 strains were predicted to grow on glycogen, six showed genetic disruptions with unknown consequences, and seven were predicted to lack this ability entirely. Variation within individuals was also found for bacteriocin classes and CRISPR-Cas genes.<br><br>CONCLUSIONS: Substantial functional diversity exists within L. crispatus, even among strains from the same individual, supporting the rationale for multi-strain vaginal probiotics. This citizen science approach enabled discovery of host-specific adaptations while ensuring participant ownership of their strains.},
}
@article {pmid42696079,
year = {2026},
author = {Mitra, S and Damini, and Devi, M and Niyogi, SG and Singh, UP and Lo, D and Bishtania, HC and Thakur, S},
title = {Unlocking the potential of bacteriophage-based therapeutic gene delivery in hepatocellular carcinoma.},
journal = {Journal of the Egyptian National Cancer Institute},
volume = {38},
number = {1},
pages = {},
pmid = {42696079},
issn = {2589-0409},
mesh = {Humans ; *Liver Neoplasms/therapy/genetics ; *Carcinoma, Hepatocellular/therapy/genetics ; *Bacteriophages/genetics ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; Animals ; CRISPR-Cas Systems ; Genetic Vectors/genetics/administration &amp; dosage ; Gene Therapy Agents ; },
abstract = {Liver cancer, mainly hepatocellular carcinoma (HCC), remains a global health burden marked by poor prognosis with limited therapeutic efficacy, and high recurrence rates. HCC remains one of the most lethal malignancies worldwide, with limited therapeutic options and high resistance to conventional treatments. Despite low therapeutic efficacy, molecular heterogeneity, treatment resistance and high recurrence rate, hepatocellular carcinoma (HCC) is still a significant health problem worldwide. These restrictions have stimulated the research of focused methods for delivering therapeutic genetic payload into cancer cells. Bacteriophages have been gaining growing attention as an emerging delivery platform due to their genetic versatility, ease of engineering, ability to be surface modified and payload targeted. In this narrative review, the therapeutic potential of engineered bacteriophages in the context of HCC therapy is critically analyzed focusing on phage display-mediated tumor targeting, phage-mediated intracellular gene delivery, TRAIL gene delivery, and CRISPR/Cas-based therapeutic strategies. It has been previously noted in the literature that phage display can be used to attach tumor-targeting ligands to the surface of a phage, which may aid in the recognition of receptors at the tumor site and promote targeted delivery to the receptor. Therapeutic application is stunted by inefficient trafficking to the cytosol, endosomal degradation, immune recognition and clearance, vector stability, manufacturing scalability and regulatory issues. In conclusion, engineered bacteriophages are a promising and versatile tool for targeted gene delivery in HCC but more mechanistic, preclinical and translational research is needed to prove their therapeutic effectiveness and clinical usefulness for this purpose.},
}
@article {pmid40923633,
year = {2026},
author = {Ramakrishnan, M and Kaul, R and Sharma, A and Ahmad, Z and Vijayakanth, V and Keerthana, K and Gao, Z and Zhou, M and Wei, Q},
title = {CRISPR RNP-Mediated Transgene-Free Genome Editing in Plants: Advances, Challenges and Future Directions for Tree Species.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {10},
pages = {7636-7656},
doi = {10.1111/pce.70176},
pmid = {40923633},
issn = {1365-3040},
support = {//The preparation of this review was supported by grants from the National Natural Science Foundation of China (32471977 and 32071848); a grant from the Natural Science Foundation of Jiangsu Province (BK20231289); the Natural Science Foundation for Distinguished Young Scholars of Nanjing Forestry University (JC2019004); the Project for Groundbreaking Achievements of Nanjing Forestry University (202211); and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors are also grateful for the Young Foreign Talent Program (Y20240114) and the support of Metasequoia Faculty Research Start-up Funding (163100028 and 163100036) at the Bamboo Research Institute, Nanjing Forestry University./ ; },
mesh = {*Gene Editing/methods ; *Ribonucleoproteins/genetics ; *Trees/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Transgenes ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR ribonucleoprotein (RNP)-mediated genome editing offers a transgene-free platform for precise genetic modification in diverse herbaceous and tree species, including rice, wheat, apple, poplar, oil palm, rubber tree and grapevine. However, its application in woody plants faces distinct challenges, notably inefficient delivery and regeneration difficulties, particularly in species such as bamboo. While some of these issues also occur in herbaceous plants, they are often significantly more complex in woody species due to factors such as intricate cell wall architecture, widespread recalcitrant genotypes and inherent limitations of current delivery platforms. This review presents the first in-depth, critical re-evaluation of recent advancements in RNP-mediated editing in woody plants, highlighting these obstacles that warrant focused attention. Unlike plasmid-based CRISPR systems, RNP editing utilises Cas9/Cas12a protein-guide RNA complexes without integrating foreign DNA. This enables a DNA-free editing strategy that simplifies regulatory approval and minimises off-target effects due to the transient presence and rapid degradation of RNPs within plant cells. While PEG-mediated protoplast transfection and particle bombardment remain the primary reported methods for RNP delivery in trees, we evaluate promising alternative strategies such as lipofection, electroporation, cell-penetrating peptides and nanoparticle-based systems for targeted RNP delivery. Despite their promise, these advanced methods remain largely untested in woody species. Finally, we outline future research directions, including the development of tree-specific RNP delivery systems and regeneration protocols to enhance efficiency and minimise cytotoxicity. These innovations are essential for unlocking the full potential of RNP-mediated genome editing in long-lived tree species. This review provides a focused and timely roadmap for expanding the application of RNP technology across diverse woody plants.},
}
@article {pmid42285341,
year = {2027},
author = {Nejatinejad, M and Maleki-Aram, A and Namavar Abibiglou, A and Vahedi, C and Panahian, A and Ebrahimi, A and Yaghoubi, R and Parsaei, H and Vafaei, S and Jafari, D},
title = {Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {592},
number = {},
pages = {121178},
doi = {10.1016/j.cca.2026.121178},
pmid = {42285341},
issn = {1873-3492},
mesh = {Humans ; *Proto-Oncogene Proteins B-raf/genetics ; *Mutation ; *Neoplasms/genetics/diagnosis ; *Polymerase Chain Reaction/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.},
}
@article {pmid42379464,
year = {2027},
author = {Zhang, Y and Lin, X and Shi, H and Chen, Z and Luo, Q and Wu, H and Zeng, T},
title = {CRISPR-Cas12a-based liquid biopsy technology: challenges in large-scale clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {592},
number = {},
pages = {121210},
doi = {10.1016/j.cca.2026.121210},
pmid = {42379464},
issn = {1873-3492},
mesh = {Humans ; Liquid Biopsy/methods ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/analysis/blood/genetics ; },
abstract = {Liquid biopsy technology, which enables the acquisition of tumor-related biomarkers in a minimally invasive and reproducible manner, has been widely applied in clinical scenarios such as early tumor detection, treatment response assessment, minimal residual disease (MRD) monitoring, and recurrence surveillance. However, its clinical utility is still limited by the extremely low abundance of clinically relevant targets, the inhibitory effects of complex matrices, and the high variability in pre-analytical stages. As a next-generation programmable nucleic acid diagnostic platform, CRISPR-Cas12a combines the unique mechanisms of sequence-specific recognition and trans-cleavage signal amplification, demonstrating significant technical advantages and translational potential in the detection of low-abundance targets in liquid biopsies. Nevertheless, a mere improvement in analytical sensitivity is insufficient to support its large-scale clinical application; the robustness of detection methods, the reproducibility of results, and clinical interpretability remain the key factors currently restricting its clinical translation. This review summarizes the mechanism of CRISPR-Cas12a. Focusing on seven representative clinical biofluids, including blood, urine, and cerebrospinal fluid, it analyzes the technical difficulties and adaptation strategies associated with different matrices, and summarizes the performance differences of detection methods alongside the most clinically rational application scenarios across various biofluids. Furthermore, it explores the primary bottlenecks this technology faces when transitioning from laboratory proof-of-concept to routine clinical application, and provides a preliminary discussion on its future development directions based on existing research. Ultimately, this review aims to provide a reference for promoting the clinical translation of CRISPR-Cas12a liquid biopsy technologies and the broader implementation of precision medicine.},
}
@article {pmid42538409,
year = {2026},
author = {Song, L and Song, Y and Nguyen, V and Xu, S and Ho, K and Mohammed, A and Shoemaker, RH and Hoang, BH and Yu, J and Uchio, E and Zi, X},
title = {The establishment of prostate-specific, SKP2 humanized mice by CRISPR knock-in method reveals neoplastic initiation and microenvironmental reprogramming.},
journal = {Oncogene},
volume = {45},
number = {37},
pages = {3984-3996},
pmid = {42538409},
issn = {1476-5594},
support = {R01CA255643//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; I01 BX005105/BX/BLRD VA/United States ; UG3 CA290368/CA/NCI NIH HHS/United States ; R01 CA260351/CA/NCI NIH HHS/United States ; I01BX005105//U.S. Department of Veterans Affairs (Department of Veterans Affairs)/ ; P30 CA062203/CA/NCI NIH HHS/United States ; UG3 CA290368//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA260351//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; Male ; *S-Phase Kinase-Associated Proteins/genetics/metabolism ; Humans ; *Prostatic Neoplasms/pathology/genetics/metabolism ; Mice ; *Tumor Microenvironment/genetics ; Gene Knock-In Techniques ; Disease Models, Animal ; Prostate/pathology/metabolism ; Mice, Transgenic ; Gene Expression Regulation, Neoplastic ; Cell Transformation, Neoplastic/genetics ; Epithelial-Mesenchymal Transition/genetics ; CRISPR-Cas Systems ; *Carcinogenesis/genetics ; Cell Movement/genetics ; },
abstract = {Genetic inactivation of SKP2 has been shown to effectively prevent cancer initiation and block tumorigenesis. However, direct in vivo evidence for SKP2 on cancer initiation and prostatic microenvironment is still lacking and a SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting SKP2. We therefore have established a prostate-specific human SKP2 knock-in mouse model driven by an endogenous mouse probasin promoter. Overexpression of hSKP2 induces PIN and low-grade carcinoma. RNA-sequencing analysis revealed significant gene expression alterations in EMT, extracellular matrix, and interferon signaling. Single-cell deconvolution showed an increase of fibroblast population and a decrease of CD8[+] T cell and B cell populations. Consistent with these results from the SKP2 humanized mouse, SKP2 protein is overexpressed in human prostatic hyperplasia, PIN and prostate adenocarcinoma compared to normal prostate tissues. Overexpression of SKP2 markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways. Inhibition of SKP2 signaling by Flavokawain A and C1 reverses EMT and affects EMT and interferon-related gene expression. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decreased viability and altered the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.},
}
@article {pmid42571853,
year = {2026},
author = {Nasir, U and Yasmeen, A and Awais, M and Latif, A and Bakhsh, A and Ahmad, N and Shahid, N and Azam, S and Rao, AQ and Shahid, AA},
title = {Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {372},
number = {},
pages = {113370},
doi = {10.1016/j.plantsci.2026.113370},
pmid = {42571853},
issn = {1873-2259},
mesh = {*Solanum tuberosum/genetics/metabolism ; *beta Carotene/metabolism ; *CRISPR-Cas Systems ; *Mixed Function Oxygenases/genetics/metabolism ; Plants, Genetically Modified/metabolism ; *Vitamin A Deficiency/prevention &amp; control/genetics ; Gene Knockdown Techniques ; *Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.},
}
@article {pmid42660118,
year = {2026},
author = {Tong, X and Visscher, M and Riemers, FM and Versluis, D and Geijsen, N and Shang, P and Tryfonidou, MA and Poramba-Liyanage, DW},
title = {CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage.},
journal = {Stem cell reports},
volume = {21},
number = {9},
pages = {103060},
doi = {10.1016/j.stemcr.2026.103060},
pmid = {42660118},
issn = {2213-6711},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Notochord/cytology/metabolism ; Cell Differentiation/genetics ; *Cell Lineage/genetics ; *SOXD Transcription Factors/genetics/metabolism ; *SOX9 Transcription Factor/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {Intervertebral disc (IVD) degeneration, a leading cause of chronic lower back pain, is associated with loss of vacuolated notochordal cells (NCs) and fibrotic remodeling of the nucleus pulposus. Emerging therapies increasingly focus on NCs, which are rare but therapeutically relevant cells for regenerating degenerated IVDs. In this study, we used CRISPR-based transactivation (CRISPRa) to direct the differentiation of human induced pluripotent stem cells (iPSCs) into the NC lineage. We tested CRISPRa-mediated activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9, coupled with single-cell sequencing of Aggrecan-2A-mScarlet reporter iPSCs. This approach identified the SOX5/6/9 combination (SOX-trio) as critical for promoting NC lineage commitment. The SOX-trio yielded the largest cell population expressing a range of genes previously associated with NC identity, including SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, and KRT18. Our study demonstrates the integration of CRISPRa with single-cell technologies as a powerful platform for investigating and enriching iPSC-derived NCs, supporting future regenerative strategies across various fields.},
}
@article {pmid42687278,
year = {2026},
author = {Han, W and Wei, P and Wang, L and Zhu, G and Xie, L and Zhu, L and He, B and Ji, X and Cao, X},
title = {Tube-Anchored Dual-Atom Nanozyme Hydrogel Microreactors with Dual-Interface Engineering for Portable Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {7426-7434},
doi = {10.1021/acssensors.6c01841},
pmid = {42687278},
issn = {2379-3694},
support = {2024TXTD11//Henan University of Technology/ ; 232300421080//Natural Science Foundation of Henan Province/ ; 242300421035//Natural Science Foundation of Henan Province/ ; 23ZX008//Key Scientific Research Project of Colleges and Universities in Henan Province/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *Hydrogels/chemistry ; DNA/chemistry ; *Nanostructures/chemistry ; Cobalt/chemistry ; Nickel/chemistry ; Colorimetry/methods ; Electrochemical Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; Limit of Detection ; Polymers/chemistry ; Indoles ; },
abstract = {DNA hydrogels are promising platforms for portable biosensing, but their combination with high-activity catalytic nanomaterials remains hindered by interfacial incompatibility and inconsistent device-level retention. Here, a tube-anchored hydrogel microreactor is developed, coupling Co-Ni dual-atom nanozymes (CoNi DANs), interfacial stabilization, and CRISPR-triggered release within a standard centrifuge tube. CoNi DANs are synthesized on a defect-rich nitrogen-doped carbon scaffold, achieving high metal loading and improved peroxidase-like activity relative to those of their single-atom counterparts. Carboxymethyl cellulose-mediated interfacial stabilization then suppresses CoNi DAN aggregation through electrostatic complementarity and polymeric steric shielding, enabling uniform dispersion of CoNi DANs within the DNA hydrogel. A polydopamine-based dual-anchoring strategy further combines covalent grafting with sequence-specific DNA hybridization, providing a chemically reinforced hydrogel-device interface. Upon target recognition, CRISPR/Cas12a trans-cleavage activity induces controlled hydrogel degradation and on-demand release of CoNi DANs for colorimetric and electrochemical dual-mode readout. Using atrazine as a proof-of-concept analyte, the platform achieves limits of detection of 6.1 and 2.1 pg mL-1 for colorimetric and electrochemical modes, respectively, with satisfactory recoveries in real samples. A smartphone-assisted digital readout module further improves the portability of the colorimetric mode. The dual-interface design, uniting material-level compatibilization with device-level dual-anchoring, provides a generalizable framework for embedding high-activity nanozymes into responsive DNA hydrogel microreactors for portable on-site biosensing.},
}
@article {pmid42687281,
year = {2026},
author = {Lu, M and Cheng, J and Guo, J},
title = {Advances in Single-Molecule Immunoassay: From Counting Strategies to CRISPR-Enhanced Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {6600-6616},
doi = {10.1021/acssensors.6c01523},
pmid = {42687281},
issn = {2379-3694},
support = {CSTB2024NSCQ-JQX0012//Natural Science Foundation of Chongqing Municipality/ ; 2023YFF0724300//National Key Research and Development Program of China/ ; 2025ZD01902700//National Science and Technology Major Project/ ; },
mesh = {Immunoassay/methods ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Single-molecule immunoassays (SMIs) overcome the sensitivity limitations of conventional bulk measurements by enabling a paradigm shift from analog to digital signal readouts, thereby facilitating highly sensitive quantification of ultra-low-abundance biomarkers for precision diagnostics. This review provides a systematic overview of recent advances in SMI technologies and the conceptual framework underlying their evolution. First, discretization strategies for single-molecule counting are classified into hard discretization, based on physical confinement, and soft discretization, based on spatiotemporal isolation, within heterogeneous and homogeneous assay systems, respectively. The fundamental mechanisms by which these strategies mitigate diffusion limitations and enhance signal-to-noise ratios are discussed. Second, the integration of SMIs with CRISPR-based diagnostic systems (CRISPR-dx) is examined, with particular emphasis on their complementary roles in target recognition and signal amplification. Finally, recent applications of SMIs in the diagnosis of oncological, neurological, infectious, and cardiovascular diseases are summarized, along with a critical discussion of current engineering challenges and future directions toward clinical translation.},
}
@article {pmid42688545,
year = {2026},
author = {Song, Z and Li, H and Zhao, Y and Li, M},
title = {[Research Progress on the Application of CRISPR/Cas System in Rapid Testing of Drug-Resistant Bacteria in Clinical Practice].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {57},
number = {4},
pages = {1212-1220},
pmid = {42688545},
issn = {1672-173X},
mesh = {*CRISPR-Cas Systems ; Humans ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects/isolation &amp; purification ; *Bacterial Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; },
abstract = {The misuse of antimicrobial agents has made antimicrobial resistance one of the major threats to global public health. Efficient and rapid detection of drug-resistant bacteria and resistance genes is crucial for controlling infection spread and safeguarding human health. However, traditional detection methods (such as culture isolation, polymerase chain reaction, etc.) exhibit significant limitations in detection speed, cost-effectiveness, and convenience, increasingly failing to meet current clinical testing demands. In this context, the CRISPR/Cas system, as an emerging molecular diagnostic technology, offers innovative solutions for this field. This review details the developmental status of CRISPR/Cas systems and their research progress in rapid clinical detection of drug-resistant bacteria. It systematically explains the working principles and efficacy of various CRISPR/Cas-based detection platforms (e.g., SHERLOCK, DETECTR, HOLMES), highlighting the system's advantages in sensitivity, specificity, efficiency, real-time capability, portability, and low cost. Additionally, the article discusses challenges faced by CRISPR/Cas systems, including direct clinical sample detection, multiplex testing, cost reduction, clinical validation, and standardization. Finally, it outlines future development directions, emphasizing technological innovation and clinical translation to establish CRISPR/Cas systems as efficient tools for drug-resistant bacteria prevention and control.},
}
@article {pmid42689566,
year = {2026},
author = {Zhang, R and Wen, Z and Zhao, Y and Cheng, L and Liu, C and Bian, S and Xu, L and Pei, R},
title = {Phosphorothioate-Enabled Cas12a Autocatalytic Network for Amplification-Free Ultrasensitive Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {25454-25462},
doi = {10.1021/acs.analchem.6c05179},
pmid = {42689566},
issn = {1520-6882},
support = {NA//Yinuosai Biotech Co., Ltd./ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; *DNA, Single-Stranded/chemistry/analysis ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; *DNA/analysis ; Limit of Detection ; *Phosphorothioate Oligonucleotides/chemistry ; *Phosphates/chemistry ; },
abstract = {Autocatalytic CRISPR/Cas systems offer a promising route toward amplification-free nucleic acid detection, yet their performance is limited by indiscriminate transcleavage of ssDNA activators and the reliance on structurally complex, thermodynamically unstable DNA architectures. A phosphorothioate (PS)-enabled Cas12a autocatalytic network (SCAN) is presented to address these challenges by leveraging the unique compatibility of F. novicida Cas12a (FnCas12a) with fully PS-modified ssDNA activators. An ssDNA mediator is engineered with fully PS-modified activator domains flanking a cleavable, unmodified spacer. The intact mediator remains sterically inactive toward Cas12a ribonucleoproteins, whereas trace-target-induced cleavage liberates split activators that trigger a self-propagating amplification network. The SCAN platform achieves ultrasensitive detection with limits of detection of 889 aM and 341 aM for ssDNA and dsDNA targets, respectively. The platform further enables amplification-free detection of Salmonella enterica and Escherichia coli in real samples, with detection limits of 30 and 20 CFU/mL, respectively. This work establishes fully PS-modified ssDNA as a robust and generalizable design element for CRISPR-based autocatalytic systems, providing a simple, flexible, and efficient strategy to enhance stability, suppress background, and improve analytical performance.},
}
@article {pmid42689567,
year = {2026},
author = {Yan, Y and Chen, M and Li, H and Zhou, Y and Chen, X and Dong, X and Liu, F and Xiao, M and Zhu, L},
title = {AND-Logic Gated Electrochemical Sensor for Separate Detection of Aflatoxin B1 and Ochratoxin A via Split-Activator CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {25286-25298},
doi = {10.1021/acs.analchem.6c03714},
pmid = {42689567},
issn = {1520-6882},
support = {22404119//National Natural Science Foundation of China/ ; JWC20250202//Teaching Reform Project of Sichuan Normal University/ ; },
mesh = {*Ochratoxins/analysis ; *Aflatoxin B1/analysis ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA, Catalytic/metabolism/chemistry ; Limit of Detection ; Food Contamination/analysis ; G-Quadruplexes ; Electrodes ; },
abstract = {To address the frequent co-occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in real food matrices, as well as the difficulty of achieving both high-specificity recognition and rapid quantification with existing detection methods, this study developed an AND logic-gated electrochemical sensor based on split-activator CRISPR/Cas12a. The sensor constructed an ordered signaling layer on the electrode interface using a DNA tetrahedron. Through DNAzyme-catalyzed reactions triggered separately by the two targets, two split DNA fragments were generated. Only when both fragments coexisted could they jointly activate the trans-cleavage activity of CRISPR/Cas12a, leading to extensive cleavage of the G-quadruplex sequence supported by the DNA tetrahedron. This cleavage caused the loss of the G-quadruplex/hemin signal-reporting group, resulting in a sharp attenuation of the electrochemical signal. By optimizing the heparin sodium concentration, a one-pot integration of the DNAzyme catalytic cycle and the CRISPR cleavage reaction was successfully achieved. The sensor achieved detection limits of 1.12 pg/mL for AFB1 and 0.26 pg/mL for OTA, which are significantly lower than international regulatory limits. For real corn flour, wheat flour, rice flour, and soybean flour samples, spike recoveries ranged from 90.99% to 107.29%, and the results were highly correlated with those obtained using a commercial enzyme-linked immunosorbent assay method (R2 > 0.996) and high-performance liquid chromatography-tandem mass spectrometry (R2 > 0.998). This work not only provides a new, ultrasensitive, and highly specific tool for copresence reporting of dual mycotoxins, but also offers an innovative paradigm for constructing logic-based biosensors designed for intelligent analysis of complex matrices.},
}
@article {pmid42689595,
year = {2026},
author = {Liu, Y and Yu, L and Shen, Y and Gan, Z and Tong, J and Zhao, F and Xiao, Y},
title = {Lanthanide Metal-Organic Frameworks Enable Target-Triggered CRISPR-Cas12a Activator Release for Ultrasensitive Non-nucleic Acid Biomarker Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {24865-24875},
doi = {10.1021/acs.analchem.6c01959},
pmid = {42689595},
issn = {1520-6882},
support = {82273895//National Natural Science Foundation of China/ ; 82572684//National Natural Science Foundation of China/ ; 2025HBBSHXF006//Hubei Provincial Postdoctoral Pioneer Talent Tracking Program/ ; WHYC202503//Wuhan Top-Notch Talent Program/ ; ZNJY202607//Hubei Provincial Clinical Research Center for Molecular Diagnostics/ ; },
mesh = {*CRISPR-Cas Systems ; *Lanthanoid Series Elements/chemistry ; Humans ; Biomarkers/blood/analysis ; Aptamers, Nucleotide/chemistry ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; *Troponin I/blood/analysis ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism ; },
abstract = {Rapid, ultrasensitive detection of cardiac troponin I (cTnI) is critical for the early diagnosis of acute myocardial infarction (AMI). However, CRISPR-Cas systems, despite their unparalleled nucleic acid detection performance, face inherent bottlenecks in protein sensing, including inefficient signal transduction, high background noise, and insufficient anti-interference capability in complex biological matrices. Herein, we report a modular fluorescence signal transduction platform integrating aptamer-functionalized magnetic beads, lanthanide metal-organic frameworks (Ln-MOFs) with high nucleic acid affinity, and CRISPR-Cas12a-mediated collateral cleavage amplification for sensitive cTnI detection. Target cTnI binding to magnetic bead-immobilized aptamers drives the formation of sandwich complexes with CRISPR activator DNA (act)-functionalized Ln-MOFs, triggering phosphate-mediated release of act. The liberated act initiates robust Cas12a trans-cleavage activity, generating an amplified fluorescence readout. This platform achieves a limit of detection (LOD) of 0.1 pg mL-1 in serum samples, with excellent specificity against off-target interfering proteins. Validation in 28 clinical serum samples demonstrates near-perfect agreement with standard clinical ELISA measurements and yields an area under the curve (AUC) of 0.995, confirming its clinical diagnostic accuracy. Furthermore, the modular design enables facile reconfiguration for diverse protein biomarkers via aptamer substitution, providing a universal strategy to expand CRISPR-Cas systems toward non-nucleic acid target detection.},
}
@article {pmid42689621,
year = {2026},
author = {Fu, H and Wang, Z and Guo, J and Wang, T and Li, X and Su, Y and Chen, Z and Yang, H and Xie, Z and Li, D},
title = {High-Sensitivity Molecular Detection of Viral Bioaerosols Using a Composite Collection System and Optimized RPA-CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {24876-24887},
doi = {10.1021/acs.analchem.6c01983},
pmid = {42689621},
issn = {1520-6882},
support = {12274197//National Natural Science Foundation of China/ ; 12304487//National Natural Science Foundation of China/ ; 2023ZDZX2071//Department of Education of Guangdong Province/ ; GJHZ20220913143207014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102618040//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20230807093808017//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20241202130558075//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2022A0505030024//Science and Technology Planning Project of Guangdong Province/ ; 2022B1515020093//Science and Technology Planning Project of Guangdong Province/ ; 2022B1515120012//Science and Technology Planning Project of Guangdong Province/ ; 2026A1515010034//Science and Technology Planning Project of Guangdong Province/ ; NA//Guangdong Provincial Intelligent Diagnosis Engineering Research Center for Molecular Instant Detection of Children?s Infectious Diseases/ ; },
mesh = {Aerosols/analysis ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Highly pathogenic airborne microorganisms pose a significant public health threat, particularly because their small particle size can support prolonged suspension and long-range transport. Here, we present an integrated platform that combines a composite bioaerosol collection system with a three-stage enrichment mechanism and recombinase polymerase amplification-CRISPR/Cas12a (RPA-CRISPR/Cas12a) detection. The platform incorporates model-based constant-temperature control, stacked optical filters, and multichannel fluorescence imaging. The temperature-control model accurately described the thermal response of the reaction module, while the optical and imaging subsystems supported stable fluorescence readout. Using aerosolized monkeypox virus (MPXV) pseudovirus as a controlled test target, the platform detected target nucleic acid in collection liquid down to 10 copies/μL under the tested conditions. These results support the feasibility of integrating aerosol enrichment with isothermal molecular detection in a compact prototype. The main contributions lie in innovations and the engineering integration of bioaerosol collection and enrichment, thermal control of biochemical reactions in molecular detection, and precision fluorescence detection techniques for molecular detection. Regarding molecular detection methods, the biochemical reaction parameters were experimentally optimized rather than introducing new biochemical reaction mechanisms. Validation with authentic clinical or environmental viral aerosols remains necessary before diagnostic or field-use claims can be made.},
}
@article {pmid42690503,
year = {2026},
author = {Indurthi, S and Kaur, G and Dutta, R and Madhu, S and Chodasani, B and Yadav, A and Singh, M and Meena, TK and Singh, G},
title = {Integrating genomics, multi-omics, CRISPR and speed breeding for stress-resilient vegetable legume improvement.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42690503},
issn = {1438-7948},
mesh = {*Fabaceae/genetics/metabolism ; *Plant Breeding ; Multiomics ; *Stress, Physiological/genetics ; Genomics ; Quantitative Trait Loci ; Genome, Plant ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Vegetable legumes are nutritionally and ecologically important crops. However, their genetic improvement has not kept pace with the increasing challenges posed by climate change due to the polygenic nature of stress tolerance, narrow genetic diversity, and the persistent gap between molecular discoveries and field-level cultivar development. Although recent reviews have examined individual genomic tools or specific stress responses, a comprehensive synthesis integrating genomics-assisted breeding, multi-omics technologies, genome editing, and speed breeding within a unified crop improvement framework has been lacking. This review addresses that gap by critically evaluating how these complementary approaches can accelerate the development of stress-resilient vegetable legumes, including pea, common bean, cowpea, faba bean, cluster bean, yard-long bean, and hyacinth bean. This review synthesizes advances in QTL mapping, genome-wide association studies, transcriptomics, metabolomics, and CRISPR-based functional genomics that have identified key regulators and pathways underlying resistance to major biotic and abiotic stresses. Rather than considering these technologies independently, the review emphasizes their convergence into a systems-level breeding framework integrating genomic discovery, functional validation, predictive breeding, and accelerated generation advancement to improve breeding efficiency. Speed breeding, enabling up to seven to eight generations annually under optimized controlled-environment experimental conditions in cowpea, is discussed as a complementary strategy with genomic selection and genome editing. The review further identifies major translational bottlenecks, including transformation recalcitrance, limited genomic resources for underutilized vegetable legumes, inadequate multi-environment validation, and fragmented omics integration, and presents an integrated systems-breeding framework to bridge the gap between gene discovery and cultivar development.},
}
@article {pmid42272442,
year = {2026},
author = {Luo, X and Qu, Y and Ye, Z and Li, Z and Zhang, Y and Luo, L and Li, S and Zhao, W and Wang, M and Bock, R and Wan, J and Tan, J},
title = {Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {50},
pages = {e76047},
pmid = {42272442},
issn = {2198-3844},
support = {BF2025302//Frontier Technologies R&amp;D Program of Jiangsu/ ; ZSBBL-KY2023-04//Zhongshan Biological Breeding Laboratory/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Oryza/genetics ; Humans ; *HMGN Proteins/genetics ; },
abstract = {RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.},
}
@article {pmid42315549,
year = {2026},
author = {Sekine, T and Shinzawa, N and Kubota, R and Kobayashi, D and Okubo, Y and Itokawa, K and Isawa, H and Amino, H and Ishino, T},
title = {Versatile, marker-free platform for life cycle-wide imaging of Plasmodium falciparum by integrating an exogenous gene cassette into a conserved intergenic locus.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42315549},
issn = {2045-2322},
support = {JPMJSP2120//Japan Science and Technology Corporation/ ; JP20wm0325018//Japan Agency for Medical Research and Development/ ; JP24wm0325074//Japan Agency for Medical Research and Development/ ; 24K02272//Japan Society for the Promotion of Science/ ; 24KK0149//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Plasmodium falciparum/genetics/growth &amp; development ; *Life Cycle Stages/genetics ; Mice ; Humans ; Genetic Loci ; Gene Editing ; Sporozoites ; Luminescent Proteins/genetics ; CRISPR-Cas Systems ; *DNA, Intergenic/genetics ; Liver/parasitology ; Malaria, Falciparum/parasitology ; },
abstract = {The creation of transgenic Plasmodium falciparum lines with robust fluorescence across the entire life cycle is essential for advancing our understanding of parasite biology, which in turn informs the development of new drugs and vaccines. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a selected intergenic locus without gene disruption. The resulting marker-free line, NF54-mCh, exhibited intense fluorescence throughout all developmental stages, including asexual and sexual blood stages, as well as mosquito (ookinete, oocyst, and sporozoite) and liver stages. NF54-mCh showed normal proliferation, gametocytogenesis, and efficient transmission to mosquitoes. The ultra-high brightness in salivary gland sporozoites allowed for the non-invasive identification of infected mosquitoes. Sporozoites remained highly infectious to humanized mouse livers, thus enabling the completion of the full life cycle. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating similar reporter lines in Plasmodium species utilized in rodent malaria models. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of malaria control strategies, including new vaccines and drugs.},
}
@article {pmid42332291,
year = {2026},
author = {Al Madhoun, A and Malik, MZ and Al-Beloushi, S and Abukhalaf, N and Miranda, L and Jacob, S and Nizam, R and John, S and George, P and Channanath, A and Bahman, F and Liao, M and Ahmad, R and Chen, W and Bekri, A and Drapeau, P and Thanaraj, TA and Al-Mulla, F},
title = {Targeting glucagon signaling in metabolic disorders, functional insights from zebrafish receptor knockouts.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {42332291},
issn = {1420-9071},
support = {RA-CB-2021-007//Kuwait Foundation for the Advancement of Sciences/ ; },
mesh = {Animals ; *Zebrafish/metabolism/genetics ; *Receptors, Glucagon/genetics/metabolism ; *Signal Transduction ; *Glucagon/metabolism ; *Metabolic Diseases/metabolism/genetics ; Protein Isoforms/genetics/metabolism ; Lipid Metabolism ; Gene Knockout Techniques ; *Zebrafish Proteins/genetics/metabolism ; Energy Metabolism ; Glucose/metabolism ; Transcriptome ; CRISPR-Cas Systems ; },
abstract = {Glucagon receptor (GCGR) signaling is essential for glucose and lipid homeostasis, making it a potential therapeutic target for metabolic disorders. Zebrafish possess two GCGR co-orthologs, GCGRa and GCGRb, however, their distinct function remain unclear. In this study we employed CRISPR/Cas9 gene editing to generate GCGRa[-]/[-], GCGRb[-]/[-], and double-knockout (GCGR[-]/[-]) zebrafish to dissect isoform-specific functions. RNA-Seq analysis was performed to characterize transcriptomic alterations, while an overfeeding protocol was used to assess metabolic tolerance, and ligand-response assays in cell lines evaluated isoform activation dynamics. Transcriptomic analysis revealed that both isoforms regulate overlapping but distinct metabolic pathways. Functional enrichment analysis linked GCGRa to lipid and energy metabolism, cholesterol biosynthesis and glucose homeostasis, through key signaling cascades such as glucagon, PPARγ and PI3K-AKT. In contrast, GCGRb loss altered fatty acid β-oxidation, GPCR signaling, and oxidative phosphorylation networks, implicating roles in metabolism and cellular stress. The GCGR[-]/[-] primarily impacted core metabolic networks including lipid, gluconeogenesis and energy metabolism, indicating complementary and overlapping functions of both receptors in maintaining hepatic metabolic homeostasis. Ligand-response assays revealed that GCGRb, but not GCGRa, is activated by both glucagon (GCGa) and glucagon like-peptide-1 (GLP1a), supporting the post-duplication receptor diversification theory. Notably, all knockouts exhibited impaired growth under high-nutrient conditions, confirming GCGR's role in diet-responsive development. This study provides the first systematic functional comparison of zebrafish GCGR isoforms, establishing zebrafish as a valuable model for investigating glucagon-based metabolic regulation and therapeutic interventions.},
}
@article {pmid42412303,
year = {2026},
author = {Khatun, M and Lundin, K and Tuuri, T and Piltonen, T and Tapanainen, JS and Salumets, A},
title = {From Gene Function to Precision Intervention: CRISPR/Cas9 and Stem Cell-Based Strategies as Emerging Disease-Modifying Approaches in PMOS.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {7},
pages = {3056-3080},
pmid = {42412303},
issn = {2629-3277},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; Animals ; Induced Pluripotent Stem Cells/metabolism ; Gene Editing/methods ; Mesenchymal Stem Cells/metabolism ; *Metabolic Syndrome/therapy/genetics ; *Precision Medicine/methods ; },
abstract = {Polyendocrine metabolic ovarian syndrome (PMOS) is a complex endocrine-metabolic disorder affecting up to 18% of women worldwide and remains the leading cause of anovulatory infertility. Despite extensive research, current treatments primarily target symptoms, including menstrual irregularities, hyperandrogenism, and metabolic dysfunction, without addressing the underlying molecular and tissue-level disturbances. Advances in multi‑omic profiling have identified disruptions across neuroendocrine, metabolic, inflammatory, and extracellular matrix pathways, alongside genetic susceptibility at loci such as DENND1A, CYP17A1, LHCGR, FSHR, IRS1, and PPARG. However, the functional roles of many variants remain unresolved. CRISPR/Cas9 gene editing enables precise interrogation of these pathways, while stem cell-based platforms, including mesenchymal stem cells (MSCs), exosomes, and gene-edited induced pluripotent stem cells (iPSCs), may serve as complementary platforms for regeneration and disease modeling. Preclinical studies demonstrate that MSCs and their derivatives modulate inflammation, restore ovarian structure, and improve metabolic parameters, while iPSC-based models enable patient-specific investigation of steroidogenic and metabolic abnormalities. Translational challenges remain, including targeted delivery, off-target effects, phenotypic heterogeneity, and regulatory considerations. Integrating CRISPR‑based functional genomics with stem cell research may shift PMOS management from symptom‑focused care to targeted, mechanism‑driven interventions that could modify the course of PMOS (Graphical Abstract).},
}
@article {pmid42681397,
year = {2026},
author = {Savash Ishanzadeh, MC and Wells, D},
title = {Clustered Regularly Interspaced Palindromic Repeats (CRISPR) Applied to Gametes and Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3038},
number = {},
pages = {447-475},
doi = {10.1007/978-1-0716-5292-3_24},
pmid = {42681397},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; Female ; *Preimplantation Diagnosis/methods ; *CRISPR-Cas Systems ; *Blastocyst/metabolism ; Fertilization in Vitro/methods ; *Germ Cells/metabolism ; Pregnancy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Preimplantation genetic testing (PGT) facilitates the identification of embryos affected by specific types of genetic abnormalities. However, PGT does not seek to treat the genetic abnormality, rather it is an embryo selection tool, employing a strategy of detection and exclusion. Until recently, the notion that mutations and aneuploidies could be corrected in gametes, or in embryos produced using in vitro fertilization (IVF), seemed improbable. However, rapid progress in the evolution of gene editing technologies may make this a realistic possibility in the near future. Not only would such an approach help to avoid the discard of human embryos, which some find challenging from ethical or religious perspectives, but it would also increase the number of embryos considered suitable for transfer, potentially leading to higher pregnancy rates than achieved in PGT cycles. This chapter considers the use of genome editing applied to human preimplantation embryos, describing a protocol that can be used to inactivate genes for research purposes, and which might, in the future, allow for the correction of pathogenic mutations.},
}
@article {pmid42681488,
year = {2026},
author = {Fernandes, LGV and Nally, JE},
title = {Gene Knockout in Leptospira spp. by CRISPR/Cas9-NHEJ (Non-Homologous End-Joining) and CRISPR-Prime Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3068},
number = {},
pages = {139-156},
pmid = {42681488},
issn = {1940-6029},
mesh = {*Leptospira/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *DNA End-Joining Repair/genetics ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; },
abstract = {Genetic manipulation of Leptospira spp. has progressed significantly in recent years. Like most prokaryotes, leptospires are unable to survive double-strand breaks (DSBs) induced by the Cas9 endonuclease, prompting the development of alternative strategies for gene knockout. We have established two systems for targeted mutagenesis of Leptospira spp.: CRISPR/Cas9-NHEJ and CRISPR-Prime Editing (PE). The CRISPR/Cas9-NHEJ approach involves coexpression of the CRISPR/Cas9 machinery alongside the DNA repair proteins LigD and Ku from Mycobacterium smegmatis, facilitating error-prone repair of DSBs that results in indel mutations. In contrast, CRISPR-PE is a DSB-free strategy that utilizes a Cas9-nickase fused to a reverse transcriptase that facilitates precise single-nucleotide edits in the genome. This expanded toolbox has placed Leptospira spp. at the forefront of bacterial genetic manipulation.},
}
@article {pmid42683944,
year = {2026},
author = {Schulman, J and Egan, R and Kandari, L and Madayiputhiya, N and Mahon, D and Tao, L and Luo, H and Condon, K and Feder, JN and Huang, D and Khetan, A},
title = {TPST Gene Knock-Out Eliminates Tyrosine Sulfation on a Recombinant Antibody Produced in CHO Cells.},
journal = {Biotechnology journal},
volume = {21},
number = {9},
pages = {e70302},
pmid = {42683944},
issn = {1860-7314},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Tyrosine/metabolism ; *Sulfotransferases/genetics/metabolism ; *Recombinant Proteins/metabolism/genetics ; *Antibodies, Monoclonal/metabolism/genetics ; Gene Knockout Techniques ; Protein Processing, Post-Translational ; CRISPR-Cas Systems ; Cricetinae ; Antibodies, Bispecific/metabolism/genetics ; },
abstract = {Tyrosine sulfation is a post-translational modification that has been reported to occur infrequently on recombinant monoclonal antibodies (mAbs). We recently demonstrated that tyrosine sulfation occurred on a bispecific antibody (bsAb) produced in Chinese hamster ovary (CHO) cells, using a multi-enzymatic approach in combination with intact mass and peptide-based mass spectrometry analysis supplemented with the use of synthetic peptides. Tyrosine sulfation needs to be controlled during the manufacturing process due to potential undesired effects, such as impact on potency and immunogenicity. Here, we report that tyrosine sulfation was not significantly inhibited by the addition of chemical inhibitors, such as sodium chlorate. Individual knockout and double knockout (DKO) of two key genes in the tyrosine sulfation pathway were carried out sequentially. Tyrosyl protein sulfotransferase 1/2 (TPST1/2) DKO by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing eliminated tyrosine sulfation while maintaining cell growth, antibody production, and overall product quality.},
}
@article {pmid42684483,
year = {2026},
author = {Wang, K and Chen, J and Bai, S and Wang, Q and Zhang, W and Qin, Z and Zhang, Z and Zhang, Z and Ma, Y},
title = {Efficient Microinjection and CRISPR/Cas9 Mediated Genome Editing in Urechis unicinctus.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {28},
number = {5},
pages = {},
pmid = {42684483},
issn = {1436-2236},
support = {42176122//National Natural Science Foundation of China/ ; 32170373//National Natural Science Foundation of China/ ; ZR2025MS368//Natural Science Foundation of Shandong Province/ ; 2022120090//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Microinjections/methods ; Caveolin 1/genetics/metabolism ; Cilia/genetics/metabolism ; *Gene Editing/methods ; Tubulin/genetics ; Base Sequence ; },
abstract = {Urechis unicinctus is an economically important aquaculture species and an emerging model for developmental and evolutionary research; however, progress in functional genetic studies in this species has remained limited by the absence of effective genome editing approaches. Here, we establish a CRISPR/Cas9-mediated genome editing platform in U. unicinctus based on an optimized microinjection procedure. Targeting the cilia-associated gene Caveolin-1, Cas9/sgRNA ribonucleoprotein complexes were delivered into oocytes, generating mosaic G0 larvae harboring insertion-deletion mutations, as confirmed by ICE analysis and cloning-based Sanger sequencing with mutation detection rates of 68.51% and 64.86%, respectively. Ciliary defects were frequently observed in edited larvae, including shortening of the circumoral ciliary ring and reduction of apical ciliary tufts, accompanied by impaired swimming performance. Whole-mount in situ hybridization further revealed altered spatial expression patterns of Caveolin-1 in edited embryos, with 78% of individuals showing detectable changes in expression patterns. To independently assess the robustness of the platform, the conserved cytoskeletal gene α-tubulin was additionally targeted, resulting in successful mutagenesis and associated ciliary abnormalities. Together, these findings establish the first CRISPR/Cas9 genome editing workflow for U. unicinctus, providing a technical framework for functional genomics in echiurans and facilitating future studies of developmental mechanisms and molecular breeding in this species.},
}
@article {pmid42686754,
year = {2026},
author = {Dinesh, RK and Wang, X and Mohammad, IA and Gunasekaran, P and Stiklioraitis, K and Villafuerte, JR and Rao, A and Hernandez-Lopez, RA and Sunwoo, JB and Cong, L},
title = {Surfaceome CRISPR activation screening uncovers ligands regulating tumor sensitivity to NK cell killing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42686754},
issn = {2041-1723},
support = {R35GM155437//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35HG011316//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35DE030054//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35 GM155437/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; 1R01GM141627//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35 DE030054/DE/NIDCR NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; Humans ; Animals ; Mice ; Ligands ; Cell Line, Tumor ; *Cytotoxicity, Immunologic ; CRISPR-Cas Systems ; Leukosialin/metabolism/genetics/immunology ; Hyaluronan Receptors/genetics/metabolism/immunology ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics/immunology ; Female ; Immunotherapy/methods ; Coculture Techniques ; },
abstract = {Natural killer (NK) cell-based immunotherapies are promising for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and "off-the-shelf" utility. However, molecular determinants governing tumor susceptibility to NK cytotoxicity remain incompletely understood. Here we employ CRISPR activation (CRISPRa) screening to identify cancer cell surface regulators of NK killing. Using a surfaceome-focused library, we screen human and murine cancer cell lines co-cultured with NK cells, identifying known and novel ligands modulating NK cytotoxicity. Screens reveal established factors including CD43 and previously uncharacterized regulators CD44, PDPN, and Siglec-1/CD169. Validation with orthogonal approaches confirm that disruption of these factors alters NK killing susceptibility in vitro and in humanized mouse models. Mechanistically, we find that CD43-mediated NK resistance operates independently of its proposed interaction with Siglec-7, and that targeting CD43 on NK cells or CAR T cells substantially enhances cytotoxic activity against leukemia. These results establish gain-of-function surfaceome screening as a powerful tool for identifying therapeutic targets for NK cell-based immunotherapy.},
}
@article {pmid41945283,
year = {2026},
author = {Chu, NH and Le, HD and Quach, NT and Pham, TKL and Ho, NA and Nguyen, THH and Tran, XK and Pham, BN and Chu, HH},
title = {CRISPR/Cas9-Mediated Knockout of the NAD-Dependent Lactate Dehydrogenases for Altered Stereospecific Lactic Acid Production in Lacticaseibacillus paracasei NC4.},
journal = {Biochemical genetics},
volume = {64},
number = {5},
pages = {7016-7031},
pmid = {41945283},
issn = {1573-4927},
support = {T&#x110;NSH0.06/22-24//Vietnam Academy of Science and Technology/ ; },
mesh = {*Lactic Acid/biosynthesis ; *CRISPR-Cas Systems ; *Lacticaseibacillus paracasei/genetics/enzymology/metabolism ; Gene Knockout Techniques ; *L-Lactate Dehydrogenase/genetics/metabolism ; *NAD/metabolism ; *Lactate Dehydrogenases/genetics/metabolism ; Stereoisomerism ; },
abstract = {The optical purity of lactic acid is a critical parameter for producing high-performance polylactic acid (PLA). To investigate the genetic basis of stereospecific lactic acid biosynthesis, the present study aimed to functionally characterize the roles of ldh1 and ldh2 in Lacticaseibacillus paracasei NC4 through targeted gene disruption. A CRISPR/Cas9 nickase-based system was employed to construct three mutant strains (Δldh1, Δldh2, and Δldh1Δldh2). Fermentation experiments were conducted under identical conditions, and the concentrations of D- and L-lactic acid were quantified using HPLC. Three mutant strains, Δldh1, Δldh2, and Δldh1Δldh2, were successfully constructed from the wild-type NC4 using the CRISPR-Cas9 system. Compared with the wild-type NC4, which produced 89.31 ± 0.21 g/L L-lactic acid and 10.74 ± 0.19 g/L D-lactic acid, the Δldh1 mutant produced 76.31 ± 2.22 g/L L-lactic acid and 7.72 ± 0.36 g/L D-lactic acid, while the Δldh2 mutant yielded 81.73 ± 0.46 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant generated 75.57 ± 2.96 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant similarly exhibited no detectable D-lactic acid formation, supporting the role of ldh2 in D-lactate biosynthesis. These results indicate that targeted deletion of ldh genes significantly alters the stereospecificity of lactic acid biosynthesis. In particular, deletion of ldh2 was sufficient to eliminate detectable D-lactic acid formation, whereas deletion of ldh1 alone did not completely abolish D-lactate production. Overall, this study provides functional genetic insight into the roles of ldh1 and ldh2 in controlling lactic acid stereospecificity in L. paracasei NC4 and establishes a genetic basis for future metabolic and process-oriented optimization of optically pure lactic acid production.},
}
@article {pmid42189436,
year = {2026},
author = {Mendoza, A and Simon, I and Hasan, S},
title = {A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.},
journal = {Biochemical genetics},
volume = {64},
number = {5},
pages = {7759-7786},
pmid = {42189436},
issn = {1573-4927},
mesh = {Animals ; *Microinjections/methods ; *Oryzias/genetics/embryology ; *Zebrafish/genetics/embryology ; CRISPR-Cas Systems ; *Embryo, Nonmammalian/metabolism ; Gene Knockdown Techniques/methods ; Female ; Morpholinos/genetics/administration &amp; dosage ; },
abstract = {Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.},
}
@article {pmid42537911,
year = {2026},
author = {Liu, K and Fan, X and Tong, Z and Zhang, J and Zhao, M and Chen, Y and Wu, C and Wang, T and Wei, S and Liu, Y and Xue, Z and Zheng, Y},
title = {Reprogrammed Komagataella phaffii for enhanced secretory expression of human lactoferrin.},
journal = {Journal of biotechnology},
volume = {419},
number = {},
pages = {1-10},
doi = {10.1016/j.jbiotec.2026.07.011},
pmid = {42537911},
issn = {1873-4863},
mesh = {*Lactoferrin/genetics/metabolism ; Humans ; *Saccharomycetales/genetics/metabolism ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Endoplasmic Reticulum/metabolism/genetics ; Bioreactors ; Vacuoles/metabolism/genetics ; },
abstract = {Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1 g/L FeCl3 was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5 L bioreactor, the secretory intact hLF titer reached 2214 mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.},
}
@article {pmid42574952,
year = {2026},
author = {Zhu, X and Zhang, W and Sun, T and Chen, L},
title = {Advances in combinatorial CRISPRi screening and applications: Decoding higher-order interactions for next-generation microbial cell factories in synthetic biology.},
journal = {Microbiological research},
volume = {313},
number = {},
pages = {128663},
doi = {10.1016/j.micres.2026.128663},
pmid = {42574952},
issn = {1618-0623},
mesh = {*Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Gene Regulatory Networks ; Bacteria/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Biological systems possess high robustness and intricate genetic redundancy, rendering traditional single-gene perturbations largely inadequate for comprehensively elucidating the true regulatory mechanisms underlying complex phenotypes. To address this, combinatorial CRISPR interference (CRISPRi) has emerged as an essential tool in systems and synthetic biology, offering reversible epigenetic control, multi-target regulation, and an absence of DNA toxicity. Unlike pooled single-gene screens, combinatorial CRISPRi facilitates the systematic dissection of buffering, synergy, and metabolic trade-offs by targeting multiple loci simultaneously. This review explores the latest advancements in pairwise and higher-order combinatorial CRISPRi screening, beginning with design strategies for multiplex guide RNA (gRNA) arrays and orthogonal systems. Computational techniques utilized for analyzing high-dimensional screening data and visualizing complex genetic networks are subsequently examined. Building upon these methodological foundations, crucial applications within microbial engineering are highlighted. Specifically, the review details the optimization of carbon flux in microbial cell factories to circumvent production bottlenecks, alongside the elucidation of protective multigenic networks against severe environmental stress. Furthermore, it addresses current obstacles, such as system noise and library construction challenges, and outlines future research directions. Ultimately, this review provides a comprehensive guide for decoding complex traits and driving rational designs of next-generation cell factories.},
}
@article {pmid41047874,
year = {2026},
author = {Ji, Y and Sun, Y and Zhou, H and Liu, Z and Sun, Z and Xu, G and Wen, H and Zheng, Z and Tu, L and Yang, Z and Zhang, Y and Liu, X and Zhou, S and Dong, X and Wang, Y and Li, C and Wan, J},
title = {Engineer the eukaryotic OMEGA-Fanzor systems for genome editing in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3160-3162},
doi = {10.1111/jipb.70049},
pmid = {41047874},
issn = {1744-7909},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics ; *Genome, Plant/genetics ; *Genetic Engineering/methods ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; },
abstract = {The activity of the eukaryotic OMEGA-Fanzor genome editing system remains limited in plants. We engineered the Fanzor nucleases SpuFz1, GtFz1, NlovFz2, and MmeFz2 in plants, with NlovFz2 being the most efficient, achieving up to 50.0% editing in regenerated rice plants, making it a promising tool for plant genome editing.},
}
@article {pmid41566884,
year = {2026},
author = {Zhang, R and Tang, X and He, Y and Wang, W and Ren, Q and Qi, Y and Zhang, Y},
title = {Enhanced exonuclease-Cas9 systems promote multiple nucleotide deletions with higher efficiency and broader targeting scope in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3316-3328},
doi = {10.1111/jipb.70155},
pmid = {41566884},
issn = {1744-7909},
mesh = {*CRISPR-Cas Systems/genetics ; *Oryza/genetics ; *Sequence Deletion/genetics ; Gene Editing/methods ; *Exonucleases/metabolism/genetics ; Plants, Genetically Modified ; },
abstract = {CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.},
}
@article {pmid41588854,
year = {2026},
author = {Zhang, C and Li, J and Li, Y and Yan, L and Yong, CSY and Li, S and He, Y and Xia, L},
title = {Coupling of both a transactivation module and a double-stranded DNA-binding domain boosts Cas12i3 variant-based cytosine and adenine editing in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3196-3207},
doi = {10.1111/jipb.70154},
pmid = {41588854},
issn = {1744-7909},
mesh = {*Gene Editing/methods ; *Oryza/genetics ; *Cytosine/metabolism ; *Adenine/metabolism ; *Transcriptional Activation/genetics ; CRISPR-Cas Systems/genetics ; Protein Domains ; *DNA/metabolism ; },
abstract = {CRISPR/Cas12i3 belongs to the type V-I Cas system, characterized by its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif. Developments of Cas12i3-mediated base editing systems for either C-to-T or A-to-G transitions will expand the editing scope and enrich the plant base editing toolkits for crop improvement. However, while the Cas12i3-based cytosine base editor (CBE) only shows very low editing efficiency in plants, its adenine base editor (ABE) has not been documented as yet. Here, we engineered a series of Cas12i3 (5M)-based CBEs (V0-V5) and ABEs (V0-V5) by fusing a deactivated dCas12i3 (5M) with a transactivation module VP64, a single-stranded DNA-binding domain Rad51, or a double-stranded DNA-binding domain HMG-D, or in combinations, and systemically evaluated their performance in rice protoplasts. Our results demonstrated that synergistic combinations of both VP64 and HMG-D outperformed other architectures and significantly boosted the efficiencies of Cas12i3 (5M)-based CBE and ABE for C-to-T and A-to-G base editing and expanded the editing window. In stable lines, in comparison to the non-fusion control, the optimized Cas12i3 (5M)-based CBE-V5 and ABE-V5 enabled up to 4.78- and 3.35-fold higher editing efficiencies, with the maximum C-to-T and A-to-G efficiencies reaching 32.35% and 38.24%, respectively, and a higher proportion of homozygous mutants in the T0 generation. Furthermore, we generated herbicide-resistant rice germplasm by using CBE-V5 and ABE-V5, demonstrating their potential for precision breeding in crops. Together, here, we report novel Cas12i3 (5M)-based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.},
}
@article {pmid41664360,
year = {2026},
author = {Liu, X and Jin, S and Xiao, Z and Ma, C and Wang, Q and Wang, H and Zhou, R and Gu, D and Xu, R and Qin, R and Li, J and Wei, P},
title = {Optimizations of Cas12a- and Cas12i-based adenine base editors for efficient precision editing in the plant genome.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3169-3171},
doi = {10.1111/jipb.70177},
pmid = {41664360},
issn = {1744-7909},
support = {//This work was supported by the Agriculture Science and Technology Major Project, the Science and Technology Major Project of Anhui Province (No. 202423110050063, No. 202423m10050002, and No. 2023n06020020), the Natural Science Foundation of China (No. 32300343, No.32572441, and No. 32570484), and Yangtze River Delta Science and Technology Innovation Community Joint Research (Basic Research) Project (No. 2024CSJZN01100)./ ; },
mesh = {*Adenine/metabolism ; *Genome, Plant/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {A strategy coupling high-activity nucleases with dimeric TadA-8e optimizes plant Cas12-based adenine base editors, boosts editing efficiency, and provides precise editors for crop breeding and genomics research.},
}
@article {pmid41814562,
year = {2026},
author = {Zhong, D and Dong, Y and Pan, H and Fu, Y and Zhao, Y and Ruan, S and Yu, W and Wang, Y and Yin, Q and Zhang, Y and Huang, Y and Shen, J and Zhang, H and Wu, Y and Xu, J and Lu, Y},
title = {CasY7: An optimized Cas12i system for enhanced genome editing in monocot crops.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3208-3219},
doi = {10.1111/jipb.70181},
pmid = {41814562},
issn = {1744-7909},
support = {CAIC (2024) 003//Construction project of Changzhou Modern Agricultural Science and Technology Innovation Center/ ; K2023001//Shanghai Agricultural Science and Technology Innovation Program/ ; 2021YFD1201300//National Key R&amp;D Program of China/ ; },
mesh = {*Zea mays/genetics ; Plants, Genetically Modified ; Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Gene Editing/methods ; Triticum/genetics ; Mutation/genetics ; *CRISPR-Associated Proteins/metabolism ; Base Sequence ; },
abstract = {The CRISPR-Cas12 family nucleases, particularly the Cas12i subtypes, are considered promising alternatives to Cas9 for genome editing in plants. We previously developed a new Cas12i variant, CasY7, which has been successfully applied in clinical trials; its performance in plants remains to be investigated. Initial testing in stable transgenic maize and rice showed that the codon-optimized CasY7 (pCasY7e1) achieved average editing efficiencies of 58.7% and 62.3% across five target sites, respectively, outperforming the typical Cpf1 (pCpf1) control that targets the same sites. To further enhance activity, we fused T5 exonuclease to CasY7 (pCasY7e2), which shifted mutation profiles toward larger deletions, and subsequently integrated an MS2 aptamer into the crRNA scaffold (pCasY7e3). The optimized pCasY7e3 system increased editing efficiencies to 87.7% in maize and 82.9% in rice-approximately 2.7-fold higher than pCpf1. We further demonstrated multiplexed editing in maize, generating biallelic dwarf mutants, and validated functionality in hexaploid wheat with editing efficiencies up to 58.8%. Overall, our comprehensive validation across 942 transgenic plants confirmed robust editing in maize, rice, and wheat, establishing CasY7 as a high-efficiency addition to the CRISPR toolkit.},
}
@article {pmid41834254,
year = {2026},
author = {Ge, F and Peng, C and Du, Y and Chen, Y and Zhao, Z and Yu, M and Feng, H and Xie, Y and Sun, S and Liu, S and Xin, B and Zhao, H and Wu, S and Bian, C and Yang, Z and Lai, J and Chen, J},
title = {Synergistic engineering of Casδ nuclease for robust genome editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3231-3242},
doi = {10.1111/jipb.70222},
pmid = {41834254},
issn = {1744-7909},
support = {2024M753551//Project funded by China Postdoctoral Science Foundation/ ; 2022YFF1002800//National Key Research and Development Program of China/ ; 32572347//National Natural Science Foundation of China/ ; Z231100003723004//Agriculture Science and Technology Major Project, the Beijing Rural Revitalization Agricultural Science and Technology Project/ ; },
mesh = {*Gene Editing/methods ; Humans ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; *Endonucleases/metabolism/genetics ; },
abstract = {Casδ is a recently identified evolutionary transitional CRISPR system characterized by its compact size (~900 amino acids), broad temperature tolerance, and guidance by a short crRNA without the requirement of a tracrRNA. However, the low editing efficiency of Casδ in eukaryotic cells limits its application. Here, we have developed a hierarchical engineering strategy to improve the genome editing activity of Casδ-1, with optimization focused on enhancing its interactions with the crRNA, the protospacer adjacent motif (PAM) duplex, the single-stranded DNA substrate, and the RNA-DNA heteroduplex. Through this strategy, we successfully generated an activity-enhanced Casδ-1 variant, designated enCasδ, which harbors 9 amino acid substitutions that synergistically augment its editing efficiency. In human cell lines, enCasδ showed 1.3- to 29.3-fold higher editing activity than the wild-type Casδ-1 across ten tested genomic loci, with an average editing efficiency of 54.6%. In addition, enCasδ also mediated robust genome editing in maize; its editing efficiency increased by an average of 5.3-fold relative to Casδ-1, and reached up to an average of 80% at the TS4 and PSY1 loci in stable transgenic lines. The overall editing performance of enCasδ was comparable to that of Streptococcus pyogenes Cas9 (SpCas9) and other Cas12 nucleases. Collectively, enCasδ represents a highly optimized Casδ-1 variant that broadens the applicability of the Casδ CRISPR system and facilitates robust genome editing in both animal cells and plants.},
}
@article {pmid41840837,
year = {2026},
author = {Lin, W and Wu, H and Kuang, H and Feng, X and Bai, M and He, F and Liang, R and Zeng, Y and Li, M and Kong, F and Liu, B and Guan, Y},
title = {Developing a robust multiplex CRISPR/Cas12i3-5M system for trait stacking in soybean.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3172-3174},
doi = {10.1111/jipb.70233},
pmid = {41840837},
issn = {1744-7909},
support = {2023YFF1000203//National Key Research and Development Program of China/ ; },
mesh = {*Glycine max/genetics ; *CRISPR-Cas Systems/genetics ; Base Sequence ; Plants, Genetically Modified ; *Quantitative Trait, Heritable ; },
abstract = {The high-efficiency multiplex gene editing technology based CRISPR-Cas12i3-5M is capable of simultaneously editing 13 target sites in soybean, and was used to generate germplasm with high oleic acid content and no beany flavor.},
}
@article {pmid41928060,
year = {2026},
author = {Cheng, Y and Li, G and Zhou, M and Mandlik, R and Wang, D and Qi, Y},
title = {Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3243-3259},
doi = {10.1111/jipb.70249},
pmid = {41928060},
issn = {1744-7909},
support = {MD-PSLA-24014//McIntire Stennis Forest Research/ ; },
mesh = {*Oryza/genetics ; *Introns/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mutation/genetics ; Base Sequence ; Plants, Genetically Modified ; },
abstract = {Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the intron-less counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV-intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.},
}
@article {pmid42021470,
year = {2026},
author = {Liu, M and Wang, Y and Zhang, L and Zhang, X and Wang, X and Liu, X and Fu, Y and Li, X and Song, Z and Liu, Y and Wang, R and Zhao, J},
title = {Optimized Cas-SF01 gene-editing toolbox shortens flowering timing in commercial maize inbred JING724.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3178-3180},
doi = {10.1111/jipb.70264},
pmid = {42021470},
issn = {1744-7909},
support = {2024-zz-087//Beijing Postdoctoral Science Foundation/ ; },
mesh = {*Zea mays/genetics/physiology/growth &amp; development ; *Flowers/physiology/genetics ; *Gene Editing/methods ; Time Factors ; Mutation/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; },
abstract = {The gene-editing tool Cas-SF01 was optimized to maximize its efficiency in maize. The Cas-SF01-TREX2 configuration was superior in enabling high-purity gene mutations. This toolkit enabled commercial maize to flower seven days earlier without yield loss, thereby securing harvests and accelerating crop breeding.},
}
@article {pmid42152501,
year = {2026},
author = {Zhou, J and Li, R and Wang, Z and Liu, S and Shi, L and Fu, X and Li, F and Zhang, J and Li, G and Zhu, J and Qian, Q and Dun, B},
title = {Engineering herbicide-resistant sorghum with CRISPR/Cas9-mediated adenine base editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3292-3294},
doi = {10.1111/jipb.70298},
pmid = {42152501},
issn = {1744-7909},
mesh = {*Sorghum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Herbicide Resistance/genetics ; *Adenine/metabolism ; Plants, Genetically Modified ; *Gene Editing/methods ; *Herbicides/pharmacology ; },
abstract = {An adenine base-editing system was established to precisely modify the sorghum SbALS gene, generating transgene-free mutant plants. These plants exhibit strong herbicide resistance, with no significant differences in agronomic traits, providing valuable germplasm for herbicide-resistance breeding in sorghum.},
}
@article {pmid42206623,
year = {2026},
author = {Bai, Y and Li, B and Peng, J and Bai, Y and Liu, S and Lai, J and Song, W},
title = {Development of fragrant broomcorn millet (Panicum miliaceum L.) via CRISPR/Cas12i.3-mediated genome editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3184-3186},
doi = {10.1111/jipb.70295},
pmid = {42206623},
issn = {1744-7909},
mesh = {*Panicum/genetics ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; },
abstract = {Co-editing the broomcorn millet PmBADH2a and PmBADH2b genes using CRISPR/Cas12i.3 generated double mutants with significantly increased 2-acetyl-1-pyrroline content, producing fragrant broomcorn millet without compromising major agronomic traits.},
}
@article {pmid42367085,
year = {2026},
author = {Zhang, R and Tang, X and Yang, X and Zhang, Y},
title = {Enhanced Cas12i3 system enables precise OsAUX3 editing for rice grain improvement.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3187-3189},
doi = {10.1111/jipb.70337},
pmid = {42367085},
issn = {1744-7909},
support = {//Agriculture Science and Technology Major Project/ ; },
mesh = {*Oryza/genetics ; Plants, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {An optimized Cas12i3 genome-editing system enables highly efficient and predictable editing of regulatory sequences in rice. Precise promoter engineering fine-tunes gene expression, improves grain size, and enhances production potential, demonstrating a powerful new approach for crop improvement through targeted regulation rather than gene disruption.},
}
@article {pmid42446212,
year = {2026},
author = {Cao, X and Liu, H and Bai, S and Wang, R and Xia, L and Sun, Y},
title = {Optimization of a hypercompact Fanzor2 system for improved genome editing performance in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3190-3192},
doi = {10.1111/jipb.70346},
pmid = {42446212},
issn = {1744-7909},
mesh = {*Genome, Plant/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; },
abstract = {Native Fanzor2 nucleases exhibit weak editing activity in plants. An optimized Fanzor2 system engineered via ωRNA optimization and structure-based protein mutagenesis achieves robust editing at recalcitrant genomic sites and enables cytosine base editing, supporting versatile crop genome modification via transformation or viral delivery.},
}
@article {pmid42619441,
year = {2026},
author = {Chen, Z and Chen, Y and Wan, G and Dong, H and Pan, T and Yang, W and Zhou, Y and Luo, B and Zhu, Y and Han, X and Lu, L and Wang, X and Lei, C and Zhao, Z and Wang, J and Ren, Y and Wan, J},
title = {Development of low-prolamin rice germplasm via CRISPR/Cas9 editing to improve eating and cooking quality.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3313-3315},
doi = {10.1111/jipb.70377},
pmid = {42619441},
issn = {1744-7909},
support = {2021YFF1000200//National Key R&amp;D Program of China/ ; 2026//Agriculture Science and Technology Major Project/ ; Y2021YJ18//Central Public-interest Scientific Institution Basal Research Fund/ ; Y2025YC02//Central Public-interest Scientific Institution Basal Research Fund/ ; CAAS-CSNCB-202302//Basic Research Center, Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025-2027//Inner Mongolia Innovation Center of Biological Breeding Technology/ ; CARS-01-05//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Oryza/genetics/metabolism ; *Cooking ; *CRISPR-Cas Systems/genetics ; *Prolamins/metabolism/genetics ; Plants, Genetically Modified ; *Seeds/genetics/metabolism ; Starch/metabolism ; },
abstract = {Simultaneous editing of two major prolamin-encoding genes in rice using a single-guide RNA suppressed prolamin accumulation and reconfigured seed storage protein composition. This targeted modification markedly improved rice eating and cooking quality while maintaining the levels of total starch, total protein, and key agronomic traits.},
}
@article {pmid42641742,
year = {2026},
author = {Chandrasekaran, S and Duraisamy, N and Jagadeesan, M and Palukuri, YK and Tamilselvan, S},
title = {Engineering CRISPR nanoplatforms to deplete cancer stem cells: Delivery checkpoints, target plasticity, and clinical viability.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {76},
number = {},
pages = {103007},
doi = {10.1016/j.nano.2026.103007},
pmid = {42641742},
issn = {1549-9642},
mesh = {*Neoplastic Stem Cells/pathology/metabolism ; Humans ; Animals ; *Nanomedicine/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/pathology ; *Nanoparticles/chemistry ; },
abstract = {Cancer stem cells (CSCs) sustain tumor initiation, therapy resistance, and relapse, yet evade durable control because they switch phenotype, enter quiescence, shelter within protective niches, resist drug efflux, and share markers with normal stem cells. Programmable CRISPR editing can disable intracellular self-renewal dependencies that antibodies and small molecules cannot reach, whereas only nanoscale carriers can confine such editing to intended cells; neither component alone solves the CSC problem. This review reframes CSC-directed CRISPR nanomedicine as an integrated design problem. We examine why target plasticity defeats static single-marker targeting; the sequential delivery checkpoints spanning blood stability, organ selection, tumor penetration, CSC recognition, endosomal escape, and productive editing; and advanced architectures including organ-selective lipid nanoparticles, biomimetic and vesicle carriers, metal-organic frameworks, and logic-gated systems. Genotoxicity, immunogenicity, incomplete depletion, manufacturing reproducibility, and absent CSC-specific clinical evidence remain limiting. Clinical viability, not imminent cure, is the realistic near-term objective.},
}
@article {pmid42681183,
year = {2026},
author = {Zhang, S and Hubbard, BP},
title = {Production of Cell Models with Differential Zygosity Using CRISPR Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {181-196},
pmid = {42681183},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Humans ; Adenine ; },
abstract = {CRISPR base editors have revolutionized the ease with which single-nucleotide gene editing can be performed in cell and organismal models. Unlike conventional CRISPR/Cas9 gene editing, which induces a double-strand DNA break and relies on endogenous homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair, base editors only induce a single-strand nick and exploit the mismatch repair (MMR) system for repair. In many instances, this results in fewer off-target effects and less cytotoxicity. Moreover, because base editors use an alternative repair mechanism, the zygosity of repair is often distinct from that of traditional HDR-based editing. Here, we provide a detailed protocol for performing gene editing using an adenine base editor (ABE) to induce a single A-G transition mutation in a gene of therapeutic relevance. We also illustrate how to sort and isolate cells with differential zygosity state, enabling the production of cell models suited to the study of both dominant and recessive mutations. This workflow can easily be adapted to the use of other variants of base editors (e.g., cytosine base editors) and can be employed in diverse cell lines.},
}
@article {pmid42681222,
year = {2026},
author = {Powell, S and Martin, SA},
title = {CRISPR-Mediated Gene Editing: Generating Gene Knockouts or Precision Base Changes in F0 Xenopus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3049},
number = {},
pages = {141-170},
pmid = {42681222},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *Xenopus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Microinjections ; Xenopus laevis/genetics ; Female ; },
abstract = {CRISPR-mediated gene editing has transformed Xenopus research by enabling targeted, heritable gene disruption in both Xenopus laevis and Xenopus tropicalis. The CRISPR/Cas9 system has allowed efficient loss-of-function analysis within days of injection, overcoming the limitations of transient morpholino knockdowns. Recent advances in CRISPR-mediated base editing technology further expands this toolkit, permitting the precision generation of single-base changes in Xenopus. High editing efficiency, external development, and large clutch size make Xenopus embryos exceptionally suited for genome manipulation and phenotype screening. CRISPR-mediated gene editing in Xenopus has reproduced classic developmental phenotypes and generated robust models of human disease, including ciliopathies, congenital heart defects, skeletal dysplasias, and neurodevelopmental disorders. Disease modeling using Xenopus CRISPR mutants has provided critical insights into conserved vertebrate pathways and the pathogenic mechanisms of human gene variants. Here, we describe the complete process of producing a gene knockout or precision base changes in F0 Xenopus: target selection, design and synthesis of sgRNA, base editor selection and synthesis of base editor mRNA, microinjection into fertilized Xenopus eggs, and genotyping to assess whether gene editing has successfully occurred.},
}
@article {pmid42681238,
year = {2026},
author = {Lun, J and Chesneau, A and Borday, C and Perron, M},
title = {Retinal Degeneration and Regeneration in Xenopus laevis Tadpoles.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3049},
number = {},
pages = {415-431},
pmid = {42681238},
issn = {1940-6029},
mesh = {Animals ; *Retinal Degeneration/genetics/physiopathology/pathology/metabolism ; *Xenopus laevis/genetics/physiology ; Larva/physiology ; *Regeneration ; Nitroreductases/genetics/metabolism ; Metronidazole/pharmacology ; *Retina/physiology ; Rhodopsin/genetics ; Animals, Genetically Modified ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; CRISPR-Cas Systems ; Ependymoglial Cells/metabolism ; Monophenol Monooxygenase/genetics ; Cell Proliferation ; },
abstract = {Xenopus laevis offers unique advantages for studying retinal regeneration due to its strong regenerative capacity and amenability to transgenesis and genome editing. We present a detailed protocol for inducing and monitoring rod photoreceptor degeneration and regeneration in Tg(rho:GFP-NTR) tadpoles, where nitroreductase expression under the rhodopsin promoter enables conditional and specific ablation of rods upon metronidazole exposure. In addition, we describe the generation of albino Tg(rho:GFP-NTR); tyr [ -/-] tadpoles using CRISPR/Cas9-mediated knockout of the tyrosinase gene, providing an alternative to existing albino Tg(rho:GFP-NTR) lines and facilitating real-time fluorescence imaging by reducing pigmentation interference. Finally, we detail methods to assess Müller glial proliferation (BrdU/Sox9 colabeling) and rod neurogenesis (BrdU pulse-chase). This manuscript provides a robust experimental model that enables the investigation of the cellular and molecular mechanisms underlying retinal regeneration in X. laevis.},
}
@article {pmid42681252,
year = {2026},
author = {Wan, W and Ning, J and Yan, A},
title = {Alanine Scanning Analysis of MexB in Multidrug-Resistant Pseudomonas aeruginosa Using the Native Type I-F CRISPR-Cas-Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3044},
number = {},
pages = {185-201},
pmid = {42681252},
issn = {1940-6029},
mesh = {*Pseudomonas aeruginosa/genetics/drug effects ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacterial Outer Membrane Proteins/genetics/metabolism ; *Gene Editing/methods ; *Membrane Transport Proteins/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The rising multidrug resistance (MDR) of the Gram-negative opportunistic pathogen Pseudomonas aeruginosa poses a global public health threat. One key resistance mechanism employed by this pathogen is the overexpression of the resistance-nodulation-cell division (RND) multidrug efflux transporters. MexAB-OprM, a prototype RND efflux pump, confers resistance to most conventional antibiotics except aminoglycosides. Molecules capable of inhibiting efflux pumps, i.e., efflux pump inhibitors (EPIs), have shown promise as therapeutic agents capable of restoring antibiotic efficacy against these superbugs by targeting MexB. Nevertheless, the structure-activity relationship governing substrate transport of MexB in the native genetic background of clinical MDR P. aeruginosa isolates is poorly characterized due to the lack of efficient genetic tools, hindering targeted EPIs development.In this chapter, we introduce a native Type I-F CRISPR-mediated precise genome editing technique that enables in situ alanine substitutions in MexB in clinical MDR P. aeruginosa genotypes. We outline detailed procedures to construct MexB alanine-substitution mutations and methods to examine the expression levels and efflux activities of the constructed MexB mutants. Methods to evaluate the effect of constructed alanine substitutions on antibiotic susceptibilities are also described. These methodologies are expected to provide advanced insights into the substrate recognition and selection mechanism of MexB in the native genetic background of clinical MDR P. aeruginosa isolates to facilitate the development of effective EPIs.},
}
@article {pmid42681339,
year = {2026},
author = {Dong, X and Weiss, RJ},
title = {Detection and Localization of Type II Membrane Proteins by Endogenous Gene Tagging and Confocal Microscopy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3020},
number = {},
pages = {49-64},
pmid = {42681339},
issn = {1940-6029},
mesh = {Microscopy, Confocal/methods ; Humans ; CRISPR-Cas Systems ; *Membrane Proteins/metabolism/genetics ; Animals ; Protein Transport ; HEK293 Cells ; },
abstract = {Type II membrane proteins are single-pass transmembrane proteins distinguished by their N-terminus facing the cytoplasmic side and C-terminus oriented toward the extracellular or luminal side. The localization of type II membrane proteins is crucial for their functional roles, molecular interactions, and biological activity. Studying their dynamics remains challenging due to limited availability of highly specific antibodies and potential artifacts introduced by overexpression systems. In this context, we outline a detailed protocol for monitoring the endogenous localization of type II membrane proteins in mammalian cells. Here, we combine CRISPR/Cas9-mediated endogenous protein tagging with confocal microscopy. This method provides a powerful tool for precisely labeling endogenous proteins and investigating the localization and dynamics of membrane proteins under physiological conditions.},
}
@article {pmid42288725,
year = {2026},
author = {Coleman, S and Tye, J and Furniss, D and Sainsbury, R and Rastogi, A and Xi, X and Murnyak, B and Bell, J and Skeate, J and Wang, M and Webber, B and Moriarity, B and Clement, K},
title = {CRISPRessoSea: streamlined analysis and comparison of pooled amplicon CRISPR screens.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42288725},
issn = {1471-2105},
support = {T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; },
mesh = {*Software ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {BACKGROUND: CRISPR genome editing enables precise modification of genomic targets but may also induce unintended edits at off-target sites with similar sequences. Pooled amplicon sequencing can assess on- and off-target editing across many samples, yet analyzing, aggregating, and visualizing results from multiple pooled experiments remains challenging. Tools to simplify and standardize these analyses are needed to provide reproducible and comparable interpretation of editing data.<br><br>RESULTS: We developed CRISPRessoSea, a software package that processes, compares, and visualizes genome editing rates from pooled amplicon sequencing experiments. The tool provides standardized workflows for analyzing editing across multiple targets and samples, supports both nuclease- and base-editing modalities, and generates clear, data-rich summaries suitable for downstream interpretation.<br><br>CONCLUSIONS: CRISPRessoSea facilitates reproducible, scalable analysis of CRISPR editing outcomes across diverse experimental designs, enabling more efficient and transparent assessment of genome editing specificity. The software is freely available at https://github.com/clementlab/CRISPRessoSea .},
}
@article {pmid42310064,
year = {2026},
author = {Davoodi, P and Atapour, M and Shahsavari, A and Kiani, R},
title = {Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42310064},
issn = {2045-2322},
support = {&#x635;/3/9/22600, (~1000$ for one year).//University of Kurdistan/ ; },
mesh = {Animals ; *Vitellogenins/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Bees/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Exons ; },
abstract = {CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.},
}
@article {pmid42485833,
year = {2026},
author = {J, MA and K, AP and A, AE},
title = {Cold-chain-free CRISPR diagnostics enabled by magnesium-tannic acid nanoencapsulation of the complete detection system.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {268},
number = {Pt 1},
pages = {115989},
doi = {10.1016/j.colsurfb.2026.115989},
pmid = {42485833},
issn = {1873-4367},
mesh = {*Magnesium/chemistry ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; DNA, Single-Stranded/chemistry/genetics ; *Metal Nanoparticles/chemistry ; Polyphenols ; },
abstract = {Cold-chain dependency remains the major barrier to the widespread deployment of CRISPR-based diagnostics, particularly in resource-limited settings, where the thermal instability of protein and nucleic acid reagents necessitates refrigerated storage and transport from manufacture to point of use. Here we report a one-step aqueous nanoencapsulation strategy that overcomes this limitation using magnesium-tannic acid metal-phenolic nanoparticles (Mg[2][+]-TA MPNs). The platform simultaneously co-encapsulates all functional components of a CRISPR-LbCas12a detection system, including the nuclease, guide RNA, and ssDNA reporter, within a single protective matrix. Magnesium ions serve as a structural coordinator of the metal-phenolic nanoparticles while maintaining biochemical compatibility with the released CRISPR system. Physicochemical characterization confirmed successful particle assembly and efficient cargo incorporation. Functional studies demonstrated that encapsulated CRISPR reagents retained greater than 70% of diagnostic activity after 8 days at 50 °C, whereas non-encapsulated controls were completely inactivated within 24 h under identical conditions. Released formulations also remained fully compatible with both fluorescence- and lateral flow-based detection formats. This approach requires no lyophilization, no specialized equipment, and no cold chain at any stage of production or storage, thereby offering a scalable and readily deployable route to thermally stable molecular diagnostics.},
}
@article {pmid42607405,
year = {2026},
author = {Liu, F and Yang, L and Zhang, J and Wang, Y and Xu, F and Ai, Y and Jiang, X},
title = {High-entropy nanozymes integrated with CRISPR/Cas12a cascade amplification for highly sensitive point-of-care detection of cardiac troponin I.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119129},
doi = {10.1016/j.bios.2026.119129},
pmid = {42607405},
issn = {1873-4235},
mesh = {*Troponin I/blood/isolation &amp; purification ; *Biosensing Techniques/methods ; Electrochemical Techniques/methods ; *Myocardial Infarction/diagnosis/blood ; Point-of-Care Systems ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Reproducibility of Results ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Myocardial infarction necessitates rapid and ultrasensitive point-of-care detection of cardiac troponin I (cTnI). In this work, we develop a HEAzyme-enabled interfacial electrocatalytic transduction strategy integrated with CHA-CRISPR/Cas12a amplification framework for ultrasensitive electrochemical detection of cTnI. Capitalizing on the cocktail effect and carbon-shell confinement, HEAzyme amplifies the electrochemical response by catalyzing the redox reaction of surface-confined methylene blue. Target recognition initiates CHA, generating abundant DNA activators for Cas12a trans-cleavage. The synergistic integration of molecular cascade amplification and interfacial electrocatalysis enables a limit of detection of 0.21 fg/mL across a broad linear range from 1 fg/mL to 100 pg/mL. It exhibits good selectivity, maintains good stability within 7 days (RSD = 3.9%), and shows good batch-to-batch reproducibility in different batches (RSD = 3.44 and 5.8% for intra and inter-batch, respectively). This work establishes an effective strategy for integrating CRISPR-based molecular amplification with HEAzyme-enabled interfacial electrocatalytic transduction, providing a promising electrochemical platform for point-of-care diagnosis of myocardial infarction.},
}
@article {pmid42612455,
year = {2026},
author = {Ke, J and Zhang, H and Chen, S and Ma, M and Tang, X and Wei, J and Deng, J and Zhai, J and Luan, T},
title = {Engineering bubble structures as Cas12a activators for highly sensitive monitoring of WRN helicase function.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119123},
doi = {10.1016/j.bios.2026.119123},
pmid = {42612455},
issn = {1873-4235},
mesh = {*Werner Syndrome Helicase/metabolism/genetics/chemistry ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/chemistry/metabolism/genetics ; R-Loop Structures ; RNA/chemistry/genetics ; DNA Replication ; },
abstract = {The Werner syndrome helicase (WRN) is a critical synthetic lethal target in microsatellite instability cancers, essential for resolving complex genomic structures like replication bubbles and R-loops. However, strategies to simultaneously discriminate WRN activity on DNA versus DNA-RNA substrates in living cells are lacking. Here, we developed a structure-specific CRISPR/Cas12a biosensing strategy to visualize WRN functional activity by engineering bubble-structure probes. These probes were rationally designed to structurally mimic DNA replication bubbles and R-loop associated DNA-RNA hybrids. Upon specific unwinding by WRN, the probes release a sequestered activator strand that triggers Cas12a trans-cleavage, effectively converting the unwinding event into an amplified fluorescent signal. This assay achieves low picomolar sensitivity (LODs: 5.6-6.0 pM) and exceptional selectivity against homologous RecQ helicases. Uniquely, this strategy enables the parallel quantification of WRN activity on both substrate types, providing insights into distinct WRN-mediated pathways for resolving genomic stress. We further demonstrated the strategy's utility by visualizing endogenous WRN dynamics in living cells and profiling the efficacy of small-molecule inhibitors. This work offers a powerful molecular toolkit for dissecting WRN biology and facilitating high-throughput drug screening in targeted cancer therapy.},
}
@article {pmid42641278,
year = {2026},
author = {Fu, R and Zhu, C and Hou, J and Wang, Z and Xianyu, Y},
title = {Click-chemistry-mediated modulation of CRISPR-Cas12a activity through activator modification.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119144},
doi = {10.1016/j.bios.2026.119144},
pmid = {42641278},
issn = {1873-4235},
mesh = {*Click Chemistry/methods ; *CRISPR-Cas Systems/genetics ; Azides/chemistry ; DNA/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Biosensing Techniques ; Cycloaddition Reaction ; Alkynes/chemistry ; Molecular Docking Simulation ; Cyclooctanes/chemistry ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; },
abstract = {Chemical modification strategies offer a promising route for spatiotemporal regulation of CRISPR-Cas12a activity in molecular diagnostics. However, existing methods involve CRISPR RNA with photolabile groups that suffer from complexity and RNA instability. To address these limitations, we report a simple and robust strategy using dibenzocyclooctyne (DBCO)-mediated click chemistry to modulate CRISPR-Cas12a activity. The copper-free strain-promoted azide-alkyne cycloaddition reaction enables CRISPR-Cas12a modulation with low toxicity, biocompatibility, and high selectivity. Utilizing azide-modified non-target DNA strand sequences at different locations to react with DBCO, we show that DBCO-modified activators can regulate CRISPR-Cas12a cleavage in three distinct states: maintain, enhance, and suppress. Mechanistic studies through cleavage kinetics and molecular docking reveal that the regulatory outcome depends on the modification position, protospacer adjacent motif composition, and DBCO concentration. We further employ asymmetric polymerase chain reaction to generate azide-modified DNA for click-chemistry-mediated modulation of CRISPR-Cas12a. This strategy could be a promising tool for regulating CRISPR-Cas12a activity in molecular diagnostics.},
}
@article {pmid42641282,
year = {2026},
author = {Wang, Y and Xu, B and Zeng, Z and Wang, H and Shi, L and Liu, W and Chen, Y and Deng, X and Chen, J and Chen, JX},
title = {Cascade-coupled colorimetric-fluorescent dual-signal detection of EGFR-positive extracellular vesicles via bifunctional MOF@Pt nanozyme integrated with a CHA-CRISPR system.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119149},
doi = {10.1016/j.bios.2026.119149},
pmid = {42641282},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; ErbB Receptors/genetics ; Colorimetry/methods ; *Extracellular Vesicles/chemistry ; Platinum/chemistry ; *Glioma/diagnosis/genetics/blood ; CRISPR-Cas Systems/genetics ; MicroRNAs/genetics ; Metal-Organic Frameworks/chemistry ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Metal Nanoparticles/chemistry ; Phthalic Acids ; },
abstract = {Extracellular vesicle (EV)-based liquid biopsy holds great promise for glioma diagnosis, but its clinical translation remains hindered by inefficient isolation of disease-relevant EV subpopulations and insufficiently integrated signal validation. Herein, we develop a platform that integrates a bifunctional nanozyme system (phosphatase-like MOF and peroxidase-like Pt) with a CHA-CRISPR/Cas12a cascade, achieving selective isolation and ultrasensitive detection of EGFR-positive glioma-derived EVs. Defective UiO-66-NH2 loaded with Pt nanoparticles and functionalized with EGFR aptamers (UiO@Pt@Apt) is immobilized on Hook strand-modified glass 96-well plates via Apt-Hook hybridization. Upon introduction of EGFR-positive EVs, they specifically bind to aptamers on UiO@Pt@Apt and induce the release of UiO@Pt@Apt-EV complexes through perturbation and destabilization of the Apt-Hook interface. After EV lysis, EV-derived miRNA-21 activates the CHA-CRISPR/Cas12a cascade to generate a fluorescent signal, while the nucleotide fragments produced by Cas12a trans-cleavage are proposed to be hydrolyzed by phosphatase-like defective UiO-66-NH2 to generate PO4[3-]. The PO4[3-] may contribute to Pt-mediated TMB oxidation, thereby supporting colorimetric signal amplification. The platform achieved a colorimetric EV detection limit of 427 particles/μL after coupling with the CHA-CRISPR system, representing a 6.9-fold improvement over the UiO@Pt@Apt system alone, and enabled a miRNA-21 detection limit of 87.0 fM. In plasma samples from 25 glioma patients and 20 healthy donors, the combined readout achieved an AUC of 0.968, showing numerically better discriminatory performance than either single readout, although the improvement was not statistically significant by DeLong analysis. This work provides a promising strategy for EV-based glioma liquid biopsy and offers a potentially adaptable framework for cascade-coupled dual-signal biosensing.},
}
@article {pmid42648078,
year = {2026},
author = {Ye, T and Xue, M and Zhou, B and Kang, S and Chen, Z and Tang, Z and Yang, S and Zhao, Q and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Biphasic spatiotemporal regulation of Cas12a substrate cleavage enables one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119152},
doi = {10.1016/j.bios.2026.119152},
pmid = {42648078},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; DNA, Single-Stranded/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Catalysis ; },
abstract = {One-pot autocatalytic CRISPR biosensing offers a promising route for signal amplification without nucleic acid pre-amplification, but its efficiency is limited by an intrinsic readout-amplification conflict: the ssDNA reporter required for signal output competes with the autocatalytic mediator required for feedback amplification. Here, we report a biphasic spatiotemporal regulation strategy (BS-Cas12a system) to resolve this substrate competition for one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets. Mechanistic studies revealed that Cas12a preferentially cleaved the ssDNA reporter over the autocatalytic circular mediator, leading to insufficient mediator linearization and impaired autocatalytic amplification. A glycerol/water biphasic system was therefore constructed to spatially delay reporter access to activated Cas12a, allowing preferential cleavage of the autocatalytic mediator and subsequent generation of additional Cas12a activators. This biphasic system achieved a 3-fold increase in the autocatalytic amplification efficiency. By integrating an aptamer-mediated target-to-activator conversion module, non-nucleic-acid recognition was programmably converted into Cas12a activation. Using sulfadimethoxine as a model target, the platform achieved a detection limit of 65 pM and showed good recoveries in fish samples. The system was further extended to Cd[2+] and thrombin detection with limits of detection of 0.65 nM and 11.63 pM, respectively. Moreover, by replacing the fluorescent reporter with a FAM/biotin-labeled reporter, a lateral-flow readout was achieved. This work provides a kinetic-regulation strategy for modular one-pot Cir DNA autocatalytic biosensing with an independent and exchangeable reporter and expands CRISPR-based detection toward diverse non-nucleic-acid targets.},
}
@article {pmid42669262,
year = {2026},
author = {Han, J and Song, Y and Ko, U and Son, SU and Lim, EK and Kim, E},
title = {One-pot dual-toehold RCA-Cas12a biosensor driven by a preassembled three-arm toehold-gated DNA template for sequence-selective miRNA liquid biopsy.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119170},
doi = {10.1016/j.bios.2026.119170},
pmid = {42669262},
issn = {1873-4235},
mesh = {*MicroRNAs/blood/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Liquid Biopsy/methods ; CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Breast Neoplasms/blood/genetics/diagnosis ; Limit of Detection ; Female ; *Endodeoxyribonucleases/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) in blood are promising liquid biopsy biomarkers, yet their short length, low abundance, and high intra-family homology hinder sensitive and specific detection. Combining rolling circle amplification (RCA) with CRISPR-Cas12a enables isothermal detection, but existing methods typically depend on auxiliary enzymes or in-assay ligation and rarely encode sequence discrimination within the template itself. Here, we report a one-pot dual-toehold RCA (dtRCA)-Cas12a biosensor driven by a preassembled three-arm toehold-gated (3TG) DNA template for ultrasensitive and selective miRNA detection. The 3TG template adopts a three-arm dumbbell conformation, eliminating the need for a ligase during the assay, and presents two target-complementary toehold domains with a Cas12a-recognition sequence. Target binding triggers strand displacement, initiating dtRCA via a single polymerase. The resulting amplicons activate Cas12a trans-cleavage for fluorescence or lateral flow assay (LFA) readouts. Crucially, a single-base mismatch within the toehold suppressed amplification, whereas a topology-matched circular template lacking the toehold gate failed to distinguish the target, demonstrating that selectivity arises from the template structure. The one-pot dtRCA-Cas12a system achieved attomolar sensitivity, detecting miR-21, miR-375, and let-7a at 2.5, 114.9, and 8.0 aM, respectively. The paper-based LFA maintained femtomolar sensitivity and enabled an instrument-light readout. In plasma, this platform discriminated breast cancer patients (n = 17) from healthy donors (n = 10) with AUC values of 0.97-0.98. Three-marker classification demonstrated robust performance in leave-one-out cross-validation and correctly classified 30 samples in an independent validation cohort, showing performance comparable to RT-qPCR. By embedding selectivity into a preassembled template, this 3TG-driven dtRCA-Cas12a platform provides a highly sensitive and specific strategy for multi-marker miRNA analysis with simplified readout.},
}
@article {pmid42680688,
year = {2026},
author = {Xhaferri, N and Biswas, S and Davies, B and Lindner, M},
title = {Kcnv2 E151X Mouse Captures Hallmarks of KCNV2-Associated Retinal Dystrophy.},
journal = {Clinical &amp; experimental ophthalmology},
volume = {},
number = {},
pages = {},
doi = {10.1111/ceo.70163},
pmid = {42680688},
issn = {1442-9071},
support = {LI 2846/5-1//Deutsche Forschungsgemeinschaft/ ; LI 2846/6-1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {BACKGROUND: KCNV2-associated retinopathy is a rare inherited retinal dystrophy caused by variants in the KCNV2 gene, leading to disrupted photoreceptor behaviour and progressive deterioration of vision. Patients have characteristic electroretinography abnormalities, including reduced cone response, delayed and reduced rod response to low light flashes and paradoxically large rod-driven response to bright flashes of light. To model this condition, we have generated a Kcnv2 E151X mouse line and assessed its structural and functional retinal features.<br><br>METHODS: We have employed CRISPR/Cas 9 gene editing technology to generate a mouse line with an early stop mutation in position E151-orthologous to the commonly encountered E143X mutation in humans-and performed a combination of immunohistochemistry and Western blot to confirm the absence of the full-length KCNV2-encoded protein, Kv8.2. Next, to assess how closely it models the human disease, we have characterised the KCNV2 mutant mouse line at histological and functional levels, via immunohistochemistry and electroretinography experiments, respectively.<br><br>RESULTS: Kcnv2 mutant mice showed markedly reduced photopic responses and reproduced the supernormal rod phenotype described in affected individuals. In the morphological context, mutant retinas demonstrated strong glial fibrillary acidic protein upregulation together with reduced cone arrestin positive cell counts and photoreceptor layers, indicating photoreceptor loss.<br><br>CONCLUSIONS: The Kcnv2 mutant mouse line replicates key functional and structural hallmarks of KCNV2-associated retinopathy. This model provides a relevant platform for mechanistic studies and preclinical evaluation of gene-based or pharmacological therapies targeting cone and rod photoreceptor dysfunction.},
}
@article {pmid42680732,
year = {2026},
author = {Galal, S and Chaltel Lima, L and Wang, N and Moury, C and Yan, G and Dai, M and Ali, S and Lebrun, JJ},
title = {In vivo CRISPR screening identifies metastasis suppressors in triple-negative breast cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42680732},
issn = {2041-1723},
mesh = {Humans ; Animals ; Female ; *Triple Negative Breast Neoplasms/genetics/pathology ; Cell Line, Tumor ; Mice ; Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; Cell Movement/genetics ; Neovascularization, Pathologic/genetics ; *Genes, Tumor Suppressor ; Neoplasm Invasiveness/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Metastatic cancer remains the leading cause of cancer-related mortality, yet tumor cell-intrinsic mechanisms restraining metastatic dissemination remain incompletely defined. Here, we perform an unbiased in vivo genome-wide CRISPR/Cas9 loss-of-function screen in a breast cancer xenograft model to identify regulators of metastatic progression. This approach uncovers clinically relevant metastasis suppressor genes (MSGs), including VPS45, CMTR2, RBSN, and NF2, whose loss enhances lung colonization. Functional validation demonstrates that depletion of these genes promotes epithelial-to-mesenchymal transition, migration, invasion, intravasation, and angiogenesis, whereas CRISPR-mediated activation suppresses metastatic spread. Integration with patient datasets reveals reduced expression in tumors and associations with advanced disease, with higher expression trending toward improved outcomes. Notably, CMTR2 loss induces vascular remodeling and intratumoral heterogeneity, supporting a role in tumor-vascular interactions. Collectively, this study identifies a network of MSGs that constrain tumor dissemination and highlights the power of in vivo CRISPR functional genomics to uncover regulators of metastatic disease.},
}
@article {pmid42681040,
year = {2026},
author = {Tiefenbacher, J and Sandrock, B},
title = {Generation of Gene Knock-Out Mutants in Ustilago maydis Using Cas9hf Nuclease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3050},
number = {},
pages = {147-161},
pmid = {42681040},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Ustilago/genetics ; *Gene Knockout Techniques/methods ; Mutation ; Basidiomycota ; },
abstract = {Gene disruption of nonessential genes became more convenient with the adaptation of the CRISPR-Cas9 system for Ustilago maydis by the group of Regine Kahmann (MPI Marburg, Germany) in 2016. In our group, we have developed the system further to create defined marker-free gene deletions. Therefore, we used the CRISPR-Cas9 system together with oligonucleotides composed of 40 nucleotides of upstream and downstream flanking regions. Here, we describe the entire way from the decision, which gene of interest should be deleted, to a marker-free U. maydis mutant strain.},
}
@article {pmid42681148,
year = {2026},
author = {Bao, X and Bornert, O and Nyström, A},
title = {Targeting, Mutagenesis, and Functional Testing Strategies for Large Collagens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3022},
number = {},
pages = {3-20},
pmid = {42681148},
issn = {1940-6029},
mesh = {Humans ; *Mutagenesis ; *Collagen Type VII/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Animals ; *Protein Engineering/methods ; *Collagen/genetics/metabolism ; *Gene Targeting/methods ; },
abstract = {Collagens comprise a homogeneous family of cell-surface or extracellular proteins. Many of them are fundamental for health, and consequently, their deficiency or dysregulation occurs in a wide range of diseases, from tissue fragility to neoplastic and fibrosing diseases, to name a few. With the advent of new omics approaches, an increasing number of variants of specific collagens have been discovered in various conditions. Some of these variants lead to a loss of expression, highlighting the need to assess the functions and loss of specific collagens in such conditions, while other variants may involve changes in amino acids of unknown consequences. Thus, with the advent of newer omics technologies, there is an increasing need to engineer specific collagens and their encoding genes. However, for larger collagens, this can be challenging because of their size and repetitive structure. Using collagen VII as an example of a large collagen, we will describe strategies for protein engineering, functional analysis, and collagen gene targeting in cells that naturally express collagens.},
}
@article {pmid42681174,
year = {2026},
author = {Su-Tobon, Q and Niu, J},
title = {CRISPR-Hybrid: Intracellular Selection of CRISPR-Associated Aptamers in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {25-45},
pmid = {42681174},
issn = {1940-6029},
mesh = {*Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Flow Cytometry ; *SELEX Aptamer Technique/methods ; Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; },
abstract = {CRISPR technologies have evolved from nuclease-based genome editing to programmable systems for transcriptional and epigenetic regulations. Emerging CRISPR systems expand editing versatility by incorporating CRISPR-associated aptamers (CAPs) into single-guide RNAs (sgRNAs), enabling recruitment of RNA-binding proteins (RBPs) fused to diverse effectors. However, the limited availability of orthogonal aptamer-RBP pairs has hindered broad application, as conventional SELEX-based aptamer discovery is time-intensive and often fails to yield aptamers functional in cells. We developed the CRISPR-Hybrid platform, an intracellular selection method that directly evolves CAPs within bacterial cells. This system links aptamer-RBP interactions to a fluorescent reporter readout, allowing fluorescence-activated cell sorting (FACS) to enrich functional variants from libraries exceeding 10[[7]] sequences. Each selection round can be completed in 2 days, enabling rapid enrichment of aptamers that retain activity in both bacterial and mammalian contexts. This protocol details construction of randomized DNA libraries, preparation of host cells, execution of intracellular selection and FACS enrichment, and recovery of aptamer sequences for downstream analysis. By providing a fast, in-cell, and physiologically relevant approach to aptamer discovery, CRISPR-Hybrid expands the repertoire of CAPs available for modular and multiplexed CRISPR editing.},
}
@article {pmid42681177,
year = {2026},
author = {Li, M and Chen, C and Tao, P},
title = {CRISPR-Engineered Bacteriophage T4 for Foot-and-Mouth Disease Nanoparticle Vaccine Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {87-100},
pmid = {42681177},
issn = {1940-6029},
mesh = {*Bacteriophage T4/genetics/immunology ; *Foot-and-Mouth Disease Virus/immunology/genetics ; Animals ; *Foot-and-Mouth Disease/prevention &amp; control/immunology/virology ; *Vaccine Development/methods ; Epitopes, T-Lymphocyte/immunology/genetics ; *CRISPR-Cas Systems ; Epitopes, B-Lymphocyte/immunology/genetics ; *Viral Vaccines/immunology/genetics ; Nanovaccines ; Nanoparticles/chemistry ; Capsid Proteins/genetics/immunology ; Protein Subunit Vaccines ; },
abstract = {Peptide-based vaccines offer a safer alternative to inactivated vaccines. However, the immunogenicity of the peptides is usually poor, and therefore, adjuvants or delivery systems are required. Bacteriophage T4, with its intrinsic immunostimulatory properties, provides a promising platform for antigen display. Here, we used CRISPR-Cas genome editing to insert a gene encoding a foot-and-mouth disease virus (FMDV) B-cell epitope (VP1130-158) into the C-terminus of the soc gene in the T4 genome. The T4 phage self-assembly system enables the display of epitopes on the capsid surface in vivo, generating VP1130-158-T4 virus-like particles. To further enhance immune activation, CD4[[+]] T-cell epitopes FMDV 3A21-35 or tetanus toxoid P2830-844 were fused downstream of the VP1 epitope. These recombinant T4 phages provide proof-of-concept for the development of safe, epitope-based FMDV vaccines.},
}
@article {pmid42681180,
year = {2026},
author = {Wang, Y and Li, X and Huang, Y and Zhong, M and Yang, H},
title = {CRISPR-Cpf1-Mediated T4 Phage Genome Editing for One-Step In Vivo Display of Heterologous Protein.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {135-145},
pmid = {42681180},
issn = {1940-6029},
mesh = {*Bacteriophage T4/genetics ; *Capsid Proteins/genetics/metabolism ; *Gene Editing/methods ; Escherichia coli/genetics ; *Genome, Viral ; *CRISPR-Cas Systems ; Recombinant Proteins/genetics ; },
abstract = {The T4 phage is a robust vector for high-density heterologous protein display. It leverages two non-essential outer capsid proteins, i.e., Soc (~870 copies) and Hoc (~155 copies). These two proteins enable the efficient display of target proteins on the capsid of T4. Here, we detail the workflow for one-step in vivo display of a heterologous protein. Specifically, this method utilizes CRISPR-Cpf1-mediated gene editing technology to insert the sequence of interest (using mCherry as an example) downstream of the Soc encoding gene within the T4 phage genome, resulting in a Soc-fused recombinant protein. This engineering method allows for endogenous expression of the Soc-mCherry recombinant protein within Escherichia coli cells during phage replication, after which the recombinant protein spontaneously assembles onto the capsid of the engineered phage. By providing a universal framework suitable for in vivo display, this approach empowers researchers to readily construct tailored T4 nanoparticles for diverse biotechnological applications.},
}
@article {pmid42681181,
year = {2026},
author = {Mahdavi-Amiri, Y and Kim, SB},
title = {Methods for Applying Prime Editing and Inverse Prime Editing in Mammalian Cell Culture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {147-161},
pmid = {42681181},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Animals ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Flow Cytometry ; Cell Culture Techniques/methods ; Moloney murine leukemia virus/genetics/enzymology ; High-Throughput Nucleotide Sequencing ; },
abstract = {Prime editing (PE) is a powerful method for introducing point mutations into the genomes of living organisms. PE utilizes a Cas9 nickase fused to an engineered Moloney Murine Leukemia Virus reverse transcriptase (MLV-RT), paired with an extended guide RNA known as pegRNA, which contains a primer binding site (PBS) and a reverse transcriptase template (RTT) complementary to the non-target strand DNA. Recently described inverse prime editing (iPE) also employs reverse transcriptase and pegRNAs; however, it utilizes an RNA template complementary to the target strand, resulting in the polymerization of target-strand DNA in the opposite direction. In this work, we provide a practical protocol for using either PE or iPE to introduce point mutation(s) or small-to-medium sized insertions and deletions in cell culture, and demonstrate how to assess editing efficiency via flow cytometry and next-generation sequencing. We include recommendations for prime editor selection and straightforward guidelines for pegRNA design, intended for researchers unfamiliar with genome editing technologies.},
}
@article {pmid42681182,
year = {2026},
author = {Golla, DA and Daniel, TC and Haugh, L and Gao, X},
title = {Constructing Drive-and-Process (DAP) CRISPR Guide RNA Arrays for Multiplexed Base- and Prime-Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {163-180},
pmid = {42681182},
issn = {1940-6029},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Cas Systems ; RNA, Transfer/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Advancements in base- and prime editing technologies in recent years have offered researchers a plethora of options for introducing precise insertions, deletions, or substitutions into targeted genomic loci. These precision editors' applications have rapidly expanded to the modeling and treatment of polygenic diseases, as well as into the growing field of functional genomics. This has created a need for compact, modular expression systems capable of producing multiple guide RNAs (gRNAs) from a single transcript while preserving high editing efficiency. To address this, we have developed the drive-and-process (DAP) array, a modular architecture composed of alternating gRNA and tRNA units. The DAP array design exploits the cell's endogenous tRNA processing machinery to cleave each tRNA from the array and release individual gRNAs, thereby enabling simultaneous editing at multiple loci following hybridization with Cas9. Here, we outline key design considerations and experimental steps required for the construction and deployment of DAP arrays as multiplex base- or prime editing tools in human cells (e.g., HEK293T). We also highlight how the DAP array can be leveraged to enable efficient processing of gRNAs along with other RNAs of similar size, such as shRNA, potentially broadening its usage in addressing complex biological questions and therapeutic applications that require coordinated genetic perturbation.},
}
@article {pmid42240879,
year = {2026},
author = {Jeong, H and Kim, H and Cho, E and Lee, HK and Kim, DY and Keum, B and Jung, C},
title = {Enhancing Next-Generation Sequencing Sensitivity with High-Recovery Adapter Ligation and Cas9-Mediated Dimer Elimination.},
journal = {Clinical chemistry},
volume = {72},
number = {9},
pages = {973-983},
doi = {10.1093/clinchem/hvag052},
pmid = {42240879},
issn = {1530-8561},
support = {//Technology Innovation Program/ ; 20009356//Ministry of Trade, Industry &amp; Energy/ ; //National Research Foundation of Korea/ ; 2021-NR061248//Korean government/ ; RS-2024-00440975//Korean government/ ; //Bio &amp; Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2022-NR067272//Ministry of Science &amp; ICT/ ; RS-2023-00259824//Ministry of Science &amp; ICT/ ; },
mesh = {Humans ; *High-Throughput Nucleotide Sequencing/methods ; *CRISPR-Cas Systems ; *Circulating Tumor DNA/genetics/blood ; Sensitivity and Specificity ; Gene Library ; Sequence Analysis, DNA/methods ; },
abstract = {BACKGROUND: Accurate detection of ultra-low-frequency variants is a major challenge in clinical liquid biopsy. In early cancer detection and minimal residual disease monitoring, Circulating tumor (ctDNA) may fall below 0.1% variant allele frequency, making sensitivity highly dependent on molecular recovery during library preparation. Losses at early steps, especially adapter ligation, permanently reduce analyzable molecules and cannot be rescued by deeper sequencing or bioinformatic refinement.<br><br>METHODS: We developed Powerful Recovery and Improved Dimer Elimination (PRIDE) next-generation sequencing NGS, a library preparation strategy that increases adapter ligation efficiency and removes adapter dimers via sequence-specific Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) cleanup. PRIDE NGS is compatible with standard clinical work flows and requires no added sequencing depth or changes to downstream bioinformatic pipelines. Performance was assessed by targeted sequencing of cell-free (cfDNA) reference standards and clinical plasma samples.<br><br>RESULTS: PRIDE NGS improved recovery and detection of low-frequency variants vs conventional preparation. In reference standards, it detected more variants at low allele frequencies, particularly below 0.1%. In clinical plasma samples, it similarly increased detection, including variants predicted to have moderate or high functional impact. These gains occurred at comparable or lower sequencing depth, indicating sensitivity improvements driven by enhanced molecular recovery.<br><br>CONCLUSIONS: By overcoming a key bottleneck in library preparation, PRIDE NGS lowers the practical detection threshold for ultra-low-frequency variants in liquid biopsy. This clinically applicable approach improves analytical sensitivity by lowering the detection limit without increasing the sequencing burden, supporting routine testing and longitudinal monitoring.},
}
@article {pmid42261770,
year = {2026},
author = {Zhang, L and Yang, C and Yao, Q and Du, X and Ding, S and Shi, Y and Sheng, C and Wang, M and Han, Y and Luo, H},
title = {Machine Learning-Enhanced Ultrasensitive Immuno-CRISPR Array Facilitates Early Diagnosis of Alzheimer's Disease by Detecting Multiple Plasma Biomarkers.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e75983},
pmid = {42261770},
issn = {2198-3844},
support = {82401859//National Natural Science Foundation of China/ ; XKTP2025B04//First-class Discipline Breakthrough Initiative of Hainan University/ ; 826MS0122//Hainan Provincial Natural Science Foundation of China/ ; 826QN0570//Hainan Provincial Natural Science Foundation of China/ ; 825QN284//Hainan Provincial Natural Science Foundation of China/ ; },
mesh = {*Alzheimer Disease/diagnosis/blood ; Humans ; *Biomarkers/blood ; *Machine Learning ; Early Diagnosis ; *Amyloid beta-Peptides/blood ; Sensitivity and Specificity ; tau Proteins/blood ; CRISPR-Cas Systems/genetics ; },
abstract = {Early and accurate diagnosis of Alzheimer's disease (AD) remains a significant challenge due to the multifactorial and dynamic nature of its pathology. Although plasma-based biomarkers such as amyloid-β (Aβ) and phosphorylated tau (p-tau) have shown promise as diagnostic indicators, current single-biomarker detection techniques lack the requisite sensitivity and specificity for early-stage diagnosis. Here, we present the development of an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) capable of concurrently detecting six core AD biomarkers, including Aβ40, Aβ42, p-tau[181], p-tau[217], p-tau[231], and p-tau[396,404]. By integrating antibody pair-based multiplex recombinase polymerase amplification (RPA) with spatially encoded CRISPR-Cas12a detection, the UCMDA achieves a detection limit of 1 fg/mL, which is 10 000-fold more sensitive than conventional ELISA. Clinical validation in a cohort of 155 plasma samples demonstrated that logistic regression (LR)-based integration of the six biomarkers significantly enhanced diagnostic performance, with the multi-biomarker model substantially outperforming single-biomarker approaches in diagnosing AD-MCI and AD. This platform offers a scalable, cost-effective, and minimally invasive strategy for early detection and disease monitoring. This work highlights the potential of CRISPR-based multiplex protein detection technologies combined with machine learning-assisted analysis to enhance the precision of diagnosing neurodegenerative disorders.},
}
@article {pmid42295796,
year = {2026},
author = {Miller, MA and Vo, N and Utkarsh, and Sokirniy, I and Pritchard, J and Freedman, BS and Medina, SH},
title = {Ultrasound-Actuated Gene Editing in Human Kidney Organoids.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e20402},
pmid = {42295796},
issn = {2198-3844},
support = {R21DK128638/GF/NIH HHS/United States ; R35GM142902/GF/NIH HHS/United States ; U01DK127553/GF/NIH HHS/United States ; U01AI176460/GF/NIH HHS/United States ; R21DK128638/DK/NIDDK NIH HHS/United States ; R35GM142902/GM/NIGMS NIH HHS/United States ; U01DK127553/DK/NIDDK NIH HHS/United States ; U01AI176460/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Organoids/metabolism ; *Kidney/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; },
abstract = {Efficient delivery of gene editing ribonucleoproteins (RNPs) into the interior of solid tissues remains a key hurdle to the clinical translation of non-viral CRISPR-Cas9 technologies. Here, we report acoustically-actuated peptide nanoemulsions (NPeps) that can be spatiotemporally guided and activated by ultrasound to ballistically deliver RNPs into cells within the bulk of dense 3D cellular structures. Using human kidney organoids as a model, we demonstrate NPep vectors improve the spatial profile of gene editing in the organoid mass relative to commercial lipofection reagents, without disruption of tissue structure or qualitative viability features. This technologic paradigm is poised to advance imaging-guided, deep tissue RNP delivery modalities to expand the clinical diagnostic and therapeutic potential of CRISPR-Cas9 editing strategies.},
}
@article {pmid42360136,
year = {2026},
author = {Ma, L and Yao, P and Wu, S and Shi, Y and Qin, L and Li, B and Zhu, J and Huang, M and Zhu, Y and Song, Y and Pang, J and Guo, Z and Wu, G and Wang, C and Xu, K and Huang, R and Kuang, Q and Qu, L and Pan, C and Xie, X and Zhu, Q and Huang, J and Lin, Q},
title = {Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e75888},
pmid = {42360136},
issn = {2198-3844},
support = {2024YFC3408200//National Key R&amp;D Program of China/ ; 2024YFF1000800//National Key R&amp;D Program of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; 20253BAC260005//Frontier Technology Program of Jiangxi Provincial Natural Science Foundation/ ; 2023-NJS-00-012//Invigorate the Seed Industry of Guangdong Province/ ; 32422050//National Natural Science Foundation of China/ ; 32401250//National Natural Science Foundation of China/ ; 2023ZT10N019//Young Elite Scientists Sponsorship Program of the China Association for Science and Technology, the Guangdong Provincial "Pearl River Talent Program" Innovation and Entrepreneurship Team Project/ ; AB24153006//Key R&amp;D Program of Guangxi Province/ ; 2025A04J7124//Science and Technology Projects in Guangzhou/ ; 2025A04J3669//Science and Technology Projects in Guangzhou/ ; 2023B10564004//specific university discipline construction project/ ; },
mesh = {Animals ; *Gene Editing/methods ; Rats ; *RNA-Directed DNA Polymerase/genetics/metabolism ; *Endogenous Retroviruses/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.},
}
@article {pmid42470685,
year = {2026},
author = {Hou, C and Yang, R and Guan, X and Zhang, J and Guo, C and Zhang, S and Pei, M and Schalper, KT and Schreiber, D and Liu, X and Liu, C},
title = {Wax-in-a-Tube: A Simple, Rapid, One-Pot Platform for Molecular Detection.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {6673-6681},
doi = {10.1021/acssensors.5c04476},
pmid = {42470685},
issn = {2379-3694},
support = {U01CA269147/CA/NCI NIH HHS/United States ; R01AI194917//National Institute of Allergy and Infectious Diseases/ ; R01EB023607/EB/NIBIB NIH HHS/United States ; },
mesh = {*DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Herpesvirus 2, Human/genetics/isolation &amp; purification ; Humans ; Rapid Diagnostic Tests ; Recombinases/metabolism ; CRISPR-Cas Systems ; },
abstract = {CRISPR technology has emerged as a powerful platform for highly sensitive and specific nucleic acid detection, particularly when coupled with isothermal amplification. However, conventional two-step CRISPR assays still require manual operations, such as shaking, centrifugation, or vortexing, that complicate the workflow and increase the risk of aerosol contamination. Here, we present a wax-in-a-tube (WIAT) platform that enables simple, rapid, one-pot recombinase polymerase amplification (RPA) and CRISPR-based detection by leveraging the phase-change properties of a molded wax separator, thereby eliminating additional manual steps and minimizing contamination risks. Using a molding approach, we directly integrated the wax separator into the reaction tube to physically partition different reaction components. The WIAT platform achieved a detection sensitivity of 10 aM for HSV-2 DNA, comparable to that of standard two-step assays. We further validated its clinical performance using HSV-2 swab samples, demonstrating results comparable to those obtained with PCR. Together, these findings establish the WIAT platform as a simple, rapid, and highly sensitive one-pot RPA-CRISPR assay with strong potential for point-of-care infectious disease detection and early surveillance.},
}
@article {pmid42486986,
year = {2026},
author = {Gao, Y and Ma, Y and Yu, K and Liu, Y and Gu, B and Tang, H and Yan, W and Yang, S and Su, J and Wang, X and Ma, X and Wang, X and Wang, F and Li, Q and Liu, M and Wang, H},
title = {Efficient and precise programmable DNA knock-in without double-strand breaks.},
journal = {Nature},
volume = {657},
number = {8130},
pages = {284-294},
pmid = {42486986},
issn = {1476-4687},
mesh = {*Gene Knock-In Techniques/methods ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Editing/methods ; *DNA/genetics ; Deoxyribonuclease I/metabolism ; Receptors, Chimeric Antigen/genetics ; Mice ; Receptors, Antigen, T-Cell/genetics ; INDEL Mutation/genetics ; },
abstract = {Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging[1,2]. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.},
}
@article {pmid42571623,
year = {2026},
author = {Zhou, M and Du, K and Jiang, M and Xu, X and Su, X and Xie, Y and Zhang, D and Sun, X and Peng, G and Xia, K and Hu, Z},
title = {FOCUS: A Dual-Mismatch crRNA Strategy Unlocks High-Fidelity One-Step SNV Detection with Cas12a.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {7335-7348},
doi = {10.1021/acssensors.6c01689},
pmid = {42571623},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; Base Pair Mismatch ; DNA/genetics ; Rapid Diagnostic Tests ; },
abstract = {CRISPR/Cas12a has emerged as a powerful tool for nucleic acid detection; however, its clinical utility is severely hampered by intrinsic limitations in single-nucleotide variant (SNV) discrimination, reliance on pre-processed single-stranded DNA (ssDNA) templates, and cumbersome multi-step workflows. Here, we report a novel molecular design principle by engineering crRNAs with site-specific dual mismatches (positions 12 and 14 relative to the PAM), which we systematically demonstrate to drastically enhance the SNV discrimination capability of Cas12a. Leveraging this breakthrough, we developed FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system), an all-in-one CRISPR sensing platform that enables isothermal detection of SNVs from double-stranded DNA (dsDNA) by integrating amplification and detection in a single reaction system. FOCUS achieved attomolar-level sensitivity (13.15 aM) and ultrafast readout (< 20 min) for distinguishing the highly homologous survival motor neuron 1 (SMN1) and SMN2 genes-the gold standard challenge for SNV genotyping in spinal muscular atrophy (SMA) diagnostics. To validate its clinical translatability, FOCUS was successfully adapted to a low-cost, equipment-free assay using lateral flow strips and UV visualization, facilitating point-of-care testing (POCT). In a comprehensive validation across 175 clinical samples, FOCUS exhibited 100% diagnostic concordance with gold-standard methods for SMA (21 samples), high-risk HPV 16/18 (27 samples), Staphylococcus aureus (20 samples), and SARS-CoV-2 (107 samples). Collectively, our study establishes a generalizable engineering strategy for Cas12a crRNAs and presents FOCUS as a robust, versatile, and field-deployable solution for precision SNV genotyping, underscoring the translational medicine value of FOCUS in molecular diagnostics.},
}
@article {pmid42608059,
year = {2026},
author = {Chen, Z and Zhou, J and Galli, M and Iohannes, SD and Clark, T and Debernardi, JM and Dubcovsky, J and Jackson, D and Gallavotti, A},
title = {The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.},
journal = {The New phytologist},
volume = {252},
number = {1},
pages = {260-275},
doi = {10.1111/nph.71508},
pmid = {42608059},
issn = {1469-8137},
support = {2424271//Division of Molecular and Cellular Biosciences/ ; 1916804//Division of Integrative Organismal Systems/ ; },
mesh = {*Zea mays/genetics/physiology/embryology ; Plants, Genetically Modified ; *Regeneration/genetics ; *Transformation, Genetic ; *Plant Leaves/physiology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Triticum/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.},
}
@article {pmid42669708,
year = {2026},
author = {Boggess, SC and Gandhi, V and Tsai, MC and Marzette, E and Teyssier, N and Chou, JY and Hu, X and Cramer, A and Yadanar, L and Shroff, K and Jeong, CG and Eidenschenk, C and Hanson, JE and Tian, R and Kampmann, M},
title = {A massively parallel CRISPR-based screening platform for modifiers of neuronal depolarization.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42669708},
issn = {2041-1723},
support = {U54 NS123746/NS/NINDS NIH HHS/United States ; 23AARF-1027616/ALZ/Alzheimer's Association/United States ; EDUC2-12730//California Institute for Regenerative Medicine (CIRM)/ ; U54 NS123746//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Humans ; *Neurons/metabolism/physiology ; Induced Pluripotent Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Calcium/metabolism ; },
abstract = {Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2 to evaluate genetic modifiers of neuronal depolarization. Using this screening method, we evaluated 1343 genes for their effect on depolarization in a human iPSC-derived neuron model, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder and Alzheimer's disease not previously described to affect neuronal depolarization. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal function in health and disease.},
}
@article {pmid42669834,
year = {2026},
author = {},
title = {Correction to "Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON)".},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e6219762},
doi = {10.1002/anie.6219762},
pmid = {42669834},
issn = {1521-3773},
}
@article {pmid42670258,
year = {2026},
author = {Ng, CF and Krishnamurthy, D and Dextre, A and Chorlay, A and Ott, M and Fletcher, DA},
title = {LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {16},
pages = {},
pmid = {42670258},
issn = {1362-4962},
support = {//Schmidt Science Fellowship/ ; //Rhodes Trust/ ; //Burroughs Wellcome Career Award/ ; //European Molecular Biology Organization/ ; DBI-1548297//National Science Foundation/ ; //Wagner Foundation/ ; 4R33AI140465-04//National Institute of Allergy and Infectious Diseases/ ; //James B. Pendleton Charitable Trust/ ; //Gordon and Betty Moore Foundation/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Kinetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; Ultraviolet Rays ; Photolysis ; Light ; },
abstract = {CRISPR diagnostics enable sensitive detection of infectious diseases, with the RNA endonuclease Cas13a providing specific, amplification-free RNA detection through collateral trans-cleavage of fluorescent reporters. However, background cleavage from unbound enzyme, contaminating nucleases, and unsynchronized initiation of reactions limits assay sensitivity and interpretability. A strategy to precisely control the onset of Cas13a catalytic activity, essentially a molecular "starting gun," would address these challenges. Here, we introduce Light-Uncaged Cas13a (LUCas), a light-controllable system that directly blocks Cas13a trans-cleavage activity using a photocleavable interfering guide RNA, even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an ~100-fold suppression of trans-cleavage activity prior to photo-uncaging, including suppression of target-independent background activity. Using measured kinetic parameters, we predict and experimentally validate the limit of detection of the LUCas system for direct detection. We further demonstrate a multiplexed detection strategy termed "temporal barcoding," enabling quantitative detection of viral co-infections in a single bulk reaction. Finally, LUCas is shown to be compatible with one-pot isothermal amplification for enhanced sensitivity and direct detection of target RNA spiked into blood plasma. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.},
}
@article {pmid42670666,
year = {2026},
author = {Mirzaee, Z},
title = {Advancing antimicrobial peptides: Mechanisms, design, and applications in the post-antibiotic era.},
journal = {Protein and peptide letters},
volume = {34},
number = {1},
pages = {19-30},
doi = {10.1016/j.ppl.2026.07.002},
pmid = {42670666},
issn = {1875-5305},
mesh = {*Antimicrobial Peptides/chemistry/pharmacology/therapeutic use ; Humans ; *Drug Design ; Protein Engineering ; Animals ; Structure-Activity Relationship ; *Anti-Bacterial Agents/chemistry/pharmacology ; Bacteria/drug effects ; *Antimicrobial Cationic Peptides/chemistry/pharmacology ; },
abstract = {The rapid emergence of multidrug-resistant and extensively drug-resistant bacteria has intensified the need for alternative antimicrobial strategies in the post-antibiotic era. Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immunity, have attracted considerable attention due to their broad-spectrum antimicrobial activity, rapid mechanisms of action, and lower propensity for resistance development. This review summarizes the structural diversity, mechanisms of action, and structure-activity relationships (SAR) of AMPs that underpin their biological activity and guide the rational design of next-generation peptide therapeutics. It further discusses recent advances in peptide engineering, peptidomimetic design, machine learning-assisted discovery, innovative production platforms, and the application of CRISPR-Cas genome editing for production host optimization. In addition, the review highlights synergistic therapeutic strategies, current clinical progress, and the expanding applications of AMPs in medicine, food preservation, agriculture, and aquaculture. Despite these advances, challenges including limited stability, potential toxicity, manufacturing costs, and regulatory barriers continue to hinder widespread clinical translation of AMP-based therapeutics. By integrating recent experimental and computational advances with current translational challenges and future perspectives, this review provides a comprehensive overview of the field and highlights key directions for the rational development and clinical translation of next-generation antimicrobial peptides to combat antimicrobial resistance.},
}
@article {pmid42671221,
year = {2026},
author = {Xu, Y and He, X and Xu, T},
title = {LAMP-CRISPR Integrated Platforms for Rapid Detection of Microbial Pathogens: Principles, Applied Strategies, and the Road to Field Translation.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag220},
pmid = {42671221},
issn = {1365-2672},
abstract = {Loop-mediated isothermal amplification (LAMP) integrated with CRISPR-Cas systems has emerged as a promising molecular diagnostic platform for the rapid detection of microbial pathogens. By combining the efficient nucleic acid amplification of LAMP with the sequence-specific recognition capability of CRISPR-Cas effectors, these platforms offer potential advantages in analytical sensitivity, specificity, operational simplicity, and field applicability. In this review, we summarize the principles, assay formats, and recent advances of LAMP-CRISPR technologies for detecting a broad spectrum of microbial pathogens, including bacterial, viral, fungal, and parasitic agents across clinical, veterinary, food safety, environmental, and agricultural applications. Representative studies are compared with attention to pathogen type, sample matrix, assay design, CRISPR-Cas system, readout format, analytical performance, and practical application. We further discuss major technical challenges that continue to hinder practical implementation, particularly complex sample pretreatment, workflow integration, carry-over contamination, reagent stability, multiplexing capability, and platform standardization. Attention is given to sample pretreatment and system-level integration, including current extraction and rapid lysis strategies, closed-tube reactions, portable readouts, and microfluidic or cartridge-based formats, which may support simplified "sample-in, answer-out" diagnostic workflows. Finally, we outline future directions for improving matrix-adapted sample processing, assay robustness, standardized validation, large-scale evaluation, and field deployment. This review provides a structured overview of current LAMP-CRISPR platforms and highlights key technological considerations for translating rapid microbial pathogen detection from laboratory research to real-world applications.},
}
@article {pmid42673787,
year = {2026},
author = {Lv, L and Zhang, Y and Fan, Y and Guo, B and Chen, Y},
title = {Bivalent aptamer-assisted CRISPR-Cas12a sensor for precise vancomycin therapeutic drug monitoring.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130504},
doi = {10.1016/j.talanta.2026.130504},
pmid = {42673787},
issn = {1873-3573},
abstract = {Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.},
}
@article {pmid42674013,
year = {2026},
author = {Liu, H and Liu, Y and Xu, Y and Wang, Z and Yu, Y and Su, G and Qu, G},
title = {Inhibitor-Regulated Cas12a Activation Enables Highly Sensitive and One-Pot Detection of Drug-Resistant Genes in River Water.},
journal = {Analytical chemistry},
volume = {98},
number = {33},
pages = {24024-24034},
doi = {10.1021/acs.analchem.6c00793},
pmid = {42674013},
issn = {1520-6882},
support = {22325606//National Natural Science Foundation of China/ ; 22576049//National Natural Science Foundation of China/ ; 2024HIAS-V001//Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences/ ; 2025ZY01044//Central Guiding Local Science and Technology Development Fund Projects/ ; },
mesh = {*Rivers/microbiology/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {Monitoring environmental drug-resistance genes (DRGs) plays a pivotal role in preventing the transmission of antimicrobial resistance, thereby reducing public health risks. In this study, a one-pot recombinase polymerase amplification (RPA)/clustered regularly interspaced short palindromic repeat (CRISPR) assay was developed for monitoring DRGs in river water. To overcome compatibility challenges between RPA and CRISPR systems, four glycosaminoglycans (heparin sodium, nadroparin calcium, dalteparin sodium, and chondroitin sulfate A sodium salt) with different molecular weights or negative charge density were evaluated as Cas-enzyme activity modulators. Among them, heparin sodium with the high molecular weight and high strong negative charge density exhibited the best performance in the one-pot DRG detection assay. In the system, CRISPR-Cas12a activity was temporarily inhibited during the RPA amplification phase. When sufficient amplicons were accumulated, Cas12a was activated for signal readout, thereby achieving orderly coupling and precise control of both reactions. To further simplify and improve the reliability of environmental DRG monitoring, a pretreatment method that can eliminate nucleic acid extraction was developed and integrated with the inhibitor-controlled one-pot platform. This assay achieved high sensitivity and specificity when it was applied to river samples, matching the performance of qPCR. The developed assay is simple to operate, has high sensitivity, and is widely adaptable, providing a robust tool for rapid antimicrobial resistance surveillance and exhibiting promise for public health management applications.},
}
@article {pmid42674033,
year = {2026},
author = {Lin, S and Chen, X and Lang, Z and Lu, B and Jia, Y and Ju, H and Cao, H},
title = {Programmable Versatile Socket: A Tight-Locking and High-Gain CRISPR/Cas12a Molecular Circuit for Ultrasensitive Sensing of Diverse Targets.},
journal = {Analytical chemistry},
volume = {98},
number = {33},
pages = {24577-24592},
doi = {10.1021/acs.analchem.6c04985},
pmid = {42674033},
issn = {1520-6882},
support = {ZDYF2026SHFZ032//Key Research and Development Project of Hainan Province/ ; 2025DNJP0208//International Cooperative Research Project/ ; 22104027//National Natural Science Foundation of China (NSFC)/ ; 22364014//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; *Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; RNA/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/genetics ; },
abstract = {CRISPR/Cas12a has emerged as an important platform for nucleic acid analysis, yet limited catalytic turnover and intrinsic nucleic acid specificity constrain its sensitivity and analyte scope. Herein, a tight-locking and high-gain Cas12a-driven strand displacement amplification (CSDA) molecular circuit is developed as a versatile socket without preamplification for programmable sensing of nucleic acid and non-nucleic acid analytes. CSDA relies on an RNA-DNA three-strand hairpin (RD-TSH) switch containing a 2-nt mismatch. RD-TSH suppresses nonspecific amplification and unintended Cas12a self-activation to ensure tight locking. Screening the number of mismatched bases in RD-TSH and molecular-level mechanistic analyses reveal a DNA breathing-driven two-step unlocking mechanism. Only complete unlocking triggers autocatalytic CSDA, allowing high-gain amplification. The sequence-independent unlocking of RD-TSH confers high orthogonality to CSDA, enabling target-specific modules to be coupled to the CSDA socket as interchangeable plugs via programmable crRNA guides, thus achieving universal detection of both nucleic and non-nucleic analytes. Using Vibrio parahaemolyticus DNA, thermostable direct hemolysin, and aflatoxin B1 as representative targets, CSDA achieved ultrasensitive detection in complex matrices with sensitivity improvements of over 5 orders of magnitude, 42-fold, and 602-fold, respectively. This plug-and-play architecture establishes CSDA as a broadly adaptable and ultrasensitive CRISPR/Cas12a sensing socket, providing a general route toward programmable sensing of diverse analyte classes and a promising strategy for more accurate integrated multitarget analytical platforms.},
}
@article {pmid42675078,
year = {2026},
author = {Tsui, W and Yang, Y and Wang, C and Li, D and Zhang, Y and Zhao, X and Wu, J and Guo, J and Wang, Y and Cheng, X and Li, X and Kotovskaya, O and Isaev, A and Ma, J and Wang, M},
title = {Dynamic balance of CRISPR-Cas immunity and resistance plasmid anti-immunity mediated by a bifunctional protein AcrIE10.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42675078},
issn = {2041-1723},
support = {W2512095, 81991531, and 32471347//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82402671//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22410710300//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 25-44-02137//Russian Science Foundation (RSF)/ ; },
mesh = {*Klebsiella pneumoniae/genetics/immunology/drug effects/metabolism ; *CRISPR-Cas Systems/immunology/genetics ; *Plasmids/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry/immunology ; Promoter Regions, Genetic ; Drug Resistance, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Despite targeting by CRISPR-Cas system, antimicrobial resistance plasmids are prevalent in clinical isolates of carbapenem-resistant Klebsiella pneumoniae which represent a major public health threat. A stable co-existence of plasmids and CRISPR-Cas systems is mediated by anti-CRISPR (Acr) proteins. Here, we report that previously identified AcrIE10 encoded by a resistance plasmid combines two functions: it inhibits CRISPR immunity by directly binding Cas7* subunit through its Acr domain, and acts as an Acr-associated (Aca) protein that self-represses the transcription of Acr locus. AcrIE10 is an example of an Aca protein that utilizes N-terminal ribbon-helix-helix (RHH) domain to specifically recognize the inverted repeat (IR) region in its own promoter. Crucially, a dimerization of AcrIE10 dimers is required for the effective binding to the IR and self-repression, while stoichiometry-dependent interaction with Cas7* facilitates transition to de-repressed state. These findings elucidate molecular mechanisms by which AcrIE10 operates as a dual functionAcr-Aca protein to achieve a delicate balance between host CRISPR-Cas immunity and plasmid anti-defense.},
}
@article {pmid42675231,
year = {2026},
author = {Rathna, ARS and Ceasar, SA},
title = {Accelerating iron biofortification in millets: progress, challenges, and future prospects.},
journal = {Planta},
volume = {264},
number = {4},
pages = {},
pmid = {42675231},
issn = {1432-2048},
mesh = {*Biofortification/methods ; *Iron/metabolism ; *Millets/genetics/metabolism ; Gene Editing ; Plant Breeding ; CRISPR-Cas Systems ; },
abstract = {Integrating conventional breeding, omics, and CRISPR-based genome editing can overcome genetic and antinutrient constraints, enabling efficient iron biofortification of millets for sustainable and nutrition-secure food systems. Iron (Fe) deficiency remains one of the most widespread forms of micronutrient malnutrition. Biofortification of staple crops has emerged as a particularly sustainable and scalable strategy to combat this issue. Millets are nutrient-dense staple cereals with exceptional nutritional quality and climate resilience. However, genetic variations and the presence of antinutrients limit Fe content in millets, which highlights the necessity of advancing biofortification strategies. This review examines the present state of multi-dimensional strategies and discusses the future prospects for efficient iron biofortification in millets. We analyzed the efforts made for Fe biofortification in millets, ranging from conventional breeding practices to next-generation molecular approaches. Recent advances in omics have enhanced understanding of Fe uptake, transport, and storage in millets. Furthermore, CRISPR/Cas-based genome editing is discussed for the regulated expression of key Fe-transporter genes and targeted knockout of genes responsible for antinutrients. A multidisciplinary approach is essential to develop high-yielding and Fe-rich millet varieties that can contribute to sustainable nutrition security.},
}
@article {pmid42677405,
year = {2026},
author = {Zhang, WY and Pei, WK},
title = {[Cellular barcoding and next-generation lineage tracing: concepts and applications].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {78},
number = {4},
pages = {731-740},
doi = {10.13294/j.aps.2026.0063},
pmid = {42677405},
issn = {0371-0874},
mesh = {*Cell Lineage/genetics ; Humans ; *DNA Barcoding, Taxonomic ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Animals ; Single-Cell Analysis ; },
abstract = {Lineage tracing is a fundamental technique for dissecting cell fate decisions and development process. With recent advances in high-throughput sequencing and single-cell sequencing technologies, cellular barcoding-based lineage tracing strategies have transitioned from low-throughput labeling methods to high-resolution, multidimensional lineage reconstruction. In this review, we systematically summarize four major barcoding paradigms: viral integration-based random integration barcodes, transposon-based random integration barcodes, recombinase-mediated DNA rearrangement (e.g., Cre-loxP), and CRISPR-Cas9-based mutation recording systems. We describe their principles, representative studies, technical advantages, and limitations. Furthermore, we discuss the core bottlenecks in terms of editing precision, integration of spatiotemporal information, and non-invasive lineage tracing, with a focus on cutting-edge advancements such as prime editing, sequential recording systems, strategies for integrating spatial transcriptomics, and epigenetic tracing. Overall, single-cell lineage tracing is evolving from clonal labeling toward the multi-dimensional integration of lineage, state, and space. In the future, the deep integration of precise gene-editing tools with high-resolution spatial omics technologies is expected to enable dynamic and systematic analysis of cellular fate trajectories, thereby providing critical technical support for research in developmental biology and regenerative medicine.},
}
@article {pmid42678547,
year = {2026},
author = {Akhtar, MS and Amin, A},
title = {CRISPR-cas systems in pharmacology: functional pharmacogenomics, drug screening, resistance, and therapeutic translation.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42678547},
issn = {1438-7948},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Pharmacogenetics/methods ; Animals ; Precision Medicine ; *Drug Discovery/methods ; },
abstract = {CRISPR-Cas9 gene-editing technology has advanced pharmacological research by enabling targeted genetic modification for disease modeling, therapeutic development, and precision medicine. This review discusses the applications of CRISPR-Cas9 in drug discovery, personalized therapy, cancer drug resistance research, genetic disorders, and antimicrobial resistance. By editing disease-associated genes, CRISPR-Cas9 supports the development of patient-specific therapeutic strategies and more accurate preclinical models. In cancer, CRISPR-Cas9 is used to investigate the target genes involved in treatment resistance, while in genetic disorders, it offers potential mutation-correcting approaches, with the most robust clinical evidence currently seen in selected hemoglobinopathies. CRISPR-based strategies also hold promise for restoring antibiotic susceptibility by targeting genes that confer antibiotic resistance. Despite these advances, clinical translation remains limited by off-target effects, delivery challenges, immune responses, long-term safety concerns, and ethical and regulatory issues. Continued improvements in editing precision, delivery systems, and governance frameworks are essential for responsible clinical integration. Overall, CRISPR-Cas9 represents a vital platform for future pharmacological innovation, but its broad clinical use may require further validation of safety, efficacy, durability, and accessibility.},
}
@article {pmid41966922,
year = {2026},
author = {Cui, J and Zhang, L and Zhou, J and Shi, T and Wu, S and Dai, T and Hao, L and Pan, J and Lai, X and Lu, W and Huang, X and Li, Z and Lai, L and Wang, X},
title = {A universal light-controlled highly sensitive one-pot CRISPR/Cas12a diagnostic based on structure-engineered crRNA.},
journal = {Trends in biotechnology},
volume = {44},
number = {9},
pages = {2699-2721},
doi = {10.1016/j.tibtech.2026.03.018},
pmid = {41966922},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics/isolation &amp; purification ; Human papillomavirus 18/genetics/isolation &amp; purification ; Polymorphism, Single Nucleotide ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has transformed molecular diagnostics through its speed and accuracy; however, one-pot formats are often limited by sensitivity and field suitability. Herein, we developed a universal light-controlled high-sensitivity one-pot CRISPR/Cas12a testing (ULTRAt) platform based on structure-engineered CRISPR RNA (crRNA) scaffolds. By incorporating photocaged 6-nitropiperonyloxymethyl groups into the crRNA stem-loop, Cas12a activity is transiently suppressed during isothermal amplification via structural modulation, enabling efficient target enrichment. Subsequent UV irradiation removes the protecting groups, restoring the native conformation and activating robust trans-cleavage. ULTRAt achieves a limit of detection of two copies of monkeypox virus per reaction with a 15-min time-to-result, representing a 100-fold sensitivity improvement over conventional assays. The platform further supports single-nucleotide polymorphism discrimination and human papillomavirus 16/18 genotyping. Analysis of 91 clinical samples demonstrates strong concordance between ULTRAt and reference qPCR and sequencing assays. Collectively, ULTRAt enables rapid, ultra-sensitive, and versatile one-pot detection, supporting near-patient diagnostics and genotyping.},
}
@article {pmid42237538,
year = {2026},
author = {Sarko, LE and Givand, D and Rattin, B and Shepley, C and Tommasi, A and Attar, A and Taylor, R and Kutler, B and Traynor, RM and Upadhyaya, A and Mnuk, M and Gehrke, C and Murren, N and Ulland, TK and Capitini, CM and Kotanchek, T and Saha, K},
title = {Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {9},
pages = {5190-5206},
doi = {10.1016/j.ymthe.2026.05.029},
pmid = {42237538},
issn = {1525-0024},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; T32 GM135119/GM/NIGMS NIH HHS/United States ; S10 RR025483/RR/NCRR NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 AG083883/AG/NIA NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *T-Lymphocytes/metabolism/immunology ; Gene Editing ; *Receptors, Urokinase Plasminogen Activator/antagonists &amp; inhibitors/genetics/immunology/metabolism ; CRISPR-Cas Systems ; Lymphocyte Activation/immunology ; Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell/genetics/metabolism ; Cell- and Tissue-Based Therapy/methods ; },
abstract = {Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations in the final product, with many cells lacking desired receptor expression or exhausted and other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via non-viral CRISPR genome editing to eliminate the T cell receptor (TCR) alpha chain (TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. In these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which triggers CAR-mediated killing within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-disialoganglioside (GD2) CAR-T cell products up to ≥99% CAR+/TCR-, favoring a memory-like phenotype. Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods.},
}
@article {pmid42427028,
year = {2026},
author = {Ginn, SL and Doroudian, F and Christina, S and Chan, OPY and Lucas, CW and Zhu, E and Yang, SF and Devanapalli, B and Klein, AH and Scott, S and Vitale, J and Cunningham, SC and Liao, SHY and Cabanes-Creus, M and Lisowski, L and Alexander, IE},
title = {Functional editing of the OTC locus by targeted integration with phenotype correction and restoration of endogenous expression patterns.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {9},
pages = {5426-5443},
doi = {10.1016/j.ymthe.2026.06.044},
pmid = {42427028},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; Hepatocytes/metabolism ; *Ornithine Carbamoyltransferase/genetics/metabolism ; Dependovirus/genetics ; *Gene Editing/methods ; Phenotype ; CRISPR-Cas Systems ; *Ornithine Carbamoyltransferase Deficiency Disease/genetics/therapy/metabolism ; Genetic Vectors/genetics/administration &amp; dosage ; Mutation ; Disease Models, Animal ; *Genetic Loci ; Liver/metabolism ; Gene Expression Regulation ; },
abstract = {Here, we report highly efficient functional repair of the ornithine transcarbamylase (OTC) locus in mutant mouse and human hepatocytes in vivo using a dual adeno-associated virus system delivering CRISPR-Cas9 editing reagents and a promoterless donor for targeted integration. The approach was mutation agnostic and targeted intronic sequences to prevent inadvertent inactivation of hypomorphic alleles. Notably, in a murine model, we corrected the metabolic defect and simultaneously achieved liver-wide restoration of physiological metabolic zonation of Otc expression by capturing native cis-acting regulatory elements. The effectiveness of this approach was confirmed using a universally configured therapeutic cassette in patient-derived primary human hepatocytes in vivo. These data provide a powerful template to guide further optimization of this approach and, given the high editing efficacy required for phenotypic effect in OTC deficiency, have broader relevance to other liver disease phenotypes.},
}
@article {pmid42556172,
year = {2026},
author = {Khare, G and Duong, A and Hanotaux, J and Shorr, R and Nampoothiri, RV and Maganti, H and Allan, DS},
title = {A scoping review of gene editing in clinical trials: identifying aspects of trial design to accelerate clinical adoption.},
journal = {Cytotherapy},
volume = {28},
number = {10},
pages = {102898},
doi = {10.1016/j.jcyt.2026.102898},
pmid = {42556172},
issn = {1477-2566},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Clinical Trials as Topic ; CRISPR-Cas Systems/genetics ; Adolescent ; Child, Preschool ; Child ; Adult ; Infant ; Leukemia/genetics/therapy ; Aged, 80 and over ; Aged ; Middle Aged ; Research Design ; Lymphoma/therapy/genetics ; },
abstract = {BACKGROUND: Initial clinical trials of gene editing have recently emerged and generated significant interest in this promising therapy. A scoping review is needed to understand aspects of study design that may accelerate further clinical translation.<br><br>METHODS: A systematic search of published clinical trials was conducted to May 22, 2025.<br><br>RESULTS: Twenty-nine published trials were identified for analysis (420 patients; median ages 6 months to 90 years). The most common gene editing technology used was CRISPR-Cas9 (23 studies, 79%). Hematopoietic cells were targeted most frequently, and leukemias and lymphomas were the most common clinical indications (8 studies; 27.5%), followed by hemoglobin disorders (6; 20.6%), solid tumors (6; 20.6%), rare genetic diseases (5; 17.2%), and others (4; 13.8%). All trials were early-phase (Phase I/II) with only 1 controlled study (29 patients). Six studies used in vivo approaches while all others performed gene editing ex vivo. Gene transfection was by electroporation (9 studies; 31.0%), viral-mediated transfection (6; 20.7%), or by lipid nanoparticles (3; 10.3%). Commonly reported outcomes included early safety and adverse events, mortality, persistence of edited cells, clinical and functional responses. Studies of leukemia/lymphoma reported rates of complete remission in 15-92% while we identified important and sustained increases in mean hemoglobin levels in studies of hemoglobin disorders. Edited cells were detected in most studies at 3 and 6 months but later timepoints were reported less frequently. While early serious adverse events were infrequently observed, reporting of longer-term outcomes was lacking.<br><br>CONCLUSION: Gene editing appears feasible and generally safe in humans although important safety outcomes such as long-term oncogenic surveillance outcomes remain to be addressed. A broad range of conditions have been treated, and most often leverages the CRISPR-Cas9 technology with transfection by electroporation. The persistence of edited cells remains ill-defined and longer-term safety outcomes are needed. Studies optimizing CART therapy for leukemia/lymphoma and treatment of hemoglobin disorders appear poised for significant clinical adoption.},
}
@article {pmid42560066,
year = {2026},
author = {Hao, Z and Zhao, Q and Zhong, Y and Li, M and Wang, C and Wang, C},
title = {Development and clinical validation of a CRISPR/Cas9-engineered reporter phage cocktail for rapid detection of Escherichia coli in urine.},
journal = {Microbiology spectrum},
volume = {14},
number = {9},
pages = {e0099626},
pmid = {42560066},
issn = {2165-0497},
mesh = {*Escherichia coli/isolation &amp; purification/genetics/virology ; *CRISPR-Cas Systems ; Humans ; *Urinary Tract Infections/diagnosis/microbiology ; *Escherichia coli Infections/diagnosis/microbiology/urine ; Sensitivity and Specificity ; *Urine/microbiology ; Genes, Reporter ; *Bacteriophages/genetics ; *Coliphages/genetics ; },
abstract = {Urinary tract infections are one of the most common infectious diseases, with Escherichia coli as the predominant pathogen. Traditional diagnostic methods fail to meet clinical demands for rapid and specific detection. Here, we developed an efficient urine E. coli detection strategy via a reporter phage cocktail. Four reporter phages (T2::Nluc, T4::Nluc, T5::Nluc, T6::Nluc) were constructed by the CRISPR/Cas9 system combined with homologous recombination. One-step growth curves, optimal multiplicity of infection, and lytic efficiency showed that the Nluc gene block insertion exerted heterogeneous effects on phages. Luminescence assays demonstrated that all five reporter phages (including previously preserved T7::Nluc) and the cocktail offered favorable limits of detection (≥10[3] CFU/mL), high specificity, and no urine matrix interference. However, single phages exhibited limited coverage among 177 clinical E. coli isolates. But the reporter phage cocktail remedies this limitation. In large-scale clinical validation, the cocktail achieved sensitivity 73.15% (63.76%-81.22%), specificity 100.00% (99.53%-100.00%), positive predictive value (PPV) 100.00% (95.44%-100.00%), and negative predictive value (NPV) 96.42% (95.18%-97.36%) (all 95% confidence interval [CI]), and excellent concordance with the gold-standard method (Kappa = 0.83, 95% CI: 0.77-0.89), greatly outperforming single reporter phages (~40.00% sensitivity). This method requires no sample pretreatment, is simple to operate, and completes detection within 4 h, significantly improving diagnostic efficiency. Accordingly, it provides a novel platform for pathogen detection and supports the clinical translation of reporter phage diagnostics.IMPORTANCEUrinary tract infections impose substantial economic and public health burdens. In this study, we successfully constructed Escherichia coli-specific reporter phages T2::Nluc, T4::Nluc, T5::Nluc, and T6::Nluc. Combined with the previously preserved T7::Nluc, these phages formed a reporter phage cocktail. Co-cultivation of this cocktail with clinical samples enabled rapid and specific detection of E. coli in clinical urine, with a significantly shortened detection time (4 h) and good concordance with the gold-standard detection method (Kappa = 0.83), effectively improving detection efficiency and accuracy. This novel pathogen detection platform, integrating specific recognition and signal amplification, not only provides a new technical approach for the rapid and accurate diagnosis of clinical urinary tract infections but also effectively promotes the coordinated improvement of infectious disease diagnosis and treatment in terms of timeliness-precision-cost.},
}
@article {pmid42599083,
year = {2026},
author = {Li, Y-y and Zhang, H and Shao, L-n and Liu, B-l and Wang, Y-m and Duan, J-q and Lai, S-Y and Xu, Z-w and Zhu, L},
title = {A rapid, sensitive, and field-deployable RAA-EsCas13d platform for detection of porcine adenovirus type 3.},
journal = {Microbiology spectrum},
volume = {14},
number = {9},
pages = {e0129126},
pmid = {42599083},
issn = {2165-0497},
support = {2024YFD1800500//Research and Application of Integrated Prevention, Control and Purification Technologies for Major Swine Epidemic Diseases/ ; 2024YFD1800102//National Key Research and Development Program of China during the 14th Five-Year Plan: Research and Development of Key Technologies for the Prevention and Control of Important Diseases in Wild Animals/ ; sccxtd-2024-08//Sichuan Pig Innovation Team of the National Modern Agricultural Industry Technology System/ ; CARS&#x2043;SVDIP//National agricultural industrial technology system Sichuan veterinary medicine innovation team special/ ; },
mesh = {Animals ; Swine ; *Adenoviruses, Porcine/genetics/isolation &amp; purification/classification ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; *Adenoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/virology/diagnosis ; CRISPR-Cas Systems ; Recombinases/genetics ; },
abstract = {Rapid, sensitive, and RADIANT-deployable nucleic acid diagnostics are essential for the prevention and control of porcine adenovirus type 3 (PAdV-3). In this study, we developed RADIANT (RAA-Cas13d DIagnostic plATform for extractioN-free Testing), an extraction-free platform capable of delivering accurate and portable detection in resource-limited settings. By systematically optimizing the reaction buffer composition, recombinase-aided amplification (RAA), T7 transcription, and CRISPR/EsCas13d-mediated cleavage were integrated into a one-pot reaction, reducing the total time-to-result to within 30 minutes. A simplified nucleic acid release step eliminated the need for laboratory-based extraction or complex heating, enhancing the accessibility of the assay. Incorporation of lyophilized reagents minimized cold-chain requirements and simplified assay preparation, enabling cost-effective storage and transport. The platform demonstrated high sensitivity in detecting PAdV-3 and offered two straightforward readout options, fluorescence under 470 nm blue/UV light and lateral flow assay (LFA), facilitating flexible interpretation under diverse RADIANT conditions. Validation with 56 clinical samples showed 100% concordance with quantitative PCR, confirming RADIANT as a rapid, user-friendly, and reliable on-site diagnostic tool for PAdV-3 detection.IMPORTANCEPorcine adenovirus type 3 (PAdV-3) is an important swine pathogen for which rapid, practical, and field-compatible diagnostic tools are still lacking. Here, we developed RADIANT, a CRISPR/EsCas13d-based platform that expands PAdV-3 detection beyond conventional laboratory workflows and supports accessible molecular testing in resource-limited settings. Its simple operation and adaptable visual readouts enhance its suitability for on-site use without compromising reliability. This study provides a valuable diagnostic approach for PAdV-3 surveillance and disease control and supports the broader application of portable CRISPR-based technologies in veterinary diagnostics.},
}
@article {pmid42653322,
year = {2026},
author = {Li, H and Zhang, X and Wang, X and Zhang, R and Liu, L and Sun, L and Yao, Z and Zhu, H},
title = {CRISPR/Cas9-Mediated Disruption of Duplicated Sizzled Genes Induces Twin-Tail-like Caudal Bifurcation in Goldfish (Carassius auratus).},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653322},
issn = {1422-0067},
support = {KJCX20261411//Beijing Academy of Agricultural and Forestry Sciences/ ; 32403015//National Natural Science Foundation of China/ ; KJCX20230216//Beijing Academy of Agricultural and Forestry Sciences/ ; 6262009//National Natural Science Foundation of China/ ; GHPT2026-16//Beijing Academy of Agricultural and Forestry Sciences/ ; PT2025-20//Beijing Academy of Agricultural and Forestry Sciences/ ; NY2401170024//Rural Development Institute/ ; GHPT2026-06//China International Science and Technology Cooperation/ ; },
mesh = {Animals ; *Goldfish/genetics/embryology ; *CRISPR-Cas Systems ; *Fish Proteins/genetics/metabolism ; Body Patterning/genetics ; Gene Expression Regulation, Developmental ; Glycoproteins ; Intercellular Signaling Peptides and Proteins ; },
abstract = {The twin-tail phenotype of goldfish represents a striking domestication-associated remodeling of the vertebrate caudal axial system and is classically linked to disruption of Chordin/BMP-mediated dorsal-ventral patterning. Although previous knockdown studies implicated sizzled (szl) in this process, genetic evidence from targeted disruption of endogenous szl loci remains limited. Here, we used CRISPR/Cas9 to mutate conserved coding regions shared by the duplicated goldfish paralogues szlA and szlB in single-tail embryos. Sanger sequencing and ICE analysis showed that szl-sgRNA2 and szl-sgRNA3 efficiently induced indels at both loci, whereas szl-sgRNA1 was ineffective. Across three independent biological replicates, twin-tail-like caudal bifurcation was observed in 44.63-48.19% of szl-sgRNA2-injected larvae, 69.47-79.61% of szl-sgRNA3-injected larvae, and 64.29-76.19% of larvae injected with the sgRNA mixture; szl-sgRNA1-injected larvae remained single-tailed. Calcein staining further revealed separation of distal caudal fin rays and partial splitting of the caudal skeletal complex in szl-edited larvae. qRT-PCR showed selective remodeling of dorsal-ventral patterning genes, including reduced chdA and eve1 expression and increased bmp2 and nog1 expression. These findings provide direct functional evidence that szl regulates median caudal patterning in goldfish and suggest that szl-dependent modulation of the Chordin/BMP network can generate twin-tail-like caudal morphology.},
}
@article {pmid42653378,
year = {2026},
author = {Santos, ARD},
title = {AI-Assisted Cross-Study Synthesis in Genome Editing: Comparing Long-Context Strategies and Uncovering Latent Contradictions in the CRISPR-Cas9 Guide RNA Prediction Literature.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653378},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Large Language Models ; *Artificial Intelligence ; Humans ; },
abstract = {Predicting CRISPR-Cas9 guide RNA efficiency and off-target activity is a precondition for precise genome editing. Computational models have progressively incorporated chromatin accessibility and epigenetic descriptors into their feature sets, yet synthesising findings from independently published studies-especially when those studies contradict one another-remains an unresolved methodological gap. Large Language Models (LLMs) have been proposed as a route to automate cross-study synthesis, but their utility depends on a constraint that receives less attention than model architecture: how much of the source text actually reaches the model at inference time. Cloud-based models process 48,000-token corpora without hardware limitations, but at the cost of data leaving the local environment and with limited reproducibility across API versions. Local RAG systems avoid the cloud dependency while fragmenting the input, discarding the global context needed to link biological arguments that are distributed across separate papers. We benchmark these strategies using a corpus of four CRISPR-Cas9 efficiency prediction studies and apply the Reduced Interaction Sampling (RIS) engine-a local sparse attention method-to retain the full sequence within the memory envelope of a laboratory server. Preserving that context uncovers three latent inconsistencies. The static epigenetic markers used in DeepCRISPR (CTCF, DNase I) show near-zero Spearman correlations with off-target cleavage (ρ≤0.07), while nucleosome positioning scores from the Block Decomposition Method reach ρ=0.388-0.423. The sequence-only Apindel model was published in June 2022 without incorporating nucleosome descriptors reported in the concurrent literature. The benchmark review by Konstantakos et al. attributed 10-20% of rank correlation to epigenetics-a figure that reflects the weak feature subset evaluated, not a ceiling on chromatin influence. These discrepancies are invisible when papers are read individually or retrieved as chunks; they become traceable only when the full corpus is processed as a single context window. An independent empirical analysis of 2000 CRISPR-Cas9 off-target cleavage events provides evidence consistent with this pattern: static epigenetic markers yield |ρ|≤0.11, whereas computed NuPoP Affinity descriptors reach r=-0.622 (p<10-210). On a 30-question cross-study synthesis benchmark (5 independent seeds), baseline accuracy is 53.33%, RAG 60.00%, and RIS (30 seeds, 3% density) 70.00% (p<0.0001, t-test vs. RAG, σ=0.00% for all configurations).},
}
@article {pmid42654749,
year = {2026},
author = {Martínez-Álvarez, S and Herrera-Espejo, S and Zarazaga, M and Höfle, U and Pachón-Ibáñez, ME and Torres, C},
title = {Virulence and Invasion Profiles of Escherichia coli Across One Health Reservoirs: Genomic Insights into High-Risk Clones and Their Defense Systems.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
pmid = {42654749},
issn = {2076-0817},
support = {PID2022-139591OB-I00//MICIU/AEI/10.13039/501100011033 and ERDF/ EU/ ; },
mesh = {Humans ; Virulence ; Animals ; *Escherichia coli Infections/microbiology/veterinary ; *Escherichia coli/genetics/pathogenicity/classification/isolation &amp; purification ; Bacterial Adhesion ; *One Health ; HEK293 Cells ; Genomics ; *Disease Reservoirs/microbiology ; Genome, Bacterial ; CRISPR-Cas Systems ; Virulence Factors/genetics ; Biofilms/growth &amp; development ; Phenotype ; },
abstract = {Escherichia coli is a genetically diverse species encompassing both commensal and pathogenic lineages capable of transitioning among various hosts. Within a One Health framework, we conducted a targeted screening of 38 E. coli strains isolated from wildlife, livestock, and food reservoirs to characterize their pathogenic potential by integrating genomic and phenotypic approaches. In vitro functional assays, including biofilm formation, surface motility, and adherence and invasion of HEK-293 epithelial cells, were statistically evaluated using the non-parametric Mann-Whitney U test. Phenotypic analyses revealed that extraintestinal pathogenic (ExPEC) and uropathogenic E. coli (UPEC) strains, particularly those belonging to the high-risk ST117 clone, exhibited significantly enhanced adherence and internalization capacities. These virulent phenotypes strongly correlated with specific genetic signatures involved in iron acquisition and epithelial invasion (chuA, fyuA, vat, and tia), underscoring that the convergence of ExPEC/UPEC determinants drives increased colonization potential. Genomic characterization further revealed that despite high virulence and widespread antimicrobial resistance, the CRISPR/Cas subtype I-E system was highly prevalent (93.8%), displaying structural variations frequently driven by insertion sequences. Spacer analyses identified limited homology to plasmids and phages, suggesting past mobilome interactions rather than active restriction of current horizontal gene transfer. Overall, these findings illustrate how phenotypic traits of high-risk clones match their genomic virulence platforms. The convergence of multidrug resistance and pathogenic fitness across human, animal, and environmental interfaces underscores the need for integrated molecular surveillance in a One Health context.},
}
@article {pmid42655688,
year = {2026},
author = {Dai, Y and Xia, L and Yang, Y and Qiao, G and Jin, X and Mao, X},
title = {Recent Advances in CRISPR/Cas Systems for Respiratory Pathogen Diagnostics.},
journal = {Viruses},
volume = {18},
number = {8},
pages = {},
pmid = {42655688},
issn = {1999-4915},
support = {HB2023111//Xuhua Mao/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Respiratory Tract Infections/diagnosis/microbiology/virology ; Bacteria/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; Viruses/genetics/isolation &amp; purification ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Fungi/genetics/isolation &amp; purification ; },
abstract = {Early, rapid, and accurate detection is essential for clinical management and epidemiological control of acute respiratory infections caused by pathogens. Traditional testing methods such as microbial culture, serological testing, and PCR are restrictive in terms of operation and logistics and are therefore not easily used in point-of-care settings. The CRISPR/Cas system is an adaptive prokaryotic immune system composed of clustered regularly interspaced short palindromic repeats and their associated proteins, which has been used as a nucleic acid diagnostic platform with programmable sequence-specific target recognition and signal-amplifying collateral cleavage activity. Existing reviews have mostly focused on the classification of Cas enzymes or amplification strategies; in this review, a pathogen-centric approach was taken, covering viral pathogens (SARS-CoV-2, influenza virus, RSV, HAdV and VZV), bacterial pathogens (Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus) and fungal pathogens (Aspergillus fumigatus and Pneumocystis jirovecii). Key technological advances, such as isothermal amplification coupling, single-vessel integrated reaction designs, amplification-free digital detection, and electrochemical biosensor integration, are evaluated for Cas9-, Cas12-, and Cas13-based systems, with their mechanistic bases outlined. The current challenges that hinder clinical translation, such as sample matrix interference, multiple signal cross-talk, crRNA off-target effects, and the lack of large-scale validation studies, are critically assessed. At the same time, future pathways for portable, integrated, and inexpensive diagnostic platforms are suggested.},
}
@article {pmid42655725,
year = {2026},
author = {Azad, MAK and Ibnat, N and Chowdhury, SS and Huq, S and Islam, S},
title = {Advances in Molecular Techniques for Detecting Sweet Potato (Ipomoea batatas (L.) Lam) Viruses: A Comprehensive Review.},
journal = {Viruses},
volume = {18},
number = {8},
pages = {},
pmid = {42655725},
issn = {1999-4915},
mesh = {*Ipomoea batatas/virology ; *Plant Diseases/virology ; *Plant Viruses/genetics/isolation &amp; purification/classification ; *Molecular Diagnostic Techniques/methods ; High-Throughput Nucleotide Sequencing ; Nucleic Acid Amplification Techniques ; Biosensing Techniques ; },
abstract = {Sweet potato (Ipomoea batatas (L.) Lam) is an important global food crop, but its production is threatened by numerous viral pathogens. More than 30 RNA and DNA viruses have been reported worldwide, making rapid and accurate detection essential for disease management, epidemiological surveillance, germplasm exchange, and resistance breeding. Although previous reviews have addressed sweet potato viruses and individual diagnostic methods, a comprehensive synthesis of emerging molecular technologies remains limited. This review addresses that gap by critically integrating recent advances from PCR-based and isothermal assays to high-throughput sequencing, CRISPR-based diagnostics, biosensors, nanotechnology, and artificial intelligence-driven detection platforms. Conventional approaches, including symptom observation, biological indexing, electron microscopy, and ELISA, have contributed to early virus identification but often lack the sensitivity, specificity, and speed needed for modern diagnostics. Molecular and isothermal techniques have substantially improved detection accuracy and enabled rapid identification and field-deployable diagnostics of diverse and mixed infections, while sequencing, CRISPR, biosensors, and AI-based platforms offer greater capacity for detecting novel and emerging viruses. This review discusses the comparative evaluation of molecular technologies for sweet potato virus detection in terms of diagnostic performance, cost-effectiveness, speed, and suitability for both laboratory and field applications, while highlighting future priorities for next-generation virus diagnostics. Integrating portable and high-throughput diagnostic platforms will strengthen virus surveillance, support virus-free planting material production, and promote sustainable sweet potato production worldwide.},
}
@article {pmid42658322,
year = {2026},
author = {Kara, G and Holcomb, M and Hijazi, AA and Ali, Y and López-Espinosa, J and Cruz-Pineda, L and Park, P and Flinn, H and Taylor, N and Galbraith, T and McMahon, L and Rostomily, R and Leonard, F and Villapol, S},
title = {Intranasal CRISPR lipid nanoparticles targeting MAPK9 attenuate neuroinflammation after traumatic brain injury.},
journal = {Biomedical microdevices},
volume = {28},
number = {3},
pages = {},
pmid = {42658322},
issn = {1572-8781},
support = {R56AG080920/AG/NIA NIH HHS/United States ; R56 AG080920/AG/NIA NIH HHS/United States ; R21NS106640/NS/NINDS NIH HHS/United States ; ission Connect 2026 grant (S.V.). (No. 026-102),//TIRR Foundation/ ; R21 NS106640/NS/NINDS NIH HHS/United States ; },
mesh = {*Brain Injuries, Traumatic/complications/pathology ; Animals ; *Nanoparticles/chemistry ; Mice ; Administration, Intranasal ; *Neuroinflammatory Diseases ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; Male ; Microglia/metabolism ; Mice, Inbred C57BL ; },
abstract = {Traumatic brain injury (TBI) induces a sustained neuroinflammatory response involving activated microglia and infiltrating myeloid cells, contributing to secondary brain damage and long-term neurological dysfunction. Modulating these inflammatory responses toward a more reparative phenotype represents a promising therapeutic strategy, but achieving targeted delivery within the injured brain remains a major challenge. Here, we developed a targeted, non-viral gene-editing platform using lipid nanoparticles (LNPs) encapsulating CRISPR-Cas12a components directed against MAPK9, a key mediator of inflammatory signaling. LNPs were functionalized with an Iba-1 antibody to enhance targeting of Iba-1 + myeloid cells following intranasal administration. In primary bone marrow-derived macrophages and primary microglia, CRISPR-mediated MAPK9 targeting reduced MAPK9 expression and suppressed pro-inflammatory activation, decreasing iNOS, NLRP3, CD80, and CCL2 while increasing the anti-inflammatory/reparative markers CD206 and Arg1. In a mouse model of TBI, intranasally delivered Iba-1-targeted CRISPR-LNPs showed preferential association with Iba-1 + cells compared with NeuN+ neurons in the injured cortex and reduced MAPK9 expression within Iba-1 + cells. CRISPR-LNP treatment attenuated microglial/macrophage activation, reduced pro-inflammatory cytokine expression, and decreased iNOS+/Iba-1 + cells while increasing CD206+/Iba-1 + cells in the peri-contusional cortex, supporting a shift toward a less inflammatory phenotype. Treatment also exhibited a favorable safety profile, with no detectable toxicity in the major organs examined. Together, these findings demonstrate that intranasal delivery of Iba-1-targeted CRISPR-LNPs enables effective MAPK9 modulation in Iba-1 + myeloid cells within the injured brain and attenuates acute neuroinflammation following TBI. This non-invasive therapeutic platform provides a promising approach for targeted modulation of neuroinflammatory responses after brain injury.},
}
@article {pmid42660250,
year = {2026},
author = {Fiaz, S},
title = {Recent trends in nucleic acid research in plants for future food security.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {154241},
doi = {10.1016/j.ijbiomac.2026.154241},
pmid = {42660250},
issn = {1879-0003},
abstract = {The agricultural production system is facing unprecedented pressure of climate change, human population, pressure on renewable and non-renewable resources, and elevation in both biotic and abiotic stress factors. The recent development in nucleic acid based technologies have transformed research in plant sciences through deployment of powerful technologies for understanding complex biological mechanisms controlling climate resilience, nutrition and yield attributes in agriculturally important crops. This editorial documented the key results of 33 articles published in this special issue, covering application of various techniques to improve desirable attributes in agriculturally important crops. Furthermore, the published literature displayed the key findings emerging through the application of CRISPR-Cas based genome editing system, integration of multi-omics, application of machine learning, RNA-based regulations and chloroplast bioengineering with higher precision, efficiency, and reliability. The innovation in plant-microbe interaction, rhizosphere engineering has revealed novel avenues of research for improving the potential of resource use efficiency and sustainability. Altogether, the research published in this special issue may play a transformative role in advancing precision breeding, stress resilience and crop performance under ever-changing climatic conditions. However, there is a requirement for continuous interdisciplinary research, embracing innovation and international collaboration for utilization of full potential of cutting-edge technologies contributing significantly to achieving food security.},
}
@article {pmid42661411,
year = {2026},
author = {Vermeulen, W and van Staden, ADP and Dicks, LMT},
title = {Nucleases and Their Inhibitors: Exploring Biological Roles, Industrial Applications, and Challenges in Heterologous Expression.},
journal = {Biotechnology journal},
volume = {21},
number = {9},
pages = {e70303},
pmid = {42661411},
issn = {1860-7314},
mesh = {Humans ; *Endonucleases/genetics/metabolism/antagonists &amp; inhibitors ; Animals ; CRISPR-Cas Systems ; },
abstract = {Nucleases hydrolyze phosphodiester bonds and participate in numerous cellular and metabolic processes. Intracellular nucleases repair nonfunctional or damaged DNA using DNA base excision repair (BER), mismatch repair (MMR), and homologous recombination (HR). Apoptotic nucleases systematically degrade cellular DNA during programmed cell death (PCD). Non-apoptotic nucleases support DNA repair and replication. Small noncoding RNAs (sncRNAs) degrade the RNA of viral particles. Extracellular and membrane-associated nucleases replenish nucleotides, especially in biofilms where cells rely on additional carbon, phosphorus, and energy. Restriction endonucleases (REs) are indispensable in recombinant DNA technology. Some genetic disorders and cancers have been treated by changing the genetic code of host cells using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system. Nucleases are also used in vaccine development. Heterologous expression of nucleases remains challenging, largely due to cytotoxicity and product instability. Some successes have been reported using the T7 promoter-based system. However, due to the formation of inclusion bodies (IBs), the nucleases were insoluble and of low activity. Refolding misfolded nucleases from IBs, tight control (sequestration) of periplasmic secretion, and coexpression with natural inhibitor proteins increased yield, purity, and biological activity. This review addresses the significance of nucleases, heterologous expression, gene regulation, activity inhibition, and product yield.},
}
@article {pmid42662772,
year = {2026},
author = {Kim, GE and Jin, HB and Kang, YJ and Park, HH},
title = {Structural insights into Cas9 inhibition by AcrIIA17 via bridge helix interaction.},
journal = {iScience},
volume = {29},
number = {9},
pages = {117218},
pmid = {42662772},
issn = {2589-0042},
abstract = {Anti-CRISPR (Acr) proteins have evolved in bacteriophages and mobile genetic elements to counteract CRISPR-Cas immune systems through diverse inhibitory mechanisms. Here, we present the crystal structure of AcrIIA17 and elucidate its mechanism of Staphylococcus aureus Cas9 (SauCas9) inhibition. AcrIIA17 adopts a previously uncharacterized protein fold and exists as a monomer in solution. Biochemical analyses reveal that AcrIIA17 inhibits SauCas9 activity in a strictly order-dependent manner, effectively suppressing DNA cleavage only when it engages Cas9 prior to single guide RNA (sgRNA) loading, whereas pre-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes are resistant to inhibition. Domain-mapping experiments demonstrate that AcrIIA17 directly binds to the bridge helix (BH) domain of SauCas9, and structure-guided mutagenesis confirms that this interaction is essential for its inhibitory function. Together, our findings identify AcrIIA17 as an Acr protein that targets the Cas9 BH domain and reveal the BH domain as a regulatory checkpoint in Cas9 activation.},
}
@article {pmid42662940,
year = {2026},
author = {Şahin, C and Formica, TM and Silva, A and Della Pelle, G and Davis, MC and Andersen, DG and Çavdar, M and Santos, L and Qiu, L and Olsen, AL and Askou, AL and Köber, M and Jimenez-Mallebrera, C and Gutiérrez, MC and Soerensen, JF and Martín, F and Bak, RO and Sendemir, A and Río, P and Cavazza, A and Benabdellah, K and Nair, RR and Luo, Y},
title = {The landscape and trajectory of global CRISPR therapeutics.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {3},
pages = {103011},
pmid = {42662940},
issn = {2162-2531},
abstract = {With the first regulatory approval in 2023 of a CRISPR-based therapy for sickle cell disease followed by the recent demonstration of an accelerated, personalized CRISPR treatment for congenital severe carbamoyl-phosphate synthetase 1 deficiency, CRISPR-Cas9 genome editing has progressed from a laboratory technology into a clinical reality in just over a decade. While enthusiasm for deploying CRISPR-Cas9 to treat a range of human genetic diseases continues to grow, broad clinical application remains constrained due to technical, regulatory, manufacturing and economic challenges. Here, scientists from the European Cooperation in Science and Technology (COST) action Genome Editing to Treat Human Diseases (GenE-HumDi) provide a comprehensive global overview of the CRISPR therapeutic landscape. We systematically analyze CRISPR-based therapeutic trials registered on ClinicalTrials.gov and the EU Clinical Trials Register, providing a thorough assessment of the current clinical activity and the trajectory of CRISPR technology as it advances toward routine clinical interventions.},
}
@article {pmid42664690,
year = {2026},
author = {Bastías, DA and Zhang, W and Gundel, PE and Mace, WJ and Prakash, S and Morozova, Y and Jáuregui, R and Maclean, PH and Sprosen, J and Johnson, RD},
title = {Fungal endophytes relieve the growth-defence trade-off of their plant hosts through the production of bioactive alkaloids.},
journal = {Plant physiology and biochemistry : PPB},
volume = {238},
number = {},
pages = {111685},
doi = {10.1016/j.plaphy.2026.111685},
pmid = {42664690},
issn = {1873-2690},
abstract = {A central paradigm in plant biology is that there is a trade-off between growth and defence. We propose that Epichloë fungal endophytes relieve this trade-off in plants by the fungal-derived production of antiherbivore alkaloids. Lolium perenne plants without (nil) and with Epichloë LpTG-3 sp. strain AR37 able (wild type (wt), ΔidtA) and unable (ΔidtM) to produce bioactive indole diterpene alkaloids were subjected to an exogenous application of gibberellin (GA) hormone followed by a challenge with Rhopalosiphum padi aphids. The GA-derived plant growth promotion increased the susceptibility to aphids in both nil plants and ΔidtM-associated plants but did not affect the aphid resistance in either wt- or ΔidtA-associated plants. GA treatment changed the composition of AR37-derived alkaloids, reduced the concentration of AR37-derived epoxyjanthitrem alkaloids in wt-associated plants and reduced the amount of mycelial biomass of AR37 variants. GA treatment generally increased expression of plant genes related to abscisic acid (ABA), auxin, cell division, cell wall and GA (e.g., YUCCA2, GA2ox3), reduced expression of photosynthesis-related genes (e.g., RBCS1), and had mixed effects on the expression of plant immunity-related genes (e.g., PR-1). GA treatment increased concentrations of ABA, salicylic acid, and jasmonic acid, and did not affect auxin concentrations. Additionally, soil derived from GA-treated plants showed increased abundance of putative bacterial taxa that included plant growth-promoting members (e.g., Bryobacter). Our findings demonstrate that the AR37-derived production of indole diterpene alkaloids was the key mechanism that relieved the growth-defence trade-off of plants.},
}
@article {pmid42665203,
year = {2026},
author = {Uti, DE},
title = {Engineered exosomes for CRISPR/Cas delivery to overcome oncogene-driven drug resistance.},
journal = {Experimental cell research},
volume = {462},
number = {2},
pages = {115159},
doi = {10.1016/j.yexcr.2026.115159},
pmid = {42665203},
issn = {1090-2422},
abstract = {Engineered exosomes are emerging as biocompatible nanocarriers for delivering CRISPR/Cas components to resistant tumor cells, enabling targeted disruption of oncogenic drivers and resistance-associated pathways. Engineered exosomes offer several delivery-platform advantages, including biocompatibility, membrane-mediated cargo protection, programmable tumor targeting, and potential tissue penetration. Selection of the CRISPR modality, Cas9 ribonucleoprotein, mRNA, base editor, or prime editor, depends on payload size, stability, editing duration, endosomal escape, and nuclear delivery requirements. Therapeutically, these systems may disrupt oncogenic drivers, inhibit resistance pathways, restore tumor-suppressor activity, and re-sensitize tumors to targeted therapy, chemotherapy, or immunotherapy. Clinical translation will require scalable manufacturing, reproducible cargo loading, standardized characterization, validated potency assays, off-target control, and clearly defined regulatory pathways. The goal of this review is to outline a realistic pathway featuring proof-of-concept research, through discoveries to the creation of manufacturable, safe, and effective exosome/CRISPR therapeutics that can trigger durable therapeutic responses in resistant malignancies.},
}
@article {pmid42666455,
year = {2026},
author = {Handelmann, CR and Skeens, E and Lisi, GP and Buck, MJ},
title = {Evaluating high-fidelity CRISPR-Cas nucleases in nucleosomal contexts using a quantitative framework.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1759382},
pmid = {42666455},
issn = {2673-3439},
support = {R35 GM158384/GM/NIGMS NIH HHS/United States ; },
abstract = {Chromatin presents a significant obstacle to CRISPR-Cas gene editing, as chromatin restricts nuclease access to DNA. Recent advances have produced a wide range of high-fidelity Cas9 and Cas12a variants with enhanced properties. However, their precision in targeting DNA within different contexts remains poorly understood. This gap limits our ability to predict and optimize Cas performance in the dynamic chromatin landscape. To elucidate how chromatin variability impacts Cas editing accuracy, we utilized GEMiNI-seq to systematically profile wild-type and engineered Cas9 and Cas12a nucleases across a range of nucleosome sequences. All nucleases showed reduced cleavage in nucleosomal DNA relative to naked DNA, with the strongest inhibition at dyad-proximal sites. Cleavage within nucleosomes was highly variable, with wtSpCas9 exhibiting up to 65-fold different activity depending on the nucleosome type. Editors with high catalytic activity (wtSpCas9, HIFIv2, LbCas12a ULTRA) consistently outperformed high-fidelity variants such as evoSpCas9, which displayed excellent specificity on naked DNA but poor performance in nucleosomal contexts. ROC and PRC analyses revealed that nucleosome sequence and orientation shape both sensitivity and specificity, with HIFIv1 emerging as the top-performing nuclease for nucleosomal targets, while evoSpCas9 excelled in exposed contexts. Our findings demonstrate that local nucleosome sequence and structure profoundly influence Cas nuclease accessibility and specificity. Variability in cleavage across nucleosome types underscores the need to consider chromatin context during target selection and nuclease design. These results provide a framework for selecting or engineering Cas editors optimized for therapeutic genome editing within chromatin.},
}
@article {pmid42667235,
year = {2026},
author = {Postell, L and Elturk, N and Leonard, H and Mycek, J and Snethen, B and de Noronha, CMC and Sharifi, HJ},
title = {Targeting Cas9 to Perform Rescue Experiments in Stably Transduced Knockout HEK293 Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {234},
pages = {},
doi = {10.3791/72172},
pmid = {42667235},
issn = {1940-087X},
mesh = {Humans ; HEK293 Cells ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Cullin Proteins/genetics/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-Cas9 gene editing technology has revolutionized molecular biology. Often, this technology is employed to delete a gene encoding a protein of interest. The resulting phenotype provides valuable insight into the protein's function. The functional importance of the target protein can be confirmed by reintroducing the protein to restore the lost function (rescue). This is typically accomplished by introducing the protein-coding cDNA in trans using an expression vector. However, in knockout cell lines that stably express the CRISPR-Cas9 system, the newly introduced expression plasmid may also be cleaved by Cas9. The protocol presented here provides a strategy to circumvent this potential barrier to rescue experiments. This approach is demonstrated using HEK293 cells in which the gene encoding the E3 ubiquitin ligase scaffold protein CUL4B was disrupted by CRISPR-Cas9. Transduction of these cells with a guide RNA (gRNA) targeting the integrated Cas9 transgene resulted in the loss of detectable Cas9 protein. Cas9 ablation enabled restoration of CUL4B expression and function following introduction of a CUL4B expression plasmid. These results provide proof of concept for a broadly applicable approach to studying protein function through rescue experiments.},
}
@article {pmid42667462,
year = {2026},
author = {da Silveira Fonseca, ML and de Souza Júnior, LC and Dos SantosNascimento, F and de Souza Ramos, AP and Santana, WS and Mascarenhas, MS and Amorim, EP and Ferreira, CF},
title = {A banana susceptibility gene underlying fusarium wilt: validation as a target for disease resistance.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42667462},
issn = {1573-4978},
mesh = {*Musa/genetics/microbiology ; *Fusarium/pathogenicity ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Roots/microbiology/genetics ; Genes, Plant ; },
abstract = {BACKGROUND: Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense, remains the principal constraint on banana production, particularly for the widely cultivated cultivar 'Prata-Ana' (AAB) in Brazil. Given the limited efficacy of conventional control strategies, susceptibility (S) genes have emerged as promising targets for developing resistant cultivars. This study investigated the expression of the banana DMR6 gene during the interaction with Foc. DMR6 was selected because it is a conserved plant susceptibility gene that negatively regulates salicylic acid-mediated immunity, making it a promising target for genome editing.<br><br>METHODS AND RESULTS: Banana plantlets were inoculated with Foc Subtropical Race 4 under controlled conditions. Temporal expression of the banana DMR6 gene was analyzed by RT-qPCR, and host defense responses were assessed by histochemical and microscopic analyses. DMR6 expression initially decreased and subsequently increased, reaching a 6.5-fold induction at 72&#xa0;h post-inoculation relative to non-inoculated controls. This expression peak coincided with spore formation and advanced vascular colonization. Although infected roots exhibited callose deposition and phenolic compound accumulation, these defense responses were insufficient to restrict pathogen progression, resulting in severe disease symptoms and a disease severity index of 80% at 90 days after inoculation.<br><br>CONCLUSIONS: The findings indicate that banana DMR6 functions as a negative regulator of plant immunity and is closely associated with susceptibility to Fusarium wilt. These results provide a molecular basis for future functional validation and support DMR6 as a potential target for precise genome editing to develop resistant banana cultivars.},
}
@article {pmid42667516,
year = {2026},
author = {Brancazio, S and Khalili, K and Jacobson, S and Kaminski, R},
title = {CRISPR-mediated excision of HTLV-1 reduces proviral loads in PBMCs from HAM/TSP patients.},
journal = {Journal of neurovirology},
volume = {32},
number = {5},
pages = {},
pmid = {42667516},
issn = {1538-2443},
support = {T32 MH079785/MH/NIMH NIH HHS/United States ; 161664-04400-02//School of Medicine/Center for Neurovirology and Gene Editing internal PI fund/ ; },
mesh = {Humans ; *Human T-lymphotropic virus 1/genetics/growth &amp; development ; *Proviruses/genetics/growth &amp; development ; Viral Load ; *Paraparesis, Tropical Spastic/virology/therapy/genetics ; *Leukocytes, Mononuclear/virology ; *CRISPR-Cas Systems ; Gene Products, tax/genetics/metabolism ; Basic-Leucine Zipper Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA, Viral/genetics/metabolism ; Genome, Viral ; Terminal Repeat Sequences/genetics ; Retroviridae Proteins ; },
abstract = {CRISPR technology is emerging as a promising therapeutic approach for eliminating chronic viral infections, such as herpesviruses and HIV. Here, for the first time, we demonstrate in vitro that CRISPR can be used to excise the HTLV-1 genome and reduce proviral loads in PBMCs from HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis) patients. Single treatment with CRISPR-RNP (ribonucleoprotein) complexes composed of two gRNAs targeting the HTLV-1 env gene and 3'LTR sequences resulted in excision of a 2613 bp segment of the proviral genome, spanning tax and HBZ genes, without detectable off-target activity. Furthermore, CRISPR treatment led to over 50% reduction in proviral loads 5 days post-electroporation. Our data indicate that CRISPR-Cas9 gene editing can be used as a therapeutic strategy to eliminate HTLV-1 DNA from infected cells and may serve as a platform for curing HAM/TSP.},
}
@article {pmid42668207,
year = {2027},
author = {Casado, A and Wellner, SM and Quirós, A and Herrero-Fresno, A and Olsen, JE and Alvarez, MA and Ladero, V},
title = {CRISPR-Cas9 mediated adiA knockout in Hafnia paralvei: Implications for agmatine production and acid stress survival in a fermented dairy matrix.},
journal = {Food microbiology},
volume = {141},
number = {},
pages = {105270},
doi = {10.1016/j.fm.2026.105270},
pmid = {42668207},
issn = {1095-9998},
mesh = {*Agmatine/metabolism ; *CRISPR-Cas Systems ; *Carboxy-Lyases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Gene Knockout Techniques ; Fermentation ; *Enterobacteriaceae/genetics/metabolism/enzymology ; *Acids/metabolism ; Hydrogen-Ion Concentration ; Cheese/microbiology ; Fermented Foods/microbiology ; },
abstract = {Agmatine, the product of the decarboxylation of arginine, catalysed by arginine decarboxylase (ADC), is a bioactive compound that functions as a neuromodulator and co-transmitter and has gained increasing attention in recent years due to its therapeutic potential, particularly for its neuroprotective properties. Members of the genus Hafnia are the main agmatine producers in dairy products. In this regard, Hafnia is considered a beneficial microorganism due to its ability to enhance cheese organoleptic properties and its emerging probiotic potential, making it relevant for functional food development, specially agmatine-enriched dairy products. This study aimed to identify the genetic basis for agmatine production in Hafnia paralvei and to assess its role in bacterial fitness. Genomic analysis of the strain H. paralvei IPLA15029 revealed the presence of two genes encoding putative ADC enzymes, adiA and speA, however, organized slightly different than those in other enterobacteria. In some bacteria, ADC exists in two forms: one involved in polyamine biosynthesis, encoded by the constitutive speA gene, and another involved in acid stress resistance, encoded by the adiA gene, which is inducible under acidic conditions. In vivo experiments under controlled pH conditions showed that agmatine accumulation occurs exclusively under acidic conditions, which also stabilize the compound by preventing its catabolism to putrescine. Gene expression analysis revealed that adiA was transcribed as a monocistronic unit, and that in these conditions, adiA is the gene responsible for agmatine production. This was confirmed by generating an adiA knockout mutant after the implementation of the CRISPR-Cas9 system, marking the first successful application of this technology in the genus Hafnia. Moreover, the adiA knockout demonstrated that the encoded arginine decarboxylase is essential for survival under severe acid stress.},
}
@article {pmid42063231,
year = {2026},
author = {Hou, Z and Zhao, Y and Sun, Z and Zhan, Y and Dai, X and Wang, H},
title = {One-Pot RAA-CRISPR/Cas12a Assay for Rapid Detection of Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV) in Shrimp Aquaculture.},
journal = {Journal of fish diseases},
volume = {49},
number = {10},
pages = {e70196},
doi = {10.1111/jfd.70196},
pmid = {42063231},
issn = {1365-2761},
support = {202205//Agriculture Research System of Shanghai, China/ ; },
mesh = {Animals ; *Densovirinae/isolation &amp; purification ; *Penaeidae/virology ; Aquaculture ; *Nucleic Acid Amplification Techniques/methods/veterinary ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods/veterinary ; },
abstract = {The infectious hypodermal and haematopoietic necrosis virus (IHHNV) represents a significant viral threat to global shrimp aquaculture, leading to considerable economic losses. In this study, we have developed a one-step, one-pot isothermal assay for the detection of IHHNV, employing recombinase-aided amplification in conjunction with clustered regularly interspaced short palindromic repeats-Cas12a (RAA-CRISPR/Cas12a). The assay is performed at a constant temperature of 37°C, achieving a detection limit of 10 copies per reaction for the fluorescence assay and 1 copy per reaction for the lateral flow dipstick (LFD) assay within a 60-min timeframe. Additionally, we evaluated the assay against four other prevalent shrimp pathogens (WSSV, DIV1, EHP, VpAHPND) and observed no cross-reactivity. This straightforward detection method exhibits high sensitivity and specificity for IHHNV, offering a promising approach for early and rapid field diagnosis.},
}
@article {pmid42322407,
year = {2026},
author = {Sigrist, R and Chen, T and Montané, MR and Gockel, P and Qiao, Y and Jönsson, M and Yang, Z and Weber, T and Ding, L and Özdemir, E and Yang, L},
title = {Heterologous production of a plant biostimulant in Streptomyces albidoflavus.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42322407},
issn = {1432-0614},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; NNF22OC0079928//Novo Nordisk Fonden/ ; NNF23OC0082882//Novo Nordisk Fonden/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Metabolic Engineering/methods ; Promoter Regions, Genetic ; Multigene Family ; Polyketide Synthases/genetics/metabolism ; Fermentation ; CRISPR-Cas Systems ; Cloning, Molecular ; Biosynthetic Pathways/genetics ; },
abstract = {Climate change-associated abiotic stresses threaten agricultural productivity, creating a need for sustainable strategies that improve plant resilience. Pteridic acids F and H (PTA-F and PTA-H), originally isolated from Streptomyces iranensis HM 35, are plant growth-promoting polyketides with reported activity under drought and salinity stress. However, reported production was extremely low (~ 0.08 and 0.02 mg/L), limiting further development and application. Here, we established a heterologous production platform for PTA biosynthesis by cloning the 68-kb type I polyketide synthase biosynthetic gene cluster using Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), followed by CRISPR-Cas9-mediated genomic integration and promoter engineering in Streptomyces hosts. Initial heterologous expression resulted in detectable elaiophylin production but not PTA, whereas BGC engineering with the strong constitutive kasOp* promoter enabled PTA production (although below the limit of quantification). Genome-scale metabolic model-guided media optimization further improved production and fed-batch fermentation yielded 1.7 mg/L PTA in J1074-PTA-kasOp* and 2.8 mg/L PTA in NBC1270-PTA-kasOp*. These titers represent a more tha n 20-fold increase compared with the native producer under comparable conditions. This work provides the first functional heterologous platform for PTA biosynthesis and demonstrates how synthetic biology and genome-scale metabolic modeling can be combined to improve production of complex plant-beneficial polyketides. KEY POINTS: • Direct BGC cloning and engineering enabled production of PTA in heterologous host. • Genome-scale metabolic models (GEMs) guided media optimization for PTA production. • Fed-batch fermentation achieved > 20-fold PTA titer improvement over native strain.},
}
@article {pmid42508370,
year = {2026},
author = {Kruisselbrink, AB and Wilpshaar, TAH and Palubeckaitė, I and Dijkland, RC and Belova, T and Cardoso, S and Wijers-Koster, PM and Briaire-de Bruijn, IH and van Zeijl, RJM and Dalebout, H and Kuijjer, ML and Mei, H and Heijs, B and Szuhai, K and Bovée, JVMG and Venneker, S},
title = {Targeting vulnerabilities in IDH mutant tumours: The model matters.},
journal = {Neoplasia (New York, N.Y.)},
volume = {80},
number = {},
pages = {101338},
pmid = {42508370},
issn = {1476-5586},
mesh = {Humans ; *Isocitrate Dehydrogenase/genetics ; *Mutation ; *Chondrosarcoma/genetics/pathology/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {INTRODUCTION: Synthetic lethal interactions with IDH1 and IDH2 (IDH) mutations were identified in non-endogenous IDH mutant (IDH[MUT]) AML and glioma models, but are absent in endogenous IDH[MUT] chondrosarcoma cell lines. The translation into successful clinical applications has remained challenging, implying artificially created models do not fully recapitulate endogenous IDH[MUT] tumour biology. The aim of this study was to elucidate if the model system is indeed an important factor to consider when studying therapeutic vulnerabilities in IDH[MUT] tumours.<br><br>METHODS: Vector-based and CRISPR-Cas9 approaches were used to introduce or revert the IDH1 mutation in chondrosarcoma cell lines. These isogenic cell line pairs were used to examine the presence of known therapeutic vulnerabilities and their underlying biological mechanisms.<br><br>RESULTS: Vector-based IDH[MUT] chondrosarcoma models showed the previously reported synthetic lethal interactions, but these treatment sensitivities were absent in the CRISPR-edited models. Interestingly, not all vector-based IDH[MUT] cell lines displayed the same therapeutic vulnerabilities. Differences in treatment response were associated with multiple factors, including IDH[MUT] protein expression and D-2-HG levels, in line with the fact that therapeutic vulnerabilities could be induced in the CRISPR-edited models by enhancing D-2-HG levels.<br><br>CONCLUSION: Our findings demonstrate that synthetic lethal interactions observed in vector-based models are often a consequence of IDH[MUT] protein overexpression and supra-physiological D-2-HG levels. These results highlight that relying on artificially created IDH[MUT] models may lead to the identification of therapeutic vulnerabilities that are not present in IDH[MUT] tumours, potentially explaining the poor translation of preclinical findings to clinical trials.},
}
@article {pmid42521833,
year = {2026},
author = {Zaalberg, A and Lacoste, A and Minnee, E and Mayayo-Peralta, I and Schuurman, K and Gregoricchio, S and van Schaik, TA and Hoekman, L and Li, D and Corey, E and Janssen, H and Lieftink, C and Prekovic, S and Proost, N and van de Ven, M and Zander, S and Altelaar, M and Nelson, PS and Beijersbergen, RL and Zwart, W and Bergman, AM},
title = {A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator.},
journal = {Oncogene},
volume = {45},
number = {36},
pages = {3797-3811},
pmid = {42521833},
issn = {1476-5594},
mesh = {Humans ; Male ; *Prostatic Neoplasms/genetics/immunology/pathology ; *Receptors, Androgen/genetics/metabolism/immunology ; *Macrophages/immunology/metabolism ; NF-kappa B/metabolism/genetics ; Cell Line, Tumor ; Signal Transduction ; CRISPR-Cas Systems ; Nitriles ; Tumor Microenvironment/immunology/genetics ; I-kappa B Kinase/genetics ; Benzamides ; Phenylthiohydantoin/analogs &amp; derivatives/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Macrophages are the most abundant immune cells in the prostate tumor microenvironment and capable of killing tumor cells, but tumor intrinsic modulators of resistance to the innate immune system are unknown. To identify genes essential for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified Androgen Receptor (AR), PRKCD, and multiple components of the NF-κB pathway (IKBKB/IKBKG/CHUK) as tumor-intrinsic essential factors to allow for macrophage-mediated killing. Mechanistically, both AR and NF-κB directly drive expression of PRKCD within cancer cells, functionally implicating all hits within one molecular pathway. Importantly, androgen deprivation and AR-inhibition both rendered tumor cells resistant to macrophage-mediated killing, which positions tumor-intrinsic AR signaling as a bona fide immunomodulatory pathway. Proteomic analyses showed a selective downregulation of the oxidative phosphorylation pathway in PRKCD- and IKBKG-KO cells, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Finally, phosphoproteomic analyses revealed that all hits perturbing macrophage-mediated tumor cell eradication, impaired ferroptosis signaling in the tumor cells, which was confirmed transcriptionally using samples from a neoadjuvant phase II clinical trial with the AR-inhibitor enzalutamide. These data reveal immune protection from macrophages as an adverse consequence of hormonal therapy in prostate cancer patients.},
}
@article {pmid42586255,
year = {2026},
author = {Ivanova, E and Ramp, P and Zimmer, N and Mund, M and Antonov, E and Schiklenk, C and Degreif, D},
title = {Inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production.},
journal = {Metabolic engineering},
volume = {98},
number = {},
pages = {102523},
doi = {10.1016/j.ymben.2026.102523},
pmid = {42586255},
issn = {1096-7184},
mesh = {Flocculation ; *Saccharomycetales/genetics/metabolism ; *Biomass ; Saccharomyces cerevisiae Proteins/genetics/biosynthesis ; CRISPR-Cas Systems ; Mannose-Binding Lectins/genetics/biosynthesis ; },
abstract = {Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation or gravity sedimentation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY® VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.},
}
@article {pmid42595042,
year = {2026},
author = {Yu, Z and Chen, K and Maimaitirexiati, G and Bai, Z and Li, S and Yu, A and Yu, T and Guo, S},
title = {A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.},
journal = {Metabolic engineering},
volume = {98},
number = {},
pages = {102525},
doi = {10.1016/j.ymben.2026.102525},
pmid = {42595042},
issn = {1096-7184},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Saccharomycetales/genetics/metabolism ; },
abstract = {Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.},
}
@article {pmid42628890,
year = {2026},
author = {Hassibian, S and Esmaelpourfarkhani, M and Alibolandi, M and Ramezani, M and Abnous, K and Dehnavi, SM and Taghdisi, SM},
title = {CRISPR/Cas12a-based tag-free fluorescent biosensor using G-quadruplex specific thioflavin T for detection of oncogenic microRNAs.},
journal = {Methods (San Diego, Calif.)},
volume = {255},
number = {},
pages = {37-47},
doi = {10.1016/j.ymeth.2026.08.006},
pmid = {42628890},
issn = {1095-9130},
mesh = {*Biosensing Techniques/methods ; *G-Quadruplexes ; *Benzothiazoles/chemistry ; Humans ; *MicroRNAs/blood/genetics ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Fluorescence ; Fluorescent Dyes/chemistry ; Endodeoxyribonucleases ; },
abstract = {MicroRNAs (miRs) are central regulators of tumor initiation and progression, and their aberrant expression patterns have been identified as clinically valuable biomarkers for the early diagnosis of malignancies and prognostic evaluation. Here, we report tag-free fluorescence biosensing platform for the detection of circulating miRs in serum, targeting microRNA-21 (miR-21) and microRNA-10b (miR-10b) as clinically relevant oncogenic markers. The assay integrates CRISPR/Cas12a-mediated signal regulation with complementary strand (CS)-mediated target recognition. In this strategy, target miRs hybridize with the CS, thereby preventing CS-mediated activation of the Cas12a-crRNA complex. As a result, Cas12a collateral cleavage is suppressed, the G-quadruplex reporter remains intact, and Thioflavin T fluorescence is enhanced. The platform demonstrated excellent sequence discrimination capability, effectively distinguishing closely related and mismatched targets. Sensitive quantification was achieved with limits of detection of 1.4 nM for miR-21 and 852 pM for miR-10b. Importantly, robust analytical performance was maintained in complex biological matrices, confirming its applicability to serum samples. Collectively, this CRISPR/Cas12a-enabled fluorescent biosensor provides a simple approach for circulating miR detection.},
}
@article {pmid42652212,
year = {2026},
author = {Vanapilli Nursimulu, T and Ali, M and Shin, JA},
title = {Exploring the Evolutionary Landscape with Targeted In Vivo Hypermutations.},
journal = {Biomedicines},
volume = {14},
number = {8},
pages = {},
pmid = {42652212},
issn = {2227-9059},
support = {Discovery Grant 04846//Natural Sciences and Engineering Research Council of Canada/ ; Operating Grant 1050460//Cancer Research Society/ ; },
abstract = {Directed evolution has revolutionized protein engineering by applying the principles of natural selection to the laboratory. However, traditional in vitro methods are quite labor-intensive, while common in vivo methods suffer from low mutation rates and high rates of off-target mutations. To address these issues, researchers have developed targeted mutagenesis tools for rapid in vivo evolution of biomolecules. In this review, we discuss recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms. We focus on three main mechanisms of hypermutation: (i) orthogonal replication, which uses error-prone replication machinery to replicate the target gene with low fidelity; (ii) CRISPR-Cas-guided mutators, where mutagenic proteins are localized to virtually any user-defined loci; and (iii) transcription-coupled mutagenesis, a simple, yet elegant tool that exploits the innate processivity of orthogonal ribonucleic acid (RNA) polymerases to guide mutagenic proteins along the target gene during transcription. We highlight key advantages of these systems, as well as some clinically- and biotechnology-relevant applications. We discuss important limitations and how they could be addressed in the future to make hypermutation tools with broad mutational spectra and windows that span entire genes with minimal off-target effects.},
}
@article {pmid42652949,
year = {2026},
author = {Hamimed, S and Merazka, R and Kamah, A and Kamah, FZ and Keroui, M},
title = {Emerging Frontiers in CRISPR-Based Strategies for the Detection and Degradation of Microplastics.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {8},
pages = {},
pmid = {42652949},
issn = {2075-1729},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-based genome engineering is reshaping how environmental contamination can be interrogated and remediated, offering a level of programmability and specificity that conventional physicochemical workflows seldom match. Microplastics polymer fragments below 5 mm that now pervade virtually every ecosystem are especially difficult to monitor and remove because of their chemical heterogeneity, sub-millimeter size, and capacity to adsorb co-pollutants. This review examines how the molecular logic of CRISPR-Cas systems is being repurposed for two complementary goals: sensitive analytical detection and microbially driven degradation of plastic particles. We first outline the biochemistry of Cas-mediated cis- and trans-cleavage that underpins isothermal, amplification-free biosensing, and then survey direct strategies, in which polymer-binding DNA (deoxyribonucleic acid) aptamers are coupled to Cas12a (CRISPR-associated protein 12a), alongside indirect strategies that read out the molecular stress signatures provoked by microplastic exposure in sentinel organisms and plastisphere communities. On the remediation side, we discuss how targeted editing, CRISPR interference, and rationally assembled microbial consortia enhance enzymatic depolymerization and redirect carbon flux toward valuable bioproducts. By integrating detection and remediation within a single conceptual framework, we identify the principal bottlenecks, aptamer selectivity in complex matrices, reagent stability under field conditions, and host metabolic burden, and outline research priorities for translating these tools from proof of concept toward deployable environmental technologies.},
}
@article {pmid42653084,
year = {2026},
author = {Du, L and Dong, Y and Yang, J and Zhang, Z and Liu, Z},
title = {DNA Methylation as a Programmable Information Layer: From Molecular Marks to Disease State Engineering.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653084},
issn = {1422-0067},
support = {12302397//National Natural Science Foundation of China/ ; },
mesh = {*DNA Methylation ; Humans ; Epigenome Editing ; *Epigenesis, Genetic ; Animals ; CRISPR-Cas Systems ; Epigenomics/methods ; },
abstract = {DNA methylation has long been regarded as a stable, maintenance-based epigenetic marker. However, this classical binary model struggles to fully explain the dynamic and situational dependence of methylation regulation at the multi-biological level. This review defines DNA methylation as a programmable information layer that systematically integrates the latest advances in three interrelated dimensions of molecular coding, disease status indication, and epigenomic engineering. At the molecular level, this paper describes how the chemical diversity of cytosine modification, the writing-erasing enzyme network, and the three-dimensional structure of chromatin jointly construct a methylated polymorphic coding system and evaluates the performance of emerging sequencing technologies in DNA integrity, reading length, modification resolution, and analytical complexity through a multidimensional scoring framework. At the cellular and clinical levels, this paper comprehensively demonstrates methylation as a quantifiable indicator of cell identity, biological aging and disease status, covering circulating free DNA biomarkers and spatial heterogeneity analysis. Critically, this paper evaluates how the clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing platform achieves causal inference and promotes the transformation of methylation from related biomarkers to functional therapeutic targets. At the same time, persistent challenges such as off-target specificity, in vivo delivery, and spatiotemporal regulation encountered in epigenetic gene editing are discussed. This review reveals the paradigm shift of DNA methylation from passive observation markers to actively engineered regulatory parameters, which has direct therapeutic application prospects.},
}
@article {pmid42653108,
year = {2026},
author = {Wang, X and Zhou, H and Lim, TS and Węgrzyn, G},
title = {Phage Therapy Enhanced by Using Engineered Bacteriophages: A Powerful Antibacterial Tool to Address the Dilemma Posed by Multidrug-Resistant Bacterial Infections.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653108},
issn = {1422-0067},
mesh = {*Phage Therapy/methods ; Humans ; *Bacteriophages/genetics/physiology ; *Bacterial Infections/therapy/microbiology ; *Drug Resistance, Multiple, Bacterial ; CRISPR-Cas Systems ; Anti-Bacterial Agents/therapeutic use/pharmacology ; Genetic Engineering ; Animals ; Synthetic Biology/methods ; Gene Editing ; Bacteria/virology ; Biofilms ; },
abstract = {The continuous slowdown in the research and development of new antibiotics and antibiotic overuse have turned the problem of antibacterial resistance into a global public health crisis. As a very promising alternative to multi-drug-resistant bacterial infection, phage therapy is receiving renewed attention. However, the inherent biological limitations of natural phages restrict their extensive clinical application. This review examines how synthetic biology can be harnessed to transform phages and to build the next generation of antibacterial therapies. We outline the main advantages of natural phages, including high host specificity, self-amplification, bactericidal activity and the ability to degrade biofilms. We also point out the bottlenecks of clinical applications of bacteriophages, such as narrow host range, rapid removal in the body and potential genetic safety risks. Moreover, we elaborate on the core synthetic biological tools used to overcome the above limitations, including CRISPR-Cas gene editing, receptor-binding protein reprogramming, functional load delivery and immunogenic regulation, and summarize the recent clinical progress and personalized treatment process. The increasing clinical evidence shows that synthetic biology can effectively overcome the inherent defects of natural bacteriophages, confirming the safety and initial efficacy of bacteriophage therapy. Engineered phages provide a practical strategy to meet the antimicrobial resistance challenge. Clinical applications of such phages will mainly depend on progress in production standardization, regulatory framework construction and scientific and reasonable joint treatment program development.},
}
@article {pmid42653124,
year = {2026},
author = {Tang, H and Li, H and Tai, Y and Yang, X and Zhang, L and Ma, Y and Cai, G and Zhao, H and Zeng, T and Ai, X and He, S and Wang, J and Gu, Z and Deng, X},
title = {Heterogeneous CRISPR/Cas9 Editing of HMOX1 Is Associated with Altered Heme-Biliverdin Metabolism and Basal Stress-Associated Transcriptional Programs in Chicken LMH Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {16},
pages = {},
pmid = {42653124},
issn = {1422-0067},
support = {2022LZGCQY016//Department of Science and Technology of Shandong Province/ ; 32472896//National Natural Science Foundation of China/ ; 2022JXCQZY05//Jiangxi Provincial Joint Research Project/ ; //the National Joint Breeding Research Project. Additional support was provided by the 2115 Talent Development Program of China Agricultural University/ ; },
mesh = {Animals ; Chickens/genetics ; *Heme Oxygenase-1/genetics/metabolism ; *Biliverdine/metabolism ; *Heme/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Transcription, Genetic ; Cell Line, Tumor ; },
abstract = {Heme oxygenase-1 (HO-1), encoded by HMOX1, catalyzes the rate-limiting step of heme degradation and generates biliverdin, carbon monoxide, and ferrous iron, thereby linking heme turnover with redox regulation and stress-associated signaling. In birds, biliverdin is retained as a major heme-derived product, but the cellular consequences of HMOX1 perturbation remain insufficiently defined. Here, CRISPR/Cas9-mediated editing was used to generate a heterogeneous HMOX1-edited population in Chicken hepatocellular carcinoma-derived cells. The selected sgRNA reduced HO-1 protein abundance by approximately 47%, and no detectable cleavage was observed at the seven predicted high-risk off-target loci examined. Compared with vector-control cells, HMOX1-edited cells exhibited intracellular heme accumulation, reduced biliverdin levels, increased oxidation-sensitive fluorescence, and reduced CCK-8 absorbance values, indicating disruption of heme-biliverdin metabolic and redox homeostasis. RNA sequencing identified 2650 differentially expressed genes, including 951 upregulated and 1699 downregulated genes. Downregulated genes were mainly enriched in immune, cytokine, MAPK/stress, and extracellular signaling-associated pathways, whereas DNA replication and cell-cycle-related genes were increased. Enrichment-term association and STRING functional-association analyses further identified a coordinated module involving IL1B, JUN, NFKBIA, IRF1, TGFB1, IL10, CCL5, and PTGS2. Independent RT-qPCR analysis confirmed selected expression trends. These findings show that heterogeneous HMOX1 editing and reduced HO-1 abundance are associated with disruption of the avian heme-biliverdin metabolic axis and coordinated remodeling of basal immune, stress, extracellular signaling, and cell-cycle-associated transcriptional programs in Chicken hepatocellular carcinoma-derived cells.},
}
@article {pmid42442654,
year = {2026},
author = {Baruah, A and Wimmer, T and Stieger, K and Ponnam, SPG},
title = {Knockdown of XRCC5 and XRCC6 activity using CRISPR/Cas9 technology enhances homology-directed DNA repair at the CHST6 locus in HEK293 cells.},
journal = {Experimental eye research},
volume = {271},
number = {},
pages = {111165},
doi = {10.1016/j.exer.2026.111165},
pmid = {42442654},
issn = {1096-0007},
mesh = {Humans ; HEK293 Cells ; *Ku Autoantigen/genetics ; *CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics ; Blotting, Western ; DNA End-Joining Repair ; Gene Knockdown Techniques ; *DNA Helicases/genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; },
abstract = {In mammalian cells, DNA double-strand breaks (DSBs) are repaired by two competing pathways-homologous recombination (HR) and non-homologous end-joining (NHEJ)-that act on the same DNA ends. Downregulation of NHEJ has been shown to enhance HR-mediated repair. Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive corneal opacity and vision loss in humans. More than 180 mutations in the CHST6 gene are linked to MCD, with over 70% occurring in exon 3, making it a promising target for genome editing. In this study, we performed in vitro editing of exon 3 of CHST6 using CRISPR/Cas9 in Human Embryonic Kidney (HEK293) cells. To promote HR, the NHEJ genes XRCC6 and XRCC5, encoding KU70 and KU80, were knocked down individually or in combination. A homologous donor template was also introduced, and HR efficiency was assessed by Western blot analysis. Results demonstrated a significant increase in HR activity following downregulation of these NHEJ components, as indicated by elevated RAD51 expression. As proof of concept, partial restoration of CHST6 protein expression was observed in edited cells compared with CHST6 knockdown controls after suppression of XRCC6 and XRCC5 along with donor template delivery. These findings suggest that targeting NHEJ to enhance HR may represent a promising therapeutic strategy for MCD.},
}
@article {pmid42649367,
year = {2026},
author = {Zahn, V and Sievers, AJ and Kersten, B and Fladung, M and Bruegmann, T},
title = {Genetic transformation and CRISPR/Cas12a-mediated gene editing of European beech (Fagus sylvatica L.) employing a transient protoplast system.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42649367},
issn = {2399-3642},
support = {2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; 2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; 2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; 2219NR359//Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources)/ ; },
mesh = {*Fagus/genetics ; *Protoplasts/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; },
abstract = {Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report a protocol for protoplast isolation from seedling leaves and demonstrate transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 ± 6.19% within distinct seasonal windows. Protoplast yield and transformation efficiency showed pronounced temporal variation throughout the year, indicating a strong seasonal influence on reproducibility of the workflow despite controlled growth conditions. A basic molecular toolkit for functional genomics and future biotechnological applications was established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). Although further optimization of transformation reproducibility and regeneration systems remains necessary, the presented protoplast platform provides a valuable foundation for transient functional assays and genome editing studies in this non-model tree species.},
}
@article {pmid42649517,
year = {2026},
author = {Peng, Z and Zhang, X and Deng, X and Hou, D and Liu, R},
title = {Drivers of the paradigm shift in norovirus diagnostics: technological innovation, contextual demands, and collaborative synergy.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42649517},
issn = {1743-422X},
mesh = {*Norovirus/isolation &amp; purification/genetics ; Humans ; *Caliciviridae Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Gastroenteritis/diagnosis/virology ; Rapid Diagnostic Tests ; Point-of-Care Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {This paper systematically reviews paradigm shifts in diagnostic technologies for norovirus. Given the substantial burden of acute gastroenteritis caused by this virus and the current lack of licensed antiviral therapies, early and accurate diagnosis is of paramount importance. Diagnostic technologies have evolved from electron microscopy and immunological assays to molecular detection methods, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) currently recognized as the gold standard for molecular diagnostics. However, RT-qPCR is constrained by complex operational procedures, the need for specialized equipment, and limited suitability for rapid point-of-care applications. Emerging technologies, including isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies, have significantly improved diagnostic sensitivity, specificity, and turnaround time. Nevertheless, challenges related to standardization, quality control, and contamination prevention remain to be addressed before their widespread implementation. Furthermore, this review proposes a stratified, scenario-adaptive diagnostic framework in which rapid immunochromatographic assays are used for preliminary screening in community settings, highly sensitive PCR-based or multiplex molecular methods are employed for clinical diagnosis in healthcare facilities, and digital PCR (dPCR) is applied for precise quantification in food safety and environmental monitoring. Looking ahead,, advances in norovirus diagnostics will increasingly rely on interdisciplinary collaboration to accelerate the development of integrated and intelligent point-of-care testing (POCT) platforms. In particular, biosensors based on molecular recognition technologies and microfluidic platforms, which offer advantages such as, simple operation, rapid processing and high stability, are among the promising approaches for achieving instrument-free, on-site "sample-in, result-out" diagnostics.},
}
@article {pmid42650019,
year = {2026},
author = {Diamantopoulos, MA and Boti, MA and Kanellopoulos, E and Scorilas, A},
title = {MicroRNAs in Breast Cancer: Biological Functions and Technologies for Experimental and Therapeutic Applications.},
journal = {Cancers},
volume = {18},
number = {16},
pages = {},
pmid = {42650019},
issn = {2072-6694},
abstract = {Breast cancer is a highly heterogeneous malignancy that remains one of the leading causes of cancer-related mortality among women worldwide. Despite significant advances in breast cancer research and therapy, disease heterogeneity, treatment resistance, and metastatic progression remain major obstacles to effective disease management. Among the molecular regulators involved in breast cancer, microRNAs (miRNAs) have been recognized as critical post-transcriptional regulators of gene expression, functioning as either oncogenes or tumor suppressors. By modulating the expression of target RNAs, miRNAs control key biological processes involved in tumor initiation and progression, including cell proliferation, apoptosis, angiogenesis, epithelial-mesenchymal transition (EMT), invasion, and metastasis. To investigate miRNA function and explore their therapeutic potential, a wide range of approaches have been developed to modulate miRNA expression. These include gain-of-function strategies, like miRNA mimics, miRNA expression vectors, and CRISPR activation (CRISPRa), as well as loss-of-function approaches, including anti-miRNA oligonucleotides (AMOs), miRNA sponges, CRISPR-Cas9-mediated gene knockout, and CRISPR interference (CRISPRi). This review provides a comprehensive overview of the biological roles of miRNAs in breast cancer and discusses current technologies for miRNA modulation, their molecular mechanisms, experimental and therapeutic applications, and associated limitations. In addition, it summarizes recent advances in miRNA delivery systems, including viral vectors, organic nanoparticles, and inorganic nanocarriers, highlighting their potential to improve delivery efficiency, target specificity, and facilitate clinical translation. Finally, the review discusses future perspectives, emphasizing the transition from single-target interventions toward network-level regulation and the integration of miRNA-based strategies into precision oncology to support the development of more effective breast cancer therapies.},
}
@article {pmid42650652,
year = {2026},
author = {Toma, EA and Enciu, O and Matache, IM and Porosnicu, AL and Calu, V and Miron, A and Delawan, M and Bydon, M and Popa, MI},
title = {Novel Therapeutic Approaches and Alternatives to Antibiotic Therapy for Drug-Resistant Intra-Abdominal Infections.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
pmid = {42650652},
issn = {2079-6382},
abstract = {Antimicrobial resistance (AMR) among pathogens involved in intra-abdominal infections (IAIs) represents a critical and escalating clinical challenge. The interconnected nature of antimicrobial resistance, spanning human medicine, veterinary practice, agricultural use and environmental reservoirs, has required coordinated international responses based on the 'One Health' principle. This study presents an update on efforts underway worldwide to develop new antibiotics, novel combined antimicrobial agents, and alternatives to classic therapies for IAIs. New antibiotics or compounds with antibacterial activity are currently in various stages of clinical trials, including several fluoroquinolones, beta-lactamase inhibitors, and polymyxin analogues. To reduce the risk of bacterial resistance, various additions to antimicrobial treatments are being explored, such as nanoparticles (NPs), antimicrobial peptides (AMPs), bacteriophages, the CRISPR/Cas system, and probiotics. Each modality offers distinct mechanisms that circumvent established resistance pathways, including multi-target membrane disruption, sequence-specific gene editing, and microbiome restoration. Current preclinical and clinical evidence is synthesized, and key translational barriers, including delivery challenges, safety concerns, regulatory complexity, and the need for IAI-specific pharmacokinetic data are critically examined. In conclusion, the convergence of novel antibiotic agents and non-traditional antimicrobial strategies reviewed herein provides the foundation for a new paradigm in the management of drug-resistant IAIs. The transition from a monotherapy-centric approach to an integrated, multi-modal treatment framework, guided by rapid diagnostics and informed by antimicrobial stewardship, will be essential to preserve therapeutic efficacy against AMR threats of the coming decades.},
}
@article {pmid42650792,
year = {2026},
author = {Luan, H and Yang, R and Qiu, W and Feng, K and Xu, W and Wang, F and Feng, W and Song, P},
title = {Construction of Engineered Escherichia coli and Optimization of Conditions for Carcinine Synthesis via Multi-Enzyme Cascade Catalysis.},
journal = {Biomolecules},
volume = {16},
number = {8},
pages = {},
pmid = {42650792},
issn = {2218-273X},
support = {No. 82304838//National Natural Science Foundation of China/ ; ZR2022MC159//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Peptide Synthases/metabolism/genetics ; Transferases (Other Substituted Phosphate Groups)/metabolism/genetics ; CRISPR-Cas Systems ; *Aminoimidazole Carboxamide/analogs &amp; derivatives ; Biocatalysis ; Bacterial Proteins ; },
abstract = {Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4'-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl2. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.},
}
@article {pmid42650936,
year = {2026},
author = {Akter, R and Ryu, SW and Lee, JH},
title = {Advancements in CRISPR/Cas Technologies for Sensitive Cancer Detection: Mechanisms, Platforms, and Clinical Translation Roadmap.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {16},
number = {16},
pages = {},
pmid = {42650936},
issn = {2075-4418},
abstract = {Early cancer detection is critical for improving patient outcomes, yet current diagnostic approaches often fail to identify malignancies when tumor-specific biomarkers are relatively scarce. Emerging CRISPR/Cas-based technologies have revolutionized the ultra-sensitive detection of cancer biomarkers in liquid biopsies, overcoming the inherent limitations of traditional diagnostic approaches such as tissue biopsies and imaging, which frequently fail to detect early-stage malignancies with sufficient sensitivity. This review explores recent advances in CRISPR/Cas diagnostics (CRISPR/Cas-Dx) that employ programmable CRISPR effectors, including Cas9, Cas12, Cas13, and Cas14. In particular, the collateral (trans-) cleavage activities of Cas12, Cas13, and Cas14 enable signal amplification for highly sensitive detection of circulating tumor DNA, microRNAs, exosomes, and other multi-omics biomarkers, often without the need for extensive nucleic acid amplification. Representative CRISPR/Cas-Dx platforms include amplification-coupled assays, amplification-free frameworks, biosensing and multiplexing capabilities, and new digital or droplet-based configurations that include artificial intelligence to improve analytical precision. These technologies demonstrate single-molecule resolution and adaptability for point-of-care testing in malignancies such as non-small cell lung, colorectal, and breast carcinoma. Finally, we outline a clinical translation roadmap encompassing manufacturability, regulatory and standardization requirements, and real-world implementation challenges. This perspective offers a blueprint for CRISPR-powered, ultra-sensitive liquid biopsy diagnostics that can enable population-scale early cancer screening and truly preventive oncology by bridging molecular insights, engineering innovation, and clinical imperatives.},
}
@article {pmid42651807,
year = {2026},
author = {Athanasopoulou, K and Daneva, GN and Michalopoulou, VI and Stamelou, MR and Tsiakanikas, P and Adamopoulos, PG},
title = {Engineering the Future of Precision Medicine: A Comprehensive Guide to RNA Therapeutics.},
journal = {Current issues in molecular biology},
volume = {48},
number = {8},
pages = {},
pmid = {42651807},
issn = {1467-3045},
abstract = {RNA therapeutics have evolved from passive genetic intermediaries into highly programmable platforms, fundamentally transforming the landscape of precision medicine. This comprehensive review examines the molecular architecture and mechanisms of established platforms in the clinical setting, including mRNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) and aptamers, alongside next-generation platforms, such as CRISPR-guided systems and circular RNAs (circRNAs). Moreover, we discuss strategies to overcome systemic delivery bottlenecks and evaluate advanced non-viral systems, emphasizing lipid nanoparticles (LNPs), polymers and tissue-specific ligand conjugates that facilitate precise intracellular targeting. Furthermore, we explore the clinical expansion of these platforms across infectious diseases, rare genetic disorders, oncology and cardiovascular conditions. Finally, we highlight how the integration of artificial intelligence (AI) and machine learning (ML) redefines the limits of individualized, programmable RNA therapies by accelerating sequence optimization and nanoparticle formulation.},
}
@article {pmid42364748,
year = {2026},
author = {Gao, QY and Han, D and Liao, XR and Lei, JJ and Qi, LJ and Huang, QY and Fu, FF and Cheng, SX},
title = {Targeting delivery systems with aptamer-conjugated hyaluronic acid for personalized assessment of treatment response to targeted cancer therapy.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153260},
doi = {10.1016/j.ijbiomac.2026.153260},
pmid = {42364748},
issn = {1879-0003},
mesh = {Humans ; *Hyaluronic Acid/chemistry ; *Neoplasms/genetics/drug therapy/pathology/therapy ; *Aptamers, Nucleotide/chemistry ; Cell Line, Tumor ; GTPase-Activating Proteins/genetics ; Cell Proliferation/drug effects ; Apoptosis/drug effects/genetics ; *Precision Medicine ; CRISPR-Cas Systems ; *Drug Delivery Systems ; Molecular Targeted Therapy ; Gene Knockout Techniques ; },
abstract = {A central objective in oncology is to identify therapeutic targets essential for the survival of proliferating malignant cells. However, a major challenge in pre-clinical research is the scarcity of robust models capable of accurately assessing therapeutic efficacy in patient-specific heterogeneous cancer cells. Here, we developed an ex vivo platform for patient-specific assessment using biomacromolecule-based targeting delivery systems for CRISPR-Cas9 mediated gene knockout and in situ mRNA profiling in patient-derived circulating malignant cells (CMCs). Using a cancer targeting vector, we conducted a personalized evaluation of the impact of Rac GTPase activating protein 1 (RACGAP1) knockout on cell growth in a cancer cell line and patient-derived heterogeneous CMCs. RACGAP1 knockout induced irreversible cytokinesis failure, leading to markedly reduced proliferation and invasion capacity, DNA damage, and increased apoptosis. Our approach establishes a promising strategy for the personalized validation of novel therapeutic targets.},
}
@article {pmid42413679,
year = {2026},
author = {Jangra, A and Tiwari, S and Chhokar, V},
title = {Pioneering the formation of 2-carboxylic anthraquinone: CRISPR/Cas9-mediated functional validation of Octaketide synthase and Polyketide reductase genes in Aloe vera.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153385},
doi = {10.1016/j.ijbiomac.2026.153385},
pmid = {42413679},
issn = {1879-0003},
mesh = {*Aloe/genetics/enzymology/metabolism ; *Anthraquinones/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *Polyketide Synthases/genetics/metabolism ; Gene Editing ; },
abstract = {Aloe vera is an authentic medical plant abundant in aromatic polyketides, including the crucial hexaketides aloenin, aloesin, and barbaloin used in pharmaceuticals, yet the enzymatic basis of their biosynthesis remains incompletely understood. While it has been suggested that octaketide synthase (OKS) initiates anthraquinone biosynthesis, heterologous expression of OKS alone consistently produces shunt polyketide products, and the mechanism underlying this derailment was uncertain. To comprehend the proposed anthraquinone biosynthesis pathway, we combined biochemical constitution, structural characterization, and CRISPR/Cas9-mediated editing of key genes in Aloe vera. The results showed that the inclusion of a PKR (polyketide reductase) altered the reaction profile and supported the formation of a product spectroscopically consistent with 2-carboxy anthraquinone (C16H1205). ESI-MS analysis detected the molecular cation in [M][+] and [M + H][+] forms (m/z 284.2936 and 285.2421, respectively), while FTIR and [1]H NMR analyses indicated the presence of characteristic anthraquinone, carboxyl, hydroxyl, and terminal methyl functionalities. The spectroscopic profile additionally distinguished the characterized product from compounds previously misannotated in the literature. Alongside, CRISPR/Cas9-based genome editing of candidate genes resulted in a significant reduction in aloin content in edited lines (OKS mutant: 2.54-fold, PKR mutant 1 and 2: 1.23 and 1.53-fold, respectively) compared to the non-edited control aloe line. Together, these findings support the involvement of tailoring enzyme ketoreductase for the efficient and appropriate formation of anthraquinones and provide functional insights into polyketide biosynthesis in aloe plants that sustain as an indigenous herb for mankind.},
}
@article {pmid42648842,
year = {2026},
author = {Lin, Z and Luo, Y and Li, H and Shao, B and Yang, X and Wang, J and Wan, Y and Song, F},
title = {CRISPR/Cas12a dual-gRNA assay enables precise detection of single-nucleotide mutations via cis-staggered-cleavage.},
journal = {Analytica chimica acta},
volume = {1420},
number = {},
pages = {345968},
doi = {10.1016/j.aca.2026.345968},
pmid = {42648842},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/genetics ; *Polymorphism, Single Nucleotide ; Methicillin-Resistant Staphylococcus aureus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Robust discrimination of single-nucleotide mutations (SNMs) remains a central challenge in nucleic acid analysis, particularly under minimal sequence constraints. Here, we report a programmable CRISPR/Cas12a sensing strategy, termed STAND (cis-staggered-cleavage-based dual-gRNA assay), that enables precise SNM discrimination through a structurally gated cleavage cascade. In this system, a primary guide RNA directs Cas12a to perform site-specific cis-staggered cleavage of double-stranded DNA, generating a predictable sticky-end intermediate. This transient structure is subsequently recognized by a secondary guide RNA via strand displacement and branch migration, which reactivates Cas12a for PAM-independent trans-cleavage of reporter substrates. This sequential, structure-mediated activation decouples target recognition from PAM constraints and converts single-nucleotide variations into amplified fluorescence signals with high fidelity, and achieves a detection limit as low as 10[1] CFU/mL. We demonstrate that STAND achieves accurate SNM discrimination in clinically relevant targets, including the nuc gene of Staphylococcus aureus and methicillin-resistant S. aureus, outperforming conventional qPCR in specificity. Owing to its modular design, minimal guide requirements, and programmable architecture, this strategy establishes a generalizable framework for high-resolution genetic analysis and molecular diagnostics.},
}
@article {pmid42649178,
year = {2026},
author = {Chen, Y and Li, H and Duan, L and Liu, Y and Yan, J and Chen, H and Yang, J},
title = {A decoupled transcription platform enables tunable and predictable gene expression in yeast.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649178},
issn = {2041-1723},
support = {32122006//National Science Foundation of China | Young Scientists Fund/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Promoter Regions, Genetic/genetics ; *Gene Expression Regulation, Fungal ; *Transcription, Genetic ; Lycopene/metabolism ; CRISPR-Cas Systems/genetics ; Lactic Acid/analogs &amp; derivatives/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; },
abstract = {Predictable control of gene expression is essential for building genetic circuits and improving metabolic pathways, but conventional promoter libraries often behave unpredictably when genes are combined. Here we develop CRISPR-Activated Promoter-based Orthogonal expression (CAPO), a quantitative platform for controlling multiple genes in yeast. CAPO uses synthetic CRISPR-activated promoters that remain silent until matching guide RNAs recruit dCas9-VPR. We tune each gene by varying guide RNA abundance with defined T7 promoters, while keeping regulatory channels orthogonal. CAPO reaches expression levels comparable to strong native yeast promoters, maintains low background activity, and preserves promoter-strength order across different genes. We apply CAPO to program broad fluorescence color outputs and to rapidly optimize lycopene and 3-hydroxypropionic acid biosynthesis. These results establish CAPO as a scalable platform for predictable engineering of eukaryotic gene networks.},
}
@article {pmid42649193,
year = {2026},
author = {Ittiprasert, W and Smout, MJ and Mann, VH and Moyle, M and Kinahan, SM and Ackerman, DN and Rivera, DN and Santarpia, JL and Carnes, EC and Mentink-Kane, MM and Costa, MR and Hokke, CH and Roestenberg, M and Bottazzi, ME and Bracken, BK and Rosa, BA and Djuranovic, S and Pickering, DA and Giacomin, PR and Watterson, D and Modhiran, N and Moescheid, MF and Grevelding, CG and Mitreva, M and Loukas, A and Brindley, PJ},
title = {Heritable transgenic schistosomes as a living platform for SARS-CoV-2 neutralizing antibody secretion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649193},
issn = {2041-1723},
support = {N66001-21-C-4013//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 107475/Z/15/Z//Wellcome Trust (Wellcome)/ ; CA164719//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; GR 1549/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Schistosoma mansoni/genetics/immunology ; *SARS-CoV-2/immunology ; Mice ; *Animals, Genetically Modified ; *Antibodies, Neutralizing/immunology ; COVID-19/immunology ; *Antibodies, Viral/immunology ; Female ; Biomphalaria/parasitology ; Angiotensin-Converting Enzyme 2 ; Humans ; Immunoglobulin G/immunology/genetics ; Immunoglobulin Fc Fragments/genetics/immunology ; },
abstract = {We report the generation and propagation of not only the first heritable transgenic schistosome line but also a line that secretes a functional therapeutic protein in vivo. Using multiplexed CRISPR/Cas-mediated homology-directed knock-in targeted to a predicted genomic safe-harbor, we inserted a VHH-IgG1 Fc (termed C5-Fc) transgene into Schistosoma mansoni eggs. Single-miracidium infections of Biomphalaria glabrata yielded parental P0 lines; serial passage through snail and mouse hosts produced an F2 cohort in which all parasites carried the C5-Fc transgene and secreted C5-Fc into the murine venous circulation. Molecular assays confirmed chromosomal insertion, germline transmission and systemic secretion. Sera from mice harboring C5-Fc transgenic worms neutralized SARS-CoV-2 in vitro with potent activity consistent with the expected ACE2-binding blockade by the C5 variable domain of heavy-chain-only antibody (VHH). These results demonstrate (i) stable, heritable transgenesis of a platyhelminth, (ii) delivery of a biologically active antibody fragment by a live helminth in a mammalian host, and (iii) feasibility of using transgenic schistosomes as sustained, single-dose protein delivery platforms. This technology and delivery system enable new experimental approaches for schistosome biology and motivate exploration of living-foundry therapeutics.},
}
@article {pmid42649204,
year = {2026},
author = {Madsen, A and Selfjord, N and Martinez-Lage, M and Loyd, AL and Kurgan, G and Ståhlberg, M and Lindgren, J and Liz Touza, J and Wigge, L and Firth, M and Nordström, K and Collin, J and Jachimowicz, D and Schiffthaler, B and Dillmann, I and Antoniou, P and Emmanouilidi, A and Hellsten, J and Forsström, J and Magnell, K and Jacobi, A and Behlke, M and Porritt, M and Madeyski-Bengtson, K and Maresca, M and Akcakaya, P},
title = {Single-cell and in vivo profiling reveal heterogeneous and organ-specific CRISPR-Cas9 off-target and translocation outcomes.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649204},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Single-Cell Analysis/methods ; *Gene Editing/methods ; Organ Specificity/genetics ; DNA Methylation ; Embryonic Stem Cells/metabolism ; Chromatin/metabolism/genetics ; *Translocation, Genetic ; Mouse Embryonic Stem Cells/metabolism ; Embryo, Mammalian/cytology/metabolism ; },
abstract = {CRISPR-Cas9 holds promise for treating genetic disease, but rare off-target mutations and structural variants remain as key safety concerns, especially at scales relevant to therapy. Here, we establish workflows to resolve Cas9 off-target activity in vitro at single-cell resolution and in vivo across different tissues. Using clonally expanded electroporated mouse embryos and embryonic stem cells, we reveal that individual cells exhibit unique off-target and translocation profiles, including events missed in bulk analyses. Integrating single-cell editing with chromatin accessibility, transcription, and DNA methylation measurements suggest that sequence-independent features modulate Cas9 access and cleavage, with preferential editing in regions characterized by open chromatin and lower methylation. In Cas9-inducible mouse models, editing analyses reveal organ-distinct off-target spectra, DNA repair pathway usage, indel patterns, and markedly varying translocation propensity between tissues. These findings demonstrate that off-target activity is heterogeneous across cells and context-dependent across organs, motivating sensitive single-cell analyses and organ-specific evaluation in preclinical development to more accurately assess risk and improve the safety of CRISPR-based genomic medicines.},
}
@article {pmid42649208,
year = {2026},
author = {Hong, M and Luan, C and Yuan, M and Huang, H and Guo, X and Meng, D and Huang, M and Xu, Y and Zhao, S and Chen, K and Chen, J and Li, D and Chen, L},
title = {High-diversity base mutagenesis via simultaneous adenine, cytosine and guanine editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42649208},
issn = {2041-1723},
support = {2024YFC3407900//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; },
mesh = {*Guanine/metabolism ; *Adenine/metabolism ; *Cytosine/metabolism ; *Mutagenesis ; Humans ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Base editors hold great promise in endogenous mutagenesis for genetic screening. However, the development of base editors that induce saturated multi-base conversions with diverse mutation spectrum is challenging. Here, we develop triple base editors (smACGs) that simultaneously mutagenize adenine, cytosine, and guanine within the same allele. Through screening and embedding engineered deaminase and alkyladenine DNA glycosylase variants in Cas9 structure, smACGmax is generated to catalyze robust triple-base conversion efficiencies of up to 41% across varied sequence contexts while maintaining low RNA off-target effects compared to previous dual-base editors. We apply smACGmax to enable high coverage (94%) of targeted HBEGF mutagenesis that identified diphtheria toxin-resistant mutations and to dissect SF3B1 variants with alternative splicing specificity via complex single, double, and triple base conversion screening. smACGmax expands base conversion capability from single and double substrates to trinucleotide level, which facilitates the generation of high-diversity and complex genetic variants, providing a useful platform for mutagenesis-based application.},
}
@article {pmid41436006,
year = {2026},
author = {Zhang, H and Chen, B and Gu, L and Wang, C and Xu, L and Ji, X and Wang, J and Wang, Z and Xiao, X and Liu, Y},
title = {A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants.},
journal = {Journal of advanced research},
volume = {87},
number = {},
pages = {947-962},
doi = {10.1016/j.jare.2025.12.015},
pmid = {41436006},
issn = {2090-1224},
mesh = {*Anti-Bacterial Agents/pharmacology ; *Homologous Recombination/drug effects/genetics ; Rec A Recombinases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/drug effects ; Microbial Sensitivity Tests ; Escherichia coli Proteins/genetics/metabolism ; Cisplatin/pharmacology ; Genome, Bacterial ; },
abstract = {INTRODUCTION: The widespread misuse and overuse of antibiotics have driven the emergence of multidrug-resistant and pan drug-resistant bacteria, constituting a formidable global health threat. Antibiotic adjuvants that potentiate the efficacy of existing antibiotics represent a particularly promising avenue to address this challenge.<br><br>METHODS: We performed a genome-wide CRISPR interference (CRISPRi) screening to identify potential targets for broad-spectrum antibiotic adjuvants, which highlighted the homologous recombination pathway as a promising candidate. To functionally validate this pathway, we employed three strategies to suppress the expression and function of recA, a key component of homologous recombination, including a CRISPRi system delivered via transconjugation, a RecX-derived peptide (RecX-20) fused to a cell-penetrating motif, and a small-molecule inhibitor cisplatin validated by surface plasmon resonance.<br><br>RESULTS: Disruption of the homologous recombination pathway not only significantly increased bacterial susceptibility to multiple classes of antibiotics, including quinolones, &#x3b2;-lactams, aminoglycosides, and nitrofurantoin, but also reduced horizontal gene transfer of antibiotic resistance. In addition, recA deficiency resulted in a cascade of physiological disruptions, including membrane damage, efflux pump dysfunction, oxidative stress imbalance and metabolic disruption. All three recA-targeting strategies enhanced the antibacterial activity, with cisplatin exhibiting the most pronounced potentiating effect both in vitro and in vivo.<br><br>CONCLUSIONS: This study reveals that the homologous recombination pathway, particularly RecA, is a viable target for the development of broad-spectrum antibiotic adjuvant. Our findings provide mechanistic insights and practical strategies to restore the effectiveness of existing antibiotics and address the growing threat of antimicrobial resistance.},
}
@article {pmid42625528,
year = {2026},
author = {van Putten, M and Linssen, M and Tanganyika-de Winter, C and Brouwers, CM and Claassens, JWC and Verwey, N and Walsh, M and Loredan Stan, T and Aartsma-Rus, A and Hohenstein, P},
title = {Four new mouse models of Duchenne muscular dystrophy with clinically relevant exon deletions in the human DMD gene.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {8},
pages = {},
doi = {10.1242/dmm.052875},
pmid = {42625528},
issn = {1754-8411},
support = {24745//AFM-Telethon/ ; 24745//AFM-T&#xe9;l&#xe9;thon/ ; //Leids Universitair Medisch Centrum/ ; //REGENXBIO/ ; },
mesh = {Animals ; *Exons/genetics ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Humans ; *Dystrophin/genetics/metabolism ; Disease Models, Animal ; *Sequence Deletion/genetics ; Mice ; Mice, Inbred mdx ; CRISPR-Cas Systems/genetics ; Base Sequence ; Muscle, Skeletal/pathology ; Male ; Mice, Inbred C57BL ; Oligonucleotides, Antisense ; },
abstract = {Variant-specific therapeutic approaches, such as exon skipping or gene editing, hold promise for the treatment of Duchenne muscular dystrophy (DMD). Translatability of preclinical studies investigating these approaches could greatly be improved through the use of humanized mouse models, as these allow preclinical testing of human-specific sequences. We developed four novel humanized mouse models of DMD with a deletion of exon 44, 45, 51 or 53 in the human DMD gene, in a mouse dystrophin-negative background (mdx mouse; exon 23 nonsense mutation). Our optimized prescreening pipeline allowed us to do so very efficiently with the CRISPR-Cas9 technology. We confirmed either complete lack of dystrophin or expression of trace levels, which led to development of muscle pathology consisting of muscle fiber degeneration and regeneration, inflammation and fibrosis in young adult mice. Intramuscular treatment with vivo-morpholinos targeting a flanking exon induced exon skipping in the DMD strains, which restored the disrupted open reading frame and, subsequently, dystrophin expression. This validates these models as valuable tools for preclinical studies investigating human sequence-specific therapeutic approaches for DMD.},
}
@article {pmid42644396,
year = {2026},
author = {Dong, Y and Xu, C and Yan, B and Mou, S and Li, C and Liu, Y},
title = {Quantitative and Targeted Regulation of Ferroptosis in Bladder Cancer: Preclinical Study.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {16},
pages = {e71333},
pmid = {42644396},
issn = {1582-4934},
support = {2021YFA0911600//National Key R&amp;D Program of China/ ; RCJC20221008092723011//Shenzhen Science and Technology Program/ ; JCYJ20220818102001002//Shenzhen Science and Technology Program/ ; },
mesh = {*Ferroptosis/genetics ; Humans ; *Urinary Bladder Neoplasms/genetics/pathology/metabolism ; Animals ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; *Beclin-1/genetics/metabolism ; Mice ; Amino Acid Transport System y+/genetics/metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics/metabolism ; Cell Proliferation ; CRISPR-Cas Systems/genetics ; Female ; },
abstract = {The activation of ferroptosis, a cell death mechanism driven by excessive ferrous ions (Fe[2+]) and lipid peroxides, has emerged as a promising target for cancer treatment. However, in the case of quantitative regulation of target genes, it remains uncertain whether ferroptosis can be induced in bladder cancer (BCa) cells without affecting normal ones. We investigated this using an innovative CRISPR-dCas9 system to upregulate and downregulate the ferroptosis-related gene BECN1 and OTUB1, respectively. We identified two genes that can affect and promote ferroptosis-related pathways, analysing their expression in bladder tissue through The Cancer Genome Atlas. Our unique CRISPR-dCas9 technology, under the control of an hTERT promoter, selectively adjusted BECN1 and OTUB1 expression exclusively in cancer cells. RT-qPCR and western blotting demonstrated significant alterations in the expression of GPX4 and SLC7A11, proteins strongly associated with ferroptosis, in BCa cells, while normal bladder cells remained unaffected. We developed a quantitative model based on synthetic biology principles to describe the regulatory relationships between the ferroptosis-related genes BECN1 and OTUB1 and their downstream targets GPX4 and SLC7A11 in bladder cancer cells. The model establishes a direct proportional relationship between BECN1 upregulation and decreased GPX4 expression, and between OTUB1 downregulation and decreased SLC7A11 expression. In vitro experiments revealed reduced viability, proliferation, migration, and invasion in UMUC-3 and T24 BCa cells. Importantly, Fer-1 and DFO rescued the viability loss, and C11-BODIPY staining confirmed increased lipid ROS accumulation, supporting ferroptosis-associated cell death following BECN1/OTUB1 regulation. In vivo xenograft experiments showed that BECN1 upregulation or OTUB1 downregulation suppressed tumour growth. Tumour-tissue immunofluorescence further showed reduced GPX4 expression in BECN1-upregulated tumours and reduced SLC7A11 expression in OTUB1-downregulated tumours, supporting suppression of the GPX4/SLC7A11 ferroptosis-protective axis in vivo. The quantitative equation derived from our data suggests that the induction of ferroptosis in bladder cancer cells can be effectively modulated by these two genes, and the experimental results also indicate our system can modulate these two genes to affect the function of BCa cells without affecting the normal cells, offering a promising new direction for the development of targeted therapy for bladder cancer.},
}
@article {pmid42644503,
year = {2026},
author = {Gakpo, JO and Gulabrai, B and Sanders, CE and Parrella, JA and Proudman, J and Berger, T and Mitloehner, F},
title = {U.S. consumers' processing of information about CRISPR-edited pork products.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2719351},
doi = {10.1080/21645698.2026.2719351},
pmid = {42644503},
issn = {2164-5701},
mesh = {Animals ; Humans ; Female ; Swine ; *Consumer Behavior ; Male ; *Gene Editing ; United States ; Adult ; Middle Aged ; Information Seeking Behavior ; Young Adult ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Adolescent ; },
abstract = {The commercialization of CRISPR gene-edited pork is advancing rapidly, following the U.S. Food and Drug Administration's approval of gene-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome (PRRS). As these products move closer to market entry, understanding how consumers seek, process, and avoid information about them is critical for developing effective communication strategies. Guided by the Risk Information Seeking and Processing (RISP) model, this study examined factors influencing information seeking, information avoidance, and information processing related to CRISPR-edited pork products among U.S. consumers (n = 2,006). Results show higher information sufficiency thresholds were associated with greater information seeking and lower information avoidance. Information seeking was strongly and positively correlated with systematic processing. Relevant channel beliefs and perceived information gathering capacities were positively associated across communication channels, suggesting the need for integrated communication approaches. Relevant channel beliefs for news media and social media were positively associated with information seeking, while stronger relevant channel beliefs for Extension were associated with lower information seeking. Respondents with some college education reported higher information seeking than those with only a high school diploma or GED, while older adults and individuals with higher education levels reported lower information avoidance. Results also showed that respondents exhibited high intentions to seek information and low tendencies to avoid information, suggesting openness to learning about CRISPR-edited pork. Participants also reported engaging more in systematic processing than heuristic processing, indicating a preference for careful and analytical evaluation of information. Findings highlight the importance of audience segmentation, multi-channel communication strategies, and evidence-based messaging to support informed public engagement with CRISPR-edited food technologies.},
}
@article {pmid42645040,
year = {2026},
author = {Amanzholova, M and Akimbekova, A and Shaizadinova, A and Sutimbekova, N and Bissenova, N and Tarlykov, P and Abeldenov, S},
title = {Multiplex RPA-CRISPR/Cas12a Assay for Rapid Detection of Class D OXA-Type Carbapenem-Resistant Acinetobacter baumannii.},
journal = {Biosensors},
volume = {16},
number = {8},
pages = {},
pmid = {42645040},
issn = {2079-6374},
support = {BR24992881//Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*Acinetobacter baumannii/genetics/isolation &amp; purification/drug effects ; Carbapenems/pharmacology ; *beta-Lactamases/genetics ; Humans ; CRISPR-Cas Systems ; Bacterial Proteins/genetics ; Rapid Diagnostic Tests ; },
abstract = {Acinetobacter baumannii is a critical WHO priority pathogen due to its multidrug resistance and high mortality in carbapenem-resistant infections. Resistance is predominantly mediated by class D carbapenemase genes blaOXA-23 and blaOXA-40, which spread rapidly via horizontal gene transfer in healthcare settings. To address the lack of a rapid assay capable of detecting both blaOXA-23 and blaOXA-40 in a single analytical workflow, we developed a multiplex two-step RPA-CRISPR/Cas12a assay. Since infections caused by strains harboring either gene require identical therapeutic management, their co-detection in a single reaction is clinically justified. Although simultaneous use of two crRNAs within a single CRISPR/Cas12a reaction is often considered technically challenging due to potential inter-crRNA competition, here it advantageously enables dual-target coverage without compromising sensitivity. The assay demonstrated high specificity with no cross-reactivity against a panel of clinically relevant bacterial species, including closely related Acinetobacter spp. Evaluation using genomic DNA extracted from 63 cultured clinical A. baumannii isolates revealed blaOXA-23 in 19 isolates (30.2%), blaOXA-40 in 28 (44.4%), and co-carriage of both genes in 9 (14.3%), with at least one resistance gene detected in 60.3% of isolates. The complete workflow was accomplished within 45 min without specialized equipment, offering a rapid, sensitive, and cost-effective solution for point-of-care molecular surveillance of carbapenem-resistant A. baumannii in clinical and resource-limited settings.},
}
@article {pmid42647707,
year = {2026},
author = {Tan, K and Sun, W and Fullwood, MJ and Jia, L and Lyu, H and Zhang, L and Dao, F},
title = {AcrPLMEvo: A Two-Stage Framework Integrating Evolutionary Profiles with Protein Language Models for Anti-CRISPR Prediction.},
journal = {IEEE journal of biomedical and health informatics},
volume = {PP},
number = {},
pages = {},
doi = {10.1109/JBHI.2026.3727674},
pmid = {42647707},
issn = {2168-2208},
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and are important regulators for controllable genome-editing applications. However, their computational identification remains challenging because Acrs are sequence-diverse, weakly conserved, and supported by limited labeled data. Here, we present AcrPLMEvo, a two-stage framework that integrates protein language model (PLM) representations with PSSM derived evolutionary profiles for low-homology Acr prediction. We systematically compared four representative PLM backbones, parameter-efficient adaptation strategies, and alternative PSSM-coupling routes. Evolutionary profiles were not universally beneficial; instead, their effects depended on both PLM backbone and the stage at which they were incorporated. A key finding was that evolutionary information was more consistently beneficial when retained at the downstream decision stage than when used only during PLM adaptation. Guided by this observation, AcrPLMEvo combines PSSM-aware DoRA adaptation of ESM-2 with frozen feature extraction and final-stage evolutionary feature reintroduction. In the matched benchmark comparison, AcrPLMEvo achieved the best overall performance among competing Acr predictors, with an AUC of 0.965 and an AUPRC of 0.778. Its predictive reliability was further supported on an independently curated external set of 44 proteins, where it correctly classified 41 proteins and produced no false positives. These results indicate that stage-consistent integration of evolutionary profiles can improve PLM-based Acr prediction and support the prioritization of low-homology Acr candidates.},
}
@article {pmid42648526,
year = {2026},
author = {Gamage, A and Herath, HMLPB and de Silva, KMN and de Silva, RM},
title = {RNA biosensors in oncology: Mechanisms, Cancer-specific applications, and a Hallmark-aligned clinical roadmap.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {121306},
doi = {10.1016/j.cca.2026.121306},
pmid = {42648526},
issn = {1873-3492},
abstract = {Cancer diagnosis continues to rely on invasive tissue sampling and static molecular assessments that cannot reflect the real time RNA alterations driving tumour progression. RNA biosensors are genetically encoded or synthetic devices that translate specific RNA markers and tumour microenvironment signals into measurable outputs offer a compelling alternative, particularly for liquid biopsy applications where non-invasive, dynamic monitoring is essential. This review systematically examines key RNA biosensor classes developed for oncology, spanning fluorescence based platforms such as aptamers, FRET probes, and molecular beacons, enzymatic and electrochemical architectures including CRISPR Cas systems and field effect transistors, metabolite responsive designs encompassing riboswitches, RNA thermometers, and reactive oxygen species sensors and sequence specific toehold switches alongside exosomal detectors. For each class, we discuss operating principles, cancer relevant applications, reported detection thresholds reaching into the zeptomolar range, and current limitations. These biosensor capabilities are mapped onto Hanahan's hallmarks of cancer, and practical clinical roadmaps are outlined for three priority applications early population screening, longitudinal therapy response and resistance monitoring, and tumour microenvironment prognostication. Convergence with microfluidic integration, AI assisted interpretation, and multiplexed nanotechnology represents the critical next step in translating these platforms from laboratory tools into routine diagnostic practice.},
}
@article {pmid42648813,
year = {2026},
author = {Zhao, M and Zhuang, Q and Wang, X and Gong, J and Chen, L},
title = {CRISPR/Cas12a-based dual intelligent sensors for home pet detection via personal glucose meters.},
journal = {Analytica chimica acta},
volume = {1420},
number = {},
pages = {345935},
doi = {10.1016/j.aca.2026.345935},
pmid = {42648813},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods/instrumentation ; Animals ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Glucose/analysis ; Magnetite Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; Rapid Diagnostic Tests ; Dogs ; },
abstract = {Against the backdrop of iterative upgrades in pet pathogen detection technology, rapid on-site testing (POCT) has become the core technology for on-site identification of deadly pet diseases. Based on the research and development of new materials, intelligent sensors with high sensitivity, fast response, and high design flexibility have demonstrated strong application value and have become an important development direction for the next-generation technology system in the field of pet pathogen detection. Herein, we report two advanced intelligent material-integrated biosensing platforms: a DNA hydrogel-encapsulated glucose amylase-based assay (RC-HGPGA) and a magnetic nanoparticles (MNPs)-based system where single-stranded DNA (ssDNA) serves as a molecular bridge to conjugate MNPs with invertase (RC-MBI). Both systems operate via a cascade reaction: recombinase polymerase amplification (RPA) of target nucleic acids first activates Cas12a nuclease, which then exerts trans-cleavage activity toward the biosensing elements. Subsequent enzymatic hydrolysis generates glucose, whose concentration is quantifiable using a commercial personal glucose meter (PGM). All experimental procedures were conducted at a constant temperature of 37 °C, eliminating the need for complex thermal cycling equipment. Our findings demonstrate that the RC-HGPGA and RC-MBI platforms achieve ultra-sensitive detection of feline panleukopenia virus (FPV) and canine distemper virus (CDV)-two clinically significant pet viruses-with limits of detection (LODs) as low as 10° copies/μL and 10[1] copies/μL, respectively, within a rapid time of 35 min. Both systems exhibit high sensitivity, excellent specificity, broad adaptability, and user-friendliness, thereby showing great potential for on-site detection of pet viruses.},
}
@article {pmid41712125,
year = {2026},
author = {Saxena, S and Saxena, S and Gupta, D},
title = {Miniaturized CRISPR: Ultra Compact Systems for In Vivo Delivery and Portable Diagnostics.},
journal = {Annals of biomedical engineering},
volume = {54},
number = {9},
pages = {2859-2872},
pmid = {41712125},
issn = {1573-9686},
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; Miniaturization ; *Gene Editing/methods ; Point-of-Care Systems ; },
abstract = {Reduced-size CRISPR systems have become a possible remedy to the delivery and size constraints of the traditional SpCas9 (~ 1368 Å). Recently described small nucleases, including Cas12f (400-700 Å) or CasX (~ 980 Å), along with designed mini-Cas9 versions, can efficiently be used in vivo to edit cells as well as to perform point-of-care diagnostics because of their lower molecular weight and less complex structures. This review will sum up progress in compact Cas protein engineering, guide RNA optimization, and delivery vector miniaturization, and point to their influence in therapeutic gene editing and portable diagnostic platforms. We additionally cover the contemporary issues of interest, such as off-target activity, delivery barriers and regulatory requirements, and future opportunities provided through AI-assisted protein design and synthetic biology. The miniaturized CRISPR technology is bound to substantially transform the translational arena of gene editing and world diagnostics.},
}
@article {pmid42504735,
year = {2026},
author = {Luong, LH and Stone, S and Bui, V and Bhattarai, B and Vu, MT and Aydin, EP and Craig, T},
title = {Lonvoguran ziclumeran: a CRISPR-CAS9-based gene therapy for the treatment of hereditary angioedema.},
journal = {Expert opinion on investigational drugs},
volume = {35},
number = {8},
pages = {545-553},
doi = {10.1080/13543784.2026.2710107},
pmid = {42504735},
issn = {1744-7658},
mesh = {Humans ; Animals ; *Genetic Therapy/methods/adverse effects ; *Angioedemas, Hereditary/therapy/genetics/physiopathology ; CRISPR-Cas Systems ; Gene Therapy Agents ; Gene Editing/methods ; Precision Medicine ; Plasma Kallikrein/genetics ; Nanoparticles ; },
abstract = {INTRODUCTION: Hereditary angioedema (HAE) is a rare genetic disorder characterized by recurrent swelling caused by dysregulation of the kallikrein-kinin pathway. Although current therapies effectively reduce attack frequency, treatment remains lifelong. Lonvoguran ziclumeran (Lonvo-z; NTLA-2002) is the first systemically administered in vivo CRISPR/Cas9 gene-editing therapy designed to provide durable suppression of plasma kallikrein through permanent disruption of the KLKB1 gene.<br><br>AREAS COVERED: This review summarizes the pathophysiology and current management of HAE, the development of Lonvo-z, its lipid nanoparticle delivery platform, and the technical advances enabling in vivo genome editing. Preclinical studies and clinical evidence, including early-phase trials and the Phase 3 HAELO study, are reviewed with emphasis on efficacy, safety and clinical implications.<br><br>EXPERT OPINION: Lonvo-z represents a major milestone in precision medicine and the clinical application of systemic genome editing. A single administration has produced sustained reductions in plasma kallikrein levels and HAE attack frequency. Although long-term follow-up is ongoing, current evidence supports its potential as the first one-time disease-modifying treatment for HAE and a landmark advance in CRISPR-based therapeutics.},
}
@article {pmid42621610,
year = {2026},
author = {Li, J and Zhang, H and Yu, H and Liang, P and Xu, S and Zhong, L and Fu, X and Zhang, Y and Wang, Y},
title = {Application of bacteriophages in the prevention and control of bacterial infectious diseases in animals.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1851321},
pmid = {42621610},
issn = {1664-302X},
abstract = {The global spread of antimicrobial resistance (AMR) has intensified the search for alternatives to conventional antibiotics in animal production systems. Bacteriophages can be engineered beyond narrow-spectrum antibacterial agents into multifunctional biological platforms that integrate direct killing, immune modulation, and antigen delivery. We summarize recent advances across livestock, poultry, and aquaculture, delineating mechanistic distinctions between lytic phage therapy, phage display-derived interventions, and engineered platforms including CRISPR-Cas-enabled theranostic systems. However, as detailed below, most evidence remains preclinical, and translational gaps are substantial. Unlike prior descriptive reviews, we analyze translational bottlenecks-host range constraints, pharmacokinetic limitations, regulatory fragmentation-and assess the existing research evidence for claimed advantages such as microbiota preservation and biofilm penetration while upfront acknowledging inconsistent experimental outcomes and inherent application limitations behind these beneficial effects. We conclude that realizing phages' therapeutic potential in veterinary medicine requires coordinated progress in synthetic biology, scalable manufacturing, and regulatory harmonization within a One Health framework.},
}
@article {pmid42624339,
year = {2026},
author = {Gao, R and Jin, H and Zhang, T and Zhang, H and Huang, P and Wang, H},
title = {CRISPR/Cas trans-cleavage activity in pathogen detection: research progress and innovations.},
journal = {Biotechnology advances},
volume = {93},
number = {},
pages = {109017},
doi = {10.1016/j.biotechadv.2026.109017},
pmid = {42624339},
issn = {1873-1899},
abstract = {The trans-cleavage activity of CRISPR/Cas systems has catalyzed significant progress in molecular diagnostics. Compared with traditional methods such as polymerase chain reaction (PCR) and its derivatives, CRISPR/Cas diagnostics are often credited with high specificity, portability, and visual readout. Among various CRISPR systems, CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13 have been extensively applied in pathogen detection owing to their distinct target-recognition and nucleic acid-cleavage mechanisms. In particular, Cas12- and Cas13-based systems exploit target-activated trans-cleavage activity for sensitive signal amplification, whereas Cas9-based diagnostic platforms generally rely on sequence-specific cis-cleavage. This review assesses the integrated CRISPR/Cas detection workflow from sample collection and processing through final result output, and systematically analyzes the intrinsic characteristics of Cas effector proteins with respect to target enrichment, reporter molecules, readout formats, sample background, and validation design. Based on a practical application-oriented framework, we analyzed the adaptability of various CRISPR/Cas systems in distinct scenarios, including point-of-care screening, quantitative laboratory testing, and multiplex pathogen identification. In addition, we highlight engineering innovations derived from mechanistic investigations of Cas9, Cas12, Cas13 and Class I CRISPR systems, discuss the specific diagnostic bottlenecks these effectors can resolve, and outline remaining challenges requiring further optimization prior to clinical translation.},
}
@article {pmid42624823,
year = {2026},
author = {Sun, J and Liu, M and Zheng, X and Ouyang, G and Chen, A and Qian, H},
title = {Partial deletion in the cuticular protein gene BmorCPR2 results in a body shape mutant in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae).},
journal = {Journal of insect science (Online)},
volume = {26},
number = {4},
pages = {},
pmid = {42624823},
issn = {1536-2442},
support = {2023-YBNY-134//Key Industrial Chain Projects of Shaanxi Provincial/ ; 2023-JC-YB-188//Basic Research Programs of the Shaanxi Provincial Science and Technology Department/ ; CARS-18-ZJ0101//the China Agriculture Research System of MOF and MARA/ ; BE2020418//Key R &amp; D plan of Jiangsu Province/ ; },
mesh = {Animals ; *Bombyx/genetics/growth &amp; development/anatomy &amp; histology ; *Insect Proteins/genetics/metabolism ; Larva/genetics/growth &amp; development/anatomy &amp; histology ; Mutation ; CRISPR-Cas Systems ; Sequence Deletion ; },
abstract = {The Bombyx mori L. (Lepidoptera: Bombycidae) is a significant economic insect used for silk production. A novel body shape mutant, stony^sunken (st^sk), that exhibits a sunken intersegmental membrane was isolated from the wild type of st^sk (WT-n08). Investigation indicated that the mutation had no significant effect on its growth and development. To elucidate the molecular mechanism underlying this body shape mutant, genetic analysis, positional cloning, and the CRISPR/Cas9 gene editing system were performed. Genetic analysis demonstrated that the mutant trait in st^sk is controlled by an autosomal recessive gene and follows Mendelian inheritance. Positional cloning showed that a putative cuticular protein gene, BmorCPR2 on chromosome 8, was the candidate gene. Sequencing analysis revealed partial deletion of BmorCPR2 exon 2 and intron 2 sequences occurred and subsequently resulted in the premature termination of gene expression. Knock-out of BmorCPR2 using the CRISPR/Cas9 gene editing system led to the sunken intersegmental membrane phenotype. These findings highlight the essential role of BmorCPR2 in silkworm cuticular formation, providing a foundation for further research on cuticular protein function.},
}
@article {pmid42624898,
year = {2026},
author = {Bindu, S and Ash, A and Sarkar, K},
title = {Advanced gene editing technologies for oncology mechanisms, applications, and clinical implementation.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42624898},
issn = {1476-5500},
support = {EMDR/SG/15/2023-5901//Indian Council of Medical Research (ICMR)/ ; },
abstract = {Advanced gene editing tools have transformed oncology by facilitating precise molecular therapies aimed at the hereditary basis of cancer. This thorough study examines the mechanisms, applications, and clinical implementation of advanced genome editing technologies in cancer treatment. This review commences with the molecular principles of genome editing and DNA repair mechanisms, systematically analyzing established technologies such as Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases, and various CRISPR/Cas systems (Cas9, Cas12, Cas13), in addition to novel advancements including base editors, prime editors, and the PASTE system. Additionally, hybrid platforms such as ARCUS, MegaTALs, and modified recombinases are examined, highlighting their amalgamation with artificial intelligence, biosensors, and synthetic biology concepts. The study outlines significant applications including functional genomics, disease modeling, synthetic lethality screening, and direct therapeutic interventions, with a specific focus on CAR-T cell engineering and immune checkpoint regulation. Applications unique to various cancer types are thoroughly examined throughout lung, breast, colorectal, hematologic, liver, pancreatic, head &amp; neck, esophageal, prostate, gastric, and brain cancers. Significant obstacles such as delivery optimization via viral and non-viral vectors, tumor-specific targeting, off-target effects, immunogenicity, and ethical issues related to germline vs somatic editing are comprehensively examined. The translational landscape is analyzed via current clinical trials, regulatory structures, and the incorporation of organoid models and patient-derived xenografts for the advancement of personalized therapies. This review highlights the transformative impact of gene editing on cancer medicine, advancing toward more accurate, effective, and personalized therapeutic approaches.},
}
@article {pmid42627520,
year = {2026},
author = {Lv, Z and Wang, G},
title = {Establishment of a CRISPR/Cas9-mediated system for targeted editing of the MFS gene in mint.},
journal = {Plant cell reports},
volume = {45},
number = {9},
pages = {},
pmid = {42627520},
issn = {1432-203X},
support = {23H010204158//Contract Research Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mentha/genetics/metabolism ; Plants, Genetically Modified ; Protoplasts/metabolism ; Oils, Volatile/metabolism ; Base Sequence ; Plant Proteins/genetics/metabolism ; Transformation, Genetic ; *Gene Editing/methods ; },
abstract = {The key message of this study is that we established a CRISPR/Cas9-mediated genome-editing system for Mentha haplocalyx "738" by optimizing protoplast transient assay and screening effective regulatory elements. Targeted knockout of the MFS gene generated edited mint plants with reduced menthofuran content, offering a strategy for quality improvement of mint essential oil. The commercial value of mint (Mentha spp.) essential oil is often diminished by the presence of undesirable metabolites, notably menthofuran, which impairs flavor and raises safety concerns. This study aimed to develop a robust CRISPR/Cas9 gene editing system for mint 738 (Mentha haplocalyx "738") and apply it to disrupt the menthofuran synthase (MFS) gene, thereby redirecting metabolic flux to enhance oil quality. We established an optimized system for high-efficiency protoplast isolation and transient transformation from young mint leaves. Key parameters for enzymatic digestion (1.5% cellulase R10, 0.2% macerozyme R-10, 3 h) and PEG-mediated transformation (40% PEG6000, 0.4 M mannitol, 0.4 M CaCl2) were systematically determined. Using this platform, we screened endogenous regulatory elements, identifying a truncated mint U6 promoter (HmU6.1-3P) and the tomato SlEF1α promoter as the most effective drivers for sgRNA and Cas9 expression, respectively. A CRISPR/Cas9 vector targeting the MFS gene was constructed and used for Agrobacterium-mediated stable transformation. The positive transgenic mint lines were obtained. Sequencing confirmed heritable mutations at the target sites within the MFS gene in multiple independent lines. The results revealed a substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.},
}
@article {pmid42627922,
year = {2026},
author = {Wang, SK and Li, Z and Shah, SH and Edwards, QA and Bouffard, R and Hines, ES and Imventarza, JA and Korte, S and Lawrence, MS and Song, E and Helmy, E and Amaya, L and Kang, NW and Myung, D and Tsai, MC and Greenleaf, WJ and Waymouth, RM and Wang, S and Wender, PA and Chang, HY},
title = {RNA delivery to the corneal endothelium using charge-altering releasable transporters.},
journal = {Science advances},
volume = {12},
number = {34},
pages = {eady8161},
pmid = {42627922},
issn = {2375-2548},
support = {R01 CA245533/CA/NCI NIH HHS/United States ; T32 EY027816/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *Endothelium, Corneal/metabolism ; Humans ; Mice ; *Gene Transfer Techniques ; *RNA/administration &amp; dosage/genetics ; Nanoparticles/chemistry ; CRISPR-Cas Systems ; },
abstract = {RNA therapies hold tremendous promise for treating genetic eye diseases. However, their development is limited by the lack of non-viral delivery platforms that can target specific ocular cell types. Here, we describe a charge-altering releasable transporter (CART) that delivers RNA selectively to the corneal endothelium, a non-regenerative cell layer whose dysfunction underlies several blinding conditions. We characterize the safety of CART-RNA nanoparticles in mice and show that they facilitate delivery of diverse RNA cargoes to the corneal endothelium, including circular RNA and CRISPR/Cas9. We verify that these nanoparticles can be redosed and apply them to achieve corneal gene editing. We further demonstrate CART transfection of corneal endothelial cells from a human donor in vitro and in a non-human primate in vivo, supporting the feasibility of clinical translation. Our findings establish CARTs as a platform for non-viral gene delivery to the eye, with the potential to treat corneal dystrophies and other vision disorders.},
}
@article {pmid42627995,
year = {2026},
author = {Ripken, L and Hoekman, TD and Willemse, M and Troost, ML and Kenyon, AN and van den Broek, WJAA and Wansink, DG},
title = {CRISPR/dCas9-mediated tuning of DMPK transcription reveals a quantitative relationship between toxic repeat RNA expression and MBNL1 activity in myotonic dystrophy.},
journal = {Human molecular genetics},
volume = {35},
number = {17},
pages = {},
doi = {10.1093/hmg/ddag079},
pmid = {42627995},
issn = {1460-2083},
support = {W.OR18-06//Prinses Beatrix Spierfonds/ ; },
mesh = {Humans ; *Myotonic Dystrophy/genetics/pathology/metabolism ; *Myotonin-Protein Kinase/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; Myoblasts/metabolism ; RNA Splicing/genetics ; Trinucleotide Repeat Expansion/genetics ; Cell Line ; Alternative Splicing/genetics ; RNA/genetics ; },
abstract = {Myotonic dystrophy type 1 (DM1) is caused by (CUG)n-expanded DMPK transcripts that sequester the splicing factor MBNL1 in the nucleus, resulting in widespread splicing abnormalities. Although significant progress has been made in understanding DM1 pathogenesis, the contribution of DMPK transcript levels to disease severity, and the variability of these levels across cell types, tissues, and patients, remains poorly understood. To investigate this in a quantitative manner, we developed isogenic human immortalized myoblast models with inducible modulation of DMPK RNA levels using CRISPR activation (CRISPRa) and interference (CRISPRi) guided by synthetic sgRNAs. CRISPRa elevated DMPK RNA levels by more than three-fold, intensifying MBNL1-dependent splicing defects. In contrast, CRISPRi reduced DMPK RNA expression by approximately 80%, partially rescuing splicing abnormalities. These changes were validated by visualizing (CUG)n foci using RNA FISH. Lowering DMPK transcript levels increased the availability of free nucleoplasmic MBNL1, whereas upregulation further depleted MBNL1, reinforcing the central role of MBNL1 sequestration in repeat RNA toxicity. Our findings demonstrate that expanded DMPK transcript levels modulate free MBNL1 concentration and alternative splicing in a dose-dependent manner, underscoring the central role of repeat RNA expression in DM1 pathogenesis. These models provide a powerful platform for dissecting variability in DMPK expression and for defining the therapeutic thresholds required for effective DMPK knockdown, thereby offering critical insights for the design and evaluation of DMPK and MBNL1-directed therapeutic strategies.},
}
@article {pmid42631783,
year = {2026},
author = {Saleem, M and Syed Khaja, AS and Ahmad, I and Alraey, Y and Azhar, MA and Khan, MS},
title = {Staphylococcus aureus biofilms: molecular mechanisms, resistance determinants, and emerging therapeutic strategies.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42631783},
issn = {1573-4978},
support = {R.G.P.2/503/46//The Deanship of Research and Graduate Studies at King Khalid University, Abha, Saudi Arabia./ ; },
mesh = {*Biofilms/drug effects/growth &amp; development ; Humans ; *Staphylococcus aureus/drug effects/genetics/pathogenicity/physiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Quorum Sensing/drug effects ; *Staphylococcal Infections/microbiology/drug therapy ; *Drug Resistance, Bacterial/genetics ; Virulence Factors/genetics ; },
abstract = {S. aureus is a significant opportunistic pathogen that causes a variety of community and healthcare-associated infections. Biofilm formation is one of its many virulence factors and contributes to persistent, recurrent, and device-associated infections through enhancing bacterial survival, immune system evasion, and resistance to antimicrobial agents. The development of biofilms is a complex, highly regulated process influenced by genetic regulators, environmental factors, and intercellular communication, leading to the formation of a structured microbial community with a protective extracellular matrix (ECM). These biofilms undergo large-scale physiological, transcriptomic, and proteomic changes, which help them survive harsh host conditions and reduce their susceptibility to immune responses and standard antibiotics. Biofilm-associated antimicrobial resistance is also facilitated by several complementary mechanisms, including limited penetration of antimicrobials, changes in bacterial physiology, persister cell formation, adaptive stress responses, and the presence of other clinically relevant microorganisms in polymicrobial biofilms. Recent evidence has also emphasized the importance of host-biofilm interactions in the establishment of chronic infections, including dysregulated inflammatory responses and immune evasion. Due to the intrinsic inefficacy of traditional antimicrobial drug treatment against mature biofilms, significant efforts have been made to develop novel anti-biofilm interventions, such as matrix-disrupting agents, quorum-sensing inhibitors, antimicrobial peptides, bacteriophages, nanotechnology-assisted delivery systems, CRISPR-Cas-based therapeutics, and rational combination therapy. This review aims to provide a comprehensive and up-to-date overview of the molecular biology of S. aureus biofilms, biofilm-associated antimicrobial resistance, interactions with the host, polymicrobial interactions, and emerging therapeutic strategies, and to highlight the ongoing challenges and future directions in the prevention and treatment of persistent biofilm-associated infections.},
}
@article {pmid42633712,
year = {2026},
author = {Kulishova, LM and Zharkov, DO},
title = {Glycosylase Base Editors: New Tools for Genome Editing.},
journal = {Biochemistry. Biokhimiia},
volume = {91},
number = {7},
pages = {1093-1113},
doi = {10.1134/S0006297926601073},
pmid = {42633712},
issn = {1608-3040},
mesh = {*Gene Editing/methods ; Humans ; *DNA Glycosylases/metabolism/genetics ; CRISPR-Cas Systems ; Animals ; DNA/genetics/metabolism ; },
abstract = {Genome editing using the CRISPR/Cas9 system has become a staple of modern genome manipulation. In its original form, editing involved introducing double-strand breaks into DNA, which can cause genomic instability. The appearance of the first base editors in 2016 expanded the range of editing technologies and enabled single-nucleotide changes to be introduced into the genome through deamination of nucleobases, bypassing the double-strand break stage. Further development of base editors involves the incorporation of additional modules, DNA glycosylases, that can remove modified or even normal nucleobases and create non-instructive apurinic/apyrimidinic sites in DNA, significantly expanding the range of available single-nucleotide substitutions. This review examines the operating principles of the glycosylase base editors, the main limitations of these genome manipulation tools, and promising areas for the development of this technology.},
}
@article {pmid42634244,
year = {2026},
author = {Gulfam, T and Li, W and Han, Z and Gulfam, Y and Li, J and Fan, Z and Zhang, H and Wang, F and Yang, J},
title = {Gene Editing in Forest Tree Breeding for Stress Resistance: From Mechanisms to Future Prospects.},
journal = {Plant, cell &amp; environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70840},
pmid = {42634244},
issn = {1365-3040},
support = {31870649//National Natural Science Foundation of China/ ; },
abstract = {Forest ecosystems face escalating threats from climate change alongside a surging demand for sustainable bioproducts. While conventional tree breeding is inherently constrained by long generation cycles, high heterozygosity, and complex genomes, CRISPR-based genome editing provides a precision framework for targeted genetic improvement. This review synthesises the fundamental principles and limitations of multiple gene-editing technologies, with a particular emphasis on CRISPR systems (Cas9, Cas12, and Cas13), in the specific context of woody perennial biology. Recent applications in key forest genera, including Populus, Pinus, and Eucalyptus, demonstrate the efficacy of these gene-editing tools in manipulating complex traits, such as rewiring phytohormone signalling networks for drought tolerance or remodelling root system architecture to combat abiotic stress. We critically evaluate persistent translational bottlenecks in forest tree genome editing, with a specific focus on recalcitrant, genotype-dependent regeneration and the multifaceted challenges of long-term field validation. Finally, we highlight how synergising CRISPR technologies with multi-omics, genomic selection, and high-throughput phenomics can accelerate the development and application of climate-resilient woody perennials.},
}
@article {pmid42634480,
year = {2026},
author = {Kawamata, M and Niwa, S and Suzuki, A},
title = {Chemical and Structural Engineering of Guide RNAs for Precision Genome Editing: From Design Principles to Clinical Applications.},
journal = {Chemical biology &amp; drug design},
volume = {108},
number = {2},
pages = {e70377},
pmid = {42634480},
issn = {1747-0285},
support = {JP18H04737//Japan Society for the Promotion of Science/ ; JP20H05041//Japan Society for the Promotion of Science/ ; JP23K18097//Japan Society for the Promotion of Science/ ; JP23K27462//Japan Society for the Promotion of Science/ ; JP25K22439//Japan Society for the Promotion of Science/ ; JP18H05102//Japan Society for the Promotion of Science/ ; JP19H01177//Japan Society for the Promotion of Science/ ; JP19H05267//Japan Society for the Promotion of Science/ ; JP20H05040//Japan Society for the Promotion of Science/ ; JP22H05634//Japan Society for the Promotion of Science/ ; JP22H04698//Japan Society for the Promotion of Science/ ; JP22H00592//Japan Society for the Promotion of Science/ ; JP23K18579//Japan Society for the Promotion of Science/ ; JP25K22908//Japan Society for the Promotion of Science/ ; JP25H00445//Japan Society for the Promotion of Science/ ; JPMXP1323015486//MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence Program/ ; //the Center for Clinical and Translational Research of Kyushu University Hospital/ ; //the Fukuoka Financial Group Enterprise Development Foundation (KYUTEC)/ ; //the Medical Research Center Initiative for High Depth Omics/ ; //the Takeda Science Foundation (to M.K., and A.S.), the Uehara Memorial Foundation/ ; //Naito Foundation/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Genetic Engineering ; Machine Learning ; *Gene Editing ; Animals ; },
abstract = {CRISPR-Cas9 has revolutionised genome editing by enabling efficient and programmable modification of defined DNA sequences, with guide RNAs (gRNAs) serving as indispensable elements that direct Cas9 to specific genomic loci. Initially regarded as auxiliary components, gRNAs are now recognized as critical determinants of editing efficiency and specificity and have attracted growing attention as independent targets for engineering. Chemical modification, sequence optimisation, and structural alteration of gRNAs have been shown to enhance on-target activity, suppress off-target effects and cytotoxicity, and even achieve allele-selective precision editing in a programmable manner. Moreover, advances in artificial intelligence and machine learning have markedly improved the predictive accuracy of gRNA design through large-scale data analysis. Despite rapid progress, a consolidated review that integrates chemical, structural, and computational advances in gRNA engineering and highlights their translational potential for therapeutic genome editing has been lacking. This review uniquely addresses that gap by presenting an integrated framework that connects molecular design principles with clinical applicability.},
}
@article {pmid42635641,
year = {2026},
author = {Singh, H and Kumar, P and Sharma, V and Singh, J and Swiecicki, WK and Jedryczka, M and Gawlowska, M and Tiwari, S},
title = {Efficient in-vitro regeneration and transformation for CRISPR/Cas9-mediated genome editing of phytoene desaturase (PDS) gene in pea (Pisum sativum L.).},
journal = {Plant cell reports},
volume = {45},
number = {9},
pages = {},
pmid = {42635641},
issn = {1432-203X},
support = {PPN/ BIN/ 2019/1/00142/U/00002//Polish National Agency for Academic Exchange (NAWA), Poland/ ; DST/INT/POL/P-45/2020//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*Pisum sativum/genetics/enzymology/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Regeneration/genetics ; *Transformation, Genetic ; *Oxidoreductases/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Plant Shoots/genetics/growth &amp; development ; },
abstract = {The present study addresses optimization of in-vitro regeneration via direct organogenesis and Agrobacterium-mediated genetic transformation, enabling efficient multiplex CRISPR/Cas9-based genome editing of the phytoene desaturase (PsPDS) gene in pea. Pea (Pisum sativum L.) is an important legume crop valued for food, plant-based protein, vegetable, and green manure. Although genome editing offers a precise and rapid strategy for crop improvement, its application in pea remains challenging due to inherent recalcitrance to in-vitro regeneration and genotype-dependent transformation. The regeneration and Agrobacterium-mediated transformation systems were optimized, and the dicotyledonary node (DCN) was identified as the preferred explant for multiplex CRISPR/Cas9-based genome editing in pea. Among three explant types (embryonic axis, DCN and nodal segment), DCN showed the highest regeneration efficiency, producing 100% shoot bud induction and 39.70 shoots per explant on MS medium augmented with 6-benzylaminopurine (BAP; 6.00 mg/L) and kinetin (1.00 mg/L). Shoot elongation and rooting efficiencies were improved using GA3 (1.00 mg/L), BAP (1.00 mg/L), IAA (0.10 mg/L), and NAA (0.5 mg/L), respectively. Manipulating explant type, Agrobacterium optical density, vacuum infiltration, acetosyringone concentration, infection time, and co-cultivation duration improved the transient transformation efficiency. We noted efficiency from 23.33% to 90.00% in DCN and from 6.66% to 93.33% in embryonic axis explants across 10 pea cultivars. Stable transformed lines generated from the DCN of cultivar Kashi Samridhi were confirmed by GUS staining and PCR. The optimized regeneration and transformation system facilitated targeted editing of phytoene desaturase (PsPDS) in pea, achieving ICE-estimated mutation frequencies of upto 97% in independent lines. The study provides a robust platform for functional genomics and accelerates the deployment of genome-editing technologies for pea improvement.},
}
@article {pmid42638006,
year = {2026},
author = {Sreekanth, D and Singh, C and Pawar, DV and Yadav, MK and Sahadeo, IK and Basavaraj, PS and Kumar, R and Mahesh, S},
title = {CRISPR/Cas-Mediated Genome Editing for Developing Herbicide Tolerant Rice: A Step-by-Step Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3017},
number = {},
pages = {47-57},
pmid = {42638006},
issn = {1940-6029},
mesh = {*Oryza/genetics/drug effects/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Herbicides/pharmacology ; *Herbicide Resistance/genetics ; Acetolactate Synthase/genetics ; Plants, Genetically Modified/genetics ; Benzoates ; Pyrimidines ; },
abstract = {Weed management in rice cultivation has predominantly relied on acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides, particularly against Echinochloa spp. (watergrass). However, these herbicides carry a high risk of resistance evolution, as evidenced by the numerous resistant biotypes reported worldwide. The emergence of herbicide resistance necessitates innovative and sustainable weed control strategies. Genome editing, particularly through the CRISPR/Cas system, provides a precise and efficient platform for introducing targeted genetic modifications to develop herbicide-tolerant (HT) rice cultivars. In this protocol, we present a step-by-step approach for generating bispyribac sodium-tolerant rice using the CRISPR/Cas-mediated editing of the ALS gene. The method encompasses guide RNA design, vector construction, transformation, selection of edited plants, and molecular confirmation of targeted mutations. This approach offers a robust framework for producing HT rice lines, potentially reducing reliance on conventional herbicide regimes and mitigating the risk of resistance development in weed populations.},
}
@article {pmid42638019,
year = {2026},
author = {Mukherjee, A and Basak, S and Singh, R and Bajani, R and Kundu, P},
title = {Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3017},
number = {},
pages = {221-239},
pmid = {42638019},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/microbiology ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; *Plant Diseases/microbiology/genetics ; *Gene Editing/methods ; Streptococcus pyogenes/genetics ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) has become integral to modern biological research, with the Streptococcus pyogenes CRISPR/Cas9 system serving as the most extensively used tool for precise, site-specific genome editing across a wide range of organisms and cell types. Compared with earlier genome-editing platforms, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 offers greater simplicity, precision, versatility, and scalability. Beyond introducing stable DNA modifications, this system can be reengineered to reversibly activate (CRISPRa) or repress (CRISPRi) the transcription of any gene by employing unique nuclease-deactivated variants of Cas9 (dCas9) fused to transcriptional activators or repressors, respectively, providing a compelling alternative to RNA interference (RNAi) and conventional overexpression techniques. In plants, CRISPR/dCas9-based programmable gene control presents an innovative and transformative framework for rewiring gene regulatory networks to study pathogenic stress-signaling pathways. Notably, its strategic use in orchestrating the simultaneous regulation of multiple defense-related genes sets the stage for developing crops with robust and quantitative disease resistance. In this chapter, we outline a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.},
}
@article {pmid42638068,
year = {2026},
author = {Yao, M and Li, T and Sun, B and Li, M and Peng, D and Wang, Y and Qiu, HJ and Zhang, D and Li, LF},
title = {[Construction and characterization of a stable Cas9-expressing monoclonal WSL cell line].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {8},
pages = {3736-3748},
doi = {10.13345/j.cjb.250933},
pmid = {42638068},
issn = {1872-2075},
support = {32372983//the National Natural Science Foundation of China/ ; ZD2025C008//the Natural Science Foundation of Heilongjiang Province/ ; },
mesh = {Animals ; Cell Line ; *African Swine Fever Virus/genetics/physiology ; *CRISPR-Cas Systems/genetics ; Swine ; Lentivirus/genetics/metabolism ; Lung/cytology ; Genetic Vectors/genetics ; Clone Cells ; },
abstract = {The molecular mechanism underlying the cellular invasion of African swine fever virus (ASFV) remains incompletely understood, particularly with respect to its key cellular receptors, which constitutes a major bottleneck in the development of effective vaccines and targeted antiviral therapies. To establish a robust experimental platform that supports efficient ASFV replication and enables genome-wide CRISPR screening for systematic identification of host factors involved in viral entry, we employed the CRISPR/Cas9 system to generate stable monoclonal cell lines expressing Cas9 protein based on the wild boar lung (WSL) cell line. Recombinant lentiviruses co-expressing Cas9 and blasticidin resistance gene were packaged via a lentiviral vector system and transduced into WSL cells. Following blasticidin selection, a polyclonal population stably expressing Cas9 was obtained and subjected to fluorescence-activated cell sorting (FACS) to derive monoclonal cell lines. Cas9 expression was determined by Western blotting. To assess the functional gene editing activity of the established clones, we introduced the lentiviruses carrying an EGFP reporter gene along with its specific single-guide RNA (sgRNA) into the monoclonal cell lines, and quantitatively evaluated the editing efficiency via flow cytometry. Furthermore, sgRNAs specifically targeting the ASFV B646L gene and the host TMEM239 gene were designed and synthesized to validate the cell line's capacity for editing both viral and host genomic loci. The results demonstrated the successful establishment of seven stable WSL-Cas9 monoclonal cell lines expressing Cas9 protein, among which clone WSL-Cas9-3# exhibited the highest editing efficiency, enabling effective genetic modification of both ASFV and host genes, while maintaining favorable genetic stability and normal growth properties. This study reports the generation of a WSL-Cas9 monoclonal cell line with high CRISPR/Cas9 editing efficiency, stable proliferation, and permissiveness for robust ASFV replication. This engineered cell line provides a reliable platform for future genome-wide functional screening to systematically identify host factors governing ASFV entry and establishes a critical technical foundation for delving into virus-host interactions.},
}
@article {pmid42640835,
year = {2026},
author = {Rahat, H and Qaiser, D and Maqsood, Q and Mehmood, T},
title = {Engineered Microbial Cellulases for Biomass Conversion: Integrating Omics, Expression Platforms, Fermentation Engineering and Enzyme Reusability.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70337},
pmid = {42640835},
issn = {1097-0290},
abstract = {The conversion of lignocellulosic biomass, which is an abundant renewable carbon source, is limited by the cost, stability, loading requirement and scale-up constraints of cellulase systems for sustainable biomanufacturing. Cellulose deconstruction is catalyzed by microbial cellulases such as endoglucanases, cellobiohydrolases, beta-glucosidases and accessory enzymes, which are used in biorefineries, food and feed processing, textiles, detergents, pulp and paper and waste valorization. This review focuses on cellulase production as a platform for biotechnology rather than as a standalone fermentation process. It connects native cellulase-producing microorganisms, omics-guided enzyme discovery, lignocellulosic substrate selection, pretreatment and inhibitor tolerance, solid-state and submerged fermentation, recombinant expression systems, enzyme engineering, downstream recovery, immobilization, reusability and industrial translation. Focus is given to the transition from conventional microbial producers to engineered platforms that combine CRISPR/Cas systems, transcriptional regulation, base and prime editing, strain improvement, promoter and secretion engineering, synthetic biology, enzyme-cocktail optimization and structure-guided or AI-assisted cellulase design. Despite the advances in cellulase yield, catalytic efficiency, thermostability and substrate specificity, the use of cellulases on a large scale is still hindered by the heterogenicity of the feedstock, catabolite repression, enzyme inhibition, downstream recovery cost and scale-up limitations. The next step will be the integration of microbial diversity, multi-omics, advanced host engineering, process intensification, low-cost recovery strategies and application-specific enzyme cocktails to create robust, economically viable cellulase platforms for sustainable biorefineries and circular bioeconomy applications.},
}
@article {pmid42642425,
year = {2026},
author = {Wu, Y and Xia, Y and Yao, Z and Chen, W and Jia, X and Liang, N and Champer, J},
title = {Finding the perfect promoter for Cas9 in homing gene drives using single cell transcriptome data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42642425},
issn = {2041-1723},
support = {32270672//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Promoter Regions, Genetic/genetics ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems/genetics ; Female ; Male ; *Transcriptome/genetics ; Single-Cell Gene Expression Analysis ; Single-Cell Analysis ; Alleles ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Gene drive can modify or suppress vector populations by spreading drive alleles. In CRISPR homing drives, regulating Cas9 expression has been effective for improving drive performance, but selecting suitable promoters is often a major challenge. Here, we evaluate 35 Cas9 constructs with distinct promoters in Drosophila melanogaster and identify associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is associated with elevated expression of the promoter-associated gene in reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. Additionally, we find that an in situ construct significantly reduces potentially harmful somatic expression. Based on these results, we propose criteria for selecting promoters, providing a rationale and guidance for optimization of homing gene drives.},
}
@article {pmid42642483,
year = {2026},
author = {Li, TT and Chen, X and Wang, F and Tang, YA and Sim, M and Xiao, L and Jin, WB and Shi, H and Ma, JY and Yang, X and Liu, Y and Sorbara, MT and Guo, CJ},
title = {Transferable genetic toolsets for nonmodel gut Clostridia enable in vivo reversible control of metabolite production.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42642483},
issn = {1546-1696},
abstract = {Gut Clostridia species, including commensal members of the Clostridiaceae and Lachnospiraceae families, maintain microbiota homeostasis and influence human health and disease; however, adequate genetic toolsets to study abundant but nonmodel gut Clostridia are lacking. Here we present a set of transferable and modular genetic toolsets that function broadly across phylogenetically diverse gut Clostridia. We first identify a panel of strong constitutive promoters that drive robust gene expression across diverse clostridial strains. We then develop an inducible promoter system that enables precise, tunable gene regulation and facilitates the implementation of CRISPR-Cas gene-deletion systems. We apply this system for targeted and reversible control of trimethylamine and deoxycholic acid production, two microbiota-derived metabolites implicated in host lipid metabolism and diseases, in mice. This robust genetic toolkit for nonmodel gut Clostridia enables functional studies to causally link microbiota genes to host physiology and disease, paving the way for therapeutic genetic engineering of microbiota.},
}
@article {pmid42642601,
year = {2026},
author = {Zhou, X and Lu, R},
title = {Dissecting Epigenetic Drug Response Mechanisms Using CRISPR Knockout Screens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3005},
number = {},
pages = {383-401},
pmid = {42642601},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Epigenesis, Genetic/drug effects ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Epigenome Editing ; *Neoplasms/genetics/drug therapy ; Gene Editing/methods ; *Antineoplastic Agents/pharmacology ; Animals ; },
abstract = {Epigenetic drugs are widely applied in cancer therapy due to their ability to modify gene expression without altering the DNA sequence. Despite their therapeutic potential, drug resistance frequently occurs, posing a significant challenge in cancer treatment. CRISPR screens have emerged as a powerful tool to address this issue by leveraging the precision of CRISPR-Cas9 gene editing to enable the systematic interrogation of genes. This approach involves the simultaneous targeting of thousands of genes to elucidate their roles in various biological processes, disease mechanisms, and drug responses, providing valuable insights into gene function and potential therapeutic targets. In cancer therapy, CRISPR screens provide a deeper understanding of cancer progression by enabling the identification of essential genes and facilitating the discovery of novel therapeutic targets when combined with epigenetic drugs. Here, we present an overview of the CRISPR screen methodology, which involves introducing guide RNAs targeting specific genes into cells, followed by phenotype selection and analysis.},
}
@article {pmid42642602,
year = {2026},
author = {Tang, Q and Liu, J},
title = {Tools for Cancer Research: CRISPR/Cas-Based Gene Editing and Auxin-Induced Degron Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3005},
number = {},
pages = {403-415},
pmid = {42642602},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Degrons ; *Indoleacetic Acids/pharmacology/metabolism ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; Gene Knock-In Techniques ; },
abstract = {CRISPR/Cas9 and auxin-degron systems represent two powerful and complementary genetic tools that have revolutionized cancer research. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein)-based gene editing system has been widely used in numerous biological fields, significantly enhancing the capacity of researchers to elucidate the underlying mechanisms of biological phenomena. The two most prevalent applications for CRISPR-mediated gene editing are knockout and knock-in. This protocol covers both single-gRNA (sgRNA) and dual-gRNA directed knockout systems. Furthermore, we describe the procedure for constructing an Auxin-Inducible Degron (AID) knock-in-mediated system to induce the rapid degradation of a target protein in cells, thereby investigating its function.},
}
@article {pmid42643124,
year = {2026},
author = {Zafar, MM and Firdous, H and Siddiqua, A and Naveed, A and Razzaq, A and Munawar, S and Ijaz, A and Anwar, Z and Ercisli, S and Jiang, X and Fei, Q},
title = {Programmable Domestication: CRISPR, Pan-Genomics and System Level Engineering for Next-Generation Crops.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
pmid = {42643124},
issn = {1467-7652},
abstract = {Global agriculture is increasingly challenged by climate instability, genetic erosion, emerging pathogens and rising food demands, exposing the limitations of conventional breeding and traditional domestication strategies. Recent advances in CRISPR-based genome editing, pangenomic, synthetic biology, artificial intelligence (AI)-assisted breeding and predictive phenomics are transforming de novo domestication from a slow evolutionary process into a programmable framework for rational crop redesign. This review synthesises recent advances in programmable de novo domestication and highlights how crop wild relatives and underutilised germplasm can be harnessed to develop resilient, climate-adaptive and sustainable crop systems. The integration of multiplex genome editing, pan-genomic variation discovery, AI-driven genomic prediction and predictive breeding enables precise engineering of key domestication traits governing plant architecture, yield potential, stress resilience and nutritional quality. Furthermore, we propose a trajectory-based framework for programmable domestication comprising Adaptive Rescue, Agronomic Refinement and Novel Chassis Engineering, which illustrates distinct evolutionary pathways, engineering complexity and crop redesign objectives. We also examine the major system level challenges that constrain programmable domestication, including cryptic genetic variation, epistasis, gene regulatory network complexity, genotype phenotype predictability, biodiversity conservation and regulatory considerations. Collectively, programmable domestication represents a transformative shift from conventional crop improvement towards system-level engineering of next-generation crops, providing a strategic foundation for enhancing global food security, agricultural sustainability and environmental resilience in the face of accelerating climate change.},
}
@article {pmid42643189,
year = {2026},
author = {Takata, M and Chikumi, H and Yoshifuji, A},
title = {Molecular Diagnostics of Infectious Diseases.},
journal = {Yonago acta medica},
volume = {69},
number = {3},
pages = {228-247},
pmid = {42643189},
issn = {0513-5710},
abstract = {With the advances in molecular biology, molecular testing has been incorporated into pathogen testing for infectious diseases. There are two main objectives for the molecular diagnosis of infectious diseases- first, to detect pathogen genes by using highly sensitive nucleic acid amplification tests, such as polymerase chain reaction and isothermal amplification, and second, to characterize the properties of the pathogen using next-generation sequencing. Polymerase chain reaction plays a key role in molecular testing. The extensive development of polymerase chain reaction methods is underway, focusing on the acceleration of reaction time (microfluidic polymerase chain reaction), quantification (real-time polymerase chain reaction, digital polymerase chain reaction), full automation, and point-of-care testing. Isothermal amplification is a method for amplifying nucleic acids at a constant temperature. Loop-mediated isothermal amplification, recombinase polymerase amplification, and nucleic acid sequence-based amplification have been developed for isothermal amplification. Isothermal amplification does not require a thermal cycler or simplified temperature control; therefore, it is suitable for point of care testing. CRISPR-based diagnostics are a new method for detecting amplified nucleic acids. CRISPR-Cas reaction proceeds at a constant temperature, it is often combined with isothermal amplification. Next-generation sequencing has a high sequence throughput to rapidly obtain large amounts of genomic information and can be used to detect novel pathogens and diagnose complex infectious diseases. It can also be used to track the sources and transmission routes of outbreaks and monitor pathogen evolution. Furthermore, next generation sequencing has enabled the analysis of microbiomes that can serve as biomarkers for diseases or disease susceptibility. In the future, the molecular diagnosis of infectious diseases will advance by overcoming these shortcomings and integrating various technologies as hybrid platforms. This review describes the developments in molecular diagnostics for the treatment of infectious diseases.},
}
@article {pmid42184634,
year = {2026},
author = {Liu, G and Cao, Z and He, Y and Zhu, X and Ali, MA and Sun, H and Zuo, Q and Niu, Y and Song, J and Han, W and Wei, W and Chen, G and Li, B and Jin, K},
title = {Establishment of an inducible knockout model for the chicken Z-chromosome-linked gene DMRT1.},
journal = {Poultry science},
volume = {105},
number = {9},
pages = {107147},
pmid = {42184634},
issn = {1525-3171},
mesh = {Animals ; Male ; *Chickens/genetics ; *Transcription Factors/genetics/metabolism ; *Gene Knockout Techniques/veterinary/methods ; *Sex Determination Processes/genetics ; Chick Embryo ; Female ; *Avian Proteins/genetics/metabolism ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; },
abstract = {Sex determination is a crucial process in animal development, regulated by complex genetic networks. In avian species, Doublesex and mab-3 related transcription factor 1 (DMRT1) plays a vital role in gonadal development and sex determination. To unravel the function of DMRT1 in chicken sex determination, establishing an inducible DMRT1 knockout model is essential. In this study, we constructed an inducible DMRT1 knockout system and verified its efficiency and effects on related genes and physiological indicators. To achieve precise genomic ablation, we screened multiple sgRNAs targeting the DMRT1 locus and integrated the optimal sequence into a doxycycline-responsive (Tet-on) CRISPR/Cas9 architecture. For in vitro experiments, vectors were delivered via cell transfection and induced with 20 µg mL[-1] doxycycline (DOX), achieving an 80% knockout efficiency. Following the administration of polyethylenimine (PEI)-encapsulated plasmids into chicken embryos, we successfully implemented the inducible system in vivo. Quantitative analysis confirmed a mosaic knockout of DMRT1 with an observed efficiency reaching 45%. Following targeted disruption, we evaluated sex-related gene and protein expression alterations via qRT-PCR and Western blot (WB). Furthermore, ELISA was performed to measure testosterone levels in male embryonic gonads across multiple developmental stages (E4.5 to E18.5). qRT-PCR analysis showed that after induction, female-related genes (CYP19A1, FOXL2, ESR1) were significantly upregulated, and male-related genes (DMRT1, SOX9, AMH) were significantly downregulated. WB results revealed increased protein expression levels of CYP19A1 and FOXL2, and decreased protein expression of SOX9 post-induction. ELISA confirmed that testosterone levels in the gonads of induced male embryos were significantly reduced compared to normal and non-induced males. The study successfully established an inducible DMRT1 knockout system in chickens. This system effectively regulates the expression of sex-related genes and reduces testosterone levels in male embryos, providing theoretical and technical support for breeding novel sex-controlled breeding materials.},
}
@article {pmid42204401,
year = {2026},
author = {Gao, ZY and Shen, TL and Cheng, CY and Sun, YD and Zhang, LM and Zhang, JP and Zhang, XB},
title = {A Dual-Viral Delivery Platform Enables Efficient Site-Specific Integration of Therapeutic-Length Genes in Human Primary Stem Cells.},
journal = {Human gene therapy},
volume = {37},
number = {17-18},
pages = {858-875},
doi = {10.1177/10430342261453040},
pmid = {42204401},
issn = {1557-7422},
mesh = {Humans ; *Lentivirus/genetics ; *Genetic Vectors/genetics/administration &amp; dosage ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Transfer Techniques ; *Gene Editing/methods ; *Genetic Therapy ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; Integrases/genetics ; },
abstract = {Site-specific integration of large genes in human primary stem cells remains a significant challenge in gene therapy, particularly for treating multiallelic diseases. Gene editing efficiency in primary stem cells is heavily influenced by the delivery strategy, which often faces issues with programmability, efficiency, and specificity. Here, we developed a dual-viral delivery system, targeted integration via virus-like particles and integrase-deficient lentivirus (TIVID). This system combines virus-like Cas9 edit particles for delivering Cas9/sgRNA ribonucleoprotein complexes and integrase-deficient lentiviral vectors for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 65% ± 5% in human induced pluripotent stem cells (iPSCs). In erythroid progenitor HUDEP2 cells, TIVID mediates precise integration of a 7.1 kb HBB-GFP cassette (from cut site to cut site) at the AAVS1 locus with 20% efficiency and stable expression. Crucially, we demonstrate that TIVID overcomes stringent packaging constraints to deliver an approximately 6 kb full-length HBB therapeutic cassette into primary human CD34[+] hematopoietic stem and progenitor cells. This platform achieved 5-10% targeted integration efficiency and preserved robust lineage-specific differentiation capacity, demonstrating its potential for treating β-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (∼50% vs. <10% at AAVS1 in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (∼20% vs. ∼1.5%). Compared with traditional electroporation delivery, TIVID offers lower early cytotoxicity, promotes predominantly mono-allelic integration, and exhibits enhanced compatibility with primary stem cells. By decoupling nuclease and donor delivery, TIVID circumvents the payload constraints of single-vector systems and the toxicity of physical transfection, providing a robust ex vivo engineering platform for complex gene replacement therapies.},
}
@article {pmid42204417,
year = {2026},
author = {Hu, J and Zhang, J and Shao, Y and Li, H and Yang, Y and Zhang, J},
title = {Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.},
journal = {Human gene therapy},
volume = {37},
number = {17-18},
pages = {849-857},
doi = {10.1177/10430342261453858},
pmid = {42204417},
issn = {1557-7422},
mesh = {Humans ; *Proto-Oncogene Proteins c-cbl/genetics ; Animals ; Roundabout Proteins ; *Receptors, Immunologic/genetics/metabolism ; Mice ; *Killer Cells, Natural/immunology/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; Cell Line, Tumor ; *Adaptor Proteins, Signal Transducing/genetics ; Xenograft Model Antitumor Assays ; *Immunotherapy, Adoptive/methods ; Female ; CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics ; *Neoplasms/therapy/genetics/immunology/pathology ; Gene Knockout Techniques ; },
abstract = {Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and uterine cancer. Our and other laboratories' studies have shown that ROBO1 CAR-NK cells exhibit promising tumor therapeutic effects. However, the results still have some limitations. Cbl-b, an E3 ubiquitin ligase, has been reported to negatively regulate NK cell activation, homeostasis, and antitumor immunity.[1] Therefore, we attempted to further enhance the antitumor activity of ROBO1 CAR-NK92 cells by knocking out Cbl-b using CRISPR/Cas9 gene-editing technology. In this study, we conjugated Cbl-b sgRNA with Cas9 protein to form ribonucleoprotein complexes, which were then delivered into ROBO1 CAR-NK92 and NK-92 cells (control cells) via electroporation. Through fluorescence-activated cell sorting, limiting dilution, and sequencing, we obtained monoclonal Cbl-b-knock-out (KO) cell lines. Both in vitro cytotoxicity assays and in vivo tumor xenograft experiments were conducted to examine whether Cbl-b knockout enhances the target cell killing and tumor suppression capacities of ROBO1 CAR-NK92 cells. In this study, monoclonal cell lines of ROBO1 CAR-NK92-Cbl-b-KO and NK92-Cbl-b-KO were successfully established. In vitro, at an effector-to-target (E:T) ratio of 0.1:1, ROBO1 CAR-NK92-Cbl-b-KO (50.55%) cells exhibited significantly higher cytolytic activity against ROBO1-positive T47D target cells after 3 h of coculture than ROBO1 CAR-NK92 (34.10%), NK92-Cbl-b-KO (22.22%), and parental NK-92 cells (3.28%). In vivo, tumor volume and weight measurements demonstrated that mice treated with ROBO1 CAR-NK92-Cbl-b-KO cells developed significantly smaller tumors than all control groups, achieving a tumor growth inhibition (TGI) rate of 32.45%, indicating enhanced antitumor efficacy conferred by Cbl-b knockout. In vitro and in vivo data confirmed that Cbl-b knockout potentiates the antitumor efficacy of ROBO1 CAR-NK92 cells. The overall cytotoxic capability ranked as follows: ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92.},
}
@article {pmid42214263,
year = {2026},
author = {Han, Y and Woo, SJ and Choi, HJ and Han, JY},
title = {Compact Cas12f enables genome editing in avian cells.},
journal = {Poultry science},
volume = {105},
number = {9},
pages = {107116},
pmid = {42214263},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *Chickens/genetics ; *CRISPR-Cas Systems ; Male ; },
abstract = {Precise genome editing in avian species has been constrained by the low delivery efficiency of conventional CRISPR nucleases, such as Cas9 and Cas12a, due to their large molecular sizes. Cas12f (also known as Cas14), a compact CRISPR nuclease, has emerged as a potential genome editing system with enhanced delivery efficiency in mammalian systems. However, its effectiveness in avian systems has not been previously validated. Here, Cas12f showed notable transfection efficiency and intracellular expression in chicken Leghorn male hepatoma (LMH) cells and primordial germ cells (PGCs), with no detectable cytotoxicity. Next-generation sequencing (NGS) revealed that Cas12f achieved locus-dependent on-target editing efficiencies, reaching up to 40% at specific loci in LMH cells. Cas12f consistently generated a deletion-dominant indel profile with minimal insertions, distinct from Cas9-mediated patterns. Off-target analysis using Sanger sequencing and Inference of CRISPR Edits (ICE) revealed a few predicted off-target candidates and no detectable off-target mutations above the detection threshold. Consistent with this observation, cross-species in silico analysis showed only a modest increase in predicted Cas12f off-target proportions with increasing genome size. These findings show that Cas12f is a compact genome editing tool in avian cells, serving as a basis for further improvement in genetic engineering and biotechnological research.},
}
@article {pmid42241751,
year = {2026},
author = {Liu, H and Cruvinel, JM and Warren, WC and Ma, W and Chen, PR},
title = {Strain-specific responses of avian influenza virus to disruption of solute carrier family 35 member A1 (SLC35A1) in chicken cells.},
journal = {Poultry science},
volume = {105},
number = {9},
pages = {107178},
pmid = {42241751},
issn = {1525-3171},
mesh = {Animals ; *Chickens ; *Influenza in Birds/virology/genetics ; Cell Line ; Virus Replication ; *Influenza A virus/physiology ; *Avian Proteins/genetics/metabolism ; CRISPR-Cas Systems ; N-Acetylneuraminic Acid/metabolism ; *Nucleotide Transport Proteins/genetics/metabolism ; Influenza A Virus, H5N1 Subtype/physiology ; },
abstract = {Avian influenza virus (AIV) poses a persistent threat to poultry health and food security, with conventional control measures offering limited protection. A promising alternative is the use of gene editing to generate host resistance by ablating viral entry receptors or cellular proteins that are required for completion of the viral life cycle. The solute carrier family 35 member A1 (SLC35A1) gene encodes a Golgi-localized CMP-sialic acid transporter that is a key step in the sialylation of glycoproteins. In this study, we used the CRISPR/Cas9 system to disrupt SLC35A1 in chicken DF-1 fibroblasts and evaluated the effect on sialic acid expression and susceptibility to different strains of AIV. Lectin staining and flow cytometry confirmed a significant reduction in α2,3-linked sialic acids in SLC35A1 knockout cells, while α2,6-linked sialic acids were absent in the cells regardless of genotype. Infection experiments with three avian influenza virus strains (H5N1/PR8, H5N2, and H7N1) revealed that SLC35A1 knockout reduced viral replication in a strain-specific manner. Knockout cells infected with H5N1/PR8 showed the greatest dependence on SLC35A1-mediated sialylation with decreased viral load at 24 hours post-infection (hpi) and 48 hpi compared to wildtype cells and no observable viral growth between the timepoints. Infection of knockout cells with H5N2 resulted in a modest decrease in viral load at both timepoints as well as absence of viral growth. On the other hand, infection of knockout cells with H7N1 resulted in decreased viral load only at 48 hpi compared to wildtype cells, but the amount of virus in knockout cultures increased from 24 hpi to 48 hpi. These results demonstrate that SLC35A1 is a key host factor that supports AIV entry via α2,3-linked sialic acids; however, viral dependency on this host factor may be confounded by strain.},
}
@article {pmid42283432,
year = {2026},
author = {Li, GH and Xu, Y and Liu, T and Ma, XY and Hou, MT and Pang, SC and Zhang, JP and Cheng, T and Zhang, XB},
title = {AviNP-Seq: A Blindspot-Free Single-Molecule Framework for Unmasking AAV Genome Heterogeneity and Determining Packaging Limits.},
journal = {Human gene therapy},
volume = {37},
number = {17-18},
pages = {887-902},
doi = {10.1177/10430342261454324},
pmid = {42283432},
issn = {1557-7422},
mesh = {*Dependovirus/genetics ; Humans ; *Genome, Viral ; *Nanopore Sequencing/methods ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Terminal Repeat Sequences ; *Viral Genome Packaging ; },
abstract = {Comprehensive recombinant adeno-associated virus characterization is essential for establishing the knowledge base required to ensure clinical safety and efficacy, yet current long-read methods suffer from library preparation biases that obscure genome integrity. We present AviNP-seq, a blindspot-free nanopore sequencing framework utilizing one-end-sufficient ligation and Cas9-ribonucleoprotein (RNP) linearization to minimize terminal selection. Applied to a 1.5-6.5 kb panel, AviNP-seq delineates a sharp packaging cliff at 5.0-5.2 kb and reveals that sequence structure modulates integrity by 2-5× at fixed lengths. It unmasks covalent head-to-tail tandems in sub-3 kb vectors, detecting them with significantly higher sensitivity than PacBio HiFi. The Cas9-RNP step boosts ligation yield ∼7-fold, providing an unbiased assessment of genome integrity (≥95% inverted terminal repeat [ITR]-to-ITR). In addition, the assay quantifies plasmid impurities down to 0.05% with linear response. By integrating integrity mapping, tandem detection, and impurity profiling into a rapid (<36 h), low-input workflow, AviNP-seq provides a robust analytical tool to guide vector design and de-risk early-stage process development.},
}
@article {pmid42612103,
year = {2026},
author = {Viskadourou, M and Workman, JN and Burke, EV and Eckley, DM and Dohr, J and Newby, GA},
title = {Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71449},
pmid = {42612103},
issn = {1940-087X},
support = {R00 HL163805/HL/NHLBI NIH HHS/United States ; DP2 OD038783/OD/NIH HHS/United States ; T32 GM148383/GM/NIGMS NIH HHS/United States ; },
mesh = {*Electroporation/methods ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/administration &amp; dosage ; *RNA, Messenger/genetics/administration &amp; dosage ; Animals ; Induced Pluripotent Stem Cells ; Fibroblasts ; Electroporation Therapies ; },
abstract = {CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. Editing outcomes are commonly evaluated in cultured primary cells, patient-derived cells, or engineered cell lines to study the impact of genetic variation or as a first step before initiating animal studies or clinical translation. Delivery of editors as mRNA together with synthetic guide RNAs into mammalian cells can improve editing efficiency relative to plasmid-based approaches and prevent issues such as DNA integration or off-target editing from sustained expression. This article presents a workflow to prepare genome editor mRNA by in vitro transcription (IVT), including co-transcriptional capping and chemically-modified nucleotides, electroporate editor mRNA and guide RNAs into primary human fibroblasts, induced pluripotent stem cells (iPSCs), or lymphoblastoid cell lines (LCLs), and quantify editing outcomes by targeted amplicon sequencing on an Illumina platform followed by analysis using CRISPResso2. This workflow enables quantitative benchmarking of guide RNAs, electroporation parameters, and editor variants, and supports downstream applications including single-cell cloning, phenotypic assays, preclinical animal studies, and therapeutic development.},
}
@article {pmid42612424,
year = {2026},
author = {Chen, C and Afshar-Saber, W and Iglesias, I and Kim, K and Lewis, B and Srinivasan, G and Chen, C and Hirsh, R and Guardado, R and Polanco, T and Swanson, A and Norabuena, E and Whye, D and Jain, A and Cai, C and Sun, L and Chopra, M and Chen, I and Iannello, G and Rozumny, B and Hanson, E and Sahin, M and Buttermore, ED},
title = {Development of a human iPSC and patient phenotyping resource for preclinical investigations of neurodevelopmental disorders.},
journal = {Stem cell research},
volume = {95},
number = {},
pages = {104073},
doi = {10.1016/j.scr.2026.104073},
pmid = {42612424},
issn = {1876-7753},
abstract = {In this manuscript, we report the development of a comprehensive resource designed to harness the transformative potential of patient-derived induced pluripotent stem cells (iPSCs) to advance the study of neurodevelopmental disorders (NDDs). Using CRISPR-Cas-mediated genome editing, the Human Neuron Core generated a repository comprising 29 isogenic iPSC pairs, two sex-matched parental control iPSC pairs, and one unmatched patient line representing six monogenic NDDs: Tuberous Sclerosis Complex, PTEN Hamartoma Tumor Syndrome, KCNQ2 Developmental and Epileptic Encephalopathy, FOXG1 Syndrome, Phelan-McDermid Syndrome, and SETBP1 Haploinsufficiency Disorder. In parallel, detailed clinical phenotyping data were collected to enable comparison of cellular phenotypes with clinical severity in future studies. This integrated collection of genetically defined iPSC lines and associated clinical data provides a powerful platform for investigating disease mechanisms and advancing iPSC-based drug discovery for NDDs.},
}
@article {pmid42613564,
year = {2026},
author = {Xu, S and G C, B and Tan, K and Wu, C},
title = {Gene Silencing by CRISPR Interference in Fusobacterium nucleatum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3055},
number = {},
pages = {67-79},
pmid = {42613564},
issn = {1940-6029},
support = {R01 DE030895/DE/NIDCR NIH HHS/United States ; R21 DE034542/DE/NIDCR NIH HHS/United States ; },
mesh = {*Fusobacterium nucleatum/genetics ; *Gene Silencing ; *CRISPR-Cas Systems ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics ; },
abstract = {Fusobacterium nucleatum is a strictly anaerobic bacterium associated with periodontal disease and several systemic conditions, including colorectal cancer and adverse pregnancy outcomes. Genetic manipulation in F. nucleatum has been limited by poor transformation efficiency and difficulty studying essential genes. To overcome these challenges, we developed a CRISPR interference (CRISPRi) system that enables efficient and reversible gene silencing without altering the genome. This system uses an inducible dCas9 and a customizable sgRNA expressed from a pCWU6-based shuttle plasmid (pZP4C). In this chapter, we present a step-by-step protocol for designing sgRNAs, constructing CRISPRi plasmids, transforming F. nucleatum ATCC 23726, and evaluating gene silencing phenotypes. We use the nonessential gene ftsW, which encodes a protein required for peptidoglycan synthesis and cell division, as a model target. This protocol is also applicable to other genetically recalcitrant F. nucleatum strains, offering a versatile tool for investigating both essential and nonessential gene functions.},
}
@article {pmid42614264,
year = {2026},
author = {Keerthi, V and Ravindran, P and Kaliyur, S and Tuttagunta, SUS and Mathpal, S and Joshi, T and Ramaiah, S and Anbarasu, A},
title = {A comprehensive review of emerging therapeutic strategies against methicillin-resistant Staphylococcus aureus.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1815573},
pmid = {42614264},
issn = {1664-302X},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the most significant multidrug-resistant bacterial pathogens responsible for a broad spectrum of infections ranging from mild skin infections to severe invasive diseases, including bacteremia, pneumonia, endocarditis, osteomyelitis, and sepsis. The rapid global dissemination of MRSA is primarily driven by the acquisition of the mecA gene encoding penicillin-binding protein 2a, which confers resistance to β-lactam antibiotics. In addition to β-lactam resistance, MRSA exhibits resistance to multiple antimicrobial classes through diverse mechanisms, including target-site mutations, efflux pumps, biofilm formation, horizontal gene transfer, and adaptive phenotypic variations. The virulence and persistence of MRSA is further enhanced by numerous virulence factors such as adhesins, toxins, immune evasion proteins, and extracellular enzymes that facilitate colonization, persistence, and host tissue damage. Biofilm formation additionally contributes to chronic infection and antibiotic tolerance. Despite the availability of conventional agents such as vancomycin, linezolid, and daptomycin, the emergence of resistant strains including vancomycin-resistant Staphylococcus aureus has significantly limited current therapeutic options. Consequently, there is an urgent need for innovative therapeutic strategies. This review comprehensively summarizes the evolution, pathogenesis, virulence mechanisms, biofilm biology, and antibiotic resistance mechanisms of MRSA, with particular emphasis on emerging therapeutic approaches. Novel strategies including antimicrobial peptides, nanomedicine, bacteriophage therapy, CRISPR-Cas systems, biomimetic nano-NETs, probiotics, monoclonal antibodies and plant-derived compounds are discussed as promising alternatives or adjuncts to conventional antibiotics. Collectively, these advances highlight the evolving landscape of MRSA management and the potential for next-generation therapeutics to overcome antimicrobial resistance challenges.},
}
@article {pmid42614947,
year = {2026},
author = {Wang, S and Kang, L and Li, M and Zhou, X and Li, B and Wang, F and Meng, J and Li, C and Yang, K},
title = {Comparative genomic analyses provide new insights into phylogenetic and functional diversification in genus Fenollaria.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1862120},
pmid = {42614947},
issn = {1664-302X},
abstract = {The genus Fenollaria has gained attention due to its associations with human prostate cancer, colorectal cancer and other diseases. The higher abundance of Fenollaria was believed to be associated with biochemical recurrence of prostate cancer but remission of colorectal cancer. Owing to the fastidious growth requirements of Fenollaria species in laboratory isolation and culture, the genomes of isolated strains is rarely available. Consequently, only limited comparative genomic studies have been conducted, leaving knowledge gap regarding the genomic diversity, distribution of functional genes, and evolutionary relationships, which hindered the understanding of ecological adaptation and mechanism exploration of Fenollaria. Here, a large-scaled genomic investigation of Fenollaria genus was performed using four high quality MAGs generated in this study and publicly available genomic data. The four MAGs were constructed from urine metagenome samples from bladder cancer patients, which were under conditions of oligotrophy and limited oxygen. Four mono-clades were revealed by phylogenomic analysis, representing for three previously described species (i.e., F. massiliensis, F. timonensis, and F. sporofastidiosus) as well as a novel proposed Fenollaria species. The divergences among these clades were also supported by genome-wide G + C content, ANI and AAI values. The functional difference between clades were revealed by the distribution of clade-specific genes in COG categories, as well as the biased distribution of ARGs, VFs, and CRISPR-Cas systems.},
}
@article {pmid42616864,
year = {2026},
author = {Chou, CW and Sinan, S and Kuo, HC and Chang, YC and Arguello, C and Sahaya, D and Russell, R and Finkelstein, IJ},
title = {Structural basis for target discrimination and activation by Cas13d.},
journal = {Science advances},
volume = {12},
number = {34},
pages = {eaec4221},
pmid = {42616864},
issn = {2375-2548},
support = {R35 GM131777/GM/NIGMS NIH HHS/United States ; },
mesh = {Cryoelectron Microscopy ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Catalytic Domain ; Models, Molecular ; Protein Binding ; Protein Domains ; },
abstract = {CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity.},
}
@article {pmid42617139,
year = {2026},
author = {Wang, X and Wu, S and Ding, Y and Ding, J and Li, P and Lian, Z},
title = {A genome-wide CRISPR knockout screen identified host genes essential for Brucella invasion and intracellular survival.},
journal = {Emerging microbes &amp; infections},
volume = {15},
number = {1},
pages = {2713322},
pmid = {42617139},
issn = {2222-1751},
mesh = {Humans ; *Macrophages/microbiology ; *Brucella/physiology/genetics ; *Brucellosis/microbiology/genetics ; Gene Knockout Techniques ; *Host-Pathogen Interactions/genetics ; THP-1 Cells ; CRISPR-Cas Systems ; Apoptosis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Microbial Viability ; },
abstract = {For Brucella spp., the ability to invade and survive within host macrophages is essential for causing chronic infections in their mammalian hosts. In this study, a genome-wide CRISPR knockout screen was performed for the first time in human THP-1 macrophages to identify host genes mediating resistance to Brucella invasion and intracellular survival. Results showed that the screening identified 35 candidate genes, 11 of which were selected to generate monoclonal knockout cell lines for functional validation. This study demonstrated that knockout of WDR4, ZNF532, or MTHFD1 significantly restricted Brucella invasion and early intracellular survival. In addition, TRAPPC2 knockout restricted Brucella invasion and, crucially, its intracellular survival throughout infection, exerting the most potent antibacterial effect. Mechanistically, TRAPPC2 deficiency suppresses Brucella infection by inhibiting autophagosome formation in macrophages. Furthermore, TRAPPC2 knockout decreases macrophage apoptosis and improves host cell viability following Brucella infection. These results provide therapeutic targets for combating Brucella infection and offer novel insights into the molecular mechanisms associated with Brucella-induced chronic infections.},
}
@article {pmid42617808,
year = {2026},
author = {Xiao, J and Liang, M and Lei, Y and Huang, Z and Guo, W and Xue, L and Sun, X and Wang, Y and Chen, K and Cao, X and Fan, J and Huang, Z and Chen, M},
title = {Research Advances in Modern Immunoassay Technologies and Novel Alternative Biotechnologies for Rapid Detection of Foodborne Pathogens and Chemical Contaminants.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100896},
doi = {10.1016/j.jfp.2026.100896},
pmid = {42617808},
issn = {1944-9097},
abstract = {The development and application of novel rapid detection technologies are critical for advancing food safety regulation. In recent years, immunology-based methods have played a pivotal role in food safety supervision due to their speed, operational simplicity, and cost-effectiveness. However, conventional colloidal gold techniques exhibit limited sensitivity for trace analytes (e.g., early-stage microbial contamination) and are primarily qualitative, thus failing to meet the quantitative detection requirements for pesticides, veterinary drugs, and food additives, which restricts their practical applicability. Additionally, the difficulty and high cost of obtaining high-quality monoclonal antibodies increase the technical barriers and commercialization costs of immunochromatographic assays. Consequently, modern immunological techniques and novel alternative biotechnologies have emerged as focal points in rapid detection research. This review examines the principles and technical characteristics of advanced immunological methods, including immunofluorescence quantitative chromatography (IF-QCT) and flow cytometry-based immunophenotyping (FCI), as well as emerging antibody-alternative technologies such as aptamers, CRISPR/Cas systems, and phage-based technologies. This review further summarizes the latest research advances of the above-mentioned technologies in the rapid detection of food safety risk factors, including pathogenic microorganisms, biotoxins, residues of pesticides and veterinary drugs, heavy metals, and other chemical contaminants. It critically analyzes their technical advantages and practical limitations, and discusses potential future directions. This review aims to provide insights and a theoretical basis for developing and applying technologies to rapidly detect food safety hazards.},
}
@article {pmid42618112,
year = {2026},
author = {Sanjay, BR and Nishanth, MAD and Vergis, J and Pollumahanti, N and Chatlod, L and Gadekar, Y and Muthukumar, M and Reddy, BP and Malik, SVS and Barbuddhe, SB and Rawool, DB},
title = {LAMP-assisted CRISPR-Cas12a platform for detection of Bacillus anthracis spores in environmental samples.},
journal = {Analytica chimica acta},
volume = {1419},
number = {},
pages = {345904},
doi = {10.1016/j.aca.2026.345904},
pmid = {42618112},
issn = {1873-4324},
mesh = {*Bacillus anthracis/isolation &amp; purification/genetics ; *Spores, Bacterial/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Soil Microbiology ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Environmental persistence of Bacillus anthracis spores sustains anthrax transmission, necessitating rapid and field-deployable detection tools. This study aimed to develop and quantitatively evaluate a LAMP-assisted CRISPR-Cas12a assay for sensitive and specific detection of B. anthracis spores in soil and meat meal matrices. Two B. anthracis-specific target genes plasmid-encoded lef and chromosomal SNP locus (CR5)- were selected. Target-specific LAMP primers and CRISPR crRNAs were designed using Primer Explorer, CRISPOR, and RNAfold platforms. The LAMP-CRISPR/Cas12a assay was optimised for reaction conditions and evaluated for analytical sensitivity and specificity using UV-inactivated spores and closely related Bacillus spp. Environmental applicability was assessed via spiking experiments in sterile soil and meat meal using the GABRI recovery method. Field validation was performed on 100 samples from anthrax-endemic regions of India, with performance compared against the WOAH-recommended lef gene-based real-time PCR. LAMP-CRISPR-Cas12a assay achieved detection limits of 10 spores/ml (lef) and 10[2] spores/ml (CR5), with no cross-reactivity against related species. In spiked matrices, spore recovery ranged from 50 to 75%, and detection sensitivity remained consistent. Field evaluation demonstrated a sensitivity of 91.70% and specificity of 100%, with near-perfect agreement (κ = 0.95) relative to real-time PCR. The assay delivered results within ∼70 min, including amplification and detection. The LAMP-assisted CRISPR-Cas12a platform provides a rapid, sensitive, and cost-effective approach for environmental detection of B. anthracis spores. The minimal equipment requirements and high diagnostic accuracy support its applicability for field-level surveillance and biosafety monitoring in resource-limited settings.},
}
@article {pmid42618126,
year = {2026},
author = {Tao, Q and Xing, Y and Yang, H and Deng, L and He, M and Cheng, Y and Wang, Q and Gao, Z and Wan, H and Li, D and Ling, S},
title = {Field-deployable RspCas13d platform for rapid and extraction-free detection of giant panda- and canine-derived Canine Distemper virus.},
journal = {Analytica chimica acta},
volume = {1419},
number = {},
pages = {345960},
doi = {10.1016/j.aca.2026.345960},
pmid = {42618126},
issn = {1873-4324},
mesh = {*Distemper Virus, Canine/isolation &amp; purification/genetics ; Animals ; Dogs ; *Ursidae/virology ; *RNA, Viral/genetics/analysis ; Nucleic Acid Amplification Techniques/methods ; *Distemper/diagnosis/virology ; Rapid Diagnostic Tests ; CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Canine distemper virus (CDV) is a highly contagious RNA virus that causes severe disease in domestic dogs and diverse wildlife species, including endangered giant pandas. Current CDV diagnosis mainly relies on laboratory-based RT-qPCR, which requires nucleic acid extraction, trained personnel, and temperature-controlled instruments, limiting its use in field surveillance and resource-limited settings. Although CRISPR-based diagnostics offer promising alternatives, chemically defined extraction-free workflows compatible with one-pot Cas13d detection remain limited. This study addresses the need for a rapid, visual, extraction-free, and low-infrastructure method for CDV detection.<br><br>RESULTS: We developed CLEAR-VISION, an integrated CRISPR diagnostic platform combining CLEAR (Chemical Lysis for Extraction-free Access to RNA) with VISION (Visual Isothermal Single-tube Integrated One-pot Nucleic acid detection). VISION integrates RPA amplification, T7 transcription, and RspCas13d-based detection into a single-tube reaction supported by a chemically defined buffer. CLEAR enabled rapid RNA release at room temperature without extraction kits or heating and was compatible with downstream one-pot detection. The PAM- and PFS-independent property of RspCas13d allowed flexible target selection, while lyophilized reagents improved storage and transportation convenience. CLEAR-VISION enabled CDV detection within 30&#x202f;min and provided dual visual readouts, including fluorescence and lateral flow assays. Clinical evaluation in the current sample set showed consistent results with RT-qPCR for samples from giant pandas, stray dogs, and pet dogs. The assay also maintained stable performance at physiological temperature (37&#x202f;&#xb0;C), reducing reliance on temperature-controlled equipment.<br><br>SIGNIFICANCE AND NOVELTY: CLEAR-VISION provides a chemically defined, extraction-free, and low-infrastructure CRISPR diagnostic workflow for rapid CDV detection. Its novelty lies in integrating room-temperature chemical lysis with single-tube RPA-T7-RspCas13d detection, lyophilized reagents, and dual visual readouts. This platform supports the potential application of CRISPR-based diagnostics for on-site CDV surveillance in domestic animals and wildlife.},
}
@article {pmid42618136,
year = {2026},
author = {Shao, LN and Zheng-Luo, and Liu, BL and Tong-Xu, and Wang, YM and Duan, JQ and Lei-Zhao, and Li, YY and Dai, YM and Jia, QR and Zhang, LY and Ling-Zhu, and Xu, ZW},
title = {A lyophilized RPA-CRISPR/Cas13d one-pot platform for rapid detection of porcine circovirus type 3.},
journal = {Analytica chimica acta},
volume = {1419},
number = {},
pages = {345832},
doi = {10.1016/j.aca.2026.345832},
pmid = {42618136},
issn = {1873-4324},
mesh = {*Circovirus/isolation &amp; purification/genetics ; Animals ; Swine ; *CRISPR-Cas Systems/genetics ; Freeze Drying ; *Nucleic Acid Amplification Techniques/methods ; DNA, Viral/genetics ; Rapid Diagnostic Tests ; },
abstract = {Rapid molecular diagnostics are essential for effective surveillance of infectious diseases in swine production systems. Here, we report SHARP (Single-step Hybrid RPA-CRISPR/EsCas13d Platform), a one-pot CRISPR-based platform for detection of porcine circovirus type 3 (PCV3). By integrating rapid nucleic acid release with CRISPR/Cas13d detection, a single-step one-pot detection system was established, enabling detection within 30 min under simplified reaction conditions. In the visual readout mode, the detection limit was 50 copies/μL, with no cross-reactivity observed against common swine viruses. After lyophilization and rehydration, the SHARP system showed consistent detection performance across 50 clinical samples, in agreement with qPCR results. These results indicate that SHARP provides a simplified workflow for rapid PCV3 detection, with potential for decentralized molecular surveillance in livestock production systems.},
}
@article {pmid42620120,
year = {2026},
author = {Brodmann, M and Baca, CF and Chandanani, J and Campbell, EA and Marraffini, LA},
title = {MtvS1 and MtvS2 Interact with RNA Polymerase to Regulate the Francisella Type V-A CRISPR-Cas System.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42620120},
issn = {2692-8205},
support = {R35 GM151879/GM/NIGMS NIH HHS/United States ; },
abstract = {Bacteria and archaea often harbor multiple CRISPR-Cas loci to defend against mobile genetic elements. Little is known, however, about whether and how different CRISPR-Cas systems are differentially regulated, in many instances due to the impossibility of studying CRISPR immunity in native hosts. Here we investigated the regulation of the endogenous type II-B and type V-A CRISPR-Cas systems present in the opportunistic human pathogen Francisella novicida U112. We found that while the type II-B system is constitutively expressed, the type V-A system is differentially expressed at stationary phase and high cell density. We identified MtvS1 and MtvS2 as factors required for this regulation, as well as for the modulation of many additional genes in stationary phase, some of which are required for Francisella virulence. Both Francisella MtvS proteins bind to RNA polymerase. MtvS1 is predicted to interact with the β' subunit of the RNA polymerase, and MtvS2 with multiple RNA polymerase subunits as well as MtvS1. We propose that MtvS1 and MtvS2 constitute noncanonical alternative sigma factors involved in the regulation of the expression of the type V-A CRISPR locus and other genes in Francisella. Last, we show that the MtvS1 homolog YgfB is required for expression of the type I-E CRISPR-Cas system in E. coli, a result that suggests a broader role in gene regulation for these alternative sigma factors.},
}
@article {pmid42620293,
year = {2026},
author = {Matrishin, CB and Haase, EM and Miles, AK and Steimer, S and Soh, D and Smardz, M and Diaz, PI and Kauffman, KM},
title = {Pervasive integrative and conjugative elements shape Porphyromonas gingivalis gene repertoires.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.08.04.741601},
pmid = {42620293},
issn = {2692-8205},
abstract = {BACKGROUND: Porphyromonas gingivalis (Pg) is an oral pathobiont that contributes to periodontal disease and has been associated with systemic health conditions. Although Pg is recognized as exhibiting extensive strain-level genomic diversity and recombination, the extent to which mobile elements contribute to this variation, and their relevance to its fitness and virulence, remain incompletely understood. Our recent study of the Pg pangenome revealed diverse accessory defense-associated genes, raising the question of whether these are carried by unrecognized mobile genetic elements (MGEs). Integrative and conjugative elements (ICEs) are large autonomous mobile elements that often encode genes for proteins beneficial to their bacterial hosts, including defense systems that protect against phage infection. To date, only one ICE, CTnPg1, has been described in Pg .<br><br>RESULTS: Here, we developed a bioinformatic approach integrating ICE prediction and curation, hallmark-gene detection, and genomic-context analysis, to investigate ICEs in Pg . We discovered that ICEs are pervasive in Pg genomes, with >90% of genomes harboring at least one ICE. We found that these elements comprise at least five distinct groups, two of which dominate and frequently co-occur in Pg genomes, inserting into distinct characteristic insertion sites. Using marker-gene analysis of enrichment-culture mini-metagenomes from subjects with periodontal disease we detected representatives of these dominant Pg ICE groups, as well as others, in recent clinical samples. We found that anti-defense and defense genes are common in Pg ICEs, and that these elements commonly encode biosynthetic gene clusters, including for menaquinone synthesis and predicted ribosomally synthesized and post-translationally modified peptides (RiPPs). In contrast to the extensive CRISPR-Cas defense targeting we observed for Pg phages, we detected no exact matches between ICE sequences and Pg CRISPR spacers.<br><br>CONCLUSION: This work establishes that ICEs are pervasive contributors to Pg 's pangenome and unique strain-level gene repertoires. Their distinct cargo profiles suggest that ICEs likely impact the virulence and ecology of Pg through the introduction and spread of advantageous traits, including expansion of Pg 's biosynthetic capacity and resistance to phage infection. This work provides a curated framework for investigating ICE diversity in Pg and establishes a foundation for expanded experimental studies of their host ranges and roles in shaping Pg 's interactions with phages, other microbes, and the human host.},
}
@article {pmid40216545,
year = {2026},
author = {Febrianti, RA and Narulita, E and Sulistyaningsih, E and Addy, HS},
title = {Innovations in Bacteriophage Genome Engineering for Combating Multidrug-Resistant Bacterial Infections.},
journal = {Foodborne pathogens and disease},
volume = {23},
number = {10},
pages = {639-647},
doi = {10.1089/fpd.2024.0194},
pmid = {40216545},
issn = {1556-7125},
mesh = {*Bacteriophages/genetics ; *Drug Resistance, Multiple, Bacterial ; *Genetic Engineering/methods ; *Phage Therapy/methods ; *Genome, Viral ; *Bacterial Infections/therapy/microbiology ; CRISPR-Cas Systems ; Humans ; Bacteria/virology ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Bacteriophage engineering is a promising strategy to address multidrug-resistant (MDR) bacterial infections that pose significant challenges to public health due to the overuse of antibiotics. Bacteria can develop resistance mechanisms, such as receptor modification and activation of antiviral defense systems, which further complicates the application of phage therapy. Additionally, long-term phage therapy can result in the production of anti-phage antibodies, which may interfere with treatment. These factors require advanced engineering techniques to improve the efficacy of phages and expand their host range. Recent advances in genome engineering methods, including CRISPR/Cas9, homologous recombination, and other synthetic biology techniques, offer promising solutions to these challenges. By modifying receptor-binding proteins and using high-yield screening methods, researchers can create phages that are better equipped to target MDR bacteria effectively. Furthermore, understanding the intricate interactions between phages and their bacterial hosts is critical to guiding these engineering efforts. Future development perspectives lie in integrating these advanced engineering techniques into clinical practice, potentially putting bacteriophages at the forefront of fighting MDR bacterial infections.},
}
@article {pmid42418712,
year = {2026},
author = {Poch, D and Mukherjee, C and Mallik, S and Todorow, V and Kuiper, EFE and Dhingra, N and Surovtseva, YV and Schlieker, C},
title = {Integrative chemical genetics platform identifies condensate modulators linked to neurological disorders.},
journal = {Molecular biology of the cell},
volume = {37},
number = {9},
pages = {ar86},
doi = {10.1091/mbc.E26-06-0256},
pmid = {42418712},
issn = {1939-4586},
mesh = {Humans ; Nuclear Envelope/metabolism ; Drug Discovery/methods ; *Nervous System Diseases/genetics/metabolism ; Molecular Chaperones/metabolism/genetics ; Ubiquitin-Protein Ligases/metabolism/genetics ; CRISPR-Cas Systems ; Animals ; },
abstract = {Dysregulation of biomolecular condensates is implicated across multiple neurological disorders. However, approaches to systematically identify their modulators remain limited. Here, we expand the utility of MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatics pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1760 bioactive compounds in a cellular DYT1 dystonia model, we validate the platform for condensate-targeted drug discovery, identifying drugs that prevent the accumulation of the MLF2 reporter into nuclear envelope condensates. In parallel, a genome-wide CRISPR/Cas9 screen correlates nuclear condensate abundance with genes implicated in microcephaly and over eight additional neurodevelopmental disorders. Machine learning and confocal imaging resolve distinct condensate phenotypes, with RNF26 deletion provoking nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable platform for identifying modulators of condensates and establishes a correlative connection between nuclear condensate accumulation and genes implicated in neurodevelopmental disorders.},
}
@article {pmid42478827,
year = {2026},
author = {Wang, H and Liu, L and Bao, C and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {A low-cost CHA-integrated CRISPR/Cas12a-based test strip platform for on-site gene detection.},
journal = {Lab on a chip},
volume = {26},
number = {17},
pages = {4679-4688},
doi = {10.1039/d6lc00394j},
pmid = {42478827},
issn = {1473-0189},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Chorionic Gonadotropin/analysis/genetics ; *Pregnancy Tests/instrumentation ; *Reagent Strips ; Female ; DNA/genetics/analysis ; },
abstract = {On-site nucleic acid detection plays a crucial role in disease diagnosis, biosafety monitoring, and food quality control. This study develops a novel nucleic acid detection platform that integrates catalytic hairpin assembly (CHA) with the CRISPR/Cas12a system and utilizes pregnancy test strips (PTS) for result visualization, addressing the limitations of existing nucleic acid detection methods in balancing sensitivity, specificity, and portability with cost and dependence on a cleanroom. The main mechanism involves the following three steps. The presence of target RNA triggers the CHA reaction, generating double-stranded DNA (dsDNA) as an activation unit. Subsequently, this unit activates the CRISPR/Cas12a system to specifically cleave the single-stranded DNA (ssDNA) that has bridged human chorionic gonadotropin (HCG) to a magnetic bead, ultimately releasing HCG that produces a visual result on the PTS. This dual-signal amplification strategy (CHA cycling and Cas12a trans-cleavage) can detect concentrations as low as 10 pM in approximately 50 min, without the need for pre-amplification of the target nucleic acid. This detection system ensures high sensitivity and specificity while effectively avoiding non-specific activation. In practical applications with transgenic maize samples, the detection results are highly consistent with those of real-time quantitative polymerase chain reaction (qPCR), validating its reliability in real-world scenarios. This innovative method offers advantages such as simple operation and low cost, providing an efficient tool for rapid nucleic acid detection while demonstrating broad potential for application in resource-limited settings.},
}
@article {pmid42611132,
year = {2026},
author = {Ramesh, S and Kumar, N and Ghosh, A},
title = {A novel needleless delivery of Cas9 ribonucleoprotein complex in Bemisia tabaci embryos for gene editing.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {9},
pages = {},
pmid = {42611132},
issn = {1573-0972},
support = {(BT/PR40767/AGIII/103/1277/2020).//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Hemiptera/genetics/embryology ; RNA, Guide, CRISPR-Cas Systems/genetics ; HSP70 Heat-Shock Proteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR/Cas9 system, particularly the Cas9-sgRNA ribonucleoprotein (RNP) complex, offers a highly efficient platform for gene editing. However, traditional microinjection methods for RNP delivery in insect embryos are labor-intensive, technically demanding, and often reduce embryo viability, especially in species with fragile, microscopic embryos. In this study, a novel, non-invasive delivery method for the RNP complex in tiny insect embryos has been optimized. Whitefly, Bemisia tabaci, an invasive insect pest of agricultural importance and vector of plant diseases, was considered as a model organism. A needleless dechorionation-permeabilization method was employed to deliver the RNP complex to the early embryonic stage of B. tabaci embryos. B. tabaci heat shock protein 70 (hsp70) gene, which interacts with the begomovirus coat protein, aiding in its internalization and successful transmission by B. tabaci in a persistent circulative manner, was targeted for knockout. Two sgRNAs were synthesized via in vitro transcription, and the Cas9-sgRNA complexes were validated by in vitro cleavage assays. The localization of the GFP-labelled Cas9-sgRNA complex confirmed successful RNP delivery, as observed through confocal microscopy. A survival rate of 18% of the embryos was recorded post-RNP delivery. Sequencing of treated embryos showed 25- and 28-nucleotide deletions in the hsp70 exon. Synthego ICE analysis revealed up to 84% gene knockout efficiency. This method enables batch processing of embryos, drastically reducing delivery time and associated costs while improving throughput. Hsp70 KO B. tabaci mutants generated in the study are expected to be incompetent begomovirus transmitters, which would help restrict the spread of the virus. Our study overcomes a key bottleneck in CRISPR/Cas delivery to small insect embryos, opening new avenues for rapid, high-throughput, and cost-effective RNP delivery methods in insect embryos. The novel non-invasive methods would be helpful in the deployment of gene editing for sustainable pest control.},
}
@article {pmid42611239,
year = {2026},
author = {Zhou, Y and Li, H and Chen, M and Ye, J and Yan, Y and Yang, L and Meng, T and Jiao, D and Wang, D and Zhu, L and Yang, X},
title = {Ligand-Engineered Mn-Cysteine as a Potent Laccase Mimic for CRISPR/Cas12a Electrochemical Biosensing of Hepatocellular Carcinoma Biomarkers.},
journal = {ACS nano},
volume = {20},
number = {32},
pages = {22787-22803},
doi = {10.1021/acsnano.6c07904},
pmid = {42611239},
issn = {1936-086X},
support = {2025ZB395//Jiangsu Provincial Department of Human Resources and Social Security/ ; KY2025014//Sichuan Normal University/ ; 2024NSFSC1129//Department of Science and Technology of Sichuan Province/ ; 22404119//National Natural Science Foundation of China (NSFC)/ ; 32501984//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Biosensing Techniques/methods ; *Laccase/chemistry/metabolism ; *Biomarkers, Tumor/analysis/blood ; *Cysteine/chemistry ; *Liver Neoplasms/diagnosis/blood ; Humans ; *Carcinoma, Hepatocellular/diagnosis/blood ; *Manganese/chemistry ; Ligands ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; MicroRNAs/analysis/blood ; alpha-Fetoproteins/analysis ; },
abstract = {Laccase is an environmentally friendly catalyst with water as the sole catalytic byproduct, yet its biomedical detection potential remains underexplored. Herein, a ligand engineering strategy was employed to synthesize Mn-cysteine nanoflowers (Mn-Cys NF) with laccase-mimicking activity via a one-pot method, using manganese (Mn) with rich valence variations as the active center and cysteine (Cys) as the ligand. Spectroscopic characterizations confirmed Cys-modulated Mn electronic structure, and theoretical calculations validated enhanced substrate adsorption and reduced reaction barriers. The specific activity of Mn-Cys NF is approximately 3.56 times that of natural laccase and exhibited excellent stability across pH, temperature, ionic strength, and organic solvent conditions. Leveraging this high-performance nanozyme, a CRISPR/Cas12a electrochemical biosensor was constructed with a DNA triangular prism interface, where a target-triggered catalytic hairpin assembly (CHA)-DNAzyme cascade regulated Cas12a cleavage to enable signal-on detection. This biosensor achieved quantification of hepatocellular carcinoma (HCC) biomarkers alpha-fetoprotein (AFP) and microRNA-122 (miRNA-122), with detection limits as low as 4.47 fg/mL and 6.21 aM, respectively. It also effectively discriminated HCC patients from healthy individuals in clinical serum samples. This work offers a ligand engineering strategy for designing high-performance laccase-mimicking nanozymes and expands the application scope of laccase nanozymes from environmental remediation to biomedical biosensing.},
}
@article {pmid42611583,
year = {2026},
author = {Chun, L and Quan, Z and Ke, M},
title = {CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {234},
pages = {},
doi = {10.3791/72662},
pmid = {42611583},
issn = {1940-087X},
mesh = {*Klebsiella pneumoniae/genetics ; *CRISPR-Cas Systems ; *Mutagenesis, Site-Directed/methods ; Plasmids/genetics ; },
abstract = {Constructing target-gene mutants with a common genetic background is crucial for elucidating gene function in antimicrobial resistance (AMR) research. Taking advantage of the single-guide RNA (sgRNA) and protospacer adjacent motif (PAM) sequence (3'-NGG) specificity of the Cas9 protein in the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system and codon degeneracy, the authors design a repair template that incorporates the desired point mutation while excluding the PAM sequence disrupted by a synonymous substitution, thereby preventing re-cleavage by CRISPR/Cas9. This method employs two available plasmids, pCasKP-apr and pSGKP-spe, offering straightforward operation and high screening specificity. As a result, the approach enables efficient generation of genetically defined mutant strains of Klebsiella pneumoniae (K. pneumoniae) and is readily adaptable to routine laboratory settings. Furthermore, the protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.},
}
@article {pmid42611961,
year = {2026},
author = {Li, C and Li, L and Chen, Y and Ou, X and Ding, Y and Li, X and Hou, W and Cheng, G},
title = {CRISPR/Cas9-Mediated Generation and Characterization of an Ent2*/CyO Drosophila melanogaster Strain.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {234},
pages = {},
doi = {10.3791/69546},
pmid = {42611961},
issn = {1940-087X},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; Female ; Male ; *Nucleotide Transport Proteins/genetics ; },
abstract = {In this study, a CRISPR/Cas9-based genome-editing approach was used to introduce mutations in the equilibrative nucleoside transporter 2 (Ent2) gene in Drosophila melanogaster. Guide RNAs targeting the coding region of Ent2 were designed and co-injected with Cas9 mRNA into w[1118] embryos. Mutant alleles were identified by Sanger sequencing and maintained as a stable Ent2*/CyO heterozygous line using a balancer chromosome. Subsequently, we evaluated body weight, climbing ability, survival rate, and the activities of superoxide dismutase (SOD) and catalase (CAT) in fruit flies at 22 °C and 25 °C, respectively. The results indicate that at both 22 °C and 25 °C, the body length and weight of Ent2*/CyO fruit flies were significantly reduced compared to the w[1118], and their development was delayed. At 22 °C, the overall lifespan of Ent2*/CyO flies was slightly longer than that of the w[1118], whereas at 25 °C, no significant difference was observed. Regarding locomotor ability, the climbing performance of heterozygous flies was significantly lower than that of the w[1118] at both temperatures, with males being more severely affected. In addition, the antioxidant enzyme activities of CAT and SOD in Ent2*/CyO fruit flies were significantly reduced, indicating a clear impairment of antioxidant capacity. These results describe the phenotypic profile of a CRISPR-generated Ent2 mutant line and demonstrate the feasibility of combining genome editing with balancer chromosome strategies in Drosophila. This study provides a methodological framework and a genetic resource for future investigations of genes associated with metabolism and environmental responses.},
}
@article {pmid42189126,
year = {2026},
author = {Wang, Z and Liu, H and Wang, X and Teng, J and Zheng, Z and Zhang, J and Xiao, W and Liang, Q and Li, J and Jia, X and Feng, X and Cui, H and Luo, M and Yang, T and Wu, L and Zhao, K and Yang, W and Li, MJ and Huang, D and Yang, J},
title = {Genome-Wide CRISPR Screen Identifies a microRNA Orchestrating Pleiotropic Resistance to Targeted Therapy and T Cell Immunity in Melanoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {47},
pages = {e15158},
pmid = {42189126},
issn = {2198-3844},
support = {2024YFC2707700//National Key Research and Development Program of China/ ; 32000640//National Natural Science Foundation of China/ ; 82422038//National Natural Science Foundation of China/ ; 82473477//National Natural Science Foundation of China/ ; 32470671//National Natural Science Foundation of China/ ; 25ZXZSSS00910//National Key Laboratory Major Special Project of Tianjin Science and Technology Plan Project/ ; 24JCZDJC00480//Natural Science Foundation of Tianjin/ ; LZYQ25H090001//Natural Science Foundation of Zhejiang/ ; 2005DKA21300//National Human Genetic Resources Sharing Service Platform/ ; OJQD2024001//Oujiang Laboratory Research Launch Project/ ; 016YFC1201703//National Key Research and Development program of China/ ; //Cancer Biobank of Tianjin Medical University Cancer Institute and Hospital/ ; 20250204//Tianjin Medical University Cancer Hospital 'Clinical-Basic' Co-PI Project/ ; //Major Project of State Key Laboratory of Experimental Hematology in 2025/ ; //Major Project of the National Key Laboratory of Drug Developability Evaluation and Systematic Transformation in 2025/ ; },
mesh = {*MicroRNAs/genetics ; *Melanoma/genetics/immunology/therapy/drug therapy ; Humans ; Animals ; *Drug Resistance, Neoplasm/genetics ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *CD8-Positive T-Lymphocytes/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Acquired resistance to both targeted therapies and immunotherapies in cancer presents major clinical challenges, yet the molecular mechanisms underlying cross-resistance remain poorly understood. We hypothesized that loss of specific microRNAs (miRNAs) could potentiate melanoma resistance to both targeted drugs and CD8[+] T cell-mediated cytotoxicity. Through genome-wide miRNA CRISPR knockout screening integrated with cellular models, longitudinal clinical samples, and in vivo experiments, we identified miR-18a as a pivotal upstream regulator of pleiotropic resistance in melanoma. We show that miR-18a deficiency drives resistance through two distinct mechanisms: derepressing AJUBA-regulated Hippo signaling during MAPK inhibition, and enhancing THBS1-CD47 interactions that impair the immunological synapse between tumor cells and CD8[+] T cells. Furthermore, hnRNP A1 plays an essential role in modulating miR-18a expression, thereby mediating cross-resistance. These findings suggest that targeting non-coding RNA vulnerabilities may represent a promising therapeutic strategy to overcome complex resistance mechanisms and improve clinical outcomes in melanoma.},
}
@article {pmid42378032,
year = {2026},
author = {Huang, J and Liu, X and Floyd, W and Haugh, W and Sun, Z and Kasiewicz, MJ and Wu, Y and Piening, B and Welle, JT and Rosales, WK and Rajamanickam, V and Kim, SY and Xu, ES and Luo, L and Ma, Y and Patel, R and Zhang, Z and Bernard, B and Redmond, WL and Urba, WJ and Bell, RB and Kirsch, DG},
title = {A tailored in vivo CRISPR screen identifies BAP1 as a potent tumor suppressor of sarcoma.},
journal = {JCI insight},
volume = {11},
number = {16},
pages = {},
doi = {10.1172/jci.insight.192686},
pmid = {42378032},
issn = {2379-3708},
support = {K22 CA248849/CA/NCI NIH HHS/United States ; R35 CA197616/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Tumor Suppressor Proteins/genetics/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Humans ; *Sarcoma/genetics/pathology/drug therapy/metabolism ; Polo-Like Kinase 1 ; Proto-Oncogene Proteins/metabolism/antagonists &amp; inhibitors/genetics ; Protein Serine-Threonine Kinases/metabolism/antagonists &amp; inhibitors/genetics ; Cell Cycle Proteins/metabolism/antagonists &amp; inhibitors/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Tumor Microenvironment/genetics ; Female ; Disease Models, Animal ; },
abstract = {Undifferentiated pleomorphic sarcoma (UPS) is one of the most common adult soft-tissue sarcomas (STSs), yet therapeutic progress remains limited because of the absence of recurrent oncogenic driver mutations. To identify tumor suppressors contributing to UPS pathogenesis, we performed a customized in vivo CRISPR/Cas9 screen in mice. This approach identified BRCA1-associated protein 1 (BAP1) as a potent tumor suppressor in STS. Integrative analyses using RNA sequencing, multiplex immunohistochemistry, and flow cytometry revealed that Bap1-deficient sarcomas exhibited a markedly immunosuppressive tumor microenvironment. Consistent with these findings, BAP1 protein expression was reduced in human UPS, whereas polo-like kinase 1 (PLK1) expression was elevated. Functional studies demonstrated that PLK1 was required for the growth and survival of Bap1-deficient sarcomas. Pharmacologic inhibition of PLK1 with volasertib significantly suppressed tumor growth in both syngeneic and autochthonous mouse models. Moreover, combining PLK1 inhibition with anti-PD-1 therapy enhanced tumor control and improved survival compared with either treatment alone. Together, these results identify PLK1 as a potential therapeutic vulnerability in BAP1-deficient sarcomas and support further evaluation of combined PLK1 inhibition and immune checkpoint blockade as a treatment strategy for a subset of STSs.},
}
@article {pmid42385356,
year = {2026},
author = {Desterke, C and Jarén, A and Francés, R and Casafont, &#xcd; and Barrachina, MD and Esplugues, JV and Mata-Garrido, J},
title = {Integrative transcriptomic and CRISPR dependency analysis identifies hepatoblastoma-specific essential genes and actionable vulnerabilities.},
journal = {Cancer genetics},
volume = {306-307},
number = {},
pages = {165-179},
doi = {10.1016/j.cancergen.2026.06.008},
pmid = {42385356},
issn = {2210-7762},
mesh = {Humans ; *Hepatoblastoma/genetics/pathology ; *Liver Neoplasms/genetics/pathology ; Gene Expression Profiling/methods ; *Genes, Essential ; *Transcriptome ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; },
abstract = {BACKGROUND: Hepatoblastoma (HB) is the most common primary liver malignancy in childhood, yet its molecular determinants, functional dependencies, and therapeutic vulnerabilities remain incompletely characterized. Integrative analyses combining transcriptomic profiling with functional genomic datasets provide a strategy to identify essential genes, biomarkers predictive of tumor behavior and treatment response.<br><br>METHODS: Differential expression analysis comparing HB tumors with normal liver was processed on training cohort. These genes were integrated with DepMap CRISPR-Cas9 dependency scores to prioritize HB-essential candidates. Elastic Net regression was used to derive a 16-gene predictive signature, which was validated in an external cohort. Single-cell RNA-seq datasets were analyzed to assess expression patterns across hepatic and tumor-associated cell populations. A supervised deep-learning classifier was trained on single-cell profiles to distinguish tumor cells from hepatocytes, and SHAP values were computed to interpret gene contributions. Drug-gene interactions were queried using curated repressive compounds from DGIdb, and approved drugs were screened for relevance in pediatric cancer clinical trials.<br><br>RESULTS: A total of 789 genes were found overexpressed in HB tumors from the training transcriptome cohort. Chronos DepMap analysis identified 73 HB-essential genes that were not essential in adult liver cancer cell lines (hepatocellular carcinoma and cholangiocarcinoma). Elastic-net tuning based on the expression of 16 HB-essential genes in the split training cohort enabled robust tumor-normal discrimination, with AUC = 0.88, specificity = 0.90, and sensitivity = 0.90 in internal validation. This performance was confirmed in an independent external cohort, achieving AUC = 0.99, specificity = 1.00, and sensitivity = 0.98. Single-cell validation further demonstrated tumor-specific enrichment of the signature. The deep-learning classifier (tumor cells vs. normal hepatocytes) reached high accuracy (AUC = 0.99; F1-score = 0.97), with SHAP analysis highlighting PEG10, GREB1, PLCB4, RHOBTB1, CRIM1, FSD1L, CORO2A, KIT, ANKRD50, HDAC11, ZNF233, SEMA7A, and FABP4 as major contributors. Six of these genes were confirmed to be absent or lowly expressed in the background liver microenvironment. Drug-gene interaction analysis identified HDAC11 as a potential therapeutic target of approved drugs used in pediatric oncology.<br><br>CONCLUSIONS: This integrative framework combining transcriptomics, CRISPR dependency mapping, machine learning, and pharmacogenomic annotation identifies clinically relevant HB-essential genes and predictive molecular signatures for tumor identity. The derived expression-based scores provide tools for patient stratification, while drug-gene mapping highlights actionable vulnerabilities on HDAC11 with pediatric approved drugs that support rational drug repurposing strategies in hepatoblastoma.},
}
@article {pmid42477347,
year = {2026},
author = {Huang, Q and Yang, D and Zhou, X and Li, G and Liu, W and Fan, X and Yuan, F and Chang, X},
title = {Incorporating AI-optimized zinc finger proteins enhances the efficiencies and targeting ranges of miniature base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42477347},
issn = {2041-1723},
support = {2022YFA0807300//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2018YFA0801400//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 2022SDXHDX0002//Science and Technology Department of Zhejiang Province/ ; 82450102//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31870927//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Zinc Fingers/genetics ; Animals ; Dependovirus/genetics ; Mice ; Humans ; Genetic Vectors/genetics ; Survival of Motor Neuron 2 Protein/genetics ; Muscular Atrophy, Spinal/therapy/genetics ; Exons/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {The therapeutic application of base editors is limited by their large sizes, which are beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress that has identified many compact CRISPR proteins, the resulting miniature base editors often exhibit reduced activities and limited targeting scope. Here, we introduce a zinc finger protein (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable ZFPs to improve efficiencies and targeting scopes of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language models to optimize ZFPs designed by modular assembly further simplifies the development of zmBEs. Leveraging these methodologies, we engineer a zmBE that effectively induces the SMN2 exon 7 T:A(6) > C:G conversion, restores the exon 7 inclusion, and improves spinal muscular atrophy in a murine model after being delivered via a single AAV vector. Our study provides a versatile platform for developing miniature base editors for in vivo therapeutic applications.},
}
@article {pmid42572954,
year = {2026},
author = {Xu, ZH and Weng, X and Zhang, MP and Lin, RM and Xu, W and Wu, H and Gao, H},
title = {An aggregation-induced electrochemiluminescence sensor for ochratoxin A detection integrating CRISPR-Cas12a and tetrahedral DNA nanostructures.},
journal = {The Analyst},
volume = {151},
number = {17},
pages = {4950-4955},
doi = {10.1039/d6an00771f},
pmid = {42572954},
issn = {1364-5528},
mesh = {*DNA Nanostructures/chemistry ; *Biosensing Techniques/methods ; *Ochratoxins/analysis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Endodeoxyribonucleases/chemistry ; },
abstract = {An aggregation-induced electrochemiluminescence (AIECL) biosensor combining CRISPR-Cas12a and tetrahedral DNA nanostructures (TDNs) is fabricated for OTA detection, with polymer dots (Pdots) serving as emitters. The sensor achieves favorable analytical performance, with a limit of detection of 0.41 pg mL[-1], showing promising applications in food security monitoring.},
}
@article {pmid42610445,
year = {2026},
author = {Arti, and Yadav, G and Mathur, J},
title = {A critical review of mechanistic insights and technological advancement for the amelioration of Pb and Cd through phytoremediation.},
journal = {International journal of phytoremediation},
volume = {},
number = {},
pages = {1-23},
doi = {10.1080/15226514.2026.2716124},
pmid = {42610445},
issn = {1549-7879},
abstract = {Heavy metal contamination is a major environmental concern due to its persistence, bioaccumulation, and long-term impacts on ecosystems and human health. Among toxic metals, cadmium (Cd) and lead (Pb) are particularly harmful because of their high toxicity and carcinogenic potential, posing serious risks to plants, animals, and humans even at low concentrations. These metals often enter soil and water through industrial activities, mining, agricultural inputs, and improper waste disposal. Conventional remediation methods, such as chemical treatment, soil excavation, and stabilization, have been used to manage contaminated sites; however, they are often costly, labour- intensive, and may cause secondary environmental pollution, creating a need for more sustainable alternatives. Phytoremediation has emerged as an eco-friendly and cost-effective approach that utilizes the natural ability of plants to absorb, accumulate, detoxify, or stabilize contaminants from soil, water, and air using solar energy. The objectives of this review are to examine the mechanisms of phytoremediation and evaluate recent advance technologies that enhance its efficiency, with a focus on plant growth-promoting microorganisms, biochar, nanomaterials, CRISPR/Cas9-based genetic engineering, isotope monitoring, and AI/ML tools. The novelty of this review lies in its integrated assessment of these emerging technologies as complementary strategies for advancing sustainable heavy metal remediation. Overall, these developments highlight the growing potential of phytoremediation as a sustainable strategy for environmental cleanup. Nevertheless, challenges related to large-scale application, plant tolerance to heavy metals, and long-term ecological sustainability remain, requiring further research to enhance its practical implementation in environmental management.},
}
@article {pmid42610742,
year = {2026},
author = {Kumar, A and Kumari, P and Mishra, S and Thakur, S and Acharya, S and Chakraborty, D and Maiti, S and Jain, N},
title = {Energetic Determinants of Expanded PAM Recognition in Engineered FnCas9.},
journal = {Biochemistry},
volume = {65},
number = {16},
pages = {2495-2509},
doi = {10.1021/acs.biochem.6c00350},
pmid = {42610742},
issn = {1520-4995},
support = {BT/RLF/Re-entry/HRD/35/2019//Department of Biotechnology, Ministry of Science and Technology, India/ ; OLP2303//Council of Scientific and Industrial Research, India/ ; },
mesh = {*Francisella/enzymology/genetics ; *Bacterial Proteins/genetics/metabolism/chemistry ; Thermodynamics ; *CRISPR-Cas Systems ; Protein Engineering ; Substrate Specificity ; Mutation ; },
abstract = {Expanding protospacer-adjacent motif (PAM) compatibility while preserving specificity remains a central challenge in CRISPR-Cas9 engineering. Francisella novicida Cas9 (FnCas9) exhibits high intrinsic specificity but is constrained by stringent PAM requirements. Here, we quantitatively examine the energetic and catalytic consequences of PAM-interacting mutations in three engineered variants, en1 (E1369R), en15 (E1603H), and en31 (G1243T/E1369R/E1449H), using a VEGFA3 DNA substrate framework. Microscale thermophoresis and isothermal titration calorimetry reveal that the engineered variants enhance binding affinity toward the canonical NGG PAM relative to wild-type FnCas9, with modest gains in binding free energy. Selected noncanonical PAM substrates, particularly TGA and TAG, also show improved binding by en15 and en31, with en31 displaying the strongest overall binding among the substrates tested. Thermodynamic profiles indicate that enhanced affinity is associated with more favorable enthalpic contributions, consistent with altered interactions at the PAM interface; however, the specific molecular contributions underlying these changes remain to be directly established. Despite improved binding, active-site titration reveals reduced fractions of catalytically competent enzyme in engineered variants, particularly en31, necessitating higher enzyme concentrations to achieve cleavage efficiencies comparable to wild-type. Cleavage assays demonstrate that en31 most effectively couples improved recognition of the tested noncanonical PAM substrates to productive catalysis, enabling robust cleavage of both TGA and TAG substrates while maintaining minimal off-target activity under the conditions examined. Together, these results suggest that PAM-interacting mutations in FnCas9 can alter the energetic coupling between DNA binding and catalytic activation within the VEGFA3 substrate framework tested, highlighting the importance of balancing substrate affinity with conformational activation in the design of high-precision genome-editing nucleases.},
}
@article {pmid42610908,
year = {2026},
author = {Liu, H and Chen, J and Xiu, L and Liu, Y and Wei, TT and Wang, Y and Shi, L and Wang, X and Li, X and Yin, K},
title = {Integrated CRISPR/Cas12a-Based Duplex Detection Platform for Species Fingerprinting.},
journal = {Analytical chemistry},
volume = {98},
number = {32},
pages = {23672-23683},
doi = {10.1021/acs.analchem.6c02717},
pmid = {42610908},
issn = {1520-6882},
support = {2025YFC3409100//National Key Research and Development Program of China/ ; 24142201300//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; 24J22800900//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; 2024ZZ2012//Shanghai Municipal Health Commission's Seed Program/ ; MDPDMT-2023-02//Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology, State Administration for Market Regulation/ ; YG2024ZD02//Interdisciplinary Program of Shanghai Jiao Tong University/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Milk/chemistry ; Cattle ; Colorimetry ; Lab-On-A-Chip Devices ; Buffaloes ; Food Contamination/analysis ; },
abstract = {Species fingerprinting is crucial to ensure food safety and human health, which requires a rapid, simple, multiplex, and field-deployable detection technique. In response, a lab-on-a-disc microfluidic chip with CRISPR/Cas12a that integrates target preamplification and signal readout enhanced by tetrahedral DNA frameworks (TDFs) has been developed in this study. This platform automates the workflow from recombinase polymerase amplification (RPA) through signal readout. In addition, a portable heating module was developed specifically for colorimetric detection, providing a complete field-deployable solution. The performance of the CRISPR/Cas12a platform was evaluated by detecting cow's milk adulteration in buffalo milk. The platform demonstrated high sensitivity, achieving detection limits of 1% (v/v) and 5% (v/v) for fluorescence and colorimetric detection within 1 h, respectively. The results agree well with those from real-time quantitative polymerase chain reaction (qPCR) in real-sample analysis. The integrated CRISPR/Cas12a-based duplex detection platform features high sensitivity and specificity, reaction automation, minimal aerosol contamination risk, and decentralized operation, which demonstrates significant potential for field-deployable species fingerprinting and risk prediction.},
}
@article {pmid42095874,
year = {2026},
author = {Quansah, E and Yang, S and Jia, Y and Yu, L and Zhang, C},
title = {The dCas9-SSAP as a promising genome editing tool in malaria parasites.},
journal = {Critical reviews in microbiology},
volume = {52},
number = {5},
pages = {864-873},
doi = {10.1080/1040841X.2026.2667183},
pmid = {42095874},
issn = {1549-7828},
mesh = {*Gene Editing/methods ; *Plasmodium/genetics ; CRISPR-Cas Systems ; Humans ; *Malaria/parasitology ; Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Measures to combat the Plasmodium parasites which cause malaria have become compromised because of reliance on a small arsenal of drugs, emerging drug resistance and the lack of effective vaccines. A promising avenue for addressing these challenges is the revolutionary gene-editing technology CRISPR-Cas9, due to its high efficiency and ease of design for genetic manipulation. The catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP)(dCas9-SSAP) is a recently emerged next-generation gene editing system added to the ever-growing CRISPR-Cas9-based technologies. While the classical Cas9-nuclease technologies are "double-strand break, damage-repair systems", the dCas9-SSAP is distinctively a "cleavage-free" editing tool. Unlimited to the Plasmodium genome, Cas9-nucleases imprint inheritable genetic scars on the subject genomes when applied. Here, we discussed the DSB genotoxicity pitfalls of existing nuclease-based editing tools, especially CRISPR-Cas9, and how the dCas9-SSAP presents a formidable option to the drawbacks within the context of Plasmodium genome editing. Then, we sought to infer a plausible mechanistic framework that could account for dCas9-SSAP-mediated genome editing. Finally, we discussed how dCas9-SSAP aligns with Plasmodium parasites' biology. This review would set the stage for continued research into the potential of this new, exciting technology in malaria parasites.},
}
@article {pmid42610144,
year = {2026},
author = {Taki, AG and Shareef, A and Arora, V and Oweis, R and Jyothi, SR and Singh, U and Sahoo, S and Chauhan, AS and Klebleeva, G and Sameer, HN and Yaseen, A and Athab, ZH and Adil, M},
title = {AI-driven CRISPR strategies in breast cancer: Organoid modeling, adaptive editing, and precision delivery.},
journal = {Iranian journal of basic medical sciences},
volume = {29},
number = {6},
pages = {823-843},
pmid = {42610144},
issn = {2008-3866},
abstract = {Triple-negative breast cancer (TNBC) is defined by profound heterogeneity, dormant metastatic reservoirs, and rapid therapy resistance. Building on our AI-Driven CRISPR Strategies in Breast Cancer framework, CRISPR-Cas9 is emerging as more than a gene-editing tool, capable of restoring circadian integrity, eliminating dormant clones, and re-programming immune surveillance. A structured PubMed, Scopus, and ClinicalTrials.gov review through 2025 integrated mechanistic, preclinical, and early clinical evidence. Beyond standard knockout, base, and prime editing, we highlight chrono-genomic repair of BMAL1/PER2, dormancy-focused synthetic-lethality screens, and genomic-collapse tactics for BRCA1-deficient tumors. Adaptive AI pipelines that iteratively refine guide RNAs and exosome-mimetic carriers, incorporating Boolean logic gates, were also evaluated for self-regulated, tumor-specific delivery. Proof-of-concept studies show that HER2 deletion, TP53 rescue, and ABCB1 silencing enhance chemosensitivity across luminal, HER2-positive, and TNBC models. Circadian restoration expands therapeutic windows and delays relapse in xenografts. Dormancy-directed CRISPR screens reveal unique vulnerabilities in disseminated tumor cells, whereas genomic collapse selectively destroys BRCA1-mutant clones. Integration with CAR-T cells and antibody-drug conjugates amplifies cytotoxicity, and transient nanoparticle or exosome systems improve solid-tumor penetration while minimizing off-target events. CRISPR-Cas9 is transitioning from a molecular scalpel to an adaptive, self-learning therapeutic ecosystem. By uniting AI-guided design, circadian reprogramming, dormancy eradication, and logic-gated delivery, the strategies detailed here define a next-generation precision-oncology paradigm capable of anticipating tumor evolution, overcoming resistance, and preventing metastatic relapse.},
}
@article {pmid42425298,
year = {2026},
author = {Sun, X and Zhang, Y and Lu, R and Dai, T and Qiu, J and Jia, X and Wang, G and Pan, Z and Xu, S and Sima, Y},
title = {Integrated mapping and gene editing identify BmCCP as a regulator of cocoon shape and spinning behaviour in Bombyx mori.},
journal = {Journal of insect physiology},
volume = {173},
number = {},
pages = {105033},
doi = {10.1016/j.jinsphys.2026.105033},
pmid = {42425298},
issn = {1879-1611},
mesh = {Animals ; *Bombyx/genetics/growth &amp; development/metabolism/physiology ; *Insect Proteins/genetics/metabolism ; Gene Editing ; Chromosome Mapping ; *Silk/metabolism ; CRISPR-Cas Systems ; Larva/growth &amp; development/genetics ; },
abstract = {The cocoon shape in Bombyx mori (silkworm) is a construction trait shaped by cocoon-spinning behaviour, but the molecular regulation pathways remain poorly understood. Here, quantitative phenotyping, bulked segregant analysis sequencing (BSA-seq), brain transcriptomics and CRISPR/Cas9 mutagenesis were combined to identify a regulator of cocoon morphogenesis and to assess potential roles in spinning behaviour. Using representative strains L6J5 and J8, which produce short oval and long peanut-shaped cocoons, respectively, showed that cocoon shape, quantified by the cocoon aspect ratio, is a quantitative trait. BSA-seq mapped the trait to a 5.04-Mb candidate interval on chromosome 4 containing 213 annotated genes. Integration of the mapping results with brain transcriptomes from individuals with extreme cocoon phenotypes identified Bombyx mori cocoon shape-correlated protein (BmCCP) as the sole overlapping candidate gene. BmCCP was more highly expressed in the brain of strain J8 than L6J5 at the wandering stage, and the locus contained multiple associated polymorphisms. CRISPR/Cas9-mediated knockout of BmCCP in strain J8 significantly increased the cocoon aspect ratio and cocoon size. In the widely used experimental strain DaZao, which has not been artificially selected for cocoon shape, BmCCP deficiency likewise increased cocoon size and significantly reduced the larval spinning rate, while crosses with J8 further supported the role of BmCCP in regulating the cocoon aspect ratio. Together, these results identify BmCCP as a regulator of cocoon morphogenesis and provide a foundation to investigate the relationship between the cocoon morphology and spinning behaviour of the silkworm.},
}
@article {pmid42435068,
year = {2026},
author = {Zhang, C and Josyula, NK and Cornejo-Corona, I and Watson, R and Cediel-Becerra, JD and Chevrette, MG and Devarenne, TP and Straight, PD},
title = {Tandem-sgRNA Provides an Effective Reverse Genetic Approach for Suppression of Streptomyces Biosynthetic Gene Clusters and Secondary Metabolism.},
journal = {ACS synthetic biology},
volume = {15},
number = {8},
pages = {3382-3395},
doi = {10.1021/acssynbio.6c00274},
pmid = {42435068},
issn = {2161-5063},
support = {GM141700/GM/NIGMS NIH HHS/United States ; },
mesh = {*Streptomyces/genetics/metabolism ; *Multigene Family/genetics ; *Secondary Metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Reverse Genetics/methods ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Bacterial biosynthetic gene clusters (BGCs) encode secondary metabolites with diverse biological activities; however, most BGC products remain uncharacterized. One approach to identifying products and their metabolism is to use reverse genetics to identify metabolite-associated phenotypes. CRISPR interference (CRISPRi) offers a promising approach to disrupt BGC functions in high-GC genomes, typical of Streptomyces species. In many of these organisms, single-guide RNA (sgRNA)-mediated CRISPRi often results in incomplete product suppression, resulting in partial phenotypes that are unsuitable for functional studies. Using Streptomyces sp. Mg1, we found that a tandem-sgRNA configuration for CRISPRi improved the efficiency of target metabolite suppression. We engineered strains to express two sgRNAs to target the same promoter region within a BGC, resulting in greater than 80% metabolite suppression across diverse secondary metabolite classes. We used tandem-sgRNA CRISPRi to identify phenotypes associated with the loss of polyketide linearmycins, the siderophore desferrioxamine, the terpene β-carotene, and an uncharacterized nonribosomal peptide synthetase (NRPS). This approach revealed that β-carotene depletion substantially reduced intrinsic cellular autofluorescence. Targeting the unknown NRPS produced developmental phenotypes and enabled the identification of the biosynthetic genes for the antibiotic lavendomycin, revealing a noncollinear organization of genes in the BGC. We suggest that tandem-sgRNA CRISPRi provides an efficient reverse genetics platform for the functional characterization of Streptomyces BGCs, enabling the correlation of metabolites with the gene function, identification of associated phenotypes, and prioritization of cryptic BGCs for natural product discovery.},
}
@article {pmid42435429,
year = {2026},
author = {Zhou, Q and Xu, B and Wang, Y and Yang, X and Wang, L and Zheng, X and Lou, Y and Zheng, M and Xu, F and Xiao, X},
title = {LAMP-Based Two-DNA-Fragment Fusion and Its Application in Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {15},
number = {8},
pages = {3300-3309},
doi = {10.1021/acssynbio.6c00187},
pmid = {42435429},
issn = {2161-5063},
support = {JS2026005//Wenzhou Medical University/ ; 22578338//National Natural Science Foundation of China/ ; LTGY24H200005//Natural Science Foundation of Zhejiang Province/ ; 2024YFC2309905//National Key Research and Development Program of China/ ; NA//Key Discipline of Zhejiang Province in Medical Technology/ ; WKJ-ZJ-26018//Medical Science and Technology Project of Zhejiang Province/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; *DNA, Bacterial/genetics/analysis ; CRISPR-Cas Systems/genetics ; *DNA/genetics ; Molecular Diagnostic Techniques ; },
abstract = {Loop-mediated isothermal amplification (LAMP) continuously generates strand-displaced single-stranded DNA intermediates, providing the possibility of assembling DNA fragments. Here, we developed a novel two-DNA-fragment fusion technique, termed fusion LAMP, which is an isothermal DNA-fusion strategy that enables the fusion of two independent DNA fragments within a single amplification reaction. By combining fusion LAMP with CRISPR/Cas13a, we further established an "AND-gate" nucleic acid detection platform, termed Fusion LAMP-Coupled CRISPR/Cas13a (FLCC), which enables concurrent detection of two targets by reading the fusion product-triggered fluorescence signals. This platform generates signals only when two targets are present simultaneously. To prove this concept, we then employed FLCC to identify the methicillin-resistant Staphylococcus aureus (MRSA). This method achieved a limit of detection of 10 copies/μL of MRSA genomic DNA and showed no cross-reactivity with closely related bacterial strains. Furthermore, we validated its feasibility by detecting 19 clinical isolates, demonstrating a simple and accurate approach for MRSA detection. Collectively, the FLCC platform ensures identifying pathogens accurately and provides a promising diagnostic approach for detecting complex genetic targets.},
}
@article {pmid42454678,
year = {2026},
author = {Feldmann, D and van Beljouw, SPB and Haagsma, AC and Kalogeropoulos, K and Muralidharan, A and Brouns, SJJ},
title = {Craspase Protease Activation Is Sensitive to Oncogenic Single-Nucleotide RNA Mismatches.},
journal = {ACS chemical biology},
volume = {21},
number = {8},
pages = {1877-1882},
doi = {10.1021/acschembio.6c00241},
pmid = {42454678},
issn = {1554-8937},
support = {101003229//European Commission/ ; OCENW.XS23.1.006//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; SUMMIT.1.004//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; 15602//KWF Kankerbestrijding/ ; 4257-00010B//Danmarks Frie Forskningsfond/ ; NNF16OC0020670//Novo Nordisk Fonden/ ; },
mesh = {Humans ; Enzyme Activation ; *RNA/genetics ; *Base Pair Mismatch ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Peptide Hydrolases/metabolism/genetics ; },
abstract = {The type III-E CRISPR-controlled protease Craspase is distinguished from other type III systems by its single-subunit RNA-guided protein complex and direct coupling of RNA recognition to protease activation without second messenger signaling, making it an attractive development platform for bioengineering and therapeutics. Here, we identify five positions within the CRISPR RNA (crRNA) of Craspase from Candidatus "Scalindua brodae" (Sb-Craspase) that are sensitive to single-nucleotide mismatches. We leverage these positions to design crRNAs that selectively target clinically relevant single-nucleotide variants (SNVs) in oncogenic RNA transcripts. Using this approach, Sb-Craspase is selectively activated by the "undruggable" KRAS G12D SNV, while the wild-type transcript does not induce protease activation. Collectively, our results establish a framework for designing crRNAs to target clinically relevant SNVs, laying the groundwork for Craspase-based diagnostics and therapeutics against otherwise intractable oncogenic mutations.},
}
@article {pmid42477478,
year = {2026},
author = {Tokgün, O and İnci, K and Gültekin, A and Çelikkaya, B and İrep, N and Akça, H and Tokgün, PE},
title = {Targeting RAB27A-mediated small extracellular vesicle secretion via CRISPR-Cas9 negatively affects proliferation and metastasis in both in vitro and in vivo SCLC models.},
journal = {Cancer gene therapy},
volume = {33},
number = {8},
pages = {1000-1013},
pmid = {42477478},
issn = {1476-5500},
mesh = {Humans ; Animals ; Cell Proliferation ; *rab27 GTP-Binding Proteins/genetics/metabolism ; Mice ; *Lung Neoplasms/pathology/genetics/metabolism ; *Small Cell Lung Carcinoma/genetics/pathology/metabolism/therapy ; *CRISPR-Cas Systems ; *Extracellular Vesicles/metabolism/genetics ; Cell Line, Tumor ; Female ; Neoplasm Metastasis ; Xenograft Model Antitumor Assays ; },
abstract = {Small cell lung cancer (SCLC) comprises 15% of lung cancers with a capacity for early and distant metastatic development, high proliferative capacity, and poor survival rates. Ionizing radiation and chemotherapy are effective against early-stage SCLC. This sensitivity wanes over time, however, making treatment difficult. Different types of neoplasms have demonstrated the pivotal role of small extracellular vesicles (sEVs) in disease progression. However, the role of sEVs development in SCLC remains unclear. In this study, the impact of sEVs secretion in SCLC cells was investigated using the CRISPR-Cas9 system to target the RAB27A. The effects of sEVs release inhibition on tumour growth and metastasis were evaluated using micro-PET-CT analysis. A reduction in cellular proliferation as a consequence of sEVs release, along with diminished expression of proteins and RNA (CD9, CD63, and Tsg101) implicated in sEVs secretion in silenced SCLC cells (p < 0.001, p < 0.0001) was detected. The suppression of sEVs release exhibited significant adverse effects on tumor development and metastatic dissemination in the in vivo tumor model. The present study suggests that the targeting of RAB27A could be a viable cancer therapy for SCLC. Targeting the exosomal pathway has the potential to enhance treatment efficacy, and SCLC may depend on sEVs secretion.},
}
@article {pmid42567105,
year = {2026},
author = {Hassannia, M and Amirifar, P},
title = {CRISPR-Cas9 gene editing approaches in colorectal cancer: Current progress and future prospects.},
journal = {Cancer treatment and research communications},
volume = {48},
number = {},
pages = {101341},
doi = {10.1016/j.ctarc.2026.101341},
pmid = {42567105},
issn = {2468-2942},
mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods ; Animals ; },
abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by genetic heterogeneity and the accumulation of mutations in key oncogenes and tumor suppressor genes. CRISPR-Cas9 technology has greatly advanced genetic research by enabling precise genome editing. This review focuses on the innovative applications of CRISPR-Cas9 in CRC research, particularly its role in identifying novel therapeutic targets, elucidating mechanisms of drug resistance, and uncovering metabolic and stem cell pathway alterations in tumorigenesis. We highlight the diverse CRISPR systems, including Cas9, Cas12, Cas13, and advanced variants such as CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), base editing, and prime editing, which have expanded gene knockout studies and enhanced our understanding of CRC. Despite these breakthroughs, challenges such as off-target effects and delivery limitations remain. Ongoing efforts to refine CRISPR technology aim to enhance its precision and clinical applicability, ultimately paving the way for more effective and personalized treatment strategies for CRC. In this review, we explore these advances and focus on the latest developments in CRISPR-based approaches for CRC treatment.},
}
@article {pmid42567230,
year = {2026},
author = {Dewar, CE and King, EFB and Rojas, F},
title = {Optimising electroporation protocols for Trypanosoma brucei using the Amaxa 4D-nucleofector system.},
journal = {Molecular and biochemical parasitology},
volume = {267},
number = {},
pages = {111767},
doi = {10.1016/j.molbiopara.2026.111767},
pmid = {42567230},
issn = {1872-9428},
mesh = {*Trypanosoma brucei brucei/genetics ; *Transfection/methods ; *Electroporation/methods/instrumentation ; Cell Survival ; Animals ; CRISPR-Cas Systems ; },
abstract = {Stable transfection of Trypanosoma brucei remains a cornerstone for functional genetic studies in this model parasite. Although the Amaxa Nucleofector II system dramatically improved transfection efficiency in both monomorphic and pleomorphic bloodstream forms, the more recent 4D Nucleofector platform offers enhanced programmability and buffer flexibility that have yet to be systematically evaluated for T. brucei. Here, we benchmark a range of 4D Nucleofector programs to determine optimal parameters for transfection efficiency, cell viability, and reproducibility in bloodstream forms. Using a CRISPR/Cas9 expressing cell line, we compare stable transfection efficiencies across programs. We further demonstrate the advantages of the 16-well Nucleocuvette™ Strip format, enabling simultaneous processing of multiple experimental conditions in 20 µL reactions, reducing DNA, cell, and reagent requirements while increasing experimental throughput. Our results provide a standardized framework for future genetic manipulation of T. brucei using the 4D-Nucleofector X Unit, facilitating robust and reproducible transfection across life-cycle stages and strains.},
}
@article {pmid42609829,
year = {2026},
author = {Al-Azzani, H and Aliouat, H and Cheng, H and Dagah, OMA and Al-Subari, MH and Zhou, W and Wu, S},
title = {Antibacterial Immunotherapy: Mechanistic Insights, Emerging Therapeutic Strategies, and Clinical Translation.},
journal = {Infection and drug resistance},
volume = {19},
number = {},
pages = {619916},
pmid = {42609829},
issn = {1178-6973},
abstract = {Antimicrobial resistance (AMR) continues to compromise the effectiveness of conventional antibacterial therapy, driving the development of therapeutic strategies that extend beyond direct antibiotic-mediated bacterial killing. Multidrug-resistant (MDR) pathogens evade treatment through diverse mechanisms, including enzymatic drug inactivation, target modification, efflux pump overexpression, biofilm formation, and persisters development. AMR results in chronic and recurrent infections, prolonged hospitalization, increased healthcare costs, and elevated morbidity and mortality, underscoring the need for innovative therapeutic approaches that target both the pathogen and the host. To bridge the dynamic interplay between bacterial pathogens and the host immune system with emerging therapeutic innovations, this narrative review first examines the biological mechanisms underlying bacterial resistance. It then explores therapeutic strategies beyond conventional antibiotics, providing an overview of current approaches and their limitations, including drug repurposing, bacteriophage therapy, and CRISPR-Cas technology. The review subsequently focuses on antibacterial immunotherapy, discussing a broad range of emerging approaches, including probiotics, monoclonal antibodies, cell-based therapies, host-directed therapies, aptamers, nanotechnology-based platforms, cytokine-based therapies, and antimicrobial peptides. An integrated overview of preclinical evidence, clinical studies, and FDA-approved therapies is presented to assess the translational potential of immunotherapy strategies in combating AMR. Scientific, regulatory, manufacturing, and implementation challenges that influence their successful translation into clinical practice are discussed throughout. By integrating the biological basis of host-pathogen interactions with emerging antibacterial therapeutics and their translational development, this review provides a comprehensive framework for evaluating innovative strategies against antimicrobial resistance. In contrast to modality-focused reviews, it offers a unified perspective that highlights the complementary roles of pathogen-targeted and host-directed interventions and identifies future opportunities to improve the prevention and management of multidrug-resistant bacterial infections.},
}
@article {pmid42379321,
year = {2026},
author = {Wei, Y and Zhu, G},
title = {Targeted gene editing of heterodisulfide reductase mediated electron bifurcation optimises heterodisulfide reductase-ferredoxin-adenosine triphosphate axis for enhanced methanogenesis in anaerobic granular sludge.},
journal = {Bioresource technology},
volume = {459},
number = {},
pages = {135281},
doi = {10.1016/j.biortech.2026.135281},
pmid = {42379321},
issn = {1873-2976},
mesh = {*Methane/biosynthesis/metabolism ; *Sewage/microbiology ; *Adenosine Triphosphate/metabolism ; *Oxidoreductases/genetics/metabolism ; Anaerobiosis ; *Ferredoxins/metabolism ; *Gene Editing/methods ; *Electrons ; CRISPR-Cas Systems/genetics ; },
abstract = {The thermodynamic bottleneck of syntrophic propionate oxidation constrains the efficiency and stability of anaerobic digestion (AD), which depends on flavin-based electron bifurcation (FBEB) mediated by heterodisulfide reductase (Hdr). Five CRISPR-Cas9 engineered Methanobacterium formicicum strains targeting Hdr, nickel homeostasis, and flavin metabolism were evaluated in 10 % and 20 % granular sludge systems to rewire electron flux toward the energy-conserving ferredoxin (Fd) reduction pathway. Within each experimental tier, results are reported against matched controls. In the enzyme-supplementation tier, the heterodisulfide reductase ABC subunit-F420-reducing hydrogenase A subunit (HdrABC-MvhA) supplement produced the highest cumulative methane yield and elevated intracellular adenosine triphosphate (ATP) to 28.31 ± 1.60 nmol/L versus 16.27 ± 0.90 nmol/L in the matched wild-type control (1.74 ± 0.10-fold; P < 0.01, n = 3). In the genome-editing tier, the Δhpt-nikR strain achieved the highest cumulative methane yield under high sludge loading. Because N[5]-methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) and A1A0-ATP synthase activities were not directly measured, and the Hdr activity increment falls within the variance of crude-extract assays, these co-occurring changes are interpreted as correlative support for a putative "Hdr-Fd-ATP" working model rather than direct demonstration of a defined energy-conservation pathway. The engineered strains enriched hydrogenotrophic methanogens (Methanobacterium, 1.08-1.13-fold) and increased the predicted genomic abundance of electron-bifurcation, CO2-reduction, and methyl-transfer pathway genes, as inferred from 16S rRNA-based functional prediction. This study provides correlative evidence that CRISPR-based metabolic engineering of methanogens can modulate the Hdr-Fd-ATP axis within heterogeneous granular sludge communities, establishing a mechanistic framework for in situ bioaugmentation strategies targeting intracellular energy-conservation bottlenecks.},
}
@article {pmid42463075,
year = {2026},
author = {Cheng, Y and Niu, S and Zhang, Z and Qian, H and Huang, Z and Jing, H and Liao, L and Man, C and Gao, H and Chen, Q and Du, L and Chen, S and Wang, F},
title = {TBC1D14 positively regulates autophagy induced by Brucella melitensis vaccine strain BA0711 in Sheep Leydig Cells.},
journal = {Microbial pathogenesis},
volume = {219},
number = {},
pages = {108664},
doi = {10.1016/j.micpath.2026.108664},
pmid = {42463075},
issn = {1096-1208},
mesh = {Animals ; *Autophagy/genetics ; *Brucella melitensis/immunology ; Male ; *GTPase-Activating Proteins/genetics/metabolism ; Sheep ; *Leydig Cells/microbiology/metabolism/immunology ; Gene Knockout Techniques ; *Brucellosis/veterinary/immunology ; CRISPR-Cas Systems ; Microscopy, Electron, Transmission ; },
abstract = {Brucella is an intracellular Gram-negative bacterium that primarily infects the host reproductive and immune systems, inducing autophagy and facilitating pathogen replication. TBC (Tre2-Bub2-Cdc16) domain-containing proteins are important in membrane trafficking, cell polarity, and signal transduction as regulators of Rab small GTPases. Previously, we demonstrated that B. melitensis M5-90 modulates the expression of miR-146b-5p, which targets TBC1D14, in RAW264.7 cells. In this study, CRISPR-Cas9 was used to generate TBC1D14-knockout (KO) Sheep Leydig cells (SLCs), and B. melitensis BA0711 treatment experiment was conducted at a multiplicity of infection (MOI) of 100. After confirming that SLCs retain autophagic activity, Western blot, autophagy flux assays, transmission electron microscopy (TEM), and RT-qPCR were performed to identify the function of TBC1D14. We found that autolysosome fluorescence was significantly enhanced in NC-SLCs compared with TBC1D14-KO-SLCs following BA0711 treatment at 8 and 12 hpi. Notably, autophagy flux in NC-SLCs remained consistently higher than that in KO-SLCs from 8 hpi onwards. Consistently, Western blot revealed a decreased LC3-II/LC3-I ratio, and TEM confirmed a reduced number of autolysosomes in KO-SLCs at 12 hpi. These results indicate that TBC1D14 positively regulates autophagy in SLCs by BA0711 stimulation. Although transcriptomic and proteomic analyses indicated activation of autophagy- and phagocytosis-related pathways during BA0711 exposure, this response was significantly attenuated in TBC1D14-KO cells. Furthermore, TBC1D14 knockout led to downregulation of phagosome-related proteins and altered chemotaxis pathways, whereas re-expression of TBC1D14 in KO cells upregulated RAB28. In conclusion, this study demonstrates that TBC1D14 positively regulates autophagy and provides insights into the response of SLCs treated by B. melitensis BA0711. Furthermore, it suggests that RAB28 may serve as a downstream effector of TBC1D14.},
}
@article {pmid42492860,
year = {2026},
author = {Lee, JH and Lee, ES and Xiang, XR and Kyung, SM and Seo, J and Lee, W and Park, HE and Shin, MK and Yoo, HS},
title = {Roles of mdh and MAP1981c in Mycobacterium avium subsp. paratuberculosis intracellular survival within bovine monocyte-derived macrophages via CRISPR interference.},
journal = {Microbial pathogenesis},
volume = {219},
number = {},
pages = {108724},
doi = {10.1016/j.micpath.2026.108724},
pmid = {42492860},
issn = {1096-1208},
mesh = {Animals ; *Mycobacterium avium subsp. paratuberculosis/genetics/pathogenicity/growth &amp; development ; Cattle ; *Macrophages/microbiology/immunology ; Paratuberculosis/microbiology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Profiling ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Microbial Viability ; Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and is capable of persisting within macrophages despite the activation of host immune defenses. Although this intracellular persistence is a key determinant of MAP pathogenicity, the bacterial factors and host responses that regulate this process remain poorly understood. In this study, we established the first CRISPR interference (CRISPRi) platform applied to bovine monocyte-derived macrophages (MDM) to evaluate the functions of MAP genes involved in intracellular survival and to perform an integrative analysis of host transcriptomic responses. MAP mutants were targeted to two genes (mdh and MAP1981c). The optimal concentration of anhydrotetracycline (ATc) was determined to be 2 μg/ml by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to 72 h. The gene expression profiles and intracellular MAP levels were investigated using RNA-seq and colony-forming units, respectively. The survival rates of the MAP mutants significantly decreased with the time course of infection in MAP-mdhKD and MAP1981cKD (KD, knockdown). RNA-seq-based gene expression profiling suggested that target gene silencing in MAP mutants led to altered expression of host genes involved in lipid metabolism, T-cell activation reduction, and antimicrobial response in bovine MDM, contributing to reduced intracellular survival of MAP. Our study demonstrates that the downregulation of mdh and MAP1981c in MAP significantly alters the host transcriptomic landscape in bovine MDM, revealing their critical roles in subverting host immune defenses for intracellular persistence.},
}
@article {pmid42576388,
year = {2026},
author = {Zhang, Y and Yang, Y and Liu, Z and Li, Y and Xue, Y and Zhang, Z and Chen, G and Liu, J and He, M and Lu, T and Zhang, Y and Zhao, D and Yang, K and Miao, L and Gao, F and Guo, Y},
title = {The DreAM-plus integrative RNA switch enhances transient AAV expression and reduces side effects of gene editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2026.08.006},
pmid = {42576388},
issn = {1525-0024},
abstract = {Uncontrolled long-term adeno-associated virus (AAV) expression prohibits therapeutic strategies that require more precise and dynamic regulation. For example, long-lasting expression of gene editors by AAV could augment off-target effects and immunogenicity. Drug-inducible RNA switches are desirable tools to achieve transient AAV expression. However, current RNA switches only target a single mechanism such as transcription or RNA splicing, exhibiting limited capacity in transgene regulation. Here, we report DreAM-plus, a multilayer RNA switch that integrates an aptamer-based poly(A) regulator (pA), a drug-elicitable alternative splicing module (DreAM), and an engineered P2A element with conditional upstream open reading frames (uORFs). The pA-DreAM concatenation enhanced gene inducibility by up to 5-fold more than pA or DreAM alone, with 1.4- to 6.3-fold further improvement by uORFs. DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, and IsDra2 TnpB) with a temporal resolution of less than 24 h, which significantly mitigated off-target effects by 1.4- to 2.8-fold. With lipid-nanoparticle-delivered pre-existing immunity in mice, DreAM-plus attenuated AAV-delivered Cas-specific CD8[+] T cell immune toxicity in the liver and heart. Therefore, the inducible RNA switches could be synergistically integrated to build sophisticated genetic cassettes for enhanced safety of AAV-mediated gene editing.},
}
@article {pmid42603302,
year = {2026},
author = {He, R and Zhang, C and Zhang, J and Zhang, K and Yin, W and Qiao, B and Zhang, L and Zhang, S and Qiao, J and Liu, Y},
title = {A One-Pot Reverse Transcriptase-Mediated, Pre-amplification-Free CRISPR/Cas12a Assay for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01202},
pmid = {42603302},
issn = {2379-3694},
support = {2025AFB825//Natural Science Foundation of Hubei Province/ ; WHPU-EIRF-2606//Wuhan Polytechnic University/ ; 2023DJC136//Department of Science and Technology of Hubei Province/ ; },
abstract = {CRISPR/Cas12a holds great promise for biosensing and diagnostics, but conventional methods suffer from low catalytic efficiency, high background, and reliance on pre-amplification. Direct detection of structured RNAs also remains challenging. Herein, we report the development of a reverse transcriptase and LNA probe (LNA-p)-mediated CRISPR/Cas12a positive feedback system (RTLC) for highly efficient, one-pot detection of both DNA and RNA. Without pre-amplification or thermal cycling, the assay achieves a 0.5 aM detection sensitivity within 27 min, exhibits single-base resolution, and allows direct detection of RNAs up to 985 nt in length. Together, RTLC is successfully validated in practical samples by detecting lncRNA HULC and miR-21, offering a robust, versatile tool for high-performance nucleic acid diagnostics.},
}
@article {pmid42604377,
year = {2026},
author = {Su, L and Mara, P and Edgcomb, V and Teske, A},
title = {From CRISPR-Cas to Argonautes: Defense systems of bacteria and archaea in the hydrothermal deep subsurface.},
journal = {ISME communications},
volume = {6},
number = {1},
pages = {ycag200},
pmid = {42604377},
issn = {2730-6151},
abstract = {Microbial defense systems are central to virus-host interactions and thus to microbial survival but remain poorly studied in microbial communities of extreme environments. Here we examine the repertoire and distribution of microbial defense genes in hydrothermally influenced deep subsurface sediments and rocks of the Guaymas Basin (Gulf of California). Restriction-modification and abortive infection systems were broadly distributed across the examined sediment depths, and clustered, regularly interspaced short palindromic repeats-Cas systems were primarily detected within temperate surficial sediments. Prokaryotic Argonaute genes were found mostly in archaeal MAGs at elevated temperatures up to 81.8°C. Overall defense gene repertoire declines downcore, as temperature increases and phylogenetic host range narrows, with phylogeny as the decisive control factor. We suggest that these defense systems, together with DNA repair mechanisms, protein maintenance activities, and RNA modification pathways, constitute a survival toolkit for the hydrothermally influenced subsurface, where energy limitation and temperature extremes select for resilient microbial communities.},
}
@article {pmid42605670,
year = {2026},
author = {Le, S and Thach, T},
title = {Structure- and deep learning-guided engineering of a size-minimized CRISPR/Cas.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70707},
pmid = {42605670},
issn = {1742-4658},
abstract = {Multidomain proteins play central roles in cellular regulation, yet their intrinsic flexibility and structural instability often hinder optimization for biotechnological applications. Here, we present an integrated structure-guided and deep learning-assisted engineering strategy that combines structure modeling with Protein Message Passing Neural Network (ProteinMPNN)-based sequence design to generate an ultracompact CRISPR activator (uCRISPRa) derived from the miniature CRISPR/Cas12f. Structural and computational analyses identified flexible, nonessential regions within both Cas12f and its single-guide RNA (sgRNA), enabling rational truncation and sequence redesign while preserving DNA-targeting capability. When delivered as mRNA encapsulated in lipid nanoparticles, uCRISPRa achieved selective activation of olfr544 among more than a thousand homologous olfactory receptor genes in skeletal muscle cells, leading to enhanced mitochondrial biogenesis. These findings demonstrate that the integration of structure-based protein engineering with deep learning sequence optimization provides a powerful framework for developing compact and efficient CRISPR effectors, offering broad potential for precise gene regulation and functional studies of complex macromolecular systems.},
}
@article {pmid42608492,
year = {2026},
author = {Majewska, Z and Jursza, G and Dolzblasz, A},
title = {Dexamethasone-inducible LhGR/pOp system: simple and flexible spatiotemporal control of gene expression.},
journal = {Planta},
volume = {264},
number = {4},
pages = {},
pmid = {42608492},
issn = {1432-2048},
support = {BPIDUB.4610.174.2022//Centre of Excellence and Applied Sport Science Research, Queensland Academy of Sport/ ; 501/73/10110/MPK 2599150000/2026//funding provided by the Department of Plant Developmental Biology/ ; },
mesh = {*Dexamethasone/pharmacology ; *Arabidopsis/genetics/drug effects ; *Gene Expression Regulation, Plant/drug effects ; Plants, Genetically Modified/genetics ; *Arabidopsis Proteins/genetics/metabolism ; },
abstract = {Our review illustrates how the dexamethasone-inducible LhGR/pOp system has been used across numerous tissues/organs and plant species, and summarizes the resources and inducer application procedures established to date. The establishment of groundbreaking molecular biology tools has enabled the rapid advancement of research based on the model plant Arabidopsis, which currently strongly benefits non-model but economically important species. Spatiotemporal control of transgene expression via the chemically inducible system GR-LhG4/pOp has proven to be a particularly powerful, universal and non-invasive experimental approach. This review characterizes the mechanism of action of GR-LhG4/pOp and synthesizes information on available GR-LhG4 transgenic lines and inductor (dexamethasone) application procedures across various plant tissues. Moreover, feasible experimental approaches are depicted that range from classical ones aiming at selected genes overexpression or silencing to highly innovative ones, like those integrating the GR-LhG4/pOp system with CRISPR-Cas for targeted ablation of specific cell types. Information about the existing GR-LhG4/pOp lines from non-Arabidopsis plants is also provided, in hope that this sophisticated but universal research tool will become more exploited in crop research.},
}
@article {pmid42609038,
year = {2026},
author = {Candelotti, AM and Garzillo, G and Bartolini, S and Di Censo, C and Peruzzi, G and Stabile, H and Fionda, C and Sciumè, G and Pietropaolo, G},
title = {Rapid and Highly Efficient CRISPR-Cas9 RNP Genome Editing in Primary ILC2s.},
journal = {European journal of immunology},
volume = {56},
number = {8},
pages = {e70254},
pmid = {42609038},
issn = {1521-4141},
support = {IG-28719//the Italian Association for Cancer Research (AIRC)-Investigator Grant "Mirella Rizzo"/ ; PNRR-MAD-2022-12375947//the Italian Association for Cancer Research (AIRC)-Investigator Grant "Mirella Rizzo"/ ; //Next Generation EU-PNRR M6C2 - Investimento 2.1 Valorizzazione e potenziamento della ricerca biomedica del SSN/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; Mice ; Humans ; },
abstract = {CRISPR-mediated gene editing enables efficient genetic manipulation of ILC2s through ex vivo or in vivo activation and Cas9 RNP delivery. This platform provides a robust approach to dissect gene function in ILC2s, with minimal manipulation.},
}
@article {pmid40945831,
year = {2025},
author = {Duan, X and Zhou, Z and Zeng, X and Chen, J and Mao, A},
title = {Single-step purification of functional Cas9 protein via the ubiquitin expression system.},
journal = {International journal of biological macromolecules},
volume = {328},
number = {Pt 1},
pages = {147590},
doi = {10.1016/j.ijbiomac.2025.147590},
pmid = {40945831},
issn = {1879-0003},
mesh = {*Ubiquitin/genetics/metabolism/chemistry ; Escherichia coli/genetics/metabolism ; *CRISPR-Associated Protein 9/isolation &amp; purification/genetics/metabolism/chemistry ; Streptococcus pyogenes/enzymology/genetics ; CRISPR-Cas Systems ; Chromatography, Affinity ; Recombinant Fusion Proteins/isolation &amp; purification/genetics ; *Gene Expression ; },
abstract = {The CRISPR/Cas9 system serves as a powerful platform for precise genome editing, with CRISPR/Cas9 ribonucleoprotein (RNP) complexes exhibiting superior editing efficiency compared to alternative delivery modalities. However, current methods for Cas9 production typically involve multiple purification steps. Here, we developed a streamlined single-step purification strategy for preparation of functional Streptococcus pyogenes Cas9 (SpCas9) with dispensable tag removal after inducible expression in Escherichia coli. Notably, N-terminal Ubiquitin (Ub) fusion preserved both accurate nuclear localization and remarkable protein stability, enabling robust production of over 8 mg/L of >95 % pure Ub-Cas9 via single metal affinity chromatography. Comprehensive endonuclease activity assays confirmed that the purified Ub-Cas9 maintained robust DNA cleavage capacity both in vitro and in vivo. Our work provides a convenient and efficient platform for production of highly purified Ub-Cas9 protein for widespread applications in gene function study.},
}
@article {pmid42323083,
year = {2026},
author = {Byrnes, C and Clarke, BA and Zhu, H and Lee, YT and Majumder, S and Kono, M and Proia, RL},
title = {1-Deoxysphingolipids require very-long-chain ceramide synthesis to induce ER stress and cytotoxicity.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {8},
pages = {113281},
pmid = {42323083},
issn = {1083-351X},
mesh = {Humans ; *Ceramides/biosynthesis/chemistry ; *Endoplasmic Reticulum Stress/drug effects ; *Sphingolipids/metabolism/chemistry ; Cell Line, Tumor ; Cell Survival/drug effects ; CRISPR-Cas Systems ; },
abstract = {Sphingolipids play key roles in cellular systems both as membrane components and as signaling molecules. Their biosynthesis, which occurs in the endoplasmic reticulum (ER), begins with the condensation of an amino acid, typically serine, and a fatty acyl-CoA. Under certain pathological conditions, alanine can be substituted for serine in the condensation reaction, producing 1-deoxysphingolipids, which lack the 1-hydroxyl group on the sphingoid base. Unlike typical sphingolipids, 1-deoxysphingolipids are unable to accept a head group modification, which alters their metabolic processing and prevents their canonical degradation. The accumulation of these "headless" 1-deoxysphingolipids causes neurotoxicity in various neurological and metabolic disorders. Here, we conducted a genome-wide CRISPR-Cas9 screen to identify pathways leading to 1-deoxysphinganine-induced toxicity in SH-SY5Y cells, a model used to study neurotoxic responses. Our top genetic hits highlighted the pathway involved in synthesizing ceramides with very-long-chain fatty acids (C22-C26). Using CRISPR-Cas9-modified SH-SY5Y cells with loss-of-function (LOF) mutations in the TECR or CERS2 genes-both critical for producing very-long-chain ceramides-we validated that this pathway was essential for 1-deoxysphinganine-mediated toxicity. Furthermore, we demonstrated that the ceramide synthesis pathway is required for 1-deoxysphinganine to trigger ER stress, as evidenced by significantly increased expression of the unfolded protein response in WT, but not TECR or CERS2 LOF mutant, SH-SY5Y cells exposed to 1-deoxysphinganine. Collectively, the data support a model in which ceramide synthase-dependent conversion of 1-deoxysphinganine to very-long-chain 1-deoxyceramide species is required for full ER-stress induction and cytotoxicity. The findings highlight potential therapeutic targets for neuropathological diseases caused by 1-deoxysphingolipid accumulation.},
}
@article {pmid42459132,
year = {2026},
author = {Wen, Z and Wei, C and Shen, M and Yu, S},
title = {Signal logic gate mediated by a controllable CRISPR/Cas12a system for simultaneous detection of DNA mutation and methylation.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {32},
pages = {6823-6830},
doi = {10.1039/d6ay01060a},
pmid = {42459132},
issn = {1759-9679},
mesh = {*DNA Methylation/genetics ; Humans ; Septins/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Promoter Regions, Genetic ; Colorectal Neoplasms/genetics/diagnosis ; DNA Mutational Analysis/methods ; },
abstract = {Early detection of both genetic mutations and epigenetic modifications is critical for cancer diagnosis, but current methods often require separate assays or suffer from bisulfite-induced DNA damage. Here, we present a controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction. The strategy converts target information into two distinct ssDNA activators (T1 for methylation and T2 for mutation) via orthogonal enzymatic cascades (GlaI- and FEN1-mediated cleavage followed by strand displacement amplification). By limiting crRNA concentrations, the CRISPR/Cas12a trans-cleavage activity produces three well-resolved fluorescence kinetic states: low (mutation only), medium (methylation only), and high (both targets), respectively, which could be distinguished using three predefined threshold values. Validation with clinical samples from colorectal cancer patients and healthy controls showed complete concordance with Sanger sequencing and qPCR. And the proposed method successfully detected Septin9 methylation in peripheral blood. This isothermal, single-tube, bisulfite-free strategy offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.},
}
@article {pmid42600477,
year = {2026},
author = {Yuan, J and Shen, M and Ding, L and Yang, X and Wu, Y and Yu, S},
title = {Orthogonal Cas13a/Cas12a cascade for one-pot amplification-free detection of miRNA-21.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130431},
doi = {10.1016/j.talanta.2026.130431},
pmid = {42600477},
issn = {1873-3573},
abstract = {MicroRNAs (miRNAs) are promising biomarkers for clinical diagnosis and disease monitoring. However, current CRISPR/Cas-based miRNA sensors generally require reverse transcription or nucleic acid amplification to improve sensitivity, which complicates the workflow and increases the risk of contamination, nonspecific amplification, and false-positive results. Herein, we developed a one pot amplification-free Cas13a/Cas12a cascade platform based on a designed dual-functional molecular bridge probe, Conv HP-3, which served as both a substrate for Cas13a-mediated trans-cleavage and an activator for Cas12a-mediated trans-cleavage, thereby linking target recognition to cascade signal amplification for miRNA-21 detection. Following the introduction of miRNA-21, Cas13a was specifically activated through target-crRNA recognition and cleaved the Conv HP-3 probe to release a Cas12a-activating DNA fragment. This fragment subsequently triggered Cas12a-mediated cleavage of the ssDNA reporter, generating a markedly enhanced fluorescence signal. This one-pot Cas13a/Cas12a cascade fluorescence biosensor enabled quantitative detection of miRNA-21 over a concentration range of 1-1000 pmol/L within 60 min, with a low detection limit of 0.66 pM. Notably, the assay achieved average recoveries ranging from 95.97% to 108.59%, with a variation between 0.6% and 1.78%, demonstrating its good accuracy and precision. This biosensing platform shows great promise for the rapid and sensitive detection of miRNAs in clinical applications.},
}
@article {pmid42600889,
year = {2026},
author = {Shiroshita, K and Stolz, A and Malouf, C and Tran, VL and Li, J and Weiss, MJ and Naldini, L},
title = {Unlocking the Curative Potential of Gene Transfer and Editing for Hematopoietic Disorders.},
journal = {Experimental hematology},
volume = {},
number = {},
pages = {105493},
doi = {10.1016/j.exphem.2026.105493},
pmid = {42600889},
issn = {1873-2399},
abstract = {Hematopoietic stem and progenitor cells (HSPCs) gene therapy may transform the therapeutic landscape for inherited hematological disorders and already offers potentially curative options for some diseases, including inborn errors of immunity and β-hemoglobinopathies. Its continued success relies on further refinement of gene transfer technologies, gene editing tools such as CRISPR-Cas, and optimized ex vivo HSPC manipulation protocols that ensure robust, long-term engraftment and clonal diversity with reduced-toxicity, non-genotoxic conditioning strategies. Here, we review recent developments and refinements in gene transfer and editing technologies for HSPCs, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar. Future directions must prioritize integrating technological innovation with the development of equitable and simplified models to reduce costs and ensure that these life-saving cellular therapies reach patients worldwide. Teaser abstract Hematopoietic stem and progenitor cells (HSPCs) gene therapy is advancing rapidly, with lentiviral gene transfer, genome editing, and emerging in vivo delivery approaches expanding the therapeutic landscape for inherited hematologic disorders. Recent clinical successes have demonstrated the potential for durable correction, while ongoing refinements continue to improve safety, efficacy, and feasibility. Key challenges remain in genotoxicity, conditioning toxicity, manufacturing scalability, and equitable global access.},
}
@article {pmid42601370,
year = {2026},
author = {Iqbal, G and Pinto, N and Pawaskar, D and Sonwane, AA and Rasal, KD and Singh, LS and Chikkathimmashetty, NN and Goswami, M},
title = {Design and transfection of CRISPR/Cas9 constructs for the myostatin gene in Labeo rohita muscle cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42601370},
issn = {2045-2322},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Transfection/methods ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Muscle Cells/metabolism ; *Cyprinidae/genetics ; },
abstract = {Myostatin (mstn) is a negative regulator of skeletal muscle growth and is considered as an important target for enhancing aquaculture production. The present study aimed to design and validate single-guide RNAs (sgRNAs) and CRISPR/Cas9 constructs for exon 1 of the mstnb gene in Labeo rohita, and to evaluate their transfection efficiency in the L. rohita dorsal muscle (LRDM) cell line at the 10th, 20th, and 30th passages. sgRNAs were designed and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector using BbsI restriction digestion and ligation. Successful insertion and correct orientation of the sgRNAs were confirmed through Sanger sequencing. LRDM cells were revived and maintained in L-15 medium supplemented with 10% fetal bovine serum. Transfection was performed at the 10th, 20th, and 30th passages. Distinct GFP-positive cells were observed at all passages for both sgRNA constructs, indicating the ability of the developed cell line to successfully express the constructs across different passages. The study successfully established CRISPR/Cas9 plasmid constructs for the mstnb gene in L. rohita and demonstrated their transfection in LRDM cell line across multiple passages. These findings provide a basis for future studies on genome editing approaches using CRISPR/Cas9 constructs in fish muscle cell lines and highlight the potential application of CRISPR/Cas9 technology for genetic engineering applications in fish muscle cells.},
}
@article {pmid42602771,
year = {2026},
author = {Hina, A and Abbasi, A and Chaudhry, A and Sanaullah, T and Arshad, M and Sarwar, HM and Sarfaraz, S and Karikari, B and Kavhiza, NJ},
title = {A review of flavonoids at the crossroads of plant defense: integrating biotic and abiotic stress tolerance through AI- and CRISPR/Cas-guided metabolic reprogramming.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1865665},
pmid = {42602771},
issn = {1664-462X},
abstract = {Flavonoids are multifunctional phenylpropanoid-derived metabolites that occupy a central position in plant adaptation to environmental stress. Beyond their established roles in antioxidant protection, they contribute to defense against pathogens and herbivores, signaling processes, and physiological acclimation to adverse environmental conditions. Although flavonoid responses to individual biotic or abiotic stresses have been extensively investigated, considerably less attention has been given to how flavonoid-associated regulatory networks function when multiple stresses occur simultaneously. This gap is particularly important because crops in agricultural systems are routinely exposed to overlapping biotic and abiotic challenges that generate distinct physiological, transcriptional, and metabolic responses. This review synthesizes current knowledge of flavonoid biosynthesis, structure-activity relationships, and the regulatory mechanisms governing flavonoid accumulation under diverse stress conditions. Particular emphasis is placed on the reorganization of flavonoid-associated networks under combined stress, including signaling crosstalk, pathway competition, metabolic trade-offs, and flux allocation that collectively shape adaptive responses. This review further evaluates how artificial intelligence can support identification of regulatory targets and pathway bottlenecks, how integration with CRISPR/Cas technologies may facilitate more precise manipulation of flavonoid biosynthesis, and how iterative Design-Build-Test-Learn (DBTL) frameworks could improve predictive flavonoid engineering through continuous integration of computational prediction and experimental validation. By integrating advances in stress biology, computational prediction, genome engineering, and iterative DBTL frameworks, this review outlines a roadmap for predictive reprogramming of flavonoid networks under combined stress and the development of crops with improved resilience to increasingly complex environmental conditions.},
}
@article {pmid42286694,
year = {2026},
author = {Xu, J and Xu, J and Cao, C and Shu, Y and Shen, L and He, X and Huangfu, Q and Sun, C and Wang, W and Wei, J and Cai, M and Wang, B and Li, A and Liu, Y and Wen, J},
title = {IDMME and IDMDE: inducible CRISPR-dCasRx platforms for spatiotemporal RNA m[5]C editing.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42286694},
issn = {1474-760X},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; 82371630//National Natural Science Foundation of China/ ; },
mesh = {*5-Methylcytosine/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA Methylation ; *RNA Editing ; Animals ; Epigenesis, Genetic ; *Gene Editing/methods ; Epigenome Editing ; Carcinoma, Renal Cell/genetics ; Cell Line, Tumor ; },
abstract = {RNA 5-methylcytosine (m[5]C) is a dynamic epigenetic mark implicated in tumorigenesis, however, existing editors lack spatiotemporal regulation and reversibility. Here, we develop abscisic acid (ABA)-inducible CRISPR-dCasRx systems for programmable m[5]C methylation (IDMME) and demethylation (IDMDE). These editors enable site-specific, low off-target m[5]C modification through ligand-dependent assembly of split effector domains. We further integrate photocaged ABA to achieve light-controlled activation. Application in renal carcinoma models shows that targeted m[5]C editing modulates transcript function and suppresses tumor growth in vitro and in vivo. This platform provides a versatile, spatiotemporally controllable approach for dissecting RNA epigenetic mechanisms and advancing RNA-based therapeutic strategies.},
}
@article {pmid42361799,
year = {2026},
author = {He, W and Huang, JW and Wang, Y and Hayward, SB and Leuzzi, G and Fu, R and Wang, S and Vaitsiankova, A and Chen, Y and Bedford, MT and Guerois, R and Ciccia, A and Xu, H},
title = {Deciphering protein mutation-phenotype linkages from CRISPR-based tiling mutagenesis screens.},
journal = {Cell systems},
volume = {17},
number = {8},
pages = {101651},
pmid = {42361799},
issn = {2405-4720},
support = {R01 CA197774/CA/NCI NIH HHS/United States ; R35 GM137927/GM/NIGMS NIH HHS/United States ; R35 GM153387/GM/NIGMS NIH HHS/United States ; R35 GM156417/GM/NIGMS NIH HHS/United States ; },
mesh = {*Mutagenesis/genetics ; Phenotype ; Mutation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-based high-throughput mutagenesis screens enable systematic mapping of mutations to phenotypes, yet deciphering mutation-phenotype links remains challenging. Here, we present ProTiler-Mut, a versatile computational framework that leverages tiling mutagenesis screens, which introduce variants across entire protein sequences, to analyze mutation effects at the levels of residues, substructures, and protein-protein interactions (PPIs). Applying ProTiler-Mut to multi-condition base-editing (BE) screens targeting DNA damage response proteins and T cell regulators, we define a separation-of-function (SoF) category beyond the conventional loss-of-function (LoF) and gain-of-function (GoF) classes, where SoF mutations show the strongest enrichment for ClinVar-annotated pathogenic variants. ProTiler-Mut also identifies candidate substructures that enable functional inference of unscreened pathogenic mutations and prioritizes candidate phenotype-associated PPIs potentially disrupted by functional variants. Using ProTiler-Mut, in cells with elevated programmed cell death 1 (PD-1) expression, we identify pathogenic GoF mutations that constitute a substructure that may disrupt mitogen-activated protein kinase (MAPK)1-RSK1 interactions and lead to MAPK activation. Finally, we show that ProTiler-Mut is applicable across different mutagenesis screening platforms. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid42372725,
year = {2026},
author = {Hu, Y and Li, Q and Li, Y and Zeng, Y and Zheng, L and Shen, J and Gao, X and Zhao, GP and Zhao, W and Dai, L},
title = {Targeted genomic editing of human gut Bacteroides species based on CRISPR-associated transposases.},
journal = {Cell systems},
volume = {17},
number = {8},
pages = {101650},
doi = {10.1016/j.cels.2026.101650},
pmid = {42372725},
issn = {2405-4720},
mesh = {Humans ; *Bacteroides/genetics ; *Gene Editing/methods ; *Transposases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Gut Bacteroides are abundant and critical to human health, yet most are genetically cumbersome, non-model microbes. A widely applicable editing tool for Bacteroides is essential for gut microbiome manipulation. Here, we develop STIB (ShCAST-based transient insertion system for Bacteroides), an efficient genome-editing tool derived from CRISPR-associated transposases that enables rapid and site-specific insertions independent of homologous recombination. By fusing a nicking homing endonuclease to the transposase and an ATPase to Cas12k, we systematically optimize STIB to minimize plasmid cointegration and achieve >97% on-target insertion. STIB exhibits broad applicability across different genomic loci in diverse Bacteroides species, including non-model species. Finally, we apply STIB to achieve species- and site-specific editing of distinct Bacteroides species within a complex synthetic gut microbiota. Overall, STIB expands the toolbox for the functional investigation and engineering of the human microbiome. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid42595755,
year = {2026},
author = {Saxena, S and Kabra, M and Abdeen, AA and Tabima, DM and Sinha, D and Rawding, PA and Zhu, M and Xie, R and Kulkarni, T and Hanstad, GM and Fernandez Zepeda, MA and Gamm, DM and Pattnaik, BR and Gong, S and Saha, K},
title = {Genome-wide CRISPR screening identifies cellular factors controlling nonviral genome editing efficiency.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42595755},
issn = {2041-1723},
support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; U19 NS132296/NS/NINDS NIH HHS/United States ; U19NS132296//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R35GM119644//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Connexin 26 ; Connexins/genetics/metabolism ; Potassium Channels, Inwardly Rectifying/genetics/metabolism ; Receptors, GABA-A/genetics/metabolism ; Mutation ; HEK293 Cells ; Genome, Human ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies.},
}
@article {pmid42597245,
year = {2026},
author = {Ye, R and Liu, M and Zhang, X and Liang, Q and Cao, Y and Zhu, C and Yu, H and Zhang, S and Wang, Y},
title = {Rapid and ultrasensitive detection of Candida tropicalis using multiple cross-displacement amplification combined with CRISPR/Cas12a.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1796886},
pmid = {42597245},
issn = {2235-2988},
mesh = {*Candida tropicalis/genetics/isolation &amp; purification ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Candidiasis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; DNA, Fungal/genetics ; Multiplex Polymerase Chain Reaction ; Rapid Diagnostic Tests ; Endodeoxyribonucleases/genetics ; CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; DNA, Ribosomal Spacer/genetics ; Bacterial Proteins ; },
abstract = {BACKGROUND: Candida tropicalis (C. tropicalis), classified as a World Health Organization "critical priority" pathogen, causes life-threatening invasive infections with high mortality due to diagnostic delays. Therefore, the development of rapid fungal detection platforms is an urgent scientific and clinical priority.<br><br>METHODS: We designed multiple cross-displacement amplification (MCDA) primers to target the internal transcribed spacer 2 (ITS II) gene of C. tropicalis for specific amplification. Subsequently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-crRNA complex bound to the amplified products, activating its trans-cleavage activity to generate a detectable fluorescent signal. Finally, clinical samples were used to validate the detection results, which were further compared with those obtained using fungal culture and multiplex polymerase chain reaction (Multiplex PCR).<br><br>RESULTS: Under optimized conditions, the C. tropicalis-MCDA-CRISPR/Cas12a assay was completed within approximately 53&#xa0;min, with a limit of detection of 30 fg of genomic DNA per reaction. The assay showed no cross-reactivity with non-C. tropicalis pathogens. Clinical validation using 128 specimens demonstrated a sensitivity of 100.0% (95% CI: 93.4-100.0%) and a specificity of 97.3% (95% CI: 90.6-99.7%) against a composite reference standard, with near-perfect agreement (&#x3ba;= 0.968).<br><br>DISCUSSION: The C. tropicalis-MCDA-CRISPR/Cas12a assay is an efficient, accurate, and practical diagnostic tool suitable for use in resource-limited laboratories.},
}
@article {pmid42597591,
year = {2026},
author = {Hermain, S and K S, H and Nimbagal, RN},
title = {Beyond chemotherapy: The rise of nucleic acid nanoformulations in personalized lung cancer therapy.},
journal = {Cancer pathogenesis and therapy},
volume = {4},
number = {5},
pages = {349-361},
pmid = {42597591},
issn = {2949-7132},
abstract = {Lung cancer remains the leading cause of cancer-related mortality worldwide, driven by complex crosstalk among genetic, molecular, and environmental factors. Conventional treatments, including immunotherapies and targeted inhibitors, face three main challenges: tumor heterogeneity, drug resistance, and systemic toxicity. Nucleic acid therapeutics (NATs) encompass a diverse array of DNA- and RNA-based tools, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), antisense oligonucleotides (ASOs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems. These tools are central to developing precision oncology approaches that operate through direct gene regulation, mutation correction, and immune system reprogramming. The clinical application of NATs currently faces three main obstacles, which include their vulnerability to enzymatic degradation, their limited ability to penetrate tissues, and their tendency to cause off-target effects. The field has progressed through the implementation of nanoformulation techniques, which utilize lipid-based polymeric and metallic carriers together with exosomes and DNA origami, and hybrid nanostructures as new platforms to enhance the stability of drugs and their cellular absorption and targeted delivery to tumors. The scientists developed functionalized nanocarriers by combining targeting ligands with materials that could respond to specific environmental changes, which allowed them to manage drug distribution and release patterns throughout the tumor microenvironment. This review focuses on establishing a direct connection between nucleic acid design and nanotechnology through an analysis of mechanistic details and progress in preclinical and clinical research, and the difficulties encountered during the progress to practical applications. The research demonstrates how artificial intelligence and bioinspired nanocarriers and multi-omics data integration create new opportunities for developing personalized adaptive nanogenetic treatment methods, which will treat lung cancer. The current advancements indicate that we are approaching a transformative era in which nanomedicine and nucleic acid therapeutics will enable safe genetic alterations of cancer through targeted therapeutic applications.},
}
@article {pmid42598860,
year = {2026},
author = {Chilamkurthy, R and Sudhakar, S and Pater, AA and Bosmeny, MS and Stabile, F and Katolik, A and Harikrishna, S and El Azzouzi, F and Turk, R and Damha, MJ and Leslie, S and Korolev, S and Pradeepkumar, PI and Gagnon, KT},
title = {CRISPR RNA architecture steers Cas9 catalysis and fidelity via a guide repeat clasp motif.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42598860},
issn = {1362-4962},
support = {R01 GM135646/GM/NIGMS NIH HHS/United States ; R01 GM154323/GM/NIGMS NIH HHS/United States ; 1R01GM135646/NH/NIH HHS/United States ; R01GM154323/NH/NIH HHS/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Biocatalysis ; Base Sequence ; },
abstract = {A widely adopted modification of CRISPR-Cas9 is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). Here we find that these guide architectures induce differential catalysis, gene editing, and specificity. Spacer sequence and RNA structural features could not predict guide architecture editing preference across 255 endogenous targets. We used cryo-EM and molecular dynamics to identify a new Cas9 structural motif, the guide repeat clasp (GRC), that checks guide RNA repeat structure and coordinates with R-loop sensing checkpoint mechanisms to help license cleavage. Limited mutagenesis of GRC residues significantly altered Cas9 editing and specificity, supporting a key role in catalysis. To further understand the role of the GRC and guide RNA repeat dynamics, we created guide repeat-truncated sgRNAs, or grtRNAs, which conferred some dgRNA properties onto sgRNA, including generally lower off-target editing for targets with PAM-proximal mismatches. dgRNAs and grtRNAs could be combined with a new high-fidelity Cas9 variant called ZiFY, rationally designed to reduce editing of targets with PAM-distal mismatches, to generate broader mismatched target discrimination. These results uncover a previously unknown mechanism that steers Cas9 catalysis and demonstrate the potential to improve Cas9 fidelity by modulating guide repeat interactions.},
}
@article {pmid42599326,
year = {2026},
author = {Musini, A and Yata, VK and Bukke, SPN and Akhila, K and Shivani, T and Narapureddy, BR and Thalluri, C and Mohamed, AOA and Adam, MDM},
title = {Phage and CRISPR based precision antimicrobials: a dual strategy against multidrug-resistant bacteria.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42599326},
issn = {1573-4978},
support = {RGP2/639/46//Deanship of Research and Graduate Studies at King Khalid University (Large Research Project)/ ; },
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacteria/genetics/drug effects/virology ; Humans ; *Phage Therapy/methods ; Anti-Bacterial Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Anti-Infective Agents/pharmacology ; },
abstract = {The rapid global emergence of multidrug-resistant (MDR) bacterial pathogens has significantly reduced the effectiveness of conventional antibiotics, creating an urgent need for alternative antimicrobial strategies. Among emerging precision therapeutics bacteriophage therapy and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems have shown to have strong potential through highly specific bacterial targeting mechanisms. Bacteriophages have the ability to replicate themselves and penetrate biofilms, and the ability of CRISPR-Cas systems to edit the genes responsible for antimicrobial resistance, virulence factors, and the mobile genetic elements that underlie bacterial resistance. The recent advancement enabled the integration of these technologies through CRISPR-armed bacteriophages, which utilize bacteriophages as delivery mechanisms for CRISPR and address the large populations of MDR bacteria. Compared to administering CRISPR and bacteriophage independently, the current data suggest that the use of these two methods synergistically will lead to greater efficacy of delivery, specific targeting of resistance determinants, decreased risk of resistance development, and minimal impact on the body's beneficial microorganisms. While the potential combination of these approaches holds great promise to help combat the issue of MDR bacteria, there are still numerous barriers to overcome in order to implement these methods which include narrow phage host range, bacterial escape mechanisms, off-target CRISPR activity, anti-CRISPR proteins, host immune responses, and unresolved manufacturing and regulatory limitations. This review critically examines bacteriophage-based antimicrobials, CRISPR-Cas therapeutic systems, and their emerging integration as CRISPR-armed phages, highlighting their comparative advantages, current limitations, and future potential as promising targeted antimicrobial approach platforms requiring further clinical validation.},
}
@article {pmid42599328,
year = {2026},
author = {Mengistu, DA and Mekasha, YT and Molla, FW},
title = {Non-Drug therapeutic strategies to combat antimicrobial resistance in livestock: a review.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42599328},
issn = {1573-4978},
mesh = {Animals ; *Livestock/microbiology/genetics ; Humans ; Probiotics/therapeutic use ; Phage Therapy ; CRISPR-Cas Systems ; *Drug Resistance, Microbial ; Anti-Infective Agents ; Fecal Microbiota Transplantation ; Anti-Bacterial Agents ; Genetic Engineering/methods ; },
abstract = {BACKGROUND: Antimicrobial resistance (AMR) represents an escalating global public health crisis, projected to cause 10 million deaths annually by 2050. Pathogens such as bacteria, viruses, fungi, and parasites evade treatments in animals and humans due to overuse and misuse of antimicrobials, particularly antibiotics, in medical and veterinary practices. This drives a worldwide surge in resistant infections, disproportionately burdening livestock health and productivity.<br><br>OBJECTIVES: This review examines the global rise of AMR and its impacts on livestock and public health, underscoring the need for non-drug therapeutic alternatives.<br><br>CONCLUSION: Promising alternatives include genetic engineering and CRISPR-Cas systems, phage therapy, probiotics, antimicrobial peptides, fecal microbiota transplantation, phytotherapy and essential oils, immunomodulators, nanotechnology, biofilm disruptors, acidifiers, vaccination strategies, and precision livestock farming. These approaches target resistance mechanisms without relying on conventional drugs. Despite challenges like knowledge gaps, regulatory barriers, financial limitations, and policy gaps, stakeholders must prioritize accelerated research, regulatory reforms, investments, and international collaboration. Rapid integration of these technologies into human and veterinary medicine is vital to mitigate AMR's health, economic, and social impacts.},
}
@article {pmid42599397,
year = {2026},
author = {Sun, J and Ni, Y and Guo, M and Yu, T},
title = {A Multiplex CRISPR-Cas12a-Based Hydrogel Microarray Platform for the Simultaneous Detection of Common Adenovirus Types in Clinical Samples.},
journal = {ACS infectious diseases},
volume = {12},
number = {8},
pages = {2815-2824},
doi = {10.1021/acsinfecdis.6c00407},
pmid = {42599397},
issn = {2373-8227},
mesh = {Humans ; *CRISPR-Cas Systems ; Hydrogels/chemistry ; Sensitivity and Specificity ; *Adenoviridae/genetics/isolation &amp; purification/classification ; *Microarray Analysis/methods ; CRISPR-Associated Proteins/genetics ; *Adenoviridae Infections/diagnosis/virology ; Rapid Diagnostic Tests ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Adenovirus infections are a leading cause of respiratory and gastrointestinal diseases, representing a significant global health challenge. Rapid and accurate detection of adenovirus types is essential for timely diagnosis and effective management. Traditional diagnostic methods, such as PCR, are often time-consuming and require complex laboratory infrastructure, limiting their application in resource-limited settings. In this study, we present a CRISPR/Cas12a-based assay integrated with a hydrogel microarray for the simultaneous detection of six common adenovirus types (1, 2, 3, 4, 7, and 14). After amplification of adenoviral DNA using recombinase polymerase amplification (RPA), the amplified DNA enters the hydrogel, where it activates the Cas12a-crRNA complex trapped within the gel. This activation leads to the cleavage of an ssDNA reporter, generating a fluorescent signal. The use of a hydrogel microarray enables efficient and multiplexed detection of adenovirus types in a single assay. The method demonstrated high sensitivity, with detection limits ranging from 10 to 50 copies/μL across the six adenovirus types. It also showed excellent specificity, with no cross-reactivity observed with other respiratory viruses. Clinical validation with 30 human adenovirus samples revealed 100% specificity and high concordance with qPCR results. This CRISPR/Cas12a-based hydrogel microarray platform offers a rapid, cost-effective, and highly specific diagnostic tool with significant potential for clinical and public health applications.},
}
@article {pmid42599816,
year = {2026},
author = {Sun, T and Yuan, A and Xie, W and Jiang, G and Peng, H},
title = {CRISPR RNA Engineering Enables Single Nucleotide Polymorphism Discrimination in Nucleic Acid Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e00004},
doi = {10.1002/anie.202600004},
pmid = {42599816},
issn = {1521-3773},
support = {22276199//National Natural Science Foundation of China/ ; 2023YFA0915102//National Key Research and Development Program of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 22306195//Youth Fund from National Natural Science Foundation of China/ ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic systems have emerged as powerful platforms for sensitive nucleic acid detection, yet their clinical reliability is frequently constrained by off-target activation and insufficient discrimination of closely related sequences. This review synthesizes current advances aimed at enhancing the specificity of CRISPR diagnostics, with particular emphasis on the pivotal role of CRISPR RNA (crRNA) engineering. We detail how structural determinants of crRNA, including spacer length optimization, intentional mismatch design, secondary-structure modulation, chemical modification, strand-displacement gating, and synergistic design frameworks, govern CRISPR-mediated target recognition and define the energetic and kinetic thresholds for accurate cleavage. Key engineering strategies encompassing computational prediction and modeling, high-throughput screening, and hybrid guide architectures are systematically examined for their capacity to elevate single-nucleotide discrimination, stabilize reaction performance, and enable robust multiplexed detection for pathogen profiling and mutation identification. Despite rapid progress, outstanding challenges persist, including interference from complex clinical matrices, lack of unified evaluation standards, and scalability barriers that hinder clinical translation. Addressing these limitations through integrated crRNA design, system-level optimization, and standardized benchmarking will be essential for realizing the promise of CRISPR diagnostics. Ultimately, these advances are poised to support ultrasensitive, highly specific, and portable point-of-care testing, thereby accelerating precision medicine and strengthening infectious disease surveillance and management.},
}
@article {pmid42600441,
year = {2026},
author = {Zhang, X and Wang, Z and Guo, Z and Wang, H and Chen, Y and Dong, Z and Zeng, L and Yu, D and Hong, M and Wang, J and Tian, G and Yang, W},
title = {A Novel CRISPR/Cas13a sensor for highly sensitive detection of DENV based on AuPt/g-C3N4@GO and Ag@NU-1000.},
journal = {Biosensors &amp; bioelectronics},
volume = {313},
number = {},
pages = {119122},
doi = {10.1016/j.bios.2026.119122},
pmid = {42600441},
issn = {1873-4235},
abstract = {Dengue virus, an arthropod-borne pathogen, poses a significant global public health concern, necessitating the development of sensitive and rapid diagnostic assays. This paper introduces a novel biosensing platform designed for the ultrasensitive detection of dengue viral RNA. The platform integrates recombinase polymerase amplification, CRISPR/Cas13a-based recognition, and electrochemiluminescence resonance energy transfer (ECL-RET). The sensor employs a highly efficient luminescent emitter, which is a ternary composite of gold-platinum nanoparticles supported on graphitic carbon nitride and graphene oxide. A suitable energy acceptor, consisting of silver-decorated metal-organic frameworks, effectively quenches the emitter's signal via resonance energy transfer. Upon the introduction of target nucleic acids, RPA rapidly generates numerous amplicons. These amplicons are specifically recognized by the CRISPR/Cas13a system, which subsequently activates the collateral cleavage activity of Cas13a. This enzymatic activity cleaves a DNA linker that tethers the acceptor to the electrode surface, leading to the release of the acceptor and a subsequent restoration of the electrochemiluminescence intensity. The recovered signal exhibits a linear proportionality to the target concentration across a broad dynamic range, spanning from 0.1 fg/mL to 100 ng/mL. The detection limits for the four dengue serotypes range from 1.09 to 2.5 fg/mL. The assay demonstrates excellent specificity, showing no cross-reactivity with Zika, Japanese encephalitis, or West Nile viruses, and also exhibits good reproducibility. By combining isothermal amplification, specific CRISPR targeting, and a sensitive luminescence readout, this work presents a straightforward and robust method for the early diagnosis of dengue and for outbreak surveillance. Furthermore, this platform can be readily adapted for the detection of other pathogens of interest.},
}
@article {pmid42109006,
year = {2026},
author = {Rajabi Zangi, A and Amiri, A and Eskandari, F and Pazooki, P and Heidari, HR and Hamishehkar, H and Javadzadeh, Y},
title = {RNA therapeutics and their delivery methods: a paradigm for haemophilia management.},
journal = {Journal of drug targeting},
volume = {34},
number = {8},
pages = {1394-1413},
doi = {10.1080/1061186X.2026.2638330},
pmid = {42109006},
issn = {1029-2330},
mesh = {Humans ; *Hemophilia A/therapy/genetics ; *RNA, Small Interfering/administration &amp; dosage ; Animals ; Nanoparticles ; RNA, Messenger/administration &amp; dosage ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Lipids/chemistry ; Drug Delivery Systems ; },
abstract = {Haemophilia management is currently undergoing a paradigm shift from traditional protein replacement to RNA modalities and their delivery. This review provides a critical analysis of RNA-based therapeutics (specifically small interfering RNA (siRNA), mRNA and CRISPR/Cas9) as a versatile paradigm distinct from DNA ones. We synthesise clinical and preclinical data to contrast these modalities: siRNA strategies (e.g. fitusiran) have indicated ∼90% reductions in bleeding rates by rebalancing haemostasis independent of factor deficiency; lipid nanoparticles (LNPs)-mRNA platforms offer tuneable, transient factor production without genomic integration risks; and CRISPR-based editing aims for permanent correction (up to 170% coagulation factor IX, FIX expression in preclinical studies) but necessitates rigorous monitoring for off-target effects. Crucially, this article dissects the non-viral delivery landscape determining the clinical viability of these cargos. We evaluate LNPs as the current clinical gold standard for hepatic delivery, contrasting them against emerging polymeric systems, aptamer conjugates and exosomes designed to overcome rate-limiting barriers such as endosomal entrapment and renal clearance. By juxtaposing the immunogenic limitations of viral vectors, we conclude that next-generation RNA therapeutics, enabled by LNPs and GalNAc-conjugation, offer a strategic pathway to overcome the durability gap observed in haemophilia A and expand treatment access to patients currently excluded by viral seroprevalence.},
}
@article {pmid42269411,
year = {2026},
author = {Zhang, Y and Ma, H and Li, W and Wang, Z and Song, Z and Qi, Y},
title = {A CRISPR/Cas12a-hyperbranched rolling circle amplification sensor for ultrasensitive visual bacteria detection.},
journal = {Talanta},
volume = {310},
number = {},
pages = {129967},
doi = {10.1016/j.talanta.2026.129967},
pmid = {42269411},
issn = {1873-3573},
mesh = {Milk/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Smartphone ; *Biosensing Techniques/methods ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Copper/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Food Microbiology ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The swift spread of pathogenic bacteria via food, air, and water poses severe risks to human health. Conventional detection methods often suffer from time-consuming operations, bulky instruments, and insufficient sensitivity for on-site screening, highlighting an urgent demand for sensitive and visual platforms. Herein, a detection platform (Cas12a-HRCA) was constructed for sensitive pathogen quantification by integrating hyperbranched rolling circle amplification (HRCA), CRISPR/Cas12a system, copper fluorescence nanoparticles (CuNPs) and smartphone-based signal readout. In this strategy, pathogen target-activated CRISPR/Cas12a precisely regulates HRCA initiation, which exponentially generates AT-TA-rich sequences. These products serve as templates for the self-assembly of the fluorescence CuNPs, achieving integrated rapid signal amplification without requiring sample preprocessing steps. Combined with smartphone-based RGB analysis, the platform enables direct, on-site quantitative readout. Cas12a-HRCA demonstrated exceptional performance for S. aureus, with a detection limit of 1 CFU/mL, high specificity, and 91%-106% reliable recovery in spiked milk samples without pre-enrichment or purification, showcasing great potential for point-of-need food safety monitoring. the detection of various pathogenic bacteria in food, clinical or environmental settings.},
}
@article {pmid42296867,
year = {2026},
author = {Xie, Z and Wu, Y and Zhou, P and Zhang, Y and Li, X and Li, Y and Cui, H},
title = {Label-free CRISPR/Cas12a biosensor based on G-triplex/Thioflavin T and GlaI-assisted strand displacement amplification for ultrasensitive DNA methylation detection.},
journal = {Talanta},
volume = {310},
number = {},
pages = {130144},
doi = {10.1016/j.talanta.2026.130144},
pmid = {42296867},
issn = {1873-3573},
mesh = {*DNA Methylation ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Benzothiazoles/chemistry ; DNA/genetics ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {DNA methylation represents a pivotal epigenetic biomarker for cancer, and precise profiling of carcinogenesis-associated methylation is essential for early diagnosis and prognostic evaluation. Herein, we report a novel label-free, sensitive DNA methylation biosensor by integrating GlaI-assisted double cascade strand displacement amplification with G-triplex-facilitated CRISPR/Cas12a (G-DCSDA/Cas12a). Instead of conventional fluorescent-quenched (FQ) probes, this assay employed G-triplex/Thioflavin T as a simple and efficient signal reporter for CRISPR/Cas12a. The methylation-specific endonuclease GlaI selectively digested methylated DNA to release free 3'-OH ends, which initiated the subsequent DCSDA and triggered Cas12a activation. The double-template cascade amplification system delivers significantly improved sensitivity in comparison with the single-template strategy. Upon activation, the trans-cleavage activity of Cas12a rapidly disrupted G-triplex/Thioflavin T complexes, generating a distinct fluorescence response. By combining the high specificity of GlaI, efficient signal amplification of DCSDA, and robust collateral cleavage of Cas12a, the G-DCSDA/Cas12a platform achieved ultrahigh sensitivity and selectivity, enabling detection of methylation levels as low as 0.005% in a background of excessive unmethylated DNA. Furthermore, this strategy was successfully integrated into a lateral flow assay (LFA), enabling visual and point-of-care testing (POCT) of DNA methylation. Importantly, the developed biosensor achieved sensitive detection of genomic DNA methylation in real samples and accurately discriminates cancer cells from normal cells, as well as between colorectal cancer tissue and adjacent normal tissue. These results highlighted the significant potential of the G-DCSDA/Cas12a platform for clinical early cancer diagnosis.},
}
@article {pmid42330823,
year = {2026},
author = {Jiang, H and Yang, J and Li, A and Mou, D and Deng, Y and Lv, X},
title = {Competitive kinetic mechanisms in one-pot isothermal amplification-CRISPR systems: From model construction to performance evaluation.},
journal = {Talanta},
volume = {310},
number = {},
pages = {130184},
doi = {10.1016/j.talanta.2026.130184},
pmid = {42330823},
issn = {1873-3573},
mesh = {Kinetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Hybridization ; },
abstract = {The urgent demand for integrating real-time molecular diagnosis with isothermal amplification and CRISPR-based detection has underscored the critical need for streamlined, single-tube one-pot methodologies. However, such integration is often hindered by temporal incompatibilities, including premature activation of early amplification products by CRISPR components, which leads to primer degradation and reduced amplification efficiency. To address this challenge, we established a dedicated quantitative competitive kinetic framework for enzyme-free isothermal amplification-CRISPR one-pot systems, using hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) as representative models. Two core inhibitory mechanisms were identified: "pre-activation-degradation inhibition", in which early CRISPR activation degrades amplification intermediates, and "substrate competition inhibition" in which hairpin probes compete with reporter probes for CRISPR trans-cleavage. Corresponding kinetic equations were derived to describe these interactions quantitatively. Systematic experimental validation of key parameters such as DNA activator concentration, reporter probe concentration, hairpin probe concentration, and ribonucleoprotein (RNP) complex concentration, confirmed the reliability and predictive accuracy of the proposed models. These results provide mechanistic insights into the factors governing one-pot HCR/CHA-CRISPR coupling and identify conditions that optimize assay performance. Overall, this study offers a theoretical foundation and experimental guidance for probe design, reaction condition optimization, and sensitivity enhancement in enzyme-free isothermal amplification-CRISPR one-pot platforms, and provides a general reference for the rational integration of isothermal amplification and CRISPR-based detection.},
}
@article {pmid42469290,
year = {2026},
author = {Relova-Hernández, E and Díaz-Bravo, AB and Cantero-Rodríguez, A and Alvarez-Arzola, R and Hernández-Álvarez, N and Carmenate, T and Rojas, G},
title = {CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42469290},
issn = {2045-2322},
mesh = {*Interleukin-2 Receptor alpha Subunit/genetics/metabolism ; Animals ; Humans ; *Interleukin-2 Receptor beta Subunit/metabolism/genetics ; Mice ; Interleukin-2/metabolism ; *Interleukin Receptor Common gamma Subunit/metabolism/genetics ; Cell Line ; Signal Transduction ; Cell Proliferation ; CRISPR-Cas Systems ; STAT5 Transcription Factor/metabolism ; Gene Knockout Techniques ; },
abstract = {Interleukin-2 (IL-2) has pleiotropic roles within the immune system. Its functional versatility depends upon the interactions with diverse arrays of receptor subunits differentially expressed on immune cell subsets. The balance between stimulation of regulatory T cells (Tregs) and effector lymphocytes determines the immunological outcome. While Tregs display constitutively a trimeric IL-2 receptor composed by alpha (also known as CD25), beta and gamma subunits, resting and memory CD8 + T cells express a dimeric beta/gamma receptor. The effects of IL-2 signaling through the trimeric receptor are routinely studied in vitro using either mouse CTLL-2 or human Kit225 lymphoid cell lines. We obtained equivalent IL-2-responsive cellular models displaying dimeric beta/gamma receptors. The purpose was achieved through genetic ablation of CD25 expression on CTLL-2 and Kit225 cells using CRISPR/Cas9 edition. The usefulness of the stable CD25-KO cell lines thus generated was illustrated by setting up proliferation assays, and characterizing a panel of IL-2-derived recombinant muteins, including agonists, super-agonists and antagonists. Problems and pitfalls of such assays, as well as the ways to mitigate them, were depicted. Stat5 phosphorylation assays based on KO cell lines were also performed. These cellular models, and the experience accumulated during their use, could contribute to the evaluation of the output of IL-2 engineering strategies developed across different laboratories.},
}
@article {pmid42497864,
year = {2026},
author = {Zhang, C and Li, T and Hao, H and Wang, G and Zhang, Y and Huang, Y and Chen, L and Wang, J and Yang, XJ and Bi, X},
title = {CRISPR-Cas9-producing probiotic bacteria for editing NOX2/gp91[phox] and treating inflammatory bowel disease.},
journal = {Cell reports. Medicine},
volume = {7},
number = {8},
pages = {102940},
doi = {10.1016/j.xcrm.2026.102940},
pmid = {42497864},
issn = {2666-3791},
mesh = {Animals ; *NADPH Oxidase 2/genetics/metabolism ; Mice ; *Probiotics/metabolism ; *Inflammatory Bowel Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; Reactive Oxygen Species/metabolism ; RAW 264.7 Cells ; *Gene Editing/methods ; Colitis/chemically induced ; Dextran Sulfate ; Humans ; Oxidative Stress ; Disease Models, Animal ; Intestinal Barrier Function ; Mice, Inbred C57BL ; },
abstract = {The primary pathogenic mechanism of inflammatory bowel disease (IBD) involves elevated levels of reactive oxide species (ROS) in the gut, leading to oxidative stress and damage to the intestinal barrier function. We engineered a non-pathogenic bacterial strain, Escherichia coli Nissle 1917 (EcN), for oral CRISPR-Cas9 delivery to edit NOX2 (encoding the gp91[phox] subunit of NADPH oxidase 2), thereby alleviating IBD symptoms by reducing ROS. EcN expressing the Cas9/sgRNA ribonucleoprotein (RNP) was encapsulated in a hydrogel (composed of hyaluronic acid, chitosan, and MgCl2), which protected EcN-RNP from degradation and increased survival from 0.07% to 12%. EcN-RNP hydrogel reduced NOX2 expression by 47% and ROS by 88% in lipopolysaccharide-induced RAW264.7 cells. Moreover, oral delivery of EcN-RNP hydrogel mitigated inflammation in dextran sodium sulfate-induced colitis mouse models. Mechanistically, the hydrogel could activate the NRF2-HO1/GPX4 pathway by inhibiting NOX2 expression, enhancing oxidative defense, and promoting glutathione accumulation. Thus, the EcN-RNP hydrogel offers a promising therapeutic strategy for IBD.},
}
@article {pmid42585941,
year = {2026},
author = {Wang, R and Pang, W and Yu, M and Dong, W and Wang, R},
title = {Evolution of CRISPR-cas detection systems: From nucleic acid recognition to omnidirectional point-of-care diagnostic applications.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {4},
pages = {117589},
doi = {10.1016/j.diagmicrobio.2026.117589},
pmid = {42585941},
issn = {1879-0070},
abstract = {Molecular diagnostic technologies play an indispensable role in modern medicine and public health. However, traditional diagnostic platforms frequently face an inherent trade-off between laboratory-grade analytical precision and the speed and operational simplicity required for point-of-care testing. In recent years, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein system has precipitated disruptive technological changes to this field. CRISPR-Cas system possesses high-fidelity target recognition capability and exhibits a distinctive trans-cleavage activity upon activation, which functions as signal amplification. This technology alleviates the inherent trade-off between sensitivity and portability. This review systematically summarizes the core molecular mechanisms of CRISPR-Cas detection platforms, addressing the differences in substrate preference and cleavage behavior among mainstream effector proteins (e.g., Cas9, Cas12, Cas13, and Cas14) and prokaryotic Argonaute (pAgo) proteins. Furthermore,this review sorts out the technological iteration path of detection platforms and presents the applications of this technology in fields such as infectious disease surveillance, cancer liquid biopsy, preliminary screening of genetic diseases, food and environmental safety, and veterinary port quarantine. Despite the challenges in quantitative accuracy and anti-interference ability, CRISPR biosensors are powerfully driving precision medicine towards decentralized, on-site, and accessible Point-of-Care Testing (POCT).},
}
@article {pmid42586052,
year = {2026},
author = {Wang, MR and Sánchez-Rivera, FJ},
title = {eVLP compound delivery breaks the prime editing efficiency ceiling.},
journal = {Cell genomics},
volume = {6},
number = {8},
pages = {101333},
pmid = {42586052},
issn = {2666-979X},
support = {P01 CA291694/CA/NCI NIH HHS/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; T32 GM136540/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {Prime editing could resolve gene variant function at scale, but the editing machinery needs to be delivered efficiently and reproducibly. Langley, Baudrier, et al.[1] show that timing of editor delivery is rate limiting and introduce PRIME-VLP, a repeated dosing strategy with engineered virus-like particles that improves editing efficiency and screening performance.},
}
@article {pmid42586245,
year = {2026},
author = {Rathore, S and Gupta, A and Shah, K and Chauhan, NS and Gupta, SK},
title = {Targeting the hallmarks of ageing: Pharmacological challenges and breakthroughs in CRISPR delivery systems and future prospects.},
journal = {Ageing research reviews},
volume = {121},
number = {},
pages = {103301},
doi = {10.1016/j.arr.2026.103301},
pmid = {42586245},
issn = {1872-9649},
abstract = {CRISPR has emerged as a next-generation gene-editing tool with the potential to target the molecular pathways associated with ageing and related disorders. It functions through RNA-guided Cas nucleases, directing DNA cleavage and utilizing the native DNA repair machinery for genetic manipulations. Advances in CRISPR technology have significantly enhanced the precision and flexibility of techniques for genome editing. The enzyme Cas9's ability to cut DNA at exact site has revolutionized genome editing by enabling accurate modifications within living eukaryotic cells. This review critically examines recent developments in CRISPR-based technologies, including Cas9, Cas12, base editing, prime editing, and CRISPR-mediated gene regulation. It highlights their rising applications in ageing research, with more emphasis on neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The review also discusses the major pharmacological and translational challenges that currently limit clinical applications, including inefficient tissue-specific delivery, off-target genome editing, immunogenicity, manufacturing complexity, and long-term safety concerns. Also, recent progress in both, viral and non-viral delivery methods are critically evaluated, including adeno-associated viruses, lentivirus vectors, lipid nanoparticles, gold nanoparticles, exosomes, electroporation, and microinjection, is thoroughly discussed to highlight their therapeutic potential and translational limitations. Current studies indicate that CRISPR-based approaches have preclinical potential for targeting important hallmarks of ageing, particularly genomic instability, telomere attrition, and mitochondrial dysfunction. Other hallmarks of ageing, such as stem cell exhaustion, epigenetic modifications, and microbiome changes, are at earlier stages of development. Overall, this review describes future strategies for developing safe, precise, and clinically translatable CRISPR-based treatments to promote healthy ageing.},
}
@article {pmid42586508,
year = {2026},
author = {Hasan, MU and Azhar, MW and Noor-Ul-Saba, and Khurshid, M and Riaz, R},
title = {Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.},
journal = {Journal of virological methods},
volume = {346},
number = {},
pages = {115447},
doi = {10.1016/j.jviromet.2026.115447},
pmid = {42586508},
issn = {1879-0984},
abstract = {The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.},
}
@article {pmid42587136,
year = {2026},
author = {Hsu, A and Chen, PJ and Li, AH and Hemez, CF and Gao, XD and Terrey, M and Nelson, C and Selvam, V and Cristian, A and McElroy, AN and Steinbeck, BJ and Mahadeshwar, GK and Pandey, S and Barsdale, Z and Chen, PZ and Sousa, AA and Sakai, HA and Silverstein, RA and Morad, I and Krueger, RK and Shen, MW and Kleinstiver, BP and Lutz, CM and Tolar, J and Blazar, BR and Osborn, MJ and Liu, DR},
title = {Mechanistic machine learning for prediction of prime editing outcomes.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42587136},
issn = {1546-1696},
support = {U01AI142756//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; RM1HG009490//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01EB022376//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R35GM118062//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {Prime editing (PE) can make specific local changes to genomic DNA in living systems but its efficient application currently requires extensive optimization of PE guide RNA (pegRNA) sequences. Here we present OptiPrime, a machine learning model of PE efficiency based on current understanding of PE mechanisms. OptiPrime achieves state-of-the-art accuracy on PE efficiency prediction and enables prediction of nicking guide RNA (PE3) and dual pegRNA (twinPE) outcomes. We validate that OptiPrime has learned the determinants of mammalian mismatch repair (MMR) and is well suited for nominating MMR-evasive silent edits that improve PE efficiency. We demonstrate the use of OptiPrime in a variety of prospective therapeutic contexts in primary human and mouse cells. Lastly, we show that OptiPrime can be used to achieve streamlined and efficient in vivo correction of a pathogenic mutation in the brain of a mouse model of KIF1A-associated neurological disorder. We provide a webserver for OptiPrime (https://optipri.me/) as a community resource.},
}
@article {pmid42589580,
year = {2026},
author = {Siddika, A and Husseiny, FE and Rousseau, J and Tremblay, JP},
title = {Prime Editing Mediated Generation and Correction of the mdx5cv Mutation Restores Dystrophin Expression in Myoblasts.},
journal = {International journal of molecular sciences},
volume = {27},
number = {15},
pages = {},
pmid = {42589580},
issn = {1422-0067},
support = {53320215//Defeat Duchenne Foundation, Canada/ ; },
mesh = {Animals ; *Dystrophin/genetics/metabolism ; *Myoblasts/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; *Gene Editing/methods ; Cell Line ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; },
abstract = {Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish dystrophin expression. Prime editing enables precise genome modification without generating double-strand DNA breaks or requiring donor DNA templates. We established an in vitro prime editing workflow to generate and subsequently correct the mdx5cv mutation in mouse C2C12 myoblasts. Following optimization of engineered prime editing guide RNAs (epegRNAs) and PAM-flexible prime editors, wild-type cells were edited, clonally isolated, and genotyped. Mutation correction was then evaluated using optimized epegRNA designs. Two rounds of prime editing introduced the mdx5cv mutation into approximately 20% of alleles in C2C12 cells creating the mdx5cv C2C12 cell line. Clonal isolation yielded five homozygous mutant clones among 59 expanded colonies. Optimization studies identified an epegRNA containing a 16 nucleotide reverse transcription template and a 10 nucleotide primer binding site (RTT16/PBS10) as the most efficient design. Correction of the pathogenic allele reached approximately 26%, whereas longer PBS lengths reduced editing efficiency. In silico off-target analysis using Cas-OFFinder identified no candidate genomic loci with fewer than three mismatches for the spacer sequences used in either mutation generation or correction, suggesting a favorable predicted specificity profile. Corrected mdx5cv C2C12 myoblasts retained their capacity to differentiate into multinucleated myotubes. Representative Western blot analysis detected dystrophin protein expression in differentiated corrected mdx5cv myotubes, consistent with successful correction of the pathogenic mutation. These findings establish a robust prime editing platform for both the generation and correction of the mdx5cv mutation and provide proof of concept that precise correction of the pathogenic mutation is associated with restoration of dystrophin expression following myogenic differentiation.},
}
@article {pmid42589608,
year = {2026},
author = {Steiman, S and Kinsella, C and Zhang, Y and Omer, S and Brown, C and Forguson, G and Elbakr, L and Pastore, S and Ivakine, EA},
title = {Restoring the Balance: CRISPRa-Driven β-Tubulin Compensation as a Strategy for Tubulinopathy Treatment.},
journal = {International journal of molecular sciences},
volume = {27},
number = {15},
pages = {},
pmid = {42589608},
issn = {1422-0067},
support = {//Nicol Family Foundation/ ; },
mesh = {*Tubulin/genetics/metabolism ; Humans ; Cilia/metabolism/genetics ; *CRISPR-Cas Systems ; Microtubules/metabolism/genetics ; *Gene Editing/methods ; Animals ; Mutation ; *Ciliopathies/genetics/therapy/metabolism ; },
abstract = {Microtubules are essential cytoskeletal components comprising alpha- and beta-tubulin proteins that facilitate organelle positioning, cell migration, division, and intracellular trafficking. Mutations in tubulin genes can lead to tubulinopathies, a class of rare genetic neurodevelopmental disorders characterized by a range of brain malformations and other clinical features. Recent studies have shown that pathogenic variants in beta-tubulin genes such as TUBB[G308S] have been found to underlie the development of ciliopathies, disorders impacting cilia, important organelles for development and cell motility. Thus, mutations in distinct tubulin genes, which present a significant hurdle for the development of therapeutic gene editing strategies and targeted therapeutics. Here, we describe the development of a mutation-independent treatment strategy based on the upregulation of non-mutated beta-tubulin isotype protein using CRISPR-Cas9 activation. By increasing the expression of various beta-tubulin proteins, we demonstrate a restoration of the microtubule network and primary cilia formation.},
}
@article {pmid42589635,
year = {2026},
author = {Kong, L and Iwabuchi, S and Li, YY and Murai, K and Korai, R and Kawaguchi, K and Nio, K and Imafuku, T and Shimakami, T and Yamashita, T and Tajima, A and Suzuki, Y and Honda, M and Hashimoto, S},
title = {Chromatin Accessibility-Guided Targeting Identifies Structurally Constrained Regions in HBV cccDNA and Suppresses Viral Replication.},
journal = {International journal of molecular sciences},
volume = {27},
number = {15},
pages = {},
pmid = {42589635},
issn = {1422-0067},
support = {17fk0310110h0001, 20fk0310110s8003, 21fk0310110s8005, 22fk0310514s0101, 23fk0310514s0102.//AMED/ ; },
mesh = {*Hepatitis B virus/genetics/physiology ; *DNA, Circular/genetics ; Humans ; *Virus Replication/genetics ; *DNA, Viral/genetics ; *Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; Hepatitis B/virology/genetics ; Hepatocytes/virology ; Genome, Viral ; },
abstract = {Covalently closed circular DNA (cccDNA) is a stable episomal form of the hepatitis B virus (HBV) genome that serves as the template for viral transcription and replication and represents a major barrier to HBV cure. Here, we investigated chromatin accessibility patterns of cccDNA in HBV-infected hepatocyte cells at single-molecule resolution. We found that most cccDNA copies exhibited limited accessibility around nucleotides 800-1000, a region overlapping the polymerase open reading frame and the pregenomic RNA transcriptional region. Notably, a small subset of cccDNA showed detectable accessibility at this site, suggesting the presence of heterogeneous chromatin states. Based on this observation, we targeted this accessibility-associated region using a CRISPR/Cas9-based approach and observed reductions in HBV DNA-related signals, including cccDNA-enriched fractions and total HBV DNA levels across complementary experimental systems. These findings suggest that chromatin accessibility profiling may provide an additional framework for identifying candidate cccDNA target regions. Our study provides a proof-of-concept for accessibility-informed HBV targeting and supports further investigation of chromatin-associated vulnerability within HBV cccDNA.},
}
@article {pmid42592160,
year = {2026},
author = {Christopher, CW and Zhou, X},
title = {In vivo CRISPR editing for cancer immunotherapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1872510},
pmid = {42592160},
issn = {1664-3224},
support = {R00 CA282989/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; *Immunotherapy/methods ; Animals ; Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; },
abstract = {Cancer immunotherapy has shown significant promise in certain patient populations, but further advancements are needed to extend its benefits to a wider range of patients. Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing has rapidly evolved in recent years, enabling its transition into direct therapeutic applications. This review summarizes recent progress in applying CRISPR systems in vivo for cancer immunotherapy, focusing on approaches that target cancer cells and the tumor microenvironment, as well as those that directly engineer immune cell populations themselves. Novel CRISPR editing platforms and strategies enabling multiplexed editing have also recently demonstrated promising impacts on driving antitumor immunity, however, the platforms investigated are still in the early stages and further investigation will be needed to robustly assess the potential for clinical translation. Future work can expand the array of therapeutic targets by incorporating data from functional genomics and must also carefully evaluate both editing modalities and delivery systems to optimize efficacy, safety, and scalability.},
}
@article {pmid42593176,
year = {2026},
author = {Kilani, H and Hamzaoui, Z and Ferjani, S and Lengliz, S and Tayh, G and Ben Zedira, A and Mosbahi, M and Kanzari, L and Ben Chehida, F and Abbassi, MS and Boutiba-Ben Boubaker, I},
title = {Whole-Genome Sequencing of Feline Uropathogens Reveals Multidrug Resistance and Zoonotic Potential in Domestic Cats in Tunisia.},
journal = {Vector borne and zoonotic diseases (Larchmont, N.Y.)},
volume = {},
number = {},
pages = {15303667261477573},
doi = {10.1177/15303667261477573},
pmid = {42593176},
issn = {1557-7759},
abstract = {BACKGROUND: Urinary tract infections (UTIs) in cats are increasingly recognized as clinically relevant conditions frequently associated with multidrug-resistant (MDR) bacteria of potential zoonotic origin, yet genomic data on feline uropathogens remain scarce in Tunisia.<br><br>METHODS: We used whole-genome sequencing to characterize seven bacterial isolates recovered from six cats with clinical signs of UTI: Mammaliicoccus lentus (n = 2), Staphylococcus schleiferi (n = 1), Mammaliicoccus sciuri (n = 1), Enterococcus faecalis (n = 1), Enterococcus casseliflavus (n = 1), and Klebsiella aerogenes (n = 1).<br><br>RESULTS: Resistome analysis revealed determinants conferring resistance to &#x3b2;-lactams (blaZ, blaCMY-132), methicillin (mecC-type), macrolides (erm(43), ermB), tetracyclines (tet(M), tet(45), tetB), fosfomycins (fosI, fosB, fosA5), and aminoglycosides (aac(6'), aph(3')-IIIa, aph(6)-Id), alongside efflux pump genes (efrA, sepA, sdrM, oqxA, KpnE/F/G), vancomycin-operon genes (vanT, vanY, vanC, vanG), and biofilm/biocide-tolerance genes (salB, qacG). Notably, M. lentus S104 carried mecC-type elements, the first such report in Tunisia, while K. aerogenes displayed an extensive MDR profile, including blaCMY-132 and fosA5. Multilocus sequence typing/ribosomal multilocus sequence typing (MLST/rMLST) identified diverse lineages, including the internationally distributed E. faecalis ST19 and the rarely reported K. aerogenes ST242. Plasmids were absent in all isolates; a Tn916/1545-type transposon occurred in E. casseliflavus, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were unevenly distributed.<br><br>CONCLUSIONS: These findings highlight companion animals as reservoirs of clinically important resistance genes, reinforcing the need for One Health AMR surveillance.},
}
@article {pmid42593847,
year = {2026},
author = {Lozano-Montalbá, L and Denis, Z and Courgnaud, V and Moreno-García, J and Majzoub, K and Sanjuán, R and Dufloo, J},
title = {Glycosphingolipids are essential entry factors for non-influenza orthomyxoviruses.},
journal = {The Journal of general virology},
volume = {107},
number = {8},
pages = {},
pmid = {42593847},
issn = {1465-2099},
mesh = {*Virus Internalization ; *Glycosphingolipids/metabolism ; Animals ; *Thogotovirus/physiology/genetics ; Glucosyltransferases/metabolism/genetics ; Virus Replication ; Cell Line ; *Orthomyxoviridae/physiology ; Humans ; CRISPR-Cas Systems ; },
abstract = {Thogotoviruses and quaranjaviruses are arthropod-borne orthomyxoviruses that circulate widely in wildlife and domestic animals and include several zoonotic members. Despite their close phylogenetic relationship to influenza viruses, the mechanisms underlying their replication remain poorly understood, and the host factors mediating viral entry are unknown. Here, we performed genome-wide loss-of-function CRISPR-Cas9 screens using replication-competent recombinant vesicular stomatitis viruses expressing thogoto- and quaranjavirus glycoproteins to identify cellular determinants of viral entry. These screens identified the glycosphingolipid (GSL) biosynthesis pathway as a key regulator of viral entry, with the upstream enzyme uridine diphosphate (UDP)-glucose ceramide glucosyltransferase (UGCG) emerging as a central host entry factor. Pharmacological inhibition of UGCG impaired thogoto- and quaranjavirus entry. The importance of GSL biosynthesis for thogotovirus replication was further validated using multiple thogotovirus isolates. Together, our findings establish GSLs as critical host entry factors for non-influenza orthomyxoviruses and identify UGCG as a potential target for antiviral intervention.},
}
@article {pmid42595700,
year = {2026},
author = {Mani, M and Poonguzhali, S and Baghyalakshmi, K},
title = {CRISPR/Cas-based genome editing for enhancing grain quality traits in rice (Oryza sativa L.).},
journal = {Plant signaling &amp; behavior},
volume = {21},
number = {1},
pages = {2713868},
pmid = {42595700},
issn = {1559-2324},
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Gene Editing/methods ; Plants, Genetically Modified ; },
abstract = {Rice is one of the most important food crops and feeds more than half of the world's population. Enhancing grain quality is currently a highly important issue since consumers are now more concerned with the taste, appearance, and nutritional value of the grain. The quality of grain in rice is complex and regulated by a multitude of genes that influence qualities such as amylase content, grain size and shape, chalkiness, aroma and nutrient content. The traditional forms of breeding, such as hybridization and marker-assisted selection, are slow and less effective since such characteristics are regulated by many genes and are influenced by environmental conditions. CRISPR/Cas genome editing has become a potent tool that enables scientists to directly and specifically edit grain quality-related genes. Important genes such as Wx (amylose), GS3, GW8, and TGW3 (grain size), Chalk5 (chalkiness), BADH2 (aroma), and nutrient-related grain size genes such as OsAAP6, OsAAP10, and OsVIT1/2, have been successfully edited to enhance the quality of rice. Newer methods, such as base editing and prime editing, enable this process to become even more precise by modifying the specific bases of DNA without cutting the DNA. CRISPR has assisted in the improvement of rice by controlling the amylose content, reducing chalkiness, enhancing aroma, and improving nutritional quality. It is more accurate and quicker than traditional breeding, and it can enhance various traits simultaneously. Nonetheless, other challenges, such as off-target effects, regulatory concerns, and acceptance by the people, still have to be overcome. Combining CRISPR with artificial intelligence and genomic selection in the future will aid in creating superior versions of rice in shorter periods of time. Overall, CRISPR/Cas genome editing is a potential method to enhance the quality of rice and secure food security in the whole world.},
}
@article {pmid42595703,
year = {2026},
author = {Mukherjee, A and Thakur, D and Ahuja, M and Nayan, P and Kumar, L},
title = {Enzyme-driven antimicrobial resistance and advancements in sustainable anti-infective strategies.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-39},
doi = {10.1080/07388551.2026.2693159},
pmid = {42595703},
issn = {1549-7801},
abstract = {Antibiotic resistance is an emerging global issue that has reduced the efficacy of antibiotics for treating life-threatening bacterial infections. Bacterial adaptive enzymatic defense mechanisms allow cells to activate or modify specific enzymes that inactivate antibiotics and support survival under antimicrobial stress. Antibiotics are predominantly inactivated through enzymatic degradation or chemical modification. Most of the β-lactamases, macrolide esterases, tetracycline-modifying enzymes, fosfomycin degrading enzymes, aminoglycoside-modifying enzymes, and other bacterial enzymes chemically modify or degrade the antibiotics making them inactive. This review covers classification and mechanisms of enzyme-mediated resistance and emphasizes the significant enzymes involved in inactivation of various antibiotic classes. It also summarizes recent biotechnological advances to combat antibiotic resistance, including β-lactamase inhibitors, phage therapy, antimicrobial peptides, and CRISPR-Cas9 systems, along with emerging therapeutic approaches and current trends in antibiotic research. A deeper understanding of enzyme-mediated resistance and the cellular intelligence driving bacterial adaptation is crucial for designing effective therapeutic strategies, preserving antibiotic efficacy, and reducing the global burden of resistant infections.},
}
@article {pmid42341757,
year = {2026},
author = {Kim, J and Kovacs, H and Wisnovsky, S},
title = {Rapid discovery of cell-surface glycosylation regulators using a lectin-based magnetic CRISPR screen.},
journal = {Cell reports methods},
volume = {6},
number = {8},
pages = {101507},
doi = {10.1016/j.crmeth.2026.101507},
pmid = {42341757},
issn = {2667-2375},
mesh = {Humans ; Glycosylation ; *Lectins/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Polysaccharides/metabolism ; *Cell Membrane/metabolism ; Flow Cytometry/methods ; Cell Line, Tumor ; *CRISPR-Cas Systems ; },
abstract = {FACS-based CRISPR screening has emerged as a potent tool for dissecting the genetic networks that regulate cell-surface glycosylation. However, existing protocols can be tedious and are not compatible with many cell models. We developed a lectin-based magnetic-activated cell sorting platform (Lec-MACS) that enables rapid identification of genes controlling expression of specific cell-surface glycans. Lec-MACS offers superior speed and multiplexability compared to FACS, while also being well-suited to studying cell models that are not amenable to flow-based sorting. We subsequently applied Lec-MACS to map genes that regulate hypersialylation in an adherent breast cancer cell line. Subsequent hit validation confirmed an unexpected link between DNA damage response signaling and cell-surface sialylation. The Lec-MACS method will expand the scope and throughput of genetic screens targeted at cell-surface glycans.},
}
@article {pmid42420316,
year = {2026},
author = {Favoino, G and Pšenka, D and Frideres, L and Volke, DC and Nikel, PI},
title = {A portable Cas6f-based system for multiplex translational repression in bacteria.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42420316},
issn = {2041-1723},
support = {NNF20CC0035580//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF18OC0034818//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF21OC0067996//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF24OC0091501//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF23OC0083631//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF24SA0100980//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF20CC0035596//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; 101082049//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*Pseudomonas putida/genetics/metabolism ; *Escherichia coli/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; *Protein Biosynthesis ; Metabolic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockdown Techniques/methods ; Synthetic Biology/methods ; },
abstract = {Engineered small RNAs (sRNAs) enable programmable gene knockdowns and support metabolic engineering and multiplex regulation in model bacteria. Still, precise, tunable, and multiplex gene repression remains a challenge in synthetic biology. Common tools can impose genetic burden, depend on host RNA factors, or do not transfer well across species. Here we present MORTISE (Multiplex, ORthogonal Translation Interference SystEm), a compact Cas6f-based platform for programmable translational repression in Gram-negative bacteria. The system functions without host Hfq or RNases and operates robustly in Escherichia coli and Pseudomonas putida. We demonstrate repression in both species using chromosomal reporter assays, with performance improving when guide and target transcription are matched and when the translation initiation region is targeted. Single-promoter multiplexing enables simultaneous knockdowns and a cloning toolbox facilitates assembly of up to nine guides in a single step. Finally, MORTISE is leveraged to boost malonyl-coenzyme A-dependent production in P. putida, supporting pathway balancing.},
}
@article {pmid42585319,
year = {2026},
author = {Dinh, DT and Smith, KM and McPhee, T and Foot, NJ and Bersten, DC and White, MA and Thomas, PQ and Robker, RL and Russell, DL},
title = {Reassessing the role of progesterone receptor isoforms PGR-A and PGR-B in female fertility.},
journal = {Science advances},
volume = {12},
number = {33},
pages = {eadz7757},
pmid = {42585319},
issn = {2375-2548},
support = {INV-001616//Bill &amp; Melinda Gates Foundation/United States ; INV-024199//Bill &amp; Melinda Gates Foundation/United States ; },
mesh = {Female ; *Receptors, Progesterone/genetics/metabolism ; Animals ; *Fertility/genetics ; Protein Isoforms/genetics/metabolism ; Mice ; CRISPR-Cas Systems ; Mice, Knockout ; },
abstract = {Progesterone is a critical reproductive hormone that acts via progesterone receptor transcriptional regulators. The short PGR-A isoform lacks a 164-amino acid amino-terminal region present in PGR-B that markedly enhances transcriptional activity. Isoform-specific mutants inactivating either PGR-A (PRAKO) or PGR-B (PRBKO) indicated that PGR-A is specifically essential for female fertility. This study revises that interpretation by showing that an inadvertent frameshift mutation in the PRAKO caused ablation of both isoforms, not the intended PGR-A isoform-specific mutation. A true PGR-A-specific mutant generated through CRISPR-Cas9 editing with a complete lack of PGR-A but retained PGR-B expression showed a phenotype indistinguishable from wild type. Thus, while the short PGR-A isoform has distinct function, it is not independently essential for female reproduction in mice, and the physiological role of these highly conserved isoforms must be reconsidered in light of this new information.},
}
@article {pmid42584997,
year = {2026},
author = {Wu, P and Zhou, X},
title = {Canon enables causal inference of downstream genes in single-cell CRISPR studies via instrumental variable analysis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {33},
pages = {e2525359123},
doi = {10.1073/pnas.2525359123},
pmid = {42584997},
issn = {1091-6490},
support = {R01HG009124//HHS | NIH (NIH)/ ; R01GM144960//HHS | NIH (NIH)/ ; R01GM126553//HHS | NIH (NIH)/ ; R01HG011883//HHS | NIH (NIH)/ ; },
mesh = {Humans ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; *Gene Regulatory Networks/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A critical analytical task in sc-CRISPR screening is identifying downstream genes influenced by perturbed target genes. Existing methods for this task primarily rely on traditional association-based analyses, which not only fall short in establishing causal relationships between genes but also suffer from high false positive rates and limited statistical power. To overcome these limitations, we introduce a causal inference-based framework that leverages the perturbation status of gRNAs in single cells as instrumental variables (IVs) to infer causal gene relationship via IV analysis. Building upon this framework, we further present Canon, a one-sample IV analysis method specifically tailored to systematically identify genes that are potentially causally influenced by perturbed target genes across diverse sc-CRISPR platforms. Canon ensures robust type I error control while maintaining high statistical power. We evaluated its performance through comprehensive simulations and real data applications. The gene-gene relationships identified by Canon provide valuable insights into the causal gene regulatory network, uncovering candidate therapeutic targets with potential relevance for cancer biology and demonstrating the transformative potential of sc-CRISPR screening to resolve causal regulatory networks at an unprecedented scale.},
}
@article {pmid42582274,
year = {2026},
author = {Liu, W and Gu, J and Xie, C and Du, B and Liu, M and Zhang, J},
title = {Rewriting CAR-T cell fate: CRISPR/Cas gene editing for solid tumor therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1910092},
pmid = {42582274},
issn = {1664-3224},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *Receptors, Chimeric Antigen/immunology/genetics ; Animals ; Tumor Microenvironment/immunology ; *T-Lymphocytes/immunology/metabolism ; T-Cell Exhaustion ; },
abstract = {Although chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematological malignancies, its therapeutic efficacy in solid tumors remains limited by several challenges, including insufficient tumor infiltration, T cell exhaustion and the immunosuppressive tumor microenvironment (TME). CRISPR/Cas, a third-generation gene editing technology developed in recent years, is characterized by its simplicity and high efficiency. This technology has demonstrated broad application potential across multiple fields and has emerged as a powerful tool for improving CAR-T cell therapy. In this review, we summarize recent advances in the application of CRISPR/Cas gene editing technology to enhance the antitumor activity of CAR-T cells against solid tumors. We also discuss the key challenges currently faced and systematically propose potential strategies for overcoming the limitations.},
}
@article {pmid42582990,
year = {2026},
author = {Ma, AJ and Brown, BH and Kim, S and Hilton, IB},
title = {Clinical translation of epigenome editing technologies.},
journal = {Current opinion in biomedical engineering},
volume = {38},
number = {},
pages = {},
pmid = {42582990},
issn = {2468-4511},
support = {R01 EB036003/EB/NIBIB NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR/Cas-based epigenome editing technologies hold great promise for identifying novel therapeutic targets, improving gene and cell therapies, and directly addressing the underlying issues in many diseases, all while minimizing risks of genotoxicity often associated with conventional genome editing. Exciting recent advances in CRISPR/Cas-based epigenome editing technologies have drastically enhanced the ability to precisely control the timing, levels, and durations of endogenous gene expression and reprogram epigenetic states in human cells. As a result, epigenome editing is now poised to unlock new biomedical discoveries and treatments for diseases driven by transcriptional and epigenetic dysregulation as well as those stemming from aberrantly repetitive genomic regions or complex genomic arrangements that are difficult to target using conventional genome editing. Additionally, the power of epigenome editors is generating new strategies to control cell fate and function, which has direct and important implications for cell therapies and regenerative medicines. Here, as the first wave of CRISPR/Cas-based epigenome editors move into clinical trials, we cover recent advances as the field looks to address pressing hurdles facing widespread clinical deployment of epigenome editing technologies including delivery, performance, and safety. For instance, the discovery of compact Cas chassis, engineering efforts to reduce effector sizes for efficient delivery, and campaigns to tailor the targeting discrimination of epigenome editors are rapidly progressing, as is research into the development of new effector domains with high specificity, robust performance, and a lack of immunogenicity and cytotoxicity. This exciting progress is quickly moving the community closer to fulfilling the promise of CRISPR/Cas-based epigenome editing as a powerful class of platform technologies for biological discoveries, biotechnological innovations, and medicines.},
}
@article {pmid42584708,
year = {2026},
author = {Skoczek, D and Hohendorff, J and Malecki, MT and Roig-Merino, A and Bak, RO and Kachamakova-Trojanowska, N},
title = {CRISPR/Cas9-based repair of a heterozygous HNF1A mutation in patient-derived hiPSCs.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {42584708},
issn = {1432-1203},
support = {2020/38/E/NZ3/00516//Narodowe Centrum Nauki/ ; 2020/38/E/NZ3/00516//Narodowe Centrum Nauki/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Hepatocyte Nuclear Factor 1-alpha/genetics ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Frameshift Mutation ; Mutation ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor's genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.},
}
@article {pmid41820594,
year = {2026},
author = {Lee, SJ and Nam, BG and Hong, SA and Jo, DH and Lee, SM and Bae, S and Kim, JH},
title = {Topical application of Cas9 ribonucleoproteins inhibits corneal neovascularization in a mouse model of alkali burn injury.},
journal = {Gene therapy},
volume = {33},
number = {4},
pages = {435-447},
pmid = {41820594},
issn = {1476-5462},
support = {2022M3A9E4017127//National Research Foundation of Korea (NRF)/ ; RS-2023-00260351//National Research Foundation of Korea (NRF)/ ; RS-2024-00467177//National Research Foundation of Korea (NRF)/ ; 18-2023-0010//Seoul National University Hospital (SNUH)/ ; GTL24021-000//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/ ; },
mesh = {Animals ; *Corneal Neovascularization/therapy/genetics ; Mice ; *Burns, Chemical/therapy ; *Genetic Therapy/methods ; Disease Models, Animal ; *Eye Burns/therapy/chemically induced ; Vascular Endothelial Growth Factor A/genetics/metabolism ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/administration &amp; dosage ; Gene Therapy Agents ; Gene Editing ; Mice, Inbred C57BL ; Administration, Topical ; *CRISPR-Associated Protein 9/genetics ; },
abstract = {Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.},
}
@article {pmid42174157,
year = {2026},
author = {Li, JX and Zhang, SM and Ma, XY and Deng, DH and Bai, PD and Zhao, JJ and Gong, A and Pang, SC and Dong, F and Wang, SD and Zhang, JP and Zhang, XB},
title = {AviTag-seq unifies nucleotide-resolution maps of CRISPR off-targets and AAV vector integrations.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42174157},
issn = {2399-3642},
support = {82402188//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Genetic Vectors/genetics ; *Dependovirus/genetics ; Mice ; *Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Liver/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Virus Integration ; },
abstract = {Comprehensive safety assessment of gene-editing therapies requires quantifying both off-target cleavage and vector integration. However, current double-strand break (DSB)-dependent assays are fundamentally limited when evaluating nickase-based editors and are hindered by tag polarity constraints. Here, we present AviTag-seq, a platform repurposing AAV Inverted Terminal Repeats (ITRs) as universal capture tags. By exploiting the ITRs' single-stranded hairpin structure, AviTag-seq overcomes polarity issues, enabling high-sensitivity detection with a single primer pair, particularly in iPSCs. Crucially, it captures off-target events from prime and base editors that evade conventional detection. In vivo, AviTag-seq outperformed DISCOVER-Seq+ in profiling Pcsk9 off-targets in mouse liver while simultaneously mapping AAV integration sites. This dual profiling revealed that, unlike in vitro, AAV vectors in vivo preferentially integrate into active gene promoters, highlighting a specific genotoxic risk for liver-directed therapies. AviTag-seq thus offers a unified, regulatory-grade solution for evaluating diverse genome-editing modalities.},
}
@article {pmid42243947,
year = {2026},
author = {Wei, Z and Xu, X and Qiao, Q and Guo, J and Wu, T and Rong, H and Ning, S and Zhu, X and Zhao, K and Ke, J and He, L and Chi, Y and Ge, Y and Cui, L and Min, X},
title = {A temporally controlled isothermal amplification-CRISPR/Cas12a platform for the rapid detection of monkeypox virus.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42243947},
issn = {1743-422X},
support = {JSJK2026D00033//Science and Technology Program Project of Jiangsu Provincial Bureau of Disease Prevention and Control/ ; MQ2025039//Scientific Research Project of Jiangsu Commission of Health/ ; M2024024//Scientific Research Project of Jiangsu Commission of Health/ ; BK20231374//Natural Science Foundation of Jiangsu Province/ ; 2023YFC2605100, 2023YFC2605104//National Key Research and Development Program of China/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Monkeypox virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Mpox, Monkeypox/diagnosis/virology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Humans ; Animals ; },
abstract = {BACKGROUND: Given the continuous threat posed by emerging and re-emerging infectious diseases worldwide, a rapid, sensitive, and practical molecular detection technology is urgently required for timely point-of-care (POC) diagnosis. Although the quantitative real-time PCR (qPCR) based technologies exhibit high sensitivity and specificity compared with traditional pathogen detection methods, they are not applicable for POC detection scenarios.<br><br>METHODS: Based on the photocontrolled principle, this study established a universal photoactivation strategy for LbCas12a by modifying four sites in the repeat region of LbCas12a crRNA with the photocleavable protecting group 6-nitropiperonyloxymethyl (NPOM). This strategy was integrated with multienzyme isothermal rapid amplification (MIRA) to develop a photocontrolled one-pot rapid detection method for monkeypox virus (MPXV), termed the temporally controlled MIRA-CRISPR/Cas12a (TC-MIRA-CRISPR/Cas12a) assay.<br><br>RESULTS: The TC-MIRA-CRISPR/Cas12a assay exhibited a 100-fold improvement in detection sensitivity over the conventional one-step assay, achieving a limit of detection (LOD) of 3.9 copies per reaction, which was comparable to stepwise detection assay. The entire assay can be completed within 40&#xa0;min, faster than the widely used qPCR. In clinical sample detection, this method showed good consistency with qPCR, with a Kappa coefficient of 0.980 (P&#x2009;<&#x2009;0.001), a sensitivity of 98.4%, and a specificity of 100%.<br><br>CONCLUSIONS: This method effectively avoids amplicon contamination while maintaining high sensitivity, and exhibits excellent universality and expandability. It enables detection of other pathogens simply by modifying the spacer sequence of crRNA, providing a novel technical approach and research perspective for POC detection of MPXV and other pathogens.},
}
@article {pmid42265600,
year = {2026},
author = {Jiang, S and Chen, F and Ma, H and Wu, S and Tang, X and Pan, X and Li, Q and Tao, A and Xu, J and Qi, J and Fang, P and Chen, J and Zhang, L},
title = {Cloning and functional verification of endogenous U6 promoters for developing an efficient CRISPR/Cas9-mediated genome editing system in kenaf (Hibiscus cannabinus L.).},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {42265600},
issn = {1471-2229},
support = {32472219//the National Natural Science Foundation of China/ ; 2023J01443//Fujian Provincial Natural Science Foundation of China/ ; CARS-16//China Agricultural Research System of MOF and MARA/ ; KFB23001//Science and Technology Innovation Project of Fujian Agriculture and Forestry University/ ; ASTIP-IBFC-01//Agriculture Science and Technology Innovation Program/ ; },
mesh = {*Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems ; *Hibiscus/genetics ; *Gene Editing/methods ; Cloning, Molecular ; *RNA, Small Nuclear/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {BACKGROUND: The U6 promoter is a critical component of the CRISPR/Cas9 system, as it drives the transcription of single-guide RNAs (sgRNAs) to enable precise genome editing. Endogenous promoters typically exhibit higher transcriptional activity than their exogenous counterparts, which can significantly enhance editing efficiency. However, the endogenous U6 promoter in kenaf (Hibiscus cannabinus L.), an important fiber crop, has not yet been characterized.<br><br>METHODS: Using the Arabidopsis U6-26 (AtU6-26) promoter as a reference, we performed a homologous sequence search and identified two candidate U6 promoters in kenaf, designated HcU6-1 and HcU6-14. Promoter fragments were amplified from the kenaf cultivar 'Fuhong 952' and cloned into a &#x3b2;-glucuronidase (GUS) reporter vector. Histochemical GUS staining assays revealed that both HcU6 promoters were transcriptionally active, with HcU6-14 showing significantly stronger expression levels compared to HcU6-1.<br><br>RESULTS: To further evaluate the utility of these promoters for genome editing, we constructed CRISPR/Cas9 vectors targeting the kenaf acetolactate synthase (ALS) gene, driven by either HcU6-14P or the exogenous cotton GbU6-9P promoter. Agrobacterium rhizogenes K599-mediated transformation was used to induce hairy roots, and mutation analysis of the ALS gene was performed via Sanger sequencing. Notably, targeted mutations in the ALS gene were detected in hairy roots transformed with the HcU6-14P-driven CRISPR/Cas9 vector, whereas no mutations were observed in roots transformed with the exogenous GbU6-9P promoter. These results demonstrate that the endogenous HcU6-14 promoter confers superior genome editing efficiency compared to the heterologous promoter, which facilitates the development of improved varieties with enhanced agronomic traits.},
}
@article {pmid42409798,
year = {2026},
author = {Zhao, C and Shih, M and Ahmed, S and Song, S and Lennon, A and Mayes, JM and Fan, MJ and Lo, PY and Perera, J and Tutar, A and Kang, A and Warren, E and McClure, R and Khan, AA and Kelley, SO and Abdrabou, AM},
title = {AI-guided CRISPR screening reveals therapeutic targets in psoriasis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42409798},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Psoriasis/genetics/drug therapy/chemically induced ; Keratinocytes/metabolism/drug effects ; Mice ; *Receptors, Interleukin-17/metabolism/genetics ; Arachidonate 5-Lipoxygenase/genetics/metabolism ; Imiquimod ; Receptors, Oxytocin/antagonists &amp; inhibitors/genetics/metabolism ; CRISPR-Cas Systems ; Hydroxyurea/pharmacology/analogs &amp; derivatives ; Clustered Regularly Interspaced Short Palindromic Repeats ; Interleukin-17 ; },
abstract = {Psoriasis affects over 125 million people globally. Biologics targeting the IL-17/IL-17RA axis are effective but require systemic administration, are costly, and are unsuitable for some patients. Developing topical small-molecule alternatives requires a better understanding of how IL-17 receptor A (IL17RA) is regulated in keratinocytes, the principal effector cells of psoriatic lesions. Here, we report a genome-wide CRISPR knockout screen for regulators of surface IL17RA in primary human epidermal keratinocytes. We prioritize hits using experimental enrichment together with VirtualCRISPR, a language-model framework trained on functional-genomics data, and validate two regulators with minimal prior connection to IL17RA: 5-lipoxygenase (ALOX5) and the oxytocin receptor (OXTR), which act through distinct cell-intrinsic mechanisms. Topical zileuton, an ALOX5 inhibitor, and cligosiban, an OXTR antagonist, suppress imiquimod-induced psoriasiform dermatitis in mice, mirroring systemic anti-IL17RA antibody efficacy. By linking AI- guided selection to genetic perturbation screening, this study provides an efficient route from candidate gene nomination to biological validation and therapeutic discovery.},
}
@article {pmid42414323,
year = {2026},
author = {Wu, X and Li, Y and Cao, Y and Zhao, Z and Lu, H and Liang, S and Lui, GCY and Chan, DPC and Hsing, IM},
title = {Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42414323},
issn = {2041-1723},
support = {16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*Polymorphism, Single Nucleotide/genetics ; Thermodynamics ; *Point-of-Care Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genotyping Techniques/methods ; DNA Primers/genetics ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Genotype ; },
abstract = {One-pot CRISPR diagnostics face a fundamental incompatibility: isothermal nucleic acid amplification enables rapid target accumulation, whereas CRISPR activation irreversibly consumes those substrates, destabilizing reaction kinetics. Here we show that reaction order can be programmed into DNA primers through thermodynamic design. Differences in primer-binding strength create two sequential amplification stages, delaying CRISPR activation until enough amplicons have accumulated without physical separation or external control. The design also introduces the protospacer adjacent motif (PAM), a short sequence required for CRISPR recognition, through the primer rather than relying on its presence in the native target, expanding target accessibility while retaining single-nucleotide discrimination. An ordinary differential equation model captures the threshold behavior and establishes a predictable framework for primer design. Building on this principle, we develop Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), which achieves attomolar sensitivity within 30 min and enables sequencing-concordant SNP genotyping and pathogen detection in a single-step microfluidic format.},
}
@article {pmid42574383,
year = {2026},
author = {Xu, A and Cates, K and Kerr, C and Wysong, K and Barna, M},
title = {Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/66881},
pmid = {42574383},
issn = {1940-087X},
mesh = {*Biotinylation/methods ; *Ribosomes/chemistry/metabolism ; Carbon-Nitrogen Ligases/chemistry/metabolism/genetics ; Biotin/chemistry ; Humans ; Ribosomal Proteins/chemistry/genetics ; CRISPR-Cas Systems ; },
abstract = {Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of specific mRNAs is key to ensuring that proteins with compartment-specific functions are produced in the ideal milieu and in appropriate quantities. However, techniques for subcellular isolation and characterization of the ribosomes that translate these mRNAs have been limited to date, such that much remains unknown about how the composition of translational machinery contributes to regulation of localized mRNA translation. Here, we demonstrate AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi), a method that combines epitope tagging, a newly developed optogenetically activated split-biotin ligase, and affinity purification to rapidly and specifically label and isolate ribosomes localized to any subcellular compartment of interest. First, CRISPR/Cas9 editing is used to fuse an AviTag peptide, a tobacco etch virus (TEV) protease cleavage site, and a FLAG epitope tag to a ribosomal protein. The split biotin ligase, fused to an organelle-targeting domain, is expressed in this cell line and is inactive under normal biotin concentrations. Upon activation by supplemental biotin and blue light illumination, the ligase biotinylates AviTagged ribosomes in the immediate vicinity, allowing for affinity purification of biotinylated ribosomes and associated proteins and mRNAs on streptavidin-coated beads. Non-denaturing elution via TEV protease cleavage yields samples suitable for downstream characterization of core ribosomal proteins, ribosome-associated proteins, and ribosome-bound mRNAs via RNA sequencing or mass spectrometry proteomics. In this protocol, we review design principles for fusing AviTag to a ribosomal protein and targeting the split biotin ligase enzyme to the organelle of interest. We demonstrate activation of the ALIBi system, cell lysis, affinity purification, and sample elution. Finally, we discuss typical results and troubleshooting.},
}
@article {pmid42574397,
year = {2026},
author = {Langille, ER and Al-Zahrani, KN and Nurtanto, J and Lee, Y and Chiu, CH and Schramek, D},
title = {Parallel In Vivo Screening of Gene Knockout and Activation in the Mouse Mammary Gland.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71115},
pmid = {42574397},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Mammary Glands, Animal/physiology/metabolism ; Female ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Forward genetics screens are routinely employed to perturb thousands of genetic elements in a pooled fashion with the goal of producing large-scale genotype-to-phenotype maps. While often carried out in cell culture systems, accumulating evidence supports that in vivo screens have the power to unveil new biology that cannot be recapitulated in vitro. However, the widespread application of this approach has been limited by two major challenges: a predominant focus on loss-of-function perturbations rather than gene activation and the significant technical hurdles of delivering complex genetic libraries to specific tissues in vivo. To overcome these challenges, we describe a simple and versatile intraductal injection strategy that enables efficient and rapid functional genomic screening in the mouse mammary gland, by generating tens of thousands of discrete epithelial clones. Furthermore, we provide all the details necessary for library generation, intraductal injection, screen deconvolution, and analysis of CRISPR-Knockout and Activation libraries for comprehensive in vivo screens. Using these tools, which we termed CRISPR-KOALA (Knockout and Activation Linked Assay), we have identified new tumor suppressors and oncogenes within the coding and non-coding genome in pooled libraries ranging from 46 loci to one-fifth of the genome. Importantly, this approach and analysis can be applied to other organs to study the biological function of any gene during homeostasis or disease.},
}
@article {pmid42574536,
year = {2026},
author = {Yedier-Bayram, O and Guvener, EA and Bagci-Onder, T},
title = {Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71175},
pmid = {42574536},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Paclitaxel/pharmacology ; *Gene Knockout Techniques/methods ; *Epigenome/genetics ; *Triple Negative Breast Neoplasms/genetics/drug therapy ; Female ; },
abstract = {Chemotherapy resistance remains a major challenge in cancer treatment, driven by cancer cells' ability to acquire adaptive properties, rewire signaling pathways, and alter chromatin structure to evade drug-induced cytotoxicity. Because these processes rely heavily on epigenetic mechanisms that regulate chromatin organization and transcriptional plasticity, epigenetic regulators have emerged as key contributors to chemotherapy resistance. To investigate resistance to paclitaxel, one of the most widely used chemotherapeutic agents in triple-negative breast cancer (TNBC), we employed an epigenome-focused knockout library (EPIKOL), a CRISPR-Cas9-based library, designed to systematically disrupt genes involved in chromatin regulation. Chemoresistant cell lines were generated through a stepwise dose-escalation protocol that recapitulates clinically relevant drug adaptation. However, these resistant cells exhibit a multidrug-resistant (MDR) phenotype, posing significant challenges for efficient viral transduction and the selection of stable cell populations. In this study, we describe key methodological steps for achieving high-efficiency lentiviral transduction and selection, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models. Following the described protocol, an epigenome-wide CRISPR screen was conducted on chemoresistant TNBC cells, and novel epigenetic regulators of chemoresistance were identified. This protocol provides a robust framework for identifying epigenetic regulators that contribute to acquired paclitaxel resistance using a CRISPR-based loss-of-function approach.},
}
@article {pmid42577382,
year = {2026},
author = {Lin, W and Shi, J and Chen, H and Chai, R and Zhu, S and Chen, L and Mo, S and Wang, Z and Li, H and Feng, Y and Zhao, L and Chen, J and Yu, G and Lu, T and Wang, J},
title = {Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1878099},
pmid = {42577382},
issn = {1664-3224},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Genetic Vectors/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; HEK293 Cells ; Animals ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Cell Engineering/methods ; CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; Cell Line ; },
abstract = {INTRODUCTION: CRISPR-Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches.<br><br>METHODS: We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9-gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules-Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope-into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days.<br><br>RESULTS: Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci.<br><br>DISCUSSION: It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.},
}
@article {pmid42577913,
year = {2026},
author = {Tenkolu, LA and Balcha, F},
title = {Molecular Diagnosis of Bacterial Meningitis in Ethiopia: A Narrative Review of Current Evidence and Implementation Gaps.},
journal = {The Canadian journal of infectious diseases &amp; medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale},
volume = {2026},
number = {},
pages = {4447180},
pmid = {42577913},
issn = {1712-9532},
abstract = {BACKGROUND: Bacterial meningitis (BM) remains a significant public health concern in Ethiopia, particularly due to diagnostic challenges posed by limited laboratory infrastructure, late patient presentation, and technical skills required for cerebrospinal fluid (CSF) sampling. Molecular diagnostic methods offer rapid, sensitive, and specific alternatives to conventional culture.<br><br>OBJECTIVE: This narrative review synthesizes available evidence on molecular diagnostics for BM in Ethiopia, evaluates their performance compared to conventional methods, and identifies barriers to implementation.<br><br>METHODS: A structured literature search was conducted in PubMed, Google Scholar, Scopus, and Web of Science (August 1977-September 2024) using the terms: Meningitis OR Bacterial meningitis AND Diagnostics OR Molecular diagnostics, including PCR, CRISPR, next-generation sequencing (NGS), and MALDI-TOF. The review used a repeatable research selection procedure, well-defined inclusion/exclusion criteria, and PRISMA reporting requirements. Studies from Ethiopia and other low-resource settings were included. After screening titles/abstracts and full texts, 66 articles were selected.<br><br>RESULTS: In Ethiopia, multiplex PCR detected bacterial DNA in 10%-22% of the CSF samples, whereas culture was positive in only 0.5%-1% of the cases, primarily due to pre-admission antibiotic use. Globally, molecular panels (e.g., BioFire FilmArray ME) show >&#x2009;90% sensitivity and specificity. Advanced techniques (CRISPR-Cas, NGS, and MALDI-TOF) have not yet been implemented in Ethiopian routine diagnostics.<br><br>CONCLUSION: Molecular methods vastly improve the detection of BM in Ethiopia, but high costs, infrastructure gaps, and shortage of trained personnel prevent widespread adoption. Molecular assays have outstanding analytical sensitivity, but culture remains the gold standard for confirmation of the viability of live pathogens. Implementation gaps require urgent targeted investment in point-of-care diagnostics and specialized transport networks.},
}
@article {pmid42578365,
year = {2026},
author = {Lv, S and Wang, Q and Gao, S and Wang, L and Zheng, L},
title = {Specific cleavage of 5' overhangs of non-target strand by Cas9 activated by target DNA binding.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42578365},
issn = {1362-4962},
support = {U23A20265//National Natural Science Foundation of China/ ; 32530084//National Natural Science Foundation of China/ ; 2408085MC072//Anhui Provincial Natural Science Foundation/ ; PA2025GDGP0026//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*DNA/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; DNA Cleavage ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; SARS-CoV-2/genetics ; *CRISPR-Associated Protein 9/metabolism ; Humans ; Base Sequence ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become a powerful genome-editing tool that uses RNA-DNA pairing to cleave target DNA with protospacer adjacent motif (PAM) sequences. While its primary function is well-studied, secondary activities remain poorly understood, causing unintended off-target effects. This study reports for the first time that Cas9 specifically cleaves the 5' overhang of the non-target strand (NTS) in target double-stranded DNA. This specific cleavage requires an additional PAM element at the NTS 5' region, is mediated by Cas9's RuvC domain, and is regulated by the HNH domain. It depends on the exact positioning of the NTS 5' end, but not on the overhang homopolymer sequence or overhang length. Adequate single-guide RNA-DNA complementarity is also essential. This discovery potentially advances our understanding of Cas9's enzymatic versatility to enhance genome-editing precision and efficacy and offers new nucleic acid detection strategies. Based on this cleavage, we developed a sensitive assay for Severe Acute Respiratory Syndrome Coronavirus 2 pseudovirus down to 2.4 copies μL-1, demonstrated extremely high sensitivity in diagnostic applications.},
}
@article {pmid42578374,
year = {2026},
author = {Zou, S and Ye, T and Fu, L and Zhou, JQ},
title = {A CRISPR/Cas9-induced blunt-end telomere system in S. pombe reveals RNase H2-dependent RNA primer removal at the terminal Okazaki fragment of lagging telomeres.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42578374},
issn = {1362-4962},
support = {2023YFA0913400//National Key Research and Development Program of China/ ; //Shanghai Academy of Natural Sciences/ ; },
mesh = {*Telomere/genetics/metabolism/chemistry ; *Schizosaccharomyces/genetics/metabolism ; *Ribonuclease H/metabolism/genetics ; *CRISPR-Cas Systems ; *RNA/metabolism/genetics ; DNA Replication/genetics ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; *DNA/genetics/metabolism ; },
abstract = {Studying the fine-scale dynamics of telomere replication has been hindered by the heterogeneity of native telomeres and the limitations of existing tools. Here, we report a highly efficient and inducible CRISPR/Cas9-mediated system for generating de novo telomeres with defined blunt ends in S. pombe. This fine setting allows for the precise dissection of post-replicative telomere end structures at near single-nucleotide resolution. Using this system, we show that the replicated leading-strand telomere is blunt-ended, while the lagging-strand counterpart contains an ∼10-nt 3' overhang resulting from RNA primer removal. By analyzing mutants deficient in ribonucleases, we found that the removal of this terminal RNA primer is specifically dependent on RNase H2, but not RNase H1. This RNase H2-dependent mechanism is essential for defining the mature structure of the lagging-strand telomere with authentic telomeric sequences. Our findings reveal a fundamental asymmetry in telomere end processing after replication and establish RNase H2 as the key enzyme responsible for resolving the terminal RNA primer in lagging-strand telomeres. This mechanism, conserved from budding to fission yeast, underscores the critical and evolutionarily ancient role of RNase H2 in defining eukaryotic telomere architecture.},
}
@article {pmid42579777,
year = {2026},
author = {Ma, Y and Deng, Y and Xu, J and Wang, S and Shi, B and Liang, K and Deng, R and Yang, H},
title = {Engineering CRISPR for Point-of-Care Tests.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01485},
pmid = {42579777},
issn = {2379-3694},
support = {2026YFHZ0080//International S and T Cooperation Program of Sichuan Province/ ; 22504094//National Natural Science Foundation of China/ ; 32271392//National Natural Science Foundation of China/ ; 32571536//National Natural Science Foundation of China/ ; 82270970//National Natural Science Foundation of China/ ; 82470967//National Natural Science Foundation of China/ ; //Sichuan University/ ; 2023NSFSC0333//Natural Science Foundation of Sichuan Province/ ; 2024NSFSC0676//Natural Science Foundation of Sichuan Province/ ; 2025ZNSFSC1041//Natural Science Foundation of Sichuan Province/ ; },
abstract = {CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.},
}
@article {pmid42580522,
year = {2026},
author = {Reis, BCC and Georget, C and Meunier, AC and de Andrade Silva, EM and Perin, C and Herbert, L and Dos Santos Lopes, N and Micheli, F and de Oliveira Mendes, TA},
title = {Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software.},
journal = {New biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.nbt.2026.07.005},
pmid = {42580522},
issn = {1876-4347},
abstract = {Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. High-resolution melting (HRM) analysis is an established approach for screening CRISPR/Cas-induced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wild-type melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.},
}
@article {pmid42580703,
year = {2026},
author = {Yamaji, M and Tabata, H and Nakamura, M and Fuse, R and Okada, T and Ohba, SI and Ohishi, T and Kawada, M and Saito, I and Sato, S and Nakanishi, T},
title = {Adenovirus Vector-Mediated In Vivo Knock-in Treatment of Neonatal Phenylketonuria Mice Using Terminally Cleaved Donor DNA.},
journal = {The journal of gene medicine},
volume = {28},
number = {8},
pages = {e70104},
pmid = {42580703},
issn = {1521-2254},
support = {JP21fk0108560//Japan Agency for Medical Research and Development/ ; JP24fk0310523//Japan Agency for Medical Research and Development/ ; JP19K06476//Japan Society for the Promotion of Science/ ; JP23K27095//Japan Society for the Promotion of Science/ ; JP24K21938//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Genetic Vectors/genetics/administration &amp; dosage ; *Gene Knock-In Techniques/methods ; Mice ; *Adenoviridae/genetics ; *Phenylalanine Hydroxylase/genetics ; *Phenylketonurias/therapy/genetics ; CRISPR-Cas Systems ; Genetic Therapy ; Disease Models, Animal ; Animals, Newborn ; Humans ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA/genetics ; },
abstract = {BACKGROUND: Adenovirus vectors (AdVs) are widely used and have an advantage of large insert capacity compared with adeno-associated virus vectors. However, AdVs have scarcely been used in genome-editing knock-in strategies because of low efficiency.<br><br>METHODS: Novel AdVs possessing a very large, 3.7&#x2009;kb donor DNA fragment and six or eight multiplex gRNA expression units were developed for CRISPR/Cas9-mediated knock-in to correct a phenylalanine hydroxylase (Pah) gene in a Pah[enu2] phenylketonuria mouse model. These AdVs were co-infected to Hepa1-6 cells or liver cells in&#xa0;vivo together with an AdV expressing either native Cas9 or Cas9 nickase (Cas9n) for double-nicking cleavage.<br><br>RESULTS: In vitro knock-in of the AdVs carrying 3.7&#x2009;kb donor DNA and six gRNA units targeting the cell genome was observed in both cases using Cas9 and Cas9n, though their efficiencies were low. Therefore, we generated AdVs carrying an additional two gRNA units that cleave the donor DNA terminus in the AdV genome via native Cas9 or Cas9 nickase. The knock-in efficiency increased approximately twofold for both vectors and reached a maximum of 8% for native Cas9 without selection. Newborn phenylketonuria model mice were intravenously administered the knock-in AdV together with the native-Cas9 AdV. Although the knock-in efficiency by homologous recombination occurred in only approximately 1% of hepatocytes, blood phenylalanine levels were reduced by up to 30%. Also, unintended fragments produced by nonhomologous end-joining were observed between the cleavage site at the terminus of the donor DNA in the AdV genome and the target site in the cell genome.<br><br>CONCLUSIONS: The knock-in efficiency of AdVs can be increased by cleaving the terminus of the donor DNA, although it would be desirable to avoid nonhomologous end-joining between double-strand break termini.},
}
@article {pmid42581041,
year = {2026},
author = {Kinney, KJ and Jia, K and Zhang, H and Schmaljohn, E and Osborne, T and Thommandru, B and Murugan, K and Sánchez-Peña, A and West, S and Chen, S and Codipilly, R and Sturgeon, M and Turk, R and McNeill, MS and Behlke, M and Jacobi, A and Cromer, MK and Rettig, G and Kurgan, GL},
title = {UNCOVERseq enables sensitive and controlled gene editing off-target nomination across CRISPR-Cas modalities and systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42581041},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Hematopoietic Stem Cells/metabolism ; DNA Breaks, Double-Stranded ; },
abstract = {The rapid expansion of CRISPR-Cas gene editing enables new therapeutic strategies but complicates assessment of unintended editing risks due to emerging modalities and unclear analytical standards. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow that sensitively identifies rare off-target events using defined inputs and analytical process controls. Using an inter-method off-target confirmation benchmarking dataset, UNCOVERseq demonstrates high analytical sensitivity (97.6%) and precision (78%), outperforming published nomination methods. We apply UNCOVERseq across 192 guide RNAs and identify six guides spanning a broad specificity range, enabling relative risk assessment across S. pyogenes Cas9, high-fidelity variants, and base editors in hematopoietic stem and progenitor cells. We further show that double-strand break nomination sites retain strong rank-order concordance with single-strand break-mediated base editing. Together, these results establish UNCOVERseq as a robust framework for informed off-target risk assessment in translational gene-editing systems.},
}
@article {pmid42086878,
year = {2026},
author = {Wang, MM and Li, Y and Ho, CEH and Yu, WSS and Coupland, SE and Park, G and Chan, ASY and Figueiredo, CR},
title = {Novel CRISPR-Cas9 BAP1 knockout pre-clinical tumor model recapitulates human melanoma tumorigenesis and immune evolution.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42086878},
issn = {2399-3642},
mesh = {Animals ; *Ubiquitin Thiolesterase/genetics ; Humans ; *Tumor Suppressor Proteins/genetics ; *Melanoma/genetics/immunology/pathology/metabolism ; *CRISPR-Cas Systems ; Mice ; *Carcinogenesis/genetics ; Mice, Knockout ; Disease Models, Animal ; Cell Line, Tumor ; Uveal Melanoma ; *Skin Neoplasms/genetics/pathology/immunology ; Uveal Neoplasms/genetics/immunology/pathology ; Female ; },
abstract = {BAP1-deficient melanocytic tumors exhibit strong immunosuppressive features and poor prognosis. Currently, no immune-competent preclinical models exist to study their tumor-immune interactions or test new immunotherapies. This limitation hinders progress in understanding how BAP1 loss drives tumor aggressiveness and immune evasion. To address this, we generate a syngeneic BAP1 knockout melanocyte tumor line using CRISPR-Cas9. We then evaluate its functional and immunological impact in immune-competent mice, including its ability to recapitulate metabolic and immunosuppressive features of human BAP1-deficient melanomas. The selected knockout clone exhibits hallmarks of aggressive skin and intraocular melanomas, including epithelioid morphology, in vivo tumorigenic potential, rapid growth, and key immunosuppressive features, mirroring those observed in human BAP1-deficient melanomas. Cross-species single-cell transcriptome analysis demonstrates strong molecular overlap between BAP1 knockout mouse tumors and high-risk (class 2) human uveal melanomas, highlighting shared pathways in lipid metabolism, transmembrane receptor signaling, and immune modulation. Gene Set Enrichment Analysis confirms that lipid metabolic reprogramming, previously described in human tumors, is also a key feature of our model, validating its ability to recapitulate human disease biology. This study introduces a syngeneic preclinical model that mimics the immunosuppressive landscape of BAP1-deficient melanocytic tumors, enabling the development and optimization of new combination immunotherapies.},
}
@article {pmid42120907,
year = {2026},
author = {Jajarmi, J and Guest, MR and Ma, LJ and Thu, KL and Chari, R and Lockwood, WW},
title = {Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42120907},
issn = {2399-3642},
support = {N/A//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; HHSN261201500003I/CA/NCI NIH HHS/United States ; HHSN261201500003I//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Promoter Regions, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Library ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Mice ; Gene Editing ; Plasmids/genetics ; },
abstract = {CRISPR-Cas9 screening relies on uniform representation of single-guide RNA (sgRNA) libraries to enable accurate gene discovery. However, technical biases during library preparation can compromise screen performance. Here we show that commonly used "all-in-one" CRISPR vectors expressing both Cas9 and sgRNAs drive unintended Cas9 protein expression in Escherichia coli during plasmid amplification. This bacterial Cas9 expression causes guide-specific toxicity, leading to selective loss of sgRNAs and highly skewed library representation. We demonstrate that this effect occurs across multiple bacterial strains and affects both targeted and genome-wide libraries, including widely used human CRISPR libraries. Mechanistically, toxicity is driven by Cas9 expression rather than plasmid size and is only partially alleviated by catalytically inactive Cas9. Importantly, replacing the EF-1α promoter with a mouse phosphoglycerate kinase promoter suppresses Cas9 expression in bacteria while preserving genome editing efficiency in mammalian cells, restoring sgRNA uniformity. These findings identify a previously unrecognized source of bias in CRISPR library preparation and provide a practical solution to improve screening fidelity.},
}
@article {pmid42213648,
year = {2026},
author = {Kaminski, PJ and Min, K and Traxler, EA and Khandros, E and Abdulmalik, O and Godfrey, B and Keller, CA and Giardine, BM and Hardison, RC and Shi, J and Blobel, GA},
title = {Dissecting polycomb complexes for enhanced fetal hemoglobin production.},
journal = {Blood},
volume = {148},
number = {7},
pages = {882-895},
doi = {10.1182/blood.2026033804},
pmid = {42213648},
issn = {1528-0020},
mesh = {*Fetal Hemoglobin/genetics/biosynthesis/metabolism ; Animals ; Humans ; Mice ; *Enhancer of Zeste Homolog 2 Protein/genetics/metabolism ; Erythroid Cells/metabolism ; CRISPR-Cas Systems ; Polycomb Repressive Complex 2/metabolism/genetics ; Histones/metabolism ; *Polycomb-Group Proteins/metabolism/genetics ; },
abstract = {Polycomb repressive complex 1 (PRC1) and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin (Hb) production, a process whose reversal is a goal for the treatment of sickle cell disease and β-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal Hb (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-CRISPR-associated protein 9 (Cas9) mutagenesis screen identified a region in EZH2 in which Cas9 induced exon 14 skipping (EZH2Δ14). EZH2Δ14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2Δ14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human β-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting nonenzymatic domains within chromatin-modifying complexes.},
}
@article {pmid42486984,
year = {2026},
author = {Meng, H and Lei, Z and Yan, Y and Wang, L and Zhang, S and Rao, X and Shao, C and Zhang, X and Chen, K and Yang, L and Liu, R and Yang, G and Shen, R and Gu, R and Wang, X and Wang, Y and Lu, S and Lv, Z and He, B and Wen, H and Li, D and Yi, C},
title = {Precise DNA base editing using AlphaFold3-based contact modelling.},
journal = {Nature},
volume = {656},
number = {8127},
pages = {463-473},
pmid = {42486984},
issn = {1476-4687},
mesh = {*DNA/genetics/chemistry/metabolism ; *Gene Editing/methods ; *Models, Molecular ; CRISPR-Cas Systems/genetics ; Adenine/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Substrate Specificity ; CRISPR-Associated Proteins/metabolism/chemistry ; Base Sequence ; Adenosine Deaminase ; Escherichia coli Proteins ; },
abstract = {Achieving high specificity in biochemical transformations is crucial for research and therapeutics. This is particularly important for genome editing, where enhancing tool specificity ensures effective and precise editing outcomes[1,2]. Current strategies are constrained by activity-specificity trade-offs, high labour intensity and low success rates[3,4]. Here we present ContactSeek, an artificial-intelligence-driven framework that uses AlphaFold3 (AF3)-predicted contact probability[5] to improve the specificity of genome editors. Using Cas9-TadA adenine base editors[6-8] as a demonstration, we mapped their genome-wide off-targets and fed the off-target DNA sequences to AF3. Among AF3 outputs, we found that contact probability was more sensitive than predicted three-dimensional structures for detecting differential interactions between on- and off-target complexes. Correlating contact probability with sequencing-based off-target signals, ContactSeek identified and ranked consensus contact regions, which are neighbouring Cas residues with consistent contact changes to DNA/guide RNA, and pinpointed specificity-determining residues within them. ContactSeek can also be applied modularly and identified key residues in the TadA8e deaminase. Targeted amplicon sequencing, genome-wide profiling, R-loop assay and RNA-sequencing together confirmed the greatly enhanced specificity; our best variant, combining two mutations of Cas9 and TadA8e, outperformed several known high-fidelity adenine base editors. ContactSeek is also generalized to Cas12a-based cytosine base editors. Collectively, our framework represents an AF3-driven model tailored for specificity improvement, establishing a paradigm for improving the precision of genome editing tools through the integration of structural and functional dimensions.},
}
@article {pmid42570142,
year = {2026},
author = {Ahad, A and Hullon, D and Singh, T and Dabiry, SM and Sakthivel, L and Padaria, J and Rekhraj, AS and Bhattacharjee, A},
title = {The Vascular Genome as a Therapeutic Target: A Systematic Review of CRISPR-based Gene Editing In Vascular Disease.},
journal = {Cardiovascular toxicology},
volume = {26},
number = {8},
pages = {},
pmid = {42570142},
issn = {1559-0259},
mesh = {Humans ; Animals ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; *Vascular Diseases/genetics/therapy/metabolism/physiopathology ; *CRISPR-Cas Systems ; Vascular Remodeling/genetics ; Genetic Predisposition to Disease ; Phenotype ; },
abstract = {Despite advances in therapy, arterial, venous, and pulmonary vascular diseases remain leading causes of morbidity and mortality. Persistent endothelial dysfunction, inflammation, oxidative stress, and maladaptive vascular remodeling continue to drive disease progression and residual risk. CRISPR/Cas9 technology offers a unique opportunity to modify the molecular pathways underlying vascular pathophysiology directly. The PRISMA 2020 guidelines guided the systematic review. The databases PubMed/MEDLINE, Embase, Web of Science, Cochrane Library, ClinicalTrials.gov, and Google Scholar were searched from their inception until September 2025 for experimental and/or clinical studies evaluating the application of CRISPR/Cas9 on vascular disease. Included were in vitro studies, animal model studies, and early-phase human studies aimed at targeting the endothelial cell regulatory pathways, inflammatory pathways, metabolic remodeling processes, and hereditary causes of vasculopathy. Seventeen studies met the inclusion criteria. CRISPR technologies targeting PCSK9, NOS3, HIF1A, NLRP3, METTL4, BMPR2, and ACTA2 were identified to enhance repair mechanisms in endothelial cells, regulate inflammation, modulate lipid metabolism, and remodel the vascular system. The human studies demonstrated sustained gene silencing effects following a single dose of CRISPR-induced in vivo editing. The use of CRISPR technology to edit cell genomes offers potential to alter disease progression in vascular medicine, with a growing body of translational evidence supporting the feasibility and durability of the approach.},
}
@article {pmid42570236,
year = {2026},
author = {Jiang, S and Tian, X and Wang, F and Han, W and She, Q and Feng, X},
title = {Recruitment of Cas3 enables DNA cleavage by the type I-A CRISPR-Cas system of Saccharolobus islandicus.},
journal = {Cell reports},
volume = {45},
number = {8},
pages = {117820},
doi = {10.1016/j.celrep.2026.117820},
pmid = {42570236},
issn = {2211-1247},
abstract = {Type I CRISPR-Cas systems constitute the most prevalent prokaryotic adaptive immune pathways and are classified into seven subtypes (I-A to I-G). These antiviral systems typically exploit a Cascade complex for RNA-guided DNA recognition and a Cas3 helicase-nuclease effector for DNA degradation, yet their diverse activation mechanisms remain not fully understood. In this study, we isolate the I-A Cascade from Saccharolobus islandicus, revealing a minimal form of Cascade lacking both Cas3 and the CRISPR-RNA maturase Cas6. Cas3 is recruited to the R-loop structure formed after Cascade binding to target DNA, which activates the effector for both cis- and trans-DNA cleavage. Strikingly, ATP not only enables the processive target degradation by Cas3 but also suppresses the trans-cleavage of the same enzyme. Together, the Sa. islandicus I-A system operates via target-dependent Cas3 recruitment-a mechanism distinct from other characterized I-A systems, thus underscoring the mechanistic diversity within type I CRISPR-Cas immunity.},
}
@article {pmid42571080,
year = {2026},
author = {Ullah, O},
title = {Breaking the growth-defense trade-off in cereal crops: CRISPR/Cas and moonlighting proteins in biotic stress resistance.},
journal = {Molecular breeding : new strategies in plant improvement},
volume = {46},
number = {8},
pages = {80},
pmid = {42571080},
issn = {1572-9788},
abstract = {Recurrent crop disease outbreaks linked to global warming pose challenges to sustainable food production. Conventional plant breeding techniques may become less effective at addressing these threats, as improving disease resistance often causes yield reduction. Amid these challenges, CRISPR/Cas-based gene editing offers targeted and tractable solutions. This review synthesizes recent approaches to uncoupling immunity from productivity in cereals. We show that susceptibility (S) gene disruption can provide resistance without activating costly defense mechanisms. We also discuss the generation of new alleles through targeted modifications that mitigate autoimmunity-associated fitness costs. Here, we propose CRISPR-mediated de-moonlighting, an approach for decoupling multifunctional protein activities. Multiplex editing of minor resistance loci, especially in polyploids such as wheat, offers long-lasting, broad-spectrum protection. These strategies converge on the manipulation of canonical moonlighting proteins, multifunctional signaling hubs, and pleiotropic regulators, which serve as regulatory nodes that link development and immunity. CRISPR-mediated precision modification of these regulators can fine-tune the growth-defense balance. Combining these approaches with systems biology, AI-driven design and advanced breeding pipelines can help develop high-yielding, disease-resistant cereals for sustainable agriculture.},
}
@article {pmid42571242,
year = {2026},
author = {Eken, JA and Havenaar, FRM and de Groen, RAL and Quinten, E and Mei, H and Sepúlveda-Yáñez, JH and Navarrete, MA and Drexler, HG and Vermaat, JSP and van Bergen, CAM and Veelken, H},
title = {Alternative oncogenic drivers and sensitivity to BTK inhibition in CRISPR/Cas gene-edited human DLBCL cell models.},
journal = {Blood neoplasia},
volume = {3},
number = {3},
pages = {100261},
pmid = {42571242},
issn = {2950-3280},
abstract = {Diffuse large B-cell lymphoma (DLBCL) can be subclassified by phenotype into germinal center B-cell-like and activated B-cell-like (ABC) subtypes and by recurrent potentially oncogenic mutations into 5 to 7 genetic clusters. In ABC-DLBCL, potentially oncogenic mutations frequently occur in genes involved in B-cell receptor (BCR) signaling and NF-κB activation. Autonomous BCR signaling acts as an alternative immunologic driver predominantly in ABC-type DLBCL that cannot be captured by either subclassification system. The relative functional contribution and interdependence of these mechanistically diverse oncogenic drivers have not been completely defined. To directly compare the effects of autonomously signaling BCR and signalosome-activating CARD11 mutations on NF-κB activation and survival of ABC-DLBCL, we reciprocally exchanged these driver mechanisms in the MYD88[L265P]-mutated ABC-DLBCL cell lines TMD8 and OCI-Ly3. Only CARD11[L251P] (not CARD11[K215N], CARD11[D230N], and CARD11[R337Q]) compensated TMD8 cells for the loss of autonomous BCR signaling, as indicated by survival of BCR knockout and conversion to complete resistance to acalabrutinib. Transduction of the TMD8 BCR rescued OCI-Ly3 cells from replacing the CARD11[L215P] variant with CARD11[WT]. The autonomous TMD8 BCR signal provided a slight growth advantage over CARD11[L251P]-driven cells in both reciprocal systems. Unsupervised clustering of genetically engineered TMD8 and OCI-Ly3 clones demonstrated tight clustering with their parental cells and only minor alterations of cellular pathways. Only the strongest signalosome-activating mutation has functional near-equivalency to an autonomously signaling BCR for NF-κB activation and growth and survival in ABC-DLBCL. Quantifying the effects of co-occurring potential NF-κB-activating mechanisms is essential to predict Bruton tyrosine kinase (BTK) inhibition sensitivity in individual ABC-DLBCL cases.},
}
@article {pmid42572397,
year = {2026},
author = {Baoling, L and Lina, S and Tong, X and Qinrui, J and Ling, Z and Zhiwen, X},
title = {A Rapid, Field-Deployable Diagnostic Platform for Getah Virus Based on RT-RAA and CRISPR EsCas13d.},
journal = {Microbial biotechnology},
volume = {19},
number = {8},
pages = {e70429},
pmid = {42572397},
issn = {1751-7915},
support = {2024YFD1800102//National Key Research and Development Program of China during the 14th Five-Year Plan: Research and Development of Key Technologies for the Prevention and Control of Important Diseases in Wild Animals/ ; 2024YFD1800500//Research and Application of Integrated Prevention, Control and Purification Technologies for Major Swine Epidemic Diseases/ ; 2021ZDZX0010-3//Sichuan Province Major Science and Technology Project for Sichuan Pigs during the 14th Five-Year Plan Period/ ; sccxtd-2024-08//Sichuan Pig Innovation Team of the National Modern Agricultural Industry Technology System/ ; },
mesh = {Animals ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; RNA, Viral/genetics ; Reverse Transcription ; *CRISPR-Cas Systems ; Temperature ; },
abstract = {The emerging zoonotic Getah virus (GETV) poses an increasing threat to both animal and human health, underscoring the need for rapid, sensitive and field-deployable diagnostic tools. In this study, we developed and optimized a rapid, one-step, visual detection (ROSVD) platform for GETV by integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-EsCas13d-mediated collateral RNA cleavage. Notably, the ROSVD assay uses a simplified sample-preparation strategy based on rapid nucleic acid release, eliminating the need for conventional nucleic acid extraction and purification. The entire workflow, including amplification and detection, is completed within 30 min at 37°C or ambient temperature (25°C) without specialized instrumentation. Detection results can be visualized directly under ultraviolet light or with a lateral flow assay. At 37°C, the ROSVD assay achieved sensitivity comparable to RT-qPCR, and evaluation of clinical specimens showed 100% concordance with RT-qPCR results. Collectively, these findings demonstrate that ROSVD is a rapid, sensitive, cost-effective and instrument-independent diagnostic platform, providing a practical solution for on-site surveillance of GETV and a versatile framework for the detection of other emerging RNA pathogens.},
}
@article {pmid42573069,
year = {2026},
author = {Fan, X and Liang, L and Wang, H and Liu, G and Tan, H and Zhang, F and Liu, L and Liu, R},
title = {A self-iterative orthogonal base-editing platform enables multiplex N-to-N diversification and genome-scale functional screening in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42573069},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 22278058//National Natural Science Foundation of China/ ; 22578048//National Natural Science Foundation of China/ ; 22208044//National Natural Science Foundation of China/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project for Young Faculty/ ; XLYC2203075//Xingliao Talent Plan/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Gene Editing/methods ; *Genome, Bacterial ; Butanols/metabolism ; Escherichia coli Proteins/genetics/metabolism ; DNA Repair/genetics ; Codon/genetics ; Gene Library ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Base editing enables precise genome modification without double-strand breaks but remains limited by narrow editing windows, DNA repair pathway biases, and restricted nucleotide diversity. Here, we report MUTATOR, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli. MUTATOR combines CWBE and ABE with iterative editing on two complementary DNA strands, thereby overcoming endogenous DNA repair constraints and expanding A-to-N and C-to-N editing outcomes across both strands. This strategy substantially expands accessible nucleotide outcomes, codon variants, and amino-acid diversity within existing editing windows relative to conventional editors. Using four gRNAs, MUTATOR facilitated four-site editing of ompR, generating 84 distinct amino-acid combinations and 252 codon combinations, with the synonymous OmpR_P160P variant increasing isobutanol production by up to 56.2%. We further applied MUTATOR to a 151-gene library encompassing transcriptional regulators, translation factors, DNA repair proteins, ribosomal components, and NAD(P)H-associated metabolic genes, identifying single and combinatorial mutations that markedly enhanced cell growth and ethanol utilization when ethanol was used as the sole carbon source. Together, these results establish MUTATOR as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.},
}
@article {pmid42573087,
year = {2026},
author = {Guy, J and Hein, E and Alexander-Howden, B and von Bock Und Polach, T and Mathieson, T and Kleinstiver, BP and Zoghbi, HY and Bird, A},
title = {Translational reading frame predicts the pathogenicity of C-terminal frameshift deletions in MeCP2.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42573087},
issn = {2050-084X},
support = {10.35802/222507//Wellcome/ ; 13669477_13669480//Rett Syndrome Research Trust/ ; DP2CA281401/NH/NIH HHS/United States ; 13800235_1380239//Simons Initiative for the Developing Brain/ ; },
mesh = {*Methyl-CpG-Binding Protein 2/genetics/metabolism ; Animals ; Humans ; *Frameshift Mutation ; Mice ; *Rett Syndrome/genetics/pathology ; Disease Models, Animal ; *Sequence Deletion ; },
abstract = {Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) removes ~100 amino acids and accounts for approximately 10% of RTT-causing mutations. Their pathogenicity is unexpected because this C-terminal domain is dispensable in mice. Analysis of pathogenic and benign human MECP2 variants reveals that some individuals with apparently typical CTDs do not develop Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human sequence data and mouse models we show that pathogenicity results from a marked reduction in MeCP2 levels and depends on the presence of a proline proline stop motif (-PPX) generated by a shift to the +2 reading frame. CTDs that shift to the +1 frame avoid this motif and are benign. Replacing the stop codon of the PPX motif with tryptophan restores MeCP2 expression and rescues RTT-like phenotypes in a CTD mouse model. An adenine base editor efficiently introduces this substitution in cultured cells. These findings define a reliable prognostic distinction between benign and pathogenic CTDs and establish a potential editing strategy for correcting disease-causing CTD mutations.},
}
@article {pmid42573653,
year = {2026},
author = {Nguyen, BD and Kolluri, SK},
title = {Genome-wide CRISPR screen reveals CGS-15943 induced heme-dependent cell death mediated by aryl hydrocarbon receptor in lung cancer cells.},
journal = {Apoptosis : an international journal on programmed cell death},
volume = {31},
number = {8},
pages = {},
pmid = {42573653},
issn = {1573-675X},
mesh = {Humans ; *Receptors, Aryl Hydrocarbon/genetics/metabolism ; *Heme/metabolism ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; Cell Line, Tumor ; *Apoptosis/drug effects ; Cell Death/drug effects ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic/drug effects ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Oxidative Stress/drug effects ; },
abstract = {Induction of programmed cancer cell death by selective aryl hydrocarbon receptor (AHR) ligands represents a promising strategy for developing novel anticancer therapeutics. In this study, we characterized the anticancer activity and underlying mechanism of the selective AHR ligand CGS-15943 in lung cancer cells. CGS-15943 potently inhibited the growth of lung cancer cell lines expressing high levels of AHR, whereas CRISPR-mediated knockout of AHR in H460 and H69AR cells markedly rescued cells from CGS-15943-induced cell death, demonstrating an essential role for AHR. To identify additional mediators of this response, we performed a genome-wide CRISPR knockout screen, which revealed eight enzymes involved in the heme biosynthesis pathway, three heme-containing enzymes, as well as AHR and its transcriptional partner ARNT, as critical determinants of CGS-15943-induced cell death. Transcriptomic analyses further showed that CGS-15943 induced AHR-dependent transcriptional programs enriched for oxidative stress and oxidized phospholipid response pathways. Together, these findings identify key components of the AHR signaling network that regulate a programmed heme-dependent cell death pathway and establish CGS-15943 as a promising lead compound for targeting AHR-positive lung cancers.},
}
@article {pmid42573697,
year = {2026},
author = {Ward, JD},
title = {Protein Depletion in Caenorhabditis elegans Using the Auxin-Inducible Degradation System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3069},
number = {},
pages = {169-185},
pmid = {42573697},
issn = {1940-6029},
mesh = {Animals ; *Indoleacetic Acids/pharmacology/metabolism ; *Caenorhabditis elegans/genetics/metabolism ; F-Box Proteins/genetics/metabolism ; Degrons ; *Proteolysis/drug effects ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; Receptors, Cell Surface/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knock-In Techniques/methods ; Arabidopsis/genetics ; },
abstract = {The auxin-inducible degradation (AID) system is a powerful tool in modern molecular genetics that allows for conditional protein depletion. In Caenorhabditis elegans, tools exist to allow for rapid, tissue-specific depletion of target proteins. The system requires tagging a gene of interest with an AID degron and a transgene expressing the Arabidopsis thaliana TIR1 F-box protein, which can form a functional SCF ubiquitin ligase with endogenous Skp and Cullin proteins. Here, I describe how to generate degron knock-ins by CRISPR, cross them to TIR1-expressing strains, and perform depletion experiments. I provide a description of our current methods and highlight alternative approaches.},
}
@article {pmid42323652,
year = {2026},
author = {Liu, X and Wang, Y and Wong, JCY and Zhang, X and Wu, C and Chen, P and Yang, Y and Liu, P and Lau, CS and Cook, M and Li, PH},
title = {Modeling Hereditary Angioedema With Personalized Expanded Potential Stem Cell-Derived Hepatocytes: A CRISPR-Validated Platform for Mutation-Specific Mechanisms and Therapeutic Innovation.},
journal = {Allergy},
volume = {81},
number = {8},
pages = {2858-2873},
pmid = {42323652},
issn = {1398-9995},
support = {//the InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government/ ; },
mesh = {Humans ; *Hepatocytes/metabolism/cytology ; *Mutation ; *Angioedemas, Hereditary/genetics/therapy/metabolism ; Cell Differentiation ; *Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; Complement C1 Inhibitor Protein/genetics/metabolism ; Models, Biological ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Precision Medicine ; },
abstract = {Hereditary angioedema (HAE) with C1 esterase inhibitor (C1INH) deficiency is caused by pathogenic SERPING1 mutations that disrupt production of the plasma protease inhibitor C1INH. However, the molecular mechanisms and consequences of patient-specific mutations remain poorly understood due to the lack of physiologically relevant human models. Here, we established a personalized, isogenic, stem-cell-derived hepatocyte platform to investigate the underlying mutation-specific mechanisms of HAE. Specifically, peripheral blood mononuclear cell (PBMC)-expanded erythroblasts from four representative HAE-C1INH-Type1 patients containing distinct point, insertion, deletion, or large fragment SERPING1 mutations were reprogrammed into expanded potential stem cells (EPSCs) and further differentiated into hepatocyte-like cells (HLCs). These HLCs exhibited appropriate transcriptional transitions, mature hepatic features, and C1INH secretion comparable to that observed in human plasma. All patient-derived HLCs demonstrated impaired C1INH secretion with mutation-specific differences in both SERPING1 transcription and intracellular accumulation. Moreover, to verify that the mutations directly drive the phenotype, we performed CRISPR/Cas9-mediated genome repair, which restored SERPING1 mRNA expression and C1INH secretion. Conversely, identical patient mutations installed into healthy EPSCs showed the same transcriptional and secretory defects, confirming sufficiency. Collectively, we have established a robust human hepatocyte model that accurately recapitulates key hepatocyte-specific aspects of HAE pathophysiology and provides a scalable foundation for investigation of future precision therapies.},
}
@article {pmid42389905,
year = {2026},
author = {Minaiyan, G and Aussel, C and Ammann, S and Cathomen, T},
title = {Genome Editing for Familial Hemophagocytic Lymphohistiocytosis: Design Principles, Challenges, and Translational Perspectives.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {702-712},
doi = {10.1177/10430342261465555},
pmid = {42389905},
issn = {1557-7422},
mesh = {Humans ; *Lymphohistiocytosis, Hemophagocytic/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; Translational Research, Biomedical ; Perforin/genetics ; Membrane Proteins/genetics ; Mutation ; CRISPR-Cas Systems ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by genetic defects in cytotoxic lymphocyte function. Current therapies can control disease activity, but transplantation of allogeneic hematopoietic stem and progenitor cells (HSPCs) remains the only curative option and is associated with substantial risks. These limitations have accelerated development of genome editing approaches enabling precise correction of disease-causing mutations in autologous cells. Familial HLH (FHL) represents a compelling target for genome editing, but successful and safe clinical translation has remained challenging. Preclinical studies demonstrate that targeted editing of key genes, such as PRF1 and UNC13D, can restore cytotoxic function in HSPCs and T cells. Translation to the clinic, however, depends on multiple factors, including the choice of target cell population, the level of functional correction required, and gene-specific constraints such as locus complexity and regulation of gene expression. In this review, we synthesize current progress in genome editing for FHL and highlight critical biological and technical barriers to clinical implementation. We propose a conceptual framework for designing genome editing strategies tailored to FHL, emphasizing the alignment of editing platform, gene architecture, and cellular context to enable effective and clinically translatable therapies.},
}
@article {pmid42393087,
year = {2026},
author = {Weickert, P and Liu, Y and Strecker, J},
title = {Prokaryotic Schlafen proteins cleave tRNAs during type III CRISPR immunity.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42393087},
issn = {2041-1723},
support = {DP2HL185100//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {*RNA, Transfer/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Schlafen nucleases restrict viral infection in mammals by cleaving self RNAs, however, their function and mechanism in prokaryotic immunity is unknown. Here, we uncover CRISPR-associated Schlafen (Cash) proteins containing a Schlafen domain fused to Csx15, an uncharacterized member of Rossmann-like nucleotide-binding sensors. Cash is activated by cyclic tetra-adenylate (cA4) produced during type III CRISPR interference and induces cell toxicity by cleaving tRNAs, primarily in the T-loop. Cryo-electron microscopy structures of Chloroflexi bacterium Cash reveal an inactive dodecamer, the formation of a filament upon cA4 binding to align catalytic interfaces, and the molecular basis of substrate recognition and cleavage in a tRNA-bound complex. We identify numerous families of prokaryotic Schlafen proteins associated with diverse antiviral defense systems and characterized by unique sensor domains. This work highlights tRNA depletion by Schlafen nucleases as an evolutionary recurring antiviral strategy and reveals mechanistic differences between Cash and human Schlafen members.},
}
@article {pmid42424999,
year = {2026},
author = {Dong, J and Li, X and Gu, T and Zhang, Y and Deng, L and Luo, X and Hou, C and Huo, D},
title = {Compartmentalization-inspired dual-chamber CRISPR sensing coupled with single-atom electrocatalysis for crosstalk-free multiplex microRNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119016},
doi = {10.1016/j.bios.2026.119016},
pmid = {42424999},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis/blood ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Electrochemical Techniques ; Nucleic Acid Amplification Techniques ; Catalysis ; Equipment Design ; },
abstract = {Compartmentalization is a hallmark of cells, enabling parallel biochemical processes to proceed with high fidelity and minimal interference. Drawing inspiration from this spatial isolation principle, we developed a compartmentalization-inspired dual-chamber sensing platform for crosstalk-minimized multiplex miRNA analysis. In each physically isolated compartment, the target miRNA directs padlock-probe ligation to form a circular template, followed by rolling-circle-extension-driven loop-mediated isothermal amplification (R-LAMP). The resulting amplicons specifically activate the corresponding CRISPR/Cas12a-crRNA complex, triggering trans-cleavage of a hairpin-DNA biogate that seals Fe-MOF nanocontainers. Gate opening releases distinct electroactive reporters (3,3',5,5'-tetramethylbenzidine, TMB; or methylene blue, MB) from their respective chambers. After the compartmentalized reactions finish, the two supernatants are combined and read out on a screen-printed electrode modified with a Co-N-C single-atom catalyst, producing two well-resolved DPV peaks for simultaneous quantification. The platform achieves femtomolar detection limits in simultaneous assays (0.87 fM for miRNA-21 and 0.72 fM for miRNA-155), a broad linear range (1 fM-100 pM), and high discrimination against non-cognate or mismatched sequences. Accurate recoveries in diluted human serum and consistent trends in cell lysates (validated by RT-qPCR) confirm practical applicability. By integrating bioinspired compartmentalization with CRISPR precision and single-atom electrocatalysis, this platform provides a generalizable route to multiplex nucleic acid diagnostics with enhanced fidelity and sensitivity.},
}
@article {pmid42431049,
year = {2026},
author = {Ji, S and Wang, B and Yan, Y and Zhang, C and Ren, H and Yang, J and Wei, X and Hou, W and Huang, M and Song, J and Wang, S and Qiu, L and Wang, H},
title = {TOPS-CRISPR: Thermally-regulated and oligonucleotide-mediated one-pot CRISPR-Cas12a assay for ultra-sensitive and rapid on-site diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {118998},
doi = {10.1016/j.bios.2026.118998},
pmid = {42431049},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; Oligonucleotides/chemistry/genetics ; *Brucella/isolation &amp; purification/genetics/pathogenicity ; *Brucellosis/diagnosis/microbiology ; Rapid Diagnostic Tests ; Temperature ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; },
abstract = {CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.},
}
@article {pmid42447864,
year = {2026},
author = {Langley, J and Baudrier, L and Curry, J and Narta, K and Todesco, HM and Potts, K and Morrissy, S and Mahoney, DJ and Billon, P},
title = {Compound delivery of eVLPs enhances prime editing for targeted genome engineering and high-throughput screening.},
journal = {Cell genomics},
volume = {6},
number = {8},
pages = {101302},
doi = {10.1016/j.xgen.2026.101302},
pmid = {42447864},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; *Virion/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Engineered virus-like particles (eVLPs) enable transgene-free ribonucleoprotein delivery for genome editing, yet optimized strategies for high-throughput applications remain unexplored. Prime editing enables precise genomic modifications but suffers from limited efficiency. Here, we present PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), which exploits eVLP-mediated editing kinetics through multiple sequential sub-saturating transductions at optimal intervals. PRIME-VLP achieves 1.5- to 2.9-fold improvements in prime editing efficiency across diverse genomic targets and cell types without increasing off-target editing, compromising cellular viability or causing transcriptional perturbations. By decoupling pegRNA and editor delivery through pegRNA-free eVLPs, PRIME-VLP enables pooled prime editing screens and circumvents transgene silencing limitations. Using a 6,000-pegRNA library targeting TP53, PRIME-VLP achieved 2.8-fold higher editing and improved reproducibility compared to conventional lentiviral delivery, identifying TP53 loss-of-function variants conferring Nutlin-3 resistance. This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics.},
}
@article {pmid42470832,
year = {2026},
author = {Azhar, M and Malviya, R and Chandra, P and Sridhar, SB and Shareef, J and Wadhwa, T},
title = {Precision prime editing of TP53 mutations for functional tumor suppression in colorectal cancer.},
journal = {Biochemical and biophysical research communications},
volume = {831},
number = {},
pages = {154311},
doi = {10.1016/j.bbrc.2026.154311},
pmid = {42470832},
issn = {1090-2104},
mesh = {*Colorectal Neoplasms/genetics/therapy ; Humans ; *Mutation ; *Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; Precision Medicine/methods ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is a major global health concern, with high mortality due to genetic heterogeneity and resistance to treatment. Tumor Protein p53 (TP53) mutations are also among the most important molecular changes that can disrupt genomic stability and facilitate tumor progression, so it is a critical target for precision-based interventions.<br><br>AIM: This review aims to discuss the future potential of prime editing as a new generation of genome engineering to identify precise approaches to correct TP53 mutations in colorectal cancer.<br><br>METHOD: A focused literature review was conducted on PubMed, Scopus, Web of Science, and Google Scholar for articles published between the years of 2010 and 2026. The keywords used in the search were CRC, TP53 mutation, prime editing, Prime Editing Guide RNA (pegRNA), CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9), and precision oncology. Studies were screened for experimental, mechanistic, and translational, and the focus was on mutation-specific editing, delivery platforms, organoid validation, clinically relevant barriers, etc. RESULTS/DISCUSSION: Prime editing is a programmable search-and-replace method that does not involve two single-stranded Deoxyribonucleic Acid (DNA) breaks, resulting in fewer Insertions/deletions (indels) and greater precision compared with traditional CRISPR-Cas9 approaches. Recent systems like Prime Editor Max (PEmax), PE5/PE5max, engineered pegRNAs, twin prime editors, PrimeDel, and PASTE have enhanced the efficiency, range, and flexibility. Hotspot and organoid studies suggest that variants of TP53, particularly R175H, R248Q/W, R273&#x202f;H/C, and R282W, can be repaired. But cargo size, delivery specificity, tumor heterogeneity, varying cargo editing efficiency, cargo recognition by the immune system, and off-target risk are all barriers to clinical translation.<br><br>CONCLUSION: Precision oncology with prime editing has the potential to be a useful tool for CRC, though optimized delivery, thorough preclinical testing, and safety monitoring will be required for therapeutic adoption.<br><br>ORIGINALITY: This review combines TP53 hotspot biology, recent breakthroughs in prime editing technology, and CRC-specific translational challenges, and provides a step-by-step approach to its clinical application in a unique way.},
}
@article {pmid42472526,
year = {2026},
author = {Liu, Z and Wang, J and Yang, Z and Huang, H and Zhang, Z and Wu, T},
title = {High-specificity gene point mutation detection by PAM-free Cas12a system with double-stranded substrate positioning-unwinding.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119043},
doi = {10.1016/j.bios.2026.119043},
pmid = {42472526},
issn = {1873-4235},
mesh = {*Point Mutation/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/genetics/chemistry ; *CRISPR-Associated Proteins/genetics ; DNA Mutational Analysis/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR/Cas12a system holds great promise for nucleic acid detection, but its strict dependence on the protospacer adjacent motif (PAM) severely limits its application in gene point mutation analysis, with fewer than 2% of known mutation sites naturally harboring adjacent PAM sequences. Herein, we developed a PAM-free Cas12a system with double-stranded substrate positioning-unwinding (dsPU-Cas12a), wherein "bubble" structures formed by unpaired base pairs release partial single-stranded target strand as a toehold, and excess auxiliary strands induce local unwinding of double-stranded DNA to facilitate R-loop formation. After optimization, the dsPU-Cas12a system achieved an ultra-low limit of detection of 0.013% for gene point mutations, with excellent linearity over the mutation abundance range of 0-10%. Furthermore, it exhibited robust feasibility and accuracy in detecting the JAK2 V617F mutation in blood samples from patients with myeloproliferative neoplasms. This simple and universal strategy overcomes the sequence limitation of Cas12a, providing a high-performance tool for clinical gene point mutation detection.},
}
@article {pmid42474386,
year = {2026},
author = {Liao, J and Su, Y and Jiang, F},
title = {CRISPR-Cas12a assay for rapid and specific detection of Shigella flexneri 2a in clinical samples.},
journal = {Journal of clinical microbiology},
volume = {64},
number = {8},
pages = {e0161525},
pmid = {42474386},
issn = {1098-660X},
mesh = {Humans ; Sensitivity and Specificity ; *Shigella flexneri/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Dysentery, Bacillary/diagnosis/microbiology ; Feces/microbiology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {Shigella flexneri 2a is the most common cause of shigellosis, a major public health concern in developing countries. Rapid and reliable diagnostic tools are critical for timely outbreak detection and management. Leveraging clustered regularly interspaced short palindromic repeats (CRISPR) technology, we developed a CRISPR-Cas12a-based assay for the rapid and specific detection of S. flexneri 2a and validated its performance using stool specimens from patients. Two guide RNAs targeting the gtrII and gtrX genes, unique markers of the S. flexneri 2a serotype, were designed to ensure specificity. Recombinase polymerase amplification (RPA) was coupled with Cas12a-mediated collateral cleavage for signal amplification, with detection by fluorescence or lateral flow. Analytical sensitivity, specificity, and clinical accuracy were compared with conventional PCR using purified DNA and 588 clinical stool specimens. The CRISPR-Cas12a assay achieved a detection limit of 10 copies/µL, comparable to PCR, and showed 100% analytical specificity without cross-reactivity to other bacteria. The isothermal reaction operated at room temperature and was completed within 1 h. Both readouts allowed visual interpretation without specialized equipment. Clinical validation of the CRISPR-Cas12a assay demonstrated a diagnostic sensitivity of 95% and specificity of 98%, comparable to PCR when evaluated using the same clinical specimens. This study provides two key advances: it establishes a CRISPR-Cas12a assay specifically targeting S. flexneri 2a, the predominant serotype, and validates it using a large clinical cohort. The assay's simplicity, speed, and high diagnostic accuracy make it a valuable tool for clinical diagnostics and field-based surveillance in resource-limited settings.IMPORTANCERapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve low-copy detection (10 copies/µL) within 1 h, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The method's simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks.},
}
@article {pmid42485703,
year = {2026},
author = {Tan, Y and He, X and Shao, E and Chen, Q and Zhang, L and Deng, S},
title = {A one-tube autocatalytic transcription-driven CRISPR cascade for ultrasensitive mRNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119049},
doi = {10.1016/j.bios.2026.119049},
pmid = {42485703},
issn = {1873-4235},
mesh = {*RNA, Messenger/genetics/analysis/isolation &amp; purification ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; *Biomarkers, Tumor/genetics/isolation &amp; purification ; *Breast Neoplasms/genetics/diagnosis ; Nucleic Acid Amplification Techniques ; Limit of Detection ; },
abstract = {The rapid identification and precise quantification of cancer biomarkers are essential for the purposes of diagnosis, classification, and therapeutic intervention. Traditional molecular diagnostic methodologies, such as polymerase chain reaction (PCR), provide considerable sensitivity; however, they depend on exponential amplification and advanced instrumentation, thereby constraining their applicability for point-of-care testing. In this study, we developed a Transcription-driven CRISPR Cascade Amplification (TCCA) for one-tube detection of mRNA at the concentration of 0.6 copies/μL within just 30 min. The target-induced assembly of a three-way junction (TWJ) facilitates the generation of a split T7 promoter, which initiates transcription and activates Cas13a collateral cleavage. The activated Cas13a subsequently cleaves cascade probes and releases new trigger strands, forming a transcription-driven cascade amplification circuit. The assay enables multiplex detection of breast cancer-associated mRNA biomarkers (HBB, KRT17, and CD55) in cell lysates, demonstrating robust performance in intricate biological matrices. Furthermore, incorporation of a multiplex OR-gated logic design enables parallel target recognition, thereby enhancing detection reliability for rapid and accurate clinical diagnostics.},
}
@article {pmid42504876,
year = {2026},
author = {Wang, P and Sayed, S and Buchholz, F},
title = {Restoring Cancer Genomes: Functional Mutation Correction as a Platform for Precision Oncology.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {663-669},
doi = {10.1177/10430342261468973},
pmid = {42504876},
issn = {1557-7422},
mesh = {Humans ; *Neoplasms/genetics/therapy/diagnosis ; *Mutation ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Genome, Human ; Animals ; },
abstract = {Cancer genome sequencing has uncovered an extensive landscape of somatic mutations. However, determining which of these alterations are biologically consequential and therapeutically actionable remains a central challenge in oncology. CRISPR-based genome editing now enables precise correction of oncogenic mutations within their endogenous genomic context. Recent work demonstrates that repairing cancer hotspot mutations restores conserved tumor-relevant transcriptional programs across diverse tumor types, revealing tumor-agnostic dependencies. Beyond therapeutic implications, this mutation-correction framework provides a scalable functional platform to stratify drivers, interrogate variants of uncertain significance, and refine precision diagnostics. This perspective discusses how programmable mutation correction advances (i) mechanistic cancer biology, (ii) personalized cancer diagnostics, and (iii) next-generation precision gene therapies. We propose that systematic reversal of cancer mutations represents a conceptual shift from observing mutational landscapes to actively testing their biological necessity.},
}
@article {pmid42508243,
year = {2026},
author = {Wei, K and Heng, H and Wang, T and Liu, Z and Wang, K},
title = {Integration of dual-output TMSD and CRISPR/Cas12a for crosstalk-free electrochemiluminescence detection of aflD gene and AFB1.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119062},
doi = {10.1016/j.bios.2026.119062},
pmid = {42508243},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry ; Humans ; Limit of Detection ; Food Contamination/analysis ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; },
abstract = {Aflatoxins are among the most toxic mycotoxins and pose a severe threat to food safety and human health. In addition to the direct monitoring of aflatoxin B1 (AFB1), simultaneous detection of its key biosynthesis gene, aflD, can effectively indicate the presence of toxin-producing strains, thereby enhancing the early screening and traceability of AFB1 contamination. Due to the significant functional differences among various biomarkers, performing multi-target analysis on a single detection interface remains challenging. Herein, we constructed a novel dual-target electrochemiluminescence (ECL) biosensor for the sequential and quantitative detection of the aflD gene and AFB1. This sensor innovatively integrates a dual-output toehold-mediated strand displacement (TMSD) and CRISPR/Cas12a trans-cleavage mechanisms to establish a dynamic "signal writing-erasing" regulation on a single ECL emitter. Specifically, aflD triggers the TMSD reaction, driving the enrichment of ferrocene (Fc)-labeled DNA at the electrode interface and quenching the ECL signal, corresponding to signal "write" (signal-off). Subsequently, AFB1 is converted via aptamer recognition into an activator DNA that initiates Cas12a trans-cleavage, leading to the removal of Fc-DNA from the interface and recovery of ECL emission, corresponding to signal "erase" (signal-on). This strategy enables cross-category detection and quantitative analysis of small-molecule toxins and nucleic acid biomarkers within a single luminescence system, effectively avoiding signal crosstalk while offering high sensitivity, high specificity, and high interfacial utilization efficiency. It provides a versatile new approach for the early warning and source tracing of contaminants in complex food matrices.},
}
@article {pmid42520408,
year = {2026},
author = {Kumar, J and Alok, A and Fox, J and Srivastava, A and Voytas, DF and Zhang, F and Kianian, SF},
title = {A novel genome editing strategy in plants using broad-host-range viral vectors derived from geminiviruses.},
journal = {Plant physiology},
volume = {201},
number = {4},
pages = {},
doi = {10.1093/plphys/kiag553},
pmid = {42520408},
issn = {1532-2548},
support = {//U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) National Plant Disease Recovery System (NPDRS)/ ; },
mesh = {*Geminiviridae/genetics ; *Genetic Vectors/genetics ; *Genome, Plant/genetics ; *Host Specificity/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with the novel genome-editing platform, opens possibilities for editing across a wide range of plant species.},
}
@article {pmid42526210,
year = {2026},
author = {Zhao, J and He, J and Du, H and Dai, C and Ma, X and Kong, D and Yue, Y and Zhang, S and Liu, K and Liu, Y and Zhang, G and Wu, Y and Wei, D},
title = {Photo-switchable CRISPR electrochemical system enables de-interferential biosensing signal output.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119067},
doi = {10.1016/j.bios.2026.119067},
pmid = {42526210},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; *Lung Neoplasms/genetics/diagnosis ; Mutation ; Limit of Detection ; Equipment Design ; Nucleic Acid Amplification Techniques ; Light ; },
abstract = {Most clustered regularly interspaced short palindromic repeats (CRISPR)-based electrochemical biosensors lack controllable switching function and are vulnerable to false signals induced by intrinsic amplification signal crosstalk. Thus, exploring strategies that mitigate intrinsic amplification crosstalk with a precise switch would be extremely useful for reliable and accurate bioanalysis. Herein, we develop a photo-switchable CRISPR/Cas12a electrochemical (PSCE) system by introducing a photocleavable (PC) linker and adopting a light-responsive strategy, which enables output of de-interference electrochemical signals and achieves highly sensitive and accurate detection of gene mutations for non-small cell lung cancer (NSCLC). The system decouples nucleic acid amplification (NAA) from signal transduction and produces unique photo-switchable response signals by light irradiation to activate Cas12a activity. This architectural design further separates signal readout from sample pretreatment, suppressing intrinsic amplification-derived signal crosstalk rather than global biological interference to deliver an ultralow limit of detection (LoD). The PSCE achieves 98.1% sensitivity, 92.7% specificity, and 98.9% overall accuracy when tested with 67 clinical samples. Moreover, the exploration of PSCE system in flexible wearable electronics and machine-learning analysis of clinical patient samples demonstrates significant application potential clinical diagnosis of mutation-associated diseases.},
}
@article {pmid42546382,
year = {2026},
author = {Qin, L and Tang, Z and Yu, Y and Liu, T and Zhang, M and Zhu, X and Hu, Q and Zhao, Z and Zhang, F and Tang, BZ and Cen, Y},
title = {DIRECTOR: DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119075},
doi = {10.1016/j.bios.2026.119075},
pmid = {42546382},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/chemistry/genetics ; Molecular Dynamics Simulation ; *MicroRNAs/genetics/analysis ; *Bacterial Proteins/chemistry/genetics ; },
abstract = {Precise regulation of Cas12a activity is crucial for expanding its application in molecular diagnostics. However, existing split crRNA systems exhibit hardly any activation efficiency at low-abundance target and lack a well-defined regulated mechanism, representing a persistent bottleneck for practical application. This work proposes a DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a (DIRECTOR) strategy. This work combines artificial intelligence-driven AlphaFold3 structure prediction, computer-powered molecular dynamics simulations with fluorescence analysis to demonstrate that the 3' terminal extension of activator acts as a spatial director, utilizing DNA-guided spatially ordered assembly of split crRNA and stabilizing key Cas12a domains, thereby activating Cas12a. Conversely, the 5' terminal extension serves as a spatial misdirector, inhibiting Cas12a activation by destabilizing the protein structure and introducing the steric hindrance to shield the catalytic center. Furthermore, the structural and energy thresholds required for effective Cas12a activation were identified. Finally, utilizing the spatial director as an energy amplification element, DIRECTOR achieves a limit of detection as low as 42.1 fM for single-target miR-155 and dual-response detection of wide-scope nucleic acids. Owing to its direct activation strategy, DIRECTOR provides mechanistic insights for affordable and programmable CRISPR molecular diagnostics.},
}
@article {pmid42546384,
year = {2026},
author = {Li, L and Wei, H and Wei, M and Yang, X and Wang, T and Min, S and Wang, Y and Wang, X and Li, M and Wang, S and Rong, Z},
title = {An integrated centrifugal microfluidic CRISPR-based diagnostics platform for multiplexed point-of-care testing of respiratory pathogens.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119083},
doi = {10.1016/j.bios.2026.119083},
pmid = {42546384},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/instrumentation ; *Point-of-Care Testing ; Equipment Design ; *CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/microbiology/virology ; Lab-On-A-Chip Devices ; Centrifugation/instrumentation ; *Microfluidic Analytical Techniques/instrumentation ; Limit of Detection ; Rapid Diagnostic Tests ; Point-of-Care Systems ; },
abstract = {Rapid, accurate, and multiplexed point-of-care testing (POCT) of respiratory pathogens is critical for clinical triage and infection control. However, existing platforms frequently necessitate trade-offs between sensitivity, throughput, and operational complexity. To address this, we developed the iCARD (integrated Centrifugal Assay on a Rotating Disc) platform, a streamlined microfluidic molecular diagnostic system based on single-step CRISPR kinetics and centrifugal microfluidics. Comprising a polymethyl methacrylate disc pre-loaded with lyophilized CRISPR/Cas13a reagents and a self-developed portable fluorescence analyzer, the platform enables automated multiplexed detection following sample loading. Furthermore, a novel resin-based pretreatment method was engineered to efficiently purify and concentrate nucleic acids from throat swabs. The iCARD system facilitates the parallel screening of six respiratory pathogens across four independent samples within 40 min. Analytical validation confirmed single-copy sensitivity (limit of detection: 0.25-1.0 copies/μL) without cross-reactivity. Clinical validation using 94 retrospective patient throat swabs demonstrated exceptional diagnostic accuracy, achieving a sensitivity of 95.5% (64/67; 95% CI: 87.64%-98.47%) and a specificity of 100.0% (27/27; 95% CI: 87.51%-100.00%). These findings demonstrate that the iCARD platform serves as a robust, high-throughput, and accurate diagnostic tool for decentralized molecular screening and epidemiological surveillance.},
}
@article {pmid42557300,
year = {2026},
author = {Du, J and Luo, Z and Xie, D and Chen, Y and Yang, M and Li, Q and Wang, L and Han, L and Zhang, Y and Li, H and Lan, Z and Shi, H and Li, Y and Cheng, Q and Dong, F and Gao, Y and Yao, Y and Cheng, T and Wei, T and Rao, S},
title = {Engineered lipid nanoparticles for in vivo and durable editing of haematopoietic stem cells within humanized mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {42557300},
issn = {2157-846X},
support = {82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82470240//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {In vivo genetic engineering of haematopoietic stem and progenitor cells (HSPCs) holds the potential to revolutionize the treatment landscape for numerous diseases. However, despite its transformative potential, it remains hindered by the difficulty in efficiently and specifically targeting quiescent human HSCs while maintaining their long-term functionality. Here, after screening 15 lipid nanoparticles (LNPs), we report an LNP that efficiently delivers reporter mRNA to human HSPCs both in ex vivo and in vivo settings when conjugated with the anti-CD34 antibody (CD34/LNP[DP]). Using CRISPR/Cas editing cargos, CD34/LNP[DP] achieves high editing efficiency in human HSPCs ex vivo. Intrafemoral administration of CD34/LNP[DP] in humanized mice results in efficient editing of the erythroid-specific BCL11A enhancer within human HSPCs, enabling the sustained long-term reactivation of fetal haemoglobin (HbF) expression in erythroid cells. In a humanized neutropaenia model harbouring an ELANE mutation, intrafemoral administration of CD34/LNP[DP] achieves robust editing, targeting exon 2 of ELANE in human HSPCs, partially restoring neutrophil development impairment under long-term observation. Collectively, CD34-targeted delivery enables in vivo HSPC modification without perturbing haematopoiesis, underscoring its suitability for clinical translation.},
}
@article {pmid42558756,
year = {2026},
author = {Sharma, N and Thakur, K and Zinta, R and Shaunak, I and Batta, S and Saini, A and Sehgal, R and Bhagta, S and Singh, B and Thakur, AK},
title = {Transgene-free genome editing in potato, a clonally propagated crop - strategies and future prospects.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {32},
number = {8},
pages = {1747-1757},
pmid = {42558756},
issn = {0971-5894},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas)-based genome editing technology has come out as very precise and effective tool for targeted modification in the gene of interest and offers unprecedented potentials in crop improvement. However, in the present regulatory framework for commercialization of genome edited crops, in many countries including India, the edited lines must be transgene-free. In India, only site directed nuclease (SDN) I and SDN II category of genome edited events which are transgene-free are permitted for commercialization. Potato is a vegetatively propagated crop, having autotetraploid genome and is highly heterozygous in nature. Removal of the transgene from potato genome of edited lines through genetic segregation, either by crossing or selfing, is not the appropriate method as the elite background of the genome gets disturbed due to heterozygous nature of the crop. Every individual seed of potato, i.e. true potato seed (TPS) behaves like a different individual than the parental line and is unable to maintain the genetic identity. In this review article, we have discussed several strategies that can be enacted for generation of transgene-free genome edited lines in potato. This article will provide deeper insight and enhance understandings about the optimum use of CRISPR as non-GMO technology in the genetic enhancement of potato and to adopt the best strategies in editing this important tuberous, clonally propagated crop.},
}
@article {pmid42559426,
year = {2026},
author = {Li, A and Cao, C and Yang, C and Liu, Y},
title = {Programmable transcriptional condensates for enhanced CRISPR-based gene regulation.},
journal = {Theranostics},
volume = {16},
number = {14},
pages = {8215-8229},
pmid = {42559426},
issn = {1838-7640},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HeLa Cells ; Transcription, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RATIONALE: Efficient gene activation or repression through programmable CRISPR-Cas9 has revolutionized molecular biology and drug development. Nonetheless, the currently available CRISPRa/i approaches are modestly potent and require multi-component delivery, which hampers the wide use of the technology in both research and therapy.<br><br>METHODS: We developed a modular CRISPR-condensate platform by appending a multivalent RNA nanostar to the 3' end of a single-guide RNA, producing a sgRNA-nanostar chimera that mediates phase separation at Cas9-bound genomic loci. The nanostar scaffold also contains MS2 stem-loops, which recruit MCP-tagged transcriptional effectors (VP64 for activation, KRAB for repression) to the condensate microenvironment at high local concentration. We examined condensate formation, genome targeting, and transcriptional output by using live-cell imaging, RT-qPCR, ChIP-seq, RNA-seq and CUT&amp;Tag in HEK293T, HeLa, U-2 OS, MDA-MB-231, as well as human iPSC cell lines.<br><br>RESULTS: The CRISPR-condensate design resulted in up to 50-100-fold target-gene activation, compared with 5-10-fold activation by direct VP64 fusion, and 20-30-fold transcriptional repression, compared with 3-5-fold repression by direct KRAB fusion, with high target specificity (12 versus 28 non-target differentially expressed genes assessed by RNA-seq). Orthogonal kissing-loop (KL) pairings enabled independent condensate systems for simultaneous activation and repression of multiplexed targets. Janus condensates containing both activating and repressive domains enabled bidirectional regulation at a single locus. The system requires delivery of only three independently expressible components-dCas9-NLS, an sgRNA-nanostar chimera bearing MS2 stem-loops (MS2SLs), and an MCP-fused effector (VP64-MCP for activation or KRAB-MCP for repression)-and showed minimal innate immune response and high cell viability.<br><br>CONCLUSIONS: The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation. This strategy makes biomolecular condensation a general principle for enhancing CRISPR gene regulation, opening up possibilities for functional genomics, cell engineering, and therapy development.},
}
@article {pmid42559496,
year = {2026},
author = {Lee, HK and Lim, J and Shepherd, S and Nguyen, MTT and Akin, L and Bacon, A and Sidavi, Y and Shurgalin, M and Cobi, A and Nikolayenko, A and Fuflyigin, V and Bashir, R and Wang, X and Cunningham, BT},
title = {A Dual-Port, Smartphone-Linked, Pocket-Size Fluorimeter for Rapid Molecular Diagnostic Assays at Point of Care.},
journal = {IEEE sensors journal},
volume = {26},
number = {14},
pages = {20538-20555},
pmid = {42559496},
issn = {1530-437X},
support = {U01 AA029348/AA/NIAAA NIH HHS/United States ; },
abstract = {We present the design, testing, and demonstration of a portable, pocket-size, smartphone-linked fluorimeter called the "VPodDuo." The instrument is capable of reading the output of several fluorescence-generating biomolecular detection assays with sensitivity that is similar to larger and more expensive laboratory-based instruments. In this work, we focus on demonstrating the capability for readout of assays used to detect target nucleic acid sequences associated with infectious pathogens and cancer with incubation times of approximately 10 min. The VPodDuo features a dual-port configuration that allows simultaneous measurements of a negative experimental control (CTRL) in parallel with the test sample. We demonstrated compatibility with several assay protocols, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, and the target recycling amplification process (TRAP). Benchmarking against three commercially available fluorimeters showed comparable detection limits for clinically relevant nucleic acid sequences, including Zika virus (ZIKV, 10[4] copies/μL), methicillin-susceptible Staphylococcus aureus (MSSA, 10[2] copies/μL), human immunodeficiency virus (HIV, 6.83 pM), a lung cancer-associated circulating tumor DNA sequence [L858R point-mutated epidermal growth factor receptor (EGFR) gene, 29.2 pM], and a microRNA biomarker associated with lung cancer (miR-375-3p, 500 pM). We further validated the VPodDuo's performance under varying ambient temperatures through in-lab simulations and real-world outdoor testing using the TRAP assay for miR-375-3p detection, demonstrating cancer-associated biomarker detection at point-of-care (POC) settings. With its compact form-factor, low cost, portability, and real-time data transmission and analytical capabilities, the VPodDuo represents a promising solution for expanding access to rapid, on-site molecular diagnostics in diverse clinical and field settings.},
}
@article {pmid42560391,
year = {2026},
author = {Uc-Chuc, MA and Aguilar-Hernández, V and Jiménez-Ramírez, IA and Brito-Argaez, L and Loyola-Vargas, VM},
title = {In vitro CRISPR/Cas9-RNP cleavage of CcYUC1 in Coffea canephora.},
journal = {Planta},
volume = {264},
number = {3},
pages = {},
pmid = {42560391},
issn = {1432-2048},
support = {1515//Secretar&#xed;a de Ciencia, Humanidades, Tecnolog&#xed;a e Innovaci&#xf3;n (Secihti)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Coffea/genetics/enzymology/metabolism ; Indoleacetic Acids/metabolism ; *Plant Proteins/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Base Sequence ; },
abstract = {An in vitro CRISPR/Cas9-RNP system efficiently cleaves CcYUC1 in Coffea canephora, establishing a foundation for DNA-free genome editing in coffee. Somatic embryogenesis depends on auxin biosynthesis and signaling; however, functional validation of candidate genes remains limited. In this study, we identified CcYUC1, a putative flavin monooxygenase gene associated with indole-3-acetic acid biosynthesis, during SE induction in Coffea canephora. CcYUC1 transcripts accumulated during the early stages of SE, suggesting a role in embryogenic induction. To establish a genome editing platform in coffee, we designed a CRISPR/Cas9 ribonucleoprotein (RNP) system targeting exon 4 of CcYUC1. In vitro cleavage assays confirmed specific and efficient digestion of the target, achieving over 80% cleavage under optimized Cas9/sgRNA conditions. These findings establish a proof-of-concept CRISPR/Cas9-RNP platform for DNA-free genome editing in coffee and provide a basis for future functional studies of auxin biosynthesis during somatic embryogenesis.},
}
@article {pmid42562481,
year = {2026},
author = {Tan, G and Qi, S and Hu, M and Wang, D and Lin, K and Wang, Y and Chen, S and Zhang, Q and Zhao, L},
title = {Understanding phage dynamics and their potential roles during soy sauce fermentation using metagenome-assembled genomes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {241},
number = {},
pages = {119707},
doi = {10.1016/j.foodres.2026.119707},
pmid = {42562481},
issn = {1873-7145},
mesh = {*Fermentation ; *Bacteriophages/genetics/classification/physiology ; *Soy Foods/microbiology/virology ; *Metagenome ; Genome, Viral ; *Food Microbiology ; Metagenomics ; },
abstract = {The composition and functional roles of phages in fermented foods have been gaining increasing attention. However, their ecological functions and underlying mechanisms in high-salt soy sauce fermentation remain largely unexplored. In this study, we investigated phage communities, their potential functions, phage-host interactions, and host defense mechanisms in two different soy sauce fermentation processes (Cantonese-type process, CP; Japanese-type process, JP) using shotgun metagenomics. A total of 823 phage species (viral operational taxonomic units, vOTUs) were identified, with the majority exhibiting a temperate lifestyle (89.19%). The most abundant family was Straboviridae (CP, 9.95%-11.39%; JP, 12.04%-13.73%), followed by Salasmaviridae (CP, 6.92%-7.94%; JP, 5.70%-7.02%). Although the phage composition differed between the two processes, the number of vOTUs was positively correlated with prokaryotic species richness, total acidity, and amino acid nitrogen content, and negatively correlated with pH. A comparative genomic analysis revealed that 91 phages were associated with 26 bacterial genomes (metagenome-assembled genomes, MAGs), with Lactococcus petauri (MAG16) and Halomonas elongata (MAG51) hosting the most phage species. An analysis of host defense mechanisms showed that all 45 bacterial MAGs harbored CRISPR-Cas type I systems, and 95.6% encoded restriction-modification systems. Functional annotation using the KEGG and CAZymes databases indicated that the phages predominantly encoded genes related to cell growth, replication, and metabolism of amino acids, carbohydrates, and nucleotides, with more genes enriched in the JP than in the CP. Additionally, auxiliary metabolic genes (e.g., pfkA, ldh, adhP, ilvE, and arcA) were identified in 29 phages. These genes are potentially involved in metabolic pathways that may be linked to flavor compound production. Together, these findings provide novel insights into the ecological and potential functional roles of phages during soy sauce fermentation.},
}
@article {pmid42563405,
year = {2026},
author = {Adil, M and Gul, I and Lu, S and Bashir, S and Razzaq, S and Lu, H and Daud, M and Iqbal, Y and Tao, Y},
title = {Crop Resilience to Combined Drought and Salinity Stress in Drylands: From Soil Processes to Genomic Solutions.},
journal = {Plant, cell &amp; environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70804},
pmid = {42563405},
issn = {1365-3040},
support = {41871079//National Natural Science Foundation of China/ ; SKLECRA2023//Chinese Research Academy of Environmental Sciences/ ; //Open Foundation of State Key Laboratory of Environmental Criteria and Risk Assessment/ ; },
abstract = {Drought and soil salinization increasingly co‑occur, threatening global food security, particularly in dryland farming systems where these stresses can interact additively, synergistically or antagonistically to reduce crop yields. This review critically distinguishes these interaction types and moves beyond single‑stress frameworks to provide a multiscale synthesis of plant responses, soil water-salt dynamics, root‑system plasticity, rhizosphere microbiome modulation, emerging breeding modelling and agronomic tools. This review provides a detailed overview of the multilevel damage from these co-occurring stresses, including osmotic shock, ionic toxicity and systemic oxidative damage, and examines plant strategies such as ion homoeostasis, osmotic adjustment and antioxidant responses. Beyond plant-centric views, the review explores epigenetic reprogramming for stress memory and the potential of rhizosphere microbiome engineering with plant growth-promoting rhizobacteria as a biological stress-alleviation strategy. It critically examines how high-throughput multiomics techniques, combined with innovative breeding methods, provide a transformative approach to decode complex tolerance traits and accelerate cultivar development. These techniques cover genomics, epigenomics, transcriptomics, proteomics and metabolomics, while the breeding methods include genomic selection and CRISPR-Cas gene editing. Despite progress, key challenges remain: validating combined stress resilience under field conditions, harnessing epigenetic inheritance and integrating microbial solutions into breeding pipelines. We propose a unified framework that merges mechanistic discovery, microbiome-assisted breeding, soil and water management and systems-level predictive modelling. This integrative approach is essential for developing climate-resilient crops to sustain dryland agriculture.},
}
@article {pmid42567578,
year = {2026},
author = {Wang, H and Bao, C and Liu, L and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {CRISPR/Cas12a and CHA-based SERS platform for ultrasensitive nucleic acid detection.},
journal = {Analytica chimica acta},
volume = {1418},
number = {},
pages = {345840},
doi = {10.1016/j.aca.2026.345840},
pmid = {42567578},
issn = {1873-4324},
mesh = {*Spectrum Analysis, Raman/methods ; *CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Silver/chemistry ; Limit of Detection ; DNA, Single-Stranded/analysis ; Metal Nanoparticles/chemistry ; },
abstract = {BACKGROUND: Highly sensitive nucleic acid detection is essential for analytical applications. Conventional methods often require complex pre-amplification procedures, limiting their practical utility in screening. Developing nucleic acid detection strategies with high sensitivity and selectivity, without target gene pre-amplification, remains a significant challenge.<br><br>RESULTS: This study integrates CRISPR/Cas12a recognition, catalytic hairpin assembly (CHA) amplification, and surface-enhanced Raman spectroscopy (SERS). CRISPR/Cas12a recognizes target nucleic acids and cleaves single-stranded DNA (ssDNA), thereby blocking the toehold-mediated strand displacement reaction (TSDR) and triggering CHA. Hairpin probe HP1 with C-Ag[+]-C structures bridges CHA, releasing Ag[+] through cyclic amplification. Ag[+] induces charge transfer and aggregation of AgNPs@4-ABT, generating strong SERS signals. The platform achieved femtomolar sensitivity and high selectivity in detecting pCaMV35S, with 96.4% accuracy in maize seeds and 100% in maize leaves.<br><br>SIGNIFICANCE: This strategy eliminates the need for pre-amplification of target genes by combining CRISPR's targeting feature, CHA, and ultrasensitive SERS detection. It demonstrates excellent performance in genetically modified organism screening, seed quality testing, and leaf sample analysis, providing a promising tool for food safety and agricultural regulation.},
}
@article {pmid42568984,
year = {2026},
author = {Sahu, A and Kumar, A and Vaidya, A and Mishra, J and Mishra, S and Prajapti, SK},
title = {Decoding the silent conversations: targeting quorum sensing to disarm bacterial pathogens in the age of antimicrobial resistance.},
journal = {RSC medicinal chemistry},
volume = {},
number = {},
pages = {},
pmid = {42568984},
issn = {2632-8682},
abstract = {Antimicrobial resistance (AMR) has emerged as a global health challenge, imposing significant clinical and economic burdens worldwide. The widespread and often indiscriminate use of antibiotics has accelerated resistance, necessitating alternative therapeutic strategies to combat microbial pathogenicity. Quorum sensing, a cell density-dependent signalling system, represents a promising target in this aspect. This review examines the molecular framework of quorum sensing across diverse microbial communities, its signalling cascades, and its role in regulating biofilm formation, efflux pump modulation and horizontal gene transfer with the quorum signalling. It further discusses quorum-sensing inhibition strategies, including natural products, synthetic compounds, quorum-quenching enzymes, and antibody-mediated and vaccine-mediated approaches. Application of CRISPR/Cas, engineered probiotic strains and nanocarrier-mediated delivery systems for quorum signalling disruption has been addressed. A key strength of this review is that it is the first to combine the underlying molecular mechanisms of quorum sensing with future translational tools such as artificial intelligence, CRISPR, engineered probiotics and nanotechnology to develop next-generation anti-virulence solutions for drug-resistant infection. While the preclinical findings have several challenges specific to the specificity and pharmacokinetic properties, strategies to resolve these considerations have been discussed. Overall, quorum signalling as a target is a major paradigm shift that may offer sustainable antimicrobial therapy to fight the global antimicrobial resistance issue. This can be achieved through a multidisciplinary approach to optimise antimicrobial therapy beyond the traditional concept of "killing".},
}
@article {pmid42569449,
year = {2026},
author = {Choi, Y and Yang, J and Kim, J and Enkhtaivan, K and Son, J and Jang, J and Lee, HY and Choi, J},
title = {Exosome-Based Liquid Biopsy in Biliary Tract Cancer: Nanotechnology-Enabled Strategies and Future Perspectives.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {613686},
pmid = {42569449},
issn = {1178-2013},
mesh = {Humans ; *Exosomes/chemistry ; *Biliary Tract Neoplasms/pathology/diagnosis ; Liquid Biopsy/methods ; *Nanotechnology/methods ; Biomarkers, Tumor/analysis ; Nanomedicine ; Biosensing Techniques ; },
abstract = {Biliary tract cancer (BTC) remains a formidable clinical challenge owing to its asymptomatic early stages, anatomical complexity, and lack of reliable and noninvasive diagnostic tools. Although traditional tissue biopsy is often limited by invasiveness and sampling bias, liquid biopsy, particularly the analysis of tumor-derived exosomes, has emerged as a promising and clinically relevant alternative for early detection and longitudinal monitoring. Exosomes are specialized extracellular vesicles that sequester diverse molecular cargo including proteins, lipids, and nucleic acids, thereby reflecting the physiological state of their parental tumor cells. However, the clinical translation of exosomal biomarkers is often hindered by technical challenges in achieving high-purity isolation and ultrasensitive detection in complex biological matrices, such as blood and bile. To address these limitations, this review provides a comprehensive overview of recent advancements in nanotechnology-enabled platforms designed to overcome these challenges. First, we examined sophisticated nanostructured systems, such as immuno-magnetic nanoparticles and microfluidic nanoVelcro chips, for high-yield exosome enrichment. Subsequently, we investigated next-generation biosensing modalities with a focus on surface-enhanced Raman scattering for label-free molecular fingerprinting and CRISPR-Cas-integrated nanosensors for amplification-free nucleic acid detection. Significant emphasis has now been placed on the integration of artificial intelligence and deep learning algorithms, which have become indispensable for deciphering complex exosomal signatures to differentiate BTC from benign conditions such as cholangitis. Finally, we discuss the emerging clinical significance of bile-derived exosomes and remaining challenges in standardizing nanomedicine-based liquid biopsies for precision oncology. These integrated platforms could potentially redefine the BTC management paradigm by bridging the gap between advanced nanomaterials and clinical diagnostics.},
}
@article {pmid42296510,
year = {2026},
author = {Zhang, ZM and Xu, C and Zhu, Y and Wei, Y and Pei, Y and Zhang, H and Pang, J and Xie, ZJ and Xu, K and Wang, J},
title = {Integrated CRISPR-Cas12a-Based Biosensors with Subwavelength Grating Microring Resonators for Ultrasensitive Mutation-Specific Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5674-5682},
doi = {10.1021/acssensors.6c00141},
pmid = {42296510},
issn = {2379-3694},
support = {62422503//National Natural Science Foundation of China/ ; U21A20454//National Natural Science Foundation of China/ ; 2022A0505030024//Natural Science Foundation of Guangdong Province/ ; 2022A1515110756//Natural Science Foundation of Guangdong Province/ ; 2022B1515020093//Natural Science Foundation of Guangdong Province/ ; 2022B1515120012//Natural Science Foundation of Guangdong Province/ ; GJHZ20220913143207014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102406013//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102618040//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20241202130558075//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; RCYX20210609103707009//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; RCYX20221008092907027//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2022YFB3207200//National Key Research and Development Program of China/ ; 2022B1515020057//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; Mutation ; Gold/chemistry ; *SARS-CoV-2/genetics/isolation &amp; purification ; Metal Nanoparticles/chemistry ; Humans ; *CRISPR-Associated Proteins ; DNA Probes/chemistry/genetics ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {The rapid and precise detection of nucleic acids is critical for identifying viral mutations, yet it presents formidable difficulties for conventional diagnostics. While established techniques such as quantitative polymerase chain reaction and next-generation sequencing involve complex workflows, emerging on-chip integrated photonic biosensing techniques are often limited by inadequate specificity and sensitivity. Here, we introduce an integrated photonic biosensing platform that synergizes the programmable recognition of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, with the superior sensitivity of subwavelength grating microring resonators. The sensor surface is functionalized with single-stranded DNA probes conjugated to gold nanoparticles. Upon target recognition, activated Cas12a cleaves the probes, releasing the nanoparticles and generating a quantifiable resonance wavelength shift. In particular, the spectral response gets further amplified by a resonance-enhanced photothermal effect. The detection of SARS-CoV-2 variants enables discrimination between wild-type, Delta, and Omicron strains. The extracted detection limit of 0.7 fM represents a four-order-of-magnitude improvement over conventional fluorescence-based CRISPR assays. Our work establishes a generalizable platform for ultrasensitive, mutation-resolved molecular diagnostics on a CMOS-compatible photonic chip, paving the way for advanced point-of-care testing and genomic surveillance.},
}
@article {pmid42391661,
year = {2026},
author = {Zheng, C and Nong, L and Luo, J and Liang, L and Wei, Y and Luo, Q and Zhang, K and Liao, X},
title = {An electrochemiluminescence biosensor based on the hairpin-mediated exponential amplification and CRISPR/Cas12a amplification for ultrasensitive detection of MMP-2.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {267},
number = {},
pages = {115948},
doi = {10.1016/j.colsurfb.2026.115948},
pmid = {42391661},
issn = {1873-4367},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Matrix Metalloproteinase 2/analysis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Humans ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Electrodes ; *Nucleic Acid Amplification Techniques/methods ; Inverted Repeat Sequences ; Limit of Detection ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {In this study, we report a novel electrochemiluminescence (ECL) biosensor for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), an important biomarker associated with tumor invasion and metastasis. The biosensor integrates hairpin-mediated exponential amplification with CRISPR/Cas12a-based trans-cleavage for dual-stage signal amplification. In this design, MMP-2 specifically cleaves a peptide sequence (GPLG↓VRGK) on the DNA hairpin probe (HP1), releasing an initiator peptide nucleic acid (PNA) that triggers hairpin-mediated exponential amplification reaction. The amplified DNA products then activate the Cas12a/gRNA complex, which induces collateral cleavage of ferrocene (Fc)-labeled probes immobilized on a DNA tetrahedron-modified PEI-Ti3C2Tx/Ru/AuNPs electrode, thereby generating a strong ECL response. The incorporation of the DNA tetrahedron nanostructure provides a well-defined three-dimensional framework that ensures ordered probe orientation, enhanced hybridization efficiency, and reduced steric hindrance on the electrode surface. This structural organization significantly improves electron transfer and signal stability compared with conventional planar immobilization. Under optimized conditions, the biosensor exhibited a broad linear range from 0.01 fM to 10 nM and an ultralow detection limit of 10 aM. It displayed high specificity against interfering proteins (thrombin, IgG, BSA, lysozyme), excellent stability, and satisfactory recoveries (96.9%-105.0%) in LO2 cell culture supernatants. Overall, this enzyme-responsive, DNA-tetrahedron-assisted, CRISPR-amplified ECL biosensor represents a robust and versatile platform for precise and rapid detection of protease activity, showing great promise for biomedical diagnostics and clinical biomarker monitoring.},
}
@article {pmid42399104,
year = {2026},
author = {Liu, Z and He, Y and Lin, X and Pal, T and Pfeilsticker, A and Liu, BM and Chen, J},
title = {Indiscriminate Trans-Cleavage Activity of CRISPR/SuCas12a2 Enables Sensitive Detection of SARS-CoV-2.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5397-5403},
pmid = {42399104},
issn = {2379-3694},
support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; R35GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *COVID-19/diagnosis/virology ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; *COVID-19 Nucleic Acid Testing/methods ; Endodeoxyribonucleases/metabolism/genetics ; Fluorescent Dyes/chemistry ; Rapid Diagnostic Tests ; },
abstract = {Sensitive detection of SARS‑CoV‑2 remains critical for controlling COVID‑19 outbreaks and guiding patient care. Although reverse transcription-polymerase chain reaction (RT‑PCR), the gold standard for detecting SARS-CoV-2, is highly sensitive, the need for specialized equipment and trained personnel limits its widespread application in low or middle-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology could overcome these limitations by providing simplicity, low cost, and high specificity. However, current CRISPR-based diagnostics can simultaneously cleave the target and fluorescence probes, as they are the same nucleic acid type (ssDNA or ssRNA), thereby reducing detection sensitivity. Herein, we developed a novel CRISPR-based viral detection method using SuCas12a2 (Cas12a2 from Sulfuricurvum sp. PC08-66), which harnesses its unique broad trans-cleavage activity and offers flexibility in selecting fluorescence probes. Using the conserved SARS‑CoV‑2 envelope gene as the model analyte, the analytical performance of the CRISPR/SuCas12a2 system for viral detection was evaluated. The CRISPR/SuCas12a2 detection workflow achieved a detection limit of 5 × 103 copies/μL for SARS-CoV-2 viral RNA. When detecting nasopharyngeal swab samples from patients, the CRISPR/SuCas12a2 system showed preliminary agreement with RT-qPCR in a set of clinical samples. Our CRISPR/SuCas12a2 system provides a flexible detection platform with simplified probe selection and enhanced compatibility, offering new insights into future portable diagnostic applications and enhancing global public health surveillance.},
}
@article {pmid42402044,
year = {2026},
author = {Kim, T and Scheeres, EC and Fiebig, A and Olive, AJ and Crosson, S},
title = {A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells.},
journal = {Infection and immunity},
volume = {94},
number = {8},
pages = {e0011726},
doi = {10.1128/iai.00117-26},
pmid = {42402044},
issn = {1098-5522},
support = {R01AI177619/NH/NIH HHS/United States ; R35GM146795/NH/NIH HHS/United States ; },
mesh = {Humans ; *Macrophages/microbiology/metabolism ; *Brucellosis/genetics/microbiology ; *Host-Pathogen Interactions/genetics ; *CRISPR-Cas Systems ; *Brucella ; Brucella abortus ; Cell Line ; },
abstract = {Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus signal at 3 hours post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support the B. abortus and B. ovis infection process at a post-entry step. Gene set enrichment analysis further identified positive regulators of mTORC1 signaling as host factors. This result was validated by genetic disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Together, these data indicate that the AKT-Ragulator-mTORC1 axis contributes to establishing a permissive intracellular niche. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that impact Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.},
}
@article {pmid42412687,
year = {2026},
author = {Pan, Y and Yang, Z},
title = {Paper Microfluidic Platform Using Multiplexed Isothermal Amplification and CRISPR/Cas12a for Aquatic Pathogen Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5893-5908},
doi = {10.1021/acssensors.6c00719},
pmid = {42412687},
issn = {2379-3694},
support = {RL-2022-041//Leverhulme Trust/ ; FF\1920\1\36//Royal Academy of Engineering/ ; NE/R013349/2//National Centre for Earth Observation/ ; NE/V010441/1//National Centre for Earth Observation/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods/instrumentation ; *Paper ; *CRISPR-Cas Systems/genetics ; *Water Microbiology ; *Bacteria/isolation &amp; purification/genetics ; *Microfluidic Analytical Techniques/instrumentation/methods ; Rapid Diagnostic Tests ; Colorimetry ; Limit of Detection ; Molecular Diagnostic Techniques ; },
abstract = {The global health threat posed by microbial contamination of aquatic systems demands feasible pathogen monitoring solutions. However, current detection methods are limited by expensive instrumentation and specialized personnel, which hinders their application in point-of-care testing (POCT). Here, we presented an integrated paper microfluidic platform for spatially multiplexed detection of pathogenic bacteria, including Salmonella, E. coli, C. perfringens, B. cereus, V. parahaemolyticus, S. aureus, and L. monocytogenes, selected due to their epidemiological significance and regulatory relevance in environmental and food safety monitoring. LAMP, RAA-CRISPR, and RPA-CRISPR assays were housed within physically isolated reaction chambers on two-layer chips. An engineered horseradish peroxidase (HRP) cascade-coupled crRNA modification system with DNA-conjugated labels was designed for colorimetric detection. Operation was enabled by solar-powered and portable hardware for incubation and imaging, coupled with a web application for quantitative analysis. Exceptional analytical performance was demonstrated, achieving an LOD of 1 CFU/mL, a dynamic range of 1-107 CFU/mL, high reproducibility (CV <5%), low batch-to-batch variation (<6%), low cost (£2.5 per test), and scalable integration, with a sample-to-answer time of 60 min. Successful field validation in diverse aquatic environments confirmed its practical feasibility, consistent with gold standard PCR (R2 = 0.98). This platform offers a promising POCT solution for public health protection and epidemic monitoring, particularly in resource-limited settings.},
}
@article {pmid42425083,
year = {2026},
author = {Nair, U and Akauliya, M and Warner, JE and Parikh, S and Ronsard, L and Ssozi, M and Rays Wahba, L and Esposito, AG and Kelley, B and McCarthy, C and Weldon, SR and Wu, L and Lingwood, D and Batista, FD},
title = {CRISPR-mediated precise large fragment insertion in zygotes enables rapid generation of humanized immunoglobulin heavy-chain mice.},
journal = {Immunity},
volume = {59},
number = {8},
pages = {2334-2350.e8},
pmid = {42425083},
issn = {1097-4180},
support = {R01 AI153098/AI/NIAID NIH HHS/United States ; R01 AI155447/AI/NIAID NIH HHS/United States ; INV-085294/GATES/Gates Foundation/United States ; R01 AI195539/AI/NIAID NIH HHS/United States ; R01 AI168114/AI/NIAID NIH HHS/United States ; INV-046626/GATES/Gates Foundation/United States ; },
mesh = {Animals ; Humans ; Mice ; *Immunoglobulin Heavy Chains/genetics ; *CRISPR-Cas Systems/genetics ; *Zygote ; Chromosomes, Artificial, Bacterial/genetics ; B-Lymphocytes/immunology ; Immunoglobulin Variable Region/genetics ; Mice, Transgenic ; V(D)J Recombination ; Somatic Hypermutation, Immunoglobulin ; },
abstract = {Current CRISPR-Cas9 methods are restricted to small genomic edits. We developed a CRISPR-guided approach that enables direct insertion of large genomic sequences into mouse zygotes. We deleted the murine 2.4-Mb immunoglobulin heavy-chain (IgH) variable (VH) locus and then precisely inserted a bacterial artificial chromosome (BAC) containing a 155-kb human VH DNA fragment flanked by 20-kb homology arms. Full-length, single-copy BAC integration occurred without ectopic recombination. Human sequences were stably transmitted and expressed VH segments that recombined with endogenous mouse sequences, and mice exhibited normal B cell development. Upon immunization, human VH-expressing B cells underwent class-switch recombination and somatic hypermutation, secreting antigen-specific antibodies. We demonstrated modular IgH humanization by replacing endogenous mouse diversity and joining (DH-JH) segments with human sequences, producing V(D)J recombination and diverse antibodies. Unlike traditional methods requiring more than a year, this approach enables the generation and validation of mice carrying large genomic insertions within 8 weeks.},
}
@article {pmid42433170,
year = {2026},
author = {Qiu, Z and Chen, J and Wu, J and Wang, J and Hu, Y and Feng, W and Zhao, J and Minamoto, T and Chen, X and Zhou, L and Liu, M and Wang, C and Lin, M and Hu, M and Zhou, X and Wang, J},
title = {Optical-Controlled One-Pot RPA-CRISPR Assay for Environmental DNA Detection of a Critically Endangered Species.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5553-5565},
doi = {10.1021/acssensors.5c04638},
pmid = {42433170},
issn = {2379-3694},
support = {ZJZX-06//Ministry of Agriculture and Rural Affairs of the People's Republic of China/ ; },
mesh = {*Endangered Species ; *DNA, Environmental/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Recombinases/metabolism ; Animals ; },
abstract = {Developing a rapid, sensitive, and field-deployable assay for on-site environmental DNA (eDNA) detection of endangered species is crucial, as current PCR-based assays are slow and expensive and rely on laboratory-based thermal cycling. Here, we adapted a previously reported optically controlled one-pot RPA-CRISPR-Cas12a (OORC) assay to detect eDNA of the critically endangered Bahaba taipingensis. The assay combines isothermal recombinase polymerase amplification (RPA) with the high specificity of a CRISPR-Cas12a trans-cleavage reaction in a single tube. The entire process is operated at a constant temperature, avoiding thermal cycling. Low-template replicate experiments conservatively redefined the OORC limit of detection as 6 copies/reaction. To support near-field application, we further integrated the assay with a portable handheld fluorescence detector capable of 365 nm photoactivation and fluorescence readout. This portable and highly sensitive workflow extends the potential of eDNA monitoring, offering a practical tool for the conservation of endangered species.},
}
@article {pmid42441514,
year = {2026},
author = {Wang, X and Zhao, S and Jiang, J and Wang, H and Encarnación, C and Zheng, L and Zhang, L},
title = {5' Dual-Overhang Short PAM-less dsDNA as Switchable Activators of Cas12a trans-Cleavage for Amplification-Free miRNA Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5753-5764},
doi = {10.1021/acssensors.6c00524},
pmid = {42441514},
issn = {2379-3694},
support = {24DX2800100//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; },
mesh = {*MicroRNAs/blood/analysis/genetics ; Humans ; *DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; },
abstract = {Precise and programmable regulation of CRISPR-Cas12a activity is essential for advancing controllable nucleic acid diagnostics, yet the structural determinants governing Cas12a activation by short PAM-less double-stranded DNA (dsDNA) remain largely unexplored. This study systematically investigates the effects of the terminal architectures of short PAM-less dsDNA on Cas12a trans-cleavage activity. By profiling a series of dsDNA constructs bearing distinct 5'/3' overhang configurations, a 5' dual-overhang motif was identified as a highly effective structural inhibitor that suppresses Cas12a activation. Kinetic fluorescence assays combined with computational structural modeling indicated that this inhibition arises from steric constraints imposed by the 5' terminal architecture. Leveraging this structure-guided regulatory mechanism, an amplification-free CRISPR-Cas12a assay was developed for the direct detection of oncogenic microRNAs miR-155 and miR-21, achieving femtomolar sensitivity without reverse transcription. The assay was further evaluated in human serum samples spiked with target miRNAs, supporting its proof-of-concept performance in a more complex matrix. Collectively, these findings highlight the potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity and provide useful insight for the design of CRISPR-based biosensing strategies.},
}
@article {pmid42462715,
year = {2026},
author = {Zhu, Y and Wang, Q and Cao, Y and Hou, L and Yang, L and Cheng, C and Ye, S and Li, W and Zhang, J},
title = {Single cell CRISPR screen identifies antagonism between Nsd1-H3K36me2 and Ezh2-H3K27me3 orchestrates pluripotency transition.},
journal = {Stem cell reports},
volume = {21},
number = {8},
pages = {103016},
doi = {10.1016/j.stemcr.2026.103016},
pmid = {42462715},
issn = {2213-6711},
mesh = {Animals ; *Enhancer of Zeste Homolog 2 Protein/metabolism/genetics ; Mice ; *Histones/metabolism/genetics ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism/cytology ; Epigenesis, Genetic ; DNA Methylation ; CRISPR-Cas Systems ; *Single-Cell Analysis/methods ; DNA Methyltransferase 3A ; DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/metabolism/genetics ; },
abstract = {The transcriptional and epigenetic landscape imposes constraints on the self-renewal capacity and lineage specification potential of both naive and primed mouse embryonic stem cells (mESCs). CRISPR/Cas9-based functional screening coupled with single-cell RNA-seq (CROP-seq) establishes relationships between gRNA-mediated knockout genotype and transcriptome phenotype, providing a powerful tool to dissect gene regulatory networks. Here, we employed CROP-seq to investigate the epigenetic regulation governing the pluripotency network in mESCs. This highly sensitive method identified key genes essential for the acquisition and exit from pluripotency, and revealed a novel role for H3K36me2 in modulating DNA methylation through regulating the expression of Dnmt1 and Dnmt3a. Specifically, loss of Nsd1-mediated H3K36me2 delayed naive state exit, whereas Ezh2 deficiency accelerated primed entry. Collectively, our findings identify an epigenetic regulatory network critical for determining mESCs' pluripotent state transitions.},
}
@article {pmid42503780,
year = {2026},
author = {Shi, JY and Wu, SL and Tan, Y and Jiang, HX and Liu, SQ and Gan, XQ and Sun, WB and Li, MY and Li, KD and Tang, AN and Zhu, LN and Cai, QL and Kong, DM},
title = {DNA Nanowire-Assisted CRISPR/Cas12a Triple Cascade Amplification for Sensitive Detection of Myeloperoxidase Activity.},
journal = {Analytical chemistry},
volume = {98},
number = {31},
pages = {22931-22942},
doi = {10.1021/acs.analchem.6c02223},
pmid = {42503780},
issn = {1520-6882},
support = {22293030//National Natural Science Foundation of China/ ; 22474063//National Natural Science Foundation of China/ ; 72474154//National Natural Science Foundation of China/ ; 82570985//National Natural Science Foundation of China/ ; 25JCLZJC00250//Tianjin Municipal Science and Technology Program/ ; 25JCLZJC00640//Tianjin Municipal Science and Technology Program/ ; 23JCJQJC00080//Science Fund for Distinguished Young Scholars of Tianjin Municipality/ ; },
mesh = {*Peroxidase/metabolism/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/metabolism ; *Nanowires/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Myeloperoxidase (MPO) is an inflammation-associated heme enzyme implicated in cardiovascular oxidative stress, but sensitive activity-based detection in complex clinical samples remains challenging. Herein, we report a CRISPR/Cas12a-based triple-cascade amplification platform for rapid and sensitive MPO activity detection. The core sensing element consists of DNA nanowires containing multiple Cas12a activator strands, tethered to magnetic beads via biotin-labeled and phosphorothioate-modified linkers. In the chloride-containing assay system, MPO-catalyzed generation of HOCl oxidatively cleaves these linkers, releasing the nanowires and activating Cas12a, which, in turn, cleaves fluorescent reporters. This design integrates three amplification stages (MPO catalysis, multiactivator release, and Cas12a trans-cleavage), achieving ultrasensitive detection without additional nucleic acid amplification. Under the optimized chloride-containing conditions, the assay achieved a detection limit of 10.20 pg/mL for MPO. A preliminary pilot analysis using human serum samples from acute coronary syndrome patients and healthy individuals showed different signal distributions, supporting the feasibility of applying the platform to complex serum matrices, although contributions from eosinophil peroxidase/HOBr-mediated probe activation cannot be excluded. The platform is readily adaptable to lateral flow assays and portable fluorescence readouts, offering versatile formats for point-of-care-compatible analysis. This work provides a sensitive CRISPR/Cas12a-based strategy for MPO activity-related hypohalous oxidant assessment under defined assay conditions and demonstrates its preliminary applicability in complex serum matrices.},
}
@article {pmid42550765,
year = {2026},
author = {Torrance, R and Orf, K and White, N and Matti, C and McKenna, AJ and Cosentino, A and Casirati, G and Albuquerque, AS and Thrasher, AJ and Genovese, P and Booth, CA and Fox, TA and Burns, SO and Morris, EC},
title = {Functional restoration of immune defects in STAT1 gain-of-function disease following stem cell gene editing.},
journal = {Blood},
volume = {},
number = {},
pages = {},
doi = {10.1182/blood.2025032242},
pmid = {42550765},
issn = {1528-0020},
abstract = {Germline gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) gene cause a dominantly inherited inborn error of immunity (IEI) characterized by chronic mucocutaneous candidiasis, autoimmunity, severe opportunistic infections and an increased risk of malignancy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is curative but is associated with increased risk of morbidity and mortality in STAT1 GOF patients compared to other IEI. To develop a curative, autologous alternative to HSCT, we evaluated gene editing strategies in STAT1 GOF model cell lines, primary T cells, and patient-derived HSCs. Universal and mutation-specific strategies using CRISPR/Cas-mediated homology-directed repair (HDR) were limited by low efficacy (<25%), poor viability, and a lack of allele-specificity. In contrast, adenine base editing corrected the recurrent and highly pathogenic p.T385M mutation with upwards of 90% efficiency in patient T cells and HSCs without significant unintended on- or off-target genomic aberrations. Gene editing functionally restored total STAT1 expression (p<0.0217), STAT1 phosphorylation (p<0.0056), interferon-stimulated gene expression (OAS1; p=0.0005) and improved IL-17 production (p<0.0001). Edited HSCs retained multilineage differentiation capacity and sustained engraftment with persistence of the corrected allele at 16 weeks in humanized immunodeficient mice. These data demonstrate efficient and precise correction of STAT1 GOF mutations by base editing, with maintenance of the correction through long-term engraftment in vivo. This represents the first application of gene editing to correct a dominant gain-of-function mutation causing immunodeficiency, with potential applicability to other genetic disorders associated with heterozygous and gain-of-function mutations.},
}
@article {pmid42551559,
year = {2026},
author = {Khosrojerdi, M and Hashemi, SA and Besharati, R and Haghbin, A and Azimian, A},
title = {CRISPR-Cas systems as precision antimicrobials: Reversing the tide of antimicrobial resistance.},
journal = {Virus research},
volume = {371},
number = {},
pages = {199782},
doi = {10.1016/j.virusres.2026.199782},
pmid = {42551559},
issn = {1872-7492},
abstract = {Antimicrobial resistance (AMR) has escalated into a global health crisis, with resistant pathogens causing over 1.2 million direct deaths annually and threatening to render modern medicine unsustainable. This review provides a comprehensive and updated synthesis of CRISPR-Cas-based antimicrobial strategies with a unique focus on: (i) critical comparison with conventional antibiotics and emerging alternatives; (ii) quantitative evaluation of delivery platforms; (iii) novel strategies including AI-optimized guide design and the ATTACK-CreTA system; (iv) comprehensive analysis of ecological risks; and (v) technology readiness level assessments for clinical translation. The CRISPR-Cas system, originally discovered as a bacterial adaptive immune mechanism, has been repurposed as a programmable precision tool to combat AMR by selectively targeting and eliminating resistance genes. We systematically evaluate the mechanistic diversity of Cas effectors, from DNA-cleaving Cas9 and Cas3 to RNA-targeting Cas13, and their application in reversing resistance phenotypes in WHO priority pathogens. We critically assess emerging delivery platforms, including engineered bacteriophages, conjugative plasmids, nanoparticles, and outer membrane vesicles, quantitatively comparing their delivery efficiency, payload capacity, and biosafety profiles. Novel strategies such as CRISPR interference (CRISPRi) for gene silencing without genomic cleavage, the ATTACK-CreTA system for enhanced bactericidal activity, and AI-driven optimization of guide RNA design are examined with appropriate caveats. We comprehensively address clinical translation challenges including immunogenicity, pharmacokinetics/pharmacodynamics, manufacturing scalability, regulatory pathways, and bacterial resistance mechanisms including anti-CRISPR proteins. No CRISPR-based antimicrobial has yet received regulatory approval, and we critically evaluate the gap between proof-of-concept and clinical utility. A detailed roadmap for clinical development is proposed. By integrating recent advances in Cas protein engineering, delivery technologies, and diagnostic applications, this review positions CRISPR-Cas systems as next-generation precision therapeutics capable of both treating resistant infections and curtailing the spread of AMR across clinical and environmental settings.},
}
@article {pmid42554845,
year = {2026},
author = {Zubair, A and Hemal, MAKP and Ahmed, A and Ahmad, F and Waheed, Y and Afghan, N},
title = {CRISPR/Cas system as a novel therapeutic strategy to combat multi-drug-resistant bacteria.},
journal = {Archives of microbiology},
volume = {208},
number = {11},
pages = {},
pmid = {42554845},
issn = {1432-072X},
mesh = {*Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Humans ; *Bacterial Infections/therapy/microbiology ; Gene Editing/methods ; },
abstract = {Antimicrobial resistance (AMR) has diminished the effectiveness of present antibiotics, posing a huge threat to global community health and economic stability. This study investigates the CRISPR-Cas framework's potential as a cutting-edge tactic to fight antimicrobial resistance. Current applications, limitations, and prospective future uses are analyzed. CRISPR antimicrobial strategies, which bring together the latest developments in gene-targeting strategies, engineered delivery platforms, and translational applications to fight multidrug-resistant pathogens. CRISPR technology is different from traditional antimicrobial treatments that target general antimicrobial resistance genes, instead allowing targets to be eliminated specifically by sequence, while retaining beneficial microbial communities, which has the potential to be a transformative precision antimicrobial treatment. Nevertheless, there is still a need for optimization of delivery systems, specificity of targets, biosafety, and regulations to ensure successful clinical translation, especially given their amazing advances. Recent research confirms that CRISPR-based mechanisms also affect different bacterial species, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, playing a key function in averting the emergence of resistance genes in these bacteria. Changes to CRISPR loci affect how resistance genes are targeted in ESKAPE pathogens, and CRISPR-Cas9 successfully lowers resistance by focusing on genes like tetM and ermB. A promising application of CRISPR-Cas systems in combating antimicrobial resistance (AMR) is the precise targeting of plasmid-borne mcr-1 resistance genes and other mobile genetic elements that facilitate the dissemination of colistin resistance. But the efficiency of CRISPR-Cas is diminished in some bacterial strains due to variations in their CRISPR loci. Enhancing transformation approaches and minimizing off-target impacts are critical challenges to confirm the precision and safety of CRISPR-based mechanisms in therapeutic applications. Advances in these areas are likely to continue to enable the development of next-generation CRISPR therapeutics for the effective management of multidrug-resistant bacterial infections.},
}
@article {pmid42556039,
year = {2026},
author = {Li, H and Du, H and Xu, R and Xue, H and Liu, Y and Cao, Y and Guo, Z and Pan, J and Liu, Y},
title = {Point-of-care detection for respiratory diseases: From samples and biomarkers to principles and applications.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130377},
doi = {10.1016/j.talanta.2026.130377},
pmid = {42556039},
issn = {1873-3573},
abstract = {Early screening can significantly reduce the severe morbidity and mortality of respiratory diseases and alleviate the burden on public healthcare systems. Point-of-care (POC) devices refer to portable instruments that meet the REASSURED criteria and can be conveniently used near the patient without professional laboratory conditions. POC devices played an important role in patient initiated early diagnosis during the COVID-19 pandemic, demonstrating great potential. From a macro-to-micro perspective, this review first comprehensively introduces clinically relevant sample types, including blood, respiratory tract and oral samples, and exhaled breath, along with the clinical significance of corresponding biomarkers (nucleic acids, proteins, gaseous molecules, extracellular vesicles, circulating tumor cells, and pathogen particles) and pre-processing methods. Subsequently, it summarizes the test principles and promising bioreceptors including base pairing-based systems (PCR, isothermal amplification, CRISPR/Cas), antibodies and antibody mimetics, and other affinity-based recognition elements, and innovatively presents portable integration platforms of biosensors from the perspective of bioreceptor compatibility. It then evaluates the application performance of transducers and corresponding optical or electrochemical portable detection devices, including SERS, e-nose, nanopore sensors, and portable GC-MS. Finally, the latest applications of computer technology and artificial intelligence tools in POC detection for respiratory diseases, spanning device design, bioreceptor screening, biomarker discovery, and diagnostic data processing, are presented by functional category. This review aims to provide a reference for the research and application of multiplex biomarker/sample/disease POC testing in respiratory diseases, and to offer perspectives on future directions for technological innovation, intelligentization, and commercial translation.},
}
@article {pmid42556347,
year = {2026},
author = {Zhou, Z and Saffarian-Deemyad, I and Shi, H and Weiss, T and Ur-Rehman, MM and Vohra, K and Skopintsev, P and Yoon, PH and Trinidad, MI and Langeberg, CJ and Kamalu, M and Amerasekera, J and Zhou, Y and Doherty, EE and Aris, KDP and Al-Sayyad, N and Thornton, BW and Weissman, RF and Wasko, KM and Esain-Garcia, I and DeTurk, EC and Savage, DF and Jacobsen, SE and Bryant, Z and Doudna, JA},
title = {Stepwise DNA-unwinding gates TnpB genome-editing activity.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.07.015},
pmid = {42556347},
issn = {1097-4164},
abstract = {TnpB is a compact RNA-guided endonuclease and an evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited, and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived, partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, H4W-L304F-V305R (Ymu1-WFR), stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.},
}
@article {pmid42557080,
year = {2026},
author = {Hu, YW and Zhang, Y and Ren, Y and Qin, H and Zhao, H},
title = {[Advances in phage therapy for pneumonia caused by Klebsiella pneumoniae].},
journal = {Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases},
volume = {49},
number = {8},
pages = {902-907},
doi = {10.3760/cma.j.cn112147-20251012-00632},
pmid = {42557080},
issn = {1001-0939},
support = {202304021301063//Shanxi Province scientific and technological achievements transformation guidance project/ ; 2023-190//Research Project Supported by Shanxi Scholarship Council of China/ ; },
mesh = {*Klebsiella pneumoniae/virology ; Humans ; *Phage Therapy/methods ; *Klebsiella Infections/therapy/microbiology ; *Pneumonia, Bacterial/therapy/microbiology ; Bacteriophages ; Animals ; Anti-Bacterial Agents/therapeutic use ; Drug Resistance, Multiple, Bacterial ; },
abstract = {Klebsiella pneumoniae (KP) has emerged as a formidable nosocomial pathogen in the era of antimicrobial resistance, with mortality from pneumonia caused by carbapenem-resistant strains exceeding 50%. Phage therapy has re-emerged as a promising alternative or adjunctive strategy for managing refractory KP infections. This review consolidates the current preclinical and clinical evidence base, outlines the molecular mechanisms of phage-host interactions, and appraises evolving therapeutic approaches. Preclinical investigations in murine pneumonia models have consistently demonstrated that intranasal or nebulization phage administration markedly reduces pulmonary bacterial burden, attenuates inflammatory lung injury, and improves survival, often exhibiting synergistic effects when combined with conventional antibiotics. Clinical case reports and small compassionate-use series have further provided preliminary yet compelling evidence supporting the safety and therapeutic promise of personalized phage formulations in critically ill patients with multidrug-resistant KP pneumonia who have exhausted standard treatment options. Mechanistically, phage tropism is mediated through the specific recognition of bacterial surface receptors-principally capsular polysaccharide and, to a lesser extent, lipopolysaccharide-by phage-encoded receptor-binding proteins, culminating in bacterial lysis. In response, KP has evolved a multilayered defensive arsenal encompassing receptor modification to impede adsorption, nucleic acid interference systems (e.g., CRISPR-Cas and restriction-modification), and abortive infection mechanisms that curtail phage propagation at the population level. To surmount the inherent limitations of narrow host range and the inevitable emergence of phage-resistant mutants, a suite of optimization strategies is under active refinement, including rationally designed phage cocktails, genetically engineered phages with extended tropism, artificial intelligence-assisted host-range prediction, and innovative delivery platforms such as hydrogel encapsulation to enhance pulmonary bioavailability. Despite ongoing challenges in mechanistic complexity, manufacturing standardization, and regulatory uncertainty, current initiatives- such as the establishment of geographically diverse phage libraries, real-time surveillance of phage resistance, and the development of phage-derived enzyme products-hold promise for establishing precision phage therapy as a viable and sustainable component of the antimicrobial stewardship armamentarium.},
}
@article {pmid42557140,
year = {2026},
author = {Zhou, X and Wang, L and Peng, X and Liu, M and Li, Y and Gui, F and Zhou, L and Deng, Z},
title = {A light-activated one-pot ERA/CRISPR-Cas12a assay for cost-effective dual-mode detection of HBV DNA.},
journal = {Talanta},
volume = {},
number = {},
pages = {130329},
doi = {10.1016/j.talanta.2026.130329},
pmid = {42557140},
issn = {1873-3573},
abstract = {Light-activatable CRISPR-Cas systems offer an effective strategy to overcome the kinetic incompatibility in one-pot nucleic acid assays; however, their practical application remains limited by high reagent cost, the lack of compatible reaction buffers, and reliance on instrument-dependent readout. In this work, we developed a cost-effective light-controlled one-pot ERA/CRISPR-Cas12a platform for rapid hepatitis B virus (HBV) DNA detection. NPOM-caged crRNA was employed to temporarily suppress Cas12a activity during amplification and to trigger target-dependent trans-cleavage by a brief 30 s UV irradiation. By replacing RPA with ERA, the per-reaction reagent cost of the one-pot assay was reduced from approximately US$6.0 to US$2.0, corresponding to a reduction of approximately 66.7%, while maintaining comparable analytical performance. In addition, a PEG-free unified buffer was established through systematic optimization, enabling efficient integration of ERA and Cas12a reactions in a sealed single-tube format. The proposed platform provided dual-mode readout through fluorescence and lateral flow assay (LFA), with a limit of detection of 1 copy μL[-1] and a total assay time of 30 min. Clinical evaluation using 79 serum samples showed a sensitivity of 100% for fluorescence readout and 96.3% for LFA, with excellent agreement with qPCR (Cohen's κ = 0.94 - 1.00). Owing to its low cost, high sensitivity, and operational simplicity, this platform represents a promising tool for rapid HBV molecular diagnosis in both centralized laboratories and resource-limited settings.},
}
@article {pmid42133510,
year = {2026},
author = {Cimolato, C and Petrelli, S and Favaro, D and Frusteri Chiacchiera, A and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Conjugative- and Phage-Mediated CRISPR-Based System Against Antimicrobial Resistant Bacteria.},
journal = {IEEE transactions on bio-medical engineering},
volume = {73},
number = {8},
pages = {2961-2971},
doi = {10.1109/TBME.2026.3693402},
pmid = {42133510},
issn = {1558-2531},
mesh = {*Bacteriophages/genetics ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; *Models, Biological ; Computer Simulation ; *Bacteria/genetics/virology/drug effects ; *Phage Therapy/methods ; },
abstract = {OBJECTIVE: Antimicrobial resistance (AMR) poses a significant threat to global health by diminishing the effectiveness of conventional antibiotics. This study aims to assess, using a systems biology approach, a potential synthetic biology-based strategy that employs engineered conjugative probiotic bacteria and bacteriophages to combat AMR, examining the implications of implementing targeted gene silencing as an alternative to direct bacterial killing.<br><br>METHODS: A comprehensive mathematical model was developed to describe the dynamics of the delivery systems (engineered conjugative probiotic bacteria and engineered phages) and the antimicrobial actuators being studied (genome cutting via CRISPR systems and AMR-gene silencing through CRISPR interference), also compared to traditional phage therapy (selection of phages capable of killing pathogens through bacterial-specific viral infection). The target population includes antibiotic-resistant bacteria competing with other probiotic bacteria for colonizing the host environment. The model explicitly incorporates parameters for mutations that affect actuator functionality and simulates their impact on overall therapeutic performance.<br><br>RESULTS: Simulations show how variations in actuator efficiency and emergence of new mutations in target pathogens affect the long-term suppression of resistance genes. Including mutational effects provides insights into system robustness and guides optimal therapeutic design choices.<br><br>CONCLUSION: The proposed modeling framework effectively captures key biological and mechanistic aspects of engineered therapies, enabling the prediction and optimization of each intervention against resistant pathogens. It highlights engineered phages and CRISPR interference as the most promising candidates for the design of new engineered biological therapeutics.<br><br>SIGNIFICANCE: This study establishes a quantitative foundation for rational design and dosage optimization in engineered phage- and bacterial-based therapies, advancing the use of synthetic biology methods to fight antimicrobial resistance.},
}
@article {pmid42405454,
year = {2026},
author = {Chen, Z and Wu, H and Chu, LT and Lin, J and Zhang, Y and Lin, X and Zeng, T},
title = {Aptamer-based CRISPR-Cas12a fluorescent biosensors for serum biomarker detection.},
journal = {The Analyst},
volume = {151},
number = {16},
pages = {4516-4534},
doi = {10.1039/d6an00123h},
pmid = {42405454},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Biomarkers/blood ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {The CRISPR-Cas12a system enables sensitive nucleic acid detection due to its programmability, trans-cleavage activity, and biocompatibility. To expand its applications beyond nucleic acid analysis, aptamers have emerged as ideal recognition elements owing to their high specificity, design flexibility, ease of modification and low cost. The integration of Cas12a with aptamers enables the conversion of target-binding signals into nucleic acid recognition signals, thereby combining molecular recognition with signal amplification for the detection of non-nucleic acid targets. This review provides a concise overview of the working mechanism and features of the Cas12a system, with particular emphasis on recent advances in Cas12a-aptamer-based fluorescent biosensors for serum biomarker detection. The advantages and limitations, current challenges, and future prospects are also discussed.},
}
@article {pmid42547606,
year = {2026},
author = {Akkoul, N and Kumar, T and Sharma, S and Sharma, V and Raja, V and Malik, TG and Wan Mohd Jaafar, WS and Gupta, AK and Hakeem, KR},
title = {CRISPR-Cas-based detection of Mycobacterium tuberculosis: current advances and translational bottlenecks.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {42547606},
issn = {1615-6102},
abstract = {Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge due to persistent diagnostic gaps. CRISPR-Cas-based diagnostics have emerged as highly sensitive and programmable platforms for nucleic acid detection, enabling rapid identification of Mtb targets, including drug-resistance-associated mutations. These systems integrate isothermal amplification, diverse Cas effectors, and multiple signal readout strategies to achieve high analytical performance. This review provides a comparative analysis of clinically evaluated CRISPR-based TB diagnostic platforms, highlighting substantial variability in assay design, performance, and translational readiness. While many platforms demonstrate strong analytical sensitivity, their implementation remains constrained by workflow complexity and limited integration into true point-of-care formats. This highlights that successful clinical translation of CRISPR-based TB diagnostics is determined more by real-world adaptability than by analytical performance alone. The current review presents a comparative analysis of CRISPR-based diagnostic platforms for tuberculosis, evaluating the variability in assay design, analytical and clinical performance, and translational readiness across currently available systems.},
}
@article {pmid42548009,
year = {2026},
author = {Chen, SY and Yang, LH and Liang, ZQ and Li, JD and Li, SD and Deng, YL and Chen, GQ and Ling, JW and Zhou, SS and Chen, G and He, RQ},
title = {CRISPR Screen Reveals Pathways and Factors Driving Tyrosine Kinase Inhibitor Resistance in Hepatocellular Carcinoma.},
journal = {Cancer medicine},
volume = {15},
number = {8},
pages = {e72028},
pmid = {42548009},
issn = {2045-7634},
support = {2025GXNSFAA069130//Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation/ ; NSFC82460783//The National Natural Science Foundation of China/ ; //Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University/ ; S202410598060X//The China Undergraduate Innovation and Entrepreneurship Training Program/ ; YCBZ2025127//The Innovation Project of Guangxi Graduate Education/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Signal Transduction ; },
abstract = {Tyrosine kinase inhibitor (TKI) resistance severely limits clinical outcomes in hepatocellular carcinoma (HCC), highlighting the urgent need to elucidate its underlying molecular mechanisms. In this study, an unbiased genome-wide CRISPR/Cas9 screening identified novel key factors related to the therapeutic responsiveness of TKI in HCC. By integrating data from 20 datasets encompassing 322 samples, a comprehensive TKI therapeutic response landscape for HCC was constructed. GO and Reactome enrichment analyses revealed that dysregulated RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways modulate TKI sensitivity, with close links to antitumor immunity. This study identified GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37 as key genes mediating TKI resistance in HCC. These six genes were found to be highly expressed in HCC and significantly associated with HCC patient prognosis. Drug sensitivity assays identified a significant association between their expression and responsiveness to TKI agents. In-house quantitative real-time PCR validated their differential expression levels in normal hepatocytes, parental HCC cells, and TKI-resistant HCC sublines. ssGSEA, TIMER2, and ESTIMATE analysis revealed that their expression modulates HCC immune infiltration. Bibliometric analysis revealed a growing focus on immunotherapy-based combination regimens to overcome TKI resistance. Ferroptosis, epithelial-mesenchymal transition and hypoxia were new research directions, which were closely related to the pathways investigated in this study. In conclusion, this study identified RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways, as well as GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37, as novel directions and targets for TKI-immunotherapy combination strategies, providing new insights for overcoming TKI resistance in HCC.},
}
@article {pmid42549139,
year = {2026},
author = {Pytlik, D and Gerovac, M and Bischler, T and Schlosser, A and Vogel, J and Schoen, C},
title = {The CRISPR/Cas-associated scaRNA modulates efeUOB expression and stress responses in Neisseria meningitidis.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag027},
pmid = {42549139},
issn = {2633-6693},
abstract = {Neisseria meningitidis is a human-adapted commensal pathogen that must continuously balance nutrient acquisition with stress tolerance. Here, we identify a type II-C CRISPR/Cas-associated small RNA (scaRNA) as a posttranscriptional regulator of the efeUOB operon and oxidative stress responses. Using in vitro RNA binding and structure probing assays, we show that the scaRNA interacts with the 5' untranslated region of efeO mRNA, leading to reduced translation of this component of the ferrous iron transporter EfeUOB. Consistent with this, efeO translational fusions demonstrate repression by the scaRNA, whereas a ΔscaRNA mutant shows increased reporter expression. We further show that meningococcal Cas9 (Nme1Cas9) is able to cleave scaRNA in vitro, but in vivo phenotypes are primarily scaRNA-dependent, indicating that Nme1Cas9 contributes, at most, indirectly to this regulation. In line with this observation, comparative proteomics revealed overlapping but distinct roles of scaRNA and Nme1Cas9 in oxidative stress adaptation, energy metabolism, and ion transport. While steady-state protein abundances did not capture all scaRNA-dependent effects, functional assays confirmed that scaRNA inactivation reduces survival under oxidative stress. Together, our results identify scaRNA-mediated repression of efeO as a novel posttranscriptional mechanism that contributes to stress adaptation in meningococci. These findings expand the functional repertoire of CRISPR-associated elements and suggest a role for small RNA-based regulation in iron-related stress adaptation in a major human pathogen.},
}
@article {pmid42549572,
year = {2026},
author = {Iwe, IA and Liu, FX and Corsano, A and da Silva, SJR and Doucet, J and Singh, S and Lamothe, G and Zayani, R and Nguyen, J and Matthews, Q and Vigar, JRJ and Bayat, P and Simchi, M and Bozovicar, K and Charania, M and Panfilov, S and Kelly, P and Cai, R and Hubbard, BP and Li, X and Mazzulli, T and Tremblay, JP and Zhao, Y and Green, AA and Li, Z and Yao, S and Pardee, K},
title = {RAPID: evaluation of Cas12a protospacer nicking and chimeric reporters for PAM-independent RNA and DNA diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549572},
issn = {1362-4962},
support = {//Precision Medicine Initiative/ ; PRMUHT2024-001//Canadian Institute of Health Research/ ; 202410MFE-531769-419793//Canadian Institute of Health Research/ ; //Ontario Graduate Scholarship/ ; 201610FDN-375469//CIHR Foundation/ ; 950-231075//Canada Research Chairs Program/ ; 950-233107//Canada Research Chairs Program/ ; RGPIN-2016-06352//NSERC Discovery/ ; N66001-23-2-4042//Defense Advanced Research Projects Agency/ ; PJT-189974//CIHR/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *RNA/analysis/genetics ; *DNA/analysis/genetics ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Genes, Reporter ; },
abstract = {CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans-cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans-cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans-cleavage activity matching conventional Cas12a. We then apply RAPID to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.},
}
@article {pmid42549575,
year = {2026},
author = {Li, Y and Han, P and Yuan, R and Bai, L and Qing, M},
title = {Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549575},
issn = {1362-4962},
support = {82072378//National Natural Science Foundation of China/ ; 22504009//National Natural Science Foundation of China/ ; CSTB2025NSCQ-LZX0126//Natural Science Foundation of Chongqing/ ; CSTB2025NSCQ-GPX0375//Natural Science Foundation of Chongqing/ ; CQYC202005015//Chongqing Talents: Exceptional Young Talents Project, China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification-CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification-CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine-tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.},
}
@article {pmid42549577,
year = {2026},
author = {Winter, E and Emiliani, F and Cook, A and Abderrahim, A and McKenna, A},
title = {BASELINE: a CRISPR base editing platform for mammalian-scale single-cell lineage tracing.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549577},
issn = {1362-4962},
support = {DP2GM149750/NH/NIH HHS/United States ; //V Foundation/ ; //Pew Biomedical Scholars/ ; },
mesh = {*Cell Lineage/genetics ; *Single-Cell Analysis/methods ; Animals ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Pancreatic Neoplasms/genetics/pathology ; },
abstract = {A cell's fate is shaped by its inherited state, or lineage, and the ever-shifting context of its environment. CRISPR-based recording technologies are a promising solution for mapping the lineage of a developing system; however, challenges remain regarding single-cell recovery, engineering complexity, and scale. Here, we introduce BASELINE, which uses base editing to generate high-resolution lineage trees in conjunction with single-cell profiling. BASELINE uses the Cas12a adenine base editor to irreversibly edit nucleotides across target arrays built from 50 synthetic target sites, which are integrated multiple times into a cell's genome. We demonstrate that BASELINE accumulates lineage-specific marks over a wide range of biologically relevant intervals, recording more than 4300 bits of information in a model of pancreatic cancer, a 50-fold increase over existing technologies. Single-cell sequencing reveals high-fidelity capture of these recorders, averaging 29 cell divisions captured per lineage, within the estimated range of mammalian development. We expect BASELINE to apply to a wide range of lineage-tracing projects in development and disease, especially those in which cellular engineering makes small, more distributed systems challenging.},
}
@article {pmid42549821,
year = {2026},
author = {Jafari, A and Manzari-Tavakoli, A and Manzari Tavakoli, M and Hashemian, SM and Jamaati, H and Akhtari, M and Tabarsi, P and Omrani, M},
title = {Theranostic innovation in infectious lung diseases: integrating biotechnology and nanotechnology for precision medicine.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2026.2714060},
pmid = {42549821},
issn = {1744-8352},
abstract = {INTRODUCTION: Introduction: Infectious lung diseases, including pneumonia, tuberculosis (TB), COVID-19, influenza, and emerging fungal infections, are major causes of illness and death worldwide. Traditional methods have serious limitations such as diagnostic delays, antimicrobial resistance, and non-targeted therapy. Theranostics offers a transformative precision medicine paradigm for pulmonary infections.<br><br>AREAS COVERED: This review looks closely at how biotechnology and nanotechnology synergistically advance theranostic strategies for infectious lung diseases. We explore biotechnological tools including CRISPR-Cas systems, non-coding RNAs (ncRNAs), and monoclonal antibodies (mAbs) for detecting specific pathogens and intervening directly. We also discuss nanotechnological platforms such as nanosensors, surface-enhanced Raman spectroscopy (SERS), and various nanocarriers (lipid nanoparticles, polymeric nanoparticles, liposomes, metallic nanoparticles, mesoporous silica nanoparticles, and biomimetic systems) for drug, gene, and vaccine delivery with better targeting, controlled release, and imaging capabilities. Integrated case studies across major diseases, including COVID-19, influenza, TB, pneumonia, COPD, and idiopathic pulmonary fibrosis, demonstrate effective theranostic applications. We also address associated challenges like safety, manufacturing, regulatory hurdles, and economic feasibility.<br><br>EXPERT OPINION: The combination of biotechnology and nanotechnology represents a paradigm shift toward personalized pulmonary medicine. Future success needs to develop smart, multi-stimuli-responsive nanoplatforms, integrating artificial intelligence for predictive modeling and treatment optimization, and establishing closed-loop theranostic systems that connect real-time diagnostics with adaptive therapies. Key priorities include standardized preclinical models, clear regulations for combination products, and health economic analyses demonstrating cost-effectiveness. Interdisciplinary collaboration among material scientists, molecular biologists, clinicians, and regulatory specialists will be essential to translate these promising platforms from bench to bedside.},
}
@article {pmid42550282,
year = {2026},
author = {Yang, L and Ji, X and Li, Z and Duan, F and Jia, Q and Zhang, S and Hu, B and Zhang, Z},
title = {Ultrasensitive electrochemiluminescence determination of Salmonella based on CRISPR/Cas12a integrated with bimetallic semiconductive metal-organic frameworks.},
journal = {Mikrochimica acta},
volume = {193},
number = {9},
pages = {},
pmid = {42550282},
issn = {1436-5073},
support = {252300420760//Natural Science Foundation of Henan Province/ ; 252300421534//Natural Science Foundation of Henan Province/ ; 254200510025//Zhongyuan Sci-Tech Innovation Leading Talents/ ; 25IRTSTHN003//Program for Innovative Research Team (in Science and Technology) in University of Henan Province/ ; },
mesh = {*Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Salmonella/isolation &amp; purification/chemistry ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; DNA, Single-Stranded/chemistry ; Semiconductors ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Bacterial Proteins ; },
abstract = {An ultrasensitive electrochemiluminescence (ECL) biosensor was established by combining CRISPR/Cas12a technique and semiconductive bimetallic-organic framework (scMOF) [[CuxNi3-x(HITP)2] (HITP = 2,3,6,7,10,11-hexaiminotriphenylene)]] emitter and employed to detect Salmonella using the allosteric probe as the recognition component. Given that CuxNi3-x(HITP)2 has demonstrated large specific surface area, both in-plane and out-of-plane charge transfer ability, narrowed band gap, and enhanced separation of holes and electrons, it can be simultaneously employed as the superior ECL emitter and bioplatform for anchoring single-strand DNA (ssDNA), thus improving the detection sensitivity toward Salmonella. The CRISPR/Cas12a-based system can specifically recognize the target sequence of Salmonella and activate the nuclease activity of Cas12a, and the activated Cas12a possesses trans-cleavage ability toward ssDNA. The CuxNi3-x(HITP)2 emitter is then released, resulting in the decline of the ECL response. The developed CuxNi3-x(HITP)2-CRISPR/Cas12a-based ECL biosensor exhibits the ultralow detection limit of 0.25 CFU mL[- 1] in the linear range from 1.0 CFU mL[- 1] to 10[6] CFU mL[- 1], significantly lower than those of reported ones. Furthermore, the developed biosensor exhibits outstanding overall biosensing properties with high selectivity, favorable reproducibility and stability, together with promising practical applicability for the determination of Salmonella in a variety of foodstuffs.},
}
@article {pmid42050729,
year = {2026},
author = {Sofianos, G and Petmezas, A and Samaras, A and Karaoglanidis, G},
title = {Resistance of Botrytis cinerea to anilinopyrimidine fungicides: A novel ARMS-PCR method for the detection of Bcpos5 mutations and characterization of resistance using CRISPR/Cas9 editing.},
journal = {Pest management science},
volume = {82},
number = {9},
pages = {8189-8198},
pmid = {42050729},
issn = {1526-4998},
support = {//General Secretariat for Research and Technology/ ; },
mesh = {*Botrytis/drug effects/genetics ; *Fungicides, Industrial/pharmacology ; *Drug Resistance, Fungal/genetics ; *Pyrimidines/pharmacology ; *Polymerase Chain Reaction/methods ; Mutation ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {BACKGROUND: Anilinopyrimidine (AP) fungicides have been widely used against Botrytis cinerea, yet their resistance mechanisms have only recently been clarified. Resistance is primarily linked to mutations G408V, L412V, and L412F in the Bcpos5 gene, whose encoded protein is localized to the mitochondria. In this study, we developed a detection method and tested the fitness of L412F/V mutants obtained by using the CRISPR/Cas9 editing technique.<br><br>RESULTS: For rapid and cost-effective mutation identification, a TETRA-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) was developed to rapidly detect the nucleotide alterations that lead to L412F and L412V mutations, producing a 702&#x2009;bp band in all isolates, with additional 470&#x2009;bp (F) or 252&#x2009;bp (V) fragments. Isolates harboring only the 702&#x2009;bp band were further digested with MlyI to confirm the mutation leading to the amino acid substitution G408V mutation (467&#x2009;bp&#x2009;+&#x2009;235&#x2009;bp). Results were validated by Sanger sequencing. Application of the assay to 170 isolates from strawberry and tomato revealed mutation frequencies of 70.2% (L412F), 8.3% (L412V), and 4.7% (G408V) within the resistant fraction of the population (n&#x2009;=&#x2009;82 resistant isolates). Furthermore, sequencing analysis revealed also a low frequency of the E407K mutation in Bcmdl1, along with evidence of additional, yet undefined, resistance mechanisms. To further characterize the mutations, the B. cinerea reference strain B05.10 was transformed with L412F and L412V alleles via CRISPR/Cas9 and homologous recombination. The resulting mutants displayed resistance to cyprodinil, and potential fitness costs were assessed in both field-derived and CRISPR/Cas9-generated isolates through measurements of mycelial growth and sporulation in vitro, and pathogenicity in planta. The L412F and L412V transformants did not differ significantly from the parental B05.10 strain in any of the evaluated fitness parameters.<br><br>CONCLUSION: Overall, the developed ARMS PCR offers a fast, cost-effective tool for resistance monitoring aiming to identify the most common mutations conferring resistance to APs, while CRISPR/Cas9 provides an efficient approach for functional validation of resistance mutations in B. cinerea. Using this approach, we confirmed that L412F and L412V mutations in Bcpos5 confer resistance to APs, while are not associated with fitness cost. &#xa9; 2026 The Author(s). Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.},
}
@article {pmid42546506,
year = {2026},
author = {Pal, P and Anand, U and Saha, SC and Sundaramurthy, S and Okeke, ES and Kumar, M and Radha, and Bontempi, E and Albertini, E and Dey, A and Di Maria, F},
title = {Retraction notice to "Novel CRISPR/Cas technology in the realm of algal bloom biomonitoring: Recent trends and future perspectives" [Environ. Res 231 (2023) 115989].},
journal = {Environmental research},
volume = {306},
number = {Pt 4},
pages = {125335},
doi = {10.1016/j.envres.2026.125335},
pmid = {42546506},
issn = {1096-0953},
}
@article {pmid42547416,
year = {2026},
author = {Zhuang, Q and Wang, F and Zhang, H and Zhang, Q and Gao, Y and Feng, Z and Gao, H and Sun, S and Lin, S and Li, S and Zhao, Q and Huang, X and Li, Q and Sheng, W and Huang, G},
title = {A cardiac-related, promoter-proximal, regulatory element shapes chromatin and JAG1 transcription.},
journal = {Life science alliance},
volume = {9},
number = {10},
pages = {},
pmid = {42547416},
issn = {2575-1077},
mesh = {*Jagged-1 Protein/genetics/metabolism ; *Chromatin/genetics/metabolism ; *Promoter Regions, Genetic/genetics ; Animals ; Transcription, Genetic ; Mice ; Humans ; Enhancer Elements, Genetic ; Gene Expression Regulation ; Apoptosis/genetics ; Cell Movement/genetics ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {The Jagged1 (JAG1) gene is essential for cardiac development, yet its tissue-specific transcriptional regulation remains poorly understood. In this study we used an integrative screening approach to identify 19 candidate enhancers within the ±100 kb region flanking the JAG1 locus, among which R7 exhibited the highest activity in dual-luciferase assays. CRISPR/Cas9-mediated deletion of R7 in AC16 cells significantly reduced JAG1 expression, decreased proliferative and migratory capacities, and increased apoptosis. Mechanistically, R7 deletion altered local chromatin contacts and reduced accessibility at CTCF-bound regions near the JAG1 promoter, accompanied by decreased H3K27ac, H3K4me3, RNA polymerase II, and SRF occupancy. These findings identify R7 as a cardiac-associated promoter-proximal regulatory element with enhancer-like activity that contributes to local chromatin organization and transcriptional activity at the JAG1 locus.},
}
@article {pmid42379397,
year = {2026},
author = {Shao, D and Wen, X and Luo, Q and Liu, X and Li, L and Men, S},
title = {Combined T-DNA and CRISPR/Cas9 mutagenesis reveals redundant developmental roles of the Arabidopsis BAG family.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {371},
number = {},
pages = {113305},
doi = {10.1016/j.plantsci.2026.113305},
pmid = {42379397},
issn = {1873-2259},
mesh = {*Arabidopsis/genetics/growth &amp; development/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Gene Expression Regulation, Plant ; Plant Growth Regulators/metabolism ; Indoleacetic Acids/metabolism ; *Molecular Chaperones/genetics/metabolism ; Germination/genetics ; },
abstract = {BAG (Bcl-2-associated athanogene) genes encode evolutionarily conserved co-chaperones that participate in proteostasis regulation, stress responses, and programmed cell death. However, their collective functions during plant development remain poorly understood. Promoter cis-element analysis revealed multiple hormone-responsive elements in promoters of Arabidopsis thaliana (Arabidopsis) BAG genes, suggesting potential involvement of BAG genes in phytohormone-mediated developmental regulation. To investigate this, we generated a bag-septuple (bag-s) mutant in which all seven Arabidopsis BAG genes were knocked out using a combination of T-DNA insertion alleles and CRISPR/Cas9-mediated mutagenesis. Phenotypic characterization revealed pleiotropic defects, including delayed seed germination, increased seed coat mucilage accumulation, reduced primary root elongation, decreased rosette diameter and plant height, and delayed leaf senescence. Consistent with the delayed leaf senescence phenotype, expression of senescence-associated genes and senescence-promoting transcription factors was downregulated in the bag-s mutant. RT-qPCR analyses further showed that genes involved in auxin biosynthesis and auxin signaling were downregulated in the bag-s mutant. Furthermore, exogenous IAA partially rescued the root elongation defect of the bag-s mutant, supporting a functional association between BAG genes and auxin-dependent root growth. Collectively, these findings indicate that BAG genes redundantly regulate seed germination, vegetative growth, auxin-related root development, and leaf senescence, providing a genetic framework for further dissecting BAG-mediated coordination of proteostasis, hormone signaling, and plant development.},
}
@article {pmid42391902,
year = {2026},
author = {Liao, W and Li, S and Wu, S and Yang, X and Liu, B and Ling, L and Zeng, Q and Zhang, J},
title = {Sensitive detection of prostate cancer antigen 3 (PCA3) in urine based upon CRISPR/Cas12a and gold nanorods (AuNRs).},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {363},
number = {Pt 2},
pages = {128309},
doi = {10.1016/j.saa.2026.128309},
pmid = {42391902},
issn = {1873-3557},
mesh = {Humans ; Male ; *Gold/chemistry ; *Nanotubes/chemistry/ultrastructure ; *Antigens, Neoplasm/urine ; *Prostatic Neoplasms/urine/diagnosis ; *CRISPR-Cas Systems ; Limit of Detection ; Spectrometry, Fluorescence ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Biomarkers, Tumor/urine ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism ; },
abstract = {Prostate Cancer Antigen 3, a type of long non-coding RNA, exhibits outstanding specificity as a biomarker for the diagnosis of prostate cancer, offering a highly effective diagnostic indicator, whereas the currently used prostate specific antigen exhibits low specificity, leading to reduced accuracy in prostate cancer diagnosis. Herein, we designed a novel fluorescent sensing platform for targeted detection of PCA3, which integrates the non-specific trans-cleavage activity of the CRISPR/Cas12a with the remarkable fluorescence quenching effect of Gold Nanorods. The Cas12a recognizes and binds to specific sequences of PCA3, thereby activating nonspecific cleavage activity, which cleaves fluorescent reporter probes adsorbed on AuNRs, thus leading to the recovery of fluorescence signals and enabling sensitive detection. The proposed fluorescent sensor exhibits excellent accuracy and convenience for the detection of PCA3 in urine, and a detection limit as low as 1.65 pM was obtained. This sensing system has achieved effective detection of clinical samples of prostate cancer and is expected to provide significant assistance in the screening and therapeutic feedback of prostate cancer in clinical diagnosis.},
}
@article {pmid42437612,
year = {2026},
author = {Li, X and Xin, C and Guo, J and Zheng, Y and Huang, F and Li, X and Guo, H and Xu, Y and Shao, W},
title = {Multiplex CRISPR-Cas9 editing of starch branching enzyme II and vacuolar invertase simultaneously enhances resistant starch content and cold-induced sweetening resistance in Solanum chacoense.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {371},
number = {},
pages = {113319},
doi = {10.1016/j.plantsci.2026.113319},
pmid = {42437612},
issn = {1873-2259},
mesh = {*beta-Fructofuranosidase/genetics/metabolism ; *Starch/metabolism ; *1,4-alpha-Glucan Branching Enzyme/genetics/metabolism ; *Solanum/genetics/enzymology/metabolism ; *CRISPR-Cas Systems ; Cold Temperature ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Potato processing suffers from a high glycemic index due to amylopectin-rich starch and from undesirable color and acrylamide formation during frying, mainly caused by cold-induced sweetening (CIS). To address both issues simultaneously, we used CRISPR-Cas9 to knock out two key genes in diploid Solanum chacoense: ScSBE II (starch branching enzyme II), which controls amylopectin biosynthesis, and ScVInv (vacuolar invertase), a central regulator of CIS. Knockout of ScSBEⅡ increased tuber fresh weight-based absolute amylose content by ∼5-fold versus wild type. Amylose proportion in total starch elevated from 22% to 54%, while amylopectin abundance declined 1.5-fold, substantially optimizing the amylose/amylopectin mass ratio of tuber starch. These lines also showed a 4-fold reduction in rapidly digestible starch (RDS), 3.5‑fold and 1.2‑fold increases in slowly digestible starch (SDS) and resistant starch (RS), respectively, and markedly improved pasting properties. Enzyme assays confirmed a 2.5‑fold reduction in ScSBE II activity. In wild‑type (WT) tubers, cold storage (4 °C, 7 d) increased ScVInv activity ∼5‑fold (to 48 μg·min[-1]·g[-1]) and reducing sugars 6‑fold (from 11 to 68 mg·g[-1]). Notably, ScVInv single‑knockout and ScSBE II/ScVInv double‑knockout lines produced chips with lighter color and much lower acrylamide than WT or ScSBE II single‑knockout lines. This dual‑gene editing strategy creates novel potato germplasm with enhanced resistant starch (health benefit) and superior processing quality (safer, visually appealing fried products).},
}
@article {pmid42543873,
year = {2026},
author = {Wang, J and Peng, Q},
title = {Innovations, Applications, and Future Trends in Veterinary Diagnostic Technologies.},
journal = {Transboundary and emerging diseases},
volume = {2026},
number = {1},
pages = {e6973879},
pmid = {42543873},
issn = {1865-1682},
support = {32470195//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Veterinary Medicine/trends/methods ; *Animal Diseases/diagnosis ; High-Throughput Nucleotide Sequencing/veterinary ; },
abstract = {Veterinary diagnostics is undergoing a significant transformation driven by technological advancements, extending its scope from the traditional confirmation of specific pathogens to the continuous, dynamic surveillance of animal population's health. This paradigm shift has the potential to enable more timely disease control, precise intervention, and enhanced public health security. Traditional clinical and laboratory diagnostic methods, such as microbial culture, serological assays, and nucleic acid-based polymerase chain reaction, form the cornerstone of the current diagnostic framework and are widely applied based on varying detection needs and practical environments. Nonetheless, the field is experiencing profound innovation. Firstly, novel detection technologies are emerging, such as digital PCR (dPCR), CRISPR-Cas-based molecular diagnostic tools, next-generation sequencing (NGS), and metagenomic sequencing. These technologies have not only achieved breakthroughs in sensitivity and specificity but, more importantly, enable the unbiased discovery of novel pathogens. Secondly, the deep integration of artificial intelligence (AI) and big data is reshaping the diagnostic pipeline. By consolidating and analyzing multimodal information streams from imaging, genomics, wearable devices, and production data, AI algorithms can provide objective, quantitative decision support, facilitating a transition from post-symptomatic diagnosis towards predictive and preventive health management. This scoping review systematically summarizes both mainstream and emerging veterinary diagnostic technologies, elaborates and discusses their advantages and limitations as well as future developmental directions, while highlighting that the combined application of multiple methods represents an optimal diagnostic strategy.},
}
@article {pmid42545527,
year = {2026},
author = {Hu, Y and Fang, F and Cui, Z and Chen, S},
title = {A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {28},
number = {4},
pages = {},
pmid = {42545527},
issn = {1436-2236},
support = {2023TD20//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2023ZD0405503//STI 2030-Major projects/ ; 2022YFD2400101//the National Key R&amp;D Program of China/ ; 32230107//National Natural Science Foundation of China/ ; 2023ZLYS02//the Key Research and Development Project of Shandong Province/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Electroporation/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA, Messenger/genetics/metabolism ; Embryo, Nonmammalian/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Green Fluorescent Proteins/genetics ; Polyglutamic Acid ; Chorion/metabolism ; Zebrafish Proteins/genetics ; },
abstract = {This study presents a streamlined electroporation-based method for efficient macromolecular delivery into zebrafish embryos. We first characterized the physical barrier posed by the chorion using TEM (Transmission Electron Microscope) and established that its removal is prerequisite for effective delivery. A systematic optimization of electroporation parameters for dechorionated embryos identified optimal conditions (e.g., poring pulse: 25 V, 20 ms; transfer pulse: 5 V, 50 ms) that ensured high embryo survival and robust eGFP mRNA transfection. Applying this protocol, we achieved targeted gene knockout by electroporation-mediated delivery of Cas9 ribonucleoproteins (RNPs) against multiple loci. Targeting the tyr locus resulted in a phenotypic albinism rate of (38.6 ± 3.30)%. Furthermore, we incorporated polyglutamic acid (PGA) to modify the RNP complexes (target tyr), which inhibited aggregation and enhanced editing efficiency to (44.45 ± 1.41)%, outperforming a commercial. electroporation system, while targeting the pigmentation-related gene slc24a5 yielded an albinism or hypopigmentation rate of (38.33 ± 2.62)%. In addition, targeting the development-associated gene ddx19b produced developmental defect phenotypes in (38.33 ± 1.88)% of embryos. The successful introduction of indels at the target site was confirmed by sequencing. Our work establishes a highly effective electroporation strategy, augmented by nanotechnology, for the delivery of mRNA and RNP complexes, enabling high-efficiency protein expression and gene editing in zebrafish embryos, with broad potential applications in aquatic biotechnology.},
}
@article {pmid42545751,
year = {2026},
author = {Haneef, S and Zhou, YJ and Bai, F},
title = {Transcriptional regulation: Efficient genetic engineering tools for non-conventional yeasts.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foag032},
pmid = {42545751},
issn = {1567-1364},
abstract = {Non-conventional yeasts are recognized as valuable hosts for producing biofuels, pharmaceuticals, and other high-value chemicals, owing to their diverse physiological traits, ability to utilize various substrates, and greater tolerance to environmental stresses compared to conventional model yeast Saccharomyces cerevisiae. To fully optimizing metabolic flux toward desired products, effective genetic engineering tools enabling precise modulation of gene expression and coordinated control of metabolic pathways are essential. In this context, we discussed classical transcriptional regulation tools like promoters, and transcription factors, alongside innovations in synthetic biology that allow metabolic engineering in non-conventional yeasts to produce higher biofuels and other useful products, promoting the development of sustainable resources, and assisting the development of innovative bio-products. It also discussed innovative programmable technologies, such as CRISPR/Cas-mediated transcriptional activation and repression, as well as dynamic regulatory systems that can fine-tune metabolic routes and balance cellular resources. Strategies for promoter engineering, transcription factor manipulation for transcriptional regulation, and metabolic rewiring were highlight as methods to boost pathway efficiency and yields. This review concluded with current challenges and future directions, focusing on integrating synthetic biology and systems biology to create robust, controllable transcriptional frameworks for next-generation yeast cell factories.},
}
@article {pmid42545979,
year = {2026},
author = {Patel, MA and Singh, M and Sinha, H and Vo, PQN and Chin, AB and Ellouzi, A and West, M and Hirukawa, A},
title = {A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In Suspension T Cells and 3D Cell Models.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/70573},
pmid = {42545979},
issn = {1940-087X},
mesh = {Humans ; *Electroporation/methods/instrumentation ; *Transfection/methods/instrumentation ; HEK293 Cells ; *RNA, Messenger/genetics/administration &amp; dosage ; *T-Lymphocytes/physiology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Microfluidic Analytical Techniques/methods/instrumentation ; },
abstract = {Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4[+] and CD8[+] T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.},
}
@article {pmid42263565,
year = {2026},
author = {Wang, Q and Wang, Y and Jia, T and Hu, X and Chen, F},
title = {Evaluation of the performance of reverse transcription-recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas12a and microfluidics for one-step detection of common HCV genotypes.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117482},
doi = {10.1016/j.diagmicrobio.2026.117482},
pmid = {42263565},
issn = {1879-0070},
mesh = {Humans ; *Hepacivirus/genetics/isolation &amp; purification/classification ; Genotype ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Hepatitis C/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Microfluidics/methods ; Rapid Diagnostic Tests ; *Genotyping Techniques/methods ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) genotyping is critical for guiding therapy, yet current methods are technically demanding and time-consuming.<br><br>AIM: To develop and evaluate a one-step, integrated assay combining reverse transcription-recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a detection on a microfluidic platform for rapid and accurate HCV genotyping.<br><br>METHODS: The assay was validated using 186 clinical samples genotyped by Sanger sequencing. The microfluidic chip enabled sequential RT-RPA amplification and CRISPR/Cas12a detection via centrifugal fluid transfer, with real-time fluorescence monitoring.<br><br>RESULTS: The assay demonstrated high concordance with Sanger sequencing (overall accuracy >98%), with sensitivities of 100% for genotypes 1b and 6a, and >97% for 2a and 3a. The limit of detection was 1 IU/mL (5 copies/mL)across major genotypes, with no cross-reactivity against other viruses.<br><br>CONCLUSION: The integrated RT-RPA-CRISPR/Cas12a-microfluidics platform offers a rapid, sensitive, and specific one-step assay for HCV genotyping, suitable for point-of-care applications in resource-limited settings.},
}
@article {pmid42289138,
year = {2026},
author = {Sata, TN and Sah, AK and Ismail, M and Sharma, G and Doloi, R and Nayak, B and Shalimar, and Venugopal, SK},
title = {Development of RPA and nested-RPA based CRISPR/Cas13a diagnostic platform for the identification of HBV DNA and HCV RNA in Indian patient cohort.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117514},
doi = {10.1016/j.diagmicrobio.2026.117514},
pmid = {42289138},
issn = {1879-0070},
mesh = {Humans ; *Hepatitis B virus/genetics/isolation &amp; purification ; *Hepacivirus/genetics/isolation &amp; purification ; India ; *Hepatitis B/diagnosis/virology ; *Hepatitis C/diagnosis/virology ; *DNA, Viral/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/genetics ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems ; Recombinases/metabolism ; Cohort Studies ; },
abstract = {BACKGROUND: Among the Indian population, hepatitis B virus (HBV) is one of the major burdens and the hepatitis C virus (HCV) chronically infects around 1% Indian population. CRISPR-based detection platforms have shown to be a novel low-cost technology with high sensitivity and specificity. In the presence of target nucleic acids, Cas13a molecule is activated to trans-cleave the fluorophore quencher (FQ)-labeled ssRNA reporter, and illuminate detectable fluorescent signals.<br><br>METHODS: Leptotrichia wadei (Lwa) cas13a was expressed and purified. Recombinase Polymerase Amplification (RPA) was implemented to produce T7 RNA polymerase appended amplicons of conserved regions of HBV and HCV at 37&#xb0;C and 42&#xb0;C respectively. Corresponding crRNAs have been designed against amplified regions and produced using In-Vitro Transcription (IVT). With T7 RNA polymerase, the RPA-amplified HBV and HCV templates are transcribed into ssRNAs, which are further used in detection assay containing expressed Cas protein, crRNA, and fluorescent probes. This detection was performed in the microplate reader in kinetic format.<br><br>RESULTS: LwaCas13a was expressed and was purified using the strep tag. Conserved regions among Indian HBV and HCV genotypes are selected as targets of detection. RPA and Nested-RPA was performed using primers against conjunct region of HBV polymerase and surface antigen and RNA-dependent RNA polymerase (RdRp) region of HCV. The detection assay was performed from 45 HBV and 30 HCV human samples, out of which it could differentiate positive and healthy samples.<br><br>CONCLUSION: This approach can be a better alternative to be used in rural India and at the same time with high sensitivity, for a rapid detection of HBV and HCV, which could be used as a novel a low-cost diagnostics platform for identification of HBV DNA and HCV RNA.},
}
@article {pmid42328982,
year = {2026},
author = {Mao, Y and Fei, X and Yang, X and Hu, C and Zhu, C and Wang, J and Ge, Y and Ye, C and Yang, S and Cheng, P and Li, Y and He, X and Hu, Z and Qi, Y},
title = {CRISPR-Cas12a-based fluorescent and visual assays for universal detection and clade discrimination of mpox virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {8},
pages = {e0010526},
pmid = {42328982},
issn = {2165-0497},
support = {2025YQZL01//Huadong Research Institute for Medicine and Biotechniques/ ; SKLADCPKFKT202512//State Key Laboratory for Animal Disease Control and Prevention/ ; },
mesh = {Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Orthopoxvirus/genetics/isolation &amp; purification/classification ; Fluorescence ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins/genetics ; Polymerase Chain Reaction/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The global mpox outbreak has highlighted critical gaps in diagnostic capabilities, particularly the need for methods that can distinguish between the high-fatality Clade I and more transmissible Clade II of the mpox virus (MPXV). Current PCR-based approaches remain reliant on laboratory infrastructure, limiting their use in resource-limited settings. There is an urgent need for a versatile diagnostic platform that can provide both accurate clade discrimination and flexible deployment across diverse healthcare environments. We developed a dual-mode detection platform by integrating recombinase-aided amplification with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a technology, creating two distinct assays: a universal assay targeting OPG034, and a Clade I-discriminatory assay targeting OPG033. The platform achieved detection sensitivities of 1 copy/reaction for both fluorescence and visual readouts with OPG034, and 10 copies (fluorescence) and 1 copy/reaction (visual) with OPG033. Both assays demonstrated high specificity, successfully distinguishing MPXV from related orthopoxviruses and common viruses. Clinical validation using 23 Clade II samples and 10 healthy controls showed that, relative to quantitative PCR (qPCR) (cycle threshold ≤37), the OPG034 fluorescence assay detected all 13 qPCR-positive samples and 2 additional positives (100% sensitivity, 80.0% specificity), while the visual assay detected 12 of 13 positives (92.3% sensitivity, 100% specificity). For Clade I-specific detection, both OPG033 fluorescence and visual assays showed 100% specificity in Clade II samples (23/23). While the clade-discriminatory capability was established through sequence-specific design, further evaluation with authentic Clade I clinical specimens is warranted to confirm typing performance. The dual-mode design provides flexibility for both laboratory and field use, advancing mpox surveillance and outbreak response.IMPORTANCEMpox is a significant zoonosis. Accurate discrimination between its highly lethal Clade I and more transmissible Clade II is critical for clinical management and outbreak control, yet current methods primarily enable only general detection. To address this, we identified novel genetic markers (OPG034 for universal detection and OPG033 for Clade I specificity) and developed a dual-mode detection platform integrating Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a with recombinase-aided amplification. Its key advantage is providing two result readouts: a sensitive fluorescence mode for laboratories and an instrument-free visual colorimetric mode for field use. The demonstrated excellent performance on clinical samples confirms that this platform meets the precision requirements of clinical laboratories while remaining suitable for resource-limited settings like field clinics. Thus, it offers a flexible and practical tool for enhancing mpox surveillance and control globally, particularly in regions with constrained medical resources.},
}
@article {pmid42372673,
year = {2026},
author = {Yang, H and Li, X and Su, Y and Guo, J and Wang, T and Wang, Z and Fu, H and Chen, Z and Xie, Z and Li, D},
title = {Enhanced stability of RPA-CRISPR-Cas12a system for respiratory pathogen detection using Trehalose-Carboxymethyl Chitosan Lyoprotectant.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117523},
doi = {10.1016/j.diagmicrobio.2026.117523},
pmid = {42372673},
issn = {1879-0070},
mesh = {Humans ; Sensitivity and Specificity ; *Chitosan/analogs &amp; derivatives/chemistry ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Trehalose/chemistry ; *Respiratory Tract Infections/diagnosis/virology/microbiology ; SARS-CoV-2/genetics/isolation &amp; purification ; Neisseria meningitidis/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Streptococcus pneumoniae/genetics/isolation &amp; purification ; Freeze Drying ; },
abstract = {PURPOSE: Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection.<br><br>MATERIALS AND METHODS: A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis, SARS-CoV-2, Streptococcus pneumoniae, and human adenovirus) using real-time fluorescence and gel electrophoresis.<br><br>RESULTS: This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR-Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non&#x2011;hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4&#xb0;C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR-preliminarily supporting the system's potential diagnostic accuracy-we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance.<br><br>CONCLUSION: The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.},
}
@article {pmid42539204,
year = {2026},
author = {Saxena, AG and Ramey, GD and Capra, JA and Conklin, BR and Macklin, BL},
title = {EXCAVATE-HT: A Bioinformatic Pipeline to Identify Targetable Genomic Variants for Allele-Specific Editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42539204},
issn = {2692-8205},
abstract = {Allele-specific CRISPR/Cas editing is a powerful tool with great potential for treating genetic diseases and for uncovering the effects of allelic diversity. By targeting commonly inherited single nucleotide polymorphisms (SNPs), a small number of gRNAs can treat many more individuals than targeting rare disease mutations. However, current tools for identifying common targetable variants and generating CRISPR guide RNAs (gRNA) have fundamental conceptual and technical limitations. Here, we introduce EXCAVATE-HT (EXtracting Common Allelic VAriants for Targeted Editing in High-Throughput) a bioinformatic tool that mines population variant data to generate CRISPR libraries targeting genomic loci for allele-specific editing. Users define their loci of interest, Cas species, and SNP frequency, then EXCAVATE-HT outputs an annotated list of allele-specific gRNAs. EXCAVATE-HT can also generate libraries of gRNA pairs to enable excision. We illustrate the use of EXCAVATE-HT to design and characterize multiple gRNA libraries for allele-specific targeting of the disease gene, Cone-Rod Homeobox (CRX). EXCAVATE-HT revealed multiple excisions that could treat >30-fold more patients than targeting a single CRX disease mutation.},
}
@article {pmid42542233,
year = {2026},
author = {Liu, Y and Feng, L and Li, J and Zhang, R},
title = {CRISPR-based live-cell DNA imaging: Technologies, biological insights and future perspectives.},
journal = {Biotechnology advances},
volume = {92},
number = {},
pages = {108999},
doi = {10.1016/j.biotechadv.2026.108999},
pmid = {42542233},
issn = {1873-1899},
abstract = {Live-cell DNA imaging provides a direct view of genome behavior in real-time and has advanced rapidly with the development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system. Here, we review progress in live-cell DNA imaging from two major directions: non-repetitive loci visualization and multicolor imaging. We also highlight biological insights enabled by these technologies, covering DNA replication, damage and repair, chromatin organization and interactions, epigenetic regulation, extrachromosomal DNA, and viral genome dynamics. We then discuss future trends in live-cell DNA imaging and its potential impact on both biotechnology and biomedical research.},
}
@article {pmid41639251,
year = {2026},
author = {Khan, A and Herring, G and Zhu, JY and Petterson, M and Lister, R},
title = {Designing and testing CRISPRi-based synthetic gene circuits in plants.},
journal = {Nature protocols},
volume = {21},
number = {8},
pages = {3528-3551},
pmid = {41639251},
issn = {1750-2799},
support = {DP240103385//Department of Education and Training | Australian Research Council (ARC)/ ; CE230100015//Department of Education and Training | Australian Research Council (ARC)/ ; GNT2035042//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Protoplasts/metabolism ; Arabidopsis/genetics ; *Gene Regulatory Networks ; *Genes, Synthetic ; *CRISPR-Cas Systems ; Triticum/genetics ; *Plants/genetics ; Brassica napus/genetics ; Bryopsida/genetics ; },
abstract = {Synthetic gene circuits are powerful tools for precisely programming gene expression and introducing novel cellular functions. However, their development and application in plants has lagged behind other systems, due mainly to the limited availability of modular genetic parts. We recently developed a CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. Using a robust and high-throughput protoplast-based dual luciferase assay, we demonstrated the development, testing and functionality of these circuits in various plant species. Here we detail the key design principles and considerations for building and testing programmable and reversible CRISPRi-based gene circuits in plants. We also provide detailed procedures for isolating protoplasts from multiple plant species, including Arabidopsis thaliana, Brassica napus, Triticum aestivum and Physcomitrium patens. Furthermore, we provide step-by-step instructions for the 96-well plate-based protoplast transfection assay for testing genetic parts and synthetic circuits, using a dual luciferase assay. The detailed descriptions of these developed systems will enhance the efficiency and reproducibility of the construction, testing, and implementation of synthetic gene circuits in a variety of plant species. This protocol enables the design and testing of CRISPRi-based gene circuits in plants within ~4 weeks.},
}
@article {pmid41933972,
year = {2026},
author = {Li, Z and Wang, X and Liu, J and Janssen, JM and Hoeben, RC and Gonçalves, MAFV},
title = {Selector adeno-associated viral vectors facilitate on-target precise genome editing and purge off-target chromosomal insertions.},
journal = {Trends in biotechnology},
volume = {44},
number = {8},
pages = {2422-2445},
doi = {10.1016/j.tibtech.2026.01.006},
pmid = {41933972},
issn = {1879-3096},
mesh = {*Dependovirus/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Humans ; CRISPR-Cas Systems ; },
abstract = {Adeno-associated viral (AAV) vectors are commonly used for genome editing owing to the proclivity with which their single-stranded genomes serve as homologous recombination (donor) substrates during programmable nuclease-assisted gene targeting. However, the highly recombinogenic nature of AAV genomes also facilitates their nonhomologous end joining at off-target chromosomal breaks ('capture') created by said nucleases, mutagens, or DNA metabolic processes. Moreover, AAV donor constructs can equally yield imprecise on-target edits resulting from end-joining recombination pathways. Here, we demonstrate that endowing AAV vectors with exogenous marker-free selectable sequences permits enrichment for cells precisely coedited at endogenous target and ATP1A1 alleles. These selector AAV vectors install ATP1A1 polymorphisms conferring resistance to the small molecule ouabain, yielding high frequencies of on-target and precisely edited cell populations. Crucially, we further report that selector AAV vectors achieve a thorough removal of heterogeneous off-target DNA species resulting from conventional AAV-based genome editing procedures.},
}
@article {pmid42035261,
year = {2026},
author = {Tan, K and Del Bosque Siller, D and Xiong, AY and Wang, AXA and McCallister, TX and Mummadi, S and St John, LA and Lee, TK and Carrillo, AN and Renshaw, DG and Zhou, RH and Lim, CKW and He, J and Fields, CJ and Hayden, MR and Gaj, T},
title = {Treatment of Huntington's disease with a pan-HTT-targeting CRISPR nuclease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {8},
pages = {4635-4655},
pmid = {42035261},
issn = {1525-0024},
support = {R01 GM141296/GM/NIGMS NIH HHS/United States ; R01 NS123556/NS/NINDS NIH HHS/United States ; U01 NS122102/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Huntington Disease/therapy/genetics/metabolism/pathology ; *Huntingtin Protein/genetics/metabolism ; Mice ; Disease Models, Animal ; *CRISPR-Cas Systems ; Humans ; Dependovirus/genetics ; *Genetic Therapy/methods ; Genetic Vectors/genetics/administration &amp; dosage ; Gene Editing ; Trinucleotide Repeat Expansion ; Gene Targeting ; Neurons/metabolism ; },
abstract = {Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene, which leads to a mutant protein that destroys neurons in the brain. Despite intense effort, there remains no approved disease-modifying therapy for HD. Here, we develop a pan-HTT-targeting CRISPR-Cas9 system that, when delivered to the striatum of R6/2 and YAC128 mice by adeno-associated virus serotype 5 (AAV5), lowered mutant HTT mRNA and protein by 55%-80% via its induction of frameshift-inducing insertion or deletion (indel) mutations in HTT exon 1. Cas9 targeting improved motor coordination and locomotor activity, decreased anxiety-like deficits, reduced clasping and weight loss, limited striatal atrophy, and decreased the formation of intranuclear inclusions immunoreactive for the mutant HTT protein. In Hu21/21 mice, which carry the wild-type human HTT gene in lieu of the mouse ortholog, Cas9 lowered the HTT protein by 44% but induced no measurable behavioral deficits and had no adverse effect on neuronal viability, though its targeting was associated with neuroinflammation. Altogether, our results demonstrate the ability of a newly developed pan-HTT-targeting Cas9 system to affect HD-related phenotypes across models and provide insights into its tolerability.},
}
@article {pmid42231480,
year = {2026},
author = {Xu, L and Liang, H and Bai, S and Xu, R and Liu, B and Xu, X and Xu, X and Liu, M},
title = {Protein arginine methyltransferase 5 is essential for virulence in Toxoplasma gondii.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {},
pmid = {42231480},
issn = {1756-3305},
support = {2021A1515011707//Guangdong Provincial Natural Science Foundation/ ; },
mesh = {*Toxoplasma/pathogenicity/genetics/enzymology ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; Animals ; Virulence ; Mice ; Mice, Inbred BALB C ; Arginine/analogs &amp; derivatives/metabolism ; *Protozoan Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Protein arginine methyltransferase 5 (PRMT5) is a key enzyme responsible for catalyzing symmetric dimethylarginine (SDMA) modifications and plays crucial roles in epigenetic regulation, transcription, and cell cycle progression in eukaryotes. Although our previous study determined the expression and cellular localization of PRMT5 in tachyzoites and bradyzoites, and confirmed its type II PRMT activity, its functional significance in Toxoplasma gondii remains entirely uncharacterized.<br><br>METHODS: This study aimed to explore the biological functions of PRMT5 in T.&#xa0;gondii. The prmt5 gene was disrupted in the type I RH strain using the clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 system. The biological roles of PRMT5 were evaluated via multiple functional assays, including plaque formation, intracellular proliferation, host cell invasion, virulence, and tachyzoite to bradyzoite conversion assays. RNA sequencing was further performed to profile transcriptomic alterations induced by prmt5 disruption.<br><br>RESULTS: Phenotypic characterization revealed that the &#x2206;prmt5 strain exhibited reduced symmetric dimethylarginine (SDMA) levels as well as severe defects in plaque formation, invasion, intracellular replication, and bradyzoite differentiation. Accordingly, the virulence of the &#x2206;prmt5 strain was dramatically attenuated, as all infected BALB/c mice survived over a 10-day period, in stark contrast to the 100% mortality observed in the wild-type control group within 10&#xa0;days. RNA-sequencing analysis uncovered the molecular basis for these phenotypes, demonstrating that prmt5 disruption leads to global transcriptional dysregulation. Specifically, we identified a significant downregulation of genes associated with motor protein function and fatty acid metabolism pathways.<br><br>CONCLUSIONS: Our research has demonstrated that PRMT5 plays a critical role in the proliferation, survival, pathogenicity, and regulation of gene expression in Toxoplasma gondii.},
}
@article {pmid42259916,
year = {2026},
author = {Zeng, J and Cheng, Z and Chen, H and Wang, Z and Thompson, J and Crosby, KT and Han, H and Singhal, A and Ngo, W and Xia, C and Rosas-Rivera, D and Zhang, Z and Kang, MH and Mao, Y and Diolaiti, ME and Lee, GC and Diffley, JFX and Song, Y and Qiu, L and Krah, NM and Murthy, N and Jackson, RN and Liu, Y and Ashworth, A and Doudna, JA},
title = {Targeting cancer-specific mutations with RNA-triggered chromatin shredding.},
journal = {Nature},
volume = {656},
number = {8126},
pages = {199-206},
pmid = {42259916},
issn = {1476-4687},
mesh = {Humans ; *Chromatin/metabolism/genetics ; *Mutation/genetics ; *Neoplasms/genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; DNA Damage ; Animals ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Genetic mutations that drive cancer often occur in tumour-suppressor proteins such as the p53 transcription factor, which is altered in 40-50% of cases[1,2]. However, current therapies often fail to target these mutations because the mutant proteins typically lack defined drug-binding pockets and restoring their endogenous function has proven challenging. Here we program Cas12a2, an RNA-guided CRISPR nuclease with trans-nucleolytic cleavage activity[3,4], to kill cancer cells selectively by targeting cancer-specific transcripts. This approach limited cell growth by inducing trans shredding of chromatin and triggering DNA-damage responses and cell death. In contrast to existing methods, RNA-guided Cas12a2 senses cellular RNA signatures, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative approach to precision disease treatments for undruggable targets.},
}
@article {pmid42331809,
year = {2026},
author = {Zhou, C and Dong, C and Zhao, W and Liang, FS},
title = {Hierarchical interplay between H3K27ac and H3K4me3 in transcriptional regulation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42331809},
issn = {2041-1723},
support = {R01GM143256//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Histones/metabolism/genetics ; *Transcription, Genetic ; RNA Polymerase II/metabolism ; Epigenome Editing ; Humans ; Promoter Regions, Genetic ; *Gene Expression Regulation ; CRISPR-Cas Systems ; Chromatin/metabolism ; RNA Stability ; Animals ; },
abstract = {H3K27ac and H3K4me3 are enriched at transcriptional start sites and have been implicated in transcription. However, how these marks concertedly regulate transcription is not fully understood. Here, we develop a dual chemically inducible CRISPR/dCas9-based epigenome editing system that enables independent, temporal and transcription stage-specific modulation of H3K27ac and H3K4me3 at a specific gene locus. Stage-specific removal of H3K4me3 impairs RNA polymerase II recruitment, increases promoter-proximal pausing, reduces productive elongation, and accelerates mRNA decay via increased m[6]A deposition. Losing both H3K27ac and H3K4me3 rapidly abolishes transcriptional activity, while preserving H3K4me3 without H3K27ac can partially sustain transcription. These findings reveal a functional hierarchy and interdependence between H3K27ac and H3K4me3 in different transcription stages at the tested gene loci. This versatile tool will contribute to the functional dissection of the temporal dynamics of chromatin modifications in gene regulation.},
}
@article {pmid42358199,
year = {2026},
author = {Gijsbertsen, M and Duarte, FM and Fuentes Manjón, A and Maduro, T and Kassem, M and van der Oost, J and Mathijssen, IMJ and van Leeuwen, JPTM and van de Peppel, J},
title = {Evaluation of Prime Editing Efficiency in Human Immortalized MSC-TERT Cells with Osteogenic Potential for Modeling FGFR2-Linked Craniosynostosis.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {191-206},
doi = {10.1177/25731599261457618},
pmid = {42358199},
issn = {2573-1602},
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism/cytology ; *Receptor, Fibroblast Growth Factor, Type 2/genetics ; *Osteogenesis/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; HEK293 Cells ; *Craniosynostoses/genetics ; Telomerase/genetics/metabolism ; Mutation ; Cell Line ; },
abstract = {Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific in vitro models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human in vitro disease model with the CRISPR-Cas9 prime editing variant, using an immortalized TERT-immortalized mesenchymal bone marrow-derived stem (MSC-TERT) cell line with osteogenic potential. MSC-TERT cells showed a higher resistance to prime editing, compared with HEK293FT cells. Addition of dnMLH1 and epegRNAs resulted in higher editing efficiencies in HEK293FT cells, but not in MSC-TERT cells. Prime editing efficiency varied between targeted loci and was found to be more efficient in nonadherent cells compared with adherent cells. Prime editing continued over 4 days in an isolated nonadherent HEK293FT culture. Our results present a foundation on the use of prime editing to establish FGFR2 mutation-specific in vitro models and their application in MSC-TERT cells.},
}
@article {pmid42413621,
year = {2026},
author = {Nan, Y and Yan, S and Zhang, L and Yang, F and Qiao, M and Xin, Y},
title = {Markerless large DNA integration in Lactococcus lactis through the coupling of homologous single-crossover and Cre/loxP system.},
journal = {Journal of biotechnology},
volume = {418},
number = {},
pages = {60-68},
doi = {10.1016/j.jbiotec.2026.07.004},
pmid = {42413621},
issn = {1873-4863},
mesh = {*Lactococcus lactis/genetics ; Plasmids/genetics ; *Integrases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Lactococcus lactis is widely used in food fermentation and has great potential as a microbial cell factory for producing high-value compounds. Currently, heterologous gene expression in this host mainly relies on plasmid-based systems, which suffer from segregational instability without antibiotic selection. To address this problem, several genome integration tools have been developed, yet most are limited to single gene insertions or leave selection markers. In this study, we developed an efficient and markerless platform for integration single genes and large DNA fragments combining a temperature-sensitive plasmid for homologous single-crossover and the Cre/loxP system for plasmid backbone excision. First, using CRISPR/Cas9 assisted ssDNA recombineering, we introduced a loxP site into the ribB gene of L. lactis NZ9000/RecT, creating the chassis strain L. lactis loxP. We then constructed a donor plasmid, pG15AribB-Up-Cat-loxP, which carries a homology arm of ribB, the chloramphenicol resistance gene cat, and a second loxP site. This donor plasmid was integrated into the chromosome next to the existing loxP site through a single-crossover event. Subsequent expression of Cre recombinase then removed the plasmid backbone, leaving only the target gene cat at the insertion site. After 99 generations without chloramphenicol selection, the integrant L. lactis IMT maintained nearly 100% genetic stability. Using this method, we next successfully markerless integrated the ∼4.8 kb crtEBI cluster, and the seven gene tagatose-6-phosphate pathway (∼7.0 kb). This work provides an efficient platform for markerless and stable large DNA integration in L. lactis for constructing microbial cell factory to produce high-value compounds.},
}
@article {pmid42456866,
year = {2026},
author = {Qu, ZL and Liu, T and Qin, TK and Yue, HM and Huang, L and Ruan, R and Wu, CS and Ye, H and Li, CJ},
title = {Establishment of a novel brain cell line from grass carp (Ctenopharyngodon idella) with high transfection efficiency and CRISPR/Cas9-mediated genome editing capacity.},
journal = {Fish &amp; shellfish immunology},
volume = {177},
number = {},
pages = {111597},
doi = {10.1016/j.fsi.2026.111597},
pmid = {42456866},
issn = {1095-9947},
mesh = {Animals ; *Carps/genetics/immunology ; Cell Line ; Transfection/veterinary ; *CRISPR-Cas Systems ; *Brain/cytology ; *Gene Editing/veterinary ; Reoviridae/physiology ; Fish Diseases/immunology/virology ; Reoviridae Infections/veterinary/immunology ; },
abstract = {Fish cell lines serve as valuable tools in aquaculture research, particularly in immunology, pathology, and toxicology. In this study, we successfully established a novel cell line derived from brain tissue of grass carp, designated CIB. This cell line has been subcultured over 100 times and exhibits a fibroblast-like morphology. Chromosomal analysis revealed that the diploid chromosome number of CIB cells is 2n = 48, while sequencing of the 18S rRNA gene confirmed the cell line's origin. Notably, CIB cells demonstrated a transfection efficiency of 62.4% with pEGFP-N3, highlighting their potential for studies involving exogenous gene expression. Furthermore, CIB cells were susceptible to grass carp reovirus genotype I (GCRV-I), as evidenced by cytopathic effects (CPE), increased synthesis of viral proteins, and accumulation of viral particles within the cells. Both viral infection and poly(I:C) stimulation significantly increased the expression of intracellular interferon-related signaling molecules. Additionally, electroporation of a gRNA-Cas9 ribonucleoprotein (RNP) complex into CIB cells achieved the first successful large-fragment gene knockout in cultured grass carp cells and generated a homozygous clonal line, thereby enhancing the antiviral response. In summary, this novel cell line represents a significant advancement for studying gene functions, host-virus interactions, and genetic engineering in teleost fish.},
}
@article {pmid42460742,
year = {2026},
author = {Lv, B and Chen, Y and Zhou, R and Liu, R and Li, D},
title = {DNAzyme-mediated synergistic activation of CRISPR/Cas12a for Cd[2+] and Pb[2+] biosensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {61},
pages = {15252-15256},
doi = {10.1039/d6cc04053e},
pmid = {42460742},
issn = {1364-548X},
mesh = {*Lead/analysis ; *DNA, Catalytic/metabolism/chemistry ; *Cadmium/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {This work reports an isothermal, one-pot assay for Cd[2+] and Pb[2+] based on DNAzyme-triggered synergistic activation of CRISPR/Cas12a.},
}
@article {pmid42479891,
year = {2026},
author = {Feng, H and Wang, Y and Zhao, J and Fan, H and Wu, T and Qu, J and Pan, S and Wang, J and Han, Z and Liang, A and Xu, T and Chen, C and Hu, C and Hu, T},
title = {Topology-Gated λ Exonuclease Enables Amplification-Free Signal Boosting.},
journal = {Journal of the American Chemical Society},
volume = {148},
number = {30},
pages = {32782-32792},
doi = {10.1021/jacs.6c11250},
pmid = {42479891},
issn = {1520-5126},
support = {23ZR1450300//Natural Science Foundation of Shanghai Municipality/ ; 23-0178-0002//National University of Singapore/ ; A-8002032-00//Ministry of Education - Singapore/ ; 22304061//National Natural Science Foundation of China/ ; 22304157//National Natural Science Foundation of China/ ; LQ23H200005//Natural Science Foundation of Zhejiang Province/ ; ZR2023MB019//Natural Science Foundation of Shandong Province/ ; 24-0994-P0001//National Centre for Infectious Disease/ ; },
mesh = {*Exodeoxyribonucleases/metabolism/chemistry ; CRISPR-Cas Systems ; *RNA, Viral/analysis ; *Exonucleases/metabolism/chemistry ; Humans ; *Bacteriophage lambda/enzymology ; MicroRNAs/analysis ; Viral Proteins ; },
abstract = {Amplification-free detection remains a fundamental challenge in CRISPR-based RNA diagnostics. Here, we identify a previously unrecognized topological property of λ exonuclease, whereby duplex substrates bearing 5' phosphates at both termini undergo a self-sustained cyclic cleavage-reforming process. This topology-gated behavior enables signal renewal without external amplification. Through systematic biochemical and structural analyses, we elucidate the underlying mechanism and establish λ exonuclease as a topology-driven signal amplifier. Then, we design a topology-gated dumbbell probe that sequesters 5' phosphates within dual RNA hairpin loops. Upon target recognition, CRISPR/Cas13 specifically cleaves the loops, exposing the hidden phosphates and thereby activating the λ exonuclease-mediated cyclic reaction. The resulting cascade, termed Topo-CRISPR (Topology-gated λ exonuclease enables CRISPR amplification-free), achieves attomolar sensitivity within 25 min without preamplification. Applied to clinical samples, Topo-CRISPR enables robust and specific detection of enterovirus RNA, miR-21, and ciR1445, demonstrating performance comparable to RT-qPCR. We further extend the Topo-CRISPR to non-nucleic-acid targets via aptamer-mediated conformational gating. This work uncovers a previously overlooked enzymatic topology, positioning λ exonuclease as a cyclic signal transducer and offering a general framework for ultrasensitive molecular sensing.},
}
@article {pmid42487445,
year = {2026},
author = {Collantes, JC and Xu, K and Ruiz-Urigüen, M and Samaniego-Villacis, AC and Stombaugh, J and Jin, S},
title = {Development and Characterization of RNA Aptamer-Mediated Modular Base Editors Containing Staphylococcus aureus Cas9 Derivatives and Novel Deaminase Orthologs.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {207-222},
doi = {10.1177/25731599261467606},
pmid = {42487445},
issn = {2573-1602},
mesh = {*Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Animals ; *Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics ; APOBEC-1 Deaminase/genetics ; HEK293 Cells ; Rats ; },
abstract = {Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform's modularity, and its feasibility for convenient screening of target-specific base editors.},
}
@article {pmid42488965,
year = {2026},
author = {Shepard, A and Minones-Moyano, E and Mork, C and Pyhtila, B and Barrangou, R},
title = {The Diversifying Distribution Trends of Maturing CRISPR Technologies by Addgene.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {184-190},
doi = {10.1177/25731599261470096},
pmid = {42488965},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {Since the advent of Cas9-based CRISPR technologies in 2012, there has been a remarkable growth in genome editing research, literature, applications, and translational impact. Much of this research has been fueled by the global dissemination of CRISPR plasmids through nonprofit distribution by Addgene, as both a repository and distributor of enabling biological material. Recently, key milestones have been reached, with over 20,000 plasmids deposited by over 1,000 labs, being distributed over 300,000 times globally. The driving trends reflect multidimensional diversification in terms of effectors (Cas9, Cas12 and beyond), editing modalities (base editing, prime editing, epigenetic modification, CRISPRi/a), and deployment across phylogenetic groups (mammalian, bacterial, plant, yeast, insects, and more). Noteworthy, guide RNA and HDR templates account for the bulk of deposits, while cloning backbones are the most requested, and lentiviral plasmids comprise the majority of expression material. The data reflect a continued diversification of the CRISPR-based toolbox, robust interest in genome editing applications across the tree of life, maturation in terms of adoption, and rising relative distribution beyond the USA and China, with Addgene continuing to play a critical role in access to equitable and disruptive technologies.},
}
@article {pmid42525182,
year = {2026},
author = {Fang, G and Zheng, S and Miao, J and Zeng, Y and Peng, Y and Zhai, Y and Guo, H and Wang, J and Dong, Y and Tian, X and Liu, M and Liu, Y and Li, X and Wang, M and Zhao, X and Du, J and Wang, Y and Dong, J},
title = {The ApoE-Null Golden Hamster: A Novel Model of Atherosclerosis.},
journal = {Cardiovascular toxicology},
volume = {26},
number = {8},
pages = {},
pmid = {42525182},
issn = {1559-0259},
mesh = {Animals ; *Atherosclerosis/pathology/genetics/metabolism/blood ; Disease Models, Animal ; *Apolipoproteins E/genetics/deficiency ; Mesocricetus ; Diet, High-Fat ; Plaque, Atherosclerotic ; *Aortic Diseases/pathology/genetics/metabolism ; Cholesterol, Dietary ; CRISPR-Cas Systems ; *Aorta/pathology/metabolism ; Male ; Phenotype ; Genetic Predisposition to Disease ; Lipids/blood ; Liver/pathology/metabolism ; Hyperlipidemias/genetics/pathology ; Animals, Genetically Modified ; Fibrosis ; },
abstract = {Atherosclerosis is a chronic, progressive arterial disease characterized by the deposition of lipids on the inner arterial walls, leading to plaque formation and serious cardiovascular events. Traditional mouse models of atherosclerosis require prolonged dietary induction to exhibit arterial lesions due to significant differences in lipid metabolism compared to humans. In contrast, Golden hamsters share a lipid metabolic profile more closely aligned with humans. In this study, we utilized CRISPR/Cas9 to generate ApoE knockout (ApoE[-/-]) hamsters using, which spontaneously developed atherosclerotic lesions in the arterial wall after 8 weeks on a standard chow diet. When fed on a high-cholesterol/high-fat diet, they exhibited even more severe aortic atherosclerosis, fatty liver, and liver fibrosis. Our findings demonstrated that the ApoE[-/-] hamster model is highly valuable tool for translational research, offering significant potential for studying hyperlipidemia and atherosclerosis in a context more relevant to human physiology.},
}
@article {pmid42525385,
year = {2026},
author = {Kim, GD and Gu, D and Park, M and Wook Chi, S},
title = {abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas genome editing.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btag568},
pmid = {42525385},
issn = {1367-4811},
abstract = {SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97,583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58,875,004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing.<br><br>The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.<br><br>SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}
@article {pmid42528148,
year = {2026},
author = {du Plessis, J and Omar, A},
title = {Patient-Derived Organoid-Based CRISPR Screens in Cancer Research: Applications, Advances, and Challenges.},
journal = {Cancer medicine},
volume = {15},
number = {8},
pages = {e72112},
pmid = {42528148},
issn = {2045-7634},
mesh = {Humans ; *Organoids/metabolism ; *Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genomics/methods ; Animals ; Precision Medicine/methods ; },
abstract = {Patient-derived organoids (PDOs) have emerged as physiologically relevant cancer models that preserve key genetic, histological, and functional features of the tumors from which they are derived. In parallel, CRISPR-based perturbation technologies have transformed functional genomics by enabling scalable interrogation of gene function. Their integration provides a powerful framework for identifying cancer dependencies, modeling oncogenic evolution, and investigating mechanisms of drug response and resistance in patient-relevant settings. This review examines how CRISPR knockout, CRISPR interference/activation, and precision editing approaches have been applied in PDO systems to uncover context-specific vulnerabilities, reconstruct mutational trajectories, and study tumor heterogeneity. We further compare pooled and arrayed screening formats and discuss what is uniquely enabled by performing CRISPR screens in organoids rather than conventional 2D models. Particular emphasis is placed on the technical and analytical constraints of organoid-based screening, including variable editing efficiency, clonal bottlenecks, biological heterogeneity, and limited scalability. We argue that the major value of organoid-based CRISPR screening lies in its ability to identify functionally actionable cancer vulnerabilities in a patient-contextualized model, while also introducing methodological challenges that must be addressed for robust clinical translation.},
}
@article {pmid42529880,
year = {2026},
author = {Zhang, W and Hang, Y and Zhan, S and Song, W and Li, B and Chen, N and Lv, M},
title = {Emerging point-of-care technologies for bacterial pathogen detection.},
journal = {Journal of Zhejiang University. Science. B},
volume = {27},
number = {7},
pages = {677-697},
pmid = {42529880},
issn = {1862-1783},
support = {32371439 and 22404026//the National Natural Science Foundation of China/ ; 24ZR1455600 and 24ZR1455900//the Natural Science Foundation of Shanghai/ ; },
mesh = {Humans ; *Point-of-Care Systems ; *Bacteria/isolation &amp; purification/genetics ; *Point-of-Care Testing ; *Bacterial Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; Immunoassay ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Bacterial infections remain a significant threat to public health worldwide, driving an urgent need for rapid, accurate, and field-deployable diagnostic techniques. Point-of-care testing (POCT) has emerged as a transformative strategy, providing timely detection, operational simplicity, and portability. Recent studies have aimed at enhancing sensitivity, specificity, multiplexing capability, and automation through the integration of molecular diagnostics with microfluidics and lab-on-chip technologies, alongside the development of low-cost, portable devices equipped with smartphone-based readout and cloud connectivity for real-time surveillance in resource-limited settings. Nonetheless, evidence-based frameworks for selecting optimal detection targets-such as genomic sequences, conserved protein epitopes, or viable whole cells-and matching them to appropriate POCT modalities remain notably underrepresented in the literature. This review systematically summarizes recent advances in POCT strategies for bacterial detection, categorized according to three major types of detection targets, including cellular phenotypic characteristics, surface antigens, and nucleic acids. We discuss the principles, advantages, limitations, and representative applications of key POCT platforms, which include microscopy-based visualization, immunoassays, isothermal amplification, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems, and microfluidic biosensors. Critical challenges, such as sample pretreatment, detection sensitivity, and operational simplicity, have been partially addressed through recent innovations. Finally, we outline the main future research directions focused on the development of integrated, automated, and intelligent POCT systems for clinical deployment.},
}
@article {pmid42530034,
year = {2026},
author = {Sharma, N and Tanwar, D and Grewal, U and Keerthi, MM and Ahlawat, YK and Pandey, N and Makvana, CG},
title = {Pectinase-Based Bioprocesses for Circular Bioeconomy and Sustainable Industrial Development.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70312},
pmid = {42530034},
issn = {1097-0290},
abstract = {Pectinases play a vital role in the degradation of pectic part of the plant cell wall and are considered in the group of hydrolytic enzymes. In the present scenario, demand of economically feasible and environment friendly techniques and approaches has significantly led the research on the microbial production, standardization of process parameters and improvements of pectinases. The development of pectinase-based bioprocesses has been found to promote sustainable industrial practices by reducing the use of chemicals, energy, and waste, thereby supporting eco-friendly production systems. The unique aspect of this review is that it explains traditional methods of pectinase production alongside novel methods, which include solid-state and submerged fermentation, recombinant DNA techniques, heterologous gene expression, protein engineering, CRISPR/Cas genome editing technologies, enzyme immobilization, and nanobiotechnological methods, which improve the production, stability, and performance of pectinases. It further demonstrates the increasing importance of the use of pectinases in sustainable manufacturing through decreasing the use of chemicals and energy, as well as reducing waste production from industry. It illustrates that innovations in microbial strain engineering, process optimization, and utilization of low-cost substrates have significantly increased the economic viability and efficiency of producing pectinases. Yet, some issues associated with commercialization, standardization of production processes, recovery, and stability of enzymes still exist. Overall, this review paper gives an overview of the recent advancements and limitations of next-generation pectinases.},
}
@article {pmid42531075,
year = {2026},
author = {Zhou, R and Zhan, Y and Sun, Y and Wang, R and Wang, T and Liu, Z and Shan, Z and Li, X and Zhang, S and Sun, N and Zhang, H and Yuan, Z and Yang, J},
title = {Molecular basis of single-mismatch-induced nuclease-to-nickase conversion in TIGR-TasH.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42531075},
issn = {1362-4962},
support = {2024ZD037//Scientific Research Program of Tianjin Municipal Education Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline/ ; 2024ZD037//Scientific Research Program of Tianjin Municipal Education Commission/ ; },
mesh = {Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *Base Pair Mismatch ; *Deoxyribonuclease I/metabolism/genetics/chemistry ; Protein Domains ; Models, Molecular ; },
abstract = {Tandem interspaced guide RNA (TIGR)-Tas systems are a distinct class of RNA-guided double-stranded DNA nucleases that employ dual-spacer guide RNAs (tigRNAs) for PAM-independent target recognition. A single mismatch between the tigRNA and target DNA can convert Salicola phage CGphi29 (Sp)TasH from a double-strand nuclease into a nickase in a position-dependent manner, but the molecular basis underlying this functional switch remains unknown. Here, we combined biochemical analyses and cryo-electron microscopy to investigate tigRNA maturation and mismatched target recognition by the Nop domain of SpTasH. We show that the Nop domain is required for pre-tigRNA processing and stabilizes the mature tigRNA through extensive interactions, thereby establishing a cleavage-competent ribonucleoprotein complex. Structural analyses of SpTasH complexes bound to substrates containing single mismatches reveal that a mismatch at the 5'-most position of spacer A is readily accommodated through Nop domain-mediated stabilization of the spacer-target heteroduplex. In contrast, a mismatch proximal to the cleavage site destabilizes the heteroduplex, preventing recruitment of the corresponding HNH domain, thereby converting the complex into a nickase. Together, these findings establish the structural basis for position-dependent mismatch recognition and reveal how Nop domain-mediated tigRNA-target stabilization enables differential responses to mismatches, providing a foundation for engineering TIGR-Tas systems for genome-editing applications.},
}
@article {pmid42532990,
year = {2026},
author = {Liu, Y and Zhao, H and Cao, X and Jiang, S and Fu, J and Xue, J and Li, C and Xu, Z and Li, M and Du, W},
title = {Rapid and quantitative measurement of bacteriophage infectivity via fully automated droplet digital PCR.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42532990},
issn = {2041-1723},
support = {XDB0810000//CAS | State Key Laboratory of Microbial Resources (State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences)/ ; 22374016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370090//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Bacteriophages/genetics/physiology/pathogenicity ; *Polymerase Chain Reaction/methods ; RNA, Ribosomal, 16S/genetics ; DNA, Viral/genetics ; Automation ; Bacteria/virology/genetics ; Phage Therapy/methods ; },
abstract = {The clinical translation of phage therapy for multidrug-resistant infections is constrained by the lack of rapid, standardized therapeutic phage selection. Here, we introduce digital phage susceptibility testing (dPhaST), an automated droplet digital PCR workflow that quantifies phage-induced DNA release as a molecular signature of lysis. By targeting conserved 16S rRNA regions, dPhaST measures lytic activity across diverse bacterial pathogens within 3 h. Across 122 phage-host combinations involving 19 bacterial strains from six species, dPhaST shows 95.9% concordance with spot tests while resolving weak and heterogeneous lytic activities that are not readily distinguished phenotypically. It remains robust during the early infection window despite phage-encoded nuclease activity and tolerates phage cross-contamination better than spot tests. The method captures defense-mediated interactions involving CRISPR-Cas and Sir2-HerA systems. In this work, we show that automated digital quantification enables rapid and mechanistically informative profiling of early phage lytic efficacy across Gram-positive and Gram-negative pathogens.},
}
@article {pmid42533766,
year = {2026},
author = {Kong, W and Fu, L and Li, Y and Pan, W and Li, N and Tang, B},
title = {Amplification-free CRISPR/Cas biosensors for point-of-care nucleic acid detection: recent advances and future perspectives.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6cc02994a},
pmid = {42533766},
issn = {1364-548X},
abstract = {Nucleic acid biomarkers are critical targets for early diagnosis of disease, public health surveillance and environmental safety. However, their low abundance and the complexity of the sample matrix pose strict requirements for the high sensitivity and portability of detection technologies. Although traditional clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems exhibit advantages such as high specificity, operational simplicity, and compatibility with mild reaction conditions, their reliance on pre-amplification steps elevates the risk of false-positive results and hinders their broader application. To overcome these limitations, amplification-free CRISPR/Cas technologies have emerged and undergone extensive development. These approaches enable highly sensitive nucleic acid detection without the need for pre-amplification and are more amenable to integration with portable devices, thereby offering promising avenues for point-of-care testing (POCT). This review systematically examines the fundamental principles and design strategies underlying amplification-free CRISPR/Cas biosensors and summarizes recent advances in detection platforms based on autocatalytic signal enhancement, nanomaterial-coupled amplification, and integrated high-sensitivity readout systems, while also outlining their practical applications in POCT settings. Furthermore, the key technical challenges and future development directions of amplification-free CRISPR technologies are discussed based on current advances in the field. These insights and perspectives aim to provide a systematic reference for further research and to facilitate the expanded application of amplification-free CRISPR/Cas systems in POCT.},
}
@article {pmid42534996,
year = {2026},
author = {Zhu, L and Huang, J and Xie, C},
title = {AI-enhanced framework for optimizing CRISPR-Cas gene editing in crop biotechnology addressing regulatory challenges and opportunities in global agricultural practices.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1770472},
pmid = {42534996},
issn = {1664-462X},
abstract = {INTRODUCTION: The integration of CRISPR Cas genome editing with artificial intelligence (AI) offers significant potential for crop biotechnology by supporting more precise and adaptive strategies for trait improvement under complex agricultural and regulatory conditions. However, the global governance of gene edited crops remains highly heterogeneous, creating major challenges for the development of frameworks that can jointly support optimization, uncertainty management, and regulatory alignment. Conventional approaches often lack the ability to account for evolving regulatory requirements and multi source uncertainties in a unified manner.<br><br>METHODS: In this paper, we introduce the Adaptive Regulatory Optimizer (ARO), an AI enhanced framework designed to support CRISPR Cas genome editing in crop biotechnology under biologically, regulatorily, and contextually constrained conditions. The ARO consists of three interconnected modules: the Manifold Constrained Gene Editor, the Agent Driven Regulatory Planner, and the Uncertainty Propagation Filter. Together, these modules embed editing decisions within biologically feasible manifolds, incorporate jurisdiction aware regulatory planning, and model interacting uncertainties associated with gene editing and deployment contexts. The The framework combines constrained optimization refinement, probabilistic uncertainty modeling, and adaptive regulatory planning to provide a structured basis for compliance aware and context sensitive decision support.<br><br>RESULTS AND DISCUSSION: Experimental results on the evaluated datasets indicate that the ARO achieves improved performance on the selected metrics relative to the compared methods, while its architecture is explicitly designed to integrate regulatory constraints into the optimization process. These findings suggest that the proposed framework provides a promising foundation for supporting more transparent, adaptive, and analytically grounded decision making in CRISPR Cas applications for crop biotechnology.},
}
@article {pmid42537394,
year = {2026},
author = {Zemmouchi, M and El-Fermawi, A and Benagdi, A and Baaziz, S and Hiram, R},
title = {Translational consistency of gene therapy strategies targeting inflammation in atrial fibrillation's management.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {202},
number = {},
pages = {119824},
doi = {10.1016/j.biopha.2026.119824},
pmid = {42537394},
issn = {1950-6007},
abstract = {Atrial fibrillation (AF) is the most common cardiac rhythm disorder. AF risk factors include pathological ageing, hypertension, obesity, diabetes, and cardiac conditions such as myocardial infarction. Chronic inflammation is a major pathophysiological profile commonly observed in AF and its risk factors. Clinical and preclinical studies have suggested that increased expression of proinflammatory biomarkers such as the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, interleukin (IL)-1β, or IL6 is associated with the development and maintenance of cardiac arrhythmias including AF. Current anti-arrhythmic and anti-inflammatory treatments are non-optimal in AF management. In parallel, mounting evidence suggests that new biotechnologies including gene therapy approaches, might help to target specific genes to prevent or promote their expression and their associated protein activity. Applied to cardiac arrhythmias, gene therapy might help to restore normal functions of ion channels, optimal calcium (Ca[2 +])-handling machinery, functional gap junctions, and efficient inflammatory signaling, known to be altered in AF. With an emphasis on the importance of gene therapy strategies targeting inflammation, this narrative review aims to: i) highlight the rationale and clinical relevance of gene therapy as an innovative strategy for the management of AF; ii) evaluate current knowledge regarding gene therapy vectors and delivery platforms applicable to cardiology and AF; iii) review emerging molecular targets explored in the context of AF gene therapy; and iv) identify promising gene-based therapeutic candidates, with a particular focus on inflammation-related pathways in AF.},
}
@article {pmid42538871,
year = {2026},
author = {Wang, X and Trypsteen, W and Anckaert, J and de Bony, E and Mestdagh, P},
title = {An Optimized Workflow for In Vitro Transcription of Single Guide RNAs Minimizes Innate Immune Activation.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {223-232},
doi = {10.1177/25731599261467974},
pmid = {42538871},
issn = {2573-1602},
mesh = {*Immunity, Innate/genetics ; *Transcription, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics/immunology ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Double-Stranded/genetics ; Workflow ; Gene Knockdown Techniques ; Sodium Chloride ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {CRISPR interference (CRISPRi) often uses single guide RNAs (sgRNAs) generated by in vitro transcription (IVT); however, IVT-derived RNAs can trigger innate immune responses that confound functional analyses. Here, we evaluate innate immune activation induced by IVT sgRNAs in a CRISPRi setting and show that enzymatic removal of the 5'-triphosphate group alone is insufficient to consistently eliminate this response. We therefore assessed modifications of IVT reaction conditions and found that supplementation with sodium chloride or urea further attenuated immune activation. Based on immune suppression, sgRNA yield, and knockdown efficiency, 0.15 M NaCl was selected for the optimized IVT condition. This condition showed a lower double-stranded RNA (dsRNA) concentration, providing direct support for reduced dsRNA by-products as a contributor to diminished immune activation. By integrating NaCl-supplemented IVT with phosphatase treatment, we establish an optimized and scalable workflow that minimizes innate immune responses while preserving sgRNA-mediated target knockdown efficiency in stable CRISPRi cells.},
}
@article {pmid40476386,
year = {2026},
author = {Su, S and Xu, Z and Suo, J and Zhou, Y and Zhang, X and Peng, X and Chen, M and Li, F},
title = {Function analysis of flightin gene in the global tortricid fruit borer Grapholita molesta using CRISPR/Cas9.},
journal = {Insect science},
volume = {33},
number = {4},
pages = {1357-1368},
doi = {10.1111/1744-7917.70079},
pmid = {40476386},
issn = {1744-7917},
support = {//the Coordinated Research Project (CRP) of the International Atomic Energy Agency/ ; //the National Key R&amp;D Program of China/ ; //the Science and Technology Partnership Program of Ministry of Science and Technology of China/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Moths/genetics/growth &amp; development/physiology/metabolism ; *Insect Proteins/genetics/metabolism ; Flight, Animal ; Larva/growth &amp; development/genetics/physiology ; Female ; Pupa/growth &amp; development/genetics ; Gene Knockout Techniques ; Male ; },
abstract = {Many tortricid moths are significant fruit borers characterized by limited flight capacity. However, some individuals within a population of tortricid species exhibit extended flight capabilities, facilitating gene flow between orchards and enabling host switching. To date, research on the proteins involved in flight among fruit borers is limited. Flightin is recognized as a flight muscle protein, yet its function remains unexplored in lepidopteran insects. In this study, quantitative polymerase chain reaction analysis revealed that the flightin gene is expressed at various developmental stages and tissues of Grapholita molesta, with the highest expression in adults and the thorax. Using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) gene editing technology, we successfully generated a homozygous flightin gene knockout strain of G. molesta. The knockout of the flightin gene resulted in contraction of indirect flight muscle fibers, irregularities in the Z-disc of the flight muscles, and a significant elongation of sarcomere length. Additionally, cumulative flight distance and flight time were significantly reduced. The larval period and preoviposition period were significantly prolonged, while larval weight, pupal weight, longevity, and fecundity were all significantly decreased. The results indicate that the flightin gene not only plays an important role in the flight capacity of G. molesta, but also has an effect on the growth and development, and reproduction of the insect, suggesting that flightin may be a potential target for pest management of G. molesta. This is the first investigation into the function of the flightin gene using CRISPR/Cas9.},
}
@article {pmid41159566,
year = {2026},
author = {Yen, PS and Verkuijl, SANR and Capriotti, P and Del Corsano, G and Yee, CKG and Hoermann, A and Inghilterra, MG and Aramburu-Gonzalez, I and Khan, MA and Vlachou, D and Christophides, GK and Windbichler, N},
title = {The nanosd integral gene drive enables population modification of the malaria vector Anopheles gambiae.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf246},
pmid = {41159566},
issn = {2160-1836},
support = {OPP1158151//Bill and Melinda Gates Foundation/ ; INV-058071//Bill and Melinda Gates Foundation/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; Female ; *Mosquito Vectors/genetics/parasitology ; *Malaria/transmission/parasitology ; Male ; *Gene Drive Technology ; CRISPR-Cas Systems ; Fertility/genetics ; *Insect Proteins/genetics ; },
abstract = {The modification of mosquito populations at scale through CRISPR-Cas9-mediated homing gene drives is a promising route for malaria vector control. Integral gene drives (IGDs) are designed to utilize the regulatory sequences of endogenous genes to reduce the size of the modification required for nuclease and effector expression. In this study, we describe the creation and characterization of the nanosd integral gene drive, which targets and is inserted into the nanos gene of the malaria vector Anopheles gambiae, and show that it achieves high rates of gene drive (98.4% in females, 99.5% in males). We find that homozygous nanosd females but not males show impaired fecundity and exhibit variable degrees of ovary underdevelopment. Transcriptomic analysis of ovaries points to decreased transcript levels of the nanos gene when harboring Cas9 and changes to other fertility-related genes. As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations that would affect vector competence, and we show that its susceptibility to Plasmodium spp. and O'nyong nyong virus infection remains similar to wild-type mosquitoes. Importantly, we find that nanosd propagates efficiently in caged mosquito populations and is maintained as a source of Cas9 after the emergence of drive-resistant alleles, whilst also mobilizing a nonautonomous antiparasitic effector modification. The nanosd gene drive shows promise as a genetic tool for malaria vector control via population modification, and we outline steps towards its further optimization.},
}
@article {pmid41706533,
year = {2026},
author = {Overton, MS and Guy, SE and Chen, X and Martsul, A and Carolino, K and Akbari, OS and Meyer, JR and Kryazhimskiy, S},
title = {Upper bound on the mutational burden imposed by a CRISPR-Cas9 gene-drive element.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf315},
pmid = {41706533},
issn = {2160-1836},
support = {HR0011-17-2-0047//DARPA/ ; R35GM153242/GF/NIH HHS/United States ; 5T32GM133351-02//Pathways in Biological Sciences NIH T32 program/ ; //Tata Institute for Genetics and Society BS/MS Fellowship/ ; R35GM153242/GM/NIGMS NIH HHS/United States ; },
mesh = {Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Mutation ; Loss of Heterozygosity ; Mutation Rate ; },
abstract = {Homing-based CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, with applications from disease eradication to species conservation. However, Cas9 alone and in a complex with guide RNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to unintended loss-of-heterozygosity (LOH) events. These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To measure how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. We found no detectable effects on the genome-wide rates of mutations or LOH events. Our power calculations suggest that CCGD or Cas9 affect these rates by less than 30%, which is much less than natural variation for these traits in yeast. A more detailed examination shows that CCGD or Cas9 may alter the lengths and genomic distributions of LOH events, but the statistical support for these effects is weak. Thus, our results demonstrate that CCGDs impose at most a weak additional mutational burden in the yeast model. Although mutagenic effects of gene drives need to be further evaluated in other systems, our results add credence to the proposition that the evolutionary risks posed by well-designed gene drives may be acceptable.},
}
@article {pmid41774761,
year = {2026},
author = {Harvey-Samuel, T and Kaur, R and Leftwich, PT and Feng, X and Gantz, V and Alphey, L},
title = {Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus.},
journal = {Genetics},
volume = {233},
number = {4},
pages = {},
doi = {10.1093/genetics/iyag054},
pmid = {41774761},
issn = {1943-2631},
support = {BBS/E/I/00007033//UK Biotechnology and Biological Sciences Research Council/ ; BBS/E/I/00007038//UK Biotechnology and Biological Sciences Research Council/ ; BBS/E/I/00007039//UK Biotechnology and Biological Sciences Research Council/ ; },
mesh = {Animals ; *Culex/genetics/virology ; *Gene Drive Technology/methods ; CRISPR-Cas Systems ; Transgenes ; Mosquito Vectors/genetics ; },
abstract = {CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied ("homed"). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (ie copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications, and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drive's efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting "split" homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimization of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.},
}
@article {pmid42083788,
year = {2026},
author = {Surender, S and Haeusser, LA and Kuhlburger, L and Tsiami, F and Dogan, NO and Maise, L and Nahnsen, S and Beck, S and Merk, DJ and Tabatabai, G},
title = {Molecular modulators of cyclin-dependent kinase 4/6 inhibitor response in experimental glioma identified through genome-wide CRISPR-Cas9 screening.},
journal = {Neuro-oncology},
volume = {28},
number = {8},
pages = {1904-1920},
doi = {10.1093/neuonc/noag093},
pmid = {42083788},
issn = {1523-5866},
support = {2019_Kolleg_14//Else Kr&#xf6;ner Fresenius Stiftung/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors/genetics ; *Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors/genetics ; Animals ; *Glioma/drug therapy/genetics/pathology ; Mice ; *Protein Kinase Inhibitors/pharmacology ; *CRISPR-Cas Systems ; *Brain Neoplasms/drug therapy/genetics/pathology ; Cell Proliferation ; Checkpoint Kinase 1/genetics/antagonists &amp; inhibitors ; Apoptosis ; Oncogene Proteins/genetics ; Cell Line, Tumor ; Cyclin E ; },
abstract = {BACKGROUND: Glioblastoma harbors frequent alterations in the retinoblastoma pathway, providing a genetic rationale for therapeutic targeting with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. The NOA-20 trial did not reveal a progression-free survival benefit of CDK4/6 inhibition plus radiation therapy in newly diagnosed, O6-methylguanine DNA methyltransferase (MGMT)-unmethylated glioblastoma. In fact, CDK4/6 inhibitor monotherapy has not demonstrated efficacy in solid tumors. We aimed at discovering response modulators to CDK4/6 inhibition, paving the way for rational combination therapies.<br><br>METHODS: We conducted genome-wide CRISPR-Cas9 screens in human glioma cell lines and stem-like cells (LN229, LN18, LNZ308, T98G, and GS-9) under CDK4/6 inhibition, employing knockout (Brunello library) and activation strategies (Calabrese library), followed by genetic and pharmacological validation of selected candidate genes in vitro and ex vivo (primary cultures) as well as the investigation of 1 functionally instructed combination therapy in vivo.<br><br>RESULTS: Loss of AMBRA1 and gain of function of CCNE1 reduced sensitivity to CDK4/6 inhibition in glioma cells, whereas disruption of checkpoint kinase 1 (CHEK1) or FAM122A resulted in synthetic lethality in combination with CDK4/6 inhibition. AMBRA1-deficient glioma cells exhibited increased sensitivity to CHK1 inhibition, revealing a context-specific vulnerability. Combined inhibition of CHK1 and CDK4/6 led to synergistic antiglioma activity in vitro, ex vivo, and in vivo.<br><br>CONCLUSIONS: Our data identify AMBRA1, CCNE1, CHEK1, and FAM122A as potential molecular modifiers of CDK4/6 inhibition response in experimental glioma and provide a biological rationale for combinatorial targeting with CDK4/6 inhibition in glioblastoma.},
}
@article {pmid42192133,
year = {2026},
author = {Elena, M and Giuliana, N and Giuseppina, R and Valeria, L and Geppino, F and Viola, C and Maria, V and Tiziana, A and Dario, A and Alessandra, P},
title = {Unveiling a novel role for p19Arf (alternative reading frame) in mESC differentiation toward the pancreatic lineage.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42192133},
issn = {2045-2322},
support = {2022R74KBC//MUR- Ministero Universit&#xe0; Ricerca-PRIN 2022/ ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; Endoderm/cytology/metabolism ; *Mouse Embryonic Stem Cells/cytology/metabolism ; *Pancreas/cytology/metabolism ; *Cell Lineage/genetics ; Gene Expression Profiling ; CRISPR-Cas Systems ; Mutation ; },
abstract = {The tumor suppressor ARF (p14 in human, p19 in mouse), has traditionally been characterized by its pivotal role in tumor surveillance. However, its involvement in an expanding range of cellular processes reveals that its functions are broader and more complex than initially appreciated. Here, we uncover a previously unrecognized role of p19ARF in endodermal differentiation, specifically in pancreatic lineage specification using an in vitro differentiation model of mouse embryonic stem cells (mESCs). Using CRISPR/Cas9-mediated mutagenesis, we show that mESCs with mutations in p19Arf are unable to efficiently differentiate towards pancreatic endoderm. Transcriptomic profiling reveals substantial alterations in gene networks associated not only with lineage commitment but also with cytoskeletal organization and cell morphology. These changes correlate with a disruption in stem cell architecture and suggest the persistence of pluripotency feature when only one copy of functional p19Arf is present. Taken together, our findings highlight a role for p19Arf in modulating endodermal differentiation while maintaining the essential cellular properties of stem cells, thus expanding its relevance beyond tumor suppression.},
}
@article {pmid42210018,
year = {2026},
author = {Malaiwong, N and Malaiwong, P and Kim, C and O'Donnell, M},
title = {FLInt 2.0: robust and customizable single-shot integration in C. elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkag138},
pmid = {42210018},
issn = {2160-1836},
support = {DP2 GM154014/GM/NIGMS NIH HHS/United States ; DP2 GM154014/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Transgenes ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Luminescent Proteins/genetics ; Gene Transfer Techniques ; },
abstract = {Transgenesis in Caenorhabditis elegans has revolutionized biological research by enabling the precise control of expression of both endogenous and exogenous genes. FLInt (Fluorescent Landmark Interference) was developed to integrate transgenes via CRISPR-Cas9 using visible changes in existing fluorescent protein expression strains. While the original FLInt method (FLInt 1.0) enabled a simple visual readout of potential transgene integration, the process was prone to false positives, leading to burdensome screening efforts. Here, we present an alternative FLInt strategy, FLInt 2.0, that reduces false positives by targeted CRISPR-Cas9 cutting of fluorescent protein landing sites in a manner which largely retains fluorescence in nonintegrative repair events but eliminates expression upon transgene integration. We demonstrate that this targeted approach maintains effective integration while significantly decreasing the proportion of false positives. Molecular and transmission analyses confirm that nonfluorescent F2 animals more reliably represent stably integrated multicopy transgenic lines. We show that integration efficiency and array transmission are influenced by DNA structure and composition, with linear DNA substrates promoting more robust array formation and insertion. We further show that multicopy transgene lines can be tailored to desired expression levels using a simple subsequent Cas9 targeting approach, reducing labor-intensive screening and increasing experimental throughput. Our strategy provides a robust, visually guided refinement of FLInt, offering a generalizable framework for improving site-specific transgene integration in C. elegans.},
}
@article {pmid42226151,
year = {2026},
author = {Huang, M and Li, X and Pan, T and Wu, D and Li, G and Wu, W},
title = {CRISPR-Cas9-mediated construction of a Streptococcus agalactiae vaccine for tilapia and evaluation of its protective efficacy.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42226151},
issn = {1746-6148},
support = {AB22013007//the Fangchenggang Science and Technology Program/ ; AA17204081-1//Guangxi Science and Technology Program/ ; },
mesh = {Animals ; *Streptococcus agalactiae/immunology ; *Fish Diseases/prevention &amp; control/microbiology/immunology ; *Streptococcal Infections/veterinary/prevention &amp; control ; *Tilapia/immunology/microbiology ; *CRISPR-Cas Systems ; *Streptococcal Vaccines/immunology ; },
abstract = {BACKGROUND: Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant Escherichia coli DH5α-ORF4-GFP (targeting GBS scpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine's protective efficacy, biosafety and regulatory effects via multi-dimensional assays.<br><br>RESULTS: The optimal regimen was single immersion at 1.5&#x2009;&#xd7;&#x2009;10[4] CFU/mL, with a maximum RPS of 73.13% and stable 65.79% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and numerical increases in globulin, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not alter intestinal microbial richness but modulated community structure, enriching beneficial taxa such as Alphaproteobacteria, suggesting a potential interaction between vaccination and gut microbiota that may contribute to enhanced host defense.<br><br>CONCLUSIONS: In conclusion, this study successfully developed an effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.},
}
@article {pmid42525171,
year = {2026},
author = {Duveneck, S and Ille, K and Melzer, S},
title = {FLC genes control flowering time to varying degrees in a Brassica napus spring cultivar.},
journal = {Plant molecular biology},
volume = {116},
number = {4},
pages = {},
pmid = {42525171},
issn = {1573-5028},
mesh = {*Brassica napus/genetics/physiology/growth &amp; development ; *Flowers/genetics/physiology/growth &amp; development ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *MADS Domain Proteins/genetics/metabolism ; Vernalization/genetics ; Seasons ; Plant Leaves/genetics ; *Genes, Plant ; Gene Expression Profiling ; CRISPR-Cas Systems ; },
abstract = {In the crop Brassica napus (oilseed rape), distinct growth types have been established that differ mainly in their vernalization requirement for flowering. The need for vernalization in Arabidopsis is controlled by the expression of the floral repressor FLOWERING LOCUS C (FLC), which also regulates cold-responsive flowering in B. napus. Notably, FLC homologs are also retained in spring oilseed rape despite its lack of vernalization requirement. To elucidate the functions of the nine BnFLC homologs in a spring type, we generated CRISPR/Cas9 knockout mutants of all homologs in the cultivar Westar. We show that the loss of BnFLC genes significantly accelerates flowering, demonstrating that BnFLC genes regulate flowering in spring types independently of vernalization. Transcriptomic analyses in leaves revealed distinct expression patterns among the BnFLC genes, with some remaining active during floral transition. Finally, no epigenetic regulation of the BnFLC homologs associated with flowering time was detected, while BnFLC.A03b carried persistent repressive marks and was constitutively silenced. Unexpectedly, additional flowering time regulators, including genes typically active in the shoot apical meristem, were expressed in leaves, with some showing altered expression and chromatin states in the mutant. These findings reveal that BnFLC genes are developmentally regulated and directly control flowering in spring oilseed rape, while also modulating the expression of other floral regulators in B. napus.},
}
@article {pmid40397277,
year = {2026},
author = {Lodewijk, GA and Kozuki, S and Guiltinan, C and Topacio, BR and Shariati, SA},
title = {Application of CRISPR-Based Epigenome Editing Tools for Engineering Programmable Embryo Models.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3048},
number = {},
pages = {211-238},
pmid = {40397277},
issn = {1940-6029},
support = {R35 GM147395/GM/NIGMS NIH HHS/United States ; },
mesh = {*Epigenome Editing/methods ; Animals ; Mice ; *CRISPR-Cas Systems ; *Embryonic Development/genetics ; Humans ; *Embryo, Mammalian/cytology ; Embryonic Stem Cells/cytology/metabolism ; },
abstract = {Stem cell-based embryo models (SEMs) have the potential to transform our understanding of early human embryogenesis. A critical step in engineering SEMs is the generation of the major cell types that compose preimplantation embryos including two primary extraembryonic lineages: (i) trophoblast cells, which are crucial for implantation and the establishment of maternal-fetal exchange, and (ii) hypoblast cells, which contribute to yolk sac formation. In addition, both cell types provide key signaling cues necessary for embryonic development. CRISPR-based epigenome editors are programmable devices that allow for efficient and precise activation (CRISPRa) or repression (CRISPRi) of cell fate-determining factors by modulating endogenous regulatory elements. Here, we present a step-by-step method to implement CRISPRa for controlling cell fate in embryonic stem cells based on our work in generation of CRISPR-programmed mouse embryo models.},
}
@article {pmid42218379,
year = {2026},
author = {Shi, Y and Yin, J and Ning, S and Yuan, J and Yang, D and Chuai, G},
title = {DeepCas12a: a hybrid deep learning framework for accurate AsCas12a efficiency prediction from sequence and epigenetic information.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {42218379},
issn = {1471-2164},
support = {2025080107//Tongji University "Medicine + X" Cross Research Program/ ; 62002265//National Natural Science Foundation of China/ ; },
mesh = {*Deep Learning ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; Convolutional Neural Networks ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR-Cas12a (Cpf1) offers distinct advantages for genome editing due to its flexible, T-rich PAM recognition. However, variable cleavage efficiency-modulated by sequence context and epigenetic features-remains a challenge, with existing tools facing challenges in modeling the high-order interactions between multimodal features. Here, we present DeepCas12a, a hybrid deep learning framework integrating Convolutional Neural Networks (CNNs) and a Vision Transformer (ViT) encoder to capture both local sequence motifs and long-range dependencies. The model fuses DNA sequence data with epigenetic profiles (DNA methylation and chromatin accessibility) in an end-to-end architecture. Benchmarked on an independent test set, DeepCas12a outperformed state-of-the-art predictors, achieving an Average Precision of 0.783, an AUC of 0.868, and a Spearman correlation of 0.630. Furthermore, interpretability analysis via saliency maps confirms the model captures biologically relevant features, including PAM specificity and seed region sensitivity, facilitating rational guide RNA design.},
}
@article {pmid42350674,
year = {2026},
author = {Navalayeu, T and Beer, N and Bebjaková, M and Kalis, RW and Hohmann, U and Stejskal, K and Krššáková, G and Fasching, N and Herzog, VA and Popitsch, N and Roitinger, E and Plaschka, C and Zuber, J and Ameres, SL},
title = {Cellular assembly and functional resilience of the mammalian RNA exosome.},
journal = {The EMBO journal},
volume = {45},
number = {15},
pages = {5423-5456},
pmid = {42350674},
issn = {1460-2075},
support = {LS23-053//Vienna Science and Technology Fund (WWTF)/ ; CoG-866166//EC | European Research Council (ERC)/ ; 10.55776/F80//Austrian Science Fund (FWF)/ ; 10.55776/DOC177//Austrian Science Fund (FWF)/ ; PhD Fellowship//Boehringer Ingelheim Fonds (BIF)/ ; },
mesh = {Animals ; Mice ; *Exosome Multienzyme Ribonuclease Complex/metabolism/genetics ; *Exosomes/metabolism ; RNA-Binding Proteins/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; *Mouse Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; RNA/metabolism ; },
abstract = {Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings define an assembly logic of of the mammalian exosome and uncover previously unrecognized plasticity in the composition and function of this essential ribonucleolytic complex.},
}
@article {pmid42462663,
year = {2026},
author = {Chen, H and Jin, Z and Duan, C and Song, Z and Cheng, Y and Chen, M and Zhang, C and Lan, Y and Shen, W and Fu, Y and Liu, R and Zheng, X},
title = {Effects of the ecpA gene on the biological characteristics and pathogenicity of avian pathogenic Escherichia coli strain FJLY68.},
journal = {Veterinary microbiology},
volume = {320},
number = {},
pages = {111145},
doi = {10.1016/j.vetmic.2026.111145},
pmid = {42462663},
issn = {1873-2542},
mesh = {Animals ; *Escherichia coli/pathogenicity/genetics ; *Escherichia coli Infections/microbiology/veterinary ; *Poultry Diseases/microbiology ; Chickens/microbiology ; Biofilms/growth &amp; development ; Virulence/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Bacterial Adhesion/genetics ; Fimbriae, Bacterial/genetics ; Chick Embryo ; *Fimbriae Proteins/genetics ; Cell Line ; CRISPR-Cas Systems ; Genetic Complementation Test ; Gene Deletion ; },
abstract = {Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis in poultry, yet the role of the ecpA gene, which encodes the major structural subunit of the Escherichia coli common pilus (ECP), remains incompletely defined in APEC pathogenesis. To investigate the role of ecpA in the biological characteristics and pathogenicity of Avian Pathogenic Escherichia coli (APEC) strain FJLY68, an ecpA deletion mutant (ΔecpA) and its corresponding complemented strain (CΔecpA) were constructed using the CRISPR/Cas9 system and verified by PCR and Sanger sequencing. Phenotypic analyses revealed that the ΔecpA mutation significantly impaired bacterial motility, biofilm formation, adherence to chicken embryonic fibroblast (DF-1) cells, and fimbriae assembly. Transcriptomic analysis identified 1720 differentially expressed genes in the ΔecpA mutant, significantly enriched in pathways associated with flagellar assembly, chemotaxis, and metabolism, consistent with the observed phenotypic changes. Although in vitro growth was unaffected, the ΔecpA mutant exhibited markedly attenuated virulence in a chick infection model, as indicated by an increased LD50, attenuated clinical signs and pathological lesions, and reduced bacterial colonisation in tissues. Full genetic complementation restored all observed defects to wild-type levels. This study identifies ecpA as a critical determinant of APEC pathogenesis, directly linking its function to bacterial motility, biofilm formation, adhesion, and in vivo virulence, and provides a theoretical basis for developing novel control strategies targeting this virulence factor.},
}
@article {pmid42470400,
year = {2026},
author = {Ang, YS and Yung, LL},
title = {Sequence Engineering of Guide DNA for Precise RNA Targeting by Cas12a.},
journal = {ACS nano},
volume = {20},
number = {30},
pages = {21115-21127},
doi = {10.1021/acsnano.6c02663},
pmid = {42470400},
issn = {1936-086X},
support = {NA//Singapore Ministry of Education Academic Research Fund Tier 1/ ; NA//Singapore Ministry of Health?s National Medical Research Council, Programme for Research in Epidemic Preparedness and Response (PREPARE)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; *RNA/metabolism/chemistry/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Base Sequence ; },
abstract = {Cas12a is highly accommodative toward noncanonical activation pathways to the extent of flipping its identity to be a DNA-guided RNA-targeting effector. A sequence engineering approach was used to systematically identify desirable guide DNA (gDNA) sequence motifs to achieve comparable RNA targeting efficiency as the canonical RNA-guided Cas12a with good selectivity down to single-nucleotide mismatch. Importantly, we introduced a split gDNA design concept with greater energetic differences arising from subtle nucleotide changes to probe the key spacer features for effective Cas12a-gDNA activation. Similar to the canonical RNA-guided activation pathway, Cas12a was found to engage actively in the "seed-like" scaffold-proximal region while the scaffold-distal region was largely hybridization-driven. We further evolved the split gDNA design to enhance the sequence selectivity by up to 21-fold compared to a single gDNA design and achieve single-nucleotide discrimination among representative let-7 family members. This study has established a gDNA sequence design framework to reprogram Cas12a as a precise RNA targeting platform.},
}
@article {pmid42480873,
year = {2026},
author = {McClain, IM and Yigit, NS and Royzen, M},
title = {Non-chromatographic purification of guide RNA for gene-editing experiments.},
journal = {Bioorganic &amp; medicinal chemistry letters},
volume = {140},
number = {},
pages = {130740},
doi = {10.1016/j.bmcl.2026.130740},
pmid = {42480873},
issn = {1464-3405},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/isolation &amp; purification/genetics/chemistry ; *Gene Editing/methods ; CRISPR-Cas Systems ; Chromatography, High Pressure Liquid ; Green Fluorescent Proteins/genetics ; },
abstract = {CRISPR-Cas12a gene editing technology is gaining momentum as a powerful tool for many biochemical and medicinal applications. The technology requires guide RNA, which is typically made using solid phase synthesis and purified by HPLC. The latter is often the most complex and time-consuming element of the synthetic process. This communication describes a non-chromatographic method for purification of synthetic RNAs. The method consists of five steps and yields target RNA in over 80% purity, which adheres to the FDA's standard for gene editing applications. The non-chromatographic RNA purification approach was applied to synthesize guide RNA targeting the GFP gene. Its purity was analyzed by analytical HPLC. Its functional fidelity was tested in CRISPR-Cas12a experiments in solution and live mammalian cells.},
}
@article {pmid42518166,
year = {2026},
author = {Kang, J and Kim, HH and Yoon, HM and Choi, Y and Heo, J and Woo, Y and Yu, S and Lee, KH and Lee, Y},
title = {Phenotypic and Whole-Genome Characterization of Enterococcus Isolates from Korean Doenjang and Meju: E. durans Edu-1 as a Food-Grade Probiotic Candidate with Epithelial Wound-Healing Activity.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {42518166},
issn = {1867-1314},
support = {2026-RISE-09-A32//the Regional Innovation System &amp; Education (RISE) program through the Gyeonggi Province RISE initiative, funded by the Ministry of Education (MOE) and the Gyeonggi Province, the Republic of Korea/ ; },
abstract = {Enterococcus species are widely distributed in traditional fermented foods and have shown probiotic potential. However, their food-grade application has been limited because the European Food Safety Authority excluded the genus from the qualified presumption of safety (QPS) list. Therefore, strain-specific safety assessment is essential. In this study, we characterized Enterococcus isolates from Korean Doenjang and Meju using an integrated phenotypic and whole-genome sequencing approach. Sixteen strains were isolated and identified/determined by 16S rRNA gene sequencing. We evaluated phenotypic safety and probiotic traits, and five candidates were further tested in HT-29 wound-healing scratch assays and a 115-gene host expression array. The two best wound-healing strains were then analyzed by whole-genome sequencing using a multi-database approach (VirulenceFinder, VFDB, ResFinder, CARD/RGI, PlasmidFinder, CRISPRCasFinder, and antiSMASH). The 16 isolates belonged to the five species, among which E. durans Edu-1 showed the highest wound closure ability (18.75 ± 1.24% at 72 h), followed by E. raffinosus Era-1 (16.71 ± 0.49%). Whole-genome sequence analysis showed that Edu-1 had no acquired transmissible antibiotic resistance and virulence factors genes and in addition, contains a plasmid-borne Bacteriocin_II family biosynthetic gene cluster, which can explain/support its broad-spectrum antimicrobial activity. In contrast, Era-1 carried tet(M) gene on the chromosome and ermB gene on an insertion-sequence-rich mobile element, although it also has a functional Type I-B CRISPR-Cas system and three glutamate decarboxylase (gadB) genes for γ-aminobutyric acid biosynthesis. These findings identify/suggest E. durans Edu-1 as a food-grade probiotic candidate with epithelial wound-healing activity, while E. raffinosus Era-1 represents a strain of biological interest for mechanistic wound-healing research rather than food application.},
}
@article {pmid42519596,
year = {2026},
author = {Hess, WR and Marchfelder, A and Randau, L},
title = {Editorial: creative CRISPR-Cas: RNA-guided functions in defence and beyond.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag026},
pmid = {42519596},
issn = {2633-6693},
}
@article {pmid42521019,
year = {2026},
author = {Dalabehera, M and Chaudhari, S and Kumar, J and Poonia, N and Subudhi, RN and Choonara, YE and Figueiras, A and Shukla, N and Sharma, N and Mascarenhas-Melo, F},
title = {Mechanistic advances in nanomedicine, nucleic acid therapies, and AI-driven research on cervical cancer.},
journal = {Journal of pharmaceutical sciences},
volume = {},
number = {},
pages = {104442},
doi = {10.1016/j.xphs.2026.104442},
pmid = {42521019},
issn = {1520-6017},
abstract = {Cervical cancer pharmacotherapy is significantly limited by physiological and cellular barriers that restrict drug access to therapeutic targets, resulting in suboptimal biodistribution, systemic toxicity, and the emergence of drug resistance. This review provides a mechanistic and biopharmaceutics-centered analysis of how advanced drug delivery systems are being engineered to overcome these limitations. We critically examine the role of nanocarriers, including lipid-based vesicles, polymeric nanoparticles, and inorganic hybrid systems, in modulating absorption, distribution, and tumor-targeting efficiency, with emphasis on their physicochemical properties and interaction with biological barriers such as the tumor microenvironment and cellular uptake pathways. In parallel, we analyze nucleic acid-based therapeutics (CRISPR/Cas systems, miRNA, and antisense oligonucleotides) from a pharmaceutical sciences perspective, focusing on delivery constraints, stability, intracellular trafficking, and their ability to modulate pharmacological response and drug resistance mechanisms. The review also discusses the integration of immunomodulatory strategies within nanodelivery platforms as a means to alter disease-related biological barriers and improve therapeutic index. Finally, we explore the emerging role of AI-assisted models in optimizing formulation design, predicting pharmacokinetic behavior, and supporting precision dosing strategies in drug development workflows. By integrating drug delivery engineering, molecular biopharmaceutics, and computational optimization, this work outlines a translational framework for overcoming key barriers in pharmaceutical intervention design for oncology applications.},
}
@article {pmid42521788,
year = {2026},
author = {Liu, H and Yuan, Z and Han, J and Zhao, Y},
title = {A dual-readout RAA-CRISPR/Cas13a diagnostic platform for rapid and sensitive detection of Eggerthella lenta.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42521788},
issn = {1436-5073},
support = {2019LJ001//Academic Promotion Program of Shandong First Medical University/ ; 2021ZDSYS27//the Key Research and Development Project of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Actinobacteria/isolation &amp; purification/genetics ; Limit of Detection ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Recombinases/metabolism ; },
abstract = {Eggerthella lenta (E. lenta) is an opportunistic anaerobic pathogen associated with severe systemic infections, yet rapid and accurate diagnostic tools remain limited. To address this challenge, we developed a highly sensitive and specific dual-readout diagnostic platform integrating recombinase-aided amplification (RAA) with the CRISPR/Cas13a system, targeting the highly conserved rsmG gene of E. lenta. The assay offers two detection modalities: a real-time fluorescence readout and a visually interpretable lateral flow strip. Analytical evaluation demonstrated that the fluorescence-based assay achieved a limit of detection (LOD) of 4.4 copies per reaction (95% CI: 3.7-5.6 copies/reaction), while the instrument-free lateral flow assay yielded an LOD of 10[4] copies per reaction. The platform exhibited exceptional specificity, showing no cross-reactivity with 10 common non-target bacterial species. Clinical validation was performed using 24 synovial fluid samples, all confirmed positive for E. lenta by Sanger sequencing. The fluorescence assay successfully detected all 24 samples, achieving a detection rate of 100% (24/24). In parallel, the lateral flow assay detected 21 of the 24 positive samples, yielding a detection rate of 87.5% (21/24). The three samples undetected by the lateral flow strip were verified as true positives by sequencing, indicating that the discrepancy was due to the lower analytical sensitivity of the strip format rather than a lack of specificity. In conclusion, this dual-mode RAA-CRISPR/Cas13a platform serves as a robust and practical tool for rapid clinical diagnosis and point-of-care (POC) triaging of E. lenta infections. The fluorescence format is optimal for high-sensitivity laboratory testing, whereas the lateral flow variant provides a deployable alternative for rapid, point-of-care screening in resource-limited environments.},
}
@article {pmid42521823,
year = {2026},
author = {Tuo, W and Wang, X and Wu, T and Zhang, S and Guo, P and Lin, X and Lin, Q and Zhai, J and Wu, S},
title = {Establishment of an efficient Agrobacterium-mediated transformation system and CRISPR/Cas9-mediated genome editing of the OvPDS1 gene in Oxalis vulcanicola 'Sunset Velvet'.},
journal = {Planta},
volume = {264},
number = {3},
pages = {},
pmid = {42521823},
issn = {1432-2048},
support = {32471958//National Natural Science Foundation of China/ ; KFB24022A//Study on the Molecular Mechanisms of Leaf Coloration in Three Sulfur Oxalis Varieties, Innovation Project of Fujian Agriculture and Forestry University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Gene Editing/methods ; *Agrobacterium/genetics ; *Plant Proteins/genetics/metabolism ; Acetophenones ; },
abstract = {This study overcomes a key technical barrier by establishing transformation and enabling first CRISPR/Cas9 editing in Oxalis, providing a platform for functional genomics and breeding. The lack of an efficient genetic transformation system has considerably hindered functional genomics studies in Oxalis vulcanicola 'Sunset Velvet'. Here, we established a stable and efficient Agrobacterium-mediated transformation system using stem segments as explants. Key parameters, including pre-culture duration, infection time, Agrobacterium cell density, acetosyringone (AS) concentration, and co-cultivation period, were systematically optimized. Under optimal conditions, the highest transient β-glucuronidase (GUS) expression rate reached approximately 9.0%, and eight stable transgenic lines were successfully obtained. CRISPR/Cas9-mediated genome editing was achieved in Oxalis for the first time. Targeted mutagenesis of OvPDS1, a gene involved in carotenoid biosynthesis, resulted in an albino phenotype, and Sanger sequencing confirmed a base substitution at the target site. Although the editing efficiency was relatively low (0.5%), this result demonstrates the feasibility of genome editing in Oxalis. This study overcomes a major technical bottleneck and provides a robust platform for functional gene analysis, trait improvement, and molecular breeding in O. vulcanicola 'Sunset Velvet' and other non-model ornamental plants.},
}
@article {pmid42522380,
year = {2026},
author = {Xue, F and Xin, Z and Wang, G and Xing, J and Han, H and Zhao, X and Song, N},
title = {Recent advances of CRISPR-based gene editing technologies and delivery strategies.},
journal = {Artificial cells, nanomedicine, and biotechnology},
volume = {54},
number = {1},
pages = {415-431},
doi = {10.1080/21691401.2026.2702869},
pmid = {42522380},
issn = {2169-141X},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; },
abstract = {CRISPR technology is a powerful tool for gene editing, in which the efficient delivery of living target cells allows it to show great clinical potential. At present, the commonly used in vivo delivery strategies mainly include biological methods (AAV, VLP, SEND) and chemical methods (LNP), which subtly deliver gene editors to living target cells safely and efficiently from different ways. However, existing delivery systems have different extents of limitations in terms of editing efficiency, immunogenicity, half-life, etc., so developing optimized delivery systems is the key to fully realizing the potential of CRISPR-Cas system for intracellular gene editing. In order to fully understand the advantages of different delivery strategies to maximize the ability to help CRISPR systems choose delivery methods, we conducted a systematic review. In this paper, we introduce the types, principles and characteristics of gene editing systems in order to understand their requirements for delivery tools. We focus on describing the type, principle, load, immunogenicity, specificity, toxicity, etc. of the delivery system, so as to fully analyse its advantages and disadvantages for the selection of different editing environments. This review aims to provide new insights to facilitate appropriate delivery systems or improve the efficacy of existing systems.},
}
@article {pmid42522597,
year = {2026},
author = {Lu, W and Yang, Q and Zhao, P and Cao, Y and Jing, Z and Zhang, N and Li, J and Li, X and Wang, X and Zhang, X and Cao, L and Gong, P},
title = {A One-Pot RPA-CRISPR/Cas12a Assay for Rapid Genus-Level Detection of Babesia spp. in Ticks and Livestock Blood Samples.},
journal = {Transboundary and emerging diseases},
volume = {2026},
number = {1},
pages = {e9289663},
pmid = {42522597},
issn = {1865-1682},
support = {2024YFD1800100//National Key Research and Development Program of China/ ; CARS-39//China Wool-sheep &amp; Cashmere-goat Research System/ ; },
mesh = {Animals ; *Babesia/isolation &amp; purification/genetics ; *Babesiosis/diagnosis/parasitology/blood ; Cattle ; *Cattle Diseases/diagnosis/parasitology/blood ; *Sheep Diseases/diagnosis/parasitology/blood ; CRISPR-Cas Systems ; *Ticks/parasitology ; Sheep ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Livestock ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Babesiosis, a globally significant tick-borne disease, poses substantial threats to livestock production and public health. Reported cases of human babesiosis in the United States increased from 1742 in 2014 to 3586 in 2023. In livestock, cattle babesiosis causes mortality, reduced meat and milk production, reproductive losses, and substantial control costs, with annual economic losses estimated at hundreds of millions of US dollars in several endemic countries. Rapid and sensitive detection methods are essential for early warning, surveillance, and control of this disease. In this study, we developed a closed-tube, one-pot assay for genus-level detection of Babesia spp. associated with cattle and sheep, based on recombinase polymerase amplification (RPA) coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. This format effectively minimizes cross-contamination risks associated with repeated tube opening in conventional assays. A three-channel signal readout system, including blue-light fluorescence visualization, ultraviolet (UV) fluorescence visualization, and lateral flow strip (LFS) readout, was integrated to enable flexible endpoint detection under different laboratory and field conditions. The assay targets a conserved region of the Babesia 18S rRNA gene and enables genus-level detection of Babesia spp. within 40 min at 37°C. The established RPA-CRISPR/Cas12a platform exhibited high analytical sensitivity, with a limit of detection of 5 copies/μL for recombinant plasmid templates, high analytical specificity against the tested nontarget pathogens, and low equipment dependency. The detection limit of the LFS format reached 50 copies/μL. Field validation using 71 pooled tick samples and 53 clinical blood samples collected from cattle and sheep yielded positive rates of 15.49% and 9.43%, respectively, with 100% concordance between this assay and conventional polymerase chain reaction (PCR) for both specimen types. In conclusion, this one-pot RPA-CRISPR/Cas12a detection platform provides a rapid, sensitive, and field-applicable molecular screening tool for genus-level detection of Babesia spp. This assay may support early warning and preliminary field monitoring of babesiosis, particularly in resource-limited settings. However, species-level confirmation should be performed by sequencing or other species-specific methods when epidemiological tracing or precise species identification is required.},
}
@article {pmid42524122,
year = {2026},
author = {Rananaware, SR and Narisetty, KV and Shah, RA and Jain, PK},
title = {CRISPR-based ex vivo gene editing of donor organs.},
journal = {Nature reviews bioengineering},
volume = {},
number = {},
pages = {},
pmid = {42524122},
issn = {2731-6092},
support = {R21 AI156321/AI/NIAID NIH HHS/United States ; R21 AI168795/AI/NIAID NIH HHS/United States ; R35 GM147788/GM/NIGMS NIH HHS/United States ; R61 AI181016/AI/NIAID NIH HHS/United States ; },
abstract = {Donor organs are frequently discarded because of concerns about quality or pathogen risk, challenges that could be mitigated through ex vivo gene editing or silencing during machine perfusion. Here, we discuss the development of CRISPR-based approaches for ex vivo gene silencing in human donor organs, from proof of concept in kidney biopsies to the challenges of organ-scale translation.},
}
@article {pmid42524609,
year = {2026},
author = {Martin, L and Bohinc, J and Recchia, A and Gritti, S and Santilli, G and Zeyland, J and Vidaković, M and Grdović, N and Benabdellah, K and Ortiz-Bueno, M and Butuner, BD and Pisaniello, L and Stilhano, R and Benati, D and Hapil, FZ and Khawaja, S and Nair, RR and Giacomelli, C and Atilla, E and Zinghirino, F and Ferrari, T and Corradi, F and Laufer, TJ and Khnykin, D and Aseguinolaza, GG and Skrbinek, M and Mlakar, T and Lapanja, T and Lainšček, D},
title = {In vivo delivery strategies for therapeutic CRISPR genome editing.},
journal = {International journal of biological sciences},
volume = {22},
number = {12},
pages = {6539-6581},
pmid = {42524609},
issn = {1449-2288},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; Epigenome Editing ; Genetic Therapy/methods ; *Gene Transfer Techniques ; },
abstract = {CRISPR-based genome and epigenome editing technologies have rapidly evolved from programmable nucleases into a diverse therapeutic toolbox encompassing conventional CRISPR systems, base editing, prime editing, RNA targeting, and epigenetic modulation. While early clinical successes relied on ex vivo manipulation of patient-derived cells, recent advances in delivery chemistry and vector engineering are enabling direct in vivo editing across multiple organs. Here, we provide a comprehensive review of delivery modalities of CRISPR systems solely in vivo that underpin their therapeutic translation. We examine how anatomical, cellular, and immunological constraints shape organ-specific editing strategies in different organ systems and we highlight key preclinical and clinical milestones that define the current translational landscape. Across indications, delivery remains a critical determinant of efficacy, safety, and scalability, governing editor exposure, tissue selectivity, and risk of unintended genomic or epigenomic perturbation. This review, authored by members of the COST Action Genome Editing to treat Human Diseases (GenE-HumDi) Network, delineates the principles guiding in vivo genome and epigenome editing and outlines the remaining barriers to durable, tissue-selective, and broadly deployable CRISPR therapeutics.},
}
@article {pmid41795918,
year = {2026},
author = {Liu, Y and Zhang, Y and Ding, H and Li, J and Gao, T and Wen, Z and Wang, Y and Wu, B},
title = {N-myristoyltransferase 1, a key gene for protein N-myristoylation, is dispensable for fertility in male mice.},
journal = {The Journal of reproduction and development},
volume = {72},
number = {4},
pages = {632-640},
doi = {10.1262/jrd.2025-064},
pmid = {41795918},
issn = {1348-4400},
mesh = {Animals ; Male ; *Acyltransferases/genetics/metabolism ; *Fertility/genetics/physiology ; *Spermatogenesis/genetics ; Mice ; Mice, Knockout ; Testis/metabolism ; Spermatozoa/metabolism ; Meiosis/genetics ; Infertility, Male/genetics ; CRISPR-Cas Systems ; },
abstract = {N-Myristoyltransferase 1 (NMT1), the predominant enzyme catalyzing myristoylation of proteins, is involved in various biological processes, including early embryonic development, immune responses, apoptosis, cellular homeostasis, tumorigenesis, and infection, with therapeutic potential in viral and parasitic infections as well as cancer. Despite the critical functions of NMT1, there have been no reports to date regarding its role in reproduction, especially in spermatogenesis. To investigate the function of NMT1 in this context, we utilized CRISPR/Cas9 technology to create a germ cell-specific Nmt1 knockout mice model for the first time. Surprisingly, male mice lacking NMT1 maintained fertility, exhibiting normal testicular structure and sperm morphology, with no significant differences in spermatogenic tubule structure or germ cell distribution compared to wild-type mice. Additionally, the Nmt1[f/f]; Stra8-Cre male mice showed no notable defects in meiosis. These findings suggest that NMT1 is not critical for spermatogenesis or male fertility in mice. However, further studies have shown that the compensatory role of NMT2 may play an unexpected role in maintaining myristoylation levels, which provides a new perspective for understanding the role of myristoylation in spermatogenesis.},
}
@article {pmid41873043,
year = {2026},
author = {Lee, SJ and Lee, GS and Kim, J and Park, KH and Go, SR and Moon, JH and Woo, EJ},
title = {Mass spectrometry based identification of AMP-O-Tris generated by Thermococcus onnurineus Cas10.},
journal = {FEBS open bio},
volume = {16},
number = {8},
pages = {1593-1601},
pmid = {41873043},
issn = {2211-5463},
support = {Korean Government MSIP RS-2022-NR071772 RS-202//National Research Foundation of Korea/ ; KGM5382632 KGM1062612 KGM1322612//Korea Research Institute of Bioscience and Biotechnology/ ; CRC22024-500//National Research Council of Science and Technology/ ; },
mesh = {*Thermococcus/metabolism/genetics ; Tandem Mass Spectrometry/methods ; *Adenine Nucleotides/metabolism/chemistry ; CRISPR-Cas Systems/genetics ; Adenosine Triphosphate/metabolism ; Chromatography, High Pressure Liquid ; Bacterial Proteins/metabolism ; Adenosine Monophosphate/metabolism ; },
abstract = {Cas10, the catalytic core of type III CRISPR-Csm systems, synthesizes cyclic oligoadenylate (cOA) second messengers to activate downstream immune responses. Although Cas10 activity is regulated by complex assembly, the nucleophile selectivity and off-pathway reactivity of isolated Cas10 remain poorly understood. Here, using HPLC separation and subsequent tandem mass spectrometry (MS/MS) analysis, we identify and structurally characterize AMP-O-Tris as a noncanonical adenylylated product generated by isolated Thermococcus onnurineus Cas10. Our results reveal that purified Cas10 exhibits relaxed nucleophile selectivity, diverting ATP turnover into nonproductive adenylylation involving buffer-derived nucleophiles. This suggests that effector complex assembly constrains Cas10 reactivity to promote efficient cOA synthesis and suppress off-pathway chemistry. Furthermore, interception of reactive intermediates by buffer-derived nucleophiles may represent a potential chemical fail-safe that limits unintended signaling when Cas10 is uncoupled from the complex. Together, our study provides mechanistic insight into Cas10 regulation and informs the development of robust type III-based diagnostic platforms. Impact statement Our study reveals that Cas10 exhibits latent catalytic flexibility when isolated, identifying a noncanonical adenylation reaction. These findings demonstrate how complex assembly constrains enzymatic specificity to prevent aberrant signaling. This mechanistic insight is crucial for improving the fidelity and design of next-generation CRISPR-based diagnostic platforms.},
}
@article {pmid42089437,
year = {2026},
author = {Wei, S and Zhang, K and Deng, S and Chen, J and Huang, X and Guo, J and Wu, Y and Guo, Y and Liang, Z},
title = {Transposase-Assisted Donor Tethering Boosts Large-Fragment HDR in Plants.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {43},
pages = {e75565},
pmid = {42089437},
issn = {2198-3844},
support = {32170410//National Natural Science Foundation of China/ ; 62572289//National Natural Science Foundation of China/ ; 202403021221020//Fundamental Research Program of Shanxi Province/ ; 2023-006//Shanxi Scholarship Council of China/ ; 202203021224002//Natural Science Foundation of Shanxi Province for the Excellent Youth/ ; },
mesh = {*Transposases/genetics/metabolism ; *Recombinational DNA Repair/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Precise insertion of large DNA fragments by homology-directed repair (HDR) remains inefficient and poorly reproducible in plants, largely due to limited donor availability at double-strand break sites. Here, we develop a transposase-assisted donor tethering strategy that improves the reliability of HDR-mediated large-fragment insertion. By fusing Cas9 to an integration-defective piggyBac variant that retains sequence-specific DNA-binding activity, donor templates are physically co-localized with Cas9-induced breaks. When combined with a transcription-coupled donor and a repair-pathway-biased Cas9 variant, this system enhances the frequency of accurate large-fragment insertions. Using this approach, we achieved efficient and precise kilobase-scale targeted gene insertions across multiple loci in both dicot and monocot species. These findings establish donor tethering as an effective strategy to improve plant HDR efficiency and provide a general framework for precise large-fragment genome insertion.},
}
@article {pmid42309993,
year = {2026},
author = {Yu, W and Chen, J and Guo, J and Yu, F and Wang, G and Lin, J and Dai, X and Tan, X and Ma, P and Wu, L and Zhang, Y and Huang, S and Lan, P and Bian, Q and Huang, X and Wei, J and Cheng, T and Zheng, X and Qiao, Y},
title = {Guide RNA reprogramming facilitates minimized tracrRNA-dependent off-target and versatile CRISPR/Cas9 engineering.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42309993},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Mice ; *Gene Editing/methods ; HEK293 Cells ; Machine Learning ; Transcriptome ; },
abstract = {While innovative, current CRISPR-Cas9 systems face safety concerns and practical hurdles, notably sequence-independent, noncanonical off-targeting. We demonstrate that the crRNA:tracrRNA duplex in guide RNAs (gRNA) is both splittable and reprogrammable. This property, however, enables endogenous RNAs with crRNA-like sequences to hijack any gRNAs, causing low-frequency yet pervasive tracrRNA-dependent off-target (TDO) effects. Using machine learning trained on high-throughput gRNA variant screens, we derive optimal gRNA-designing rules and engineer crRNA variants mismatched to the human/mouse transcriptomes, thereby minimizing TDO. By leveraging splittability and reprogrammability, we develop reprogrammable tracrRNAs for CRISPRa-based mRNA detection and redesign scaffolds to curb PAM-less Cas9-mediated "self-editing". We further create a separately expressed gRNA (segRNA) platform featuring split tracrRNAs and non-repetitive tandem crRNAs, enabling multiplexed editing of up to six genes and functional enhancer annotation in stem cells. Our findings uncover a previously overlooked off-target mechanism and offer versatile strategies to enhance the safety and utility of CRISPR systems.},
}
@article {pmid42310027,
year = {2026},
author = {Busquets, O and Li, H and Syed, KM and Jerez, PA and Dunnack, J and Lo Bu, R and Verma, Y and Pangilinan, GR and Martin, A and Straub, J and Du, Y and Simon, VM and Poser, S and Bush, Z and Diaz, J and Sahagun, A and Gao, J and Hong, S and Hernandez, DG and Levine, KS and Pochet, N and Booth, EO and Blanchette, M and Bateup, HS and Rio, DC and Blauwendraat, C and Hockemeyer, D and Soldner, F},
title = {iSCORE-PD: an isogenic stem cell collection to research Parkinson's disease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42310027},
issn = {2041-1723},
support = {1R56NS128015//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS138402//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS133140//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 5F31NS129265//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; P30 CA013330/CA/NCI NIH HHS/United States ; P30 CA013330/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Parkinson Disease/genetics/pathology ; Mutation ; Female ; Gene Editing ; alpha-Synuclein/genetics ; *Induced Pluripotent Stem Cells/metabolism ; Whole Genome Sequencing ; Cell Line ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Genetic Variation ; },
abstract = {Genome-edited human pluripotent stem cells (hPSCs) provide a powerful platform to study complex diseases such as Parkinson's disease (PD). Here, we describe iSCORE-PD, an isogenic collection of 65 genome-edited hPSC lines carrying disease-causing or high-risk variants in 11 PD-linked genes (SNCA, PRKN, PINK1, DJ1/PARK7, LRRK2, ATP13A2, FBXO7, DNAJC6, SYNJ1, VPS13C, and GBA1). All lines are derived from a well-characterized female hESC line and subjected to extensive quality control. Whole-genome sequencing reveals that genetic variation between lines, largely confined to non-coding regions, is minimal relative to inter-individual differences in patient-derived hiPSCs, with most variation arising from random mutations acquired during cell culture rather than genome-editing-induced off-target effects. Including multiple independently derived clones per mutation can control for this random genetic drift. Our systematic approach ensures high quality of this publicly available iSCORE-PD resource, highlights the advantages of prime editing over conventional CRISPR/Cas9 methods, and establishes best practices for generating disease-modeling hPSC collections.},
}
@article {pmid42315520,
year = {2026},
author = {Chen, S and Hsiao, S and Xie, T and Chen, D and Chen, N and Jiang, J and Li, J and Wu, Y and Liao, J},
title = {Deep learning-guided engineering of SpuFz1 and rational miniaturization of ωRNA enables efficient genome editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42315520},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Gene Editing/methods ; Mice ; *Deep Learning ; *Protein Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; HEK293 Cells ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; *RNA/genetics ; Mutation ; },
abstract = {Advancing the performance of programmable genome editing nucleases remains a key challenge in expanding their research and therapeutic applications. Here, we introduce a scalable deep learning-guided protein engineering framework for improving nuclease activity without requiring experimental training data. As a demonstration, we apply this strategy to SpuFz1, a compact Fanzor nuclease of eukaryotic origin, identifying and validating beneficial mutations that produces a multi-mutant variant with an 11.6-fold increase in editing efficiency. In parallel, we use comparative sequence analysis to design and experimentally validate a 75-nt ultrashort ωRNA scaffold, reducing guide RNA length by 79% while maintaining activity. Integration of these optimized components yields enFanzor, a compact genome editing system that achieves editing efficiencies up to 81.9% in mammalian cells, with strong editing performance in both human hematopoietic stem and progenitor cells (HSPCs) and mouse embryos. The outperforming variant developed through this strategy also supports robust CBE and ABE activity. Notably, the shortened ωRNA not only improves nuclease editing specificity but also leads to a substantial increase in base editing efficiency. Together, this work demonstrates the power of combining AI-guided protein optimization with rational RNA design, and establishes a generalizable strategy for engineering next-generation genome editing tools.},
}
@article {pmid42333443,
year = {2026},
author = {},
title = {RNA-Triggered Chromatin Shredding Hits Cancer's Hardest Targets.},
journal = {Cancer discovery},
volume = {16},
number = {8},
pages = {OF1},
doi = {10.1158/2159-8290.CD-NW2026-0072},
pmid = {42333443},
issn = {2159-8290},
mesh = {Animals ; Humans ; *Chromatin/genetics/metabolism ; *Neoplasms/genetics/therapy ; Mice ; CRISPR-Cas Systems ; Mutation ; *RNA/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {RNA-triggered chromatin shredding may offer a new way to attack cancers driven by mutations that have resisted conventional drugs, two new studies show. In mouse models, upon activation by a target transcript, the CRISPR enzyme Cas12a2 can selectively eliminate tumor cells carrying mutations in TP53, MYC, and other hard-to-drug cancer genes.},
}
@article {pmid42511810,
year = {2026},
author = {Zaman, W and Ayaz, A},
title = {Beyond Permanent Genome Editing: Molecular Delivery Strategies for RNA Editing and Epigenome-Editing Therapeutics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {14},
pages = {},
pmid = {42511810},
issn = {1422-0067},
mesh = {Humans ; *Epigenome Editing/methods ; *RNA Editing ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; },
abstract = {Reversible genetic medicines are emerging as controllable alternatives to permanent genome editing by enabling programmable modulation of RNA sequence, transcript abundance, chromatin state, and gene expression without irreversible genomic alteration. However, reversibility is not a single binary property: transient editor exposure, decay of the molecular effect, recovery of cellular function, and clinical capacity to stop, redose, or counteract treatment may diverge. This review therefore distinguishes mechanistic, functional, and clinical reversibility while examining targeted delivery systems for RNA-editing and epigenome-editing therapeutics. Key payloads include ADAR-recruiting oligonucleotides, CRISPR-Cas13 RNA editors, guide RNAs, chemically modified RNAs, editor-encoding mRNAs, dCas9 transcriptional regulators, DNA methylation editors, histone-modifying systems, and CRISPRoff-like platforms. We evaluate extracellular and intracellular delivery barriers, including nuclease degradation, immune recognition, renal clearance, liver uptake, cellular entry, endosomal escape, cytoplasmic release, nuclear localization, chromatin access, editing-window duration, off-target activity, immunogenicity, repeat-dosing feasibility, manufacturing, quality control, potency assays, and regulatory translation. Overall, delivery systems for reversible genetic medicines should be judged by tissue selectivity, functional editing, duration of action, reversibility after treatment withdrawal, safety, manufacturability, and clinical controllability.},
}
@article {pmid42511812,
year = {2026},
author = {Liu, R and Cong, S and Gao, Y and Xu, J and Shi, X},
title = {Application of CRISPR-Cas9-Based Gene Editing Technology in Inherited Liver Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {14},
pages = {},
pmid = {42511812},
issn = {1422-0067},
support = {82300674//National Natural Science Foundation of China/ ; 2024BSL005//Liaoning Normal University Doctoral Research Start-up Project/ ; },
mesh = {Humans ; *Liver Diseases/genetics/therapy ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; },
abstract = {Inherited liver diseases are predominantly caused by monogenic mutations, and the vast majority of these conditions currently lack curative treatment options. Although liver transplantation may be used for patients with end-stage disease, it faces numerous challenges, including donor organ shortage, immune rejection, and the need for lifelong immunosuppression. In recent years, CRISPR-Cas9-based gene editing technology has advanced rapidly, offering transformative hope for the treatment of these diseases. This review systematically elucidates the working principles and technical advantages of the CRISPR-Cas9 system and its derived tools (base editing and prime editing), summarizes recent applications of these technologies in the treatment of hereditary liver diseases, and discusses the prospects and challenges of their clinical translation, aiming to provide a theoretical reference for future research in this field.},
}
@article {pmid42514096,
year = {2026},
author = {Wu, Y and Huang, X and Gu, Q and Li, Y and Zhou, Y and Sun, L and Wang, X},
title = {Characterization of CRISPR Loci and Antimicrobial Resistance in Foodborne Listeria monocytogenes Isolates.},
journal = {Microorganisms},
volume = {14},
number = {7},
pages = {},
pmid = {42514096},
issn = {2076-2607},
support = {2025KJ29//Nanjing Customs/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) are widespread in bacterial and archaeal genomes as an adaptive immune system against invading mobile genetic elements. This study investigated the distribution of CRISPR loci and their potential association with antimicrobial resistance (AMR) in 40 foodborne Listeria monocytogenes isolates. CRISPR analysis showed that 18 isolates harbored CRISPR Locus 1, five carried Locus 2, and five possessed both loci. Antimicrobial susceptibility testing against seven antimicrobial agents indicated that most isolates were highly susceptible to the tested agents. Specifically, all isolates were susceptible to gentamicin, ampicillin, penicillin, and tetracycline, whereas resistance was observed in a small subset of isolates: four were resistant to chloramphenicol, five to levofloxacin, and four to ciprofloxacin. Statistical analysis showed no statistically significant association between the presence of CRISPR loci and antimicrobial susceptibility phenotypes. These findings provide baseline information on CRISPR locus distribution and antimicrobial susceptibility profiles in foodborne L. monocytogenes isolates. Further studies based on larger isolate collections, whole-genome sequencing, and characterization of associated cas genes are needed to clarify the potential role of CRISPR-Cas systems in AMR evolution in this species.},
}
@article {pmid42514545,
year = {2026},
author = {Huiban, FA and Galović, V and Tripon, MR and Orlović, S and Tulcan, C and Camen, D},
title = {Nanotechnology-Enabled CRISPR Delivery: Emerging Opportunities in Agriculture and Forest Biotechnology.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {14},
pages = {},
pmid = {42514545},
issn = {2223-7747},
abstract = {Genome editing is one of the key technologies in contemporary plant biotechnology, which has been revolutionized using CRISPR/Cas systems that offer rapid, flexible, and precise options to improve agricultural characteristics, enhance stress tolerance, and accelerate breeding of crops and trees. Despite the significant benefits of CRISPR/Cas systems, their use is restricted by difficulties in genome-editing materials into plant cells. The conventional approaches include Agrobacterium-mediated transformation, particle bombardment and PEG-mediated transfection; these have contributed significantly to advancements in the field; however, dependent on specific plants and requiring tissue cultures, these methods lead to random transgene insertion and poor transformation efficiency. In addition, nanotechnology represents a novel method of delivering CRISPR cargos into plant cells using minimal invasiveness and potentially without DNA. This review provides a synopsis of the most employed CRISPR/Cas systems within plants, comparing the traditional delivery mechanisms and the various nanotechnological delivery vehicles, such as lipid nanoparticles, carbon nanotubes, DNA nanostructures, mesoporous silica nanoparticles, magnetically responsive nanoparticles and green nanomaterials. This review discusses the present challenges of delivery efficacy, biocompatibility, cargo integrity, and regulatory issues, and provides suggestions for future research directions regarding nanotechnology-assisted genome editing for precision breeding, sustainable agriculture, production of crops tolerant to climate conditions, and forest biotechnology.},
}
@article {pmid42514579,
year = {2026},
author = {Janeeshma, E and Das, S and Bouzroud, S and Sarraf, M and Akhtar, N and Mousavi, H and Ibrahimova, U and Fujita, M and Hasanuzzaman, M},
title = {Reactive Oxygen Species in Crop Plants: Production, Detoxification, Signaling, and Molecular Cross-Talk.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {14},
pages = {},
pmid = {42514579},
issn = {2223-7747},
abstract = {Although excess generation of reactive oxygen species (ROS) is harmful for plants, at mild concentrations, they are able to positively regulate the metabolic pathway and signaling cascades of the cell. The mechanisms functional under the ROS-induced stress tolerance need to be decoded for the development of new abiotic stress-tolerant crop varieties with a clear vision. This study details ROS generation, roles of enzymatic antioxidants as well as non-enzymatic antioxidants, and ROS-induced cellular events. Emphasis is given to crucial topics such as cyclin-dependent kinases and mitogen-activated protein kinases signaling mechanisms, calcium-mediated cellular cross-talk, and activation or inactivation of various transcription factors. Introduction of different genetic and molecular approaches to manipulate the ROS pathway helps to find out novel recombinant plant varieties. Selection of specific biotechnological tools, appropriate omics analysis and implementation of CRISPR/Cas 9 in crop plants assist the designing of mutants. This review highlights the beneficial roles of oxidative stress, spotlighting the molecular mechanisms that support the physiological, morphological and biochemical modifications of plant cells.},
}
@article {pmid42516529,
year = {2026},
author = {Nikravan, M and Heidari, H and Khoshnood, S and Ghafourian, S and Kazemian, H},
title = {Molecular Profiling of CRISPR-Cas System, Virulence Traits, and Antimicrobial Resistance in Enterococcus faecalis Clinical Isolates.},
journal = {International journal of microbiology},
volume = {2026},
number = {},
pages = {5903066},
pmid = {42516529},
issn = {1687-918X},
abstract = {INTRODUCTION: Enterococcus faecalis is responsible for life-threatening enterococcal infections. This study is aimed at investigating virulence factors, antimicrobial resistance patterns, and molecular characteristics of clinical E. faecalis isolates.<br><br>MATERIALS AND METHODS: A total of 42 E. faecalis isolates were collected. Antimicrobial resistance and the minimum inhibitory concentrations (MICs) of vancomycin and gentamicin were determined using standard microbroth dilution method. The presence of virulence, antibiotic resistance, and CRISPR-Cas genes was investigated by polymerase chain reaction (PCR). Biofilm formation was also assessed, and the genetic diversity of the isolates was analyzed using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR).<br><br>RESULTS: The highest resistance rates were observed for gentamicin and ampicillin, and all isolates were high-level gentamicin resistant (HLGR) (MICs &#x2265;&#x2009;500&#x2009;&#x3bc;g/mL). Vancomycin resistance was detected in 15 isolates (35.7%) (MICs &#x2265;&#x2009;32&#x2009;&#x3bc;g/mL). Thirty-nine isolates (92.8%) were biofilm producers. The efaA gene was the most frequently detected (95.2%), followed by esp (69%), gelE (66.6%), ace (66.6%), and asa1 (57.1%). The aac(6 &#x2009;['])-Ie-aph(2 &#x2009;[&#x2033;])-Ia gene was detected in all isolates (100%), followed by ermB (78.6%), ermA (38.1%), ermC (23.8%), and vanA (19%). CRISPR3 was the most frequently detected locus (80.95%), followed by CRISPR1 (50%), CRISPR1-cas csn1 (23.8%), and CRISPR2 (9.5%). The CRISPR3-cas csn1 gene was not detected in any isolate. High heterogeneity was observed among the isolates, with 35 different ERIC types identified.<br><br>CONCLUSION: This study demonstrated notable resistance traits and high genetic diversity among clinical E. faecalis isolates, but no statistically significant association was found between CRISPR-Cas genes and phenotypic or genotypic resistance.},
}
@article {pmid42517263,
year = {2026},
author = {Hu, X and Xu, P and Shen, M and Chai, X and Tan, J and Sun, X and Yang, L and Wang, Q and Tan, F},
title = {A CRISPR-Cas12a and Quantum-Dot Lateral-Flow Assay for Rapid Species-Level Detection of Trichophyton rubrum.},
journal = {Mycoses},
volume = {69},
number = {7},
pages = {e70208},
pmid = {42517263},
issn = {1439-0507},
support = {22Y11905700//Program of the Science and Technology Commission of Shanghai Municipality "Science and Technology Innovation Action Plan" Medical Innovation Research Special Project/ ; SHDC12026111//Shanghai Shen Kang Clinical Technology Promotion Program/ ; YXX2024-KF01-04//The Center for Basic Research and Innovation of Medicine and Pharmacy (MOE)/ ; 2026ms03//Xi'an Municipal Health Commission General Research Project/ ; },
mesh = {Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Quantum Dots ; Rapid Diagnostic Tests ; *Arthrodermataceae/isolation &amp; purification/genetics ; *Tinea/diagnosis/microbiology ; *Trichophyton/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; Real-Time Polymerase Chain Reaction ; DNA, Fungal/genetics ; },
abstract = {BACKGROUND: Accurate species-level diagnosis of dermatophytes, particularly Trichophyton rubrum, is important for guiding targeted antifungal therapy and improving the management of recurrent or atypical superficial mycoses. However, routine microscopy has only moderate sensitivity and cannot resolve fungal species; fungal culture is time-consuming, and real-time quantitative PCR (qPCR) requires costly instrumentation.<br><br>OBJECTIVES: To develop and clinically evaluate CRI-RUB, a CRISPR-Cas12a-based assay integrating recombinase-aided amplification (RAA) and a quantum-dot (QD) fluorescent lateral-flow strip for rapid species-level detection of T. rubrum.<br><br>PATIENTS/METHODS: Analytical sensitivity and specificity were assessed using plasmid standards and a panel of common cutaneous fungi. A total of 140 clinical specimens were tested by both CRI-RUB and TaqMan qPCR. Discrepant or grey-zone qPCR results (Ct 33-34), together with a subset of concordant cases, were further arbitrated by internal transcribed spacer (ITS) sequencing to establish a composite reference standard.<br><br>RESULTS: CRI-RUB achieved a visual limit of detection of 2.3&#x2009;&#xd7;&#x2009;10[1] copies/&#x3bc;L and showed no cross-reactivity with other common cutaneous fungi. Among the 140 clinical specimens, CRI-RUB showed a sensitivity of 96.1% (73/76), specificity of 100.0% (64/64), positive predictive value of 100.0% (73/73), negative predictive value of 95.5% (64/67) and Cohen's &#x3ba; of 0.96 compared with the composite reference standard, indicating excellent concordance. The complete workflow required approximately 85&#x2009;min.<br><br>CONCLUSIONS: CRI-RUB provides a rapid, sensitive, and specific approach for species-level detection of T. rubrum. By combining CRISPR-Cas12a detection with QD-based lateral-flow readout, this assay shows potential for near-patient or point-of-care dermatophyte diagnosis.},
}
@article {pmid42510959,
year = {2026},
author = {Gubaidullin, N and Ospankulova, G and Gajimuradova, A and Syzdykova, A and Zhumalin, A and Dairova, K and Konysbayeva, D and Gorbulya, V and Makangali, K},
title = {ROS-Centered Transcriptomic Regulatory Networks Linking Salinity Stress, Antioxidant Defense and Processability Traits in Salicornia spp.},
journal = {Current issues in molecular biology},
volume = {48},
number = {7},
pages = {},
pmid = {42510959},
issn = {1467-3045},
support = {BR22883587//the ministry of agriculture of Kazakhstan/ ; },
abstract = {Salinity stress affects not only the survival and productivity of halophytic plants, but also the composition, structure and processability of their biomass. In Salicornia spp., salt-induced regulation of ion transport, osmotic adjustment, reactive oxygen species signaling, antioxidant defense, and cell wall remodeling can directly influence residual salinity, water retention, texture, extractability, drying behavior, and oxidative stability of plant biomass. However, most existing transcriptomic studies of Salicornia and related halophytes have focused mainly on salt tolerance mechanisms, whereas the connection between stress-regulated molecular networks and processing-related biomass traits remains insufficiently systematized. This review addresses this gap by proposing a mechanistic framework that links salinity perception, ROS-mediated signaling, ABA and MAPK pathways, antioxidant gene families, transcription factor networks and processing-oriented quality traits. Special attention is given to enzymatic antioxidant systems, including SOD, CAT, APX, POD and components of the ascorbate-glutathione cycle, as well as to non-enzymatic defense mechanisms involving ascorbate, glutathione, phenolic compounds, carotenoids, proline and glycine betaine. The review also discusses the regulatory roles of WRKY, DREB/CBF, NAC, bZIP and MYB transcription factor families as molecular control points connecting salinity stress responses with downstream metabolic and structural traits. Network-based approaches, including WGCNA, pathway signatures and transcript panels, are considered more informative than single-gene markers for predicting complex quality traits in Salicornia biomass. In addition, recent genomic and computational strategies, including CRISPR/Cas-mediated functional validation, GWAS, genomic selection, multi-omics integration and AI-assisted modeling, are discussed as emerging tools for candidate-gene prioritization and predictive assessment of stress-dependent biomass quality. Overall, this review shifts the interpretation of Salicornia transcriptomics from a descriptive salt-tolerance model toward a mechanistic and application-oriented framework for improving halophytic raw materials for food, feed and bioprocessing applications.},
}
@article {pmid42511636,
year = {2026},
author = {Zhan, Y and Luo, T and Sun, Y},
title = {Efficient Gene Editing in Fish Primary Germline Stem Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {14},
pages = {},
pmid = {42511636},
issn = {1422-0067},
support = {2022YFD2400101//The National Key R&amp;D Program of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Transfection/methods ; Oryzias/genetics ; *Stem Cells/metabolism/cytology ; *Germ Cells/metabolism/cytology ; *Eels/genetics ; Carps/genetics ; },
abstract = {Genome editing by the CRISPR/Cas9 system is widely used for production of gene-modified animals, including fish. However, efficient gene editing in fish cultured cells, in particular germline stem cells (GSCs), is challenging, likely due to the difficulty in transfecting these cells. The ricefield eel (Monopterus albus), a sequential hermaphroditic species, is a freshwater fish of significant economic value in China. In this work, we report a simple method that can achieve high gene editing efficiency in primary GSCs of fish species, including ricefield eel. High transfection efficiency (50-95%) is achieved in fish GSCs by using a microchannel-based cell transfection system. Gene editing efficiency of up to 60% in primary ricefield eel GSCs is achieved using an integrated CRISPR/Cas9 vector strategy. High gene editing efficiency is also achieved in gibel carp (Carassius gibelio) GSCs and the medaka spermatogonial stem cell line SG3, demonstrating the general applicability of our method. Our data suggest that efficient transfection is key to high gene editing efficiency in fish cultured GSCs. This study establishes an efficient and reliable gene editing system for fish GSCs, which may facilitate the creation of new germplasm of genetically difficult-to-breed fish by combining GSC transplantation with gene editing techniques.},
}
@article {pmid42480477,
year = {2026},
author = {Toishigawa, K and Magoori, K and Sato, H and Edahiro, T and Ureshino, H and Shindo, T and Suzuki, R and Sakuma, T and Yamamoto, T and Okada, M and Ichinohe, T},
title = {Platinum TALEN-mediated nonviral gene editing facilitates clinical-scale production of cancer antigen-reactive T cells.},
journal = {Cytotherapy},
volume = {28},
number = {9},
pages = {102911},
doi = {10.1016/j.jcyt.2026.102911},
pmid = {42480477},
issn = {1477-2566},
mesh = {Humans ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism ; *Gene Editing/methods ; *Antigens, Neoplasm/immunology/genetics ; *T-Lymphocytes/immunology ; *Receptors, Antigen, T-Cell/genetics ; CRISPR-Cas Systems/genetics ; *Neoplasms/immunology/therapy ; },
abstract = {BACKGROUND AIMS: Recently, target-genome editing has emerged as a next-generation tool for the clinical development of designed cellular products. Although Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) is the most frequently adopted nuclease for therapeutic genome editing, its widespread use is still hampered by potential off-target effects and high patent royalties. Platinum transcription activator-like effector nuclease (TALEN) is a modified TALEN that harbors non-repeat-variable di-residue (non-RVD) variations and confers higher efficiency than conventional TALENs lacking non-RVD variations.<br><br>METHODS: In this study, using Platinum TALEN targeting T-cell receptor (TCR) gene loci and a single-stranded DNA homology-directed repair (HDR) template, we aimed to produce TCR-replaced human T cells reprogrammed to recognize a cancer antigen and kill cancer cells.<br><br>RESULTS: This system can reproducibly produce TCR-engineered T cells from &#x223c;50 mL of peripheral blood on a clinical scale. The resulting genome-edited T cells retain na&#xef;ve/na&#xef;ve-like and memory phenotype cells in both CD4+ and CD8+ fractions and exhibit efficient cytolytic activity against cancer cell lines in vitro.<br><br>CONCLUSION: In conclusion, Platinum TALEN-mediated nonviral genome editing facilitates the replacement of the endogenous TCR with a desired TCR and can be applied to the clinical manufacturing of therapeutic T-cell products.},
}
@article {pmid42510814,
year = {2026},
author = {Zhang, X and Lu, C and Hu, Z and Geng, X and Li, G and Cai, J},
title = {Complete Genome Analysis of Pectobacterium brasiliense BS1113, a Causal Agent of Cigar Tobacco Soft Rot, with Phenotypic Characterization of Virulence and Copper Tolerance.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510814},
issn = {2073-4425},
support = {This research was funded by the Major Research Project of Fuyang Normal University (2025FSKJ31), the Doctoral Talent Introduction Project of Fuyang Normal University (2020KYQD0031), the Major Project of Anhui Huatuo Academy of Traditional Chinese Medicine//Fuyang Normal University/ ; },
mesh = {*Copper/metabolism/toxicity ; *Plant Diseases/microbiology/genetics ; *Nicotiana/microbiology ; Virulence/genetics ; *Genome, Bacterial ; Phenotype ; Whole Genome Sequencing ; },
abstract = {Background:Pectobacterium brasiliense-mediated soft rot severely threatens the production of diverse cash crops worldwide and brings severe yield reduction risks. A virulent strain BS1113 was separated from diseased cigar tobacco plants collected in Yunnan, yet its virulence regulatory genes and copper resistance-related genetic background have not been fully analyzed so far. This study aims to decipher the genomic features of BS1113 and clarify its pathogenic and copper-tolerant characteristics via whole-genome sequencing, comparative genomics and indoor phenotype verification. Methods: Hybrid sequencing strategies combining Illumina short reads and PacBio long reads were adopted to obtain the complete circular genome sequence of strain BS1113. Subsequent comparative genomic analysis and multiple phenotypic identification experiments were conducted to characterize its genetic architecture and physiological traits. Results: Genome assembly results showed that the circular chromosome of BS1113 spans 4,916,962 bp with a GC content of 51.96%, which encodes a total of 4369 functional protein-coding genes. Genomic comparison revealed that BS1113 completely lacks the T3SS gene cluster, while it conserves intact T2SS, T6SS and I-F CRISPR-Cas systems; the chromosomal copper resistance operon copRSAB was also detected in this isolate. Pathogenicity tests validated that BS1113 satisfies all criteria of Koch's postulates on cigar tobacco hosts. In addition, BS1113 displayed prominent tolerance against eight mainstream copper bactericides widely used for tobacco disease management. Conclusions: This research generates the first complete high-quality genome of P. brasiliense isolated from cigar tobacco hosts. The genomic data explain the infection mechanism of this pathogen independent of intact T3SS, and also reveal the genetic basis supporting its persistent survival under long-term copper fungicide pressure in field cultivation environments.},
}
@article {pmid42510829,
year = {2026},
author = {Yoshino, H and Fukuda, I and Enokida, H and Seki, N and Goto, Y},
title = {MEK1 as a Synthetic Lethal Target with Cabozantinib in Renal Cell Carcinoma: Insights from CRISPR/Cas9 Screening.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510829},
issn = {2073-4425},
support = {24K12458//Japan Society for the Promotion of Science/ ; 24K11347//Japan Society for the Promotion of Science/ ; 24K12641//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *Pyridines/pharmacology ; *Carcinoma, Renal Cell/genetics/drug therapy/pathology ; *Anilides/pharmacology ; Animals ; *Kidney Neoplasms/genetics/drug therapy/pathology ; *MAP Kinase Kinase 1/genetics/antagonists &amp; inhibitors ; CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Synthetic Lethal Mutations ; Xenograft Model Antitumor Assays ; Protein Kinase Inhibitors/pharmacology ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; },
abstract = {Background/Objectives: Cabozantinib is a tyrosine kinase inhibitor that primarily targets MET. It has become an important drug in the treatment of renal cell carcinoma (RCC); however, many patients do not respond to cabozantinib treatment and there is no effective next-line therapy. In this study, we identified molecular-targeted drugs that exhibit synergistic effects with cabozantinib using CRISPR/Cas9 screening. Methods: A kinome-wide synthetic lethal CRISPR/Cas9 screen was used to identify target molecules using 786-o RCC cells. A library was generated, and treatment with vehicle or cabozantinib was carried out, followed by next-generation sequencing to identify candidate genes. A combination index based on the Chou-Talalay method was used to evaluate the synergistic effect of cabozantinib through cell viability assays. Xenograft assays were conducted to determine the effect in vivo. Results: CRISPR/Cas9-based screening revealed four genes (MEK1, DCLK1, DYRK3, and FGFR1) that were candidates for synthetic lethality by cabozantinib in RCC cells. We focused on MEK1 because the MEK1 inhibitor cobimetinib has been approved for melanoma treatment. In a cell proliferation assay using 786-o and A498 RCC cells, the combination of cobimetinib and cabozantinib exhibited a synergistic effect. A xenograft assay also revealed a significant synergistic effect of cobimetinib and cabozantinib. Conclusions: CRISPR/Cas9 screening identified MEK1 as a candidate for a synthetic lethal target with cabozantinib in RCC. The combined inhibition of MET/VEGFR and MEK1 suppressed compensatory MAPK reactivation and downregulated the PI3K-Akt pathway, including the survival-associated genes PPP2R3B and ATF6B, and produced significant tumor growth suppression in vivo. These findings highlight the potential of cabozantinib plus cobimetinib, an already-FDA-approved MEK inhibitor, as a readily translatable combination strategy to overcome cabozantinib resistance in RCC.},
}
@article {pmid42510848,
year = {2026},
author = {Zafar, MA and Harling, LC and Li, Y and Celik, NB and Mukherjee, SK and Rizzo, J and Prendergast, A and Elefteriades, JA},
title = {A Preliminary Zebrafish Model of ACTA2 Deficiency Reveals Increased Larval Phenotype Burden and Suggests Reduced Adult Mutant Survival.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510848},
issn = {2073-4425},
support = {N/A//Masone Family/ ; Walter-Benjamin Scholarship//German Research Foundation/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Actins/genetics/deficiency ; Phenotype ; Larva/genetics ; Mutation ; *Zebrafish Proteins/genetics/deficiency ; Disease Models, Animal ; Genotype ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: ACTA2 encodes smooth muscle alpha-actin and is one of the most common genetic causes of inherited non-syndromic thoracic aortic aneurysm and dissection. Although murine models have provided important mechanistic insight, complementary vertebrate systems may enable more rapid in vivo phenotyping and future therapeutic screening.<br><br>METHODS: Utilizing a CRISPR/Cas9-based approach, we developed zebrafish acta2 mutant models and assessed phenotypes at larval and adult stages. At 3 days post-fertilization, larvae were screened by brightfield microscopy for edema, axis defects, hemorrhage, and a composite endpoint (any phenotype) under basal conditions and after exposure to 0.2 mM epinephrine. Phenotype occurrence was summarized as group-specific proportions across a 2 &#xd7; 2 design defined by genotype (wild-type vs. acta2-deficient mutant) and treatment (DMSO control vs. epinephrine), with prespecified pairwise comparisons using Pearson's chi-squared tests. Pooled fish-level logistic regression models were also fit for each endpoint. Adult follow-up was performed by genotyping all available acta2 fish at the facility and tracking staggered tank-level cohorts longitudinally.<br><br>RESULTS: Initial gross morphologic assessment did not reveal overt external phenotypic differences between heterozygous or homozygous acta2 mutant larvae and wild-type controls. In larval analyses, the mutant genotype was associated with a greater burden of adverse phenotypes than treatment exposure. For the composite endpoint, mutant larvae demonstrated higher proportions than normal larvae under both control (28.3% vs. 17.4%, p = 0.007) and epinephrine conditions (26.9% vs. 17.4%, p = 0.023), whereas epinephrine did not significantly alter composite phenotype frequency within either genotype. Similar genotype-associated trends were observed for axis defects and hemorrhage. In pooled logistic regression, acta2 deficiency was associated with increased odds of axis defects (OR 2.64, 95% CI 1.48-4.89, p = 0.001), hemorrhage (OR 2.40, 95% CI 1.09-5.68, p = 0.036), and the composite endpoint (OR 1.88, 95% CI 1.19-3.00, p = 0.008), whereas epinephrine exposure did not demonstrate a consistent independent effect across endpoints. Adult staggered follow-up showed greater attrition in homozygous mutant cohorts than in wild-type or heterozygous cohorts, with aggregated losses of 24%, 12%, and 3%, respectively.<br><br>CONCLUSIONS: This preliminary zebrafish acta2 model demonstrated that acta2 deficiency is associated with increased adverse larval phenotype burden and suggested reduced long-term persistence of homozygous mutant fish in adulthood. The strongest signal in the current study was genotype-associated phenotype burden rather than a robust epinephrine-dependent effect. These findings support the feasibility of zebrafish-based ACTA2 phenotyping while highlighting the need for more specific vascular endpoints, refined longitudinal follow-up, and future variant-specific modeling.},
}
@article {pmid42259495,
year = {2026},
author = {Chandrapalan, T and Kwong, RWM},
title = {DMT1 regulates systemic trace metal handling and developmental outcomes: Insights from targeted mutagenesis in zebrafish.},
journal = {Comparative biochemistry and physiology. Toxicology &amp; pharmacology : CBP},
volume = {308},
number = {},
pages = {110597},
doi = {10.1016/j.cbpc.2026.110597},
pmid = {42259495},
issn = {1532-0456},
mesh = {Animals ; *Zebrafish/genetics/metabolism/growth &amp; development ; *Cation Transport Proteins/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; Solute Carrier Family 11, Member 2 ; *Trace Elements/metabolism ; Mutagenesis ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {Divalent metal transporter 1 (DMT1) is thought to be the primary route for non-heme iron absorption in vertebrates, but its systemic role remains poorly understood. Using CRISPR-Cas9 gene editing, we generated a DMT1 knockout (dmt1[-/-]; KO) zebrafish mutant line to examine the developmental and physiological consequences of DMT1 loss. Phenotypic and hematological assessments were performed alongside measurements of whole-body and tissue-specific metal concentrations. Further, to identify potential compensatory pathways during DMT1 loss, the expression profile of candidate metal transporters or ion channels (hcp1, zip4, zip8, zip14, and ecac) was quantified using droplet digital PCR (ddPCR). DMT1 KO larvae exhibited delayed development, anemia, and broad disruption in multiple trace metals (iron, zinc, manganese, cobalt, and selenium). Gene expression analysis during early development revealed higher hcp1 mRNA abundance in the mutant, suggesting a possible compensatory response to maintain metal homeostasis during DMT1 loss. Although viable to adulthood, the mutants had persisting iron dysregulation and red blood cell abnormalities. This study provides the first in vivo evidence of the physiological role of DMT1 in multi-metal balance in fish and offers new insight into compensatory mechanisms underlying DMT1 deficiency.},
}
@article {pmid42288133,
year = {2026},
author = {Abrahms, ZN and Majdi, M and Madsen, SM and Morton, CJ and Sen, AK and Fried, NB and Sawyer, LE and Cegielski, ER and Spezzano, SJ and Jones, JA},
title = {Genome-based optimization of psilocybin and N,N-dimethyltryptamine biosynthetic pathways in E. coli using CRISPR-associated transposases.},
journal = {Metabolic engineering},
volume = {97},
number = {},
pages = {102490},
doi = {10.1016/j.ymben.2026.102490},
pmid = {42288133},
issn = {1096-7184},
mesh = {*Escherichia coli/genetics/metabolism ; *Psilocybin/biosynthesis/genetics ; *Metabolic Engineering/methods ; *Genome, Bacterial ; *CRISPR-Cas Systems ; *Biosynthetic Pathways/genetics ; },
abstract = {Stable, high-level biosynthesis of complex natural products requires precise control of heterologous pathway expression, yet transcriptional architectures optimized on plasmids often fail when transferred to the chromosome. Here, we present ePathIntegrate, a genome-centric pathway engineering strategy that leverages CRISPR-associated transposases (CASTs) to integrate and rebalance multigene metabolic pathways in Escherichia coli. Direct genomic transfer of plasmid-optimized psilocybin and N,N-dimethyltryptamine (DMT) pathways resulted in a loss of productivity, driven by context-dependent promoter behavior. To address this, we developed and characterized a library of mutant T7 promoters that restore mid-range transcriptional control on the genome. Applying ePathIntegrate enabled re-optimization of both pathways, yielding genome-encoded strains that achieve 1.88 g/L psilocybin and 1.62 g/L DMT in fed-batch bioreactors. Whole-genome sequencing of CAST-mediated strains further revealed (i) precise on-target integration, (ii) some off-target pathway integrations, and (iii) small mutations in a subset of strains, highlighting both the power and limitations of CAST-mediated strain engineering.},
}
@article {pmid42297823,
year = {2026},
author = {Adler, KM and Xu, H and Gladstein, AC and Irizarry-Negron, VM and Robertson, MR and Doerig, KR and Petrov, DA and Winslow, MM and Feldser, DM},
title = {Tumor suppressor genotype influences the extent and mode of immunosurveillance in lung cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42297823},
issn = {2041-1723},
support = {R01-CA262619-04//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-279698-01)//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; *Lung Neoplasms/genetics/immunology/pathology ; Genotype ; *Genes, Tumor Suppressor ; Humans ; *Immunologic Surveillance/genetics ; Immunoediting, Cancer ; Antigens, Neoplasm/immunology/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {The impact of cancer driving mutations on immunosurveillance throughout tumor development remains poorly understood. To better understand the contribution of tumor genotype to immunosurveillance, we generated and validated lentiviral-based vectors that create increasingly immunogenic neoantigens. This vector system is compatible with autochthonous Cre-regulated cancer models, CRISPR/Cas9-mediated somatic genome editing, and tumor barcoding. Here, we show that in the context of oncogenic KRAS-driven lung cancer and strong neoantigen expression, tumor suppressor genotype dictates the degree of immune cell recruitment, positive selection of tumors with neoantigen silencing, and tumor outgrowth. By quantifying the impact of 11 commonly inactivated tumor suppressor genes on tumor growth across neoantigenic contexts, we show that the growth-promoting effects of tumor suppressor gene inactivation correlate with increasing sensitivity to immunosurveillance. Importantly, some genotypes also dramatically changed sensitivity to immunosurveillance independently of their growth-promoting effects. We propose a model of immunoediting in which tumor suppressor gene inactivation works in tandem with neoantigen expression to shape tumor immunosurveillance and immunoediting such that the same neoantigens uniquely modulate tumor immunoediting depending on the genetic context.},
}
@article {pmid42479086,
year = {2026},
author = {Ma, L and Zhao, J and Chen, Y and Wang, Z and Yan, B and Liu, Z and Ma, N},
title = {RI-augmented TdT-CRISPR fluorescent biosensor for sensitive quantification of DNA breakage in CT-irradiated human sperm.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {29},
pages = {6090-6094},
doi = {10.1039/d6ay01260d},
pmid = {42479086},
issn = {1759-9679},
mesh = {Humans ; *Spermatozoa/radiation effects/metabolism ; *Biosensing Techniques/methods ; Male ; *DNA Damage ; *DNA ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Featuring a linear range of 0.001-0.4 nM and a limit of detection of 0.12 pM, the RI-enhanced TdT-CRISPR sensor precisely detects radiation-induced trace DNA damage in sperm, outperforming the conventional DFI method.},
}
@article {pmid42482463,
year = {2026},
author = {Yu, J and Wang, J and Yang, Y and Sun, L and Hu, X and Nie, T and Yang, X and Wang, X and Li, C and You, X},
title = {Multifaceted effects of galU deletion on phenotype and virulence of Pseudomonas aeruginosa in vitro and in vivo.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2707803},
doi = {10.1080/21505594.2026.2707803},
pmid = {42482463},
issn = {2150-5608},
mesh = {*Pseudomonas aeruginosa/pathogenicity/genetics/drug effects ; Virulence ; Animals ; Pseudomonas Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; *Gene Deletion ; *Bacterial Proteins/genetics/metabolism ; Phenotype ; O Antigens/genetics/biosynthesis ; Polymyxins/pharmacology ; Quorum Sensing ; Microbial Sensitivity Tests ; Mice ; CRISPR-Cas Systems ; },
abstract = {Pseudomonas aeruginosa is a widespread Gram-negative opportunistic pathogen in environmental and hospital settings, frequently causing respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD), and ventilator-associated pneumonia. In our previous study, a galU-deleted clinical P. aeruginosa was found to exhibit increased susceptibility to polymyxins. The galU gene plays an important role in the biosynthesis of lipopolysaccharide (LPS) O-antigen. Here, we systematically evaluated the effects of galU deletion on the phenotype and virulence of P. aeruginosa PAO1. A galU deletion mutant was successfully constructed in P. aeruginosa PAO1 by CRISPR/Cas9, and the complementation was accomplished by pUCP18 plasmid carrying wild-type galU. The changes in phenotype, virulence, and pathogenicity were systemically studied. The results revealed that knockout of galU led to the loss of O-antigen, which affected growth, virulence, and pathogenicity through various ways in P. aeruginosa, and significantly affected the susceptibility of P. aeruginosa to polymyxins. Mechanism study suggested the involvements of quorum sensing, Entner-Doudoroff pathway, and tyrosine metabolism on bacterial virulence and antibiotic susceptibility changes after galU deletion. galU and the related pathways may serve as effective targets for the treatment of P. aeruginosa infection, providing a theoretical basis for the development of novel antibacterial drugs.},
}
@article {pmid42508244,
year = {2026},
author = {Chen, W and Cao, L and Zhu, X and Kang, W and Qiu, Y and Nie, Z and Huang, Y and Lei, C},
title = {Proteolysis-triggered CRISPR/Cas activation via cascaded enzyme switches for viral protease detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119073},
doi = {10.1016/j.bios.2026.119073},
pmid = {42508244},
issn = {1873-4235},
abstract = {Sensitive analysis of viral proteases is crucial for early infection diagnosis and antiviral drug development; however, developing activity-based assays with high sensitivity and broad adaptability remains a significant challenge. To address this, we developed a cascade signal amplification strategy that enhances protease detection sensitivity through rational coupling of two modular, protease-responsive enzyme switches. Upon recognition of the target protease, this upstream switch triggers the activation of a downstream, protease-responsive CRISPR/Cas12a effector, which converts the specific proteolytic event into an amplified fluorescence signal. This cascaded enzymatic amplification generates a robust signal output, achieving an order-of-magnitude improvement in detection sensitivity compared to single-stage CRISPR/Cas12a assays. The modular nature of the enzyme switches renders the system highly expandable, allowing for the specific detection of diverse viral proteases. We demonstrated the platform's utility in complex biological samples by sensitively monitoring 3C protease activity within enterovirus 71-infected cells. Furthermore, dose-dependent inhibition analysis using the HRV 3C protease inhibitor rupintrivir validated the system's potential for evaluating antiviral drug efficacy. Collectively, this work establishes a versatile and modular analytical platform for the sensitive detection and functional study of viral proteases.},
}
@article {pmid42508886,
year = {2026},
author = {Tian, S and Tang, X and Zhang, S and Yao, Y and Bi, R and Li, H and Sun, X and Chen, Z},
title = {Construction of the CRISPR/Cas12a-digital integrated immunoassay technology for CD44 detection.},
journal = {Analytica chimica acta},
volume = {1417},
number = {},
pages = {346010},
doi = {10.1016/j.aca.2026.346010},
pmid = {42508886},
issn = {1873-4324},
mesh = {Humans ; Immunoassay/methods ; *Hyaluronan Receptors/blood/analysis ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; },
abstract = {Digital immunoassay allows for the detection of proteins at fg/mL levels by leveraging the principles of Poisson distribution. Nevertheless, the inherent Poisson noise restricts digital sensitivity to targets exceeding 100 molecules, rendering standard digital assays incapable of detecting targets below this threshold. To overcome this constraint, we introduce an integrated CRISPR/Cas12a-digital immunoassay technology. In this system, the CRISPR/Cas12a machinery first acts as a molecular amplifier, converting each target protein into multiple enzymatic reporters. These enzyme molecules are then individually quantified via a digitized readout system, enabling ultrasensitive protein detection. By harnessing the catalytic amplification of CRISPR/Cas12a, the method achieves detection of proteins at copy numbers below 100, effectively surpassing the conventional sensitivity barrier of digital immunoassays. We validated this approach through highly selective and accurate detection of CD44 protein. A linear response was observed across a concentration range of 0.05 to 5 fg/mL, conforming to the calibration model: P(X > 0) = 0.1191c + 0.0036. The limit of detection was determined to be 0.018 fg/mL, equivalent to approximately 36 molecules of CD44. The method was further applied to quantify CD44 in plasma samples from colorectal cancer patients, demonstrating its strong potential for clinical use in early cancer diagnosis and treatment monitoring.},
}
@article {pmid42510584,
year = {2026},
author = {Yang, JW and Kim, HS and Kim, YH},
title = {Redox Regulation of Plant-Root-Knot Nematode Interactions: From ROS-Mediated Immunity to Sustainable Resistance.},
journal = {Antioxidants (Basel, Switzerland)},
volume = {15},
number = {7},
pages = {},
pmid = {42510584},
issn = {2076-3921},
abstract = {Root-knot nematodes (RKNs; Meloidogyne spp.) are among the most destructive plant parasites, causing severe yield losses in diverse crops. Reactive oxygen species (ROS), particularly superoxide radicals (O2•[-]) and hydrogen peroxide (H2O2), are central regulators of plant-RKN interactions. This review synthesizes current molecular, biochemical, genetic, transcriptomic, and translational evidence showing that the outcome of infection is determined by the spatiotemporal regulation of H2O2 rather than by ROS abundance alone. In resistant interactions, nematode perception activates PTI-associated signaling through selected cell-surface receptor complexes, including some BAK1/SERK3-associated pathways, together with BIK1, Ca[2+] signaling, and RBOHD/F, generating a sustained oxidative activity associated with salicylic acid-dependent immune signaling and reduced H2O2-scavenging capacity and coupled to hypersensitive response, lignin and callose deposition, and feeding site restriction. In susceptible interactions, RKNs deploy ROS-targeting effectors such as Mi-CRT, MjTTL5, CATLe, Mj-NEROSs, and CMII to suppress ROS production, enhance antioxidant scavenging, or weaken SA-dependent defense. Evidence from a cyst-nematode system suggests that RBOH-derived ROS can restrict excessive cell death around syncytia; whether an analogous lower-redox requirement exists in RKN-induced giant cells remains unresolved. Finally, redox-based strategies, including CRISPR/Cas editing, host-induced gene silencing, chemical priming, and biocontrol, are discussed as promising approaches for durable and sustainable nematode resistance.},
}
@article {pmid42510769,
year = {2026},
author = {Wyman, SK and Romero, Z and Heo, SJ and Navarrete, M and Krishnappa, N and Kohn, DB and Martin, DIK and Walters, MC and Boffelli, D},
title = {Oligonucleotide Synthesis Errors Are a Source of Untoward Variation in HDR-Mediated Gene Editing.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510769},
issn = {2073-4425},
support = {TRAN1-09292//California Institute for Regenerative Medicine/ ; CLIN1-11497//California Institute for Regenerative Medicine/ ; CLIN2-11722//California Institute for Regenerative Medicine/ ; HL151319//National Heart Lung and Blood Institute/ ; OT3HL147741//National Heart Lung and Blood Institute/ ; },
mesh = {Humans ; *Oligonucleotides/genetics ; *Recombinational DNA Repair ; High-Throughput Nucleotide Sequencing ; Anemia, Sickle Cell/genetics/therapy ; *Gene Editing ; beta-Globins/genetics ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Background/Objectives: Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. Methods: We performed deep sequencing of ssODNs from three manufacturers and amplicons from edited hematopoietic stem/progenitor cells. Results: We find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign β-globin variants, while the less frequent frameshift deletions are predicted to generate β-thalassemia-like alleles. Conclusions: Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs.},
}
@article {pmid42510803,
year = {2026},
author = {Gonzales, PR},
title = {Advances in Functional Genomics for Human Health.},
journal = {Genes},
volume = {17},
number = {7},
pages = {},
pmid = {42510803},
issn = {2073-4425},
mesh = {Humans ; *Genomics/methods ; Genome, Human ; },
abstract = {Cytogenomics, including karyotyping, FISH, chromosomal microarrays, and optical genome mapping, has yielded significant results for clinical phenotypes in constitutional and cancer genetics, including intellectual disability, autism spectrum disorders, dysmorphic features, and hematological and solid-tissue neoplasia. However, some of these assays have yielded results of unclear significance because the abnormalities detected were often located in intergenic regions of the genome. Because these abnormalities are within the "dark matter" of the genome, their clinical significance has been a matter of speculation. However, functional genomics can explore the clinical implications of such abnormalities more robustly, whether the abnormalities disrupt topologically associating domains (TADs), delete regulatory regions, etc. Some human genetic diseases associated with these intergenic abnormalities and characterized by functional genomics include preaxial polydactyly (SHH gene), Pierre Robin syndrome (SOX9), and 5q14.3 microdeletion syndrome (MEF2C). While functional genomics is a broad research topic, this review focuses on prior and current efforts to leverage functional genomics within the intergenic regions for human health.},
}
@article {pmid41805199,
year = {2026},
author = {Marín-Sanz, M and Berlanga-Torres, JA and Guzmán-López, MH and Sánchez-León, S and Vallés, MP and Castillo, AM and Barro, F},
title = {Engineering ultra-low-gliadin wheat for celiac disease using an integrated RNAi, CRISPR, and doubled haploid strategy.},
journal = {Journal of experimental botany},
volume = {77},
number = {14},
pages = {4417-4434},
doi = {10.1093/jxb/erag131},
pmid = {41805199},
issn = {1460-2431},
support = {QUAL21_023 IAS//Junta de Andaluc&#x131;&#x301;a/ ; //Conexi&#xf3;n TRIGO, grant number 202490E049)./ ; },
mesh = {*Triticum/genetics/metabolism ; *Gliadin/genetics/metabolism ; *RNA Interference ; Haploidy ; *Celiac Disease/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Glutens ; },
abstract = {The growing prevalence of gluten-related disorders in humans has driven the development of wheat varieties with reduced immunogenic gluten. This study aimed to integrate RNA interference (RNAi) and CRISPR genome editing within a doubled haploid (DH) platform to overcome challenges of gene redundancy and polyploidy in wheat gliadins. We generated DH lines from crosses between RNAi and CRISPR lines and elite wheat cultivars, enabling stable fixation of multiple genetic modifications in a single generation. Deep sequencing analysis of α-gliadin amplicons was conducted using a custom bioinformatics pipeline optimized for complex, repetitive gene families. Gluten protein profiles were evaluated using RP-HPLC and R5 monoclonal antibody. Several DH lines presented >70% reduction in immunogenic epitopes in α-gliadins, with lines outperforming both parents. Editing frequency was influenced by sgRNA efficiency and parental background. Silencing and editing combined led to nearly depleted gliadins in some lines, often with compensatory increases in other storage proteins linked to bread-making quality, such as high-molecular-weight glutenin subunits. Kernel and specific weight traits were largely maintained. This work demonstrates that combining RNAi and CRISPR in a DH platform enables efficient, heritable reduction of immunogenic gluten, providing a viable strategy for breeding wheat lines safer for individuals with gluten-related disorders.},
}
@article {pmid41928454,
year = {2026},
author = {Tietz, SM and Brenner, K and Moyo, T and Young, PR and Vivier, MA},
title = {Optimized protocol for efficient generation, confirmation, transformation, and CRISPR editing of grapevine hairy roots.},
journal = {Journal of experimental botany},
volume = {77},
number = {14},
pages = {4352-4374},
doi = {10.1093/jxb/erag165},
pmid = {41928454},
issn = {1460-2431},
support = {//South Africa Wine/ ; UID120460//National Research Foundation (NRF)/ ; },
mesh = {*Vitis/genetics/growth &amp; development ; *Plant Roots/genetics/growth &amp; development ; *Transformation, Genetic ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Hairy root cultures (HRCs) are powerful tools in plant biotechnology but show variable establishment efficiencies, limiting broader applications. Here, we present a standardized and optimized reference methodology for the routine generation, multiplication, and maintenance of HRCs across diverse grapevine genotypes. Our workflow evaluated three Rhizobium strains, seven grapevine cultivars (three Vitis vinifera cultivars; four Vitis rootstock hybrids), multiple explant types, infection protocols, co-cultivation times, growth media types, and anti-browning agents. The resulting protocol was effective for all grapevine genotypes and, with minor adjustments, also yielded HRCs from two other important South African plant species, namely Sutherlandia frutescens and Aspalathus linearis. Useful molecular tools were developed for transformation and selection of HRCs, including universal multiplex primers for confirmation of transformation, tested antibiotic resistance markers (kanamycin and hygromycin), and fluorescent reporters (DsRed and eyGFPuv), with DsRed found to be particularly versatile. To test the system, we overexpressed the VviMYBA1 transcription factor gene, leading to increased anthocyanin accumulation and red pigmentation in HRCs. Additionally, we achieved CRISPR/Cas9 editing of the VviPUB19 gene, the first report of CRISPR-edited grapevine HRCs. Gene editing combined with HRCs can facilitate rapid gene function studies, offering an efficient alternative or pre-screening system to whole-plant transformations, that could support advanced functional genomics and biotechnological applications in grapevine.},
}
@article {pmid42215861,
year = {2026},
author = {Liu, F and Zhang, X and Yang, Y},
title = {A comparison between CARLIN and DNA Typewriter in CRISPR-mediated lineage tracing.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42215861},
issn = {1471-2105},
support = {NCI U01-CA253553/CA/NCI NIH HHS/United States ; R01-CA251950/NH/NIH HHS/United States ; NCI U01-CA253553/CA/NCI NIH HHS/United States ; R01-CA251950/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Cell Lineage/genetics ; },
abstract = {BACKGROUND: CARLIN and DNA Typewriter are two major breakthroughs in CRISPR-based lineage tracing technology. It is essential to understand the potential and performance of these methods in lineage tracing, which provides important guidance on experimental design.<br><br>RESULTS: In this study, we systematically compare these two strategies using a unified stochastic simulation framework with known ground-truth lineages. By explicitly modeling CRISPR editing dynamics, barcode evolution, and cell division processes, the framework enables quantitative benchmarking of lineage reconstruction accuracy across diverse experimental parameter regimes. Both methods are evaluated using multiple accuracy metrics, including Robinson-Foulds accuracy and triplet accuracy, allowing a comprehensive assessment of lineage reconstruction performance under various editing probabilities, sampling depths, and lineage lengths.<br><br>CONCLUSIONS: DNA Typewriter consistently outperforms CARLIN in lineage reconstruction accuracy when sufficient numbers of recording targets are used, particularly in more cell divisions. Sequential and ordered recording in DNA Typewriter substantially reduces ambiguity in lineage inference compared to unordered CRISPR barcode editing. CARLIN's lineage-recording potential exhausts rapidly under continuous induction, limiting its effectiveness in long-term lineage tracing. Triplet accuracy provides a more permissive and informative metric than Robinson-Foulds accuracy, especially under partial sampling scenarios.},
}
@article {pmid42233593,
year = {2026},
author = {Zhao, W and Zheng, Z and Li, R and Xie, H and Yu, H and Zhang, Y and Wu, Y and Yang, Y and Zhang, Z and Gao, H and Li, Y and Zhang, K},
title = {In Situ Amplified Mutational mRNA Imaging Using a Spatially Confined CRISPR Nanoplatform.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {31},
pages = {e7088080},
pmid = {42233593},
issn = {1521-3773},
support = {22377110//National Natural Science Foundation of China/ ; 82402749//National Natural Science Foundation of China/ ; U23A20531//National Natural Science Foundation of China/ ; 22122409//National Natural Science Foundation of China/ ; 252300421073//Natural Science Foundation of Henan Province/ ; 252102311025//Science and Technology Department of Henan Province/ ; 2025SGAQZ-MS-03//State Key Laboratory of Metabolic Dysregulation &amp; Prevention and Treatment of Esophageal Cancer/ ; 261111313300//Henan Provincial Key Research and Development Program/ ; },
mesh = {*RNA, Messenger/genetics/analysis ; Humans ; Mutation ; *CRISPR-Cas Systems/genetics ; },
abstract = {Highly sensitive spatial analysis of RNA mutations is essential for understanding cellular heterogeneity and disease mechanisms. Herein, we developed an integrated CRISPR/Cas13a-based nanoprobe system for rapid detection of RNA in tissue sections (Integrated CRISPR/Cas13a-based RNA Rapid Detection, InCasRD). Unlike conventional "always-on" probes that rely on accumulated probe hybridization, InCasRD leverages the trans-cleavage activity of Cas13a to achieve spatially confined signal amplification and a high signal-to-background ratio (SBR). Using InCasRD, we achieved imaging of multiple target RNAs in tumor cells within 0.5 h of incubation, including mRNA (survivin), microRNA (miR-21), and circular RNA (circ1785). Furthermore, the engineered InCasRD system enabled mapping of RNA mutations, such as the EGFR L858R and ovarian tumor domain (OTUD) single-nucleotide variant (SNV, 23439980 G>T), in tumor tissue sections, thereby facilitating clear tumor boundary delineation. Collectively, InCasRD is a powerful, one-step tool for in situ RNA analysis with potential for diagnosis and precision medicine.},
}
@article {pmid42335403,
year = {2026},
author = {Luo, H and Gao, J and Huang, X and Fang, Y and Huang, T and Xia, Y and Yu, Z and Cao, C and Xiong, Z},
title = {Thermo-Responsive Living Microspheroids Enable a Regenerative Living Disk-Drive System for DNA Data Storage.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {42},
pages = {e73806},
pmid = {42335403},
issn = {1521-4095},
support = {53330200321//Tsinghua University/ ; },
mesh = {*DNA/chemistry/genetics ; *Temperature ; Plasmids/genetics ; *Information Storage and Retrieval/methods ; CRISPR-Cas Systems ; },
abstract = {DNA offers exceptional information density and long-term stability, yet its practical deployment is limited by destructive readout and the absence of a reusable, physically addressable architecture that connects nanoscale molecular information with macroscale device-level data organization. Here, we present a regenerative Living Disk-Drive system based on thermo-responsive engineered living memory microspheroids (ELMMs), in which data-encoded bacteria are encapsulated as discrete, file-level living storage units. Each ELMM contains a clonal bacterial population carrying both an information plasmid, which encodes 26 × 26 pixel icon payloads and one- to three-color intracellular fluorescent retrieval indices, and a help plasmid that enables CRISPR-Cas12a/λ-Red rewriting of the data sequence and retrieval tag. A lyophilized ELMM database forms the Living Disk, which is coupled to an Optical Retriever and desktop-scale Living Drive for closed-loop retrieval, regeneration, and database replenishment. Released bacteria regrow for downstream readout or rewriting, while a fraction is re-encapsulated into new ELMMs. The tested system retains retrieval, regrowth, and sequence recovery after four months of ambient dry storage and 13 lyophilization-rehydration cycles. Model-based performance estimates are reported only as theoretical architecture-level bounds. These results establish an experimentally bounded yet extensible architecture for physically manageable and regenerative DNA memory.},
}
@article {pmid42372505,
year = {2026},
author = {Wang, R and Pan, Q and Huang, Y and Dai, W and Ping, Y and Jin, Q},
title = {Versatile hollow Ca[2+]-phenolic nanoparticles for intracellular delivery of diverse bioactive molecules and CRISPR-Cas9 genome editing.},
journal = {Biomaterials},
volume = {335},
number = {},
pages = {124407},
doi = {10.1016/j.biomaterials.2026.124407},
pmid = {42372505},
issn = {1878-5905},
mesh = {Humans ; *Nanoparticles/chemistry/ultrastructure ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Calcium/chemistry ; *Phenols/chemistry ; },
abstract = {The development of versatile nanoplatforms capable of universally encapsulating diverse bioactive molecules holds significant promise in biomedicine. In this study, size-tunable hollow Ca[2+]-tannic acid (TA) nanoparticles (HCT NPs) are synthesized as universally applicable drug nanocarriers by simply adding TA into amorphous calcium carbonate nanoparticles. The formation of HCT NPs is identified as a surface-protected self-etching process. A wide range of hydrophobic and hydrophilic small-molecule drugs, metal ions, and biomacromolecules including proteins and nucleic acids can be encapsulated in HCT NPs for efficient intracellular delivery. HCT NPs show rapid and efficient endosomal escape, which is crucial for maintaining the bioactivity of biomacromolecules. Remarkably, a wide array of cargo proteins, spanning different molecular weights and isoelectric points can be delivered into the cytosol by HCT NPs without compromising their bioactivities. The therapeutic potential of HCT NPs for intracellular cargo delivery is exemplified by cytosolic delivery of Cas9 plasmids and Cas9 ribonucleoprotein (RNP) for CRISPR-Cas9 genome editing both in vitro and in vivo. The facile and ultrafast synthesis, versatile cargo encapsulation capabilities, efficient cell uptake and endosomal escape, and excellent biocompatibility make HCT NPs a prominent candidate for intracellular delivery of diverse bioactive molecules, particularly in therapeutic applications such as genome editing.},
}
@article {pmid42390232,
year = {2026},
author = {Duvenage, L and Chetty, A and Thomson, DD and Ballou, ER and Govender, NP and Rappleye, CA and Hoving, JC},
title = {Tools for genetic manipulation of the endemic fungal pathogen Emergomyces africanus and application of a fluorescent reporter strain in infection models.},
journal = {mSphere},
volume = {11},
number = {7},
pages = {e0018026},
pmid = {42390232},
issn = {2379-5042},
support = {209293/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 310933/Z/24/Z/WT_/Wellcome Trust/United Kingdom ; AI148561/NH/NIH HHS/United States ; },
mesh = {Animals ; Green Fluorescent Proteins/genetics ; *Genes, Reporter ; Mice ; Plasmids/genetics ; Macrophages/microbiology ; Disease Models, Animal ; *Mycoses/microbiology ; *Ascomycota/genetics/pathogenicity ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Emergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa, which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterization of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of Histoplasma capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall α(1,3)-glucan by targeting the transcripts of the α-glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus.<br><br>IMPORTANCE: Emergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognized in recent years, and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (i) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (ii) methods for knocking down gene expression in E. africanus, and (iii) the permanent disruption of genes through CRISPR/Cas9 gene editing.},
}
@article {pmid42492264,
year = {2026},
author = {Shen, H and Nourmohammadi, S and Zhou, Y and Harata-Lee, Y and Qu, Z and Wang, W and Yool, AJ and Adelson, DL},
title = {Target pathway validation for Compound Kushen Injection active against cancer cells.},
journal = {Phytomedicine : international journal of phytotherapy and phytopharmacology},
volume = {159},
number = {},
pages = {158598},
doi = {10.1016/j.phymed.2026.158598},
pmid = {42492264},
issn = {1618-095X},
abstract = {BACKGROUND: Traditional Chinese medicines are based on complex mixtures of natural products and their multi-target mechanism of action. Therefore the discovery and validation of targets and mechanisms have always been challenging. In previous studies, using transcriptomic methods and Compound Kushen Injection (CKI) as a model drug, we identified multiple pathways and candidate target genes for validation, through which CKI exerts its pharmacological effects.<br><br>PURPOSE: This study aimed to demonstrate the involvement of multiple genetic targets in different pharmacological activities for natural products in Compound Kushen Injection.<br><br>METHODS: In this study, we selected eight key genes from four candidate pathways and used CRISPR/CAS technology to knock out these genes in four cell lines, validating their role in CKI activity.<br><br>RESULTS: Although the sensitivity of different cell lines to gene knockout varied, overall, it led to reductions in various cellular activities. After the addition of CKI, we observed that, except for the minor impact of CDKN1A gene knockout on the effect of CKI, knocking out the other genes significantly affected the pharmacological efficacy of CKI in different assays. Among them, knockout of MYD88 and NFkB genes enhanced the efficacy of CKI. At the same time, we found that the genes IL24 and CYP1B1 play a crucial role in CKI inhibition of tumour cell migration, and the CYP1A1 gene is critical for the cell cycle arrest induced by CKI.<br><br>CONCLUSIONS: These findings validate the results of our previous transcriptomic analysis and further demonstrate the complexity of pharmacological mechanisms of multi-target synergistic action of natural product mixtures.},
}
@article {pmid42492655,
year = {2026},
author = {Patel, S and Panchal, J and Patel, A and Chauhan, H and Sharma, K and Sabara, P and Vahora, S and Shrimali, M and Shekh, S and Thakor, A and Mohapatra, S and Hati, S},
title = {Unravelling the Resistome of Carbapenem-Resistant E. coli from Bovine Mastitis via Whole-Genome Sequencing.},
journal = {Veterinary journal (London, England : 1997)},
volume = {},
number = {},
pages = {106792},
doi = {10.1016/j.tvjl.2026.106792},
pmid = {42492655},
issn = {1532-2971},
abstract = {Carbapenem-resistant Escherichia coli (CREC) poses a growing threat to public health, particularly when emerging from animal reservoirs such as dairy cattle. This study aimed to characterize CREC isolates recovered from bovine mastitis cases in Gujarat, India, using a combination of phenotypic antibiotic susceptibility testing and whole-genome sequencing (WGS). Out of 130 confirmed E. coli isolates from 790 mastitic milk samples, 33 (25.38%) were resistant to imipenem. Of these, nine exhibited multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant (PDR) phenotypes. WGS was performed on four representative isolates (SKN144, SKN685, SKN687, SKN926), revealing genome sizes ranging from 4.7 to 5.4Mb and GC content between 50.4% and 50.8%. Annotation identified numerous resistance determinants, including carbapenemase genes (blaNDM, blaOXA-48, blaTEM, blaCMY, blaCTX-M), aminoglycoside-modifying enzymes (APH, AAC), macrolide resistance genes (mphA, ermB), and multiple efflux pump systems (AcrAB-TolC, EmrAB, MdtEF-TolC). Functional genes associated with replication, repair, stress response, and mobile genetic elements (integrases, transposases, CRISPR-Cas) were also detected, indicating high genomic adaptability. Phenotypic testing revealed alarming resistance to key antimicrobials, including ampicillin (56.15%), amikacin (55.38%), ceftazidime (53.08%), and colistin (79.23%, including intermediate strains). Subsystem analysis highlighted metabolic versatility, defence mechanisms, and virulence-associated pathways. Phylogenetic analysis indicated that all isolates clustered within the same clade, suggesting possible clonal dissemination within the bovine population. The presence of CRISPR-Cas elements, integrases, and transposases suggests ongoing horizontal gene transfer and genome plasticity. These findings underscore the alarming prevalence of CREC in dairy environments and the urgent need for enhanced AMR surveillance, prudent antibiotic stewardship, and implementation of a One Health approach to prevent zoonotic transmission. This study contributes valuable genomic insights into livestock-associated CREC and highlights their close genomic parallels with high-risk human clinical clones.},
}
@article {pmid42493436,
year = {2026},
author = {Wang, Y and Song, H and DU, Y and Xu, T},
title = {[CRISPR-Cas9 activation screening identifies candidate chemokine regulators of ter-tiary lymphoid structure formation in bladder cancer].},
journal = {Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences},
volume = {58},
number = {4},
pages = {707-715},
pmid = {42493436},
issn = {1671-167X},
mesh = {Animals ; Mice ; *Urinary Bladder Neoplasms/genetics/pathology/immunology ; Humans ; *Chemokines/genetics/metabolism ; Cell Line, Tumor ; Mice, Inbred C57BL ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Chemokine CCL20/genetics ; },
abstract = {OBJECTIVE: To identify the cytokine genes influencing the formation of tertiary lymphoid structures (TLS) through CRISPR-Cas9 library screening, and to discover potential key regulatory molecules, providing new targets for enhancing the efficacy of bladder cancer immunotherapy.<br><br>METHODS: Based on a mouse whole-genome library, 44 chemokine-related genes were identified, and an single-guide RNA (sgRNA) library targeting these genes was designed and constructed, with three sgRNAs assigned to each gene. Using a lentiviral packaging system, the library plasmids were used to transfect HEK293T cells to generate a lentiviral library, which was then used to infect the mouse bladder cancer cell line MB49. Purinomycin selection was performed to obtain the MB49-mCherry cell line stably over-expressing chemokines. The cells were inoculated into the peritoneal cavity of C57BL/6 mice to establish a bladder cancer xenograft model, and tumor growth was monitored. Three weeks later, tumor tissue was excised, genomic DNA was extracted for high-throughput sequencing, and sgRNA enrichment was analyzed to screen for differentially expressed cytokine genes. Concurrently, immunohistochemical staining was performed to detect TLS markers CD20 and CD3, and the number, distribution, and maturity of TLS were assessed. The selected candidate genes were validated individually in vivo to further confirm their impact on TLS formation.<br><br>RESULTS: We successfully constructed a cytokine gene library containing 132 sgRNAs, covering 44 chemokine genes. Following lentiviral infection, we obtained the MB49-mCherry cell line, which stably expressed the library, and isolated dead Cas9-positive monoclonal cell lines via flow cytometry to ensure the homogeneity and reproducibility of subsequent experiments. Intratumoral tumor experiments in mice revealed that the number of TLS cells in the experimental group was significantly higher than in the control group, primarily distributed at the tumor margins. High-throughput sequencing results showed that, compared with the control group, in the experimental group, Cxcl16 sgDNA was significantly enriched, while Ccl20 and Cx3lc1 sgDNA levels decreased compared with baseline (P < 0.05). Further validation of the individual roles of each factor via intraperitoneal injection revealed that the number of TLSs in tumors decreased in the group treated with the CX3CL1 chemokine, suggesting that CX3CL1 might negatively regulate TLS formation. Immunohistochemical results showed that in the CX3CL1-treated group, the aggregation of CD20-positive B cells and CD3-positive T cells in the tumor tissue was reduced, and the TLS structure was incomplete.<br><br>CONCLUSION: Through CRISPR-Cas9 library screening combined with in vivo validation, this study successfully identified CX3CL1 as a potential negative regulator of TLS formation in bladder cancer. High CX3CL1 expression was associated with a reduction in TLS numbers, suggesting that it might exert an inhibitory role in the immune microenvironment of bladder cancer. This finding provides new clues and research directions for understanding the molecular mechanisms of TLS formation in bladder cancer. However, whether CX3CL1 can serve as an immunotherapeutic target remains to be further validated through clinical specimen analysis, multidimensional mechanistic investigation, and immunotherapy response correlation studies.},
}
@article {pmid42494101,
year = {2026},
author = {Liu, K and Ren, C and Liu, X and Qi, X and Liu, Y and Ma, S and Zhang, S and Wong, XY and Wang, X and Hu, T and Hu, C},
title = {Shared CRISPR arrays underpin type I-A/I-B coexistence.},
journal = {Protein &amp; cell},
volume = {},
number = {},
pages = {},
doi = {10.1093/procel/pwag050},
pmid = {42494101},
issn = {1674-8018},
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes, yet how multiple CRISPR-Cas subtypes coexist and coordinate within a single genome remains unclear. Comparative genomic analysis revealed that nearly one-third of type I-A CRISPR-Cas3 systems are adjacent to a type I-B system, often sharing a single CRISPR array. Using Thermococcus siculi RG-20 (Tsi) as a model, we show that purified TsiCas6a and TsiCas6b independently recognize and cleave the shared pre-crRNA, producing mature crRNAs with comparable efficiency. Plasmid interference assays further demonstrated that crRNAs produced by either Cas6a or Cas6b enzyme could guide both type I-A and type I-B interference complexes. This interchangeability shows that crRNAs generated by either Cas6a or Cas6b can be loaded into, and function with, both type I-A and type I-B interference complexes. Structural modelling revealed distinct but complementary recognition strategies for Cas6a and Cas6b, and mutational analysis of their RNA-binding residues impaired pre-crRNA cleavage and abolished interference activity. Together, these results uncover a shared-array logic in which Cas6a/Cas6b-compatible processing routes a single pre-crRNA to multiple type I effectors-providing a potential mechanism for subtype co-existence, a plausible explanation for array-less (or "orphan") interference modules, and an evolutionary bet-hedging strategy that prevents Acrs from shutting down immunity wholesale.},
}
@article {pmid42494113,
year = {2026},
author = {Ohtomo, M and Takarabe, S and Namiki, T and Kawata, Y and Kaneko, R and Ozawa, M and Yamada, Y and Mori, H and Kageyama, A and Kamoshita, M and Terakawa, J and Ito, J},
title = {Generation and Characterization of a Rdh1-iCre Line to Study Uterine Glandular Biology.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {4},
pages = {e70068},
pmid = {42494113},
issn = {1526-968X},
support = {JP21K09512//Japan Society for the Promotion of Science/ ; JP24K01950//Japan Society for the Promotion of Science/ ; JP25KJ2187//Japan Society for the Promotion of Science/ ; JP22H04922//Japan Society for the Promotion of Science/ ; JP25K22429//Japan Society for the Promotion of Science/ ; //Azabu University/ ; },
mesh = {Animals ; Female ; *Uterus/metabolism ; Mice ; *Alcohol Oxidoreductases/genetics/metabolism ; *Integrases/genetics/metabolism ; Pregnancy ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {The uterus is an essential organ for fetal development in most mammals. Uterine glands, highly conserved structures in the mammalian uterus, play critical roles in the establishment and maintenance of pregnancy and have been implicated in the pathogenesis of uterine diseases, including endometrial cancer and endometriosis. Previous studies have shown that Retinol dehydrogenase 1 (Rdh1) is specifically expressed in the glandular epithelium (GE) from the onset of gland formation through adulthood. In this study, to develop a GE-specific Cre driver line, we generated Rdh1-iCre mice by introducing an improved Cre recombinase (iCre) into the Rdh1 locus using the CRISPR/Cas9 system. To evaluate the utility of this model, Rdh1-iCre mice were crossed with ROSA26-H2B-mCherry reporter mice, and Cre-dependent reporter expression was analyzed. Robust mCherry fluorescence was observed throughout the uterine glands at 2 weeks after birth, coinciding with the active elongation and branching of the GE. These results demonstrate that the Rdh1-iCre mouse line is a valuable and highly efficient tool for investigating the physiological roles of uterine glands during development and pregnancy, as well as their contribution to the progression of GE-derived uterine diseases.},
}
@article {pmid42494486,
year = {2026},
author = {Wu, W and Wang, R and Li, J and Zhang, Z and Yao, W and Zhang, N and Xu, W},
title = {Engineering climate-resilient horticultural crops: advances in transcriptional regulation, genome editing, and synthetic networks.},
journal = {Horticulture research},
volume = {13},
number = {8},
pages = {uhag119},
pmid = {42494486},
issn = {2662-6810},
abstract = {Abiotic stresses-particularly cold, drought, and salinity-pose significant threats to the productivity and sustainability of horticultural crops. Recent studies have revealed conserved and species-specific regulatory mechanisms that allow plants to adapt dynamically to these environmental constraints. This review synthesizes advances in understanding key transcription factor families-such as CBF/DREB, NAC, MYB, WRKY, and bHLH-that orchestrate stress-responsive gene networks and modulate physiological processes, including osmotic regulation, antioxidant defense, and ionic homeostasis. We also discuss the emerging roles of chromatin remodeling, DNA methylation, histone modifications, and noncoding RNAs in conferring transcriptional plasticity and stress memory. Beyond endogenous pathways, we evaluate transgenic strategies, CRISPR/Cas-based genome editing, and synthetic gene circuits for engineering abiotic stress tolerance. Particular attention is given to trade-offs between growth and defense, challenges in horticultural crop transformation, and gaps in field translation. We further examine the regulatory role of secondary metabolites-such as flavonoids and salicylic acid-as biochemical interfaces between signal transduction and adaptive responses. Finally, we propose a forward-looking roadmap integrating multi-omics, ideotype design, and precision breeding toward climate-resilient horticultural systems.},
}
@article {pmid42494498,
year = {2026},
author = {Banik, I and Coppé, JP},
title = {CRISPRing through time: How cutting-edge technology is revolutionizing life sciences and medicine.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {3},
pages = {103003},
pmid = {42494498},
issn = {2162-2531},
abstract = {Given the plethora of emerging technologies, none have truly captured the minds as CRISPR. From the groundbreaking research, the ultimate battle of the prizes and patents to a number of books, the science of CRISPR continues to be significant in the biomedical field. For many decades now, the emergence of synthetic biology as an intervention to correct diseases has become the foundation of biomedical research. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genetic editing has become a common place for routine investigation of scientific hypotheses in pre-clinical settings. More recently, CRISPR-based diagnostic testing kits for SARS-CoV-2 have showcased a translational output. Furthermore, a technological landmark was achieved when the Food and Drug Administration (FDA) approved the first CRISPR-based gene therapy (exa-cel) to edit erythroid specific enhancer region of BCL11A in hematopoietic stem cells, introduced in patients suffering from sickle cell anemia to achieve durable remission. In this review, we provide a snapshot into the most important milestones along the journey of CRISPR from its discovery in bacteria to its usage in precision medicine. The intervention of machine learning tools has now intertwined complex biology with high-throughput scalable outputs. Given the vast amount of information on CRISPR, we try to pin down key take-home messages for scientists as well as non-scientist readers. This review article attempts to understand why and how CRISPR remains significant and seamlessly integrates in the emerging era of new technologies.},
}
@article {pmid42496566,
year = {2026},
author = {Krishna, TPA and Harikrishnan, D and Veena, M and Maharajan, T and James, M and Udhayakumar, M and Arockiam Jeyasundar, PGS and David, SJ and Dineshkumar, R and Rajan, R and Rathinapriya, P},
title = {Next-Generation Metabolic Engineering of Capsaicinoids Biosynthesis in Chilli Pepper: Bridging Genomic Insights to Biotechnological Applications.},
journal = {Biotech (Basel (Switzerland))},
volume = {15},
number = {3},
pages = {},
pmid = {42496566},
issn = {2673-6284},
abstract = {Chilli peppers (Capsicum species) have been widely used around the world because of their economic value and distinctive sensory characteristics. They contain abundant functional metabolites, especially a group of vanillylamide compounds belonging to the family of capsaicinoids, which have been exploited for medicinal, nutritional, agricultural, and cosmetic uses. The demand for capsaicinoid molecules is increasing day by day due to their high economic value and wide range of applications. Therefore, increasing bioactive metabolites, especially capsaicinoids in chilli peppers, is a major priority in the current scenario. Multi-omics approaches such as genomics, transcriptomics, proteomics, and metabolomics have substantially contributed to understanding the complex regulatory networks governing capsaicinoid biosynthesis. Key structural genes, transcription factors, and signaling pathways involved in the phenylpropanoid and branched-chain fatty acid pathways have been identified, providing valuable targets for metabolic engineering in chilli pepper. Despite these advances, the integration of genetic modification approaches for the targeted enhancement of capsaicinoid production remains limited in chilli pepper. Recent developments in biotechnology, particularly CRISPR/Cas-mediated genome-editing, enable the precise genetic modification of metabolic pathways and regulatory networks in plants. Therefore, it can contribute to the precise modification of key genes involved in the capsaicinoid biosynthesis pathway, offering potential strategies to enhance the capsaicinoid content in chilli pepper. However, CRISPR/Cas-mediated genome editing in chilli pepper is still in its early stages. There are currently no reports available on the successful enhancement of capsaicinoid content in chilli peppers through CRISPR/Cas-mediated genome editing. To date, no comprehensive review has evaluated the CRISPR-Cas-mediated genome-editing approaches for capsaicinoid metabolic engineering in chilli pepper. This review critically evaluates the recent advances in CRISPR/Cas-mediated metabolic engineering in chilli peppers, with particular emphasis on regulatory genes involved in capsaicinoid biosynthesis. Furthermore, multi-omics approaches are expected to complement these strategies by enabling the identification of key regulatory genes, the optimization of genome-editing targets, and the prediction of metabolic outcomes for enhanced capsaicinoid production. Overall, this review provides insights into improving capsaicinoid accumulation in chilli peppers through advanced genome-editing technologies.},
}
@article {pmid42497250,
year = {2026},
author = {Zhou, Z and Yang, Y and Zhou, F and Liang, K and Gong, T and Zhou, X and Li, J and Luo, J and Li, J and Yang, J},
title = {Energy-assisted CRISPR cleavage and probiotic vesicle signaling platform: Microbiome reprogramming for homeostasis.},
journal = {Science advances},
volume = {12},
number = {30},
pages = {eaef1760},
pmid = {42497250},
issn = {2375-2548},
mesh = {*Probiotics ; *Homeostasis ; *Microbiota/genetics ; *Streptococcus mutans/genetics/metabolism ; Adenosine Triphosphate/metabolism ; *CRISPR-Cas Systems ; Quorum Sensing ; *Extracellular Vesicles/metabolism ; Humans ; Signal Transduction ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {Pathogenic infections drive microbial dysbiosis and persistent inflammation, posing therapeutic challenges due to difficulties in precise pathogen eradication and microbiome restoration. Although CRISPR-based therapeutics enable pathogen-specific antibacterial targeting, their effectiveness in treating pathogenic infections is constrained by difficulties in navigating complex microbial ecosystems, penetrating pathogenic barriers, sustaining energy-intensive intracellular cleavage, and, critically, restoring microbial balance after pathogen clearance. Here, we engineer a probiotic vesicle-synergized CRISPR platform by encapsulating gtfB-targeting CRISPR plasmids within hybrid extracellular vesicles from probiotics and pathogenic Streptococcus mutans. The pathogen-derived vesicle component enables targeted uptake by S. mutans, facilitating intracellular cleavage of the virulence gene gtfB. Vesicle-carried endogenous adenosine triphosphate (ATP) boosts CRISPR activity, amplifying targeted DNA cleavage for potent and selective pathogen elimination. Probiotic-derived vesicle components further remodel quorum-sensing networks and immunity, restoring microbial homeostasis. This probiotic vesicle-based strategy integrates ATP-enhanced CRISPR cleavage with microbiome and immune modulation, offering a next-generation therapeutic paradigm for microbiome-associated diseases.},
}
@article {pmid42497869,
year = {2026},
author = {Terkelsen, T and Yumiceba, V and Kim, J and Melo, US and Axelgaard, E and Febbraro, F and Gunnarsson, AV and Balachandran, S and Dahl-Jessen, M and Christensen, R and Peters, BA and Asan, and Thelle, T and Nyegaard, M and Bak, RO and Denham, M and Spielmann, M and Jensen, UB},
title = {Position effect at the SOX3 locus by an interchromosomal insertion causes hereditary spastic paraplegia.},
journal = {American journal of human genetics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ajhg.2026.07.001},
pmid = {42497869},
issn = {1537-6605},
abstract = {Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.},
}
@article {pmid42500818,
year = {2026},
author = {Li, A and Zhang, X and Li, S and Wang, Y and Xu, C and Lv, C and Zhao, M and Liu, Y and Ding, M and Cao, C},
title = {ETTAS: a modular aptamer-recruited platform for programmable translational activation.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42500818},
issn = {1362-4962},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; A2503021//Shenzhen Medical Research Fund/ ; A2303071//Shenzhen Medical Research Fund/ ; 82403183//National Natural Science Foundation of China/ ; 82303113//National Natural Science Foundation of China/ ; 82360603//National Natural Science Foundation of China/ ; 82560157//National Natural Science Foundation of China/ ; 82300871//National Natural Science Foundation of China/ ; 82474383//National Natural Science Foundation of China/ ; JCYJ20250604180714019//Shenzhen Science and Technology Program/ ; RCJC20221008092723011//Shenzhen Science and Technology Program/ ; JCYJ20240813140522029//Shenzhen Science and Technology Program/ ; RCBS20231211090747077//Shenzhen Science and Technology Outstanding Innovative Talent Training/ ; },
mesh = {Humans ; *Aptamers, Nucleotide/genetics/metabolism ; *Protein Biosynthesis/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; *CRISPR-Cas Systems ; PTEN Phosphohydrolase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; },
abstract = {Precise enhancement of endogenous protein synthesis offers a reversible therapeutic strategy without permanent genomic modification. However, existing Cas13-mediated translational activation systems are limited by modest potency and restricted modular expandability. Here, we developed the Enhanced Targeted Translational Activation System (ETTAS), a modular RNA-guided platform that combines dCas13a, the SINEB2 translational activation element, and an independently recruitable aptamer-mediated auxiliary module. Systematic ortholog screening identified dCas13a as the most effective scaffold for SINEB2-mediated translational activation, whereas direct tandem duplication of SINEB2 elements impaired rather than enhanced activity. To overcome this architectural limitation, we used aptamer-mediated recruitment to spatially separate target recognition from auxiliary activation. A binding-validated, non-interfering dCas13a-binding aptamer enabled construction of a dual-module system in which an aptamer-recruited SINEB2 element enhanced translation without altering target mRNA abundance or stability. Compared with the previously reported dCasRx-SINEB2 system, ETTAS produced stronger reporter activation, stronger endogenous induction of P53 and PTEN, and greater antiproliferative and pro-apoptotic effects in bladder cancer cells. Proteomic analyses showed selective target protein upregulation with limited global perturbation. In vivo, dual-AAV delivery of ETTAS activated endogenous P53 and suppressed tumor growth. ETTAS establishes a programmable framework for modular post-transcriptional upregulation of endogenous proteins.},
}
@article {pmid42500819,
year = {2026},
author = {Wang, W and Chen, Y and Li, Z and Zhang, L and Gui, K and Wu, Y and Yin, N and Han, X and Zhang, Y and Lu, R and Zhang, Z and Wang, L and Xie, G},
title = {A structural accessibility principle for LbuCas13a activation by noncontiguous DNA.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42500819},
issn = {1362-4962},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; CSTB2024NSCQ-MSX0521//Natural Science Foundation of Chongqing/ ; CSTB2024NSCQ-MSX1223//Natural Science Foundation of Chongqing/ ; 2024M763898//China Postdoctoral Science Foundation/ ; 2024CQBSHTB3005//Chongqing Postdoctoral Special Funding Project/ ; },
mesh = {*DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; Molecular Dynamics Simulation ; },
abstract = {CRISPR-Cas13a is mainly known as an RNA-guided RNA endonuclease. Recent studies show that Leptotrichia buccalis Cas13a (LbuCas13a) can interact with DNA substrates too, without PAM or PFS constraints, but current understanding of DNA-mediated activation is largely based on continuous target strands. Here, we define a structural accessibility principle for LbuCas13a activation by noncontiguous DNA. We show that activation occurs only when overhang positioning creates an accessible protein-DNA interface. Outer overhangs near the crRNA repeat-adjacent side restore strong trans-cleavage activity by stabilizing key LbuCas13a-DNA contacts, whereas distal outer overhangs support only weak activation. In contrast, inner overhangs cause steric mismatch, destabilize the complex, and block formation of an active conformation. Molecular modeling and molecular dynamics simulations support this structure-dependent rule. Noncontiguous DNA also broadens the single-nucleotide discrimination window of LbuCas13a and enables accurate IDH1 R132H detection in glioma tissues. We further develop a one-step APE1-activated CRISPR-LbuCas13a reaction (ACROSS) for sensitive APE1 detection. Because activated LbuCas13a cleaves RNA reporters but not DNA-triggering products, ACROSS preserves the activating structure and supports stable signaling in vitro, in live cells, and in breast cancer serum samples.},
}
@article {pmid42501080,
year = {2026},
author = {Gençoğlu, HS and Aydemir, E and Ayaz, F},
title = {Bacterial immune systems.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {8},
pages = {},
pmid = {42501080},
issn = {1572-9699},
mesh = {*Bacteria/immunology/virology/genetics ; Bacteriophages/immunology/physiology ; CRISPR-Cas Systems ; Immunity, Innate ; Adaptive Immunity ; },
abstract = {Bacterial immune systems encompass the multi-layered defense mechanisms that bacteria develop against bacteriophages and mobile genetic elements, such as plasmids. This review covers bacterial innate defense systems (surface defenses, superinfection exclusion, restriction-modification, abortive infection, and toxin-antitoxin systems), CRISPR-Cas-mediated adaptive immunity, and the escape strategies used by phages to overcome these defenses (genome modifications, anti-restriction proteins, and anti-CRISPR factors). Emerging evidence also highlights the role of outer membrane vesicles (OMVs) in anti-phage defense and their translational potential as vaccine and delivery platforms. In this context, a better understanding of bacterial defense systems contributes to the development of biotechnology and medical applications such as CRISPR technologies, diagnostic approaches, and phage therapy.},
}
@article {pmid42503339,
year = {2026},
author = {Yuan, YL and Dai, JL and Xiao, L and Jiang, JG},
title = {Advances in engineering microalgae for heterologous terpenoid synthesis: A review.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108993},
doi = {10.1016/j.biotechadv.2026.108993},
pmid = {42503339},
issn = {1873-1899},
abstract = {Terpenoids are a class of natural products widely distributed in living organisms, with isoprene as their fundamental structural unit. However, traditional plant extraction and chemical synthesis methods are often limited by low product purity, difficult separation, and complex synthetic steps, making it challenging to meet the demands of large-scale production. Conventional hosts such as Escherichia coli and Saccharomyces cerevisiae are utilized for terpenoid synthesis due to their advantages of short growth cycles and controllable cultivation conditions. Nevertheless, the complexity of terpenoid biosynthetic pathways poses significant challenges for these hosts in producing structurally complex terpenoids. In contrast, microalgae as photosynthetic microorganisms, possess well-developed endogenous terpenoid metabolic pathways, abundant precursor pools, and subcellular structures and regulatory mechanisms similar to those of plants, demonstrating significant advantages in the heterologous production of complex terpenoids. This review systematically summarizes recent advances in the production of heterologously synthesized terpenoids in eukaryotic microalgae, ranging from monoterpenes to triterpenes, and provides an in-depth analysis of key engineering strategies, including MEP/MVA pathway regulation, gene expression optimization, subcellular compartmentalization, and cultivation process intensification. In addition, the application potential of advanced tools such as CRISPR/Cas, microalgae-microorganism co-culture, and artificial intelligence is introduced. Finally, the major bottlenecks faced by microalgae as a sustainable green cell factory for terpenoid production are briefly analyzed, and future research directions are proposed.},
}
@article {pmid42503939,
year = {2026},
author = {Yang, XP and Gao, YQ and Huang, L and Zhao, LJ and Xiao, Y and Li, L and Zhang, TS},
title = {[Advances in research on the molecular mechanisms and gene therapy of hereditary hearing impairment].},
journal = {Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]},
volume = {60},
number = {7},
pages = {1138-1149},
doi = {10.3760/cma.j.cn112150-20251230-01243},
pmid = {42503939},
issn = {0253-9624},
support = {82560219//National Natural Science Foundation of China/ ; 2025S129//Kunming Medical University 2025 Master's Degree Education Innovation Fund Project/ ; },
mesh = {Humans ; *Genetic Therapy ; Connexin 26 ; *Hearing Loss/genetics/therapy ; Animals ; Connexins ; },
abstract = {Hereditary hearing impairment represents a significant etiology of language and social dysfunction in both children and adults, primarily caused by genetic factors. To date, over 150 genes have been identified in association with this disorder. The pathogenic mechanisms involve multiple molecular levels, including abnormalities in hair cell cytoskeleton and stereociliary structure, dysfunction of intercellular gap junctions (e.g., GJB2, GJB6), dysregulation of ion channels and transporters (e.g., SLC26A4, KCNQ4), alterations in extracellular matrix composition, and disruption of intracellular signaling pathways. In recent years, research has expanded to investigate the role of the inner ear immune microenvironment in this condition, with emerging evidence suggesting that immune dysregulation may contribute to disease initiation and progression. Therapeutically, novel strategies such as adeno-associated virus (AAV)-based gene replacement therapy, CRISPR/Cas-mediated gene editing systems, and lipid nanoparticle (LNP)-delivered mRNA therapeutics have demonstrated partial restoration of auditory function in animal models of hereditary hearing impairment involving genes such as TMC1, OTOF, and GJB2, with some approaches having advanced to clinical trial stages. This article systematically summarizes recent advances in the molecular mechanisms, immune microenvironment involvement, and gene therapy strategies for hereditary hearing impairment, delineates the research trajectory from gene discovery and mechanistic elucidation to therapeutic development, and discusses future translational research directions and clinical challenges.},
}
@article {pmid42507199,
year = {2026},
author = {Bhadra, M and Sachan, M and Nara, S},
title = {Recent advances in nanozyme assisted miRNA biosensing for disease diagnosis.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42507199},
issn = {1436-5073},
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; Humans ; *Nanostructures/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; },
abstract = {MiRNAs have emerged as key biomarkers for early disease detection and therapeutic monitoring; yet, their ultrasensitive and specific detection confronts challenges due to low expression levels in biological fluids and high sequence homology, therefore it demands innovative sensing strategies. Enzyme mimicking nanostructures or nanozymes, enable manifold signal amplification and ultrasensitive analyte detection. In recent years, nanozymes, mainly oxidases and peroxidases, have been used as signal amplifiers in miRNA sensors due to their detectable catalytic products at low concentrations. Considering the increasing use of nanozymes in miRNA detection, this dedicated review presents the latest advancements in the field of miRNA diagnostics and unravels the contributions of nanozymes in miRNA sensing. This work highlights two major approaches commonly used for miRNA detection in sensors; (i) assisted through nucleic acid amplification, and (ii) amplification-free approaches. It then comprehensively underpins nanozymes as signal amplifier in both aforementioned strategies through diverse sensing modalities such as colorimetry, electrochemical, SERS, and chemiluminescent. It further presents the enhancement of sensitivity through integration of nanozymes with latest technologies like machine learning based approaches, CRISPR-Cas, or towards designing point of care sensors. Lastly, the review explores the challenges in translating nanozyme-derived miRNA sensing platforms to clinical settings.},
}
@article {pmid42507484,
year = {2026},
author = {Liang, Y and Qi, X and Gao, S and Wang, Y and Kan, G and Valentovich, LN and Guo, J and An, Y},
title = {Multi-level precise regulation of gene transcription in the yeast Saccharomyces cerevisiae based on light-sensitive CRISPR/Cas systems.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42507484},
issn = {1362-4962},
support = {32571457//National Natural Science Foundation of China/ ; JYTYB2024044//Scientific Research Projects of Liaoning Provincial Department of Education/ ; LJKZ0660//Scientific Research Projects of Liaoning Provincial Department of Education/ ; //Liaoning Provincial Academic Leadership Support Program/ ; //Liaoning Provincial Enterprise Sci-Tech Specialist Assignment Program/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcription, Genetic ; *Gene Expression Regulation, Fungal ; Luminescent Proteins/genetics/metabolism ; Light ; Optogenetics/methods ; Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Red Fluorescent Protein ; },
abstract = {Regulation of gene transcription based on clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool for constructing synthetic gene circuits in Saccharomyces cerevisiae. The current CRISPR-based regulatory approaches primarily focus on inhibiting the binding of dCas9 protein to single guide RNA (sgRNA) or blocking target site recognition. However, these regulation strategies are often at a single level, and their sensitivity still needs to be improved. In this study, the gene regulatory approaches at the translational and post-translational levels were integrated with optogenetic control patterns to attain very sensitive multi-level precision regulation of the dCas9 protein, thereby facilitating flexible regulation of transcription levels of target genes. This strategy was used to regulate the transcription levels of fluorescent proteins, resulting in up to 2.58-fold increase in the fluorescence intensity of mCherry compared to that without regulation. This CRISPR-based multi-level optogenetic system should be extremely helpful in understanding gene regulatory networks and in designing robust genetic circuits for synthetic biology.},
}
@article {pmid42479319,
year = {2026},
author = {Eldemir, ME and Karaca, AN and Akçelik, N and Akçelik, M},
title = {CRISPR rewired: from adaptive immunity to a global virulence control network in Salmonella.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {8},
pages = {},
pmid = {42479319},
issn = {1573-0972},
mesh = {Virulence/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Bacterial ; *Salmonella enterica/genetics/pathogenicity/immunology ; *Adaptive Immunity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; Genomic Islands ; Biofilms/growth &amp; development ; },
abstract = {The Type I-E CRISPR/Cas system in Salmonella enterica is increasingly hypothesized to function as a condition-dependent regulatory interface rather than exclusively as an adaptive immune module. Rooted in its evolutionary origins within mobile genetic elements such as casposons, this system reflects a functional transition toward influencing bacterial pathogenesis. A central hypothesis suggests that CRISPR components-specifically Cascade, Cas3, and Cas6-are integrated into core regulatory networks governing pathogenicity islands, biofilm formation, and oxidative stress adaptation. This regulatory control likely operates through a programmed deviation from the traditional immunity paradigm, where suboptimal PAM recognition or partial sequence complementarity allows Cascade to bind DNA without licensing Cas3 for lethal cleavage. Consequently, the machinery may facilitate transcriptional modulation through steric hindrance, acting as a natural CRISPR interference mechanism. Coordinated by global regulators like H-NS and LeuO in response to environmental cues such as pH fluctuations, this system effectively functions as a molecular rheostat. Collectively, these hypotheses offer a conceptual framework for novel translational strategies, including anti-CRISPR-based therapeutics and engineered evolutionary trap concepts.},
}
@article {pmid42482458,
year = {2026},
author = {Hao, J and Xie, J and Ma, K and Li, X and Chen, X and Bao, G and Hu, J and Li, G},
title = {A novel acinetobacter phage reveals altered virulence traits in phage-resistant strains.},
journal = {Virulence},
volume = {},
number = {},
pages = {2707728},
doi = {10.1080/21505594.2026.2707728},
pmid = {42482458},
issn = {2150-5608},
abstract = {Phage therapy represents a promising alternative for combating bacterial infections. This study employed an A. baumannii isolate harboring the I-F CRISPR-Cas system as a host to isolate phage and evaluate its biological characteristics. Phage-resistant mutants were screened using a double-layer agar plate assay, and the underlying molecular mechanisms were identified through whole-genome sequencing, followed by validation via gene knockout. Transcriptome sequencing was subsequently applied to alterations in the global regulatory networks of these mutants. Our results demonstrate the successful isolation of a novel myovirus, stable at 40-50 ℃, which was successfully isolated and found to utilize the capsule as its adsorption receptor. Whole-genomic analysis confirmed its taxonomic distinction from currently published phages. Investigation into the primary resistance mechanism revealed that the capsule loss, due to an insertional mutation in the UDP-glucose 4-epimerase encoding gene galE. This conclusion was further validated through targeted gene knockout of galE. This defect concurrently attenuated bacterial virulence, as demonstrated by significantly reduced lethality in the Galleria mellonella infection model and enhanced susceptibility to serum killing, while concurrently enhancing the capacity for biofilm formation. Transcriptomic profiling indicated that the ΔgalE significantly upregulated multiple biofilm-associated genes and remodeled the transcriptomic-wide regulatory. Furthermore, the combination of carbenicillin or ceftazidime with the phage exhibited a synergistic effect in vitro, effectively inhibiting biofilm formation and suppressing the emergence of phage resistance. Overall, this work characterizes a novel phage and delineates the host's biological network changes triggered by phage resistance, offering valuable insights for developing phage-based antimicrobial strategies.},
}
@article {pmid42482984,
year = {2026},
author = {Ma, C and Zhang, J and Jiang, Y and Li, X},
title = {Hybridization chain reaction-assisted CRISPR/Cas12a strategy for rapid and visual detection of Haemophilus influenzae.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1844708},
pmid = {42482984},
issn = {2235-2988},
mesh = {*Haemophilus influenzae/genetics/isolation &amp; purification ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Haemophilus Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; Colorimetry/methods ; *Nucleic Acid Hybridization/methods ; *Molecular Diagnostic Techniques/methods ; Benzidines ; },
abstract = {Haemophilus influenzae (H. influenzae) is a major pathogen causing community-acquired pneumonia in children, posing a serious threat to children's health. Rapid and convenient testing is needed for effective treatment. Traditional detection methods, such as bacterial culture and qPCR are cumbersome to operate, and require sophisticated instrumentation. Here, we developed a visual CRISPR/Cas detection platform that integrates the hybridization chain reaction (HCR) and horseradish peroxidase (HRP)-catalyzed 3,3',5,5'-Tetramethylbenzidine (TMB) colorimetric change, called Vi-CasHCP. The single-base recognition capability of CRISPR/Cas12a improves the specificity, while the efficiency of HCR shortens the detection time and improves the sensitivity. The peroxidase-like activity of HRP catalyzes the oxidation of TMB to oxTMB, resulting in a blue color change. Vi-CasHCP achieved a detection limit of 11.8 CFU/mL in 70 min, after Recombinase polymerase amplification. Clinical validation using 50 respiratory samples showed complete diagnostic agreement with qPCR, with 100% sensitivity, specificity, PPV, and NPV. Therefore, this platform offers rapid detection, requires no complex instruments, and has high sensitivity, providing fast and accurate diagnostic support for clinical practice, and is particularly suitable for resource-limited settings.},
}
@article {pmid42484920,
year = {2026},
author = {Dhall, RK and Rana, N and Kaur, G and Mandyal, SS},
title = {Genomics assisted breeding for mildew resistance in cucumber: from gene discovery to future innovations.},
journal = {Plant molecular biology},
volume = {116},
number = {4},
pages = {},
pmid = {42484920},
issn = {1573-5028},
support = {BT/Ag/CoE/PAU-GSKIG/2020-21//Ministry of Science and Technology, Department of Biotechnology, Government of India/ ; },
mesh = {*Disease Resistance/genetics ; *Cucumis sativus/genetics/microbiology ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding/methods ; *Genomics/methods ; Quantitative Trait Loci/genetics ; Genome, Plant ; Ascomycota ; Chromosome Mapping ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; },
abstract = {Cucumber is an economically important vegetable crop cultivated worldwide, but its productivity is severely affected by destructive foliar diseases particularly powdery mildew and downy mildew. These pathogens cause significant yield and quality losses and the continuous emergence of new races makes disease management increasingly challenging. Conventional approaches including cultural, biological and chemical control often provide limited and short-term effectiveness. Therefore, the development of host plant resistance remains the most sustainable and environmentally sound strategy for long-term disease control. Recent advances in cucumber genomics and molecular breeding have enabled the identification of resistance-associated loci through SNP genotyping, QTL mapping, genome-wide association studies and marker-assisted selection. Furthermore, multi-omics approaches such as transcriptomics, proteomics and metabolomics combined with innovative technologies like CRISPR/Cas-mediated genome editing, genomic selection and speed breeding are transforming resistance breeding. Therefore, by integrating advanced molecular tools with omics-driven insights, this review aims to accelerate genetic gains and facilitate the development of durable, broad-spectrum mildew-resistant cucumber cultivars for sustainable and resilient production systems.},
}
@article {pmid42485704,
year = {2026},
author = {Chen, J and Zheng, H and Guan, L and Kanaherarachchi, A and Munusamy, S and Kong, J and Zhou, S and Jahani, R and Guan, X},
title = {Chemical additives-enhanced CRISPR/Cas12a-based RNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {312},
number = {},
pages = {119048},
doi = {10.1016/j.bios.2026.119048},
pmid = {42485704},
issn = {1873-4235},
abstract = {The CRISPR/Cas12a system has revolutionized molecular diagnostics due to its RNA-guided trans-cleavage activity, enabling programmable and highly accurate nucleic acid detection. However, most Cas12a-based assays are optimized for DNA targets, while direct RNA detection constrained by limited sensitivity, typically at the nanomolar level. Existing strategies to improve the performance of RNA analysis often rely on additional DNA activators or complex auxiliary systems. Here, we report a simple yet effective chemical additive-based strategy that overcomes these limitations. This chemical additives-enhanced CRISPR/Cas12a-based RNA detection (CARD) enables femtomolar-level RNA detection using only a single crRNA, without the need for DNA activators, reverse transcription, or strand-displacement reactions. Notably, this approach can be adapted to single-stranded DNA, enabling ssDNA detection at attomolar levels. Collectively, CARD provides a straightforward, amplification-free, and highly sensitive diagnostic framework that might be readily extended to other CRISPR/Cas systems for ultrasensitive nucleic acid diagnostics.},
}
@article {pmid42486091,
year = {2026},
author = {Regmi, M and Ma, K and Bi, C and Zhang, X and Zhao, D and Yu, L and Yang, H and Wang, N and Yang, C},
title = {Base editing for precision therapeutics.},
journal = {Cell genomics},
volume = {},
number = {},
pages = {101298},
doi = {10.1016/j.xgen.2026.101298},
pmid = {42486091},
issn = {2666-979X},
abstract = {Base editing (BE), the precise installation of single-nucleotide changes in DNA or RNA without inducing double-strand breaks, holds substantial therapeutic promise for correcting single-nucleotide variants, which constitute more than half of the known pathogenic genetic variants. Recent advances have improved base editor specificity, efficiency, and delivery, enabling clinically oriented procedures. Clinically, BE has shown early success or strong translational promise in sickle cell disease, β-thalassemia, leukemia (via CAR T and epitope engineering), hypercholesterolemia (PCSK9 and ANGPTL3), alpha-1-antitrypsin deficiency, and glycogen storage disease type Ia. Key remaining challenges include bystander editing within the activity window, residual off-target DNA and RNA editing, delivery constraints (payload size, tissue targeting, and redosing limits), immunogenicity, and the need for durable long-term safety evidence across relevant cell types and disease contexts. Continued technological refinements, careful preclinical validation, and rigorous clinical assessment will be essential to fully realize BE's transformative potential in precision medicine.},
}
@article {pmid42486099,
year = {2026},
author = {Savulescu, J and Porsdam Mann, S and Gyngell, C and Schaefer, GO},
title = {Ethics of gene therapy.},
journal = {Med (New York, N.Y.)},
volume = {},
number = {},
pages = {101225},
doi = {10.1016/j.medj.2026.101225},
pmid = {42486099},
issn = {2666-6340},
abstract = {CRISPR-Cas systems, base editing, and prime editing have made precise genetic interventions possible, and several approved therapies now treat monogenic disorders that were previously untreatable. Heritable genome editing remains ethically contested. We argue that heritable interventions should not be treated as a single category subject to uniform prohibition. We distinguish three targets: catastrophic monogenic disorders, polygenic risk reduction, and non-disease trait enhancement. For catastrophic monogenic conditions in which preimplantation selection cannot yield unaffected embryos, heritable editing is permissible, and the duty of beneficence toward future persons may require it. When the alternative is certain severe suffering or early death, the expected benefits clearly outweigh the risks. For polygenic interventions, current scientific uncertainty makes clinical application premature: predictive validity remains insufficient and pleiotropic effects are poorly understood. For enhancement, the case is weaker still. Some of its benefits are positional; the risks of social stratification are significant; and the evidence base is absent. We conclude that governance frameworks should permit what the evidence supports under stringent safeguards and prohibit what it does not. The central ethical questions concern welfare, not appeals to nature or abstract notions of dignity. Where the evidence warrants it, failing to pursue heritable gene therapy responsibly may itself be an ethical failure. We outline a translational pathway for ethical germline gene editing.},
}
@article {pmid42486981,
year = {2026},
author = {Shu, X and Wang, R and Zhou, X and Cheng, F and Ma, J and Li, Z and Li, X and Wu, T and Wu, A and Xue, Q and Liu, C and Zhao, H and Cao, X and Wang, L and Zhang, S and Zhang, Y and Li, M},
title = {CRISPR-Cas regulates expression of embedded anti-phage defence systems.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {42486981},
issn = {1476-4687},
abstract = {Bacteria utilize diverse defence systems to protect against harmful foreign DNA such as bacteriophages[1,2], but how these systems coordinate with each other remains poorly understood. Here we uncover CRISIS (CRISPR-supervised immune system), a widespread regulatory paradigm whereby type I CRISPR-Cas loci embed and transcriptionally modulate diverse innate defences. Small non-canonical CRISPR RNA (crRNA)-like RNAs guide the I-C CRISPR-associated complex for antiviral defence (Cascade) effector complex to inhibit promoters of diverse immune cassettes-including composite multi-system clusters-enabling their basal expression for antiviral activity while mitigating fitness costs associated with hyperactivation, such as host growth impairment or exclusion of beneficial plasmids. When CRISPR-Cas is compromised by mutation or anti-CRISPR proteins, there is a burst in transcription of these embedded defence systems, leading to higher-level innate immunity at the expense of host fitness. Together, adaptive CRISPR-Cas systems orchestrate diverse innate immune systems into a layered defence network, comprising a prokaryotic 'immunity guard' strategy.},
}
@article {pmid42490001,
year = {2026},
author = {Feng, L and Fang, J and Xu, Y and Luo, L and Hong, B and Wang, J and Ma, Y},
title = {Functional Activity of the Lysis Protein E From Phage ID52: Dependence on SecB for Efficient Host Cell Lysis.},
journal = {Biotechnology journal},
volume = {21},
number = {7},
pages = {e70271},
pmid = {42490001},
issn = {1860-7314},
support = {XZ202601ZY0032//Science and Technology Projects of Xizang Autonomous Region, China/ ; Grant No. NERCGM-OF-20250301//Opening Foundation of National Engineering Research Center of Genetic Medicine, China/ ; 2022A1515010716//the Natural Science Foundation of Guangdong Province/ ; },
mesh = {*Bacteriophages/genetics/metabolism ; *Escherichia coli/virology/genetics/metabolism ; *Viral Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics ; *Bacteriolysis ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; *Escherichia coli Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Single gene encoded phage lysis proteins offer a promising strategy for bacterial ghost production, yet their host-dependent regulatory mechanisms remain poorly understood. Here, we investigated the lysis protein E from phage ID52 (ID52-E), which exhibits stronger lytic activity than φX174 E. By screening ID52-E-resistant mutants, we identified a four-base insertion in secB as the genetic alteration associated with lysis resistance in BL21, and CRISPR-Cas9-mediated secB disruption confirmed that SecB is required for ID52-E-mediated lysis. Proteomic analysis revealed altered protein expression in resistant mutants despite preserved bacterial morphology. Co-immunoprecipitation and biolayer interferometry supported an apparent interaction between SecB and ID52-E, with an apparent KD of 3.541 × 10[-] [8] M under the tested 1:1 fitting model. Molecular docking, molecular dynamics simulations, mutagenesis, lysis assays, and binding measurements further implicated SecB Ala145 as a key interface residue. Together, these findings identify SecB as a host factor that facilitates ID52-E-mediated bacterial lysis and provide mechanistic insight for improving bacterial ghost production.},
}
@article {pmid42490334,
year = {2026},
author = {Lv, Y and Kulsoom, and Jia, L and Wang, Z and Wang, F},
title = {Overcoming Liquid Biopsy Barriers: Nucleic Acid Biosensors Integrating DNA Nanotechnology and CRISPR-Cas System for Cancer Precision Theranostics.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01673},
pmid = {42490334},
issn = {2379-3694},
abstract = {Liquid biopsy holds immense potential for the early detection of cancer, yet its clinical utility is hindered not by the lack of available tumor-associated biomarkers but by the inadequate sensitivity and clinical robustness of current molecular diagnostic tools. Nucleic acid-based biosensors have emerged as highly programmable platforms, enabling the detection of low-abundance cancer biomarkers such as microRNAs (miRNAs), circulating tumor DNA (ctDNA), and messenger RNAs (mRNAs) in complex biological fluids. Leveraging advances in DNA nanotechnology, CRISPR-Cas-mediated RNA sensing, and chemically engineered nucleic acid analogues, these biosensors achieve attomolar-level detection through nanoscale spatial confinement and enzyme-assisted signal amplification strategies. However, their clinical translation is hindered by biological sample variability, nonspecific amplification, probe degradation, and poor reproducibility. This review analyzes the core design principles of three major biosensor categories: functional DNA nanostructures, CRISPR-Cas-based sensing systems, and synthetic analogues (PNAs, SNAs). It elucidates their structural and enzymatic optimization mechanisms, distinguishes analytical from clinical sensitivity, and addresses key liquid biopsy challenges. Finally, it outlines promising strategies for clinical translation, including microfluidic integration, artificial intelligence-assisted data analysis, and theranostic nanostructures combining diagnosis with targeted therapy. This review provides a comprehensive theoretical and technical framework for the rational design of next-generation nucleic acid biosensors and offers critical insights to bridge the gap between nanoscale engineering innovation and clinical translation, ultimately advancing the development of minimally invasive and precise cancer theranostics in precision oncology.},
}
@article {pmid42491328,
year = {2026},
author = {Hussain, A and Mojgani, N and Siddiqui, MF and Khan, MI and Ali, SA},
title = {Exploring the emerging role of CRISPR-Cas systems in probiotic development.},
journal = {Engineering microbiology},
volume = {6},
number = {3},
pages = {100279},
pmid = {42491328},
issn = {2667-3703},
abstract = {Modification of the gut microbiota by beneficial microbes can enhance an organism's lifespan, giving rise to the concept of probiotics. Probiotics are live microorganisms that provide health benefits when taken in sufficient amounts. Owing to their outstanding health benefits, probiotics have experienced rapid expansion and gained interest for the development of new applications. The exploration of microbial applications via genetic modification is currently of great interest to researchers. Genetic engineering using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has received considerable attention and has established applications. Owing to these enhanced properties, the CRISPR-Cas system is currently used in medicine, agriculture, food, and biotechnology. Considering the adaptive immune system in bacteria, this genetic tool is used to alter the microbial genome. Lactic acid bacteria (LAB) are widely recognized for their probiotic potential, and over 40% of LAB species contain the CRISPR-Cas system. The rising demand for probiotics and their expanding applications necessitate the enhancement of their existing characteristics. The CRISPR-Cas system, recognized for its precision, accuracy, and speed, has enabled researchers to modify the genomes of probiotics, thereby enhancing their beneficial attributes. This system can enhance probiotic properties through additive, subtractive, or modulatory mechanisms. Various approaches have been developed to improve probiotic functionalities using the CRISPR-Cas system, such as substituting slow promoters with efficient alternatives, eliminating undesirable components, boosting metabolism, and increasing tolerance levels. Furthermore, CRISPR-engineered probiotics have emerged as next-generation probiotics with enhanced properties and advanced applications across diverse fields, including the food, medicine, agriculture, and pharmaceutical sectors.},
}
@article {pmid42424384,
year = {2026},
author = {Källstig, E and Ruchti, E and Raman, M and Asadzadeh, J and Schneider, BL and McCabe, BD},
title = {Highly frequent undesired insertional mutagenesis during Drosophila genome editing.},
journal = {PLoS genetics},
volume = {22},
number = {7},
pages = {e1012192},
doi = {10.1371/journal.pgen.1012192},
pmid = {42424384},
issn = {1553-7404},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutagenesis, Insertional/genetics ; Genome, Insect ; Recombinational DNA Repair/genetics ; Genetic Vectors/genetics ; },
abstract = {CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing designs. To avoid these events, we have generated a novel HDR template vector that enables animals with these undesired insertions to be identified and excluded. Our results suggest that HDR based genome edited animals must be carefully screened for unwanted vector template genomic integration in order to avoid misleading interpretations of genome editing outcomes.},
}
@article {pmid42475178,
year = {2026},
author = {Farhangmehr, S and Braunschweig, U and Wu, M and Nabeel-Shah, S and Brown, KR and Fine, JL and Moffat, J and Blencowe, BJ},
title = {A genome-wide functional analysis of conserved intronic regions reveals essential roles for speckle-associated retained introns.},
journal = {Cell reports},
volume = {45},
number = {7},
pages = {117696},
doi = {10.1016/j.celrep.2026.117696},
pmid = {42475178},
issn = {2211-1247},
abstract = {Hundreds of human introns harbor extended regions of high evolutionary conservation that have not been previously characterized. A survey of these sequences reveals that they are associated with intron retention and enriched in genes that function in RNA processing, chromatin remodeling and neuronal biology. Using a dual CRISPR-Cas editing approach, we targeted 2,600 of these regions for deletion and observed that a subset of these perturbations affects cell growth. Many of these "fitness" sequences affect intron retention and expression levels of their host genes. Deletions in nuclear speckle-associated retained introns in the FNBP4 and DDX5 genes further cause downstream effects on cell growth-related genes and intron retention, respectively. The intronic deletion in DDX5 additionally results in the accumulation of R-loops overlapping retained introns of speckle-proximal genes. Overall, the results highlight critical and multifaceted roles of highly conserved intronic sequences in the control of gene regulation, R-loop resolution, and cell growth.},
}
@article {pmid42477308,
year = {2026},
author = {Sikandar Zaman, M and Azeem, A and Nouman, A and Khalid, A and Ghafoor, S and Zia Ul Haq, M and Aslam, MT and El-Beltagi, HS},
title = {CRISPR-Cas systems for enhancing chilling tolerance in rice: recent advances and future prospects.},
journal = {Biologia futura},
volume = {},
number = {},
pages = {},
pmid = {42477308},
issn = {2676-8607},
abstract = {Rice (Oryza sativa L.) is an important staple crop in global food security and highly vulnerable to chilling stress, which greatly affects growth, development, and yield. The conventional breeding methods for enhancing chilling tolerance face numerous problems due to the polygenic nature of chilling tolerance and genetic complexities. The present review discusses the use of CRISPR-Cas genome editing technologies as an accurate and effective approach to increasing chilling tolerance in rice. We initially describe the physiological effects of chilling stress, such as membrane fluidity impairment, inhibition of photosynthesis, nutrient imbalance, and oxidative injury, and summarize major molecular pathways and genetic materials involved in chilling tolerance. The review then outlines the recent developments in CRISPR-Cas systems, including the modes of delivery (Agrobacterium-mediated transformation, protoplast transfection, and ribonucleoprotein techniques) and how they apply to rice genome editing. The precise examination of CRISPR-based functional genomics has shown that cold-responsive genes (OsMYB30, OsWRKY76, OsAnn3, OsPRP1, and OsKASI-2) are selectively manipulated, thus contributing to a clearer understanding of their functional roles in stress signaling, membrane stability, and antioxidant defense. Moreover, we also discuss recent CRISPR strategies, including multiplex editing, transcriptional reprogramming (CRISPRa/i), and omics-guided fine-tuning of gene networks. Synthesizing latest advancements, current review establishes a conceptual framework to overcome translational challenges in CRISPR-mediated improvement of complex traits in oilseed crops, through integrating the pivotal aspects of genotype-specific delivery, multi-gene network design, field validation, and the evolving regulatory landscape. The review concludes with a reflection of gaps in research and future opportunities, with a discussion on how integrated CRISPR technologies can be used to enhance the development of climate-resistant rice varieties.},
}
@article {pmid42477873,
year = {2026},
author = {Chen, Y and Gao, XH and Vichas, A and Wang, J and Golhar, R and Neuhaus, I},
title = {ALPINE: a scalable pipeline for comprehensive classification of gene-editing outcomes from long-read amplicon sequencing.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {7},
pages = {},
pmid = {42477873},
issn = {1367-4811},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *High-Throughput Nucleotide Sequencing/methods ; *Software ; Humans ; *Sequence Analysis, DNA/methods ; DNA Repair ; },
abstract = {SUMMARY: CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing supporting both PacBio HiFi and Oxford Nanopore platforms. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Dual-target benchmarking on simulated datasets demonstrated high accuracy for transgene integration events. Independent validation on public crosslinked-HDR dataset confirmed ALPINE's integration detection capabilities, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling.<br><br>AVAILABILITY: ALPINE is available under MIT license at https://github.com/Maggi-Chen/ALPINE and https://doi.org/10.5281/zenodo.20272510. All analysis scripts and visualization code used in this manuscript are available at https://github.com/Maggi-Chen/ALPINE-manuscript-analysis. Simulated datasets are deposited at Zenodo (https://doi.org/10.5281/zenodo.20260865). Public dataset PRJNA913199 is available through NCBI SRA.},
}
@article {pmid42478499,
year = {2026},
author = {Du, Y and Shi, Y and Chen, D and Wang, S and Su, J and Liu, K and Zhang, H and Wang, F},
title = {Gene Editing-Driven Engineering of Microbial Systems and Metabolites for Precision Medicine and Sustainable Environmental Remediation.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e74410},
doi = {10.1002/smll.74410},
pmid = {42478499},
issn = {1613-6829},
support = {BJ-2023-118//National High Level Hospital Clinical Research Funding and Fundamental Research Funds for the Central Universities/ ; T2322025//National Natural Science Foundation of China/ ; 82272161//National Natural Science Foundation of China/ ; 22125701//National Natural Science Foundation of China/ ; 52372274//National Natural Science Foundation of China/ ; 22388101//National Natural Science Foundation of China/ ; 2024YFA0919300//National Key R&amp;D Program of China/ ; 20240101175JC//Natural Science Foundation of Jilin Province, China/ ; XF012022C0200//Xiangfu Lab Research Project/ ; },
abstract = {The engineering of microorganisms is undergoing a fundamental paradigm shift, transitioning from the construction of static cell factories to the programming of dynamically responsive living materials. However, translating molecular interventions into robust macroscopic functions requires overcoming distinct microbial-specific barriers, including delivery bottlenecks and genetic stability. In this review, we establish a unified Edit-Reprogram-Functionalize conceptual framework that systematically delineates transient genetic regulation from permanent genomic engineering. We critically examine the evolutionary trajectories of five foundational technologies: plasmid engineering, CRISPR-Cas systems, base editors, prime editors, and enzyme engineering. Rather than analyzing these toolsets in isolation, we map their convergence into an integrated engineering continuum that drives the precise synthesis of two distinct output classes: engineered living microbial materials and robust microbial metabolite-derived materials. By evaluating representative breakthroughs-from ultrasound-actuated bacterial therapeutics to ultra-tough, biosynthesized protein composites-through the strict lens of host-dependent constraints, we reveal the mechanistic principles governing successful preclinical translation. Finally, we propose an actionable roadmap centered on systemic miniaturization, closed-loop control, and multi-scale integration, providing a definitive blueprint for the next generation of precision medicine, advanced biomanufacturing, and ecological remediation.},
}
@article {pmid42248880,
year = {2026},
author = {Wang, X and Jowsey, WJ and Cheung, CY and Dickerhof, N and Chapman, CL and Taka, JRH and Hampton, MB and Bashiri, G and Gardner, PP and Fineran, PC and Cook, GM and Jackson, SA and McNeil, MB},
title = {Genome scale CRISPRi reveals both shared and strain-specific vulnerabilities in genetically diverse drug-resistant strains of Mycobacterium tuberculosis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42248880},
issn = {2041-1723},
support = {20/459//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 22/323//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 22/156//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 23/228//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/metabolism/isolation &amp; purification ; Bacterial Proteins/genetics/metabolism ; DNA-Directed RNA Polymerases/genetics ; Mutation ; *Genome, Bacterial ; Rifampin/pharmacology ; *Drug Resistance, Bacterial/genetics ; Antitubercular Agents/pharmacology ; Humans ; Genotype ; Amino Acyl-tRNA Synthetases/genetics/metabolism ; CRISPR-Cas Systems ; Microbial Sensitivity Tests ; Sulfur/metabolism ; Drug Resistance, Multiple, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {The global health burden caused by Mycobacterium tuberculosis is aggravated by the emergence and spread of drug resistance. Mutations that cause drug resistance can have collateral effects that increase the vulnerability of downstream pathways to inhibition. Here, using genome scale CRISPR interference we identified collateral effects associated with different drug-resistant genotypes of M. tuberculosis. We demonstrate that drug resistance generated shared vulnerabilities in several overlapping functional pathways. Most drug-resistant strains were more sensitive to tRNA synthetase knockdowns than the parental drug-sensitive strain, highlighting the potential of tRNA synthetases as high-value drug targets. Additionally, the rifampicin-resistant mutant RpoB(S450L) had increased sensitivity to the dysregulation of sulphur metabolism due to transcriptional dysregulation. This increased vulnerability did not translate to all rpoB genotypes but was linked to predicted effects on transcriptional dynamics. Amongst clinical isolates, non-synonymous mutations in sulphur metabolism genes have evolved in a geographic lineage specific manner to mitigate fitness costs associated with the collateral phenotypes of drug resistance. Combined, our findings highlight the power of functional genomics in pinpointing highly vulnerable drug targets across drug-resistant strains.},
}
@article {pmid42270637,
year = {2026},
author = {Seidel, S and Zwaans, A and Regalado, S and Choi, J and Shendure, J and Stadler, T},
title = {SciPhy: A Bayesian phylogenetic framework using sequential genetic lineage tracing data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42270637},
issn = {2041-1723},
mesh = {Bayes Theorem ; *Phylogeny ; Animals ; *Cell Lineage/genetics ; Mice ; *Software ; Computer Simulation ; CRISPR-Cas Systems ; },
abstract = {CRISPR-based lineage tracing offers a promising avenue to decipher single-cell lineage trees, especially in organisms not amenable to microscopy. Sequential genome editing records not only genetic edits but also the order in which they occur. To leverage this enriched information, we introduce SciPhy, a simulation and inference tool implemented in BEAST 2. SciPhy utilizes a Bayesian phylogenetic approach to jointly estimate time-scaled phylogenies and cell population parameters. After validation on simulated data, we use simulated and real data from a monoclonal cell culture to benchmark SciPhy against existing methods and find that it consistently reconstructs more accurate phylogenies. Compared to UPGMA, SciPhy additionally reports uncertainty and proliferation rates. Our second example applies SciPhy to murine gastruloids, demonstrating its ability to model time-varying population dynamics in early development. Together, these results establish a phylodynamic framework for the quantitative analysis of lineage tracing data. SciPhy's codebase is publicly available at https://github.com/azwaans/SciPhy .},
}
@article {pmid42473674,
year = {2026},
author = {Ananda, H and Sahana, SR and Murthy, SR and Kunnath, AN and Prashant, A and Kaifi, JT and Kiran, PK and Suvilesh, KN},
title = {Genomic innovations in cancer prevention, diagnosis, prognosis and precision therapeutics.},
journal = {Frontiers in genetics},
volume = {17},
number = {},
pages = {1828450},
pmid = {42473674},
issn = {1664-8021},
abstract = {Cancer research has undergone a transformative change with the advent of high-throughput genomic technologies. Advances in next-generation sequencing accelerated the identification of somatic and germline alterations that drive tumorigenesis enabling the transition from traditional histology-based cancer classification to molecularly informed precision oncology. Large-scale sequencing initiatives and clinical genomic profiling facilitated the development of companion diagnostic assays and targeted therapies. Beyond targeted therapies, genomic innovations have also catalyzed the emergence of novel therapeutic strategies including immunogenomics-driven immunotherapies, RNA-based therapeutics, cancer vaccines and genome editing technologies based on CRISPR-Cas systems. This review summarizes the major technological developments in cancer genomics, including sequencing platforms, transcriptomic profiling, liquid biopsy, and functional genomic screening, and highlights the utility of these innovations in discovery of actionable biomarkers and next-generation therapeutic strategies. Collectively, these advances underscore the central role of genomic technologies in driving the evolution of precision oncology toward more personalized and effective cancer treatment strategies.},
}
@article {pmid42263439,
year = {2026},
author = {Guo, B and Li, Y and Shi, M and Zhang, J and Wu, Q and Wu, X and An, R and Wang, F},
title = {A triplex-readout CRISPR-Cas12a multimodal biosensing platform for point-of-care detection of avian influenza H5N1.},
journal = {Journal of hazardous materials},
volume = {514},
number = {},
pages = {142562},
doi = {10.1016/j.jhazmat.2026.142562},
pmid = {42263439},
issn = {1873-3336},
mesh = {*Influenza A Virus, H5N1 Subtype/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Point-of-Care Systems ; Animals ; *Influenza in Birds/diagnosis/virology ; Rapid Diagnostic Tests ; RNA, Viral/analysis ; Humans ; },
abstract = {The highly pathogenic avian influenza A (H5N1) virus poses a significant zoonotic threat to public and animal health. Conventional detection methods often face limitations in complexity, time, and equipment requirements. In this study, we report a rapid and lightweight-equipment triplex diagnostic platform for H5N1 detection, integrating locked nucleic acid (LNA)-assisted target recognition, toehold-mediated strand displacement for signal amplification, and the high specificity of the CRISPR-Cas12a system. The developed assay achieved a detection limit of 3.7 × 10[2] copies/μL for H5N1 pseudovirus RNA within a 60-minute core detection process, and exhibited excellent specificity without cross-reactivity to other influenza subtypes or coronaviruses. Moreover, it successfully identified H5N1 in clinical swab samples, yielding consistent results across three independent readout formats: fluorescence, lateral flow strip, and a portable glucose meter. With advantages of low cost, rapid operation, and multimodal verification capability, this diagnostic system is well suited for point-of-care screening in resource-limited settings and represents a promising tool for frontline outbreak response.},
}
@article {pmid42320852,
year = {2026},
author = {Tadokoro, T and Liu, N and Olson, EN},
title = {Precision modification of heart failure signaling by CRISPR-Cas9 base editing.},
journal = {Journal of molecular and cellular cardiology},
volume = {217},
number = {},
pages = {89-93},
pmid = {42320852},
issn = {1095-8584},
mesh = {Humans ; *Heart Failure/genetics/therapy/metabolism ; *Signal Transduction/genetics ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; },
abstract = {Heart failure remains a leading cause of morbidity and mortality worldwide, and current therapies largely focus on symptom management and slowing disease progression rather than correcting the underlying molecular abnormalities. Recent advances in genome editing technologies have created new opportunities to treat heart failure. Among these approaches, CRISPR-Cas9 base editing has emerged as a particularly promising strategy because it enables precise nucleotide conversions without introducing double-strand DNA breaks and demonstrates relatively high efficiency in vivo. While correction of disease-causing mutations by CRISPR-Cas9 base editing represents an important application of genome editing, an alternative strategy is to directly modulate key signaling pathways that drive cardiac dysfunction. Protein kinase C alpha (PKCα) functions as a key regulator of cardiac contractility and pathological remodeling. Precision editing of phosphorylation sites that control PKCα stability or activation may therefore represent an effective strategy to suppress maladaptive kinase signaling in cardiomyocytes. This concept of "precision signaling modification" may provide a broadly applicable therapeutic approach for heart failure. Similar strategies may also be applicable to other signaling molecules, including Ca[2+]/calmodulin-dependent protein kinase II delta (CaMKIIδ), and illustrate the broader potential of signaling-focused genome editing approaches. Despite these advances, several challenges remain for clinical translation, including efficient delivery of genome editing components to the adult heart, long-term safety, and potential immune responses. Continued advances in delivery technologies and genome editing platforms may ultimately enable durable, potentially one-time therapeutic interventions for heart failure.},
}
@article {pmid42470562,
year = {2026},
author = {Zhong, S and Leng, Y and Yang, S},
title = {Targeted Gene Editing in Wheat During Haploid Production via Wide Hybridization with Transgenic Maize Expressing Cas9 and Guide RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3029},
number = {},
pages = {313-329},
pmid = {42470562},
issn = {1940-6029},
mesh = {*Triticum/genetics ; *Zea mays/genetics ; *Gene Editing/methods ; *Haploidy ; Plants, Genetically Modified/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Hybridization, Genetic ; Genome, Plant ; },
abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system is an efficient and versatile genome engineering tool, which has been widely used for targeted mutagenesis and gene functional characterization in various organisms. This system is simple because it only requires a Cas9 enzyme serving as a nuclease and guide RNA (gRNA) containing a 20-nt sequence matching the target gene. Delivery of a vector expressing Cas9 and gRNA or the preassembled Cas9/gRNA complex as a ribonucleoprotein (RNP) into plant cells for gene targeting are usually via the biolistic- or Agrobacterium-mediated approach. However, most wheat genotypes suffer from low efficiency of callus induction and plant regeneration from explants receiving the vector or RNP delivered by the biolistic- or Agrobacterium-mediated transformation method, limiting the application of genome editing systems in many commercially grown wheat varieties. Here, we describe a stepwise protocol for targeted gene editing in wheat via wide hybridization with transgenic maize expressing Cas9 and gRNA. A binary vector expressing Cas9 and gRNA is constructed and used for Agrobacterium-mediated transformation to generate transgenic maize plants, which are used to pollinate emasculated spikes of wheat varieties. After fertilization, the maize chromosomes enter the transient hybrid zygote and the transgene (T-DNA) on a maize chromosome expresses the Cas9 enzyme and gRNA, which forms an RNP complex to edit the target gene in wheat genome. After several cell divisions, maize chromosomes in the hybrid zygote are eliminated, resulting in formation of haploid wheat embryos with the target gene edited, which can be rescued by embryo culture technique to produce haploid plants. Doubled haploid (DH) wheat plants with homozygous gene mutations are developed by chromosome doubling through colchicine treatment of the haploid plants. The wheat × maize hybridization combined with the CRISPR/Cas9 system provides a one-step approach for generating DH lines with the target gene edited from any wheat genotypes of interest.},
}
@article {pmid42470563,
year = {2026},
author = {Shi, C and Zhang, X and Zhao, Z and Sun, MX},
title = {In Vivo Rice Haploid-Induction System by an Egg Cell-Specific Peptidase Knockout.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3029},
number = {},
pages = {331-344},
pmid = {42470563},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Haploidy ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Peptide Hydrolases/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; *Plant Proteins/genetics ; },
abstract = {Doubled haploid (DH) technology is a fast and convenient approach for crop breeding and genetic research. Currently, in vitro anther culture is the main method for rice haploid production. However, genotype dependence remains a major problem in the anther culture of most rice subspecies or cultivars for haploid induction. In this chapter, we describe a protocol for in vivo haploid induction in rice using egg cell-specific peptidase (ECS) knockout lines, including mutation of ECS using CRISPR-Cas9 system, selection of homozygous Osecs mutants, and identification of haploids in the offspring of Osecs by flow cytometry. The ECS mediated maternal in vivo haploid-induction system is a convenient, time-saving and labor-saving technique to produce rice DH lines.},
}
@article {pmid42472629,
year = {2026},
author = {Kimura, K and Tsukamoto, M and Shishida, K and Sugisaki, H and Katahira, J and Tanaka, M and Kuwamura, M and Kol, A and Okada, M and Iijima, M and Nakanishi, M and Sugiura, K and Hatoya, S},
title = {Red blood cell differentiation using canine-induced pluripotent stem cells.},
journal = {Stem cells translational medicine},
volume = {15},
number = {8},
pages = {},
pmid = {42472629},
issn = {2157-6580},
support = {JP18H02349//JSPS KAKENHI/ ; 22J14623//JSPS KAKENHI/ ; 22H02525//JSPS KAKENHI/ ; 23K23790//JSPS KAKENHI/ ; 26K01900//JSPS KAKENHI/ ; },
mesh = {Animals ; Dogs ; *Cell Differentiation ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Erythrocytes/cytology/metabolism ; Glycophorins/metabolism/genetics ; CRISPR-Cas Systems ; Green Fluorescent Proteins/metabolism ; },
abstract = {BACKGROUND: Red blood cell (RBC) transfusions are essential for treating various medical conditions, but global demand is difficult to meet due to a dwindling donor pool and compatibility issues. Pluripotent stem cells (PSCs) offer a promising alternative of blood dependent on volunteer donors for RBC production, and dogs serve as an excellent model for translational research due to their physiological and genetic similarities to humans.<br><br>METHODS: Canine induced pluripotent stem cells (ciPSCs) were differentiated toward hematopoietic and erythroid lineages. Differentiated cells were evaluated for hematopoietic marker expression, hemoglobinization, colony-forming capacity, enucleation, and hemoglobin gene expression. Glycophorin A (GYPA)-enhanced green fluorescent protein (EGFP) reporter ciPSC lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing to visualize GYPA expression during differentiation.<br><br>RESULTS: This study introduces a protocol for RBC differentiation using ciPSCs. We achieved generation of hemoglobinized RBCs, progressing through polychromatic and orthochromatic erythroblast-like stages. CiPSC-derived hematopoietic cells/RBCs were confirmed to have immature characteristics as determined by limited colony-forming capacities, low enucleation, and embryonic and fetal hemoglobin gene expression. Additionally, we created GYPA-EGFP reporter ciPSC lines using CRISPR-Cas9-mediated genome editing, enabling real-time visualization of GYPA expression. This innovation confirmed GYPA as a viable surface marker for ciPSC-derived RBCs.<br><br>CONCLUSION: Our findings mark an initial step toward establishing a canine PSC-based erythroid differentiation system, providing a foundation for future improvements and exploration of applications for canine PSC-derived RBCs.},
}
@article {pmid42464358,
year = {2026},
author = {Farooq, A and Rafique, A and Han, E and Park, SM and Kim, HS and Kim, MJ and Hussain, A and Sheeraz Ahmad, M and LaPointe, G},
title = {Interactions of antiphage defense systems in the ESKAPE pathogen plasmids.},
journal = {Mobile DNA},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13100-026-00410-2},
pmid = {42464358},
issn = {1759-8753},
support = {ALLRP 566176-2021//Natural Sciences and Engineering Research Council of Canada/ ; },
abstract = {BACKGROUND: The global rise of multidrug resistant (MDR) ESKAPE pathogens represents a serious threat to antimicrobial therapy. While phage therapy has re-emerged as a promising alternative, its effectiveness may be compromised by bacterial defense systems, particularly those encoded on plasmids. Comprehensive surveillance of the distribution, diversity, and mobilome context of plasmid-encoded defense systems in ESKAPE pathogens remains key to the design of effective phage therapies.<br><br>RESULTS: We analyzed 7,330 dereplicated plasmids from ESKAPE pathogens to characterize the prevalence, diversity, and co-occurrence of plasmid-encoded antiphage defense systems. Conjugative plasmids, especially from Enterobacter spp. and K. pneumoniae, harbored the highest prevalence and diversity of defense systems. Defense-positive plasmids showed larger sizes, higher GC content, and frequent co-occurrence of resistance genes, especially from &#x3b2;-lactam, aminoglycoside, and sulfonamide classes, along with transposable elements such as IS6, IS3, and Tn3. Random forest and correlation analyses confirmed TEs and ARGs as dominant predictors of defense system occurrence. Network analysis revealed structured and partially conserved interactions among defense genes, TEs, and ARGs. RM and CBASS systems were frequently linked to beta-lactam and aminoglycoside resistance genes, as well as TEs such as IS6 and IS3. Recurrent associations such as RM-IS6, RM-IS1380, CBASS-IS3 and RM-OXA suggest shared horizontal transfer mechanisms.<br><br>CONCLUSIONS: Plasmid-encoded antiphage defense systems in ESKAPE pathogens are widespread, structured, and linked to ARGs and mobile genetic elements. These findings highlight the contribution of plasmids to the dissemination of phage-resistance traits, underscore the importance of the mobilome in shaping phage-resistance landscapes in multidrug-resistant pathogens, and support the incorporation of plasmid defense profiling into phage therapy design.},
}
@article {pmid42465601,
year = {2026},
author = {Kumar, S and Singh, RM and Gadhave, KR},
title = {CRISPR-Cas systems for plant virus management: detection, surveillance, and host resistance.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1804262},
pmid = {42465601},
issn = {1664-462X},
abstract = {The CRISPR-Cas system has transformed genome manipulation by enabling precise and programmable modification of genetic material. Initially developed as a genome-editing tool, CRISPR technologies have expanded from fundamental research to applied use across plant, animal, and microbial systems due to their simplicity, accuracy, and versatility. In agriculture, CRISPR-Cas9 has progressed from crop improvement to host-directed strategies conferring resistance against a broad range of plant viruses. Concurrently, the discovery of additional Cas effector proteins, particularly Cas12a and Cas13a, has enabled highly sensitive nucleic acid-based diagnostic platforms supporting rapid, field-deployable pathogen detection. Here, we present a focused synthesis integrating CRISPR-mediated host resistance engineering with CRISPR-based diagnostic surveillance within a unified framework for plant virus management. Unlike previous reviews that treat these domains independently, we emphasize their convergence in enabling early detection, real-time surveillance, and targeted intervention across the disease cycle. Cas12a-based systems, currently the most widely implemented, have been coupled with isothermal amplification and visual readouts for rapid virus detection, whereas Cas13a-based platforms offer direct RNA targeting with potential for simplified workflows, although they remain less developed. We examine key design considerations, performance characteristics, and limitations of these platforms, including challenges related to sensitivity, multiplexing, and field deployment. Finally, we highlight future directions, including vector-based detection, multiplex diagnostics, and integration of CRISPR technologies into scalable surveillance systems. Collectively, this review positions CRISPR-based genome editing and diagnostics as complementary components of a next-generation strategy for plant virus detection, surveillance, and management.},
}
@article {pmid42467248,
year = {2026},
author = {Zhong, H and Ma, Q and Wei, J and Yang, A and Liu, Y and Zhen, D},
title = {CRISPR/cas-based biosensors for point-of-care testing: a comprehensive review of signal readout strategies.},
journal = {Archives of microbiology},
volume = {208},
number = {10},
pages = {},
pmid = {42467248},
issn = {1432-072X},
support = {202510555097//National College Students Innovation and Entrepreneurship Training Program/ ; 82503565//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Humans ; Colorimetry/methods ; Rapid Diagnostic Tests ; Point-of-Care Systems ; Electrochemical Techniques/methods ; },
abstract = {The CRISPR/Cas system has emerged as a transformative tool for nucleic acid detection, offering significant potential for point-of-care testing (POCT). However, translating CRISPR/Cas-based assays into practical POCT devices critically depends on the development of portable, sensitive, and user-friendly signal readout modalities. This review systematically compares four major readout modalities: fluorescence, electrochemical, colorimetric, and distance‑based readout, analyzing their mechanisms, analytical performance, and practical limitations. Key challenges, including sample preparation, amplification-free detection, multiplexing, and commercialization barriers, are critically assessed. Finally, future perspectives are proposed: integrating microfluidics with smartphone‑based readout, leveraging artificial intelligence and the Internet of Things for automated signal interpretation and cloud connectivity, and establishing regulatory pathways for clinical translation. This review aims to provide actionable insights for researchers developing next‑generation CRISPR diagnostics and to accelerate the transition from laboratory prototypes to deployable POCT devices.},
}
@article {pmid42469036,
year = {2026},
author = {Portell-Montserrat, J and Höpfler, M},
title = {Nascent peptides emerge as regulators of mRNA stability.},
journal = {Trends in cell biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tcb.2026.06.010},
pmid = {42469036},
issn = {1879-3088},
abstract = {Mobile genetic elements and their hosts engage in continuous evolutionary conflict. Marino et al. recently uncovered an unusual anti-CRISPR mechanism: the phage protein AcrVA2 triggers translation-coupled mRNA degradation by recognizing nascent Cas12. The findings suggest that nascent peptides may signal an underappreciated layer of gene regulation across the kingdoms of life.},
}
@article {pmid42469498,
year = {2026},
author = {Khan, T and Abro, AA and Zulfiqar, U and Alotaibi, MS and Asadullaeva, D and Allaberdiev, R and Tang, X and Fan, G},
title = {CRISPR-enabled functional genomics for bolstering plant tolerance to abiotic and biotic stress; a comprehensive review.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42469498},
issn = {1438-7948},
mesh = {*Stress, Physiological/genetics ; *CRISPR-Cas Systems ; Drought Resistance ; *Gene Editing/methods ; *Genomics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Climate change intensifies abiotic stresses including salinity, drought, and extreme temperatures alongside biotic threats such as pathogens and insect pests, collectively undermining global crop productivity and food security. Salinity and drought alone affect 20-50% of irrigated soils, with projections indicating that nearly half of global farmland could become saline by mid-century. Conventional breeding and earlier genome editing tools zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (ZFNs, TALENs) are constrained by genetic diversity limitations, technical complexity, and slow trait deployment. The CRISPR-Cas9 system has emerged as a transformative platform offering superior precision, efficiency, scalability, and affordability for crop improvement. This review systematically examines how CRISPR-Cas9 enables targeted engineering of stress tolerance in major crops (rice, wheat, maize, tomato, barley) through gene knockout and knock-in strategies. Key applications include editing transcription factors (ART1, DRO1, OsDST) for drought and salinity tolerance, modifying transporter genes (OsHMA2, OsNramp5) for heavy metal detoxification, and disrupting susceptibility genes (MLO, OsERF922, CsLOB1) for broad-spectrum disease and pest resistance. Beyond direct editing, we highlight emerging synergies with functional genomics, multi-omics integration, and high-throughput phenotyping to accelerate target discovery and validation. A central focus is placed on nanobiotechnology-enabled CRISPR delivery systems, including lipid nanoparticles (LNPs), exosomes, and engineered nanocarriers that overcome the plant cell wall barrier a major bottleneck in plant genetic transformation. These platforms enable efficient, genotype-independent delivery of ribonucleoprotein (RNP) complexes, facilitating DNA-free editing for sustainable crop protection. By integrating CRISPR-based precision with advances in nanodelivery and molecular breeding, this review outlines a road-map for developing climate-resilient, high-yielding, and nutritionally enhanced crops to safeguard global agricultural sustainability.},
}
@article {pmid42470537,
year = {2026},
author = {Sisay, T and Berhan, A and Mihrete, K and Hunie, E and Bizuye, A},
title = {Genomic regulation of the diphtheria toxin gene and Its implications for molecular diagnostics and surveillance in low-resource settings.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42470537},
issn = {1573-4978},
mesh = {*Diphtheria Toxin/genetics/metabolism ; *Corynebacterium diphtheriae/genetics/pathogenicity ; Humans ; *Diphtheria/diagnosis/genetics/microbiology ; Genomic Islands ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; Genome, Bacterial ; DNA-Binding Proteins ; },
abstract = {Corynebacterium diphtheriae remains a significant, though often underestimated, public health concern, particularly in low- and middle-income countries. The pathogenicity of the disease is primarily determined by diphtheria toxin (DT), which is produced by the tox gene, a bacteriophage-associated element, and is tightly regulated by the iron-dependent transcriptional repressor DtxR, encoded by the dtxR gene. Despite extensive investigation into the molecular biology of DT, its regulation within the broader genomic organization, as well as its implications for diagnostic methods and surveillance strategies, have not yet been fully elucidated. This review consolidates existing evidence regarding the genomic context and molecular regulation of the tox gene, encompassing chromosomal organization, variability in GC content, genomic islands, and mechanisms of horizontal gene transfer. Significant attention is focused on lysogenic conversion mediated by corynephages and regulatory pathways responsive to iron. We also evaluate both established and novel molecular diagnostic approaches, including PCR, real-time PCR, sequencing technologies, and isothermal amplification methods like loop-mediated isothermal amplification (LAMP). Recent genomic discoveries, including pan-genome variation, CRISPR-Cas mechanisms, and the emergence of non-toxigenic tox-bearing strains are analyzed in relation to diagnostic precision and epidemiological surveillance. Understanding the genomic regulation and evolutionary dynamics of toxin production is essential for improving diagnostic accuracy and strengthening surveillance systems, particularly in resource-limited settings where diphtheria is often underdiagnosed and underreported.},
}
@article {pmid41570009,
year = {2026},
author = {Tay, YL and Thomson, SB and Hnatova, S and Ng, S and Teo, SR and McCallum, R and Sim, B and Tarantini, L and Tai, FL and Bollati, V and Loh, M and Hayden, MR and Leavitt, BR and Pouladi, MA},
title = {Silencing of human HTT by targeted CRISPR/dCas9-mediated epigenetic editing.},
journal = {Journal of Huntington's disease},
volume = {15},
number = {3},
pages = {399-407},
doi = {10.1177/18796397251415368},
pmid = {41570009},
issn = {1879-6400},
mesh = {Humans ; *Huntingtin Protein/genetics ; *DNA Methylation/genetics ; *Gene Silencing ; *Huntington Disease/genetics/therapy ; DNA Methyltransferase 3A ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; Epigenome Editing ; DNA (Cytosine-5-)-Methyltransferases/genetics ; },
abstract = {BackgroundGene silencing is widely recognized as a promising therapeutic approach for dominant monogenic disorders. Current silencing strategies, many of which are transient, utilize RNA interference. Gene silencing may also be achieved through directed epigenetic editing using a CRISPR/dCas9 effector fused to DNA methyltransferase 3A (dCas9-DNMT3A). We used this system to direct DNA methylation to HTT, the causal gene underlying the autosomal dominant neurodegenerative disorder Huntington's disease, to assess the translational potential of this strategy for treating a genetic neurological disease.ObjectiveTo characterize the regulatory effect of targeted dCas9-DNMT3A-mediated DNA methylation at HTT.MethodsWe exploited DNA methylation profiles of high and low HTT-expressing tissues and targeted hypomethylated regions of HTT associated with high levels of HTT expression.ResultsDe novo DNA methylation of loci within defined upstream, promoter, intragenic and downstream regions of HTT resulted in robust, acute silencing of HTT. The best long-term silencing of HTT, which persisted up to 30 days, was observed when targeted DNA methylation was directed to the 5'UTR and promoter regions of HTT.ConclusionsHTT gene silencing may be achieved via targeted de novo DNA methylation within hypomethylated regulatory regions at the HTT locus. DNA methylation editing may be an attractive therapeutic approach for Huntington disease due to its potential for long-term silencing and reversibility.},
}
@article {pmid42045650,
year = {2026},
author = {Saha, A and Ocampo, RF and Wright, JT and Taylor, DW and Palermo, G},
title = {Molecular mechanisms and biotechnology applications of CRISPR-Cas12a.},
journal = {Nature reviews. Molecular cell biology},
volume = {27},
number = {8},
pages = {601-616},
pmid = {42045650},
issn = {1471-0080},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Animals ; *Bacterial Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; },
abstract = {CRISPR-Cas12a is a versatile RNA-guided nuclease that has rapidly gained prominence for its dual functionality in genome editing and nucleic acid detection. In this Review, we discuss the structural, biochemical and mechanistic features of Cas12a that underpin its autonomous processing of the guide RNA and indiscriminate cleavage of single-stranded DNA, which enable Cas12a applications ranging from gene therapy to rapid diagnostics. We discuss key allosteric regulators and functional modules that orchestrate Cas12a activity, focusing on the core regulatory structural elements that control maturation of the guide RNA, target specificity, and both cis-cleavage and trans-cleavage activities, including the determinants of off-target cleavage. We provide a comparative analysis of Cas12a and the widely used Cas9, which further illuminates the distinctive attributes of Cas12a, and discuss recent advances in the characterization of its orthologues and in the development of engineered variants that expand its capabilities. Collectively, we present a comprehensive understanding of Cas12a and its increasing impact on biotechnology, therapeutics and molecular diagnostics.},
}
@article {pmid42190664,
year = {2026},
author = {Zhang, H and Zhang, Z and Wang, P and Xu, T and Chen, X and Zhao, Y and Lin, S and Cai, W and Ren, P and Luo, C and Zhang, P and Wang, Y and Hou, S and Zhao, Y and Zeng, H and Liu, Z and Wang, C and Gao, Z and Feng, Y and Pan, D and Zeng, Z},
title = {Uncovering spatially resolved functional genomics with CRISPR screen sequencing.},
journal = {Cell},
volume = {189},
number = {15},
pages = {4594-4618.e48},
doi = {10.1016/j.cell.2026.04.049},
pmid = {42190664},
issn = {1097-4172},
mesh = {*Genomics/methods ; Animals ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Macrophages/metabolism/immunology ; Intercellular Adhesion Molecule-1/metabolism/genetics ; CD8-Positive T-Lymphocytes/metabolism/immunology ; Hyaluronan Receptors/metabolism ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Spatial omics has advanced our understanding of tissue-level biology, yet tools to systematically link gene functional perturbations to spatial phenotypes and signaling pathways remain limited. To address this, we developed spatial CRISPR screen sequencing (SPAC-seq), a high-throughput spatial CRISPR screen platform, and TARDIS (target prioritization toolkit for perturbation data in spatial omics), a statistical spatial perturbation analysis toolkit. Using SPAC-seq and TARDIS, we linked gene perturbations to spatial phenotypes and pathways, uncovering how Icam1 loss in tumor cells promotes metastasis via immune suppression and macrophage polarization. In CD8[+] T cells, we revealed Cd44's role in regulating spatial phenotypes by interacting with Spp1 on macrophages. We also demonstrated the model of the transcription factor-chemokine receptor axis coupling cell states with chemotaxis. SPAC-seq and TARDIS provide an effective framework to study spatially resolved functional genomics and pathways across diverse biological and disease contexts.},
}
@article {pmid42192532,
year = {2026},
author = {Hwang, HY and Yi, H and Gwon, Y and Jeon, E and Kim, D},
title = {High-fidelity genome and prime editing enabled by the AI-designed openCRISPR-1.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42192532},
issn = {1756-994X},
support = {RS-2025-00521074//National Research Foundation of Korea/ ; RS-2025-02214578, HR22C1363, RS-2024-02507183//Korea Health Industry Development Institute (KHIDI)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Genome, Human ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Associated Protein 9/genetics ; },
abstract = {BACKGROUND: RNA-guided nucleases such as CRISPR-Cas9 systems have revolutionized genome engineering by enabling programmable DNA modifications. Although structure-guided and evolution-derived high-fidelity Cas9 variants improve target specificity, they often compromise on-target activity or constrain guide RNA (gRNA) design.<br><br>METHODS: We performed head-to-head comparisons of OpenCRISPR-1 and Cas9 in human cells using amplicon sequencing, multiplex Digenome-seq, and off-target validation by targeted sequencing. Editing activity was assessed across 28 endogenous loci in HEK293T cells and further evaluated in human induced pluripotent stem cells (iPSCs) and MRC-5 fibroblasts. To test clinically relevant delivery, Cas9 and OpenCRISPR-1 ribonucleoproteins were delivered using engineered virus-like particles (eVLPs). We also generated OpenCRISPR-based prime editors, OpenCRISPR-PE2 and OpenCRISPR-PE7, and compared them with PE2max and PE7 using pegRNAs and engineered epegRNAs.<br><br>RESULTS: Here, we show that OpenCRISPR-1, an AI-designed, Cas9-like nuclease, retains Cas9-level editing efficiency across multiple genomic loci while significantly reducing off-target mutations. Using multiplex Digenome-seq and targeted deep sequencing, OpenCRISPR-1 exhibits up to a 553-fold reduction in off-target mutations compared to Cas9 and achieves off-target indices that match or surpass those of high-fidelity Cas9 variants. OpenCRISPR-1 also sustains robust editing across diverse gRNA formats (GX19, gX19, and gX20), highlighting its enhanced versatility. Furthermore, converting OpenCRISPR-1 into a prime editor yields comparable editing efficiencies while lowering the relative specificity ratio by up to 97%.<br><br>CONCLUSIONS: These findings establish generative AI-guided protein design as a powerful strategy to overcome the specificity-efficiency trade-off, expanding the genome editing toolkit for both research and therapeutic use, and ushering in a new era of rational protein design.},
}
@article {pmid42218140,
year = {2026},
author = {Di Bernardo, M and Kern, RS and Cepeda Diaz, AK and Mallar, A and Choi, SJ and Nutter-Upham, A and Lourido, S and Blainey, PC and Cheeseman, I},
title = {Brieflow: an integrated computational pipeline for high-throughput analysis of optical pooled screening data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42218140},
issn = {2041-1723},
support = {GM126930//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 000955563//National Science Foundation (NSF)/ ; Data Science Internship Program//Massachusetts Life Sciences Center (MLSC)/ ; UROP Program//Massachusetts Institute of Technology (MIT)/ ; R01HG009283//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AI144369//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Computational Biology/methods ; *High-Throughput Screening Assays/methods ; CRISPR-Cas Systems ; Reproducibility of Results ; Humans ; *Genomics/methods ; Phenotype ; Software ; Large Language Models ; Pooled Testing ; },
abstract = {Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate Brieflow's capabilities through reanalysis of a CRISPR-Cas9 screen encompassing 5072 fitness-conferring genes, processing more than 70 million cells with multiple phenotypic markers. To accelerate biological interpretation, we additionally present MozzareLLM, a framework leveraging large language models to identify biological processes within phenotypic clusters and prioritize gene candidates for experimental validation. Our combined analysis recovers coherent biological modules missed by existing analytical approaches, including five core mitochondrial sub-programs absent from the original study. The modular design and open-source implementation of Brieflow facilitates the integration of new analytical components while ensuring computational reproducibility and improved performance for the use of high-content phenotypic screening in biological discovery.},
}
@article {pmid42230636,
year = {2026},
author = {Snetkova, V and Galan, C and Lopez, R and Rios, AR and Kudo, T and Dorighi, K and Warming, S and Haley, BJ},
title = {A tunable Cas12a platform for single-cell perturbation screening and CRISPRi.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42230636},
issn = {2041-1723},
mesh = {*Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Degrons ; HEK293 Cells ; Gene Knockout Techniques/methods ; Transcriptome ; Animals ; Endodeoxyribonucleases ; },
abstract = {Single-cell perturbation (Perturb-seq) screens have primarily relied on Cas9 for inducing loss-of-function phenotypes, whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a's guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery during single-cell RNA sequencing library preparation. To overcome the self-processing constraint, we optimized pre-crRNA expression vectors and established a degron-based, enhanced Cas12a system for gene knock-out. As demonstrated across cell types, target genes, and with a minimized guide RNA library, this platform allows for accurate detection of pre-crRNAs and gene editing-induced effects on the transcriptome in single cells. Additionally, we show that HyperLbCas12a outperforms other existing variants for multiplexed gene suppression. While the rapid reversibility of this repressor highlights specific kinetic constraints for degron-based single-cell recording, the system provides a potent, modular tool for contexts requiring tunable, transient silencing. Together, this suite of technologies greatly expands the possibilities for future Perturb-seq efforts and broader application of Cas12a for genetic disruption at scale.},
}
@article {pmid42236706,
year = {2026},
author = {Zhu, L and Nguyen, LT and Bell, AG and Krebel, T and Gillmann, KM and Cao, Q and Oatman, H and Hariri, J and Möglich, A and Myhrvold, C and Toettcher, JE},
title = {Multimodal control of Cas13d activity through domain insertion at an allosteric hotspot.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42236706},
issn = {2041-1723},
support = {R01GM144362//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Domains ; Blue Light ; HEK293 Cells ; Sirolimus/pharmacology/analogs &amp; derivatives ; },
abstract = {CRISPR-Cas13d RNA nucleases are powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of RfxCas13d tools that can be multimodally controlled by either light or small molecule addition. By screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain, we identify an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. We show that the same allosteric hotspot can be exploited to generate an OptoCas13d-on with an inverted light response and a ChemoCas13d that is activated by rapamycin analogs, enabling knockdown of endogenous mRNA and protein targets. Overall, our study shows that engineered allostery can produce stimulus-controlled Cas13d variants to modulate RNA with high spatial and temporal precision.},
}
@article {pmid42236735,
year = {2026},
author = {Turocy, J and Jerabek, S and Hur, W and Kim, J and Xu, S and Zhao, Q and Xu, J and Robles, A and Liu, X and Treff, N and Marin, D and Hadjantonakis, AK and Egli, D},
title = {Asymmetric attrition and secondary chromosome destabilization after double-strand breaks in human embryonic development.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42236735},
issn = {2041-1723},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *Embryonic Development/genetics ; *Chromosomal Instability/genetics ; Centromere/genetics/metabolism ; Chromatids/metabolism ; Telomere/genetics/metabolism ; DNA Repair ; Spindle Apparatus/metabolism ; CRISPR-Cas Systems ; },
abstract = {DNA repair in human embryos is poorly understood, and double-strand breaks (DSBs) can cause chromosome loss. We show that chromosomal alterations relative to an induced DSB are asymmetric: acentric arms show complementary gains and losses, while centric arms are biased toward losses. Centromeric to the cut site secondary breakage and attrition is extensive. In contrast, break sites at acentric arms are conserved with no secondary breakage. These differences reflect differential forces at the mitotic spindle. Telomeric arms detach from the pro-metaphase spindle while centric truncated chromosomes lag during anaphase, suggesting that the DSB impedes sister chromatid separation. Secondary breakage near the centromere concordant with extensive attrition at the DSB site indicates a DSB can destabilize a chromosome without end-joining of sister chromatids. These results highlight the risks of chromosomal-scale changes in CRISPR-Cas9 genome editing and show that a single DSB can destabilize a human embryo chromosome independent of fusion-breakage cycles.},
}
@article {pmid42251067,
year = {2026},
author = {Gopal, N and Abay, T and Payne, C and Gomez, M and Rogers, MM and Fofanah, IU and Kallon, TPMS and Kamara, MS and Suk, HJ and Sandi, JD and Brock-Fisher, T and Stachler, E and Allan-Blitz, LT and Roach, DJ and Paye, MF and Wilkason, C and Grant, DS and Ozonoff, A and Sabeti, PC},
title = {Rapid development and field evaluation of a portable CRISPR-based assay for Mpox during the 2025 Sierra Leone outbreak.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42251067},
issn = {2041-1723},
mesh = {Sierra Leone/epidemiology ; Humans ; *Disease Outbreaks ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems/genetics ; *Hemorrhagic Fever, Ebola/epidemiology/diagnosis/virology ; *Ebolavirus/genetics/isolation &amp; purification ; },
abstract = {The large 2025 Mpox clade IIb outbreak in Sierra Leone underscores the urgent need for portable, low-cost diagnostics in decentralized settings. While CRISPR-based assays offer high sensitivity and flexibility, their deployment during active outbreaks remains limited. Here we show the rapid development and field evaluation of Mpox SHINE, a CRISPR-Cas13 assay that integrates lyophilized reagents, ambient-temperature lysis, and automated fluorescence detection on the portable DxHub device. The assay achieves analytical sensitivity down to 10 copies/µL. Clinical validation in Sierra Leone, using 56 clinical specimens, confirms complete concordance with qPCR, demonstrating 100% sensitivity and 100% specificity. Crucially, Mpox SHINE also detects the virus directly from unextracted lesion swabs while maintaining 100% sensitivity and specificity. The mean time-to-result is fast, averaging 11.4 minutes for extracted samples and 27.9 minutes for unextracted samples. These findings demonstrate that CRISPR-based diagnostics translate quickly from genomic sequence to clinically validated, deployable tools within a single outbreak window.},
}
@article {pmid42269486,
year = {2026},
author = {Popsuj, S and Kalsang, T and Kim, K and Drummond, E and Manekar, P and Munagapati, P and Oleti, M and Sato, H and Vickery, I and Gigante, ED and Stolfi, A},
title = {Validated CRISPR/Cas9 guide RNAs targeting neurodevelopmental genes in the tunicate Ciona robusta.},
journal = {Differentiation; research in biological diversity},
volume = {150},
number = {},
pages = {100973},
doi = {10.1016/j.diff.2026.100973},
pmid = {42269486},
issn = {1432-0436},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Neurodevelopment/genetics ; *Ciona/genetics/growth &amp; development ; Mutagenesis ; Transcription Factors/genetics ; Gene Expression Regulation, Developmental ; *Ciona intestinalis/genetics ; },
abstract = {The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of developmental and cell biology. With a larval CNS composed of just over 200 neurons and sensory cells, it has also emerged as a model organism for neurobiology and the development of the nervous system. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona as an important technique used to interrogate gene function in diverse biological processes, validated single-guide RNAs (sgRNAs) have yet to be validated for several key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates to scores generated by different predictive algorithms, observing a modest but potentially improved correlation with predictions based on a newer algorithm. Based on these results, we recommend considering both scoring algorithms in combination, for improved predictive value for Ciona.},
}
@article {pmid42276496,
year = {2026},
author = {Wang, F and Zhang, M and Chen, S and Fu, S and Huai, M and Liu, M and Meng, G and Dong, C},
title = {CRISPR/Cas9-mediated disruption of Cmpks1 reveals its role as a key regulator of carotenoid biosynthesis and metabolic adaptation in Cordyceps militaris.},
journal = {International journal of biological macromolecules},
volume = {372},
number = {},
pages = {152987},
doi = {10.1016/j.ijbiomac.2026.152987},
pmid = {42276496},
issn = {1879-0003},
mesh = {*Cordyceps/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Carotenoids/metabolism ; Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics/metabolism ; *Polyketide Synthases/genetics/metabolism ; Oxidative Stress ; *Adaptation, Physiological/genetics ; Light ; Mutation ; },
abstract = {Cordyceps militaris, a renowned edible mushroom, produces orange-yellow fruiting bodies (FBs), primarily due to carotenoid accumulation. However, genetic mechanisms and functional roles underlying carotenoid biosynthesis remain poorly understood. Here, we identified Cmpks1, a light-induced gene encoding a reducing type I polyketide synthase, as a key regulator of pigment biosynthesis. Transcription of Cmpks1 was CmWC-1-dependent and upregulated during FB development. CRISPR/Cas9-mediated loss-of-function mutants of Cmpks1 exhibited stable albino phenotypes but retained FB differentiation. In addition to abolishing carotenoid biosynthesis, the disruption of Cmpks1 increased sensitivity to high light and oxidative stress, indicating its role in redox homeostasis. Metabolomic profiling of the ΔCmpks1 mutant, including significantly reduced ergothioneine and elevated cordycepin, revealed extensive metabolic reprogramming, coupled with activation of compensatory survival mechanisms. These findings elucidate the genetic mechanisms governing pigment formation that influence the quality of Cordyceps products, offering new insights into the role of metabolites in fungal morphogenesis and stress adaptation.},
}
@article {pmid42276945,
year = {2026},
author = {Meng, D and Zhang, Y and Zou, S and Wang, J and Tian, C and Gao, T and Liu, J and Yuan, X and Chen, S and Qiao, Y and Ma, N and Chang, H and Gao, X},
title = {Cas9-PALB2 fusion protein enhances CRISPR/Cas9 mediated gene knock-in efficiency.},
journal = {Journal of bioscience and bioengineering},
volume = {142},
number = {3},
pages = {187-195},
doi = {10.1016/j.jbiosc.2026.05.004},
pmid = {42276945},
issn = {1347-4421},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; *Fanconi Anemia Complementation Group N Protein/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; *Recombinant Fusion Proteins/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {Over the past decade, CRISPR-based technologies have revolutionized our capacity to manipulate genomes, thereby reshaping the landscape of functional genomics research. Among the CRISPR toolkit, CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing with predefined mutations, rendering it an indispensable tool for gene functional analysis, disease model construction, and the development of gene therapy strategies. Nevertheless, despite the robust efficiency of CRISPR/Cas9 in mediating gene knockouts, HDR-dependent gene knock-in (KI) remains a major bottleneck due to its inherently low efficiency. Herein, we report that the co-expression of PALB2 with the CRISPR/Cas9 nuclease could trigger an enhanced HDR effect. Specifically, the fusion of Cas9 with PALB2 elevated KI efficiency by approximately 1.7-fold in human HEK293T cells. Furthermore, this Cas9-PALB2 fusion strategy exhibited cross-cell-type efficacy, demonstrating its broad applicability beyond a single cell line. Notably, the combined application of the Cas9-PALB2 fusion system and Nocodazole further boosted KI efficiency to a remarkable 25.5%. Collectively, these findings establish the Cas9-PALB2 fusion as a highly potent and versatile strategy to augment HDR-mediated KI efficiency, highlighting its substantial potential for widespread utilization in applications that demand high-fidelity genome editing.},
}
@article {pmid42379534,
year = {2026},
author = {Lummerstorfer, M and Xue, Z and Zheng, D and König, L and Brunner, L and Lächelt, U},
title = {Lipid nanoparticles for Cas9 ribonucleoprotein delivery: design and evaluation of ionisable oligoamine-lipidoids.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {224},
number = {},
pages = {107596},
doi = {10.1016/j.ejps.2026.107596},
pmid = {42379534},
issn = {1879-0720},
mesh = {*Nanoparticles/chemistry/administration &amp; dosage ; *Lipids/chemistry/administration &amp; dosage ; *Ribonucleoproteins/administration &amp; dosage/chemistry ; *CRISPR-Associated Protein 9/administration &amp; dosage/chemistry ; Humans ; *Amines/chemistry/administration &amp; dosage ; CRISPR-Cas Systems ; Gene Editing ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are the most advanced RNA delivery technology and are used with CRISPR-RNA in multiple clinical in vivo genome editing trials. By contrast, systemic delivery of Cas9 ribonucleoproteins (RNPs) - despite their high intrinsic efficiency - has lagged, largely due to a lack of mature delivery systems, and RNA‑optimised LNPs cannot readily be translated to RNPs. Differences arise from the pH-sensitive protein and cargo-specific optimal lipid compositions. With regard to the fundamental ionisable lipid component, comparatively less optimisation has been carried out for Cas9-RNPs than for RNA. In this work, C12-200, developed as a potent ionisable lipidoid for RNA-LNPs and also well-suited for Cas9-RNPs, served as the lead structure. Using an analogue synthesis strategy, 17 alternative C12-lipidoids were generated from different oligoamine precursors with structural differences, including the number of nitrogens (2 to ≈40), architecture (linear, branched or containing an N-heterocycle), and separating alkyl spacers (ethyl, propyl) between ionisable groups. Employing the different lipidoids in analogous LNP formulations enabled a systematic assessment at relevant stages of Cas9-RNP delivery and the identification of structure-activity relationships. Two C12-lipidoids with piperazine ring, ethyl spacers and three (C12-AEP) or four nitrogens (C12-BAEP) were identified as the most effective, exhibiting potencies comparable to or exceeding C12-200 in the in vitro knockout model. This study reports a systematic evaluation of ionisable oligoamine-lipidoids in Cas9-RNP-LNP formulations, highlights critical delivery bottlenecks, and provides recommendations for the design of potent candidates.},
}
@article {pmid42406843,
year = {2026},
author = {Morianou, I and Phillimore, L and Khatri, BS and Marston, L and Gribble, M and Burt, A and Bernardini, F and Hammond, AM and Nolan, T and Crisanti, A},
title = {Engineering resilient gene drives for sustainable malaria control by predicting, testing and overcoming target site resistance in Anopheles gambiae.},
journal = {PLoS biology},
volume = {24},
number = {7},
pages = {e3003879},
pmid = {42406843},
issn = {1545-7885},
mesh = {Animals ; *Anopheles/genetics ; *Gene Drive Technology/methods ; Female ; *Mosquito Control/methods ; *Malaria/prevention &amp; control/transmission ; Mosquito Vectors/genetics ; *Insecticide Resistance/genetics ; Alleles ; CRISPR-Cas Systems ; },
abstract = {CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito, Anopheles gambiae. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance, and the likelihood of resistance emerging at natural population scales remains poorly defined. Here, we present a pipeline to quantify the evolutionary space for resistance, enabling accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our approach to stress-test a best-in-class suppression gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, including a novel type of partially resistant alleles that can perturb drive-invasion dynamics. Integrating experimentally derived resistance rates with population genetic modeling shows that single-target suppression drives are unlikely to be robust at natural mosquito population sizes, even at highly constrained loci. Here, we engineer and validate multiplexed gene drives in Anopheles gambiae, that target multiple conserved sites, actively removing resistant alleles. Our models predict that such gene drives could supress large natural mosquito populations in the field.},
}
@article {pmid42454502,
year = {2026},
author = {Madhusudan, S and Eskici, N and Gomez-Sanchez, C and Pulli, K and Vaaralahti, K and Yellapragada, V and Conway, JRW and Wang, Y and Raivio, T},
title = {CRISPR activation of DLX5 drives neural progenitors to the GnRH cell fate.},
journal = {Journal of molecular endocrinology},
volume = {77},
number = {1},
pages = {},
doi = {10.1530/JME-26-0040},
pmid = {42454502},
issn = {1479-6813},
mesh = {*Gonadotropin-Releasing Hormone/metabolism ; Humans ; *Homeodomain Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Neurons/metabolism/cytology ; Animals ; Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; Fibroblast Growth Factor 8/metabolism/genetics ; Mice ; Hypothalamic-Pituitary-Gonadal Axis ; *Cell Lineage/genetics ; Transcription Factors ; },
abstract = {Gonadotropin-releasing hormone (GnRH) neurons regulate the hypothalamic-pituitary-gonadal (HPG) axis and are required for puberty onset and reproductive competence. However, the transcriptional regulators governing GnRH neuron specification and migration remain poorly defined. The homeodomain transcription factor DLX5 is expressed in fetal human GnRH neurons, its expression precedes that of GNRH1 in human pluripotent stem cell (hPSC)-derived GnRH neurons, and in mice, it serves as a guidance cue for GnRH neuron migration. We hypothesized that DLX5 may act as an upstream regulator of human GnRH neuron fate specification and migratory capacity. Using CRISPR activation, we upregulated DLX5 during FGF8b-directed differentiation of hPSCs to GnRH neurons via dual SMAD inhibition and Notch inhibition, as previously described. DLX5 activation increased neural progenitor motility (P < 0.001), upregulated FGF8 (P < 0.05), and induced GABAergic markers, including GAD1 and GAD2. Notably, DLX5 activation induced GNRH1 in the absence of exogenous FGF8b (P < 0.05), suggesting that in GnRH neurons, DLX5 regulates FGF8. When combined with exogenous FGF8b, DLX5 activation produced distinct neuronal patterning accompanied by upregulation of extracellular matrix genes, such as SPARC, which has been implicated in neurite outgrowth. Collectively, these data indicate that activation of DLX5 promotes GnRH neurogenesis from hPSCs, by driving GABAergic fate, inducing FGF8, and remodeling the extracellular matrix.},
}
@article {pmid42458955,
year = {2026},
author = {Kigaru, A and Ateka, EM and Pappu, HR and Nganga, EM and Murori, R and Toili, ME and Runo, S},
title = {Enhanced Rice Yellow Mottle Virus Resistance via CRISPR/Cas9-Targeted Mutagenesis of the Rice eIF(iso)4G Gene.},
journal = {Molecular plant pathology},
volume = {27},
number = {7},
pages = {e70312},
pmid = {42458955},
issn = {1364-3703},
support = {//African Union Commission/ ; },
mesh = {*Oryza/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Plant Viruses/physiology/pathogenicity ; *Mutagenesis/genetics ; *Eukaryotic Initiation Factor-4G/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Genes, Plant ; Mutation/genetics ; },
abstract = {Rice is a staple crop primarily recognised for its high content of carbohydrates and proteins. Rice yellow mottle disease (RYMD) is a destructive disease affecting rice fields in sub-Saharan Africa and is caused by the rice yellow mottle virus (RYMV). Development of virus-resistant genotypes is a highly recommended and effective approach to controlling RYMV. A genetic approach that exploits recessive mutations in susceptibility (S) genes may enhance resistance to the virus. Reports indicate that most rice genotypes grown in Kenya are vulnerable to RYMV infection. Genome editing has shown promise in enhancing agronomic traits in crops. We obtained enhanced resistance to RYMV in the Indica rice cv. IR2793-80-01 using the CRISPR-Cas9 system. The eIF(iso)4G susceptibility gene was targeted because natural mutations in this gene confer recessive resistance to RYMV. A Cas9-OseIF(iso)4G-gRNA-expressing vector targeting the eIF(iso)4G gene was introduced into rice calli via Agrobacterium-mediated transformation. Ten T2 homozygous mutant plant lines were assessed for their reaction to RYMV, and infection was significantly reduced. There were no significant differences in the agronomic characteristics between the T2 mutant lines and the wild-type plants. CRISPR/Cas9-mediated knockout alleles of the eIF(iso)4G gene conferred enhanced resistance to RYMV, which may be classified as partial. Findings underscore the need to embrace precise editing strategies, such as prime editing, to generate superior resistance alleles. Overall, the study provides an alternative resistance enhancement strategy that can create knockout resistance alleles that can be incorporated into breeding programmes for RYMV resistance.},
}
@article {pmid42459963,
year = {2026},
author = {Dasgupta, R and Das, K},
title = {CasPINS: an integrated web-based platform for CRISPR/TALEN gRNA design, primer generation, and indel decomposition analysis.},
journal = {Bioinformatics advances},
volume = {6},
number = {1},
pages = {vbag189},
pmid = {42459963},
issn = {2635-0041},
abstract = {Genome editing researchers currently navigate multiple disconnected tools for guide RNA (gRNA) design, primer generation, and editing analysis-a fragmented workflow that introduces errors and limits reproducibility. CasPINS (Cas-Primer-Indel Suite) addresses this gap as an open-source, unified platform integrating the complete genome editing computational workflow into a single interactive web application accessible without programming expertise. The platform supports 90+ species, 14 CRISPR-Cas variants, TALEN design, and six editing modes. Primer design integrates with Ensembl and NCBI databases relative to cut sites, while indel quantification utilizes Non-Negative Least Squares (NNLS) decomposition of Sanger chromatograms with maximum signal extraction and R 2 -corrected conservative modes. Benchmarking demonstrates strong concordance with established tools, including a 68.8% recovery of CHOPCHOP gRNAs and 67.2% of CRISPOR gRNAs across five human benchmark genes, alongside an algorithmic agreement within 2.6 percentage points on gold-standard TIDE data. Ultimately, a step-count analysis shows that CasPINS significantly streamlines usability, reducing discrete user actions from 25 to 8 steps compared to the traditional sequential-tool pipeline.},
}
@article {pmid42460575,
year = {2026},
author = {Li, M and Huang, D and Xu, C and Fang, M and He, P and He, Y and Wang, X and Xu, Z},
title = {Thermally Unlocked One-Pot RPA-CRISPR Cas12b Assay Integrated with the Centrifugal Microfluidic Chip for Multiplex Detection of Porcine Viruses.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c02126},
pmid = {42460575},
issn = {1520-6882},
abstract = {Highly contagious porcine viruses, represented by African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus (PRV), inflict severe economic losses on the swine industry and pose significant threats to global food security. Consequently, developing rapid, convenient, and efficient point-of-care testing (POCT) methods is essential for viral disease control. Although recombinase polymerase amplification (RPA) coupled with CRISPR/Cas systems demonstrates significant POCT potential, its practical application is currently restricted by operational complexity and limited throughput. Herein, a thermally unlocked one-pot RPA-CRISPR Cas12b assay integrated with the centrifugal microfluidic chip (TORCH) platform was developed in this paper. In this strategy, a thermal gating switch was utilized to physically isolate CRISPR reagents from the RPA during the initial phase, effectively addressing the inherent incompatibility in one-pot reactions. By employing the centrifugal microfluidic chip with a portable device, a highly integrated workflow enables fully automated processing ranging from sample lysis to multiplexed detection. Validated using pseudovirus-spiked porcine blood samples, TORCH successfully achieved multiplexed detection of ASFV, PRV, and PRRSV with the limits of detection as low as 0.5 copies/μL, while exhibiting exceptional resistance to interference and robust reagent stability. Overall, TORCH stands as a robust and user-friendly diagnostic solution, holding significant potential for early warning intervention and decentralized biosecurity control in resource-scarce environments.},
}
@article {pmid42462008,
year = {2026},
author = {Skopintsev, P and Esain-Garcia, I and DeTurk, EC and Yoon, PH and Zhou, Z and Weiss, T and Kamalu, M and Chamraj, A and Loi, KJ and Langeberg, CJ and Boger, RS and Nisonoff, H and Karp, HM and Chen, LX and Shi, H and Vohra, K and Banfield, JF and Cate, JHD and Jacobsen, SE and Doudna, JA},
title = {Structure and evolution-guided design of minimal RNA-guided nucleases.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6808},
pages = {313-318},
doi = {10.1126/science.aed6123},
pmid = {42462008},
issn = {1095-9203},
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Directed Molecular Evolution/methods ; DNA/chemistry ; *Gene Editing ; Models, Molecular ; Protein Conformation ; *Protein Engineering/methods ; RNA/chemistry ; *RNA, Guide, CRISPR-Cas Systems/chemistry ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; },
abstract = {The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.},
}
@article {pmid42462085,
year = {2026},
author = {Effah, CY and Li, X and Zhang, Q and Ding, L and Drokow, EK and Zhang, X and Wu, Y},
title = {Integrating CRISPR/Cas Biosensors with Advanced Platforms: A Holistic Path Toward Preamplification-free, Multiplexed, and Continuous Molecular Monitoring.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00860},
pmid = {42462085},
issn = {2379-3694},
abstract = {The paradigm of molecular diagnostics has been transformed by the repurposing of CRISPR-Cas systems from being gene-editing tools to nucleic acid detection engines with remarkable specificity and programmability. Both the SHERLOCK and DETECTR platforms have shown high sensitivity and specificity; however, the requirement of a pre-amplification step to achieve clinically relevant detection limits adds another layer of complexity and cost and is also a potential source of contamination, precluding their use as true point-of-care (POC) tools. The next frontier for CRISPR diagnostics will be the design of biosensors that enable preamplification-free, multiplex, and continuous direct detection of targets. Achieving this goal will involve the very close integration of CRISPR biology with nano-biotechnology, microfluidics, orthogonal Cas enzyme systems, and artificial intelligence (AI). This review aims to provide a comprehensive overview of recent advancements and strategic thinking related to this integration. This review discusses how nanomaterials facilitate signal generation and transduction, how microfluidics automates, multiplexes, and miniaturizes "all-in-one" devices, and how orthogonal CRISPR systems can enable robust multiplexing. We will also probe into the emerging application of AI to accelerate guide RNA design and optimize the performance of CRISPR biosensors. Furthermore, the roles of orthogonality and nanomaterials in real-time, continuous molecular monitoring will be assessed. The review will finally discuss the transformative future applications of high-throughput biomarker discovery and theranostics potential through massively parallelized CRISPR sensing.},
}
@article {pmid42462700,
year = {2026},
author = {Gao, Y and Wang, B},
title = {Programmable cell killer: CRISPR-Cas12a2 eliminates cells via RNA identity.},
journal = {Molecular cell},
volume = {86},
number = {14},
pages = {2662-2664},
doi = {10.1016/j.molcel.2026.06.029},
pmid = {42462700},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; },
abstract = {In a recent issue of Nature, Scholz et al.[1] apply the RNA-triggered DNA shredding activity of CRISPR-Cas12a2 in eukaryotic cells to enable programmable elimination of yeast and human cells expressing target transcripts with single-nucleotide resolution specificity and non-detectable off-target activity.},
}
@article {pmid42462716,
year = {2026},
author = {Hong, A and Liu, M and Truta, A and Talaie, A and Smith, GR and Bondy-Denomy, J},
title = {Gabija restricts phage circularization and DNA replication.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2026.06.016},
pmid = {42462716},
issn = {1934-6069},
abstract = {Anti-bacteriophage systems such as restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable the identification of invaders. How Gabija, a highly prevalent nuclease-helicase antiphage system, limits phage replication while executing self- vs. non-self-discrimination remains unknown. Here, we show that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting a temperate lambda-like phage in Pseudomonas aeruginosa, Gabija prevents phage genome circularization and subsequent replication. DNA end-binding complexes, including a phage exonuclease and a single-stranded DNA (ssDNA)-annealing protein or GamMu dimers that prevent loading of the host repair complex RecBCD, are necessary and sufficient to license phage and plasmid sensitivity to Gabija. Mutant escape phages lacking these DNA end-binding proteins become protected from Gabija by RecBCD translocation activities. RecBCD activity on the bacterial genome, presumably whenever it is linearized, also prevents Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes the circularization and replication of linear DNA devoid of RecBCD as a mechanism to identify and antagonize foreign invaders.},
}
@article {pmid42463576,
year = {2026},
author = {Li, Q and Wang, H and He, Y and Hong, G and Bao, G},
title = {Gene editing of hematopoietic stem cells: applications and advances.},
journal = {International journal of hematology},
volume = {},
number = {},
pages = {},
pmid = {42463576},
issn = {1865-3774},
abstract = {Allogeneic hematopoietic stem cell transplantation remains the standard treatment for various hematologic genetic disorders resulting from single or multiple genes. However, this strategy is hindered by two main problems: failure to find a matching donor and the risk of graft-versus-host disease (GVHD) after transplantation. Recent advances in gene editing, particularly nucleases exemplified by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and related derivative tools, have overcome the limitations posed by the poor specificity of traditional gene modification techniques. A robust groundwork has been established for developing efficient, precise, and diverse gene editing strategies, facilitating the clinical application of ex vivo modified autologous hematopoietic stem cells (HSCs). In contrast, autologous HSC transplantation does not have the previously mentioned problems associated with allogeneic transplantation. Consequently, gene editing involving ex vivo genetic modification of HSCs and subsequent reinfusion in a single patient has emerged, with related research progressing from investigation into fundamental mechanisms and proof-of-concept studies to clinical trials.},
}
@article {pmid42463755,
year = {2026},
author = {Nevard, K and Gonzalez, E and Harvey-Samuel, T and Sanders, C and Homem, RA},
title = {CRISPR-Cas9 mediated knockout of the white gene in the bluetongue virus vector, Culicoides sonorensis (biting midge).},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42463755},
issn = {2045-2322},
mesh = {Animals ; *Ceratopogonidae/genetics/virology ; *CRISPR-Cas Systems ; *Bluetongue virus ; Female ; *Insect Vectors/genetics/virology ; *Gene Knockout Techniques ; *Insect Proteins/genetics ; Gene Editing ; },
abstract = {Culicoides biting midges are small blood feeding insects responsible for the transmission of important arthropod-borne viruses (arboviruses) such as bluetongue virus (BTV), Schmallenberg virus (SBV) and epizootic hemorrhagic disease virus (EHDV), which cause major losses to livestock production worldwide. Culicoides sonorensis is the primary vector of BTV in North America and one of the few Culicoides species to be colonised and reared in artificial conditions. Gene editing technology has been used to explore virus-vector interactions in other vector groups, particularly within mosquitoes. Despite the availability of a reference genome since 2018, to date there have been no reports of gene editing in C. sonorensis. Here, we report the first instance of gene editing in C. sonorensis, achieved by intrathoracic injection of adult females with Cas9 and sgRNAs targeting the white gene. We generated heritable mutations in the white gene which produced both white eye and red eye phenotypes and went on to establish a homozygous knockout line carrying a single mutation. We observed gene editing efficiencies of up to 12.3%, making this an efficient protocol for genetic manipulation of Culicoides biting midges, opening the door to functional genomics studies and the development of control strategies in these important and understudied disease vectors.},
}
@article {pmid42463825,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Ibnaouf, K and Arsad, N},
title = {Reversing cancer cell behavior using AI-guided CRISPR and quantum nanobiology: a systems-based approach to epigenetic reprogramming.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42463825},
issn = {1476-5462},
abstract = {Treatment effectiveness is hindered by the phenotypic plasticity of cancer and the genetic complexity of tumors. However, CRISPR-Cas-based medicines face challenges with specificity, off-target effects, and tumor heterogeneity adaptability. This work investigates the possible combination of quantum biological processes, artificial intelligence, and nanomaterials to improve CRISPR gene editing and modulate or reverse selected malignant phenotypes. Quantum machine learning (QML) can be used to simulate quantum processes like electron tunneling in DNA repair and spin-dependent enzyme activity. To enable exact tumor phenotypic reversal, these models will be combined with optimization approaches powered by AI to direct CRISPR editing in oncogenic signaling networks. Graphene, gold nanoparticles, and lipid-based vectors are some of the nanomaterials that will be used as carriers to effectively and deliver CRISPR systems in a biocompatible manner to the cancer microenvironment. We hypothesize that selected homeostatic gene-expression states may be partially restored in experimental cancer models through the integration of quantum-informed AI, CRISPR gene alteration, and nanomaterial delivery. This integrated strategy could support future cancer therapies that move beyond tumor suppression toward controlled modulation of malignant cell states, although substantial preclinical and clinical validation remains necessary.},
}
@article {pmid42464282,
year = {2026},
author = {Zheng, W and Wang, M and Tu, Q and Bian, X and Zhang, Y and Wang, X},
title = {Establishment of SRLC: a multiplex genome editing technology for Saccharomyces cerevisiae and its application in metabolic engineering of malonyl-CoA pathway.},
journal = {Microbial cell factories},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12934-026-03068-w},
pmid = {42464282},
issn = {1475-2859},
support = {2023YFC3402003//National Key R&amp;D Program of China/ ; },
abstract = {The development of advanced genome engineering tools is crucial for optimizing metabolic pathways in Saccharomyces cerevisiae and achieving efficient biomanufacturing. This study proposes an enhancing multiplex genome editing strategy in S. cerevisiae by employing Escherichia coli-derived single-stranded annealing proteins (SSAPs) combined with S. cerevisiae-derived homologous recombinases (Rad51 and Rad52). The strategy utilizes an SSAP-Rad-Linearized CRISPR (SRLC) platform, which supports efficient simultaneous editing of multiple genomic loci without constructing complex multi-gRNA expression vectors. Co-overexpressing Rad51/Rad52 and E. coli SSAP proteins significantly enhances homologous recombination (HR), allowing precise multi-locus genome editing mediated by short homologous arms. Furthermore, SRLC employs a linearized CRISPR-Cas system to stimulate homologous recombination and enable counter-selection in S. cerevisiae, thereby improving precise multiplex genome editing efficiency. We applied SRLC to engineer the malonyl-CoA metabolic pathway in S. cerevisiae. Through a single round of editing and screening, we constructed a chassis strain with 9 targets simultaneously modification and achieved a 9.6-fold increase in intracellular malonyl-CoA. Using this chassis, 3-hydroxypropionic acid production increased 4.5-fold relative to wild-type S. cerevisiae. This platform offers a robust and scalable tool for S. cerevisiae manipulation and a practical pathway-engineering strategy for building for malonyl-CoA-derived factories.},
}
@article {pmid42115602,
year = {2026},
author = {Larrosa-Godall, M and Shackleford, L and Edgington, MP and Leftwich, PT and Luk, JCY and Southworth, J and Rosell, S and Creasey, JT and Aked, JM and Nevard, K and Dodds, A and Mckee, M and Adedeji, E and Gonzalez, E and Ang, JXD and Anderson, MAE and Alphey, L},
title = {Integrating multiplexing into confineable gene drives effectively overrides resistance in Anopheles stephensi.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42115602},
issn = {2041-1723},
support = {INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, BBS/E/I/00007039//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; INV-008549/GATES/Gates Foundation/United States ; },
mesh = {Animals ; *Anopheles/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Insecticide Resistance/genetics ; Malaria/transmission/prevention &amp; control ; *Mosquito Control/methods ; },
abstract = {Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance due to end-joining repair is a significant challenge to the deployment of these systems. The use of multiple sgRNAs has the potential to solve this issue. Here we perform experimental crosses to assess the homing and cutting efficiency of both classical (e.g. four adjacent sgRNAs all in one construct) and additive (e.g. separate constructs each expressing a single sgRNA) multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We find resistance alleles at one sgRNA target site can be mitigated by the presence of the additional sgRNAs with either strategy, and do not significantly reduce the homing efficiency for either strategy, validating their effectiveness. Further modelling using parameters derived from the strains generated indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.},
}
@article {pmid42154535,
year = {2026},
author = {Ziegler, M and Günter, C and Alecu, JE and Xue, X and Kim, HM and Saffari, A and Davies, AK and Sahin, M and Ebrahimi-Fakhari, D},
title = {CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators.},
journal = {JCI insight},
volume = {11},
number = {14},
pages = {},
doi = {10.1172/jci.insight.202204},
pmid = {42154535},
issn = {2379-3708},
mesh = {Humans ; *Membrane Proteins/metabolism/genetics ; *Vesicular Transport Proteins/metabolism/genetics ; CRISPR-Cas Systems ; *Autophagy-Related Proteins/metabolism/genetics ; *Neurons/metabolism ; Protein Transport/genetics ; *Adaptor Protein Complex 4/genetics/deficiency/metabolism ; trans-Golgi Network/metabolism ; *Spastic Paraplegia, Hereditary/genetics/metabolism ; Loss of Function Mutation ; },
abstract = {Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the "druggable genome," in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.},
}
@article {pmid42156946,
year = {2026},
author = {di Lillo, A and Tavella, S and Iannelli, F and Crisafulli, G and Gioia, U and Trastus, LA and Cabrini, M and d'Adda di Fagagna, F},
title = {Site-specific DNA double-strand break induces local transcription in cis and protein expression.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42156946},
issn = {2399-3642},
support = {21762//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 22458//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 30471//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Transcription, Genetic ; Humans ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; },
abstract = {The DNA damage response is a complex network of pathways that cells activate to safeguard genome integrity following DNA damage, including DNA double-strand breaks. We and others previously reported that RNA polymerase II, together with components of the preinitiation complex, is recruited to exposed DNA ends. This results in the assembly of a fully competent transcriptional apparatus and the synthesis of damage-induced long non-coding RNAs, which are necessary for full DNA damage response activation. Thus, DNA double-strand breaks could act as transcriptional promoters. Whether such DNA breaks, generated upstream of an open reading frame lacking a transcriptional promoter and followed by a polyadenylation signal, can induce the transcription of a coding RNA that is subsequently translated into a protein product remains unknown. Here, taking advantage of the CRISPR/Cas9 technology, we generate a sequence-specific double-strand break upstream of a promoter-less, and therefore silent, reporter gene in two distinct cellular systems. In both cell models, a DNA double-strand break is sufficient to trigger the expression of polyadenylated transcripts and a protein product. Collectively, our results demonstrate that DNA double-strand breaks can act as functional promoters capable of driving protein synthesis, revealing an additional mechanism through which DNA damage can regulate gene expression.},
}
@article {pmid42191711,
year = {2026},
author = {Feng, X and Ding, J and Liu, Y and Lopez Del Amo, V and Gantz, VM and Chen, XX and Champer, J and Liu, F},
title = {Self-limiting population suppression gene drive design in the West Nile vector mosquito, Culex quinquefasciatus.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42191711},
issn = {2041-1723},
support = {82372289//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82202559//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270672//National Natural Science Foundation of China (National Science Foundation of China)/ ; MS25C140016//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Culex/genetics/virology ; Female ; *Mosquito Vectors/genetics/virology ; *Gene Drive Technology/methods ; West Nile virus ; Male ; CRISPR-Cas Systems ; *West Nile Fever/transmission/virology ; Mosquito Control/methods ; Alleles ; },
abstract = {Culex quinquefasciatus is a major vector of West Nile virus and other pathogens, yet genetic population suppression tools for this species remain limited. Here, we develop a self-limiting, CRISPR-based suppression gene drive system targeting doublesex, close to the male-determining locus, promoting male transmission. A recoded dsxM sequence converts females into sterile intersexes, preventing population-level spread. The drive achieves super-Mendelian inheritance (~ 71%) and generates resistance alleles that are fully or partially dominant female sterile. Single-release cage trials show extended but self-limiting population suppression. Population modeling of this RIDD (Release of Insects carrying a Dominant-sterile Drive) system further indicates that repeated releases can substantially reduce fertile female numbers at low release ratios and intrinsic growth rates, outperforming non-drive strategies under comparable conditions. Together, these results establish a self-limiting suppression gene drive platform for Culex, providing a confined and sustainable framework for vector population control.},
}
@article {pmid42192102,
year = {2026},
author = {Gong, Y and Shi, S and Li, L and Qian, Y and Lu, T and Zhang, Z and Jiang, L and Liu, G and Cui, M and Li, S and Li, Z and Lin, H},
title = {PIWIL3-piRNA pathway controls rabbit oogenesis and embryogenesis via broad regulation of the transcriptome and proteome.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42192102},
issn = {2041-1723},
mesh = {Animals ; Female ; *Oogenesis/genetics ; *Piwi-Interacting RNA/metabolism/genetics ; *Argonaute Proteins/metabolism/genetics ; *Embryonic Development/genetics ; Rabbits ; Oocytes/metabolism ; *Transcriptome/genetics ; *Proteome/metabolism/genetics ; *RNA, Small Interfering/metabolism/genetics ; Humans ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Fertility/genetics ; },
abstract = {Female infertility often arises from oogenic defects, yet the underlying molecular mechanisms remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis, but its role in mammalian female fertility remains unclear, partly due to reliance on mouse models lacking PIWIL3. PIWIL3 exits in most other placental mammals and is highly expressed in human oocytes, but its function remains largely unexplored. Here, we show that rabbit PIWIL3 closely resembles its human counterpart and is the predominant PIWI protein in oocytes. Using CRISPR-Cas9 knockout, we demonstrate that PIWIL3 is essential for female fertility in rabbits, its loss leads to severe defects in oogenesis. Embryos lacking maternal PIWIL3 arrest by the 8-cell stage. Mechanistically, PIWIL3 binds ~18-nucleotide piRNAs, supports piRNA biogenesis, and regulates transcriptomic, proteomic, and transposable element dynamics during oocyte maturation and early embryogenesis. These findings establish PIWIL3 as an essential regulator of female fertility in non-rodent mammals, potentially including humans.},
}
@article {pmid42202890,
year = {2026},
author = {Li, L and Zhao, N and Ding, K and Zuo, L and Gan, Y and Liu, Y and Kong, L and Zhang, X and Zhao, Y},
title = {Development of a CRISPR/RspCas13d-based on-site rapid detection system for GII Norovirus.},
journal = {Journal of virological methods},
volume = {345},
number = {},
pages = {115421},
doi = {10.1016/j.jviromet.2026.115421},
pmid = {42202890},
issn = {1879-0984},
mesh = {*Norovirus/genetics/isolation &amp; purification ; Humans ; Sensitivity and Specificity ; *Caliciviridae Infections/diagnosis/virology ; Feces/virology ; Rapid Diagnostic Tests ; *Gastroenteritis/virology/diagnosis ; RNA, Viral/genetics ; DNA Primers/genetics ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Noroviruses (NoVs) are major cause of acute viral gastroenteritis and a serious public health concern. Current detection methods are limited in rapidity, equipment requirements, or sensitivity. In this study, we developed a rapid, sensitive, and specific detection assay for GII NoV by combining RT-RAA, T7 transcription, and the RspCas13d system. The RspCas13d protein was expressed and purified. RT-RAA primers and crRNA were designed against the conserved region of GII Nov. The assay was optimized and evaluated for specificity, sensitivity, and clinical performance. Results showed that the RT-RAA-RspCas13d method exhibited high specificity without cross-reactivity to other common enteric viruses. The limit of detection was 5 copies/μL. In clinical fecal samples, the assay showed high consistency with RT-qPCR. This method is rapid, simple, sensitive, and specific, providing a reliable tool for the rapid on-site detection of GII NoV.},
}
@article {pmid42270039,
year = {2026},
author = {Krishnamurthy, KA and Xiao, R and Rutten, MGS and Bos, T and Bleeker, A and Zhang, M and de Vries, HI and Koster, M and Huijkman, N and Smit, M and Kloosterhuis, N and Boer, T and Schomakers, B and van Weeghel, M and van de Sluis, B and Wolters, JC and Bakker, BM and Oosterveer, MH},
title = {Hepatocyte-specific Cas9-mediated editing of G6pc and Slc37a4 elicits comparable biochemical and regulatory responses between glycogen storage disease (GSD) type Ia and Ib mice.},
journal = {Molecular metabolism},
volume = {110},
number = {},
pages = {102393},
pmid = {42270039},
issn = {2212-8778},
mesh = {Animals ; Mice ; *Glycogen Storage Disease Type I/metabolism/genetics ; *Hepatocytes/metabolism ; Liver/metabolism/pathology ; *Glucose-6-Phosphatase/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems ; Male ; Female ; Humans ; Disease Models, Animal ; Proteomics ; *Antiporters/genetics/metabolism ; },
abstract = {BACKGROUND/OBJECTIVE: Glycogen storage disease type I (GSD I) is an autosomal recessive inborn error of carbohydrate metabolism. Patients with GSD type Ia and Ib exhibit overlapping and distinct symptoms and complications. Notably, GSD Ia patients show more severe hypertriglyceridemia and higher risk of hepatic tumors than GSD Ib patients.<br><br>METHODS: Given the liver's pivotal role in these processes, this study utilized hepatocyte-specific CRISPR/Cas9-mediated somatic gene editing to explore the pathophysiological and biochemical adaptations in hepatic GSD Ia and Ib side-by-side. Additionally, hepatic histology, transcriptomics, and proteomics analysis was performed.<br><br>RESULTS: Compared to controls, hepatic GSD Ia and Ib mice showed hepatomegaly, fasting hypoglycemia, hyperlactatemia, and increased uric acid in plasma, which was somewhat more pronounced in GSD Ia than Ib. Both GSD I subtypes showed similar reductions in hepatic acetyl-CoA precursor pool enrichment and increases in de novo biosynthesis of hepatic stearate and oleate. Interestingly, only GSD Ia mice showed mildly elevated plasma triglyceride and hepatic phosphate sugars. Metabolic changes were reflected at the transcriptomic and proteomic levels, with largely similar responses between GSD Ia and Ib livers. Moreover, altered mRNAs and protein levels related to nucleotide-binding oligomerization domain (NOD) signaling pathways, infection and inflammation, liver disease, and chemical carcinogenesis were somewhat more pronounced in hepatic GSD Ia than in GSD Ib mice.<br><br>CONCLUSIONS: Overall, the metabolic disturbance was more severe in hepatocyte-specific GSD Ia than in GSD Ib mice, consistent with the clinical phenotype in patients. The metabolic disorders and specific metabolites, genes, and proteins identified in this study provided new insights into the pathophysiological and biochemical phenotypes of GSD I subtypes in the liver.},
}
@article {pmid42455481,
year = {2026},
author = {Marques, BS and Mendes, M and Alves, JL and Pais, A and Vitorino, C},
title = {Decoding and Overcoming Temozolomide Resistance Through CRISPR/Cas Technologies.},
journal = {Molecular diagnosis &amp; therapy},
volume = {},
number = {},
pages = {},
pmid = {42455481},
issn = {1179-2000},
support = {2022.06174.PTDC//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UID/PRR/00313/2025//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UID/00313/2025//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; },
abstract = {Intrinsic and acquired resistance to temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma (GBM), is highly common and results in poor clinical outcomes. This review highlights the emerging dual role of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) technologies in addressing this challenge. First, it examines genome-wide CRISPR screens that revealed DNA damage repair networks, stress adaptations, stemness maintenance, and tumor heterogeneity as key drivers of resistance to TMZ. Second, it examines CRISPR/Cas-based strategies, including targeted gene disruption and epigenetic silencing of O[6]-methylguanine-DNA methyltransferase (MGMT), to restore TMZ sensitivity. Finally, it explores CRISPR/Cas-engineered brain tumor models. Alongside these approaches, CRISPR/Cas technologies highlight the value of decoding the multifactorial basis of TMZ resistance and guiding rational therapeutic strategies. Continued refinement of CRISPR/Cas tools may ultimately contribute to more effective treatments for GBM.},
}
@article {pmid42455488,
year = {2026},
author = {Wu, H and Jiang, F and Tian, S},
title = {Research progress on detection technologies for Mycoplasma pneumoniae.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {42455488},
issn = {1435-4373},
abstract = {Mycoplasma pneumoniae (M. pneumoniae) is a primary pathogen responsible for community-acquired pneumonia (CAP), particularly prevalent among children and adolescents. The recent global resurgence of infection cases, coupled with the rapid dissemination of macrolide-resistant M. pneumoniae (MRMP), underscores the critical clinical need for rapid and precise diagnostic technologies. This article systematically reviews the evolutionary trajectory of detection technologies, covering the transition from traditional culture and serological testing to modern molecular diagnostic techniques. It critically analyzes emerging platforms, including isothermal amplification, CRISPR/Cas-based diagnostics, microfluidic chips, and biosensors. It explores their potential in facilitating point-of-care testing (POCT) and simultaneous antimicrobial resistance profiling. Despite continuous technological advancements, challenges remain regarding the differentiation between active infection and colonization, as well as the balancing of cost-effectiveness. The future of M. pneumoniae diagnostics lies in the deep integration of multidisciplinary biotechnologies with artificial intelligence, aiming to construct intelligent "sample-to-answer" solutions to optimize clinical antimicrobial stewardship.},
}
@article {pmid42455881,
year = {2026},
author = {Gao, X and Hao, J and Lu, S and Wang, S and Sun, Y and Ke, X and Gao, X and Su, Y and Sun, Y and Tian, Y and Yan, W and Wang, J and Zheng, Z and Hai, R and Zhang, Q and Wang, J and Hu, W and Wang, G},
title = {Genome-wide CRISPR screen reveals PEX11B as a host restriction factor against ORFV through membrane fluidity regulation.},
journal = {PLoS pathogens},
volume = {22},
number = {7},
pages = {e1013767},
pmid = {42455881},
issn = {1553-7374},
mesh = {Animals ; Sheep ; *Membrane Fluidity ; *Host-Pathogen Interactions ; *Parvoviridae Infections/genetics/virology/metabolism/veterinary ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Virus Replication ; Male ; Clustered Regularly Interspaced Short Palindromic Repeats ; Antigens, Differentiation ; },
abstract = {Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.},
}
@article {pmid42457405,
year = {2026},
author = {Hong, JF and Zou, QL and Xie, XY and Jiang, YP and Wang, SY and Ling, X and Zhou, C and Cai, X and Yang, YX and Chen, Y and Sun, W and Chen, B and Qiao, L},
title = {Engineered promoter system enables high-efficiency transgenic CRISPR editing in Malaria transmitting mosquito Anopheles sinensis.},
journal = {Zoological research},
volume = {47},
number = {4},
pages = {1045-1058},
doi = {10.24272/j.issn.2095-8137.2026.040},
pmid = {42457405},
issn = {2095-8137},
mesh = {Animals ; *Anopheles/genetics ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; Female ; *Gene Editing/methods ; Malaria/transmission ; *Mosquito Vectors/genetics ; },
abstract = {The binary CRISPR/Cas9 system deployed through crosses of transgenic lines facilitates efficient mutagenesis, but its application in non-model insects remains limited by the scarcity of validated species-specific regulatory elements. In the malaria vector Anopheles sinensis, we screened three germline-biased promoters (Asvasa2, Aszpg, Asnanos) for Cas9 expression, and found that Asvasa2 drove the highest editing efficiency with respect to target site mutagenesis. For gRNA transcription, comparative analysis identified AsU6-1 as the most active of four endogenous U6 promoters. Crossing stable transgenic lines harboring these components yielded F 1 progeny with complete germline editing penetrance at the Aswhite locus, a phenotype inherited in the F 2 generation. Quantitative sequencing of F 1 ovaries confirmed near-saturation (>99%) targeted mutagenesis using the optimal Asvasa2/ AsU6-1 combination, whereas alternative promoters showed markedly lower mutagenesis efficiency. Functional validation through knockout of Asdsx- F, a key sex differentiation regulator, efficiently induced complete female-to-male sexual reversal and sterility. This study provides a foundational genetic toolkit for genome engineering in this vector species, as well as an effective reference for binary transgenic manipulation in non-model insects.},
}
@article {pmid41307817,
year = {2026},
author = {Zhang, F and Lu, Q and Qian, X and Xing, Y and Wang, W},
title = {Development of Knockout Cardiac Muscle Cell Lines Using Integrase-Deficient Lentivirus-Mediated CRISPR/Cas9 Gene Editing.},
journal = {Biochemical genetics},
volume = {64},
number = {4},
pages = {5394-5413},
pmid = {41307817},
issn = {1573-4927},
support = {82101314 to Y. X.; 81772559 to W. W//National Natural Science Foundation of China (NSFC) grants/ ; },
mesh = {*Lentivirus/genetics ; *CRISPR-Cas Systems ; *Myocytes, Cardiac/metabolism/cytology ; Animals ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Integrases/genetics/deficiency ; Cell Line ; Humans ; TRPM Cation Channels/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Vectors ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a highly efficient genome editing tool that can genetically disrupt genes and genetic elements, making it a timely, cost-effective, and powerful tool for studying gene function. The success of gene editing depends on the ability to introduce CRISPR components, including guide RNA (gRNA) and Cas9 nuclease, into the target cell, which is challenging in numerous difficult-to-transfect cell types, such as cardiomyocytes. Lentiviral vectors (LVs) are among the primary delivery methods for the CRISPR/Cas9 system as they can stably maintain robust expression in various dividing and non-dividing cells. However, stably integrated LVs consistently express CRISPR/Cas9 components at high levels, rendering them susceptible to off-target effects. New-generation integrase-deficient LV (IDLV) offers an attractive alternative approach for delivering CRISPR/Cas9 components. This study constructed transient receptor potential cation channel mucolipin subfamily member 1 gene knockout models in H9C2 cell lines using IDLVs. Strategies for gRNA design and screening, the IDLV packaging process, CRISPR delivery, and knockout validation are outlined. These protocols will assist researchers in the application of CRISPR technology to study gene function in mammalian cells.},
}
@article {pmid42020342,
year = {2026},
author = {He, Z and Liu, M and Zhang, N and Yan, J and Li, F and Zhao, P and Guo, C},
title = {A CRISPR-Cas9 screen identifies LAPTM4A (lysosomal protein transmembrane 4 alpha) as a key host barrier against PRRSV infection.},
journal = {Autophagy},
volume = {22},
number = {8},
pages = {1882-1902},
doi = {10.1080/15548627.2026.2664607},
pmid = {42020342},
issn = {1554-8635},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Autophagy/genetics ; Lysosomes/metabolism ; *Porcine respiratory and reproductive syndrome virus/physiology ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/genetics/metabolism ; Humans ; Host-Pathogen Interactions/genetics ; Virus Replication ; Signal Transduction ; Ubiquitination ; HEK293 Cells ; Sequestosome-1 Protein/metabolism ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) manipulates host intracellular processes, particularly macroautophagy/autophagy and lysosomal function, to facilitate its replication and spread. However, the precise host factors and molecular mechanisms by which PRRSV remodels the autophagy-lysosome axis remain poorly defined. Here, we performed a CRISPR-Cas9 knockout screen targeting 1,332 genes involved in protein degradation, metabolism, and vesicular trafficking, and identified LAPTM4A (lysosomal protein transmembrane 4 alpha) as a critical antiviral factor involved in the lysosomal pathway. A yeast two-hybrid screen identified LAPTM4A as an interactor of PRRSV GP5 (glycoprotein 5). Mechanistically, GP5 recruits the E3 ubiquitin ligase NEDD4 and the autophagy receptor SQSTM1/p62 to promote K63-linked polyubiquitination of LAPTM4A, leading to its autophagic degradation. This selective degradation activates the AMPK-ULK1-MAP1LC3/LC3 signaling cascade, initiating autophagy while facilitating MTOR-lysosome colocalization, thereby suppressing TFEB nuclear translocation and transcription of lysosome-related genes. The resulting incomplete autophagic flux enhances viral replication. Additionally, in terms of host defense, LAPTM4A maintains lysosomal homeostasis by restraining excessive autophagy through AMPK-ULK1-LC3 signaling and promoting TFEB-dependent lysosomal gene expression by impairing the binding of RPTOR/raptor to MTOR, thus providing broad antiviral protection against multiple RNA viruses. Collectively, our findings identify LAPTM4A as a central regulator of lysosome-autophagy homeostasis and reveal a viral strategy that dismantles this defense axis to facilitate infection.Abbreviations: ATG5: autophagy related 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; Baf A1: bafilomycin A1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DMVT library: protein degradation, metabolism, and vesicular trafficking library; LAPTM4A: lysosomal protein transmembrane 4 alpha; MAGeCK: model-based analysis of genome-wide CRISPR-Cas9 knockout; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NC: negative control; PAMs: porcine alveolar macrophages; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PRRSV: porcine reproductive and respiratory syndrome virus; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.},
}
@article {pmid42283463,
year = {2026},
author = {Sugiokto, FG and Liu, Y and Li, R},
title = {Proteomic screening identifies HNRNPA2B1 as an epigenetic repressor of Epstein-Barr virus reactivation.},
journal = {Journal of virology},
volume = {100},
number = {7},
pages = {e0061326},
doi = {10.1128/jvi.00613-26},
pmid = {42283463},
issn = {1098-5514},
mesh = {Humans ; *Herpesvirus 4, Human/physiology/genetics ; *Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism/genetics ; *Epigenesis, Genetic ; Proteomics/methods ; *Virus Activation/genetics ; Histone Demethylases/metabolism/genetics ; Promoter Regions, Genetic ; Gene Expression Regulation, Viral ; Trans-Activators/genetics/metabolism ; Virus Latency/genetics ; *Epstein-Barr Virus Infections/virology/genetics/metabolism ; Histones/metabolism ; Immediate-Early Proteins/genetics/metabolism ; Chromatin Immunoprecipitation ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Cell Line, Tumor ; },
abstract = {Epstein-Barr virus (EBV) establishes lifelong persistent infection in over 90% of the world's population. The virus persists as an episome in the host cells during latency and periodically reactivates through transcriptional activation of the immediate-early (IE) genes. While epigenetic regulation is central to maintaining viral latency, the host factors that enforce repression at these promoters remain incompletely defined. Here, we employed a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation coupled with mass spectrometry (enChIP-MS) approach to identify proteins associated with the promoter of EBV IE gene ZTA. This approach revealed an enrichment of multiple heterogeneous nuclear ribonucleoproteins and identified HNRNPA2B1 as a potential regulator of EBV ZTA gene expression. Functional analyses across multiple EBV+ cancer cell models demonstrated that HNRNPA2B1 acts as a restriction factor for EBV lytic reactivation. Depletion of HNRNPA2B1 led to increased expression of IE and downstream lytic genes, enhanced RNA polymerase II recruitment to the ZTA and RTA promoters, and elevated the proportion of cells entering the lytic cycle. Conversely, enforced expression of HNRNPA2B1 suppressed EBV lytic reactivation. Mechanistically, HNRNPA2B1 enhances repressive viral chromatin states by facilitating recruitment of the histone demethylase LSD1 to EBV IE gene promoters, thereby limiting the activating histone H3 lysine 4 trimethylation. Together, these findings identify HNRNPA2B1 as a key epigenetic regulator of EBV latency and link RNA-binding proteins to epigenetic control of viral reactivation.IMPORTANCEThis study identifies HNRNPA2B1 as a previously unrecognized host factor that promotes Epstein-Barr virus (EBV) latency through direct regulation of viral chromatin at immediate-early gene promoters. By integrating locus-specific chromatin proteomics with functional and mechanistic analyses, our work reveals how an RNA-binding protein HNRNPA2B1 recruits a histone-modifying enzyme to control EBV reactivation. These findings provide new insights into host-virus interactions that control EBV latency and reactivation and highlight the role of RNA-binding proteins in chromatin regulation that may be broadly relevant to other latent DNA viruses.},
}
@article {pmid42313514,
year = {2026},
author = {Xu, Y and Kou, S and Huang, X and Cui, D and Guo, Y and Sun, F},
title = {CRISPR-Based Programmable RNA-Responsive Protein Materials.},
journal = {ACS macro letters},
volume = {15},
number = {7},
pages = {1005-1012},
doi = {10.1021/acsmacrolett.6c00237},
pmid = {42313514},
issn = {2161-1653},
mesh = {Pseudomonas aeruginosa ; *CRISPR-Cas Systems ; Hydrogels/chemistry ; *RNA, Viral/analysis/genetics ; Biofilms ; },
abstract = {With the rapid expansion of RNA biology and associated biotechnologies, smart materials with programmable RNA responsiveness offer immense opportunities for biosensing, diagnostics, and therapeutics. Here, we present a programmable RNA-responsive protein material system leveraging CRISPR-Cas7-11, an RNA-guided protease complex. By immobilizing the protease complex and cleavable payload proteins onto protein scaffolds via SpyTag/SpyCatcher chemistry, we developed two platforms: (1) synthetic spider-silk fibers and (2) protein hydrogels. These materials enable sequence-specific RNA detection, triggering the controlled release of payloads such as GFP or the biofilm-degrading enzyme PslG. Applications demonstrated include viral RNA sensing and Pseudomonas aeruginosa detection with targeted biofilm degradation.},
}
@article {pmid42424186,
year = {2026},
author = {Yang, Y and Pan, Q and Liu, M and Zeng, L and Zou, H and Liu, S and Fu, S and Xu, Y and Dai, Z and Fang, S and Pan, Y},
title = {Pan-Cancer Liquid Biopsy and Treatment Monitoring via a Split crRNA-Activated Label-Free CRISPR/Cas12a Platform for Ultrasensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {28},
pages = {20968-20977},
doi = {10.1021/acs.analchem.6c02601},
pmid = {42424186},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/blood/genetics/analysis ; Liquid Biopsy/methods ; Limit of Detection ; *Neoplasms/diagnosis ; Biosensing Techniques/methods ; Cell Line, Tumor ; },
abstract = {Liquid biopsy based on circulating microRNAs (miRNAs) holds great promise for cancer diagnosis and treatment monitoring. However, the development of detection methods that are sensitive, specific, cost-effective, and compatible with diverse biofluids remains a challenge. Here, we report a sensitive and label-free detection platform, termed SCAN (Split crRNA-Activated CRISPR/Cas12a and Amplification Network), that integrates split CRISPR/Cas12a with catalytic hairpin assembly (CHA) for isothermal miRNA analysis. In this design, the target miRNA, serving as an alterable spacer RNA (sRNA), assembles with a conserved repeat RNA (rRNA) to reconstitute a functional full-length crRNA, activating the trans-cleavage activity of Cas12a. This cleaves a blocker probe and releases an initiator strand, which subsequently triggers a CHA cascade. The CHA reaction generates abundant G-quadruplex (G4) structures that bind specifically to N-methylmesoporphyrin IX (NMM), yielding a strong turn-on fluorescence signal. The optimized "signal-on" model achieved a detection limit of 2 fM for miR-21, offering approximately 5 orders of magnitude higher sensitivity than the basic split CRISPR/Cas12a system. The platform exhibited excellent specificity, capable of single-base mismatch discrimination, and could be readily adapted for detecting miR-128, miR-27a, and miR-155 through simple exchange of the double-stranded DNA activator. Importantly, by employing the label-free G4/NMM reporter, the cost of the signaling module was reduced by more than 45-fold compared to conventional dual-labeled probes. The SCAN platform reliably quantified miR-21 overexpression in colon cancer cell lines and robustly differentiated plasma samples from patients with multiple cancer types (colorectal, lung, cervical, breast, and thyroid cancers) from healthy individuals. Furthermore, it demonstrated utility in tracking treatment response through noninvasive urine analysis in prostate cancer and bladder cancer. This work establishes a sensitive, specific, low-cost, and versatile biosensing platform for miRNA-based liquid biopsy, holding strong potential for clinical diagnostic applications.},
}
@article {pmid42424600,
year = {2026},
author = {Li, X and Gao, X and Gu, T and Li, Q and Wang, L and Deng, F and Guo, M and Huo, D and Hou, C},
title = {Tribos: A Modular Hairpin-Enhanced CRISPR/Cas12a Biosensor for Ultrasensitive Detection of HER2 Protein.},
journal = {Analytical chemistry},
volume = {98},
number = {28},
pages = {21055-21063},
doi = {10.1021/acs.analchem.6c03581},
pmid = {42424600},
issn = {1520-6882},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Female ; Breast Neoplasms/diagnosis ; Limit of Detection ; Aptamers, Nucleotide/chemistry/genetics ; Molecular Docking Simulation ; *Bacterial Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Inverted Repeat Sequences ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of human epidermal growth factor receptor 2 (HER2) is critical for early breast cancer screening and personalized therapy. This study constructed a target-triggered, hairpin-enhanced CRISPR/Cas12a biosensor named "Tribos" for ultrasensitive HER2 detection. The system integrates an aptamer hairpin switch (HAS), HAS-allosterically triggered rolling circle amplification (RCA), and a hairpin-enhanced CRISPR/Cas12a fluorescence reporter module. Taking advantage of Cas12a's high affinity for stem-loop structures, we designed a double-stem-loop reporter probe (DS-FQ) and validated its trans-cleavage enhancement mechanism via molecular docking. Under optimal conditions, Tribos exhibited a linear range from 10 fg/mL to 10 ng/mL, with a limit of detection as low as 1.08 fg/mL. In clinical validation with 29 breast cancer patients and 13 healthy controls, the sensor achieved a sensitivity of 82.76% and a specificity of 100%, which were highly consistent with clinical diagnoses and ELISA results, and it effectively distinguished different HER2 expression levels. The modular design of Tribos offers a new strategy for high-performance CRISPR diagnostics and lays a foundation for next-generation molecular diagnostic technologies based on nucleic acid conformational regulation.},
}
@article {pmid42451761,
year = {2026},
author = {Yilmaz, I and Yoğurtçu, BM and Aisida, S and Ezer, EB},
title = {Next-Generation Strategies to Encounter Antimicrobial Resistance (AMR): From Lariocidin to Gene Editing and Nanotechnology-Based Approaches.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {13},
pages = {},
pmid = {42451761},
issn = {1420-3049},
mesh = {*Gene Editing/methods ; *Anti-Bacterial Agents/pharmacology/chemistry ; *Nanotechnology/methods ; *Drug Resistance, Bacterial/drug effects/genetics ; Humans ; Biofilms/drug effects ; CRISPR-Cas Systems ; Nanoparticles/chemistry ; },
abstract = {The escalation of antimicrobial resistance (AMR) represents a serious global threat to public health, with AMR-associated mortality estimated to increase by 70% by 2050. As pathogens evolve through enzymatic inactivation, target modification, efflux-mediated clearance, biofilm formation, and broader genetic adaptation, conventional therapies are increasingly compromised, while the antibiotic development pipeline remains critically constrained by high discovery and development costs, weak commercial incentives, and the escalating complexity of resistance mechanisms. This review comprehensively synthesizes advanced pharmacological and biotechnological innovations designed to circumvent these entrenched resistance mechanisms. We highlight the development of novel therapeutic classes, particularly lariocidin, which disrupts bacterial protein synthesis via a previously unexploited ribosomal-binding site. Moreover, we critically evaluate molecular interventions, emphasizing CRISPR/Cas-based gene silencing and genome editing as precise tools to neutralize specific resistance determinants, such as the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). Concurrently, we explore the integration of engineered nanoparticles to revitalize existing antimicrobials by overcoming biofilm barriers, improving drug solubility, and enabling targeted delivery. Collectively, mastering the evolving AMR landscape requires a multidimensional framework that seamlessly integrates these novel molecular targets with advanced rapid diagnostics and robust international governance.},
}
@article {pmid42452230,
year = {2026},
author = {Song, S and Fan, X and Zhang, N and Lin, N and Wang, G},
title = {Molecular Crosstalk Between Flowering Time and Drought Adaptation in Cereal Crops.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {13},
pages = {},
pmid = {42452230},
issn = {2223-7747},
support = {32501990//National Natural Science Foundation of China/ ; 242300421572//Natural Science Foundation of Henan Province/ ; 242300421571//Natural Science Foundation of Henan Province/ ; },
abstract = {Increasingly frequent and severe drought events restrict global agricultural productivity. As sessile organisms, cereal crops have evolved phenotypic plasticity, drawing on drought escape (DE) and drought avoidance (DA) strategies to balance survival and reproduction. While the mechanisms governing photoperiodic flowering and drought responses are well characterized individually, their molecular intersection remains poorly understood. This review summarizes recent advances in the crosstalk between these two pathways. We highlight the divergent roles of core genetic hubs, such as florigen regulation, GIGANTEA (GI), DELLA proteins, and dual-function transcription factors (e.g., ZmCCT, Ghd7, Ppd-H1), and the breeding-selected alleles, including Green Revolution variants, that can partly uncouple stress tolerance from developmental penalties, though trade-offs often remain. Furthermore, we examine the internal networks driving this crosstalk, including circadian clock phase shifts, sugar and energy signaling through the trehalose-6-phosphate (T6P)-SNF1-related protein kinase 1 (SnRK1) module, and the antagonistic balance within phytohormone networks centered on abscisic acid (ABA). Finally, we propose that integrating epigenetic stress memory, systemic root-to-shoot signaling, and targeted CRISPR/Cas promoter engineering provides a useful conceptual framework for breeding climate-resilient, yield-stable crops.},
}
@article {pmid42452277,
year = {2026},
author = {Kim, HJ and Chae, J and Han, SJ and Kim, JH and Chung, YS and Karthik, S and Heo, JB},
title = {AI-Guided DNA-Free and Genotype-Independent Genome Editing for Soybean Improvement.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {13},
pages = {},
pmid = {42452277},
issn = {2223-7747},
abstract = {Soybean is a strategic crop for global protein and vegetable oil supply chains; however, genetic improvement remains constrained by genotype-dependent regeneration, variable transformation efficiency, and regulatory concerns regarding stable transgene integration. This review synthesizes emerging DNA-free and genotype-independent genome-editing frameworks for soybean, where genotype independence is defined as the ability to recover fertile, non-chimeric edited plants across elite germplasm. We critically examine the soybean genome-editing toolbox, including CRISPR-Cas9, Cas12a, multiplex editing systems, base editing, and prime editing, and discuss persistent bottlenecks associated with target selection, off-target assessment, editability, and plant recovery. Particular emphasis is placed on artificial intelligence (AI)-assisted approaches that integrate genomic, epigenomic, chromatin-accessibility, and multi-omics datasets to improve target prioritization, guide RNA design, off-target prediction, and locus- and genotype-specific editability assessment. We further evaluate DNA-free genome-editing technologies, including CRISPR-Cas ribonucleoproteins, transient RNA-based systems, and nanocarrier-mediated delivery platforms, highlighting their potential to generate non-integrative edits while reducing prolonged nuclease exposure. In addition, we discuss regeneration reprogramming strategies based on developmental regulators and morphogenic modules, including BBM-WUS, GRF-GIF, de novo meristem induction, and somatic embryogenesis, as enabling technologies for overcoming cultivar-dependent regeneration barriers. Importantly, this review proposes an integrated AI-to-field framework that connects target discovery, editability prediction, DNA-free editing, regeneration reprogramming, phenotypic validation, and breeding deployment into a unified soybean improvement pipeline. We further highlight emerging opportunities in multi-omics-guided target discovery, genotype-aware prediction models, regeneration-aware editing strategies, and closed-loop machine-learning systems that continuously improve editing decisions through experimental feedback. Collectively, these convergent innovations provide a practical foundation for accelerating the development of climate-resilient, nutritionally enhanced, and industry-ready soybean cultivars.},
}
@article {pmid41872727,
year = {2026},
author = {Sivaprakasam, M and Jeanpierre, AR and Mohammed, S and Srinivasan, R and Mohanty, AK},
title = {Zebrafish and CRISPR-A synergistic approach to decipher and cure human diseases.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {6},
pages = {1167-1179},
doi = {10.1002/ame2.70141},
pmid = {41872727},
issn = {2576-2095},
mesh = {Animals ; *Zebrafish/genetics ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Genetic Diseases, Inborn/therapy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Rapidly emerging infectious and genetic diseases demand robust vertebrate models to investigate pathogenesis and accelerate therapeutic discovery. Zebrafish (Danio rerio) offer substantial translational value owing to their conserved physiology, optical transparency, rapid reproduction, and the presence of orthologs for approximately 70% of human genes and approximately 82% of disease-associated genes. The integration of CRISPR/Cas9 technology has transformed zebrafish research, enabling efficient generation of targeted knockouts, knockins, and high-throughput mutagenesis screens. This synergy supports mechanistic dissection and modeling of cardiovascular, oncologic, viral, and other genetic disorders. Despite these advantages, rigorous allele validation, consideration of paralog redundancy, maternal contribution, and off-target analysis remain essential to ensure translational accuracy. This review summarizes current applications, methodological advances, limitations, and best-practice recommendations for combining zebrafish models with genome editing to improve understanding and treatment of human diseases.},
}
@article {pmid42185265,
year = {2026},
author = {Li, XQ and Li, XY and Chen, WF and Xu, ZY and Liu, ZQ and Wang, Y and Zhang, JY and Gu, YY and Yao, L and Tan, YF and Chen, XJ and Deng, B and Wang, KH and Xu, JQ and He, MJ and Geng, ZH and Fan, KY and Zhang, ZC and Wang, L and Xiang, AY and Pan, HT and Hu, ZB and Xie, YL and Wang, C and Zhou, PH and Li, QL},
title = {A frameshift variant in FAM129C contributes to achalasia through B cell responses against the GABAA receptor.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42185265},
issn = {2041-1723},
support = {82570629//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82500618//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Esophageal Achalasia/genetics/immunology/pathology ; *Frameshift Mutation ; Mice ; *B-Lymphocytes/immunology/metabolism ; Disease Models, Animal ; Humans ; Female ; Esophageal Sphincter, Lower/immunology/pathology ; Male ; CRISPR-Cas Systems ; Autoantibodies/immunology ; Lymphocyte Activation ; Mice, Inbred C57BL ; },
abstract = {Achalasia is a rare esophageal motility disorder of poorly understood etiology. Here, we perform a large trio-based whole-genome sequencing study of achalasia and identify a recessively inherited frameshift variant in FAM129C (p.Ala454fs). A CRISPR/Cas9-engineered Fam129c-mutant mouse model recapitulating key features of achalasia was established, including growth retardation, elevated lower esophageal sphincter (LES) pressure, and selective loss of inhibitory neurons. Multi-omic analyses revealed substantial B cell expansion and activation within the LES, accompanied by enhanced humoral immune responses. Time-course experiments demonstrated that B cell accumulation preceded overt neuronal loss, while B cell depletion via anti-CD20 antibodies or intravenous immunoglobulin treatment partially rescued the phenotypes. Further protein profiling and cell-based assays suggested that the GABAA receptor may represent one potential neuronal antigen targeted by circulating autoantibodies. Together, these findings identify FAM129C as a genetic contributor to achalasia and support a neuroimmune mechanism in which B cell activation and autoantibody-mediated responses contribute to inhibitory neuronal injury. These results provide important insights into achalasia pathogenesis and highlight the potential of immunomodulatory strategies for disease intervention in the early stage.},
}
@article {pmid42225068,
year = {2026},
author = {Fenoglio, S and Yu, Y and Tepper, J and Grove, L and Bejnood, A and Meier, SR and Choi, AH and Wu, HJ and Devault, A and Liu, S and Shen, B and Khendu, T and Stowe, H and Uijttewaal, ECH and Zhang, M and Haines, BB and Wilker, E and Huang, A and Schramek, D and Elling, U and Pan, X and Andersen, JN and Teng, T},
title = {Temporal control of sgRNA library activation unlocks large-scale in vivo CRISPR screens.},
journal = {Cell reports methods},
volume = {6},
number = {7},
pages = {101470},
doi = {10.1016/j.crmeth.2026.101470},
pmid = {42225068},
issn = {2667-2375},
mesh = {Humans ; Animals ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Reproducibility of Results ; },
abstract = {CRISPR-StAR (stochastic activation by recombination) is an inducible pooled screening system that activates gene knockout after tumor engraftment and provides matched internal controls for guide-level normalization. In this study, we explore the scalability and reproducibility of this approach for in vivo cancer screens. Through barcode-embedded sequencing and the development of a Bayesian analysis pipeline, we screened a 30,000-sgRNA library in A549 xenografts, achieving reproducible dropout and enrichment phenotypes using just ∼30 tumors. Across additional xenograft models, single tumors yielded reliable functional annotation for ∼1,000 genes. Comparing in vivo and in vitro screens uncovered tumor suppressor effects detectable only in vivo; for example, KMT2C and KMT2D knockouts produced contrasting growth and transcriptional programs. Together with our R analysis package, we show that CRISPR-StAR enables scalable in vivo dependency mapping that complements in vitro resources and reduces animal use by up to 7-fold versus conventional dropout screens, improving methodological rigor at genome-scale clonal resolution.},
}
@article {pmid42391025,
year = {2026},
author = {Bakhtiyari, N and Masoudi-Sobhanzadeh, Y and Farajnia, S and Kumar, S},
title = {An interpretable deep learning framework uncovers features governing CRISPR-Cas9 genome-editing efficiency.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {7},
pages = {},
doi = {10.1093/bioinformatics/btag483},
pmid = {42391025},
issn = {1367-4811},
support = {//Princess Margaret Cancer Foundation, Canada Research Chair Program/ ; //Terry Fox Research Institute/ ; 7130//Drug Applied Research Center, Tabriz University of Medical Sciences/ ; },
mesh = {*Deep Learning ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome ; *Computational Biology/methods ; *Gene Editing/methods ; },
abstract = {MOTIVATION: CRISPR-Cas9 genome-editing efficiency is strongly influenced by the sequence composition and positional context of single-guide RNAs (sgRNAs). Although numerous deep learning-based models have been developed to predict Cas9 efficiency from sgRNA sequences, most operate as black boxes, offering limited insight into the sequence determinants underlying Cas9 activity. In addition, previous studies often overlook how the positional context of sequence motifs within sgRNAs influences their effects on Cas9 binding or cleavage.<br><br>RESULTS: We introduce DeepCC9, an interpretable machine learning framework that combines explicit sequence feature extraction with a residual block-based deep architecture to improve interpretability and identify composition- and position-based motifs governing Cas9 genome-editing efficiency. We applied this method to multiple Cas9 variant datasets, achieving superior predictive performance compared with existing methods while enabling direct interpretation of sequence motifs and their positional effects. Our analysis uncovered 74 sequence motifs enriched or depleted at specific positions within sgRNAs and strongly associated with Cas9 efficiency, providing mechanistic insight into sequence features that influence guide performance. Together, these results establish DeepCC9 as a generalizable and interpretable framework for modeling sequence-function relationships and advancing the understanding of the sequence determinants underlying CRISPR-Cas9 genome editing.<br><br>The authors have implemented their algorithm in the Python programming language (version 3.X), which is accessible using (https://zenodo.org/records/20073890).},
}
@article {pmid42450220,
year = {2026},
author = {Haratau, JIC and Niculescu, LS and Barbalata, T and Sanda, GM and Fuior, EV and Sasson, S and Sima, AV and Stancu, CS and Toma, L},
title = {Longstanding Transcriptional Activation of APOA1 and PON1 in Human Hepatocytes by CRISPR/dCas9 Technology: Transcriptomic Profile and Crosstalk with Endothelial Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450220},
issn = {1422-0067},
support = {PNRR-III-C9-2022-I8-197//EU - PNRR Programme/ ; PN-IV-P6-6.1-CoEx-2024-0029//Ministry of Education and Research, CCCDI - UEFISCDI/ ; },
mesh = {Humans ; *Apolipoprotein A-I/genetics/metabolism ; *Aryldialkylphosphatase/genetics/metabolism ; *Hepatocytes/metabolism ; Human Umbilical Vein Endothelial Cells/metabolism ; *Transcriptional Activation ; *Transcriptome ; *CRISPR-Cas Systems ; Oxidative Stress ; Tumor Necrosis Factor-alpha ; Endothelial Cells/metabolism ; Gene Expression Profiling ; },
abstract = {Apolipoprotein A1 (APOA1) and paraoxonase 1 (PON1) are key proteins of high-density lipoproteins (HDL). The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 longstanding upregulation in hepatocytes that can be further used to decipher the mechanisms of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. The functionality of the secreted APOA1/PON1 was evaluated as the capacity of the conditioned medium (CM) from transfected HuH-7 to modulate the oxidative and inflammatory stress in TNFα-activated primary human umbilical endothelial cells (HUVEC). The results showed that: (1) a robust, longstanding upregulation (46 days) of endogenous APOA1/PON1 was obtained after CRISPR/dCas9 transfection and antibiotics selection; (2) APOA1/PON1 upregulation led to a modified transcriptomic profile and increased the expression of several antioxidant genes in transfected hepatocytes as demonstrated by RNAseq analysis; (3) secreted APOA1/PON1 were functional as demonstrated by the CM ability to reduce the levels of reactive oxygen species and inflammatory markers (VCAM-1, MCP-1) in TNFα-activated HUVEC. In conclusion, we achieved an experimental model of successful longstanding upregulation of endogenous APOA1 and PON1 in human hepatocytes. The targeted proteins are secreted in a functional form and can be used for deciphering their complex mechanism of protective action in various pathological conditions.},
}
@article {pmid42450255,
year = {2026},
author = {Mais, R and Kumar, A and Ahmetaj, A and Burgos-Crespo, G and Sanchez, MM and Roxas, DC and Dcosta, C and Ilyas, A and Hadjiargyrou, M and Zanganeh, S},
title = {Nanoengineering Systems for Gene Therapy: Mechanisms, Modalities, and Future Directions.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450255},
issn = {1422-0067},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Nanotechnology/methods ; Animals ; CRISPR-Cas Systems ; Nanostructures/chemistry ; Gene Transfer Techniques ; },
abstract = {Nanotechnology has become an important platform in the fields of gene therapy and genome editing, providing delivery strategies that address persistent therapeutic challenges by improving the precision, efficiency, and safety of genetic modifications. This review highlights the central role of nanomaterials in overcoming persistent barriers to genetic interventions, including inefficient delivery, instability of genetic cargo, and off-target effects. Specifically, we emphasize the combined use of nanomaterials with clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems, which can improve editing specificity and therapeutic efficacy. Beyond the classical CRISPR/Cas9 platform, this review also discusses next-generation modalities such as base editors, Cas13, prime editing, and the recently described Tandem Interspaced Guide RNA and TIGR-associated protein (TIGR-Tas) system, while considering their therapeutic potential and distinct delivery challenges. By using nanomaterials, the stability and intracellular delivery of genome-editing systems are improved, enabling more effective treatments for genetic disorders and acquired diseases such as cancer and infectious diseases. In addition, nanocarriers provide controlled release, protection from degradation, and better biocompatibility, thereby improving the safety and reliability of gene-editing therapies. Despite these advances, important translational challenges remain, including immunotoxicity, large-scale manufacturing, and regulatory integration. Overall, the continued convergence of nanotechnology and genome engineering may support the development of personalized medicine strategies that adapt genetic engineering tools for patient-specific applications.},
}
@article {pmid42450278,
year = {2026},
author = {Podralska, M and Górska, A and Kaczmarek, M},
title = {Genome Editing Approaches in Flax (Linum usitatissimum L.): From Tools to Trait Improvement.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450278},
issn = {1422-0067},
support = {DHR.hn.070.1.2026//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Flax/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Genome editing, particularly CRISPR/Cas-based systems, has emerged as a key tool for functional genomics and trait improvement in flax (Linum usitatissimum L.), an important fiber and oilseed crop. This review focuses specifically on flax as an emerging target species and distinguishes experimentally validated applications from approaches adapted from model plants. Recent progress includes the characterization of endogenous U6 promoters, which improved guide RNA expression and contributed to enhanced genome editing performance under optimized conditions. Reported studies demonstrate efficient targeted mutagenesis in flax; however, editing outcomes remain strongly dependent on genotype, construct design, and regeneration capacity, and stable homozygous edited lines are still limited. Target genes include pathways involved in lignin and cellulose biosynthesis, fatty acid metabolism, and stress responses, influencing fiber quality, oil composition, and stress adaptation. Despite current bottlenecks such as low homologous recombination efficiency and regeneration constraints, base editing, prime editing, and multiplex CRISPR systems provide promising avenues for precision breeding in flax.},
}
@article {pmid42450173,
year = {2026},
author = {Effah, SN and Barrera, SC and Urturi Ortiz, N and Dampier, W and Nonnemacher, MR and Wigdahl, B},
title = {CRISPR/Cas9-Based Genome Editing: Understanding Differences in DNA Repair Pathways, Profiles, and Outcomes.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450173},
issn = {1422-0067},
support = {MH110360/MH/NIMH NIH HHS/United States ; MH092177/MH/NIMH NIH HHS/United States ; MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *DNA Repair/genetics ; Animals ; Genetic Therapy/methods ; },
abstract = {Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.},
}
@article {pmid42447097,
year = {2026},
author = {Zhang, Y and Xing, J and Zhang, H and Kong, J and Nang, SC and Zhang, M and Pan, Y and Zhai, Y and Yuan, L and Zhao, J and Wu, H},
title = {Disruption of rcnB modulates colistin susceptibility in Acinetobacter baumannii AB5075.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2697100},
pmid = {42447097},
issn = {2150-5608},
mesh = {*Colistin/pharmacology ; *Acinetobacter baumannii/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Bacterial Proteins/genetics/metabolism ; Microbial Sensitivity Tests ; Drug Resistance, Bacterial ; Drug Resistance, Multiple, Bacterial/genetics ; Oxidative Stress ; CRISPR-Cas Systems ; },
abstract = {Acinetobacter baumannii AB5075 is a clinically relevant multidrug-resistant (MDR) isolate that poses a major therapeutic challenge. Although colistin has been reinstated as a last-resort antibiotic against MDR Gram-negative infections, the rapid emergence of colistin resistance threatens its clinical utility. Here, we employed a CRISPR-Cas9-based genome editing system to generate an A. baumannii AB5075 ΔrcnB mutant and uncovered a previously underappreciated role of rcnB in modulating colistin susceptibility. Loss of rcnB markedly potentiated colistin-mediated killing through multiple associated changes, including compromised membrane integrity, impaired oxidative stress defenses, and reduced efflux pump activity. Transcriptomic profiling further revealed that rcnB deletion reshaped global stress-response networks, including suppression of fatty acid biosynthesis and reactive oxygen species (ROS)-detoxifying pathways, alongside altered metal ion and sulfur metabolism during colistin exposure. Collectively, our findings suggest that rcnB may contribute to colistin susceptibility of colistin resistance and provide mechanistic insights that may inform the development of targeted strategies to enhance colistin efficacy against MDR A. baumannii.},
}
@article {pmid42449928,
year = {2026},
author = {Poonooru, R and Park, KE and Schmelzle, A and Telugu, BP},
title = {Functional Inactivation of PAX4 Results in Disrupted Endocrine Pancreas Development and Neonatal Diabetes in Pigs.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42449928},
issn = {1422-0067},
support = {W81XWH-22-1-0017//Congressionally Directed Medical Research Programs/ ; },
mesh = {Animals ; *Paired Box Transcription Factors/genetics/metabolism ; *Islets of Langerhans/metabolism/pathology/growth &amp; development ; Swine ; *Homeodomain Proteins/genetics/metabolism ; Animals, Newborn ; CRISPR-Cas Systems ; *Diabetes Mellitus/genetics/metabolism/pathology ; Humans ; },
abstract = {Variants in the human PAX4 gene are associated with both monogenic and complex forms of diabetes, yet their pathogenic effects remain difficult to define in models that accurately mimic human islet architecture and neonatal metabolic transitions. Here, we created a porcine PAX4 loss-of-function model using CRISPR/Cas9 cytidine deaminase base editing to introduce a premature stop codon in the PAX4 coding sequence. PAX4 knockout piglets developed severe hyperglycemia within 24 h of birth, followed by rapid postnatal clinical deterioration and uniform death by day 3. Biochemical analysis showed significant diabetic decompensation, including electrolyte imbalances, hyperosmolality, azotemia, dyslipidemia, and metabolic acidosis. Gross and histological examinations revealed notable pancreatic hypoplasia with preservation of exocrine tissue. Single-nucleus RNA sequencing and immunohistochemistry demonstrated an almost complete loss of insulin- and somatostatin-producing β- and δ-cells, respectively, with relative preservation of glucagon-expressing α-cells. Overall, these results establish PAX4 as a crucial factor in pancreatic endocrine development and postnatal glucose regulation in a large-animal model. This platform offers a human-relevant system for studying diabetes-associated PAX4 variants and for testing regenerative and gene-based therapies for insulin-deficient diabetes.},
}
@article {pmid42450131,
year = {2026},
author = {Samoń, M and Przyborowski, M},
title = {Wheat's Up with CRISPR-Cas-Current Advances, Obstacles and Perspectives.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450131},
issn = {1422-0067},
support = {Dotacja Celowa task 4.1//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Triticum/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Genetic Engineering/methods ; },
abstract = {The emergence of CRISPR-Cas editing systems-comprising clustered regularly interspaced short palindromic repeats and associated Cas proteins-marked a breakthrough in genetic engineering, owing to the simplicity, efficiency, and adaptability of the method. Despite continuous improvements and the incorporation of innovative discoveries to develop reliable, fine-tuned tools, the effective application of CRISPR-Cas technology in cereals remains challenging. This review provides a technically oriented overview of CRISPR-Cas-mediated genome editing in wheat (Triticum aestivum L.), one of the world's fundamental crops. While focusing on established solutions and progressive methodological modifications, we also discuss pertinent topics, including plant genetic transformation, prospective innovations, and compliance considerations.},
}
@article {pmid41168295,
year = {2026},
author = {Yeo, JH and Lee, S and Kim, S and Min, JG and Gopalappa, R and Oh, HC and Kim, HK and Nam, EJ and Kim, HH},
title = {High-throughput evaluation of in vitro CRISPR activities enables optimized large-scale multiplex enrichment of rare variants.},
journal = {Nature biomedical engineering},
volume = {10},
number = {7},
pages = {1410-1430},
pmid = {41168295},
issn = {2157-846X},
support = {RS-2022-NR070713, 2018R1A5A2025079, RS-2022-NR067326, RS-2022-NR067345, RS-2023-00260968//National Research Foundation of Korea (NRF)/ ; 2021R1I1A1A01047269//National Research Foundation of Korea (NRF)/ ; RS-2023-NR076625//National Research Foundation of Korea (NRF)/ ; 2024-22-0165//Yonsei University/ ; 22B-000-0101//Seoul National University Hospital (SNUH)/ ; RS-2024-00467177//Korea Drug Development Fund (KDDF)/ ; 1730158, 1540112, 1541349, 1826967, 2138811, 2112167, 2100237, 2120019, 1419152, 1743354, 2027170//National Science Foundation (NSF)/ ; 6-2019-0166//Yonsei University | Yonsei University College of Medicine (YUCM)/ ; },
mesh = {RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Base Sequence ; Genetic Variation ; },
abstract = {Previous high-throughput evaluations of CRISPR activities for a large number of target and guide RNA sequences were based on measuring insertion-deletion frequencies rather than cleavage efficiencies. Here we develop two high-throughput in vitro methods, Cut-seq1 and Cut-seq2, to evaluate Cas9 cleavage efficiency for tens of thousands, or even hundreds of thousands, of guide RNA-target pairs. These methods reveal low correlations between in vitro cleavage efficiencies and insertion-deletion frequencies in cells, yet high concordances in protospacer adjacent motif compatibility. Using the resulting large datasets of in vitro cleavage efficiencies, we develop DeepCut, a set of deep learning models that can identify optimized single-guide RNAs that can selectively cleave specific sequences, even in the presence of similar noise sequences. Using these optimized single-guide RNAs, we develop a method, CLOVE-seq (which stands for cleavage for large-scale optimized variant enrichment sequencing), to enrich rare variants in a multiplexed manner by Cas9-mediated specific cleavage of noise or rare variant sequences. Our methods can enhance the understanding of CRISPR nuclease activities and could be used to detect a large number of rare variants in various biomedical contexts.},
}
@article {pmid41947506,
year = {2026},
author = {Park, JC and Song, Y and Choi, HW and Sung, J and Jin, H and Gwak, W and Yoo, K and Lee, S and Park, J and Kim, J and Jo, HJ and Koo, J and Jeong, Y and Lee, KH and Kee, SJ and Kim, H},
title = {Viral Infection-Inspired Autonomous Detection of Fusion-Competent Viruses for Screening and Environmental Surveillance.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {40},
pages = {e21241},
doi = {10.1002/adma.202521241},
pmid = {41947506},
issn = {1521-4095},
support = {2021R1A2C2013961//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; 2022R1C1C2007002//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; RS-2024-00345402//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; RS-2023-00209955//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; Institutional 2E33111//Korea Institute of Science and Technology (KIST)/ ; RS-2024-00396818//Korea Institute of Planning and Evaluation for Technology in Food, Agricultural and Forestry (IPET) through High-Risk Animal Infectious Disease Control Technlogy Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)/ ; 6634-332//The Korea Disease Control and Prevention Agency (KDCA)/ ; RS-2025-25409767//The Materials &amp; Components Technology Development Program funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea)/ ; },
mesh = {*Environmental Monitoring/methods ; Humans ; RNA, Viral/analysis ; SARS-CoV-2/isolation &amp; purification/genetics ; CRISPR-Cas Systems ; Virus Internalization ; Respiratory Syncytial Viruses/isolation &amp; purification ; Influenza A virus/isolation &amp; purification/genetics ; Liposomes/chemistry ; },
abstract = {The persistent burden of respiratory viruses requires rapid, simple, and robust screening and environmental surveillance technologies that enable widespread and frequent testing. Importantly, these technologies should be based on infectivity-relevant signals, as RNA detection alone has limited correlation with transmission risk. Here, we present a membrane fusion-mediated platform that autonomously detects viruses by recapitulating the native viral entry mechanism. Fusogenic vesicles selectively fuse with fusion-competent viral particles, triggering encapsulated CRISPR-Cas13a components to generate fluorescent signals upon recognition of the released viral RNA. Through an autonomous workflow and accelerated signal generation within a confined vesicle, our platform achieves one-step detection of viruses within 2 min. The assay robustly detects three major respiratory viruses, with analytical sensitivities down to 5 TCID50/mL for RSV and 50 TCID50/mL for SARS-CoV-2 and IAV. Clinical validation with 100 nasopharyngeal samples achieved 91.7% sensitivity. Remarkably, the sprayable format enables large-area surveillance of surface contamination-like luminol revealing hidden bloodstains, it makes invisible viral threats visible. This approach establishes an intuitive real-time detection platform, extending beyond clinical specimens to encompass environmental threats.},
}
@article {pmid42086608,
year = {2026},
author = {Cao, J and Liu, Z and Chen, X and Lan, Z and Liu, L and Zhai, Y and Wang, W and Xue, C and Cheng, H and Yao, Y and Cheng, T and Rao, S},
title = {Engineered dCas12f1-SAM enables robust transcriptional activation and gain-of-function screening in primary human cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42086608},
issn = {2041-1723},
support = {82370118, 82470240 and 82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Transcriptional Activation/genetics ; T-Lymphocytes/metabolism/immunology ; *CRISPR-Cas Systems/genetics ; Transcription Factors/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; Interleukin-2/genetics/metabolism ; Kruppel-Like Transcription Factors/genetics/metabolism ; LIM-Homeodomain Proteins/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Receptors, Chimeric Antigen/metabolism/genetics ; },
abstract = {Despite considerable powers, the application of CRISPR activation (CRISPRa) screens in primary human cells remains a formidable challenge. Here, we develop dCas12f1-SAM, a compact SAM-based transcriptional activation platform, that outperforms existing systems in both immortalized cell lines and primary human T cells and hematopoietic stem/progenitor cells (HSPCs). Using dCas12f1-SAM, we perform a pooled CRISPRa screen targeting 1559 human transcription factors (TFs) in primary human T cells and identify multiple positive regulators of IL-2 expression. We further implement a single-cell CRISPRa screen via our miCROP-seq construct, resolving how these genetic perturbations reshape T cell activation dynamics and drive functionally distinct cellular states. Among the top-ranking genes, we spotlight KLF12 and LHX5, whose overexpression significantly improves antigen-specific responses of chimeric antigen receptor T (CAR-T) cells. Collectively, these findings establish dCas12f1-SAM as a robust transcriptional activation tool, highlighting its potential to advance applications in cellular engineering and immunotherapy.},
}
@article {pmid42103719,
year = {2026},
author = {Bourgeois, W and Rice, HE and Wenge, DV and Perner, F and Yue, H and Regalado, BD and Wan, G and Schroeder, JC and Sommerschield, A and Hatton, C and Singh, S and Singh, S and Bijpuria, S and McKeever, BM and Miller, WH and Safer, JF and Iqbal, S and Perry, JA and Fischer, ES and Doench, JG and McGeehan, GM and Cutler, JA and Armstrong, SA},
title = {CRISPR base editor screening identifies spectrum of MEN1 mutations impacting menin inhibitors in clinical trials.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103719},
issn = {2041-1723},
support = {Chromatin Collaborative award//St. Jude Children's Research Hospital/ ; P50 CA206963/CA/NCI NIH HHS/United States ; R01 CA259273/CA/NCI NIH HHS/United States ; NIH 5 P30 CA06516//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P01 CA066996/CA/NCI NIH HHS/United States ; CA206963//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 511811315//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 2021-EKEA.111//Else Kr&#xf6;ner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation)/ ; CA066996//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; CA259273//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; PE 3217/4-1, PN: 517204983//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Proto-Oncogene Proteins/genetics/antagonists &amp; inhibitors/chemistry/metabolism ; Humans ; Histone-Lysine N-Methyltransferase/genetics/metabolism/antagonists &amp; inhibitors ; *Mutation ; Nucleophosmin ; Myeloid-Lymphoid Leukemia Protein/genetics/metabolism ; Animals ; Drug Resistance, Neoplasm/genetics ; Nuclear Proteins/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; },
abstract = {Menin inhibitors have entered clinical trials for histone lysine methyltransferase 2 A (KMT2A)-rearranged and nucleophosmin 1 (NPM1)-mutant acute leukemias and are demonstrating promising activity. CRISPR base editor screening previously predicted several MEN1 (menin) mutations that have arisen in patients receiving SNDX-5613 and confer resistance. The extent to which MEN1 mutations will impact each menin inhibitor is mostly unknown. Here we show that CRISPR base editor screens can be leveraged to profile the MEN1 mutations that may impact five different menin inhibitors in clinical trials. We identify shared (M327I/V/T, G331D) and inhibitor-specific (C334R, E368K/V, V372A) resistance mutations. Co-crystal structures of menin bound to each menin inhibitor suggest resistance mechanisms related to how each inhibitor engages the KMT2A binding pocket of menin. Orthogonal in vitro and in vivo MEN1 mutation generation under therapeutic pressure suggest the MEN1 mutations identified with CRISPR base editor screening are likely to arise and impact all menin inhibitors.},
}
@article {pmid42103787,
year = {2026},
author = {Cipria, D and Baccega, T and Rizzo, M and Quarato, P and Reschigna, A and El Khoury, R and Cappelluti, MA and Ammann, S and Poeta, VM and Conti, M and Valsoni, S and Spinelli, P and Merelli, I and Cathomen, T and Casucci, M and Lombardo, A},
title = {Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103787},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; *Gene Editing/methods ; T-Lymphocytes/metabolism ; Epigenome Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; Receptors, Antigen, T-Cell/genetics ; Transgenes ; Gene Silencing ; Receptors, Chimeric Antigen/genetics ; },
abstract = {The parallel disruption of multiple genes coupled with targeted transgene insertion offers a powerful strategy for more effective and precise cell engineering. However, such orthogonal editing involves the induction of multiple DNA breaks, raising safety concerns related to the risks of chromosomal translocations. Here, we present a polyfunctional CRISPR-Cas9-based strategy that enables both transgene insertion and epigenetic silencing at distinct genomic loci in a single treatment without inducing reciprocal chromosomal translocations. This is accomplished through an optimized all-in-one epigenome editor equipped with a catalytically active Cas9, whose endonuclease activity is selectively disabled at epigenetically silenced loci using truncated gRNAs. As a proof of concept, we demonstrate that this platform enables efficient multi-locus editing, including functional replacement of the endogenous TCR with a tumor-selective one, targeted insertion of a prototypic CAR with either a selectable marker or an immunomodulatory receptor into a TCR locus or a ubiquitously expressed gene, and durable, multiplexed epigenetic silencing of clinically relevant genes in primary human T cells. Polyfunctional editing establishes a versatile and safe framework for orthogonal editing, broadening the scope of genome and epigenome engineering in cancer immunotherapy and beyond.},
}
@article {pmid42120395,
year = {2026},
author = {Morency, C and Rousseau, GM and Morneau, Z and Moineau, S},
title = {Phage satellites induced by virulent phages are mobilized by natural competence leading to phage resistance in a new host.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42120395},
issn = {2041-1723},
mesh = {*Streptococcus thermophilus/virology/genetics ; *Bacteriophages/genetics/pathogenicity/physiology ; CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Virulence ; Genome, Viral ; Prophages/genetics ; },
abstract = {A phage satellite (PS) typically resides within repeat regions (attL and attR sites) of a bacterial genome. Its genome ranges from 7 to 20-kb and includes genes encoding an integrase along with regulatory and DNA replication functions. However, it lacks genes associated with viral structural proteins. Streptococcus thermophilus (S.t.) is extensively used to produce yogurt and specialty cheeses. Intriguingly, the majority of S.t. strains harbor a PS while very few possess a complete prophage, suggesting that PSs may confer advantages to their hosts. In this study, we showed that PSs of S.t. can excise from the bacterial chromosome, at a very low rate, without any phage interaction. Furthermore, we found that they can also be induced by virulent phages. By leveraging CRISPR-Cas9, we selected S.t. cells devoid of any PS (delta-PS strain). Then, we mobilized a PS from one strain to a delta-PS strain, using only natural competence, bypassing the need for a helper phage. The resulting strain exhibited increased resistance to virulent phages. Through the isolation of phage mutants escaping the resistance phenotype, we pinpointed a specific phage protein responsible for the induction of a PS. Lastly, we demonstrated that a PS can be significantly induced by a virulent phage, which, in turn, greatly promotes its transfer and specific integration into new cells through natural competence. Our study introduces a novel natural approach to develop phage-resistant strains.},
}
@article {pmid42140952,
year = {2026},
author = {Wang, S and Hou, S and Luo, C and Zhang, H and Jin, Y and Zhang, R and Zhao, Y and Xiong, X and Guo, R and Wang, C and Bao, Y and Wen, L and Pan, D and Ye, Y and Zeng, Z and Gao, Z},
title = {Arid3b suppresses CD8 + T cell infiltration and function in microsatellite-stable colorectal cancer via Runx3.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42140952},
issn = {2041-1723},
support = {92478117//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92374116//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32470664//National Natural Science Foundation of China (National Science Foundation of China)/ ; T2321001//National Natural Science Foundation of China (National Science Foundation of China)/ ; L248043//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {*Core Binding Factor Alpha 3 Subunit/genetics/metabolism/immunology ; Animals ; *Colorectal Neoplasms/genetics/immunology/pathology/metabolism ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; Tumor Microenvironment/immunology/genetics ; Humans ; Mice ; *DNA-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Lymphocytes, Tumor-Infiltrating/immunology ; CRISPR-Cas Systems ; Microsatellite Repeats ; *Transcription Factors/genetics/metabolism ; },
abstract = {Microsatellite-stable/proficient mismatch repair (MSS/pMMR) colorectal cancer (CRC) is characterized by a cold tumor microenvironment, with limited CD8[+] T cell infiltration and poor responsiveness to immune checkpoint inhibitors (ICIs). Here, using an in vivo CRISPR/Cas9 screen in a CMT93 cell-derived murine tumor model, we identify Arid3b as a key negative regulator of CD8[+] T cell infiltration and antitumor activity. Genetic ablation of Arid3b in CD8[+] T cells significantly enhances their intratumoral accumulation and promotes robust tumor control. Mechanistically, Arid3b deficiency upregulates Runx3, driving a tissue-resident memory-like phenotype and effector function. Notably, the benefits conferred by Arid3b deficiency are abrogated upon Runx3 deletion, indicating a RUNX3-dependent mechanism. Together, targeting ARID3B could offer a promising strategy to reshape the tumor microenvironment and sensitize MSS CRC to immunotherapy.},
}
@article {pmid42264060,
year = {2026},
author = {Zaki, HF and Bishri, J and Abdul Muqtadir, M and Abu-Zaid, A},
title = {Correcting photoreceptor diseases at their source: CRISPR strategies for cone-rod dystrophy and achromatopsia.},
journal = {Experimental eye research},
volume = {270},
number = {},
pages = {111119},
doi = {10.1016/j.exer.2026.111119},
pmid = {42264060},
issn = {1096-0007},
mesh = {*Color Vision Defects/therapy/genetics ; Humans ; Animals ; *Genetic Therapy/methods ; *Cone-Rod Dystrophies/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Disease Models, Animal ; },
abstract = {BACKGROUND: Cone-rod dystrophy (CORD) and achromatopsia (ACHM) are inherited retinal dystrophies for which conventional adeno-associated virus (AAV) gene augmentation has important limitations, particularly in autosomal-dominant gain-of-function CORD and recessive ACHM. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offers the potential for one-time, mutation-specific gene correction or allele ablation. This systematic review summarizes preclinical evidence on CRISPR/Cas9-based approaches for CORD and ACHM, focusing on editing efficiency, phenotypic rescue, and safety.<br><br>METHODS: This review followed PRISMA guidelines. PubMed, Google Scholar, and ScienceDirect were searched through June 2025 for original experimental studies using CRISPR/Cas9 in CORD or ACHM animal models or human-derived cell lines. Dual independent screening and data extraction were performed. Outcomes related to editing efficiency, structural or functional rescue, and safety were synthesized narratively.<br><br>RESULTS: Four studies were included: three targeting CORD and one targeting ACHM. In vivo studies used AAV-delivered SaCas9 to disrupt GUCY2D (or murine orthologs) in mouse and macaque photoreceptors, achieving approximately 8-45% on-target editing in mice and approximately 13% in macaques. Although ablation alone reduced retGC1 expression, it did not improve retinal function; however, a dual-AAV "ablate-and-replace" strategy preserved outer nuclear layer thickness for up to 24 weeks in CORD6 mice. In vitro, PROM1 correction in patient-derived iPSCs restored CD133 expression, and SpCas9-HiFi-mediated PDE6C correction in ACHM iPSCs achieved approximately 80% editing efficiency while preserving pluripotency and showing no detectable off-target effects. Safety data were limited, with immune responses assessed in only one primate study.<br><br>CONCLUSIONS: CRISPR/Cas9 shows promising preclinical efficacy for CORD and ACHM, particularly allele-specific ablate-and-replace strategies for CORD and precise HDR-based correction for ACHM. However, the available evidence remains limited, underscoring the need for expanded safety assessment, non-human primate studies, and standardized functional outcomes measures before clinical translation.},
}
@article {pmid42284763,
year = {2026},
author = {Liu, L and Wang, H and Shi, L and Zhang, C and Zhang, L and Lv, L and Wang, Y},
title = {Development of super Vδ2 T cells for relapsed/refractory acute myeloid Leukemia via non-viral site-specific integration.},
journal = {International immunopharmacology},
volume = {185},
number = {},
pages = {116981},
doi = {10.1016/j.intimp.2026.116981},
pmid = {42284763},
issn = {1878-1705},
mesh = {Humans ; *Leukemia, Myeloid, Acute/therapy/immunology ; Animals ; *Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; Receptors, Chimeric Antigen/genetics ; *T-Lymphocytes/immunology/transplantation ; NK Cell Lectin-Like Receptor Subfamily K/genetics/metabolism ; Mice ; Cell Line, Tumor ; CD3 Complex/genetics ; CRISPR-Cas Systems ; Cytotoxicity, Immunologic ; Mice, SCID ; },
abstract = {The efficacy of chimeric antigen receptor (CAR)-T cell therapy in relapsed/refractory acute myeloid leukemia (R/R AML) is limited by tumor heterogeneity, antigen evasion, and treatment-related toxicities. Gamma delta (γδ) T cells mediate antitumor activity independent of MHC by sensing stress-induced ligands. A prominent mechanism involves NKG2D ligand (NKG2DL) recognition, which is highly upregulated in malignancies but generally low or restricted expression in healthy tissues under homeostatic conditions. In human peripheral blood, the Vδ2 subset represents the predominant population. Vδ2 T cells transduced with the NKG2D-CD3ζ construct, which incorporates into the natural γδ TCR/CD3 complex, preserve innate phosphoantigen recognition while acquiring potent NKG2DL-directed cytotoxicity, enabling dual-pathway tumor recognition. These cells are termed "Super Vδ2 T cells." We successfully generated TRAC-specific integrated Super Vδ2 T cells using CRISPR/Cas9 technology, achieving 90-93% CAR[+] expression. In vitro assays demonstrated that the engineered "Super Vδ2 T cells" exhibited potent cytotoxic activity against multiple AML targets, including cell lines and primary R/R AML blasts, in contrast to their negligible toxicity on monocytes. In vivo, Super Vδ2 T cells demonstrated substantial tumor reduction without graft-versus-host disease (GvHD) reaction. Collectively, our data demonstrated that Super Vδ2 T cells represent a viable allogeneic therapy for AML.},
}
@article {pmid42439627,
year = {2026},
author = {Park, SH and Hong, J and Hwang, W and Kim, M and Yu, HJ and Bae, T and Lee, HK and Lee, JY and Lee, YC and Park, CK and Hur, JK},
title = {CRISPRi-Mediated Epigenetic Suppression of TERT Reduces Cell Growth in Non-Small-Cell Lung Cancer Cells.},
journal = {Cells},
volume = {15},
number = {13},
pages = {},
pmid = {42439627},
issn = {2073-4409},
support = {RS-2021-NR056589; RS-2023-00261114; RS-2025-02218918//Ministry of Science and ICT/ ; RS-2025-16063805//the Korea US Collaborative Research Fund/ ; 2023R1A6C101A009//the Ministry of Education/ ; RS-2023-00260529, RS-2026-25493418//National Research Foundation of Korea/ ; },
mesh = {Humans ; *Telomerase/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; *Lung Neoplasms/genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Epigenesis, Genetic ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Neoplastic ; Cell Survival/genetics ; Epigenome Editing ; },
abstract = {TERT, the catalytic subunit of telomerase, is aberrantly activated in most cancers and represents an attractive therapeutic target. However, conventional TERT-targeting strategies, including chemical inhibitors and siRNA, are limited by several issues, such as insufficient efficacy and off-target effects. In this study, we investigated whether dCas9-KRAB-mediated CRISPR interference (CRISPRi) could overcome the limitations by transcriptional repression of TERT without DNA cleavage. We first assessed the efficacy of the dCas9-KRAB system by applying it to H1299 non-small-cell lung cancer cells and observed reduction in TERT expression up to approximately 80% and significant decreases in cell viability and growth. Transcriptome-wide analysis showed limited detectable changes in non-target-gene expression under the conditions tested. Together, the results suggest that dCas9-KRAB-mediated CRISPRi could serve as a proof-of-principle approach for targeted repression of TERT in cancer cells with limited detectable effects on non-target-gene expression.},
}
@article {pmid42439976,
year = {2026},
author = {Mundada, AR and Badikol, AR and Mangu, K},
title = {CRISPR-Cas9-based therapies for Huntington's disease and Friedreich's ataxia: mechanisms, advances, and future perspectives.},
journal = {Neurogenetics},
volume = {27},
number = {1},
pages = {},
pmid = {42439976},
issn = {1364-6753},
mesh = {*Friedreich Ataxia/therapy/genetics ; Humans ; *Huntington Disease/therapy/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods/trends ; *Gene Editing/methods ; Animals ; Trinucleotide Repeat Expansion ; },
abstract = {Huntington's disease (HD) and Friedreich's ataxia (FRDA) are progressive inherited neurodegenerative disorders caused by trinucleotide repeat expansions but characterized by distinct pathogenic mechanisms. HD arises from a coding-region CAG expansion in the HTT gene that produces toxic gain-of-function effects of mutant huntingtin (mHTT), whereas FRDA results primarily from intronic GAA repeat expansion in FXN, leading to epigenetic repression and frataxin deficiency. The emergence of CRISPR-based genome engineering has created new opportunities to address these diseases at their genetic origin. This review examines current CRISPR therapeutic strategies for HD and FRDA, including allele-specific editing, transcriptional suppression, repeat excision, epigenetic reactivation, and emerging precision editing approaches such as base editing and prime editing. We compare the molecular rationale, preclinical outcomes, and translational limitations associated with each approach while highlighting how disease architecture influences therapeutic design. Although preclinical studies demonstrate promising restoration of cellular phenotypes and functional improvement, significant barriers remain. Efficient delivery to the central nervous system and cardiac tissue, control of editing duration, immune responses, off-target activity, and emerging concerns regarding on-target genomic instability continue to limit clinical translation. Recent advances in delivery engineering, non-viral systems, and programmable editing platforms suggest that future therapeutic success will depend on integrating disease-specific biology with increasingly precise and controllable genome engineering technologies. Ethical and regulatory concerns remain substantial, particularly regarding informed consent in the context of cognitive decline and the irreversibility of genomic modification.},
}
@article {pmid42441240,
year = {2026},
author = {Yu, W and Huang, X and Hu, Y and Chen, S},
title = {Beyond adaptive immunity: Functional diversity of the type III-A CRISPR-Cas system in Mycobacterium tuberculosis.},
journal = {Cell insight},
volume = {5},
number = {4},
pages = {100342},
pmid = {42441240},
issn = {2772-8927},
abstract = {CRISPR-Cas systems are best known as prokaryotic adaptive immune pathways that defend against invading genetic elements. Mycobacterium tuberculosis (Mtb) harbors a type III-A CRISPR-Cas system that is structurally conserved yet exhibits little evidence of ongoing spacer acquisition. Nevertheless, its interference machinery remains functional, and increasing evidence suggests that this system has evolved roles beyond canonical adaptive immunity. Accumulating studies indicate that this system is deeply integrated into cellular regulatory networks by governing stress responses, metabolic adaptation, and host-pathogen interactions. Mechanistically, the Mtb type III-A CRISPR-Cas system operates through transcription-dependent target recognition and cyclic oligoadenylate (cOA)-mediated signal amplification, in which the ancillary ribonuclease Csm6 serves as a key effector. Functionally, CRISPR-associated proteins influence antibiotic susceptibility, oxidative stress resistance and host immune responses, and may even act as secreted immunomodulatory factors. In this review, we summarize current understanding of the genomic organization, regulatory mechanisms, and non-canonical functions of the Mtb type III-A CRISPR-Cas system, with particular emphasis on its emerging roles in stress adaptation and host immune regulation.},
}
@article {pmid42442865,
year = {2026},
author = {Zheng, J and Zhang, W and Conrad, M},
title = {Ferroptosis induction via genetic approaches - CRISPR/Cas9-based disruption on key anti-ferroptotic genes.},
journal = {Methods in cell biology},
volume = {209},
number = {},
pages = {91-103},
doi = {10.1016/bs.mcb.2026.05.002},
pmid = {42442865},
issn = {0091-679X},
mesh = {*Ferroptosis/genetics/drug effects ; Humans ; *CRISPR-Cas Systems/genetics ; *Phospholipid Hydroperoxide Glutathione Peroxidase/genetics ; Amino Acid Transport System y+/genetics ; Cell Line, Tumor ; Gene Knockout Techniques/methods ; },
abstract = {Unlike apoptosis, necroptosis, or pyroptosis which are executed by dedicated proteins, ferroptosis is a distinct form of regulated cell death driven by lipid peroxidation downstream of metabolic dysfunction. In most physiological settings, the cyst(e)ine/glutathione/glutathione peroxidase 4 (GPX4) axis constitutes the central anti-ferroptotic machinery, and disruption of this axis is usually sufficient to trigger ferroptosis. For in vitro studies, commonly employed ferroptosis inducers include erastin, which blocks cystine uptake by targeting system xc[-], and (1S,3R)-RSL3, which inhibits GPX4 activity. However, both compounds exhibit off-target effects - erastin can activate voltage-dependent anion channels in mitochondria, whereas (1S,3R)-RSL3 affects other selenoproteins in addition to GPX4. Thus, genetic approaches to induce ferroptosis provide a valuable complement to chemical inducers by excluding off-target concerns. Here, we describe an efficient CRISPR/Cas9-based strategy to generate SLC7A11- and GPX4-knockout HT1080 cells. These knockout lines require routine culture in medium supplemented with β-mercaptoethanol or liproxstatin-1, while withdrawal of these supplements readily induces ferroptosis.},
}
@article {pmid42444979,
year = {2026},
author = {Yang, M and Song, Y and Wang, Z and Chao, K and Li, L and Zhang, X and Duan, X and Yu, C and Xue, R and Zhao, J},
title = {Delivery Systems for Therapeutic Genome Editing: Challenges, Innovations, and Future Perspectives.},
journal = {MedComm},
volume = {7},
number = {7},
pages = {e70791},
pmid = {42444979},
issn = {2688-2663},
abstract = {Therapeutic genome editing has advanced rapidly with the development of diverse programmable nucleases, from zinc-finger nucleases and transcription activator-like effector nucleases to clustered regularly interspaced short palindromic repeats (CRISPR)-based systems such as base and prime editors. Despite these breakthroughs, clinical translation remains constrained by the challenge of achieving safe, efficient, and tissue-specific delivery. Viral vectors, particularly adeno-associated viruses, have enabled durable editing in selected organs but are limited by their restricted cargo capacity, immunogenicity, and complex manufacturing. Nonviral platforms, most notably ionizable lipid nanoparticles, have demonstrated remarkable efficacy for hepatic targets, with clinical trials reporting up to 93% protein knockdown after a single dose. An expanding set of emerging modalities, including virus-mimicking nanosystems, cell-derived extracellular vesicles, cell-penetrating peptides, and intelligent-responsive multifunctional scaffolds, further enriches the delivery toolbox by supporting transient expression and programmable targeting across diverse editors and tissues. Parallel advances in high-throughput barcoded screening and machine learning are accelerating vector optimization, while rational chemical modification of payloads improves in vivo stability and specificity. This review provides a comprehensive overview of current and emerging delivery systems for genome editing, highlighting key innovations, unresolved challenges, and interdisciplinary strategies poised to unlock broader therapeutic potential.},
}
@article {pmid42445839,
year = {2026},
author = {Kruglova, NA and Borovikova, SE and Shepelev, MV},
title = {Production of virus-like particles with AsCas12a nuclease and CMV-driven crRNA for mammalian genome editing.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1735339},
pmid = {42445839},
issn = {2673-3439},
abstract = {CRISPR/Cas genome editing tools represent a promising technology for biomedicine with significant therapeutic potential for numerous human diseases. However, efficient delivery of these tools into primary cells, particularly in the form of ribonucleoprotein (RNP) complexes, remains a critical bottleneck that limits clinical translation. Virus-like particles (VLPs) derived from human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) have emerged as promising delivery vehicles for RNP complexes, yet their activity is limited by suboptimal nuclease and guide RNA packaging. Previously, we generated NanoMEDIC VLPs incorporating the AsCas12a nuclease with CMV-driven crRNA, which demonstrated substantially enhanced editing efficiency over SpCas9-VLPs with U6-driven gRNA. Here, we describe a detailed protocol for a small-scale production of AsCas12a-VLPs using three distinct transfection methods [cationic lipids, polyethyleneimine (PEI), and calcium-phosphate] and a large-scale production of VLPs using calcium-phosphate transfection. We show that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies, reaching up to 60% of CXCR4 knockout in Jurkat T cells.},
}
@article {pmid42120643,
year = {2026},
author = {Cui, H and Peng, J and Song, J and Yang, Y and Hao, X},
title = {An ultrasensitive CRISPR-strand displacement amplification biosensor achieves piRNA-54265 detection and imaging in colorectal cancer cells.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {14},
pages = {4613-4621},
pmid = {42120643},
issn = {1618-2650},
support = {20252BAC250153//Natural Science Foundation of Jiangxi Province/ ; 22277047//National Natural Science Foundation of China/ ; 82160631//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Biosensing Techniques/methods ; *Colorectal Neoplasms/genetics/diagnosis ; *Piwi-Interacting RNA/analysis/genetics ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Cell Line, Tumor ; *RNA, Small Interfering/analysis/genetics ; },
abstract = {PIWI-interacting RNAs (piRNAs) are well-recognized as promising diagnostic biomarkers for cancer, yet their quantitative detection remains a great challenge owing to their short sequences, low cellular abundance, high degradation susceptibility, and significant sequence homology among family members. Herein, we developed an ultrasensitive and highly specific biosensor for the detection of piRNA-54265-a colorectal cancer (CRC)-associated piRNA-by integrating strand displacement amplification (SDA) with the CRISPR/Cas12a system. After systematic optimization, the biosensor exhibited remarkably enhanced amplification efficiency and target specificity, achieving an ultra-low limit of detection (LOD) of 57.54 aM for piRNA-54265. Notably, this CRISPR-SDA platform enabled accurate discrimination of CRC cells from other cancer cells via high-fidelity intracellular imaging of piRNA-54265 and also realized reliable detection of the target in complex biological matrices with favorable recovery. Benefiting from its simple sequence design, user-friendly operation, and isothermal reaction conditions, the developed biosensor not only overcomes the inherent technical bottlenecks in piRNA detection but also shows great potential for applications in cellular imaging and early clinical diagnosis of CRC.},
}
@article {pmid42130163,
year = {2026},
author = {Nomura, C and Kanzaki, H and Kanzaki, E and Shimizu, M and Oikawa, K and Utsushi, H and Ito, K and Sugimura, Y and Terauchi, R and Abe, A},
title = {Fine-tuning quantitative agronomic traits by manipulating gene copy number in rice.},
journal = {The New phytologist},
volume = {251},
number = {4},
pages = {1609-1616},
doi = {10.1111/nph.71258},
pmid = {42130163},
issn = {1469-8137},
support = {KAKENHI JP22K20584//Japan Society for the Promotion of Science/ ; KAKENHI JP23K26882//Japan Society for the Promotion of Science/ ; JPJ007097//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Dosage ; *Quantitative Trait, Heritable ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Genes, Plant ; Phenotype ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Although plant pan-genome studies have revealed extensive copy number variations, their phenotypic consequences remain poorly understood. Here, we manipulated the copy number of OsMADS18 in rice (Oryza sativa) cv 'Hitomebore' using the CRISPR/Cas9 system. We established rice lines harboring one to three tandem copies of OsMADS18, as identified by quantitative PCR and sequencing. The presence of one to three OsMADS18 tandem copies was reflected in stepwise increases in transcript levels and concomitant agronomic trait values. These results demonstrate that manipulating gene copy number can fine-tune important quantitative traits, providing a novel breeding strategy for crop improvement.},
}
@article {pmid42155537,
year = {2026},
author = {Albeladi, HA and Al-Zahrani, MH and Alghamdi, RA},
title = {Modulating claudin-2 with CRISPR-Cas9 to improve photodynamic therapy outcomes in colorectal cancer.},
journal = {Tissue &amp; cell},
volume = {102},
number = {},
pages = {103586},
doi = {10.1016/j.tice.2026.103586},
pmid = {42155537},
issn = {1532-3072},
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/drug therapy/therapy ; *Photochemotherapy ; Apoptosis/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; Reactive Oxygen Species/metabolism ; *Claudin-2/metabolism/genetics ; Cell Movement/drug effects/genetics ; Chlorophyllides ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Porphyrins/pharmacology ; Claudins ; },
abstract = {Claudin-2 (CLDN2) is a tight junction protein that is overexpressed in colorectal cancer (CRC) and is associated with chemoresistance. Photodynamic therapy (PDT) is an emerging treatment that utilizes a photosensitizer (in this case, chlorin e6 [Ce6]) and light to generate cytotoxic reactive oxygen species (ROS). This paper investigated the influence of the combination of Ce6-PDT and CRISPR-Cas9-mediated CLDN2 knockout (KO) on the relative metabolic activity of the CRC cell line. CRISPR-Cas9 was used to produce HCT116 cells with CLDN2 KO. Ce6 was placed on the cells, and the red laser (659 nm, 6 J/cm[2]) was used to illuminate the cells. The relative metabolic activity, migration, Apoptosis, cell cycle, and ROS generation, gene expression, protein expression were measured by MTT assay, wound healing assay, flow cytometry, DCFH-DA method, RT-PCR, western blot and bioinformatics, respectively. PDT significantly decreased the relative metabolic activity and/or migration, more in CLDN2KO cells (p < 0.0001) than in the WT. The CLDN2KO cells had a high level of Apoptosis (46.56 ± 2.05%), compared to the WT (26.03 ± 6.72%), the p = 0.0072. The production of ROS was also increased to 779.51 % in CLDN2KO cells, which is higher than the production in WT cells at 767.10 %. Upregulation of P53 and BAX following PDT was greatly enhanced, and BCL2 expression was significantly reduced as compared to wild-type groups. Coexistence of Ce6-PDT with CLDN2 KO enhances the Apoptosis of the CRC cell line. The Western blot results showed a decrease in ZO-1 and Occludin proteins after Ce6-PDT. Bioinformatics analysis demonstrated that increased CLDN2 expression in CRC, associated with multiple genes and implicated in various cellular pathways. Although the initial results are encouraging regarding the possibility of synergy, further studies are needed to determine its effectiveness and safety in clinical practice. The results of this research suggest the possibility of a therapeutic approach aimed at enhancing the effectiveness of CRC treatment through genetic regulation with the aid of PDT.},
}
@article {pmid42284917,
year = {2026},
author = {Zhao, D and Peng, W and Liu, Z and Zhou, Q and Wu, L and Zhou, Y and Ran, F},
title = {A sensitive detection of C-reactive protein based on the combination of CRISPR/Cas13a, MNPs and RNase H.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {280},
number = {},
pages = {117609},
doi = {10.1016/j.jpba.2026.117609},
pmid = {42284917},
issn = {1873-264X},
mesh = {*C-Reactive Protein/analysis ; Humans ; *Biosensing Techniques/methods ; *Ribonuclease H/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; *Magnetite Nanoparticles/chemistry ; },
abstract = {C-reactive protein (CRP) is a potential risk factor for disease. Here, developed a rapid and accurate fluorescence biosensor for detecting CRP, which contributes to early diagnosis and timely treatment of diseases. The CRP binds with the aptamer resulting in the probe 1 (P1) releasing from the complex of aptamer/P1/magnetic nanoparticles (MNPs). After magnetic separation, the free P1 hybridized with the RNA (P2) modified on the MNPs, leading to the P2 being multiple-turnover cut by ribonuclease H (RNase H). The formed free RNA can specifically bind with the crRNA and the tans-cleavage activity of CRISPR/Cas13a was triggered, leading to the RNA reporter containing a dye and quencher pair being cleaved and generating the fluorescence signal. This developed fluorescent biosensor takes full advantage of the synergy of aptamer, RNase H, MNPs and CRISPR/Cas13a. Here, the developed fluorescent biosensor exhibits excellent sensitivity and specificity towards the detection of CRP with a linear range from 10 pg/mL to 200 ng/mL. The detection limit is low down to 7.5 pg/mL. Additionally, this method successfully detected the CRP in human serum samples with satisfactory recoveries. Therefore, this developed biosensor will offer a valuable tool for the rapid diagnosis of CRP-related diseases.},
}
@article {pmid42341421,
year = {2026},
author = {Le, HT and Nghi, NB and My, VD and Vu, HA and Thanh, MC and Tri, BM and Niem, VVT and Phuong, HA},
title = {Functional characterization of PIK3CA E545A mutation in MCF-7 breast cancer cells reveals enhanced proliferation and resistance to Alpelisib.},
journal = {Biochemical and biophysical research communications},
volume = {829},
number = {},
pages = {154189},
doi = {10.1016/j.bbrc.2026.154189},
pmid = {42341421},
issn = {1090-2104},
mesh = {Humans ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists &amp; inhibitors/metabolism ; Cell Proliferation/drug effects/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology ; Female ; *Drug Resistance, Neoplasm/genetics/drug effects ; MCF-7 Cells ; *Mutation ; *Thiazoles/pharmacology ; CRISPR-Cas Systems ; },
abstract = {PIK3CA mutations are central oncogenic drivers in hormone receptor-positive, HER2-negative breast cancer; however, the functional and therapeutic relevance of noncanonical variants remains incompletely defined. The E545A mutation, increasingly reported in specific patient populations, has not been systematically investigated. We generated an isogenic MCF-7 cell model harboring the PIK3CA E545A mutation using CRISPR/Cas9-mediated homology-directed repair to delineate its phenotypic and pharmacological consequences. E545A induced a robust gain-of-function phenotype, characterized by a mesenchymal-like morphological transition with reduced circularity and decreased cell size. This structural shift was accompanied by enhanced tumor cell fitness, including accelerated proliferation kinetics, increased metabolic activity, and significantly elevated clonogenic capacity compared with wild-type controls. Notably, growth trajectories showed sustained divergence between mutant and control cells across all time points, indicating a stable proliferative advantage. Importantly, E545A conferred diminished sensitivity to the PI3Kα inhibitor Alpelisib. Mutant cells retained migratory capacity under treatment and exhibited a pronounced, time-dependent increase in IC50, consistent with adaptive resistance. Collectively, these findings identify E545A as a functionally active and therapeutically consequential PIK3CA variant. Our study expands the current understanding of PIK3CA-driven oncogenic diversity beyond canonical hotspot mutations and underscores the need for variant-resolved stratification to improve the efficacy of PI3K-targeted therapies.},
}
@article {pmid42342973,
year = {2026},
author = {Duan, M and Meng, B and Zhou, L and Wu, L and Tong, X and Huang, D and Yin, H and Liu, ZJ and Zhang, Y},
title = {Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {7},
pages = {1062-1074},
pmid = {42342973},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *DNA/chemistry/metabolism/genetics ; Models, Molecular ; Mutation ; *Alicyclobacillus/enzymology/genetics ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; },
abstract = {The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for N4CNNN and N4RNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.},
}
@article {pmid42432276,
year = {2026},
author = {Tian, Y and Li, M and Liu, C and Liu, T and Zhang, X and Liu, Q},
title = {Simultaneous detection of multiple foodborne pathogens using a CRISPR/Cas12a-based pump-free microfluidic chip.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {42432276},
issn = {1618-2650},
abstract = {The development of microfluidic chips for nucleic acid detection provides efficient technical support for monitoring food safety. With the increasing maturity of CRISPR technology, it has the advantages of high specificity and high sensitivity in the detection of single or multiple nucleic acids. In this study, a microfluidic biosensor based on the CRISPR/Cas12a system was constructed using a pump-free microfluidic chip as the carrier, with a focus on the rapid, simultaneous detection of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Cronobacter sakazakii. For each target pathogenic bacterium, two replicate channels for each pathogen were established, along with corresponding negative and positive controls, which effectively ensured the reliability and repeatability of the detected results and successfully achieved the simultaneous high-sensitivity, high-specificity, and high-accuracy detection of multiple foodborne pathogens. The detection sensitivity of the sensor for S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii was as low as 10[3] CFU/mL, 10[3] CFU/mL, 10[2] CFU/mL, and 10[3] CFU/mL, respectively. This integrated CRISPR/Cas12a sensor chip has the advantages of the simultaneous efficient detection of multiple pathogens, parallel verification, and control settings, and the detection results can be visualized by fluorescence, indicating broad application prospects in the field of on-site rapid nucleic acid analysis.},
}
@article {pmid42434562,
year = {2026},
author = {Liang, Z and Li, Z and Li, C and Zhao, Y and Liu, J and Zhang, J},
title = {Advances in gene editing tools for four typical Gram-positive bacteria.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1882312},
pmid = {42434562},
issn = {1664-302X},
abstract = {Gram-positive bacteria serve as important chassis microorganisms in synthetic biology, industrial fermentation, and probiotic development. The rapid advancement of gene editing technologies has provided critical technical support for the iterative construction and functional validation of engineered strains. However, due to factors such as cell wall structure, differences in genetic backgrounds, and tool compatibility, the development and editing efficiency of gene editing systems for Gram-positive bacteria still face many challenges. This review focuses on four representative Gram-positive bacterial species-Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, and Corynebacterium glutamicum-and traces the evolution and current state of their editing tools, from traditional homologous recombination to CRISPR-Cas9, base editors, and large-fragment integration tools. On this basis, we summarize the common challenges and corresponding strategies concerning host repair capacity, tool compatibility, and inherent limitations of editors in these four bacterial species, and propose recommendations for tool selection based on different application scenarios. This review aims to provide a technical reference for gene editing studies of the above-mentioned bacterial species. Although the conclusions cannot be directly extended to all Gram-positive bacteria, the common issues summarized here may inform the development of gene editing tools for other Gram-positive bacteria.},
}
@article {pmid42434939,
year = {2026},
author = {Wang, S and Hasan, R},
title = {CRISPR Biosensing for Environmental Monitoring: Workflow Design and Performance Benchmarking.},
journal = {Environmental science &amp; technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.6c02006},
pmid = {42434939},
issn = {1520-5851},
abstract = {CRISPR-based biosensing has rapidly emerged as a promising platform for environmental monitoring due to its high specificity, programmability, and compatibility with portable readouts. However, translation from biomedical diagnostics to environmental matrices remains challenging because of diverse sample types, complex inhibitors, and the breadth of biological and chemical targets. This Review provides a comprehensive analysis of CRISPR-based sensing technologies tailored for environmental contaminant detection, spanning both biological and chemical targets. We systematically evaluate published studies across target classes, Cas effectors, recognition mediators, sample matrices, pretreatment strategies, preamplification or signal-gain approaches, readout modalities, and reported performance metrics. To support practical implementation, we summarize a five-step experimental framework for environmental CRISPR sensing. We then propose a decision-guided design flowchart that links monitoring goals and matrix constraints to the selection of effectors, mediator-enabled transduction routes, pretreatment modules, amplification strategies, readouts, and validation controls. We further benchmark reported detection limits by normalizing units and comparing trends across preamplification-aided versus preamplification-free designs and by contextualizing performance against relevant regulatory or guideline thresholds when available. Across the literature, most studies rely on spiked-matrix validation, highlighting the need for broader nonspiked real environmental sample testing and more transparent reporting of sampling, pretreatment, and performance evaluation. Finally, we advocate standardized data reporting, including consistent units, workflow metadata, and matrix-matched validation, to enable cross-study comparison and accelerate the deployment of CRISPR-based sensors for real-world environmental monitoring.},
}
@article {pmid42435220,
year = {2026},
author = {Khan, MA and Durand, A and Skouri-Panet, F and Benzerara, K and Cassier-Chauvat, C and Chauvat, F and Ouchane, S},
title = {Targeted genome editing of the non-model cyanobacterium Cyanothece PCC 7425 via CRISPR/Cas12a.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-026-13959-y},
pmid = {42435220},
issn = {1432-0614},
support = {ANR-19-CE44-0017//Agence Nationale de la Recherche/ ; ANR-19-CE44-0017//Agence Nationale de la Recherche/ ; },
abstract = {Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of some cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece sp. PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a-mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of the ΔsodB mutant further confirmed its targeted deletion. Overall, CRISPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece sp. PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity, and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. KEY POINTS: • Rapid and efficient CRISPR/Cas12a editing established in Cyanothece sp. PCC 7425. • Fully segregated knockout mutants obtained after a single selection round. • Platform for exploring the biotechnological potential of Cyanothece sp. PCC 7425.},
}
@article {pmid42436385,
year = {2026},
author = {Vásquez-Herrera, L and Vallejos, OP and Acevedo-López, J and Campos-Gajardo, S and Piña-Iturbe, A and Tichy-Navarro, D and Seeram, D and Uhlemann, AC and González, PA and Kalergis, AM and Moreno-Switt, AI and Bueno, SM},
title = {Whole genome sequence-based comparative genomics reveals preliminary genomic features of Salmonella enterica subsp. enterica serovar Enteritidis phage type 1 and phage type 4 strains from EnteroBase.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-026-05329-5},
pmid = {42436385},
issn = {1471-2180},
support = {N&#xb0; 21251772//ANID National Doctoral Fellowship/ ; N&#xb0; 1240971//Fondo Nacional de Desarrollo Cient&#xed;fico y Tecnol&#xf3;gico/ ; N&#xb0; 1231851//Fondo Nacional de Desarrollo Cient&#xed;fico y Tecnol&#xf3;gico/ ; N&#xb0; 1231905//Fondo Nacional de Desarrollo Cient&#xed;fico y Tecnol&#xf3;gico/ ; ICN 2021_045//Millennium Institute of Immunology and Immunotherapy/ ; ECOS220027//ECOS-ANID/ ; },
abstract = {BACKGROUND: Whole Genome Sequencing (WGS) enables detailed characterization of circulating and emerging bacterial strains. Although tens of thousands of Salmonella genomes have been acquired over the years, analyses of the genomic differences between strains of different phage types are scarce.<br><br>RESULTS: We compared two Salmonella enterica subsp. enterica serovar Enteritidis (SEn) phage types, namely phage types 1 and 4 from available databases, using bioinformatic tools and nanopore sequencing of a Chilean PT1 strain. Comparisons between the two phage types show very low genomic divergence and high genomic sequence similarity. Single nucleotide polymorphism (SNP) searches identified SNPs specific to each phage type. Although a translocated region was identified in the Chilean PT1 strain analyzed in this study when compared to the genome of a PT4 strain, this was not present in the genomes of other PT1 strains, suggesting a local strain-specific rearrangement. Further analyses yielded no differences in the CRISPR-Cas locus, but a slight difference was observed in Gifsy-2 prophage detection and DNA modification systems between PT1 and PT4 strains.<br><br>CONCLUSIONS: Our findings provide insights into the genomic differences between SEn strains of two different phage types, serving as a basis for future genomic studies, yet further analyses with more diverse geographical locations collected over a longer time span are essential to validate these differences with the potential to establish molecular markers for strain identification and characterization in the context of epidemiological surveillance as a complement to WGS when this technique is not available.},
}
@article {pmid42437521,
year = {2026},
author = {Asemoloye, MD},
title = {Enhancing the Secretion Systems: Genetic Engineering of Super Bioagents for Effective Plant Disease Control.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70303},
pmid = {42437521},
issn = {1097-0290},
abstract = {The escalating threat of plant diseases to global agriculture and food security necessitates innovative and sustainable control strategies. Conventional biological control agents (BCAs), while environmentally friendly, often suffer environmental challenges and secretion of limited/poor antimicrobial compounds. Advances in CRISPR/Cas genome editing, protease engineering, and synthetic biology have enabled precise modifications that improve pathogen targeting and secretion efficiency. Interest should now be shifted on development of "Super Bioagents (SBs)" with enhanced secretion systems (SSs) for plant disease suppression against changing environmental factors. This will create sustainable ecofriendly alternative to chemical pesticides. This review explores a detailed overview of molecular mechanisms of microbial SSs and the potentials of SBs as a frontier in plant disease management. While there are still challenges in mass deployment of BCAs in sustainable agriculture, this review is guided by the hypothesis that rational, quantitative engineering of microbial SSs can transform conventional BCAs into integrated SBs. It synthesizes current advances within a systems‑level bioengineering framework linking secretion efficiency, regulation, and field performance. It further explores possible integration of SBs in plant-microbiome interactions to further enhance their adaptability and effectiveness. Finally, the review dives into recent breakthroughs, current challenges, and future directions for SBs development and application as next-generation plant disease control agents.},
}
@article {pmid42438474,
year = {2026},
author = {Ravendran, S and Fammé, S and Noer, MG and Skov, TW and Mikkelsen, NS and Lee Schneller, J and Dorset, SR and Wolff, JH and Møller, AMJ and Haslund, D and Revenfeld, ALS and Holm, M and Mogensen, TH and Møller, BK and Mikkelsen, JG and Bak, RO},
title = {AAV vector production in suspension cells using PEI transfection and sodium butyrate with orthogonal assessment of function and quality.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {3},
pages = {201787},
pmid = {42438474},
issn = {3117-387X},
abstract = {Adeno-associated virus (AAV) vectors are widely used in gene therapy, yet academic in-house production remains dominated by labor-intensive adherent cell workflows with limited scalability. Here, we describe an AAV vector production platform using suspension cells in orbital shaking Erlenmeyer flasks, based on polyethyleneimine (PEI) transfection and sodium butyrate supplementation. Following systematic evaluation of transfection conditions, this approach yields vectors with performance comparable to a commercial production kit. Vector quality was interrogated using orthogonal methodologies, including two-dimensional ddPCR, mass photometry, and nanopore sequencing, enabling comparative assessment of genome packaging, capsid composition, and vector heterogeneity. Functional validation was performed by in vitro transduction of K562 cells and primary human CD34+ hematopoietic stem and progenitor cells, as well as in vivo gene delivery to mouse liver and heart. Across assays, vectors produced using this protocol demonstrated comparable genome integrity and transgene expression. Comparative purification analysis revealed that iodixanol density gradient purification resulted in higher proportions of full capsids and reduced producer-cell-derived impurities relative to PEG 8000 precipitation. Together, this work establishes a scalable suspension-based AAV production workflow and demonstrates the value of orthogonal quality assessment combined with functional validation for robust vector benchmarking.},
}
@article {pmid42438737,
year = {2026},
author = {Faleiros, CA and Gonçalves, OS and Nunes, AT and Pires, CS and Poleti, MD and Fukumasu, H},
title = {Host breed and geography shape the antiviral defense landscape of the bovine rumen microbiome.},
journal = {ISME communications},
volume = {6},
number = {1},
pages = {ycag162},
pmid = {42438737},
issn = {2730-6151},
abstract = {The rumen microbiome represents a complex, phage-rich ecosystem where microbial survival depends on both metabolic cooperation and antiviral defense. However, global and breed-associated variations in rumen prokaryotic immune systems remain poorly understood. Here, we performed the most comprehensive profile to date of antiviral defense systems (DS) in the rumen, analyzing 6530 microbial genomes and metagenome-assembled genomes (MAGs) from diverse cattle breeds and geographic regions. In this global dataset, we identified >90 000 DS, the most abundant of which were restriction-modification, PDC-S01, deoxyribonucleic acid modification systems (DMS_other), AbiE and SoFic, with variations influenced by both host the lineage and geographic region. A more in-depth analysis was performed using two complementary antiviral annotation frameworks for Nellore cattle (Bos indicus) from Brazil. Data exhibited a remarkably enriched antiviral defense repertoire, with over 15 632 DS encoded across 547 high-quality MAGs. These systems were densely clustered in dominant rumen lineages, such as Prevotella, and positively correlated with prophage abundance, consistent with virus-host coevolution. Notably, we also detected viral contigs encoding both antiviral defense and anti-defense genes, underscoring the arms race between the phages and their microbial hosts. Metatranscriptomic data from North America and Oceania revealed high expression levels of toxin-antitoxin modules, clustered regularly interspaced short palindromic repeats components, and restriction enzymes, suggesting a basal level of antiviral activity. These findings reveal the rumen as an antiviral innovation hotspot, highlighting microbiome resilience with implications for ecology, adaptation, and phage-based interventions.},
}
@article {pmid42439519,
year = {2026},
author = {Iyer, MS and Hagström, E and Näslund, K and Andersson, SGE},
title = {Harnessing endogenous CRISPR-Cas9 for inducible genetic engineering of Apilactobacillus kunkeei.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0072826},
doi = {10.1128/aem.00728-26},
pmid = {42439519},
issn = {1098-5336},
abstract = {UNLABELLED: Despite substantial advances in bacterial genome engineering, functional genetic analysis remains challenging in many non-model bacterial species, particularly among host-associated gram-positive bacteria. The fructophilic species Apilactobacillus kunkeei has been investigated for more than two decades and is a dominant member of the honeybee microbiome, where it contributes to pathogen resistance and colony fitness. Nevertheless, the mechanistic investigation of this ecologically important species has remained limited despite its growing probiotic relevance. To enable functional genomics in this organism, we developed an inducible genome-engineering platform that leverages its endogenous Type II-A CRISPR-Cas9 system. The system uses a sakacin-responsive dual-plasmid initiator-effector design in which phage-derived recombineering genes and a single-guide RNA are coordinately expressed, while DNA cleavage is mediated by natively expressed Cas9. Using this approach, we achieved scarless deletion of individual genes, including targets as large as ~25 kb, gene replacement with a fluorescent reporter, C-terminal epitope tagging, and precise nucleotide substitutions, with editing efficiencies approaching 100%. Both plasmids can be readily cured following modification, allowing recovery of clean mutant genotypes. We further demonstrate that endogenous Cas9 can be repurposed for CRISPR interference using a single, self-contained plasmid to enable targeted transcriptional repression. Together, this work establishes a robust strategy for genetic manipulation of A. kunkeei and expands the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria.<br><br>IMPORTANCE: Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria.},
}
@article {pmid42135407,
year = {2026},
author = {Bazick, HO and James, LM and Zylka, MJ},
title = {Nickase NmCas9 unsilences paternal Ube3a in a mouse model of Angelman syndrome without causing AAV vector integration.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42135407},
issn = {2045-2322},
support = {631904//Simons Foundation/ ; 1R01NS109304/NS/NINDS NIH HHS/United States ; 1R01NS109304/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Mice ; *Dependovirus/genetics ; *Angelman Syndrome/genetics/therapy/metabolism ; Genetic Vectors/genetics ; Disease Models, Animal ; Neurons/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; Gene Editing/methods ; Male ; Virus Integration ; CRISPR-Cas Systems ; },
abstract = {Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of maternal UBE3A. In neurons, the paternal (pat)UBE3A allele is silenced by a long non-coding antisense transcript called Ube3a-ATS. Previous genome-editing approaches used active nucleases to unsilence patUbe3a by disrupting Ube3a-ATS. However, these methods create DNA double-strand breaks (DSBs) and promote integration of adeno-associated virus (AAV) vector genomes, both of which raise potential safety concerns. Here, we found that a nickase Neisseria meningitidis Cas9 variant (nNmCas9-D15A) disrupted Ube3a-ATS transcription when targeted to the non-template strand and unsilenced patUbe3a in cultured mouse neurons without generating DSBs or causing AAV integration. Intracerebroventricular delivery of AAV9-nNmCas9-D15A in AS model mice potently and durably reduced Ube3a-ATS and elevated Ube3a throughout the cerebral cortex and hippocampus for at least 6 months. Further, this vector restored UBE3A expression in ~ 87% of cortical neurons, which compares favorably to previously reported efficiencies with active Cas9, dead Cas9, and zinc finger nuclease vectors. These results demonstrate that nNmCas9 is a highly effective and potentially safer genome editor for the treatment of AS.},
}
@article {pmid42303099,
year = {2026},
author = {Zhou, Z and Dong, S and Li, S and Tan, X and Zhou, Z and Fan, L and Liang, J and Li, G},
title = {CRISPR/Cas12a and nanocomposite-based electrochemical/ colorimetric parallel dual-channel aptasensor for highly sensitive LDL detection.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {75},
number = {},
pages = {102981},
doi = {10.1016/j.nano.2026.102981},
pmid = {42303099},
issn = {1549-9642},
mesh = {*Nanocomposites/chemistry ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Lipoproteins, LDL/analysis/blood ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Colorimetry/methods ; Graphite/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Atherosclerotic cardiovascular disease (ASCVD) remains a leading global health threat, necessitating precise monitoring of low-density lipoprotein (LDL) as a key risk biomarker for assessing ASCVD risk. Herein, an electrochemical/colorimetric dual- channel aptasensor was developed by integrating nitrogen-doped reduced graphene oxide-Hemin-trimanganese tetroxide nanoparticles (NrGO-Hemin-Mn3O4 NPs) with the CRISPR/Cas12a system. The CRISPR/Cas12a system introduces a powerful signal amplification cascade: a single target binding event activates the trans-cleavage of numerous ssDNA probes, translating into a highly amplified electrical and optical response. The NrGO-Hemin-Mn3O4 NPs serves as a conductive redox probe and exhibits superior peroxidase-like activity through the synergistic effect between Hemin and Mn3O4. Mechanistically, surface-bound single-stranded DNA (ssDNA) initially induces steric hindrance, which obstructs electron transfer and suppresses the enzyme-mimicking performance of the NrGO-Hemin-Mn3O4 NPs. Upon the target LDL binding, the released activator DNA triggers the trans-cleavage activity of Cas12a to degrade the ssDNA, thereby restoring both the electroactivity and catalytic performance of the probe. Experimental results demonstrated that the dual-channel aptasensor achieved a wide linear range from 0.01 to 1000 nM with a detection limit of 0.01 nM, demonstrating that CRISPR integration is pivotal for achieving high sensitivity in complex biological matrices. This dual-channel strategy offers a sensitive, intuitive tool for early clinical screening of ASCVD diseases.},
}
@article {pmid42420735,
year = {2026},
author = {Rijal, S and Zhang, R and Tian, XJ},
title = {Harnessing CRISPRi Competition to Develop Multimodule Controllers for Resource-Aware Circuit Design.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {305-318},
pmid = {42420735},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; },
abstract = {Cellular resource limitations give rise to resource competition, undermining the modularity and predictability of engineered genetic circuits. In systems containing positive feedback, such competition can drive Winner-Takes-All (WTA) dynamics, resulting in severe imbalances in resource allocation across circuit modules. In this chapter, we present an experimental implementation of a Negatively Competitive Regulatory (NCR) controller based on CRISPR interference (CRISPRi) in dual self-activation (DSA) circuits. We describe a detailed workflow for chromosomal integration of a tunable dCas9 expression cassette, as well as the design of self-activation modules and module-specific guide RNAs that induce self-repression through competition for limiting dCas9. This architecture introduces effective negative feedback to the more active module while reallocating resources to the less active one, thereby promoting balanced module activity. Finally, we provide guidelines for quantitatively assessing the regulation of resource competition between DSA modules using the NCR strategy. Overall, these guidelines demonstrate how CRISPRi can be leveraged to implement NCR strategy in gene circuits, thereby enhancing circuit modularity and predictability.},
}
@article {pmid42423795,
year = {2026},
author = {Ata, A and Topuz Ata, D},
title = {A comprehensive review of CRISPR-Cas9-mediated genome editing in Leishmania strains: methodologies, applications, challenges and future directions.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42423795},
issn = {1573-4978},
mesh = {*Leishmania/genetics ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; Animals ; Genome, Protozoan ; Leishmaniasis/parasitology/genetics ; Humans ; },
abstract = {Genome editing employing CRISPR-Cas9 has rapidly transformed experimental research in Leishmania, providing opportunities to investigate the genetic factors responsible for parasite survival, response to drugs and pathogenic traits. This review provides a comprehensive synthesis of CRISPR-based systems implemented across Leishmania species, spanning Cas9-mediated gene deletion, precise genome editing, endogenous locus tagging and pooled screening strategies. Furthermore, we highlight the emergence of Cas variants and next-generation CRISPR systems which expand the range of targetable genomic regions, improve editing precision and reduce the need for generation of double-strand DNA breaks (DSBs). Particular emphasis is placed on conditional and inducible genome-editing platforms, cytosine base-editing technologies, and recently developed CRISPR-based approaches such as prime editing, CRISPR activation/interference and Cas12-associated implementations. This review also discusses the principal biological and technical constraints influencing CRISPR-based studies in Leishmania, including genome plasticity, multicopy gene families, required genes, guide RNA design limitations and off-target considerations. Notably, the review also addresses CRISPR-Cas implementations in sand-fly vector biology, drawing on a foundational study in Phlebotomus papatasi. Through systematic compilation of published studies into comparative tables, we evaluate the strengths, limitations, experimental utility, delivery strategies, experimental workflows and representative applications of major CRISPR platforms. Together, these advances highlight the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania, while offering a consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research.},
}
@article {pmid42424815,
year = {2026},
author = {Tang, Q and Zhang, Y and Garza, DR and Ruan, C and Liu, B and Rocha, U and Shen, P and Wei, Y and Deng, Y and Zhang, J and Richnow, HH},
title = {Virus-mediated fate of antimicrobial resistance genes in livestock manure anaerobic digestion.},
journal = {Water research},
volume = {305},
number = {},
pages = {126401},
doi = {10.1016/j.watres.2026.126401},
pmid = {42424815},
issn = {1879-2448},
abstract = {Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.668 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions established by CRISPR-Cas spacer, tRNA and homology matches revealed 889 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 18 ARG transduction events. Further analysis showed that the relative abundance of vOTUs assigned to the reduction role (4.11% ± 3.19%) was substantially higher than that of reproduction (0.72% ± 0.64%) and transduction (0.19% ± 0.30%), demonstrating that, among viral processes, lysis outweighs transduction in contributing to ARG abundance reduction in AD. Furthermore, an antiviral defense system (ADS) catalogue (GADSC_LMAD), derived from 2760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS, with ADS prevalence in ARB (7.8 ± 6.0 per MAG), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The resulting CRISPR-Cas immune network with expressed spacers targets foreign ARG-carrying sequences (primarily plasmids and ICEs), suggesting a mechanism that restricts horizontal gene transfer (HGT) via conjugation and transformation, despite shielding ARB from phage lysis. Collectively, these findings highlight that viral communities significantly contribute to ARG reduction through phage lysis relative to transduction, while the ADS-mediated arms race, despite protecting ARB, constructs a biological firewall that potentially limits HGT of ARGs. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.},
}
@article {pmid42424969,
year = {2026},
author = {Samad, MA and Ahmad, I and Jabir, NR and Rehan, M and Zaidi, SK and Al-Abbasi, F and Tabrez, S},
title = {Role of long non-coding RNAs in therapeutic resistance and clinical applications in cancer.},
journal = {European journal of medicinal chemistry},
volume = {317},
number = {},
pages = {119090},
doi = {10.1016/j.ejmech.2026.119090},
pmid = {42424969},
issn = {1768-3254},
abstract = {Cancer is one of the leading causes of mortality worldwide and is recognized as a complex, multifactorial disease with no clearly defined etiology for its onset and progression. Long non-coding RNAs (lncRNAs) are widely distributed across the human body and play varied roles in regulating cellular processes. In recent years, they have gained the attention of the scientific community as key regulators of cancer due to their diverse functional roles and complex regulatory mechanisms. Aberrant expression of lncRNAs contributes to tumor progression, functioning as oncogenes that modulate various pathways through different mechanisms. Early technologies could not study lncRNAs effectively and considered it as "junk" RNA. Studies using gene-expression analyses, functional experiments, and animal-based models have shown that dysregulated lncRNAs are implicated in the maintenance of cancer stem cells (CSCs) and in driving therapeutic resistance. Additionally, lncRNAs have shown promise as valuable biomarkers for cancer diagnosis, prognosis, predicting patient outcomes, and guiding treatment strategies. Moreover, therapeutic strategies targeting lncRNAs, such as antisense oligonucleotides (ASOs), RNA interference (RNAi), exosome-based delivery systems, nanomedicine, virus-mediated therapy, and CRISPR-Cas technologies, have opened new avenues for cancer treatment. This review highlights the diverse roles of lncRNAs in therapeutic resistance and emphasizes their clinical potential as diagnostic and prognostic tools and emerging therapeutic strategies.},
}
@article {pmid42425355,
year = {2026},
author = {V, MS and Chaudhary, N and Hasan, M and Kumar, A and Tripathi, MK},
title = {Filamentous fungi as microbial cell factories for lignocellulosic biomass valorization: A comprehensive review.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153415},
doi = {10.1016/j.ijbiomac.2026.153415},
pmid = {42425355},
issn = {1879-0003},
abstract = {The transition toward a sustainable bioeconomy requires efficient conversion of lignocellulosic biomass (LCB), the most abundant renewable biological macromolecular resource on Earth, into fuels, chemicals, and other high-value products. However, the complex architecture of cellulose, hemicellulose, and lignin imparts significant recalcitrance, limiting biomass deconstruction and industrial utilization. Although recent reviews have examined fungal biorefineries, lignocellulolytic enzymes, or fungal strain engineering separately, an integrated synthesis linking lignocellulosic biomass characteristics, fungal deconstruction mechanisms, hydrolysate utilization, and cell-factory engineering remains limited. This review presents an integrated framework for lignocellulosic biomass valorization using filamentous fungi as microbial cell factories. We examine biomass composition, recalcitrance, and pretreatment strategies, followed by the fungal macromolecular machinery responsible for biomass deconstruction, including cellulases, hemicellulases, lignin-active oxidoreductases, and auxiliary activity enzymes. Particular emphasis is placed on the regulatory networks and engineering strategies that govern fungal performance, including transcription factor engineering, promoter engineering, metabolic rewiring, heterologous pathway engineering, RNA interference, and CRISPR-Cas-based genome editing. The review further discusses the conversion of lignocellulose-derived hydrolysates into biofuels, organic acids, industrial enzymes, and other high-value compounds, together with emerging advances in co-culture fermentation, downstream processing, and integrated biorefinery design. Collectively, this review highlights how the integration of fungal enzymatic systems, strain engineering, and process-level innovations can overcome biomass recalcitrance and improve lignocellulosic bioconversion efficiency. These insights provide a framework for developing robust fungal platforms for the sustainable production of high-value bioproducts from renewable biomass.},
}
@article {pmid42426280,
year = {2026},
author = {Grigg, S and Shembrey, C and Fareh, M and Blombery, P and Corn, JE and Seymour, JF and Casan, JML},
title = {CRISPR in clinical oncology: translational advances from molecular diagnostics to therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {},
number = {},
pages = {},
pmid = {42426280},
issn = {1759-4782},
abstract = {Cancer care is increasingly driven by molecular classification, yet many key oncogenic drivers remain undruggable, and intrinsic or acquired resistance to treatment frequently limits durable clinical benefit. CRISPR-Cas technologies provide a modular, programmable platform to interrogate and directly manipulate cancer biology via sequence-specific targeting of DNA or RNA and have advanced from experimental tools to the early stages of clinical translation. In this Review, we outline how CRISPR-enabled functional genomics approaches can reveal unexpected cancer dependencies and resistance mechanisms. We discuss emerging applications of CRISPR-based diagnostics in oncology that convert precise nucleic acid sequence recognition into rapid mutation detection. We also discuss applications of CRISPR in therapeutic strategies ranging from ex vivo immune cell engineering to nascent in vivo interventions that directly target tumour-related sequences such as fusion junctions or single-nucleotide variants. Finally, we highlight technological and regulatory challenges, including effective delivery of the editing machinery to cells in vivo, safety and platform-level regulatory frameworks, that will determine the clinical utility of CRISPR-based diagnostics and therapies in oncology.},
}
@article {pmid42427176,
year = {2026},
author = {Łakomy, W and Myślińska, M and Tarnawska, E and Rogóż, W and Kulig, K and Owczarzy, A and Maciążek-Jurczyk, M},
title = {Biotechnological strategies to combat antibiotic resistance.},
journal = {Polimery w medycynie},
volume = {56},
number = {1},
pages = {41-51},
doi = {10.17219/pim/218777},
pmid = {42427176},
issn = {0370-0747},
mesh = {Humans ; Antimicrobial Peptides/pharmacology ; Phage Therapy ; *Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; *Drug Resistance, Microbial ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Nanoparticles ; Nanotechnology ; Animals ; },
abstract = {This article aims to present the current state of knowledge on four major biotechnological antimicrobial strategies and to evaluate their potential clinical applications in the context of increasing antibiotic resistance. Approaches such as phage therapy, CRISPR-Cas9 gene editing, nanoparticles, and antimicrobial peptides (AMPs) may significantly contribute to limiting the spread of resistance genes. Particular attention is given to advances in genetic engineering that enable precise targeting and elimination of resistance determinants, as well as to the therapeutic potential of the microbiome. A literature review of studies published between 2010 and 2025 was conducted using the following keywords: antimicrobial resistance, phage therapy, CRISPR-Cas9, AMPs, and nanotechnology. Both review articles and original studies, including preclinical and clinical data, were considered. Phage therapy demonstrates high efficacy against antibiotic-resistant pathogens, particularly in the form of phage cocktails and genetically engineered phages. Antimicrobial peptides exhibit broad-spectrum activity and can be structurally optimized to improve stability and selectivity. CRISPR-Cas9 systems enable targeted elimination of resistance genes or direct disruption of pathogen genomes, while nanotechnology facilitates drug delivery, biofilm penetration, and bactericidal activity, particularly through metal-based nanoparticles. Notably, all approaches show potential for synergistic use with conventional antibiotics. Biotechnological treatment strategies may become a key component in combating antibiotic resistance. However, their clinical implementation requires further research, comprehensive safety evaluation, regulatory development, and integration into medical practice. Advances in these areas could significantly reduce the global burden of infectious diseases.},
}
@article {pmid42428244,
year = {2026},
author = {Zhang, X and Shi, H and Yang, J and Du, L and Zhang, X and Li, X},
title = {The application of CRISPR gene-editing technology in influenza prevention and control.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1844919},
pmid = {42428244},
issn = {2673-3439},
abstract = {Influenza A virus (IAV) and influenza B virus (IBV) remain major global public health threats because of their rapid antigenic evolution and efficient human-to-human transmission. In contrast, influenza C virus (ICV) and influenza D virus (IDV) generally exhibit narrower host ranges and milder pathogenicity, yet their potential for interspecies transmission and zoonotic spillover still warrants attention. Conventional prevention strategies, such as inactivated and live-attenuated vaccines, suffer from prolonged development timelines and diminished efficacy against rapidly evolving viral strains. However, antiviral drugs are increasingly limited by the rapid emergence of drug-resistant variants. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene-editing technology has emerged as a promising platform for influenza prevention and control owing to its programmability and precise targeting capability. In this paper, we summarize recent advances in CRISPR-based strategies for influenza prevention and control. The RNA-targeting CRISPR-associated protein 13 (Cas13) system can recognize conserved viral RNA sequences and suppress replication across influenza subtypes, whereas the DNA-targeting CRISPR-associated protein 9 (Cas9) system can edit host susceptibility genes and thereby reduce cellular permissiveness to infection. In addition, lipid nanoparticle (LNP)-based delivery systems have become important tools for improving the in vivo delivery and expression of CRISPR components by enhancing targeting efficiency and reducing immunogenicity. CRISPR-based diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK), further expand the clinical utility of this technology by enabling rapid and sensitive detection of influenza viruses. Despite these advances, substantial challenges remain, including delivery inefficiency, off-target activity, long-term safety concerns, and the risk of viral escape. With continued technological refinement and careful translational development, CRISPR may become a versatile tool for influenza prevention, diagnosis, and therapy.},
}
@article {pmid42429865,
year = {2026},
author = {Preetam, S and Rath, P and Al-Enazi, NM and Sharaf, AAM and Jumah, JB and Govindarajan, RK and Goud, P and Thiruvengadam, M and Mathivanan, K},
title = {Engineering extracellular vesicle biogenesis for therapeutic gene delivery: emerging genetic programming strategies and translational prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429865},
issn = {1573-4978},
mesh = {Humans ; *Extracellular Vesicles/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Engineering/methods ; Animals ; },
abstract = {Extracellular vesicles (EVs) have emerged as promising biological nanocarriers for gene therapy due to their intrinsic ability to transport nucleic acids, proteins, and lipids between cells. Advances in EV biology have revealed complex regulatory mechanisms governing vesicle biogenesis, cargo sorting, secretion, and uptake, offering multiple opportunities for therapeutic engineering. Concurrently, modern genetic technologies, including the CRISPR-Cas9 genome editing system and synthetic biology tools, have enabled precise manipulation of EV composition and functionality. This review integrates current knowledge of EV biogenesis with emerging genetic engineering strategies to transform EVs into programmable gene delivery systems. We discuss recent advances in genetic tools for studying EV dynamics, methods for engineering EV cargo and targeting specificity, and the application of EV platforms for RNA and genome-editing therapies. Furthermore, key challenges related to vesicle heterogeneity, large-scale production, and clinical translation are examined. Finally, we highlight future perspectives on programmable EV therapeutics and their potential role in next-generation precision medicine.},
}
@article {pmid42429888,
year = {2026},
author = {Thevendran, R and Maheswaran, S and Lee, SY},
title = {Development of attenuated and inactivated Dengue strains using advanced gene editing tools.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429888},
issn = {1573-4978},
support = {FRGS/1/2023/SKK06/AIMST/03/2//Ministry of Higher Education, Malaysia/ ; },
mesh = {Humans ; *Dengue Virus/genetics/immunology/pathogenicity ; *Dengue Vaccines/immunology/genetics ; *Dengue/prevention &amp; control/immunology/virology/genetics ; *Gene Editing/methods ; Vaccines, Attenuated/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Vaccine Development/methods ; },
abstract = {Dengue fever remains a persistent viral threat, affecting millions of families every year, turning a simple mosquito bite into a potentially life-threatening emergency. This disease remains a constant burden on our global healthcare system, demanding innovative solutions to protect worldwide communities. While many researchers discuss general treatments, preventions and modern medical interventions, there is often a lack of focus on how current, cutting-edge molecular and genetic tools are employed to engineer dengue strains as vaccine candidates. Therefore, in this paper, we explore the recent genetic strategies, such as targeted virulent gene deletions, CRISPR-Cas inactivation, and viral codon deoptimization approaches used to attenuate or inactivate Dengue viruses specifically. Assays and techniques used in validating Dengue viral attenuation or inactivation are also discussed in detail, highlighting the importance of the balance between safety and immunogenicity for Dengue vaccine uses. The article also briefly elaborates the complex biological challenges and safety concerns that centre on Dengue vaccine developments. By bridging the gap between advanced genetics and public health, this review provides readers with a comprehensive understanding of how modern genetics is paving the way for the next generation of safe and effective Dengue vaccines.},
}
@article {pmid42431872,
year = {2026},
author = {Kaya, NH and Abukhalaf, M and Fuentes, G and Taubenheim, J and Hentschel, U and Tholey, A and Fraune, S},
title = {c-JUN controls microbial colonization via selective phagocytosis in the sea anemone Nematostella.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42431872},
issn = {2041-1723},
support = {CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Sea Anemones/microbiology/immunology/genetics/metabolism ; *Phagocytosis/immunology ; Immunity, Innate ; Vibrio/immunology ; *Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology ; Microbiota/immunology ; CRISPR-Cas Systems ; Lysosomes/metabolism ; },
abstract = {Innate immunity is traditionally viewed as a broad defense system with limited specificity. However, increasing evidence suggests that innate immune cells can discriminate between distinct microbial partners. How such specificity arises in early-diverging animals remains unclear. Here, we identify in the sea anemone Nematostella vectensis a selective host innate immune mechanism mediated by nematosomes, motile multicellular bodies that differentially process bacterial cells. Nematosomes preferentially engulf non-native Vibrio isolates while showing reduced uptake of native host-associated strains. We identify the transcription factor cJUN as a key regulator of this process. CRISPR/Cas9-mediated knockout of cJUN reduces nematosome abundance, impairs lysosomal response, alters microbiome assembly, and increases susceptibility to bacterial infection. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit sophisticated innate immunity mechanisms for microbiome regulation. Our findings support the idea that immune specificity can arise through repurposing deeply conserved pathways and may have deep evolutionary origin.},
}
@article {pmid42163455,
year = {2026},
author = {Xu, Z and Qiu, S and Tan, Y and Kuang, Y and Yang, C and Yan, F and Zhou, X and Zhou, H},
title = {Optimized tRNA processing and TREX2-SpCas9 fusion enable high-efficiency multiplex genome editing in plants.},
journal = {Plant communications},
volume = {7},
number = {7},
pages = {101921},
doi = {10.1016/j.xplc.2026.101921},
pmid = {42163455},
issn = {2590-3462},
mesh = {*Gene Editing/methods ; *Oryza/genetics ; *RNA, Transfer/genetics/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Genome, Plant/genetics ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; },
abstract = {Multiplex genome editing is a powerful approach for dissecting gene networks and engineering complex traits in crops because it enables the simultaneous modification of multiple genomic loci. However, achieving high editing efficiency across multiple targets remains a significant challenge. To address this, we developed an optimized CRISPR system for rice that combines a monomeric TREX2-SpCas9 fusion with a novel array of tRNA-based gRNA processing elements. The TREX2-SpCas9 fusion significantly enhanced editing performance, resulting in higher editing efficiency, larger deletions, and increased mutation frequencies compared with wild-type SpCas9 and other exonuclease fusions. By systematically evaluating 38 endogenous rice tRNA genes, we identified 13 high-performing candidates, including tRNA[Leu-1] and tRNA[Pro-1], that outperformed the widely used tRNA[Gly] and tRNA[Met] elements, enabling highly efficient processing of multiplexed gRNA arrays. Incorporating these top-performing tRNAs into our system enabled simultaneous editing of up to 29 OsCPK genes in a single rice plant. Furthermore, we demonstrated the cross-species applicability of this platform in the dicot Nicotiana benthamiana using transient expression, where rice-derived tRNA elements facilitated high-efficiency editing. This optimized multiplex gene-editing system provides a robust, scalable platform for accelerating plant functional genomics and engineering complex agronomic traits.},
}
@article {pmid42240651,
year = {2026},
author = {Ma, SH and Yu, G and Park, S and Sung, H and Jeong, UJ and Kim, HH and Cho, J and Yoon, SS},
title = {Adapting prime editing with split prime editors in Escherichia coli and its application to Staphylococcus aureus genome editing.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42240651},
issn = {1432-0614},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *Methicillin-Resistant Staphylococcus aureus/genetics ; *Staphylococcus aureus/genetics ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics/enzymology ; },
abstract = {Prime editing is a precise and rapid genome-editing technique that modifies short DNA sequences using tailored guide RNAs. To implement this technique in bacteria, we used Prime Editor 2 (PE2) with the DeepPrime gRNA design tool and assessed its gene-editing efficiency in Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) cells. Our findings indicate that a split PE2, comprising a reverse transcriptase and two Cas9 nickase domains, exhibited gene-editing efficiency comparable to that of the intact PE2. The efficiency observed in E. coli was significantly affected by the target sites, edit type, and the presence of exonucleases. In MRSA, which serves as a model to evaluate the applicability in non-model bacterial species, Streptococcus pyogenes PE2 (SpPE2) exhibited superior performance relative to Staphylococcus aureus PE2 (SaPE2). Furthermore, the split SpPE2 lacking the reverse transcriptase successfully induced the intended mutation in MRSA. This study demonstrates the feasibility of prime editing within bacterial systems.},
}
@article {pmid42310097,
year = {2026},
author = {Fajardo, AF and Gowda, CP and Johnson, E and Petroni, R and Tomar, VS and Liu, Z and Andres Blanco, M and Janssen, J and Elcheva, IA and Lanza, M and Fuchs, SY and Spiegelman, VS},
title = {In vivo CRISPR knockout screen identifies Polr1a as a key driver and a potential therapeutic target for melanoma metastasis.},
journal = {Oncogene},
volume = {45},
number = {29},
pages = {2978-2987},
pmid = {42310097},
issn = {1476-5594},
support = {CA243167//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; CA288849//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; CA304343//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Melanoma/genetics/pathology ; Humans ; Animals ; Cell Movement/genetics ; Mice ; Cell Line, Tumor ; *DNA-Directed RNA Polymerases/genetics/metabolism ; Neoplasm Metastasis ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Identification and characterization of novel mechanisms driving melanoma metastases and ways to target them are paramount for the development of effective treatment modalities. Here, we employed in vivo CRISPR knockout screening targeting the genes associated with poor prognosis to identify Polr1a as a potent driver of melanoma metastasis. High Polr1a levels correlate with increased metastasis and reduced survival in patients. Polr1a inhibition suppressed migration, invasion, and the ability of melanoma cells to colonize lungs. Ribo-seq analysis revealed that Polr1a is involved in regulating the non-canonical NF-κB pathway. Indeed, targeting Polr1a decreased levels of RelB and p52 and suppressed non-canonical NF-κB transcriptional activity; this suppression was responsible for the effects of Polr1a on melanoma cell migration. Accordingly, pharmacological inhibition of Polr1/Polr1a suppressed cell migration, tumor growth, and metastases. We discuss the potential utilization of Polr1 inhibitors for neoadjuvant treatment of melanoma.},
}
@article {pmid42366688,
year = {2026},
author = {Wu, J and Yan, J and Li, C and Liu, X and Zhao, K and Wu, T},
title = {CRISPR/Cas12a-Enhanced Cascade Amplification for Ultra-sensitive DNA Ligase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20113-20121},
doi = {10.1021/acs.analchem.6c00404},
pmid = {42366688},
issn = {1520-6882},
mesh = {*DNA Ligases/analysis/metabolism ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {DNA ligases are essential enzymes for maintaining genomic integrity, serving as critical biomarkers for the early diagnosis of various malignancies. However, current detection paradigms are often hindered by laborious workflows, high costs associated with chemical modifications, and insufficient sensitivity for low-abundance targets. In this study, we developed an integrated, label-free detection system where DNA ligase serves as a molecular gatekeeper to initiate CRISPR/Cas12a activity. This strategy exploits the discovery that nicked activators exhibit significantly attenuated affinity for the Cas12a-crRNA ribonucleoprotein complex, whereas ligase-mediated repair restores backbone continuity to create a high-affinity intact activator. Upon this ligation-gated activation, the system triggers a subsequent circular DNA-mediated autocatalytic cascade, exponentially amplifying the initial enzymatic signal. Through this dual-stage amplification, we achieved an ultimate limit of detection (LOD) of 2.59 × 10[-6] U/mL. Notably, the platform can reach the analytical sensitivity of established methods in as little as 30 min (LOD of 6.12 × 10[-5] U/mL), significantly compressing the diagnostic time frame. The system demonstrates high selectivity against diverse physiological interferents and has been successfully validated for quantifying endogenous DNA ligase in MC38 tumor cell extracts. This innovative ligase-gated CRISPR cascade provides a modular and robust framework for rapid clinical diagnostics and advanced enzymology research.},
}
@article {pmid42381600,
year = {2026},
author = {Dong, P and Gao, Y and Zhao, W and Fan, L and Wang, Y},
title = {A real-time microfluidic surveillance system for multiplex detection of heavy metal contamination in wastewater.},
journal = {Lab on a chip},
volume = {26},
number = {14},
pages = {4229-4234},
doi = {10.1039/d6lc00331a},
pmid = {42381600},
issn = {1473-0189},
mesh = {*Wastewater/chemistry/analysis ; *Metals, Heavy/analysis ; *Water Pollutants, Chemical/analysis ; *Microfluidic Analytical Techniques/instrumentation ; *Lab-On-A-Chip Devices ; Limit of Detection ; CRISPR-Cas Systems ; },
abstract = {Water pollution, particularly from heavy metals, poses a critical threat to ecosystems and human health. This study integrates the CRISPR-Cas12a system with MOF-based bio-barcode technology to create a platform for the rapid, real-time and on-site detection of multiple heavy metal ions, demonstrating exceptional sensitivity and selectivity. The detection limits for Cu[2+], Pb[2+], and Hg[2+] are 0.26 nM, 0.06 nM, and 0.80 nM, respectively. Inductively coupled plasma-mass spectrometry analysis of real water samples confirmed the high accuracy and reliability of this method. Furthermore, a mobile phone-assisted portable device paired with a microfluidic chip facilitates real-time, rapid multi-channel metal ion detection in resource-limited settings.},
}
@article {pmid42393911,
year = {2026},
author = {Zhu, L and Liao, L and Huang, Y and Nie, Z and Lei, C},
title = {Label-Free Electrochemical CRISPR Platform Gated by Allosteric Transcription Factors for Ultrasensitive Small-Molecule Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20617-20627},
doi = {10.1021/acs.analchem.6c02971},
pmid = {42393911},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Transcription Factors/metabolism/chemistry/genetics ; Allosteric Regulation ; *Tetracycline/analysis ; Animals ; Copper/analysis ; Milk/chemistry ; *CRISPR-Cas Systems ; G-Quadruplexes ; DNA Probes/chemistry ; Parabens/analysis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; },
abstract = {The highly sensitive analysis of small-molecule targets holds profound significance across diverse fields, ranging from clinical diagnosis and environmental monitoring to food safety. Herein, we developed a label-free electrochemical CRISPR platform gated by allosteric transcription factors (aTFs) for the ultrasensitive detection of various small molecules. In this system, the specific binding of target analytes to their cognate aTFs induces the release of programmable DNA adaptors, which subsequently trigger Cas12a to trans-cleave DNA probes anchored to the electrode surface. Consequently, the truncated DNA probes serve as initiators to form electroactive G-quadruplex/hemin complexes in situ via terminal deoxynucleotidyl transferase (TdT)-mediated elongation, generating a robust electrochemical response signal. Using TetR as a model aTF, this integrated electrochemical CRISPR biosensor achieved tetracycline detection with picomolar sensitivity. Furthermore, the versatility of this platform was demonstrated by extending its application to p-hydroxybenzoic acid and copper ions through the simple substitution of the aTF modules. The practical utility of the assay was further demonstrated by the robust detection of tetracycline in complex matrices such as milk. Ultimately, this study not only provides a novel strategy for constructing universal, label-free electrochemical CRISPR platforms but also paves the way for the sensitive detection of low-abundance non-nucleic acid targets.},
}
@article {pmid42397942,
year = {2026},
author = {Tong, Z and Huang, Z and Liu, J and Su, J and Zhu, R and Xiong, R and Yang, Y and Xie, W and Xiao, R},
title = {Heterojunction-Enhanced Interfacial Evanescent-Tunable Fiber Optic Probe for Amplification-free CRISPR/Cas12a-Based Rapid and Ultrasensitive Detection of MPXV.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20429-20441},
doi = {10.1021/acs.analchem.6c02129},
pmid = {42397942},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Gold/chemistry ; *Fiber Optic Technology ; Zinc Oxide/chemistry ; *Optical Fibers ; Metal Nanoparticles/chemistry ; Surface Plasmon Resonance ; Limit of Detection ; },
abstract = {Conventional polymerase chain reaction (PCR)-based detection methods suffer from time-consuming procedures, reliance on specialized equipment, and difficulty in achieving early viral diagnosis. In this study, interferometric fiber-optic sensing is integrated with the CRISPR/Cas12a system for the first time. With sensitivity further enhanced by immobilizing ZnO@Au on the fiber surface, the platform enables rapid, amplification-free detection of monkeypox virus (MPXV) at the single-molecule level. Whispering-gallery modes (WGMs) excited in the fiber probe provide high sensitivity to ambient refractive-index changes, while the ZnO@Au layer induces localized surface plasmon resonance (LSPR) and coupled plasmon-waveguide resonance (CPWR) on the fiber surface. By controlling the AuNPs occupancy on ZnO, the LSPR and CPWR absorption peaks can be tuned to match the demodulation spectral band. Moreover, the ZnO-Au heterojunction further strengthens the LSPR, thereby improving the sensitivity of the fiber probe. The resulting sensing probe achieves amplification-free detection of plasmid targets from both MPXV subtypes down to 10° copies/μL, with the entire assay completed within 9 min. The detection capability was validated using real clinical MPXV samples, showing complete agreement with qPCR results. The strategy proposed in this work offers a feasible approach for early and rapid viral detection.},
}
@article {pmid42420723,
year = {2026},
author = {Kim, G and Kim, HJ and Seo, SW},
title = {Construction of a Tl-CRISPRi Genetic Circuit in Bacteria for Translation-Level Gene Knockdown.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {47-57},
pmid = {42420723},
issn = {1940-6029},
mesh = {*Gene Knockdown Techniques/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Protein Biosynthesis ; *Bacteria/genetics ; Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Regulatory Networks ; Genetic Engineering/methods ; },
abstract = {The Tl-CRISPRi system, which harnesses the specific RNA-binding activity of CRISPR-dCas13, has been recently developed for translation-level gene knockdown in bacteria. By introducing spacers complementary to the translation initiation region of the mRNA, dCas13 can be directed to block the ribosome and inhibit the translation of that mRNA. Here, we discuss how to construct the Tl-CRISPRi genetic circuit and implement this system for gene knockdown. This chapter describes how to design spacer sequences and install them into the guide RNA expression plasmid. Also, we describe how to mutate the handle of gRNA to achieve tunable knockdown of a target gene. By following the method described in this chapter, we anticipate that a precise and controllable knockdown of a target gene in bacterial cells can be performed in a programmable manner.},
}
@article {pmid42420724,
year = {2026},
author = {Santos-Moreno, J},
title = {Design of CRISPRi-Based Synthetic Gene Circuits in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {59-83},
pmid = {42420724},
issn = {1940-6029},
mesh = {*Gene Regulatory Networks ; *Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Bacteria/genetics ; *Genes, Synthetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; Gene Expression Regulation, Bacterial ; Escherichia coli/genetics ; },
abstract = {Synthetic gene circuits are key elements of engineered biological systems that allow us to control and program cellular behavior. Yet, circuit design can be challenging to newcomers due to the numerous design choices and the abundance and variety of parameters that can influence circuit performance. While transcription factors have dominated the circuit construction toolbox for two decades, CRISPRi-based tools offer important benefits-especially for large circuits-but also require unique design considerations. Here I provide a detailed guide for designing CRISPRi circuits in bacteria, using the CRISPRlator, the first CRISPRi oscillator, as an example that illustrates the design process.},
}
@article {pmid42420725,
year = {2026},
author = {Park, D and Shin, W and Kang, H and Kim, J},
title = {Design of Conditional Guide RNAs for the Logical Regulation of Gene Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {85-107},
pmid = {42420725},
issn = {1940-6029},
mesh = {Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR interference (CRISPRi) is an RNA-guided regulator that silences gene expression by binding to its cognate DNA target, halting transcription in both prokaryotic and eukaryotic cells. Recent advances in RNA synthetic biology have endowed CRISPR guide RNAs (gRNAs) with conditional functionality: these so-called conditional guide RNAs (cgRNAs) fold into strong hairpins that block their activity until a specific trigger RNA is present. Upon introduction of the cognate trigger RNAs, the hairpin structure unfolds, allowing the activated cgRNA to direct transcriptional repression with large dynamic ranges, minimal crosstalk, expanded tunability, and logic-gated signal processing. Furthermore, cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression. This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli.},
}
@article {pmid42420731,
year = {2026},
author = {Pujar, A and Sharma, A and Kushwaha, M},
title = {Intercellular CRISPRi for Distributed Genetic Circuits.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {229-243},
pmid = {42420731},
issn = {1940-6029},
mesh = {*Gene Regulatory Networks ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacteria/genetics ; Synthetic Biology/methods ; Escherichia coli/genetics ; },
abstract = {Microbial communities and multicellular organisms employ diverse strategies for allocation of available resources, achieved through task distribution among specialized cells. Drawing inspiration from nature, several synthetic multicellular circuits have been recently constructed where a larger circuit is distributed into several cells in order to reduce the burden on individual cells. Here, we describe the implementation of multicellular logic-gate circuits in bacterial co-cultures that combine DNA messaging with CRISPRi regulation. Leveraging the easily programmable and information-dense DNA molecules, our system is composed of sender bacteria that transmit DNA messages encoding guide RNAs and receiver bacteria that receive them and express the guide RNAs to regulate transcription by CRISPR interference. We demonstrate several functional multicellular circuits representing digital logic gates that operate on timescales comparable to small molecule signaling: NOT, YES, AND, and AND-AND-NOT. The receiver cells process the inputs received to perform computations and generate a logical output.},
}
@article {pmid42340184,
year = {2026},
author = {Kawai-Harada, Y and You, S and Scarborough, T and Siraj, N and Yedla, J and Rennells, T and Walton, SP and Chan, C and Harada, M},
title = {Generation of Cellular Biofactories for the Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing.},
journal = {ACS biomaterials science &amp; engineering},
volume = {12},
number = {7},
pages = {3821-3831},
doi = {10.1021/acsbiomaterials.6c00782},
pmid = {42340184},
issn = {2373-9878},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism/genetics/chemistry ; HEK293 Cells ; },
abstract = {Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods relying on transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we present a synthetic-biology-based biomaterial manufacturing platform that uses CRISPR-Cas9 genome editing to generate stable HEK293T cell lines for continuous production of surface-functionalized EVs. A fusion construct encoding mCherry-C1C2 was site-specifically integrated into the AAVS1 safe-harbor locus, enabling consistent and heritable expression of EV membrane proteins without repeated transfection. Engineered cells produced EVs with uniform size (120-130 nm), preserved canonical markers (CD63 and ALIX), and enhanced surface-display efficiency compared with transiently transfected controls. These vesicles exhibited robust cellular uptake and maintained structural and functional stability for over 25 passages (∼3 months), confirming durable genome-encoded production. Overall, this platform eliminates batch-to-batch variability inherent to transient systems and provides a genetically defined route to biofunctional nanomaterial fabrication. This approach links genetic design to nanoscale surface functionality, establishing a versatile foundation for reproducible biomanufacturing of engineered EVs for biomaterial, therapeutic, and diagnostic applications.},
}
@article {pmid42416053,
year = {2026},
author = {Xia, C and Lian, M and Ma, B and Yu, H and Zhang, R and Wen, L and Wang, X and Zhao, Y and Ouyang, Z and Ye, Y and Feng, X and Wu, H and Lai, L},
title = {Development of a BM7G(TKO/hCD46/hCD55/hTHBD/hEPCR) donor pig with endogenous promoter-driven transgenes for xenotransplantation.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1827497},
pmid = {42416053},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Promoter Regions, Genetic ; Animals, Genetically Modified ; Humans ; Swine ; Galactosyltransferases/genetics ; *Transgenes ; *Thrombomodulin/genetics ; *Membrane Cofactor Protein/genetics ; Graft Rejection/immunology/genetics/prevention &amp; control ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Heterografts ; Mixed Function Oxygenases/genetics ; Graft Survival ; N-Acetylgalactosaminyltransferases ; },
abstract = {INTRODUCTION: Xenotransplantation holds promise for addressing the organ shortage crisis. Multi-genetic modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing multi-gene modification strategies face challenges: transposon-based transgenic technology may lead to unstable expression, while exogenous promoters used in site-specific integration strategies are susceptible to epigenetic silencing, making it difficult to maintain long-term, stable expression levels. Therefore, developing a donor pig model capable of achieving stable and long-lasting multi-gene expression is a critical need in the field.<br><br>METHODS: CRISPR-Cas9 technology was used to knockout three major glycan antigen genes (GGTA1, CMAH, &#x3b2;4GalNT2) to eliminate hyperacute rejection. Subsequently, four human protective genes (hCD55, hCD46, hTHBD, hEPCR) were site-specifically integrated into the porcine Rosa26 safe-harbor locus. Their expression was driven by the porcine endogenous Rosa26 promoter and the THBD core promoter, respectively, to ensure long-term stable and tissue-specific expression. Furthermore, the selection marker gene was efficiently removed using the Cre/loxP system.<br><br>RESULTS: The three glycan antigens were completely absent at both cellular and tissue levels in BM7G genetically modified pigs. What's more, four protective proteins were stably expressed in vascular endothelial cells and major organs such as the heart, liver, and kidneys. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR were specifically expressed in the vascular region. In-vitro functional assays confirmed that BM7G porcine vascular endothelial cells significantly reduced the binding of human antibodies, effectively inhibited complement-dependent cytotoxicity, and decreased the formation of thrombin-antithrombin (TAT) complexes.<br><br>CONCLUSION: In summary, by combining the knockout of xenoantigens with the use of endogenous promoters to drive the expression of multiple human protective genes, we successfully constructed a seven-gene modified pig model with low immunogenicity and synergistic protective functions. This provides an important donor resource for preclinical research in xenotransplantation.},
}
@article {pmid42416071,
year = {2026},
author = {Akula, S and Wernersson, S and Hellman, L},
title = {Immunity: defense against infections essential for all living organisms.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1840774},
pmid = {42416071},
issn = {1664-3224},
mesh = {Animals ; Humans ; *Adaptive Immunity ; Immunity, Innate ; Host-Pathogen Interactions/immunology ; },
abstract = {All organisms need protection against infection. Bacteria are often primarily seen as infectious agents, but they also need protection against bacterial viruses, so-called bacteriophages. To this end, bacteria have developed very complex defense systems, including apoptosis-like mechanisms, restriction enzymes, and even adaptive-type mechanisms involving immunological memory of immune responses through a system called CRISPR-Cas. An earlier dominating view was that adaptive immunity in eukaryotes only exists in jawed vertebrates, as their immune system includes the classical and highly variable immunoglobulins (Igs) and T-cell receptors (TCR). However, other types of variable molecules, which may be involved in immunity, have also been identified in insects, snails, lancelets, plants, sea urchins, and jawless fishes. Interestingly, fishes without jaws, such as the hagfish and lamprey, have a very complex adaptive immunity built on lymphocyte-like cells and variable lymphocyte receptors (VLRs). Notably, the variability of these VLRs has been estimated to be in the same range as Igs and T-cell receptors. This illustrates that very diverse strategies have been used to create an adaptive immune system in different organisms, indicating potent convergent evolution. Vertebrate immunity includes both adaptive and non-adaptive components, which work closely together to form a very powerful immune system for defense against infections. In contrast to adaptive immunity, the majority of the non-adaptive innate defense mechanisms, such as pattern recognition receptors, antimicrobial peptides (AMPs), iron-binding proteins, the complement system, and lysozymes, can be traced back to early eukaryotes. Immunity of invertebrates seems to rely almost entirely on innate defense mechanisms, while the presence of complex adaptive mechanisms in invertebrates, such as the VLRs of jawless fishes and Igs and TCR of jawed vertebrates, is questionable. This review summarizes old and recent findings of importance for our understanding of how immunity became an integrated part of all living organisms, from bacteria to humans, and the very different strategies that different organisms use in the protection against infection.},
}
@article {pmid42416743,
year = {2026},
author = {Raj D, D and Maurya, AK and Singh, J and Kumar G, M and Ramani, A and Khurana, AK and Purwar, S and Biswas, D},
title = {Off-Target activity as a Translational Barrier in Programmable Gene-Editing Strategies for Nontuberculous Mycobacteria: Narrative Review.},
journal = {Maedica},
volume = {21},
number = {2},
pages = {495-503},
pmid = {42416743},
issn = {1841-9038},
abstract = {OBJECTIVES: To review the clinical and translational implications of off-target activity associated with clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches in nontuberculous mycobacteria (NTM) and discuss current strategies aimed at specificity and safety.<br><br>MATERIALS AND METHODS: The relevant published literature on the application of CRISPR-Cas systems, including Cas9, Cas12a and CRISPR interference (CRISPRi), in NTM research was reviewed. Particular attention was given to off-target mechanisms, mycobacteria-specific genomic challenges, computational predictions, experimental detection methods, high-fidelity nucleases and delivery optimisation approaches.<br><br>RESULTS: Nontuberculous mycobacteria infections often require prolonged treatment and are frequently associated with relapse and rising antimicrobial resistance, particularly in Mycobacterium abscessus infections. CRISPR-based technologies provide advantages in precision diagnostics, functional genomics and therapeutic development; however, high guanine-cytosine (GC) content, repetitive PE/PPE gene families, mismatch tolerance and unique DNA repair mechanisms contribute considerably to off-target effects. Emerging high-fidelity nucleases, guide RNA optimisation, artificial intelligence (AI)-assisted prediction platforms and alternative editing systems demonstrate considerable potential for improving editing specificity and translational safety.<br><br>CONCLUSIONS: Advances in nuclease engineering, computational modelling, delivery systems, and genome-wide validation approaches may improve therapeutic precision and diagnostic reliability. Addressing these challenges through interdisciplinary innovation will be essential for the future clinical integration of CRISPR-based antimycobacterial strategies.},
}
@article {pmid42417038,
year = {2026},
author = {Abdullah, N and Lewis, J and Arumugam, P},
title = {Genome-wide CRISPR/Cas9 screening reveals lipid metabolism and inflammatory signalling as modulators of ganoderic acid DM cytotoxicity.},
journal = {Journal of genetics},
volume = {105},
number = {},
pages = {},
pmid = {42417038},
issn = {0973-7731},
mesh = {Humans ; *Triterpenes/pharmacology ; *Lipid Metabolism/drug effects/genetics ; Signal Transduction/drug effects ; *CRISPR-Cas Systems/genetics ; *Inflammation/genetics/metabolism ; Cell Line, Tumor ; Sterol Regulatory Element Binding Proteins/genetics/metabolism ; *Melanoma/genetics/drug therapy/pathology/metabolism ; NF-kappa B/genetics/metabolism ; Cholesterol/biosynthesis ; },
abstract = {Ganoderic acid DM (GA-DM), a triterpenoid derived from Ganoderma lucidum, exhibits anti-cancer and anti-diabetic activities, but the underlying mechanisms of action remain unclear. To identify genetic modulators of the GA-DM response, we conducted a genome-wide CRISPR/Cas9 knockout screen in human melanoma cells. The screen revealed key roles for genes regulating lipid metabolism and inflammatory signalling, particularly those involved in the SREBP (sterol regulatory element-binding protein) and NF-jB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathways, in the cellular response to GA-DM. While loss of genes involved in the regulation of cholesterol biosynthesis conferred resistance to GA-DM, disruption of genes involved in ubiquitin-mediated proteolysis and the Hippo pathway sensitised cells to GA-DM. Inflammatory genes enriched at later time points suggests that a delayed cellular response contributes to cytotoxicity. Our findings propose a mechanistic model wherein GA-DM perturbs lipid and inflammatory pathways to exert cytotoxic effects and highlight potential targets to enhance its therapeutic efficacy. This work demonstrates the utility of functional genomics in elucidating the mechanisms of action of natural products and guiding rational drug development.},
}
@article {pmid42417866,
year = {2026},
author = {Tang, Y and Zhang, L and Wang, W and Yang, X},
title = {An integrated signal amplification strategy based on catalytic hairpin assembly and hybridization chain reaction for driving a CRISPR/Cas12a biosensor toward ultrasensitive detection of microRNAs.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42417866},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Humans ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; DNA Probes/chemistry/genetics ; },
abstract = {A novel biosensing platform is proposed that integrates catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cascade isothermal amplification with the CRISPR/Cas12a system, enabling ultrasensitive detection of microRNAs (miRNAs) targets. Within this platform, two modules are integrated: a target recognition and signal amplification module constructed by the cascade of CHA and HCR, and a signal transduction module in which the CRISPR/Cas12a system acts in concert with DNA probes loaded onto gold nanoparticles (AuNPs). This design achieves cascaded amplification from target recognition to signal output, thereby conferring high signal gain. Experimental results demonstrate that the proposed biosensor had high sensitivity toward the target miRNA, with a detection limit as low as 37 fM. Moreover, it shows remarkable single-base discrimination capability, effectively distinguishing sequences with single-nucleotide mismatches. Notably, the sensor maintains stable and reliable performance in complex biological matrices, including serum samples and lysates from various tumor cells. This strategy effectively couples signal amplification with the CRISPR system, achieving both high sensitivity and specificity, making it a useful tool for miRNA detection and early cancer screening.},
}
@article {pmid42420722,
year = {2026},
author = {Irvine, TCT and Bailey, AM and Gorochowski, TE},
title = {A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {33-45},
pmid = {42420722},
issn = {1940-6029},
mesh = {*Genome, Bacterial ; Escherichia coli/genetics ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The insertion of large genetic circuits and metabolic pathways into bacterial genomes is becoming increasingly common within the field of synthetic biology due to the improved robustness and stability that come with genome integration. CRISPR-associated transposases (CASTs) enable RNA-guided DNA insertion without introducing double-stranded breaks and have been shown to function across diverse bacterial species. Here, we present an improved tool called pSPIN-GG and supporting protocols for simplified CAST-based genome engineering. The pSPIN-GG system includes Golden Gate-compatible promoter, guide, and cargo modules for simple assembly, a green fluorescent protein dropout cassette for rapid verification of guide replacement, and a set of tested sites within the Escherichia coli BL21 chromosome to enable gene dosing of genetic cargoes. These refinements support accelerated library construction, reduce assembly and screening burden, and expand the accessibility of CAST systems for multiplexed bacterial genome engineering.},
}
@article {pmid42415313,
year = {2026},
author = {Solanki, M and Yousuf, F and Srivastava, A and Vaikuntapu, PR and Molla, K and Neeraja, CN and Thakur, V and Barbadikar, KM and Sundaram, RM and Monhannath, G and Mangrauthia, SK},
title = {Powering Genome Editing in Rice by Harnessing Promising Gene Resources: A Comprehensive Roadmap.},
journal = {Physiologia plantarum},
volume = {178},
number = {4},
pages = {e71005},
doi = {10.1111/ppl.71005},
pmid = {42415313},
issn = {1399-3054},
support = {NASF/CRISPR-Cas-7003/2017-18//Indian Council of Agricultural Research/ ; NASF/BGAM-9021/2022-23//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; Plant Breeding/methods ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The imprecise breeding methods including recombination breeding, physical/chemical mutagenesis, and marker-assisted breeding have been extensively utilized for trait improvement of rice crop. Despite tremendous progress made through these breeding methods, the critical issues, such as linkage drag, unintended phenotype, and longer duration of time required to breed a cultivar, have been the major limitations. Among the new breeding technologies, genome editing (GE) has become the most promising approach because of its specificity, precision, and speed. Despite its transformative potential, genome editing continues to face several limitations in crop improvement. These include well-recognized policy challenges, such as biosafety regulations and intellectual property constraints, alongside technical barriers like inefficient tissue culture and transformation systems. Additionally, researchers remain constrained by the limited availability of precise gene information necessary for accurate targeted editing and effective trait enhancement. This review presents an analysis of genes that regulate abiotic and biotic stresses, yield, grain quality and nutrition, plant architecture, nutrient absorption and use efficiency, and other agronomically important traits of rice. The trait-wise probable target genes for genome editing have been discussed in detail. This review will serve as a ready reckoner for rice researchers and funding agencies.},
}
@article {pmid41115768,
year = {2026},
author = {Jordan, AJ and Balmforth, C and Craig, N and Dhaun, N and Baker, A and Dweck, MR and Newby, DE},
title = {Ribonucleic acid and gene therapies in cardiovascular disease: clinical applications, delivery challenges and emerging precision tools.},
journal = {Heart (British Cardiac Society)},
volume = {112},
number = {15},
pages = {828-837},
doi = {10.1136/heartjnl-2024-325280},
pmid = {41115768},
issn = {1468-201X},
mesh = {Humans ; *Cardiovascular Diseases/therapy/genetics ; *Genetic Therapy/methods ; *RNA, Small Interfering/therapeutic use ; *Precision Medicine/methods ; Gene Therapy Agents ; Gene Editing/methods ; *RNAi Therapeutics/methods ; Gene Transfer Techniques ; CRISPR-Cas Systems ; },
abstract = {Cardiovascular diseases remain a leading cause of global mortality despite advancements in pharmacotherapies, with current treatments facing challenges related to efficacy, tolerability and patient adherence. In response, advanced therapies, such as RNA and gene therapies, have emerged as a promising alternative for addressing both acquired and monogenic cardiovascular conditions. This review explores the current landscape of RNA and gene therapies for cardiovascular disease, focusing on RNA-based therapeutics such as small-interfering RNAs (siRNAs), antisense oligonucleotides and clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9)-based gene editing systems. Recent European Medicines Agency and Food and Drug Administration-approved RNA therapies, including patisiran, vutrisiran and inclisiran, which employ lipid nanoparticle delivery systems, highlight the clinical potential of siRNAs for targeting hepatic molecular pathways. Emerging CRISPR-Cas9 technologies are poised to address genetic mutations at their source, offering permanent correction of pathogenic variants and the potential to treat a broad range of hereditary cardiovascular conditions. Together, these therapies represent a major leap forward in precision medicine, offering long-lasting therapeutic effects and improved patient care and adherence. However, many challenges remain, particularly in targeting such therapies to cardiac tissues and optimising delivery systems. This review discusses the current state of the art in cardiovascular RNA and gene therapies, including current evidence, delivery challenges and the current landscape of gene and RNA therapies in phase I clinical trials and beyond.},
}
@article {pmid42224782,
year = {2026},
author = {Dong, Z and Liu, Y and Wu, X and Ban, W and Zhao, L and Liu, X and Ding, J},
title = {Rapid multiplex detection of Echinococcus granulosus and Echinococcus multilocularis using a one-pot RPA-assisted CRISPR-Cas12a/Cas13a assay in a portable multi-tube device.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118857},
doi = {10.1016/j.bios.2026.118857},
pmid = {42224782},
issn = {1873-4235},
mesh = {Animals ; *Echinococcus multilocularis/isolation &amp; purification/genetics ; *Echinococcosis/parasitology/diagnosis ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems ; *Echinococcus granulosus/isolation &amp; purification/genetics ; Dogs ; Rapid Diagnostic Tests ; DNA, Helminth/genetics/isolation &amp; purification ; Limit of Detection ; Humans ; Equipment Design ; },
abstract = {Echinococcosis, caused by Echinococcus granulosus and Echinococcus multilocularis, remains a significant zoonotic threat, particularly in pastoral regions where rapid environmental surveillance is essential yet technically constrained. Here, we report a rapid and integrated one-pot recombinase polymerase amplification -assisted, orthogonal CRISPR-Cas12a/Cas13a platform for rapid and specific discrimination of these two species in environmental samples. Coupled with a simplified NaOH-based DNA extraction method, the assay enables a streamlined workflow completed within 60 min, achieving a detection limit of as low as 1 copy/μL without observable cross-reactivity. To facilitate point-of-care deployment, we further developed a low-cost, miniaturized handheld device capable of parallel analysis of up to eight samples with dual-target readout. The platform was validated using field samples, including canine feces, pasture grass, and vegetables, demonstrating complete agreement with quantitative PCR results, with 100% sensitivity and specificity. This integrated CRISPR-based biosensing system provides a robust and field-deployable solution for on-site echinococcosis surveillance and offers a scalable framework for multiplex environmental pathogen detection.},
}
@article {pmid42247945,
year = {2026},
author = {Fan, Z and Yin, X and Ma, M and Du, B and Liu, Z and Xu, J and Liu, B and Tong, Z},
title = {Cas12a2-based multiplexed screen-printed electrode electrochemiluminescence biosensor for amplification-free SARS-CoV-2 detection in aerosols.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118885},
doi = {10.1016/j.bios.2026.118885},
pmid = {42247945},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation/methods ; *SARS-CoV-2/isolation &amp; purification/genetics ; Humans ; Aerosols/analysis ; *COVID-19/diagnosis/virology ; Electrochemical Techniques/instrumentation/methods ; RNA, Viral/analysis/genetics ; Luminescent Measurements/instrumentation ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry ; Electrodes ; Limit of Detection ; *Endodeoxyribonucleases/chemistry ; Equipment Design ; Rapid Diagnostic Tests ; Bacterial Proteins ; },
abstract = {Airborne transmission of respiratory viruses poses a severe public health threat, urgently requiring portable and sensitive techniques for viral aerosol monitoring. CRISPR-Cas12 technology has brought extensive innovations to the field of nucleic acid detection. Among them, Cas12a2 exhibits unique RNA-triggered trans-cleavage activity, showing prominent advantages in the amplification-free detection of respiratory RNA viruses. Herein, we developed an amplification-free and electrode-modification-free electrochemiluminescence biosensing platform based on screen-printed electrodes by integrating the specific recognition capability of Cas12a2 and the synergistic activation effect of multiple crRNAs. The optimized Cas12a2-based system achieves ultrasensitive detection of SARS-CoV-2 RNA with a low limit of detection of 76 aM. Furthermore, we constructed a stable viral aerosol generation and collection device and successfully validated the practical capability of the proposed platform for SARS-CoV-2 aerosol detection. This rapid and portable detection strategy offers a promising alternative for on-site monitoring of airborne pathogens and further expands the application scope of CRISPR biosensing technology in viral detection.},
}
@article {pmid42250350,
year = {2026},
author = {Wang, Q and Choi, S and Heo, W and Kim, MW and Park, S and Park, SJ and Shin, J and Hyun, KA and Kim, J and Lim, CS and Jung, HI},
title = {Electrochemical-sensor-assisted lab-in-a-cartridge (EC-LIC) for on-site detection of SARS-CoV-2 with a self-contained heating system.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118899},
doi = {10.1016/j.bios.2026.118899},
pmid = {42250350},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation &amp; purification/genetics ; Humans ; *COVID-19/diagnosis/virology ; *Biosensing Techniques/instrumentation ; *Electrochemical Techniques/instrumentation ; Rapid Diagnostic Tests ; Equipment Design ; Limit of Detection ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Heating/instrumentation ; COVID-19 Nucleic Acid Testing/instrumentation ; },
abstract = {Rapid and accurate detection of respiratory viruses is essential for controlling disease transmission and enabling effective public health responses, particularly in resource-limited settings. In this study, we present an electrochemical-sensor-assisted lab-in-a-cartridge (EC-LIC) platform for on-site detection of SARS-CoV-2 featuring a self-contained chemical heating system. The device incorporates rotational and gravity-driven fluid handling along with exothermic heating using calcium oxide and a flameless ration heater to generate controlled temperature gradients. Coupled with a CRISPR-Cas13a-based electrochemical sensor, the system enables direct detection of the SARS-CoV-2 N gene without nucleic acid amplification, achieving high sensitivity and specificity. Integrated with a handheld electrochemical reader, the EC-LIC operates as a fully automated sample-to-answer system, completing the assay within 40 min over a wide dynamic range from 1.0 × 10° to 1.0 × 10[5] fg/mL with a limit of detection as low as 1.21 × 10[-1] fg/mL. Clinical validation using samples from 102 individuals (60 positive and 42 negative) demonstrated a sensitivity of 98% and a specificity of 90%. These results establish the EC-LIC as a robust nucleic acid detection platform for rapid clinical screening and early epidemic response.},
}
@article {pmid42269453,
year = {2026},
author = {Ren, K and Yu, C and Wu, L and Ma, J and Xu, X and Jia, L and Yang, Q},
title = {Construction of an amplification-free dual-mode sensor based on CRISPR/Cas12a-mediated and dual-mode integrated reporter FU for ultrasensitive detection of non-nucleic acid target deoxynivalenol.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118879},
doi = {10.1016/j.bios.2026.118879},
pmid = {42269453},
issn = {1873-4235},
mesh = {*Trichothecenes/analysis/isolation &amp; purification/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Colorimetry/methods ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Sensitive and accurate detection of deoxynivalenol (DON) is crucial for public health. The CRISPR/Cas12a system exhibits high specificity and efficiency in biosensing, but challenges remain in non-nucleic acid detection, such as reliance on multiple reporters for dual-mode signal output and low detection sensitivity without amplification. In this study, leveraging the magnetic and fluorescence quenching properties of Fe3O4/Au/PDA and the fluorescence/catalytic capabilities of UiO-66-NH2, we developed a multimodal integrated reporter (FU) as Fe3O4/Au/PDA-ssDNA-UiO-66-NH2, enabling dual-mode signal output via a single reporter. In the presence of DON, the DON-Ab-aDNA complex activates CRISPR/Cas12a, which then indiscriminately cleaves the single-stranded DNA in FU, releasing free UiO-66-NH2. Consequently, the fluorescence signal of UiO-66-NH2 is restored while it catalyzes TMB to produce a blue color reaction. The CRISPR/Cas12a-based fluorescence-colorimetric dual-mode biosensor (CrisprFU) achieved a colorimetric limit of detection (LOD) for DON of 2.15 × 10[-3] ng/mL (detection range: 2-100 ng/mL) and a fluorescence LOD of 7.96 × 10[-4] ng/mL (detection range: 0.5-40 ng/mL). Successful application in real samples demonstrated average recovery rates of 97.04%-104.4% for fluorescence detection and 96.4%-101.8% for colorimetric detection, confirming its practical potential. Furthermore, by replacing the recognition antibody, the CrisprFU system can be extended to detect other analytes.},
}
@article {pmid42269938,
year = {2026},
author = {Wei, E and Tang, Y and Lei, Y and Liu, S and Xu, B and Du, B and Wang, Y and Liao, Y and Wang, Y and Zhao, J},
title = {Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.},
journal = {Journal of microbiological methods},
volume = {247},
number = {},
pages = {107584},
doi = {10.1016/j.mimet.2026.107584},
pmid = {42269938},
issn = {1872-8359},
mesh = {*Bacillus cereus/genetics/metabolism ; *CRISPR-Cas Systems ; Pyrethrins/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Bacterial Proteins/genetics ; Probiotics ; Gene Deletion ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Bacillus cereus GW-01, an efficient degrader of β-cypermethrin (β-CY), has a high safety profile and probiotic potential for regulating intestinal flora and fermented foods, which is difficult to genetically engineer for modification due to its restrictive modification system. This study successfully developed a CRISPR/enCas12f-based genome editing system, first selecting the plcR gene for proof-of-concept validation with 100% knockout efficiency. Subsequently, this system was utilized to delete the virulence gene nheABC in GW-01, yielding a safer probiotic strain. Compared with the wild-type strain GW-01, the probiotic-related indicators of the ΔnheABC mutant, including cell surface hydrophobicity, auto-aggregation ability and biofilm formation ability, were 80%, 90% and 2.9 (OD595), respectively. There were no significant differences in these indicators between the mutant and the wild type. Meanwhile, the ΔnheABC mutant still maintained a high β-cypermethrin degradation efficiency of 80% at the concentration of 30 μg/mL. This work facilitates functional genomic research and genetic modification of Bacillus cereus GW-01. The established CRISPR/enCas12f system enables targeted gene deletion to explore gene functions and phenotypic mechanisms, and paves the way for its development into safe probiotics and excellent microbial chassis.},
}
@article {pmid42308857,
year = {2026},
author = {Li, L and Guo, X and Yang, X and Qin, S and Wang, W},
title = {High-sensitivity and portable detection of oral pathogens based on CRISPR/Cas13a combined with exonuclease-assisted cycling amplification and lateral flow assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118935},
doi = {10.1016/j.bios.2026.118935},
pmid = {42308857},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Ribosomal, 16S/genetics/isolation &amp; purification ; Rapid Diagnostic Tests ; *Bacteria/isolation &amp; purification/genetics/pathogenicity ; Limit of Detection ; Exonucleases/chemistry ; *Mouth/microbiology ; },
abstract = {Infectious diseases caused by oral pathogens represent a significant threat to human health. Current diagnostic technologies for oral pathogens lack the characteristics of speed, sensitivity, and convenience, making it difficult to meet the needs of rapid testing in laboratories and on-site. Consequently, the development of novel high-sensitivity and high-specificity pathogen analysis methods and sensing systems is imperative. In this study, we established a high-throughput CRISPR/Cas13a method for identifying pathogenic bacteria 16S rRNA, which we combined with isothermal enzyme cycling amplification technology (CRIE) to improve sample detection resolution, sensitivity, and speed. Furthermore, based on the characteristics of dopamine catalyzed by G4/hemin to form polydopamine and combined with CRIE, we developed lateral flow assay (CRIEC) for simple, portable, and rapid detection of pathogenic bacteria. Preliminary experiments were performed to verify its analytical performance and application potential. The obtained data may lay a basic foundation for the subsequent research and clinical application in the field of oral pathogen detection.},
}
@article {pmid42323937,
year = {2026},
author = {Zhang, Y and Li, L and Li, Y and Zeng, Y and Liu, H and He, M},
title = {Functionalized carbon nanotube-assisted dual-mode CRISPR/Cas12a detection of hepatitis C virus via catalytic assembly circuit-driven Y-shaped dsDNA activators.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118946},
doi = {10.1016/j.bios.2026.118946},
pmid = {42323937},
issn = {1873-4235},
mesh = {*Nanotubes, Carbon/chemistry ; *Hepacivirus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis C/virology/diagnosis ; *RNA, Viral/genetics/isolation &amp; purification/analysis ; Colorimetry/methods ; Limit of Detection ; Hydrogen Peroxide/chemistry ; Catalysis ; DNA/chemistry ; Endodeoxyribonucleases/chemistry ; CRISPR-Associated Proteins/chemistry ; },
abstract = {Hepatitis C virus (HCV) is a major etiological agent of liver diseases and remains a serious global health threat. Herein, we report a dual-modal HCV biosensing platform for ultrasensitive HCV RNA detection by integrating near-infrared fluorescence and colorimetric readouts. In this system, the presence of HCV RNA initiates a catalytic assembly circuit (CAC) that forms a Y-shaped DNA structure, exposing two double-stranded DNA activators with complete protospacer adjacent motif (PAM) to trigger CRISPR/Cas12a nuclease activity. The combination of CRISPR/Cas12a-driven signal amplification and hemin-binding aptamer-functionalized single-walled carbon nanotubes (HeApt-SWCNTs) enables highly sensitive target quantification. Upon exposure to hydrogen peroxide (H2O2), ferric ion in hemin catalyzes a Fenton-like reaction, generating hydroxyl radicals (·OH) that quench SWCNT fluorescence and oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to its blue oxidized form (oxTMB). In the presence of target HCV RNA, CRISPR/Cas12a-mediated HeApt cleavage inhibits ·OH generation, resulting in SWCNT fluorescence recovery and suppresses TMB oxidation. Under optimal conditions, detection limits of 0.23 fM and 4.1 fM are achieved for the fluorescence and colorimetric modes, respectively. This integrated CAC-Cas12a-HeApt-SWCNTs (CCHS) biosensing strategy offers high specificity, dual-mode signal reliability, and broad potential for early diagnosis of HCV and other RNA viruses.},
}
@article {pmid42330664,
year = {2026},
author = {Zhao, J and Xu, H and Fei, S and Xu, C and Yin, W and Wei, Q and Lin, J and Liu, G and Feng, S and Gao, F and Wang, Y},
title = {Smartphone-integrated RPA-CRISPR/Cas12a detection system with microneedle sampling for early point-of-care diagnosis of potato late blight.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118943},
doi = {10.1016/j.bios.2026.118943},
pmid = {42330664},
issn = {1873-4235},
mesh = {*Solanum tuberosum/microbiology/parasitology ; *Plant Diseases/microbiology/parasitology ; Smartphone ; *Phytophthora infestans/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; Point-of-Care Systems ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/instrumentation ; Plant Leaves/microbiology ; Equipment Design ; Needles ; },
abstract = {Potato late blight, caused by the oomycete pathogen Phytophthora infestans (P. infestans), is one of the most devastating diseases threatening global potato production. Conventional plant disease detection methods rely on a labor-intensive and time-consuming workflow and require bulky and expensive benchtop equipment, limiting their in-field applications. Here, we report a portable RPA-CRISPR/Cas12a-based diagnostic platform integrated with a polyvinyl alcohol (PVA) microneedle (MN) patch, which allows rapid in-field sampling, and smartphone-based fluorescence acquisition and analysis to detect P. infestans in potato at the early stage. The PVA MN enables leaf sampling rapidly within 1 min, and yields efficient DNA extraction of 56.3 ± 4.2 ng/mg, which is ∼3-fold higher than the traditional CTAB method (18.1 ± 2.1 ng/mg). The RPA-CRISPR/Cas12a isothermal assay achieved specific detection of P. infestans with no cross-reactivity against closely-related species Phytophthora sojae or Phytophthora capsici. The smartphone-based point-of-care test (POCT) system demonstrates a detection limit of 4 pg/μL for P. infestans genomic DNA, which is comparable to that acquired with commercial laboratory equipment. The method enables early-stage diagnosis of potato late blight as early as Day 2 post-inoculation, with detection rates of 37.5% on Day 2 and 75% on Day 3, prior to the development of visible symptoms on leaves. This portable "sample-to-result" platform provides a promising strategy for rapid, field-deployable early diagnosis and surveillance of plant disease.},
}
@article {pmid42330669,
year = {2026},
author = {Zheng, X and Li, H and Yao, S and Wang, X and Wang, J and Yin, C and Wang, J and Zhao, C},
title = {Multiple DNA cycle amplification-assisted one-pot isothermal Cas12a for ultrasensitive nucleic acid detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118953},
doi = {10.1016/j.bios.2026.118953},
pmid = {42330669},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; Gold/chemistry ; *COVID-19/diagnosis/virology ; Metal Nanoparticles/chemistry ; Limit of Detection ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Proteins ; Colorimetry ; *COVID-19 Nucleic Acid Testing/methods ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; DNA ; Bacterial Proteins ; Coronavirus Nucleocapsid Proteins ; Phosphoproteins ; Spike Glycoprotein, Coronavirus ; },
abstract = {Rapid and ultrasensitive nucleic acid detection is essential for environmental monitoring and biomedical diagnostics. Here, we report a modular one-pot isothermal platform that integrates catalytic hairpin assembly (CHA), rolling circle amplification (RCA), and CRISPR-Cas12a to construct a self-reinforcing multilayer DNA circuit (CRC). In this system, Cas12a cis-cleavage generates short DNA fragments that recursively activate CHA and RCA, forming a self-sustained cascade amplification loop, while trans-cleavage enables real-time fluorescence signal readout. Using this one-pot platform, ultralow detection limits of 62 aM and 58 aM were achieved for the SARS-CoV-2 S and N genes, respectively, with a total assay time ranging from 20 to 120 min depending on the required sensitivity. Furthermore, functionalizing single-stranded DNA probes on gold nanoparticles (AuNPs) allowed the cleaved DNA to restore fluorescence of fluorophore-quencher reporters, and freeze-thaw-induced AuNP aggregation produced visible colorimetric changes and measurable photothermal signals, enabling trimodal readout without sophisticated instruments. The system demonstrated effective discrimination in controlled experiments, indicating its potential suitability for point-of-care applications. This integrated, one-pot, and scalable platform provides a versatile strategy for fast, sensitive, and multimodal nucleic acid detection applicable to diverse targets.},
}
@article {pmid42333535,
year = {2026},
author = {Labun, K and Rio, O and Dahal-Koirala, S and Komisarczuk, AZ and Valen, E and Haapaniemi, E},
title = {SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling.},
journal = {Nucleic acids research},
volume = {54},
number = {W1},
pages = {W145-W153},
doi = {10.1093/nar/gkag409},
pmid = {42333535},
issn = {1362-4962},
support = {331912//Norges Forskningsr&#xe5;d/ ; 190290//Kreftforeningen/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; *Software ; Recombinational DNA Repair ; Computational Biology/methods ; Alleles ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of "blocking" silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard "correction-only" strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.},
}
@article {pmid42342132,
year = {2026},
author = {Eskandani, NA and Mirzaee, D and Ramezani Farani, M and Hatami, A and Hatami, M and Ghasemzaei, M and Ghoreishian, SM and Hwang, SK and Huh, YS},
title = {Light-controlled CRISPR-dCas9 epigenome editing: advanced drug-delivery strategies and oncology applications.},
journal = {Advanced drug delivery reviews},
volume = {236},
number = {},
pages = {115921},
doi = {10.1016/j.addr.2026.115921},
pmid = {42342132},
issn = {1872-8294},
mesh = {Humans ; *Epigenome Editing/methods ; *Neoplasms/genetics/therapy/drug therapy ; *Drug Delivery Systems ; Animals ; *CRISPR-Cas Systems/genetics ; Light ; Optogenetics/methods ; Epigenesis, Genetic ; },
abstract = {Cancer is increasingly recognized as a disease of the dysregulated epigenome; however, current epi-drugs are blunt, systemically toxic instruments. Catalytically dead CRISPR nucleases (dCas9) linked to chromatin effectors have now made it possible not only to write and erase epigenetic marks at specified loci without double-strand breaks but also to add an element of optogenetics, or reversible and light-encoded control over the timing and localization of the editors. In this review, the technological underpinnings of light-controlled CRISPR-dCas9 epigenome editing, which include architectures of dCas9 scaffold and guide, blue-to-near-infrared photoswitches, and high-gain epigenetic effector designs, are synthesized, and viral, non-viral, and stimuli-responsive delivery platforms, which have to be co-optimized with clinical light interfaces, are discussed. We then outline four functional routes by which opto-epigenome editors may be used therapeutically in cancer: tumor suppressor reactivation; oncogene and super-enhancer repression with metabolic rewiring; control of cancer stem cell differentiation; and immunomodulation of the tumor microenvironment. Lastly, a translational roadmap is defined in terms of preclinical model tiers, biomarker strategies, regulatory and manufacturing factors, and future directions, including NIR and bioluminescent actuation, implantable μLED devices, and AI-guided closed-loop illumination. Together, these aspects constitute design principles for advancing light-addressable epigenome editors toward first-in-human studies and for integrating them into combination regimens as a new class of precision cancer therapeutics.},
}
@article {pmid42379043,
year = {2026},
author = {Zheng, Y and Tian, X and Wang, J and Zhang, Y and Liu, T and Huang, J and Guo, Y and Liang, S and Wang, C},
title = {CRISPR/Cas12a-based dual-modal signal platform using MIL-101(Fe) for colorimetric and electron spin resonance detection of HPV-16 nucleic acid.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118976},
doi = {10.1016/j.bios.2026.118976},
pmid = {42379043},
issn = {1873-4235},
mesh = {*Human papillomavirus 16/isolation &amp; purification/genetics ; Humans ; Colorimetry/methods ; *CRISPR-Cas Systems/genetics ; Electron Spin Resonance Spectroscopy/methods ; *Biosensing Techniques/methods ; *DNA, Viral/genetics/isolation &amp; purification/analysis ; *Papillomavirus Infections/virology/diagnosis ; *Metal-Organic Frameworks/chemistry ; Iron/chemistry ; Limit of Detection ; },
abstract = {Human papillomavirus (HPV) infection is a leading cause of cervical cancer and other malignancies, underscoring the urgent need for accurate and rapid early diagnostic strategies. Herein, we report a dual-mode colorimetric and electron spin resonance (ESR) method for the qualitative detection of HPV-16, based on the integration of the CRISPR/Cas12a system with a metal-organic framework (MOF). A peroxidase-mimicking iron-based MOF, designated MIL-101(Fe), was conjugated to magnetic beads via a single-stranded DNA linker to serve as a signal probe. Upon recognition of the target nucleic acid, MIL-101(Fe) catalyzes the decomposition of hydrogen peroxide to generate hydroxyl radicals, which oxidize a chromogenic substrate to produce a visible color change. Meanwhile, the generated radicals are captured by a spin trap and detected by ESR spectroscopy. The assay enables sensitive and rapid detection of HPV-16, with clear discrimination even in mixtures containing both HPV-16 and HPV-18. Importantly, when evaluated with clinical specimens, the method achieved 100% sensitivity and specificity. Overall, this work provides a feasible and promising strategy for ultrasensitive nucleic acid detection and offers a new avenue for advancing CRISPR-based multimodal diagnostic platforms toward practical applications.},
}
@article {pmid42402242,
year = {2026},
author = {Wang, F and He, C and Lin, Y and Zhou, X},
title = {One-Tube RPA-CRISPR-Cas13a assay with rational design for single-molecule detection of waterborne viruses in drinking water treatment.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118983},
doi = {10.1016/j.bios.2026.118983},
pmid = {42402242},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; *Drinking Water/virology ; *Norovirus/isolation &amp; purification/genetics ; *Rotavirus/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Water Purification ; Limit of Detection ; Water Microbiology ; Humans ; Rapid Diagnostic Tests ; },
abstract = {The global rise in waterborne viral infections has created an urgent need for portable, highly efficient environmental virus detection technologies. CRISPR-based nucleic acid detection coupled with isothermal amplification (e.g., Recombinase Polymerase Amplification, RPA) shows great promise for field applications. However, most reported designs fail to achieve the single-molecule sensitivity, which significantly limits their practical applications. To bridge the gap, we proposed a rational design strategy for the RPA primer and the CRISPR-Cas13a crRNA, suggesting that sensitivity can be enhanced by simplifying the secondary structure of the crRNA spacer region. Subsequently, we established a portable, one-tube CRISPR-Cas13a bioassay to detect two major waterborne viruses, achieving ultrasensitive detection limits of 5/8 aM for norovirus and 2/3 aM for rotavirus within 40 min. Thereafter, seasonal sampling across different treatment stages of a drinking water treatment plant was conducted, and water samples were analyzed using the one-tube CRISPR-Cas13a bioassay in comparison with qPCR and dPCR, revealing a positive detection rate of 15.79% (6/38) for the one-tube CRISPR-Cas13a bioassay, 18.42% (7/38) for qPCR, and 15.79% (6/38) for dPCR. The assay's modular design allows for broad applicability to other pathogens by simply modifying the target nucleic acid sequence, offering high sensitivity and specificity. This innovation paves the way for deployable point-of-care testing and large-scale spatiotemporal virus monitoring.},
}
@article {pmid42411270,
year = {2026},
author = {Baldenweck, L and Berg, N and Djisalov, M and Efremov, V and Novakovic, Z and Auger, S and Vidic, J},
title = {Isothermal amplification techniques for rapid bacterial detection: alternatives to culturing and PCR-based methods.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6ay00756b},
pmid = {42411270},
issn = {1759-9679},
abstract = {Rapid identification of bacteria and their virulence factors is essential for global public health. Isothermal amplification has become a cornerstone of point-of-care diagnostics, enabling genetic testing to be faster, simpler, and more accessible than culturing or polymerase chain reaction (PCR). This review examines recent advances in some of the most commonly used isothermal amplification methods for bacterial detection: SDA, LAMP, HDA, RPA, RCA, and NASBA. The integration of isothermal amplification with the CRISPR/Cas system or microfluidic devices is also highlighted as an advanced gene detection technology. We present various readout methods used to detect gene amplification products or processes, including colorimetric, fluorescent, electrochemical, and quartz microbalance techniques. These integrated approaches can detect very small amounts of bacterial DNA, in under an hour, providing rapid, sensitive, versatile, and portable tools for health control.},
}
@article {pmid42411453,
year = {2026},
author = {Ono, Y and Peterka, M and Love, M and Khan, A and Bowers, F and Bhandari, A and Gordon, E and Ball, JS and Hammond, C and Tyler, CR and Rees, S and Bohlooly-Y, M and Maresca, M and Scholpp, S},
title = {Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42411453},
issn = {2050-084X},
support = {BB/X008401/1//UK Research and Innovation/ ; BB/X001458/1//UK Research and Innovation/ ; DA 8438235/WT_/Wellcome Trust/United Kingdom ; NC/X001407/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)/ ; 29317//Versus Arthritis Senior fellowship/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Genome ; *Mutagenesis, Insertional/methods ; *DNA/genetics ; Deoxyribonuclease I/metabolism ; *Gene Editing/methods ; },
abstract = {CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase- and a nuclease-based PE to perform programmed short DNA substitutions and insertions at various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA.},
}
@article {pmid42412324,
year = {2026},
author = {Adiga, U and Vasishta, S and Adiga, S and Augustine, AJ},
title = {Targeting the Gut-Heart Axis in Atherosclerosis: Microbial Metabolites, Molecular Mechanisms, and Precision Therapeutics.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {42412324},
issn = {1867-1314},
abstract = {Despite advances in lipid-lowering and anti-inflammatory medications, atherosclerotic cardiovascular disease (ASCVD) continues to be the leading cause of morbidity and mortality worldwide. Recent studies have identified the gut microbiota as a key modulator of cardiovascular health via the gut-heart axis. This review investigates the molecular processes by which microbial metabolites affect atherogenesis. Proatherogenic substances like trimethylamine-N-oxide (TMAO), which are produced from dietary precursors through gut microbial and hepatic metabolism, aggravate foam cell production, platelet aggregation, and vascular inflammation. Short chain fatty acids (SCFAs), such as butyrate and propionate, have been shown to protect against atherosclerosis by activating G-protein-coupled receptors, regulating gene expression, and improving endothelial function. Additionally, secondary bile acids, tryptophan derivatives, and phenylacetylglutamine have emerged as important microbial metabolites involved in vascular disease. The review also summarizes various therapeutic strategies such as use of probiotics, prebiotics, postbiotics, precision microbiome editing (using bacteriophages and CRISPR-Cas systems), and fecal microbiota transplantation (FMT) for targeting gut-heart axis. Multi-omic systems combined with artificial intelligence can now detect disease-specific microbial signatures, improving risk stratification and paving the way for precision microbiome-based therapeutics. However, challenges such as determining causality, regulatory intricacies, and inter-individual variability in host-microbiome interactions remain. Despite these obstacles, the gut-heart axis provides a disruptive paradigm in preventive cardiology by emphasizing tailored microbiome therapies as a complement to traditional ASCVD care.},
}
@article {pmid42412819,
year = {2026},
author = {Tziony, I and Orenstein, Y},
title = {CROP: a feature-independent context-aware method for CRISPR-Cas9 frameshift prediction.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {Supplement_1},
pages = {},
pmid = {42412819},
issn = {1367-4811},
support = {358/21//Israel Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *Frameshift Mutation ; *Software ; *Computational Biology/methods ; Prediction Algorithms ; },
abstract = {MOTIVATION: The CRISPR-Cas9 complex has revolutionized genome-editing technologies. By designing a 20 nt-long guide RNA, a Cas9 nuclease can be guided to cleave almost any genomic target site (followed by NGG). The cleavage induces double-stranded DNA breaks, which are then repaired by cellular pathways. Accurate CRISPR-Cas9 repair-outcome prediction is essential for designing guide RNAs with desired genomic effects, such as gene knockout. A central challenge is quantifying the rate of frameshifts, i.e. repair-outcomes that lead to a change in the local length that is not a multiple of three. Previous methods for frameshift-rate prediction were trained on only a few experimental or cellular contexts, mostly relied on manually defined microhomology features, and were limited by sparse features and class labels.<br><br>RESULTS: We developed CROP, a feature-independent context-aware repair-outcome prediction method. By aggregating specific repair outcomes as &#x394;length classes, CROP overcomes class sparsity. We designed CROP to work with variable input sequence lengths and output classes to utilize multiple datasets simultaneously. We benchmarked CROP against state-of-the-art repair-outcome prediction methods over 18 datasets, which we curated and standardized from various studies. Across all datasets, CROP outperformed all competing methods in frameshift-rate prediction. We performed cross-experiment and cross-cellular frameshift-rate predictions to investigate the generalizability of repair mechanisms. Finally, we show that CROP learned microhomology principles from raw sequences without explicit feature engineering, establishing an end-to-end architecture for CRISPR-Cas9 repair-outcome prediction that learns from multiple datasets.<br><br>CROP is available at https://github.com/OrensteinLab/CROP.},
}
@article {pmid42413622,
year = {2026},
author = {Yu, Q and Waheed, A and Hanioui, M and Ma, P and Wang, X and Liu, K and Li, X and Xue, Z and Zhang, G and Zhao, M},
title = {Engineering a tyrosine-auxotrophic Escherichia coli chassis for residue-specific in vivo DOPA incorporation into mussel foot protein mimics.},
journal = {Journal of biotechnology},
volume = {418},
number = {},
pages = {47-56},
doi = {10.1016/j.jbiotec.2026.07.003},
pmid = {42413622},
issn = {1873-4863},
abstract = {Mussel foot proteins (Mfps) achieve exceptional marine adhesion through post-translational conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Recombinant production, however, is limited by poor solubility, low yields, and insufficient DOPA incorporation. We generated a genetically stable tyrA-deficient Escherichia coli chassis using CRISPR/Cas, thereby abolishing endogenous tyrosine biosynthesis and enabling residue-specific in vivo incorporation of exogenously supplied DOPA into mussel foot protein (MFP) mimics through selective pressure incorporation (SPI). A two-stage cultivation strategy decoupled biomass accumulation from DOPA-dependent protein synthesis, yielding 18.01 mg L[-1] of purified FP1. Residue-specific DOPA incorporation was verified by the characteristic + 16 Da shift in the [M-H][-] ion, accompanied by the loss of the tyrosine signal following exogenous DOPA supplementation in M9 medium. FP3 was largely soluble (65%) in crude extracts, while FP5 expression remained minimal. Fusion to thioredoxin (TrxA) and magnetoreceptor protein (MagR) further enhanced FP3 solubility to 90% and 85%, respectively. Proteins expressed in minimal M9 medium displayed exceptional shear stability, with viscosity fluctuations limited to ±2.2%, reflecting preserved catechol chemistry and structural integrity. This integrated strategy overcomes recurring trade-offs between DOPA incorporation, solubility, and yield, providing a basis for the potentially scalable production of functional, catechol-rich Mfps. Collectively, these findings support the development of next-generation mussel-inspired adhesives and catechol-based biomaterials.},
}
@article {pmid42413656,
year = {2026},
author = {Zhang, J and Shi, X and Ding, Y and Zheng, J and Li, H and Zhao, H and Zhang, T and Xing, Q and Zhao, C and Yao, S and Wang, J},
title = {Dynamic bidirectional diffusion-controlled multi-enzyme system for one-pot viral detection.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.07.019},
pmid = {42413656},
issn = {2090-1224},
abstract = {INTRODUCTION: Integrating isothermal amplification with CRISPR-based detection in a single reaction vessel holds significant promise for rapid and sensitive point-of-care virus diagnostics. However, conventional one-pot methods often suffer from mutual inhibition between amplification and CRISPR-Cas reactions, which compromises detection performance and limits their practical application.<br><br>OBJECTIVES: This study aims to overcome the inhibitory interactions between isothermal amplification and CRISPR-based detection by developing an integrated reaction system that enables efficient multi-enzyme coordination within a single tube.<br><br>METHODS: We designed a dynamic bidirectional diffusion-controlled RPA/CRISPR-Cas12a multi-enzyme system based on a dual-phase separation strategy. In this system, glycerol was used to modulate viscosity and accelerate the RPA reaction, while sucrose was introduced to create a density gradient that enables spatial separation. This configuration effectively coordinates the activities of multiple enzymes within one reaction vessel. Furthermore, a 3D-printed nucleic acid extraction device was integrated to simplify sample preparation and enhance overall detection efficiency.<br><br>RESULTS: The developed system achieved single-copy sensitivity and completed detection within 30&#xa0;min, exhibiting over 100-fold higher sensitivity than conventional one-pot assays. The method was further validated by detecting Norovirus in both clinical and food samples, confirming its robustness and accuracy.<br><br>CONCLUSION: The proposed dual-phase RPA/CRISPR-Cas12a system provides a simple, rapid, and highly sensitive platform for nucleic acid detection. Its operational simplicity, compatibility with low-resource settings, and potential for home-based diagnostics highlight its strong applicability for decentralized virus detection.},
}
@article {pmid41930411,
year = {2026},
author = {Tyagi, DS and Banoo, H and Jha, DK and Meena, V and Joon, R and Agrwal, K and Yadav, P and Kumar, A and Satbhai, SB and Long, T and Pandey, AK},
title = {CRISPR/Cas9 Editing of the Wheat Iron Sensor TaHRZ1 Confirms Its Conserved Role in Iron Homeostasis and Allocation in Grains.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {8},
pages = {4975-4991},
doi = {10.1111/pce.70516},
pmid = {41930411},
issn = {1365-3040},
support = {//NABI-CORE/ ; },
mesh = {*Triticum/genetics/metabolism ; *Homeostasis ; *Iron/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems/genetics ; Phylogeny ; Plants, Genetically Modified ; *Seeds/metabolism/genetics ; Arabidopsis/genetics ; Amino Acid Sequence ; Edible Grain/metabolism/genetics ; },
abstract = {Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum L. (wheat) HRZ homoeologs referred to as TaHRZ1 and TaHRZ2, were identified by BLAST searches using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode for proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain of TaHRZ1, coupled with devlopmental regulators GRF4-GIF1 chimeric protein, achieved 6.4%-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved Fe partitioning compared to the non-edited plants. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.},
}
@article {pmid42010781,
year = {2026},
author = {Yue, K and Liang, X and Wang, Y and Jiang, N and Hu, Y and Su, J and Lu, C and Gao, H and Gai, X and Guo, W},
title = {A field-deployable RPA-CRISPR/Cas12a dual-mode assay for rapid detection of Fusarium oxysporum in Nicotiana tabacum.},
journal = {Pest management science},
volume = {82},
number = {8},
pages = {7610-7619},
doi = {10.1002/ps.70825},
pmid = {42010781},
issn = {1526-4998},
support = {2024530000241006//Yunnan Academy of Tobacco Agricultural Sciences/ ; 202405AD350100//Yunnan Applied Fundamental Research Projects/ ; 110202401006(JY-06)//China National Tobacco Corporation (CNTC)/ ; },
mesh = {*Fusarium/isolation &amp; purification/genetics ; *Nicotiana/microbiology ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Fusarium oxysporum is a devastating soil-borne pathogen that causes severe economic losses in tobacco and other crops, necessitating rapid and accurate detection methods for effective disease management. Herein, we developed a dual-mode RPA-Cas12a platform incorporating both lateral flow dipstick (LFD) and fluorescence-based detection (FBD), targeting the CYP51C gene to enable rapid and equipment-facilitated detection of F. oxysporum under isothermal conditions.<br><br>RESULTS: The entire assay from sample to result can be completed within 68&#x2009;min. The platform offers two complementary detection formats. The LFD mode provides visual qualitative results with a detection limit of 360 copies, making it ideally suited for rapid on-site screening. In contrast, the FBD mode achieves a detection limit of 3.6 copies, demonstrating 100-fold higher sensitivity and enabling precise quantitative analysis.<br><br>CONCLUSION: The assay successfully identified F. oxysporum in both artificially inoculated and field-collected tobacco samples, showing high concordance with fluorescence intensity. This work provides a sensitive, rapid, and practical diagnostic tool for the on-site detection and monitoring of tobacco root rot caused by F. oxysporum. &#xa9; 2026 Society of Chemical Industry.},
}
@article {pmid42092617,
year = {2026},
author = {Richter, E and Klöhn, M and Nocke, MK and Friedrich, ME and Todt, D and Steinmann, E and Brüggemann, Y},
title = {Development of a CRISPR-Cas13-based antiviral strategy against hepatitis E virus.},
journal = {JHEP reports : innovation in hepatology},
volume = {8},
number = {7},
pages = {101885},
pmid = {42092617},
issn = {2589-5559},
mesh = {*Hepatitis E virus/genetics/drug effects ; Humans ; *CRISPR-Cas Systems ; Virus Replication/drug effects/genetics ; *Antiviral Agents/pharmacology ; *Hepatitis E/virology/therapy ; Genome, Viral ; },
abstract = {BACKGROUND &amp; AIMS: Effective antiviral drugs remain unavailable for many clinically relevant pathogens, including the hepatitis E virus (HEV). This study aimed to evaluate the CRISPR/Cas13d system as a potential antiviral strategy against HEV.<br><br>METHODS: We developed a reporter assay to screen CRISPR RNAs (crRNAs) targeting conserved regions of the HEV genome and tested their antiviral activity in human hepatoma cells using a robust HEV cell culture model. HEV replication was assessed using a subgenomic replicon, infectious particle production was quantified by immunofluorescence and titration assays. A bioinformatic analysis was performed to identify a minimal set of crRNAs capable of broadly targeting circulating human pathogenic HEV strains.<br><br>RESULTS: A crRNA screen identified multiple functional crRNAs targeting HEV-3, with ORF1-targeting crRNAs significantly reducing viral capsid expression (p <0.01) and the number of HEV-infected cells (p <0.01). Cas13d-mediated targeting led to robust reduction of HEV replication and markedly lowered infectious virus production in vitro (p <0.001). Bioinformatic analysis revealed that just three distinct crRNAs could cover &#x223c;94% of known HEV genomes with zero mismatches, while four crRNAs achieved complete coverage.<br><br>CONCLUSIONS: Our findings demonstrate that CRISPR/Cas13d can target HEV replication and viral progeny production in vitro. The identification of a minimal crRNA set capable of broadly targeting circulating HEV strains suggests that the CRISPR/Cas13d system may offer an antiviral strategy to address challenges related to viral evolution and treatment escape.<br><br>IMPACT AND IMPLICATIONS: This study establishes CRISPR/Cas13d as a proof-of-concept antiviral strategy against hepatitis E virus (HEV), demonstrating suppression of viral replication and particle production in vitro. By identifying a minimal set of broadly effective crRNAs, we provide a framework for targeting diverse HEV variants and buffering against viral evolution. These findings highlight the potential of CRISPR-based systems as innovative antiviral strategies.},
}
@article {pmid42115764,
year = {2026},
author = {Lv, G and Li, Y and Chen, J and Wu, Z and Wu, W and Wu, X and Lin, H and Wang, T},
title = {CRISPR/Cas9-mediated knockout of ZmHMA3 reveals its essential role in zinc homeostasis and high-zinc stress tolerance in maize.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42115764},
issn = {2045-2322},
support = {2023YFD1201105//National Key Research and Development Program of China/ ; },
mesh = {*Zea mays/genetics/metabolism/physiology ; *Zinc/metabolism/toxicity ; *Homeostasis ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Plant Leaves/metabolism/genetics ; Gene Knockout Techniques ; Plant Roots/metabolism/genetics ; *Adenosine Triphosphatases/genetics/metabolism ; Oxidative Stress ; Plants, Genetically Modified ; },
abstract = {Excessive Zn is toxic to maize (Zea mays L.). The heavy metal ATPase gene ZmHMA3 is associated with heavy metal transport, but its function in maize tolerance to high Zn stress has not been fully characterized. In this study, CRISPR/Cas9 technology was used to generate zmhma3 knockout mutants to investigate its function under high Zn stress. High Zn stress significantly induced the expression of ZmHMA3 in maize leaves and roots. Phenotypic analysis showed that, compared to the WT plants, the zmhma3 mutants exhibited significantly reduced tolerance to excessive Zn, manifested as severe growth inhibition, impaired root structure, decreased activity of key antioxidant enzymes (CAT, POD, SOD), and aggravated membrane damage. Furthermore, the mutants accumulated significantly higher levels of Zn in both roots and leaves, accompanied by disordered subcellular Zn distribution, indicating disrupted intracellular Zn homeostasis. Our results demonstrate that ZmHMA3 is a key positive regulator in maize's response to high Zn stress, likely by coordinating Zn compartmentalization and alleviating oxidative damage. This study provides new genetic and physiological insights into the molecular mechanisms of Zn stress tolerance in maize and offers a potential target for breeding new maize varieties tolerant with improved high-Zn-efficiency.},
}
@article {pmid42302780,
year = {2026},
author = {Ueki, H and Tomita, Y and Duong, C and Mitake, H and Kiso, M and Furusawa, Y and Zhao, D and da Silva Lopes, TJ and Wu, L and Feng, H and Yamayoshi, S and Fukuyama, S and Yamashita, M and Ozawa, M and Ikawa, M and Yoshida, N and Watanabe, T and Kawaoka, Y},
title = {A CRISPR knockout mouse library for functional genomics in influenza research.},
journal = {Cell},
volume = {189},
number = {14},
pages = {4471-4488.e7},
doi = {10.1016/j.cell.2026.05.032},
pmid = {42302780},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Mice ; *Influenza A virus/genetics ; *Orthomyxoviridae Infections/genetics/virology ; *Genomics/methods ; Mice, Knockout ; Cytoskeletal Proteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Guanine Nucleotide Exchange Factors/genetics/metabolism ; Humans ; RNA, Small Interfering/metabolism/genetics ; Host-Pathogen Interactions/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; Mice, Inbred C57BL ; },
abstract = {Functional validation of host factors in whole-animal models is a major bottleneck in virology; it hinders the translation of data from in vitro studies into a deeper understanding of the viral life cycle and pathogenesis. To address this challenge, we developed a systematic in vivo screening platform for influenza A virus. This platform comprises a library of 84 CRISPR-Cas9-generated gene-modified mouse lines targeting host factors prioritized from the literature and in vitro small interfering RNA (siRNA) screening studies. Using this resource, we identified 17 host factors whose genetic ablation conferred resistance to influenza A virus infection. Further studies of two of these factors, Arhgef28 and Lasp1, revealed distinct protective mechanisms against influenza A virus. We offer this mouse library to the research community as a powerful platform for studying virus-host interactions in a physiologically relevant context.},
}
@article {pmid42409605,
year = {2026},
author = {Huang, YW and Hu, CC and Cho, YH and Tsai, CH and Lin, NS and Hsu, YH and Dinesh-Kumar, SP},
title = {Efficient CRISPR-Cas9 delivery and transgene-free multiplex genome editing in plants using cymbidium mosaic virus-derived vectors.},
journal = {The Plant journal : for cell and molecular biology},
volume = {127},
number = {1},
pages = {e71031},
pmid = {42409605},
issn = {1365-313X},
support = {NSTC 114-2313-B-005-052-MY2//National Science and Technology Council/ ; //Ministry of Education in Taiwan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Nicotiana/genetics ; Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transgenes/genetics ; Potexvirus ; },
abstract = {Virus-induced genome editing (VIGE) has become a useful method by enabling transient delivery of gene-editing reagents; however, many viral systems face limitations in cargo size, host range, or reliance on transgenic Cas9-expressing plants. In this study, we developed a cymbidium mosaic virus (CymMV)-based VIGE platform that enables simultaneous expression of Streptococcus pyogenes Cas9 (SpCas9) and one or more guide RNAs (gRNAs) from a single viral RNA. In Nicotiana benthamiana, this system induced editing in the Phytoene desaturase (PDS) gene, with indel rates exceeding 50% within 6 days after inoculation, outperforming traditional delivery methods by about fivefold. Notably, over 80% of regenerated plants contained targeted mutations, and 82% of these were both transgene- and virus-free, including tetra-allelic knockouts directly in the M0 generation. Adding a Ruby-based visual counterselection marker enabled rapid, reliable identification of transgene-free, edited plants without antibiotic selection. When adapted to Phalaenopsis aphrodite orchids, the platform efficiently edited the PaPDS gene, achieving a 47% indel frequency at 20 days post-inoculation, with visible bleaching in leaf tissue from inoculated protocorm-like bodies. Additionally, expressing multiple gRNAs from a single CymMV replicon enabled multiplex editing in orchid tissues, demonstrating the system's versatility for complex, polyploid crops. Our findings broaden the use of VIGE in orchids and provide a reliable framework for precision plant breeding.},
}
@article {pmid42409731,
year = {2026},
author = {Shangguan, YT and Xie, LL and Liu, WB and Gu, RX and Xu, YX and Wang, M and Wang, JX},
title = {[Novel CD6-targeted CAR-T cell therapy for T-cell acute lymphoblastic leukemia: a safe and efficient strategy to prevent fratricide through gene editing].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {47},
number = {5},
pages = {433-441},
doi = {10.3760/cma.j.cn121090-20260130-00068},
pmid = {42409731},
issn = {0253-2727},
support = {82341213, 82570279, 82570278//National Natural Science Foundation of China/ ; 23JCYBJC01060//Tianjin Natural Science Foundation Project/ ; },
mesh = {Humans ; *Immunotherapy, Adoptive ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; *Gene Editing ; Receptors, Chimeric Antigen ; *Antigens, CD ; *Antigens, Differentiation, T-Lymphocyte ; CRISPR-Cas Systems ; T-Lymphocytes ; Receptors, Antigen, T-Cell ; },
abstract = {Objective: To explore a novel strategy that addresses the dual challenges of fratricide and on-target off-tumor toxicity in current chimeric antigen receptor T-cell (CAR-T) therapy for T-cell acute lymphoblastic leukemia (T-ALL) and to develop a safe and efficacious anti-T-ALL CAR-T product by identifying a new target and compatible gene-editing approach. Methods: Public single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze bone marrow cells extracted from patients with T-ALL and healthy donors, evaluating the differential expression profiles of CD6 and CD7. In investigating the endogenous role of CD6 in CAR-T cells, the CRISPR/Cas9 RNP system was first employed in a CD19 CAR-T model to evaluate the impact of CD6 knockout on the phenotype and activation status of CAR-T cells. Subsequently, CD6-knockout, CD6-targeted CAR-T cells (6KO-6CAR) were constructed, and their functional activities were evaluated. Results: scRNA-seq analysis revealed that CD6 is broadly expressed in T-ALL. Compared with the traditional target CD7, which is also expressed in a subset of normal hematopoietic stem/progenitor cells and myeloid cells, CD6 exhibits a more restricted expression profile, exhibiting superior safety characteristics. Studies on the CD19 CAR-T model indicated that CD6 knockout enables CAR-T cells to maintain a superior functional state: their baseline activation level (CD25 expression) was reduced (P<0.05) while generating a higher proportion of TNF-α(+)IFN-γ(+) cells (P<0.05) upon antigen stimulation. The further constructed 6KO-6CAR cells exhibited potent specific activation (significantly upregulated CD107a expression level, all P<0.001) and cytotoxicity (all P<0.05) against multiple CD6(+) T-ALL cell lines (MOLT-4, CCRF-CEM, and Jurkat) in vitro. Conclusion: CD6 is a novel therapeutic target for T-ALL with high coverage and a favorable safety profile, and knocking out endogenous CD6 globally optimizes the intrinsic functional state of CAR-T cells. Constructing 6KO-6CAR based on the CRISPR/Cas9 technology addresses fratricide in CAR-T cells while enhancing their antitumor functionality, thereby providing a novel immunotherapy regimen with safety and clinical translational potential for relapsed/refractory T-ALL.},
}
@article {pmid42409995,
year = {2026},
author = {Goudarzi, F and Salehipour, P and Modarressi, MH and Hosseini, M},
title = {A "turn-on" CRISPR-mediated method using enhanced fluorescent bimetallic DNA nanoclusters for EGFR mutation detection in non-small cell lung cancer.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-61115-3},
pmid = {42409995},
issn = {2045-2322},
support = {981234//University of Tehran/ ; },
abstract = {An affordable, precise detection of mutations is critical for guiding targeted cancer therapies and improving patient outcomes. Epidermal growth factor receptor (EGFR), a protein on the surface of cells that regulates growth and division, is frequently mutated in non-small cell lung cancer (NSCLC). Early identification of these mutations enables clinicians to select the most effective tyrosine kinase inhibitors, thereby enhancing treatment response and survival rates. Recent studies have focused on developing CRISPR-based detection strategies incorporating nanomaterials to achieve more accurate results. In this study, we present a CRISPR-based "turn-on" detection platform that leverages the cleavage of a novel enhanced bimetallic DNA nanocluster to measure EGFR exon 19 deletion in non-small cell lung cancer (NSCLC). The system is innovatively designed using guide RNAs (gRNAs) rationally derived from the normal EGFR gene, enabling the determination of exon 19 deletion through CRISPR-Cas activation in samples containing the normal and mutant. Upon recognition of the normal EGFR gene, the Cas12a enzyme induces cleavage of the Spermiform-designed Ag/Au DNA nanocluster and fluorescence quenching. At the same time, fluorescence signal retention depends on mutation frequency, with higher mutation frequencies resulting in greater or "turn-on" fluorescence signals. This approach achieves a detection limit (LOD) of approximately 0.35 nM, which is capable of detecting about 1.5% mutation, offering a cost-effective, label-free diagnostic tool and a promising strategy for future detection of deletion-related subtypes in PCR products by targeting normal sequences. The integration of bimetallic nanocluster-based reporters with CRISPR precision provides an emerging platform for next-generation molecular diagnostics targeting EGFR and other clinically relevant mutations.},
}
@article {pmid42410413,
year = {2026},
author = {Fan, Q and Stevanie, S and Frielingsdorf, S and Neubauer, P and Lenz, O and Gimpel, M},
title = {Genomically integrated orthogonal translation system in Escherichia coli enables production of functional modified [NiFe]-hydrogenases.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {42410413},
issn = {1475-2859},
mesh = {*Hydrogenase/genetics/metabolism/biosynthesis ; *Escherichia coli/genetics/metabolism ; Cupriavidus necator/genetics/enzymology ; CRISPR-Cas Systems ; *Protein Biosynthesis ; Plasmids/genetics ; Gene Editing ; },
abstract = {The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.},
}
@article {pmid41419637,
year = {2026},
author = {Meng, X and Reis, N and Bassik, MC and Pașca, SP},
title = {CRISPR screens in human neural organoids and assembloids.},
journal = {Nature protocols},
volume = {21},
number = {7},
pages = {3127-3147},
pmid = {41419637},
issn = {1750-2799},
mesh = {Humans ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Neurodevelopment ; *Neurons/cytology/metabolism ; Induced Pluripotent Stem Cells/cytology ; *Gene Editing/methods ; Cell Differentiation ; },
abstract = {Studying the molecular mechanisms underlying the assembly of the human nervous system remains a significant challenge. The ability to generate neural cells from pluripotent stem cells, combined with advanced genome-editing techniques, provides unprecedented opportunities to uncover the biology of human neurodevelopment and disease. Organoids and assembloids enable the in vitro modeling of previously inaccessible developmental processes, such as the specification and migration of human neurons, including the integration of cortical interneurons from the ventral into the dorsal forebrain. Here, we present a detailed protocol that combines pooled CRISPR-Cas9 screening with neural organoid and assembloid models and illustrate how it can be applied to map hundreds of disease genes onto cellular pathways and specific aspects of human neural development. Our protocol outlines key steps, from planning and optimizing genetic perturbations to designing readouts for neuronal generation and migration, conducting the screening and validating candidate genes. The screening experiments take ~3 months to complete and require expertise in stem cell culture and neural differentiation, genetic engineering of human induced pluripotent stem cell lines, fluorescence-activated cell sorting and next-generation sequencing and analyses. The integration of genetic screening and human cellular models constitutes a powerful platform for investigating the mechanisms of human brain development and disease, paving the way for the discovery of novel therapeutics.},
}
@article {pmid41832622,
year = {2026},
author = {Yuan, C and Chen, F and Gao, X and Yusufu, A and Lu, D and Wu, X and Ni, L},
title = {UBE2M Identified by CRISPR Screening as a Key Regulator of Cisplatin-Induced Acute Kidney Injury via the p53 Pathway.},
journal = {Endocrine, metabolic &amp; immune disorders drug targets},
volume = {26},
number = {},
pages = {e18715303410982},
pmid = {41832622},
issn = {2212-3873},
mesh = {*Cisplatin/toxicity ; *Acute Kidney Injury/chemically induced/genetics/metabolism/pathology ; Animals ; *Ubiquitin-Conjugating Enzymes/genetics/metabolism ; *Tumor Suppressor Protein p53/metabolism/genetics ; Signal Transduction/drug effects/physiology ; Humans ; *CRISPR-Cas Systems ; *Antineoplastic Agents/toxicity ; Mice ; Apoptosis/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {INTRODUCTION: Acute kidney injury caused by cisplatin (Cis-AKI) is a major limitation in its clinical use, primarily due to the lack of effective therapeutic targets to mitigate nephrotoxicity. Although several molecular pathways are involved in Cis-AKI, identifying reliable and actionable therapeutic targets has been challenging. Through a CRISPR-based genome-wide screening approach, UBE2M was identified as a novel gene involved in cellular survival during cisplatin-induced stress. However, its expression, biological function, and underlying mechanism in Cis-AKI have not been thoroughly investigated. This study aims to identify key therapeutic targets for Cis- AKI and investigate the role of UBE2M in this condition.<br><br>METHODS: A CRISPR-Cas9 genome-wide screening approach was employed to identify key genes involved in cisplatin-induced renal tubular epithelial cell injury. UBE2M, identified as a critical survival factor, was further investigated using both gain- and loss-of-function strategies to explore its biological function and underlying regulatory mechanisms in the Cis-AKI model.<br><br>RESULTS: CRISPR screening identified UBE2M as a key regulator of cellular survival in Cis-AKI, and subsequent validation experiments confirmed its suppression in cisplatin-induced renal injury models. UBE2M overexpression alleviated apoptosis and renal injury by reducing p53 activation. In contrast, UBE2M knockdown exacerbated these effects, leading to increased apoptosis and renal injury.<br><br>DISCUSSION: This study reveals that UBE2M is a critical regulator of cisplatin-induced renal tubular epithelial cell injury. By regulating the p53-mediated apoptotic pathway, UBE2M protects against Cis-AKI.<br><br>CONCLUSION: UBE2M could serve as a novel therapeutic target for the prevention and treatment of cisplatin-induced nephrotoxicity.},
}
@article {pmid42135732,
year = {2026},
author = {Xiao, WT and He, JY and Yang, D and Xun, Y},
title = {Construction of a novel signature based on CRISPR-Cas9 screening for prognostic prediction in breast cancer.},
journal = {BMC cancer},
volume = {26},
number = {1},
pages = {},
pmid = {42135732},
issn = {1471-2407},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/mortality/pathology ; Female ; Prognosis ; *CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; Transcriptome ; },
abstract = {BACKGROUND: Breast cancer (BC) is a highly heterogeneous malignancy and remains the leading cause of cancer-related mortality among women worldwide. Although advances in molecular classification and targeted therapies have improved outcomes for certain subtypes, robust prognostic biomarkers applicable across clinical contexts are still lacking. The CRISPR-Cas9 system offers a powerful platform for identifying cancer cell vulnerabilities and may facilitate the development of clinically relevant prognostic models.<br><br>METHODS: We integrated genome-wide CRISPR-Cas9 screening data from the DepMap database with transcriptomic and clinical data from TCGA and GEO datasets to identify BC cell survival-dependent genes (CSDGs). CSDGs prognostic signature was constructed using univariate Cox regression, LASSO, and stepwise multivariate Cox regression analyses. The model was validated in internal and external cohorts. Functional enrichment analyses, including GO, KEGG, WGCNA, and GSEA, were performed to explore the biological mechanisms underlying the signature. Random forest analysis and functional experiments were conducted to investigate the role of key gene in CSDGs signature.<br><br>RESULTS: A total of 1,622 CSDGs were identified, and a nine-gene prognostic CSDGs signature (BRD4, CHORDC1, COPZ1, HNRNPC, NUP43, RAD1, RBBP8, TUBA1B, and VPS28) was developed. This signature effectively stratified patients into high- and low-risk groups with significantly different overall survival, and its robustness was confirmed across multiple internal and external cohorts. High-risk patients exhibited a significant association with multiple adverse clinical features. A nomogram that combined the risk score with clinical variables showed robust predictive performance, and its C-index surpassed those of individual predictors, underscoring the enhanced accuracy of the integrated model. Functional analyses revealed enrichment of oncogenic pathways (e.g., MYC targets, G2/M checkpoint, mTORC1 signaling) in high-risk patients, while low-risk patients exhibited immune and hormone response signatures. CHORDC1 was identified as the most critical gene in the model. Knockdown of CHORDC1 significantly inhibited proliferation, migration, and invasion of BC cells. Transcriptomic profiling further linked CHORDC1 to oncogenic pathways, including EMT, mTORC1 signaling, and TNF-&#x3b1;/NF-&#x3ba;B signaling activation.<br><br>CONCLUSION: We developed a CRISPR-Cas9 screening-based prognostic signature for BC that effectively stratifies patient risk and demonstrates robust predictive performance across cohorts. CHORDC1 was identified as a key oncogenic driver, promoting tumor progression via pathways such as EMT and mTORC1 signaling, highlighting its potential as a therapeutic target. These findings may contribute to the development of personalized prognostic tools and therapeutic strategies in BC.},
}
@article {pmid42235446,
year = {2026},
author = {Olfati Sumar, M and Mohammadi, F and Khoshbin, Z and Abbasi Ghaeni, F and Abnous, K and Taghdisi, SM},
title = {A CRISPR-driven aptasensor for colorimetric monitoring of lead (II) ion assisted by rolling circle amplification process: Effective in controlling food and health safety.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {362},
number = {},
pages = {128167},
doi = {10.1016/j.saa.2026.128167},
pmid = {42235446},
issn = {1873-3557},
mesh = {*Colorimetry/methods ; *Lead/analysis ; *Aptamers, Nucleotide/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Food Safety/methods ; Food Contamination/analysis ; },
abstract = {Herein, an efficient colorimetric aptasensor has been introduced for highly sensitive measurement of lead (II) ions (Pb[2+]) by integrating the advantages of clustered regularly interspaced short palindromic repeats (CRISPR) system, the rolling circle amplification (RCA) process, and the catalytic activity of gold nanoparticles (AuNPs) for the first time. The presence of Pb[2+] inactivates the CRISPR system, making it unable to cleave the complementary sequence (CS) on the surface of ferrofluids (FFDs) and the formation of RCA product. By trapping AuNPs inside the RCA mass and subsequent magnetic separation of FFDs, the supernatant color remains yellow after adding 4-nitrophenol (4-NP). In the absence of Pb[2+], the supernatant color changes to colorless, due to the activation of CRISPR-Cas12a and the lack of large DNA structures. The colorimetric aptasensor can monitor Pb[2+] ions in the concentration ranges of 0.1 pM-20 nM and 20 nM-800 nM with a detection limit of 0.024 pM. It can also quantify Pb[2+] in the biological, cosmetic, and marine samples.},
}
@article {pmid42284937,
year = {2026},
author = {Yang, Q and Cao, Y and Yuan, J and Lu, W and Guo, Y and Sun, X and Wang, X and Li, X and Li, J and Zhang, N and Cao, L and Gong, P},
title = {A rapid and specific strategy for detecting Orientobilharzia turkestanicum, a water-associated schistosome of veterinary and environmental concern.},
journal = {Water research},
volume = {303},
number = {},
pages = {126262},
doi = {10.1016/j.watres.2026.126262},
pmid = {42284937},
issn = {1879-2448},
mesh = {Animals ; Cattle ; *Schistosoma/isolation &amp; purification ; *Water/parasitology ; CRISPR-Cas Systems ; Environmental Monitoring ; },
abstract = {Orientobilharzia turkestanicum (O. turkestanicum) is a water-associated schistosome parasite widely distributed in pastoral regions of Asia and Europe, where freshwater systems act as key interfaces linking livestock hosts, snail intermediate hosts, and environmental transmission pathways. Despite its strong environmental dependency, effective surveillance of O. turkestanicum in water-related settings remains limited by conventional diagnostic approaches that are time-consuming, equipment-dependent, and poorly suited for field-based monitoring. In this study, a rapid and sensitive detection assay based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (RPA-CRISPR/Cas12a) was developed for on-site identification of O. turkestanicum. The ITS1-5.8S region was selected as the molecular target to enable reliable species discrimination from closely related schistosomes, particularly Schistosoma japonicum (S. japonicum). The assay operates under isothermal conditions at 37 °C and allows result interpretation through visual fluorescence and lateral flow strip (LFS) readouts without the need for sophisticated instrumentation. The established RPA-CRISPR/Cas12a assay exhibited high analytical specificity, showing no cross-reactivity with a range of non-target parasites and bacterial species. Sensitivity evaluation using serially diluted standard plasmids demonstrated analytical limit of detection of 0.16 copies/μL by visual fluorescence and 160 copies/μL by LFS. Field applicability was validated using snail samples, cattle feces, liver, intestinal tissues and simulated infected water samples, yielding an overall concordance rate of 97.55 % compared with qPCR. Collectively, these results indicate that the proposed RPA-CRISPR/Cas12a assay provides a practical and field-deployable tool for livestock and water-associated transmission environment of O. turkestanicum and offers a useful framework for improving field surveillance and risk assessment of livestock schistosomiasis in endemic regions.},
}
@article {pmid42300772,
year = {2026},
author = {Chen, J and Le, Y and Yang, L and Qin, H and Yang, W},
title = {Dual mode analysis of lead ions in regulation of intestinal flora via split-DNA re-assembly catalyzed colorimetric reaction and trans-cleavage activity of Cas12a/crRNA.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {26},
pages = {5441-5448},
doi = {10.1039/d6ay00799f},
pmid = {42300772},
issn = {1759-9679},
mesh = {*Colorimetry/methods ; *Lead/analysis/blood ; Humans ; *Gastrointestinal Microbiome/drug effects ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; DNA, Catalytic/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; Limit of Detection ; Biosensing Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Chronic exposure to lead ions (Pb[2+]) disrupts intestinal flora homeostasis, underscoring the need for sensitive and practical detection methods. Herein, we report a dual-mode analytical strategy combining split-DNA re-assembly-driven colorimetric reaction with CRISPR/Cas12a trans-cleavage activity for Pb[2+] analysis. A bifunctional magnetic probe is constructed, where Pb[2+]-specific DNAzyme recognition triggers cleavage and release of crRNA, which subsequently activates Cas12a to generate fluorescence by cleaving a fluorophore-quencher reporter. Concurrently, the remaining magnetic bead-anchored trigger initiates split G-quadruplex re-assembly, catalyzing a visible colorimetric reaction. Under optimized conditions, the fluorescence mode achieves a detection limit of 1.03 fM with a linear range of 5 fM to 100 pM, while the colorimetric mode offers a limit of 62.1 fM from 100 fM to 500 pM. Both modes exhibit excellent specificity against competing metal ions. The platform's clinical feasibility is validated using human serum samples, showing strong correlation with ICP-MS, satisfactory recoveries (97.1-103.4%), and good repeatability (CV = 4.1%). Magnetic separation effectively minimizes matrix interference, making the assay suitable for complex biological specimens such as intestinal contents and fecal samples. This dual-mode design integrates fluorescence for precise quantification and colorimetry for equipment-free visual detection, holding great promise for point-of-care testing, early risk assessment of lead-induced gut microbiota dysbiosis, and environmental monitoring.},
}
@article {pmid42400707,
year = {2026},
author = {Wang, Y and Chen, M and Wang, Y and Yu, Y},
title = {Simultaneous Detection of Human Norovirus GI, GII and Hepatitis A Virus Using CRISPR-Cas12a-Based RT-RPA and Lateral Flow Strip Method.},
journal = {Food and environmental virology},
volume = {18},
number = {3},
pages = {},
pmid = {42400707},
issn = {1867-0342},
support = {22N31900700//Plan of Action for Scientific and Technological innovation of Science and Technology Commission of Shanghai Municipality/ ; 31601570//National Natural Science Foundation of China/ ; },
mesh = {*Norovirus/isolation &amp; purification/genetics/classification ; Humans ; *Hepatitis A virus/isolation &amp; purification/genetics/classification ; CRISPR-Cas Systems ; Shellfish/virology ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Animals ; *Caliciviridae Infections/virology ; Sensitivity and Specificity ; Hepatitis A/virology ; },
abstract = {Human norovirus (HuNoV) and hepatitis A virus (HAV) are highly prevalent and contagious foodborne pathogens that pose a significant threat to global public health. Current molecular detection methods such as RT-qPCR and RT-ddPCR are highly sensitive and specific but time-consuming, require specialized equipment, and are unsuitable for on-site detection. We developed a multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay coupled with CRISPR-Cas12a and lateral flow detection for rapid, simultaneous identification of HuNoV GI, GII, and HAV. Through rigorous in silico design and experimental validation, we optimized primer pools and crRNAs to ensure broad genotype coverage and high specificity. Using 2 µL of input per target per 50 µL reaction, the assay achieved limits of detection of 10[1] copies/µL (2 × 10[1] copies/reaction) for HAV, 10[3] copies/µL (2 × 10[3] copies/reaction) for GI HuNoV, and 10[2] copies/µL (2 × 10[2] copies/reaction) for GII HuNoV, with a total assay time of 50 min from purified RNA to final readout. No cross-reactivity occurred with other common foodborne viruses. Validation using total RNA extracted from shellfish digestive glands artificially spiked with RNA standards provided preliminary evidence supporting the feasibility of the method under laboratory conditions. This portable system shows strong potential as a rapid multiplex molecular detection platform.},
}
@article {pmid42401475,
year = {2026},
author = {Luo, G and Wei, L and Wang, Q and Guo, Y and Liu, Y and Wang, J and Deng, Y and Li, S and Nie, L and He, N and Chen, Z},
title = {A dual-modal RPA-CRISPR/Cas12a biosensor for rapid and ultrasensitive detection of Staphylococcus aureus in bloodstream infections.},
journal = {Analytica chimica acta},
volume = {1416},
number = {},
pages = {345800},
doi = {10.1016/j.aca.2026.345800},
pmid = {42401475},
issn = {1873-4324},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; Humans ; *Nucleic Acid Amplification Techniques ; *Staphylococcal Infections/blood/diagnosis/microbiology ; Limit of Detection ; Electrochemical Techniques ; *CRISPR-Cas Systems/genetics ; Recombinases/metabolism ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Bloodstream infections (BSIs) caused by Staphylococcus aureus (S. aureus) require rapid and accurate diagnosis to guide effective antimicrobial therapy and improve patient outcomes. However, current diagnostic methods often struggle to balance speed, sensitivity, portability, and cost.<br><br>RESULTS: After systematic optimization of reaction parameters (including primer design, reaction temperature and time, buffer composition, and probe concentration), the fluorescence assay demonstrated high specificity and completed detection within 35&#x202f;min. Under optimized conditions (1.0&#x202f;&#x3bc;M HS-ssDNA-MB and 10&#x202f;min CRISPR incubation), the electrochemical sensor achieved a detection limit of 138 copies/mL, with a wide linear dynamic range from 4.37&#x202f;&#xd7;&#x202f;10[0] to 10[5]copies/&#x3bc;L, and showed high specificity against non-target pathogens. Furthermore, the platform demonstrated reliable performance in spiked artificial blood samples, with recovery rates ranging from 100.7% to 107.3%. This dual-modal recombinase polymerase amplification (RPA)-CRISPR/Cas12a biosensor combines the rapid amplification capability of isothermal methods with the high specificity of CRISPR-based detection.<br><br>SIGNIFICANCE: This platform provides both a high-throughput fluorescence detection mode and a portable, low-cost electrochemical detection mode, offering an efficient and flexible solution for the rapid point-of-care diagnosis and large-scale screening of BSIs caused by S. aureus, with promising potential for clinical translation.},
}
@article {pmid42402276,
year = {2026},
author = {Pradhan, RR and Pati, S and Samal, SK},
title = {Artificial intelligence and CRISPR-based approaches for targeted delivery of bacteriophages.},
journal = {International journal of pharmaceutics},
volume = {},
number = {},
pages = {127155},
doi = {10.1016/j.ijpharm.2026.127155},
pmid = {42402276},
issn = {1873-3476},
abstract = {The rapid emergence of multidrug-resistant (MDR) bacteria has increased interest in bacteriophage therapy as a promising alternative to conventional antibiotics. Bacteriophages are host-specific bacterial viruses that selectively infect and destroy pathogenic bacterial strains. Recent developments in artificial intelligence (AI) and CRISPR-based technologies offer innovative approaches to address challenges such as narrow host range, rapid immune clearance, phage instability, bacterial resistance, and biofilm penetration barriers. By integrating AI-driven structural modeling with CRISPR-mediated genome editing, these methods enable the targeted delivery of bacteriophages. This review focuses on next-generation approaches that combine AI-assisted phage identification, host prediction, and therapeutic optimization with CRISPR-based genome engineering for targeted phage delivery and improved safety. Overall, this review highlights the potential of AI- and CRISPR-assisted phage therapy for the treatment of MDR bacterial infections. This review provides a systematic overview of bacteriophage biology, life cycle, and mechanisms of action, highlighting the influence of phage morphology on therapeutic performance, recent advances, current clinical and preclinical studies, and future perspectives. Although phage therapy shows considerable potential against MDR bacterial infections, several challenges related to delivery, safety, and clinical translation remain. The integration of AI and CRISPR technologies may improve phage selection, targeting specificity, and therapeutic performance. Continued research, clinical validation, and regulatory development will be essential for translating these advances into practical antimicrobial therapies.},
}
@article {pmid42402524,
year = {2026},
author = {Verupanda, S and Chakraborty, A and Shrivastava, M and Pati, SK and Nandi, D},
title = {Harnessing CRISPR-Cas technology to enhance rice resilience under abiotic and biotic stress.},
journal = {Journal of applied genetics},
volume = {},
number = {},
pages = {},
pmid = {42402524},
issn = {2190-3883},
abstract = {The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) genome-editing technology has become an effective and accurate tool for crop development, enabling targeted changes to genes linked to stress tolerance, agronomic performance, and yield. CRISPR-Cas-based technologies are increasingly used in rice (Oryza sativa L.), one of the world's most significant staple crops, to mitigate the negative impacts of disease stress and climate change on productivity. Abiotic factors, including drought, salinity, heat, and cold, as well as biotic challenges such as rice blast, bacterial blight, sheath blight, and insect pests, have a significant impact on rice cultivation and cause substantial yield losses globally. Recent advances in CRISPR/Cas9, base editing, and prime editing have enabled precise manipulation of stress-responsive genes, facilitating the development of climate-resilient and disease and pest-resistant rice varieties. This review summarizes the current progress in CRISPR-Cas-mediated rice improvement, highlighting key genes and molecular pathways involved in tolerance to abiotic and biotic stresses. It also discusses emerging approaches such as transgene-free editing via ribonucleoprotein (RNP) delivery, and high-fidelity Cas variants that enhance editing efficiency and minimize off-target effects. Overall, CRISPR-Cas-based genome editing represents a promising and efficient approach for accelerating the development of high-yielding, climate-resilient, and stress-tolerant rice cultivars, thereby contributing significantly to sustainable rice production and global food security under changing environmental conditions.},
}
@article {pmid42405245,
year = {2026},
author = {XiuJuan, W and Faisal, M and Muhammad, S and Aslam, A and Razzaq, MK and Masroor, A and Yefang, S and Quronfulah, AS and Al-Malki, MA and Osman, HES},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas genome editing transforming crop stress tolerance for global food security.},
journal = {PeerJ},
volume = {14},
number = {},
pages = {e21450},
pmid = {42405245},
issn = {2167-8359},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems ; *Food Security ; Plant Breeding/methods ; Plants, Genetically Modified ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Plant ; Climate Change ; },
abstract = {Climate change increasingly threatens global crop productivity by intensifying drought, salinity, temperature extremes, and biotic stresses. Developing climate-resilient cultivars has therefore become a central objective in modern crop breeding programs. Conventional breeding approaches are often limited by complex trait inheritance and long selection cycles, particularly for polygenic stress-adaptive traits. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) genome editing genome editing provides a precise and efficient platform for targeted manipulation of genes controlling stress tolerance, yield stability, and adaptive performance. This review synthesizes recent advances in CRISPR mediated improvement of resilience to major abiotic stresses (drought, salinity, heat, and cold) and biotic stresses (fungi, bacteria, viruses, and insects) across important cereal, legume, and horticultural crops. Emphasis is placed on the editing of transcription factors, signaling regulators, susceptibility genes, and redox-associated pathways that enhance physiological and molecular stress adaptation. Furthermore, the integration of CRISPR with genomics, transcriptomics, proteomics, metabolomics, genome-wide association studies, high-throughput phenotyping, and artificial intelligence-driven prediction tools is accelerating precision breeding strategies. Despite remaining challenges related to off-target effects, delivery systems, and regulatory frameworks, genome editing represents a transformative approach for advancing climate-resilient crop development and sustainable agricultural production.},
}
@article {pmid42405767,
year = {2026},
author = {Zhuang, S and Luo, W and Lan, B and Liu, Y},
title = {Strategic Design and Engineering of CRISPR/Cas-Powered Sensing Platforms for Enhanced Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01710},
pmid = {42405767},
issn = {2379-3694},
abstract = {Rapid and accurate nucleic acid detection is fundamental to effective disease management. While PCR remains the gold standard, its requirement for sophisticated instrumentation limits its application in point-of-care settings. CRISPR-Cas systems have emerged as a disruptive diagnostic technology, leveraging the programmable specificity and unique trans-cleavage activity of Cas effectors to revolutionize biosensing. This review systematically evaluates the evolution of CRISPR-Cas-powered sensing platforms, categorized by their signal transduction modalities. We first discuss the expanding biochemical landscape of Cas nucleases, highlighting recent discoveries where conventional boundaries of Cas9, Cas12, and Cas13 have been transcended to enable versatile DNA/RNA targeting. Subsequently, we provide a comprehensive analysis of four primary sensing architectures: (1) Fluorescence-based platforms, exploring diverse strategies from target and signal amplification with dual-labeled ssDNA probes to nanomaterial-based probes; (2) Naked-eye visual platforms, encompassing both solid-phase lateral flow assays and solution-phase colorimetric strategies that facilitate rapid, instrument-free screening; (3) Electrochemical biosensors, which transduce biological recognition events into measurable electrical parameters, offering high sensitivity and seamless integration with miniaturized electronics; and (4) Electronic and Optoelectronic systems, including field-effect transistors and plasmonic sensors, which offer high-sensitivity, label-free detection. Despite significant progress, the translation of CRISPR-Dx from laboratory proof of concepts to clinical reality faces several bottlenecks. We critically analyze current challenges, including the need for integrated "sample-to-answer" workflows, high-throughput multiplexing, and digital quantification. Finally, we envision future trends such as AI-assisted signal processing and wearable sensing interfaces. By bridging the gap between molecular biology and advanced engineering, CRISPR-powered platforms are poised to make precision molecular diagnostics universally accessible.},
}
@article {pmid42405957,
year = {2026},
author = {Soto-Serrano, A and Vincze, T and Roberts, RJ and Krych, L and Mahony, J and Deptula, P},
title = {Comparative genomics and methylome profiling of Pseudolactococcus laudensis reveal signatures of niche adaptation and strain-level variation in mobile genetic elements and phage defence.},
journal = {Microbial genomics},
volume = {12},
number = {7},
pages = {},
pmid = {42405957},
issn = {2057-5858},
mesh = {*Bacteriophages/genetics ; Genome, Bacterial ; Milk/microbiology ; *Interspersed Repetitive Sequences ; Genomics/methods ; Animals ; Gene Transfer, Horizontal ; DNA Methylation ; Adaptation, Physiological/genetics ; *Lactococcus/genetics/virology ; Phylogeny ; },
abstract = {Pseudolactococcus laudensis (formerly named Lactococcus laudensis) is an emerging lactic acid bacterium first isolated from raw milk in 2015 and subsequently detected in vegetables and dairy mesophilic starter cultures. Despite its recurrent isolation from diverse environments, the genetic basis of its niche adaptation, horizontal gene transfer and phage defence remains unexplored. Here, we perform the first comparative genomic and epigenomic analysis of P. laudensis using complete genomes of a plant-derived isolate (MCRI-603), a milk isolate (DSM 28961) and 20 strains from a Danish dairy mesophilic starter culture. Genomes were annotated and analysed using pangenomics, Clustering of Orthologous Genes and methylome profiling. Average nucleotide identity, pangenome and Clustering of Orthologous Genes analyses revealed niche-associated structure: dairy starter strains formed a tight cluster, while the plant isolate MCRI-603 and milk isolate DSM 28961 were more similar to each other than to the starter culture group. The pangenome comprised 4,946 genes, with 1,396 core genes. Dairy starter strains showed markedly elevated numbers of insertion sequences, pseudogenes, plasmids and genomic islands relative to MCRI-603, which was plasmid-free and carried very few insertion sequence elements or genomic islands. DSM 28961 displayed pseudogene count similar to the dairy starter strains but markedly fewer transposases. These patterns are consistent with a plant-associated origin of P. laudensis and progressive dairy specialization via mobile genetic element acquisition. The P. laudensis mobilome was found to carry key niche-related traits. Lactose utilization operons were plasmid-encoded, whereas exopolysaccharide-encoding loci, opp oligopeptide transport systems and several defence loci, including clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), were consistently encoded within chromosomal integrative elements. All strains harboured prophage-like elements, including putatively intact prophages in 13 of them, and ~67% of 238 predicted antiphage systems resided on mobile genetic elements, underscoring their central role in phage defence. Restriction-modification systems dominated the defensome, and three strains encoded CRISPR-Cas systems (including type III-A and type I-C), indicating a higher prevalence than has been reported for Lactococcus lactis and Lactococcus cremoris, where CRISPR-Cas has rarely been observed. Methylome analysis identified 43 distinct motifs, of which 25 were novel. The P. laudensis methylome was overwhelmingly dominated by N[6]-methyladenine, and most motifs were short, non-palindromic and largely associated with type III restriction-modification systems and some type I and II subtypes. Nearly all strains exhibited distinct methylation profiles, including those isolated from the same dairy starter culture, highlighting extensive epigenetic diversification in dairy environments. Altogether, the data reveals a highly dynamic genomic and epigenomic landscape in P. laudensis, greatly shaped by mobile genetic elements, and provides a foundation for future work in this species and other Pseudolactococci.},
}
@article {pmid42406963,
year = {2026},
author = {Carballar-Lejarazú, R and Dong, Y and Pham, TB and Tushar, T and Stillinger, D and Nguyen, DN and Winokur, L and Tavadia, M and Tao, M and Dimopoulos, G and James, AA},
title = {Long-term stability and performance of Cas9/guide RNA-based gene drives in anopheline mosquitoes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {28},
pages = {e2605739123},
doi = {10.1073/pnas.2605739123},
pmid = {42406963},
issn = {1091-6490},
support = {AI170692//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; INV-043645/GATES/Gates Foundation/United States ; NA//University of California, Irvine, Malaria Initiative/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; *CRISPR-Cas Systems ; Malaria/prevention &amp; control ; },
abstract = {Gene-drive population modification strategies are being developed to control the transmission by anopheline mosquitoes of the parasites that cause human malaria. These approaches are designed to reduce disease prevalence and incidence by spreading dominant antiparasite effector genes throughout vector populations. The strains must sustain drive and parasite suppression properties over extended periods of time to have an epidemiological impact. Three gene-drive strains, AcTP13 and AcTP43 in Anopheles coluzzii and AgTP13 in Anopheles gambiae, carrying autonomous Cas9/guide RNA-based drive systems linked to multiple antiparasite effector genes were remarkably stable in all A. coluzzii replicates over a 2-y (35 generation) period in laboratory cage trials. Two of three A. gambiae replicates performed equally well. Stability was assessed as a function of population dynamics (size), molecular integrity of the gene-drive cassettes, maintenance of drive efficiency (gene conversion), generation and accumulation of mutant drive-resistant target alleles, drive system-generated off-target effects, and effector gene parasite suppression activity. All lines met stability requirements with the exception of one AgTP13 cage replicate that was affected by drive-resistant target-site mutations. Notably, all strains retained parasite suppression activity and high drive efficiencies throughout the duration of the trials. These results support the further development and deployment of these strains for malaria control.},
}
@article {pmid42407157,
year = {2026},
author = {Mu, Y and Yang, Y and Niu, Y and Yan, Z and Lv, X and Li, X},
title = {CRISPR-based diagnostics for ESKAPE drug-resistant bacteria: From proof-of-concept to point-of-care.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130240},
doi = {10.1016/j.talanta.2026.130240},
pmid = {42407157},
issn = {1873-3573},
abstract = {The ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, represent a major challenge in the antimicrobial resistance (AMR) crisis. These pathogens are progressively acquiring pan-resistance and spreading beyond traditional healthcare settings, yet conventional diagnostic methods remain ill-equipped for point-of-care (POC) deployment due to slow processing times and limited adaptability. CRISPR/Cas systems feature programmable target specificity and intrinsic signal amplification, enabling rapid and accurate nucleic acid detection. Despite these advantages, the translation of CRISPR-based assays for ESKAPE pathogens from proof-of-concept to practical POC tools faces major challenges. Current research is fragmented, and key trade-offs between sensitivity, multiplexing performance and operational simplicity have not been fully addressed. This review offers a critical assessment of the field, moving beyond a simple summary of existing studies to analyze how various CRISPR systems (Cas9, Cas12, Cas13, and Cas14) and amplification strategies address the demands of POC testing. We identify key barriers to clinical application, particularly sample preparation, multiplex detection, reagent stability, and discuss emerging solutions such as microfluidic integration, lyophilized reagents development, and artificial intelligence-driven data interpretation. By focusing on the central question of how to transition from benchtop research to bedside application, this review provides a strategic framework for advancing next-generation CRISPR diagnostics capable of rapid, precise, and real-time detection of drug-resistant ESKAPE pathogens in the fight against AMR.},
}
@article {pmid42409192,
year = {2026},
author = {Shamim, S and Singh, AP and Sharma, H and Gohri, S and Taumar, D and Chaudhary, V},
title = {Programmable gene modulation networks for Parkinson's disease using nanotechnology enabled CRISPR/Cas brain delivery.},
journal = {International journal of pharmaceutics},
volume = {},
number = {},
pages = {127151},
doi = {10.1016/j.ijpharm.2026.127151},
pmid = {42409192},
issn = {1873-3476},
abstract = {Parkinson's disease is a progressive neurodegenerative disorder driven by interconnected molecular pathways, including α-synuclein aggregation, mitochondrial dysfunction, impaired proteostasis, and neuroinflammation. Current therapies are primarily symptomatic and have not consistently demonstrated prevention of disease progression. This review introduces Programmable gene modulation networks, a systems-level framework that integrates CRISPR/Cas technologies with nanotechnology-enabled brain delivery for precision intervention in Parkinson's disease. Advanced CRISPR modalities, including CRISPR interference, activation, base editing, prime editing, and epigenetic editing, are evaluated for reversible and targeted modulation of disease-relevant gene networks. Non-viral nanocarrier platforms, such as lipid nanoparticles, polymeric systems, and exosome-mimetic vesicles, are discussed for overcoming blood-brain barrier limitations and improving brain-specific delivery. The review further emphasizes translational challenges, including delivery efficiency, off-target effects, long-term safety, manufacturing scalability, and regulatory considerations. By integrating molecular network biology, programmable gene regulation, and translational decision-making, this review provides a roadmap for developing next-generation disease-modifying therapies for Parkinson's disease.},
}
@article {pmid42409502,
year = {2026},
author = {Wang, X and Gu, Z and Xu, Q and Dong, M and Duan, N and Wang, Z and Wu, S},
title = {A dual-mode electrochemical-colorimetric aptasensor based on HCR-CRISPR/Cas12a cascade amplification for MTGase detection.},
journal = {Food research international (Ottawa, Ont.)},
volume = {240},
number = {},
pages = {119676},
doi = {10.1016/j.foodres.2026.119676},
pmid = {42409502},
issn = {1873-7145},
mesh = {*Transglutaminases/analysis ; *Colorimetry/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Limit of Detection ; Triticum ; },
abstract = {Microbial transglutaminase (MTGase) is widely used in food processing, but its potential food safety risks cannot be neglected. MTGase-treated wheat products show immunoreactivity, which may induce celiac disease and damage intestinal barrier function. Therefore, trace detection of MTGase is of great significance. In this study, an electrochemical-colorimetric dual-mode aptasensor for MTGase was developed based on HCR-CRISPR/Cas12a cascade amplification. The system employs high-affinity aptamers for specific recognition of MTGase and significantly enhances the detection signal through HCR-Cas12a cascade amplification, achieving highly sensitive responses. The platform incorporates MXene@AuNPs and Co-Fc-MOF composite materials, where MXene@AuNPs provides excellent conductivity and a stable loading interface, and Co-Fc-MOF offers both electrochemical activity and peroxidase-like activity, enabling synchronous electrochemical and colorimetric signal output. Experimental results demonstrate a wide linear range of 0.5-2000 ng/mL, with a detection limit of 0.12 ng/mL for the electrochemical mode and 0.14 ng/mL for the colorimetric mode. The platform exhibits excellent stability and reproducibility, and achieves satisfactory recovery in real sample analysis. This dual-mode sensing strategy efficiently integrates aptamer recognition, bifunctional nanomaterials, and cascade amplification, providing a rapid and reliable method for trace MTGase detection in food matrices, and offering guidance for the development of multi-mode aptasensor.},
}
@article {pmid41588790,
year = {2026},
author = {Favaratto, L and da Silva, ML and Buss, DS and Quadros, OF and Tapia-Tussell, R and Ventura, JA and Fernandes, AAR and Fernandes, PMB},
title = {The regulatory frameworks surrounding CRISPR-edited papaya and their impact on international commerce.},
journal = {Journal of the science of food and agriculture},
volume = {106},
number = {11},
pages = {6262-6270},
pmid = {41588790},
issn = {1097-0010},
support = {# 404972/2021-7 and 441499/2023-6//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; # 1132/2024//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Esp&#xed;rito Santo/ ; #301052/25-5 and #307905/2020-9//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; },
mesh = {*Carica/genetics/virology/immunology/growth &amp; development ; *Plant Diseases/virology/economics/genetics ; *Gene Editing/economics/legislation &amp; jurisprudence ; *Commerce/legislation &amp; jurisprudence/economics ; CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics/virology/immunology ; Plant Proteins/genetics/metabolism/immunology ; Fruit/genetics/virology/economics ; },
abstract = {The papaya tree (Carica papaya L.), native to the Americas, is cultivated in tropical regions and holds substantial economic importance, with an estimated export volume of 365 000 t in 2023. However, diseases caused by viruses, fungi, bacteria, and nematodes can lead to severe losses. Among the more than 38 known viral diseases affecting papaya, only a few poses serious threats to cultivation, notably Papaya Ringspot, Papaya Mosaic, and Papaya Sticky Disease (PSD). Emerging technologies, particularly CRISPR/Cas9 gene editing, offer promising avenues to enhance plant resistance. This study examines regulatory paradigms in key papaya-producing and importing countries, highlighting the need for international regulatory harmonization to reduce trade barriers and improve market access for CRISPR-edited cultivars. We demonstrate the feasibility of CRISPR-based genome editing in papaya (Carica papaya L.) by targeting phytoene desaturase as a proof-of-concept marker gene and β-1,3-glucanase, a resistance gene identified through proteomic profiling of host-pathogen interactions during infection by the papaya meleira virus (PMeV and PMeV2) complex. This virus complex causes PSD, a major threat to papaya production, rendering the fruit commercially unviable due to negative effects on texture and flavor as well as inhibiting the formation of benzyl isothiocyanate (BITC), and the fruits become susceptible to fruit flies, which are quarantine pests. Despite extensive traditional breeding efforts, resistant papaya genotypes have yet to be identified, underscoring the need for innovative approaches. However, translating advancements into commercial applications remains challenging due to the diverse and often inconsistent regulatory frameworks governing genome-edited crops across different jurisdictions. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.},
}
@article {pmid42116141,
year = {2026},
author = {Ford, H and Dalvie, NC and Lorgeree, TR and Barry, RM and Sibel, AN and Ahlmark, RA and Narayanan, H and Sunday, BC and Whittaker, CA and Acharya, R and Elenberger, CM and Love, JC},
title = {Deletion of low-essentiality, secretion-associated genes enhances recombinant protein production in Komagataella phaffii.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {42116141},
issn = {1475-2859},
mesh = {*Recombinant Proteins/biosynthesis/genetics ; *Saccharomycetales/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Deletion ; Antibodies, Monoclonal/biosynthesis/genetics ; *Genes, Essential ; },
abstract = {BACKGROUND: Komagataella phaffii (K. phaffii) is used to manufacture biologic medicines, food proteins, reagents, and materials. Despite its increasing prevalence, further improvements to its productivity would enhance its economic and operational benefits. Genomic engineering represents one approach to increase its cell-specific productivity. We hypothesized that combining the metrics for the relative essentiality of genes with biological inference for relevance to protein secretion could identify genes that, when disrupted, would improve specific productivity in the resulting strains.<br><br>RESULTS: The essentiality of genes in K. phaffii (NRRL Y-11430) were predicted through a genome-wide knockout screen using CRISPR-Cas9. Based on the results from this screen, we selected and subsequently disrupted the least essential genes from two gene groups heavily associated with secretion, namely those relating to the cell wall and vacuolar transport. Strains of K. phaffii with single gene disruptions from these gene sets showed significantly improved production of a monoclonal antibody (mAb). These strains exhibited no discernible differences in growth or apparent profiles of host cell proteins when compared to the parental strain. The best-performing strains consistently showed 2-3x enhancements in specific productivity and titers across scales (3-150 mL), culture formats (plates, flasks, bioreactors), and processing operations (batch and fed-batch).<br><br>CONCLUSIONS: This study demonstrates how combining data on gene essentiality and prior knowledge of biological pathways related to a phenotypic trait of interest (here protein secretion) can inform strain engineering to enhance the trait. This study expands the catalog of genetically engineered strains of K. phaffii with improved productivity. These strains support the long-term goal of achieving low-cost, high-volume production of recombinant proteins using this host. Further engineering of these strains and optimization of fermentation processes could enable volumetric productivities comparable to those of other established hosts used to produce mAbs and other complex recombinant proteins.},
}
@article {pmid42148575,
year = {2026},
author = {Kang, B and Kim, J-Y and Oh, S and Kweon, J and Park, H and Kim, M and Choi, I-G and Chang, IS},
title = {A CRISPR interference system for tunable gene expression integrated with a promoter library for Eubacterium callanderi KIST612, an acetogen of functional diversity and versatility.},
journal = {Microbiology spectrum},
volume = {14},
number = {7},
pages = {e0377925},
pmid = {42148575},
issn = {2165-0497},
support = {RS-2021-NR060081//National Research Foundation of Korea/ ; RS-2023-00249146//National Research Foundation of Korea/ ; },
mesh = {*Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Bacterial ; Gene Library ; *CRISPR-Cas Systems ; Metabolic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; },
abstract = {UNLABELLED: Acetogens are key biocatalysts for carbon-neutral biorefineries, yet their metabolic engineering is limited by the lack of tunable transcriptional regulation systems. Here, we developed a synthetic promoter library for Eubacterium callanderi KIST612 and integrated it with a CRISPR interference (CRISPRi) system to establish precise and scalable gene regulation. Motif analysis of 3,109 putative native promoters revealed conserved and semi-conserved -35 and -10 elements, which were used to construct a promoter library spanning a >20-fold dynamic range of transcriptional strengths. The system was validated by knockdown of pyrF, where promoter strength directly determined repression efficiency (R[2] = 0.92), with high-strength promoters achieving near-complete gene silencing. Application to lactate dehydrogenase (ldh) revealed that increasing promoter strength progressively reduced lactate production from 93.3% to 0.0% of control. This study establishes a versatile synthetic promoter-CRISPRi platform tailored for acetogens, enabling precise control of gene expression and mechanistic dissection of redox metabolism.<br><br>IMPORTANCE: Transitioning to a carbon-neutral economy requires biocatalysts that can efficiently convert waste-derived substrates into valuable products. Acetogens are industrially relevant organisms for gas fermentation, but the lack of genetic toolkits tailored to their physiology has constrained metabolic engineering. We present the first synthetic promoter-CRISPRi platform specifically optimized for Eubacterium callanderi KIST612, a model acetogen with high industrial potential. This system provides tunable and predictable regulation of gene expression, extending from mild repression to a near-complete knockdown that could alternate gene deletion systems. This system could be used for not only advancing fundamental understanding of acetogen physiology but also providing a broadly applicable genetic toolbox for precision engineering of sustainable microbial biorefineries.},
}
@article {pmid42148800,
year = {2026},
author = {Zheng, J and Wen, Z and Li, Y and Zou, W and Ge, P and Peng, H},
title = {Rapid detection of Enterococcus faecalis using RPA-CRISPR/Cas12a-assisted technology.},
journal = {Microbiology spectrum},
volume = {14},
number = {7},
pages = {e0016826},
pmid = {42148800},
issn = {2165-0497},
support = {2023A1515011733//Natural Science Foundation of Guangdong Province/ ; 82272364, 82330072//National Natural Science Foundation of China/ ; },
mesh = {*Enterococcus faecalis/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Gram-Positive Bacterial Infections/microbiology/diagnosis ; Rapid Diagnostic Tests ; Recombinases ; CRISPR-Associated Proteins/genetics ; },
abstract = {Enterococcus faecalis (E. faecalis) is an opportunistic pathogen capable of causing various life-threatening infections, including urinary tract infections, bloodstream infections, infective endocarditis, and meningitis. As a major etiological agent of healthcare-associated infections (HAIs), its global prevalence continues to rise, a trend closely linked to the increasing problem of multidrug resistance driven by overuse of antibiotics. Therefore, rapid and accurate detection is essential for timely treatment and improved prognosis. In this study, the pheS gene of E. faecalis was rapidly amplified using recombinase polymerase amplification (RPA), and detection was achieved via a CRISPR/Cas12a system. The Cas12a-crRNA complex specifically recognized the amplification product and triggered nonspecific cleavage of a single-stranded DNA (ssDNA) reporter, generating a fluorescent signal that could be quantified in a real-time PCR system or visualized directly under ultraviolet (UV) light. After optimization of key parameters-including RPA primers, reaction conditions, crRNA sequence, and the crRNA/Cas12a combination-the assay achieved a limit of detection (LOD) of 10[-2] ng/μL within a short turnaround time, and showed no cross-reactivity with other common pathogen detection results from clinical isolates and spiked samples were fully consistent with those obtained through PCR/qPCR, confirming high reliability. In summary, the RPA-CRISPR/Cas12a detection method established in this study is sensitive, specific, and reliable. Its simplicity, minimal equipment requirements, and cost-effectiveness make it a promising tool for rapid clinical detection of E. faecalis.IMPORTANCEEnterococcus faecalis is a major opportunistic pathogen responsible for severe healthcare-associated infections, with rising prevalence linked to antibiotic resistance. Rapid and accurate detection is critical for timely treatment and infection control. Conventional methods are often time-consuming or require complex laboratory infrastructure, limiting their use at the point of care. This study developed a rapid detection assay by integrating recombinase polymerase amplification with the CRISPR/Cas12a system, targeting the pheS gene of E. faecalis. The method is sensitive and specific, providing visual results under UV light within a short turnaround time. It offers a simple, cost-effective, and requires minimal equipment, suitable for clinical and resource-limited settings, potentially improving diagnostic efficiency and supporting antimicrobial stewardship.},
}
@article {pmid42155443,
year = {2026},
author = {Liang, WW and Mueller, SJ and Hart, SK and Wessels, HH and Méndez-Mancilla, A and Sookdeo, A and Choi, O and Caragine, CM and Corman, A and Lu, L and Kolumba, O and Williams, B and Sanjana, NE},
title = {Essential lncRNAs in the human transcriptome.},
journal = {Cell genomics},
volume = {6},
number = {7},
pages = {101253},
doi = {10.1016/j.xgen.2026.101253},
pmid = {42155443},
issn = {2666-979X},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Transcriptome/genetics ; Gene Expression Profiling/methods ; CRISPR-Cas Systems/genetics ; Neoplasms/genetics ; Apoptosis/genetics ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Cell Cycle/genetics ; },
abstract = {Mammalian genomes host a diverse array of RNAs, including protein-coding and noncoding transcripts. However, the functional roles of most long noncoding RNAs (lncRNAs) remain elusive. Using RNA-targeting CRISPR-Cas13 screens, we probed how the loss of ∼5,500 lncRNAs impacts cell fitness across five human cell lines and identified 788 lncRNAs with context-specific or broad essentiality. We confirm their essentiality through individual perturbations and find that the majority of essential lncRNAs operate independently of their nearest protein-coding genes. Using transcriptome profiling in single cells, we discover that loss of essential lncRNAs impairs cell cycle progression and drives apoptosis. Many essential lncRNAs demonstrate dynamic expression across tissues during development. Using ∼9,000 primary tumors, we pinpoint those lncRNAs whose expression in tumors correlates with survival, yielding new biomarkers and potential therapeutic targets. This transcriptome-wide survey of functional lncRNAs advances our understanding of noncoding transcripts and demonstrates the potential of transcriptome-scale noncoding screens with Cas13.},
}
@article {pmid42214793,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {CRISPR/Cas9 system-mediated p21 knockout impairs the MITF signaling pathway.},
journal = {Journal of biotechnology},
volume = {417},
number = {},
pages = {81-90},
doi = {10.1016/j.jbiotec.2026.05.014},
pmid = {42214793},
issn = {1873-4863},
mesh = {*Microphthalmia-Associated Transcription Factor/metabolism/genetics ; Animals ; *Signal Transduction/genetics ; *Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Melanogenesis ; Gene Knockout Techniques/methods ; Melanins/metabolism ; Cell Line, Tumor ; },
abstract = {The CRISPR/Cas9 method facilitates targeted disruption of gene sequences, providing a reliable means to analyze gene-dependent regulatory pathways. This study aims to investigate melanogenesis in p21-knockout B16F1 cells generated by the CRISPR/Cas9 system. The mutation was confirmed by DNA Sanger sequencing, which identified frameshift-inducing indels in the p21 locus. The protein structure of p21 in KO cells was predicted by the α-Fold2 and ChimeraX models. The expression level of the p21 gene was completely reduced in RT-PCR and qPCR assays. Notably, while p21-knockout cells exhibited significantly reduced SA-β-galactosidase activity, this was not indicative of cellular rejuvenation. Instead, it correlated with a loss of melanocytic functionality, as evidenced by the concurrent decrease in melanin synthesis and collagen production. Western blotting and immunofluorescence analyses were performed to examine cell cycle and melanogenesis-associated proteins in p21-deficient cells. Loss of p21 resulted in reduced expression of p21, phosphorylated p21, p53, acetylated p53, CDK2, Cyclin D, Cyclin E, MITF, TRP-1, TRP-2, TYR, and p-ERK. Collectively, these findings indicate that p21 is essential for maintaining MITF-driven melanogenic signaling.},
}
@article {pmid42284907,
year = {2026},
author = {Xiao, S and Zhou, L and Tian, Z and Zhou, S and Kong, J and Zhang, X},
title = {CRISPR-Cas12a-regulated photo-ATRP for ultrasensitive detection of lipopolysaccharide.},
journal = {Food chemistry},
volume = {522},
number = {},
pages = {149992},
doi = {10.1016/j.foodchem.2026.149992},
pmid = {42284907},
issn = {1873-7072},
mesh = {*Lipopolysaccharides/analysis ; *Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Limit of Detection ; *Bacterial Proteins/genetics/chemistry ; Food Contamination/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Beverages/analysis ; },
abstract = {Lipopolysaccharide (LPS), a primary virulence factor produced by Gram-negative bacteria, demands rapid and ultrasensitive detection for safeguarding food safety and supporting clinical diagnosis. This study developed a novel electrochemical biosensing platform by integrating the programmable recognition of CRISPR-Cas12a with the high-gain signal amplification of photoinduced ATRP. In this mechanism, the binding of LPS to its aptamer regulated Cas12a activity, which controlled an N3-DNA initiator probe. This probe, in turn, guided in-situ photopolymerization via click chemistry, generating a dramatically enhanced signal. The sensor exhibited a linear range from 10 fg/mL to 1 ng/mL with a detection limit of 2.48 fg/mL, along with high selectivity, reproducibility, and stability. Tests in spiked beverage samples showed high recovery rates and strong anti-interference capability. This work not only achieves ultrasensitive LPS detection but also provides a new approach for extending CRISPR-based biosensing to non-nucleic acid targets.},
}
@article {pmid42297231,
year = {2026},
author = {Fatima, SW},
title = {Cellular plasticity and epigenetic instability in cancer: Mechanistic insights and functional dissection with CRISPR-based epigenome editing.},
journal = {Cancer letters},
volume = {656},
number = {},
pages = {218679},
doi = {10.1016/j.canlet.2026.218679},
pmid = {42297231},
issn = {1872-7980},
mesh = {Humans ; *Epigenome Editing ; *Cell Plasticity/genetics ; *Neoplasms/genetics/pathology/therapy ; *Epigenesis, Genetic ; Tumor Microenvironment/genetics ; *CRISPR-Cas Systems ; Animals ; Gene Expression Regulation, Neoplastic ; DNA Methylation ; },
abstract = {Cellular plasticity is a fundamental driver of tumor heterogeneity, cancer stemness, immune evasion, therapeutic resistance, and disease progression. In malignancies such as breast cancer and glioblastoma, tumor cells undergo reversible phenotypic transitions between proliferative, stem-like, invasive, and drug-tolerant states in response to intrinsic regulatory programs and extrinsic signals from the tumor microenvironment. These adaptive dynamics are governed by complex interactions among signaling pathways, transcriptional networks, chromatin remodeling, DNA methylation, histone modifications, non-coding RNAs, and immune-mediated microenvironmental cues. Such epigenetic instability enables stochastic and therapy-induced shifts between alternative cellular states, thereby contributing to tumor evolution, metastasis, resistance to targeted therapies, and variable responses to immunotherapy. Understanding the mechanisms that govern epigenetic plasticity remains a central challenge in cancer biology. Recent advances in CRISPR/dCas9-based epigenome editing have provided powerful experimental tools for investigating the functional consequences of locus-specific chromatin modifications without altering the underlying DNA sequence. Catalytically inactive Cas9 (dCas9) fused to epigenetic effector domains, including DNMT3A, TET1, KRAB, and p300, enables targeted modulation of gene expression programs implicated in cell-state transitions, lineage specification, and adaptive resistance. These technologies offer a versatile platform for interrogating causal relationships between chromatin states and cellular phenotypes and for modeling mechanisms of tumor adaptation. This review examines the molecular basis of epigenetic plasticity in cancer, evaluates current CRISPR-based epigenome editing strategies, and discusses their application in studying tumor heterogeneity, microenvironment-driven adaptation, immune escape, and therapy resistance. This study highlights emerging opportunities and persistent challenges associated with epigenome editing, including delivery barriers, durability of epigenetic modifications, context-dependent biological responses, and translational limitations. Collectively, these approaches provide valuable experimental frameworks for dissecting the regulatory logic of cancer cell plasticity while informing future therapeutic development.},
}
@article {pmid42303162,
year = {2026},
author = {Durairaj, S and Durairaj, S and Krishnan, S and Raju, A},
title = {CRISPR Cas9 revolutionizing genetic engineering and therapeutic applications.},
journal = {Journal of biotechnology},
volume = {417},
number = {},
pages = {246-264},
doi = {10.1016/j.jbiotec.2026.06.012},
pmid = {42303162},
issn = {1873-4863},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; *Genetic Engineering/methods ; },
abstract = {Genetic engineering has been transformed by CRISPR-Cas9 technology, offering high precision and adaptability in biological research and therapeutic innovation. Originating from a bacterial defense system, CRISPR-Cas9 enables targeted DNA editing through guide RNA-directed Cas9 nuclease activity, allowing gene modification, mutation correction, and disease mechanism analysis. This has opened new avenues in personalized medicine and gene therapy, particularly for cancer and inherited disorders, alongside applications in agriculture. In oncology, CRISPR-Cas9 demonstrates strong potential in oncogene targeting, immune cell engineering, and CAR-T-based immunotherapy, supported by substantial preclinical success. Delivery efficiency is enhanced through systems such as exosomes, liposomes, and nanoparticles, improving stability and tumor targeting. However, clinical translation remains constrained by off-target effects, delivery limitations, and ethical concerns in human genome editing, particularly germline modification. CRISPR shows therapeutic promise for muscular dystrophy, sickle cell disease, and cystic fibrosis. Emerging platforms including base editing, prime editing, and dCas9-based epigenome editing enable precise genome and gene regulation without double-strand breaks, reducing toxicity and expanding therapeutic scope in cancer and genetic diseases. Regulatory frameworks remain heterogeneous, affecting translation. The United States leads in approvals and clinical progress, the European Union emphasizes safety and ethics, China shows rapid expansion in clinical trials, and India remains in early stages due to regulatory and infrastructure constraints. Public perception influences adoption, shaped by misinformation and limited awareness. Persistent gaps in long-term safety, clinical efficacy, and population diversity remain challenges. Overall, CRISPR-Cas9 represents a transformative but carefully regulated platform for advancing biotechnology and medicine.},
}
@article {pmid42394824,
year = {2026},
author = {Wu, Y and Cai, H and Wu, Q and Wu, J and Hu, J and Huang, E and Li, Z and Liang, S and Hu, X and Dai, J and Liao, R},
title = {The CRISPR-Cas toolkit for mosquito-borne virus surveillance: detection, tracing, and discovery.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1873187},
pmid = {42394824},
issn = {2235-2988},
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; *Mosquito-Borne Diseases/virology ; *Epidemiological Monitoring ; *Culicidae/virology ; *Mosquito Vectors/virology ; *Viruses/genetics/isolation &amp; purification/classification ; *Virus Diseases/virology/transmission/diagnosis ; },
abstract = {Mosquito-borne virus surveillance increasingly requires rapid, distributed detection of co-circulating pathogens, serotypes, and lineages across clinical and vector-sampling sites. CRISPR-Cas platforms offer a programmable toolkit for this purpose, but their readiness differs substantially across surveillance functions. Here, we review CRISPR-Cas methods for mosquito-borne virus surveillance across detection, tracing, and discovery-supporting targeted screening. Detection is the most advanced application: selected Cas12- and Cas13-based assays for dengue, Zika, chikungunya, West Nile, Japanese encephalitis, and related mosquito-associated viruses report sub-hour workflows, portable readouts, and targeted serotype- or lineage-marker discrimination. However, performance remains assay-, target-, and sample-matrix-dependent, and validation in pooled mosquito samples and field settings is still limited. Tracing currently relies mainly on validated portable amplicon-sequencing workflows, whereas CRISPR-aided sample-preparation methods such as DASH, FLASH, RAPID-DASH, and Cas9-targeted enrichment remain transferable opportunities for host depletion or target enrichment rather than established mosquito-borne virus genomic-surveillance workflows. For discovery-oriented surveillance, multiplex CRISPR-Cas systems such as CARMEN can support targeted screening of known or near-neighbor viruses represented by predesigned crRNAs, while metagenomic next-generation sequencing remains necessary for divergent or previously unknown viruses. Across these functions, CRISPR-Cas programmability may accelerate parts of assay redesign, but practical retargeting still requires compatible amplification primers, effector-specific target constraints, cross-reactivity assessment, and analytical revalidation. Routine surveillance use will require integrated demonstrations with clinical and pooled-vector samples, comparison against established molecular and sequencing methods, cost validation, and regulatory evidence.},
}
@article {pmid42395789,
year = {2026},
author = {Abdallah, NM and Mohammed, MR and Al Haideri, H and Alahmari, AS and Alwutayd, KM and Zarah, RK and Hamdi, H and Al Masoudi, LM and Al Thagafi, NT and Althobaiti, AT and Khormi, MA and Binshaya, AS and Abalkhail, A and Soliman, MKY},
title = {Green synthesis of ZnO/Fe3O4 nanocomposites from Citrus reticulata peel: antibacterial activity against MDR Acinetobacter baumannii, CRISPR-Cas gene modulation, and anticancer potential.},
journal = {RSC advances},
volume = {},
number = {},
pages = {},
pmid = {42395789},
issn = {2046-2069},
abstract = {The emergence of multidrug-resistant (MDR) Acinetobacter baumannii necessitates the development of alternative antimicrobial strategies. In this study, ZnO/Fe3O4 nanocomposites (NCs) were green-synthesized using Citrus reticulata peel extract and evaluated for antibacterial, CRISPR-Cas gene modulation, anticancer, and antioxidant activities. Phytochemical profiling by GC-MS and HPLC confirmed a terpene- and polyphenol-rich composition supporting nanoparticle formation and stabilization. The synthesized NCs were characterized by UV-vis, FTIR, and XRD analyses, supporting the formation of crystalline ZnO and Fe3O4 phases, while TEM revealed nanoscale morphology (55.64 ± 24.2 nm) with hydrodynamic size of ∼181.3 nm and a zeta potential of +2.64 mV. Fifteen clinical A. baumannii isolates were identified, among which nine exhibited multidrug-resistant (MDR) profiles. CRISPR-associated genes were screened in the MDR isolates, and four isolates harboring the target genes were selected for further molecular analyses. The ZnO/Fe3O4 NCs exhibited antibacterial activity with inhibition zones ranging from 17 to 24 mm and MIC values of 250-500 µg mL[-1]. TEM analysis of treated bacteria demonstrated severe structural damage, including membrane disruption and cytoplasmic leakage. Furthermore, sub-MIC exposure resulted in downregulation of CRISPR-associated genes (Cas1: 0.61-0.98; Csy1: 0.54-0.95; Csy3: 0.70-0.83). Cytotoxicity assays revealed selective antiproliferative effects against Caco-2 colorectal cancer cells (IC50 = 60.7 µg mL[-1]) compared to normal Vero cells (IC50 = 254.66 µg mL[-1]), accompanied by increased apoptosis (15.8%) and G2/M cell cycle arrest (39.7%). Additionally, the NCs exhibited concentration-dependent antioxidant activity, reaching up to 69.44% (DPPH), 76.12% (ABTS), 62.24% (H2O2 scavenging), 56.13% (metal chelation), and 63.52% (reducing power). Overall, these findings demonstrate that green-synthesized ZnO/Fe3O4 NCs are multifunctional nanomaterials with promising antibacterial, anticancer, and antioxidant properties.},
}
@article {pmid42398757,
year = {2026},
author = {Wang, X and Dong, W and Shen, R and Yu, X and Zhang, Y and Wang, W and Yin, X and Hu, Y and Peng, X and Yang, G and Rao, Q and Deng, X and Wang, R and Tang, F and Huang, Y and Jin, Z and Cai, Q and Xu, H and Tang, Y and Du, D},
title = {Development of optimized fluorogenic DNA aptamers for a portable one-pot CRISPR-Cas12a platform for rapid and sensitive detection of monkeypox virus and chikungunya virus.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.07.003},
pmid = {42398757},
issn = {2090-1224},
abstract = {INTRODUCTION: The recent global outbreaks of monkeypox virus (MPXV) and chikungunya virus (CHIKV) underscore the urgent need for rapid, accessible, and cost-effective diagnostic methods. Conventional CRISPR/Cas fluorescence assays rely on trans-cleavage of ssDNA/RNA reporters labeled with expensive fluorophores and quenchers, which limits widespread application.<br><br>OBJECTIVES: This study aims to develop and optimize a label-free, fluorogenic DNA aptamer-based reporter for a portable, one-pot Cas12a detection system capable of highly sensitive detection of MPXV and CHIKV directly from clinical specimens.<br><br>METHODS: We evaluated commonly used ssDNA aptamers for their fluorescence emission upon Thioflavin T (ThT) binding and their cleavage efficiency by Cas12a. Through systematic mutagenesis targeting G-rich regions, we enhanced fluorescence emission. Additionally, poly-A linkers were introduced between G-rich motifs to promote Cas12a cleavage efficiency. Circular dichroism (CD) spectroscopy confirmed G-quadruplex (G4) formation in the aptamers. The assay's sensitivity and specificity were assessed using simulated clinical samples, followed by validation with actual clinical specimens. The performance of direct detection from simulated clinical samples was compared to qRT-PCR. A battery-powered heating-pad, a mini-centrifuge, and a flashlight were used to validate its POCT applicability.<br><br>RESULTS: We designed and optimized a cost-effective, stable fluorogenic ssDNA aptamer that specifically binds to ThT. The aptamer ThT-3-5.1 exhibited the highest fluorescence enhancement and cleavage efficiency by Cas12a. Leveraging this aptamer, we developed a rapid, portable, one-pot detection platform (ROD-ThT) capable of detecting as few as 1 copy/reaction of MPXV and CHIKV nucleic acids within 35&#xa0;min. Validation with clinical samples confirmed the assay's reliability without the need for nucleic acid purification.<br><br>CONCLUSION: Our simple, efficient, portable, and affordable ROD-ThT platform holds great promise for disease diagnostics and management, particularly in resource-limited settings.},
}
@article {pmid42399052,
year = {2026},
author = {Kim, J and Kovacs, H and Wisnovsky, S},
title = {CRISPR screens to identify and characterize ligands for glycan-binding proteins.},
journal = {Methods in enzymology},
volume = {732},
number = {},
pages = {219-263},
doi = {10.1016/bs.mie.2026.03.005},
pmid = {42399052},
issn = {1557-7988},
mesh = {Ligands ; *Polysaccharides/metabolism ; Humans ; *Lectins/metabolism/genetics ; Flow Cytometry/methods ; *CRISPR-Cas Systems ; Protein Binding ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cell surface glycans regulate key biological processes including immune signaling, cell communication, and pathogen recognition. Glycan-driven signaling is primarily mediated by glycan-binding proteins (lectins), whose functions depend on the identity and presentation of their glycoprotein ligands. However, identifying ligands for glycan-binding proteins remains challenging due to the structural complexity of carbohydrates and the importance of cellular context in determining binding specificity. Here, we describe a fluorescence-activated cell sorting (FACS)-based pooled CRISPR screening workflow for systematic identification of genetic factors that regulate lectin binding in living cells. The protocol covers lentiviral transduction of pooled sgRNA libraries and phenotypic selection of high- and low-lectin-binding populations by flow cytometry. Genomic DNA extraction, sequencing, and computational sgRNA enrichment analysis enable identification of genes influencing ligand biosynthesis and presentation. Subsequent analysis of these genetic factors can provide a comprehensive view of the structural determinants that govern lectin-glycan binding. The approach is compatible with CRISPR knockout, interference, and activation strategies, allowing broad interrogation of both loss- and gain-of-function effects. Key considerations for maintaining library coverage, optimizing sorting parameters, and performing robust statistical analysis are highlighted to maximize screening performance. Overall, this workflow offers a scalable framework for mapping glycan ligand landscapes in health and disease.},
}
@article {pmid42399613,
year = {2026},
author = {Wei, R and Wang, S and Li, Y and Li, N and Pan, W and Tang, B},
title = {DNAzyme-Enhanced CRISPR/Cas12a Cascade Enables Isothermal, One-Pot RNA Diagnostics.},
journal = {ACS applied materials &amp; interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.6c05456},
pmid = {42399613},
issn = {1944-8252},
abstract = {While integrating DNAzymes with Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems offers a promising route to enhance CRISPR/Cas12a-based molecular diagnosis via enzyme-coupled cascade amplification, their implementation in simple, specific, and sensitive nucleic acid detection remains challenging, largely due to reliance on complex, multistep workflows. Here, we report an RNA-triggered DNAzyme circuit integrated with CRISPR/Cas12a that serves as a universal nucleic acid preamplifier, enabling one-pot and homogeneous detection. The catalytic activity of DNAzyme, initially suppressed by a complementary blocker strand, was restored upon the recognition of the target analyte. The activated DNAzyme then cleaved a hairpin-shaped substrate, liberating multiple activators that triggered a secondary CRISPR/Cas amplification reaction. This cascade generated a visible red band signal on a lateral flow assay via the collateral cleavage of a reporter. By employing the DNAzyme as a signal amplifier, the system efficiently converted a single RNA molecule into numerous initiators, breaking the one-to-one activation relationship between the target and Cas12a ribonucleoprotein and thereby greatly enhancing the detection sensitivity. Additionally, the system exhibited high programmability and universality, as a biosensor for a given target could be easily constructed by simply customizing the corresponding region of the blocker strand that is complementary to the target sequence. This integrated cascade system enables efficient signal amplification within a simple one-pot format and holds significant promise for practical applications.},
}
@article {pmid42400295,
year = {2026},
author = {Liang, D and Guo, H and Wei, J and Zhu, F and Zhang, Y and Green, J and Ji, Y and Jin, H and Zhang, X and Gui, H and Dan, H and Liu, Y and Zhang, Y and Wang, H and Jiang, Y and Geng, L and Lv, J and Cai, W and Song, W and Kelliher, T and Chen, X and Egger, R},
title = {A Robust Framework for Maize Elite Line Genome Editing Through Enhanced HI-Edit via LbCas12a Activity Optimization.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70715},
pmid = {42400295},
issn = {1467-7652},
abstract = {Haploid induction coupled with genome editing (HI-Edit) enables direct modification of commercial crop varieties, bypassing the need for trait introgression or direct transformation of elite lines with CRISPR machinery. However, its widespread application has been constrained by low haploid editing rates (HER), the proportion of haploids carrying edits within the short window between double fertilization and uniparental chromosome elimination. Here, we report substantial improvements in maize HI-Edit efficiency through three complementary strategies: (1) driving an optimized LbCas12a variant (LbCas12aV) using promoters that are highly active in sperm cells and early zygotes; (2) applying a post-pollination heat treatment; and (3) fusing LbCas12aV with the UBA2 domain (ubiquitin-associated domain-2 of Arabidopsis thaliana RAD23) to enhance protein stability during haploid induction. Post-pollination heat treatment alone increased HER to 19.1% (up to 12-fold improvement depending on the target site), providing a simple and effective method to boost the yield of edited doubled haploid (DH) plants. UBA2 fusion improved HER by 6-fold at the Waxy1 (Wx1) locus and 4.5-fold at the Glossy2 (Gl2) locus under normal conditions. Strikingly, combining UBA2 fusion with heat treatment raised the average HER to 25% across multiple events targeting Wx1, with the highest HER reaching 33%. Collectively, these findings demonstrate that increasing CRISPR-Cas protein abundance and modulating environmental conditions can overcome key bottlenecks in HI-Edit. We establish a robust, scalable framework that is readily transferable to other crops for elite-line genome editing.},
}
@article {pmid42400407,
year = {2026},
author = {Wan, L and Zhou, J and Yu, L and Huang, X and Fu, J and Xiao, F and Jia, N and Zhang, Y and Chen, M and Feng, Z and Wang, Y},
title = {A One-Pot CRISPR-Cas12b Assay for Rapid Detection of Human Adenovirus Serotypes 3 and 7.},
journal = {Journal of medical virology},
volume = {98},
number = {7},
pages = {e71032},
doi = {10.1002/jmv.71032},
pmid = {42400407},
issn = {1096-9071},
support = {7262007//Beijing Natural Science Foundation/ ; L234047//Beijing Natural Science Foundation-Changping Innovation Joint Fund Project/ ; XZDX-2025-002//Research Foundation of Capital Institute of Pediatrics/ ; BJRID2025-009//Beijing research center for respiratory infectious diseases project/ ; BJRID2026-011//Beijing research center for respiratory infectious diseases project/ ; 2024-0040//Pathogen spectrum and host marker analysis in respiratory tract infection of children/ ; MEKLCEPP/SXMU-202412//Key Laboratory of Coal Environmental Pathogenicity and Prevention (Shanxi Medical University), Ministry of Educatio/ ; },
mesh = {Humans ; *Adenoviruses, Human/isolation &amp; purification/genetics/classification ; *CRISPR-Cas Systems ; *Adenovirus Infections, Human/diagnosis/virology ; Serogroup ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; China ; *Molecular Diagnostic Techniques/methods ; Respiratory Tract Infections/virology/diagnosis ; },
abstract = {Human adenovirus (HAdV) is a leading cause of acute respiratory tract infections (ARTIs) in children. The high prevalence of HAdV serotypes 3 and 7 in regions such as China presents a significant public health challenge. Here, we propose a one-pot assay that integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b for the detection of HAdV-3 and HAdV-7, termed HAdV-MCDA-One. In this system, MCDA provides exponential target amplification, while the collateral cleavage activity of Cas12b enables secondary signal amplification. The entire reaction is performed isothermally at 60°C in a single tube, providing a fluorescent readout within 50 min, making the assay suitable for point-of-care testing (POCT). Leveraging the single-base recognition capability of CRISPR-Cas12b, the assay demonstrates high specificity, with no cross-reactivity observed against the other 13 identified pathogens. The limit of detection was determined to be 1.59 copies per reaction using target plasmids. Moreover, when evaluated with 96 clinical pharyngeal swabs, the assay showed 100% concordance with quantitative PCR (qPCR), confirming its clinical reliability. These results demonstrate HAdV-MCDA-One as a rapid and robust tool for HAdV-3 and HAdV-7 detection, with significant potential for clinical diagnosis and public health surveillance.},
}
@article {pmid42400607,
year = {2026},
author = {Liu, F and Jiang, T and Tanwir, SE and Ardi, WH and Huo, H},
title = {CRISPR/Cas9-mediated DFR disruption suggests coordinated changes in flavonoid flux and development in Petunia × hybrida.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42400607},
issn = {1432-203X},
support = {2019-67013-29236//National Institute of Food and Agriculture/ ; FLA-MFC-006387//USDA HATCH/ ; },
mesh = {*Petunia/genetics/metabolism/growth &amp; development/enzymology ; *Flavonoids/metabolism ; *CRISPR-Cas Systems/genetics ; *Alcohol Oxidoreductases/genetics/metabolism ; Gene Expression Regulation, Plant ; Anthocyanins/metabolism ; *Plant Proteins/genetics/metabolism ; Flowers/metabolism/genetics ; Pigmentation/genetics ; Phenotype ; Plant Leaves/metabolism/genetics ; Plants, Genetically Modified ; Chlorophyll/metabolism ; Carotenoids/metabolism ; Oxidoreductases/metabolism ; },
abstract = {Loss of DFR function in petunia alters pigment metabolism and reduces organ size, suggesting previously underexplored associations among flavonoid biosynthesis, plastidial pigments, and development. Dihydroflavonol 4-reductase (DFR) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase. While DFR's role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated DFR-edited lines in the commercial Petunia × hybrida cultivar 'Carmine Velour'. The edited lines showed a spectrum of floral pigmentation loss that was broadly consistent with the representative editing patterns inferred from Sanger sequencing, supporting the major contribution of DFR-A to corolla anthocyanin accumulation. Beyond pigmentation, dfr mutants exhibited unexpected reductions in floral dimensions (20-40%), leaf biomass (30-50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35-60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, indicating organ-specific associations between DFR disruption and growth-related traits. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: chalcone synthase A (CHSA) and chalcone isomerase A (CHIA) were downregulated while the competing branch enzyme flavonol synthase (FLS) was upregulated almost twofold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A (PORA), encoding a key chlorophyll biosynthetic enzyme, was severely suppressed by 60-75%, suggesting a possible connection between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses suggested coordinated variation, with floral anthocyanin content positively associated with leaf chlorophyll and carotenoid levels across genotypes. These findings support the view that DFR acts as a functionally important metabolic node whose disruption is associated with effects across pigment classes and organ types, with implications for precision trait engineering in floriculture.},
}
@article {pmid42400669,
year = {2026},
author = {Tamrakar, VK and Sharma, K and Singh, P and Bhargava, A and Thakur, P and Negi, SS},
title = {Development of a CRISPR/dCas9-based membrane-assisted colorimetric assay for detection of high-risk HPV16 and HPV18: a proof-of-concept study.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42400669},
issn = {1573-4978},
support = {Extramural Grant IIPR-2030-3960/F1.//Indian Council of Medical Research/ ; },
mesh = {*Colorimetry/methods ; *Human papillomavirus 18/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Human papillomavirus 16/genetics/isolation &amp; purification ; Humans ; *Papillomavirus Infections/diagnosis/virology ; Proof of Concept Study ; Genotype ; Female ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; Genotyping Techniques/methods ; },
abstract = {BACKGROUND AND AIMS: Persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, is the leading cause of cervical cancer. While molecular diagnostics offer high sensitivity, their deployment in decentralized settings remains limited. This study presents a proof-of-concept CRISPR/dCas9-based membrane-assisted detection platform for HR-HPV genotyping.<br><br>METHODS: A membrane-based assay integrating recombinase polymerase amplification (RPA) with CRISPR/dCas9-mediated sequence-specific recognition was developed. FAM-labeled amplicons were captured by immobilized dCas9-sgRNA ribonucleoprotein complexes and detected via antibody-mediated colorimetric readout.<br><br>RESULTS: The assay enabled specific detection of HPV16 and HPV18 using genotype-specific sgRNAs, producing visually interpretable signals on a nitrocellulose membrane. No signal was observed in negative controls, demonstrating high analytical specificity. Semi-quantitative signal assessment confirmed clear differentiation between positive and negative samples.<br><br>CONCLUSION: This study demonstrates the feasibility of a CRISPR/dCas9-based membrane-assisted detection system for HR-HPV genotyping. While not yet configured as a fully integrated lateral flow device, the platform provides a foundation for future development of simplified, point-of-care molecular diagnostics.},
}
@article {pmid41981786,
year = {2026},
author = {Munir, F and Zaheer, U and Asad, M and Yang, G},
title = {Knockout of Ku70 and Ku80 elevates homology-directed repair efficiency in Plutella xylostella.},
journal = {Insect molecular biology},
volume = {35},
number = {4},
pages = {434-447},
doi = {10.1111/imb.70039},
pmid = {41981786},
issn = {1365-2583},
support = {2024L3004//Fujian Province Science and Technology Project/ ; 2024-P-002//Fuzhou-Xiamen-Quanzhou National Independent Innovation Demonstration Zone RNAi Microbial Pesticide Collaborative Innovation Platform Project/ ; 2024I0007//the External Cooperation Project of Fujian Province/ ; KRA16001A//fund of the "111" program/ ; 2023J02009//Fujian Province Natural Science Foundation/ ; W2533068//Overseas Young Researcher Project of National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Moths/genetics/metabolism ; *Ku Autoantigen/genetics/metabolism ; Female ; Male ; Phylogeny ; Amino Acid Sequence ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Sequence Alignment ; },
abstract = {The suppression of Ku70 and Ku80 has been verified to increase homology-directed repair (HDR) efficiency in fruit fly, silkworm and mosquito, but not in other insects. In this study, PxKu70 and PxKu80 were identified from the Plutella xylostella genome. Domain analysis revealed that PxKu70 contained three conserved domains: Ku N, Ku and Ku C, while PxKu80 comprised the Ku N, Ku and Ku PK bind domains. Phylogenetic analysis and multiple-sequence alignment indicated strong conservation of both proteins among lepidopteran insects. RT-qPCR analysis showed that PxKu70 and PxKu80 were highly expressed in adult stages, particularly in reproductive tissues such as the ovary and testis, suggesting their role in maintaining genomic stability during gametogenesis. Two homozygous knockout lines (ΔPxKu70 and ΔPxKu80) were successfully generated through CRISPR/Cas9-mediated genome editing. These knockout lines remained viable and fertile without observable fitness effects. A donor construct carrying an EGFP cassette designed for insertion at the PxKmo locus was generated to assess HDR-mediated integration. The HDR insertion rate was significantly elevated in both knockout lines compared with the wild-type. These findings demonstrate that suppression of either PxKu70 or PxKu80 can enhance HDR in P. xylostella, offering an effective approach for precise genome editing in lepidopteran species.},
}
@article {pmid42062284,
year = {2026},
author = {Sun, J and Noss, S and Smolen, C and Bhavana, VH and Banerjee, D and Das, M and Giardine, B and Prabhu, A and Amor, DJ and Pope, K and Lockhart, PJ and Girirajan, S},
title = {Functional impact of genetic background on variable expressivity in neurodevelopmental disorders.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42062284},
issn = {2041-1723},
support = {R01 GM121907/GM/NIGMS NIH HHS/United States ; R21 NS122398/NS/NINDS NIH HHS/United States ; GM121907//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; NS122398//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {*Neurodevelopmental Disorders/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Forkhead Transcription Factors/genetics/metabolism ; Phenotype ; Neurons/metabolism ; Chromatin/metabolism ; Signal Transduction/genetics ; Neural Stem Cells/metabolism ; Neurodevelopment ; Cell Proliferation ; CRISPR-Cas Systems ; Animals ; },
abstract = {Disease-associated variants can lead to variable phenotypic outcomes in neurodevelopmental disorders, but the biological mechanisms underlying this variability remain poorly understood. Here, we develop a framework to investigate this phenomenon using the 16p12.1 deletion as a paradigm of variable expressivity. Using induced pluripotent stem cell models from affected families and CRISPR-edited lines with the 16p12.1 deletion, we find that the deletion and rare variants in the genetic background jointly influence chromatin accessibility and expression of neurodevelopmental genes. Cellular analyses identify family-specific phenotypes, including altered inhibitory neuron production and neural progenitor cell proliferation, which correlate with head-size variation. CRISPR activation of individual 16p12.1 genes variably rescue these defects by modulating key developmental signaling pathways. Integrative analyses further identify regulatory hubs, including transcription factors FOXG1 and JUN, as mediators of these effects. Our study provides a functional framework for investigating how individual genetic architectures contribute to phenotypic variability in neurodevelopmental disorders.},
}
@article {pmid42091648,
year = {2026},
author = {Chandrasekaran, J and Suthanthiram, B and Selvaraj, EP and Swaminathan, S and Chandran, SA and Ramasamy, S},
title = {Targeting a conserved functional motif in the PDS gene enables efficient CRISPR/Cas9 editing in banana.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42091648},
issn = {2045-2322},
support = {DST/WOS-A/LS-202/2021(G)//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Musa/genetics ; *Oxidoreductases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Base Sequence ; *Gene Editing ; Conserved Sequence ; },
abstract = {Incomplete editing and chimeric phenotypes are major challenges in CRISPR/Cas9-mediated genome editing of polyploid crops. In this study, a single guide RNA (gRNA) was designed to target a conserved dinucleotide-binding motif within exon 3 of the phytoene desaturase (PDS) gene in 'Grand Naine' banana. The gRNA was carefully selected for GC content, guanine residues near the PAM, and predicted secondary structure to enhance Cas9 cleavage efficiency. Agrobacterium-mediated transformation of embryonic cell suspensions produced 102 putative transgenic plants, all exhibiting altered phenotypes, with 91% displaying albino and 9% pale green coloration, indicating efficient PDS gene knockout and absence of chimerism. Sequencing confirmed tri-allelic editing, with all edited plants consistently showing two identical and one distinct mutation. Notably, small in-frame deletions of two to six amino acids within the conserved motif were sufficient to abolish PDS function, confirming its critical role in carotenoid biosynthesis. This strategy is adaptable to clonally propagated polyploid crops, providing a practical framework for achieving high-efficiency, uniform genome edits and supporting the development of precise, non-chimeric CRISPR/Cas9 editing approaches.},
}
@article {pmid42229614,
year = {2026},
author = {Kalmotia, V},
title = {Enhancing statistical accuracy in gene perturbation studies.},
journal = {Bio Systems},
volume = {266},
number = {},
pages = {105819},
doi = {10.1016/j.biosystems.2026.105819},
pmid = {42229614},
issn = {1872-8324},
mesh = {Humans ; *Gene Expression Profiling/methods ; *Single-Cell Analysis/methods ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems/genetics ; Genomics/methods ; },
abstract = {Accurately analysing gene expression changes in high-throughput perturbation studies remains a challenge due to confounding technical factors. This paper evaluates and extends the SCEPTRE (Single-Cell PerTurbation screens via Conditional REsampling) framework, originally introduced by Barry et al. (2021), demonstrating its applicability to high-multiplicity-of-infection (MOI) CRISPR screens. By leveraging a resampling-based methodology, our approach effectively adjusts for sequencing biases, reducing false discoveries while maintaining statistical power.},
}
@article {pmid42335458,
year = {2026},
author = {Yang, L and Zhou, Y and Li, H and Chen, X and Zhang, S and Zhu, L},
title = {An Entropy-Driven Autocatalysis-Regulated Signal-On CRISPR/Cas12a Biosensor Supported by DNA Triangular Prism Scaffold.},
journal = {Analytical chemistry},
volume = {98},
number = {26},
pages = {19636-19651},
doi = {10.1021/acs.analchem.6c01471},
pmid = {42335458},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *DNA/chemistry ; *Entropy ; *MicroRNAs/blood/analysis ; *CRISPR-Cas Systems/genetics ; *alpha-Fetoproteins/analysis ; Humans ; Electrochemical Techniques ; Limit of Detection ; DNA Nanostructures ; Carcinoma, Hepatocellular/diagnosis/blood ; },
abstract = {Accurate and sensitive detection of low-abundance biomarkers in complex matrices remains challenging due to the inherent trade-off between amplification efficiency and background suppression in conventional biosensing strategies. Herein, a synergistic amplification strategy was constructed by integrating an entropy-driven autocatalysis (EDAC), a signal-on CRISPR/Cas12a assay, and a DNA triangular prism (DTP) interface. In this strategy, EDAC achieved exponential signal amplification through the recycling of target molecules and reaction byproducts, and its output strands simultaneously served as specific inhibitors of CRISPR/Cas12a. Based on this mechanism, the signal-on CRISPR/Cas12a assay strictly coupled signal generation to the presence of the target, thereby fundamentally circumventing the high background interference inherent to conventional signal-off modes. As a rigid three-dimensional interfacial scaffold, DTP provided high-density and well-ordered nucleic acid assembly sites, reduced steric hindrance through a solution-like microenvironment, suppressed nonspecific adsorption, and efficiently initiated downstream hybridization chain reaction for robust electrochemical readout via methylene blue intercalation. With hepatocellular carcinoma-associated biomarkers alpha-fetoprotein and microRNA-122 as model targets, the biosensor achieved detection limits as low as 11.37 fg/mL and 18.13 aM, respectively. In clinical serum sample assays, the biosensor showed strong agreement with the classical ELISA method, with an area under the curve value of 1.00, demonstrating its promising potential for the diagnosis of hepatocellular carcinoma. With its modular architecture and adaptable recognition elements, this strategy establishes a versatile framework for ultrasensitive biosensing and holds promise for clinical translation in early disease diagnosis.},
}
@article {pmid42342147,
year = {2026},
author = {Yuan, J and Ma, Y and Li, J and Chen, X and Wen, Y and Wu, R and Hang, X and Huang, H and Du, S and Wang, Y and Yan, QG and Huang, X and Zhao, F and Yi, Z and Cao, SJ and Zhao, Q},
title = {CXCL8 is associated with aflatoxin B1-triggered injury and caspase-3 activation in porcine kidney epithelial PK15 cells: integrated transcriptomics and CRISPR/Cas9 knockout.},
journal = {Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association},
volume = {215},
number = {},
pages = {116236},
doi = {10.1016/j.fct.2026.116236},
pmid = {42342147},
issn = {1873-6351},
mesh = {Animals ; *Aflatoxin B1/toxicity ; Swine ; *Caspase 3/metabolism/genetics ; *Interleukin-8/genetics/metabolism ; *Epithelial Cells/drug effects/metabolism ; Cell Line ; *Kidney/drug effects/cytology/metabolism ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Gene Knockout Techniques ; *Transcriptome/drug effects ; Reactive Oxygen Species/metabolism ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent food- and feed-borne mycotoxin, and growing evidence indicates that the renal epithelium is a vulnerable target. However, host determinants that modify epithelial susceptibility remain poorly defined. Here, we investigated AFB1-triggered injury in porcine kidney epithelial PK15 cells and examined the contribution of CXCL8. PK15 cells were exposed to AFB1 (0-32 μM) to define dose-dependent cytotoxicity, and sub-IC50 conditions (4 and 8 μM for 24 h) were used for mechanistic analyses; RNA sequencing at 4 μM for 24 h was used as an exploratory screen to prioritize candidate susceptibility factors. CXCL8 emerged as the most strongly induced transcript and was subsequently evaluated using CRISPR/Cas9-mediated knockout. CXCL8 deficiency attenuated AFB1-induced loss of viability, reduced Annexin V/PI-positive cells, and alleviated mitochondrial ultrastructural injury. In parallel, CXCL8 knockout decreased ROS accumulation, partially restored intracellular GSH and the BCL2/BAX transcriptional ratio, and reduced caspase-3 induction and cleavage. Collectively, these data support CXCL8 as an AFB1-inducible susceptibility factor associated with oxidative stress amplification and caspase-3 activation in PK15 cells, while indicating that the upstream regulatory axis and the precise downstream signaling route require further validation in physiologically relevant renal models.},
}
@article {pmid42387926,
year = {2026},
author = {Van Heurck, R and Hammar, E and Marconi, C and Korff, C and Abramowicz, M},
title = {[Targeted therapy for ultra-rare diseases].},
journal = {Revue medicale suisse},
volume = {22},
number = {969},
pages = {1216-1220},
doi = {10.53738/REVMED.2026.22.969.48835},
pmid = {42387926},
issn = {1660-9379},
mesh = {Humans ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Oligonucleotides, Antisense/administration &amp; dosage ; *Molecular Targeted Therapy/methods ; Gene Therapy Agents ; CRISPR-Cas Systems ; },
abstract = {Nano-rare diseases, affecting fewer than 30 individuals worldwide, are mostly genetic and severe, with no effective treatments available. Nucleic acid-based therapies, such as antisense oligonucleotides, allow for the targeted modulation of gene expression. Successes like nusinersen and ultrapersonalized treatments (for example, Milasen) highlight their potential. Other approaches, including viral gene therapy and CRISPR-Cas9, enable the addition or correction of genes. However, major challenges remain, including high costs, difficulties in conducting clinical trials, and inadequate regulatory frameworks, especially for "N-of-1" therapies. International initiatives are emerging to facilitate access to these innovative treatments in Europe and Switzerland.},
}
@article {pmid42388033,
year = {2026},
author = {Sultana, H and Mohanty, S and Solomon, AD and Iqbal, MY and Wani, AK and Kumar, V and Khattri, A},
title = {AI in Genomics: From Variant Calling to Multi-Omics Integration.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {48},
number = {7},
pages = {e70160},
doi = {10.1002/bies.70160},
pmid = {42388033},
issn = {1521-1878},
mesh = {Multiomics ; *Genomics/methods ; *Artificial Intelligence ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Machine Learning ; },
abstract = {Artificial intelligence (AI) strategies are revolutionizing genomics by extracting complex patterns that traditional statistical pipelines are likely to miss. This mini-review aims to provide a concise overview of how AI is transforming major genomic technologies including variant calling, gene expression analysis, single-cell transcriptomics, CRISPR-Cas9 optimization, and multi-omics integration. In genome sequencing, machine learning variant callers greatly improve the accuracy and the rate at which single nucleotide and structural variants are called. In bulk RNA-Seq, AI augmented quantification, denoising, and differential expression modules complement the highly established STAR-featureCounts-DESeq2 pipeline, revealing subtle signals in big data sets. In single cell transcriptomics, deep learning approaches enhance batch correction, automate cell type annotation, and track developmental trajectories, hence clarifying cellular heterogeneity. AI-assisted guide RNA design, outcome prediction, and nuclease engineering enable more efficient CRISPR-Cas9 editing, reducing experimental cycles, and off-target effects. Finally, integrated platforms that combine genomic, transcriptomic, epigenomic, proteomic, and metabolomic layers provide an integrative view of cellular regulation and disease mechanisms. The review also covers current limitations, sparsity of data, model bias, privacy, and the need for standardized benchmarks and offers future directions in the form of interpretable models, collaborative learning, and open science practices. Together, these developments render AI an indispensable partner to unravel genomic complexity and accelerate precision medicine applications.},
}
@article {pmid42389571,
year = {2026},
author = {Kohabir, KAV and Rietveld, AWJ and Nooi, LO and Beijer, RE and van Dongen, JE and Linthorst, J and Wolthuis, RMF and Jonges, M and Welkers, MRA and Segerink, LI and Sistermans, EA},
title = {Toward point-of-care and amplification-free detection of human cytomegalovirus using CRISPR-Cas12a.},
journal = {iScience},
volume = {29},
number = {7},
pages = {116494},
pmid = {42389571},
issn = {2589-0042},
abstract = {Human cytomegalovirus (hCMV) is a herpesvirus that establishes lifelong latency in myeloid cells, posing health concerns particularly in fetal development and in immunocompromised individuals. Point-of-care (PoC) detection of hCMV DNA in liquid biopsies supports timely diagnosis and proper mitigation. However, ultra-low concentrations and high fragmentation rates, challenge primer-based preamplification methods. We present a proof-of-concept amplification-free CRISPR-based assay, exploiting the inherent specificity and signal-amplification of Cas12a and improving signal using a combinatorial approach. Optimizing Cas12a's trans-cleavage activity and multiplexing hCMV loci, significantly increased detection sensitivity in-bulk. Additionally, we found that AsCas12a trans-cleaves cytosine-rich reporters 4× more efficiently than conventional probes, further improving assay kinetics to reach a femtomolar limit of detection. Translating these optimizations to a microfluidic assay enables sensitive detection even if additional measures may be needed for quantitative, single molecule measurements. Our assay opens avenues toward PoC detection in low-resource settings, supporting effective and affordable infection management.},
}
@article {pmid42390428,
year = {2026},
author = {Shinoda, H and Makino, A and Yoshimura, M and Iida, T and Yamazaki, D and Minagawa, N and Kogo, Y and Sano, T and Jinnai, M and Hishiki, T and Ohya, H and Nishimasu, H and Watanabe, R},
title = {Single-Molecule Characterization of CRISPR-Cas12a for Amplification-Free Genetic Testing.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c00936},
pmid = {42390428},
issn = {1520-6882},
abstract = {CRISPR-Cas-based genetic testing has gained considerable attention for rapid and accurate diagnosis of infectious and genetic diseases. Cas12a, an RNA-guided nuclease that targets DNA, has been widely applied for DNA detection; however, its detailed enzymatic properties have not been fully elucidated. Here, we performed a systematic single-molecule analysis of Cas12a using microchamber arrays to biophysically characterize its catalytic behavior including protospacer adjacent motif (PAM) specificity, mismatch tolerance, and reaction kinetics and to evaluate the feasibility and inherent challenges of amplification-free DNA detection. Cas12a exhibited high activation efficiency toward target DNA, demonstrating the feasibility of amplification-free DNA detection with a detection limit of 104 aM within 15 min. However, Cas12a also exhibited substantial nonspecific cross-reactivity with genome-length targets, particularly mammalian genomic DNA, identifying off-target activation as a major challenge for reliable diagnostic application. Together, these findings provide a quantitative biophysical characterization of Cas12a-based amplification-free DNA detection and highlight the need to improve its target specificity for future reliable application of Cas12a in amplification-free molecular diagnostics.},
}
@article {pmid42390463,
year = {2026},
author = {Mir, YB and Manzoor, T and Mushtaq, D and Najar, AH and Kawoosa, F and Bhatia, D and Qadri, SM and Siraj, F and Ahmad, SM},
title = {Diagnostic challenges in re-emerging rickettsioses: why current tools fall short.},
journal = {Clinical microbiology reviews},
volume = {},
number = {},
pages = {e0002026},
doi = {10.1128/cmr.00020-26},
pmid = {42390463},
issn = {1098-6618},
abstract = {SUMMARYRickettsial diseases, encompassing scrub typhus, spotted fever group rickettsioses, and typhus group rickettsioses, represent a significant and escalating public health threat worldwide, particularly in the Asia-Pacific and sub-Saharan African regions. Despite their high morbidity and potential for fatal outcomes if left untreated, these infections remain notoriously underdiagnosed due to their nonspecific clinical presentation, which frequently overlaps with other acute undifferentiated febrile illnesses (AUFIs) such as dengue, malaria, and leptospirosis. This review evaluates the evolving diagnostic landscape, highlighting the severe limitations of conventional methods: the Weil-Felix test lacks necessary specificity, while the gold standard indirect immunofluorescence assay (IFA) is primarily retrospective due to delayed seroconversion. Molecular diagnostics, particularly multiplex polymerase chain reaction (mPCR), have emerged as a critical advancement, enabling early, species-specific identification during the acute phase of infection when doxycycline therapy is most effective. We further explore the paradigm shift toward syndromic molecular panels, such as the TaqMan Array Card (TAC), which facilitate simultaneous screening for multiple AUFI pathogens. Emerging platforms, including digital PCR (dPCR) for absolute quantification and CRISPR-Cas-based point-of-care (POC) systems (SHERLOCK and DETECTR), offer promising solutions for low-resource settings. Finally, this review underscores the necessity of integrating molecular surveillance within a One Health framework and utilizing artificial intelligence (AI) to address technical and implementation barriers. Overcoming these challenges is essential for transforming rickettsial diagnosis from a reactive to a proactive strategy, ultimately reducing the global burden of these neglected zoonoses.},
}
@article {pmid42391043,
year = {2026},
author = {Lin, S and Jiang, S and Tang, L and Kumbhakonam, SK and Dingman, JC and Lee, JY and Yang, R and Frudakis, T and Kirchner, M and Xu, S and Tao, C and Wang, X and Russo, JJ and Zhang, X and Chen, Q and Zhang, S},
title = {De novo direct sequencing of small therapeutic RNAs by layer-by-layer intensity-resolved mass spectrometry.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42391043},
issn = {1362-4962},
support = {R01 HG012853/HG/NHGRI NIH HHS/United States ; R41 HG013624/HG/NHGRI NIH HHS/United States ; R41 HG014125/HG/NHGRI NIH HHS/United States ; U24 HG011735/HG/NHGRI NIH HHS/United States ; RM1 HG011563/HG/NHGRI NIH HHS/United States ; R01 HD092431/HD/NICHD NIH HHS/United States ; R01 ES032024/ES/NIEHS NIH HHS/United States ; },
mesh = {*Sequence Analysis, RNA/methods ; *High-Throughput Nucleotide Sequencing/methods ; *RNA, Small Interfering/genetics/chemistry/therapeutic use ; MicroRNAs/genetics/chemistry ; Algorithms ; *Mass Spectrometry/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; },
abstract = {The rapid growth of RNA-based therapeutics demands accurate sequencing of all RNA species, including minor and modified variants. Conventional LC-MS/MS typically confirms only a predefined target sequence rather than determining RNA sequences de novo from the analyzed sample, thereby overlooking coexisting impurities and modifications. Here, we present 3D NGMS-Seq, a three-dimensional next-generation mass spectrometry-based sequencing platform for de novo direct sequencing of mixed RNA samples with essentially 100% sequence accuracy. This method incorporates MS intensity into traditional 2D mass-retention time (tR) analysis and introduces a nested algorithm that aligns ladder fragment intensities with parent RNA abundances for computational separation. Controlled acid hydrolysis produces RNA ladder fragments, which are segregated into mass-intensity-tR layers. Within each layer, short reads are generated de novo by sequentially base-calling each nucleotide, canonical or modified, from mass differences between adjacent ladder fragments and subsequently assembled into full-length RNA sequences. Guided by hydrolysis kinetics and statistical modeling, 3D NGMS-Seq accurately sequences synthetic siRNA, miRNA, and CRISPR/Cas9 sgRNAs, reveals unexpected low-abundance RNA impurities, and resolves subtle methylation ambiguities (Um versus mU; Am versus mA), while providing a quantitative profile of each RNA's relative abundance and site-specific modifications. By enabling direct, unbiased sequencing of heterogeneous RNAs without prior sequence knowledge, 3D NGMS-Seq addresses key limitations of current RNA analysis and provides a powerful tool to aid small RNA drug development, quality control, and regulatory validation.},
}
@article {pmid42392238,
year = {2026},
author = {Chen, S and Pi, C and Zhang, B and Wu, X and Gao, L and Chen, X},
title = {Technology-driven revolution in CO2 fixation: From natural pathways to programmable Biosystems.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108964},
doi = {10.1016/j.biotechadv.2026.108964},
pmid = {42392238},
issn = {1873-1899},
abstract = {The escalating atmospheric CO2 concentration, exceeding 430 ppm since the pre-industrial era, presents a critical threat to global climate stability. Moving beyond mere carbon capture, this review synthesizes cutting-edge advancements in technology-driven CO2 fixation, focusing on microbial conversion systems. It begins by examining inherent limitations of natural pathways like the Calvin-Benson-Bassham cycle, constrained by low energy efficiency (<1%) and enzymatic inefficiencies of RuBisCO. The discussion then progresses to engineering native pathways and de novo design of synthetic routes (e.g., rGly, CETCH, THETA cycles), which demonstrate superior thermodynamic and kinetic properties for efficient carbon conversion. CRISPR-Cas systems' revolutionary impact, overcoming genetic barriers in carbon-fixing microorganisms. These tools enable precise metabolic rewiring and conversion of heterotrophic chassis into synthetic autotrophs. Furthermore, the convergence of microbiology with electrochemistry and materials science is detailed, highlighting innovative platforms like microbial electrosynthesis and semi-artificial photosynthetic systems. These biohybrid technologies create synergistic interfaces where microbes utilize electrons from electrodes or artificial materials to drive efficient CO2 reduction into multicarbon compounds, addressing critical energy supply challenges. The review analyzes the transition from natural pathway optimization to custom artificial system construction, underscoring a paradigm shift from isolated improvements to deeply integrated approaches. This new paradigm fuses metabolic engineering, synthetic biology, electrochemistry, and nanomaterials, guided by AI-aided design and modeling. The conclusion emphasizes that seamless integration of microbial capabilities, advanced materials, and artificial intelligence is pivotal for advancing CO2 fixation toward precision, high efficiency, and carbon negativity, laying the essential foundation for sustainable carbon-negative biomanufacturing and contributing meaningfully to global carbon neutrality goals.},
}
@article {pmid42392979,
year = {2026},
author = {Pant, DC and Lone, MA and Parameswaran, J and Ma, F and Ziak, N and Dutta, P and Wang, Z and Pun, D and Verma, S and Hornemann, T and Jiang, J},
title = {Deletion of exon 2 in ALS-linked Sptlc1 causes lethality in homozygous mice but not in heterozygotes.},
journal = {Life science alliance},
volume = {9},
number = {9},
pages = {},
pmid = {42392979},
issn = {2575-1077},
mesh = {Animals ; *Amyotrophic Lateral Sclerosis/genetics ; *Exons/genetics ; Mice ; Heterozygote ; Homozygote ; Disease Models, Animal ; Humans ; *Sequence Deletion/genetics ; Mutation ; Genes, Lethal ; CRISPR-Cas Systems ; Male ; Gene Knock-In Techniques ; },
abstract = {Mutations in the human SPTLC1 gene have recently been linked to early-onset amyotrophic lateral sclerosis (ALS), characterized by global atrophy, motor impairments, and symptoms such as tongue fasciculations. All known ALS-linked SPTLC1 mutations cluster within exon 2, and a specific variant, c.58G>T, results in exon 2 skipping. However, it is unclear how the exon 2 deletion affects SPTLC1 function in vivo and contributes to ALS pathogenesis. Leveraging the high genomic sequence similarity between mouse and human SPTLC1, we created a novel knock-in mouse model with a CRISPR/Cas9-mediated deletion of exon 2 in the endogenous murine Sptlc1 locus. Although heterozygous mice did not develop motor defects or ALS-like neuropathology, homozygous mutants died prematurely. These findings provide valuable insights into SPTLC1 exon 2 biology and serve as a useful resource for future mechanistic studies.},
}
@article {pmid42140622,
year = {2026},
author = {Lin, YC and Lee, YJ and Li, CX and Chang, HC and Kowalczyk, M and Haque, M and Dragoiescu, A and Losier, TT and Taylor, JA and Rousseaux, MWC and Huang, WH},
title = {CRISPR screening identifies TRIM27 as a destabilizer of the Smith-Magenis syndrome protein RAI1.},
journal = {Genetics},
volume = {233},
number = {3},
pages = {},
doi = {10.1093/genetics/iyag121},
pmid = {42140622},
issn = {1943-2631},
support = {RGPIN-2020-04094//Natural Sciences and Engineering Research Council of Canada Discovery/ ; //Canada First Research Excellence Fund/ ; //Fonds de recherche du Qu&#xe9;bec awarded/ ; //McGill University/ ; },
mesh = {Humans ; Animals ; *Smith-Magenis Syndrome/genetics/metabolism/pathology ; Mice ; Ubiquitination ; *Trans-Activators/metabolism/genetics ; Protein Stability ; *Transcription Factors/metabolism/genetics ; HEK293 Cells ; Neurons/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Proteolysis ; *DNA-Binding Proteins/metabolism/genetics ; },
abstract = {The nervous system is highly sensitive to alterations in the dosage of genes crucial for neurodevelopment, as exemplified by retinoic acid-induced 1 (RAI1). A 50% change in RAI1 gene copy number, resulting in either reduced or increased protein levels, leads to distinct neurodevelopmental disorders. RAI1 haploinsufficiency causes Smith-Magenis syndrome (SMS), whereas RAI1 duplication underlies Potocki-Lupski syndrome. We recently demonstrated that restoring Rai1 levels can improve SMS-related disease phenotypes in mice. However, despite protein stability being a major determinant of protein abundance, there are currently no therapeutic approaches to modulate RAI1 protein stability. Here, we performed a forward CRISPR screen in human cells to identify post-translational regulators of RAI1 steady-state levels and identified tripartite motif-containing 27 (TRIM27) as a destabilizer of RAI1. We show that RAI1 degradation occurs primarily through the ubiquitin-proteasome system, with TRIM27 interacting with RAI1 and enabling TRIM27-dependent lysine(K)48- and K63-linked RAI1 ubiquitination. Finally, in SMS mouse primary neurons, we demonstrate that knocking down TRIM27 partially rescues SMS-associated morphological phenotypes. Our findings provide the first mechanistic insight into RAI1 proteostasis and highlight TRIM27 as a potential therapeutic target for SMS, highlighting the potential of manipulating ubiquitin-mediated proteostasis to restore gene dosage altered by copy number variations.},
}
@article {pmid42382580,
year = {2026},
author = {Pan, S and Wang, X and He, J and Peng, N},
title = {RecN: A tunable switch for DNA repair choice and stress tolerance in Zymomonas mobilis.},
journal = {Biodesign research},
volume = {8},
number = {2},
pages = {100088},
pmid = {42382580},
issn = {2693-1257},
abstract = {Engineering polyploid industrial microorganisms is hindered by their intrinsic capacity to repair induced mutations, limiting the efficiency of genome editing and directed evolution. Using the ethanologenic bacterium Zymomonas mobilis- a polyploid alphaproteobacterium that exhibits exceptionally efficient microhomology-mediated end joining (MMEJ)- we demonstrate that RecN is essential for MMEJ and homologous recombination (HR) in vivo. Strikingly, a specialized mutant RecN-K35A, with strongly impaired ATP hydrolysis, specifically blocks MMEJ while leaving HR fully intact. The physiological importance of RecN-mediated MMEJ is highlighted by the cell elongation phenotype and increased stress sensitivity observed in the RecN-K35A mutant. Based on this connection, we developed a high-phosphorus cultivation strategy that increases cellular DNA content and significantly enhanced ethanol fermentation efficiency under industrial stress conditions. In summary, this work defines RecN as a key ATP-dependent effector of MMEJ and positions it as a potential engineering target for modulating DNA repair pathway choice and stress tolerance in Z. mobilis. Moreover, the essential role of RecN in both HR and MMEJ suggests that RecN-deficient polyploid strains could facilitate directed evolution by preventing repair of newly introduced mutations, offering a new strategy for strain improvement.},
}
@article {pmid42385699,
year = {2026},
author = {Pandey, S and Burman, N and Henriques, WS and Wiegand, T and Zahl, T and Nyquist, H and Spreeuw, T and Buyukyoruk, M and Wiedenheft, B},
title = {Identification and structure determination of a type III-Bv CRISPR complex that post-translationally modifies an associated toxin.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2026.06.002},
pmid = {42385699},
issn = {1878-4186},
abstract = {Cas7-family proteins form the scaffolds of multi-subunit CRISPR RNA-guided surveillance complexes. To explore how Cas7 diversification expands CRISPR function, we identified Cas7 fusion proteins linked to diverse accessory domains, including a type III-B variant (III-Bv) in which a Cas7 homolog (Cmr1) is fused to the MntA antitoxin and encoded adjacent to a HEPN-family toxin. Structures reveal that the core Cas proteins assemble into a stable surveillance complex in the absence of crRNA, whereas incorporation of the Cmr1-MntA fusion is crRNA-dependent. Target RNA recognition triggers conformational changes that expose the Cas10 cyclase active site and promote cyclic oligoadenylate synthesis. Biochemical analyses show that the CRISPR-associated MntA is enzymatically active and AMPylates the associated HEPN protein. Together, these findings establish the structural basis for assembly of a type III-Bv surveillance complex containing an enzymatically active toxin-antitoxin module.},
}
@article {pmid42387043,
year = {2026},
author = {Dalal, B and Reena, R and Baloda, A and Saini, S and Sharma, P and Sharma, A},
title = {Nitric oxide and abscisic acid: two intimate collaborators regulating plant defense against drought.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {42387043},
issn = {1615-6102},
abstract = {Drought represents one of the most pervasive and intensifying abiotic stresses under changing climate regimes severely constraining agricultural productivity, ecosystem stability, and global food security. Water deficit disrupts cellular homeostasis, reduces photosynthetic efficiency, and induces excessive accumulation of reactive oxygen species (ROS), resulting in oxidative damage. To survive under such conditions, plants employ a diverse array of adaptive responses, including osmotic adjustment, antioxidant defense, hormonal signalling, and stress-responsive gene regulation. Among the key signalling molecules involved in drought tolerance, nitric oxide (NO) and abscisic acid (ABA) have emerged as pivotal signalling molecules orchestrating a wide spectrum of physiological and molecular responses under drought. NO functions as a versatile signalling molecule that regulates redox homeostasis, enhances antioxidant activity, and promotes the accumulation of osmoprotectant. ABA maintains drought perception by inducing stomatal closure, and activating stress-responsive pathways. Co-application of NO and ABA regulates seed germination, root-shoot growth, and stomatal movement, thereby improving relative water content (RWC), membrane stability index (MSI), and photosynthetic efficiency while reducing oxidative stress markers such as malondialdehyde (MDA) and hydrogen peroxide (H2O2). This comprehensive review navigates through a clear and integrative overview of the mechanistic role of NO and ABA, and at the molecular level, NO and ABA modulate drought tolerance through transcriptional regulation, mRNA-level control, and translational modification of stress-responsive genes. Additionally, emerging strategies, including plant-growth promoting rhizobacteria (PGPR), marker-assisted selection (MAS) with QTL mapping, and genome editing tools such as CRISPR/Cas systems, offer promising approaches for enhancing drought tolerance and developing climate-resilient crop varieties.},
}
@article {pmid42387193,
year = {2026},
author = {Xu, H and Hu, X and Chen, R and Huang, S and Wang, L and Yu, L and Li, X and Zhu, X},
title = {Triple-amplification electrochemiluminescence aptasensor integrating single-atom nanozyme catalysis with CRISPR-Cas12a/HCR cascade for zearalenone detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {7},
pages = {},
pmid = {42387193},
issn = {1436-5073},
support = {81773894//NSFC/ ; 2023J01152//Natural Sciences Foundation of Fujian Province/ ; },
mesh = {*Zearalenone/analysis/chemistry/blood ; *Electrochemical Techniques/methods ; Luminescent Measurements/methods ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; *CRISPR-Cas Systems ; Catalysis ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; DNA/chemistry ; Electrodes ; },
abstract = {Single-atom nanozymes (SANs) are emerging as interfacial catalysts that can modulate surface-confined reactive oxygen species (ROS) generation at the electrode/solution boundary. Herein, an interface-driven electrochemiluminescence (ECL) aptasensor was constructed for the ultrasensitive detection of zearalenone (ZEN), a mycotoxin of significant concern. Oxidase-mimetic Co-N/C SANs (Co-SAC@NC) immobilized on a glassy carbon electrode catalyze the reduction of dissolved O2 to ·OH and O2·[-], followed by in-situ generation of ROS, producing a 13-fold enhancement of luminol ECL without the addition of external H2O2. A ferrocene-labelled reporter DNA (Fc-DNA) tethered to the Co-SAC@NC surface quenches luminol ECL by trapping holes at the electrode interface; upon target binding, a magnetic-bead-supported HCR-CRISPR-Cas12a cascade is triggered, trans-cleaving the Fc-DNA and thus restoring the native ECL intensity. The concentration-dependent interfacial cleavage affords a linear range of 0.3-200 ng/mL and a LOD of 0.087 ng/mL (S/N = 3) for the determination of ZEN. This work establishes a modular interfacial amplification platform with potential for generalization by marrying SANs catalysis with a CRISPR-Cas12a/HCR nucleic acid cascade for advanced ECL bioanalysis.},
}
@article {pmid42387220,
year = {2026},
author = {Yaseen, Z and M, M},
title = {Overcoming Immunological Barriers in MSC-Derived Insulin-Producing Cells through CRISPR-Based Hypoimmunogenic Engineering and Translational Perspectives for Type 1 Diabetes.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {42387220},
issn = {2629-3277},
abstract = {Mesenchymal stromal cell (MSC)-derived insulin-producing cells (IPCs) represent an emerging strategy for β-cell replacement in type 1 diabetes mellitus (T1DM) owing to their differentiation potential, intrinsic immunomodulatory properties, and lower tumorigenic risk compared with pluripotent stem cell-derived platforms. However, accumulating evidence indicates that differentiation-associated immunogenicity, context-dependent immune recognition, and recurrent autoimmune responses may substantially limit long-term graft survival and therapeutic durability following transplantation. This review critically examines the immunological barriers associated with MSC-derived IPCs, including altered MHC expression, susceptibility to alloimmune and autoimmune-mediated rejection, and potential reactivation of autoreactive immune memory. We discuss the application of CRISPR-based hypoimmunogenic engineering strategies targeting antigen presentation pathways, NK-cell activation, and immune checkpoint modulation to generate more immune-evasive MSC-derived IPCs while preserving β-cell functionality. By integrating insights from T1DM immunopathogenesis, MSC biology, genome editing, and translational immunology, we propose a framework linking immune engineering with controlled differentiation, functional maturation, and long-term safety evaluation. In parallel, we comparatively position MSC-derived IPCs alongside clinically advancing iPSC-derived β-cell platforms to highlight their distinct translational niche, including potential advantages related to safety, immunomodulatory capacity, manufacturing accessibility, and scalability, while acknowledging the superior functional maturity and clinical progression currently demonstrated by iPSC-derived systems. Finally, we discuss key translational challenges, including genomic stability, immune-evasion durability, GMP-compliant manufacturing, and the need for rigorous functional and immunological benchmarking prior to clinical application of hypoimmunogenic MSC-derived IPC therapies in T1DM.},
}
@article {pmid42387274,
year = {2026},
author = {Yang, H and Zhu, L and Zhi, J and Zhong, Q},
title = {Targeting Bacterial Quorum Sensing: Insights into Quorum Sensing Inhibitors and Innovative Antimicrobial Strategies for Enhancing Food Safety and Combating Antibiotic Resistance.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c17992},
pmid = {42387274},
issn = {1520-5118},
abstract = {Antibiotic resistance transmission and the increasing diversity of antibiotic resistance phenotypes pose growing threats to food safety and public health. Foodborne bacteria employ quorum sensing (QS) to regulate virulence expression and biofilm formation, enhancing pathogenicity and drug resistance. Therefore, targeting QS is regarded as a promising strategy to control bacteria. Quorum sensing inhibitors (QSIs) are highly promising for addressing bacterial resistance, as they do not rely on the direct killing of bacteria but rather on attenuation of bacterial spoilage effects in food by disrupting their group behavior. This review focuses on natural and synthetic compounds with QSI activity, elaborating their mechanisms and potential as antimicrobial agents. Additionally, the review proposes innovative antimicrobial strategies, including nanotechnology-based delivery systems, combination with phage, CRISPR-Cas technology, and multitargeted approaches cooperated with existing QSIs. These integrated strategies are designed to overcome challenges, providing novel methodologies for controlling bacterial contamination and infections while holding broad application prospects.},
}
@article {pmid42049733,
year = {2026},
author = {Nguyen, JT and Huang, L and Levine, H and Lu, M and Bleris, L},
title = {Rewiring miR-22/SNAI1 via CRISPR-based edge editing destabilizes the epithelial phenotype.},
journal = {NPJ systems biology and applications},
volume = {12},
number = {1},
pages = {},
pmid = {42049733},
issn = {2056-7189},
support = {2114192//National Science Foundation (NSF)/ ; 2029121//National Science Foundation (NSF)/ ; CA283330//National Institute of Health/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *Snail Family Transcription Factors/genetics/metabolism ; *Epithelial-Mesenchymal Transition/genetics ; Humans ; Phenotype ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Signal Transduction/genetics ; Transforming Growth Factor beta/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Epithelial-to-Mesenchymal Transition (EMT) is a critical biological process by which cells acquire enhanced migratory and invasive properties. A key signaling pathway involved in EMT phenotypes includes transforming growth factor β (TGFβ) and transcription factors (TFs) such as SNAIL, ZEB, and TWIST. Additionally, microRNAs (miRNAs) - small, non-coding molecules that regulate gene expression by targeting mRNA transcripts - directly regulate genes central to the EMT process. Notably, miR-22 has been identified as a significant regulator of EMT through direct inhibition of EMT drivers like SNAI1 and indirect regulation of upstream genes. In this study, we performed CRISPR-based network rewiring by selectively removing an edge-the connection between two nodes-to investigate its impact on EMT dynamics. Specifically, we disrupted the connection between miR-22 and SNAI1 without affecting other interactions involving miR-22 or SNAI1 and examined the resulting effects on EMT. We demonstrate that the removal of the miR-22 target site from the SNAI1 gene renders cells more sensitive to TGFβ-mediated EMT. This finding highlights the unique advantage of edge-specific perturbation by ablating the direct regulatory connection between miR-22 and SNAI1. We demonstrate that all measured downstream effects on EMT can be attributed to this single interaction, independent of miR-22's influence on other targets or indirect pathways. More generally, our results underscore the importance of CRISPR-mediated edge ablation for exploring the interactions that govern biological networks and highlight an underexplored opportunity to develop edge-based therapeutic modalities.},
}
@article {pmid42106859,
year = {2026},
author = {Su, N and Xu, C and Liu, W and Xue, M and Huang, Z and Jiang, N and Meng, Y and Zhou, Y and Fan, Y and Zheng, Y},
title = {Establishment and application of a detection method for chinese rice-field eels rhabdovirus (CrERV) using the RPA-CRISPR/Cas12a System.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42106859},
issn = {1743-422X},
support = {No. 2024XT0602//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; No. 2023TD46//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; No.YFICG2025001//Chinese Academy of Fishery Sciences Yangtze River Fisheries Research Institute/ ; grant numbers 2023YFD2402800//National Key Research and Development Program/ ; },
mesh = {Animals ; *Eels/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Fish Diseases/diagnosis/virology ; Reproducibility of Results ; *Rhabdoviridae/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; China ; Rapid Diagnostic Tests ; },
abstract = {The Chinese rice-field eel rhabdovirus (CrERV) is an emerging pathogen that causes hemorrhagic disease in Chinese rice-field eels (Monopterus albus), leading to epidemic outbreaks, mass mortality, and considerable economic losses in aquaculture. Thus, the development of rapid and reliable diagnostic tools for on-site detection is urgently needed to address this issue. In this study, we established an RPA-CRISPR/Cas12a-based assay for CrERV detection, which exhibited superior sensitivity, specificity, and stability. The assay achieved a detection limit of 10[1] copies/µL. Specificity testing confirmed the absence of cross-reactivity with five other major aquatic viruses, including Grass carp reovirus (GCRV-II), Spring viraemia of carp virus (SVCV), Largemouth bass virus (LMBV), Cyprinid herpesvirus 2 (CyHV-2), and White spot syndrome virus (WSSV). Reproducibility analysis showed intra- and inter-assay coefficients of variation below 10%. Analysis of the 26 clinical samples showed that the RPA‑CRISPR/Cas12a assay achieved a higher positivity rate (23.08%, 6/26) compared to qRT‑PCR (15.38%, 4/26), providing preliminary evidence for its diagnostic potential in detecting CrERV. Collectively, these findings indicate that the RPA-CRISPR/Cas12a platform is a highly sensitive, specific, and user-friendly tool for rapid CrERV surveillance in aquaculture settings.},
}
@article {pmid42381030,
year = {2026},
author = {Senu, E and Ikechukwu, SC and Aboagye, C and Agyen, GK and Kamara, S and Tamakloe, VCKT and Nouwati, DD and Diawuo, HY and Doku, EE and Akpobi, S and Asori, M and Effah, A},
title = {Knowledge and perception as determinants of CRISPR application for infectious diseases in Sub-Saharan Africa: a multi-country study in Ghana, Nigeria, and Sierra Leone using regression and structural equation models.},
journal = {Journal of health, population, and nutrition},
volume = {},
number = {},
pages = {},
doi = {10.1186/s41043-026-01380-0},
pmid = {42381030},
issn = {2072-1315},
abstract = {BACKGROUND AND AIM: Infectious diseases, particularly acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV), remain a major global health burden, especially in low- and middle-income countries, despite advances in antiretroviral therapy. Emerging genome-editing technologies, such as CRISPR/Cas systems, hold promise for transforming the diagnosis and treatment of infectious diseases. However, little is known about how these technologies are understood and perceived by individuals in high-burden regions, where significant implementation challenges may limit their adoption. This study assessed knowledge, perceptions, and the potential application of CRISPR technology for infectious disease control in Ghana, Nigeria, and Sierra Leone.<br><br>METHODS: This multi-center cross-sectional study was conducted among 300 participants, including students, healthcare professionals, and researchers across the three countries. Data was collected using a well-structured questionnaire and analyzed using logistic regression and structural equation modeling (SEM) to identify the predictors of potential CRISPR applications.<br><br>RESULTS: The study revealed a significant gap, such that the majority (87.0%) of the participants demonstrated inadequate knowledge, with 70.7% exhibiting poor perception of CRISPR technology. Major barriers to implementation included funding constraints (81.7%) and inadequate infrastructure (62.3%). Adjusted multivariable logistic regression identified inadequate knowledge (aOR: 3.90; p&#x2009;<&#x2009;0.0001) and poor perception (aOR: 1.96; p&#x2009;=&#x2009;0.0060) as independent predictors of low CRISPR application potential. Structural equation modeling confirmed that knowledge significantly enhances perception (&#x3b2;&#x2009;=&#x2009;0.55), and both constructs jointly influence the potential application of CRISPR gene editing technology.<br><br>CONCLUSION: There is a critical deficit in knowledge and perception regarding CRISPR gene editing in Sub-Saharan Africa, which significantly hinders its potential application for infectious diseases. Comprehensive educational strategies and capacity building are essential to foster the adoption of CRISPR technology, especially in resource-limited settings.},
}
@article {pmid42381438,
year = {2026},
author = {Cheng, L and Liu, D and Zhang, H and Lin, H and Zhang, P and Dong, J and Li, C and Ren, F and Ge, X and An, S and Yang, F and Liu, J and Fu, J and Tang, F and Wang, F and Liu, T and Pan, H and Zou, L and Zhou, J and Ma, H and Rong, S},
title = {Synergistic CRISPR-Cas and Nanozyme-Based Fluorescent Sensors for Ultrasensitive Heavy Metal Detection.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/10408347.2026.2684551},
pmid = {42381438},
issn = {1547-6510},
abstract = {Heavy metal pollution poses a serious threat to ecological balance and human health. Traditional detection methods, such as atomic absorption spectrometry and inductively coupled plasma mass spectrometry, suffer from complex procedures, high costs and susceptibility to interference, making them unable to meet the requirements of on-site rapid detection. To address this bottleneck, this review focuses on the innovative solution of the synergistic detection strategy combining nanozymes and the CRISPR-Cas systems. The core advantages of this strategy are as follows: it leverages the excellent specific recognition and signal amplification capabilities of the CRISPR-Cas system to achieve a precise response to heavy metal trigger signals; meanwhile, it utilizes the high stability and enzyme-mimicking catalytic activity of nanozymes to convert signals into readable outputs, thereby constructing an efficient detection platform. We elaborate on how this synergistic mechanism enables high-sensitivity detection without sample pretreatment and remarkably improves analytical performance, with a wider linear range and a lower detection limit. Numerous studies have demonstrated that this combined strategy lays a solid foundation for developing portable, high-sensitivity heavy metal detection devices suitable for complex matrices such as food, water and blood samples, holding great promise for field on-site detection applications.},
}
@article {pmid42108505,
year = {2026},
author = {Pratumkaew, P and Wattanapanitch, M and Viprakasit, V and Kheolamai, P and Issaragrisil, S},
title = {Induced pluripotent stem cell-based modeling of hemolytic anemia in patients with compound heterozygous KLF1 mutations reveals defective erythroid differentiation.},
journal = {Stem cell research &amp; therapy},
volume = {17},
number = {1},
pages = {},
pmid = {42108505},
issn = {1757-6512},
mesh = {Humans ; *Kruppel-Like Transcription Factors/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology/cytology ; *Cell Differentiation ; *Mutation/genetics ; *Anemia, Hemolytic/genetics/pathology/metabolism ; *Erythroid Cells/metabolism/pathology ; Female ; Heterozygote ; CRISPR-Cas Systems ; Male ; },
abstract = {BACKGROUND: Transfusion-dependent hemolytic anemia caused by compound heterozygosity due to mutations in the erythroid Krüppel-like factor 1 (KLF1) gene is a rare and severe blood disorder. The clinical manifestations of the patient are mainly related to erythroid cells. Moreover, the roles of the identified KLF1 mutations in the pathophysiology of this disease remain unclear due to the lack of an appropriate study model. The advent of genome editing technology combined with the generation of patient-specific induced pluripotent stem cells (iPSCs) may provide a better understanding of the molecular mechanisms underlying this disease in an in vitro system and offer a novel therapeutic approach in the future.<br><br>METHODS: KLF1-mutant iPSCs were generated from patients with compound heterozygosity of KLF1 mutations, and the mutation was corrected through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system together with a single-stranded oligodeoxynucleotide donor template (ssODN). The obtained iPSC lines were differentiated towards erythroid cells, and the disease-related phenotypes were examined.<br><br>RESULTS: Erythroid cells derived from KLF1-mutated iPSCs had lower proliferative capacity, showed delayed maturation, and expressed lower level of the KLF1-related gene, CD44. These results were consistent with some of the phenotypes observed in the patients. After CRISPR/Cas9 gene editing, the corrected iPSCs retained pluripotency, exhibited a normal karyotype, and had undetectable off-target mutations. Importantly, some of the defects were partially restored after genetic correction of the KLF1 gene.<br><br>CONCLUSIONS: KLF1-iPSCs presented disease-related phenotypes of compound heterozygous KLF1 mutations, which could be mediated by gene editing through CRISPR/Cas9 and ssODN. This study offers a useful strategy for studying the underlying disease mechanisms of rare diseases, which could be applied to the development of novel treatments for inherited blood disorders in the future.},
}
@article {pmid42371202,
year = {2026},
author = {Bayramoğlu, Z},
title = {Effects of CRISPR technology on agricultural sustainability: global applications and turkish perspective.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {42371202},
issn = {1573-9368},
mesh = {*Plants, Genetically Modified/genetics/growth &amp; development ; *Agriculture/methods ; *Crops, Agricultural/genetics/growth &amp; development ; *Gene Editing/methods ; Turkey ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions-environmental, economic, social and policy, and scientific and technological-we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms-particularly through producer cooperatives-that allow smallholder farmers to benefit from edited varieties.},
}
@article {pmid42371672,
year = {2026},
author = {Yuan, G and Gao, Z and Qi, Y and Zhang, Y and Tian, X and Zhao, P and Feng, X and She, Q},
title = {CREAT: A CRISPR-Based Genome Trimming Strategy for Systematic Identification of Dispensable Regions and Rapid Genome Reduction.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e76042},
doi = {10.1002/advs.76042},
pmid = {42371672},
issn = {2198-3844},
support = {2023YFC3402003//National Key R &amp; D Program of China/ ; FX//National Key R &amp; D Program of China/ ; 2020YFA0906800//National Key R &amp; D Program of China/ ; QS//National Key R &amp; D Program of China/ ; ZR2024QC306//Department of Science and Technology of Shandong Province/ ; GY//Department of Science and Technology of Shandong Province/ ; 2023KJ009//Department of Education of Shandong Province/ ; XF//Department of Education of Shandong Province/ ; 25-1-1-250-zyyd-jch//Qingdao Natural Science Foundation/ ; SKLMTFCP-2023-05//SKLMT Frontiers and Challenges Project/ ; },
abstract = {The construction of minimal-genome microbes offers an ideal platform for understanding fundamental biological processes and synthetic biology, yet the research is hindered by incomplete lists of essential genes in microbes and by multiple rounds of genome trimming with a trial-and-error nature. To address this, we introduce CREAT (CRISPR-based genome trimming with a multi-homology-arm template)-a streamlined approach that integrates CRISPR-targeted genome cleavage and homology arm walking to classify essential from non-essential genomic subregions, thus providing the basis for predicting essential genes in a given organism. These essential genes were then assembled into synthetic gene cassettes for one-step replacement of the targeted non-deletable genomic regions for further genome trimming. Eight consecutive rounds of CREAT genome trimming achieved a 20.8% reduction in genome size in Saccharolobus islandicus. Furthermore, Cas9-based CREAT genome trimming was developed for Bacillus subtilis and Escherichia coli, with efficiency greatly enhanced by the λ-Red recombinase in the latter. Together, this iterative application of CREAT provides a scalable and generally applicable strategy for rapidly constructing minimal genomes across diverse microorganisms.},
}
@article {pmid42378111,
year = {2026},
author = {Liu, X and Chen, T and Li, X and Li, Q and Zou, G and Fan, W and Liu, C},
title = {Engineering Guide RNAs for CRISPR-Based Biosensors.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01763},
pmid = {42378111},
issn = {2379-3694},
abstract = {CRISPR-Cas systems, with their programmable nucleic acid-targeting capabilities, represent an ideal platform for constructing next-generation, highly sensitive biosensors. However, the clinical translation of these platforms is hindered by key limitations inherent to native single-guide RNAs (sgRNAs), including insufficient stability, potential immunogenicity, and off-target effects. To address these challenges, engineering sgRNAs has emerged as a central strategy to overcome such barriers and enhance overall biosensor performance. In this review, we provide a systematic overview of the field, beginning with the classification, molecular mechanisms, and structural features of representative CRISPR-Cas effector proteins to establish their foundational role as sensing elements. We then examine the specific limitations of native sgRNAs in biosensing applications. Building on this analysis, we highlight recent advances in sgRNA engineering strategies, which encompass three major approaches, including chemical modifications, structural remodeling, and modular functional integration. Furthermore, we review the integration of these engineered sgRNAs into advanced biosensor platforms, including microfluidic paper-based devices, centrifugal platforms, wearable patches, microneedles, and point-of-care testing (POCT) systems, and present a comparative table summarizing their performance in terms of detection signals, limits of detection, and other key metrics. Finally, we discuss persistent challenges such as the fine control of off-target effects, in vivo delivery bottlenecks, and system robustness in complex environments, and outline future directions toward amplification-free, multiplexed, and clinically translatable CRISPR-based biosensors. Overall, the engineering of sgRNAs offers a powerful means to systematically enhance the stability, specificity, and reliability of CRISPR-based biosensors, thereby accelerating their practical deployment in clinical diagnostics.},
}
@article {pmid42379562,
year = {2026},
author = {Del Giovane, S and Migliorelli, D and Paoletti, S and Bagheri, N and Altug, H and Burr, L},
title = {CRISPR-Cas-Based Platform for Single-Step Quantification of Monoclonal Antibodies at Point-of-Care.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c04532},
pmid = {42379562},
issn = {2379-3694},
abstract = {We report a streamlined, cost-effective point-of-care platform for the quantitative detection of monoclonal antibodies for therapeutic drug monitoring, addressing the limitations of bulky, complex, and expensive instrumentation required for standard analytical techniques. In contrast to conventional immunoassays that rely on primary/secondary antibodies for quantification, our method leverages a DNA circuit for the recognition of the target monoclonal antibody coupled with CRISPR-Cas12a signal amplification. This assay is integrated into a microfluidic chip that enables a single-step workflow, eliminating the multiple incubations and reagent addition steps typical of laboratory methods. The platform demonstrates quantitative detection of a model anti-hemagglutinin antibody within a 60-min sample-to-answer timeframe, in the nanomolar range, with a chip manufacturing cost below 1€ and reagent stability confirmed at -20 °C for over one month. This proof-of-concept illustrates the potential of simplified CRISPR-based assays for decentralized rapid protein quantification in clinical settings.},
}
@article {pmid42379944,
year = {2026},
author = {Cui, T and Li, B and Cai, B and Wang, H and Li, W},
title = {Precision gene editing: From proof-of-concept to curative therapies.},
journal = {Trends in molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molmed.2026.06.004},
pmid = {42379944},
issn = {1471-499X},
abstract = {Gene therapy is evolving from gene addition to precise genome editing, enabling the direct correction of disease-causing mutations. Breakthrough technologies, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) nucleases, base editors, prime editors, and CRISPR-associated transposases are reshaping the therapeutic landscape. This review covers the progression of precision editing technologies and their clinical applications, spanning from ex vivo therapies to in vivo treatments targeting vital organs. The rise of personalized medicine, highlighted by therapies, such as carbamoyl phosphate synthetase 1 editing, underscores the shift toward N-of-1 medicine for rare diseases. Clinical trial progress, delivery and accessibility challenges, and the role of AI in optimizing editing tools and predicting outcomes are also discussed. These innovations are transforming genetic medicine, offering the promise of safer, more durable, and personalized cures.},
}
@article {pmid42380749,
year = {2026},
author = {Kurt, IC and Guner, H and Erdem, ZA and Can, O and Gumustop, I and Sirin, A and Erol, I and Kotil, ES and Ortakci, F},
title = {Genomic evidence of ecological flexibility and cross-niche CRISPR spacerome targeting phage-plasmid hybrids in Latilactobacillus curvatus.},
journal = {BMC genomics},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12864-026-13098-8},
pmid = {42380749},
issn = {1471-2164},
support = {MGA-2024-45355//Bilimsel Ara&#x15f;t&#x131;rma Projeleri Birimi, &#x130;stanbul Teknik &#xdc;niversitesi/ ; },
abstract = {BACKGROUND: Latilactobacillus curvatus is a lactic acid bacterium with a remarkable ability to persist in diverse niches, including fermented foods and gut. Despite its industrial and potential probiotic relevance, the genomic underpinnings of its cross-niche adaptability remain poorly characterized.<br><br>METHODS: We conducted a species-contextualized comparative genomic analysis of 53 L. curvatus strains from food and gut isolates. This analysis integrated pangenome structure, metabolic repertoire, CRISPR-Cas immunity profiles, and mobilome analysis. Additionally, binding mode predictions and dynamics simulations were used to evaluate the theoretical binding energies of bacteriocins to the BamA target.<br><br>RESULTS: Phylogenomics revealed a polyphyletic population structure, indicating that long-term evolution is not strictly niche-specific. In contrast, genome-wide similarity showed clustering by isolation source, highlighting horizontal gene transfer (HGT) as a plausible contributor to niche adaptation. We identified a highly active mobilome, encompassing diverse plasmids, IS elements, and multiple intact prophages, reflecting high genomic plasticity characteristic of a multihabitat lifestyle. CRISPR-Cas systems were widespread, and analysis of 2,029 spacers revealed a broad immune repertoire targeting mobile genetic elements represented in fermented food, gut, and environmental datasets. We also identified spacer matches to phage-plasmid hybrid-like elements, highlighting the diversity of mobile genetic elements associated with the L. curvatus spacerome.<br><br>CONCLUSION: Our study reveals genomic features consistent with ecological flexibility in L. curvatus, including high genomic plasticity and a broad CRISPR spacer repertoire. Rather than demonstrating strict niche-specific evolution or a causal mechanism for cross-niche persistence, these findings support the hypothesis that this species has experienced diverse interactions with mobile genetic elements across multiple ecological contexts.},
}
@article {pmid42210583,
year = {2026},
author = {Slaufova, M and Karakaya, T and Di Filippo, M and Kündig, T and Beer, HD},
title = {Self-Assembled Skin Equivalents with Monoclonal CRISPR/Cas9-Modified N/TERT-1 Keratinocytes: A Cutting-Edge Model for Human Skin and its Diseases.},
journal = {Advanced healthcare materials},
volume = {15},
number = {25},
pages = {e71283},
doi = {10.1002/adhm.71283},
pmid = {42210583},
issn = {2192-2659},
support = {2360/2024//Vontobel Foundation/ ; 2025/09//Wolfermann-N&#xe4;geli Foundation/ ; #23C201//Jubil&#xe4;umsstiftung Swiss Life, Novartis Foundation for medical-biological Research/ ; //Monique Dornonville de la Cour Foundation/ ; LF-OC-23-001156//LEO Foundation/ ; 310030_197426/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Humans ; *Keratinocytes/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Skin/metabolism ; Cell Line ; *Skin Diseases/pathology/metabolism ; CARD Signaling Adaptor Proteins/genetics/metabolism ; Inflammasomes/metabolism ; Interleukin-1beta/metabolism ; },
abstract = {Human skin is a complex organ consisting of multiple cell types and serves as an essential barrier against environmental stressors. Due to ethical considerations and interspecies differences, in vitro human skin equivalents (SEs) are increasingly used to complement or replace animal models in mechanistic, pharmacological, and disease-modeling studies. Scaffold-free full-thickness SEs, in which fibroblasts generate their own extracellular matrix, are particularly attractive because they provide high structural stability even during extended culture. However, the use of genetically defined keratinocyte populations in these SEs has remained limited. Here, scaffold-free full-thickness SEs incorporating wild-type, polyclonal or monoclonal CRISPR/Cas9-modified N/TERT-1 keratinocytes, generated via electroporation, are established. Monoclonal N/TERT-1 keratinocytes with targeted knockout (KO) of the crucial inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) form a differentiated epidermis but fail to secrete the proinflammatory cytokines interleukin (IL)-1β and IL-18 upon inflammasome activation, indicating complete functional ablation of inflammasome signaling in the 3D model. Moreover, SEs generated with gasdermin A (GSDMA)-KO N/TERT-1 keratinocytes illustrate the feasibility of analyzing genes induced during keratinocyte differentiation under physiological conditions. These results establish scaffold-free full-thickness SEs with monoclonal genetically modified N/TERT-1 keratinocytes as a robust and reproducible human skin model for mechanistic studies and future disease-modeling applications.},
}
@article {pmid42230849,
year = {2026},
author = {Gao, P and Feng, W and Zhao, X and Shen, J and Wang, X and Wu, X and Zhou, W and Wang, Y and Chen, J and An, W},
title = {Constructing EGF mRNA-Enriched Extracellular Vesicles Based on the AAVS1 Safe Harbor Site to Promote Skin Wound Healing.},
journal = {Advanced healthcare materials},
volume = {15},
number = {25},
pages = {e04092},
doi = {10.1002/adhm.202504092},
pmid = {42230849},
issn = {2192-2659},
support = {//National Vaccine and Serum Institute/ ; },
mesh = {*Wound Healing ; *Epidermal Growth Factor/genetics/metabolism ; Animals ; *Extracellular Vesicles/metabolism ; Humans ; *RNA, Messenger/metabolism/genetics ; Rats ; *Skin/metabolism/pathology/injuries ; Cell Proliferation ; Cell Movement ; Fibroblasts/metabolism ; Cell Line ; *Dependovirus/genetics ; CRISPR-Cas Systems/genetics ; Rats, Sprague-Dawley ; },
abstract = {Skin wound healing is a complex biological process that requires the coordinated regulation of cell proliferation, migration, and extracellular matrix (ECM) remodeling. Epidermal growth factor (EGF) plays a key role in this process, but its clinical application is limited by its rapid degradation at the wound site. Extracellular vesicles (EVs), as natural nanocarriers, can protect nucleic acids from degradation and enhance their bioavailability. In this study, using CRISPR/Cas9 technology, we site-specifically integrated the EGF gene carrying the TPA signal peptide into the AAVS1 safe harbor site of 293F cells, generating a cell line that stably secretes EVs enriched in EGF mRNA. Characterization and in vitro and in vivo functional evaluation of these engineered EVs (293F-EGF-EV) demonstrated that they significantly promoted fibroblast proliferation and migration and inhibited excessive collagen production. In a rat skin defect model, 293F-EGF-EV promoted wound recovery. High-concentration 293F-EGF-EV focused on "high-quality repair," such as promoting angiogenesis, hair follicle regeneration, and epidermal structural remodeling. Low-concentration 293F-EGF-EV favored "high-efficiency closure", such as reducing scar area. This study offers new insights into skin wound treatment.},
}
@article {pmid42304391,
year = {2026},
author = {Gorbenko, F and Sala, I and Lee, YY and van de Venn, L and Yeh, CD and Tálas, A and Karvelis, T and Druteika, G and Bechter, LV and Vykhlyantseva, I and Schröder, MS and Gvozdenovic, A and Schwank, G and Siksnys, V and Corn, JE},
title = {Directed evolution of compact RNA-guided nucleases for enhanced activity in mammalian cells.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42304391},
issn = {1474-760X},
mesh = {Humans ; *Directed Molecular Evolution ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: RNA-guided nucleases enable DNA editing and offer promise for treating genetic diseases, particularly when used for precise sequence replacement. However, many of the most effective enzymes, such as Streptococcus pyogenes Cas9, are too large for delivery using vectors like adeno-associated virus. This has prompted interest in smaller alternatives from the Cas12f and TnpB families. Yet, these nucleases often show low activity in mammalian cells, limiting their utility.<br><br>RESULTS: We use directed evolution in human cells to select variants with greatly improved activity. The resulting variants, Cas12f1Super and TnpBSuper, exhibit up to 11-fold increase in editing efficiency without increased off-target effects. When tested as a base editor, Cas12f1Super shows up to tenfold improvement relative to the previously engineered CasMINI, suggesting utility beyond nuclease-related activities.<br><br>CONCLUSIONS: These compact and efficient genome editors expand the current toolkit and hold promise for both research and therapeutic use in mammalian systems.},
}
@article {pmid42366709,
year = {2026},
author = {Naqvi, RF and Ali, M and Zuberi, SA and Aamir, N and Rafique, S},
title = {Nanocarrier-Based Gene Delivery Systems: Mechanisms, Clinical Translation, and Future Perspectives.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70289},
pmid = {42366709},
issn = {1097-0290},
abstract = {Gene therapy holds revolutionary potential for managing genetic disorders, cancers and infectious illnesses. However, one of the biggest challenges is delivering DNA or RNA into targeted cells and in the safe and effective way. In this review, nano carrier-based approaches for gene delivery are critically examined, focusing on both viral and non-viral systems. The advancement of CRISPR-Cas genome editing, machine learning-assisted nanocarrier optimization, and biologically inspired delivery systems is being quickly pushed forward in this area. In this review, a comparative analysis of gene delivery systems is being provided, and the key challenges to clinical translation are being pointed out. In addition, expert opinions on future research directions are being offered, with a heavy focus on the development of multifunctional, precisely targeted, and easily scalable delivery systems that can be integrated with next-generation therapeutic technologies.},
}
@article {pmid42366824,
year = {2026},
author = {Tang, H and Xing, Y and Lu, G and Shen, J and Zhou, Q},
title = {CRISPR/Cas12a Technology Combined with Immunochromatographic Strips for the Portable Detection of SFTS Bunyavirus.},
journal = {Journal of microbiology and biotechnology},
volume = {36},
number = {},
pages = {e2603014},
doi = {10.4014/jmb.2603.03014},
pmid = {42366824},
issn = {1738-8872},
mesh = {Sensitivity and Specificity ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/methods ; *Chromatography, Affinity/methods ; *CRISPR-Cas Systems ; Humans ; *Phlebovirus/isolation &amp; purification/genetics ; RNA, Viral/genetics ; Molecular Diagnostic Techniques/methods ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; },
abstract = {Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by SFTS virus (SFTSV), is a widely distributed infection with significant mortality. Diagnosis in resource-limited settings remains challenging. For rapid and convenient diagnosis, we developed a portable rapid diagnosis method that combines Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a technology with immunochromatographic test strips. The SFTSV RNA was amplified by reverse transcription loop-mediated isothermal amplification (RT-LAMP). The homologous target sequence and single stranded DNA (ssDNA) reporter gene were cleaved by CRISPR/Cas12a in parallel, and ssDNA probes labeled with FAM fluorescein and biotin were captured by an immunochromatographic strip. Finally, the signal on the immunochromatographic strips became visible to the naked eye. Based on CRISPR/Cas12a, a rapid SFTSV detection method was developed, featuring simplicity, rapidity, low cost, and ease of use. The method was applied for the nucleic acid detection of SFTSV in 40 clinical serum samples and compared with RT-polymerase chain reaction (PCR). The new method showed 100% sensitivity and 100% specificity with a detection agreement rate of 100%. The minimum detection limit of the method was 2.5 copies/μL, and no cross-reactivity with nucleic acids from other common pathogens was observed. Detection can be completed within 80 min, and results are observable with the naked eye. For the analysis of clinical samples, the method exhibits good detection performance and thus provides an attractive option for the nucleic acid detection of SFTSV in point-of-care and resource-limited medical settings.},
}
@article {pmid42369443,
year = {2025},
author = {Meerman, JJ and de Bruijn, VMP and Luechtefeld, T and van Engelen, JGM and Kienhuis, AS and Heusinkveld, HJ},
title = {Including genetic susceptibility towards Parkinson's disease in NAM-based hazard and risk assessment of pesticides: a semi-systematic review.},
journal = {NAM journal},
volume = {1},
number = {},
pages = {100036},
pmid = {42369443},
issn = {3050-6204},
abstract = {Parkinson's disease (PD) prevalence has been steeply increasing over the last years. Environmental pollution, including certain pesticides, is considered a significant contributor to this rise. Certain genetic factors may pose an individual or subpopulation at increased risk to develop pesticide-induced PD. To support New Approach Methodologies (NAMs)-based hazard and risk assessment while considering known sources of interindividual variability, the aim of the current work was to identify biological factors related to increased susceptibility towards pesticide-induced PD onset and progression. To this end, a semi-systematic literature search was performed using Sysrev. We identified 61 relevant records. Potential gene-environment interactions were identified for several genes related to toxicokinetics, mitochondrial functioning, neurotransmission and proteostasis. These genes, except those related to toxicokinetics, have been described in Adverse Outcome Pathways (AOPs) leading to parkinsonian motor symptoms. Untargeted (epi)genetic analyses identified potential new targets. NAMs offer the opportunity to include human-relevant genetic interindividual variability in toxicological hazard and risk assessment. For example, an additional test condition with mutations of interest for each Key Event (KE) could be included in a test strategy. Whole-organism models combined with CrispR-CAS techniques are suitable to study the effects of specific mutations on PD risk. Induced pluripotent stem cells (iPSCs) are promising to study interindividual variability in disease susceptibility based on material from genetically diverse donors. Since additional experiments are resource-intensive, further research is required to establish the need and way to include genetic susceptibilities in a regulatory context.},
}
@article {pmid42370783,
year = {2026},
author = {Wang, R and Liu, X and Li, H and Wang, Y and Zhang, J and Jin, B and Zhang, Y and Yin, H and Li, Y},
title = {Effect of BRD0539 on Gene Editing and Mosaicism Rate in Porcine Gene Editing Embryos by CRISPR/Cas9.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {61},
number = {7},
pages = {e70255},
doi = {10.1111/rda.70255},
pmid = {42370783},
issn = {1439-0531},
support = {32272881//National Natural Science Foundation of China/ ; 2024AH040078//Anhui Province Higher Education Institutions Scientific Research Project/ ; 202513b10050016//Anhui Provincial Science and Technology Commissioner Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Myostatin/genetics ; *Gene Editing/veterinary/methods ; *Mosaicism/veterinary ; Swine/embryology ; Embryo, Mammalian ; Parthenogenesis ; Microinjections/veterinary ; Female ; *Sus scrofa/genetics/embryology ; },
abstract = {Microinjection is a common method for generating gene-edited animals; however, persistent Cas9 activity post-cleavage often results in mosaic embryos due to editing occurring in different blastomeres. This study investigated whether co-injecting the CRISPR/Cas9 system with the small-molecule Cas9 inhibitor BRD0539, or supplementing it in the culture medium, could reduce mosaicism while maintaining editing efficiency in porcine parthenogenetic activation embryos targeting the myostatin (MSTN) gene. The findings are as follows: Co-injection of 10 or 100 μM BRD0539 with Cas9 mRNA: sgRNA significantly reduced gene editing efficiency (28.5% ± 11.6% and 33.8% ± 4.1%, respectively, vs. 86.9% ± 4.5% in control, p < 0.05). Supplementing the culture medium with 10 or 50 μM BRD0539 also reduced both editing efficiency (20.8% ± 12.4% and 47.7% ± 14.6%, respectively, vs. 85.6% ± 4.8%) and mosaicism rate (25.0% ± 15.9% and 12.5% ± 12.5%, respectively, vs. 87.1% ± 7.8%, p < 0.05). Immunofluorescence revealed sustained Cas9 protein expression up to 48 h post-injection. Crucially, short-term addition of 10 μM BRD0539 to the culture medium between 24 and 48 h post-activation significantly reduced mosaicism (32.6% ± 7.5% vs. 78.7% ± 9.6%, p < 0.05) without compromising editing efficiency. Furthermore, this treatment did not adversely affect cleavage rates, blastocyst development, total cell number. These results demonstrate that transient inhibition of Cas9 activity with 10 μM BRD0539 during a critical window effectively reduces mosaicism in microinjected porcine embryos, offering a promising strategy to enhance the efficiency of generating non-mosaic gene-edited livestock.},
}
@article {pmid42371108,
year = {2026},
author = {Das, A and Rajput, VD and Pal, S and Banerjee, S and Adak, MK},
title = {Integrative transcriptional regulatory networks governing cereal root responses to heavy metals and drought.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42371108},
issn = {1432-203X},
support = {NTA Ref. No.: 211610058334//University Grants Commission/ ; },
mesh = {*Plant Roots/genetics/physiology/drug effects/metabolism ; *Edible Grain/genetics/physiology/drug effects/metabolism ; *Metals, Heavy/toxicity ; *Gene Regulatory Networks ; Gene Expression Regulation, Plant/drug effects ; Droughts ; Stress, Physiological/genetics ; Drought Resistance ; Transcription Factors/metabolism/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Cereal root systems perceive the onset of drought and heavy metal toxicity, which rapidly triggers signal transduction and extensive transcriptional reprogramming that underpins plant stress tolerance. This review discusses the transcriptional basis of root responses to abiotic stresses, emphasizing key regulatory genes and networks that orchestrate hormone signaling, redox dynamics, ion homeostasis, and structural modifications. Key transcription factor families-NAC, WRKY, bZIP, DREB, ARF, and MYB-serve as the nexus between the early perception and adaptive outputs such as controlled root growth, suberization, aerenchyma formation, and metal sequestration. Integrative transcriptomic, proteomic, metabolomic, and chromatin data in rice, wheat, maize, and millets highlight cell-type- and zone-specific regulatory programs, with single-cell and spatial omics uncovering modules obscured in bulk datasets. We discuss how CRISPR/Cas editing, cis-element engineering, and root-specific promoters refine core regulators, and how quantitative trait loci (QTL)/genome-wide association study (GWAS) advance breeding for drought and metal tolerance. Emerging concepts encompass multi-stress omics, high-throughput root phenomics, and artificial intelligence-driven network modeling, and collectively enable targeting of core transcriptional regulatory nodes. Overall, emerging knowledge supports targeted engineering of transcriptional regulators to develop resilient cereal root systems, contributing to sustainable yields and improved stress tolerance in real-world agroecosystems.},
}
@article {pmid41319305,
year = {2026},
author = {Point, V and Achache, W and Laudouze, J and Sepulveda Ramos, E and Maziero, M and Crauste, C and Canaan, S and Santucci, P},
title = {Mycobacterial cell division arrest and smooth-to-rough envelope transition using CRISPRi-mediated genetic repression systems.},
journal = {FEBS open bio},
volume = {16},
number = {7},
pages = {1271-1292},
doi = {10.1002/2211-5463.70172},
pmid = {41319305},
issn = {2211-5463},
support = {//Institut des sciences biologiques/ ; ANR-24-CE15-2633//Agence Nationale de la Recherche/ ; AMX-19-IET-006//Initiative d'Excellence d'Aix-Marseille Universit&#xe9; - A*MIDEX - IM2B/ ; //FEBS Excellence Award/ ; Project n&#xb0;ANRS0358//Agence Nationale de Recherches sur le Sida et les H&#xe9;patites Virales/ ; //Aix-Marseille Universit&#xe9;/ ; },
mesh = {Mycobacterium smegmatis/genetics ; Cell Division/genetics ; *CRISPR-Cas Systems/genetics ; Gene Silencing ; *Nontuberculous Mycobacteria/genetics ; Mycobacterium abscessus/genetics ; Bacterial Proteins/genetics ; },
abstract = {The genetic basis underlying nontuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9Sth1-mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use Mycobacterium smegmatis (M. smeg) and Mycobacterium abscessus (M. abs) as NTM model species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.},
}
@article {pmid42082826,
year = {2026},
author = {Kazerani, M and Cagiral, U and Tabatabaei, SZ and Akalper, RN and Bora, U and Babashah, S and Ozhan, G and Totonchi, M},
title = {A Novel BIRC6 Variant Impairs Apoptotic Regulation in Familial Premature Ovarian Insufficiency: Functional Validation in a CRISPR/Cas9 Zebrafish Model.},
journal = {Reproductive sciences (Thousand Oaks, Calif.)},
volume = {33},
number = {6},
pages = {1170-1188},
pmid = {42082826},
issn = {1933-7205},
mesh = {Animals ; Female ; Zebrafish ; *Primary Ovarian Insufficiency/genetics/metabolism/pathology ; Humans ; *Apoptosis/genetics ; Disease Models, Animal ; *CRISPR-Cas Systems ; Pedigree ; *Inhibitor of Apoptosis Proteins/genetics/metabolism ; Adult ; *Zebrafish Proteins/genetics/metabolism ; Mutation, Missense ; },
abstract = {Premature ovarian insufficiency (POI), characterized by ovarian dysfunction before age 40, remains idiopathic in over 50% of cases, underscoring the urgent need to elucidate its genetic underpinnings. Here, we report a consanguineous Iranian family with five females diagnosed with POI, exhibiting elevated gonadotropins, undetectable anti-Müllerian hormone, and bilateral ovarian atrophy. Whole Exome Sequencing identified a novel homozygous missense variant in BIRC6 (NM_016252.4: c.11266C > T; p.Arg3756Cys), a gene encoding an apoptosis regulator. Segregation analysis confirmed an autosomal recessive inheritance pattern, with homozygosity exclusively in affected individuals. Functional studies in a CRISPR/Cas9-generated birc6[del12] zebrafish model recapitulated POI phenotypes: homozygous females exhibited reduced fecundity, aberrant oocyte morphology, and elevated embryonic death. Transcriptional analysis revealed dysregulation of apoptotic (badb, sortilin, apc) and fertility-related (nanos1, vtg1) genes, alongside unaltered estradiol levels, implicating apoptosis-driven follicular atresia rather than endocrine dysfunction. Compensatory increase in egg production in mutants mirrored human POI progression, where initial irregular cycles culminate in follicular depletion. This study establishes BIRC6 as a novel POI candidate, links its anti-apoptotic function to ovarian homeostasis, and highlights zebrafish as a tractable model for dissecting POI mechanisms. Our findings expand the genetic landscape of infertility and suggest therapeutic potential for apoptosis modulation in fertility preservation.},
}
@article {pmid42092133,
year = {2026},
author = {Scholz, P and Thompson, J and Crosby, KT and Fauth, T and Krah, NM and Schlauderaff, G and Back, R and Berkheimer, ZA and Jolley, A and Sombroek, D and Medert, R and Zurek, C and Dmytrenko, O and Wilson, E and Schut, FT and Rutter, J and Zhang, X and Krohn, M and Jackson, RN and Beisel, CL and Liu, Y},
title = {RNA-triggered cell killing with CRISPR-Cas12a2.},
journal = {Nature},
volume = {655},
number = {8121},
pages = {230-239},
pmid = {42092133},
issn = {1476-4687},
mesh = {Humans ; *Cell Death/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; DNA/metabolism/genetics/chemistry ; DNA Breaks, Double-Stranded ; *Endodeoxyribonucleases/metabolism/genetics ; *Gene Editing ; RNA/metabolism/genetics ; Saccharomyces cerevisiae/genetics/cytology ; Bacterial Proteins ; },
abstract = {Selectively eradicating target cells on the basis of their genetic or transcriptional identity remains important in basic research, medicine, biotechnology and agriculture[1-3]. For applications involving bacteria, CRISPR nucleases offer promising options due to their ability to enact RNA-guided counterselection[4-7]; however, using these same nucleases for counterselection in eukaryotes has proven much more restrictive[8-14]. Here we show that Cas12a2, a recently discovered type V CRISPR nuclease, exhibits RNA-triggered DNA shredding[15,16], and enables programmable and sequence-specific elimination of yeast and human cells expressing a target transcript. Triggering Cas12a2 elicits rampant double-stranded DNA breaks in trans, leading to cell death. Cell killing can be activated by a wide range of target transcripts, with no observed off-target activation. Leveraging this approach, we selectively eliminate cells that harbour human papillomavirus, cells that failed to undergo gene editing, or cells that encode a prevalent oncogenic point mutation in KRAS. These findings expand the CRISPR toolbox to allow the selective elimination of eukaryotic cells on the basis of their transcriptional profile.},
}
@article {pmid42353483,
year = {2026},
author = {Xu, J and Cheng, F and Fang, J and Cao, K and Li, G and Luo, W and Hu, D and Zhang, J and Hu, Q},
title = {Advances in Fish Gene Editing.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {12},
pages = {},
pmid = {42353483},
issn = {2076-2615},
support = {CARS-47//China Agriculture Research System/ ; 2025LZGC010, 2024CXPT071-1//Department of Science and Technology of Shandong Province/ ; B24YQ0010, B25H1QC08//Hainan Seed Industry Laboratory/ ; BRESG-JB202502//Basic Research Project of State Key Laboratory of Mariculture Biobreeding and Sustainable Goods/ ; QDLYY-2024012//Blue Seed Industry Innovation Project of Qingdao Institute of Blue Seed Industry/ ; TSQNZ20240843//Taishan Scholars Program/ ; },
abstract = {Fish represent the most species-rich group within the phylum Chordata, possessing exceptional nutritional and ornamental value. Global aquaculture, particularly finfish farming, is experiencing rapid expansion worldwide, and fish serve as crucial model organisms for vertebrate developmental biology and functional genomics research. However, traditional breeding methods are plagued by limitations such as low precision and lengthy breeding cycles. Currently, gene editing technologies represented by the CRISPR/Cas system, base editing, and prime editing have provided revolutionary tools for dissecting gene function, modeling human diseases, targeted trait improvement, and ecological adaptation studies. This review describes the evolutionary history of gene editing technology, compares gene delivery strategies in fish embryos, and highlights landmark applications in key areas, including gene function research, aquaculture breeding, ornamental fish coloration regulation, and human disease model construction. Finally, we propose that innovation should be pursued while ensuring biosafety and regulatory compliance, to promote the transformation of fish gene editing toward large-scale and safe application.},
}
@article {pmid42353704,
year = {2026},
author = {Boyanova, L and Boyanova, LY and Medeiros, J and Dimitrov, G and Hadzhiyski, P and Gergova, R and Markovska, R},
title = {Some Newer Antibiotics Active Against Helicobacter pylori and Anaerobic Bacteria and the Potential Benefits of Their Wider Availability in More Countries: A Narrative Review.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42353704},
issn = {2079-6382},
abstract = {It is crucial to consider newer antibiotics with activity against anaerobes and Helicobacter pylori, given their healthcare importance, and the constantly growing antibiotic resistance/multidrug resistance, which complicates the therapy. The aim of this review was to emphasize certain recently approved or still-under-investigation antibiotics with potential benefits for treating Clostridioides difficile infections (CDIs), other anaerobic infections, and those caused by H. pylori, covering recent data from articles published primarily in 2020-2026. Given the limited number of antibiotics for treating CDI and fidaxomicin nonavailability in many countries, it is necessary to conduct more extensive laboratory and clinical studies of promising antibiotics such as ibezapolstat, delafloxacin, lascufloxacin, omadacycline, eravacycline, ridinilazole, and CRS3123. Against Bacteroides fragilis group species, delafloxacin and eravacycline showed good activity. Research on rifasutenizol for bacterial vaginosis, sarecycline and nadifloxacin for acne vulgaris and amixicile for periodontal diseases needs to be expanded. For H. pylori infection, delafloxacin, sitafloxacin, nemonoxacin, zoliflodacin, and rifasutenizol may improve the suboptimal success of most eradication regimens. However, more efforts, in coordination between medical, scientific, manufacturing, and government representatives, should ensure wider access to and research on the newer antibacterials. Establishing more research groups, careful examination of market issues, and additional approaches, such as nanomaterials, efflux pump inhibitors, phage therapy, and CRISPR-Cas systems, should be beneficial. Notwithstanding the difficulties, there are many opportunities to promote research on and potential use of newer antibiotics which show advantages over the older antibacterials, and to make them available to numerous countries and patients worldwide.},
}
@article {pmid42353780,
year = {2026},
author = {Zhu, C and Wang, Y and Zhu, M and Chen, G and Wang, F and Li, B and Xu, Z and Wang, G and Xu, J and Lu, X and Wang, Y and Jin, S},
title = {High-Density CRISPR/Cas12a-Mediated Multiplex Genome Editing Reveals Genome Instability in Allotetraploid Cotton.},
journal = {Genes},
volume = {17},
number = {6},
pages = {},
pmid = {42353780},
issn = {2073-4425},
support = {XL202401//Xinjiang Talent Development Fund/ ; 2025SNGGGCC-BT06//Tianshan Talents Program for Rural Development Leaders/ ; },
mesh = {*Gossypium/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genomic Instability ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Tetraploidy ; },
abstract = {BACKGROUND: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing.<br><br>METHODS: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA-crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon.<br><br>RESULTS: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65-24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations.<br><br>CONCLUSIONS: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes.},
}
@article {pmid42353789,
year = {2026},
author = {Wu, Q and Sun, J and Yang, S and Zhang, M and Yang, D and Xue, H and Wu, H and Guo, Y and Li, S and An, Y},
title = {TaKMT-7A Gene Positively Regulates Spike Number in Wheat.},
journal = {Genes},
volume = {17},
number = {6},
pages = {},
pmid = {42353789},
issn = {2073-4425},
support = {2024CXPT072//Department of Science and Technology of Shandong Province/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Quantitative Trait Loci/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Chromosome Mapping ; Phenotype ; Edible Grain/genetics/growth &amp; development ; CRISPR-Cas Systems ; Genes, Plant ; Frameshift Mutation ; },
abstract = {Wheat (Triticum aestivum L.) is a crucial global food crop that plays a central role in agricultural production and food security. The spike number per unit area (SN) is one of the three component factors of grain yield. In this study, we combined the UG-Map with 27 environments of a recombinant inbred line (RIL) population, and mapped a quantitative trait locus (QTL) for SN, QSn-7A-9048, in which the meta-QTL interval contains only one candidate gene, TraesCS7A02G-364700 (TaKMT-7A). Using the CRISPR/Cas9 system, we generated two homozygous mutant lines, aa-1 and aa-2 of TaKMT-7A, which resulted in frameshift mutations, leading to the premature termination of the translation process. The SN values for the wild type (WT), aa-1, and aa-2 were 4.48, 3.43, and 3.48, respectively. Compared with the WT, the SN of the two mutant lines significantly decreased, and no significant differences for grain number per spike (GNS) and thousand-grain weight (TGW) were detected. We also obtained two overexpression (OE) lines of TaKMT-7A, OE-1 and OE-2. The SN values for the negative control (NC), OE-1, and OE-2 were 2.31, 3.33, and 3.00, respectively. Compared with NC, the SN values in the OE lines significantly increased. The phenotypes of the knockout (KO) lines and OE lines demonstrate that TaKMT-7A acts as a positive regulator of SN in wheat. We performed RNA-Seq analysis using young tiller buds from the WT and aa-1 mutant lines at the tillering stage, and a total of 2315 differentially expressed genes (DEGs) were identified. We screened 22 wheat genes, of which 18 orthologous genes have previously been cloned and are associated with branching in rice and Arabidopsis. These genes included nitrogen transporter, amino metabolism, auxin transporter, auxin homeostasis, auxin response, auxin biosynthesis, strigolactone biosynthesis, and repress gibberellin responses. These genes may represent potential downstream targets of TaKMT-7A.},
}
@article {pmid42353791,
year = {2026},
author = {Rodriguez, SH and Yokota, T},
title = {Building CRISPR-Based Gene-Editing Platforms for Personalized Medicine: The Next Step in Interventional Genetics.},
journal = {Genes},
volume = {17},
number = {6},
pages = {},
pmid = {42353791},
issn = {2073-4425},
mesh = {Humans ; *Precision Medicine/methods/trends ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; },
abstract = {Recent advances in CRISPR technology have expanded beyond traditional double-strand break-based genome editing to include base editors and prime editors, enabling precise and programmable sequence modifications. This evolution marks a shift from conventional mutation correction toward platform-based therapeutic systems capable of targeting a broad spectrum of pathogenic variants. Such versatility holds promise for addressing a substantial proportion of known disease-causing mutations in rare monogenic disorders. This review discusses the technological progression of CRISPR systems, highlighting the principles, applications, and limitations of emerging editing modalities. We will explore their translation into personalized gene therapies, emphasizing delivery challenges, off-target safety, and the need for regulatory innovation. The paper will also introduce the concept of interventional genetics, an emerging medical framework linking genomic diagnosis directly to therapeutic intervention through adaptive gene-editing platforms. Finally, we will outline strategies for establishing unified, scalable, and regulatory-ready editing platforms that can accelerate the clinical implementation of individualized therapies for rare diseases.},
}
@article {pmid42354828,
year = {2026},
author = {Flores, AI and Morales-Cedeño, LR and Loeza-Lara, PD and Schoebitz, M and Orozco-Mosqueda, MDC and Santoyo, G},
title = {Engineering Plant-Associated Microorganisms for Bioremediation and Sustainable Agriculture.},
journal = {Microorganisms},
volume = {14},
number = {6},
pages = {},
pmid = {42354828},
issn = {2076-2607},
abstract = {As food demand increases, agricultural practices have evolved, prompting increased exploration of sustainable ecological techniques and utilization of plant-associated microorganisms. In this context, plant fitness has been enhanced by plant growth-promoting microorganisms (PGPM), which stimulate growth through direct mechanisms, such as improved nutrient availability and phytohormone production, as well as indirect mechanisms, including protection against phytopathogens and suppression of soil-borne diseases. However, these innate capabilities of PGPM can be further improved through genomic modification or editing. This article reviews advances in the genomic engineering of plant-beneficial microorganisms as tools to enhance their positive effects on crop performance and environmental remediation. The genetic modification strategies analyzed here include random mutagenesis, targeted genome editing (such as CRISPR-Cas), gene over-expression, genome shuffling, RNA interference, metabolic pathway engineering, and synthetic biology approaches. These tools have enabled the optimization of functions, such as nitrogen fixation, phosphate solubilization, secondary metabolite production, biocontrol, stress tolerance, and bioremediation. However, we propose expanding the discussion of their regulation and use in various countries. Additionally, these modifications must be efficient and safe for the beneficial microbiota associated with the target crop, as well as for humans, animals, and the environment, all of which depend on sustainable agricultural practices.},
}
@article {pmid42359401,
year = {2026},
author = {Gain, H and Banerjee, J},
title = {Cis-regulatory elements in CAMTA-mediated stress signalling: mechanisms and prospects for CRISPR-based crop improvement.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1864496},
pmid = {42359401},
issn = {1664-462X},
abstract = {Enhancements in crop resilience strategies that maintain production are essential to address the challenges posed by climate change and increasing food consumption. Calcium-dependent signaling networks are essential for plant responses to abiotic and biotic stressors, with calmodulin-binding transcription activator (CAMTA) transcription factors serving as crucial regulators within this framework, as these factors govern gene expression through specific cis-regulatory elements located in promoter regions. Recent investigations have expanded to include CAMTA-binding motifs as the stress-responsive cis-regulatory modules across several plant species under examination. These findings indicate that CAMTA-associated cis-elements, comprising CGCG motifs and ABA-responsive regions, facilitate the integration of environmental signals that influence transcription. Cis-regulatory elements (CREs), such as promoters, enhancers, silencers, and insulators, control the exact timing and location of stress-responsive gene expression in plants. Recent breakthroughs in genome editing have enabled the direct manipulation of these cis-regulatory areas, facilitating precise control over gene expression. This work presents an overview of CAMTA structures, their interaction with promoter cis-regulatory regions, and the potential for promoter cis-element engineering to enhance agricultural performance under diverse settings. It emphasizes CRISPR-based strategies for precise CRE modifications and highlights the role of CAMTA in identifying stress-responsive regions. This establishes the foundation for the advancement of next-generation stress-resilient crops, which will ensure food security.},
}
@article {pmid42360551,
year = {2026},
author = {Pramanik, S and Debnath, B and Chakraborty, A and Islam, A and Mullick, S and Chaudhary, P and Nath, R and Chellappan, DK and Mondal, M and Ashique, S},
title = {Targeting mtDNA to Modulate Mitochondrial Dysfunction in Neurodegenerative Diseases.},
journal = {Molecular neurobiology},
volume = {63},
number = {1},
pages = {},
pmid = {42360551},
issn = {1559-1182},
mesh = {Humans ; *Neurodegenerative Diseases/genetics/therapy/pathology ; *DNA, Mitochondrial/genetics/metabolism ; Animals ; *Mitochondria/genetics/metabolism/pathology ; Gene Editing ; },
abstract = {Mitochondrial dysfunction is a common pathological feature of neurodegenerative diseases namely Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Although these disorders are primarily driven by disease-specific genetic and proteopathic mechanisms, increasing evidence suggests that secondary mitochondrial DNA (mtDNA) damage and heteroplasmy shifts may exacerbate bioenergetic failure and neuronal vulnerability. Distinguishing primary disease mechanisms from downstream mtDNA alterations is critical to accurately evaluate emerging therapeutic strategies. Recent advances in mtDNA-targeted genome editing have enabled the direct manipulation of mitochondrial genomes. Mitochondrially targeted zinc finger nucleases and TALENs can selectively alter mutant mtDNA to induce heteroplasmy shifts, whereas DddA-derived cytosine base editors allow precise base editing without double-strand breaks. However, each platform has distinct limitations related to the target scope, off-target risk, design complexity, and delivery efficiency. The application of CRISPR/Cas-based systems to mammalian mtDNA remains constrained by the unresolved challenges in guiding RNA import. This review critically examines mitochondrial dysfunction and mutant mtDNA accumulation in neurodegenerative diseases. It also evaluates current and emerging mtDNA-editing techniques, and highlights key translational barriers. We highlighted that mtDNA-targeted interventions can be a promising approach for disease-modifying or adjunctive strategies, rather than curative approaches.},
}
@article {pmid42361119,
year = {2026},
author = {Dupzyk, AJ and Waldman, BS and Zengel, J and Zanini, F and Carette, JE},
title = {Dissecting the host determinants of orthoflavivirus infection using QIC-seq.},
journal = {PLoS pathogens},
volume = {22},
number = {6},
pages = {e1014279},
doi = {10.1371/journal.ppat.1014279},
pmid = {42361119},
issn = {1553-7374},
mesh = {Animals ; Humans ; *Host-Pathogen Interactions ; *Flavivirus/genetics ; CRISPR-Cas Systems ; *Flavivirus Infections/virology/genetics ; Dengue Virus/genetics ; Membrane Proteins/genetics/metabolism ; West Nile virus/genetics ; Hexosyltransferases ; },
abstract = {Orthoflaviviruses are genetically related, yet cause distinct disease patterns ranging from hepatitis and vascular shock syndrome to encephalitis and congenital abnormalities. There is an incomplete understanding of the cellular pathways co-opted by orthoflaviviruses, and differences in host response to infection may underlie the diverse pathologies caused. We present a single-cell approach (Quantification of Infection and CRISPR guide sequencing; QIC-seq) that combines CRISPR/Cas9 knockout with virus-inclusive transcriptomics to systematically compare host factor requirements and host transcriptional response to orthoflaviviral challenge. Using a CRISPR library focused on select ER-proteostasis genes, we show that dengue and yellow fever viruses are strictly dependent on subunits of the oligosaccharyltransferase complex, while the more distantly related West Nile and Langat viruses are dependent on components of the ER-associated degradation machinery. Our data further shows virus-induced upregulation of interferon-stimulated genes, and activation of the unfolded protein response. QIC-seq enables quantitative comparisons of viral host factor utilization, which may inform development of host-directed antiviral therapies.},
}
@article {pmid42362581,
year = {2026},
author = {Chen, J and Huang, L and Chen, H and Li, X and Lin, X and Guo, C and Liu, X and Fu, G and Chen, Y and Liu, L},
title = {Potential role of a CRISPR-Cas-activated toxin-antitoxin system in bacterial immunity.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74930-z},
pmid = {42362581},
issn = {2041-1723},
support = {32371346//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32301007//National Natural Science Foundation of China (National Science Foundation of China)/ ; 324B2056//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023J01023//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; 2024J011007//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; 2023J05008//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; },
abstract = {CRISPR-Cas and toxin-antitoxin systems can serve as antiviral defense mechanisms in prokaryotes. In typical toxin-antitoxin systems, toxin activation can limit phage propagation by inducing growth arrest or reduced cellular fitness, while the antitoxin neutralizes toxin activity. Here, we study potential functional synergy between a CRISPR-Cas13a system and a type II toxin-antitoxin module (HicAB) from a Leptotrichia bacterium, when heterologously expressed in E. coli, as well as in biochemical and structural analyses. We show that the antitoxin HicB exhibits toxic properties, and Cas13a directly activates HicB, triggering growth inhibition and conferring protection against bacteriophages. Structural analyses reveal that Cas13a binding promotes the spatial proximity of HicB tetramers, likely enabling its activation. The toxin HicA competitively binds to HicB, thereby inhibiting Cas13a-mediated HicB activation. Importantly, both CRISPR RNA and HicB independently suppress HicA toxicity. Structural evidence indicates that CRISPR RNA forms a hetero-tetradecameric complex with HicAB, occluding HicA's active site and neutralizing its toxic function. Thus, our findings indicate functional synergy between distinct bacterial immune strategies.},
}
@article {pmid41057262,
year = {2026},
author = {Char, SN and Liu, H and Birchler, JA and Wang, K and Yang, B},
title = {CRISPR-Cas9 Toolkit for Maize: Vector Design, Construction, and Analysis of Edited Plants.},
journal = {Cold Spring Harbor protocols},
volume = {2026},
number = {7},
pages = {pdb.prot108659},
doi = {10.1101/pdb.prot108659},
pmid = {41057262},
issn = {1559-6095},
mesh = {*Zea mays/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.},
}
@article {pmid41318237,
year = {2026},
author = {Shin, SW and Lee, GM and Lee, JS},
title = {CRISPR screen-based mammalian cell engineering for complex biotherapeutics.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1817-1820},
doi = {10.1016/j.tibtech.2025.11.010},
pmid = {41318237},
issn = {1879-3096},
mesh = {Animals ; Humans ; *Cell Engineering/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mammals ; },
abstract = {The rise of complex biotherapeutics has introduced bottlenecks in production using mammalian cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based screens enable unbiased discovery of engineering targets that mitigate biomanufacturing-relevant constraints. This forum gives an overview of recent advances and remaining challenges in applying CRISPR screening to build robust, modality-specific cell factories.},
}
@article {pmid41421891,
year = {2026},
author = {Durán-Vinet, B and Stanton, JL and Jeunen, GJ and Pochon, X and Zaiko, A and Gemmell, NJ},
title = {CRISPR as a next-generation environmental biosurveillance tool for air, land, and water.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1872-1891},
doi = {10.1016/j.tibtech.2025.11.014},
pmid = {41421891},
issn = {1879-3096},
mesh = {*Environmental Monitoring/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Air Microbiology ; Water Microbiology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based environmental biosurveillance (CRISPR-eBx) offers a portable, specific, sensitive, and cost-effective platform for detecting organisms from environmental nucleic acids. Applications are broad, ranging from pathogen detection to monitoring invasive and endangered species across a range of environmental sources, including water, soil, and air. However, if CRISPR-eBx is to be deployed for novel biological/gene targets and environmental sources, key challenges must be addressed. This review synthesizes recent developments at the intersection of CRISPR technology, computational science, synthetic biology, and biosurveillance. We highlight promising innovations and identify knowledge gaps to present a strategic road map for establishing CRISPR-eBx as a next-generation, frontline biosurveillance solution.},
}
@article {pmid41513118,
year = {2026},
author = {Amieva, R and Román, LR and Coronado, M and Powell, J and Hassan, MA and Hemphill, A and Boubaker, G and Pfarrer, C and Ortega-Mora, LM and Collantes-Fernández, E and Horcajo, P},
title = {NcROP24 loss attenuates Neospora caninum virulence and alters rhoptry organization.},
journal = {International journal for parasitology},
volume = {56},
number = {7},
pages = {104770},
doi = {10.1016/j.ijpara.2026.104770},
pmid = {41513118},
issn = {1879-0135},
mesh = {*Neospora/pathogenicity/genetics ; Animals ; Female ; Cattle ; *Coccidiosis/parasitology/veterinary ; Virulence ; *Protozoan Proteins/genetics/metabolism ; Pregnancy ; Mice ; Macrophages/parasitology ; Host-Parasite Interactions ; Cattle Diseases/parasitology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a leading cause of abortion and economic loss in cattle worldwide. Despite its veterinary significance, the molecular mechanisms underlying parasite virulence and host-pathogen interaction remain poorly understood. In particular, the contribution of rhoptry proteins, key secretory effectors involved in host cell invasion and immune modulation, has yet to be fully elucidated. Here, we investigate NcROP24, a previously understudied rhoptry protein whose expression correlates with isolate virulence. Using CRISPR/Cas9, we generated NcROP24 knock-out mutants (NcΔROP24) by deleting all three genomic copies and confirmed loss of expression with a single-copy insertion of a selectable marker DHFR-TS. In a pregnant mouse model, NcΔROP24 parasites displayed markedly reduced congenital transmission, higher neonatal survival, and lower maternal brain parasite burdens compared to wild-type controls, demonstrating significant attenuation of systemic and vertical infection. Also, in bovine monocyte-derived macrophages, NcΔROP24 tachyzoites showed impaired intracellular growth. Dual RNA-seq of infected macrophages revealed that NcΔROP24 loss prevents the parasite from reprogramming key host metabolic and degradative pathways, instead promoting a stress-induced, lipogenic state that favours clearance. Concurrently, parasites lacking NcROP24 upregulated stress-associated transcripts and downregulated additional secreted effectors, indicating a shift away from aggressive proliferation. Together, these findings establish NcROP24 as a key factor of N. caninum pathogenicity. By defining its role in host-pathogen interactions, our work highlights NcROP24 as a promising target for next-generation vaccines or therapeutics against bovine neosporosis.},
}
@article {pmid41593356,
year = {2026},
author = {Wan, Y and He, Y and Chen, X and Wang, S and Zhou, G and Ying, X and Zhang, H},
title = {Effective delivery of genome editor to cervical cancer targeting Mcl1 for cancer therapy.},
journal = {Cancer gene therapy},
volume = {33},
number = {4},
pages = {378-389},
pmid = {41593356},
issn = {1476-5500},
mesh = {Humans ; Female ; *Uterine Cervical Neoplasms/genetics/therapy/pathology ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists &amp; inhibitors ; Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Editing/methods ; Apoptosis ; Genetic Therapy/methods ; Cell Line, Tumor ; Extracellular Vesicles/metabolism/genetics ; },
abstract = {CRISPR/Cas9 represents a transformative advancement in precision therapies, offering the promise of more effective and targeted treatment options. However, there are still limitations (including off-target editing as well as unsatisfied delivery tool) which obstruct the wide application of CRISPR/Cas9. Here, an endogenic artificial extracellular vesicles (EVs) system is engineered for effective delivery of Cas9 ribonucleoprotein (RNP). We demonstrated that the endogenic Cas9 RNP were sorted by the Lamp2b and delivered by the artificial EVs, which could markedly inhibit the growth of cervical cancer cells by inducing cell apoptosis. Moreover, artificial endogenic EVs[RNP] (Cas9-Mcl1) could result in remarkable antitumor effects in animal models of cervical cancer through suppressing Mcl1 expression. Our findings indicate that the artificial EVs delivery strategy could deliver Cas9 RNP effectively to inhibit cancer progression, which might be a promising treatment.},
}
@article {pmid41662723,
year = {2026},
author = {De Silva Weligodage, H and Goenaga, R and Gutierrez, OA and Brown, JK},
title = {Development of a Recombinase Polymerase Amplification-CRISPR/Cas12a Detection System for Cacao Mild Mosaic Virus.},
journal = {Phytopathology},
volume = {116},
number = {7},
pages = {1173-1184},
doi = {10.1094/PHYTO-10-25-0319-R},
pmid = {41662723},
issn = {0031-949X},
mesh = {*Cacao/virology ; *Plant Diseases/virology ; Recombinases/metabolism/genetics ; *Caulimovirus/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; DNA Primers/genetics ; Plant Leaves/virology ; Sensitivity and Specificity ; },
abstract = {Plant viruses that cause minimal to no disease symptoms may not support readily detectable virus levels. Such viruses are of concern when they persist in plant germplasm collections or in breeding populations because they can provide an inoculum that can be spread and potentially cause outbreaks in susceptible plant species. The mealybug-transmitted cacao mild mosaic virus (CaMMV) causes symptomatic and asymptomatic infection of cacao trees that varies seasonally. The virus accumulates to low levels in leaves and petioles of at least some cacao genetic groups, which has confounded reliable CaMMV detection. Here, a multiplex recombinase polymerase amplification (RPA) assay was developed to increase the reliability of CaMMV detection. Three RPA primers were designed to amplify two regions of the movement protein (mp) gene of CaMMV, yielding fragments of 362 and 284 bp. To increase the detection sensitivity and specificity of CaMMV, two guide RNAs (20 bp) targeting both the CaMMV RPA amplicons were designed to activate Cas12a-mediated collateral cleavage of a fluorescent reporter. An RPA detection efficiency of 100% was achieved with respect to six known CaMMV mp variants, and the analytical sensitivity ranged from approximately 3 to 40 detectable CaMMV genomes. No signal was observed when cloned cacao-infecting badnavirus sequences or virus-free cacao were used as the template, indicating that this assay is highly specific for CaMMV.},
}
@article {pmid41792998,
year = {2026},
author = {Shin, EJ and Choi, Y and Jeon, EJ and Lee, KI and Lee, KJ and Son, YJ and Son, MJ and Kim, S and Cho, SW and Lee, JY},
title = {Targeted KEAP1 disruption enhances antioxidant defense and mesenchymal stromal cell therapy for chronic limb-threatening ischemia.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3949-3961},
doi = {10.1016/j.ymthe.2026.03.005},
pmid = {41792998},
issn = {1525-0024},
mesh = {*Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Animals ; *Mesenchymal Stem Cells/metabolism/cytology ; Humans ; Mice ; *Mesenchymal Stem Cell Transplantation/methods ; NF-E2-Related Factor 2/metabolism/genetics ; Oxidative Stress ; *Antioxidants/metabolism ; Disease Models, Animal ; Gene Editing ; *Chronic Limb-Threatening Ischemia/therapy/metabolism/genetics ; *Ischemia/therapy/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Reactive Oxygen Species/metabolism ; },
abstract = {Chronic limb-threatening ischemia (CLTI) is a severe vascular disorder characterized by tissue hypoxia and oxidative stress that limit the efficacy of regenerative therapies. Mesenchymal stem/stromal cells (MSCs) hold promise for CLTI treatment through paracrine angiogenic and immunomodulatory signaling, yet their survival and function are compromised in the reactive oxygen species-rich ischemic microenvironment. Here, we utilized CRISPR-Cas9 to generate a targeted knockout of Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of the antioxidant transcription factor NRF2, in human bone marrow-derived MSCs. KEAP1 editing activated the NRF2 pathway, reduced intracellular oxidative stress, and reprogrammed redox and paracrine gene networks. Edited MSCs exhibited enhanced viability, sustained secretion of proangiogenic cytokines, and improved tissue perfusion and arteriogenesis in a murine model of CLTI. These findings establish KEAP1 gene editing as a permanent, integration-free strategy to augment MSC resistance and therapeutic efficacy in oxidative ischemic environments.},
}
@article {pmid41814651,
year = {2026},
author = {Tao, D and Xu, B and Li, S and Liu, H and Wei, Y and Cao, X and Shi, S and Wang, Y and Jiang, R and Zhang, Y and Zhao, C and Ruan, J and Fu, L and Huang, X and Li, X and Zhao, S and Xie, S},
title = {Structural mining and engineering of metagenome-derived Cas12a orthologs expands the CRISPR genome editing and multiplex diagnostics toolkit.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {4104-4120},
doi = {10.1016/j.ymthe.2026.03.011},
pmid = {41814651},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Associated Proteins/genetics/chemistry ; *Metagenome ; Mice ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Swine ; Pathology, Molecular/methods ; },
abstract = {CRISPR-Cas12a is a compact, RNA-guided nuclease widely deployed in genome editing and molecular diagnostics, yet its broader utility is limited by suboptimal cis-cleavage efficiency and incompletely defined trans-cleavage behavior. To overcome these constraints, we developed an artificial intelligence-guided structural discovery pipeline powered by AlphaFold2, which identified 1,261 previously uncharacterized Cas12a orthologs. From this set, 21 structurally conserved but sequence-divergent candidates were selected for biochemical characterization. Using structure-informed engineering, we generated PcuCas12a MAX, a high-fidelity variant that achieves genome-editing efficiencies in human cells comparable to the benchmark AsCas12a Ultra while retaining robust activity in murine and porcine systems. In addition, four orthologs (LcoCas12a, FcaCas12a, EsoCas12a, and Mac2Cas12a), when paired with specifically engineered CRISPR RNAs, exhibited distinct single-stranded DNA trans-cleavage signatures. These properties enabled construction of a multiplex CRISPR sensor capable of simultaneously detecting multiple nucleic acid targets. Together, these findings expand the Cas12a endonuclease repertoire and enhance its utility in genome engineering and next-generation diagnostics.},
}
@article {pmid41814652,
year = {2026},
author = {Iyer, S and Daman, K and Sun, Y and Tutto, A and Holbrook, SE and Joynt, AT and Yan, J and Ambegaokar, P and Guo, D and Liu, P and Stauffer, JE and Maitland, SA and Lee, SM and Xiao, Y and Huang, HC and Zhu, LJ and Gallagher, TL and Cox, GA and Keeler, AM and Siegwart, DJ and Emerson, CP and Wolfe, SA},
title = {SORT LNPs encapsulating Cas9 mRNA achieve efficient editing in skeletal muscle in a dystrophic mouse model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3885-3902},
pmid = {41814652},
issn = {1525-0024},
support = {UH3 TR002668/TR/NCATS NIH HHS/United States ; R01 HL150669/HL/NHLBI NIH HHS/United States ; R01 HL170629/HL/NHLBI NIH HHS/United States ; R01 CA269787/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; R37 AI147868/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Muscle, Skeletal/metabolism ; Disease Models, Animal ; *RNA, Messenger/genetics/administration &amp; dosage ; *Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; Genetic Therapy/methods ; *Muscular Dystrophies, Limb-Girdle/genetics/therapy ; Liposomes ; },
abstract = {Limb girdle muscular dystrophy (LGMD) is the fourth most common type of muscular dystrophy. Gene editing holds promise for treating neuromuscular disorders such as LGMD, but clinical translation remains challenging due to lack of complementary delivery tools for skeletal muscle. Lipid nanoparticles (LNPs) offer a promising platform for transient delivery of gene-editing reagents as mRNA or ribonucleoprotein complexes (RNPs) to skeletal muscle, but editing efficiencies remain modest. While lipid compositions have been optimized to improve delivery to muscle, the impact of cargo type on editing efficiency, biodistribution, and immune response has not been evaluated. Here, we demonstrate that selective organ targeting (SORT) LNPs encapsulating optimized Cas9 cargo facilitate efficient, local delivery to skeletal muscle. Using an LGMDR7 mouse model harboring a mutation in TCAP as a proof-of-concept target, we show that LNP cargo type impacts LNP size, delivery to neighboring muscle groups, and editing efficiency. RNP and mRNA LNPs also provoked distinct innate and adaptive immune responses upon repeated dosing. The optimized SORT LNP platform resulted in 40% restoration of Telethonin expression in treated muscle. Overall, these findings offer valuable insights for the continued development of LNP-based gene-editing reagents to facilitate disease-modifying interventions for neuromuscular diseases.},
}
@article {pmid41877484,
year = {2026},
author = {Ralu, M and Guiraud, S and Dastidar, S and Galbiati, P and Sadaoui, E and Mazed, F and Amor, F and de Cian, A and Richard, I and Mamchaoui, K and Ronzitti, G and Tedesco, FS and Amendola, M},
title = {CRISPR-Cas9-mediated upregulation of utrophin ameliorates Duchenne muscular dystrophy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3903-3923},
doi = {10.1016/j.ymthe.2026.03.025},
pmid = {41877484},
issn = {1525-0024},
mesh = {*Muscular Dystrophy, Duchenne/genetics/therapy/metabolism/pathology ; *Utrophin/genetics/metabolism ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; MicroRNAs/genetics ; Genetic Therapy/methods ; Disease Models, Animal ; Up-Regulation ; Dependovirus/genetics ; Gene Editing ; Muscle, Skeletal/metabolism/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Gene Therapy Agents ; Gene Expression Regulation ; Mice, Inbred mdx ; Binding Sites ; },
abstract = {Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulating utrophin, a dystrophin paralog, is a promising gene therapy approach. Here, we present a CRISPR-Cas9-based strategy to enhance utrophin expression by disrupting repressor binding sites. Using a Cas9/guide RNA (gRNA) ribonucleoprotein complex, we disrupted several such sites in DMD myoblasts and identified microRNA Let-7c binding site as effective in relieving repression of the UTRN gene. Interestingly, Cas9-generated insertions or deletions (indels) were as effective as the complete removal of Let-7c binding site in upregulating UTRN expression, with minimal off-target effects. In a three-dimensional tissue-engineered human skeletal muscle model of DMD, this editing strategy resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, in mdx mice, local or systemic delivery of recombinant adeno-associated viruses encoding Cas9 and gRNA targeting the Let-7c binding site resulted in utrophin upregulation and amelioration of muscle histopathology and function. These findings provide the foundations for a mutation-independent, potentially universal gene-editing therapeutic strategy for DMD.},
}
@article {pmid41935953,
year = {2026},
author = {Fumagalli, M and An, D and Simula, L and Combe, C and Aziez, L and Simoni, Y and Alves-Guerra, MC and Valentini, A and Marchais, M and Vermare, A and Moraly, J and Manni, S and Quadraccia, MC and Quintarelli, C and De Angelis, B and Bercovici, N and Donnadieu, E and Pendino, F},
title = {An in vivo CRISPR screen unveils promising target genes to improve CAR-T cell efficacy in a solid tumor model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3976-4000},
doi = {10.1016/j.ymthe.2026.04.009},
pmid = {41935953},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; Disease Models, Animal ; *Neoplasms/therapy/genetics/immunology ; Xenograft Model Antitumor Assays ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *T-Lymphocytes/immunology/metabolism ; },
abstract = {CAR-T cell therapies are revolutionizing the treatment of refractory or relapsed hematological malignancies, but many patients do not achieve durable responses, and these therapies remain ineffective against solid tumors. Therapeutic failure is closely associated with a poor persistence of CAR-T cells in patients, highlighting the need to identify strategies promoting in vivo expansion. Although numerous gene-editing strategies have been proposed, comparative studies to identify the most effective ones are still lacking. Here, using a focused CRISPR-knockout library targeting 50 selected gene candidates, we developed a competitive screening that revealed ZC3H12A, SOCS1, PTPN2, and CDKN2A as the most robust targets to improve persistence of EGFR CAR-T cells in human lung tumor-bearing mice. Surprisingly, disruption of other genes previously reported to improve CAR-T cell efficacy in other preclinical models-MED12, PRDM1, and BATF-had a detrimental effect in this context. These results suggest that some gene-editing strategies can yield beneficial, neutral, or even deleterious effects on CAR-T cell persistence, depending on specific conditions. Altogether, these findings highlight the importance of performing context-specific evaluations of genetic modifications to accelerate the clinical translation of the most promising editing strategies for optimizing CAR-T cell therapies.},
}
@article {pmid41981915,
year = {2026},
author = {Truong, LB and Li, S and Domkofski, C and Lin, J and Co, C and Halwasia, S and Kuefner, M and Hu, Y and Mao, HQ and Wang, TH and Zhu, S},
title = {Messenger RNA and guide RNA distributions in lipid nanoparticles impact gene-editing efficiency in vivo.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3962-3975},
pmid = {41981915},
issn = {1525-0024},
support = {R01 AI183336/AI/NIAID NIH HHS/United States ; R01 CA260628/CA/NCI NIH HHS/United States ; R01 CA293906/CA/NCI NIH HHS/United States ; },
mesh = {*Nanoparticles/chemistry ; *RNA, Messenger/genetics/chemistry ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Humans ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are among the most advanced non-viral vectors for CRISPR-based gene-editing therapeutics. Co-packaging of messenger RNA (mRNA) and guide RNA (gRNA) inherently produces heterogeneous payload distributions. The impact of this heterogeneity on editing performance remains unclear. Here, we utilize cylindrical illumination confocal spectroscopy (CICS) for single-particle interrogation of ALC-0315 and DLin-MC3-DMA LNPs prepared by three different mixing methods. CICS resolves four distinct subpopulations: co-encapsulated (50.7%-60.4%), gRNA only (30.0%-36.5%), mRNA only (2.0%-3.4%), and empty (4.2%-13.8%), and it uncovers broad, particle-to-particle variability in RNA copy number within each class. Structure-function analysis reveals that LNP formulation and mixing processes influence payload distribution, resulting in a negative correlation between the fraction of empty LNPs and RNA loading per particle. We further investigated the correlation between these quality attributes and therapeutic performance. In mice, ALC-0315 LNPs carrying higher cargo loads (9.8 vs. 8.0 mRNA copies and 25.4 vs. 20.3 gRNA copies per co-encapsulated particle) yielded 1.5-fold higher in vivo editing activity (55.4% vs. 36.3% insertions and deletions [indels]) despite nearly identical biophysical characteristics including LNP size and RNA encapsulation. These results establish payload distribution as a potential determinant of gene-editing potency and demonstrate single-particle CICS as a powerful tool for rational design of multi-component nucleic acid-delivery systems.},
}
@article {pmid42015511,
year = {2026},
author = {He, L and Fu, Y and Wang, Z and Zhou, Q and Sun, H and Wang, H},
title = {In vivo systematic detection of the outcomes of CRISPR-Cas9-mediated DNA repair in skeletal muscle stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3924-3948},
doi = {10.1016/j.ymthe.2026.04.032},
pmid = {42015511},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscle, Skeletal/cytology/metabolism ; *Stem Cells/metabolism/cytology ; *DNA Repair ; Dependovirus/genetics ; Genetic Vectors/genetics ; DNA End-Joining Repair ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRSIPR-associated protein 9 (Cas9) has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profile CRISPR-Cas9 editing outcomes at 95 loci using our established CRISPR-Cas9/adeno-associated virus (AAV)9-single guide RNA (sgRNA) system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR-Cas9-mediated editing in vivo largely align with those observed in vitro. In addition to the anticipated small editing insertions or deletions (indels), such as microhomology-mediated end joining (MMEJ)-mediated deletions and non-homologous end joining (NHEJ)-mediated templated insertions, we uncover a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by 3D chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo, we identify their distinct roles in regulating the large on-target modifications. Together, our work systematically profiles CRISPR-Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR-Cas9-based gene therapies.},
}
@article {pmid42031729,
year = {2026},
author = {Moon, HC and Herschl, MH and Sclip, A and Tran, VQ and Pawluk, A and Konermann, S and Hsu, PD},
title = {A combinatorial domain screening platform reveals epigenetic effector interactions for transcriptional perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42031729},
issn = {2041-1723},
mesh = {Humans ; *Epigenesis, Genetic ; DNA Methylation ; Epigenome Editing ; *Transcription, Genetic ; Protein Domains/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetic regulation involves the coordinated interplay of diverse proteins. To systematically explore these combinations, we present COMBINE (combinatorial interaction exploration), a high-throughput platform that tests over 50,000 pairs of epigenetic effector domains up to 2,094 amino acids in length for their ability to modulate endogenous human gene transcription. COMBINE reveals diverse synergistic interactions between epigenetic protein domains, including a potent KRAB-L3MBTL3 fusion that increases the effective targeting window, enhances gene silencing in dose-limited conditions, and enables robust dual-directional CRISPR perturbation. Inducible screening shows DNA methylation modifiers are essential for epigenetic memory, with distinct combinations driving long-term repression and activation. This systematic analysis of pairwise domain interactions advances our understanding of epigenetic crosstalks and the development of next-generation epigenome editing tools. More broadly, COMBINE offers a generalizable platform to functionally characterize combinatorial biological processes at scale.},
}
@article {pmid42049824,
year = {2026},
author = {Galindo-González, L and Dupras, AA},
title = {CRISPR-Cas12a fluorescence assays identify weedy Amaranthus species.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42049824},
issn = {2045-2322},
support = {N-000470//Canadian Food Inspection Agency/ ; },
mesh = {*Amaranthus/genetics/classification ; *CRISPR-Cas Systems ; Fluorescence ; *Plant Weeds/genetics/classification ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {Tall Waterhemp (Amaranthus tuberculatus) and Palmer Amaranth (Amaranthus palmeri) populations have developed multiple-herbicide resistance and impact the production of important crops, including corn, soybean and cotton. Morphological plasticity of these species, their persistence in the soil seed bank and their presence as contaminants during trade require efficient and sensitive methodologies to support their identification. Cas12 enzymes can be directed to a specific genomic region by a crRNA, cutting the DNA double strand and exhibiting collateral enzymatic activity on free-floating single-stranded DNA. This characteristic can be used to generate single-stranded oligonucleotide reporters bearing a fluorophore and a quencher, which will produce fluorescence when cut by Cas12. We designed CRISPR-Cas12 fluorescent assays to differentiate A. tuberculatus, A. palmeri and A. palmeri`s sister species (A. watsonii) from other Amaranthus species. Our assays for identifying the A. palmeri + A. watsonii clade and A. tuberculatus were 100% accurate when presented with blind samples of 14 Amaranthus species. Fluorescence could be detected using a blue light filter on a transilluminator within minutes. A preliminary recombinase amplification step to increase the limit of detection, and the Cas12 reaction, can be performed at room temperature and within an hour once DNA has been isolated.},
}
@article {pmid42055327,
year = {2026},
author = {Mazur, CM and Kotsalidis, PE and George, M and Whalley, T and Sato, T and Doench, JG and Surface, LE and Wein, MN},
title = {Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {6},
pages = {113075},
pmid = {42055327},
issn = {1083-351X},
mesh = {*Osteocytes/cytology/metabolism ; Animals ; Mice ; *Microtubule-Associated Proteins/genetics/metabolism ; Cell Differentiation/genetics ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics ; Extracellular Matrix Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Osteocytes play critical roles in bone, making them attractive targets for therapeutics aimed at improving bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here, we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61[high] cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule-binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify the role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.},
}
@article {pmid42134180,
year = {2026},
author = {Lee, H and Jeon, BJ and Jang, HS and Choi, JY and Jang, G},
title = {Non-surgical deep uterine embryo transfer combined with electroporation-based genome editing enables scalable production of CD163-Knockout pigs.},
journal = {Theriogenology},
volume = {263},
number = {},
pages = {117977},
doi = {10.1016/j.theriogenology.2026.117977},
pmid = {42134180},
issn = {1879-3231},
mesh = {Animals ; *Embryo Transfer/veterinary/methods ; *Electroporation/veterinary/methods ; *Gene Editing/veterinary/methods ; Female ; Swine/genetics ; *Antigens, CD/genetics/metabolism ; Animals, Genetically Modified ; *Antigens, Differentiation, Myelomonocytic/genetics/metabolism ; CD163 Antigen ; *Receptors, Cell Surface/genetics ; CRISPR-Cas Systems ; *Gene Knockout Techniques/veterinary ; Pregnancy ; Electroporation Therapies ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) remains the most economically devastating viral disease in the global swine industry, causing annual losses exceeding $600 million in the United States alone. While CD163-knockout (KO) pigs have demonstrated complete resistance to the PRRS virus (PRRSV) infection, conventional production methods relying on microinjection-based genome editing and surgical embryo transfer present significant bottlenecks for industrial-scale application. Here, we demonstrate that the integration of electroporation-based CRISPR-Cas9 delivery with non-surgical deep intrauterine embryo transfer enables scalable production of CD163-KO pigs. Our approach achieved successful pregnancies and live births of genome-edited piglets without requiring surgical intervention or specialized microinjection expertise. Genotyping analysis revealed complete biallelic KOs in a subset of offspring, while others exhibited mosaic patterns reflecting the stochastic nature of electroporation-mediated editing. These findings establish a technically accessible and scalable pipeline for producing gene-edited pigs. By eliminating the need for complex surgical facilities and specialized microinjection equipment, this approach provides a practical and field-applicable strategy that can be readily implemented in standard commercial swine farms, representing a critical step toward the widespread deployment of genome-edited livestock in global agriculture.},
}
@article {pmid42319782,
year = {2026},
author = {Liu, Z and Li, M and Wang, J and Chen, B},
title = {Copper-only Superoxide dismutase 6 contributes to reactive oxygen species regulation, genotoxic stress tolerance, and virulence in Candida albicans.},
journal = {Medical mycology},
volume = {64},
number = {7},
pages = {},
doi = {10.1093/mmy/myag063},
pmid = {42319782},
issn = {1460-2709},
support = {2023YXNS187//Development Plan of Jining/ ; 202411000427//Medical and Health Science and Technology Development Project of Shandong, China/ ; SDYWZGKCJHLH2023079//Shandong medical staff science and technology innovation plan project/ ; },
mesh = {*Candida albicans/pathogenicity/genetics/enzymology ; *Reactive Oxygen Species/metabolism ; Virulence ; Animals ; Oxidative Stress ; *Superoxide Dismutase/genetics/metabolism ; Mice ; Candidiasis/microbiology ; *DNA Damage ; Disease Models, Animal ; Gene Expression Profiling ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; },
abstract = {Superoxide dismutase (SOD) is a major antioxidant enzyme that protects cells against reactive oxygen species (ROS)-mediated oxidative stress. The identification of a fungal-specific copper-only SOD family in Candida albicans (C. albicans) has revealed a previously unrecognized component of fungal oxidative stress defence, yet the role of SOD6 remains unclear. Here, we generated a sod6Δ/Δ mutant in C. albicans using a transient CRISPR/Cas9 approach and examined the contribution of SOD6 to virulence, ROS homeostasis, oxidative stress resistance, and genome maintenance. Compared with the wild-type (WT) strain, the sod6Δ/Δ mutant showed attenuated virulence in both the Galleria mellonella (G. mellonell) infection model and a murine systemic candidiasis model. This virulence defect occurred without detectable alterations in hyphal formation or other major pathogenicity-associated traits. Instead, the mutant accumulated higher intracellular and extracellular ROS levels and displayed increased sensitivity to hydrogen peroxide, zeocin, and camptothecin (CPT), consistent with impaired oxidative stress adaptation and genome maintenance. Transcriptomic analysis further revealed downregulation of genes involved in DNA replication and repair, with dpb4, which encodes a subunit of DNA polymerase epsilon required for DNA replication and genome stability, showing the greatest reduction. Together, these findings identify SOD6 as an important determinant of oxidative stress adaptation, genome maintenance, and pathogenic fitness in C. albicans.},
}
@article {pmid42349956,
year = {2026},
author = {Dutta, S and Pal, A and Srivatsan, SG},
title = {Chemoenzymatic labeling of RNA using terminal uridylyl transferase and bioorthogonal click chemistry.},
journal = {Methods in enzymology},
volume = {731},
number = {},
pages = {223-252},
doi = {10.1016/bs.mie.2026.05.007},
pmid = {42349956},
issn = {1557-7988},
mesh = {*Click Chemistry/methods ; *RNA Nucleotidyltransferases/metabolism/chemistry ; Azides/chemistry ; Uridine Triphosphate/chemistry/analogs &amp; derivatives ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics ; *RNA/chemistry ; Staining and Labeling/methods ; *Nucleotidyltransferases/metabolism ; CRISPR-Cas Systems ; Schizosaccharomyces pombe Proteins ; },
abstract = {The ability of native and engineered nucleic acid-processing enzymes to incorporate clickable nucleotide substrates has greatly advanced bioorthogonal labeling of nucleic acids, overcoming the limitations of conventional solid-phase oligonucleotide (ON) synthesis. In this chemoenzymatic approach, template-dependent polymerases routinely enable the incorporation of nucleotides bearing small reactive handles. The resulting nucleic acids undergo chemoselective reactions, such as azide-alkyne cycloaddition, inverse-electron-demand Diels-Alder, or Staudinger ligation, to install desired functionalities. Alternatively, the promiscuity of template-independent transferases, such as terminal uridylyl transferase (TUTase), provides access to site-specific labeling of RNA ONs at the 3'-end. In this methods chapter, we detail protocols for incorporating azide-modified UTP analogs into short RNA ONs and highly structured CRISPR guide RNAs (sgRNAs) using the terminal uridylyl transferase SpCID1. We describe methods to control the enzyme's incessant incorporation behavior and enable subsequent click functionalization of the RNAs. Finally, we demonstrate remodeling of the CRISPR system via synthesis of azide-modified sgRNAs, which when complexed with dCas9, recruit azide groups to specific gene targets for post-hybridization functionalization.},
}
@article {pmid42351104,
year = {2026},
author = {Zhu, Y and Bi, Z and Zhang, Z and Zhang, M and Du, Q and Hu, M and Zhou, T and Fan, Y and Zhang, S and Wang, G and Liu, G},
title = {Virus-like particles in cancer immunotherapy: bridging human and veterinary medicine through one health.},
journal = {Journal of nanobiotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12951-026-04703-9},
pmid = {42351104},
issn = {1477-3155},
support = {2024M753586//the China Postdoctoral Science Foundation/ ; 2026JB09//the Central Public-interest Scientific Institution Basal Research Fund/ ; 32473001//the National Natural Science Foundation of China/ ; 2023YFD1800700//the National Key Research and Development Program of China/ ; },
abstract = {Virus-like particles (VLPs) are engineered nanoplatforms that mimic viral structures, offering high immunogenicity, biocompatibility, and functional versatility for cancer immunotherapy. While widely explored in human oncology as nanovaccines and targeted delivery systems for chemo-/immuno-therapeutics and genetic payloads (e.g., mRNA, siRNA, and CRISPR/Cas systems), their potential in veterinary oncology remains underexploited. This review synthesizes recent advances in VLP design, including scaffold engineering, antigen display, cargo encapsulation, and surface functionalization, and discusses the mechanistic basis of VLP-induced antitumor immunity, encompassing dendritic cell activation, adaptive immune amplification, and tumor microenvironment remodeling. Importantly, we highlight the emerging role of companion animals with spontaneous tumors-such as lymphoma, melanoma, and mammary carcinoma-as immunocompetent translational models within the One Health framework. Comparative oncology reveals striking parallels in oncogenic pathways, immune landscapes, and therapeutic responses, supporting the use of canine and feline cancers as biologically relevant intermediates between murine studies and human clinical trials. We provide an evidence-based assessment of representative VLP platforms, evaluate their translational readiness, and examine cross-species opportunities for shared target development, biomarker discovery, and regulatory convergence, while also addressing species-specific biological and technical limitations. Finally, we propose a forward-looking roadmap that prioritizes manufacturing standardization, biomarker development, comparative validation, precision engineering, and emerging technologies such as AI-guided design and tumor-on-chip systems. Collectively, we position One Health as an operational strategy to accelerate the bidirectional translation of VLP-based immunotherapies for both human and veterinary cancer patients.},
}
@article {pmid42352260,
year = {2026},
author = {Kuroda, T and Yokota, T},
title = {RNA Therapeutics Targeting Skeletal Muscle: Emerging Antisense and Gene-Modifying Strategies.},
journal = {Biomolecules},
volume = {16},
number = {6},
pages = {},
pmid = {42352260},
issn = {2218-273X},
mesh = {Humans ; *Oligonucleotides, Antisense/therapeutic use/genetics ; *Muscle, Skeletal/metabolism/drug effects ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Animals ; *Genetic Therapy/methods ; RNA, Small Interfering/therapeutic use/genetics ; Morpholinos/therapeutic use ; },
abstract = {RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides-eteplirsen, golodirsen, viltolarsen, and casimersen-have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies-including base and prime editing-offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships.},
}
@article {pmid42352295,
year = {2026},
author = {McGill, LP and Banas, KH and Tiesi, G and Kmiec, EB},
title = {Genotypic and Phenotypic Diversity as a Function of CRISPR-Directed Gene Knock-Out of NRF2 in Pancreatic Adenocarcinoma Cells, a Feasibility Study.},
journal = {Biomolecules},
volume = {16},
number = {6},
pages = {},
pmid = {42352295},
issn = {2218-273X},
mesh = {Humans ; *NF-E2-Related Factor 2/genetics/metabolism ; *Pancreatic Neoplasms/genetics/pathology ; Cell Line, Tumor ; Phenotype ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Drug Resistance, Neoplasm/genetics ; *Adenocarcinoma/genetics/pathology ; Genotype ; Feasibility Studies ; Gemcitabine ; Deoxycytidine/analogs &amp; derivatives/pharmacology ; *Carcinoma, Pancreatic Ductal/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Survival/drug effects ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) presents unique treatment challenges, often due to the development of anti-cancer drug resistance. Previously, we demonstrated that CRISPR-directed gene ablation disabled the master regulator gene NRF2, a transcription factor known to control drug resistance in squamous cell carcinoma tumor cells, and restored chemosensitivity. In this short study, we evaluated a broad range of CRISPR/Cas9 molecules for their capacity to elicit similar responses in PDAC cells. Synthetic single guide RNAs (sgRNAs) were designed to target multiple functional domains encoded by NRF2. These molecules were delivered to cells via nucleofection, with outcomes analyzed by genotypic, phenotypic, and functional assays. We observed targeting efficiencies ranging from 25% to 100% with a high level of random insertions and deletions (indels). sgRNAs targeting exons 2, 3 and 4 demonstrated a high degree of genotypic, phenotypic and functional outcomes. Targeted disruption of exons 3 and 4 reveals significant loss of cell viability while overcoming drug resistance through the restoration of sensitivity to gemcitabine (>1.75 μM). Our study identifies domain-specific sites within NRF2 that, when disabled, restore sensitivity to gemcitabine, potentiating a more in-depth analysis of this novel augmentative therapeutic approach.},
}
@article {pmid42353201,
year = {2026},
author = {Walflor, HSM and Medeiros, LCS},
title = {Machine Learning for CRISPR-Based Diagnostics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353201},
issn = {1422-0067},
mesh = {Humans ; *Machine Learning ; *CRISPR-Cas Systems ; Deep Learning ; Predictive Learning Models ; Classification Algorithms ; },
abstract = {CRISPR-based diagnostics now detect viral, bacterial, and cancer-associated nucleic acids with sensitivities approaching quantitative PCR; however, their translation to decentralized care rests on computational design and interpretation that current datasets cannot sustain. Pandemic-era Cas12a assays reached 95% positive predictive agreement against reverse transcription quantitative PCR (RT-qPCR) at 10 copies/μL, and deep neural networks now design Cas13 detection assays spanning 1933 vertebrate-infecting viruses, ranking candidate guides at Spearman correlations of 0.69 to 0.84 across internal and external validation. Generative deep-learning systems improve single-nucleotide discrimination two- to three-fold, computer vision classifies lateral flow outputs at 96.5% accuracy, and multi-biomarker fusion reaches an area under the receiver operating characteristic curve (AUC) of 0.998 in lung cancer detection. These results mask a narrow data foundation. Cas13a guide prediction still draws from a single screening library of 19,209 guide-target pairs, Cas12a has one published diagnostic model, and signal classifiers almost uniformly validate on single-site cohorts. This review synthesizes mechanistic constraints, predictive and generative models, and point-of-care classifiers, and maps the path beyond this data ceiling. Evolutionary pretraining on RNA corpora and lab-in-the-loop agents that convert model failure into targeted data acquisition define the route forward.},
}
@article {pmid42353287,
year = {2026},
author = {Kim, SJ and Nam, YH and Joo, EY and Park, J and Park, S and Jung, SC and Jin, DK},
title = {Systemic AAV-hGCDH Gene Therapy Alleviates Glutaric Acid Accumulation and Attenuates Chronic Brain Vacuolation in a Novel Mouse Model of Glutaric Aciduria Type I.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353287},
issn = {1422-0067},
support = {75440-1//Inha University/ ; },
mesh = {Animals ; *Dependovirus/genetics ; *Genetic Therapy/methods ; *Glutaryl-CoA Dehydrogenase/deficiency/genetics/metabolism ; Mice ; Disease Models, Animal ; *Amino Acid Metabolism, Inborn Errors/therapy/genetics/metabolism/pathology ; *Brain Diseases, Metabolic/therapy/genetics/metabolism/pathology ; *Brain/pathology/metabolism ; *Glutarates/metabolism ; Genetic Vectors/genetics/administration &amp; dosage ; Gene Therapy Agents ; Male ; CRISPR-Cas Systems ; Liver/metabolism ; },
abstract = {Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder caused by glutaryl-CoA dehydrogenase (GCDH) deficiency, leading to the accumulation of neurotoxic metabolites that can cause both acute encephalopathic crises and progressive, insidious brain injury. Current management primarily relies on a protein-restricted diet, which remains therapeutically insufficient and burdensome for patients, highlighting the need for disease-modifying therapies. In this study, we established a novel GA1 mouse model using CRISPR/Cas9 technology and evaluated the preclinical efficacy of systemic recombinant adeno-associated virus (rAAV)-mediated gene therapy. Under standard dietary conditions without high-lysine challenge, our GA1 model exhibited sustained cerebral and hepatic glutaric acid (GA) accumulation and distinct chronic vacuolation in the hippocampus and cerebellum, mirroring the insidious-onset GA1 phenotype. Five-week-old mice received a single intravenous injection of rAAV-hGCDH using either rAAV2/8 or rAAV2/9 serotypes. Systemic rAAV-mediated gene therapy significantly reduced GA accumulation and attenuated chronic neuropathological changes in this GA1 mouse model for both serotypes. Our findings support the hypothesis that peripheral metabolic correction may play an important role in preventing the chronic neuropathological changes associated with GCDH deficiency. However, further investigation using tissue-specific expression systems is required to definitively delineate the relative contributions of hepatic versus central GCDH restoration to the observed neuroprotection.},
}
@article {pmid42353300,
year = {2026},
author = {Afifi, N and Colussi, D and Perez-Leal, O},
title = {CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353300},
issn = {1422-0067},
support = {1R21HG012241-03/NH/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Gene Editing/methods ; *Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology.},
}
@article {pmid42353328,
year = {2026},
author = {Jan, R and Iqbal, S and Ali, S and Almalki, MA and Alfredan, M and Ibrahim, RIH and Asaf, S and Kim, KM},
title = {Early Flowering (ELF) Gene Integrates Vegetative Growth, Flowering Regulation, and Reproductive Development in Arabidopsis thaliana.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353328},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics/growth &amp; development ; *Flowers/genetics/growth &amp; development ; *Arabidopsis Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Reproduction/genetics ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Germination/genetics ; Gene Editing ; },
abstract = {Early flowering-related factors play pivotal roles in coordinating plant growth and reproductive development. In this study, we investigated the biological function of early flowering gene (ELF) in Arabidopsis thaliana using CRISPR/Cas9-mediated genome editing and construction of overexpression approaches. Two independent ELF overexpression (OE-ELF) and genome-edited (ge-elf) lines were generated and systemically analyzed. ELF overexpression significantly enhanced early seedling performance, increasing germination rate and seedling fresh weight by up to 8.7%, while genome-edited lines exhibited a marked reduction. Root growth was strongly promoted in OE-ELF plants, with root length increase of 85% and 75%, whereas ge-elf lines showed a reduction of up to 48%. At later developmental stages, OE-ELF plants displayed enhanced vegetative growth, including increased leaf length (32%), leaf area (91%), and accelerated flowering (21% earlier than wild type). In contrast, ge-elf delayed flowering by up to 25% and resulted in compact plant architecture. Reproductive development was severely compromised in ge-elf plants, which exhibited malformed inflorescences, reduced pollen germination, shortened silique (45%), and a drastic decrease in seed number per silique (70%). Conversely, OE-ELF plants showed increased silique number and seed per silique. Molecular analysis revealed that ELF positively regulates key flowering-related genes, including FLC, SOC1, AP1, and LFY, which correlated strongly with growth and reproductive traits. Our results demonstrate that ELF functions as a central regulator integrating vegetative growth, floral development, male fertility, and seed production in Arabidopsis thaliana.},
}
@article {pmid42087135,
year = {2026},
author = {Ouyang, M and Wang, J and Luo, X and Tian, R},
title = {CaRPOOL: a pooled calcium‑recording CRISPR screening platform identifies CCR7 as a modulator of cellular osmomechanosensing.},
journal = {Cell communication and signaling : CCS},
volume = {24},
number = {1},
pages = {},
pmid = {42087135},
issn = {1478-811X},
support = {2024YFA0919800//National Key Research and Development Program of China/ ; A2303039//Shenzhen Medical Research Fund/ ; 2023B151502007//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *Receptors, CCR7/metabolism/genetics ; *Calcium/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Calcium Signaling ; *CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Cells must continuously sense and respond to environmental changes by translating physical and chemical cues into intracellular signals. However, systematic discovery of genes governing these sensory processes has been limited by the transient nature of signaling events and the low throughput of measurement assays. Here, we present CaRPOOL, a pooled, high‑throughput genetic screening platform that integrates the calcium‑activity recorder CaMPARI2 with CRISPR interference (CRISPRi), enabling stable capture of transient calcium signals for genome‑scale functional screening. Using osmomechanical stimulation as a model, we demonstrate that CaMPARI2 photoconversion faithfully reports stimulus‑dependent calcium responses and supports pooled fluorescence‑activated cell sorting (FACS)-based screening. A CRISPRi library targeting membrane‑associated genes identified both known and previously uncharacterized regulators of osmomechanosensing, including the chemokine receptor CCR7. Mechanistic analyses revealed that CCR7 promotes osmomechanical calcium signaling through a PIEZO1‑dependent Gαs-cAMP-PKA pathway, establishing it as a GPCR regulator of osmomechanical response. Notably, osmotic stress upregulated CCR7 expression in immune cell lines and enhanced osmomechanical responsiveness, suggesting a role in osmomechanical adaptation. Together, these findings introduce a broadly applicable platform for high‑throughput discovery of genes controlling dynamic signaling responses and reveal a GPCR-ion channel crosstalk mechanism in osmomechanotransduction with potential implications for immune cell mechanoadaptation.},
}
@article {pmid42258637,
year = {2026},
author = {Wei, G and Huang, Z and Wang, S and Zheng, F and Yao, J and Jin, Q and Zhang, Y and Luo, H and Zhang, X and He, Y and Zhou, M and Gu, M and Mao, C and Wang, Z},
title = {Improving low-phosphate tolerance via tissue-specific CRISPR/Cas9 knockout to balance growth and stress responses in rice.},
journal = {The Plant cell},
volume = {38},
number = {6},
pages = {},
doi = {10.1093/plcell/koag170},
pmid = {42258637},
issn = {1532-298X},
support = {2021YFF1000402//National Key Research and Development Program of China/ ; LR24C150001//Natural Science Foundation of Zhejiang Province, China/ ; 92581113//National Natural Science Foundation of China/ ; 32170262//National Natural Science Foundation of China/ ; 226-2024-00102//Fundamental Research Funds for the Central University/ ; },
mesh = {*Oryza/genetics/physiology/growth &amp; development/metabolism ; *Phosphates/metabolism/deficiency ; *CRISPR-Cas Systems/genetics ; Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Gene Knockout Techniques ; Plant Proteins/genetics/metabolism ; Plant Roots/genetics/metabolism ; },
abstract = {Balancing growth and stress responses is critical for improving crop stress tolerance. Inorganic phosphate (Pi) deficiency reduces agricultural yields. Plants have evolved a Pi-starvation response (PSR) network that coordinates growth and responds to fluctuating environmental Pi levels. Null mutations or whole-plant knockdown of PSR repressor genes, such as PHOSPHATE2 (OsPHO2) and OsSPX (Syg1, Pho81, XPR1) family genes, enhance Pi absorption and transfer but disrupt Pi homeostasis, inhibiting growth and reducing yields. To overcome this, we developed a CRISPR/Cas9 tissue-specific knockout (TSKO) system for efficient, vascular-specific somatic knockout of OsPHO2 in rice (Oryza sativa) cv. "Nipponbare" across several generations. The plants showed moderately increased Pi concentrations, maintained Pi homeostasis in hydroponic culture, and increased effective tiller number and grain yield in a Pi-deficient paddy. Vascular-specific OsPHO2 knockout moderately increased OsPHO2-repressed, vascular-expressed, Pi-starvation-induced signaling in roots and alleviated disordered PSR in roots and leaves. Vascular-specific knockout of OsPHO2 or OsSPX1/2 in the Zhonghua 11 background gave similar results. Field trials confirmed the enhanced low-Pi tolerance of TSKO plants in a Pi-deficient paddy and these plants showed normal growth in a Pi-sufficient paddy. This highlights the utility of improving rice low-Pi tolerance via a tissue-specific CRISPR/Cas9 knockout, provides insights into the role of vascular tissues in PSR, and offers a promising spatial-targeting strategy for crop improvement.},
}
@article {pmid42347182,
year = {2026},
author = {Feng, C and Yin, J},
title = {Advances in CRISPR-Cas for Diagnosis and Treatment of Klebsiella pneumoniae.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42347182},
issn = {2076-0817},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Klebsiella Infections/diagnosis/therapy/drug therapy/microbiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Gene Editing/methods ; Drug Resistance, Bacterial ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant pathogen for both hospital-acquired and community-acquired infections, characterized by its strong epidemic potential and high mortality rate, posing a severe threat to global public health. CRKP spreads widely across the globe through the horizontal transfer of plasmid-mediated resistance genes such as *blaKPC*, *blaNDM*, and *blaOXA-48*. The clinical treatment options for this bacterium are limited, and its resistance has been increasing year by year, urgently necessitating the development of new antimicrobial drugs or alternative strategies. In recent years, the CRISPR-Cas system has shown great potential in the diagnosis and treatment of CRKP, including rapid detection and identification, gene editing, antimicrobial strategies, and resistance inhibition. For instance, CRISPR-Cas12a/13a can be used for the rapid detection and identification of CRKP, while CRISPR-Cas9/Cas3 can target resistance genes to reverse the resistance of strains. With the advancement of delivery and biotechnologies, the CRISPR-Cas system is expected to become an important tool against drug-resistant CRKP. This review focuses on the application of the CRISPR-Cas system in the detection and treatment of CRKP, analyzing its technical advantages, limitations, and future development directions.},
}
@article {pmid42348283,
year = {2026},
author = {Ma, W and Shi, S and Ding, G and Ye, X and Wang, S and Xu, F},
title = {CRISPR/Cas9-mediated mutation of BnaA5.JAR1 alleviates boron deficiency stress by enhancing calcium-pectin cross-linking in rapeseed.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {6},
pages = {e71006},
doi = {10.1111/tpj.71006},
pmid = {42348283},
issn = {1365-313X},
support = {32372805//National Natural Science Foundation of China/ ; 31972483//National Natural Science Foundation of China/ ; 2022YFD1900705//National Key Research and Development Program of China/ ; },
mesh = {*Pectins/metabolism ; *Boron/deficiency/metabolism ; *Calcium/metabolism ; *Brassica napus/genetics/metabolism/physiology ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; Mutation ; Oxylipins/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Boron (B) is an essential micronutrient critical for plant growth and reproductive development, primarily through its role in the cell wall. Calcium (Ca[2+]) similarly stabilizes cell wall architecture by forming cross-links with de-esterified pectin. In Arabidopsis thaliana, B deficiency rapidly induces JASMO0NATE RESISTANT 1 (JAR1) and jasmonic acid (JA) accumulation, which negatively regulates growth. However, whether and how JAR1-mediated JA signaling modulates cell wall B and Ca[2+] partitioning to confer B-deficiency tolerance remains unclear. Here, we characterized the function of BnaA5.JAR1 in rapeseed (Brassica napus L.) under B deficiency. Pharmacological inhibition of JA biosynthesis with ibuprofen partially alleviated B-deficiency-induced shoot growth inhibition. BnaA5.JAR1 transcript levels were rapidly and strongly induced by B deprivation. CRISPR/Cas9-mediated knockout of BnaA5.JAR1 enhanced tolerance to B deficiency, whereas overexpression increased sensitivity, despite unchanged leaf B concentrations. Notably, mutant lines maintained robust tolerance throughout the reproductive stage, effectively rescuing the 'flowering without seed setting' phenotype characteristic of B deficiency. Under B limitation, these knockout lines retained higher B and Ca[2+] concentrations in alkali-soluble pectin, exhibited a lower degree of pectin methylesterification, and maintained thinner, more structurally normal cell walls compared with overexpression lines. Exogenous Ca[2+] mitigated B deficiency symptoms without increasing leaf B concentration. Collectively, these findings establish BnaA5.JAR1 as a negative regulator of B-deficiency tolerance and demonstrate that suppressing its activity enhances Ca[2+]-mediated cell wall stabilization across vegetative and reproductive stages, offering a targeted molecular strategy for breeding B-efficient rapeseed cultivars.},
}
@article {pmid42348614,
year = {2026},
author = {Lee, S and Song, J and Kim, Y},
title = {Broad-spectrum antifungal activity and genome-guided characterization of Paenibacillus polymyxa CACC1094 isolated from the bovine rumen.},
journal = {PloS one},
volume = {21},
number = {6},
pages = {e0350885},
pmid = {42348614},
issn = {1932-6203},
mesh = {Animals ; Cattle ; *Antifungal Agents/pharmacology/metabolism ; *Paenibacillus polymyxa/genetics/isolation &amp; purification/metabolism ; *Rumen/microbiology ; Phylogeny ; *Genome, Bacterial ; Multigene Family ; },
abstract = {While Paenibacillus polymyxa is widely recognized for its biocontrol capabilities, most characterized strains originate from soil or rhizosphere environments, leaving animal-associated populations largely unexplored. In this study, we report the isolation of P. polymyxa strain CACC1094 from the bovine rumen and its genome-guided characterization to investigate its biosynthetic potential and antifungal activity. Whole-genome sequencing yielded a complete circular chromosome of 5.55 Mb with a GC content of 45.36%, comprising 5,099 coding sequences, 39 rRNA genes, and 111 tRNA genes. Comparative phylogenomic analysis placed CACC1094 within the P. polymyxa species complex, clustering most closely with the rumen-associated strain ND24 (ANI: 98.31%) and strain 188 (ANI: 96.92%), while forming a distinct branch within the species. Genome mining identified 13 biosynthetic gene clusters, including those associated with fusaricidin and tridecaptin biosynthesis, a paenicidin-like lanthipeptide cluster, and a hybrid NRPS-PKS cluster. In dual-culture assays, CACC1094 showed broad in vitro antifungal activity against multiple plant-pathogenic fungi and oomycetes, as well as selected fungal and yeast pathogens of clinical and veterinary relevance. LC-QTOF/MS analysis of culture supernatants confirmed the production of fusaricidin A and fusaricidin B, providing direct experimental validation of genome-derived predictions. Additionally, genome annotation revealed a complete CRISPR-Cas adaptive immune system, suggesting an integrated ecological strategy that combines antimicrobial biosynthesis with defense against mobile genetic elements. Together, these findings identify CACC1094 as a rumen-associated P. polymyxa strain with broad-spectrum antifungal activity and experimentally validated fusaricidin production, highlighting its potential as a source of antifungal metabolites for agricultural and veterinary applications.},
}
@article {pmid41944382,
year = {2026},
author = {Guan, X and Guo, C and Zhang, J and Yang, R and Ramachandra, Y and Hou, C and Pei, M and Zhang, S and Schalper, KT and Liu, X and Wu, Q and Bulsara, KR and Liu, C},
title = {SDS-CRISPR for Single-Nucleotide Variant Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {36},
pages = {e75149},
doi = {10.1002/advs.75149},
pmid = {41944382},
issn = {2198-3844},
support = {U01AI148306/GF/NIH HHS/United States ; R01AI194917/GF/NIH HHS/United States ; //UConn Research Excellence Program award/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Glioma/genetics/diagnosis ; Isocitrate Dehydrogenase/genetics ; High-Throughput Nucleotide Sequencing/methods ; Mutation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR-Cas12a system offers a promising platform for simple and sensitive nucleic acid diagnostics, including tumor-associated variant detection and infectious agent identification. However, its intrinsic mismatch tolerance limits its ability to accurately detect single-nucleotide variants (SNVs). Here, we introduce Structure-Disruption-Sensitive CRISPR (SDS-CRISPR), a programmable CRISPR-Cas12a approach that achieves highly precise allele discrimination. Guided by AlphaFold3 modeling and bioinformatic analysis, we uncover how split structural design and ionic modulation reconfigure Cas12a conformations, elucidating the structural basis of SNV discrimination in SDS-CRISPR. We apply SDS-CRISPR to detect IDH1[WT] and IDH1[R132H] alleles with attomole sensitivity and 0.01% variant frequency. To facilitate intraoperative use, we combine SDS-CRISPR with a lateral-flow strip and an artificial intelligence-assisted smartphone reader, enabling on-site detection within 20 min. Clinical validation with 43 glioma tissue samples shows high concordance with immunohistochemistry, while plasma cfDNA testing demonstrates mutation fractions consistent with next-generation sequencing. Beyond glioma, SDS-CRISPR generalizes across molecular targets, discriminating microRNA isoforms and identifying HIV-1 drug-resistance mutations. Together, these results establish SDS-CRISPR as a universal, mechanistically informed, and clinically actionable framework for precision molecular diagnostics.},
}
@article {pmid42069060,
year = {2026},
author = {Cobos-Figueroa, L and Pintor-Poveda, A and Mir, C and Melamed, D and Admon, A and Lauzurica, P and Lorente, E},
title = {Innovative CRISPR/Cas9-Based Strategy for Allele-Specific HLA Peptidome Analysis Using a Pan-HLA Antibody.},
journal = {Molecular &amp; cellular proteomics : MCP},
volume = {25},
number = {6},
pages = {101578},
pmid = {42069060},
issn = {1535-9484},
mesh = {Humans ; Alleles ; *CRISPR-Cas Systems/genetics ; *Peptides/metabolism ; *Antibodies/metabolism/immunology ; *Proteomics/methods ; Tandem Mass Spectrometry ; Cell Line ; *HLA Antigens/genetics/metabolism ; Proteome/metabolism ; },
abstract = {Human leukocyte antigen (HLA) immunopeptidomics is restricted by the limited availability of allele-specific antibodies and by potential artifacts introduced by HLA overexpression systems. To address these challenges, we developed a CRISPR/Cas9-based strategy that selectively deletes undesired classical class I alleles while preserving a single endogenous allele, thereby enabling allele-resolved peptidome profiling with a pan-HLA class I antibody. As a proof of concept, we edited JY cells to eliminate HLA-B∗07:02 and HLA-C∗07:02 while retaining HLA-A∗02:01 (ΔBC clones). Peptide-HLA complexes were immunoprecipitated from WT and ΔBC clones using either the pan-HLA class I antibody W6/32 or the A∗02:01-specific antibody PA2.1, followed by nanoLC-MS/MS and computational HLA assignment. Deletion of HLA-B and HLA-C alleles caused an expected ∼55% reduction in total class I surface expression. Despite this, W6/32 immunoprecipitation from ΔBC clones recovered a comparable peptide yield to PA2.1 in WT cells. Binding predictions showed that most peptides identified in ΔBC clones using W6/32 were assigned to HLA-A∗02:01, with near-complete loss of HLA-B∗07:02- and HLA-C∗07:02-derived peptides. Sequence logo analysis confirmed the canonical A∗02:01 motif across conditions. The ΔBC W6/32 immunopeptidome exhibited a high degree of overlap (∼88%) with the WT PA2.1 repertoire, supporting the specificity and fidelity of the approach. These findings establish CRISPR-based editing of HLA alleles as a viable strategy for allele-specific immunopeptidome analysis using pan-HLA antibodies, supporting its potential application beyond this proof-of-concept system, reducing reliance on allele-specific reagents and facilitating the study of underrepresented HLA alleles.},
}
@article {pmid42148916,
year = {2026},
author = {Zhou, S and Gu, T and Deng, L and Wang, X and Zhu, S and He, X and Yang, M and Huo, D and Hou, C},
title = {One-step digestion-ligation-activation universal strategy for ultrasensitive detection of DNA methylation.},
journal = {The Analyst},
volume = {151},
number = {13},
pages = {3744-3754},
doi = {10.1039/d5an01270h},
pmid = {42148916},
issn = {1364-5528},
mesh = {*DNA Methylation ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; CpG Islands ; DNA Restriction Enzymes/metabolism ; DNA/chemistry/genetics ; },
abstract = {DNA methylation and its associated methyltransferases play pivotal roles in epigenetic regulation and are regarded as important biomarkers for early cancer diagnosis; however, the development of a universal platform capable of sensitively detecting both targets remains challenging. Herein, we report a one-step digestion-ligation-activation universal strategy integrating rolling circle extension-assisted loop-mediated isothermal amplification with CRISPR/Cas12a (DL-RLAMP/Cas12a) for ultrasensitive methylation analysis. In this design, a padlock probe hybridizes at CpG sites and undergoes simultaneous digestion and ligation, enabling methylation-dependent circular DNA formation. The use of methylation-sensitive restriction endonucleases (HhaI/BstUI) ensures selective survival of methylated or methyltransferase-modified targets, which subsequently initiate RLAMP amplification to generate abundant double stem-loop DNA structures for Cas12a-mediated trans-cleavage signal amplification. By integrating multistep reactions into a single workflow, the proposed strategy achieves efficient cascade amplification while maintaining an ultralow background signal. The assay discriminates methylation levels down to 0.1% and enables sensitive detection of CpG methyltransferase (M.SssI) with a detection limit of 6.92 × 10[-4] U mL[-1]. Furthermore, the platform demonstrates applicability in cellular methylation analysis and methyltransferase inhibitor screening. This DL-RLAMP/Cas12a system provides a versatile and highly sensitive analytical framework for epigenetic biomarker detection and holds promise for early disease diagnostics.},
}
@article {pmid42214710,
year = {2026},
author = {Yang, X and Sang, R and Hutvagner, G and Hewitt, AW and Li, D and Li, Y and Deng, W},
title = {An optimised lipid nanoparticle platform enables efficient CRISPR/Cas9 genome editing in hard-to-transfect cells.},
journal = {Acta biomaterialia},
volume = {218},
number = {},
pages = {432-444},
doi = {10.1016/j.actbio.2026.05.044},
pmid = {42214710},
issn = {1878-7568},
mesh = {Humans ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; HEK293 Cells ; *Gene Editing/methods ; *Transfection/methods ; Cell Line, Tumor ; Liposomes ; },
abstract = {CRISPR/Cas9 is a powerful tool for genome editing and functional gene studies, but its therapeutic potential is often hampered by inefficient transfection, particularly in hard-to-modify cell types. In this study, we developed and optimised a lipid nanoparticles (LNPs) platform that enhances CRISPR/Cas9-mediated genome editing across diverse cell types, including those that are difficult to modify using commercially available lipid-based delivery agents. Our engineered LNPs exhibit consistent particle size below 100 nm, low polydispersity and high encapsulation efficiency. Using this platform, GFP knockout in HEK-293 cells reached 78.7%, and maintained consistent efficiency after lyophilisation and reconstitution. Knockout of the LCN2 gene in MDA-MB-231 cells resulted in a 90.8% reduction in mRNA expression, outperforming the 51.1% reduction achieved using CRISPRMAX Lipofectamine, and functional assays confirmed that LCN2 disruption significantly inhibited cell proliferation and migration. Co-delivery of CRISPR/Cas9 and a GFP HDR template enabled precise knock-in, achieving >20% efficiency in HEK-293 cells and >8% in MSCs and DC2.4 cells, significantly outperforming Lipofectamine 3000 transfection reagent. Given the rapid expansion of CRISPR applications in biomedical research, our LNP-based delivery system represents a promising non-viral platform with broad potential for therapeutic applications, particularly in hard-to-modify cell types. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 is a transformative gene-editing technology for functional genomics and therapeutic development; however, its widespread application is constrained by the lack of safe and efficient delivery systems, particularly for hard-to-transfect cell types. Lipid nanoparticles (LNPs) represent a promising non-viral delivery strategy, yet their transfection efficiency varies substantially across cell lines due to differences in cellular membrane properties. Here, we developed and optimized an LNP platform that enables highly efficient Cas9/sgRNA-mediated gene knockout and knock-in across multiple challenging cell types, consistently outperforming commercial transfection reagents. The findings establish this LNP system as a versatile and effective non-viral platform for CRISPR/Cas9-mediated genome editing, overcoming key limitations of existing commercial lipid-based delivery agents in hard-to-modify cell types and offering a promising strategy for future clinical translation.},
}
@article {pmid42344907,
year = {2026},
author = {Oh, JH and Yang, LF and Tanaka, E and de Brito Monteiro, L and Wi, D and Archambault, AS and Klein Geltink, RI},
title = {Versatile electroporation protocols enable reproducible CRISPR-RNP delivery across multiple primary mouse cells of the hematopoietic lineage.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1820963},
pmid = {42344907},
issn = {1664-3224},
mesh = {Animals ; *Electroporation/methods ; Mice ; *CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism ; *Ribonucleoproteins/genetics ; Macrophages/metabolism ; Cell Lineage ; *Gene Editing/methods ; CD8-Positive T-Lymphocytes/metabolism ; Gene Transfer Techniques ; Mice, Inbred C57BL ; Cell Differentiation ; Electroporation Therapies ; Cells, Cultured ; },
abstract = {Genetic engineering of primary hematopoietic cells is essential for mechanistic immunology studies, however the development of cell-based therapies yet remains constrained by two major factors: the fragility of many primary lineages and challenges of viral delivery platforms that are costly, time-intensive, and biologically confounding. Here, we optimized scalable CRISPR-Cas9 electroporation workflows using the ExPERT platform across three primary mouse hematopoietic cell types: OT-I CD8[+] T cells, bone marrow-derived macrophages (BMDMs), and hematopoietic stem/progenitor cells (HSPCs). In activated OT-I CD8[+] T cells, two electroporation programs supported high-efficiency mRNA and RNP delivery with minimal impact on cell viability or proliferative capacity, with subtle activation-state-dependent sensitivity at higher energy settings. Extending optimization to myeloid and stem cell lineages, we found that BMDMs maintained high viability following electroporation, and a high-performing electroporation program supported robust RNP delivery and efficient target gene knockout while preserving macrophage differentiation. In HSPCs, the same program enabled consistent RNP delivery, sustained viability, and reproducible gene knockout. Together, these findings establish ExPERT electroporation as a robust, reproducible, and modular platform for genome editing across primary mouse hematopoietic lineages, lowering barriers to rapid genetic perturbation for both discovery and translational applications. This manuscript has been posted on BioRxiv (https://www.biorxiv.org/content/10.64898/2026.01.27.702081v1).},
}
@article {pmid42345443,
year = {2026},
author = {Fiori, S and Adragna, C and Malvicini, E and Basini, T and Galgano, D and Scarpa, E and Jovic, S and Schmacke, NA and Hornung, V and Sallusto, F and Bruno, L and Lanzavecchia, A and Albanese, M},
title = {CRISPR/Cas9-Mediated Gene Knockout Reveals a Nonredundant Role for p16[INK4A] in Controlling TCR-Dependent and Independent CD8 T Cell Expansion.},
journal = {European journal of immunology},
volume = {56},
number = {6},
pages = {e70224},
pmid = {42345443},
issn = {1521-4141},
support = {//Joachim Herz Foundation/ ; 885539/ERC_/European Research Council/International ; 28809//MFAG AIRC/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/immunology ; *CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation/genetics ; *Receptors, Antigen, T-Cell/immunology/metabolism ; Gene Knockout Techniques ; Lymphocyte Activation/immunology ; },
abstract = {The possibility of enhancing T cell function by deleting specific genes represents a long-sought goal in preclinical studies and ultimately for clinical applications. Using CRISPR/Cas9 genome editing, we report that, in human cytotoxic CD8 T cell clones, the cell cycle checkpoint gene CDKN2A, encoding p16[INK4A], plays a nonredundant role in controlling T cell receptor (TCR)-dependent and independent cell expansion. Deletion of CDKN2A dramatically enhanced antigen-driven and homeostatic proliferation, while preserving effector functions. In contrast, the deletion of other cell cycle inhibitors (CDKN1B, CDKN2C, and CDKN2D), alone or in combination, had no impact on T cell proliferation. We also report that mediator complex subunit 12 (MED12) and the E3 ubiquitin ligase CBL-B deletions did not affect proliferative capacity of CD8 T cell clones. Interestingly, deletion of the negative regulator of Ras signaling, RASA2, increased antigen sensitivity and cytotoxic activity, while not improving in vitro expansion. Collectively, these findings reveal a unique and critical nonredundant role for p16[INK4A] in regulating CD8 T cells. Deletion of CDKN2A offers a promising strategy to enhance CD8 T cell expansion ex vivo, thereby improving TCR discovery pipelines and, potentially, therapeutic applications.},
}
@article {pmid42345496,
year = {2026},
author = {Schubert, AJ and Meng, Q and Hoffmann, J and Rau, J and Abele, F and Greensmith, R and Cordero, C and Ibel, A and Mandler, JM and Eckardt, KU and Halbritter, J and Dighe, AS and Tan, X and Hwang, DY and Petzold, T and Enghard, P and Kaminski, MM},
title = {CRISPR-Based Assay for Point-of-Care Pharmacogenetic CYP2C19 Genotyping.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00841},
pmid = {42345496},
issn = {2379-3694},
abstract = {Pharmacogenetic testing enables personalized drug dosing by accounting for genetic variation in drug metabolism. Although there is increasing evidence that genetically guided dosing improves therapeutic efficacy and reduces adverse effects, clinical implementation remains limited as genotyping methods suffer from slow turnaround times and are not optimized for point-of-care use. Here, we present a rapid, multiplexed CRISPR-based assay for genotyping the key CYP2C19 polymorphisms *2, *3, and *17. These variants can alter enzyme activity, impacting the metabolism of drugs such as clopidogrel and mavacamten. Our assay combines isothermal amplification with a dual guide RNA detection strategy, where LwaCas13a selectively detects mutant alleles and LbaCas12a identifies wild-type alleles. In a validation study of 110 participants, the assay showed high concordance with Sanger sequencing, the current gold standard, with variant-specific accuracies of 97.3% (*2), 100% (*3), and 99.1% (*17), demonstrating robust performance across a diverse clinical cohort. The workflow supports both column-based DNA extraction and a simplified, crude extraction protocol. Genotyping results are obtained through either fluorescence detection or multi-analyte lateral-flow readouts, allowing visual interpretation of genotypes with minimal equipment. In conclusion, our findings show that CRISPR-based genotyping can deliver accurate CYP2C19 pharmacogenetic testing, facilitating broader adoption of genotype-guided drug dosing. Furthermore, the programmability of CRISPR-Cas systems enables adaptation to other pharmacogenetic targets relevant to drug metabolism.},
}
@article {pmid42345883,
year = {2026},
author = {Li, X and Han, X and Han, Q and He, X and Huang, Y and Liu, A},
title = {Advancements in Nanomaterial-Based Biosensors for Neuropsychiatric and Neurodegenerative Diagnostics: From Biomarker Discovery to Clinical Translation.},
journal = {Biosensors},
volume = {16},
number = {6},
pages = {},
pmid = {42345883},
issn = {2079-6374},
support = {82401622//National Natural Science Foundation of China/ ; ZR2023QH131//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Biosensing Techniques ; Humans ; Biomarkers/analysis ; *Neurodegenerative Diseases/diagnosis ; *Nanostructures ; *Mental Disorders/diagnosis ; },
abstract = {Nanobiosensors, with their unique physicochemical properties, are transformative tools for diagnosing and monitoring neurodegenerative diseases and mental disorders. This article systematically reviews the latest progress of nanomaterial systems and integrated sensing modalities in neurological disease diagnosis. First, we clarify the multiple functional roles of nanomaterials in biosensors, including signal amplification, interface optimization, and spatial positioning, and compare the applicable scenarios of various sensing principles based on different nanomaterials. Second, we evaluate the design and integration strategies of molecular recognition elements (antibodies, nucleic acid aptamers, molecularly imprinted polymers, and CRISPR-Cas systems) and discuss their synergistic integration mechanisms for improving detection performance. In terms of detection targets, we focus on three applications: high-sensitivity quantification of established protein biomarkers, real-time monitoring of dynamic neurochemicals (dopamine, serotonin, glutamate), and emerging liquid biopsy targets such as exosomal cargo and circulating microRNAs. Finally, to address the core challenges of biofouling, sensitivity-selectivity trade-offs, and multiplex detection in complex matrices, we propose three breakthrough directions for next-generation diagnostics: deep integration of multimodal and multiplexing platforms, closed-loop chemical brain-computer interfaces (cBCIs), and AI-driven predictive diagnostic models, collectively enabling a transition from passive detection to active sensing and intervention for precise, rapid, and non-invasive neurological disease management.},
}
@article {pmid42345886,
year = {2026},
author = {Hong, S and Park, CS and Been, KW and Kang, S and Hong, J and Kim, JW and Hur, JK},
title = {Sequential CRISPR-EspCas9-Mediated Wild-Type Depletion Enhances the Detection Sensitivity of Rare Mutations for Canine Liquid Biopsy Application.},
journal = {Biosensors},
volume = {16},
number = {6},
pages = {},
pmid = {42345886},
issn = {2079-6374},
support = {RS-2023-NR076663//National Research Foundation of Korea/ ; RS-2021-NR056589//National Research Foundation of Korea/ ; RS-2023-00261114//National Research Foundation of Korea/ ; RS-2025-02218918//National Research Foundation of Korea/ ; RS-2024-00468036//National Research Foundation of Korea/ ; 2023R1A6C101A009//Korea Basic Science Institute/ ; },
mesh = {Animals ; Dogs ; Female ; Cell Line, Tumor ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; *Liquid Biopsy/veterinary ; Mutation ; },
abstract = {One of the major obstacles in early cancer detection in dogs is the limited sensitivity in detecting circulating tumor DNAs (ctDNAs) with low abundances. Standard next-generation sequencing (NGS) without error correction typically achieves detection limits around ~1% mutant allele frequency (MAF). We sought to improve the detection sensitivity using a sequential CRISPR-EspCas9 enrichment strategy in which iterative in vitro cleavage (IVC) was combined with PCR amplification to selectively deplete wild-type DNA and enrich rare tumor mutations. Applying the strategy to genomic DNA and cell-free DNA mimics from canine mammary gland tumor cell lines demonstrated that IVC enrichment enabled the detection of cancer-associated PIK3CA H1047R mutations that were undetectable by conventional Sanger sequencing. To evaluate detection sensitivity, we characterized enrichment using synthetic templates for PIK3CA H1047R and other cancer-related mutations, BRAF V596E, and KRAS G12C. We observed that three iterations of sequential IVC achieved ~160, ~15, and ~2.2-fold enrichment for PIK3CA H1047R, BRAF V596E, and KRAS G12C, respectively. Under the present synthetic-template conditions, the analytical LOD reached 0.001% MAF for PIK3CA and 0.01% MAF for BRAF, whereas KRAS showed only modest enrichment and remained practically limited under the current guide design. Together, the results show that the CRISPR-EspCas9 IVC strategy enables selective enrichment of low-frequency single-nucleotide mutant alleles. We anticipate that the finding could be utilized to develop a highly sensitive veterinary liquid biopsy application with further optimization and validation using canine plasma cfDNA.},
}
@article {pmid42346156,
year = {2026},
author = {Rimskaya, B and Kropocheva, E and Ponomareva, I and Karchemkina, L and Lisitskaya, L and Gelfenbein, D and Ulashchik, E and Shmanai, V and Kulbachinskiy, A and Mazunin, I},
title = {Activity of DNA- and RNA-Guided Prokaryotic Argonautes in Human Mitochondria.},
journal = {Cells},
volume = {15},
number = {12},
pages = {},
pmid = {42346156},
issn = {2073-4409},
support = {25-24-00078//Russian Science Foundation/ ; },
mesh = {Humans ; *Mitochondria/metabolism/genetics ; *DNA, Mitochondrial/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Neurospora crassa/genetics ; CRISPR-Cas Systems ; },
abstract = {Precise manipulation of mitochondrial DNA (mtDNA) by CRISPR-Cas systems remains challenging, largely due to inefficient import of guide RNAs, motivating the exploration of alternative programmable nucleases. Here, we show that prokaryotic Argonaute nucleases (pAgos) of various classes can be efficiently targeted to human mitochondria. Using the Su9 mitochondrial targeting sequence from Neurospora crassa, we achieved robust mitochondrial import of four pAgos-DecAgo, CbuAgo, KmaAgo and RslAgo. As a functional readout of their activity in cells, we targeted the single-stranded D-loop region, which plays a central role in mtDNA replication and maintenance, reasoning that cleavage at this site was expected to potentially result in a reduction in mtDNA copy number. Of the four enzymes, only RNA-guided DecAgo induced a pronounced reduction in mtDNA levels, decreasing copy number approximately fivefold within 48 h. Unexpectedly, this effect occurred independently of exogenous guides, suggesting that DecAgo may utilize endogenous mitochondrial guide RNAs. These findings identify DecAgo as an active nuclease in human mitochondria and reveal a previously unrecognized mode of targeting, highlighting the need to further investigate the underlying mechanism and the potential role of endogenous guide molecules, as well as improving targeting specificity.},
}
@article {pmid42347171,
year = {2026},
author = {Syrym, N and Yespembetov, B and Kokanov, S and Nakhanov, A and Bulatov, Y and Abdimukhtar, A and Toleukhan, A and Serikbay, Y and Terebay, A and Anarbekova, A and Tileukhanov, K and Alpysbayeva, S and Sarmykova, M and Yerzhigit, B and Zinina, N and Suleimenov, M and Abdykalyk, A},
title = {Bacteriophage-Based Therapeutics for Bacterial Sexually Transmitted Infections: From Biological Barriers to Translational Strategies.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42347171},
issn = {2076-0817},
support = {AP23489672//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {Humans ; *Bacteriophages/physiology/genetics ; *Phage Therapy/methods ; *Sexually Transmitted Diseases, Bacterial/therapy/microbiology ; Biofilms ; Anti-Bacterial Agents/therapeutic use ; },
abstract = {Bacterial sexually transmitted and sexually associated infections remain a major global health concern, increasingly complicated by antimicrobial resistance and the limited effectiveness of existing therapies. In this context, bacteriophage-based and phage-derived approaches have re-emerged as potential alternative antibacterial strategies. This narrative review examines their applicability across key bacterial pathogens associated with sexually transmitted infections, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Treponema pallidum and biofilm-associated bacterial vaginosis, with a particular focus on pathogen-specific biological barriers. Available evidence indicates that the success of phage-based interventions is strongly dependent on factors such as intracellular localisation, structural characteristics of the bacterial envelope and the presence of polymicrobial biofilms. While phage-derived platforms, including endolysins, depolymerases and engineered phages, demonstrate antibacterial activity in experimental settings, their effectiveness is uneven across different pathogens. Biofilm-associated infections appear more accessible to these approaches, whereas intracellular and structurally atypical bacteria are currently considered more challenging targets based on available mechanistic and experimental evidence. These observations highlight the need for pathogen-specific engineering strategies and delivery systems. Overall, phage-based therapeutics in this field should be considered within a framework that integrates biological constraints with targeted antimicrobial design.},
}
@article {pmid41793486,
year = {2026},
author = {Wu, J and Zhang, Y and Guo, H and Zhang, Y and Zhang, Y},
title = {CRISPR/Cas9 knockout of DDX5 facilitates foot-and-mouth disease virus replication in PK-15 cells.},
journal = {Archives of virology},
volume = {171},
number = {4},
pages = {},
pmid = {41793486},
issn = {1432-8798},
support = {23JRRA551, 24JRRA012//the Natural Science Foundation of Gansu Province/ ; 2023M733819, 2024M763620//China Postdoctoral Science Foundation Funded Project/ ; 24CXNA030//Technology innovation guidance program of Gansu Province/ ; },
mesh = {*Foot-and-Mouth Disease Virus/physiology/genetics ; *Virus Replication ; Animals ; Cell Line ; *CRISPR-Cas Systems ; *DEAD-box RNA Helicases/genetics/metabolism ; Swine ; Gene Knockout Techniques ; *Foot-and-Mouth Disease/virology/genetics ; Immunity, Innate ; Cytokines/genetics ; },
abstract = {Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that is controlled mainly by the use of inactivated vaccines, but vaccine production is limited by inefficient cell culture systems. The RNA helicase DDX5 has been implicated in viral replication, but its role in FMDV infection remains unclear. Here, we generated a DDX5-knockout PK-15 cell line using CRISPR/Cas9 to investigate its impact on FMDV replication. DDX5 knockout cells exhibited enhanced FMDV replication, with increased viral protein expression, RNA levels, and titers compared to wild-type cells. Meanwhile, RNA sequencing (RNA-seq) analysis indicated that DDX5 knockout suppressed key proinflammatory cytokines (CXCL2/8/14, CCL2/4/5) and impaired IFN-α/β and ISG (ISG15/20, IRF3, IFIT3) responses postinfection. RT-qPCR was performed to determine the expression level of differentially expressed genes, and the results were consistent with the RNA-seq data. Altogether, the results of this study suggest that DDX5 restricts FMDV replication by modulating host innate immunity. The DDX5 knockout cell line provides a useful model for studying FMDV pathogenesis and improving vaccine development.},
}
@article {pmid41876983,
year = {2026},
author = {Chen, X and Qin, Y and Dong, S and Jia, C and Li, Y and Liu, Y and Zhao, Q and Zhou, Q},
title = {Nanoparticle-based biosensor integrated with CRISPR/Cas12b platform for sensitive and visual identification of hepatitis B virus pregenomic RNA in chronic hepatitis B patients.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41876983},
issn = {1471-2180},
support = {82460140//the National Natural Science Foundation of China/ ; Qian Ke He Support [2023] General 242//the Guizhou Provincial Key Technology R&amp;D Program/ ; Zhu Ke He (2024) 2-34//the Program of Scientific and Technological of Guiyang City/ ; Qiankehe Platform Talent ZSYS [2025]040//Guizhou Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Mechanism and Clinical Application Research/ ; LMS25H200006//the Natural Science Foundation of Zhejiang Province/ ; 2025KY1182//the Medical Scientific Research Foundation of Zhejiang Province/ ; },
mesh = {Humans ; *Hepatitis B virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *Hepatitis B, Chronic/virology/diagnosis/blood ; *RNA, Viral/genetics/blood ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Metal Nanoparticles/chemistry ; *Molecular Diagnostic Techniques/methods ; Gold/chemistry ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Chronic hepatitis B (CHB) represents a leading driver of hepatocellular carcinoma and end-stage liver disease. Serum hepatitis B virus pregenomic RNA (HBV-pgRNA) has emerged as a new bioindicator strongly related to the efficacy and prognosis of CHB treatment. Seeking ultrasensitive, rapid, highly specific, and straightforward HBV-pgRNA detection, we constructed an innovative CRISPR-HBV-pgRNA platform through the integration of a CRISPR/Cas12b system with loop-mediated isothermal amplification (LAMP). Then, we interpreted the detection results via either real-time fluorescence (RTF) or a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB). METHODS: Herein, the AuNPs-based biosensor used was manufactured following our design. The unique LAMP primers and guide RNA (gRNA) were designed against the HBV-pgRNA gene, ensuring optimized diagnostic conditions: Reaction temperature and time. Both assay sensitivity and specificity were validated, and the feasibility was validated via clinical specimens from patients having chronic HBV infection. RESULTS: The developed AuNPs-based biosensor was successfully fabricated. Primers LAMP and gRNA were specifically designed to target the HBV pgRNA sequence. The integrated assay protocol, comprising RNA extraction (45 min), RT-LAMP amplification (30 min), CRISPR/Cas12b cleavage (5 min), and visual readout (2 min), was completed in 85 min without reliance on costly instrumentation. The method achieved a detection limit of 10 copies/reaction and demonstrated no cross-reactivity with other tested pathogens. CONCLUSIONS: The CRISPR-HBV-pgRNA assay is a powerful diagnostic tool and exhibits considerable potential for POC testing for the evaluation of chronic HBV infection status and antiviral drug efficacy, especially for resource-limited regions.},
}
@article {pmid41902966,
year = {2026},
author = {Wu, Y and Li, Y and Zhang, H and Wu, L and Wei, Y and Wu, K and Yang, Y and Chen, QH},
title = {TdT cascaded CRISPR/Cas12a integrated MOF-on-MOF nanozyme for ultrasensitive electrochemical detection of acetamiprid.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41902966},
issn = {1436-5073},
support = {No. 2023KF005//Open Research Fund of State Key Laboratory of Virology and Biosafety/ ; 82272960//National Natural Science Foundation of China/ ; },
mesh = {*Neonicotinoids/analysis/chemistry ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Electrodes ; DNA, Single-Stranded/chemistry ; },
abstract = {An electrochemical biosensor was developed for the detection of acetamiprid (ACE) by integrating a terminal deoxynucleotidyl transferase-(TdT)-cascaded CRISPR/Cas12a with a NH2-MIL-88@PCN-224 nanozyme. ACE triggered the release of a DNA primer, initiating TdT-catalyzed generation of long poly-T strands. These strands activated the trans-cleavage activity of Cas12a, leading to the hydrolysis of phosphate-modified ssDNA on the electrode. Consequently, the adsorption of the nanozyme onto the electrode inversely correlated with the ACE concentration. The nanozyme efficiently catalyzed the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB), generating a measurable electrochemical signal. This synergistic combination of nucleic acid amplification and nanozyme catalysis achieved exceptional sensitivity, with a wide linear range from 100 fM to 10 nM and a LOD of 9.7 fM. The biosensor demonstrated high specificity, satisfactory stability and reproducibility, and was successfully applied to detect ACE in real food samples, showing excellent consistency (Pearson’s r = 0.99) with HPLC-MS results. This work provides a powerful and reliable platform for monitoring pesticide residues in food.},
}
@article {pmid41912740,
year = {2026},
author = {Aggarwal, F and Jatwani, S and Jadhav, RR and Yadav, R and Sarma, SJ and Khare, D},
title = {A potassium titanate whisker-assisted method for genetic transformation of Phanerochaete chrysosporium using a Cas9-sGFP expression vector.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {4},
pages = {},
pmid = {41912740},
issn = {1573-0972},
mesh = {*Phanerochaete/genetics/metabolism ; *Transformation, Genetic ; Genetic Vectors/genetics ; *Titanium/chemistry ; Green Fluorescent Proteins/genetics ; CRISPR-Cas Systems ; Hygromycin B/pharmacology/analogs &amp; derivatives ; *CRISPR-Associated Protein 9/genetics ; Genes, Reporter ; },
abstract = {Phanerochaete chrysosporium, a model white-rot fungus, plays a central role in lignin degradation but has remained recalcitrant to genetic manipulation due to inherently low transformation efficiencies. In this study, we present a novel whisker-assisted transformation method employing potassium titanate whiskers combined with freeze–thaw pretreatment, L-ornithine supplementation, and sonication to enable efficient DNA delivery into P. chrysosporium. Using the Cas9-sGFP construct as a reporter system, transformants were selected on hygromycin and validated through PCR and fluorescence microscopy. Our protocol consistently generated hygromycin-resistant transformants, achieving a reproducible transformation efficiency of ~ 0.26%, outperforming Agrobacterium-mediated transformation and comparable to shockwave-based approaches. The successful integration and functional expression of the sGFP reporter confirmed the stability and reliability of this approach. This study provides the first evidence of whisker-mediated delivery and stable integration of exogenous DNA into the genomic DNA of a white-rot fungus. By providing a simple, cost-effective, and reproducible transformation strategy, this work addresses a longstanding bottleneck in fungal biotechnology. The method provides a scalable alternative for CRISPR-based genome engineering in ligninolytic fungi and unlocks opportunities for metabolic engineering of P. chrysosporium, including targeted gene disruption, overexpression of ligninolytic enzymes, and expression of heterologous biosynthetic pathways for the sustainable production of industrially valuable compounds.},
}
@article {pmid41931156,
year = {2026},
author = {Kumar, VK and Thamodaran, V},
title = {A genome-edited isogenic human embryonic stem cell model of pompe disease recapitulates cardiac and skeletal muscle pathology.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {41931156},
issn = {1573-4978},
mesh = {Humans ; *Glycogen Storage Disease Type II/genetics/pathology/metabolism ; *Human Embryonic Stem Cells/metabolism ; Cell Differentiation/genetics ; alpha-Glucosidases/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Muscle, Skeletal/pathology/metabolism ; Cell Line ; Induced Pluripotent Stem Cells/metabolism ; Myocardium/pathology/metabolism ; Glycogen/metabolism ; Myocytes, Cardiac/metabolism/pathology ; Lysosomes/metabolism ; },
abstract = {BACKGROUND: Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene, leading to acid alpha-glucosidase deficiency and pathological glycogen accumulation, primarily in cardiac and skeletal muscle. While enzyme replacement therapy (ERT) has improved clinical outcomes, its limited efficacy especially in skeletal muscle underscores the need for improved disease models and novel therapeutic strategies. Induced pluripotent stem cells (iPSCs) from Pompe patients have facilitated mechanistic studies; however, their utility is restricted by limited patient sample availability. METHODS: To address this limitation, we employed CRISPR-Cas9 genome editing to disrupt GAA in a well-characterized human embryonic stem cell (hESC) line, BJNhem20, thereby generating a Pompe disease model independent of patient material. RESULTS: The edited hESC line exhibited markedly reduced GAA enzymatic activity while maintaining pluripotency and trilineage differentiation potential. Upon directed differentiation, cardiac and skeletal muscle cells displayed pronounced lysosome and glycogen accumulation. CONCLUSIONS: These findings demonstrate that genome-edited hESC for Pompe disease can recapitulate key pathological features, providing a robust and scalable platform for disease modelling and therapeutic screening. This approach offers a valuable alternative to patient-derived iPSCs for studying rare genetic disorders and for the development of targeted interventions.},
}
@article {pmid41952234,
year = {2026},
author = {Manosalva, I and Charles-Alfred, M and Torres, M and Roca Paixao, JF and Spicuglia, S},
title = {Validation of a CD81-based flow cytometry assay to assess dCas9 silencing activity.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {41952234},
issn = {1756-0500},
mesh = {*Flow Cytometry/methods ; Humans ; *Gene Silencing ; *Tetraspanin 28/genetics/metabolism ; Cell Line, Tumor ; Reproducibility of Results ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {OBJECTIVE: The efficiency of CRISPR interference (CRISPRi) depends on functional dCas9 activity, yet practical and reproducible validation of dCas9-expressing cell lines remains limited. Here, we describe a simple and reproducible assay to assess dCas9 functionality using a single sgRNA targeting the nonessential and ubiquitously expressed surface protein CD81. RESULTS: We evaluated this approach in multiple hematological and solid tumor cell lines expressing the dCas9-KRAB-MeCP2 repressor complex. In all tested models, CD81 targeting resulted in a consistent reduction of surface protein levels, quantified by flow cytometry. This assay provides a rapid and quantitative functional readout of dCas9 activity without the need for reporter constructs or transcriptional assays. The CD81-targeting sgRNA and validated cell lines are made available to support reproducibility and technical standardization in CRISPRi experiments. This strategy can be readily implemented in any laboratory using CRISPRi-based approaches.},
}
@article {pmid41957080,
year = {2026},
author = {Hwang, RW and Khalil, Y and Baumann, E and Huang, J and Charlebois, C and Rukhlova, M and Renner, TM and Liu, Z and Jezierski, A and Kærn, M and Costain, WJ},
title = {Accelerated reprogramming of hiPSCs into functional brain endothelial-like cells using multiplexed CRISPR activation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41957080},
issn = {2045-2322},
support = {OISB-NRC Scholarship//Ottawa Institute for Systems Biology/ ; NBR1-129//National Research Council Canada/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Endothelial Cells/cytology/metabolism ; *Cellular Reprogramming/genetics ; Cell Differentiation/genetics ; Blood-Brain Barrier/cytology/metabolism ; *Brain/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcription Factors/genetics/metabolism ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) offer a renewable and scalable source for generating brain endothelial cells (BECs), enabling the development of in vitro blood-brain barrier (BBB) models that closely reflect human physiology. In this study, we demonstrate a streamlined differentiation strategy for producing hiPSC-derived BEC-like cells (iBECs) using multiplex CRISPR/dCas9 activation (CRISPRa) to transcriptionally reprogram iPSCs by selectively and simultaneously upregulating BEC transcription factors (ERG, ETV2, FLI1) and BEC genes (CLDN5, CDH5, PECAM1, KDR). We observe that this approach significantly accelerates differentiation from 13 to five days while maintaining BBB characteristics. CRISPRa iBECs exhibit high transendothelial electrical resistance (TEER ~ 400 Ω·cm2), low transcellular permeability (< 0.027 × 10[-]3 cm/min), and expression of key BBB markers including Claudin-5, ZO-1, CDH5, PECAM1, Occludin, and GLUT-1, as well as receptor-mediated transporters TfR1, IGF1R, and TMEM30A. Our study demonstrates a novel CRISPRa-directed differentiation strategy that not only accelerates BEC differentiation but also demonstrates the utility of CRISPR gene regulation as an option in modifying the phenotype of iPSC-derived cells.},
}
@article {pmid41968299,
year = {2026},
author = {Liu, Y and Yin, X and Jin, P and Zhang, H},
title = {CRISPR/Cas9-based knockout of BnaLYK compromises pattern-triggered immunity and resistance to Sclerotinia sclerotiorum in Brassica napus.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41968299},
issn = {1471-2229},
support = {KJ2021A0189//Anhui Provincial Department of Education Scientific Research Project/ ; 32472531//National Natural Science Foundation of China/ ; 2024YFD1400800 Subject 3 (2024YFD1400803)//National Key Research and Development Program of China/ ; },
mesh = {*Brassica napus/immunology/genetics/microbiology ; *Ascomycota/physiology ; *Plant Diseases/microbiology/immunology/genetics ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; *Plant Immunity/genetics ; *Plant Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Gene Knockout Techniques ; },
abstract = {Rapeseed (Brassica napus) is severely threatened by Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot (SSR). The lack of fully resistant cultivars, combined with the genetic complexity of rapeseed as an allopolyploid species, has hindered the identification of major resistance genes for molecular breeding strategies. This study identifies the involvement of BnaLYK, a LysM receptor-like kinase, in disease resistance and immune signaling pathways. The two homologs, BnaA05.LYK and BnaC05.LYK, function as orthologs of AtLYK1 in B. napus. Both genes were induced upon S. sclerotiorum infection and were localized to the plasma membrane. Heterologous expression of either gene in the atlyk1 mutant restored chitin-triggered reactive oxygen species (ROS) burst, callose deposition, and defense gene expression. CRISPR/Cas9-mediated knockout of BnaLYK significantly compromised resistance against S. sclerotiorum, characterized by increased necrotic lesion formation and decreased expression levels of defense-related genes. BnaLYK was shown to be indispensable for chitin- and peptidoglycan-induced pattern-triggered immunity, regulating ROS production, callose accumulation, and defense gene expression. Our findings demonstrate BnaLYK is essential for chitin-induced immunity and basal resistance against S. sclerotiorum in rapeseed, which underscore the conserved functionality of LysM receptors in polyploid crops and offer a promising target for breeding SSR-resistant rapeseed varieties.},
}
@article {pmid41998240,
year = {2026},
author = {Buhl, N and Pfister, ED and Oliveira, DV and Turetti, F and Lurz, E and Baumann, U and Di Donato, N and Illig, T and Skawran, B and Andersson, ER and Mašek, J and Stalke, A},
title = {Improved functional JAG1 and NOTCH2 variant testing in patients with clinical or suspected Alagille syndrome using new low-Notch activity cells.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {41998240},
issn = {1432-1203},
mesh = {Humans ; *Alagille Syndrome/genetics/diagnosis/metabolism ; *Jagged-1 Protein/genetics/metabolism ; *Receptor, Notch2/genetics/metabolism ; Mutation, Missense ; HEK293 Cells ; Signal Transduction/genetics ; Female ; CRISPR-Cas Systems ; Male ; },
abstract = {The autosomal dominant multisystemic Alagille Syndrome (ALGS) is an important cause of pediatric cholestasis. ALGS is associated with pathogenic variants in JAGGED1 (JAG1) or NOTCH2, ligand and receptor components of the Notch-signaling pathway, respectively. The detected missense variants are commonly classified as variants of uncertain significance, hindering ALGS diagnosis. To overcome this issue, we have developed a CRISPR/Cas9-engineered Low-Notch activity (LNA) cells allowing for selective testing of JAG1-NOTCH2 signaling activity. We tested this approach on 9 patients with pediatric hepatopathies with phenotypes ranging from the full clinical ALGS spectrum to isolated neonatal cholestasis and atypical ALGS abnormalities who carried 5 JAG1 and 3 NOTCH2 missense variants of interest. Additionally, western blot analyses revealed an effect on protein expression for two JAG1 missense variants, one of which had altered glycosylation, potentially indicating pathogenic effects. For this JAG1 and one NOTCH2 de novo missense variant, luciferase-based Notch reporter activity was significantly reduced in LNA cells, while no change was observed in commonly used HEK293T, Huh7, or Hep2G cells. These findings allow independent confirmation of recently classified c.53T>G p.(Leu18Arg) in JAG1, and a re-classification of c.1235G>T p.(Cys412Phe) in NOTCH2 as likely pathogenic based on ACMG criteria. Collectively, we provide evidence that selective testing of JAG1-NOTCH2 interaction in the newly developed CRISPR-engineered cells, combined with a glycosylation assay, enables robust functional evaluation of ALGS-associated JAG1 and NOTCH2 variants.},
}
@article {pmid42000804,
year = {2026},
author = {Tamrakar, VK and Sharma, K and Singh, P and Khan, R and Bhargava, A and Thakur, P and Negi, SS},
title = {Probable hindrance of visible colour due to excess biotin with CRISPR-dCas9-sgRNA lateral flow assay detection of HPV16 and HPV18: a negative finding.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42000804},
issn = {2045-2322},
support = {IIRP-2023-3960//Indian Council of Medical Research/ ; },
mesh = {*Biotin/chemistry ; *Human papillomavirus 16/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Human papillomavirus 18/genetics/isolation &amp; purification ; Streptavidin/chemistry ; Colorimetry/methods ; Rapid Diagnostic Tests ; *Papillomavirus Infections/diagnosis/virology ; Color ; },
abstract = {To develop a CRISPR-dCas9-based lateral flow assay (LFA) for the detection of human papillomavirus 16 (HPV16) and human papillomavirus 18 (HPV18) genotypes for point-of-care molecular diagnosis. A CRISPR-dCas9-based LFA was planned to be developed by immobilizing biotin-tagged dCas9-sgRNA assembly on streptavidin-coated nitrocellulose membranes. The assay protocol involved the sequential addition of biotinylated HPV16 and HPV18 PCR amplicons, streptavidin-alkaline phosphatase conjugate, and BCIP/NBT substrate for colorimetric detection. No colour development was observed in the experimental setup, in contrast to the positive control (biotin, streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrate). The failure was attributed to steric hindrance caused by excess biotin present on both the dCas9-sgRNA complex and the HPV PCR amplicons, leading to competition for streptavidin binding sites, improper binding configurations, disrupted protein folding, and interference with biotin-streptavidin interactions. The study demonstrates that using multiple biotinylated molecules in CRISPR-dCas9-based LFAs can lead to assay failure due to competitive binding and steric hindrance. The results advocate the use of two different molecule for immobilization and signal generation and emphasize the necessity for independent optimization of binding and reporting components to establish a functional CRISPR-dCas9-based LFA platform.},
}
@article {pmid42001168,
year = {2026},
author = {Lei, L and Tan, L and Chen, Y and Peng, X and Wang, Y and Liao, F and Yuan, Y and Chen, Y and Wang, K and Huang, X and Zhan, Y and Wang, N and Duan, D and Yang, Y and Wang, A},
title = {A crRNA/Cas12a complex-driven rapid and visual detection method for four porcine diarrhea viruses.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42001168},
issn = {1746-6148},
support = {2021NK1030//Research and development of protein particle vaccines for African Swine Fever and other major viral diseases/ ; },
mesh = {Animals ; Swine ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Porcine epidemic diarrhea virus/isolation &amp; purification/genetics ; Transmissible gastroenteritis virus/isolation &amp; purification/genetics ; Sensitivity and Specificity ; Deltacoronavirus/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/veterinary/methods ; Coronavirus Infections/veterinary/diagnosis/virology ; Rotavirus/isolation &amp; purification/genetics ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus-A (PoRV) G9 are major swine pathogens primarily responsible for gastrointestinal diseases, particularly affecting lactating piglets and resulting in significant economic losses, especially in China. This study reports a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, enhances diagnostic accuracy through targeted gene editing. In this approach, the nucleic acids of the four viruses are amplified in parallel by LAMP and subsequently detected in four singleplex CRISPR–Cas12a reactions performed in separate tubes, with the incorporation of fluorescent reporter probes and a lateral flow dipstick assay establishing a visual detection system capable of separately identifying each of the four viruses. RESULTS: It enables the visual detection of viral genomes from as low as 1 copy/µL without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.9% concordance with RT-qPCR for PoRV G9. CONCLUSIONS: Offering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its potential role in enhancing biosecurity and disease management in veterinary contexts.},
}
@article {pmid42018089,
year = {2026},
author = {Satpathy, MM and Kumar, B and Thomas, P and Abhishek, and Singh, KP and Vinodhkumar, OR},
title = {Development of a high-copy target enhanced multiplexed crRNA-based, amplification-free detection assay (HiTECT) for Brucella spp.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42018089},
issn = {1573-4978},
mesh = {*Brucella/genetics/isolation &amp; purification ; *Brucellosis/diagnosis/microbiology/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Animals ; Humans ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; },
abstract = {BACKGROUND: Brucellosis, a zoonotic disease caused by Brucella species, continues to pose major health challenges for humans and livestock, along with significant economic losses. Traditional serological tests are widely used but are limited, as they cannot reliably distinguish between active infection and past exposure. Molecular tools such as PCR and real-time PCR offer high sensitivity and specificity; however, their dependence on sophisticated equipment and trained personnel restricts their use outside well-equipped laboratories. More recently, isothermal techniques such as LAMP and RPA have emerged as faster, simpler alternatives, and their integration with CRISPR-based platforms has further enhanced detection capabilities. Nevertheless, these methods still face challenges, including the requirement for pre-amplification and the risk of contamination, which limit their practical application in field conditions. METHODS AND RESULTS: In this study, we developed an amplification-free CRISPR-Cas12a assay—HiTECT (High-copy Target Enhanced CRISPR Test)—targeting the high-copy IS711 insertion element for thermocycler free rapid detection of Brucella spp. Using a multiplexed crRNA strategy, HiTECT enabled robust visual detection without nucleic acid amplification. Under optimal conditions (100 nM LbCas12a with a 1:1 Cas12a–crRNA ratio) and employing three crRNAs, fluorescence was significantly enhanced. The assay demonstrated an analytical sensitivity of 461.71 ag/µL of genomic DNA (0.92 fg/ reaction) and approximately 6*10[4] CFU/mL for Brucella suis 1330. HiTECT was found very specific as it could detect all 16 field isolates and four reference Brucella strains, with no cross-reactivity to any of the non Brucella bacterial species, and showed substantial concordance with real-time PCR (κ = 0.65). CONCLUSION: HiTECT provides a robust, amplification-free, field-deployable platform for rapid and sensitive detection of Brucella spp., offering clear advantages for resource-limited settings.},
}
@article {pmid42032034,
year = {2026},
author = {Yasui, R and Suzuki, S and Fujii, T and Yabu, Y and Maeda, Y and Murata, S and Kataoka, K and Tanaka, T},
title = {Highly efficient genome editing using CRISPR/Cas9 ribonucleoprotein in the marine oleaginous diatom Fistulifera solaris.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42032034},
issn = {2045-2322},
support = {JPNP17005//the New Energy and Industrial Technology Development Organization/ ; 24H00392//a JSPS KAKENHI Grant-in-Aid for Scientific Research A/ ; JPJS00420230003//JSPS Program for Forming Japan's Peak Research Universities (J-PEAKS)/ ; },
mesh = {*Diatoms/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Adenine Phosphoribosyltransferase/genetics ; Mutation ; Genetic Engineering/methods ; *Gene Editing/methods ; },
abstract = {Microalgae are promising hosts for sustainable aviation fuel production due to their rapid growth and their advantage of not competing with agricultural crops. The oleaginous diatom Fistulifera solaris is notable for its exceptionally high lipid content. However, genetic engineering tools for this species remain limited, partly due to its allodiploid genome structure. Here, we report the successful development of a CRISPR/Cas9 genome editing method using ribonucleoprotein (RNP) complexes in F. solaris. We first targeted the adenine phosphoribosyl transferase (apt) genes as a selectable marker. Delivery of RNPs via particle bombardment to conserved regions of the two homoeologous apt genes resulted in 87% biallelic editing efficiency. Next, we demonstrated multiplex genome editing by co-targeting the apt genes and diadinoxanthin de-epoxidase (dde) genes as representative targets for improving valuable compound production in diatoms. Among 27 co-edited clones, 85% showed mutations in at least one dde homoeolog, and 63% exhibited biallelic mutations in both genes. This study demonstrates the applicability of RNP-mediated genome editing in diatoms. Furthermore, the success of this method suggests its broader applicability to non-model diatom species, providing a valuable tool for genome engineering in diatoms.},
}
@article {pmid42032156,
year = {2026},
author = {Yu, M and Xiao, Y and Zeng, B},
title = {Optimization of CRISPR/Cas9-based multi-gene editing system in Aspergillus oryzae and its application in α-amyrin biosynthesis.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {5},
pages = {},
pmid = {42032156},
issn = {1573-0972},
support = {32200606//the National Natural Science Foundation of China/ ; 20213AAG02020//the Project of the Department of Science and Technology of the Jiangxi Province/ ; 2021YFA1301302//the National Key Research and Development Plan of China/ ; },
mesh = {*Aspergillus oryzae/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Promoter Regions, Genetic ; Metabolic Engineering/methods ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Aspergillus oryzae, with its high protein secretion capacity, post-translational modification capabilities, and safety, is a promising host for producing natural products and recombinant proteins. However, the lack of efficient genetic tools and precise genome modification methods has significantly slowed progress in the metabolic engineering of A. oryzae. Although the CRISPR/Cas9-mediated genome editing system has been applied in A. oryzae, the low efficiency of gene disruption and heterologous gene integration limits its widespread application as a chassis cell for industrial strain development. In this study, the CRISPR/Cas9-mediated genome editing system in A. oryzae RIB40 was optimized to significantly improve its editing efficiency. By evaluating the effect of promoters on sgRNA expression, it was determined that the Ao (Up338)5SrRNA promoter effectively enhances the gene disruption efficiency. Utilizing the Ao (Up338)5SrRNA promoter, a multiplex gene editing system based on the tRNAGly-sgRNA array was developed, achieving dual-gene disruption efficiency of 78.84% and triple-gene disruption efficiency of 41.15%. Additionally, when the ratio of the genome-editing plasmid to the circular donor DNA was 1:3, the site-specific integration efficiency of the exogenous gene reached 61.36%. Based on the developed CRISPR/Cas9 genome editing system, the heterologous α-amyrin synthetic gene (CADDS) was precisely integrated into the wA locus to achieving the biosynthesis of α-amyrin in Aspergillus oryzae. Furthermore, acetyl-CoA supply was improved by knocking out the competing ethanol metabolic pathway, resulting in an engineered strain that produced 0.69 mg/g of α-amyrin.},
}
@article {pmid42032748,
year = {2026},
author = {Vondracek, K and Lee, MB and Liu, T and Lee, S},
title = {Rapid screening of genome edited strawberry (Fragaria ×ananassa) regenerants using high-resolution melting analysis followed by Amplicon sequencing.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {42032748},
issn = {1756-0500},
support = {#2022-51181-38328-0//National Institute of Food and Agriculture/ ; },
mesh = {*Fragaria/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Mutation ; *High-Throughput Nucleotide Sequencing/methods ; Plants, Genetically Modified/genetics ; Sequence Analysis, DNA/methods ; },
abstract = {OBJECTIVE: Plant transformation frequently results in large quantities of regenerant plant materials which must undergo screening prior to advancement into subsequent experiments. In-depth sequencing of such quantities for detection of editing events is costly, and preliminary selection of mutant lines based on phenotypic impacts can cause significant delays. This study aimed to develop a rapid, high-throughput workflow for early detection and genotyping of CRISPR/Cas9-mediated edits in strawberry. RESULTS: High-resolution analysis reliably identified lines containing distinct mutation profiles, and subsequent Amplicon sequencing genotyping confirmed the presence of editing events at the targeted sites, with editing efficiencies varying among subgenomes of octoploid strawberry. This workflow offers a scalable, low-cost solution for early detection and prioritization of genome-edited cultivated strawberry lines.},
}
@article {pmid42046128,
year = {2026},
author = {Du, W and Zhang, T and Guo, L and Zheng, Y and Zhang, H and Zhu, X and Tang, D and Hu, H and Chen, L and Liu, C},
title = {Deep learning-driven prediction of on-target activity, off-target risk, and repair outcomes in CRISPR/Cas9: current landscape and multi-scale perspectives.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {42046128},
issn = {1479-5876},
support = {SZSM202411026//Guangdong Provincial and National Key Clinical Specialty Construction Project and National Key Clinical Specialty Construction Project, Sanming Project of Medicine in Shenzhen/ ; 82371901//National Natural Science Foundation of China/ ; 82471919//National Natural Science Foundation of China/ ; },
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing ; Prediction Algorithms ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a transformative tool in genome editing, playing a pivotal role in enabling precise genetic engineering. Achieving high on-target efficiency while minimizing off-target activity is critical for translating CRISPR/Cas9 into reliable experimental and therapeutic applications. Conventional off-target detection methods are labor-intensive and cost-prohibitive, limiting their scalability. The integration of artificial intelligence has markedly reduced detection costs and substantially increased throughput. Early shallow learning models in the CRISPR/Cas9 domain, although effective in basic classification tasks, exhibited limited feature representation and poor generalization. With advances in algorithms and computational power, deep learning architectures have significantly improved off-target prediction accuracy. However, a critical blind spot remains, most current models operate predominantly at the sequence level, overlooking the downstream functional consequences of genome edits. This review summarizes the current landscape of AI-driven CRISPR/Cas9 prediction methods and proposes a forward-looking “three-layer framework” that integrates molecular, cellular, and tissue dimensions. By linking nucleotide-level edits to protein alterations, cellular functional changes, and tissue-specific responses, this framework aims to bridge the gap between sequence-based predictions and phenotypic outcomes, thereby advancing the precision and translational potential of CRISPR/Cas9 technologies.},
}
@article {pmid42068385,
year = {2026},
author = {Wang, J and Huang, T and Zhao, W and Hu, M and Liu, M and Song, Z and Xing, L and Wang, N},
title = {Development of a one-tube RPA/Cas12a platform for dual-mode detection of Vibrio alginolyticus.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {5},
pages = {},
pmid = {42068385},
issn = {1573-0972},
support = {SF2350//Lianyungang Social Development Project Key R&amp;D Programme Funding/ ; KD2024KYJJ151//Nanjing Medical University Kanda College Key Projects/ ; },
mesh = {*Vibrio alginolyticus/genetics/isolation &amp; purification ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Rapid Diagnostic Tests ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; *Vibrio Infections/diagnosis/microbiology/veterinary ; DNA-Binding Proteins/genetics ; Recombinases ; Transcription Factors/genetics ; DNA Primers/genetics ; Animals ; Endodeoxyribonucleases ; },
abstract = {Vibrio alginolyticus is a halophilic marine bacterium that threatens aquaculture and food safety. Rapid and reliable detection is vital for early intervention and outbreak prevention. Recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a offers a sensitive, specific, and isothermal alternative suitable for portable detection. We developed a one-tube RPA/Cas12a assay for rapid and sensitive V. alginolyticus detection, which integrates fluorescence and lateral flow strip (LFS) readouts for flexible visualization. An optimal primer pair (F1/R1) and crRNA2 targeting the toxR gene enabled efficient amplification and Cas12a-mediated trans-cleavage. All key components—RPA mixture, Cas12a, and crRNA—were essential for signal generation. Reaction optimization achieved a detection limit of 0.747 copies/µL for fluorescence and 100 copies/µL for LFS. Specificity assays confirmed the exclusive detection of V. alginolyticus without cross-reactivity to other Vibrio spp. or marine bacteria. The method validation was carried out using multiple types of samples, including artificially contaminated clinical samples, aquatic products, as well as clinical isolated of V. alginolyticus, demonstrating robustness. This dual-mode system simplifies operation, minimizes contamination, and enables field-deployable, real-time monitoring of V. alginolyticus in aquaculture environments, offering a practical, rapid, and instrument-free tool for pathogen surveillance and coastal biosecurity.},
}
@article {pmid42095660,
year = {2026},
author = {Xie, Q and Wang, Q and Noettger, S and Gosálbez, G and Betzler, AC and Volcic, M and Kmiec, D and Krebs, S and Graf, A and Gülensoy, D and Weidinger, G and Sparrer, KMJ and Kirchhoff, F},
title = {Replication-competent SIVcpz CRISPR screen identifies barriers to successful cross-species transmission.},
journal = {Journal of virology},
volume = {100},
number = {6},
pages = {e0031426},
pmid = {42095660},
issn = {1098-5514},
support = {101054456/ERC_/European Research Council/International ; KI 548/21-1, VO 2829/2-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Simian Immunodeficiency Virus/genetics/physiology ; Humans ; Animals ; *Virus Replication/genetics ; *CRISPR-Cas Systems ; *Simian Acquired Immunodeficiency Syndrome/transmission/virology ; HIV-1/genetics ; Pan troglodytes ; },
abstract = {UNLABELLED: Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M (major) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1, and TMEM173. These hits only partially overlapped with those identified in analogous HIV-1-based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain-containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1) restrict replication of the analyzed SIVcpz strains but not HIV-1 group M strains in primary human CD4[+] T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that at least some HIV-1 group M strains overcame during adaptation for pandemic spread.<br><br>IMPORTANCE: Four independent transmission events of simian immunodeficiency viruses from chimpanzees and gorillas to humans gave rise to human immunodeficiency virus type 1, but only one led to the global AIDS pandemic. Understanding which adaptations allowed the pandemic HIV-1 M strains to spread efficiently in humans remains a key question in virus evolution and public health. In this study, we engineered replication-competent SIVcpz constructs carrying more than 1,500 single-guide RNAs to identify antiviral genes in Cas9-expressing cells. This approach revealed several cellular factors that restrict SIVcpz but not the pandemic HIV-1 M strains analyzed in primary human T cells. These findings provide new insights into antiviral defense mechanisms and the adaptations that most likely contributed to the efficient spread of HIV-1.},
}
@article {pmid40481608,
year = {2025},
author = {Liu, H and Sun, N and Liu, Z and Li, J and Zhang, X},
title = {Knockout of bcas3 gene causes neurodevelopment defects in zebrafish.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {34},
pmid = {40481608},
issn = {0717-6287},
support = {2023YFC2706302//National Key R&amp;D Program of China/ ; 81000079, 81170165, and 81870959//National Natural Science Foundation of China/ ; 2016QYTD02//Program for HUST Academic Frontier Youth Team/ ; },
mesh = {Animals ; *Zebrafish/genetics ; Neurodevelopment/genetics ; *Zebrafish Proteins/genetics ; Gene Knockout Techniques ; Disease Models, Animal ; *Neurodevelopmental Disorders/genetics ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {BACKGROUND: Neurodevelopmental disorders manifest in early childhood and are characterized by cognitive deficits, intellectual disabilities, motor disorders, and social dysfunction. Mutations in BCAS3 gene are associated with syndromic neurodevelopmental disorders in humans, while the detailed pathological mechanism is still unknown. METHODS: CRISPR/Cas9 technology was used to generate a bcas3 knockout zebrafish model. To investigate the effects of bcas3 on development, morphological evaluations were conducted. Locomotor behaviors, including performance in the light-dark test, novel tank test, mirror test, shoaling test, and social test, were assessed through video tracing and quantitative analysis of movement parameters. Transcriptome sequencing analysis was used to identify dysregulated pathways associated with development process. Additionally, Acridine Orange staining was employed to evaluate apoptosis. Western blot and real-time RT-PCR were used to analyze the expression levels of genes. RESULTS: Bcas3 knockout zebrafish exhibited early larval phenotypes resembling clinical features of patients with BCAS3 mutations, including global delayed development at early embryonic development, microcephaly and reduced body length. Behavior analysis revealed abnormal motor dysfunction, such as social impairment, increased anxiety and heightened aggression. Notably, human BCAS3 rescued the developmental defects and motor disorders in bcas3 knockout larvae. Transcriptomic analysis identified substantial downregulation of genes related to embryonic development and startle response, brain development and neuron migration in bcas3 knockout zebrafish, such as rpl10, cyfip2, erbb3b, eya4a, nr2f1b, prkg1b and ackr3b. Additionally, increased apoptosis was observed in bcas3 knockout zebrafish, which was further confirmed by Acridine Orange staining and a decreased Bcl2/Bax ratio in western blot analysis. The increased apoptosis observed in the brain of bcas3 knockout larvae could contribute to the developmental and locomotor deficits. CONCLUSION: The bcas3 knockout zebrafish model recapitulates the clinical features observed in patients with BCAS3 mutations. Our results suggest that increased apoptosis may underlie the developmental deficits and motor disorders in these patients. The bcas3 knockout zebrafish model provides a valuable tool to identify dysregulated molecular targets for therapeutic intervention during the early stages of disease progression.},
}
@article {pmid41258590,
year = {2025},
author = {Wang, X and Xia, C and Zhang, X},
title = {Genetic screening identifies Ube2v1 as a suppressor of immunoglobulin class switch recombination in CH12F3 cells.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {101},
pmid = {41258590},
issn = {1573-4978},
support = {31900655//National Natural Science Foundation of China/ ; },
mesh = {*Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Animals ; *Immunoglobulin Class Switching/genetics ; Ubiquitination ; B-Lymphocytes/metabolism/immunology ; CRISPR-Cas Systems ; Mice ; Cell Line ; Genetic Testing/methods ; Humans ; },
abstract = {BACKGROUND: Antibodies are essential mediators of adaptive immunity, providing defense against pathogens and serving as critical tools in therapeutics of infection and cancer. Antibody diversification, including class switch recombination (CSR), is tightly regulated by ubiquitination, a post-translational modification, that modulates protein stability and functional activity. METHODS AND RESULTS: Using CRISPR/Cas9 screening in B cells, we investigated the role of ubiquitin-related enzymes in CSR. Validation experiments with LentiCRISPR-sgAID stable cell lines, which showed significant inhibition of CSR, confirmed that it works in the CSR model CH12F3 cell. Screening of 35 E2 ubiquitin-conjugating enzymes and 85 deubiquitinases (DUBs) identified Ube2v1, an E2 enzyme, as a potent suppressor of CSR. Specifically knockdown of Ube2v1 significantly enhanced CSR efficiency, whereas its overexpression inhibited CSR without affecting germline transcripts (GLTs) or activation-induced cytidine deaminase (AID). Intriguingly, although Ube2v1 canonically functions with Ube2n to mediate polyubiquitination, overexpression and knockdown of Ube2n had no detectable effect on CSR. CONCLUSIONS: Our CRISPR screening identified multiple components of ubiquitin pathway that regulate CSR and established Ube2v1 as a novel inhibitor. Ube2v1 functions independently of expression of GLTs and AID as well as its canonical partner Ube2n, revealing a non-canonical role. These findings underscore the complexity of post-translational regulation of humoral immunity and suggest Ube2v1 as a potential therapeutic target for modulating antibody responses.},
}
@article {pmid41286760,
year = {2025},
author = {Wu, G and Ren, Y and Wang, Y and Zhao, Y and Wu, Y and Sun, H and Sun, Z and Wang, R and Du, Z},
title = {Detection of rifampin-resistant Mycobacterium tuberculosis using CRISPR/Cas14a-enabled molecular techniques.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1813},
pmid = {41286760},
issn = {1471-2334},
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/isolation &amp; purification ; *Rifampin/pharmacology ; Humans ; Bacterial Proteins/genetics ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; DNA-Directed RNA Polymerases/genetics ; *Tuberculosis, Multidrug-Resistant/diagnosis/microbiology ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S/genetics ; *Molecular Diagnostic Techniques/methods ; *Antitubercular Agents/pharmacology ; },
abstract = {BACKGROUND: The emergence of drug-resistant Mycobacterium tuberculosis (MTB) strains highlights the urgent need for precise and timely diagnostic methods to prevent prolonged and complex treatment regimens. This study aims to develops a CRISPR/Cas14a-based assay for accurate identification of MTB and rifampin-resistant MTB (RR-MTB) strains. METHOD: The 16 S rDNA sequence and rifampin resistance-determining region (RRDR) of rpoB gene were chosen for the detection of MTB and RR-MTB, respectively. Several sgRNAs were designed for each target and evaluated for their performance. The platform was then systematic optimized by adjusting the concentrations of different components, followed by the evaluation of its sensitivity for the detection of MTB and RR-MTB. The system’s efficacy was further validated through a double-blind test on 16 clinical MTB isolates, and the results were compared with genomic sequencing. RESULTS: Through a meticulous screening process, we identified optimal single-guide RNAs (sgRNAs) capable of distinguishing MTB from nontuberculous mycobacteria (NTM) and the eight predominant mutation types associated with rifampin resistance. Our refined CRISPR/Cas14a platform demonstrated a remarkable sensitivity, with a limit of detection (LOD) of 200 copies/µL for MTB and 2 copies/µL for RR-MTB, respectively. This platform demonstrated a 100% accuracy rate in identification of RR-MTB using clinical MTB isolates. CONCLUSIONS: The CRISPR/Cas14a-based platform we developed exhibited superior performance for the detection of MTB and RR-MTB, with significant implications for the diagnosis and management of tuberculosis, particularly in regions with high prevalence of drug-resistant strains.},
}
@article {pmid41354677,
year = {2025},
author = {Rangaraj, A and Kaur, H and Mejia, L and Hansen, J and Biswas, A and Tuteja, G},
title = {Characterization of a cis-regulatory element upstream of matrix metalloproteinase-9.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {1463},
pmid = {41354677},
issn = {2045-2322},
support = {R01 HD096083/HD/NICHD NIH HHS/United States ; R01HD096083//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; },
mesh = {Humans ; *Matrix Metalloproteinase 9/genetics/metabolism ; Female ; *Enhancer Elements, Genetic ; Pregnancy ; Placenta/metabolism/cytology ; Gene Expression Regulation ; Cell Line ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Dysregulated enhancer activity disrupts gene expression, contributing to disease. However, the structural and functional complexity of enhancers hinders their characterization. Here, we investigate a cis-regulatory element upstream of matrix metalloproteinase-9 (MMP9), a gene implicated in cancer, cardiovascular disease, inflammation, and pregnancy complications. Using luciferase assays and CRISPR-Cas9 mediated knockout in a human placental cell line, we define a one kilobase segment that enhances MMP9 expression. Further dissection reveals two activating sub-segments and, unexpectedly, one repressive sub-segment. Molecular assays suggest transcription factors that mediate these opposing effects. This work adds to the growing understanding that enhancers can integrate both activation and repression, revealing a more complex regulatory architecture than previously appreciated. Together, these findings underscore the importance of enhancer dissection for understanding gene regulation across tissues and diseases.},
}
@article {pmid41392248,
year = {2025},
author = {Liang, J and Liang, T and Wei, C and Li, L and Li, Y and He, S and Liao, Z and Cui, L},
title = {CRISPR/Cas9-engineered Bacillus subtilis chassis for tailored chitooligosaccharide production from marine waste chitosan.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {36},
pmid = {41392248},
issn = {1475-2859},
support = {2023GXNSFAA026505//Natural Science Foundation of Guangxi Province/ ; gxkllst-20241009//Guangxi Key Laboratory of Longevity Science and Technology/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism ; *Chitosan/metabolism ; Glycoside Hydrolases/genetics/metabolism ; *CRISPR-Cas Systems ; *Chitin/analogs &amp; derivatives/biosynthesis ; Fermentation ; Oligosaccharides ; Bacterial Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: This study establishes a sustainable bioprocess for converting chitosan from marine waste into high-value chitooligosaccharides (COSs), offering an eco-friendly alternative to conventional methods that often generate chemical waste. We achieved heterologous production of chitosanase in an engineered Bacillus subtilis chassis by knocking out its endogenous chitosanase, leveraging the dual advantages of this bacterium as a robust synthetic biology platform and an industrial microorganism. RESULTS: The endogenous chitosanase gene (BsCsn) in Bacillus subtilis WB800N was deleted via CRISPR/Cas9-mediated editing, generating the chassis strain B. subtilis WB800N ΔBsCsn. A codon-optimized GH46 chitosanase (CsnA) from Streptomyces coelicolor, fused to the AprE signal peptide, was then expressed in this host. Response surface methodology optimized the fermentation process, enabling a high extracellular CsnA activity of 540.08 ± 6.20 U/mL, in a 5-L bioreactor under DO-stat-controlled fed-batch conditions. This process achieved a productivity of 11.25 U/(mL·h) and a carbon conversion efficiency of 1682.86 U/g glycerol. Furthermore, MALDI-TOF MS analysis confirmed that CsnA produces COSs with defined degrees of polymerization (DP2-DP4). CONCLUSION: This integrated platform enables the upcycling of marine waste into high-value COSs, establishing B. subtilis as an eco-efficient cell factory and providing a valuable framework for the heterologous expression of other chitosanases in this host.},
}
@article {pmid41405764,
year = {2025},
author = {Mansoor, MJ and Al-Taie, SF and Al-Khafaji, ZA and Alkhathami, AG and Renuka, JS and Panigrahi, R and Negi, H and Jassal, P and Mustafa, YF and Hamzah, HF},
title = {Overcoming barriers in CAR-NK immunotherapy: CRISPR-Driven advances in checkpoint editing and allogeneic design.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {4},
pmid = {41405764},
issn = {1438-7948},
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *CRISPR-Cas Systems ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology ; Immunotherapy ; Tumor Microenvironment ; },
abstract = {Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are emerging as an exciting avenue in cancer immunotherapy due to their potent cytotoxicity to malignant cells and lower risk of graft-versus-host disease (GvHD) than conventional T cell therapies. The new technology of CRISPR/Cas9 genome editing has significantly expedited the engineering of CAR-NK cells by enabling easy, multiplex, and precise changes to enhance their efficacy, persistence, and specificity to tumors. This review focuses on the incorporation of CRISPR technology into CAR-NK cell development. It examines uses of knockout of inhibitory checkpoint genes (CISH, PD-1, and TGFBR2), as well as knock-in of CAR into safe genomic locations and multiplex editing of CAR-NK cells to improve cytotoxicity against cancer while resisting suppression from the tumor microenvironment (TME). We further explore immuno-cytokine armoring strategies by knock-in of IL-15 or IL-12, to ensure prolonged proliferation and survival of NK cells, and investigate CRISPR-mediated knockouts of immune inhibitors like NKG2A and TIGIT, to evade immune strategies used by the tumor to evade immune destruction. Furthermore, CRISPR-mediated upregulation of the homing receptor enhances NK cell tumor infiltration, addressing a major obstacle in treating solid tumors. It is significant to mention the progress in generating off-the-shelf products, which is a key step supporting the pursuit of allogeneic therapies. While substantial progress has been made, challenges remain related to optimizing CRISPR delivery, off-target effects, and enhancing in vivo persistence. Future directions of CAR-NK studies will likely capitalize on next-generation genome editing tools and synthetic biology for the development of tunable and logic-gated CAR-NK cells. Overall, this review illustrates the revolutionary capacity of combining CRISPR technology with CAR-NK immunotherapy to develop next-generation programmable and efficacious treatments for hematologic and solid malignancies.},
}
@article {pmid41430619,
year = {2025},
author = {Le Reun, J and Salvioli, Z and Croux, C and Esque, J and André, I and Bordes, F},
title = {A workflow to explore elongase diversity and extend the repertoire of fatty acids produced by Yarrowia lipolytica.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {18},
pmid = {41430619},
issn = {1475-2859},
mesh = {*Yarrowia/metabolism/genetics/enzymology ; *Fatty Acid Elongases/metabolism/genetics ; *Fatty Acids/biosynthesis/metabolism ; Substrate Specificity ; Workflow ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Fatty acids display highly diverse structures that confer these molecules unique chemical properties and distinct physiological functions. Identifying the substrate specificity of enzymes active on fatty acids is crucial, both for understanding their function in natural organisms and for developing efficient cell factories to produce original fatty acids. However, these enzymes are often membrane-bound and/or act on esterified substrates and studying them in vitro is thus challenging. This is why in vivo characterization of these enzymes’ specificity is an interesting approach. Herein, we harness the industrially relevant oleaginous Yarrowia lipolytica as a chassis for characterizing heterologous enzymes active on fatty acids, which can be used to diversify its fatty acid composition. As a case study, we investigated fatty acid elongases (ELO) responsible for the synthesis of very long-chain fatty acids (> 20 carbons), which are specific of given chain lengths and/or unsaturation patterns. Despite their interest, investigation and utilization of these membrane enzymes remain largely underexplored. RESULTS: We developed a workflow for characterizing heterologous elongases in Y. lipolytica, addressing several limitations to increase throughput. First, we set up a strain engineering strategy to easily integrate the ELO cassettes into targeted loci using CRISPR-Cas9, where screening of homologous recombination events is facilitated by fluorescence. We demonstrated that the native elongase YlELO2, responsible for the elongation of saturated and monounsaturated fatty acids up to 26 carbons, has to be inactivated to avoid functional redundancy and finely characterize heterologous elongase specificity. As it is an essential gene, we designed an optimized strategy for YlELO2 Knock-Out by a Knock-In of the elongase cassette. We then miniaturized cultures and fatty acid extraction in 96-well plates format. Using this workflow, we characterized seven human elongases on endogenous fatty acids and on five exogenous polyunsaturated fatty acids in a single series of experiments. CONCLUSION: We have developed tools and methods to characterize elongase specificity, from strain design to fatty acid production and analysis. Applicable to any fatty acid–modifying enzymes, these methodological developments will be useful to expand the repertoire of enzymes usable in Y. lipolytica and pave the way to produce new original fatty acids in this chassis.},
}
@article {pmid41508022,
year = {2026},
author = {Fan, T and Zhou, B and Chen, H and Liu, LP and Ni, Y and Zhang, W and Ye, L and Chen, Y and Zhang, D and Yang, S and Bai, Y and Liu, F and Zhi, C and Xu, G and Zheng, B and Lu, S and Qiu, C and Ding, Z and Chen, Y and Jiang, Y},
title = {Novel serum small extracellular vesicle miRNAs with multi-target RCA-CRISPR sensor for liver cancer detection.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {144},
pmid = {41508022},
issn = {1479-5876},
support = {21310051//Startup Fund from Shenzhen Bay Laboratory/ ; 2019156//Development and Reform Commission of Shenzhen Municipality/ ; },
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Liver Neoplasms/blood/diagnosis/genetics ; *Extracellular Vesicles/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/blood/genetics ; *Biosensing Techniques ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reproducibility of Results ; },
abstract = {BACKGROUND: Detecting liver cancer (LC) remains a significant challenge in clinical practice. Small extracellular vesicle (sEV) miRNAs show promise as non-invasive biomarkers for LC detection, yet their diagnostic potential remains largely unexplored. This study aimed to identify specific sEV miRNA signatures for LC detection and develop a novel synchronized multi-miRNA detection platform to enhance diagnostic efficiency and sensitivity. METHODS: High-throughput sequencing was conducted across four distinct cohorts: normal controls (NC), hepatitis B virus (HBV) patients, liver cirrhosis patients, and LC patients. This sequencing process identified miRNAs with differential expression, followed by RT-qPCR validation in serum sEV miRNAs from LC patients and NC. An innovative detection method, RCA-CRISPR, was introduced, combining rolling circle amplification (RCA) with CRISPR/Cas12a (RCA-CRISPR) for quick and sensitive miRNAs detection. RESULTS: Sequencing results showed a consistent elevation of hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p across all cohorts. RT-qPCR validations confirmed significant upregulation of these miRNAs in serum sEVs from LC patients, and the combined three-miRNA panel exhibited high diagnostic accuracy (p = 0.0003; AUC = 0.81). The RCA-CRISPR method demonstrated a detection limit of 3.12 pM for simultaneous multi-target miRNA detection, highlighting its exceptional sensitivity. CONCLUSIONS: Our study identifies hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p as promising sEV miRNA biomarkers for LC detection. The developed RCA-CRISPR sensor provides a robust tool for multi-miRNA analysis, potentially advancing non-invasive LC diagnostics. Future validation in larger, prospectively collected cohorts is essential to establish the clinical utility and performance of this biomarker panel and RCA-CRISPR sensor.},
}
@article {pmid41530441,
year = {2026},
author = {Li, W and Sun, Y and Ye, M and Liang, Y and Ouyang, J and Xu, W and Su, Y and Huang, X and Nie, D and Ouyang, S},
title = {Rapid visual detection of Treponema pallidum using the RPA-CRISPR/Cas12a system.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5120},
pmid = {41530441},
issn = {2045-2322},
mesh = {*Treponema pallidum/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; },
abstract = {Syphilis, caused by Treponema pallidum, is a sexually transmitted infection that has re-emerged globally over the past decade, posing significant public health challenges. Conventional diagnostic methods are limited by lengthy processing times, operational complexity, and moderate sensitivity, highlighting the urgent need for rapid, sensitive, and user-friendly detection strategies. In this study, we developed a visual detection platform for T. pallidum DNA by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. The assay can be completed within one hour, with results directly interpreted via fluorescence readout. It demonstrated a detection limit as low as 11.34 copies/µL and high specificity, accurately distinguishing T. pallidum without cross-reactivity with common blood-borne pathogens, including HIV, HBV, HCV, and DENV. The clinical sample verification showed a consistency rate of 96.6% with the actual diagnosis. To enhance suitability for point-of-care applications, the RPA-CRISPR/Cas12a system was further adapted to a lateral flow assay (LFA) format, achieving a detection sensitivity of 5.56 × 10[2] copies/µL while minimizing reliance on specialized instrumentation. Overall, this platform provides a rapid, sensitive, and robust approach for point-of-care syphilis diagnosis and offers a reference framework for detecting other pathogenic organisms.},
}
@article {pmid41535756,
year = {2026},
author = {Park, HR and Park, S and Jun, JM and Shin, YJ and Hwang, Y and Jeong, KY and Kim, MY and Kim, ST and Park, S and Yoo, YH and Lee, E and Park, G and Kim, SG and Park, SK},
title = {CRISPR/Cas9 editing of β-Conglycinin subunits reduces IgE binding in soybean [Glycine max (L.) Merr.].},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {265},
pmid = {41535756},
issn = {1471-2229},
support = {PJ016784022024//Agricultural Research Program/ ; },
mesh = {*Glycine max/genetics/immunology/metabolism ; *Seed Storage Proteins/genetics/metabolism/immunology ; *Globulins/genetics/metabolism/immunology ; *Soybean Proteins/genetics/metabolism/immunology ; *Immunoglobulin E/metabolism/immunology ; *Antigens, Plant/genetics/metabolism/immunology ; *CRISPR-Cas Systems ; *Gene Editing ; Protein Subunits/genetics ; Food Hypersensitivity/immunology ; Allergens/genetics/immunology ; Plants, Genetically Modified ; },
abstract = {BACKGROUND: Soybean [Glycine max (L.) Merr.] is a major source of plant-based protein, yet the seed storage protein β-conglycinin (7 S globulin) is a prominent allergen. The αʹ, α, and β subunits contain IgE-binding epitopes, and their high sequence similarity enables simultaneous genome editing. The development of soybean lines with reduced β-conglycinin-specific IgE-binding capacity could enhance food safety for individuals with soy allergies. RESULTS: We employed CRISPR/Cas9 to disrupt the αʹ (Glyma.10G246300) and α (Glyma.20G148300, Glyma.20G148400), subunit genes and to target the β subunit genes (Glyma.20G146200, Glyma.20G148200) of β-conglycinin, generating four edited lines: SP1 (αʹ-null), SP2 (αʹα-null), SP3 (β-null), and SP4, which shows an αʹα-edited genotype and a β subunit-null protein phenotype. SDS-PAGE and DNA sequencing confirmed complete or near-complete loss of the targeted proteins across the T0 to T6 generations, demonstrating stable inheritance of the edited seed protein profiles. IgE immunoblotting and inhibition ELISA using pooled sera from soy-allergic individuals revealed distinct IgE-binding inhibition profiles among the edited lines. At the highest inhibitor concentration, SP4 showed the lowest IgE-binding inhibition (70.0%) compared with the wild type (87.7%), whereas SP1-SP3 exhibited inhibition values similar to or only slightly lower than those of the wild type. CONCLUSIONS: CRISPR/Cas9-mediated elimination of β-conglycinin subunits reduces IgE binding to soybean seed proteins and yields lines with stably inherited seed protein phenotypes. These results highlight the potential of targeted genome editing to generate soybean lines with reduced β-conglycinin-specific IgE recognition, supporting the application of precise genome modification in crop improvement for safer soy-based foods.},
}
@article {pmid41547741,
year = {2026},
author = {Wei, Y and Jiang, J and Gao, Y and Zhu, Y and He, W and Wu, F and Xie, H and Chen, L and Cai, Q and Zhang, J},
title = {Accelerated breeding for early-maturing and aromatic glutinous restorer lines with CRISPR/Cas9-mediated targeted editing for hybrid rice.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {284},
pmid = {41547741},
issn = {1471-2229},
support = {2024R1054//the Fundamental Research Project of Fujian Provincial Research Institute for Public Welfare, China/ ; CARS-01-08//the National Rice Industry Technology System of Modern Agriculture for China/ ; XTCXGC2021001//he "5511" Collaborative Innovation project for High-quality Development and Surpasses of Agriculture between Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2024NZ029027//Key program of Science and Technology in Fujian province, China/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified ; Edible Grain/genetics ; Mutation ; },
abstract = {Hybrid rice breeding depends on the development of elite parental lines with favorable traits such as grain quality, heading time, and plant architecture. However, improving restorer lines through conventional breeding is time-consuming and labor-intensive. Here, we employed a multiplex CRISPR/Cas9 editing strategy to simultaneously target Wx, Badh2, and Se14 in the elite restorer line FH676, aiming to generate glutinous, aromatic, and early-maturing lines. Through Agrobacterium-mediated transformation, we obtained Wx/Badh2 double mutants (Dm) and Wx/Badh2/Se14 triple mutants (Tm). Grain quality analysis revealed significantly reduced amylose content and enhanced aroma content in the edited lines, consistent with Wx and Badh2 knockouts. The triple mutants also exhibited significantly earlier heading compared to the wild type. The early-maturing Tm lines and the Dm lines achieved grain yields of 39.2 ~ 39.4 g and 42.9 ~ 43.0 g per plant, respectively, both exceeding yields of conventional glutinous cultivars used at present. To evaluate hybrid performance, we crossed Tm and Dm lines with two sterile lines: LX (aromatic) and NX (glutinous). The LX/Tm hybrid headed 4.8 days earlier than its wild-type counterpart, with no reduction in plant height or yield. The NX/Tm hybrid showed an advance of ~ 6.3 days in heading but a 6.2 ~ 7.0% reduction in yield due to decreased grain number per panicle. The Se14 knockout likely relieves repression of RFT1 expression during the floral transition under long-day conditions. Variations in phenotypic response across different maternal backgrounds suggest epistatic interactions affecting the dosage response of Se14. In summary, Se14 is a promising target for engineering early-maturing hybrid rice. The edited FH676 lines provide valuable germplasm resource for developing early-maturing, aromatic, and glutinous hybrids through CRISPR-based genome editing.},
}
@article {pmid41549083,
year = {2026},
author = {Khayer, A and Ye, P and Eti, FS and Sakif, TI and Azad, RB and Ali, J and Gupta, DR and Asuke, S and Pan, Q and Moni, MA and Kang, H and Islam, T},
title = {Field pathogenomics and evolutionary conservation unveil CRISPR-targetable susceptibility genes for wheat blast resistance.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5677},
pmid = {41549083},
issn = {2045-2322},
support = {32261143468//National Natural Science Foundation of China/ ; OFANS project (Project Code No: FT-92-FNS/21)//Krishi Gobeshona Foundation/ ; Grant Code: V0156.01//Bill and Melinda Gates Foundation/ ; },
mesh = {*Triticum/genetics/microbiology ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; *Magnaporthe/pathogenicity ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; Genes, Plant ; Evolution, Molecular ; Ascomycota/pathogenicity ; Plant Proteins/genetics ; Transcriptome ; Gene Expression Profiling ; Host-Pathogen Interactions/genetics ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), threatens global wheat production, yet durable resistance mechanisms remain elusive. Current strategies relying on race-specific resistance genes or fungicides are vulnerable to pathogen evolution and inefficacy. Here, we investigated field-derived transcriptomes from the 2016 Bangladesh wheat blast epidemic, a catastrophic event devastating all local varieties to identify host susceptibility (S) genes co-opted by MoT. By analyzing RNA-seq data from infected and healthy plants across geographically distinct regions, we pinpointed 273 consistently upregulated wheat genes, enriched in defense-related pathways. Ortholog analysis with rice, a model for blast resistance, identified three conserved susceptibility (S)-gene candidates: TaSULTR3-3B (an ortholog of a rice bacterial blight susceptibility gene), TaSTP3-4D (associated with stripe rust), and TaMLO1-5A (a wheat powdery mildew susceptibility gene). While all three candidates exhibited significant expression correlation with M. oryzae Triticum (MoT) effectors in field-derived samples, in planta spike assays revealed distinct expression dynamics. Only TaMLO1-5A was significantly upregulated in the susceptible cultivar BARI Gom 26 following MoT inoculation, with no induction observed in the resistant cultivar S-615 (carrying Rmg8). Conversely, TaSULTR3-3B and TaSTP3-4D did not show significant induction under the specific conditions and time points of the in planta spike assays. This discrepancy potentially arises from tissue-specific regulation (spike vs. leaf), environmental variations, or differences in sampling time points between the field and greenhouse experiments. Disruption of such S genes, validated in other cereals for durable resistance, offers a transformative strategy to engineer non-race-specific wheat blast resilience. Our findings shift the paradigm from transient resistance genes to foundational susceptibility networks, proposing CRISPR-based editing of the candidate gene as an actionable target. This approach, resilient to pathogen evolution, could preempt epidemics in climate-vulnerable regions, safeguarding global wheat security. By bridging field pathogenomics and evolutionary genomics, we provide a roadmap for sustainable disease management in an era of expanding fungal threats.},
}
@article {pmid41620642,
year = {2026},
author = {Kumar, S and Murugan, B and Das, M and Sanan-Mishra, N and Sahoo, L},
title = {Geminiviral-CR-gRNA expressed in cowpea efficiently edited MYMV and MYMIV genome to provide resistance against cowpea yellow mosaic disease without hampering plant growth and yield.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41620642},
issn = {1471-2229},
mesh = {*Vigna/genetics/virology/growth &amp; development ; *Begomovirus/genetics ; *Plant Diseases/virology/genetics ; Plants, Genetically Modified ; *Disease Resistance/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing ; *Genome, Viral ; *Geminiviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cowpea is an economically important grain legume widely cultivated in Africa, Latin America, and Southeast Asia. In Southeast Asia, two of the most devastating viral diseases affecting cowpea are cowpea golden mosaic and severe leaf curl disease, both caused by Mungbean yellow mosaic India virus (MYMIV). Despite the availability of various molecular breeding strategies to manage viral infections, progress in cowpea improvement remains limited due to the lack of resistant germplasm, the absence of a reliable transformation system, and the restricted availability of efficient tools for viral gene inactivation. RESULTS: In this study, we employed CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the common region (CR) of the single-stranded DNA-A component of legume-infecting geminiviruses, using cowpea as a test system. Transgenic cowpea plants expressing Cas9 and a guide RNA (gRNA) targeting the CR of MYMV/MYMIV were evaluated for resistance to yellow mosaic disease (YMD). Agrobacterium tumefaciens strain EHA105 carrying pXSE901B-Cas9 and CR-gRNA cassettes was used to generate the transgenic plants. PCR and Southern blot analyses confirmed the integration of transgenes into the cowpea genome. Transgenic lines in the T1 and T2 generations were tested for YMD resistance via agroinfiltration using MYMV and MYMIV agroinfectious clones. Accumulation of AV2 and AC2 transcripts was drastically reduced in T2 lines, which also displayed either no or minimal mosaic symptoms. Mutation analysis of the viral genome revealed frameshift mutations near the PAM region of the targeted CR sequence, with editing frequencies of 28%, 34%, 22%, and 33% in MYMV/MYMIV-infected cowpea lines #L2, #L4, #L7, and #L11, respectively. The transgenic cowpea plants exhibited a normal phenotype and did not show any yield reduction under greenhouse conditions. CONCLUSION: To the best of our knowledge, this is the first report of transgenic cowpea plants stably expressing a geminiviral common region (CR)–targeting gRNA via the CRISPR/Cas9 system, leading to efficient editing of the MYMV/MYMIV genome and conferring durable resistance to Yellow Mosaic Disease without adversely affecting plant growth or yield. These findings demonstrate the potential of CRISPR/Cas9 as a precise and robust platform for developing virus-resistant cowpea and other legume crops.},
}
@article {pmid41634544,
year = {2026},
author = {Velangani, HG and Ghosh, A and Singh, S and Kiran, S},
title = {Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41634544},
issn = {1471-2164},
support = {[EMDR/SG/15/2024-01-03223 (E-File No. 222379)]//Indian Council of Medical Research/ ; FILE NO. CRG/2023/007695//ANRF, India/ ; },
mesh = {*DNA, Single-Stranded/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; },
abstract = {The usefulness of genome editing lies in the ability to induce any desirable change in the target genome. It is most efficiently achieved using ssDNA (single-stranded DNA) as the HDR template and CRISPR-Cas9 targeted DNA breaks. However, the low efficiency of HDR integration is a challenge for achieving efficient edits. Among different HDR templates, ssDNA has the highest efficiency in inducing repair after CRISPR-induced DNA breaks. Several methods are used to generate the ssDNA-HDR template. However, each method has limitations in terms of feasibility for different ssDNA types, efficiency, time required, expenses incurred, etc. Often, these factors are overlooked, confusing users regarding the most appropriate method for generating ssDNA. This study describes and compares methods for generating ssDNA and outlines considerations for designing an efficient ssDNA template. Most frequently used methods are PCR-based, utilizing modified primers (phosphorylation, biotinylation, or phosphorothioate-based) or those utilizing the IVT-RT (In vitro transcription- reverse transcriptase) method. Asymmetric PCR and M13-based methods of ssDNA generation have been used as non-PCR methods. The IVT-RT method is widely adopted as it provides a middle-ground in yield and ease. The advantages and shortcomings of these methods, based on reported studies and our own results, are discussed.},
}
@article {pmid41701371,
year = {2026},
author = {Yi, F and Li, Z and Jiang, F and Zhang, Z and Chen, Y and Zhao, Z and Deng, X and Chen, H and Xu, S and Tao, Y},
title = {An electrochemiluminescence biosensor governed by a CRISPR-actuated electrostatic gate for ultrasensitive aflatoxin B1 detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {163},
pmid = {41701371},
issn = {1436-5073},
support = {22264018//National Natural Science Foundation of China/ ; 20232BAB203019, 20232BAB216118//Jiangxi Provincial Natural Science Foundation/ ; 202410412021, 202410412246, 202410412114//the College Students' Innovation and Entrepreneurship Training Program Project of Jiangxi Province/ ; },
mesh = {*Aflatoxin B1/analysis/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Static Electricity ; *CRISPR-Cas Systems ; Limit of Detection ; Aptamers, Nucleotide/chemistry/genetics ; Food Contamination/analysis ; Electrodes ; Nucleic Acid Hybridization ; },
abstract = {Aflatoxin B1 (AFB1), a potent mycotoxin, poses a critical threat to global food safety, demanding analytical methods with exceptional sensitivity. Here, we introduce a homogeneous electrochemiluminescence (ECL) biosensor that operates on a novel CRISPR-actuated electrostatic gating mechanism. The core of our strategy relies on controlling the access of ECL reporters to a positively charged electrode surface (PAH-ITO). In the absence of AFB1, cationic Ru(phen)32+ reporters are electrostatically repelled from the electrode, resulting in a low background signal. The presence of AFB1 triggers a CRISPR/Cas12a enzymatic cascade, which activates its trans-cleavage activity to release a highly anionic hybridization chain reaction (HCR) scaffold from magnetic beads (MB). This scaffold serves as a nanocarrier, capturing the Ru(phen)32+ reporters and, by virtue of its strong negative charge, shuttling them to the electrode through potent electrostatic attraction. This action effectively “opens” the electrostatic gate, switching on a robust ECL signal. By synergistically integrating the high specificity of the aptamer-CRISPR system with the immense signal amplification of the HCR scaffold, all under the control of a charge-dominant switch, our biosensor achieves an outstanding limit of detection of 0.121 fg/mL and a broad linear range from 1 fg/mL to 100 pg/mL. Its successful application in spiked food samples validates its practicality and robustness, presenting a powerful new paradigm for designing minimal-background, high-gain ECL sensors for mycotoxin determination.},
}
@article {pmid41721389,
year = {2026},
author = {Umashankar, P and Choi, B and Nygård, Y},
title = {Towards the development of a CRISPR-Cas9 based kill switch for Saccharomyces cerevisiae.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41721389},
issn = {1475-2859},
mesh = {*Saccharomyces cerevisiae/genetics/growth &amp; development ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {BACKGROUND: Advancements in synthetic genetic circuits have enabled programmable and condition-dependent control of microbial cell growth. CRISPR-Cas9-based kill switches, genetic systems that program cells to lose viability in response to specific conditions, have recently been demonstrated for bacterial cell factories but not yet in yeast. RESULTS: In this study, we present a foundational demonstration for a CRISPR-based kill switch in Saccharomyces cerevisiae, CRISPR KiSS. The CRISPR KiSS employs inducible CRISPR targeting essential genes to elicit growth inhibition. The activation of the KiSS system is achieved through conditional expression of a guide RNA (gRNA) upon anhydrotetracycline (ATc) induction, thereby activating CRISPR-mediated gene disruption. We demonstrate that targeting the essential genes (ERG13, PGA3, TPI1 or CDC19) leads to severe growth inhibition upon ATc induction. Still, the current set up does not allow complete killing of the cells due to system inactivation, e.g. escape from CRISPR based cutting. We studied reasons for system inactivation and substantially improved the system by simultaneous expression of two different gRNAs. Sequencing escape mutants revealed mutations in both the gRNA sequences and target genes as potential sources of system inactivation. CONCLUSIONS: This work highlights the potential of harnessing a CRISPR-based kill switch in S. cerevisiae. Cells expressing the system were able to escape growth inhibition through mutations and further optimization of the KiSS system is still needed for it to be used in various cell factory applications.},
}
@article {pmid41739318,
year = {2026},
author = {Joshi, D and Kshatri, P and Tiwari, A and Bhardwaj, U and Patel, K},
title = {CRISPR/Cas9 in Cancer Therapy: Precision Genome Editing Approaches Targeting Hematological Malignancies and Solid Tumors Through Cellular, Biochemical, and Molecular Mechanisms.},
journal = {Cell biochemistry and biophysics},
volume = {84},
number = {2},
pages = {1759-1788},
pmid = {41739318},
issn = {1559-0283},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; *Hematologic Neoplasms/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Being extremely precise in terms of genetic material modifications, CRISPR/Cas9 technology has very rapidly become the cornerstone in the field of precision oncology. This review is focused on the great potential offered by CRISPR/Cas9 in terms of cancer treatment, emphasizing its cellular, biochemical, and molecular mechanisms of action. We provide a short historical background and description of its operational principles, followed by an in-depth analysis of how CRISPR/Cas9 reprograms oncogenic signaling networks by selectively modifying cancer-associated genes such as KRAS, MYC, BRAF, and EGFR, and restoring the function of tumor suppressors including TP53, RB1, and PTEN. The review further explores its ability to remodel cellular pathways involved in apoptosis, DNA repair, and cell-cycle regulation, alongside its modulation of key biochemical cascades. We analyse the technology’s application to epigenetic modifications and the regulation of non-coding RNAs as arising therapeutic targets. It also considers the deployment of CRISPR/Cas9 across various cancers, including haematological malignancies such as leukaemia and lymphoma and solid tumors such as breast, lung, and colorectal cancer, where it is being contextualized to disease-specific outcomes and limitations. To overcome delivery issues, recent advances in various viral vectors (AAV, lentivirus) and non-viral methods such as lipid nanoparticles, polymeric nanotechnologies, exosomes and magnetic nanoparticles are explained. In addition, findings from completed and ongoing clinical trials that demonstrate the clinical translation of CRISPR are presented. Lastly, important issues are considered, including immune reactions, off-target effects, and integration with precision oncology. When combined, these viewpoints provide a thorough understanding of the current state of CRISPR/Cas9 and its potential to revolutionize cancer treatment in the future.},
}
@article {pmid41742232,
year = {2026},
author = {Deng, H and Wang, J and Meng, F and Ma, C and Li, J and Wang, J and Wang, X and Zhao, C and Zhang, Y and Wang, R and Chen, N},
title = {Combining computer-aided enzyme design and chromosomal integration for plasmid-free biosynthesis of 1,5-pentanediol in Escherichia coli.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41742232},
issn = {1475-2859},
support = {22JCQNJC01310//the Natural Science Foundation of Tianjin/ ; 2023YFD13000700//the National Key Research and Development Program of China/ ; 2021ZDSYS10//the Key Research and Development Program of Shandong Province/ ; },
mesh = {*Escherichia coli/metabolism/genetics ; *Glycols/metabolism ; Plasmids/genetics ; Nocardia/genetics/enzymology ; *Metabolic Engineering/methods ; Alcohol Dehydrogenase/metabolism/genetics ; CRISPR-Cas Systems ; Computer-Aided Design ; Biosynthetic Pathways ; Pentanes ; },
abstract = {BACKGROUND: 1,5-Pentanediol (1,5-PDO) is a high-value chemical with broad uses in polymer, cosmetic, and pharmaceutical industries. Although diverse biosynthetic pathways have been constructed, current recombinant strains typically rely on plasmid-based overexpression, which necessitates antibiotics and hinders industrial-scale production. RESULTS: We developed a robust, plasmid-free Escherichia coli platform for de novo 1,5-PDO synthesis by integrating pathway genes (davB, davA, gabT, yahK, car, sfp and yqhD) into the chromosome of a lysine-hyperproducing strain via CRISPR/Cas9. Screening of carboxylic acid reductases identified Nocardia iowensis CAR-Ni as the most effective, yielding a base strain (D13) that produced 0.672 g/L 1,5-PDO. Integrated analysis confirmed the alcohol dehydrogenase (ADH)-mediated reduction of 5-hydroxypentanal (5-HP) as an underappreciated bottleneck. We subsequently screened ten endogenous ADHs and selected YjgB for computational optimization. Docking-guided saturation mutagenesis at position E205 yielded the variant YjgB(E205C), which exhibited a 3.34-fold increase in in vitro activity, reduced 5-HP accumulation, and elevated the titer to 0.935 g/L. Enhancing NADPH supply by integrating pntAB further raised the shake-flask titer to 1.5 g/L. In a 5-L fed-batch bioreactor, the final strain (D91) achieved 12.1 g/L 1,5-PDO (yield of 0.225 mol/mol glucose) without antibiotics or inducers. To our knowledge, this is the highest reported 1,5-PDO titer in E. coli. CONCLUSION: This study establishes a scalable, sustainable biosynthetic platform through synergistic metabolic engineering and computational enzyme optimization.},
}
@article {pmid41746436,
year = {2026},
author = {Chen, N and Sun, X and Liang, S and Cao, C and Lai, X and Song, C and Yan, Z and Ge, C},
title = {Single particle mediated CRISPR/Cas13a ultrasensitive direct detection of miRNA-21 at femtomolar levels without nucleic acid amplification.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {},
pmid = {41746436},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics ; Humans ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; DNA Probes/chemistry/genetics ; },
abstract = {A novel biosensing platform has been developed that couples the collateral cleavage activity of the CRISPR/Cas13a system with single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) using gold nanoparticle (AuNP)-DNA reporter probes. In the presence of target microRNA-21, Cas13a is activated and specifically cleaves RNA bases within the DNA-RNA hybrid linkers on AuNPs, preventing their hybridization with biotinylated capture probes on magnetic beads. As a result, the cleaved AuNPs remain in the supernatant and are directly quantified by sp-ICP-MS. The number of detected AuNPs correlates linearly with the concentration of miRNA-21 (y = 0.2537logCmiRNA + 0.2477 with a correlation coefficient of R2 = 0.9978), enabling a detection limit as low as 68 fM with excellent single-base mismatch discrimination. The assay demonstrated high recoveries (97–108%) and reproducibility in spiked human serum samples, confirming its reliability in complex matrices. This amplification-free strategy combines the high specificity of CRISPR/Cas13a with the single-particle sensitivity of sp-ICP-MS, providing a robust, quantitative, and versatile platform for miRNA detection with promising potential for early cancer diagnostics and precision medicine.},
}
@article {pmid41762386,
year = {2026},
author = {Duran, T and Karaselek, MA and Dagdelen, B and Kuccukturk, S and Guner, SN and Keles, S and Reisli, I},
title = {CRISPR-Cas9-based gene editing as a proof-of-concept approach in an inborn error of immunity caused by a DCLRE1C variant.},
journal = {Immunologic research},
volume = {74},
number = {1},
pages = {},
pmid = {41762386},
issn = {1559-0755},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Severe Combined Immunodeficiency/genetics/therapy ; *Endonucleases/genetics ; DNA-Binding Proteins/genetics ; *Nuclear Proteins/genetics ; Interleukin-2 Receptor alpha Subunit/metabolism/genetics ; },
abstract = {Hypomorphic DCLRE1C variants impair T and B cell development, leading to combined immunodeficiency (CID) or leaky severe combined immunodeficiency (SCID). Current treatment options, such as allogeneic hematopoietic stem cell transplantation (aHSCT), are associated with significant risks, highlighting the need for alternative therapeutic strategies. In this study, we report the first a proof-of-concept CRISPR-Cas9–mediated correction of a hypomorphic DCLRE1C variant (c.194 C > T; p.T65I) in CD4 + helper T (Th) cells using CRISPR-Cas9 gene-editing technology. CD4 + Th cells were isolated, and the variant region was edited with sgRNA and donor DNA. Gene editing efficiency was confirmed by Sanger sequencing, revealing successful restoration of the target region to its wild-type sequence. Functional analyses showed a significant increase in CD25 activation and Artemis protein expression post-editing, although DCLRE1C mRNA levels remained unchanged. The approximately 6–8% increase in CD25 expression was statistically significant but did not reach healthy control levels. These findings suggest that CRISPR-Cas9 –mediated gene editing may enable precise correction and induce measurable cellular-level functional changes, supporting biological feasibility rather than therapeutic efficacy. This study provides a foundation for future research on HSCs and underscores the potential role of CRISPR-Cas9–based approaches in the treatment of inborn errors of immunity (IEIs) associated with DCLRE1C variants.},
}
@article {pmid41764471,
year = {2026},
author = {Chen, H and Luo, W and Ma, N and Li, S},
title = {CRISPR/Cas9-mediated B2m knockout paves the way for allogeneic basal cell transplantation.},
journal = {Respiratory research},
volume = {27},
number = {1},
pages = {},
pmid = {41764471},
issn = {1465-993X},
support = {2023B111105006//Guangdong Key Area R&amp;D Program Key Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/physiology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice ; *beta 2-Microglobulin/genetics/deficiency ; Transplantation, Homologous/methods ; Mice, Knockout ; *Gene Knockout Techniques/methods ; Cells, Cultured ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Autologous transplantation of basal cells (BCs) has shown promise in treating respiratory diseases, but disease-specific subpopulations among BCs probably diminish the treatment efficacy. An alternative approach involves generating universal and healthy BCs, which offers a potentially more efficient and accessible solution for avoiding using abnormal BCs. However, such hypoimmunogenic BCs have not yet been transplanted into the airways of immunocompetent animals. METHODS: Before producing hypoimmunogenic BCs, the predominant transplantation antigen in BCs was explored through RT-qPCR and flow cytometry to identify the key target of CRISPR/Cas9‐mediated editing. The proliferation and expression of specific markers of BCs were evaluated after gene editing by CCK‐8 and RT‐qPCR, respectively. These gene‐edited BCs and wild‐type (WT) BCs, which were both derived from the same BALB/c mouse, were subsequently allogeneically transplanted into C57BL/6 mice with polidocanol‐induced airway injury to evaluate the differentiation and immune response in the recipient mice via immunehistological staining. RESULTS: In the present study, it was demonstrated that major histocompatibility complex class I (MHC-I) is the predominant transplantation antigen in BCs. The hypoimmunogenic BCs were generated through editing beta‐2 microglobulin (B2m) participating in the encoding of MHC‐I. The knockout of B2m in BCs did not affect their proliferation or the expression of specific markers in vitro. Both WT BCs and B2m‐edited BCs (B2m‾ BCs) successfully differentiated into ciliated and secretory cells in the tracheas following allogeneic transplantation and did not elicit an immune response during the 26‐day observation period in the tracheas. However, WT BCs, compared to B2m‾ BCs, induced severe lung injury by provoking an immune response in the lower airways and alveolar regions, as indicated by increased infiltration of CD45+ immune cells, epithelial cell shedding in the bronchi, and obvious alveolar hyperemia with collapse. CONCLUSIONS: The hypoimmunogenic BCs generated through CRISPR/Cas9-mediated B2m gene editing retained their ability to differentiate and maintained viability in the allogeneic respiratory system, supporting the potential application in the cell regeneration therapy of airway diseases.},
}
@article {pmid41766009,
year = {2026},
author = {Vargas-Reyes, M and Alcántara, R and Alfonsi, S and Peñaranda, K and Petrelli, D and Spurio, R and Pajuelo, MJ and Milon, P},
title = {Versatile and portable Cas12a-mediated detection of antibiotic resistance markers.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41766009},
issn = {2045-2322},
support = {C-004-2021-2//Universidad Peruana de Ciencias Aplicadas/ ; D43TW001140//Forgarty International Center/ ; PE501079419-2022//PROCIENCIA - CONCYTEC/ ; },
mesh = {*Escherichia coli/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; Genetic Markers ; Microbial Sensitivity Tests ; *Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results readout (Fluorescence vs. FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.},
}
@article {pmid41770417,
year = {2026},
author = {Melamed, J and Barnoy, S},
title = {Understanding the public's intention to adopt CRISPR-Cas9: the effect of beliefs, knowledge, and innovativeness.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {41770417},
issn = {1432-1203},
mesh = {Humans ; Adult ; *CRISPR-Cas Systems ; Female ; Male ; *Gene Editing ; Young Adult ; Middle Aged ; *Intention ; Surveys and Questionnaires ; *Health Knowledge, Attitudes, Practice ; },
abstract = {CRISPR-Cas9 is a gene editing technology with wide-ranging medical potential and significant ethical implications. This study examined how personality traits, familiarity with CRISPR, knowledge about CRISPR, and beliefs concerning its applications are connected to the public’s willingness to adopt this technology, drawing on Rogers’ Diffusion of Innovations framework. A sample of 500 young adults aged 20–45 completed questionnaires assessing innovativeness as well as familiarity, knowledge, beliefs, and willingness to adopt CRISPR-Cas9 for therapeutic and non-therapeutic purposes. Results showed that only 24% of participants were familiar with CRISPR and that knowledge levels were generally low. Based on Rogers’ typology, 18.8% were identified as innovators, and 26.8% as early adopters, and only 3.2% as laggards. Beliefs were strongly associated with willingness to adopt CRISPR-Cas9 (r = .63, p < .001), moderating the weak associations of personality traits with adoption intentions. These findings suggest that beliefs are an important factor in CRISPR-Cas9 adoption and they appear to have more influence on willingness to adopt CRISPR-Cas9 than knowledge and innovativeness in this sample. Efforts to promote informed public discussion on CRISPR should be made, with an emphasis on ethical aspects alongside scientific information. This is critical for the responsible adoption of this technology.},
}
@article {pmid41772313,
year = {2026},
author = {Li, Y and Ye, Z and Zhao, C and Tan, Y and Chen, J and Wu, Z and Zhang, Y and Guo, H and Cheng, Y and Wang, R and Wang, J and Wang, D},
title = {Integrated on-site detection of Fusarium temperatum based on a droplet digital CRISPR-based platform.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {},
pmid = {41772313},
issn = {1436-5073},
support = {2024A1515011281//Guangdong Basic and Applied Basic Research/ ; 2023C043-5, 2022C044-9//Technology Research and Development Project from the Development and Reform Commission in Jilin Province of China/ ; },
mesh = {*Fusarium/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Zea mays/microbiology ; DNA, Fungal/genetics/analysis ; Rapid Diagnostic Tests ; Smartphone ; Microscopy, Fluorescence/methods ; },
abstract = {Fusarium temperatum (F. temperatum) is a fungus whose infection can cause various diseases in maize plants, leading to premature death, and F. temperatum mycotoxin poses a serious threat to human and animal health. Rapid and early on-site detection of F. temperatum infection facilitates the prevention of disease progression, which is an unmet need. In this study, a droplet digital CRISPR-Cas12a-based platform (DD-Cas), combined with a rapid extraction procedure, was developed for amplification-free on-site detection of F. temperatum. The DD-Cas assay can rapidly detect F. temperatum genomic DNA in infected maize samples with high sensitivity (102 CFU/mL) and specificity. Furthermore, we developed a smartphone-based fluorescence microscope integrating the heating module, that could accurately detect infected samples within 30 min, enabling low-cost point-of-care testing (POCT). This platform can avoid cross-contamination and amplification bias, thus having great potential for on field detection of pathogenic bacteria in agriculture.},
}
@article {pmid41775739,
year = {2026},
author = {Li, X and Wang, S and Qiu, Z and Sun, R and Wang, T and Ren, X and Lv, B and Ma, X and Cheng, L and Liu, Y and Jiang, J},
title = {Construction and initial validation of key gene network for progesterone resistance in endometrial cancer based on genome-wide CRISPR screening.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41775739},
issn = {2045-2322},
support = {2023M731847//China Postdoctoral Science Foundation/ ; 2022YFC2704304//National Key Technology Research and Development Programme of China/ ; },
mesh = {Female ; *Endometrial Neoplasms/genetics/drug therapy/pathology ; Humans ; Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Regulatory Networks ; *Drug Resistance, Neoplasm/genetics ; Mice ; Gene Expression Regulation, Neoplastic/drug effects ; Progestins/pharmacology ; *Progesterone/pharmacology ; Apoptosis/drug effects ; Endometrium/abnormalities ; Uterine Diseases ; },
abstract = {Endometrial carcinoma, a prevailing malignancy of the female reproductive system, exhibits escalating incidence and a trend towards early onset. Hormone therapy serves as a primary choice for fertility preservation and is also considered for advanced and recurrent cases. However, a considerable number of patients fail to respond favorably to progestin treatments. We employed CRISPR/Cas9 technology to establish a comprehensive human genome library in the Ishikawa cell line. Subsequent exposure to medroxyprogesterone was followed by high-throughput sequencing, and differential gene expression and enrichment analyses were conducted using Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) Robust Rank Aggregation (RRA) and MAGeCK Maximum-Likelihood Estimation (MLE) algorithms. An iterative data intersection approach was employed, utilizing sequenced data from progestin-resistant cell lines, to identify pivotal genes associated with progestin resistance. The top 10 identified genes were functionally validated in our previously established progestin-resistant cell model through Cell Counting Kit-8 (CCK-8) assays and apoptosis detection. The progestin-resistant gene NNMT and the progestin-sensitive gene SOX17 were validated in vivo in xenograft mouse models. The constructed library exhibited high quality, meeting sequencing standards. Employing RRA and MLE algorithms, we identified 332 and 829 negative selection genes, as well as 3438 and 5098 positive selection genes. Enrichment analysis implicated pathways linked to DNA and RNA synthesis, metabolism, and related processes. After multiple data intersections, we identified a total of 5 genes promoting progestin resistance and 20 genes inhibiting resistance, with functional experiments confirming their roles. Employing CRISPR/Cas9 technology enables the construction of a relatively reliable network of pivotal genes associated with progestin resistance in endometrial carcinoma. Processes involving DNA and RNA synthesis, metabolism, and related mechanisms appear to significantly impact the progestin sensitivity of endometrial carcinoma.},
}
@article {pmid41792204,
year = {2026},
author = {Kim, Y and Jun, Y and Han, J and Choi, S and Kim, H and Lee, M and Kim, SL and Kang, SH and Suh, EJ and Park, SR and Mo, Y},
title = {CRISPR/Cas9-mediated mutagenesis of SMXL4 alters plant height and yield-related traits in rice (cv. Samkwang).},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41792204},
issn = {2045-2322},
support = {RS-2024-00322166//Rural Development Administration/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Mutagenesis ; Plants, Genetically Modified ; Gene Editing ; Phenotype ; },
abstract = {Recent climate change and frequent extreme weather events during the maturation period of rice exacerbate lodging and threaten stable production. Samkwang, a widely cultivated rice variety in Korea, is particularly vulnerable to lodging due to its tall stature. To improve lodging tolerance while preserving Samkwang’s elite genetic background, we identified an SMXL4-edited line (smxl4) with reduced culm length and stable growth from a CRISPR/Cas9-edited Samkwang population. The biological function of SMXL4, a clade Ⅳ member of the SMXL (SUPPRESSOR OF MAX2 1-LIKE) family, has not been well characterized in rice. Compared to Samkwang, the smxl4 plants showed reduced plant height, internode length, panicle length, grain number per panicle, and grain weight, while panicle number per plant increased. Transcriptome profiling of elongating internodes at booting and heading stages revealed upregulation of genes associated with cell wall remodeling and defense responses in smxl4 relative to Samkwang. These findings highlight the broad involvement of SMXL4 in rice growth and development and provide insights for breeding lodging tolerant rice cultivars.},
}
@article {pmid41803678,
year = {2026},
author = {Jiang, Q and Zeng, X and Zhang, Q and Yang, F and Lv, T and Zhang, Y and Wang, J and Li, F and Xu, D},
title = {Development and application of a rapid detection system for Aspergillus fumigatus based on ERA/CRISPR-Cas12a.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41803678},
issn = {1471-2180},
support = {2024AH051688, 2024AH051225//the University Natural Science Research Key Projects in Anhui Province/ ; },
mesh = {*Aspergillus fumigatus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; Rapid Diagnostic Tests ; *Aspergillosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/genetics ; Sputum/microbiology ; Limit of Detection ; },
abstract = {Aspergillus fumigatus (AF) is the predominant pathogen implicated in invasive aspergillosis (IA) in humans; therefore, prompt and accurate detection is critical for the effective prevention and management of IA. This study developed a rapid detection system targeting the AF-specific anxC4 gene by integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a. The reaction proceeds at a stable temperature of 37 °C, with amplification and detection systems separately positioned in the tube lid and bottom, respectively, effectively minimizing aerosol contamination typically associated with product transfers. To enhance sensitivity, the One-Pot method was optimized. Consequently, the fluorescence detection limit reached 1 fg/µL, and the sensitivity of the test strip reached 10 fg/µL, with no cross-reactivity observed against other fungi. Detection of AF was completed within 60 min, and results were visually displayed through fluorescence signals and nucleic acid test strips. Clinical practicality was further evaluated using aspergillosis samples, which demonstrated satisfactory performance. Pure culture results confirmed that out of 62 sputum samples, 32 were positive and 30 negative. Evaluation of 62 clinical samples using the One-Pot ERA-CRISPR/Cas12a system demonstrated sensitivity and specificity rates of 93.75% and 93.33%, respectively, via fluorescence detection, and 90.63% sensitivity and 96.67% specificity using lateral flow strips.},
}
@article {pmid41808028,
year = {2026},
author = {Grubben, J and Bijsterbosch, G and Visser, RGF and Schouten, HJ},
title = {Influence of gRNA efficiency and inversion size on the frequency of CRISPR/Cas9-induced chromosomal inversions in tomato protoplasts.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41808028},
issn = {1471-2229},
support = {GSGT.2019.016//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; TU18048//Top Sector Agri and Food/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Protoplasts/metabolism ; *Chromosome Inversion/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 enables induction of chromosomal inversions from hundreds of base pairs to millions of base pairs, but the factors influencing inversion frequency are not well understood. Prior reports differ in species, detection methods, and delivery strategies, making direct comparisons difficult. We addressed this by introducing a normalisation strategy based on a reference guide RNA (gRNA) as an internal standard and testing inversion sizes spanning kilobases to tens of megabases in tomato protoplasts. RESULTS: Tomato (Solanum lycopersicum) protoplasts were transfected with constructs encoding a fixed “reference” gRNA and a second “variable” gRNA positioned at increasing genomic distances, creating potential inversions from 1 kilobase to 37.5 megabases. Using the reference gRNA to normalise across samples, we found that up to ~ 1 megabase, inversion frequency tracked the cutting efficiency of the less efficient gRNA, consistent with gRNA activity being a major contributor within the chromosome tested. For these intervals, the inversion frequencies reached up to 1.24% when both gRNAs were efficient. Above ~ 1 megabase, inversion frequencies declined sharply despite efficient cutting, suggesting a size-dependent barrier to inversion formation; for example, 37.5 megabase inversions occurred at substantially lower frequency (up to 0.18%) despite efficient gRNAs. Because each interval corresponds to a distinct genomic location. Inversions were only observed when both gRNAs were active, and large deletions were more frequent than inversions when dual breaks were induced. CONCLUSIONS: In our experiments, the gRNA cutting efficiency was a major determinant of inversion frequency in our experiment up to ~ 1 megabase, while locus-specific genomic context may also contribute., Larger inversions may be limited by an additional, size-dependent constraint. These findings inform the design of edits aimed at reverting breeding-relevant inversions (for example, those linked to resistance loci) and suggest that achieving high efficiency for multi-megabase inversions will require strategies that overcome spatial or repair-related constraints. The internal reference gRNA normalises sample-to-sample variability in DNA delivery and Cas9 activity, enabling direct comparison of the performance of different gRNAs across samples on a shared, ratio-based scale. This ratio-to-reference strategy may likewise be used to benchmark gRNA performance and edit yields (inversions, deletions, translocations, and base/prime edits) across transfections in plant protoplasts, and may be extended to additional cell systems beyond plants.},
}
@article {pmid41817895,
year = {2026},
author = {Prins, TJ and Lai, TJ and Li, T and Fisher, A and Eldred, BSC and Mostafavi, R and Liau, LM and Chong, RA and Nghiemphu, PL and Cloughesy, TF and Nathanson, DA and Lai, A},
title = {MGMT downregulation by CRISPR/Cas13 RNA-guided RNA targeting enhances glioma cell sensitivity to TMZ chemotherapy.},
journal = {Journal of neuro-oncology},
volume = {177},
number = {1},
pages = {},
pmid = {41817895},
issn = {1573-7373},
support = {P50 CA211015/CA/NCI NIH HHS/United States ; P50 CA211015-01A1/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; Temozolomide ; *Tumor Suppressor Proteins/metabolism/genetics ; *Glioma/drug therapy/metabolism/genetics ; *Antineoplastic Agents, Alkylating/pharmacology ; *DNA Modification Methylases/metabolism/genetics ; *DNA Repair Enzymes/metabolism/genetics ; *Brain Neoplasms/drug therapy/metabolism/genetics ; Down-Regulation ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Dacarbazine/analogs &amp; derivatives/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Drug Resistance, Neoplasm ; RNA, Messenger/metabolism ; Cell Survival/drug effects ; },
abstract = {BACKGROUND: Current standard of care for glioblastoma involves fractionated radiotherapy administered with Temozolomide (TMZ), a DNA-alkylating agent. Inhibition of the DNA repair enzyme, O[6]-methylguanine-DNA methyltransferase (MGMT), promotes sensitivity to TMZ, particularly in tumors that repress MGMT mRNA transcription through promoter methylation. Novel strategies to inhibit MGMT are a promising avenue to improve therapeutic outcomes to TMZ. We hypothesized that CRISPR-Cas13-mediated RNA regulatory silencing of MGMT mRNA enhances response of immortalized and primary patient-derived gliomaspheres to TMZ in vitro. METHODS: We utilized the Cas13x and Cas13d variants to target MGMT mRNA in the MGMT-expressing LN18 glioma cell line and in two patient-derived gliomasphere lines (GS104, GS081). Cas13-guide RNA ribonucleoproteins were delivered via lipofection, and stable knockdown was achieved using a lentiviral all-in-one system. MGMT mRNA and protein downregulation were assessed by RT-PCR and Western blot, respectively. Cell viability and chemosensitivity to TMZ were evaluated using MTT assays. RESULTS: Both Cas13x and Cas13d systems, directed by specific guide CRISPR RNAs, achieved rapid and potent knockdown of MGMT mRNA and protein in all tested cell lines. This downregulation of MGMT expression led to an increase in the cytotoxic effects of TMZ, sensitizing previously resistant glioma cells and patient-derived gliomaspheres to standard chemotherapy. The lentiviral Cas13d system established stable chemosensitization in gliomasphere models. CONCLUSION: CRISPR-Cas13-mediated targeting of MGMT mRNA is an effective strategy for overcoming TMZ resistance in in vitro glioblastoma models. This RNA regulatory editing approach offers a proof-of-principle for CRISPR mediated therapeutics in patients with MGMT unmethylated gliomas.},
}
@article {pmid41839945,
year = {2026},
author = {Yanguas-Casás, N and Pedrosa, L and Horcajo, B and Gómez, S and Garcia-Grande, A and Muñoz-Viana, R and Fernández-Miranda, I and Pérez-Aguilera, M and Torres-Ruiz, R and Rodríguez-Perales, S and Sánchez-Beato, M},
title = {Splice-site mutations in POU2AF1 are associated with B-cell lymphomagenesis and therapeutic response.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839945},
issn = {2045-2322},
support = {POSTD18029SANC//Fundaci&#xf3;n Cient&#xed;fica de la Asociaci&#xf3;n Espa&#xf1;ola Contra el C&#xe1;ncer/ ; iPFIS predoctoral fellowship (IFI18/0004)//ISCIII-MINECO AES-FEDER, Plan Estatal I+D+I 2014-2020/ ; PEJ-2020-TL/BMD-19530//Plan de Empleo Juvenil de la CM/ ; PEJ-2023-AI/SAL-GL-28806//Plan de Empleo Juvenil de la CM/ ; B2017/BMD-3778//Direcci&#xf3;n General de Universidades e Investigaci&#xf3;n de la Consejer&#xed;a de Educaci&#xf3;n e Investigaci&#xf3;n de la Comunidad de Madrid (CM)/ ; PI17/00272, PI20/00591, PI23/01587//Spanish Ministry of Economy and Competence (MINECO) and Instituto de Salud Carlos III (ISCIII), ISCIII-MINECO AES-FEDER/ ; },
mesh = {Humans ; Cell Line, Tumor ; *RNA Splice Sites/genetics ; *Lymphoma, B-Cell/genetics/pathology/drug therapy ; Cell Proliferation/genetics ; *Mutation ; Cell Movement/genetics ; *Trans-Activators/genetics ; Point Mutation ; CRISPR-Cas Systems ; },
abstract = {BOB.1, encoded by POU2AF1, is one of many factors regulating physiological B-cell maturation in the germinal center. Recently, several studies have described recurrent mutations in a three-nucleotide region in the POU2AF1 splice site in the two most common B-cell non-Hodgkin lymphomas: diffuse large B-cell lymphoma and, more frequently, follicular lymphoma. In this study, we introduced a C→G mutation at the + 1 position of the POU2AF1 splice site in two B-cell lymphoma cell lines (WSU-NHL and SUDHL4) using CRISPR/Cas9 gene editing. Our results demonstrate how point mutations in the POU2AF1 splice site decreased BOB.1 expression levels. The mutation did not produce significant changes in cell proliferation, migration, or invasiveness, but did affect cell morphology, aggregation, and cell survival in a cell-line-dependent manner. Lastly, we found that the POU2AF1 mutation c.16 + 1G > C increased BCR activation, especially in SUDHL4 cells, downregulated oxidative phosphorylation (OxPhos) metabolism, and modified therapy sensitivities in both cell lines. Mutated B-cells were more sensitive to the BTK inhibitor ibrutinib. In conclusion, mutations in the POU2AF1 splice site impact B-cell lymphomagenesis at multiple levels and represent a potential therapeutic target for patients with tumors harboring this mutation.},
}
@article {pmid41839984,
year = {2026},
author = {Taguchi, YH and Turki, T},
title = {Gene and cell line efficiency of CRISPR computed by tensor decomposition in genome-wide CRISPR-Cas9 knockout screens.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839984},
issn = {2045-2322},
support = {Deanship of Scientific Research, IPP: 70-611-2025//King Abdulaziz University/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Cell Line ; Algorithms ; },
abstract = {Genome-wide CRISPR-Cas9 knockout screens are often used to experimentally evaluate gene function. However, the efficacy of individual sgRNAs targeting unique genes varies and is difficult to integrate. In this study, tensor decomposition (TD) was used to integrate multiple sgRNAs and sgRNA profiles simultaneously. Thus, TD can discriminate between essential and non-essential genes with the performance comparative to that of Joint analysis of CRISPR/Cas9 knockout screens (JACKS), a type of SOTA that previously outperformed various other SOTA. In addition, although TD uses simple linear algebra, it can achieve good performance even without control samples, without which JACKS cannot be performed. Moreover, because raw and logarithmic values can achieve similar performances through TD for the largest dataset among the tested datasets, taking logarithmic values as has been done frequently, which is questioned. In conclusion, TD is the first method that can integrate multiple sgRNAs attributed to single a target and sgRNA profiles at the beginning simultaneously and can achieve a performance comparable to that of JACKS.},
}
@article {pmid41888883,
year = {2026},
author = {Yuan, G and Deng, S and Dai, Z and Hofstad, BA and Pomraning, KR},
title = {Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41888883},
issn = {1475-2859},
mesh = {*Aspergillus niger/genetics ; *Cytosine/metabolism ; *Gene Editing/methods ; *Adenine/metabolism ; CRISPR-Cas Systems ; },
abstract = {Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger. For the first time, we constructed an ABE in A. niger, achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.},
}
@article {pmid41961403,
year = {2026},
author = {Sun, J and Hu, Z and Yang, Y},
title = {An ATMT-CRISPR/Cas9 system for genome editing in Monascus purpureus.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {4},
pages = {},
pmid = {41961403},
issn = {1573-0972},
mesh = {*Monascus/genetics/metabolism ; *CRISPR-Cas Systems ; *Agrobacterium tumefaciens/genetics ; *Gene Editing/methods ; Transformation, Genetic ; Homologous Recombination ; Genome, Fungal ; Gene Knockout Techniques ; Protoplasts ; Biosynthetic Pathways/genetics ; Fungal Proteins/genetics ; },
abstract = {Monascus purpureus is a filamentous fungus of significant economic value in the food and pharmaceutical industries, capable of producing a diverse array of secondary metabolites. Although CRISPR/Cas9 systems have been extensively utilized in filamentous fungi, predominantly employing protoplasts as recipients, the genetic manipulation of M. purpureus remains challenging due to the inherent difficulties associated with protoplast preparation. In addition, it has been reported that the Cas9 protein may demonstrate toxicity to cells. To overcome this limitation, this study developed a CRISPR/Cas9 gene-editing system based on Agrobacterium tumefaciens -mediated transformation (ATMT). This system utilizes M. purpureus spores as recipients and employs a homologous recombination strategy to achieve stable, site-specific integration of the Cas9 expression cassette into non-coding regions of the host genome, thereby avoiding the complexity of protoplast preparation and the uncertainty of random integration events. System evaluations indicate that the stable expression of Cas9 protein has no significant adverse effects on the nutritional growth, reproductive development, or characteristic pigment biosynthesis of M. purpureus. The system demonstrated high efficacy in single-gene editing, achieving a knockout efficiency of 74% for the key pigment biosynthetic pathway gene pksPT. Although efficiency decreased when performing double-gene and triple-gene editing (4.8% and 1.7%, respectively), this study successfully validated the system’s potential for multi-gene genome engineering. The ATMT-CRISPR/Cas9 system established herein demonstrates the feasibility of genetic manipulation of M. purpureus, providing a methodological foundation with the potential to facilitating functional genomic studies and the targeted regulation of secondary metabolism in this industrially important fungus.},
}
@article {pmid42000863,
year = {2026},
author = {Piñeiro-Silva, C and Bermejo-Álvarez, P and García-Purriños, FJ and Gadea, J},
title = {Gene editing of the GJB2 locus in porcine embryos using CRISPR/Cas9 and cytosine base editors: toward a model of congenital deafness.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42000863},
issn = {2045-2322},
support = {22065/PI/22//Fundaci&#xf3;n S&#xe9;neca-Agencia de Ciencia y Tecnolog&#xed;a de la Regi&#xf3;n de Murcia/ ; 22545/PDC/24//Fundaci&#xf3;n S&#xe9;neca-Agencia de Ciencia y Tecnolog&#xed;a de la Regi&#xf3;n de Murcia/ ; 23031/GERM/25 supported by FSRM/10.13039/100007801//Fundaci&#xf3;n S&#xe9;neca-Agencia de Ciencia y Tecnolog&#xed;a de la Regi&#xf3;n de Murcia/ ; R-496/2022//Universidad de Murcia predoctoral fellowship/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Connexin 26/genetics ; *Cytosine/metabolism ; Swine ; *Deafness/genetics/congenital ; *Connexins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; Mutation ; Disease Models, Animal ; Oocytes/metabolism ; },
abstract = {Mutations in the GJB2 gene, which encodes Connexin 26 (Cx26), are responsible for the majority of cases of non-syndromic congenital hearing loss in humans. While murine GJB2 knockout models have provided mechanistic insight, anatomical and physiological differences limit their translational relevance. Pigs represent a valuable large-animal model because their auditory anatomy and maturation closely resemble those of humans. This study compared two genome-editing approaches to disrupt GJB2 in porcine oocytes before fertilization: (1) electroporation with CRISPR/Cas9 ribonucleoprotein and (2) microinjection with cytosine base editor (BE3) and single-guide RNAs (sgRNAs). Electroporation produced high mutation rates (70–90%) across three concentrations of Cas9/sgRNA but yielded mostly heterozygous or mosaic blastocysts, with limited homozygous knockouts (< 4%). BE3 achieved precise cytosine-to-thymine conversions that introduced premature stop codons, reaching up to 47% total editing and 20% homozygous nonsense alleles. However, blastocyst formation declined at higher component concentrations. Overall, BE3 produced more predictable mutations than conventional CRISPR/Cas9, although embryo developmental competence was dose-dependent. Both methods effectively targeted GJB2 and demonstrated feasibility of pre-fertilization genome editing in porcine oocytes. These findings establish the groundwork for generating GJB2-deficient pigs as translational models of Cx26-related congenital deafness and for future evaluation of gene-therapy strategies in a large-animal system.},
}
@article {pmid42012544,
year = {2026},
author = {Shen, W and Xiao, R and Li, J and Zheng, S and Wang, C and Hao, R and Yin, J},
title = {CRISPR-based dual-mode lateral flow assay driven by magnetic SERS tags for highly sensitive detection of respiratory viruses.},
journal = {Mikrochimica acta},
volume = {193},
number = {5},
pages = {},
pmid = {42012544},
issn = {1436-5073},
support = {82372348//National Natural Science Foundation of China/ ; },
mesh = {*Spectrum Analysis, Raman/methods ; *SARS-CoV-2/isolation &amp; purification/genetics ; Humans ; *Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; Gold/chemistry ; Colorimetry/methods ; Rapid Diagnostic Tests ; Limit of Detection ; CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; },
abstract = {Effective epidemic control hinges on rapid point-of-care detection of respiratory viruses, but the sensitivity of current screening technologies remains inadequate. Here, we developed a CRISPR-activated, colorimetric Surface-Enhanced Raman Scattering (SERS) dual-mode nucleic acid lateral flow assay (LFA) that enables highly sensitive, flexible, and simultaneous detection of two common respiratory viruses, influenza A (H1N1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By coupling recombinase polymerase amplification with CRISPR for sensitive amplification and specific target recognition, viral targets activate Cas13 trans-cleavage to efficiently cleave reporter probes. In parallel, a dual-functional magnetic SERS tag (Fe3O4@Au/Au-SA) was introduced, which captures two biotinylated reporter molecules through streptavidin modification, and generates strong and stable SERS signals through built-in hotspot effects. The resulting CRISPR-magnetic SERS-LFA enables rapid qualitative screening of H1N1 and SARS-CoV-2 through reduced colorimetric signal intensity on two test lines and accurate quantification via SERS signal changes. The establish method achieves a detection limit of 7–9 copies/µL for the two target viruses and shows good agreement with quantitative reverse transcription polymerase chain reaction (qRT-PCR) in validation with 74 clinical samples. Both the sensitivity and specificity for clinical samples reach 100%, highlighting its potential for field deployment.},
}
@article {pmid42015157,
year = {2026},
author = {Yang, P and Ma, R and Zeng, J and Li, L and Peng, J and Zhou, M and Qu, M and Li, X and Lai, T and Zhou, W and Wu, Y and Lu, Y and Zhang, Y},
title = {Simple and rapid profiling of tumor EVs for differential diagnosis of NSCLC via orthogonal barcode-driven CRISPR/Cas12a.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42015157},
issn = {1477-3155},
support = {82172374//National Natural Science Foundation of China/ ; 2023GDRC005//Chongqing Science and technology joint major funding program/ ; 2022CDJYGRH-010//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2021M693730//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/diagnosis ; *Lung Neoplasms/diagnosis ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; Biomarkers, Tumor/blood ; Diagnosis, Differential ; Cell Line, Tumor ; },
abstract = {Tumor-derived extracellular vesicles (tEVs), a class of nanoscale vesicles actively released by malignant cells, have emerged as attractive biomarkers for non-invasive cancer diagnosis. However, their clinical translation remains challenging due to low abundance, molecular heterogeneity, and the requirement for multiplexed surface marker discrimination. Here, we report a dual aptamer-mediated CRISPR/Cas12a-assisted sensing platform (DA-CAS) for rapid and orthogonally programmable dual-marker profiling of tEVs, enabling differential diagnosis of non-small cell lung cancer (NSCLC) using only 10 µL of plasma within 100 min. The DA-CAS system integrates proximity ligation-based dual-marker recognition with hyperbranched rolling circle amplification (HRCA) to generate programmable DNA barcodes, which selectively trigger Cas12a trans-cleavage in an orthogonal manner. Using EpCAM and PD-L1 as representative surface markers, the platform achieves subtype-specific detection of NSCLC-derived tEVs with minimal background activation and a detection limit as low as 75 particles/mL. Moreover, a portable lateral flow readout enables accurate, instrument-free visual detection at concentrations down to 406 particles/mL. Under the condition of free-ultracentrifugation, clinical validation using a cohort of 45 plasma samples demonstrated a sensitivity of 97%, specificity of 88%, and overall diagnostic accuracy of 96%, outperforming conventional ELISA assays and multi-marker serum panels in both analytical sensitivity and subtype resolution. In addition, this platform demonstrated preliminary potential for discriminating between benign and malignant lung diseases and for dynamically monitoring radiotherapeutic responses. The orthogonal barcode design effectively eliminates inter-channel crosstalk and enzymatic interference, enabling orthogonal dual-target recognition with high subpopulation specificity. Overall, DA-CAS provides a robust, rapid, and point-of-care-compatible strategy for tEV-based cancer diagnostics, offering strong translational potential for non-invasive tumor profiling and dynamic immune status monitoring.},
}
@article {pmid42031935,
year = {2026},
author = {Hao, D and Xu, X and Li, P and Liu, Y and Liu, G and Deng, S},
title = {A method for CRISPR/Cas9-induced genetic barcoding and lineage tracing in sheep.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42031935},
issn = {2045-2322},
support = {2023-PT180-01//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Sheep/genetics/embryology ; *Cell Lineage/genetics ; DNA Transposable Elements/genetics ; *Gene Editing/methods ; *DNA Barcoding, Taxonomic/methods ; Embryonic Development/genetics ; },
abstract = {Sheep development involves continuous dynamic processes in which cells propagate, differentiate and orchestrate. However, the method for tracing cell fate during sheep (Ovis aries) embryogenesis, cell differentiation and tissue regeneration remains largely undeveloped. Here, we developed a CRISPR/Cas9-based lineage barcode recording method that directly acts on target cells. With this method, several contiguous CRISPR/Cas9 targeting arrays were synthesized and introduced into multiple loci in the sheep genome using the PiggyBac transposon vector to form the barcode region. Cas9 integrated at the Rosa26 gene locus was used to generate edits in the barcode region at multiple timepoints during early sheep embryonic development. We detected multiple integrated barcodes (intBCs) that were stably inherited in the developing embryos, confirming that the method can generate heritable clonal markers. The method could enable lineage tracing in sheep when combined with single-cell sequencing technologies. Our method establishes a foundation for ruminant lineage tracing technology by combining PiggyBac transposons and CRISPR/Cas9 gene editing tools, providing a new platform for analyzing sheep embryonic development, organ regeneration, and disease mechanisms.},
}
@article {pmid42050530,
year = {2026},
author = {Liao, X and Zhou, J and Shan, Y and Li, X and Peng, Y},
title = {CRISPR/Cas12a-assisted visual and on-site detection of porcine circovirus type 2.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42050530},
issn = {1746-6148},
support = {2024NSFSC0761//the Science and Technology Foundation of Sichuan Province, China/ ; 2021R52041//High-level Talents Special Support Plan of Zhejiang Province/ ; ZYGD23027//the 1&#xb7;3&#xb7;5 Project for Disciplines of Excellence, West China Hospital of Sichuan University/ ; },
mesh = {*Circovirus/isolation &amp; purification/genetics ; Animals ; Swine ; *CRISPR-Cas Systems ; *Circoviridae Infections/veterinary/diagnosis/virology ; *Swine Diseases/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; DNA, Viral ; },
abstract = {BACKGROUND: Porcine circovirus type 2 (PCV2) is a globally prevalent viral pathogen that causes substantial economic losses in the swine industry. Rapid and accurate on-site diagnosis is critical for controlling the spread of PCV2. In recent years, RNA-guided CRISPR/Cas12a nucleases combined with recombinase polymerase amplification (RPA) have emerged as a promising approach for nucleic acid detection. This study aimed to develop a novel RPA-CRISPR-based method for the rapid and sensitive detection of PCV2 in field settings. RESULTS: We designed and optimized CRISPR RNAs (crRNAs) targeting conserved regions of the PCV2 Cap and Rep genes. Upon recognition of the target sequence, the Cas12a nuclease was activated to cleave a single-stranded DNA-fluorophore quencher (ssDNA-FQ) reporter, generating a fluorescent signal detectable either by a fluorescence detector or via visual readout. The entire procedure was performed at 37 °C and completed within one hour. The assay achieved a detection limit as low as 10 copies/µL and showed no cross-reactivity with other major porcine viruses. Furthermore, a rapid-release reagent was used to replace conventional DNA extraction from serum samples, facilitating efficient on-site detection. The assay was validated using clinical samples, and the results showed strong concordance with those obtained by PCR. CONCLUSIONS: The RPA-CRISPR-based assay developed in this study is highly sensitive and specific, enabling detection of PCV2 within one hour. Its simplicity, rapidity, and ease of use in the field offer significant practical advantages, making it a valuable tool for the on-site diagnosis of PCV2. This method represents a promising alternative for the early and rapid detection of PCV2 infections and holds potential for contributing to the prevention and control of the disease in the swine industry.},
}
@article {pmid40506596,
year = {2025},
author = {Sajjad, MW and Muzamil, F and Naqvi, RZ and Amin, I},
title = {QBEmax redefines the precise base editing in crop plants.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {127},
pmid = {40506596},
issn = {1438-7948},
mesh = {*Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Genome, Plant ; Cytidine Deaminase/genetics/chemistry/metabolism ; *Gene Editing ; },
abstract = {Hu et al.‘s new study, published in Nature Biotechnology, introduces QBEmax; a tiny, conformationally sound editing tool with a cytidine deaminase buried within a looping permuted Cas9 (cpCas9). Supported by molecular dynamics models and AlphaFold3 structural predictions, this unique internal fusion creates a structurally protected complex that improves editing accuracy and lowers typical artifacts such as indels and impure base conversions (Hu et al. Nature Biotechnology:1-7, 2025). High precision editing (up to 99.8% purity), lower indels, and lower off target impacts well suit imminent plant transformation events. Its tiny, stable architecture and wider editing window at PAM sites increase its ability for precise and adaptable trait change in complex plant genomes.},
}
@article {pmid40999513,
year = {2025},
author = {Lee, S and Yu, Y and Kim, DH and Bock, M and Kim, Y and An, SB and Choi, H and Shin, HE and Hwang, DY and Han, I},
title = {Enhanced disc regeneration through CRISPR/Cas9-mediated SOX9 and TGFβ1 coexpression in tonsil-derived mesenchymal stromal cells.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {501},
pmid = {40999513},
issn = {1757-6512},
support = {RS-2024-00347403//National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT: Ministry of Science and ICT)/ ; RS-2023-KH141187//Korean Cell-Based Artificial Blood Project funded by the Korean government (The Minis-try of Science and ICT, The Ministry of Trade, Industry and Energy, the Ministry of Health &amp; Welfare, the Ministry of Food and Drug Safety)/ ; },
mesh = {*SOX9 Transcription Factor/genetics/metabolism ; *Transforming Growth Factor beta1/genetics/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; Animals ; Rats ; Humans ; *Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Regeneration ; Rats, Sprague-Dawley ; *Palatine Tonsil/cytology/metabolism ; *Intervertebral Disc/physiology/metabolism ; Male ; Cell Differentiation ; Chondrogenesis ; },
abstract = {BACKGROUND: Intervertebral disc (IVD) degeneration, a primary cause of chronic low back pain, currently lacks treatments that target its underlying pathological mechanisms. Tonsil-derived mesenchymal stromal cells (ToMSCs) have shown promise for IVD regeneration; however, their therapeutic potential is limited by the harsh microenvironment of degenerated discs. This study investigated whether ToMSCs engineered to co‐overexpress SOX9 and TGFβ1 using a tetracycline‐off (Tet‐off) regulatory system could enhance extracellular matrix (ECM) restoration and reduce inflammation in degenerative IVDs. METHODS: We used CRISPR/Cas9 technology to generate ToMSCs that express SOX9, TGFβ1, or both factors under Tet-off regulation. Gene expression was confirmed by Western blot and qRT-PCR analyses. In vitro studies assessed chondrogenic differentiation capacity, while in vivo assessments were performed using a rat tail needle puncture model of IVD degeneration. After administering the CRISPR-engineered ToMSCs, we monitored mechanical allodynia with the von Frey test over six weeks. Therapeutic outcomes were evaluated through T2‐weighted MRI and histological analysis. RESULTS: In vitro experiments showed that ToMSCs co-expressing SOX9 and TGFβ1 exhibited superior chondrogenic differentiation compared to cells expressing a single factor. In vivo studies demonstrated that dual-factor expressing ToMSCs significantly improved disc hydration (as confirmed by MRI), enhanced ECM synthesis—particularly aggrecan and type II collagen—and reduced inflammation compared to single-factor treatments. These improvements were accompanied by reduced mechanical allodynia, indicating functional recovery. CONCLUSION: Our study demonstrates that ToMSCs engineered to co-express SOX9 and TGFβ1 effectively promote IVD regeneration by enhancing ECM production and reducing inflammation. This dual-factor approach represents a promising therapeutic strategy for treating degenerative disc disease and warrants further investigation for clinical application.},
}
@article {pmid41003970,
year = {2026},
author = {Kanafi, MM and Moazzami, R},
title = {Leveraging CRISPR/Cas9 To Overcome Hypoxic Barriers in Regenerative Dentistry.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {1},
pages = {82-86},
pmid = {41003970},
issn = {2629-3277},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Dental Pulp/cytology/metabolism ; *Stem Cells/metabolism/cytology ; Animals ; *Dentistry/methods ; Cell Hypoxia ; *Regeneration ; *Regenerative Medicine/methods ; Oxidative Stress ; Gene Editing/methods ; },
abstract = {Dental pulp stem cells (DPSCs) have gained increasing attention as a valuable cell source for regenerative dentistry owing to their accessibility, high proliferative potential, and capacity for multilineage differentiation. Despite these advantages, their therapeutic efficacy is substantially compromised by pathological hypoxia, a common feature of injured or poorly vascularized oral tissues. Hypoxic stress not only impairs DPSC survival but also diminishes their regenerative capacity, creating a major barrier to effective clinical translation. Addressing this limitation is therefore essential to harness the full therapeutic potential of DPSCs. Recent advances in genome-editing technologies, particularly the CRISPR/Cas9 system, have created novel opportunities to enhance the resilience of DPSCs against hypoxic stress. By enabling precise genetic modifications, CRISPR offers a powerful platform to reprogram cellular pathways associated with oxygen deprivation, oxidative stress, and apoptosis. Current preclinical investigations have focused on key targets such as HIF1α, PHD2, NRF2, BAX, and VEGF, exploring their modulation through CRISPR-mediated activation, inhibition, or knockout strategies. Upregulation of HIF1α and VEGF has demonstrated the ability to enhance angiogenesis and promote cell survival in oxygen-deficient microenvironments. Similarly, activation of NRF2 improves antioxidant defense mechanisms and mitigates oxidative damage, while suppression of pro-apoptotic genes such as BAX increases overall viability. Collectively, these strategies represent a multifaceted approach to strengthening DPSC performance in adverse conditions. The integration of CRISPR/Cas9 into regenerative dentistry represents a paradigm shift in addressing hypoxia-induced barriers to stem cell therapy. While early findings are promising, several critical challenges remain, including the potential for off-target effects, the need for stable and long-term genetic modifications, and concerns regarding biosafety and ethical considerations. Robust preclinical validation and carefully designed translational studies will be required before CRISPR-engineered DPSCs can be considered for clinical application. In summary, CRISPR/Cas9-based modulation of hypoxia-responsive pathways offers a transformative strategy to enhance the therapeutic efficacy of DPSCs. By improving survival, stress tolerance, and angiogenic potential under hypoxic conditions, this approach may significantly expand the clinical applicability of stem cell–based interventions in dentistry. Continued research is essential to ensure the safety, reliability, and long-term benefits of this promising therapeutic avenue.},
}
@article {pmid41171582,
year = {2026},
author = {Feger, M and Tsapara, A and Hülße, S and Rausch, S and Barholz, M and Stournaras, C and Föller, M},
title = {Chorein Regulates Key Osteoblast Genes in UMR-106 Cells.},
journal = {Cell biochemistry and biophysics},
volume = {84},
number = {1},
pages = {1245-1252},
pmid = {41171582},
issn = {1559-0283},
mesh = {*Osteoblasts/metabolism/cytology ; Humans ; *Vesicular Transport Proteins/genetics/metabolism/antagonists &amp; inhibitors ; Cell Line ; Fibroblast Growth Factor-23 ; Fibroblast Growth Factors/metabolism/genetics ; PHEX Phosphate Regulating Neutral Endopeptidase/metabolism/genetics ; Cell Differentiation ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Animals ; *Gene Expression Regulation ; },
abstract = {Chorein is an endoplasmic reticulum protein expressed in many cell types. Loss-of-function mutations of the gene encoding chorein (VPS13A) are the cause of chorea-acanthocytosis, a rare and severe neurodegenerative disease with chorea-like movements, loss of mental function, progressive muscle weakness and misshaped erythrocytes (acanthocytes). Chorein regulates diverse cellular functions including the cytoskeleton, apoptosis, Ca2+ entry, or autophagy. Since its role in bone is enigmatic, we aimed to explore the function of chorein in osteoblasts. To this end, we generated UMR-106 osteoblast-like cells with stable chorein knockdown (KD) using a CRISPR/Cas9-based approach and compared them to cells undergoing CRISPR/Cas9 with a non-targeting sequence (NT). Gene expression was assessed by qPCR and protein by Western blotting and ELISA. Gene and protein expression of chorein and fibroblast growth factor 23 (FGF23), an osteoblast-derived hormonal regulator of phosphate metabolism, were decreased in KD compared to NT cells. Moreover, FGF23 regulator Phex was down- and Galnt3 was up-regulated in KD compared to NT cells. The expression of further genes regulating osteoblast and osteoclast differentiation was affected by chorein knockdown. Taken together, chorein is expressed in UMR-106 osteoblasts and modulates the expression of various genes affecting osteoblast and osteoclast differentiation and function.},
}
@article {pmid41250247,
year = {2025},
author = {Qian, Y and Hui, F and Niu, W and Wang, D and Hao, Y and Meng, Q and Ren, S and Kong, D and Gong, H and Wu, J and Chen, K and Alariqi, M and Gao, J and Li, Z and Jin, S},
title = {Double-stranded DNA deaminase DddA[E1347A] can increase the efficiency and targeting range of cytidine base editors.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {391},
pmid = {41250247},
issn = {1474-760X},
support = {2024M761134//The China Postdoctoral Fund/ ; KY2020YC0002//The China Tobacco Hunan Industrial Co., Ltd. Research Project/ ; 2023YFF1000204//The National Key R&amp;D Program of China/ ; 32272128//The National Science Foundation of China/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; },
mesh = {*Cytidine/metabolism/genetics ; *Gene Editing/methods ; *Cytidine Deaminase/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; *Cytosine Deaminase/genetics/metabolism ; DNA ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {BACKGROUND: Cytidine base editors (CBEs) consist of a single-strand specific cytidine deaminase fused to Cas9 nickase, enabling efficient C-to-T conversion across diverse organisms. Enhancing editing range and efficiency of these tools is essential for expanding their applications. RESULTS: In this study, we report that fusing a double-stranded DNA-specific cytosine deaminase DddAE1347A to CBEs significantly improves editing activity and broadens the editing window in cell lines, embryos, tobacco, and cotton. Compared to BE4max, the optimized DddAE1347A-BE4max exhibits up to a 93- fold increase in editing efficiency, achieving up to 52% efficiency at C14 and C15 in cell lines. Further investigation reveals that DddAE1347A is compatible with various Cas9 variants (SpCas9, SpaCas9, and Nme2Cas9) and deaminase variants (rA1, A3G, and A3A). Additionally, we demonstrate that cytosine deaminases with single-stranded DNA activity fail to enhance the CBE system. In contrast, various DddA variants can improve CBE editing activity at PAM-proximal cytosine positions, highlighting the modularity of fusion between DddAs and CBEs. CONCLUSIONS: These findings suggest that the double-stranded DNA-specific cytosine deaminase protein can act as an engineered fusion module in the CBE system, altering the performance (window/efficiency) of CBEs.},
}
@article {pmid41381576,
year = {2025},
author = {Palavesam, A and Karthik Raj, BN and Madan, N and Sri, SSL and Babitha, R and Tirumurugaan, KG},
title = {CRISPR-Cas12a-based rapid detection of Babesia gibsoni and Ehrlichia canis in dogs using fluorometer platform.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {402},
pmid = {41381576},
issn = {2045-2322},
mesh = {Animals ; Dogs ; *Ehrlichia canis/genetics/isolation &amp; purification ; *Babesia/genetics/isolation &amp; purification ; *Dog Diseases/diagnosis/microbiology/parasitology ; *Ehrlichiosis/diagnosis/veterinary/microbiology ; *Babesiosis/diagnosis/parasitology ; *CRISPR-Cas Systems/genetics ; *Fluorometry/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; },
abstract = {Canine babesiosis and canine monocytic ehrlichiosis are important tick-borne diseases caused by Babesia gibsoni (B. gibsoni) and Ehrlichia canis (E. canis) in dogs. Early diagnosis is important for effective clinical management, as these infections can result in other complications if untreated. This study aimed to develop a CRISPR-Cas12a-based detection assay for B. gibsoni and E. canis in a fluorometer platform. Custom designed guide RNAs (gRNAs) were synthesised with the spacer sequence targeting the B. gibsoni18S rRNA and E. canis p43 genes, located 20 bp downstream of the PAM site (5’-TTTV- 3’). Following PCR amplification of the short fragments encompassing the above target regions, the specific gRNA binding the LbaCas12a-gRNA complex initiated the collateral cleavage of FAM- labelled AT rich ssDNA probe for detection using a fluorometer and Biotin-digoxigenin (DIG) labelled GT-rich ssDNA probe for visual detection in LFA.The limit of detection (LOD) using the fluorometer based detection platform was 6 × 108and 6 × 109 for B. gibsoni and E. canis respectively. The LOD was comparable to that of real-time PCR, but more sensitive than point-of-care methods such as LFA, indicating its potential applicability in veterinary clinical settings.},
}
@article {pmid41437324,
year = {2025},
author = {Jun, Y and Han, J and Kim, Y and Choi, S and Kim, SL and Oh, JH and Suh, EJ and Kang, SH and Park, HR and Jeong, HC and Park, SR and Mo, Y},
title = {Phenotypic and transcriptomic characterization of OsSWEET14-edited rice (cv. Samkwang) with enhanced bacterial blight resistance.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1771},
pmid = {41437324},
issn = {1471-2229},
support = {RS-2022-RD010034//Rural Development Administration/ ; RS-2024-00322166//Rural Development Administration/ ; },
mesh = {*Oryza/genetics/microbiology/immunology ; *Xanthomonas/physiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; Phenotype ; *Transcriptome ; Plants, Genetically Modified ; *Plant Proteins/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) poses a serious threat to rice production. The pathogen promotes infection by targeting effector binding elements in the promoter regions of rice susceptibility genes such as SWEET (Sugars Will Eventually be Exported Transporters) genes. Previous studies have shown that natural or induced variations in SWEET genes can effectively enhance BB resistance in rice. However, the effects of variations in SWEET genes on disease resistance and agronomic performance vary depending on the Xoo strains and the genetic background of rice cultivars, highlighting the need for precise evaluations in breeding applications. RESULTS: In this study, we developed a CRISPR/Cas9-edited OsSWEET14 knockout line (SK-s14) in the background of the elite Korean rice cultivar Samkwang. The SK-s14 line exhibited enhanced resistance against the Xoo strain KACC10859. Transcriptomic analysis revealed that defense-related genes, including pathogenesis-related genes and salicylic acid signaling genes, were downregulated in SK-s14 relative to Samkwang under Xoo inoculation. These results suggest that the enhanced resistance may result from pathogen starvation due to restricted sugar efflux to apoplast, rather than from classical defense activation. Field evaluations showed that SK-s14 plants headed 1–7 days earlier, had longer panicles (+ 8.0%), and exhibited modest reductions in grain weight (-3.0%) and grain fertility (-4.4%) compared to Samkwang. CONCLUSIONS: Our results show that knocking out OsSWEET14 in the Samkwang background enhances BB resistance against the KACC10859 strain, likely through a pathogen starvation mechanism. Despite a modest grain yield reduction (-7.2%) observed under field conditions, SK-s14 represents a promising breeding material for disease-prone rice growing regions.},
}
@article {pmid41513714,
year = {2026},
author = {Antunes, M and Moura, F and Sebastian, IR and Alves, P and Gomes-Alves, P and Escandell, JM},
title = {Streamlined rAAV HeLaS3 producer cell line generation via GS selection.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {4658},
pmid = {41513714},
issn = {2045-2322},
support = {UID/04462: iNOVA4Health//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; 10.55776/W1224//Austrian Science Fund/ ; },
mesh = {*Dependovirus/genetics ; *Glutamate-Ammonia Ligase/genetics/metabolism ; *Genetic Vectors/genetics ; Humans ; Animals ; CRISPR-Cas Systems ; HeLa Cells ; Cricetulus ; CHO Cells ; Gene Knockout Techniques ; },
abstract = {The high cost and complexity of manufacturing recombinant adeno-associated virus vectors continue to limit the broader application of gene therapies, which offer life-changing potential for individuals affected by genetic diseases. Although stable producer cell lines represent a scalable and cost-effective alternative to transient transfection methods, their development is often delayed by inefficient selection strategies and extended timelines. In this study, we present a novel application of the glutamine synthetase-based selection system - commonly used in CHO cells - to a HeLaS3-based rAAV production platform. By generating glutamine synthetase-knockout HeLaS3 cells via CRISPR-Cas9 and applying glutamine deprivation under serum-free conditions, we significantly streamlined the PCL generation process, reducing the timeline to approximately two months while maintaining rAAV productivity (>1x1011 vg/mL) and product quality (~70% full capsids). This work establishes a robust and scalable workflow for rAAV manufacturing, with the potential to enhance accessibility and reduce viral vector production costs for applications in gene therapy.},
}
@article {pmid41746183,
year = {2026},
author = {Liu, S and Ding, Z and Lu, X and Liu, Z and Ma, W and Xu, H and Zhang, H and Dai, X and Shen, M and Huang, Y and Gao, M and Bao, J and Chen, M},
title = {Deoxyribonucleic Acid Activator-Triggered Entropy-Driven Catalysis-Modulated CRISPR/Cas12a-Based Portable Biosensor for Simultaneous Detection of Multiple Pathogenic Bacteria.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4311-4324},
doi = {10.1021/acssensors.5c03012},
pmid = {41746183},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; Pseudomonas aeruginosa/isolation &amp; purification ; Bacterial Proteins/genetics ; beta-Lactamases/genetics/analysis/metabolism ; *Methicillin-Resistant Staphylococcus aureus/isolation &amp; purification ; Catalysis ; *Klebsiella pneumoniae/isolation &amp; purification ; Lab-On-A-Chip Devices ; *DNA/chemistry ; Rapid Diagnostic Tests ; Limit of Detection ; },
abstract = {Rapid and sensitive detection of antibiotic-resistant bacteria (ARB) remains a critical challenge in clinical and public health settings. This study describes the successful construction of a portable DNA activator-triggered entropy-driven catalysis-modulated CRISPR/Cas12a-based sensor (PSDA) for the ultrasensitive and rapid detection of multiple ARB. This PSDA platform utilizes a CRISPR/Cas12a-mediated signal transduction strategy, in which a target-specific DNA activator initiates an entropy-driven dynamic DNA network for signal amplification. To further enhance detection performance, a 3D-printed microfluidic chip device with a smartphone-based readout system has been integrated into the sensor, using green-emitting Zn2GeO4:Mn persistent luminescent nanoparticles as a novel molecular beacon for fluorescence enhancement. This platform enables the simultaneous detection of methicillin-resistant Staphylococcus aureus, carbapenem-resistant Pseudomonas aeruginosa, and Klebsiella pneumoniae carbapenemase 2 (KPC-2)-expressing Klebsiella pneumoniae (KPC-2 KP) with a broad dynamic range (1-10[7] CFU/mL), an ultralow detection limit (1 CFU/mL), and rapid analysis (∼45 min). The assay results are also highly consistent with those of conventional plate counting methods (95.48-115.15%). Overall, this study presents a cost-effective, rapid-response biosensing platform for the simultaneous detection of multiple ARB, with direct applications in clinical diagnostics, food safety monitoring, and environmental surveillance.},
}
@article {pmid42136104,
year = {2026},
author = {Purman, C and Lu, C and Modi, A and Vijaykumar, V and Flister, MJ and den Hollander, AI and Kadri, S and Stender, JD},
title = {Optimization of Genome-Wide CRISPR Screens Using Dual-Guide RNA Infection with Cas9 Electroporation (DICE).},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {141-150},
doi = {10.1177/25731599261448141},
pmid = {42136104},
issn = {2573-1602},
mesh = {Humans ; *Electroporation/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lentivirus/genetics ; *Gene Editing/methods ; HEK293 Cells ; THP-1 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Single-guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression, yet is limited by scalability and robustness. Here, we introduce dual guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in Interferon-gamma-activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.},
}
@article {pmid42160542,
year = {2026},
author = {Liu, S and Hu, Y and Zheng, M and Zhang, F and Yu, Z and Sun, Q and Tang, D and Weng, Z and Ye, Z},
title = {A CRISPR/Cas12a-MXene Nanozyme Platform for Universal Detection of Trace DNA.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4345-4356},
doi = {10.1021/acssensors.5c03702},
pmid = {42160542},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; Colorimetry/methods ; *Biosensing Techniques/methods ; Proto-Oncogene Mas ; Methicillin-Resistant Staphylococcus aureus/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; Limit of Detection ; Bacterial Proteins/genetics ; *DNA, Bacterial/analysis/genetics ; *Endodeoxyribonucleases/genetics/chemistry ; *DNA/analysis ; Nitrites ; Transition Elements ; },
abstract = {The instrument-free detection of specific DNA sequences is critical for point-of-care diagnostics yet remains challenging. To address this, we developed a universal colorimetric biosensing platform integrating the CRISPR/Cas12a system with MXene nanozymes. Target recognition by the Cas12a/crRNA complex triggers trans-cleavage of single-stranded DNA inhibitors, restoring the peroxidase-like activity of MXenes to produce a visible signal. The platform achieved a detection limit of 132 copies/μL for the methicillin-resistant Staphylococcus aureus mecA gene. By incorporating an exponential circular DNA (CirDNA) amplification strategy and rationally designed crRNAs for single-base discrimination, sensitivity was further enhanced, enabling detection of the B-raf proto-oncogene V600E mutation down to 109 aM with 0.5% variant allele frequency. The platform also showed excellent reproducibility and high recovery rates in simulated clinical samples. This work provides a low-cost, label/instrument-free, and highly sensitive universal approach through the synergistic combination of CRISPR/Cas12a programmability, MXene nanozyme activity, and CirDNA amplification.},
}
@article {pmid42163774,
year = {2026},
author = {Urbaitis, T and Trinkuniene, L and Lenkaite, I and Petrauskyte, M and Krasauskas, R and Stitilyte, M and Sabaliauskas, M and Sasnauskas, G and Tamulaitiene, G and Young, JK and Siksnys, V and Gasiunas, G},
title = {A Potent CRISPR-Cas12l Double-Strand Break Gene Editor.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {126-140},
doi = {10.1177/25731599261448428},
pmid = {42163774},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA Breaks, Double-Stranded ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, Armatimonadota, was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.},
}
@article {pmid42213084,
year = {2026},
author = {Wang, XY and Wang, JX and Li, QN and Pang, XZ and Yang, QF and Zhu, LN and Kong, DM},
title = {A Review of Activator Strand Engineering Strategies for Smart CRISPR/Cas12a Diagnostics.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4224-4244},
doi = {10.1021/acssensors.6c00469},
pmid = {42213084},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA/genetics ; Biosensing Techniques/methods ; *Genetic Engineering/methods ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a transformative tool in molecular diagnostics and biosensing, leveraging its high-efficiency DNA-targeting and unique trans-cleavage activity. However, its practical deployment is hindered by persistent challenges such as elevated background signals, constrained target versatility, and insufficient controllability. The activator strand (AS), serving as the molecular trigger for Cas12a activation, presents a promising engineering target to systematically enhance system performance. This review comprehensively summarizes recent advances in AS-driven regulation of the CRISPR/Cas12a system, focusing on four core engineering strategies: terminal modification engineering, split activator design, PAM (protospacer adjacent motif) engineering and regulation, and topological conformation engineering. By redesigning AS architecture, introducing allosteric control, and refining spatial assembly, these approaches significantly improve detection sensitivity, specificity, and versatility. AS engineering has effectively mitigated background interference, expanded target scope to include non-nucleic acid analytes, and enabled precise conditional activation of Cas12a. We further discuss current challenges and future directions, aiming to guide the development of next-generation CRISPR diagnostic systems with enhanced robustness, programmability, and adaptability for real-world applications.},
}
@article {pmid42215315,
year = {2026},
author = {Zhang, J and Liang, S and Sun, Y and Zhan, C and Xue, G and Zhou, X},
title = {Glycine/PVP-Enabled One-Pot CRISPR/Cas13a Detection of Vibrio parahaemolyticus by Tetrahedron-Mediated Electrochemistry.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4980-4990},
doi = {10.1021/acssensors.6c00902},
pmid = {42215315},
issn = {2379-3694},
mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics ; *Electrochemical Techniques/methods ; *Glycine/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Povidone/chemistry ; Limit of Detection ; },
abstract = {Vibrio parahaemolyticus is a major foodborne pathogen widely distributed in aquatic environments and seafood supply chains, necessitating rapid and ultrasensitive detection strategies adaptable to diverse testing scenarios. Here, we present a glycine/ PVP- and tetrahedron-integrated one-pot CRISPR sensing platform (termed GPT-CRISPR) for robust and ultrasensitive nucleic acid detection. The platform introduces chemical regulation into a multienzyme one-pot RAA-CRISPR/Cas13a network, where glycine may help reduce nonspecific Cas13a background activity, possibly through weak competitive interactions, while polyvinylpyrrolidone (PVP) may enhance reaction compatibility through macromolecular crowding and spatial shielding. This coordinated microenvironment enables stable amplification and CRISPR activation within a single closed vessel while minimizing background interference. Upon target recognition, activated Cas13a cleaves uracil-containing, surface-immobilized DNA tetrahedra, translating molecular recognition into amplified electrochemical signals. This transduction strategy enables quantitative detection with a linear dynamic range of 1.5 to 3 × 10[3] copies μL[-1] and a limit of detection of 0.38 copies μL[-1]. The same chemically regulated one-pot CRISPR framework remains compatible with fluorescence and lateral flow readouts. The assay operates under isothermal conditions and delivers results within 30 min without complex sample preparation. Validation across real-world samples demonstrates robustness in complex matrices. Collectively, GPT-CRISPR integrates chemical stabilization of a one-pot CRISPR framework with electrochemical transduction, defining a robust sensing architecture with adaptable readout capability.},
}
@article {pmid42263201,
year = {2026},
author = {Sun, Y and Wang, R and Sun, H and Chen, J and Liang, C and Zhang, Z and Feng, Y and Chen, L and Wang, X},
title = {Modular CRISPR-Cas12a-Activated Gold Nanoparticle Assay for Rapid Visual Detection of Hepatocellular Carcinoma-Related miRNAs.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4991-5002},
doi = {10.1021/acssensors.6c00916},
pmid = {42263201},
issn = {2379-3694},
mesh = {*MicroRNAs/genetics/analysis ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Carcinoma, Hepatocellular/genetics/diagnosis ; Humans ; *Liver Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Colorimetry/methods ; *CRISPR-Associated Proteins/metabolism ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {MicroRNAs (miRNAs) are emerging biomarkers for early hepatocellular carcinoma (HCC) detection, but most CRISPR-Cas12a assays rely on reverse transcription and preamplification and often lack simple visual readouts. Here, we develop a reverse transcription-free, cleavage-guided strategy that converts miRNA recognition into visual outputs. In the presence of the target miRNA, a scaffold RNA forms an active crRNA that triggers Cas12a trans-cleavage using a preformed dsDNA activator, enabling two readout formats. In a homogeneous non-crosslinking colorimetric assay (mC-NCA), Cas12a cleavage regulates π-π-stacking-mediated gold nanoparticle (AuNP) aggregation to produce a rapid visual colorimetric response. In a heterogeneous versatile lateral flow assay (mC-vLFA), the cleavage of partially hybridized DNA-magnetic bead probes generates a target-dependent test line while maintaining a built-in control line. Using miRNA-21 and miRNA-122 as targets, mC-NCA shows calculated limits of detection (LODs) of 1.62 and 1.64 fM with a linear range of 50 fM-500 pM, whereas mC-vLFA shows calculated LODs of 1.59 and 1.40 fM with a linear range of 10 fM-10 nM. In a preliminary clinical evaluation, both formats show good agreement with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and clinical assessment while enabling faster detection with minimal instrumentation. Overall, this dual-readout platform couples RT-free CRISPR-Cas12a miRNA recognition with cleavage-directed visual signal transduction, enabling rapid and low-instrument-dependence CRISPR diagnostics for miRNAs.},
}
@article {pmid42272131,
year = {2026},
author = {Peng, CL and Kamau, WS and Freeman, J and Hill, ZJ and Esvelt, KM},
title = {Increasing the Effective Gene Drive Homing Rate by Targeting the Haploinsufficient Spermatogenesis Gene Klhl10.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {167-176},
doi = {10.1177/25731599261452112},
pmid = {42272131},
issn = {2573-1602},
mesh = {Animals ; *Spermatogenesis/genetics ; Male ; Mice ; *Haploinsufficiency/genetics ; CRISPR-Cas Systems ; Frameshift Mutation ; Female ; DNA End-Joining Repair/genetics ; Spermatozoa/metabolism ; *Gene Targeting/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-based gene drives represent a powerful new technology for limiting disease transmission and controlling invasive populations. These systems rely on homology-directed repair (HDR) to "drive" a genetic element through a population. However, mammals tend to favor non-homologous end joining (NHEJ), which generates mutations that halt further drive propagation. Here, we describe the experimental characterization of a putative target locus for a gene drive system targeting the haploinsufficient spermatogenesis gene Klhl10 in the laboratory mouse. Using a newly designed "coding sequence cassette," we introduce downstream guide RNAs within the gene, ensuring that sperm undergoing NHEJ are selectively removed from the population. As a proof of principle, we demonstrate that targeting Klhl10 with constitutively expressed LbCas12a results in strong selection against frameshift-containing sperm, validating the core purification mechanism required for this drive strategy. Unexpectedly, we also observed that female offspring lacked most frameshift mutations, suggesting a previously unrecognized role for Klhl10 in oogenesis or early embryonic development.},
}
@article {pmid42290195,
year = {2026},
author = {Michalski, MN and Diegel, CR and Zhong, ZA and Marshall, ME and Foxa Wiartalla, GE and Stevens, PD and Suino-Powell, K and Blazer, LL and Adams, JJ and Melcher, K and Sidhu, SS and Angers, S and Williams, BO},
title = {Clarifying Frizzled 2 function in development through genetically validated mouse models.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {7},
pages = {},
doi = {10.1242/dmm.052410},
pmid = {42290195},
issn = {1754-8411},
support = {K08DE031039/DE/NIDCR NIH HHS/United States ; K08DE031039//National Institute of Health/ ; /DE/NIDCR NIH HHS/United States ; //Van Andel Research Institute/ ; },
mesh = {Animals ; *Frizzled Receptors/metabolism/genetics/deficiency ; Phenotype ; Gene Deletion ; Mice, Knockout ; Mice ; Reproducibility of Results ; Integrases/metabolism ; *Embryonic Development/genetics ; CRISPR-Cas Systems/genetics ; Extremities/embryology ; Base Sequence ; Embryo, Mammalian/metabolism ; Models, Animal ; },
abstract = {Wnt receptors of the Frizzled (Fzd) family are widely considered to exhibit substantial functional redundancy, complicating efforts to therapeutically target individual receptors. Fzd2 was believed to be functionally redundant with Fzd1 and Fzd7, based on previously published global knockout mouse studies. By contrast, homozygosity for a Fzd2 global knockout mouse allele developed by the International Mouse Phenotype Consortium (IMPC) has been reported to cause embryonic lethality, suggesting that Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, conditional deletion models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 conditional deletion model does not eliminate Fzd2. We have generated a new conditional model to address the contradictory previous studies and allow tissue-specific studies of Fzd2. We successfully inserted two loxP sites around the Fzd2 gene and confirmed that subsequent Cre-mediated recombination creates a Fzd2 null allele. Global deletion of Fzd2 in this model does not cause embryonic lethality while limb-specific deletion causes limb shortening. This work supports the hypothesis that Fzd2 regulates limb development and emphasizes the importance of thoroughly validating newly generated mouse models.},
}
@article {pmid42299032,
year = {2026},
author = {Porenta, D and Benne, N and Sijts, A and Broere, F and Mastrobattista, E},
title = {Induction of SpCas9-Directed Immune Responses Using Lipid Nanoparticles and Identification of SpCas9-Derived T Cell Epitopes in C57BL/6 Mice.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {151-166},
doi = {10.1177/25731599261456887},
pmid = {42299032},
issn = {2573-1602},
mesh = {Animals ; Mice, Inbred C57BL ; Mice ; *Epitopes, T-Lymphocyte/immunology/genetics ; *Nanoparticles/chemistry/administration &amp; dosage ; Lipids/chemistry ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/immunology/genetics/metabolism ; Gene Editing/methods ; Female ; Interferon-gamma ; Liposomes ; },
abstract = {A major goal of clinically oriented CRISPR-Cas9-based applications is safe and effective in vivo gene editing (knockout or correction) with precise targeting. Substantial efforts have been devoted to the preclinical development of novel drug delivery platforms that enable efficient, targeted delivery. However, the immune responses induced by CRISPR-Cas9 treatment are often overlooked. Preexisting immunity to clinically relevant Cas9 proteins has already been established as a consequence of natural exposure to Cas9-bearing bacteria, which may implicate the safety and efficacy of CRISPR-Cas9-based therapies. Naturally, CRISPR-Cas9 therapies should be nonimmunogenic to avoid amplifying existing Cas9-specific immunity, especially cytotoxic T cell responses. Nonviral delivery systems, such as lipid nanoparticles (LNPs), are widely regarded as less immunogenic than more traditionally used viral vectors, even though LNPs are suitable as a vaccination platforms. In this study, we investigate the induction of SpCas9-directed immunity in C57BL/6 mice upon repeated dosing of LNPs encapsulating Cas9-coding mRNA in two different settings: (1) a vaccination-resembling setting using intramuscularly administered adjuvanted LNPs, and (2) a therapy-resembling setting using intravenously injected, liver-targeting LNPs. In both settings, Cas9-specific T cell responses were detected by evaluating increased total IFN-γ levels upon ex vivo restimulation of isolated splenocytes. However, undetectable Cas9-reactive antibodies induced in the therapeutic setting emphasize the discrepancy between humoral and cellular responses. To improve future monitoring of Cas9-specific T cell responses, we report six Cas9-derived epitopes recognized by CD8[+] T cells, as well as a CD4[+] T cell polypeptide carrying one of the CD8[+] T cell epitopes that induced strong IFN-γ production ex vivo. This work is intended to facilitate the preclinical monitoring of Cas9-specific T cell responses in C57BL/6 mice and support the development of safe CRISPR-Cas9-based therapies.},
}
@article {pmid42328786,
year = {2026},
author = {Xu, C and Yang, Q and Niu, X and Ke, A},
title = {Structure basis for single-strand nucleic acid targeting by IscB and variants.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42328786},
issn = {1362-4962},
support = {R35GM118174/NH/NIH HHS/United States ; S10OD023603/NH/NIH HHS/United States ; //Brookhaven National Laboratory&#x2500;Laboratory for BioMolecular Structure/ ; KP1607011//DOE Office of Biological and Environmental Research/ ; //Yale CryoEM Resource/ ; },
mesh = {*DNA, Single-Stranded/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Gene Editing/methods ; Models, Molecular ; Nucleic Acid Conformation ; Humans ; },
abstract = {Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid (ssNA) targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.},
}
@article {pmid42329468,
year = {2026},
author = {Ahmad, S and Aftab, MN and Ghalib, M and Aslam, MS},
title = {Overcoming cellular secretion bottlenecks: advanced secretion engineering and molecular tailoring for next-generation microbial α-amylases with enhanced industrial performance.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {7},
pages = {},
pmid = {42329468},
issn = {1573-0972},
mesh = {Bacillus subtilis/genetics/enzymology/metabolism ; *alpha-Amylases/metabolism/genetics/chemistry ; *Metabolic Engineering/methods ; Aspergillus niger/genetics/enzymology/metabolism ; Escherichia coli/genetics/metabolism ; Recombinant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Industrial Microbiology ; Pichia/genetics/enzymology ; Carbohydrate Binding Modules ; Protein Engineering ; Synthetic Biology ; Saccharomycetales ; },
abstract = {α-amylases are indispensable industrial biocatalysts, yet their recombinant production faces significant biochemical and cellular bottlenecks. Recent scientific advancement shifts the paradigm from traditional cloning toward a design-parameter framework, where host selection predominantly Bacillus subtilis, Pichia pastoris, and Aspergillus niger is dictated by secretion capacity, folding landscapes, and metabolic compatibility. While Escherichia coli remains a common host, its lack of efficient extracellular secretion often leads to inclusion body formation and metabolic stress. Advanced strategies are being employed to overcome these limits, including signal peptide optimization, chaperone co-expression, and the fusion of carbohydrate-binding modules (CBMs) to enhance raw-starch degradation. Furthermore, how rational design, aided by artificial intelligence and molecular dynamics simulations, enables the engineering of hyper-thermostable and alkaline-tolerant variants capable of withstanding extreme industrial processing conditions. The advent of CRISPR-Cas technology has further revolutionized the field, allowing for precise genome editing and metabolic rewiring to achieve record-breaking enzyme titers, such as 102,893 U/mL in engineered B. subtilis. By balancing transcriptional levels with enhanced secretion pathways and stress-mitigation systems, modern synthetic biology provides the tools to tailor α-amylases for specific needs, ranging from biofuel production to high-purity malto-oligosaccharide synthesis. Therefore, this comprehensive analysis underscores that reconciling host biology with enzyme biochemistry is essential for meeting global industrial demands.},
}
@article {pmid42330666,
year = {2026},
author = {Ling, H and Su, N and Huang, L and Wang, M and Ren, L},
title = {Cleavage-responsive DNA/AgNCs enable accelerated Cas12a trans-cleavage for rapid multigene methylation diagnosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {311},
number = {},
pages = {118929},
doi = {10.1016/j.bios.2026.118929},
pmid = {42330666},
issn = {1873-4235},
abstract = {DNA methylation, particularly 5-methylcytosine (5 mC), is a key epigenetic modification involved in the regulation of gene expression and genomic stability, and has emerged as a promising biomarker for early cancer screening and molecular stratification. CRISPR-Cas12a systems have been increasingly exploited to convert methylation-associated sequence information into detectable signals owing to their programmability and collateral cleavage-mediated signal amplification. However, many CRISPR-based assays remain constrained by the high background and relatively slow response kinetics of conventional fluorophore-quencher reporters. To overcome these limitations, we developed a cleavage-responsive DNA-templated silver nanocluster (DNA/AgNC) reporter that translates Cas12a trans-cleavage activity into a green-to-red ratiometric fluorescence shift. In this design, the AgNC-templating DNA scaffold itself serves as an enzymatically cleavable signal transducer, rather than relying on a terminal fluorophore-quencher pair. Compared with a representative F-ssDNA-Q reporter, the DNA/AgNC reporter exhibited stronger apparent association with Cas12a and an approximately two-fold improvement in apparent catalytic efficiency. When incorporated into an MSRE-RPA-Cas12a workflow, the platform achieved a limit of detection of 74.5 aM, while completing the Cas12a reporting step within 30 min. Coupling this assay with a miniaturized optoelectronic device further enabled spatially resolved profiling of five genomic loci, with relative errors of approximately 5%. Overall, this strategy establishes a ratiometric reporter format for CRISPR-based DNA methylation profiling and offers potential for point-of-care epigenetic biosensing.},
}
@article {pmid42333809,
year = {2026},
author = {Hou, S and Bi, W and Liu, W and Nie, H and Jiang, J and Zeng, L},
title = {Beyond DNA editing: how Cas13 redefined programmable RNA manipulation and what still limits its therapeutic promise.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42333809},
issn = {1362-4962},
support = {2024ZD0541500//Noncommunicable Chronic Diseases-National Science and Technology Major Project/ ; 82222038//National Natural Science Foundation of China/ ; 01-SWKJYCJJ06//Outstanding Young Talents of National Defense Biotechnology/ ; 2023XRC01//High-Level Talent Program Cultivation Project of Army Medical University/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA Editing/genetics ; *RNA/genetics/metabolism ; *Gene Editing/methods ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The Type VI Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13 system, evolved from prokaryotic immunity, has become a versatile, programmable RNA-targeting platform with broad biotechnological potential. Guided by CRISPR RNA (crRNA), Cas13 cleaves single-stranded RNA via higher eukaryotic and prokaryotic nucleotide-binding domains, exerting specific (cis) and nonspecific (trans) collateral cleavage, which enables ultrasensitive nucleic acid detection while introducing cytotoxicity risks in eukaryotic cells. Diversification of Cas13 subtypes, including compact variants, enhances targetability and delivery compatibility, and inhibitory strategies (anti-CRISPR proteins, crRNA mimicry/degradation) enable activity modulation for improved safety. Building on mechanistic foundations, Cas13 is repurposed for targeted RNA knockdown, nucleic acid diagnostics, live-cell RNA imaging with catalytically inactive variants, programmable RNA base editing through deaminase fusions, splicing regulation, epitranscriptomic editing of multiple RNA chemical marks, interaction mapping of RNA-protein and RNA-RNA networks, and translational control, with preliminary clinical translation in antiviral therapies, pathogenic transcript correction, and cancer therapy. Furthermore, Cas13-integrated diagnostics and functional genomics are accelerating biomarker discovery and personalized treatment. Nevertheless, successful clinical translation hinges on overcoming critical bottlenecks, including tissue-specific delivery, mitigation of collateral cytotoxicity, and management of host immunogenicity. This review synthesizes Cas13 classification, structure-function principles, regulatory inhibitors, application modalities, and translational challenges to inform next-generation engineering and responsible deployment of RNA-targeted technologies.},
}
@article {pmid42334452,
year = {2026},
author = {Lin, Y and Liou, B and Fannin, V and Adler, S and Mayhew, CN and Hammonds, JE and Hu, YC and Tchieu, J and Zhang, W and Zhao, X and Beres, RL and Setchell, KDR and Kaynak, A and Qi, X and Feldman, RA and Sun, Y},
title = {Patient-specific midbrain organoids with CRISPR correction recapitulate neuronopathic Gaucher disease phenotypes and enable evaluation of novel therapies.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42334452},
issn = {2050-084X},
support = {R21HD1027881//National Institute of Health/ ; R21OD033660//National Institute of Health/ ; R01NS138309//National Institute of Health/ ; R01NS103931//National Institute of Health/ ; Center of Pediatric Genomics Award//Cincinnati Children's Hospital Medical Center/ ; Research Innovation Pilot Funding Program Award//Cincinnati Children's Hospital Medical Center/ ; 2UL1TR001425-05A1, CHMC-CTSA 00003827/NH/NIH HHS/United States ; },
mesh = {*Gaucher Disease/genetics/therapy/pathology ; Humans ; *Organoids/pathology/metabolism ; *Mesencephalon/pathology ; *Glucosylceramidase/genetics/metabolism ; Phenotype ; *CRISPR-Cas Systems ; Mutation ; Induced Pluripotent Stem Cells ; Dopaminergic Neurons ; },
abstract = {Neuronopathic Gaucher disease (nGD) is a lysosomal storage disorder caused by GBA1 mutations, leading to defective acid β-glucosidase (GCase) and accumulation of glycosphingolipid substrates, causing inflammation and neurodegeneration. Patients with nGD manifest severe neurological symptoms, but current animal models fail to fully recapitulate the human condition, posing a major barrier to the development of effective therapies targeting the brain. To bridge this gap, we have developed midbrain-like organoids (MLOs) from human induced pluripotent stem cells of nGD patients with GBA1[L444P/P415R] and GBA1[L444P/RecNcil] mutations to model nGD brain pathogenesis. These nGD MLOs exhibited GCase deficiency, resulting in diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, mirroring nGD pathology. GBA1 mutation correction mediated by CRISPR/Cas9 restored GCase activity, normalized lipid substrate levels, and rescued dopaminergic neuron function, confirming the causal role of GBA1 mutations during early brain development. Using this novel platform, we further evaluated therapeutic strategies, including SapC-DOPS nanovesicles delivering GCase, AAV9-GBA1 gene therapy, and substrate reduction therapy with GZ452, a glucosylceramide synthase inhibitor currently under clinical investigation. These treatments either restored GCase activity, reduced lipid substrate accumulation, improved autophagic and lysosomal abnormalities, or ameliorated dysregulated genes involved in neural development. These patient-specific, 3D neural models offer a transformative, physiologically relevant platform for unraveling disease mechanisms and accelerating the discovery of therapies for patients with nGD.},
}
@article {pmid42334517,
year = {2026},
author = {Zhang, XL and Luo, JN and Cao, Y and Zhao, JX and Zhang, Y and Xu, SF and Zhuang, LN and He, J},
title = {Generation of transgenic pigs with targeted insertion of a wildtype copy of human PKD2 gene.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42334517},
issn = {1573-4978},
support = {2021YFA0805902//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Animals, Genetically Modified/genetics ; Swine/genetics ; *TRPP Cation Channels/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; *Polycystic Kidney, Autosomal Dominant/genetics/metabolism ; Female ; Disease Models, Animal ; Kidney/metabolism/pathology ; },
abstract = {BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD), primarily driven by PKD1 or PKD2 mutations, is a prevalent hereditary nephropathy for which large-animal models capturing human renal anatomy and disease tempo remain urgently needed. This study aimed to establish a site-specific human PKD2 (hPKD2) transgenic porcine model as a versatile platform capable of supporting two temporally distinct applications: longitudinal evaluation of whether sustained PKD2 overexpression is sufficient to induce renal pathology, and-should overexpression prove phenotypically silent-future crossbreeding-based functional rescue of PKD2-knockout lines.<br><br>METHODS AND RESULTS: A CRISPR/Cas9-mediated, homology-recombination-independent strategy was employed to target full-length hPKD2 cDNA into the porcine pH11 safe harbor locus. Somatic cell nuclear transfer yielded five F0 transgenic founders, and natural mating of two founders with wild-type sows produced four F1 transgenic offspring, confirming stable germline transmission. Quantitative real-time PCR and whole-genome sequencing validated single-copy, site-specific transgene integration at the designated locus. Robust hPKD2 mRNA and FLAG-tagged polycystin-2 (PC-2) expression were detected in renal and other tissues across both generations. During the initial 12-month monitoring period, serum blood urea nitrogen and creatinine levels remained within normal ranges and no gross histological abnormalities were evident. However, given that these biomarkers are insensitive to early-stage renal impairment, extended observation with more comprehensive phenotyping is required before definitive conclusions regarding renal function can be drawn.<br><br>CONCLUSIONS: A transgenic porcine model with stable, single-copy hPKD2 integration at the pH11 safe harbor locus was successfully generated and shown to permit germline transmission. This platform provides a foundation for long-term investigation of PKD2 overexpression pathophysiology and, alternatively, for functional complementation of PKD2-deficient models, thereby advancing both mechanistic and translational ADPKD research.},
}
@article {pmid42337238,
year = {2026},
author = {Shi, LD and Kolody, BC and Wang, S and Valentin-Alvarado, LE and Lei, S and Sachdeva, R and Banfield, JF},
title = {Jumbo circular extrachromosomal elements of methane-oxidizing archaea with variably extensive metabolic and defense gene repertoires.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74423-z},
pmid = {42337238},
issn = {2041-1723},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; INV-037174/GATES/Gates Foundation/United States ; },
abstract = {Archaeal extrachromosomal elements (ECEs) are arguably the least well understood of all genetic elements, and few have >200 kbp (jumbo) genomes. Here, we report circular, jumbo ECEs with genomes of up to 535 kbp in length that associate with anaerobic methane-oxidizing Methanoperedens archaea. Notably, a 409-kbp genome related to jumbo ECEs is integrated into a subset of the ~4.2 Mbp Methanoperedens chromosomes at the tRNA-Asp genes. This represents the largest integrative element in Archaea and supports the jumbo ECE-host association. Multiple genome alignments and phylogenetic analyses suggest that the large ECE sizes developed by extensive DNA acquisition from Methanoperedens. The newly identified ECEs encode, and in some cases express, metabolic genes such as tetrahydromethanopterin S-methyltransferase exclusively involved in methane metabolism, and genes for nitrogen and sulfur compound transformations. Also encoded are defense systems, some of which are absent in hosts, such as hybrid Type I/Type III-A CRISPR-Cas systems. In contrast to viruses and plasmids, they have host-like replication machinery and occur at stable copy ratios of 1.44 ± 0.24:1 to the host. Overall, our results reveal a spectrum of jumbo ECEs of Methanoperedens, ranging from plasmid-like to minichromosome-like.},
}
@article {pmid42339381,
year = {2026},
author = {Bashir, T and Rashid, R and Malik, AR and Sundouri, AS and Bhat, KM and Mir, MA and Rehman, MU and Masood, M and Tehleelah, },
title = {Reframing fruit biofortification for sustainable micronutrient security: from crop innovation to nutritional impact.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1853898},
pmid = {42339381},
issn = {1664-462X},
abstract = {Micronutrient malnutrition, commonly referred to as "hidden hunger, " remains a persistent global health challenge, particularly in regions with limited dietary diversity. Although agricultural intensification has substantially improved caloric availability, it has not ensured adequate micronutrient density in food systems, highlighting the urgent need for nutrition-sensitive crop improvement strategies. Biofortification has emerged as a sustainable and cost-effective approach to enhance the micronutrient content of food crops through agronomic, genetic, and biotechnological interventions. While biofortification research has predominantly focused on staple cereals and legumes, horticultural fruit crops have received comparatively limited attention despite their widespread consumption, high consumer acceptance, and natural richness in bioactive compounds. This review advances beyond existing overviews by providing a critical and comparative evaluation of agronomic, conventional breeding, and biotechnological approaches for fruit crop biofortification, with particular emphasis on their effectiveness, scalability, limitations, and translational potential. Current advances aimed at enhancing iron, zinc, iodine, selenium, and provitamin A concentrations are comprehensively synthesized. Special attention is given to the physiological and molecular mechanisms regulating micronutrient uptake, transport, accumulation, and storage in fruit tissues. In addition, advanced biotechnological tools, including CRISPR/Cas-mediated genome editing, are critically assessed in relation to biosafety, regulatory considerations, and practical applicability. Evidence from major fruit crops, including apple, banana, mango, pomegranate, strawberry, and papaya, demonstrates that integrated biofortification strategies can improve micronutrient density while maintaining fruit yield and quality. Importantly, this review addresses a major knowledge gap by linking crop-level nutrient enhancement with micronutrient bioavailability and human nutritional outcomes, emphasizing the influence of food matrix interactions and nutrient absorption efficiency. Key constraints, including genotype × environment interactions, postharvest nutrient instability, climate-driven variability, and limited clinical validation, are also discussed. Finally, a systems-level framework integrating plant science, human nutrition, postharvest biology, and policy perspectives is proposed to support the large-scale adoption of nutrition-sensitive fruit biofortification. Collectively, fruit crop biofortification represents a promising strategy for improving global micronutrient security and advancing sustainable food systems.},
}
@article {pmid42340480,
year = {2026},
author = {Guo, Z and Yang, Z and Liu, Z and Zheng, G and Zou, S},
title = {Fgf8, a gene knockout that leads to intermuscular bones-reduced of crucian carp (Carassius auratus), acts as a potentially regulatory factor in osteogenic development.},
journal = {Fish physiology and biochemistry},
volume = {52},
number = {4},
pages = {},
pmid = {42340480},
issn = {1573-5168},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Carps/genetics/growth &amp; development ; *Fibroblast Growth Factor 8/genetics/metabolism ; *Osteogenesis/genetics/physiology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; },
abstract = {Although fibroblast growth factor 8 (FGF8) is a critical regulator of skeletal morphogenesis in vertebrates, its specific role in the formation and development of intermuscular bones (IBs) in teleost fish remains insufficiently characterized. In this study, we generated double mutants (fgf8a[+/-] + fgf8b[+/-]) in diploid Chongming crucian carp (Carassius auratus) using CRISPR/Cas9-mediated gene editing. Compared to wild-type fish, the double mutants exhibited significantly reduced IB number (p < 0.01), demonstrating a synergistic role of fgf8a and fgf8b in IB formation. Notably, reduced number of IBs did not compromise overall growth, muscle architecture, or reproductive performance. Integrated transcriptomic and metabolomic analyses revealed that the reduced IB phenotype was linked to modifications in relevant signaling pathways and a concomitant upregulation of metabolites beneficial for muscle quality and health. Our findings highlight the crucial role of fgf8 in regulating IB formation and development in crucian carp, providing insights into the genetic mechanisms underlying this process in teleosts.},
}
@article {pmid42342668,
year = {2026},
author = {Mitsuda, Y and Sugaya, M and Ishikawa, J and Nagahata, N and Okazaki, S and Hiraizumi, M and Kato, K and Gootenberg, JS and Abudayyeh, OO and Osawa, T and Yamashita, K and Nishimasu, H},
title = {Structural mechanism of SAM-AMP and SAM-AMP2 synthesis by the type III-D2 CRISPR effector complex.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42342668},
issn = {2041-1723},
support = {25H00436//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *S-Adenosylmethionine/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Escherichia coli/metabolism/genetics/virology ; Adenosine Triphosphate/metabolism ; Escherichia coli Proteins/metabolism/genetics/chemistry ; },
abstract = {The type III-D2 CRISPR-Cas system comprises multiple Cas subunits and a CRISPR RNA, and is likely an evolutionary intermediate between the well-studied type III-A and III-E systems. Here we show that the type III-D2 complex synthesizes two distinct second messengers, SAM-AMP and SAM-AMP2, from S-adenosylmethionine (SAM) and ATP in response to target RNA recognition. We determined cryo-electron microscopy structures of the type III-D2 effector complex in different functional states, providing mechanistic insights into target RNA cleavage and second messenger synthesis. The structures reveal how SAM and ATP are recognized by the Cas10 subunit within the effector complex. Furthermore, our biological data suggest that both SAM-AMP and SAM-AMP2 act on the CorA ancillary effector, inducing growth arrest of infected bacterial cells and thereby conferring immunity. Thus, our study establishes the type III-D2 system as a unique anti-phage defense mechanism that employs both SAM-AMP and SAM-AMP2 as second messengers, expanding the repertoire of second messenger strategies in bacterial defense systems and highlighting the remarkable functional diversity of CRISPR-Cas systems.},
}
@article {pmid42342872,
year = {2026},
author = {Van Der Straeten, D and Bulut, M and Cao, D and Aharoni, A and Bouis, H and Granell, A and Gruissem, W and Lindberg Møller, B and Martin, C and Puchta, H and Sreenivasulu, N and Tissier, A and Tripathi, L and Van Montagu, M and Fernie, AR},
title = {Genetic technologies to enhance crop nutritional value under climate change.},
journal = {Nature},
volume = {654},
number = {8120},
pages = {877-891},
pmid = {42342872},
issn = {1476-4687},
mesh = {Humans ; *Biofortification/methods ; *Climate Change ; CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/metabolism/chemistry ; *Gene Editing/methods ; Metabolic Engineering ; Micronutrients/deficiency/analysis ; Nutritive Value ; },
abstract = {At present, more than 700 million people live with caloric hunger, and more than two billion suffer from micronutrient deficiencies, known as 'hidden hunger'. From an agricultural viewpoint, three major objectives need to be worked towards simultaneously to achieve zero hunger (the United Nations Sustainable Development Goal 2): (1) enhanced yield; (2) higher vitamin and mineral density to sustain recommended daily intake (multi-biofortification); and (3) enhanced climate-change resilience. Although the Green Revolution increased global calorie production, it exacerbated hidden hunger by prioritizing high yield over nutritional quality. Stress from global climate change has been shown to reduce the densities of several micronutrients. CRISPR-Cas, which allows genome editing with extremely high precision, has emerged as a groundbreaking breeding technology that has already been adopted by many countries. Here we examine how CRISPR-Cas-based approaches could be used to achieve biofortification targets by enhancing micronutrient densities to the levels necessary to alleviate dietary vitamin and mineral deficiencies. Given the limited time frame available to achieve zero hunger, we argue that CRISPR-Cas technologies should be combined with metabolic engineering based on transformation and other technologies. We also consider untapped resources beyond metabolic pathways and current CRISPR-Cas methodologies to address one of the most important societal issues of the twenty-first century.},
}
@article {pmid42342925,
year = {2026},
author = {Gong, Q and Liu, H and Nie, Q and Tong, J and Li, W and Pak, M and Mei, Y and He, K and Wang, D and Wang, NN},
title = {GmENDO-like 1, a structurally divergent homolog of S1/P1-type endonuclease, modulates the vegetative-to-reproductive phase transition in soybean.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42342925},
issn = {1432-203X},
support = {24JCQNJC01940//Natural Science Foundation of Tianjin/ ; 2023ZD040710207//Biological Breeding-National Science and Technology Major Project/ ; 32070317//National Natural Science Foundation of China, Grant/ ; 32270359//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/enzymology/physiology/growth &amp; development ; *Plant Proteins/genetics/metabolism/chemistry ; Amino Acid Sequence ; Gene Expression Regulation, Plant ; *Endonucleases/genetics/metabolism/chemistry ; Plant Leaves/genetics/physiology/growth &amp; development ; Gene Knockout Techniques ; Plants, Genetically Modified ; Flowers/genetics/physiology ; Plant Senescence/genetics ; Reproduction/genetics ; Phylogeny ; CRISPR-Cas Systems ; Phenotype ; Mutation ; },
abstract = {GmENDO-like 1, a structurally divergent S1/P1 endonuclease homolog, modulates soybean vegetative-to-reproductive transition; its knockout delays flowering and accelerates leaf senescence. Soybean is a monocarpic crop in which the transition from vegetative to reproductive growth triggers the onset of senescence in vegetative tissues. In this study, we identified a soybean homolog of the S1/P1-type endonuclease gene, GmENDO-like 1, which was significantly upregulated during leaf senescence. Sequence analysis revealed that GmENDO-like 1 has substantial deletions at both the N- and C-termini, resulting in the loss of key residues essential for the active center of canonical S1/P1-type nucleases. Specifically, among the nine conserved amino acids responsible for metal ion coordination, one tryptophan (Trp), two histidines (His), and one aspartic acid (Asp) residue are absent. Although these deletions result in a significant rearrangement of the trinuclear metal-binding center, GmENDO-like 1 remarkably retains intrinsic catalytic activity. Using CRISPR/Cas9-mediated genome editing, we generated two knockout mutant lines of the GmENDO-like 1 gene. Phenotypic analysis revealed a markedly delayed flowering time and an obviously precocious leaf senescence in the GmENDO-like 1 mutant plants. These observations were further corroborated by the altered expression levels of flowering- and senescence-associated marker genes. Both the 100-seed weight and per-plant yield of the mutants were significantly decreased. Collectively, these findings establish GmENDO-like 1 as a functional, evolutionarily divergent nuclease and an essential modulator regulating the vegetative-to-reproductive phase transition in soybean.},
}
@article {pmid42343762,
year = {2026},
author = {Han, N and Xiao, Y and Zhang, J and Guo, L and Li, Q and Yang, P and Li, X and Lin, X and Xu, L and Zhang, H},
title = {[Research progress of the CRISPR-Cas system in the detection and elimination of antibiotic resistance genes].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {5},
pages = {2051-2068},
doi = {10.13345/j.cjb.250847},
pmid = {42343762},
issn = {1872-2075},
support = {2023-YJ-zd1//the Key Research and Development Program of Health and Healthcare of Fuzhou First General Hospital/ ; 2024-YJ-ZK04//the Fujian Province Clinical Key Specialty Construction Research Project/ ; 2024J011257//the General Program of Fujian Provincial Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *Gene Editing/methods ; },
abstract = {The abuse of antibiotics has led to increasingly severe antibiotic resistance, which poses a serious threat to global public health security. Therefore, the rapid detection and effective elimination of antibiotic resistance genes are crucial for controlling the spread of drug-resistant bacteria. Conventional methods for detecting antibiotic resistance genes, such as PCR, quantitative real-time PCR, and isothermal amplification, have shortcomings including time-consuming procedures, labor intensiveness, high costs, and poor specificity. Therefore, there is an urgent need to find new solutions to combat bacterial resistance, making the development of novel and efficient technologies for detecting and eliminating antibiotic resistance genes. In recent years, gene editing technology has become a research hotspot. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins) consisted of the CRISPR-Cas system. As one of the adaptive immune systems in prokaryotes, it possesses unique sequence targeting ability and high sensitivity, which enable it to accurately recognize and cleave target nucleic acid sequences. Thus, it shows great potential in the detection and elimination of antibiotic resistance genes. This review summarizes the classification, structures, and action mechanisms of CRISPR-Cas systems, elaborates on the application of different CRISPR-Cas systems in the detection and elimination of antibiotic resistance genes, and finally discusses the challenges faced by this technology and its future development directions, aiming to provide new ideas for addressing antibiotic resistance in pathogenic bacteria.},
}
@article {pmid42343773,
year = {2026},
author = {Chen, Y and Jin, W and Ma, X and Feng, R and Yang, Y},
title = {[YTHDF1 enhances the expression of exogenous proteins in the CHO-K1-GS[‒/‒] cell line].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {5},
pages = {2219-2232},
doi = {10.13345/j.cjb.250823},
pmid = {42343773},
issn = {1872-2075},
support = {2025-2-177//the Lanzhou Science and Technology Program/ ; 26YFFA026//the Gansu Science and Technology Program/ ; 31920250053//the Fundamental Research Funds for the Central Universities/ ; 2025CXZX-242//the "Innovation Star" Project for Postgraduate Students in Gansu Province/ ; 25FNNA002//the Gansu Province Research-Production Integration Sci-Tech Project Empowerment Initiative/ ; },
mesh = {Animals ; CHO Cells ; Cricetulus ; *Glutamate-Ammonia Ligase/genetics ; *RNA-Binding Proteins/genetics/metabolism ; Cricetinae ; Recombinant Proteins/genetics/biosynthesis ; Humans ; *RNA Splicing Factors/genetics ; CRISPR-Cas Systems ; },
abstract = {YTH domain family protein 1 (YTHDF1) is an RNA-binding protein and belongs to the reader modified by N[6]-methyladenosine (m[6]A). It recognizes and binds to m[6]A modifications on RNA through specific domains, thereby performing corresponding biological functions. This study aims to explore the effects of YTHDF1 on the expression levels of exogenous proteins in the Chinese hamster ovary (CHO) cell line and evaluate its application potential in the optimization of the expression system of CHO cells. Firstly, the glutamine synthetase (GS) gene of CHO-K1 cells was knocked out by CRISPR/Cas9 to obtain the CHO-K1-GS[‒/‒] cell line. The enhancement effects of YTHDF1 overexpression on the expression of exogenous proteins in CHO-K1-GS[‒/‒] cells were further analyzed. Western blotting, qPCR, and fluorescence microscopy observations showed that the overexpression of YTHDF1 significantly up-regulated the expression levels of human serum albumin, single-chain antibody, and green fluorescent protein in cells. Moreover, this process did not have a significantly negative impact on the long-term proliferation and survival rate of the cells. In addition, the experiment with the translation inhibitor cycloheximide (CHX) confirmed that YTHDF1 mainly enhanced the protein expression by promoting translation in cells. This study demonstrates that YTHDF1 can enhance the cell's ability to synthesize exogenous proteins by promoting the translation process of mRNA. This strategy provides a new theoretical basis and technical direction for constructing efficient cell lines for producing biopharmaceuticals.},
}
@article {pmid42343789,
year = {2026},
author = {Han, C and Li, Y and Zhang, H},
title = {[Advances in genome editing technologies in Komagataella phaffii and their applications in biomanufacturing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {6},
pages = {2414-2438},
doi = {10.13345/j.cjb.250908},
pmid = {42343789},
issn = {1872-2075},
support = {1012050205238420//the Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; Recombinant Proteins/genetics/biosynthesis ; Metabolic Engineering/methods ; },
abstract = {Komagataella phaffii is widely recognized as a premier host for the production of recombinant proteins and value-added metabolites, owing to its low background secretion of endogenous proteins, strong capacity for heterologous protein secretion, and robust growth and metabolic performance under industrially relevant fermentation conditions. In recent years, rapid progress in genome editing technologies and synthetic biology toolkits has markedly improved the precision and efficiency of gene function interrogation, metabolic pathway reconstruction, and dynamic regulation in K. phaffii, thereby continuously strengthening its performance as a microbial cell factory. Consequently, beyond its established roles in producing recombinant proteins, industrial enzymes, and vaccine antigens, K. phaffii has also demonstrated substantial potential for the biosynthesis of natural products, biopharmaceutical molecules, and emerging biomaterials. This review systematically summarizes the evolution of genome engineering technologies in K. phaffii, spanning the transition from conventional recombination-based methods to next-generation precision editing tools. We highlight recent advances, optimization strategies, and engineering practices of CRISPR/Cas and related systems in this host. Moreover, in light of emerging research trends, we discuss key challenges and opportunities associated with improving editing efficiency, enabling rapid assembly of complex metabolic pathways, and accelerating industrial translation, thereby providing a reference for the rational engineering of Komagataella phaffii and its broader applications in synthetic biology and biomanufacturing.},
}
@article {pmid42343816,
year = {2026},
author = {Wang, B and DU, C and Gege, R and Zhao, Q and Wang, H and Li, B and Wang, J and Mei, J and Zhang, S and Bao, F},
title = {[CRISPR/Cas9-mediated knockout of the Soc gene in T4 bacteriophage and mutant construction].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {6},
pages = {2839-2848},
doi = {10.13345/j.cjb.250870},
pmid = {42343816},
issn = {1872-2075},
support = {32260893//the National Natural Science Foundation of China/ ; 2022MS03019//the Natural Science Foundation of Inner Mongolia Autonomous Region/ ; NJYT23094//the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region/ ; 2023YFXZ0002//the Science and Technology Project of Inner Mongolia Autonomous Region/ ; BR250101//the Basic Scientific Research Fund of Directly Affiliated Universities in Inner Mongolia Autonomous Region/ ; },
mesh = {*Bacteriophage T4/genetics ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; Escherichia coli/genetics/metabolism ; Mutation ; },
abstract = {To address the antigen display limitations of current vaccine carriers, we engineered the T4 bacteriophage into a high-capacity platform. The T4 bacteriophage has advantages such as structural stability, high loading capacity, and easy production. However, the presence of high-copy endogenous Soc protein on its capsid surface severely restricts the effective display of exogenous proteins. To overcome the aforementioned spatial limitations, we employed the CRISPR/Cas9 system to precisely knockout the Soc gene of T4 bacteriophage. We successfully achieved the knockout of the Soc gene by co-transferring the three plasmid systems-pCas, pTargetF-sgRNA, and pMD19-T-Soc-arm-into Escherichiacoli TG1, inducing the expression of Cas9 with L-arabinose, and then infecting the engineered bacteria with the wild-type T4 bacteriophage (named T4 WT). The PCR, SDS-PAGE, and sequencing results confirmed a Soc gene-deficient T4 bacteriophage mutant strain (named T4ΔSoc) was successfully constructed. This mutant strain had comparable growth, thermal stability, and pH stability to the wild type, and maintained complete infectivity. After continuous passage for five generations, it remained stable in terms of genome, protein composition, and phage plaque phenotype, with no occurrence of revertant mutations. In conclusion, T4ΔSoc is stable both genetically and phenotypically, effectively breaking through the limitation of the restricted display space of T4 WT. This study provides a reliable vector tool and technical foundation for constructing an efficient and stable antigen display and vaccine delivery platform and has good application potential in vaccine research and the construction of targeted delivery systems.},
}
@article {pmid41454999,
year = {2025},
author = {Vadakkan, K and Raphael, R and Korattil, TT and Mapranathukaran, VO and Ramadas, V and Kizhakkepeedika, RD},
title = {A Critical Review of Quorum-Sensing Inhibition Strategies in Klebsiella Pneumoniae.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {103},
pmid = {41454999},
issn = {1432-0991},
abstract = {Klebsiella pneumoniae is an antibiotic-resistant microbe that causes pneumonia, urinary tract infections, bloodstream infections, and liver abscesses. Its pathogenicity is fueled by the polysaccharide capsule, lipopolysaccharides, and quorum-sensing-regulated biofilm formation, which enhance immune evasion and antibiotic resistance. The rise of multidrug-resistant and hypervirulent pathogens poses a significant challenge to treatment. Targeting quorum sensing with quorum quenching is a promising method for disrupting bacterial interaction and reducing virulence without increasing resistance. This review examines the pathogenesis of K. pneumoniae, the significance of quorum sensing, and emerging therapeutics, including quorum-sensing inhibitors and advanced techniques such as CRISPR-Cas and computational drug development, while emphasizing the need for further investigation.},
}
@article {pmid41762232,
year = {2026},
author = {Deepika, and Sharma, S and Kumar, P and Rathore, V and Chauhan, A and Kumar, A and Dogra, RK and Handa, A},
title = {Insights into physiological, biochemical and molecular mechanisms of abiotic stress tolerance in Persian walnut (Juglans regia L.).},
journal = {Protoplasma},
volume = {263},
number = {4},
pages = {1099-1116},
pmid = {41762232},
issn = {1615-6102},
abstract = {Walnut (Juglans regia) is an economically and nutritionally valuable tree species that often encounters diverse abiotic stresses such as drought, salinity, cold, heat and heavy metal toxicity. These stresses adversely affect its growth, productivity and survival by altering physiological functions, disturbing cellular homeostasis and triggering oxidative damage. In response, J. regia deploy a multifaceted adaptive system comprising morphological changes, biochemical adjustments and intricate molecular signaling pathways. The review aims to analyze current knowledge on the physiological, morphological changes observed under stress with biochemical defense mechanism. These include the crucial antioxidant defense system (increase in Superoxide Dismutase (SOD), Peroxidase (POD) and Catalase (CAT) activity), the accumulation of protective soluble solutes and amino acids and the biosynthesis of secondary metabolites through the Methylerythritol Phosphate (MEP) pathway involved in mitigating oxidative stress caused by Reactive Oxygen Species (ROS). Crucially, we synthesize the understanding of molecular regulation that underpins stress adaptation. This encompasses stress-responsive gene expression including Jr (Juglans regia) VHAG1 (V-ATPase H+ transporting ATPase subunit G1), JrDREB (Dehydration-Responsive Element Binding protein), JrRD29 (Responsive to Dehydration 29), transcriptional regulation by myeloblastosis (MYB), Dehydration-responsive element-binding proteins (DREB) and WRKY-TF families and their interaction with hormonal (abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene) which play a pivotal role in signal transduction and stress tolerance. These mechanisms employed by walnut under abiotic stresses, highlighting key genetic and hormonal pathways that can be targeted for the development of stress-resilient cultivars and ensuring sustainable production under changing climatic conditions. Recent advancements in genomics, transcriptomics and proteomics got attention that provides new insights into the regulatory networks and candidate genes conferring stress resilience. Furthermore, it explores biotechnological approaches for improving stress tolerance, highlighting the prospects of latest high-throughput techniques, including molecular breeding, genetic engineering, Next-Generation Sequencing (NGS), microRNA (miRNA)-based regulation and Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas) gene editing. This integrated review connects multi-level stress response mechanisms, highlighting potential molecular markers and biotechnological interventions for accelerating walnut improvement and ensuring sustainable production under changing climatic conditions.},
}
@article {pmid41820648,
year = {2026},
author = {Hanif, N and Naveed, M and Salah Ud Din, M and Aziz, T and Shami, A and Al-Joufi, FA},
title = {Genome-wide characterization and comparative analysis of Bacillus paranthracis MBBL1 reveals genomic plasticity features.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {41820648},
issn = {1438-7948},
support = {PNURSP2026R31//Princess Nourah Bint Abdulrahman University/ ; },
abstract = {Bacillus paranthracis is a member of the Bacillus cereus group with close phylogenetic proximity to pathogenic species, yet its genomic diversity and pathogenic potential remain poorly understood. In this study, we present a comprehensive whole-genome analysis of B. paranthracis strain MBBL1, an environmental isolate, to explore its genomic architecture, evolutionary relationships, and potential virulence traits. Comparative genomic and pangenome analyses revealed that MBBL1 clusters closely with other B. paranthracis strains while maintaining measurable genomic similarity to B. cereus and B. anthracis. Notably, the genome lacks plasmids and known serotype markers, distinguishing it from many pathogenic members of the group. Despite this, MBBL1 harbors multiple chromosomally encoded virulence-associated genes, mobile genetic elements, and genomic islands, indicating considerable genomic plasticity. The presence of an active Type I CRISPR-Cas system may contribute to genome stability and the absence of plasmids. Pathogenicity prediction suggests a low but detectable potential for human pathogenicity. Collectively, these findings highlight the importance of environmental Bacillus strains as reservoirs of hidden virulence traits and provide a valuable genomic framework for future functional and risk assessment studies.},
}
@article {pmid41964738,
year = {2026},
author = {Karwa, P and Kharul, A and Parekar, V and Labhade, S and Sakle, N and Bhole, R and Kapare, H},
title = {Next-Generation Therapies for Genetic Diseases: The Synergy of CRISPR and Gene Therapy.},
journal = {Biochemical genetics},
volume = {},
number = {},
pages = {},
pmid = {41964738},
issn = {1573-4927},
abstract = {The introduction of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has changed the game of gene therapy and promises surgical precision and efficacy in the process of addressing genetic disorders in humans. Gene therapy that commonly assumes insertion of functional DNA with the help of viral vectors has developed or rather undergone adaptation with the introduction of CRISPR-Cas mechanisms that make it possible to edit the genome, correct or regulate it and silence. It is a critical study that reviews mechanistic disparities among traditional and CRISPR bases types of gene therapy, based on benefits, shortcomings, and states of improvement till date, as far as clinical development is concerned. It talks about their historical evolution, molecular principles, delivery modes, and therapeutic promise of the CRISPR systems such as Cas9, Cas12, Cas13, base editing, and prime editing. It is of significant value to mention the application of CRISPR as a treatment in monogenic illnesses such as 2-thalassemia and Duchenne muscular dystrophy as well as the future use of CRISPR in complex and multifactorial diseases. Also, safety concerns, ethical issues, and delivery translational problems are discussed by the review and they are paramount to successful clinical translation of CRISPR-based therapeutics. The discussion highlights the revolutionary possibility of CRISPR in precision medicine and predetermines its expansion in the future healthcare genomic practice.},
}
@article {pmid42105238,
year = {2026},
author = {Wang, DM and Tiruppathi, C},
title = {Protocol to generate endothelial cell-specific knockout mouse models using Cas9/Cdh5-Cre mice coupled with sgRNA.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104551},
pmid = {42105238},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Endothelial Cells/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice, Knockout ; Integrases/genetics/metabolism ; Cadherin 5 ; *Cadherins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD/genetics/metabolism ; *Gene Knockout Techniques/methods ; },
abstract = {The vascular endothelium is a critical regulator of vascular homeostasis and tissue fluid balance, and mouse models are essential for studying these processes in vivo. Here, we present a protocol to generate adult endothelial cell (EC)-specific gene knockout (KO) mouse models. We describe steps for Cas9-active and Cdh5-Cre-positive (Cas9/Cdh5-Cre) mouse line generation, single guide RNA (sgRNA) design for vector construction, plasmid DNA generation, and liposome preparation. We then detail procedures for liposome/plasmid complex injection, lung harvest, homogenization, protein quantification, and verification with western blotting. For complete details on the use and execution of this protocol, please refer to Wang et al.[1].},
}
@article {pmid42163310,
year = {2026},
author = {Jiang, Y and Caban, KM and Peitzsch, M and Herrmann, C and Mayr, D and Stöckl, JB and Fröhlich, T and Mayerhofer, A and Müller-Taubenberger, A and Welter, H},
title = {Knockout of filamin A in KGN granulosa tumor cells impairs proliferation, cell cycle progression, migration, and cytoskeletal organization under mechanical stress.},
journal = {Biological research},
volume = {59},
number = {1},
pages = {},
pmid = {42163310},
issn = {0717-6287},
mesh = {*Filamins/genetics/metabolism ; Humans ; Female ; *Cell Movement/physiology ; *Cell Proliferation/physiology ; *Granulosa Cell Tumor/pathology/metabolism/genetics ; *Cytoskeleton/physiology ; Stress, Mechanical ; *Cell Cycle/physiology ; Cell Line, Tumor ; Immunohistochemistry ; *Ovarian Neoplasms/pathology ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Cell Adhesion ; },
abstract = {BACKGROUND: Filamin A (FLNA) is an actin-binding protein that regulates mechanosensitivity and functions as an intracellular signaling scaffold in various cell types. It has also been implicated in tumor growth. We recently reported FLNA expression in human ovarian granulosa cells and in KGN cells, a granulosa cell tumor (GCT) line.<br><br>RESULTS: Immunohistochemistry analysis of 51 GCT samples revealed heterogeneous FLNA expression, with approximately 20% showing weak, 18% strong, and the majority moderate expression. We therefore conducted functional studies in KGN cells using CRISPR/Cas9 gene editing. A proteomic approach revealed marked changes in protein abundance upon FLNA depletion: proteins with increased abundance were predominantly related to adhesion, cytoskeletal organization, regulation of cell shape, and lipid metabolic process, whereas those with decreased abundance were associated with DNA replication, cell division, and cell cycle regulation. FLNA-knockout cells showed enlarged cell sizes, reduced proliferation, and slightly affected steroidogenesis. Disruption of FLNA further reduced migration velocity, altered actin cytoskeletal alignment under flow, and modified expression of genes involved in cytoskeletal architecture, adhesion, and mechanosensing under shear stress.<br><br>CONCLUSIONS: Our results identify crucial roles of FLNA in shaping the cellular architecture, motility, and proliferation of KGN cells. Consequently, alterations in FLNA expression may influence intracellular signaling, and responsiveness to mechanical cues in both physiological and pathological contexts.},
}
@article {pmid42172127,
year = {2026},
author = {Hong, JP and Nussenzweig, MC},
title = {Protocol for an in vivo CRISPR screen for germinal center B cells in mice using ecotropic retrovirus.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104586},
pmid = {42172127},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Germinal Center/cytology/immunology ; *B-Lymphocytes/metabolism/cytology ; *Retroviridae/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The lack of an in vitro model that recapitulates germinal center (GC) B cell biology necessitates the use of animal models for genetic studies. Here, we present a protocol for an in vivo CRISPR screen for GC B cells in mice using an ecotropic retrovirus. We describe steps for constructing a single guide (sgRNA) library and performing a genetic screen in a mouse model of protein immunization, including procedures for sequencing and data analysis.},
}
@article {pmid42213768,
year = {2026},
author = {Hassan, AZ and Zhang, X and Ward, HN and Billmann, M and Bajjali, S and Brown, KR and Bhojoo, U and Chan, K and Lin, K and Costanzo, M and Andrews, B and Boone, C and Moffat, J and Myers, CL},
title = {Orobas: A computational approach for scoring and analysis of quantitative chemical-genetic interactions from CRISPR-Cas9 screens.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104594},
pmid = {42213768},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; },
abstract = {We present Orobas, a computational approach for transforming raw read count data from CRISPR-Cas9 chemical-genetic screens into quantitative interaction scores. We describe steps for computing differential interaction scores with statistical tests that account for multiple CRISPR guides per gene. We then outline approaches for post-processing differential log2-fold-change scores across multiple screens, incorporating normalization to reduce technical artifacts and correct batch effects.},
}
@article {pmid42213769,
year = {2026},
author = {Li, Z and Roberts, A and Nimse, U and Vu, L and Fei, J},
title = {Protocol for generating endogenous degron tags in essential transcription factors in human iPSCs via CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104602},
pmid = {42213769},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Degrons ; *Gene Editing/methods ; Gene Knock-In Techniques/methods ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Precise genome engineering in human pluripotent stem cells remains inefficient, limiting endogenous fluorescent tagging needed to study phase-separated membraneless nuclear compartments. Here, we present a protocol for generating precise knockin alleles of essential genes in human induced pluripotent stem cells (iPSCs) via CRISPR-Cas9 editing. We describe steps for combining transient p53 inhibition, optimized transfection conditions, and fluorescence-activated cell sorting. This protocol enables recovery of viable edited clones that would otherwise be lost due to editing-induced stress or essential gene perturbation. For complete details on the use and execution of this protocol, please refer to Li et al.[1].},
}
@article {pmid42226724,
year = {2026},
author = {Jong, U and Lim, D and Lee, SH},
title = {Prime editing updates: technological evolution, methodological expansion, and delivery strategies for in vivo applications.},
journal = {BMB reports},
volume = {59},
number = {6},
pages = {313-320},
pmid = {42226724},
issn = {1976-670X},
mesh = {*Gene Editing/methods/trends ; CRISPR-Cas Systems/genetics ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Prime editing is a next-generation genome editing technology that enables precise base substitutions, insertions, and deletions at target genomic loci without inducing double-strand breaks or requiring exogenous donor DNA. This system operates through a fusion protein composed of Cas9 nickase and reverse transcriptase together with prime editing guide RNA; it has emerged as a precise genome editing platform that overcomes the limitations of conventional double-strand break-inducing CRISPR-Cas9 systems. Since first reported in 2019, diverse methodological improvements from PE1 to PE7 have been achieved, leading to rapid advances in editing efficiency, expansion of the editable target range, correction of large genomic regions, and development of in vivo delivery technologies. In this review, we comprehensively discuss the fundamental working mechanism of prime editing, its methodological evolution, recent expansion strategies, and delivery platforms for therapeutic applications, and provide perspectives for future development. [BMB Reports 2026; 59(6): 313-320].},
}
@article {pmid42264452,
year = {2026},
author = {Li, Y and Du, H and Chen, J and Xiao, J and Ma, Y and Zhang, Y and Wang, Q},
title = {The development of RPA-CRISPR/Cas12a assay for the detection of Pseudomonas plecoglossicida.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
doi = {10.1093/femsle/fnag067},
pmid = {42264452},
issn = {1574-6968},
support = {T2023328//Shanghai Agricultural Science and Technology Innovation Project/ ; //MOF/ ; },
mesh = {*Pseudomonas/genetics/isolation &amp; purification ; Animals ; *Fish Diseases/microbiology/diagnosis ; *CRISPR-Cas Systems ; *Pseudomonas Infections/veterinary/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Perciformes/microbiology ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; Recombinases/metabolism ; },
abstract = {Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA-CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl-1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA-CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.},
}
@article {pmid42265371,
year = {2026},
author = {Su, F and Dong, Y and Guo, R and Xie, H and Zhang, Y and Liu, J and Cao, X and Xie, H and Zhou, M and Sun, X and Wang, M and Zhang, J and Zhu, JK},
title = {Efficient prime editors for heritable multiplex precision genome editing in soybean.},
journal = {Nature plants},
volume = {12},
number = {6},
pages = {1252-1263},
pmid = {42265371},
issn = {2055-0278},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Glycine max/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; CRISPR-Cas Systems ; Genetic Engineering/methods ; RNA Editing ; },
abstract = {Prime editing (PE) is limited by low efficiency in dicot plants. Here we develop an optimized PE system for soybean, GmPEplus, by deleting the RNase H domain, introducing a V223A substitution within the reverse transcriptase domain, inserting a viral nucleocapsid protein between Cas9 and reverse transcriptase, and co-expressing a dominant-negative engineered allele of the endogenous GmMLH1. GmPEplus achieves editing efficiencies of up to 81.3% in stable transgenic lines. Subsequently, we show that nicking the non-edited strand using an additional sgRNA via the tRNA processing system enhances editing efficiency, and that optimizing its expression with an independent AtU6 cassette boosts efficiency by 13.1-fold. Importantly, a Csy4-mediated multiplex PE system (CMMPE) is established for simultaneous editing of 2-12 genes in soybean hairy roots and up to 3 genes in stable transgenic lines. GmPEplus and CMMPE offer powerful, versatile tools for precise, multiplex and heritable genome editing in soybean breeding.},
}
@article {pmid42267397,
year = {2026},
author = {Xie, X and Meng, X and Han, B and Zhang, X and Qiu, M and Zhang, J and Zhao, F and Jiang, Y and Zhang, X},
title = {A Near-Infrared Light-Driven Photoelectrochemical Biosensor Based on CRISPR/Cas12a for Highly Sensitive Detection of Pseudomonas fluorescens in Dairy Products.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {24},
pages = {19094-19104},
doi = {10.1021/acs.jafc.6c01081},
pmid = {42267397},
issn = {1520-5118},
mesh = {*Pseudomonas fluorescens/genetics/isolation &amp; purification/radiation effects ; *Biosensing Techniques/methods/instrumentation ; *Electrochemical Techniques/methods/instrumentation ; *Dairy Products/microbiology/analysis ; CRISPR-Cas Systems ; Infrared Rays ; Food Contamination/analysis ; Gold/chemistry ; Limit of Detection ; },
abstract = {Pseudomonas fluorescens (P. fluorescens) is a psychrotrophic bacterium causing dairy spoilage. Plate count and PCR methods, as conventional detection standards, are either time-consuming or dependent on expensive instruments, restricting widespread application. Herein, a near-infrared (NIR)-driven photoelectrochemical (PEC) biosensor based on CRISPR/Cas12a was constructed by using SH-ssDNA/AuNPs/AgBiS2 as the working electrode. AgBiS2 possesses broad-spectrum absorption and an appropriate bandgap for efficient NIR utilization. In the presence of P. fluorescens, the CRISPR/Cas12a system was activated by the LAMP amplification products of the target DNA to perform the trans-cleavage activity toward the SH-ssDNA modified on the AuNPs/AgBiS2 electrode surface. This cleavage released SH-ssDNA from the electrode, reducing steric hindrance and accelerating interfacial electron transfer, thereby enhancing the PEC signal. The constructed biosensor achieved sensitive detection of P. fluorescens with a limit of detection (LOD) of 1 CFU/mL in the range of 10[1]-10[8] CFU/mL, offering a reliable strategy for detecting the psychrotrophic bacteria in dairy products.},
}
@article {pmid42274413,
year = {2026},
author = {Liu, L and Wu, M and Sun, X and Lu, X and Yuan, C and Li, CY},
title = {Hierarchical Amplification-Interlinked CRISPR-Cas14a Luminescent Biosensor Coupled with Portable Photonic Crystal Biochip-Boosted Time-Delayed Signaling for the Diagnosis of Pediatric Mycoplasma pneumoniae Pneumonia.},
journal = {Analytical chemistry},
volume = {98},
number = {24},
pages = {18288-18298},
doi = {10.1021/acs.analchem.6c03088},
pmid = {42274413},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pneumonia, Mycoplasma/diagnosis ; *Mycoplasma pneumoniae/isolation &amp; purification/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Luminescent Measurements ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Luminescence ; Rapid Diagnostic Tests ; },
abstract = {Mycoplasma pneumoniae pneumonia (MPP) is a respiratory infection that readily propagates within pediatric populations, and thus, the development of a robust assay approach for its timely diagnosis is of clinical importance. In this contribution, we build a hierarchical amplification-interlinked CRISPR-Cas14a luminescent biosensor coupled with portable photonic crystal biochip-boosted time-delayed signaling to fulfill this need. To first perform highly sensitive detection, an initial DNA walker module and a later rolling circle amplification module are integrated to construct a hierarchical amplification, which is then utilized to interlink CRISPR-Cas14a systems. To further improve applicability in complicated biosamples, a time-delayed signaling actualized by afterglow luminescence is introduced to circumvent background autoluminescence from biological media. After employing a photonic crystal self-assembled from polystyrene nanospheres to manufacture a portable biochip, the naturally attenuated afterglow luminescence is dramatically boosted. Leveraging these strategies, our biosensing platform achieves a limit of detection as low as 2.27 fM for the M. pneumoniae sequence while maintaining good specificity. Moreover, the luminescent biosensor permits precise analysis of targets in throat swab samples from a pediatric cohort (n = 250) comprising severe and mild MPP patients as well as healthy controls, based on which a convolutional neural network-implemented deep learning model is finally established for accurate automated disease stratification, thereby holding great potential as an efficient and promising diagnostic tool.},
}
@article {pmid42299927,
year = {2026},
author = {Xia, Q and Guo, R and Xiao, J and Zeng, L and Huang, Y and Li, J and Chen, X and Huang, T and Xiao, B and Miao, C and Liu, W and Liang, QL and Lau, CH and Zhu, H},
title = {A rapid, multiplex, one-pot CRISPR/Dx system for visual detection of influenza A, influenza B, and respiratory syncytial viruses.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {24},
pages = {5052-5060},
doi = {10.1039/d6ay00425c},
pmid = {42299927},
issn = {1759-9679},
mesh = {*Influenza B virus/isolation &amp; purification/genetics ; *Influenza A virus/isolation &amp; purification/genetics ; Humans ; *CRISPR-Cas Systems ; *Respiratory Syncytial Viruses/isolation &amp; purification/genetics ; Rapid Diagnostic Tests ; Respiratory Syncytial Virus Infections/diagnosis/virology ; },
abstract = {The detection of Influenza A Virus (IAV), Influenza B Virus (IBV), and Respiratory Syncytial Virus (RSV) presents significant diagnostic challenges due to the high similarity of clinical symptoms with other respiratory infections, leading to the need for multiplexed, rapid testing. Herein, we have established a one-pot, multiplex CRISPR/Dx detection system based on reverse transcription-recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a. It completes the detection within 30 minutes at a constant temperature of 40 °C, with the ability to detect 10 copies per µL of IAV, 10 copies per µL of IBV, and 8 copies per µL of RSV. No cross-reactivity between these respiratory viruses was observed. When equipped with our customized miniature device, it allows visual fluorescence readout without specialized equipment. Compared with conventional RT-qPCR and two-tube RT-RPA-CRISPR/Cas12a approaches, this one-pot detection system offers a simplified workflow and shorter detection time and enables visual detection, making it especially suitable for point-of-care testing and field deployment. In essence, our CRISPR/Dx system provides a novel and practical molecular diagnostic strategy for rapid and multiplex detection of respiratory pathogens to improve patient management, rational antiviral use, and epidemic control.},
}
@article {pmid42325637,
year = {2026},
author = {Adegoke, SC and Karim, MA and Jr, MC and Yao Yawlui, IS and LaJeunesse, D},
title = {Advancements in Technologies Targeting Horizontal Gene Transfer(?)Routes to Control Drug Resistance Evolution.},
journal = {ACS bio &amp; med chem Au},
volume = {6},
number = {3},
pages = {210-236},
pmid = {42325637},
issn = {2694-2437},
abstract = {The global rise of multidrug-resistant (MDR) bacteria poses a major public health crisis, threatening the effectiveness of modern medicine. Traditional antibiotic development struggles to keep pace with bacterial evolution, largely due to the rapid dissemination of antibiotic resistance genes via horizontal gene transfer (HGT). HGT mechanisms both canonical and noncanonical enable bacteria to acquire resistance traits defining species and even special challenges. In this review, we cover the current understanding of HGT in spreading antibiotic resistance and explore possible strategies to control HGT and slow the spread of antimicrobial resistance. Recent advances highlight the potential of synthetic competence inhibitors, advanced oxidation processes (AOPs), CRISPR-Cas technologies, gene drives, and antiplasmids to disrupt horizontal gene flow and mitigate resistance evolution. Despite promising laboratory results, challenges remain in translating these approaches into clinical and environmental applications. Blocking HGT could complement antimicrobial stewardship programs and traditional antibiotic therapies by curbing the emergence of new resistant strains at their genetic roots. By targeting the foundational mechanisms of resistance acquisition, these strategies offer a proactive pathway to extend the efficacy of existing antibiotics and prevent a "postantibiotic" era. Ongoing research into bacterial pathogenesis, genome defense systems, and innovative gene-editing technologies will be critical to developing effective, scalable solutions for managing MDR infections worldwide.},
}
@article {pmid42326420,
year = {2026},
author = {Alzahrani, AJ},
title = {Bacteriophage therapy against multidrug resistant bacterial infections demonstrates clinical advances and engineering innovations between 2020-2026.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1865548},
pmid = {42326420},
issn = {1664-302X},
abstract = {PURPOSE: The global crisis of antimicrobial resistance has reached critical levels, with multi-drug resistant (MDR) bacterial pathogens threatening to render conventional antibiotics ineffective. This mini review synthesizes recent evidence from 2020 to 2026 on bacteriophage therapy against MDR bacteria, examining clinical applications, engineering advances, mechanistic insights, and emerging technologies.<br><br>METHODS: A comprehensive literature search was conducted across Embase, Scopus, and Cochrane Library databases, supplemented by PubMed, Google Scholar, and ArXiv searches, focusing on phage therapy for MDR bacterial infections published between 2020 and 2026.<br><br>RESULTS: Recent developments include expanded clinical experience through specialized phage centers, sophisticated genetic engineering techniques including CRISPR-based systems, successful compassionate-use programs, and innovative combination therapies with antibiotics. Clinical reports demonstrate safety and preliminary efficacy signals in selected refractory infections, though randomized controlled data remain limited. Engineering advances have produced phage-delivered CRISPR antimicrobials, hybrid delivery platforms, and synthetic phage particles that expand therapeutic capabilities.<br><br>CONCLUSION: While challenges remain in regulatory standardization, scalable manufacturing, and resistance management, the field has demonstrated significant progress toward clinical translation. The convergence of synthetic biology, personalized medicine approaches, and growing clinical evidence positions phage therapy as a viable complementary strategy in the fight against MDR bacterial infections.},
}
@article {pmid42328080,
year = {2026},
author = {Singh, AP and Haider, S and Sawarkar, A and Karki, S and Atta, K and Al-Zharani, M and Barasarathi, J and Rebouh, NY and Ahmed, ZFR},
title = {Programmable nanocarriers for precision plant engineering: converging nanotechnology, CRISPR, and next-generation breeding.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1845328},
pmid = {42328080},
issn = {1664-462X},
abstract = {The convergence of nanotechnology and genome editing in plant sciences is redefining modern precision breeding through efficient, transgene free, tissue culture independent pathways for genetic improvement in crops. Conventional breeding and transgenic tools are limited to genotype dependency, inefficient gene delivery, unpredictable transgene insertions, thereby restricting their application in elite germplasm. Nanoparticles-mediated gene delivery systems have revolutionized the genetic transformation in plants through targeted and transgene free delivery of CRISPR/Cas ribonucleoproteins (RNPs), DNA, and RNA into plant cells, while minimizing genome interference. Nanocarriers are the engineered delivery systems wherein the material component is a nanoparticle. DNA-free delivery refers to the absence of exogenous DNA during editing, whereas transgene free plants are those that do not retain integrated foreign DNA after regeneration. Firstly, this review summarizes current progress in designing nanocarriers, including lipid, polymeric, mesoporous silica nanoparticles, carbon-based nanoparticles, layered double hydroxides, and DNA-based nanoparticles; harnessing the function of their physicochemical traits in modulating plant cellular uptake, cargo stability, controlled delivery, and tissue specific targeting in plants. Secondly, the broad-spectrum roles of nano particles in genome editing, crop protection via RNA interference, organelle-targeted modifications are discussed, stressing transgene free approaches to mitigate somaclonal variation and regulatory concerns to foster public acceptance. The integration of nano-mediated delivery with speed breeding, meristem transformation, multiplexed editing in elite germplasm is proposed as an approach for prompt trait stacking and validation. Thirdly, the collaborative roles of experts in the field of nanotechnology, plant breeding, plant physiology, and agronomy are mentioned for mitigating multifaceted climatic effects and glitches. Moreover, current challenges including nanotoxicity, scalability and field translation, regulatory concerns, and public perception are also discussed. While nanocarrier mediated delivery shows strong potential for improving plant genome engineering, current evidence is largely confined to controlled experimental systems, and significant challenges remain before routine integration into breeding pipelines becomes feasible.},
}
@article {pmid42328450,
year = {2026},
author = {Corno, C and Costantino, M and Pettinari, P and Mirra, L and Stucchi, S and Arrighetti, N and Perta, N and Di Muccio, G and Robin, M and Beretta, GL and Corna, E and Carenini, N and Cleris, L and Colombo, D and Luison, E and Ciniselli, CM and Lecchi, M and Verderio, P and Figini, M and Linder, S and Tosi, D and La Teana, A and D'Arcy, P and Perego, P},
title = {USP18 Impacts Cisplatin Resistance in Ovarian Cancer Cells by Modulating DNA Repair.},
journal = {International journal of biological sciences},
volume = {22},
number = {11},
pages = {5780-5798},
pmid = {42328450},
issn = {1449-2288},
mesh = {Female ; *Cisplatin/therapeutic use/pharmacology ; Humans ; *Ovarian Neoplasms/metabolism/drug therapy/genetics ; *DNA Repair/genetics/drug effects ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Animals ; *Ubiquitin Thiolesterase/metabolism/genetics ; Mice ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use/pharmacology ; DNA Damage ; },
abstract = {Deubiquitinases (DUBs) are proteases with emerging roles in cancer, yet their contribution to drug resistance in ovarian cancer remains underexplored. Ovarian cancer patients often fail to benefit from platinum-based therapy, highlighting the need to identify novel factors driving drug resistance. Thus, we performed a CRISPR/Cas9 screen targeting the DUB family to identify genes essential for cisplatin-resistant ovarian carcinoma cell survival. CRISPR/Cas9 DUB knockout screens, preclinical pharmacology approaches, RNA sequencing, proteomic analyses, computational tools, surface plasma resonance were applied. We identified USP18 as a survival factor in cisplatin-resistant ovarian cancer cells. USP18 expression was elevated at the mRNA and protein levels across five cisplatin-resistant variants. Knockdown and CRISPR/Cas9 editing of USP18 sensitized cells to cisplatin, coinciding with impaired repair of cisplatin-induced DNA damage. Enhanced sensitivity to cisplatin was evident from studies in mice. RNA-seq of USP18 RNA interfered and edited cells revealed the modulation of pathways including DNA repair. A peptide-based USP18 inhibitor suppressed growth of cisplatin-resistant cells, supporting USP18 role in sustaining their growth. We identified USP18 as a novel mediator of cisplatin resistance in ovarian cancer, acting through DNA repair modulation. Targeting USP18 may offer a therapeutic strategy to improve outcomes in platinum-resistant ovarian cancer.},
}
@article {pmid42328522,
year = {2026},
author = {Wang, W and Zheng, S and Xiang, G and Feng, T and Peng, Y and Wu, Y and Du, X and Zhu, P and Ru, Y and Zhang, J and Wu, S and Han, H and Huang, T and Cao, G and Zheng, H and Zhao, Y and Wang, H and Zhang, R},
title = {Robust production of heavy-chain-only antibodies in mice by CRISPR/Cas mediated in situ modification of IgH locus.},
journal = {National science review},
volume = {13},
number = {11},
pages = {nwag270},
pmid = {42328522},
issn = {2053-714X},
}
@article {pmid42328613,
year = {2025},
author = {Selim, HMRM and Gomaa, FAM and Alshahrani, MY and Aboshanab, KM},
title = {Role of CRISPR-Cas system as a new approach in fighting the antimicrobial resistance of bacterial and viral pathogens.},
journal = {Infectious diseases &amp; immunity},
volume = {5},
number = {2},
pages = {127-137},
pmid = {42328613},
issn = {2693-8839},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Caspase (Cas) system acts as a natural defense of bacteria against invasion by mobile genetic elements, such as plasmids, transposons, and bacteriophages. The review discusses the different classes and types of CRISPR-Cas systems in terms of principles of their action, limitations, and future perspectives. Also, the role of the CRISPR-Cas system as a new arsenal in fighting multidrug-resistant pathogens and clinically relevant pathogenic viruses and up-to-date clinical trials have been discussed and highlighted. Moreover, the utilization mode, regulation, and the link of CRISPR-Cas to quorum sensing for targeting cell chromosome or antimicrobial-resistant gene(s) of some clinically relevant pathogens, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa have been discussed. The information provided in this review can act as a platform for researchers for the future use of the CRISPR-Cas system as a smart antimicrobial agent for combating infections caused by life-threatening pathogens, particularly those with limited therapeutic options.},
}
@article {pmid41061927,
year = {2026},
author = {Sun, H and Zou, J and Tu, S and Luo, D and Xiao, R and Du, Y and Xiong, C and Xie, S and Liu, H and Jin, M and Chen, H and Zhou, H},
title = {Genome-wide CRISPR screen identifies STK11 as a critical regulator of sialic acid clusters important for influenza A virus attachment.},
journal = {Journal of advanced research},
volume = {85},
number = {},
pages = {1207-1222},
doi = {10.1016/j.jare.2025.09.059},
pmid = {41061927},
issn = {2090-1224},
mesh = {Animals ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Virus Replication ; Swine ; *Influenza A virus/physiology ; CRISPR-Cas Systems ; *N-Acetylneuraminic Acid/metabolism ; *Virus Attachment ; AMP-Activated Protein Kinase Kinases ; Dogs ; Mice ; *Orthomyxoviridae Infections/virology/genetics ; Host-Pathogen Interactions ; Humans ; },
abstract = {INTRODUCTION: Swine influenza virus (SIV) is a highly contagious respiratory pathogen in pigs that causes substantial economic losses in the pig industry. Importantly, pigs act as "mixing vessels" for diverse influenza A viruses (IAVs), facilitating the emergence of novel pandemic strains through reassortment, which represents a continuous global public health threat. IAV replication relies heavily on host cellular machinery, underscoring the importance of elucidating virus-host protein interactions for the development of targeted antiviral therapeutics.<br><br>OBJECTIVES: This study aims to identify host genes required for SIV replication via a genome-wide CRISPR screen and elucidate the mechanism by which STK11 modulates viral replication.<br><br>METHODS: A pig genome-scale CRISPR knockout (PigGeCKO) screen was performed in newborn pig trachea (NPTr) cells to identify host genes required for SIV replication. Candidate genes were further validated by generating knockout cell lines using CRISPR/Cas9-mediated gene editing, followed by assessing their impact on IAV replication. The specific lifecycle stage regulated by STK11 and its mechanistic role in viral attachment were determined via Western blotting, confocal microscopy, transmission electron microscopy, and stimulated emission depletion (STED) imaging. In vivo validation of STK11 knockdown effects on IAV replication was conducted in BALB/c mice treated with STK11-targeting siRNA, with outcomes evaluated by survival analysis, body weight monitoring, lung viral titers quantification, immunofluorescence, and histopathology.<br><br>RESULTS: STK11 promotes replication of different IAV subtypes in vitro, and STK11 knockdown significantly suppresses SIV replication in vivo. Mechanistically, STK11 depletion impairs viral attachment by altering the organization of sialic acid clusters, mediated through reduced intracellular actin stress fibers via inhibition of RhoA signaling pathway.<br><br>CONCLUSION: We identify STK11 as a novel regulator of IAV attachment and elucidate its mechanistic role in facilitating viral entry. These findings highlight the potential of STK11 to serve as an ideal antiviral target against IAV infection.},
}
@article {pmid41692276,
year = {2026},
author = {Piseddu, I and Endres, R and Lanzl, F and Hammann, L and Bérouti, M and Thaler, M and Fahr, L and Fischer, H and Varlamova, V and Gärtig, J and Nixdorf, D and Layritz, P and Marx, C and Hörth, C and Witte, C and Bulut, A and Illig, D and Senz, AM and Holdt, L and Regel, I and Gottschlich, A and Subklewe, M and Mayerle, J and Anz, D and Kobold, S and Linder, A and Hornung, V},
title = {STING Ablation in T Cells Is Required for the Efficacy of STING Agonists in CAR-T Cell Immunotherapy of Pancreatic Cancer.},
journal = {Gastroenterology},
volume = {171},
number = {1},
pages = {50-65},
doi = {10.1053/j.gastro.2026.01.031},
pmid = {41692276},
issn = {1528-0012},
mesh = {Animals ; *Pancreatic Neoplasms/therapy/immunology/pathology/genetics/metabolism ; STING Protein ; *Membrane Proteins/agonists/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; Humans ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; Cell Line, Tumor ; cGAS-STING Signaling Pathway ; Mice ; Xenograft Model Antitumor Assays ; T-Cell Exhaustion ; Tumor Microenvironment/immunology ; Interferon-gamma/metabolism ; *T-Lymphocytes/immunology/metabolism/transplantation/drug effects ; Tumor Necrosis Factor-alpha/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Signal Transduction ; Female ; Immunologic Memory ; },
abstract = {BACKGROUND &amp; AIMS: Chimeric antigen receptor (CAR) T cells have shown great potential in hematological cancers, but lack efficacy in solid tumors, highlighting the need for novel strategies. Stimulator of interferon genes (STING) activation was shown to inflame the tumor microenvironment, but combination of STING agonists and CAR-T cells might be limited by detrimental outcomes of T cell-intrinsic STING activation. In this study, we evaluated the potential of combining STING agonists and CAR-T cells in the context of pancreatic cancer.<br><br>METHODS: We assessed the synergy of CRISPR-Cas9-edited CAR-T cells and the STING agonist diABZI within a T cell exhaustion model in vitro and both xenograft and syngeneic mouse models in vivo.<br><br>RESULTS: Combination of STING-ablated CAR-T cells and diABZI resulted in enhanced cancer cell killing, increased CAR-T cell proliferation, reduced exhaustion, and expansion of an effector-memory phenotype in vitro. Mechanistically, superior CAR-T cell functionality required genetic ablation of STING in CAR-T cells and was dependent on cancer cell-intrinsic STING signaling on STING-agonistic treatment. Moreover, we identified a synergistic feedback loop comprising the T cell-secreted cytokines interferon-&#x3b3; and tumor necrosis factor, which prime STING signaling within cancer cells, thereby potentiating the outcomes of cancer cell-intrinsic STING activation in inducing ameliorated CAR-T cell states. Ultimately, we could demonstrate that combination of STING deficient CAR-T cells and diABZI was able to provide enhanced tumor control in both xenograft and syngeneic mouse models. This was accompanied by increased intratumoral CAR-T cell numbers and reprogramming of the tumor microenvironment in vivo.<br><br>CONCLUSIONS: Our findings suggest that STING deficient CAR-T cells stand to benefit from STING agonists to improve CAR-T cell therapy for immune-deprived cancers such as pancreatic cancer.},
}
@article {pmid41886459,
year = {2026},
author = {Yang, M and Chen, G and Xiao, J and Zhang, X and Hu, Z and Zhou, B},
title = {Protocol for enhancing Cas9 efficiency and fidelity through structure-guided phosphate-locking loop engineering.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104452},
pmid = {41886459},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Phosphates/chemistry/metabolism ; DNA/genetics/chemistry/metabolism ; *Protein Engineering/methods ; },
abstract = {The phosphate-locking loop (PLL), stabilizing Cas9-DNA interactions, is a key target for optimizing efficiency and specificity. Here, we present a protocol for enhancing Cas9 efficiency and fidelity through structure-guided PLL engineering. We describe steps for identifying PLL engineering targets through sequence alignment and structural analysis, constructing variants via inverse PCR, evaluating efficiency using amplicon sequencing, and assessing specificity through Genome-wide Unbiased Identification of DSBs Evaluated by sequencing (GUIDE-seq (GUIDE-seq). This protocol provides a generalizable framework for Cas9 engineering across orthologs. For complete details on the use and execution of this protocol, please refer to Yang et al.[1].},
}
@article {pmid41903144,
year = {2026},
author = {Zhou, Z and Zhu, S and Pan, D and Lee, HY and Liu, N},
title = {Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104468},
pmid = {41903144},
issn = {2666-1667},
mesh = {Humans ; K562 Cells ; CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; Genome, Human/genetics ; },
abstract = {The composite transposon SINE-VNTR-Alu (SVA) is hominid-specific and composed of five parts, but the regulatory mechanism of SVA transcription is still unclear. Here, we present a protocol to identify SVA regulators using genome-wide screening in human K562 cells. We describe steps for constructing an SVA-GFP reporter and performing genome-wide CRISPR-Cas9 screening in human K562 cells to identify genes that control SVA transcription. This protocol provides a representative procedure of genome-wide screening for transposon regulators. For complete details on the use and execution of this protocol, please refer to Zhou et al.[1].},
}
@article {pmid41957359,
year = {2026},
author = {Wang, Y and Zhao, Y and Hu, J and Wang, Z and Pattarayan, D and Li, S and Zhang, Y and Wang, X and Wang, Y and Xie, W and Zhang, M and Yang, D},
title = {CRISPR activation screens identify oncogenic lncRNAs that are susceptible to CDK4/6 inhibitor treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41957359},
issn = {2041-1723},
support = {R01CA255196//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01CA222274//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA255196/CA/NCI NIH HHS/United States ; R01CA272866//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA282704/CA/NCI NIH HHS/United States ; R01CA282704//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists &amp; inhibitors/metabolism/genetics ; *Cyclin-Dependent Kinase 6/antagonists &amp; inhibitors/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; *Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; Piperazines/pharmacology/therapeutic use ; Cell Line, Tumor ; Pyridines/pharmacology/therapeutic use ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Proliferation/drug effects/genetics ; Animals ; Estrogen Receptor alpha/metabolism/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The roles of long non-coding RNAs (lncRNAs) in tumorigenesis and therapeutic response remain largely unknown. Here we perform genome-wide and focused CRISPR activation screens to identify lncRNAs regulating palbociclib response in breast cancer cells. A synchronized two-stage proliferation screen not only characterizes tumor growth-regulating lncRNAs, but also reveals a strong negative correlation between lncRNA-mediated regulation of tumor proliferation and CDK4/6 inhibitor sensitivity. By integrating CRISPRa screen results with drug response data from 815 cancer cell lines, we identify and functionally validate that TENM3-AS1, LINC01117, and ENSG00000226706 can increase breast cancer sensitivity to CDK4/6i while promoting tumor proliferation. In breast cancer patients, all three lncRNA signatures are associated with CDK4/6 inhibitor response. Mechanistically, we have shown that lncRNA TENM3-AS1 is a potential ERα-interacting lncRNA, and its regulation of CDK4/6 inhibitor sensitivity is dependent on ERα expression. Our integrated strategy characterizes oncogenic lncRNAs as potential therapeutic biomarkers for CDK4/6 inhibitor treatment in cancer.},
}
@article {pmid41986330,
year = {2026},
author = {Nguyen, QV and Lan, YJ and Chang, JC and Shih, HA and Faustine, J and Chen, CC and Ho, SY and Cheng, CW and Chao, TL and Lin, S},
title = {Genome-wide CRISPR screens in primary human natural killer cells identify countermeasures against immunosuppressive environment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41986330},
issn = {2041-1723},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *CRISPR-Cas Systems ; Dinoprostone/metabolism ; Tumor Microenvironment/immunology/genetics ; Interleukin-2/metabolism ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Serine-Threonine Kinases/genetics ; Signal Transduction ; },
abstract = {Natural killer (NK) cells are promising effectors for cancer immunotherapy, but their efficacy is limited by immunosuppressive tumor microenvironments. To uncover strategies for enhancing NK cell function, we establish a CRISPR loss-of-function screening platform for primary human NK cells by combining BaEVRless-pseudotyped lentiviral transduction of sgRNA libraries with Cas9 protein electroporation. This platform enables genome-scale interrogation of gene function in non-transformed NK cells. Kinome-focused and genome-wide screens identify key regulators of NK cell proliferation, cytotoxicity, and resistance to prostaglandin E2 (PGE2)-mediated suppression. STK17B deletion enhances NK cell expansion, while loss of CCDC53 boosts degranulation and cytotoxicity. We also uncover the CRL5 complex-including RNF7, UBE2F, and CISH-as critical inhibitors of IL-2 signaling and effector function under PGE2 stress. These findings establish a scalable platform for CRISPR-based functional genomics in primary NK cells and reveal engineering targets to enhance NK cell persistence and efficacy in tumor microenvironments.},
}
@article {pmid42012982,
year = {2026},
author = {Djamshidi, M and Tanida, R and Heshmatzad, K and Krowicki, H and Hill, A and Yang, Y and Riabowol, K},
title = {Protocol for enhancing CRISPR-Cas9 genome editing using histone deacetylase inhibition and engineered virus-like particle delivery.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104493},
pmid = {42012982},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Histone Deacetylase Inhibitors/pharmacology ; *Gene Editing/methods ; Humans ; *Virion/genetics ; },
abstract = {We present a 10-fold faster, accurate, and more efficient (FAME)-CRISPR-Cas9 gene editing workflow utilizing histone deacetylase inhibitor (HDACi)-mediated chromatin relaxation and engineered virus-like particle (eVLP) delivery of Cas9. We describe steps for optimizing HDACi concentration, euchromatinization timing, and Cas9 delivery/expression to improve CRISPR-Cas9 editing efficiency and efficacy. This protocol can eliminate the need for single-cell cloning and reduce experimental timelines up to 10-fold while minimizing HDACi-mediated toxicity. For complete details on the use and execution of this protocol, please refer to Djamshidi et al.[1].},
}
@article {pmid42025580,
year = {2026},
author = {Shukla, SK and Singh, A and Yadav, R and Kumar, A},
title = {Advances in molecular diagnostic strategies during the SARS-CoV-2 pandemic.},
journal = {Expert review of molecular diagnostics},
volume = {26},
number = {4},
pages = {293-308},
doi = {10.1080/14737159.2026.2665263},
pmid = {42025580},
issn = {1744-8352},
mesh = {Humans ; *COVID-19/diagnosis/epidemiology/virology ; *SARS-CoV-2/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; Pandemics ; COVID-19 Testing/methods ; COVID-19 Nucleic Acid Testing/methods ; Rapid Diagnostic Tests ; Pandemic Preparedness ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: The SARS-CoV-2 pandemic provided critical insights into pandemic preparedness. The community spread can be slowed down or contained through effective, rapid, and robust diagnosis of infected individuals.<br><br>AREA COVERED: During the pandemic, substantial advances were made in developing rapid and cost-effective diagnostic approaches. Self-collected gargle samples offer clear advantages over conventional NSP/OPS methods by reducing reliance on trained personnel and personal protective equipment. Colorimetric assays further improve accessibility, enabling rapid, instrument-free, and visually interpretable detection at low cost. CRISPR-based diagnostics present a promising alternative to RT-PCR, facilitating scalable mass screening with reduced technical dependence. Concurrently, optimization of RT-PCR workflows-particularly through minimization of pre-PCR steps-can enhance speed and affordability. The integration of digital technologies and artificial intelligence further leverages diagnostic capabilities. Despite this, improved regulatory frameworks and resilient supply chains are critical for ensuring scalable, equitable access, and effective pandemic preparedness.<br><br>EXPERT OPINION: Global efforts were made to develop sensitive, rapid, cost-effective, and noninvasive technologies to identify the pandemic virus; however, variations in sensitivity/specificity and limited sample size validation hampered their utility in routine diagnostics. The COVID-19 pandemic has ended, but global efforts are still needed to combat the early infection of subsequent waves or similar disease waves.},
}
@article {pmid42033729,
year = {2026},
author = {He, Z and Chen, S and Huang, A and Jiang, H},
title = {Protocol for the generation of DDT signaling reporter cell line for CRISPR screening.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104523},
pmid = {42033729},
issn = {2666-1667},
mesh = {*Signal Transduction/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Cells respond to perturbations through signaling pathways that often induce characteristic transcriptional changes. Here, we present a protocol for generating a double death trap (DDT) reporter that converts pathway activity into a binary survival-death outcome. The DDT reporter employs puromycin resistance and FKBP12(F36V)-ΔCaspase9 constructs driven by pathway-specific response elements. We describe the steps for DDT reporter plasmid construction, cell line generation, and genome-wide CRISPR screening in DDT cells. We further detail procedures for next-generation sequencing (NGS) sample preparation, sequencing, and downstream analysis. For complete details on the use and execution of this protocol, please refer to He et al.[1].},
}
@article {pmid42033731,
year = {2026},
author = {Sarmah, H and Iannello, G and Wantroba, R and Wu, C and Idiarte, J and Munoz, A and Déry, O and Rubio de la Torre, E and Lebayle, E and Seminara, S and Sirabella, D and Huynh, S and Issa, R and Kissner, M and Corneo, B},
title = {Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104518},
pmid = {42033731},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Microfluidics/methods ; Electroporation ; *Cell Separation/methods ; },
abstract = {Genetically engineered human induced pluripotent stem cells (hiPSCs) are vital for disease modeling and drug discovery, yet generating clonal lines efficiently post-editing remains challenging. Here, we present a protocol to generate clonal hiPSC lines after gene editing using either electrostatic droplet- or microfluidics-based sorting platforms. We describe steps for culturing hiPSCs, CRISPR-RNP electroporation, single-cell sorting, and expansion of gene-edited clones. Using this protocol, we generated over 100 clonal lines across seven knock-in/knock-out experiments, demonstrating broad utility and reproducibility. For additional details on the use and execution of this protocol, please refer to Patel et al.[1].},
}
@article {pmid42044055,
year = {2026},
author = {Li, X and Gao, X and Dong, J and Gu, T and Li, Q and Wang, L and Deng, F and Hou, J and Hou, C and Huo, D},
title = {Aptamer Nanoswitch-Mediated Lock-Expose Mechanism Enables Highly Sensitive In Vitro Detection and Precise In Situ Membrane Imaging of HER2 Protein.},
journal = {ACS sensors},
volume = {11},
number = {5},
pages = {4014-4023},
doi = {10.1021/acssensors.6c00371},
pmid = {42044055},
issn = {2379-3694},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/metabolism/analysis/genetics ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; Cell Line, Tumor ; Limit of Detection ; *Biosensing Techniques/methods ; Neoplastic Cells, Circulating ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Breast Neoplasms ; },
abstract = {Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer correlates with high aggressiveness, an increased recurrence rate, and poor survival, holding significant diagnostic value. In this work, a HER2-specific aptamer (Apt) was engineered into a Apt nanoswitch (hApt) and revealed the interaction of HER2-aptamers through molecular docking and quantified the binding energy and dynamic behavior through molecular dynamics simulations. A lock-expose mechanism was designed by combining the target-induced conformational switch of hApt with template-prefabricated rolling circle amplification (rRCA), forming a T-hApt-rRCA sandwich structure. Coupled with CRISPR/Cas12a and fluorescent probes, this sensor enabled highly sensitive detection, with a linear range of 10 fg/mL to 10 ng/mL and a limit of detection of 1.42 fg/mL. Using HUVEC, A549, MCF-7, and SK-BR-3 cell lines to model HER2 heterogeneity in circulating tumor cells (CTCs) enabled in situ imaging, differentiation, and quantitative detection of membrane HER2 expression, thereby providing direct visualization of expression levels and highlighting the translational promise of this approach. The accurate detection and clear differentiation between the eight healthy samples and the twenty-eight breast cancer patient samples further underscore the practical applicability of this sensing strategy.},
}
@article {pmid42066814,
year = {2026},
author = {Nie, H and Zheng, Z and Fan, W and Yan, M and Shao, W and Meng, Y and An, D and Zhao, S and Yuan, L and Yang, J and Wang, H},
title = {Homozygous IbGBSS1 knockouts in hexaploid sweet potato enable amylose-free starch without a yield trade-off.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {369},
number = {},
pages = {113172},
doi = {10.1016/j.plantsci.2026.113172},
pmid = {42066814},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/metabolism/growth &amp; development ; *Starch Synthase/genetics/metabolism ; *Amylose/metabolism ; *Starch/metabolism ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Homozygote ; Polyploidy ; CRISPR-Cas Systems ; },
abstract = {Sweet potato (Ipomoea batatas) is a major crop and an important industrial starch source; however, its hexaploid genome has hindered the generation of complete gene knockouts. Because the amylose-to-amylopectin ratio determines starch functionality, the production of amylose-free (waxy) starch is of considerable interest for food, pharmaceutical, and industrial applications. It was hypothesized that the complete knockout of all six alleles of IbGBSS1, which encodes granule-bound starch synthase I, would abolish amylose biosynthesis without compromising plant growth or yield. CRISPR/Cas9 mutagenesis combined with the Hi-TOM high-throughput mutation detection platform was used to generate homozygous Ibgbss1 mutants with confirmed edits across all alleles. These mutants contained < 1% amylose and exhibited normal growth and unchanged yield under both greenhouse and field conditions. Physicochemical analyses showed that amylose-free starch displayed larger granules, an altered amylopectin chain-length distribution (reduced DP 6-12 and enriched > DP 36), and numerous surface pores. Differential scanning calorimetry indicated increased gelatinization onset and peak temperatures, along with higher gelatinization enthalpy. Transcriptome analysis revealed broad reprogramming of starch and sucrose metabolism, accompanied by increased accumulation of glucose, fructose, and sucrose in storage roots. These results demonstrate that IbGBSS1 is essential for amylose biosynthesis and establish a strategy for generating complete multi-allelic knockouts in hexaploid sweet potato. Amylose-free germplasm was obtained without a yield penalty, providing potential for food and industrial applications.},
}
@article {pmid42080968,
year = {2026},
author = {Yang, Y and Arro, J and Zou, C and Oravec, M and Reisch, B and Zhong, GY},
title = {First brassinosteroid-based dwarf mutant discovered and characterized in grapevine.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42080968},
issn = {1432-2242},
mesh = {*Vitis/genetics/growth &amp; development ; *Brassinosteroids/biosynthesis ; Phenotype ; Chromosome Mapping ; *Cytochrome P-450 Enzyme System/genetics ; Mutation ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; CRISPR-Cas Systems ; Genetic Association Studies ; },
abstract = {In this study, we investigated the genetic control of dwarfism in naturally occurring dwarf mutant lines of grapevines. Through trait-segregation and marker-trait association analyses, we identified a major locus on Chromosome 14 tightly associated with the dwarf trait. Subsequently, we conducted a bulked RNA-seq analysis, fine mapped the dwarf trait and identified VviBR6OX1, a cytochrome P450 enzyme involved in brassinosteroid synthesis, as a candidate gene for the observed dwarfism. RNA-seq sequence analyses revealed two in-frame deletions in the gene: a 12-bp deletion in exon 1 and a 9-bp deletion in exon 4. A survey of the two indels in Vitis germplasm suggested that the 9-bp deletion is most likely the cause of dwarfism in the mutant. We recreated similar dwarf grapevines by knocking out VviBR6OX1 using CRISPR/Cas9 gene editing and confirmed VviBR6OX1's role in controlling vine architecture. Additionally, we observed several vines with an extreme compact dwarf phenotype and determined that the compact dwarfing phenotype was a result of simultaneous editing of a second BR6OX gene, VviBR6OX2. The discovery of BR-related dwarfism in grapevine provides an important genetic avenue for developing desirable vine architecture for various breeding purposes.},
}
@article {pmid42084357,
year = {2026},
author = {Stone, S and Elsharkawy, A and Patterson, LD and Natekar, JP and Jiang, H and Viktoria Hyddmark, E and Camarillo, J and Zhao, G and Kumar, M},
title = {A Novel Humanized Lethal Mouse Model of SARS-CoV-2-Associated Disease.},
journal = {Journal of medical virology},
volume = {98},
number = {5},
pages = {e70959},
doi = {10.1002/jmv.70959},
pmid = {42084357},
issn = {1096-9071},
mesh = {Animals ; *Disease Models, Animal ; Humans ; *COVID-19/pathology/virology ; Mice ; *SARS-CoV-2/pathogenicity ; *Angiotensin-Converting Enzyme 2/genetics/metabolism ; Mice, Inbred C57BL ; Lung/pathology/virology ; Mice, Transgenic ; Female ; Gene Knock-In Techniques ; Brain/pathology/virology ; Viral Load ; CRISPR-Cas Systems ; Cytokines ; },
abstract = {Mice are valuable small animal models for studying SARS-CoV-2 pathogenesis. Ancestral SARS-CoV-2 strains do not efficiently utilize murine Ace2, rendering wild-type mice resistant to infection. Although human ACE2 transgenic models such as K18-hACE2 have provided critical insights, they express multiple copies of both murine and human ACE2, and random transgene insertion can result in non-physiological receptor expression. To overcome these limitations, we employed a human ACE2 knock-in (hACE2-KI) model in which the murine Ace2 coding sequence is replaced with human ACE2 using CRISPR/Cas9 technology, generating an mAce2-null background. This design allows human ACE2 expression under endogenous regulatory control while eliminating murine Ace2 expression, thereby providing a more physiologically relevant platform to investigate SARS-CoV-2 pathogenesis and evaluate therapeutic and preventive strategies. In this study, SARS-CoV-2-associated disease was evaluated and compared among hACE2-KI, K18-hACE2 and C57BL/6J mice. Mice were intranasally inoculated with 10[5] plaque-forming units of SARS-CoV-2 lineages B.1 or B.1.351. Both hACE2-KI and K18-hACE2 mice developed severe disease after SARS-CoV-2 infection. Following infection with B.1, both K18-hACE2 mice and hACE2-KI mice exhibited significant weight loss and mortality, with high viral loads detected in the lungs and brain. hACE2-KI mice infected with SARS-CoV-2 B.1.351 also showed significant weight loss and viral loads, resulting in high mortality. The pathology and inflammatory response within the lungs and brain of infected hACE2-KI mice revealed robust expression of viral nucleocapsid protein, histopathological changes, and upregulated cytokine and chemokine responses. Together, these findings demonstrate that the hACE2-KI knock-in mouse model supports robust SARS-CoV-2 replication and mimics severe COVID-19 disease.},
}
@article {pmid42096138,
year = {2026},
author = {Zhang, Q and Li, Y and Li, Y and Han, L and Yan, J and Zhan, M and Liu, T and Ke, P and Wang, Q and Huang, X},
title = {Anti-CRISPR Protein Regulates CRISPR/Cas12a Fusogenic-Nanovesicle-Based Platform for Extracellular Vesicle-Encapsulated Non-Nucleic Acid Target In-Vesicle Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {26},
pages = {e6795217},
doi = {10.1002/anie.6795217},
pmid = {42096138},
issn = {1521-3773},
support = {2024ZD0533400//National Key Research and Development Program of China/ ; 2024ZD0533401//National Key Research and Development Program of China/ ; 0720240230//Guangdong Special Support Program Health and health talents List Project of Provincial Health Commission/ ; 2024A1515030043//Natural Science Foundation of Guangdong Province/ ; 2026A1515010500//Natural Science Foundation of Guangdong Province/ ; SZ2022QN09//"Young Talents Program" of Guangdong Academy of Traditional Chinese Medicine/ ; SKLKY2024B0011//State Key Laboratory of Traditional Chinese Medicine Syndrome Projects/ ; 2023A03J0755//Guangzhou Science and Technology Plan Projects/ ; 2023B110008//Guangdong Provincial Clinical Research Guangdong Provincial Clinical Research Center for Laboratory Medicine/ ; 2025JY-A1004//Guangdong Provincial Clinical Research Guangdong Provincial Clinical Research Center for Laboratory Medicine/ ; //Science and Technology Innovation Center of Guangzhou University of Chinese Medicine/ ; },
mesh = {*Extracellular Vesicles/chemistry/metabolism ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Aptamers, Nucleotide/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {The detection of non-nucleic acid targets encapsulated in extracellular vesicles (EVs) faces two major challenges: (1) difficulties in efficient isolation and the risk of content degradation, and (2) the low abundance of target molecules encapsulated in EVs always leads to failed signal transduction and inadequate output signal intensity. To overcome these limitations, we propose a high-efficiency in-vesicle analysis strategy that integrates targeting probe delivery and regulation by protein signal amplification. By applying aptamer-mediated membrane fusion and "locked-activated" CRISPR-Cas12a-AcrVA1 (LACA) for protein signal regulation, we fabricated a yly12-aptamer-functionalized self-assembled nanovesicle which encapsulate LACA-system (yly12-lipo@Cas12a nanovesicle) as an in-vesicle bioanalytical platform. Leveraging the high specificity of the aptamer and the regulatory function of AcrVA1 in selectively modulating Cas12a activity, the platform enables highly specifiec and sensitive detection, offering advantages of simple operation and versatility across platforms within only 2.5 h. Clinical analysis demonstrated effective differentiation between patients and healthy controls, yielding high diagnostic performance with an AUC of 0.965. The proposed platform shows great potential for EV-carrying protein biomarker analysis and has broad prospects for the disease's diagnosis in clinical settings.},
}
@article {pmid42017197,
year = {2026},
author = {Wafer, R and Tandon, P and Minchin, J},
title = {A quantitative in vivo CRISPR-imaging platform identifies regulators of hyperplastic and hypertrophic adipose morphology in zebrafish.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42017197},
issn = {2050-084X},
support = {BB/X009467/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; REA PhD Studentship/BHF_/British Heart Foundation/United Kingdom ; },
mesh = {Animals ; *Zebrafish/genetics ; *Adipose Tissue/metabolism/pathology ; Hyperplasia/genetics ; Humans ; Hypertrophy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Zebrafish Proteins/genetics/metabolism ; Adipocytes ; CRISPR-Cas Systems ; },
abstract = {Adipose tissues exhibit a remarkable capacity to expand, regress, and remodel in response to energy status. The cellular mechanisms underlying adipose remodelling are central to metabolic health. Hypertrophic remodelling - characterised by the enlargement of existing adipocytes - is associated with insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, hyperplastic remodelling - in which new adipocytes are generated - is linked to improved metabolic outcomes. Despite its clinical importance, the regulation of hypertrophic and hyperplastic adipose morphology remains poorly understood. Here, we integrate human transcriptomic data with a quantitative CRISPR-imaging platform in zebrafish to identify regulators of adipose morphology. We developed an image-based phenotyping pipeline that captures lipid droplet size, number, and spatial patterning, and applied generalised additive modelling to quantify hyperplastic versus hypertrophic morphology signatures. Using this platform, we conducted an F0 CRISPR screen targeting 25 candidate genes and identified three that induced hypertrophic morphology (txnipa, mmp14b, and foxp1b) and an additional candidate that altered total adiposity (kazna). For functional validation, we generated stable loss-of-function alleles for both zebrafish foxp1 paralogues. Spatial analysis along the anterior-posterior axis revealed that foxp1b mutants display developmental hypertrophy but profoundly blunted adaptive responses to high-fat diet (~68% reduction across all spatial zones), while foxp1a mutants show normal baseline morphology but disrupted spatial patterning of diet-induced hypertrophy. Together, these findings establish a scalable CRISPR-imaging platform for in vivo genetic screening of adipose morphology and reveal distinct roles for Foxp1 paralogues in developmental patterning and adaptive responses to dietary challenge in adipose tissue.},
}
@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {Animals ; *Chromatin/metabolism/genetics ; *Gene Editing/methods ; Mice ; Cell Differentiation ; *Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells/metabolism/cytology ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}
@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}
@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/analysis/isolation &amp; purification/blood ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; DNA/chemistry/genetics ; Nucleic Acid Hybridization ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; Inverted Repeat Sequences ; Transcription, Genetic ; Catalysis ; Bacterial Proteins ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}
@article {pmid41747626,
year = {2026},
author = {Yoo, DH and Bayarsaikhan, D and Lee, J and Im, YS and Bayarsaikhan, G and Kang, HA and Lee, B and Kim, YO},
title = {Generation and characterization of SOX17-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103943},
doi = {10.1016/j.scr.2026.103943},
pmid = {41747626},
issn = {1876-7753},
mesh = {Humans ; *SOXF Transcription Factors/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Endoderm/cytology/metabolism ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) reporter line in which EGFP was inserted in-frame at the C-terminus of the endogenous SOX17 locus using CRISPR/Cas9-mediated homologous recombination. The targeted clone, KSCBi017-A-4, was isolated by puromycin selection and validated by PCR and Sanger sequencing. This SOX17-EGFP hiPSC line retains a normal karyotype and pluripotency and displays specific EGFP expression upon directed definitive endoderm differentiation. This reporter line provides a reliable tool for monitoring SOX17 expression during human endoderm specification.},
}
@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Water/chemistry ; *Gravitation ; *Waxes/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Microspheres ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}
@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Endopeptidases/genetics/pharmacology/metabolism ; *Probiotics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Saccharomyces boulardii/genetics/metabolism ; *Listeria monocytogenes/drug effects ; Bacteriophages/genetics/enzymology ; Listeriosis/prevention &amp; control/microbiology ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}
@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Candida/genetics/pathogenicity ; Humans ; Candidiasis/microbiology ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}
@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}
@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; *Disease Models, Animal ; Female ; Sheep/genetics ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.<br><br>METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.<br><br>RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}
@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; *Plant Breeding/methods ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Crops, Agricultural/genetics ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}
@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; *Cell Proliferation/genetics ; HEK293 Cells ; Apoptosis/genetics ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}
@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Bombyx/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Insect ; *Agriculture/methods ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}
@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}
@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*Hordeum/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Ascomycota/pathogenicity ; *Plant Proteins/genetics ; *Plant Diseases/microbiology/genetics ; Alleles ; *Microbiota/genetics ; Mutation ; Plant Roots/microbiology/genetics ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}
@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/genetics/therapy ; *Brain Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}
@article {pmid41752921,
year = {2026},
author = {Panov, J and Elbert, A and Rosenthal, DS and Levi, M and Chumakov, K and Andino, R and Brodsky, L and Kaphzan, H},
title = {Spacio-Linear Screening for Ligand-Docking Cavities in Protein Structures: SLAM Algorithm.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {2},
pages = {},
pmid = {41752921},
issn = {2075-1729},
support = {R01 DK127830/DK/NIDDK NIH HHS/United States ; N/A//Tauber Foundation/ ; },
abstract = {Identifying structurally similar ligand-binding sites in unrelated proteins can facilitate drug repurposing, reveal off-target effects, and deepen our understanding of protein function. A number of tools were developed for structural screening, but many of them suffer from limited sensitivity and scalability. Using a data bank of crystallized protein structures, we aimed to discover novel protein targets for a ligand by leveraging a known ligand-binding query protein with a resolved structure. Here, we present SLAM (Spacio-Linear Alignment of Macromolecules), a novel alignment-based algorithm that detects local 3D similarities between ligand-binding cavities or protein-exposed surfaces of query and target proteins. SLAM encodes spatial substructure neighborhoods into short linear sequences of physicochemically annotated atoms, then applies pairwise sequence alignment combined with distance-correlation scoring to identify high-fidelity structural matches. Benchmarking using the Kahraman-36 dataset demonstrated that SLAM outperforms the state-of-the-art ProBiS algorithm in true-positive rate for predicting ligand-docking compatibility. Furthermore, SLAM identifies candidate ligands that may inhibit functionally critical domains of CRISPR-Cas proteins and predicts novel binding partners of toxic per- and polyfluoroalkyl Substance (PFAS) compounds (PFOA, PFOS) with plausible mechanistic links to toxicity. In conclusion, SLAM is a robust computationally efficient and flexible structural screening tool capable of detecting subtle physicochemical compatibilities between protein surfaces, promising to accelerate target discovery in pharmacology and elucidate protein-ligand interactions in environmental toxicology.},
}
@article {pmid41753780,
year = {2026},
author = {Palanisamy, V and Bosilevac, JM and Barkhouse, DA and Velez, SE and Dass, SC},
title = {Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants.},
journal = {Microorganisms},
volume = {14},
number = {2},
pages = {},
pmid = {41753780},
issn = {2076-2607},
support = {2020-67017-30776//USDA-NIFA/ ; },
abstract = {Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage-bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction-modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria-phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms.},
}
@article {pmid41754535,
year = {2026},
author = {Li, H and Wang, R and Li, J and Duan, W and Liang, Y and Sun, Q and Zhou, J and Zhang, Y},
title = {SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754535},
issn = {1999-4915},
support = {GJJKJ-2024-ZY//National Disease Control and Prevention Administration Public Health Talent Training Support Project/ ; ZDGWNLJS25-36//National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID)/ ; 2021YFC2302003//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Virus Replication ; Mice ; Humans ; *Coxsackievirus Infections/virology/immunology ; Viral Load ; *Host-Pathogen Interactions ; *Enterovirus/physiology/genetics ; Cell Line, Tumor ; },
abstract = {Coxsackievirus A16 (CVA16), a major etiological agent of hand, foot, and mouth disease, is increasingly contributing to neurological complications, with no vaccines or virus-specific antivirals currently available. To identify CVA16-restricting host factors, we investigated the role of the interferon-stimulated gene shiftless (SHFL), previously implicated in the control of other RNA viruses. Using CRISPR-Cas 9, we generated SHFL knockout rhabdomyosarcoma cells and assessed viral replication, cytopathic effects, and replication stage dynamics. We evaluated disease progression and tissue injury in neonatal mice infected with a mouse-adapted CVA16 strain. SHFL expression was strongly induced during CVA16 infection and was inducible by exogenous interferon-β treatment, and its loss markedly increased infectious virus production, accelerated early replication, and exerted severe cytopathic effects. In vivo, SHFL deficiency led to rapid weight loss, pronounced neurological signs, increased viral burden across multiple tissues, and uniform mortality, together with high viral loads and extensive pathological damage in the central nervous system, lungs, and skeletal muscle. Transcriptomic analyses revealed SHFL-dependent modulation of adhesion- and mitogen-activated protein kinase-related pathways. Overall, our results suggest SHFL as a key determinant of host resistance to CVA16, acting mainly at the post-transcriptional stage to limit viral spread and tissue injury, and highlight SHFL-linked pathways as promising host-directed antiviral targets.},
}
@article {pmid41754561,
year = {2026},
author = {Wupori, K and Garnett, L and Bello, A and Strong, JE},
title = {CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754561},
issn = {1999-4915},
support = {8//Genomics Research and Development Initiative/ ; CSSP-2022-CP-2546//Canadian Safety and Security Program/ ; },
mesh = {Humans ; *Hemorrhagic Fevers, Viral/diagnosis/virology ; *Point-of-Care Systems ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ebolavirus/genetics/isolation &amp; purification ; Point-of-Care Testing ; },
abstract = {Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.},
}
@article {pmid41755633,
year = {2026},
author = {Aguilar-González, A and Martos-Jamai, I and Ramos-Hernández, I and Molina-Estévez, FJ and Villao, NV and Puig-Serra, P and Rodríguez-Perales, S and Torres, R and Labun, K and Sánchez-Martín, RM and Díaz-Mochón, JJ and Martín, F},
title = {A novel Dual-guide CRISPR-Cas13 strategy improves specificity for single-nucleotide variant detection.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755633},
issn = {1362-4962},
support = {MCIN)/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation/ ; PID2022-141065OB-I00//European Union Next Generation/ ; CV20-77741//Consejer&#xed;a de Econom&#xed;a y Conocimiento/Project/ ; PI21/00298//Instituto de Salud Carlos III/ ; PI24/00888//Instituto de Salud Carlos III/ ; RD21/0017/0004//Instituto de Salud Carlos III/ ; RD24/0014/0005//Instituto de Salud Carlos III/ ; PI-0236-2024//Consejer&#xed;a de Salud y Familias/ ; PIP-0004-2025//Consejer&#xed;a de Salud y Familias/ ; GeneHumdi-CA21113//European Cooperation in Science and Technology/ ; FPU22/03455//Spanish Ministry of Science, Innovation and Universities/ ; RHJ-0053-2025//Consejer&#xed;a de Salud y Familias, Junta de Andaluc&#xed;a/ ; //European Social Fund/ ; //Universidad de Granada/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *SARS-CoV-2/genetics ; *Polymorphism, Single Nucleotide/genetics ; *COVID-19/diagnosis/virology/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; Sensitivity and Specificity ; RNA, Viral/genetics ; },
abstract = {The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13's heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs-dcrRNA and dtracrRNA-which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform's potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.},
}
@article {pmid41755634,
year = {2026},
author = {Hu, Z and Liu, Y and Han, Y and Li, M and Deng, K and Lu, X and Huang, Y and Liang, C and Wang, Y and Fu, Y and Xu, A},
title = {CRISPR/Cas9 screening with destabilized bicistronic fluorescent protein reporter revealed PABPN1 as a hub of regulators for alternative polyadenylation.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755634},
issn = {1362-4962},
support = {2022YFA1103900//National Key Research and Development Program of China/ ; 32470586//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 31930084//National Natural Science Foundation of China/ ; 32500472//National Natural Science Foundation of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; },
mesh = {*Polyadenylation ; Genes, Reporter ; Humans ; *CRISPR-Cas Systems ; *Poly(A)-Binding Protein I/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; DEAD-box RNA Helicases/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Cell Proliferation ; HEK293 Cells ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; },
abstract = {Alternative polyadenylation (APA) is intricately intertwined with diverse biological processes. Efficient approaches for screening the regulatory factors of specific APA events are essential to elucidate their regulation mechanisms. Here, we first engineered a destabilized bicistronic fluorescent protein reporter (dBFPR) to enhance the sensitivity of APA detection. Then, we developed a robust high-throughput screening platform for APA regulators by integrating CRISPR/Cas9, dBFPR, and fluorescence-activated cell sorting. With this method, we successfully screened the library of RNA binding proteins and found that PTBP1, ELAVL1, and DDX3X play significant roles in regulating APA and promoting cell proliferation through interaction with PABPN1, suggesting that PABPN1 is an important hub for APA regulation.},
}
@article {pmid41755886,
year = {2026},
author = {Kaniganti, S and Saini, H and Chaitanya, AK and Hegde, N and Shah, P and Magar, ND and Rijal, R and Kaushik, JJ and Nanda, D and Sachan, S and Kumar, A and Bhoite, R and Jamedar, HR},
title = {CRISPR/Cas Genome Editing and Its Applications in Cereal Crop Improvement.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {2},
pages = {e70133},
pmid = {41755886},
issn = {2575-6265},
abstract = {CRISPR/Cas-based genome editing has emerged as a transformative tool for precise genetic improvement of cereal crops. Recent advances in CRISPR technologies, including Cas9, Cas12, Cas13, base editing, and prime editing, have enabled targeted modification of genes and regulatory elements controlling yield, stress tolerance, and grain nutritional quality in major cereals such as rice, wheat, maize, and barley. This review summarizes current progress in CRISPR-mediated genome editing systems, delivery strategies, and representative applications in cereal crop improvement. Emphasis is placed on how genome editing reprograms enzymatic activities and biological pathways underlying complex agronomic traits rather than acting through single-gene effects. The review also discusses challenges related to trait complexity, regulatory considerations, and prospects for translating genome-edited cereal crops from laboratory research to field-level application. Collectively, this review highlights the potential of CRISPR/Cas genome editing as a powerful approach for developing high-yielding, resilient, and nutritionally improved cereal crops.},
}
@article {pmid41757335,
year = {2026},
author = {Shafiq, T and Khan, N and Kausar, T and Ahmed, W and Zhang, Z and Liang, Y and Duan, L},
title = {Red Blood Cell-Derived Extracellular Vesicles for Gene and RNA Therapeutics: Biological, Engineering, and Translational Challenges.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {579975},
pmid = {41757335},
issn = {1178-2013},
mesh = {Humans ; *Erythrocytes/chemistry/cytology ; Animals ; *Genetic Therapy/methods ; *Extracellular Vesicles/chemistry ; *RNA/genetics ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene therapy has great prospects of DNA/RNA manipulations and protein modulations. Its use in clinic is, however, stifled by risks of immunogenicity, low target specificity, and adverse effects. The red blood cell (RBC-EVs) extracellular vesicles can serve as a solution to this issue since they are biocompatible, long-term stable, and with low immunogenicity. RBC-EVs permit the accurate delivery of therapeutic cargo to space and time, thus minimizing systemic toxicity. This review presents the most recent developments on the expansion of the use of RBC-EVs to encapsulate the components of mRNA and CRISPR-Cas. Through the addition of the means to address these deficiencies, including stimulus-sensitive release mechanisms (eg, pH- or light-activated systems) and tissue-selective targeting approaches, RBC-EVs can be applied to enable the precise application in genetic diseases, inflammatory diseases, and cancer. Such innovations have the potential to overcome the clinical need and enable the biological complexity of mRNA- and CRISPR-Cas-based agents to provide a powerful delivery platform. Moreover, the review also demonstrates the unprecedented benefits of red blood cell EVs, which include immune evasion, scalability, and universal loading capacity, which can establish them as the next-generation delivery vehicles. Red blood cell EVs have the potential to increase the efficacy of precision medicine by increasing its feasibility. Lastly, we note the potential and translational issues in the provision of red blood cell EV-based mRNA and CRISPR-Cas therapeutic delivery of gene therapy.},
}
@article {pmid41757451,
year = {2026},
author = {Molina-Márquez, A and Kelterborn, S and Hegemann, P and Pérez-Rodríguez, M and Vigara, J and León, R},
title = {Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70811},
pmid = {41757451},
issn = {1399-3054},
support = {2019-110438RB-C22//Agencia Estatal de Investigaci&#xf3;n-MCIN/AEI/10.13039/501100011033/ ; PID2022-140995OB-C21//Agencia Estatal de Investigaci&#xf3;n-MCIN/AEI/10.13039/501100011033/ ; 426566805//German Research Foundation (DFG)/ ; //Hertie Foundation/ ; },
mesh = {*Carotenoids/metabolism ; *Oxidoreductases/genetics/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; *Microalgae/genetics/metabolism ; Gene Knockout Techniques ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.},
}
@article {pmid41758321,
year = {2026},
author = {Pathak, A and Singh, J and Swati, and Dwibedi, V},
title = {Deciphering microbial biofilm: mechanism, infection, and advanced approaches for control.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41758321},
issn = {1874-9356},
abstract = {Microbial biofilms are densely organised microbial communities that adhere to biotic and abiotic surfaces, encased within an extracellular polymeric substance (EPS). Microorganisms within these biofilm structures gain enhanced protection, versatility, and resistance to external stresses, antibiotics, and host immune systems. The biofilm formation follows a series of steps, including initial microbial adherence, microcolony establishment, EPS production, regulation by quorum sensing (QS), and dispersal. This flexibility enables biofilm survival in multiple environments, such as medical devices and natural systems, posing serious challenges in healthcare, agricultural, and industrial sectors. The review focuses on the mechanisms involved in biofilm formation and discusses the role of EPS in promoting biofilm stability and resistance to antimicrobials. It addresses biofilm-associated infections in medical environments, such as chronic wounds, cystic fibrosis, urinary tract infections (UTIs), and complications with implanted medical devices. The capacity of biofilm-forming microorganisms to evade immune responses and persist through extended antibiotic use highlights the urgent demand for novel therapeutic approaches. The discussion includes emerging strategies for biofilm control, including anti-biofilm agents, QS inhibitors, enzymatic treatments, and innovative combination therapies combining antibiotics with biofilm-disrupting agents. Emerging technologies, like antimicrobial peptides (AMPs), CRISPR-Cas systems, nanotechnology, and bioelectric therapies, present innovative biofilm disruption and removal approaches. This paper discusses the effectiveness of natural products, plant-derived compounds, and bacteriophage therapies for mitigating biofilm-associated infections linked to biofilms. The review examines the dynamic challenges posed by biofilms, particularly their role in chronic and device-related infections, which contribute to significant healthcare complications. The study highlights the significance of adopting new approaches to overcome biofilm-induced antimicrobial resistance (AMR) and improve therapeutic outcomes. Furthermore, this paper discusses the promising potential of emerging technologies, such as nanomaterials, QS interference, and biofilm-specific antimicrobial agents, in enhancing biofilm control and prevention measures across clinical, industrial, and environmental domains.},
}
@article {pmid41758644,
year = {2026},
author = {Adami, A and Garza, R and Dorazehi, F and Douse, CH and Jakobsson, J},
title = {Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104398},
pmid = {41758644},
issn = {2666-1667},
mesh = {Humans ; *Gene Silencing ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retroelements/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&amp;RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al.[1].},
}
@article {pmid41759295,
year = {2026},
author = {Molina, CE and Knight, AL and Lisi, GP},
title = {Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a.},
journal = {Physical biology},
volume = {23},
number = {2},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
support = {MCB 2143760//NSF/ ; },
mesh = {Thermodynamics ; *CRISPR-Cas Systems ; Kinetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *DNA/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Clustered regularly interspaced short palindromic repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged for genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and the DNA cleavage mechanism. In this review, we examine the thermodynamic and kinetic properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.},
}
@article {pmid41759376,
year = {2026},
author = {Xu, Y and Wu, Y and Zheng, H and Zhao, J and Chen, J and Liu, S and Han, M and Li, F and Zhou, F and Zhang, X and Cao, Y and Zhang, H and Zhang, C},
title = {CRISPR-based metabolic screening identifies PLCE1 as a pivotal regulator of oncolytic viral antitumor immunity via tumor immune microenvironment remodeling.},
journal = {Biochemical and biophysical research communications},
volume = {810},
number = {},
pages = {153505},
doi = {10.1016/j.bbrc.2026.153505},
pmid = {41759376},
issn = {1090-2104},
mesh = {*Tumor Microenvironment/immunology ; Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/immunology/physiology ; Humans ; Cell Line, Tumor ; Mice ; *Colorectal Neoplasms/therapy/immunology/genetics ; *CRISPR-Cas Systems ; },
abstract = {As a promising cancer immunotherapeutic agent, oncolytic viruses (OV) can specifically kill tumor cells and elicit systemic antitumor immune responses. However, the intrinsic resistance of tumors to oncolytic virotherapy severely limits its therapeutic efficacy. This study identified phospholipase C epsilon 1 (PLCE1) as a key negative regulator of OV antitumor effects via CRISPR-Cas9 metabolic gene screening in MC38 colorectal cancer model. PLCE1 inhibitor U-73122 enhanced OV infection efficiency and immunogenic cell death in vitro. In vivo, U-73122 combined with OV synergistically reduced tumor volume and prolonged survival. The combination therapy has been shown to remodel the tumor immune microenvironment, leading to an increase in CD45[+] immune cells and CD8[+] T cells, including naïve subsets, and a decrease in FOXP3[+] Treg cells. This shift promotes T cell activation by modulating relevant genes and signaling pathways. This study provides a novel target for optimizing OV immunotherapy.},
}
@article {pmid41759529,
year = {2026},
author = {Cao, Z and Yu, S and Peng, J and Barrett, DR and Liu, Y and Sussman, JH and Chen, C and Thadi, A and Liu, L and Alikarami, F and Xu, J and Carroll, MP and Tan, K and Bernt, KM and Shi, J},
title = {CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {968-985.e7},
pmid = {41759529},
issn = {1097-4164},
support = {R01 CA262260/CA/NCI NIH HHS/United States ; U01 CA243072/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/pathology/metabolism/drug therapy ; Animals ; *CRISPR-Cas Systems ; *Genomics/methods ; Mice ; Mutation ; Single-Cell Gene Expression Analysis ; Gene Expression Regulation, Leukemic ; Gene Regulatory Networks ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.},
}
@article {pmid41759621,
year = {2026},
author = {Yan, X and Chen, M and Yang, S and Guo, Y and Dai, Y and Chen, Y and Zhong, H and Ma, T and Zha, D and He, Y and Li, B and Jia, X and Guo, L and Hu, J and Wei, Y and Chen, X},
title = {Mitochondrial genome editing tools: prospects in animal breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2026.02.018},
pmid = {41759621},
issn = {1673-8527},
abstract = {Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.},
}
@article {pmid41759757,
year = {2026},
author = {Schmidt, GE and Weaver, EA and Kim, TH},
title = {CRISPR-based functional analysis of chicken IRF9 reveals distinct modulation of dsRNA stimulated innate immune pathways.},
journal = {Developmental and comparative immunology},
volume = {177},
number = {},
pages = {105577},
doi = {10.1016/j.dci.2026.105577},
pmid = {41759757},
issn = {1879-0089},
mesh = {Animals ; *Chickens/immunology/genetics ; Immunity, Innate/genetics ; *Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics/metabolism ; *Avian Proteins/genetics/metabolism ; Interferon Type I/metabolism ; *RNA, Double-Stranded/immunology ; Signal Transduction/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {The chicken immune system is distinct from mammalian models due to its reduced immune gene repertoire, yet it retains the ability to mount a highly effective immune response. In mammals, interferon regulatory factor 9 (IRF9) is a key transcriptional regulator of the type I interferon (IFN) pathway, stimulating the expression of hundreds of antiviral genes. Although IRF9 was previously thought to be absent in chickens, current chicken reference genome annotation (bGalGal1.mat.broiler.GRCg7b) lists a putative chicken IRF9. To investigate the function of this gene in chickens, we utilized a clustered regularly interspace short palindromic repeats (CRISPR) based transcriptional modulation platform to elucidate the role of the putative chicken IRF9 in the innate immune response. We analyzed the transcriptomes of IRF9 repressed cells stimulated with double stranded RNA at 0, 0.5, 1, and 6 h post-stimulation. Gene set enrichment analysis revealed that IRF9 repression resulted in the enrichment of pathways associated with regulating the type I IFN response, including the retinoic acid inducible gene I like (RIG-I like) receptor pathway and the Toll-like receptor pathway. Furthermore, concurrent transcriptional repression of type I IFN modulator IRF7 with transcriptional activation of IRF9 failed to rescue the expression of downstream IFN-stimulated genes. These results suggest chicken IRF9 plays a distinct regulatory role from canonical mammalian IRF9 in the type I IFN response and demonstrate a need for functional evidence-based classification of chicken IRFs.},
}
@article {pmid41761908,
year = {2026},
author = {Perry, TN and Mais, CN and Sanchez-Londono, M and Steinchen, W and Plitzko, PA and Randau, L and Pausch, P and Innis, CA and Bange, G},
title = {Structural basis of Cas8-independent Cas3 recruitment in Type I-F2 CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41761908},
issn = {1362-4962},
support = {//Deutsche Forschungsgemeinschaft/ ; 260989694//DFG/ ; 324652314//DFG/ ; 405858511//DFG/ ; 3869//DFG/ ; //Inserm/ ; 5342-2023//Research Council of Lithuania/ ; //Marburg University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics/ultrastructure ; Cryoelectron Microscopy ; Models, Molecular ; DNA/chemistry/metabolism/genetics ; *DNA Helicases/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the Type I-F2 system represents the most compact Type I CRISPR-Cas variant, distinguished by the complete absence of both large (Cas8) and small (Cas11) subunits. In other Type I systems, Cas8 is essential for protospacer adjacent motif (PAM) recognition and for triggering Cas3 recruitment, while Cas11 stabilizes the Cascade backbone and guides the nontarget DNA strand during R-loop formation. To elucidate how I-F2 executes interference in their absence, we determined the cryo-electron microscopy structure of the I-F2 Cascade bound to target DNA and Cas3. Our structure reveals that Cas5 alone mediates PAM sensing, while Cas7 subunits directly recruit Cas3, which adopts a helicase-loaded conformation compatible with DNA engagement. We show how the helicase and C-terminal domains of Cas3 capture the displaced nontarget strand to initiate directional unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference without canonical large and small subunits and emphasize the mechanistic diversity among closely related Type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the hypercompact I-F2 system for genome editing and biotechnological applications.},
}
@article {pmid41762821,
year = {2026},
author = {Golla, DA and Sun, C and Haugh, L and Straub, N and Gao, X},
title = {Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.},
journal = {Current opinion in biotechnology},
volume = {99},
number = {},
pages = {103470},
pmid = {41762821},
issn = {1879-0429},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Prokaryotic Cells/metabolism ; Animals ; },
abstract = {Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.},
}
@article {pmid41762887,
year = {2026},
author = {Yu, Y and Sun, S and Song, X and Xiong, Z and Song, Z and Peng, C and Zhang, J and Ai, L},
title = {crRNA-engineered CRISPR/Cas12a system coupled with RPA for ultrasensitive detection of Lactiplantibacillus plantarum.},
journal = {Food chemistry},
volume = {509},
number = {},
pages = {148595},
doi = {10.1016/j.foodchem.2026.148595},
pmid = {41762887},
issn = {1873-7072},
mesh = {*CRISPR-Cas Systems ; *Lactiplantibacillus plantarum/genetics/isolation &amp; purification ; Probiotics/analysis ; *RNA, Bacterial/genetics ; Bacterial Proteins/genetics/metabolism ; },
abstract = {The growing probiotic industry requires rapid and precise strain detection methods. Here, a one-pot fluorescence platform integrating RPA with an enhanced CRISPR/Cas12a system (termed RPA-ECas12a) was developed for the detection of Lactiplantibacillus plantarum. Through rational 5'end DNA extension of the crRNA, an optimal variant (5'crRNA10) was identified, which increased the trans-cleavage catalytic efficiency of Cas12a by 33% (3.6 × 10[8] M[-1] s[-1]) compared to the wild-type crRNA. The resulting RPA-ECas12a platform detected L. plantarum with a limit of detection of 1.3 CFU/mL, a linear range from 10[1] to 10[7] CFU/mL, and excellent precision (CVs < 10%). The entire detection was completed within 45 min. The platform demonstrated high selectivity and robustness when applied to commercial probiotic powders, yogurts and other complex food matrices. This work not only provides a sensitive and rapid detection tool for probiotic authentication but also offers a generalizable crRNA-engineering strategy to enhance the performance of CRISPR/Cas12a in diagnostic.},
}
@article {pmid41762975,
year = {2026},
author = {Guo, L and Cui, K and Yang, Y and Dong, S and Chen, Y and Liu, K and Lei, X and Duan, B and Zhao, Y and Lv, X and Bai, R and Zheng, M},
title = {Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens.},
journal = {Poultry science},
volume = {105},
number = {5},
pages = {106681},
pmid = {41762975},
issn = {1525-3171},
mesh = {Animals ; *Eimeria/isolation &amp; purification/classification ; *Chickens ; *Coccidiosis/veterinary/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Poultry Diseases/diagnosis/parasitology ; Sensitivity and Specificity ; Feces/parasitology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Reproducibility of Results ; Rapid Diagnostic Tests ; },
abstract = {Chicken coccidiosis, caused by infection with one or more of the seven Eimeria spp., is a major challenge in global poultry production. Rapid and accurate identification at the species level is critical for guiding targeted treatment strategies, minimizing antibiotic misuse, and mitigating disease transmission. In this study, we developed a point-of-care testing (POCT) platform, E-MRC12a (Eimeria-Multiplex RAA-CRISPR/Cas12a), which integrates multiplex recombinase-aided amplification (RAA) with CRISPR/Cas12a technology for the simultaneous detection of all seven Eimeria species in chicken fecal samples. Key assay parameters were optimized to balance detection performance and operational cost. The system was comprehensively evaluated for its sensitivity, specificity, reproducibility, and field applicability. E-MRC12a enables visual, one-pot detection of as few as 1 oocyst/μL. The process from sample loading to result interpretation required 1 h, while the total time from initial sample processing to final result readout was approximately 2 h. The assay exhibited high specificity with no cross-reactivity among Eimeria species, and demonstrated 100% concordance with conventional diagnostic methods in clinical validation. This rapid, field-deployable platform provides a species-specific coccidiosis diagnostic solution, supporting epidemiological surveillance and multivalent anticoccidial vaccine development.},
}
@article {pmid41762997,
year = {2026},
author = {Yu, D and Ren, H and He, P and Li, W and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Stage-aware quantification of the SARS-CoV-2 3CL[pro] biomarker via CsPbBr3@COF-LZU1@AuNP electrochemiluminescence and Cas13a amplification.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129593},
doi = {10.1016/j.talanta.2026.129593},
pmid = {41762997},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; Electrochemical Techniques/methods ; Gold/chemistry ; Luminescent Measurements/methods ; *COVID-19/diagnosis/virology ; Metal Nanoparticles/chemistry ; Metallocenes/chemistry ; CRISPR-Cas Systems ; Ferrous Compounds/chemistry ; Titanium/chemistry ; Limit of Detection ; Oxides/chemistry ; Biomarkers/analysis ; Nucleic Acid Amplification Techniques/methods ; Calcium Compounds ; },
abstract = {Direct, activity-based quantification of the SARS-CoV-2 main protease (3CL[pro]) remains challenging in complex matrices. Here we report a water-compatible electrochemiluminescent (ECL) biosensor that integrates a CsPbBr3@COF-LZU1 emitter, a peptide-DNA conformational switch, and CRISPR/Cas13a-assisted amplification to convert protease activity into a robust optical "turn-on" signal. The covalent organic framework physically stabilizes perovskite nanocrystals in aqueous media and, together with a sparse Au nanoparticle layer, supports assembly of ferrocene-terminated reporters that impose an ultralow baseline via near-field/redox quenching. Target-specific cleavage unlocks an initiator that drives entropy-mediated T7 promoter formation and transcription, producing RNA activators that switch on Cas13a collateral cleavage; removal or distancing of ferrocene from the emitter restores photon output. Under optimized conditions the sensor exhibits a broad log-linear response from 10 to 10[8] aM with an ultralow detection limit of 4.31 aM, high analytical selectivity against common interferents, tight fabrication-to-fabrication precision (inter-electrode RSD ∼3%), and practical robustness (≈97% signal retention over 12 h, ≥90% after 7 days at 4 °C, and ∼92% after 120 ECL cycles). Stepwise ECL, cyclic voltammetry, and impedance analysis confirm layer-by-layer assembly and the intended mechanism of ferrocene-mediated quenching and Cas13a-driven recovery. Applied directly to minimally processed pharyngeal swab eluates, the platform resolves cohort-level differences across disease stages and captures the expected attenuation of 3CL[pro] activity in late-stage specimens, supporting stage-aware quantification in real clinical samples. The modular design-reprogrammable at the protease-cleavable motif, promoter template, and crRNA-points to a general route for sensitive, selective, and water-stable ECL assays of enzymatic activity with translational potential.},
}
@article {pmid41763219,
year = {2026},
author = {Aravind, KM and Del Vecchio, D},
title = {Resource competition shapes CRISPR-mediated gene activation.},
journal = {Cell systems},
volume = {17},
number = {3},
pages = {101511},
doi = {10.1016/j.cels.2025.101511},
pmid = {41763219},
issn = {2405-4720},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Transcriptional Activation/genetics ; Humans ; },
abstract = {CRISPR-mediated gene activation (CRISPRa) allows concurrent transcriptional control of many genes and is widely used in genome-wide screening, bioproduction, and therapeutics. Multi-gene control is possible due to the sequence specificity by which guide RNAs (gRNAs) recruit dCas9 and an activator protein to target genes. Still, the optimization of CRISPRa systems remains difficult. Here, we show that, despite sequence specificity, different gRNAs interfere with each other by competing for dCas9 and the activator protein. This competition breaks modularity and hinders CRISPRa. We also discover that gene activation is biphasic, wherein increased level of a gRNA leads to target repression instead of activation. We introduce a chemical reaction-network model that captures these effects and use it for improving the dynamic range of CRISPRa. Our results demonstrate that CRISPRa is not as modular or scalable as previously thought and establish a predictive modeling tool that enables systematic design and optimization of multi-gRNA CRISPRa systems. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid41763555,
year = {2026},
author = {Shi, Y and Yuan, Y and Qin, L and Zhou, F and Wu, G and Li, B and Yao, P and Shi, M and Ma, L and Wang, Y and Zhang, Y and Wang, C and Wang, X and Huang, B and Chen, J and Xiang, Z and Lin, Q and Huang, J},
title = {Optimizing linker length of base editors for precise crop breeding and gene therapy.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {6},
pages = {1125-1137},
doi = {10.1016/j.jgg.2026.02.021},
pmid = {41763555},
issn = {1673-8527},
mesh = {Humans ; *Oryza/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Plant Breeding ; *Gene Editing/methods ; },
abstract = {Base editing enables efficient nucleotide conversions without inducing DNA double-strand breaks (DSBs) or requiring exogenous donor DNA templates. However, its broader editing window often causes bystander editing, increasing the risk of unintended mutations. In this study, we find that linker length significantly influences the editing window, and base editors with a 7-amino-acid linker reduce bystander editing by an average of 54.4% across 13 endogenous genomic sites in both rice and human cell lines. We further develop an optimized strategy by modulating the linker length between various deaminases and Cas9 nickases, which effectively reduces bystander editing across multiple applications, including functional studies, precise crop breeding, and correction of pathogenic variants. Our work reveals that shortening the linker enhances the specificity of base editing, addressing a key safety concern for its agricultural and therapeutic applications.},
}
@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation &amp; purification/genetics ; *Food Microbiology/methods ; *Bacillus/isolation &amp; purification/genetics ; Nucleic Acid Amplification Techniques ; Food Contamination/analysis ; Humans ; DNA, Bacterial/genetics ; CRISPR-Cas Systems ; Animals ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}
@article {pmid41764730,
year = {2026},
author = {D'Souza, LJ and Young, JN and Coffman, H and Petrow, EP and Bhattacharya, D},
title = {A genome-wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {2},
pages = {},
doi = {10.1093/jimmun/vkaf354},
pmid = {41764730},
issn = {1550-6606},
support = {R01AI129945//National Institutes of Health (NIH)/ ; P30CA023074//Research, Innovation &amp; Impact (RII) of the University of Arizona and National Cancer Institute/ ; S10 OD028466/GF/NIH HHS/United States ; },
mesh = {Animals ; *Plasma Cells/immunology/metabolism ; Mice ; Humans ; CRISPR-Cas Systems ; Myeloid Differentiation Factor 88/genetics/metabolism ; *Multiple Myeloma/immunology/genetics ; Vacuolar Proton-Translocating ATPases/genetics/metabolism ; Cell Line, Tumor ; },
abstract = {Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo. These data reveal novel regulators that link plasma cell secretory capacity and lifespan.},
}
@article {pmid41766140,
year = {2026},
author = {Hartig, AM and Dai, W and Zhang, K and Rottinghaus, AG and Moon, TS and Parker, KM},
title = {Genetic Markers Remain Detectable in Genetically Engineered Microbes Biocontained with a CRISPR Kill Switch.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {10},
pages = {7983-7994},
doi = {10.1021/acs.est.6c00321},
pmid = {41766140},
issn = {1520-5851},
mesh = {*Escherichia coli/genetics ; Genetic Markers ; *CRISPR-Cas Systems ; *Microorganisms, Genetically-Modified/genetics ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Biocontainment strategies, such as kill switches, have been developed to avoid the unintended proliferation of genetically engineered microbes (GEMs) intended for open-release environmental applications. However, the presence of GEM DNA after successful biocontainment presents new environmental risks and challenges for monitoring. In this study, we investigated whether biocontainment using a CRISPR-Cas9 kill switch, which causes double-strand breaks in target genes essential for GEM growth, could resolve this challenge in a model Escherichia coli GEM. Surprisingly, the escape rates of the GEM as determined by CRISPR-targeted gene abundances were as high as 10[-1.6] to 10[-1.0] in LB media, despite the escape rates measured by colony forming units (cfu) being only 10[-6.2] under the same condition. This discrepancy suggested that the CRISPR-Cas9 kill switch prevents colony growth while still leaving a large fraction of target genes intact for detection by molecular methods. Within 1 h after biocontainment, these target genes remained predominantly inside an intact cell membrane and were resistant to degradation by DNase, though degradation was observed in river water over multiple days. Overall, a detailed understanding of the impact of the biocontainment mechanism on both the GEM and its DNA is needed to minimize unintended environmental risks.},
}
@article {pmid41766608,
year = {2026},
author = {Yin, J and Wen, H and Zeng, J and Sun, Y and Chen, X and Jin, L and Song, Y and Xia, S},
title = {CRISPR-based genome editing in human embryos: a review of efficiency, safety, and ethical implications.},
journal = {Biology of reproduction},
volume = {114},
number = {6},
pages = {1775-1790},
doi = {10.1093/biolre/ioag056},
pmid = {41766608},
issn = {1529-7268},
mesh = {Humans ; *Gene Editing/ethics/methods ; Animals ; *CRISPR-Cas Systems ; *Embryo, Mammalian ; },
abstract = {Programmable gene editing tools, particularly CRISPR/Cas9 and its advanced derivatives (base and prime editors), have revolutionized biomedical research and offer unprecedented potential for studying human embryogenesis and correcting monogenic diseases. This review systematically examines the evolution and challenges of these technologies in human embryos and mammalian models. We trace key methodological advancements, from initial studies hampered by low efficiency and mosaicism to refined strategies like RNP delivery and base editing that improved precision. A critical shift occurred with the discovery that CRISPR/Cas9 can cause severe on-target damage, such as large deletions and chromosomal loss, redirecting the field's focus toward safety. We present a comparative analysis of editing efficiencies across species (human, mouse, primate, pig, cow, and rabbit) and tools (Cas9, BEs, and PEs), consistently demonstrating the superiority of RNP for precise editing. Fundamental barriers to clinical translation are discussed, including the trade-off between efficiency and mosaicism, persistent off-target effects, and profound ethical concerns. The review concludes that while somatic gene therapy advances rapidly, heritable genome editing remains premature due to unresolved risks. Future progress depends on developing safer editors, understanding on-target consequences, and adhering to rigorous ethical standards.},
}
@article {pmid41766888,
year = {2026},
author = {Li, X and Zhao, Y and Guo, X and Bai, Y and Wang, J},
title = {Characterization and diversity of defense systems in Providencia pathogen.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1755933},
pmid = {41766888},
issn = {1664-3224},
mesh = {*Providencia/genetics/immunology/pathogenicity ; Phylogeny ; Genome, Bacterial ; CRISPR-Cas Systems ; *Enterobacteriaceae Infections/microbiology/immunology ; },
abstract = {INTRODUCTION: Providencia species are emerging opportunistic pathogens associated with multidrug-resistant infections, yet their molecular defense mechanisms against phage or mobile genetic elements remain poorly characterized.<br><br>METHODS: We present a comprehensive pan-genomic analysis of antiviral defense systems across 73 complete genomes (or chromosomes) of Providencia stuartii (n = 31) and Providencia rettgeri (n = 42), using DefenseFinder and CRISPRCasFinder. We further expanded analysis of contig/scaffold assemblies to confirm conservation of core defense profiles across assembly types. BacMGEnet was employed to derive spacer-MGE interaction networks. Phylogenetic reconstruction and gene gain and loss modeling were performed to assess evolutionary patterns. To validate functionality, we experimentally tested the anti-phage activity of Gabija and Septu in heterologous E. coli assays, including point mutation analysis of conserved residues.<br><br>RESULTS: We reveal a diverse and complex defense repertoire dominated by restriction-modification systems and CRISPR-Cas Class 1 Type I-F, with significant contributions from toxin-antitoxin, GAPS2, PsyrTA, and Mokosh systems. Notably, defense genes are non-randomly distributed, often clustering into genomic islands suggestive of horizontal acquisition. Expanded analysis confirms conservation of core defense profiles across assembly types, supporting the utility of lower-quality data when complete genomes are scarce. Comparative analysis uncovers species-specific differences, with P. rettgeri harboring a higher abundance of non-CRISPR systems. BacMGEnet-derived spacer-MGE interaction networks further highlight species-specific dynamics, dense, hub-driven networks in P. stuartii versus sparser networks in P. rettgeri. Correlation analysis indicates potential associations between specific defense systems and virulence or antibiotic resistance genes. Phylogenetic reconstruction and gene gain and loss modeling further highlight dynamic evolutionary patterns. Both Gabija and Septu systems conferred robust, phage-specific protection; point mutations in conserved residues (GajA E465K and PtuB H53K) abolished defense.<br><br>DISCUSSION: Our findings unveil a multi-layered, modular immune architecture in Providencia, providing crucial insights into its genome plasticity, phage resistance, and adaptation in clinical environments. This work establishes a foundation for understanding the role of defense systems in the evolution and pathogenicity of the Providencia genus.},
}
@article {pmid41769381,
year = {2026},
author = {Mandal, S and Baloch, AR and Yuan, X and Chen, J and Saribas, AS and Zhu, Y and Zhang, D and Jaijyan, D and Xu, J and Hossain, R and Sisto, I and Wang, H and Yang, X and Li, Q and Hu, W},
title = {Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102939},
pmid = {41769381},
issn = {2590-0064},
support = {R01 AI145034/AI/NIAID NIH HHS/United States ; R01 AI174301/AI/NIAID NIH HHS/United States ; },
abstract = {The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.},
}
@article {pmid41771837,
year = {2026},
author = {Schoger, E and Kim, R and Bleckwedel, F and Peralta, TM and Priesmeier, L and Fischer, JA and Stengel, L and Rocha, C and Santos, GL and Lutz, S and Boileau, E and Baumgarten, N and Schulz, MH and Dieterich, C and Müller, OJ and Cyganek, L and Cabrera-Orefice, A and Eberl, H and Maack, C and Streckfuss-Bömeke, K and Pavez-Giani, MG and Doroudgar, S and Sossalla, S and Zelarayán, LC},
title = {Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {41771837},
issn = {2059-3635},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Humans ; Animals ; Fibrosis/genetics/pathology ; CRISPR-Cas Systems/genetics ; *Cellular Reprogramming/genetics ; *Transcription Factors/genetics ; Fibroblasts/metabolism/pathology ; Dependovirus/genetics ; Mice ; },
abstract = {Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.},
}
@article {pmid41771871,
year = {2026},
author = {Becerra, B and Wittibschlager, S and Patel, ZM and Kutschat, AP and Delano, J and Che, E and Tauber, A and Wu, T and Starrs, M and Horstmann, CS and Müller, S and Whittaker, MN and Sylvander, E and Lehner, M and Love, MI and Kleinstiver, BP and Jankowiak, M and Bauer, DE and Seruggia, D and Pinello, L},
title = {Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41771871},
issn = {2041-1723},
mesh = {Alleles ; Humans ; Antigens, CD19/genetics/metabolism ; PAX5 Transcription Factor/genetics/metabolism ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid/genetics ; Mutation ; Trans-Activators/genetics/metabolism ; Nucleotides/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.},
}
@article {pmid41772232,
year = {2026},
author = {Tinoco, AI and Henderson, CF and Meier, EK and Swinhoe, N and Cleves, PA},
title = {Efficient genome editing using CRISPR-Cas9 in reef-building corals.},
journal = {Nature protocols},
volume = {21},
number = {6},
pages = {2851-2879},
pmid = {41772232},
issn = {1750-2799},
support = {2128073//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Anthozoa/genetics ; *CRISPR-Cas Systems ; Coral Reefs ; RNA, Guide, CRISPR-Cas Systems/genetics ; Microinjections ; },
abstract = {Coral reefs are one of the most biodiverse and productive ecosystems on Earth. However, corals are currently under threat from increasing ocean temperatures driven by climate change. Despite the known importance of these fragile ecosystems, our understanding of the molecular mechanisms driving ecologically important traits has been constrained by a lack of genetic tools for functional characterization. To address this limitation, we have developed straightforward and efficient methods to genetically modify corals and study gene function throughout various life history stages using CRISPR-Cas9-based mutagenesis. In this protocol, we first describe how to spawn and collect gametes from the coral Acropora millepora during seasonal spawning events. Next, we describe a method for microinjection of one-cell coral zygotes with CRISPR-Cas9 reagents. We include considerations about effective single-guide RNA design, methods for identifying successfully injected animals, strategies for rearing mutant larvae and juveniles, and methods for the detection and quantification of genomic modifications. This protocol is currently the only way to perform gene editing in corals and takes ~2-4 weeks to complete and has been successfully applied to study genes controlling heat tolerance in coral larvae and skeleton formation in coral juveniles. These technical advances set the foundation for a new field using reverse genetics to study ecologically important traits in corals, such as the establishment of symbiosis and its breakdown upon heat stress.},
}
@article {pmid41772360,
year = {2026},
author = {Guo, Y and Yu, Z and Fan, S and Zhu, M and Ci, L and Yang, X and Chen, Y and Li, Q and Wang, N and Wang, J and Ye, S and Wang, J and Sun, R and Shen, R},
title = {A Bioluminescence Reporter Mouse Strain for In Vivo Imaging of IFNγ Cell Localization and Function.},
journal = {Immunology},
volume = {178},
number = {3},
pages = {428-438},
doi = {10.1111/imm.70127},
pmid = {41772360},
issn = {1365-2567},
support = {24141901100//Science and Technology Innovation Plan of Shanghai Science and Technology Commission/ ; 24YF2732000//Science and Technology Innovation Plan of Shanghai Science and Technology Commission/ ; 2025NS04//Shanghai Laboratory Animal Research Center/ ; 2025NS05//Shanghai Laboratory Animal Research Center/ ; },
mesh = {Animals ; *Interferon-gamma/genetics/metabolism/immunology ; Mice, Inbred C57BL ; *Luminescent Measurements/methods ; Mice, Transgenic ; Mice ; *Genes, Reporter ; Luciferases/genetics ; Disease Models, Animal ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Humans ; Promoter Regions, Genetic ; },
abstract = {Interferon gamma (IFNγ) is a pivotal inflammatory mediator and immune regulator, but its in vivo spatiotemporal dynamics and functional roles in inflammation and carcinogenesis remain incompletely understood. Here, we developed a C57BL/6J- Ifng-2A-luciferase knock-in mouse strain using CRISPR/Cas9-mediated homology-directed repair, enabling real-time bioluminescence imaging (BLI) of IFNγ-expressing cells by inserting a luciferase cassette under the endogenous Ifng promoter. The validation confirmed that this model is capable of directly detecting Poly(I:C) -induced transient IFNγ, enhancing intratumoral IFNγ signals upon anti-PD-1/CTLA-4 therapy, and dynamically tracking IFNγ expression during imiquimod-induced psoriasis. This transgenic mouse model provides a powerful tool for non-invasive, longitudinal tracking of IFNγ-expressing cells, offering novel insights into IFNγ-mediated immune regulation in inflammation and cancer. It holds promise for identifying IFNγ-related therapeutic targets and predicting responses to immunotherapies.},
}
@article {pmid41772759,
year = {2026},
author = {Donega, S and Gorospe, M and Harries, LW and Ferrucci, L},
title = {Loss of Splicing Homeostasis as a Hallmark of Aging.},
journal = {Molecular and cellular biology},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10985549.2026.2627235},
pmid = {41772759},
issn = {1098-5549},
abstract = {Alternative splicing is a fundamental mechanism that ensures accurate gene expression, supports cellular adaptability, and expands protein diversity beyond the limits of a fixed gene pool. With aging, splicing fidelity weakens, contributing to decline in RNA homeostasis and disrupting essential cellular functions, including mitochondrial oxidative phosphorylation, genome stability, and immune regulation, and in turn accelerating tissue and organ dysfunction. Evidence from senescent cells, aged tissues, and model organisms shows that altered levels of splicing factors and increased RNA polymerase II elongation rates impair co-transcriptional splicing and promote mis-spliced isoforms that reinforce senescence and drive pathology. Dysfunction of RNA-binding proteins further contributes to aberrant splicing, linking splicing defects to age-related diseases such as atherosclerosis, osteoarthritis, sarcopenia, and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Therapeutic strategies to correct splicing defects, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, ADAR-mediated editing, and RNA aptamers, can restore a homeostatic balance of mRNA isoforms. However, major challenges remain, including distinguishing adaptive physiological from pathological splicing 'noise' and achieving targeted delivery to tissues. Despite these obstacles, RNA splicing dysregulation represents a promising avenue to extend health span by reestablishing homeostatic RNA programs, and reinforces the idea that "transcriptomic instability" is a hallmark of aging.},
}
@article {pmid41773016,
year = {2026},
author = {Park, H and Yun, J and Lee, K and Kim, JH and Park, JH and Park, YJ and Park, JH and Lee, H and Kim, MG},
title = {Functional decoupling of crRNA enables customizable CRISPR diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773016},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2025-16063091//National Research Council of Science and Technology/ ; RS-2024-00411137//National Research Council of Science and Technology/ ; CRC22024-500//National Research Council of Science and Technology/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and cis-cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas cis-cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA-complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that cis-cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.},
}
@article {pmid41773018,
year = {2026},
author = {Yang, T and Tang, M and Xu, L and Jiang, L and Jiang, L and Zou, Y and Wang, J and Liu, Z and Chen, F and Ban, Y and Ren, W and Cheng, W},
title = {A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773018},
issn = {1362-4962},
support = {U24A20751//National Natural Science Foundation of China/ ; 82372334//National Natural Science Foundation of China/ ; 82502827//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0022//Chongqing Education Commission/ ; CSTB2024NSCQ-QCXMX0006//New Chongqing Youth Innovative Talents Project/ ; //National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Phosphorothioate Oligonucleotides/chemistry ; Human papillomavirus 18/genetics ; Human papillomavirus 16/genetics ; RNA, Messenger/genetics/metabolism ; HeLa Cells ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.},
}
@article {pmid41773916,
year = {2026},
author = {Sui, Z and Chen, B and Zhao, J and Deng, R and Xu, J},
title = {Pronounced Fluorescence Polarization Enhancement Driven by RPA-CRISPR/Cas12a Induced Nucleoprotein Assembly for Salmonella Analysis in Animal-Derived Food Matrices.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7822-7831},
doi = {10.1021/acs.analchem.5c08279},
pmid = {41773916},
issn = {1520-6882},
mesh = {Animals ; *Fluorescence Polarization/methods ; *Salmonella/isolation &amp; purification/genetics ; *Food Microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleoproteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; Food Contamination/analysis ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Endodeoxyribonucleases ; },
abstract = {Salmonella is one of the most hazardous foodborne pathogens, posing a serious threat to public health and food safety worldwide. Conventional recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection assays predominantly rely on the trans-cleavage of fluorescent reporters; however, such signal-generation modes are inherently susceptible to photobleaching, signal drift, and fluctuation, thereby compromising quantitative accuracy and long-term signal stability in practical pathogen detections. To overcome these limitations, we developed a trans-cleavage-independent fluorescence polarization (FP) sensing platform for the rapid and quantitative detection of Salmonella. Unlike conventional reporter-cleavage-based readouts, the proposed system exploits target-induced nucleoprotein assembly to achieve direct, physical signal amplification. In this design, a FAM-labeled forward primer serves as an intrinsic molecular reporter, while exonuclease I (Exo I) selectively degrades unincorporated primers, effectively suppressing background interference. Upon recognition of Salmonella genomic DNA, RPA produces rigid double-stranded amplicons that restrict fluorophore rotational freedom, and subsequent crRNA-guided Cas12a binding further increases molecular size and hydrodynamic volume, resulting in a stepwise enhancement of FP signals. The assay exhibits excellent linearity over a concentration range of 3 × 10[1]-3 × 10[6] CFU mL[-1], with an ultralow detection limit of 5 CFU mL[-1]. In addition, it demonstrates outstanding photostability, reproducibility, and high specificity against nontarget bacteria. Importantly, reliable Salmonella detection was achieved in complex food matrices, including meat, eggs, and dairy products, with consistently high recoveries and strong tolerance to matrix interference, offering a promising alternative to conventional fluorescence-intensity-based CRISPR diagnostics in complex food systems.},
}
@article {pmid41774834,
year = {2026},
author = {Chen, Z and Lin, H and Yoon, C and Huang, H and Kim, Y and Meng, C and Jang, H and Xie, Z and Li, L and Liu, Y and Kim, JS and Zhang, H},
title = {Bismuthene-Based Nanoplatform for Synergistic Thermogenetic CRISPR and Photothermal Cancer Therapy.},
journal = {Nano letters},
volume = {26},
number = {10},
pages = {3407-3416},
doi = {10.1021/acs.nanolett.5c06006},
pmid = {41774834},
issn = {1530-6992},
mesh = {*Photothermal Therapy/methods ; Animals ; Humans ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; *Nanostructures/chemistry ; *Triple Negative Breast Neoplasms/therapy/genetics/pathology ; Mice ; Hyperthermia, Induced ; },
abstract = {Overcoming tumor thermotolerance within clinically safe temperature ranges remains a central limitation of photothermal therapy (PTT). Here we report a closed-loop therapeutic nanoplatform that integrates topologically enhanced photothermal conversion with thermally gated CRISPR/Cas9 regulation. Rationally engineered hexagonal bismuthene nanodiscs exhibit strong near-infrared responsiveness, enabling mild hyperthermia (∼45 °C) that activates a heat-sensitive CRISPR switch targeting CDK7. The resulting disruption of the CDK7-HSP70 stress axis lowers the thermal resistance threshold and reprograms tumor adaptation, thereby amplifying photothermal efficacy and promoting immunogenic cell death. In triple-negative breast cancer models, this gene-thermal feedback achieves >93% tumor inhibition with minimal systemic toxicity. This work establishes a genetically programmable, thermogenetic nanomaterial paradigm that links material design with gene logic for next-generation precision cancer therapy.},
}
@article {pmid41776280,
year = {2026},
author = {Shirai, Y and Kao, JA and Kumar, T and Matsuda, N and Nakagawa, R and Nureki, O and Extavour, CG and Daimon, T},
title = {HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41776280},
issn = {2399-3642},
support = {20H02999, 20K21311, 22K19179, 24H00511, 24K21869//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21J20658//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Overseas Research Fellow//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Long-Term Fellow//Human Frontier Science Program (HFSP)/ ; JPJ008000//Cabinet Office, Government of Japan/ ; IOS-2220747//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Gene Knock-In Techniques/methods ; *Tribolium/genetics/embryology ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; *Oligodeoxyribonucleotides/genetics ; DNA, Single-Stranded/genetics ; },
abstract = {Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.},
}
@article {pmid41777069,
year = {2026},
author = {Zamperin, G and Palumbo, E and Castellan, M and Marciano, S and Fusaro, A and Monne, I},
title = {Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas-based rRNA depletion system.},
journal = {Veterinaria italiana},
volume = {62},
number = {2},
pages = {},
doi = {10.12834/VetIt.3908.38985.2},
pmid = {41777069},
issn = {1828-1427},
mesh = {Animals ; *CRISPR-Cas Systems ; *Metagenomics/methods ; *RNA, Ribosomal/genetics ; *Zoonoses/virology ; Genome, Viral ; RNA, Viral/genetics ; },
abstract = {Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.},
}
@article {pmid41779781,
year = {2026},
author = {Ju, X and Dong, L and Liu, T and Zhang, F and Sun, X and Schwoerer, MP and Ren, W and Gong, M and Ploss, A and Qin, W and Wu, X and Wang, L and Ding, Q},
title = {EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2529289123},
pmid = {41779781},
issn = {1091-6490},
support = {2023YFC2306900//National Key Research and Development Plan of China/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; 82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82522053//MOST | National Natural Science Foundation of China (NSFC)/ ; 20251080029//Tsinghua University Dushi Program/ ; Not applicable//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; Not applicable//High Meadows Environmental Institute/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {*Virus Replication ; Humans ; *Hepatitis E virus/physiology/pathogenicity/genetics ; *Y-Box-Binding Protein 1/metabolism/genetics ; Animals ; *Hepatitis E/virology/metabolism/pathology/genetics ; *Eukaryotic Initiation Factors/metabolism/genetics ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; },
abstract = {Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.},
}
@article {pmid41781609,
year = {2026},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ[E]-RNAP complex.},
journal = {Nature},
volume = {653},
number = {8113},
pages = {288-296},
pmid = {41781609},
issn = {1476-4687},
mesh = {*Bacterial Proteins/chemistry/metabolism/ultrastructure ; *CRISPR-Associated Proteins/metabolism/chemistry/ultrastructure ; CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA/metabolism/chemistry ; *DNA-Directed RNA Polymerases/metabolism/chemistry/ultrastructure ; Gene Expression Regulation, Bacterial ; Holoenzymes/chemistry/metabolism/ultrastructure ; Models, Molecular ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; *Sigma Factor/chemistry/metabolism/ultrastructure ; *Transcription Initiation, Genetic ; Transcription, Genetic ; },
abstract = {In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors[1-3]. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity[1,4,5]. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ[E])[6], although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ[E] system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ[E]-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ[E]-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ[E]-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}
@article {pmid41781627,
year = {2026},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, HC and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologues drive RNA-guided transcription.},
journal = {Nature},
volume = {653},
number = {8113},
pages = {277-287},
pmid = {41781627},
issn = {1476-4687},
mesh = {*Escherichia coli/genetics/enzymology/metabolism ; *CRISPR-Cas Systems/genetics ; Sigma Factor/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Promoter Regions, Genetic/genetics ; *Transcription, Genetic ; Gene Expression Regulation, Bacterial ; Transcriptional Activation ; Escherichia coli Proteins/metabolism/genetics ; Transcription Initiation, Genetic ; },
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)[1]. Although the seven σ factors in Escherichia coli have been extensively characterized[2], Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ[E]) whose precise roles are unknown, pointing to additional layers of regulatory potential[3]. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ[E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ[E] homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σ[E] was dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ[E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ[E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology[4,5].},
}
@article {pmid41782368,
year = {2026},
author = {Chuecos, MA and Park, SH and Bhakta, MM and Too-Chiobi, U and Betancourth, D and Cao, M and De Giorgi, M and Walkey, CJ and Tiwari, A and Godin, B and Assini, JM and Palmer, DJ and Ng, P and Boffa, MB and Koschinsky, ML and Bao, G and Lagor, WR},
title = {Cytosine base editing of LPA in transgenic mice averts large deletions.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {6},
pages = {3334-3352},
pmid = {41782368},
issn = {1525-0024},
support = {R01 HL169761/HL/NHLBI NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; S10 OD030414/OD/NIH HHS/United States ; U42 OD026645/OD/NIH HHS/United States ; P50 HD103555/HD/NICHD NIH HHS/United States ; R01 HG011459/HG/NHGRI NIH HHS/United States ; R01 DK124477/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice, Transgenic ; Mice ; *Gene Editing/methods ; *Lipoprotein(a)/genetics ; Genetic Vectors/genetics/administration &amp; dosage ; Dependovirus/genetics ; *Cytosine/metabolism ; Adenoviridae/genetics ; Humans ; *Sequence Deletion ; CRISPR-Cas Systems ; },
abstract = {Lipoprotein(a) (Lp(a)) is a genetically determined causal risk factor for cardiovascular disease, with approximately 20% of the population exhibiting elevated levels. While there are promising drugs in development, there are currently no approved therapies specifically designed to lower Lp(a) levels. For high-risk individuals with extreme levels of Lp(a), liver-directed genome editing could be an effective one-time solution. Genome editing approaches such as CRISPR and TALENs can reduce Lp(a) in LPA-transgenic mouse models, but they frequently induce large and potentially harmful genomic deletions. Here, we report the first application of TadA-derived cytosine base editing (CBE), delivered via helper-dependent adenovirus (HDAdV) and adeno-associated virus (AAV) vectors, to introduce premature stop codons into LPA. This strategy produced robust and durable lowering of circulating apolipoprotein(a) (apo(a)) in LPA-transgenic mice. Using SMRT-seq with single-molecule unique molecular identifiers, we quantified deletion events and found that CBE did not induce large deletions when targeting a single LPA site and produced only a small fraction (<4%) of large deletions when editing across multiple sites. In contrast, CRISPR-Cas9 cutting of LPA resulted primarily in large deletions. These findings demonstrate that CBE enables sustained reduction of circulating apolipoprotein(a) in an LPA-transgenic mouse model while largely preserving genomic integrity.},
}
@article {pmid41783940,
year = {2026},
author = {Berti, M and Ceriotti, S and Santi, L and Alberti, G and Beretta, S and Degl'Innocenti, S and Ruatti, C and Savoia, EO and Jofra-Hernandez, R and De Ponti, G and Bolamperti, S and Villa, I and Galeotti, F and Romano, A and Visigalli, I and Norata, R and Rocchi, M and Cristofori, P and Cossutta, M and Consiglieri, G and Tucci, F and Santorelli, L and Grumati, P and Ronfani, L and D'Adamo, P and Giustina, A and Angelozzi, M and Settembre, C and Mortellaro, A and Scala, S and Sanvito, F and Volpi, N and Aiuti, A and Bernardo, ME and Crippa, S},
title = {Development and characterization of a model of mucopolysaccharidosis type IVA for evaluating therapies targeting bone disease.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {2},
pages = {},
pmid = {41783940},
issn = {1754-8411},
support = {20228H9T82//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; P20223MF7X_001//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; CN_00000041 - CUP G83C22000270001//NextGenerationEU/ ; TTAGTXEKFA//Else Kr&#xf6;ner-Fresenius-Zentrum f&#xfc;r Ern&#xe4;hrungsmedizin/ ; TELE-MB//Fondazione Telethon/ ; TELE-AA//Fondazione Telethon/ ; //Ospedale San Raffaele/ ; },
mesh = {Animals ; Disease Models, Animal ; *Mucopolysaccharidosis IV/therapy/complications/pathology/blood ; *Bone Diseases/therapy/complications/pathology/blood ; Glycosaminoglycans/metabolism ; Chondroitinsulfatases/metabolism/deficiency/genetics ; Keratan Sulfate/urine/metabolism ; Chondroitin Sulfates/urine/metabolism ; CRISPR-Cas Systems/genetics ; Biomarkers/metabolism/blood ; Bone and Bones/pathology ; Humans ; },
abstract = {Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.},
}
@article {pmid41784267,
year = {2026},
author = {Wang, Z and Wu, Y and Wang, Z and Zhang, S and Liu, H and Nie, Y and Chen, K and Huang, Y and Zhou, Y and Cao, Y and Sun, L and Hao, R},
title = {crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41784267},
issn = {1362-4962},
support = {//Prevention and Control of Emerging/ ; 2025ZD01903403//National Science and Technology Major Project/ ; L234051//Beijing Municipal Natural Science Foundation/ ; L246011//Beijing Municipal Natural Science Foundation/ ; 2024-03-18//Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission/ ; 2025ZD01903403//Prevention and Control of Emerging and Major Infectious Diseases-National Science/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *Endodeoxyribonucleases/metabolism/genetics ; Klebsiella pneumoniae/genetics ; Nucleic Acid Conformation ; Bacterial Proteins ; },
abstract = {CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.},
}
@article {pmid41784340,
year = {2026},
author = {Zhou, C and Cheng, T and Zhou, J and Zhang, B and Liu, L and Jiang, G and Li, W and Wang, C},
title = {Synthetic Biofilms for Green Membranes: Engineering Low-Energy Filtration Systems.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {12},
pages = {9357-9366},
doi = {10.1021/acs.est.5c15661},
pmid = {41784340},
issn = {1520-5851},
mesh = {*Biofilms ; *Membranes, Artificial ; Filtration ; Biofouling ; Water Purification ; Ultrafiltration ; },
abstract = {Membrane filtration is a key technology to modern water purification, yet its sustainability is compromised by biofouling, which increases energy consumption and ecological impacts. Conventional control strategies often struggle to balance efficacy and environmental footprint. In this study, an inducible, engineered quorum-quenching (QQ) bacterium was constructed via a genomic integration strategy, thereby achieving control over the biofilm structure in membrane filtration biofouling layers. By using the clustered regularly interspaced short palindromic repeat (CRISPR-Cas) targeted gene editing technology, the engineered bacteria that were constructed to express aiiO under l-ribose induction have achieved the regulation of biofilms. Validation using a gravity-driven membrane ultrafiltration system (UF-GDM) model showed that the engineered bacterium effectively reduced extracellular polymeric substances (EPS) components, increased the hydrophilic porosity of the residual biofilm, and decreased its stickiness. This approach reduced transmembrane pressure by 64.5%, increased total organic carbon (TOC) removal by 13.2%, and extended membrane lifespan by 16.1%. A technical-economic analysis indicates that the 100,000 m[3]/day treatment plant achieves an annual net profit increase of 31.52%, reaching 1.55 × 10[7] CNY, while reducing its net carbon footprint by 27.43%, with an annual net reduction of 2.96 × 10[5] kg CO2eq. This study provides a novel solution strategy for achieving biofouling resistance and sustainable, low-energy operation in membrane filtration processes, which contributes to the broader application and adoption of this technology.},
}
@article {pmid41785043,
year = {2026},
author = {Xu, X and Zhang, R and Hai, G and Wang, Y and Zheng, H and Cui, P and Kou, B and Jin, X and Peng, J},
title = {DNA Logic-Gated CRISPR/Cas13a and PNTs-Hemin Biomimetic Nanozyme for Ratiometric Detection of BRCA1 and circROBO1.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7580-7589},
doi = {10.1021/acs.analchem.5c07475},
pmid = {41785043},
issn = {1520-6882},
mesh = {*Hemin/chemistry ; *BRCA1 Protein/analysis/genetics ; *Biomimetic Materials/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Nanotubes, Peptide/chemistry ; Biomimetics ; Gold/chemistry ; *DNA/chemistry ; Electrochemical Techniques ; Logic ; },
abstract = {Although nanozyme-based biosensors show great promise for the early diagnosis of cancer, their application is often limited by poor catalytic activity at neutral pH and susceptibility to matrix interference. This study involved the construction of a biomimetic nanozyme through coordination-driven self-assembly on peptide nanotubes. This rigid framework enables the periodic arrangement of histidine residues to achieve precise axial coordination with the iron center of the hemin molecule, effectively mimicking the active site and catalytic microenvironment of natural horseradish peroxidase. Consequently, PNTs-hemin exhibits peroxidase activity 2.7 times that of free hemin under near-physiological conditions. To ensure detection specificity, an AND logic gate design was integrated, triggering CRISPR/Cas13a-mediated trans-cleavage only when both targets (BRCA1 and circROBO1) are present simultaneously. Furthermore, combining the nanozyme with graphdiyne-supported gold nanoparticles resulted in the formation of a cascade catalytic system that produced a ratiometric electrochemical reading (IFc/Ihemin). This effectively corrects for environmental fluctuations and false positive signals. This study presents an effective strategy that combines the specificity of molecular logic gates with biomimetic catalysis. This opens up new avenues for the precise diagnosis of multiple targets in complex biological samples.},
}
@article {pmid41785318,
year = {2026},
author = {Weber, LI and Timpen, LE and Egger-Hörschinger, AS and Schöpf, P and Ayhan, ND and Demmel, D and Hotze, M and Zhang, Y and Mehrabi, M and Puglisi, K and Stefan, E and Ghaffari-Tabrizi-Wizsy, N and Ramos-Pittol, JM and Kwiatkowski, M and Hartl, M},
title = {Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2524159123},
pmid = {41785318},
issn = {1091-6490},
support = {P33662//Austrian Science Fund (FWF)/ ; },
mesh = {Humans ; *Colorectal Neoplasms/genetics/metabolism/pathology ; *Wnt Signaling Pathway/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; *Nerve Tissue Proteins/genetics/metabolism ; Cell Line, Tumor ; beta Catenin/metabolism/genetics ; Promoter Regions, Genetic ; Gene Silencing ; *Repressor Proteins/genetics/metabolism ; Animals ; Intracellular Signaling Peptides and Proteins ; },
abstract = {Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.},
}
@article {pmid41785636,
year = {2026},
author = {Xue, S and Sun, H and Hou, X and Li, N and Xue, L and Dai, E and Wan, J},
title = {An off-target exonuclease activity in AsCpf1 undermines CRISPR diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {303},
number = {},
pages = {118578},
doi = {10.1016/j.bios.2026.118578},
pmid = {41785636},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/analysis ; DNA/genetics/chemistry ; Exodeoxyribonucleases/chemistry/genetics ; Humans ; *Exonucleases/chemistry/genetics ; },
abstract = {The extensive utilization of CRISPR-Cas systems in molecular diagnostics stems from their crRNA-guided trans-cleavage capabilities. However, AsCpf1-based detection systems frequently exhibit unexplained sensitive variations. This research reveals that AsCpf1 maintains a crRNA-independent function, similar to exonuclease I, when utilized in standard buffers containing Mg[2+]. From a structural perspective, this exonuclease activity is independent of the RuvC domain-mediated canonical trans-cleavage activity. It is predicted by structural modeling to be potentially localized within the WED-PI domain. In the context of diagnostics, the effective target concentration is diminished by AsCpf1-mediated degradation of the free 3' ends of target DNA, which impairs detection sensitivity. To mitigate this interference, we demonstrate that 3' end capping effectively restores detection performance. This approach was validated in a CRISPR-EXPAR-based microRNA biosensor, which exhibited approximately 10-fold improvement in sensitivity following 3' end capping. Overall, this investigation characterizes a previously unidentified exonuclease activity within the AsCpf1 system and establishes practical design criteria to improve the robustness and accuracy of CRISPR-based diagnostic tools.},
}
@article {pmid41785880,
year = {2026},
author = {Petersen, A&#xd8; and Damholt, B and Grove, M and Hink, J and Marotte-Hurbon, T and Söderqvist, J and Troy, A and Zdravkovic, M and Bayer, L and Brunner, K and Bryde, T and Clube, J and Gencay, YE and Gram, A and Haaber, JK and Hallström, B and Jasinskytė, D and Pascal, R and Petersen, M and Semsey, S and Torio, AS and Turcu, IC and Smrekar, F and Taur, Y and Satlin, MJ and Sommer, MOA and van der Helm, E and Grøndahl, C},
title = {Safety, recovery, and pharmacodynamics of CRISPR-Cas therapeutic SNIPR001: a phase 1, randomised, double-blind, first-in-human, dose-escalation study.},
journal = {The Lancet. Microbe},
volume = {7},
number = {4},
pages = {101257},
doi = {10.1016/j.lanmic.2025.101257},
pmid = {41785880},
issn = {2666-5247},
mesh = {Humans ; Adult ; Double-Blind Method ; Male ; Middle Aged ; Female ; Young Adult ; Adolescent ; Aged ; *Escherichia coli/virology/drug effects ; *Bacteriophages/genetics ; Feces/microbiology ; Healthy Volunteers ; },
abstract = {BACKGROUND: Patients with haematological cancer who receive stem-cell transplantation are at risk of bloodstream infections, often caused by multidrug resistant gut pathogens such as Escherichia coli. SNIPR001 is a cocktail of four CRISPR-Cas-armed bacteriophages that reduce colonisation of E coli in the gastrointestinal tract in animal models and is designed to not affect other members of the commensal microbiota. We aimed to investigate the safety and tolerability of SNIPR001 in healthy participants.<br><br>METHODS: In this randomised, placebo-controlled, double-blind, first-in-human, dose-escalation trial conducted at a single centre (Medpace Clinical Pharmacology Unit; Cincinnati, OH, USA), we sequentially enrolled healthy participants (aged 18-65 years) with more than 10[7]E coli colony-forming units per gram of stool into cohorts 1, 2, and 3, pending a safety review of the previous enrolment group where applicable. Participants in each cohort were randomly assigned to treatment or placebo using a unique three-digit participant identification number. Participants were orally administered 10[8] plaque-forming units (PFU) per dose (cohort 1), 10[10] PFU per dose (cohort 2), and 10[12] PFU per dose (cohort 3) of SNIPR001 or placebo (phosphate-buffered saline buffer), twice daily for 7 days. All personnel, except for a pharmacy staff member who prepared both SNIPR001 and placebo vials, were masked to the administered dose and assignment; masking was ensured by fully covering the surface of each vial. Participants were followed up to day 187. The primary outcome was the incidence and severity of adverse events and medically attended adverse events from the first administration of the study drug until 4 weeks after the last dose administration on day 35 of the study. Recovery and biodistribution of SNIPR001 in faeces, blood, and urine; pharmacodynamics, including the ability of SNIPR001 to reduce E coli levels in stool (assessed using a linear mixed-effects model); and microbiome composition (using Bray-Curtis dissimilarity) were secondary outcomes. Primary safety analyses were assessed per-protocol (ie, all enrolled participants who received at least one administration of the study drug). This trial was conducted under an Investigational New Drug application from the US Food and Drug Administration, is registered with ClinicalTrials.gov (NCT05277350), and is closed to new participants.<br><br>FINDINGS: The trial was carried out between March 24, 2022, and Nov 30, 2022. 36 eligible participants were randomly assigned to receive SNIPR001 or placebo in cohorts 1 (six assigned to 10[8] PFU per dose and two assigned to placebo), 2 (six to 10[10] PFU per dose and two to placebo), and 3 (12 to 10[12] PFU per dose and eight to placebo). The mean age of participants was 42&#xb7;1 years (SD 13&#xb7;8), with 14 (39%) female participants and 22 (61%) male participants. During the trial and 4-week follow-up period, only mild and moderate adverse events were observed, with most adverse events occurring in the placebo group (13, six, one, and nine for participants receiving either placebo or SNIPR001 at 10[8], 10[10], and 10[12] PFU twice a day, respectively). The number of participants who had adverse events was not significantly higher in treatment groups than in the placebo group (p=0&#xb7;94, one-sided Fisher's exact test). The most frequently reported adverse events were headaches and diarrhoea. No grade 3-4 adverse events were reported and no serious adverse events were reported in the SNIPR001 dose groups. During and after the dosing period, the gut microbiota composition did not significantly differ between the treatment and placebo groups (p>0&#xb7;05, two-sided Mann-Whitney U test of Bray-Curtis distances, false discovery rate [FDR]-corrected). Functional SNIPR001 was recovered from stool samples in concentrations proportional to the administered dose but was not meaningfully detected in plasma (only one sample) or urine (only one sample). SNIPR001 was undetected in all samples 6 months after the last dosing, which is a favourable pharmacokinetic property and meets regulatory expectations. We observed the largest reduction in E coli levels compared with placebo 2 weeks after treatment initiation at day 14 (78%; -0&#xb7;65 log10 [SE 0&#xb7;64] for 10[12] PFU SNIPR001 twice-daily group), according to a linear mixed-effects model for the highest dose population; however, this change was not statistically significant (p=0&#xb7;811, linear mixed-effects model, FDR-corrected).<br><br>INTERPRETATION: This first-in-human study of SNIPR001 supported its safety, tolerability, and restriction to the gastrointestinal tract, while not systemically disrupting the gut microbiome. These results justify further clinical development of SNIPR001 in an ongoing phase 1b/2a trial.<br><br>FUNDING: Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) and SNIPR Biome.},
}
@article {pmid41787916,
year = {2026},
author = {Saboor, M and Jasem Alblooshi, M and Adel Alkaabi, A and Ramazan Soozaei, F and Hamad Alketbi, M},
title = {CRISPR in Thalassemia: Global Research Trend Analysis.},
journal = {Hemoglobin},
volume = {50},
number = {2},
pages = {141-155},
doi = {10.1080/03630269.2026.2634815},
pmid = {41787916},
issn = {1532-432X},
mesh = {Humans ; *Thalassemia/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; *beta-Thalassemia/genetics/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; beta-Globins/genetics ; Bibliometrics ; },
abstract = {β-Thalassemia is a prevalent inherited disorder of β-globin chains. The clustered regularly interspaced short palindromic repeats (CRISPR) genome editing system has emerged as a potential curative strategy. We conducted a bibliometric analysis to map global research trends in CRISPR-based thalassemia research. Original and review research articles were retrieved from the Scopus database using the search terms [TITLE-ABS-KEY ('βeta thalassemia' OR 'β thalassemia' OR thalassemia*) AND TITLE-ABS-KEY ('gene edit*' OR crispr* OR 'clustered regularly interspaced short palindromic repeats')] AND [LIMIT-TO (DOCTYPE, 're') OR LIMIT-TO (DOCTYPE, 'ar')] for analysis. Bibliometric mapping and network visualization were performed using VOSviewer to analyze publication trends, authorship networks, international collaborations, keyword clusters, and citation metrics. Major CRISPR-based therapeutic strategies for thalassemia were reviewed to place experimental and clinical developments within a translational framework. The analysis demonstrates a clear transition from foundational genomic studies to translational applications, with leading contributions from the United States and China. Two dominant therapeutic strategies have emerged: direct correction of the HBB gene in hematopoietic stem cells and fetal hemoglobin reactivation via BCL11A repression. The latter strategy culminated in regulatory approval of exagamglogene autotemcel (Casgevy). Advances in base editing, prime editing, and strategies to improve engraftment are expected to enhance the precision and long-term efficacy of next-generation approaches. Clustered regularly interspaced short palindromic repeats-based research on thalassemia continues to expand, supported by extensive international collaboration and growing clinical translation. Future large-scale implementation will require advances in bioprocess engineering, cost reduction for ex vivo manufacturing, and adaptable treatment models for diverse healthcare systems.},
}
@article {pmid41787951,
year = {2026},
author = {Chatla, K and Roper, B and Ayalew, L and Ko, P and Lippold, S and Doma, M and Camperi, J},
title = {Assessing mRNA and sgRNA Quality for Cell and Gene Therapy Applications Using Nanopore Direct RNA Sequencing.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7452-7461},
doi = {10.1021/acs.analchem.5c06819},
pmid = {41787951},
issn = {1520-6882},
mesh = {*RNA, Messenger/genetics/analysis ; Humans ; *Sequence Analysis, RNA/methods ; *Genetic Therapy ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Nanopores ; },
abstract = {Recent advances in RNA technology have enabled the development of diverse therapeutics spanning vaccines, immunotherapies, and genome-editing platforms. Ensuring clinical efficacy and safety requires precise characterization and control of RNA critical quality attributes (CQAs). Nanopore direct RNA sequencing (NDRS) has emerged as a powerful single-molecule analytical approach capable of simultaneously resolving sequence and structural features consistent with regulatory expectations. In this study, we establish NDRS as a comprehensive, multiattribute analytical platform by integrating novel strategies to assess key CQAs in a single assay. Following workflow optimization, NDRS accurately determined full-length mRNA sequences and evaluated transcript integrity. Notably, we developed the first sequencing-based method for quantifying 5' capping efficiency directly from native RNA molecules. Additionally, we demonstrated, for the first time, full-length sequencing of 100-nucleotide single-guide RNA (sgRNA) molecules by incorporating a 5' RNA oligo adapter, enabling complete identity verification. Quantitative results for poly(A) tail length, integrity, and capping efficiency were consistent with established orthogonal techniques, including chromatography and mass spectrometry. Moreover, functional correlation studies with Cas9 mRNA and sgRNA used in CRISPR-Cas9 editing revealed that increased mRNA degradation led to decreased knockout efficiency. Together, these findings position NDRS as a versatile and unified analytical platform for comprehensive characterization of mRNA and sgRNA, supporting quality assurance, comparability, and control in the development and manufacturing of next-generation RNA therapeutics.},
}
@article {pmid41789142,
year = {2026},
author = {Xu, C and Zeng, C and Wang, M and Wei, X and Song, M and Liu, X and Wang, W and Chen, Q and Ji, X and Luo, P and Ma, L and Sun, Y and Gou, H and Zhu, Z and Li, X and Lv, YX and Liu, P and Zhu, JK},
title = {mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo.},
journal = {Innovation (Cambridge (Mass.))},
volume = {7},
number = {3},
pages = {101151},
pmid = {41789142},
issn = {2666-6758},
abstract = {Programmable epigenetic editors (EEs) that achieve long-term gene expression modulation without altering the DNA sequence hold immense therapeutic potential. However, the clinical translation of current CRISPR-based epigenome editors is impeded by substantial challenges, particularly their large molecular size, which limits efficient in vivo delivery. Here, we report the rational design and engineering of compact, mRNA-delivered EEs (CRISPR OFF-EE) using Streptococcus pyogenes Cas9 (SpCas9), intein-split-SpCas9, or the smaller Cas-SF01 (a Cas12i3 variant). Combined with optimized mRNA architecture and lipid nanoparticle (LNP) delivery, a single intravenous LNP administration of the optimized OFF-EE V2 mRNA, along with selected guide RNAs (gRNAs) targeting Pcsk9 in mice, resulted in an ∼83.2% reduction in circulating PCSK9 levels and a corresponding ∼51.4% reduction in low-density lipoprotein cholesterol (LDL-C) levels, persisting for at least 180 days. SF01-based EEs showed higher specificity with fewer off-target methylation events than SpCas9-based counterparts. Our optimized LNP formulation also demonstrated a favorable safety profile with predominantly liver-tropic activity. These findings establish a robust and versatile platform for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered, engineered mRNA editors.},
}
@article {pmid41791393,
year = {2026},
author = {Rapp, J and Verhülsdonk, A and Garcke, A and Stadelmann, A and Farke, N and Troßmann, F and Kronenberger, T and Alvarado, A and Petras, D and Link, H},
title = {The metabolome of an E. coli CRISPRi library identifies benefits of minimal metabolite levels and targets for engineering.},
journal = {Cell systems},
volume = {17},
number = {4},
pages = {101518},
doi = {10.1016/j.cels.2025.101518},
pmid = {41791393},
issn = {2405-4720},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolome/genetics ; Metabolic Networks and Pathways/genetics ; *Metabolic Engineering/methods ; Tandem Mass Spectrometry/methods ; Chromatography, Liquid/methods ; CRISPR-Cas Systems/genetics ; Liquid Chromatography-Mass Spectrometry/methods ; Metabolomics/methods ; Polyisoprenyl Phosphates/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Sesquiterpenes/metabolism ; },
abstract = {Metabolite concentration changes can have broad consequences on the function and robustness of metabolic networks. Here, we measured the metabolome response of 1,515 CRISPR interference (CRISPRi) E. coli strains targeting all genes in the iML1515 metabolic model. Metabolites that are hardly measurable in wild-type E. coli accumulated in specific CRISPRi strains, indicating that they are normally maintained at low levels. We confirmed metabolite accumulation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated putative reference spectra for 102 metabolites for which no MS[2] data had previously been available. We show that minimal metabolite levels are beneficial because they (1) enable substrate level regulation of enzyme activity, (2) prevent competitive inhibition, and (3) suppress side reactions. However, minimal metabolite pools also limit flux through engineered pathways. For example, low levels of farnesyl diphosphate (frdp) constrained a synthetic carotenoid pathway, and we show that the knockdown of octaprenyl diphosphate synthase (IspB) increased frdp levels and carotenoid production. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid41791397,
year = {2026},
author = {Low, SJ and O'Neill, MT and Fernando, JA and Kerry, WJ and Prestedge, J and Wild, N and Chahal, S and Pollock, GL and Papadakis, G and Krysiak, M and Williams, E and Azzato, F and Tran, T and Fairley, C and Bradshaw, C and Chen, MY and Lim, CK and Williamson, DA and Pasricha, S},
title = {CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.},
journal = {The Lancet. Microbe},
volume = {7},
number = {4},
pages = {101289},
doi = {10.1016/j.lanmic.2025.101289},
pmid = {41791397},
issn = {2666-5247},
mesh = {Humans ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Sexually Transmitted Diseases/diagnosis/microbiology ; Treponema pallidum/genetics/isolation &amp; purification ; Neisseria gonorrhoeae/genetics/isolation &amp; purification ; Chlamydia trachomatis/genetics/isolation &amp; purification ; *Point-of-Care Systems ; Simplexvirus/genetics/isolation &amp; purification ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Herpesvirus 2, Human/genetics/isolation &amp; purification ; },
abstract = {BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV).<br><br>METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves.<br><br>FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82&#xb7;5% (95% CI 74&#xb7;0-88&#xb7;7) for T pallidum and 94&#xb7;4% (90&#xb7;2-97&#xb7;0) for pan-HSV; LODs were 6&#xb7;2 copies per &#x3bc;L for T pallidum and 7&#xb7;8 copies per &#x3bc;L for HSV. Reflex testing gave sensitivities of 97&#xb7;0% (91&#xb7;1-99&#xb7;3) for HSV-1 and 96&#xb7;0% (89&#xb7;7-98&#xb7;7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80&#xb7;0% (74&#xb7;0-84&#xb7;9) for N gonorrhoeae and 73&#xb7;0% (65&#xb7;5-79&#xb7;3) for C trachomatis, with a LOD of 3&#xb7;9 copies per &#x3bc;L for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63&#xb7;1% (55&#xb7;1-70&#xb7;4); however, this was dependent on sample type, with a sensitivity of 85&#xb7;7% (46&#xb7;7-99&#xb7;5) in genital samples and 61&#xb7;2% (52&#xb7;8-68&#xb7;9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0&#xb7;90 for T pallidum and 0&#xb7;99 for pan-HSV in the TP-HSV assay, with values of 0&#xb7;99 for HSV-1 and 0&#xb7;97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0&#xb7;90 for N gonorrhoeae and 0&#xb7;85 for C trachomatis, with a value of 0&#xb7;72 obtained for gyrA S91F in the reflex cNG-gyrA assay.<br><br>INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings.<br><br>FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.},
}
@article {pmid41791700,
year = {2026},
author = {Xiang, Z and Guo, K and Xi, J and Xu, S and Duan, J and Wen, S and Liu, Z and Wang, X and Zhao, P and Zhang, X},
title = {CRISPR/Cas9-mediated knockout of SPI51 reveals an essential role of protease inhibitors in silk fiber formation.},
journal = {Journal of insect physiology},
volume = {170},
number = {},
pages = {104962},
doi = {10.1016/j.jinsphys.2026.104962},
pmid = {41791700},
issn = {1879-1611},
mesh = {Animals ; *Bombyx/genetics/metabolism ; CRISPR-Cas Systems ; *Silk/chemistry/metabolism ; Gene Knockout Techniques ; *Fibroins/metabolism ; *Protease Inhibitors/metabolism ; *Insect Proteins/genetics/metabolism ; Spectroscopy, Fourier Transform Infrared ; X-Ray Diffraction ; },
abstract = {Silkworm silk is a natural protein fiber composed mainly of fibroin and sericin, with protease inhibitors representing an additional abundant constituent. However, the impact of protease inhibitors on the structure and properties of silk fibers has not yet been studied. In this study, we focused on the Kunitz-type protease inhibitor SPI51, the most abundant protease inhibitors of cocoon. CRISPR/Cas9 editing was used to generate a homozygous mutant of SPI51 (SPI51[KO]), resulting in premature translation termination at the 33rd amino acid. After knocking out SPI51, the mechanical properties of silk were significantly reduced compared with those of the wild type. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) results revealed that this deterioration was associated with significantly reduced β-sheet content and lower crystallinity. Morphological observations showed that the fibroin area of SPI51[KO] silk was significantly smaller than that in the wild type. Further Western blot analysis showed that fibroin heavy chain (Fib-H), fibroin light chain (Fib-L), and fibrohexamerin (P25) were decreased after knocking out SPI51, which resulted in a reduction of silk fibroin layer and affected structure and mechanical properties. Our results provide valuable insights into the balance between proteases and protease inhibitors in the silk gland and reveal for the first time the roles of the protease inhibitor in silk protein synthesis and the structural and mechanical properties of silk fibers.},
}
@article {pmid41793179,
year = {2026},
author = {Dong, K and Hu, H and Wang, H and Zheng, Z and Cheng, S and Shu, W and Liu, R and Xin, X and Huang, S and Qian, D and Xiao, X and Fu, Q and Wang, H},
title = {A Single-Enzyme Activated CRISPR-Cas12a Nano System via Subtly Balanced dsDNA for Kinetic-Gated UDG Detection and Spatiotemporal Cellular Imaging.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {28},
pages = {e23400},
pmid = {41793179},
issn = {2198-3844},
support = {2023YFC2705400//The National Key Research and Development Plan/ ; 82472965//National Natural Science Foundation project/ ; W2521097//National Natural Science Foundation project/ ; 2024EIA002//Hubei Provincial Central Government-Guided Local Science and Technology Fund Development Project/ ; 2025AFD286//Hubei Provincial National Natural Science Foundation/ ; 2025XHYN047//Free Innovation Pre-research Fund of Union Hospital/ ; },
mesh = {*Uracil-DNA Glycosidase/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *DNA/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Kinetics ; Humans ; Excision Repair ; *Endodeoxyribonucleases/genetics/metabolism ; *Nanotechnology/methods ; },
abstract = {Uracil-DNA glycosylase (UDG) is a key enzyme in base excision repair and an important biomarker for genomic stability and disease. In many reported sensing systems, uracil excision is coupled to signal generation through additional downstream BER processing steps, resulting in an indirect readout of UDG activity. Here, we report a single-enzyme activated CRISPR-Cas12a nanosystem driven by a subtly balanced double-stranded DNA (dsDNA) substrate. This dsDNA serves as a kinetic gatekeeper that maintains Cas12a in an inert state until UDG-mediated uracil excision disrupts the balance, lowering the energy barrier for crRNA invasion and initiating Cas12a trans-cleavage. This conformationally gated mechanism directly converts a uracil excision event into an amplified CRISPR response without requiring sequential enzymatic processing. The system achieves a 1840-fold discrimination ratio and an ultralow detection limit of 5 × 10[-7] U/mL. Furthermore, a genetically encoded variant enables nuclear localization of Cas12a and dsDNA sensors for in situ imaging of endogenous UDG. The platform visualizes UDG dynamics across distinct cell cycle phases, realizing spatiotemporal mapping of repair activity in living cells. This work introduces a new activation paradigm for CRISPR-Cas12a via subtly balanced dsDNA and establishes a generalizable strategy for precise molecular sensing in complex biological environments.},
}
@article {pmid41793913,
year = {2026},
author = {Li, X and Liu, L and Luo, C and Chen, Z and Shu, B},
title = {Efficient CRISPR/Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa).},
journal = {Plant physiology and biochemistry : PPB},
volume = {232},
number = {},
pages = {111195},
doi = {10.1016/j.plaphy.2026.111195},
pmid = {41793913},
issn = {1873-2690},
mesh = {*Agrobacterium tumefaciens/genetics ; *Plants, Genetically Modified/genetics ; *Fragaria/genetics/growth &amp; development/physiology ; *CRISPR-Cas Systems/genetics ; Regeneration/genetics ; },
abstract = {Recently, an Agrobacterium-mediated CRISPR/Cas9 editing system was successfully applied in a gene function analysis, highlighting its great value for improving strawberry genetics. However, the resulting low transformation rates and long regeneration cycles have limited its extensive application. Based on the biological characteristics of crown branching, an Agrobacterium tumefaciens-mediated CRISPR/Cas9 gene editing system was developed to increase the transformation rate and decrease the regeneration time of cultivated strawberry. Two single guide (sg)RNAs were designed for the strawberry anthracnose-related transcription factor, WRKY (FxaC_17g55530), and its alleles. These sgRNAs were inserted into pKSE401G using pCBC-DT1T2; sgRNAs for subtilisin-like protease (FxaC_22g21540) were designed and cloned in a similar manner. After 10 days of co-cultivating plantlets (without media supply of carbon) and GV3101, 65 (61.9%) and 72 (68.6%) GFP-positive calluses for the two genes were respectively obtained from the crown of 105 plantlets. The positive calluses were removed from the crown and placed on Murashige and Skoog media containing 3 mg/L thidiazuron and 0.2 mg/L indole-3-butyric acid. After 50-80 days, 3-5 positive shoots were obtained from different positive calluses for each gene. The three T0 lines for FxaC_17g55530 and FxaC_22g21540 were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2. Overall, a quick and effective CRISPR-Cas 9 gene editing system was developed for cultivated strawberry, highlighting the applicability of gene editing in breeding and gene function analysis.},
}
@article {pmid41794282,
year = {2026},
author = {Xue, Z and Lan, J and Zhao, Y and Yu, P and Liu, L and Lu, B and Yang, F},
title = {A novel rat model harboring two BDNF gene mutations exhibiting autism-like behaviors and cognitive impairments.},
journal = {Neuropharmacology},
volume = {291},
number = {},
pages = {110911},
doi = {10.1016/j.neuropharm.2026.110911},
pmid = {41794282},
issn = {1873-7064},
mesh = {Animals ; *Brain-Derived Neurotrophic Factor/genetics/metabolism ; *Disease Models, Animal ; Male ; Rats ; Social Behavior ; Receptor, trkB/metabolism/genetics ; *Autism Spectrum Disorder/genetics ; Hippocampus/metabolism ; Mutation ; Cognition Disorders/genetics ; *Autistic Disorder/genetics ; Rats, Sprague-Dawley ; Flavones/pharmacology ; Prefrontal Cortex/metabolism ; CRISPR-Cas Systems ; },
abstract = {Autism spectrum disorder (ASD) is a type of neurodevelopmental disorder that occurs most frequently in early childhood, affecting approximately 1% of the global population. Currently, the elusive nature of the pathological mechanisms underlying ASD precludes the existence of a definitive, effective treatment approach. In this study, we have successfully generated a novel ASD rat model utilizing CRISPR/Cas9 technology, offering a promising platform for further investigation and potential therapeutic interventions. The model is characterized by two crucial point mutations occurring at key enzyme cleavage sites of brain-derived neurotrophic factor (BDNF), thereby causing disruptions in enzyme cleavage processes. The phenotypes of this rat model faithfully recapitulate the salient deficits frequently encountered in ASD patients, exhibiting impairments in social behavior, cognition, and anxiety, along with neuronal abnormalities with key brain regions, notably the hippocampus (HPC) and medial prefrontal cortex (mPFC). Through preliminary RNA-seq analysis, we found changes in gene expression patterns related to synapses and neuronal excitability in these areas, providing new insights into the pathogenesis of ASD. Furthermore, our utilization of 7,8-dihydroxyflavone (7,8-DHF), a robust enhancer for the upregulation of both BDNF and TrkB mRNA and simultaneously activates the BDNF-TrkB signaling pathway, appears to strengthen the BDNF-TrkB signaling cascade. This intervention modifies firing patterns of neuronal spikes and synaptic transmission, which may contribute to the amelioration of ASD-like social interaction behavior exhibited in BDNF[met/leu] rats. Our research not only deepens our understanding of the pathogenesis of ASD, but also present encouraging avenue for early intervention strategies and treatments.},
}
@article {pmid41794473,
year = {2026},
author = {Safenkova, IV and Kamionskaya, MV and Serchenya, TS and Sviridov, OV and Dzantiev, BB and Zherdev, AV},
title = {CRISPR/Cas12a and fork-shaped probe enhance LAMP-LFT integration for equipment-free detection of Listeria monocytogenes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {230},
number = {},
pages = {118592},
doi = {10.1016/j.foodres.2026.118592},
pmid = {41794473},
issn = {1873-7145},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Food Microbiology/methods ; *CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Metal Nanoparticles/chemistry ; Limit of Detection ; },
abstract = {Listeria monocytogenes is an important foodborne pathogen associated with high mortality rates, especially among vulnerable populations, and therefore requires diagnostic methods that are not only highly sensitive and rapid but also suitable for use in resource-limited settings. In this study, we developed an isothermal amplification assay integrated with a lateral flow test (LFT) for reliable detection of L.monocytogenes. Two assay formats were designed and compared: (1) loop-mediated isothermal amplification (LAMP) with LFT detection of fluorescein- and biotin-labeled amplicons, and (2) LAMP combined with CRISPR/Cas12a, using LFT to detect a cleaved fork-shaped enhanced probe labeled with three fluoresceins. Both LFT formats utilized a common conjugate of gold nanoparticles and anti-fluorescein antibodies (anti-FAM), but differed in the test zone immobilization strategy: streptavidin for LAMP, and anti-FAM for LAMP-CRISPR/Cas12a. Among 12 tested (primer - label) combinations, the most effective was identified, but the sensitivity of the LAMP-LFT format was limited by high signal variability. In contrast, the LAMP-CRISPR/Cas12a assay, targeting LAMP amplicons with guide RNA, achieved a detection limit of 0.9 copies/reaction-representing > 20,000-fold improvement in detectable DNA concentration compared with LAMP-LFT-and comparable to fluorescence-based detection techniques. The LAMP-CRISPR/Cas12a-LFT assay was first reported to detect L.monocytogenes cells following thermal lysis (10 min at 95 °C), with a single-cell detection limit (0.2 cells/reaction in buffer, 1 cells/reaction in spiked milk) and an analysis time of 80 min. These results demonstrate the potential of the approach for sensitive, equipment-free detection of foodborne pathogens in complex food matrices.},
}
@article {pmid41795185,
year = {2026},
author = {Wang, Y and Liu, X and Xuan, W and Huang, W and Zhu, Y and Mao, C and Liu, Y},
title = {Inhalable lipid nanoparticles for macrophage-specific STING gene editing to ameliorate pulmonary fibrosis.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {6},
pages = {3353-3372},
pmid = {41795185},
issn = {1525-0024},
mesh = {Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; STING Protein ; Mice ; *Membrane Proteins/genetics/metabolism ; Disease Models, Animal ; *Gene Editing/methods ; *Macrophages, Alveolar/metabolism ; *Lipids/chemistry ; Humans ; *Macrophages/metabolism ; cGAS-STING Signaling Pathway ; CRISPR-Cas Systems ; Administration, Inhalation ; *Idiopathic Pulmonary Fibrosis/therapy/genetics/metabolism/pathology ; Liposomes ; },
abstract = {Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease with limited therapeutic options. The stimulator of interferon genes (STING) signaling pathway, particularly in alveolar macrophages, has been identified as a critical driver of fibrosis. However, achieving efficient and selective drug delivery to these pathogenic macrophages in the distal lung represents the major hurdle that hinders its clinical translation. To overcome this, we employed a systematic orthogonal screening strategy to develop a macrophage-targeted lipid nanoparticle (LNP) platform. Our optimized formulation, mCas9/gSting@DOPS, demonstrated an over 7-fold greater macrophage expression efficiency compared with commercial formulations and was engineered for precise in vivo Sting1 gene editing. This system leverages surface phosphatidylserine for selective uptake and encapsulates a CRISPR-Cas9 mRNA payload. Following inhalation, LNPs selectively accumulated in target macrophages within a murine model of pulmonary fibrosis. This targeted delivery resulted in effective Sting1 gene disruption, suppression of downstream STING signaling, and reduced secretion of pro-fibrotic cytokines. Functionally, treatment with mCas9/gSting@DOPS LNPs significantly attenuated collagen deposition, alleviated alveolar collapse, and remodeled the fibrotic immune microenvironment. Notably, this therapeutic approach prolonged survival without evidence of systemic toxicity. Our findings establish that our orthogonally optimized LNP platform enables potent and clinically viable molecular therapy for IPF by efficiently targeting pulmonary macrophages.},
}
@article {pmid41795439,
year = {2026},
author = {Zhu, L and Yang, C and Bernards, R and Wang, C},
title = {CLIM-TIME links genetic cancer drivers to immune landscapes.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1263-1265},
doi = {10.1016/j.cell.2026.01.014},
pmid = {41795439},
issn = {1097-4172},
mesh = {Humans ; *Neoplasms/genetics/immunology/therapy ; Immunotherapy ; Animals ; Tumor Microenvironment/immunology ; CRISPR-Cas Systems ; },
abstract = {Immunotherapy resistance is associated with immune-privileged microenvironments, yet the interacting role of tumor-intrinsic genetics remains unclear. In this issue of Cell, Wang et al. introduce CLIM-TIME, a spatially resolved in vivo CRISPR screening platform linking loss of tumor suppressor genes to distinct metastatic immune architectures and divergent responses to immunotherapy.},
}
@article {pmid41796459,
year = {2026},
author = {Matsuoka, T and Oda, K and Iwashita, K and Watanabe, J},
title = {Identification of DeuA, an Aspergillus oryzae-derived deuterolysin-like metalloprotease, as the predominant thermostable protease in soy sauce.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {6},
pages = {445-449},
doi = {10.1016/j.jbiosc.2026.02.010},
pmid = {41796459},
issn = {1347-4421},
mesh = {*Aspergillus oryzae/enzymology/genetics ; *Soy Foods/microbiology ; *Metalloproteases/genetics/metabolism/chemistry ; Fermentation ; Enzyme Stability ; *Peptide Hydrolases/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; Temperature ; Hot Temperature ; CRISPR-Cas Systems ; },
abstract = {The thermostable proteases present in soy sauce can degrade proteins in processed foods, such as boiled eggs and fish cakes, leading to undesirable textural changes in the product. In this study, we identified DeuA, a deuterolysin-like metalloprotease from Aspergillus oryzae, as the major contributor to thermostable protease activity during soy sauce fermentation. Using CRISPR/Cas9-based genome co-editing, we generated deuA-knockout mutants and mutants of the related gene deuB, which encodes a deuterolysin-like metalloprotease, and evaluated their enzymatic activity levels under solid-state culture conditions mimicking soy sauce koji fermentation. The ΔpyrGΔdeuA strain exhibited a marked reduction in thermostable protease activity, with residual activity barely detectable in both the koji extracts and the final soy sauce, whereas knockout of deuB had no significant effect. These results indicate that DeuA is the predominant contributor to thermostable protease activity in soy sauce. The knockout of deuA did not affect other key brewing parameters such as the nitrogen or sugar contents, indicating the potential of this gene as a target for strain improvement. Our findings establish DeuA as an essential thermostable protease in soy sauce and provide a foundation for the development of brewing strains with improved industrial applicability that will not affect the textural stability of processed foods.},
}
@article {pmid41796733,
year = {2026},
author = {Lin, J and Wang, Y and Zeng, B and Chen, Z and Lin, X and Zeng, T},
title = {CRISPR-Cas12a/Cas13a in cancer molecular diagnosis.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {587},
number = {},
pages = {120934},
doi = {10.1016/j.cca.2026.120934},
pmid = {41796733},
issn = {1873-3492},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; *Molecular Diagnostic Techniques/methods ; Biomarkers, Tumor/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Cancer remains a leading cause of global mortality, with early diagnosis being pivotal for improving treatment outcomes. Traditional tissue biopsy is limited by its invasiveness, inability to capture tumor heterogeneity, and failure to support dynamic monitoring. Liquid biopsy has emerged as a non-invasive alternative, enabling the analysis of circulating tumor biomarkers (e.g., ctDNA, miRNAs, exosomes) in bodily fluids. However, current liquid biopsy technologies (e.g., NGS, ddPCR) suffer from high costs, complex workflows, poor standardization, and insufficient sensitivity for low-abundance biomarkers. The CRISPR-Cas systems, particularly Cas12a and Cas13a, have revolutionized molecular diagnostics due to their programmable sequence recognition, robust signal amplification via trans-cleavage/collateral cleavage activity, and compatibility with point-of-care testing (POCT). Cas12a targets DNA molecules, enabling sensitive detection of gene mutations and DNA methylation, while Cas13a specifically recognizes RNA, facilitating direct analysis of miRNAs and viral RNAs. Additionally, these systems have been extended to non-nucleic acid biomarkers (e.g., proteins, exosomes) through signal conversion strategies. This review summarizes the latest advances in CRISPR-Cas12a/Cas13a-based biosensors for cancer molecular diagnosis, including the detection of gene mutations, epigenetic modifications, miRNAs, tumor-associated viruses, and non-nucleic acid biomarkers. We critically analyze current challenges (e.g., PAM dependence, matrix interference, multiplexing limitations, clinical validation gaps) and discuss future perspectives, such as engineering PAM-less Cas variants, integrating nanotechnology, microfluidics, and artificial intelligence/artificial intelligence (AI), and advancing clinical standardization. This review aims to provide a comprehensive reference for the development and clinical translation of CRISPR-based cancer diagnostic technologies.},
}
@article {pmid41797538,
year = {2026},
author = {Fang, M and Yap, J and Fei, M and Gong, M and Li, N and Lu, Y and Yu, M and Xu, Y and Wu, F and Gao, H and Sun, D},
title = {LysR-type regulator LrhA promotes CRISPR-Cas immunity in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41797538},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 31930003//National Natural Science Foundation of China/ ; 2020C02031//Key Research and Development Program of Zhejiang Province/ ; LHDMY23H160003//Natural Science Foundation of China/ ; YS2022005//Natural Science Foundation of China/ ; 2026C02A1080//Zhejiang Lingyan Research and Development Program/ ; LMRY26H200010//Joint Funds of the Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/virology/immunology/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; *Transcription Factors/metabolism/genetics ; Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics ; Operon ; },
abstract = {The CRISPR-Cas defense system safeguards prokaryotes against foreign genetic elements. Its activity is determined by the combined effects of adaptation and interference. However, the dynamic regulation of these two processes remains not fully understood. In this study, we identify the LysR-type transcriptional regulator LrhA, which is differentially expressed in various Escherichia coli strains, as a novel CRISPR-Cas activator that plays a critical role in modulating host defense levels. In a representative strain expressing a high level of LrhA, the regulator enhances CRISPR-Cas-mediated adaptive immunity against bacteriophage infection by promoting cas gene transcription through direct interaction with the promoter of the cas operon. Moderate activation of cas genes by weakly expressed LrhA in another representative strain efficiently accelerates the clearance of horizontally transferred CRISPR-targeted plasmids by enhancing spacer acquisition via interference-driven adaptation. This divergence, likely a result of genome evolution, suggests that adaptive immunity is optimized with intermediate transcription levels of cas genes by triggering positive feedback between adaptation and interference. Collectively, our findings highlight the crucial role of LrhA in fine-tuning host defense responses.},
}
@article {pmid41799755,
year = {2026},
author = {Vadrot, N and Moulin, M and Ferreiro, A and Richard, P and Buendia, B},
title = {LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2.},
journal = {International journal of medical sciences},
volume = {23},
number = {3},
pages = {741-757},
pmid = {41799755},
issn = {1449-1907},
mesh = {Cell Proliferation/genetics ; Cell Differentiation/genetics ; Animals ; *Myocytes, Cardiac/metabolism/pathology ; Mice ; CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics/metabolism ; Humans ; *Membrane Proteins/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; Myocardium/pathology ; Cell Line ; Rats ; *Aging/genetics/pathology ; Gene Knockdown Techniques ; MEF2 Transcription Factors/metabolism/genetics ; },
abstract = {Haploinsufficiency of Lap2 alpha (LAP2a), a nuclear partner of Lamins A/C, has been associated with cardiac disease in rare cases, but LAP2a function remains largely unknown. To investigate the functional role of LAP2a in cardiomyocytes, we generated clones of embryonic myocardium-derived H9C2 cells in which LAP2a expression was specifically reduced through gene editing of the LAP2a gene Tmpo by CRISPR-Cas9. Downregulation (+/-) and absence (-/-) of LAP2a expression led to a decreased proliferation capacity of cardiomyocytes in vitro. Upon differentiation, the expression of myocardial markers (alpha cardiac Actin 1/Actc1, cardiac Troponin T2/Tnnt2, Myosin-2/Myh2 and Myosin-7/Myh7) was higher in LAP2a -/- cells compared to LAP2a +/- or LAP2a +/+ cells, with consistently higher expression of their upstream regulator Mef2c in LAP2a-devoid cells. These results suggest that LAP2a promotes cardiomyocyte proliferation and negatively modulates cardiomyocyte differentiation, through mechanisms including Mef2c regulation. Accordingly, normal protein expression of LAP2a was downregulated upon cardiomyocyte differentiation, contrary to LAP2b and a LAP2b-related shorter isoform. The latter tended to increase upon differentiation in all cells, most significantly in the LAP2a -/- clone. In postnatal mouse hearts, LAP2a levels were higher in the right than in the left ventricle, and lowest in the septum. The LAP2a:LAP2b ratio was much lower in murine hearts than in H9C2 cells, and decreased significantly upon ageing, specifically in the left ventricle. Finally, our data show that expression of the nuclear envelope proteins LEMD2 and Lamin A might be influenced by LAP2a upon cardiac differentiation. Our results show that LAP2 expression is finely regulated upon cardiac differentiation in vitro and is dependent on age and heart compartment in vivo. They contribute to clarifying the potential impact of genetic LAP2a defects and their connection with heart disease, possibly including reduced cardiomyoblast proliferation, increased cardiomyocyte differentiation and altered nuclear envelope remodelling.},
}
@article {pmid41800529,
year = {2026},
author = {Yan, J and Wang, S and Xiong, S and Luo, X and Li, Y and Deng, X},
title = {Cas12a Trans-Cleavage of Hairpins Triggers a CHA Cascade for an Ultrasensitive SERS Aptasensor.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2828-2839},
doi = {10.1021/acssensors.6c00041},
pmid = {41800529},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Associated Proteins/chemistry/metabolism ; Limit of Detection ; DNA/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/chemistry/metabolism/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Silver/chemistry ; },
abstract = {Ultrasensitive detection of non-nucleic acid biomarkers using CRISPR/Cas12a remains a major challenge due to the lack of intrinsic signal amplification. Moreover, linear DNA reporters fail to maintain efficient downstream signal amplification after trans-cleavage, while pre-amplification procedures often lead to nonspecific signals, thereby compromising assay accuracy, particularly in complex biological matrices. Here, a highly SERS aptasensor is developed by harnessing CRISPR/Cas12a-driven trans-cleavage of hairpin substrates to trigger catalytic hairpin assembly (Cas12a-CHA), achieving robust cascade signal amplification. Target recognition is converted into customizable DNA triggers that precisely activate Cas12a, while a thymine-rich DNA/RNA reporter with dT5 motifs facilitates high enhances trans-cleavage efficiency, sustaining continuous CHA cycles. The integration of AuNF@4-MBA@Ag@H2 SERS nanotags that generate abundant plasmonic hotspots, the system provides significantly enhanced Raman readout. Benefiting from synergistic molecular amplification and nanostructure engineering, the aptasensor achieves an ultralow detection limit of 1.97 × 10[-17] g/mL, nearly 20,000-fold higher sensitivity than traditional sandwich assays, along with a broad dynamic range and high specificity. Furthermore, it exhibits excellent uniformity, reproducibility, stability, and recovery in spiked serum samples, using FGF2 used as a representative biomarker to validate its performance, highlighting great potential for clinical diagnostics and real-world applications.},
}
@article {pmid41802204,
year = {2026},
author = {de Paula, JA and de Araújo, MRB and Sousa, EG and Prates, FD and Castro, DLC and Fonseca, PAS and Brenig, B and Felice, AG and Pacheco, LGC and Viana, MVC and Azevedo, VAC and de Castro Soares, S},
title = {Clonal clusters of multidrug-resistant Brazilian Corynebacterium striatum strains reveal putative virulence traits.},
journal = {Journal of applied microbiology},
volume = {137},
number = {4},
pages = {},
doi = {10.1093/jambio/lxag070},
pmid = {41802204},
issn = {1365-2672},
support = {88887.950984/2024-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 32001010068P4//Federal University of Minas Gerais/ ; 311249/2023-0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; APQ-01323-15//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais/ ; },
mesh = {Brazil ; Virulence/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; *Corynebacterium/genetics/pathogenicity/drug effects/isolation &amp; purification/classification ; Phylogeny ; Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; *Corynebacterium Infections/microbiology ; Virulence Factors/genetics ; },
abstract = {OBJECTIVES: This study presents the comparative analysis of 26 multidrug-resistant (MDR) C. striatum strains isolated in Brazil.<br><br>METHODS: Additional genomes from international sources were incorporated. The analyses encompassed in vitro antimicrobial susceptibility testing and an in silico workflow for genomic similarity comparison, phylogenetic reconstruction, genomic clustering, pangenome analysis, mobilome content, virulence prediction, and functional annotation of unique proteins and putative virulence clusters.<br><br>RESULTS: Strong in silico evidence of clonality among several Brazilian isolates was obtained at the same time that some strains consistently indicated a divergent genomic profile. There are 196 unique coding sequences (CDSs) across the Brazilian IHPs. Of particular interest, strain IHP2030 carried an exclusive fimbria, sharing less than 50% similarity with other fimbriae in the dataset. Yet, structural predictions suggested conservation of key structural domains typically associated with fimbrial proteins. Mobilome content analysis revealed that IHPs strains were overall similar, differing primarily in the number of insertion sequences and in the presence or absence of CRISPR-Cas defense systems. Regarding virulence, an exclusive cluster in IHP2050 and IHP2060 suggests adaptive advantages associated with their respective environments of isolation.<br><br>CONCLUSION: This study reveals a complex genomic landscape among Brazilian MDR C. striatum strains, marked by clonal dissemination alongside strain-level genetic variation in accessory genomes, mobilome composition, and virulence-associated gene repertoires, providing genomic evidence of diversification within hospital-associated lineages.},
}
@article {pmid41802999,
year = {2026},
author = {Hao, M and Zhou, M and Pan, F and Liu, T and Li, Y and Su, N and Ashfaq, A and Song, M and Wang, H and Wang, W and Liu, J and Li, C and Fu, L and He, P and Hu, Q and Mei, D and Cheng, H},
title = {Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70221},
pmid = {41802999},
issn = {1744-7909},
support = {2025BEA003//the Major Program (JD) of Hubei Province/ ; CAAS-CSNCB-202303//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025AFB468//Hubei Provincial Natural Science Foundation of China/ ; CARS-12//Earmarked Fund for China Agriculture Research System/ ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.},
}
@article {pmid41803012,
year = {2026},
author = {Cao, L and Na, D and Cheng, J and Zhao, L and Ye, Q and Tan, WS},
title = {The Innovative Multi-Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression in CHO Cells.},
journal = {Biotechnology and bioengineering},
volume = {123},
number = {6},
pages = {1503-1517},
doi = {10.1002/bit.70185},
pmid = {41803012},
issn = {1097-0290},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Antibodies, Monoclonal/genetics/biosynthesis ; *Tyrosine/biosynthesis/metabolism/genetics ; *Metabolic Engineering/methods ; Recombinant Proteins/genetics/biosynthesis ; Cricetinae ; CRISPR-Cas Systems ; },
abstract = {The production of complex biologics in Chinese hamster ovary (CHO) cells is constrained by the lack of selection systems capable of coordinating multiple transgenes. Conventional single-marker systems have low saturable thresholds that limit enrichment efficiency, while multi-auxotrophic platforms often impose metabolic burdens. Here, we present a rationally designed tyrosine-auxotrophic system that overcomes these limitations by establishing a high-threshold cooperative selection mechanism. This is achieved through the reconstruction of an essential pathway comprising pterin-4α carbinolamine dehydratase 1 (PCBD1), phenylalanine hydroxylase (PAH), and quinoid dihydropteridine reductase (QDPR). We generated a triple-knockout CHO host via CRISPR/Cas9, wherein survival under tyrosine deprivation became strictly dependent on the balanced co-expression of all three rescue genes. This architecture creates a selection pressure that is not saturable by any single gene, enabling efficient co-enrichment. Applied to monoclonal antibody (mAb) production, the system enriched triple-positive populations to 97.49%, resulting in significantly enhanced homogeneity and coordinated upregulation of antibody chain expression. Optimized pools achieved titers of 0.35 g/L in fed-batch and 1.60 g/L in perfusion cultures without tyrosine feeding. Consequently, pathway reconstitution rewired central metabolism, reducing byproducts and enhancing biosynthesis. This antibiotic-free multi-marker platform establishes a new paradigm for stringent multigene co-expression, advancing CHO cell engineering for next-generation biologics.},
}
@article {pmid41803127,
year = {2026},
author = {Zhou, R and Liu, Y and Zhang, Q and Yin, Z and Tong, J and Zhang, C and Zhang, L and Li, X and Zhao, Y and Zhang, S and Liu, Z and Chen, W and Ji, N and Zhang, H and Li, Z and Yin, H and Zuo, S and Wei, Y},
title = {Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41803127},
issn = {2041-1723},
support = {2022YFC3400400//National Science Foundation of China | Key Programme/ ; 23ZYCGSY00750//Tianjin Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; },
mesh = {Cryoelectron Microscopy ; Protein Domains ; *Bacteriophages ; CRISPR-Cas Systems ; Escherichia coli/genetics/virology/metabolism ; Catalytic Domain ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.},
}
@article {pmid41803497,
year = {2026},
author = {Perez Taboada, V and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafen proteins mediate phage defence.},
journal = {Nature microbiology},
volume = {11},
number = {4},
pages = {1037-1048},
pmid = {41803497},
issn = {2058-5276},
support = {R00AI171893//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1T32GM156737-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Escherichia coli/virology/genetics/metabolism ; RNA, Transfer/metabolism ; *Bacterial Proteins/metabolism/genetics ; *Coliphages/physiology ; *Ribonucleases/metabolism/genetics ; Humans ; *Enterobacteriaceae/genetics/enzymology/virology ; *Bacteriophages ; Escherichia coli Proteins ; },
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here we demonstrate that prokaryotic Schlafen nucleases are widespread antiviral effectors that protect bacteria from bacteriophages and are fused to a diverse array of phage-sensing domains. We expressed seven Enterobacterales Schlafen systems in Escherichia coli, identifying two that confer defence against coliphages. We focused on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrated that it recognizes tail assembly chaperones of T5-like phages. Upon activation, the Schlafen nuclease cleaves both E. coli and phage-encoded tRNAs and restricts T5 phage by reducing its burst size. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}
@article {pmid41803672,
year = {2026},
author = {Shi, L and Yang, X and Wu, M and Zhao, C and Wu, J and Zuo, E},
title = {A programmable platform enabling targeted chromosome substitution and cross-species stability profiling.},
journal = {Protein &amp; cell},
volume = {17},
number = {6},
pages = {528-542},
pmid = {41803672},
issn = {1674-8018},
support = {32371549//National Natural Science Foundation of China/ ; 32101223//National Natural Science Foundation of China/ ; 82101872//National Natural Science Foundation of China/ ; CAAS-CSIAF-202401//Innovation Program of Chinese Academy of Agricultural Sciences/ ; CAAS-BRC-AFIS-2025-03//Innovation Program of Chinese Academy of Agricultural Sciences/ ; CAAS-SCAB-202301//Innovation Program of Chinese Academy of Agricultural Sciences/ ; },
mesh = {Animals ; Humans ; Mice ; Male ; *CRISPR-Cas Systems ; *Y Chromosome/genetics ; Species Specificity ; },
abstract = {Chromosome substitution strains (CSS) are critical tools for dissecting complex traits, although iterative breeding steps and intraspecific compatibility requirements limit conventional approaches. Here, we developed a Targeted chromosome Elimination And Microcell-mediated chromosome transfer platform (TEAM) for chromosome replacement combining CRISPR/Cas9-mediated chromosome elimination with microcell-mediated chromosome transfer (MMCT). Using this approach, we substituted the endogenous mouse Y chromosome (chrY) with either the mouse or human Y chromosome. Intraspecies substitutions yielded karyotypically stable embryonic stem cells that supported development into adult males. By contrast, in interspecies CSS, human chrY displayed severe instability and progressive DNA damage. Despite partial transcription of human chrY genes, recipient animals exhibited systemic inflammation, high rates of neonatal death, and poor growth. Reduced CENP-A levels were observed at human chrY centromeres, leading to segregation errors, micronuclei formation, and widespread chromosome rearrangements. This technology enables programmable construction of chromosome substitution models for investigating chromosomal function, genome evolution, and synthetic karyotype design in mammals.},
}
@article {pmid41804827,
year = {2026},
author = {Parada, F and Cabedo-Díaz, P and Cerda, A and Osorio-Navarro, C and Toledo, JA and Villalobos-González, L and Handford, M and Pimentel, P},
title = {CRISPR/dCas9-Mediated BRL3 Activation Enhances Growth and Metabolic Resilience Under Osmotic Stress in Nicotiana tabacum.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70816},
doi = {10.1111/ppl.70816},
pmid = {41804827},
issn = {1399-3054},
support = {3240290//ANID-FONDECYT Postdoctoral Project/ ; 3210631//ANID-FONDECYT Postdoctoral Project/ ; 1231417//ANID-FONDECYT Regular Project/ ; RF23F0002//ANID Fortalecimiento de Centros Regionales Project/ ; NCN2024_047//ANID-Millennium Science Initiative Program/ ; },
mesh = {*Nicotiana/drug effects/growth &amp; development/metabolism ; *Osmotic Pressure ; CRISPR-Cas Systems ; *Brassinosteroids/metabolism ; *Genetic Vectors/pharmacology ; Transcriptional Activation ; Plant Leaves/anatomy &amp; histology/drug effects ; Plants, Genetically Modified/growth &amp; development/metabolism ; },
abstract = {Brassinosteroids (BRs) are crucial plant hormones that influence growth and stress adaptation. However, the specific function of the BR receptor BRL3 under osmotic stress remains largely unexplored outside Arabidopsis thaliana. In this study, we used a CRISPR/dCas9-based transcriptional activation (CRISPRa) system to upregulate the Nicotiana tabacum BRASSINOSTEROID INSENSITIVE-LIKE 3 receptor (NtBRL3) and assessed its impact on osmotic stress tolerance. Synthetic activation vectors were constructed using Loop Assembly, featuring dCas9-6TAL-VP128 modules driven by either a constitutive (CaMV35S) or ABA-inducible (SlAREB) promoter, paired with dual sgRNAs targeting the NtBRL3 promoter. Transient Agrobacterium-mediated transformation followed by PEG treatment was used to impose osmotic stress. RT-qPCR confirmed a 3- to 4-fold activation of NtBRL3 transcripts in CRISPRa-infiltrated leaves. The stress-inducible SlAREB promoter produced the strongest improvements, yielding nearly four-fold higher leaf biomass and a five-fold increase in root biomass relative to PEG-stressed controls. Both constructs reduced malondialdehyde (MDA) accumulation, indicating diminished oxidative damage, and modulated osmoprotectant balance, including reduced root proline and increased total soluble solids, particularly under SlAREB-driven activation. Histological segmentation revealed promoter-dependent anatomical remodeling, with NtBRL3-activated plants exhibiting a higher frequency of enlarged leaf cells and expanded tissue domains, consistent with brassinosteroid-mediated structural plasticity. Collectively, these findings demonstrate that CRISPR/dCas9-mediated transcriptional activation of NtBRL3 enhances osmotic stress resilience in tobacco through coordinated biomass recovery, oxidative stress mitigation, osmolyte homeostasis, and tissue remodeling. This transient, non-integrative CRISPRa approach provides a robust synthetic biology framework for dissecting BR signaling and engineering stress-tolerant crops.},
}
@article {pmid41805130,
year = {2026},
author = {Yu, W and Yuan, L and Zhou, W and He, L and Huang, X and Yu, J and Deng, J and Zhang, T and Hu, Y and Zhang, Y and Chen, S},
title = {Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41805130},
issn = {1362-4962},
support = {2021YFA1300901//National Key R&amp;D Program of China/ ; 2022YFA1303500//National Key R&amp;D Program of China/ ; GZNL2024A01024//Guangzhou National Laboratory/ ; //National Key Research and Development Program of China/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/metabolism/drug effects ; *Cyclic GMP/analogs &amp; derivatives/metabolism ; *CRISPR-Cas Systems/genetics ; Oxidative Stress/genetics ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Stress, Physiological/genetics ; Promoter Regions, Genetic ; },
abstract = {Mycobacterium tuberculosis (Mtb) possesses a type III-A CRISPR-Cas system and has anti-plasmid immune activity. However, whether this system exerts other additional functions remains to be characterized. Here, we investigated the in vivo roles of the Mtb CRISPR-Cas system. We show that this system is transcriptionally dependent and exhibits limited ability to counteract exogenous nucleic acids, primarily through the Csm6 protein rather than the Cas10 HD domain. We further demonstrate that this system plays a role in mitigating oxidative stress and antibiotic treatment, a function mainly mediated by the Cas10 HD domain. Importantly, through transposon library screening, we identified oligoribonuclease (Orn) as a regulatory protein of the Mtb CRISPR-Cas system. Deletion of the orn gene resulted in elevated c-di-GMP levels. A subsequent biotin-labeled c-di-GMP pull-down assay identified the transcriptional regulator Rv3058. Knockdown of rv3058 significantly increased cas6 promoter activity, and its transcriptional repressor function was directly modulated by c-di-GMP. This regulatory pathway enhances stress defense by activating multiple protective pathways, including DNA repair, cell envelope maintenance, and iron homeostasis regulation. Together, we conclude that the regulation of the CRISPR-Cas system by Orn-mediated c-di-GMP contributes to oxidative and antibiotic stress responses in Mtb.},
}
@article {pmid41805294,
year = {2026},
author = {Anfang, M and Yahya, RH and Caldararu, O and Ben Yaakov, S and Landau, U and Berman, A and Hu, Y and Belew, ZM and Crocoll, C and Xu, D and Nour-Eldin, HH and Mayrose, I and Shani, E},
title = {Targeting redundant gene families: A multiplexed, tissue-specific CRISPR toolbox for Arabidopsis genetic screens.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117055},
pmid = {41805294},
issn = {2211-1247},
mesh = {*Arabidopsis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Plant ; *Multigene Family ; *CRISPR-Cas Systems/genetics ; Organ Specificity/genetics ; *Genetic Testing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Genome-scale targeted CRISPR libraries for forward genetic screens in plants are powerful tools for functional analysis, but they suffer from limited spatial control, single sgRNA design, and poor handling of genetic redundancy. We develop multiplexed CRISPR libraries in which each construct contains two sgRNAs that simultaneously target multiple members of a gene family. The libraries can also function at the cell-type-specific and tissue levels. A double-barcoding strategy enables efficient tracking and identification of sgRNA combinations at the plant level without individually sequencing each line. Using this platform, we generate over 1,000 Arabidopsis lines that express sgRNAs targeting 707 transporter genes across 114 gene families involved in nutrient uptake. The multiplexed design increases gene coverage and editing efficiency, underscoring its improved targeting capability to reveal hidden phenotypes. This toolbox provides a scalable resource for multi-targeted genome editing and spatially precise forward genetic screens in plants.},
}
@article {pmid41805349,
year = {2026},
author = {Han, F and Xu, Y and Wang, W and Li, Z and Zhang, Z and Du, R and Xu, Q},
title = {Methylobacterium as a Dual-Function Platform: Advances in C1-Based Biomanufacturing and Plant-Associated Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {11},
pages = {8936-8947},
doi = {10.1021/acs.jafc.5c08026},
pmid = {41805349},
issn = {1520-5118},
mesh = {*Methylobacterium/metabolism/genetics ; Metabolic Engineering ; *Carbon/metabolism ; *Plants/microbiology/metabolism ; },
abstract = {One-carbon (C1) substrates are promising feedstocks for microbial bioproduction. Methylobacterium, known for its exceptional C1 utilization capacity, has emerged as a model microbial chassis for sustainable biomanufacturing. In this review, we first outline the C1 assimilation pathways in Methylobacterium and underscore its potential for producing valuable native metabolites. Furthermore, we then survey the genetic tools available for engineering this genus, including plasmid-based methods, transposon mutagenesis, homologous recombination, and CRISPR/Cas systems. Notably, recent advances in metabolic engineering have significantly expanded its biosynthetic scope, enabling the biosynthesis of diverse non-native compounds. Beyond its biomanufacturing potential, Methylobacterium also serves as a versatile plant growth-promoting bacterium, enhancing plant health and productivity through hormone synthesis, nutrient mobilization, stress mitigation, and induced systemic resistance. Collectively, this work highlights the dual potential of Methylobacterium as a sustainable microbial cell factory for biomanufacturing and a beneficial bioinoculant for agriculture.},
}
@article {pmid41806318,
year = {2026},
author = {Sun, J and Yang, X and Jiang, W and Ji, C and Wu, Y and Sun, H and Liu, X and Yamamoto, M and Tsukamoto, T and Nomura, S and Zhao, J and Ruan, Y and Li, H and Wang, X},
title = {In Vivo CRISPR Screening Identifies the Glutamate Receptor GRIA2 as Promoting Peritoneal Metastasis of Gastric Cancer via Calcium-Dependent β-Catenin Activation.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {28},
pages = {e21746},
pmid = {41806318},
issn = {2198-3844},
support = {ZY2024-003//Scientific Research Foundation of Zhongshan Hospital/ ; },
mesh = {*Stomach Neoplasms/genetics/pathology/metabolism ; Humans ; Animals ; *Peritoneal Neoplasms/secondary/genetics/metabolism ; Mice ; *beta Catenin/metabolism/genetics ; *Receptors, AMPA/genetics/metabolism ; *Calcium/metabolism ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 beta/metabolism/genetics ; Cell Movement/genetics ; CRISPR-Cas Systems/genetics ; Wnt Signaling Pathway/genetics ; },
abstract = {Peritoneal metastasis is the most lethal manifestation of gastric cancer, with a median survival of less than one year, highlighting the need for new therapeutic targets. Through an in vivo genome-wide CRISPR/Cas9 screen, we identified GRIA2, an AMPA-type glutamate receptor subunit, as a key driver of peritoneal metastasis. GRIA2 promotes gastric cancer cell migration, invasion, stemness, and adhesion to mesothelial cells in a glutamate-dependent manner. Mechanistically, glutamate activates GRIA2, enhancing its interaction with GSK-3β and inducing calcium influx, inhibiting GSK-3β kinase activity and stabilizing β-catenin, thereby activating the Wnt/β-catenin signaling pathway. Single-cell RNA sequencing revealed that cancer-associated fibroblasts are the primary source of glutamate in the peritoneal microenvironment, which establishes a paracrine axis that enhances GRIA2-driven metastasis. Pharmacological inhibition of AMPA receptors with NBQX and Selurampanel suppressed peritoneal metastasis in both cell line-derived and patient-derived organoid xenograft (PDOX) mouse models. In clinical analysis, GRIA2 expression in peritoneal metastases correlated with the levels of β-catenin and phosphorylated GSK-3β (serine 9), with high GRIA2 expression predicting poor prognosis. These findings suggest that GRIA2 is a novel therapeutic target, and AMPA receptor antagonists are promising agents for treating gastric cancer peritoneal metastasis.},
}
@article {pmid41806413,
year = {2026},
author = {Jeong, Y and Lee, J and Choi, S and Shin, D and Jang, S and Son, SU and Kang, T and Jung, J and Hwang, J and Lim, EK},
title = {On-site detection of airborne foodborne pathogens using a field-deployable recombinase polymerase amplification and CRISPR/Cas12a cleavage activity assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118571},
doi = {10.1016/j.bios.2026.118571},
pmid = {41806413},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; Food Microbiology ; CRISPR-Cas Systems/genetics ; Listeria monocytogenes/isolation &amp; purification/genetics/pathogenicity ; Staphylococcus aureus/isolation &amp; purification/genetics/pathogenicity ; *Nucleic Acid Amplification Techniques/instrumentation ; *Air Microbiology ; Recombinases/chemistry ; Humans ; Rapid Diagnostic Tests ; Bacillus cereus/isolation &amp; purification/genetics/pathogenicity ; Food Contamination/analysis ; Limit of Detection ; *Bacteria/isolation &amp; purification/genetics/pathogenicity ; Salmonella enteritidis/isolation &amp; purification/genetics ; Endodeoxyribonucleases ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {With the global increase in single-person households, the demand for meal kits is increasing, leading to the development of large-scale food production systems and complex supply chains. However, under the influence of global warming, these systems can be susceptible to food contamination, particularly by airborne foodborne bacteria. Conventional methods for detecting airborne bacteria involve complex, time-consuming, and labor-intensive processes, which limit their applicability for field use and rapid food hygiene surveillance. In the present study, we developed a field-deployable diagnostic platform by combining recombinase polymerase amplification with CRISPR/Cas12a cleaVage Activity (RCCVA assay) for the rapid and sensitive identification of airborne foodborne bacteria. Airborne bacteria were collected using a self-developed electrostatic air sampler and analyzed using a portable isothermal amplification device. The RCCVA assay was designed to detect four major foodborne pathogens: Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus. The limit of detection was measured as 274.9, 4.5, 9.5, and 28.5 culture-forming units (CFU)/mL, respectively, within 45 min. This platform enables early on-site detection of airborne pathogens within approximately 1 h (for the analytical phase) and shows potential for real-time monitoring in food processing environments, thereby contributing to improved public health and food safety.},
}
@article {pmid41806414,
year = {2026},
author = {Kim, H and Kim, D and Han, H and Lee, C and Roh, YH and Han, TS and Lim, EK and Park, J and Ahn, JK and Kang, T and Jung, J and Lee, CY},
title = {On-site microRNA detection with 'off-the-shelf' glucose meter empowered by chimeric probe connecting CRISPR/Cas13a activation to kinases-driven glucose phosphorylation.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118568},
doi = {10.1016/j.bios.2026.118568},
pmid = {41806414},
issn = {1873-4235},
mesh = {*MicroRNAs/blood/genetics/isolation &amp; purification/analysis ; Humans ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems/genetics ; Phosphorylation ; *Glucose/metabolism ; *Stomach Neoplasms/genetics/blood/diagnosis ; Limit of Detection ; Point-of-Care Systems ; Biomarkers, Tumor/blood ; },
abstract = {MicroRNAs (miRNAs) are promising biomarkers for cancer diagnosis due to their stability in body fluids and disease-specific expression profiles. However, current detection methods suffer from limitations including cumbersome workflows, heavy instrumentation for signal readout, or vulnerability in minimizing instrumentation. To address these challenges, we describe a novel point-of-care miRNA detection platform executable with "off-the-shelf", personal glucose meter (PGM), termed 'KEY-FACT (Kinases Ensemble-driven glucose phosphorYlation upon Fuel-Aided CRISPR acTivation)'. Upon recognition of target miRNA, a fuel-assisted toehold-mediated strand displacement reactions liberate guide RNAs (gRNAs) to activate Cas13a to cleave a chimeric reporter probe, producing 2',3'-cyclic adenosine monophosphates (cAMP). Subsequent dephosphorylation and kinases ensemble-mediated phosphorylation/dephosphorylation cycles lead cAMP to consume a large amount of glucose. A user can immediately measure resulting glucose level change with PGM on the spot. This strategy allows sensitive, prompt detection of miR-135b, a gastric cancer (GC) biomarker, with a limit of detection (LOD) of 1.4 pM within 2 h. KEY-FACT is specific to the target miRNA and is applicable to body fluids such as human serum with dilution (95.2% < recovery rates <104.3%, coefficients of variation ≤13%). Owing to its simple probe design, KEY-FACT was readily expanded to detect another GC biomarker, miR-21, with comparable sensitivity (LOD = 1.5 pM). The proposed platform fulfills minimal instrumentation and thus enables cost-effective, field-deployable analysis, paving the way for practical, on-demand miRNA diagnostics.},
}
@article {pmid41806830,
year = {2026},
author = {Escobar, M and Malik, SA and Srinivasa, MA and Mendez-Sosa, MA and Miller, JM and Lydon, SL and Luong, SN and Mathew, PR and Abouleisa, RRE and Chakravarty, S and Pathan, S and Mohamed, TMA and Ghanta, RK and Hilton, IB},
title = {CRISPR-Cas-based activation of PPARGC1A boosts endogenous mitochondria and enhances cardiac function after myocardial infarction.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {6},
pages = {3320-3333},
pmid = {41806830},
issn = {1525-0024},
support = {R01 HL166280/HL/NHLBI NIH HHS/United States ; R01 HL163258/HL/NHLBI NIH HHS/United States ; R01 HL147921/HL/NHLBI NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; R15 HL168688/HL/NHLBI NIH HHS/United States ; 25TPA1463933/AHA/American Heart Association-American Stroke Association/United States ; 917025/AHA/American Heart Association-American Stroke Association/United States ; R01 HL174616/HL/NHLBI NIH HHS/United States ; },
mesh = {*Myocardial Infarction/genetics/therapy/metabolism/physiopathology ; Humans ; *Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Myocytes, Cardiac/metabolism ; Energy Metabolism ; Mice ; *Mitochondria/metabolism/genetics ; Transcriptional Activation ; *Mitochondria, Heart/metabolism/genetics ; Disease Models, Animal ; Gene Expression Regulation ; },
abstract = {Insufficient energy supply due to impaired mitochondria has emerged as a key pathological factor in the development of heart failure (HF) after myocardial infarction (MI). Unfortunately, no current therapeutic strategies directly augment myocardial energy production. While mitochondrial biogenesis is orchestrated by the activity of multiple genes, activation of PPARGC1A, a key regulator, can increase cellular mitochondria; however, supraphysiological levels of PPARGC1A result in adverse tissue remodeling and heart dysfunction. CRISPR activation (CRISPRa) technologies present a unique opportunity to address these shortcomings, as they enable tunable control over endogenous target gene expression. Here, we demonstrate that transcriptional activation of PPARGC1A using CRISPRa increases cellular mitochondria in human cell types. This effect is mediated through the activation of transcriptional programs driving mitochondrial biogenesis, mitochondrial function, and cellular bioenergetics. These activated transcriptional programs synergize to increase ATP production and reserve capacity in human cardiomyocytes. CRISPRa targeting of PPARGC1A in vivo increases cardiac mitochondria to recover heart ejection fraction in an acute MI model. Furthermore, CRISPRa acts on the adult human heart to increase PPARGC1A protein and cellular mitochondria, elevating mitochondrial function in both normal and HF-diagnosed hearts. These results provide the first proof of concept that endogenous gene activation via CRISPRa can improve heart function after MI.},
}
@article {pmid41807051,
year = {2026},
author = {Gur Dedeoglu, B and Noyan, S and İlhan, KNK},
title = {Non-coding RNAs regulation in breast cancer pathogenesis.},
journal = {Epigenomics},
volume = {18},
number = {4},
pages = {493-512},
pmid = {41807051},
issn = {1750-192X},
mesh = {Humans ; *Breast Neoplasms/genetics/pathology/metabolism ; Female ; *RNA, Untranslated/genetics ; *Gene Expression Regulation, Neoplastic ; Epigenesis, Genetic ; Piwi-Interacting RNA/genetics ; },
abstract = {Breast cancer represents a molecularly heterogeneous disease shaped by complex genetic, epigenetic, and transcriptional dysregulation. Non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) as well as small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), and small nuclear RNAs (snRNAs), have emerged as key epigenetic regulators that integrate multiple layers of gene control. Through interactions with chromatin-modifying enzymes, RNA-binding proteins, and signaling effectors, ncRNAs modulate transcriptional activity, chromatin accessibility, and post-transcriptional stability of target genes. miRNAs predominantly act as post-transcriptional repressors, whereas lncRNAs and circRNAs exert transcriptional and epigenetic control via scaffolding, miRNA sponging, and chromatin remodeling; some circRNAs even encode functional peptides. Aberrant ncRNA expression contributes to proliferation, metastasis, metabolic reprogramming, immune evasion, and therapeutic resistance, with distinct expression signatures associated with triple-negative, HER2-positive, and hormone receptor - positive breast cancers. Owing to their stability and detectability in plasma and exosomes, ncRNAs hold promise as minimally invasive biomarkers for early detection and disease monitoring. Moreover, therapeutic strategies targeting ncRNAs, such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based editing, and ncRNA-derived vaccines, are advancing toward clinical translation. Collectively, ncRNAs redefine the epigenetic landscape of breast cancer, offering a framework for integrated diagnostic and therapeutic approaches in precision oncology.},
}
@article {pmid41807709,
year = {2026},
author = {Jansson-Fritzberg, L and Chica, B and Latrick, C and Olland, A and Dementiev, A and White, A and Kutter, S and Lemercier, JN and Wolk, S},
title = {Mechanistic basis for improved activity of Engineered AsCas12a.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41807709},
issn = {2399-3642},
mesh = {*Protein Engineering ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; DNA/metabolism ; DNA Cleavage ; Substrate Specificity ; R-Loop Structures ; },
abstract = {CRISPR-associated proteins (Cas) are central to gene editing, forming nuclease complexes with guide RNA to enable precise genome modification. Among numerous Cas variants, Cas9 and Cas12a are the most extensively studied. While much is known about the genomic substrates for these enzymes, less is known about the determinants of the DNA cleavage activity. Wild-type Cas12a exhibits higher intrinsic specificity than Cas9, minimizing off-target activity, but lower overall potency. Recent protein engineering has sought to improve both parameters. Here, we shed light on the structural and mechanistic basis by which an engineered AsCas12a variant achieves high potency while retaining its hallmark specificity. We show that reduced protein-DNA interactions facilitate more rapid R-loop formation, thereby enhancing cleavage activity. These results provide mechanistic insight into Cas12a function and highlight strategies for designing genome-editing nucleases with optimal balance between efficiency and specificity.},
}
@article {pmid41807728,
year = {2026},
author = {Godsil, M and Wei, N and Meeske, AJ},
title = {Conditional activation of Cas13 enforces lysogeny in a native type VI-A CRISPR host.},
journal = {Nature microbiology},
volume = {11},
number = {4},
pages = {920-928},
pmid = {41807728},
issn = {2058-5276},
support = {R35 GM142460/GM/NIGMS NIH HHS/United States ; FAIN2235762//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; R35GM142460//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Lysogeny/genetics ; Prophages/genetics/physiology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA, Viral/genetics ; Listeria monocytogenes/virology ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems present a barrier to prophage acquisition by restricting invading phages or by inducing autoimmune cleavage of integrated prophage DNA. The RNA-sensing type VI CRISPR nuclease Cas13 mediates non-specific RNA cleavage upon recognition of phage lytic transcripts, but how this system influences the temperate phage life cycle remains unknown. Here we report that the Listeria seeligeri type VI-A CRISPR system restricts the lytic cycle of temperate phages but tolerates prophage acquisition and interferes with prophage induction through a non-abortive mechanism. During attempts at induction, Cas13 activation forces prophage re-integration, thus maintaining lysogeny. We also find that during polylysogenic induction, Cas13 acts specifically, restricting only the targeted phage, in contrast to its behaviour during lytic replication. Our findings show that Cas13 elicits a unique response to each stage of the temperate phage life cycle, enabling type VI CRISPR hosts to acquire potentially beneficial prophages while mitigating lysis.},
}
@article {pmid41808208,
year = {2026},
author = {Zhang, Y and Wang, M and Bi, C and Li, M},
title = {Targeted native long-read sequencing of DNA methylation alterations following CRISPR-Cas9-induced double-strand breaks in human cells.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {41808208},
issn = {1756-0500},
support = {BAS/1/1080-01-01//King Abdullah University of Science and Technology/ ; },
mesh = {Humans ; *DNA Methylation/genetics ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems/genetics ; 5-Methylcytosine/analogs &amp; derivatives/metabolism ; *Sequence Analysis, DNA/methods ; snRNP Core Proteins/genetics ; Epigenesis, Genetic ; Genomic Imprinting ; Nanopore Sequencing ; },
abstract = {OBJECTIVES: CRISPR-Cas9 nucleases are widely used to introduce targeted DNA double-strand breaks (DSBs) for genome engineering, but the long-term impact of these lesions on local epigenetic information remains poorly characterized. In a companion research article, we used Cas9-assisted targeted nanopore sequencing (CTS) to reveal that CRISPR-Cas9-induced DSBs can disrupt local epigenetic maintenance across multiple genomic contexts and cell systems. Here, we present a structured description of the raw and minimally processed datasets underlying the study. These datasets provide base-resolution measurements of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at the differentially methylated regions (DMRs) of several imprinted loci, two heterochromatic regions, a cancer-associated promoter epimutation region, and the SNRPN DMR at early/late passages of a clonal line. They enable re-analysis and methodological benchmarking of DSB-associated epigenetic instability.<br><br>DATA DESCRIPTION: We provide aligned BAM files and per-CpG methylation calls for multiple genomic contexts under both CRISPR-targeted and non-targeting control conditions. Specifically, the collection includes: (i) imprinted loci in human embryonic stem cells (hESCs), including small nuclear ribonucleoprotein polypeptide N (SNRPN), paternally expressed 10 (PEG10), and KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), (ii) heterochromatic regions in hESCs, including urothelial cancer associated 1 (UCA1), and cysteine rich C-terminal 1 (CRCT1)), (iii) the epimutation locus of MutL homolog 1 (MLH1) in RKO cells, and (iv) the DMR of SNRPN locus in early- and late-passage derivatives of a single hESC clone. For each collection, there is a dataset that includes both the raw aligned Nanopore sequencing reads (BAM) deposited in the NCBI Sequence Read Archive (SRA) and the corresponding processed per-CpG 5mC/5hmC matrices deposited in Zenodo. All higher-level analyses in the research article-such as DMR calling, haplotype-resolved analyses, and structural variant (SV) characterization-are fully reproducible using these deposited data. Additional processed analyses are comprehensively documented in the companion article and are therefore not duplicated here. Together, these datasets offer a rich resource for benchmarking long-read methylation analysis workflows and further investigation of DSB-associated epigenetic instability across diverse genomic contexts.},
}
@article {pmid41808396,
year = {2026},
author = {Zargul, A and Liu, H and Zhang, W and Wang, H and Liu, J and Chen, C},
title = {Advances in Pathogen Detection by Nanosensors: Biorecognition Strategies, Signal Amplification, and Platform Engineering.},
journal = {ACS nano},
volume = {20},
number = {11},
pages = {9007-9050},
doi = {10.1021/acsnano.5c22148},
pmid = {41808396},
issn = {1936-086X},
mesh = {*Biosensing Techniques/methods ; *Nanotechnology/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Bacteria/isolation &amp; purification ; },
abstract = {The escalating global threat of infectious diseases, compounded by antimicrobial resistance (AMR), calls for improved diagnostic strategies. Conventional pathogen detection techniques─culture, enzyme-linked immunosorbent assay (ELISA), and microscopy─remain hindered by prolonged turnaround times, suboptimal sensitivity for low-abundance analytes, and operational intricacy. Nanosensor technologies have emerged as powerful enablers of rapid, ultrasensitive, and field-deployable diagnostics. This review delineates the convergence of three transformative domains: (1) advanced biorecognition strategies─including monoclonal antibodies, aptamers, bacteriophages, antimicrobial peptides, molecularly imprinted polymers, and lectins─that confer high-fidelity molecular selectivity within complex biological matrices; (2) multimodal signal amplification technologies, encompassing nanomaterial-enhanced mechanisms, enzymatic cascades, and isothermal nucleic acid amplification that drive detection down to the single-cell and femtomolar regimes; and (3) integrated platform engineering, uniting clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems, artificial intelligence (AI), and microfluidics to achieve multiplexed, real-time, point-of-care deployment. Advances are critically evaluated through standardized performance metrics─limit of detection, assay time, specificity, and operational simplicity─to reveal both synergistic opportunities and enduring translational bottlenecks. Collectively, these developments define a strategic framework for next-generation nanosensor diagnostics poised to revolutionize infectious disease surveillance and enable precision-guided therapeutic intervention.},
}
@article {pmid41808567,
year = {2026},
author = {Ding, X and Liu, Y and Luo, L and Cai, Y and Wang, C and Jin, J and Chen, Y},
title = {Review Genomic Hotspot Mining and Characterization for Stable Expression of Therapeutic Protein in Chinese Hamster Ovary Cells.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {1241-1247},
doi = {10.1021/acssynbio.5c00776},
pmid = {41808567},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; Cricetulus ; Cricetinae ; CRISPR-Cas Systems/genetics ; *Recombinant Proteins/genetics/metabolism/biosynthesis ; Promoter Regions, Genetic ; Chromosome Mapping ; },
abstract = {The development of rCHO cell lines that stably express therapeutic proteins is crucial for pharmaceutical protein industrial production. In this study, a systematic method was established to identify genomic hotspots for exogenous protein expression in CHO cells and construct stable recombinant CHO cell strains. Four stable monoclonal cell lines (1b7, 1d2, 2d9, and 2f7) were obtained by using the lentiviral random integration reporter gene. Chromosome mapping analysis found four stable integration sites: chr1_0 (7,30,83,299-7,32,45,508 bp) in 1b7, chr1_0 (17,69,68,187-17,69,68,191 bp) in 1d2, chr3 (4,08,81,262-4,08,99,858 bp) in 2d9, and chr5 (1,69,77,575-1,70,61,744 bp) in 2f7. Based on these sites, we developed recombinant CHO cells capable of long-term stable expression of foreign proteins through the combined application of CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange. Utilizing "promoter capture technology", all screened LP cell monoclonal lines can express exogenous proteins, with the entire construction process completed in just 2∼3 weeks.},
}
@article {pmid41809894,
year = {2025},
author = {Yang, P and Khoshandam, M and Bhia, I and Raji, S and Soltaninejad, H and Hosseinkhani, S and Sani, M and Hamidieh, AA and Sheykhhasan, M},
title = {Integrating CRISPR/Cas technology with clinical trials: Principles, progress and challenges.},
journal = {Asian journal of pharmaceutical sciences},
volume = {20},
number = {6},
pages = {101068},
pmid = {41809894},
issn = {2221-285X},
abstract = {CRISPR represent a groundbreaking genome-editing technology that has revolutionized genetic modification. This innovative tool offers an unparalleled revolution in the future treatment of genetic disorders, neurological diseases, infectious diseases and cancer. Despite the rapid expansion of CRISPR applications, its clinical use in humans is still relatively limited, with only 69 active clinical trials and 6 completed studies reported so far. This review examined current clinical trials and their processes in addressing various diseases via the CRISPR/Cas system. While earlier literatures have focused mainly on delivery methods and materials for CRISPR/Cas9, our review emphasized innovative targeting conditions and approaches for novel and functional therapeutic designs. In addition, we reviewed recent research to increase the efficiency of CRISPR editing in the management of genetic disorders and cancer, while exploring their future challenges and potential. This review provided a unique perspective on the advancement of CRISPR technology. By addressing these aspects, we aim to contribute to ongoing efforts to improve CRISPR-based therapies and expand their clinical applications, ultimately striving to transform the future of medical treatment.},
}
@article {pmid41810060,
year = {2026},
author = {Shen, Z and Liu, Y and Hao, Y and Bo, Y and Dai, X and Wang, S and Xia, T and Su, X and Liu, H},
title = {Advances in Double-Stranded DNA Targeting Technologies.},
journal = {Exploration (Beijing, China)},
volume = {6},
number = {1},
pages = {20250065},
pmid = {41810060},
issn = {2766-2098},
abstract = {Double-stranded DNA (dsDNA) serves as a fundamental repository of genetic information and plays a pivotal role in the diagnosis and therapeutic management of diseases. However, the inherent stability of the DNA double helix under physiological conditions presents a challenge in accessing internal bases. To address this, various molecular targeting technologies have been developed, offering high specificity while destabilizing the DNA structure. This review provides a comprehensive overview of current dsDNA targeting tools, such as hybridization probes, modified nucleic acid probes, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, Argonaute proteins (Agos), and the lambda exonuclease-pDNA system (λ Exo-pDNA), and some cutting-edge molecular tools. It delves into the mechanisms behind these technologies. It highlights their applications in diverse areas, including in vitro detection, in situ imaging, gene editing, and their integration with artificial intelligence (AI)-driven tools. Additionally, the review compares these techniques, discusses future technological opportunities, and identifies challenges in integrating these tools into diagnostic and therapeutic practices. By providing a holistic view of these rapidly evolving technologies, this review aims to fill a gap in the current literature and explore the future potential of dsDNA targeting innovations.},
}
@article {pmid41810315,
year = {2026},
author = {Guo, Z and Hu, R and Wang, J and Zhou, M and Zhu, K and Xu, Y},
title = {Research Progress on Point-of-Care Testing Technology for Mycoplasma Pneumonia.},
journal = {International journal of general medicine},
volume = {19},
number = {},
pages = {584824},
pmid = {41810315},
issn = {1178-7074},
abstract = {Mycoplasma pneumoniae (MP) is a significant respiratory pathogen in children, often causing refractory and severe pneumonia. Sensitive, rapid, and portable diagnostic tools are crucial for guiding clinical management. Although traditional methods like culture, ELISA, and PCR are widely used, they suffer from drawbacks such as lengthy turnaround times, complex procedures, or reliance on laboratory equipment. Consequently, Point-of-care testing (POCT) technologies, valued for their speed, portability, and ease of use, have emerged as a key research focus for MP diagnosis. This review systematically summarizes advancements in POCT platforms, covering: (1) immunological methods; (2) molecular biology methods; and (3) biosensor technologies. Their sensitivity, specificity, and clinical performance are comparatively analyzed. Colloidal gold immunochromatography delivers results within 15 minutes but exhibits limited sensitivity. Molecular methods like LAMP and CRISPR-coupled systems achieve single-copy detection limits via isothermal amplification and gene editing, with processing times under 1 hour. Biosensors enable high-sensitivity automated detection through integrated signal amplification and microfluidics. Despite these advantages, POCT development faces challenges including cost-sensitivity tradeoffs, standardization barriers, and sample matrix interference. Future directions encompass multi-modal detection, AI-assisted interpretation, multiplex pathogen screening, and dynamic drug resistance gene monitoring. These innovations will expand POCT device deployment in primary care and home settings. This will ultimately improve effectiveness in controlling respiratory infections.},
}
@article {pmid41810550,
year = {2026},
author = {Rahmanian, M and Khoshandam, M and Mousazadeh, M and Yang, P and Soltaninejad, H and Karami Dehkordi, P and Sadeghizadeh, M and Hedayati Goudarzi, MT and Azimi, AH and Sheykhhasan, M},
title = {CRISPR in Medicine: A Systematic Review of Clinical Trials and Therapeutic Applications.},
journal = {Human gene therapy},
volume = {37},
number = {5-6},
pages = {170-182},
doi = {10.1177/10430342251409714},
pmid = {41810550},
issn = {1557-7422},
mesh = {Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Gene Editing/methods ; Clinical Trials as Topic ; *Neoplasms/therapy/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9 (CRISPR/Cas9) technology has become a revolutionary tool in medicine, offering substantial potential for treating a wide range of diseases, including hematological disorders, cancers, genetic conditions, and ophthalmological diseases. This systematic review evaluates the efficacy, safety, and applicability of CRISPR/Cas9 in clinical trials. A comprehensive search of the PubMed, Scopus, Web of Science, and Cochrane databases was conducted. All studies, up to November 2024, meeting the eligibility criteria assessing the application of CRISPR for the treatment of diseases were included. A quality assessment of the included studies was conducted using the Cochrane risk of bias tool. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement for systematic reviews and meta-analyses was followed, and a total of 17 studies were included. This systematic review of CRISPR/Cas9 technology focused on its effectiveness and safety across various diseases. In nonmalignant hematological disorders, CRISPR successfully treated β-thalassemia and sickle cell disease, resulting in high transfusion independence and the elimination of disease crises. In malignant hematological disorders, B-cell acute lymphoblastic leukemia, CRISPR-engineered chimeric antigen receptor T (CAR-T) cells achieved an 83.3% complete remission rate. Furthermore, CRISPR-based CAR-T cells showed promising results in B-cell non-Hodgkin's lymphoma. In oncology, lung cancer and other solid tumors are among the diseases that have been safely engineered using CRISPR gene editing technology. For genetic disorders, CRISPR improved vision in retinal degeneration and reduced symptoms in hereditary angioedema and transthyretin amyloidosis with mild side effects. The results demonstrated CRISPR's potential across a wide range of conditions. In conclusion, the findings underscore the potential role of CRISPR/Cas9 technology across a wide range of diseases. However, challenges remain, including optimizing delivery systems, minimizing off-target effects, addressing immunogenicity concerns, and ethical considerations.},
}
@article {pmid41811192,
year = {2026},
author = {Shen, Y and Yeung, AT and Ditchfield, P and Korn, E and Clements, R and Chen, X and Wang, B and Huang, Z and Sheen, M and Jarman, PA and Han, C},
title = {A genome-wide MAGIC kit for recombinase-independent mosaic analysis in Drosophila.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41811192},
issn = {2050-084X},
support = {R24 OD031953/OD/NIH HHS/United States ; R24OD031953//NIH Office of the Director/ ; },
mesh = {Animals ; *Drosophila/genetics ; *Mosaicism ; *Crossing Over, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Recombinases/metabolism ; CRISPR-Cas Systems ; Recombination, Genetic ; Genome, Insect ; },
abstract = {Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes, deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions.},
}
@article {pmid41811196,
year = {2026},
author = {Vieira, CSD and Wang, W and Sanchez-Valdez, F and Lim, J and White, BE and Souza, CGS and Tarleton, RL and Paes, MC and Nogueira, NPA},
title = {Glycosomal Phosphoenolpyruvate Carboxykinase CRISPR/Cas9-Deletion and Its Role in Trypanosoma cruzi Metacyclogenesis and Infectivity in Mammalian Host.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {6},
pages = {e71672},
pmid = {41811196},
issn = {1530-6860},
support = {00x0ma614//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior (CAPES)/ ; 88887.311601/2018-00-2629/2018//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior (CAPES)/ ; E26/010.001706/2019//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; E26/010.100623/2018//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; E26/211.815/2021//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; 402419/2022-7//Conselho Nacional Pesquisa (CNPq) SWE (Sandu&#xed;che no Exterior)/ ; },
mesh = {*Trypanosoma cruzi/pathogenicity/genetics/enzymology/growth &amp; development ; Animals ; *CRISPR-Cas Systems ; Mice ; *Chagas Disease/parasitology ; *Phosphoenolpyruvate Carboxykinase (ATP)/genetics/metabolism ; *Microbodies/enzymology/metabolism ; Mitochondria/metabolism ; Glucose/metabolism ; *Protozoan Proteins/genetics/metabolism ; Energy Metabolism ; },
abstract = {Trypanosoma cruzi, the causative agent of Chagas disease, possesses glycosomes-unique organelles that house key metabolic enzymes, several of which are promising therapeutic targets. Among them, phosphoenolpyruvate carboxykinase (PEPCK) plays a central role in succinic fermentation, the main pathway for NAD[+] regeneration within the organelle. Using CRISPR/Cas9 editing, the PEPCK gene was disrupted in T. cruzi, producing single-allele knockout epimastigotes (TcPEPCK-sKO) with reduced PEPCK expression and enzyme activity. In a high glucose environment, PEPCK disruption impaired glucose consumption and mitochondrial respiration, particularly oxidative phosphorylation, reducing dependence on mitochondrial ATP production when glucose was supplied. To compensate, pyruvate phosphate dikinase was upregulated, increasing alanine production, possibly to maintain redox balance in glycosomes. Despite this metabolic adaptation, the growth of TcPEPCK-sKO epimastigotes was partially reduced compared with non-deleted parasites. In contrast, under low glucose conditions, PEPCK activity was not critical for mitochondrial bioenergetics, ATP production, or proliferation. Although TcPEPCK-sKO epimastigotes exhibited a minor reduction in growth in high glucose medium, their differentiation (metacyclogenesis) and invasion were severely compromised. However, once inside the host cell, TcPEPCK-sKO amastigotes increased their replication, leading to enhanced trypomastigote production. The same was observed in in vivo infection, where TcPEPCK-sKO infection in IFNγ-deficient mice caused uncontrolled parasitemia and severe pathology, highlighting the critical role of PEPCK in host-pathogen interactions. However, an intact immune system effectively contained TcPEPCK-sKO infection. Taken together, our findings demonstrate that glycosomal PEPCK is crucial for coupling glycolysis to mitochondrial bioenergetics, enabling the parasite differentiation within the insect vector and controlling the infection of mammalian host cells.},
}
@article {pmid41811507,
year = {2026},
author = {Thakur, MK and Pandey, S and Singh, SK and Singh, SK and Singh, A},
title = {Hybrid seed production: new paradigms and challenges in the twenty-first century.},
journal = {Planta},
volume = {263},
number = {4},
pages = {},
pmid = {41811507},
issn = {1432-2048},
mesh = {*Seeds/genetics/growth &amp; development ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Hybridization, Genetic ; Hybrid Vigor ; Plants, Genetically Modified/genetics ; Genomics ; },
abstract = {Hybrid seed technology future depends on integrating advanced genomics, AI-driven breeding, and enabling policies to sustainably delivery climate-resilient, high-performing hybrids with broad accessibility and equitable benefits worldwide. Hybrid seeds, which exploit heterosis, have driven agricultural productivity gains since the 1920s. Understanding the genetics and molecular biology of hybrid generation led to the development of modern hybrid systems. With time, modern hybrid systems integrated advanced genomic tools such as CRISPR/Cas, marker-assisted selection (MAS), and genomic selection (GS) with established technologies like cytoplasmic male sterility (CMS), restorer-of-fertility (Rf) systems, and chemical hybridizing agents (CHAs) for better hybrid production in a shorter time. Moreover, the integration of emerging approaches leveraging artificial intelligence and machine learning (AI/ML) for trait prediction, multi-parent populations to expand genetic diversity, and epigenetics to engineer climate-resilient hybrids with enhanced stress tolerance is also being explored. However, regulatory hurdles, such as divergent global policies for genetically modified (GM) hybrids, intellectual property (IP) disputes, and restricted germplasm exchange under access-and-benefit-sharing frameworks like the Nagoya Protocol, hinder innovation. Climate change exacerbates both biotic and abiotic stresses, disrupts production zones, and threatens pollinator-dependent crops, while socio-economic barriers limit the adoption of smallholder farming. Case studies of different crops demonstrate the success of hybrids, yet gaps in scalability and accessibility persist. Overall, realizing the potential of hybrid technology hinges on sustained collaboration across scientific, industrial, and policy domains to overcome technical, environmental, and socio-economic constraints. This review examines various techniques for hybrid production that incorporate genomics, future advancements, and synergies between synthetic biology, automation, and predictive breeding, as well as policies that strike a balance between intellectual property protection and germplasm accessibility for hybrid seed production.},
}
@article {pmid41811974,
year = {2026},
author = {Baba, S and Oncul, O and Aktas, Z},
title = {CRISPR-Cas based plasmid design for multidrug-resistant Klebsiella pneumoniae isolates.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
pmid = {41811974},
issn = {1574-6968},
support = {TDK-2020-36552//Istanbul University/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; *Plasmids/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; *CRISPR-Cas Systems ; beta-Lactamases/genetics ; Klebsiella Infections/microbiology ; Humans ; Imipenem/pharmacology ; Gene Editing/methods ; Bacterial Proteins/genetics ; },
abstract = {Antimicrobial resistance is a major global health concern that requires innovative therapeutic strategies. This study aimed to address this challenge by designing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (CRISPR-Cas)-based plasmid systems for potential genome editing applications in multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined according to European Committee on Antimicrobial Susceptibility Testing guidelines. All isolates (n = 5) were resistant, with MIC ranges of 4-128 μg/ml for imipenem, 8-64 μg/ml for meropenem, and 8-256 μg/ml for ertapenem. Resistance gene analysis revealed blaOXA-48-like and blaCTX-M-15 in all isolates, while blaNDM-1 was detected in one isolate. Two CRISPR-based plasmid systems, CRISPR-Cas9 and CRISPR-assisted cytidine deaminase, were designed. Target genes were amplified by polymerase chain reaction, and guide RNA (gRNA) sequences were designed from selected regions. Apramycin (50 μg/ml) was identified as a suitable selection marker. The pSGKP-AmpR(Pro)-ApmR plasmid was successfully constructed, whereas Cas9 and APOBEC constructs could not be cloned. Overall, this study highlights technical challenges in developing CRISPR-based tools for MDR K. pneumoniae and emphasizes the need for isolate-specific plasmid design and gRNA optimization.},
}
@article {pmid41812798,
year = {2026},
author = {Huang, G and Zhuge, B and Du, X and Wang, M and Zong, H},
title = {TSA-ultrasound synergy enhances CRISPR-Cas9 gene editing efficiency in diploid yeast.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107449},
doi = {10.1016/j.mimet.2026.107449},
pmid = {41812798},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems/genetics ; *Hydroxamic Acids/pharmacology ; Diploidy ; *Gene Editing/methods ; *Candida/genetics/drug effects ; Sonication/methods ; Chromatin ; Gene Knockout Techniques ; },
abstract = {The CRISPR-Cas9 system is invaluable for microbial engineering. However, its efficiency remains limited in numerous microorganisms, especially in polyploid yeasts where the compact chromatin structures pose significant physical barriers, which are major constraints in microbial engineering. To address this limitation, we developed and validated a standardized synergistic protocol using the diploid industrial yeast Candida glycerinogenes as a model. This protocol combines TSA-induced chromatin decondensation with brief, low-intensity ultrasonication to enhance Cas9 accessibility. Key parameters were systematically optimized, and 200 nM Trichostatin A (TSA) (10 h) followed by 200 W sonication (3 min) were established as the optimal condition set. Validation results showed that the protocol more than doubled single-gene knockout efficiencies (GPD1, TRP1) compared with conventional methods. Importantly, it enabled complex edits that were previously unattainable in C. glycerinogenes, including the precise deletion of a 7.8-kb fragment and the editing of an 11.4-kb region for functional genomics. In summary, this study establishes a simple and effective workflow that overcomes chromatin-based barriers in the polyploid industrial yeast C. glycerinogenes, providing a practical tool for genetic engineering and functional genomics in this and potentially other recalcitrant yeasts.},
}
@article {pmid41812941,
year = {2026},
author = {Wang, M and Niu, D and Zhang, Q and Tang, Y and Zhao, Y and Chen, F},
title = {CRISPR-based correction of apolipoprotein E4 in Alzheimer's disease: Therapeutic strategies and macromolecular delivery innovations.},
journal = {International journal of biological macromolecules},
volume = {354},
number = {},
pages = {151352},
doi = {10.1016/j.ijbiomac.2026.151352},
pmid = {41812941},
issn = {1879-0003},
mesh = {Humans ; *Alzheimer Disease/genetics/therapy/metabolism ; *Apolipoprotein E4/genetics ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Blood-Brain Barrier/metabolism ; *Genetic Therapy/methods ; Exosomes/metabolism ; },
abstract = {Alzheimer's disease (AD) is the leading cause of dementia worldwide, with substantial unmet clinical needs. The apolipoprotein E4 (APOE4) allele is the strongest genetic risk factor for late onset AD, with each copy increasing risk approximately two- to three-fold, and homozygous carriers facing up to a 10- to 15-fold higher risk compared to APOE3 carriers. APOE4 contributes to diverse pathogenic mechanisms including lipid dysregulation, neuroinflammation, synaptic dysfunction, and vascular compromise. The precise, allele-specific correction of APOE4 therefore holds transformative therapeutic potential. CRISPR-based genome editing technologies, including nuclease disruption, base editing, and prime editing, offer unprecedented opportunities to directly modify APOE4 at its genomic source. Here, we review mechanistic underpinnings of APOE4 pathology, summarize current gene editing platforms for APOE4 correction, evaluate relevant in vitro and in vivo model systems, and assess delivery strategies with an emphasis on nanoparticle and exosome based approaches. We highlight recent breakthroughs in exosome mediated APOE4 editing while addressing ongoing technical hurdles in allele specificity and translational barriers such as Cas nuclease immunogenicity, limited delivery efficiency across the blood brain barrier (BBB), and concerns over long term genomic safety. This review concludes that overcoming BBB constraints remains the most significant challenge for clinical translation, and that innovations in exosome and nanoparticle based delivery platforms represent the most promising strategies for advancing CRISPR therapeutics for AD.},
}
@article {pmid41812962,
year = {2026},
author = {Yogi, D and Shashikala, T and Subramanian, G and Kumar, A and Kaninika, V and Manamohan, M and Jithesh, MN and Jha, GK and Asokan, R and Ashok, K},
title = {First report of CRISPR prime editing in a globally significant non-model organism, the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).},
journal = {Methods (San Diego, Calif.)},
volume = {250},
number = {},
pages = {36-45},
doi = {10.1016/j.ymeth.2026.03.001},
pmid = {41812962},
issn = {1095-9130},
mesh = {Animals ; *Spodoptera/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The power of CRISPR/Cas9 mediated genome editing has been harnessed in different facets of entomological research, particularly useful in developing genetic pest management strategies. The edits thus obtained are robust and results in a loss-of-function of the target gene. Recently the development of newer editing approach called Prime editing is yet another addition in the insect editing tool-box. In this regard, the prime editing offers a transformative approach to precise genome manipulation by enabling targeted insertions, deletions, and nucleotide substitutions without double-strand break or donor template. While its application has been explored in mammalian system and plants, its deployment through the delivery of ribonucleoprotein complex (RNP) has been demonstrated for the first-time in the globally significant pest, Spodoptera frugiperda. Using a Cas9 (H840A)-reverse transcriptase fusion protein (PE2) and a customized prime editing guide RNA (pegRNA), we targeted exon 3 of the Tryptophan 2,3-dioxygenase (SfTO) gene to introduce a premature stop codon. Recombinant PE2 protein was expressed in E. coli, purified, and validated functionally through RT-PCR. The Ribonucleoprotein complex was microinjected into G0 eggs and subsequent genotyping revealed successful edits, including perfect and imperfect prime edits, as well as unintended mutations. Phenotyping revealed the mutants with altered eye pigmentation, and chromatographic analysis confirmed disruption in ommochrome biosynthesis, validating functional consequences of Prime editing. This study provides a foundational proof-of-concept for Prime editing in insect pests, opening new avenues for functional genomics and designing next-generation pest management strategies.},
}
@article {pmid41813543,
year = {2026},
author = {Qiao, JH and Gao, Q and Wang, XB},
title = {Virus-induced genome editing: toward crop breeding applications.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2026.01.007},
pmid = {41813543},
issn = {1878-4372},
abstract = {CRISPR-Cas-based genome editing has revolutionized precise genome manipulation in plants, yet its practical application is still constrained by the inefficient delivery of editing reagents across different genotypes. Plant viruses are promising vehicles for delivering genome-editing components, bypassing plant transformation and/or tissue culture. Virus-induced genome editing (VIGE) has provided powerful tools for achieving heritable edits in model plants such as Arabidopsis thaliana and Nicotiana benthamiana. VIGE has now progressed from proof-of-concept to practical applications in agricultural crops. Notably, a recent breakthrough in VIGE in tiller has successfully achieved heritable genome editing in hexaploid wheat. This review outlines the latest advances in VIGE across diverse plant species, highlights its potential for crop improvement, and discusses future research directions.},
}
@article {pmid41813774,
year = {2026},
author = {Guo, J and Shi, S and Xie, S and Jing, L and Wang, C and Tao, D},
title = {An advanced rapid-visual CRISPR assay for detecting porcine reproductive and respiratory syndrome virus.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41813774},
issn = {2045-2322},
support = {(2022-021)//the SAAS Program for Excellent Research Team/ ; No.2023BBB170//the Key Research and Development Program of Hubei Province, China/ ; },
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics/isolation &amp; purification ; Animals ; Swine ; *CRISPR-Cas Systems ; RNA, Viral/genetics ; Sensitivity and Specificity ; *Porcine Reproductive and Respiratory Syndrome/diagnosis/virology ; },
abstract = {The porcine reproductive and respiratory syndrome virus (PRRSV) remains a significant threat to the global swine industry, underscoring the urgent need for innovative diagnostic methods to detect and manage outbreaks effectively. We developed a novel CRISPR-based fluorescence assay for the highly sensitive detection of PRRSV-2. By combining reverse transcription-recombinase polymerase amplification (RT-RPA) with multiple-crRNA CRISPR/Cas13a system and single-stranded RNA-fluorescently quenched reporters (RQ-5U), our assay achieved a significant 28-fold increase in sensitivity compared to existed CRISPR/Cas13a-based PRRSV-2 detection methods. This multiple crRNA strategy allows detecting as low as 6 copies/µL of PRRSV-2 RNA, significantly improving the detection limit. Moreover, our method's accuracy in detecting simulated PRRSV-2 clinical samples matches that of quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate that this visual, sensitive, and specific nucleic acid detection method holds great promise for enhancing the diagnosis and management of PRRS in the swine industry.},
}
@article {pmid41813887,
year = {2026},
author = {Tou, CJ and Xie, K and Ferreira da Silva, J and Kalailingam, P and Amar-Lewis, E and Rufino-Ramos, D and Sawyer, W and Eller, ML and Starzyk, J and Majumdar, I and Wang, J and Lee, D and Yang, S and Meis, RJ and Dahl, GA and Li, J and Shan, R and Artzi, N and Musolino, PL and Wu, H and Kleinstiver, BP},
title = {Immune evasive DNA donors and recombinases license kilobase-scale writing.},
journal = {Nature},
volume = {653},
number = {8114},
pages = {576-586},
pmid = {41813887},
issn = {1476-4687},
mesh = {Humans ; Animals ; Mice ; *Gene Editing/methods ; *Recombinases/metabolism ; *Genome, Human/genetics ; *DNA/immunology/genetics/metabolism ; Female ; Immunity, Innate ; DNA, Single-Stranded/genetics/immunology/metabolism ; DNA, Circular/genetics/immunology/metabolism ; Male ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Genome-editing technologies that use recombinases to insert kilobase-scale DNA sequences into mammalian genomes canonically require large double-stranded DNA (dsDNA) donors[1,2]. However, dsDNA molecules evoke problematic and toxic innate immune responses, limiting integration efficiencies and generally constraining applicability to ex vivo or immune-deficient contexts. By harnessing mechanisms of integrative prokaryotic viruses and mobile genetic elements, here we demonstrate that recombinases are compatible with immune evasive circular single-stranded DNA molecules optimally bearing a partial-duplex region that reconstitutes the recombinase recognition sequence. This approach, which we term integration through nucleus-synthesized template addition of large lengths (INSTALL), is compatible with diverse protein and RNA-guided recombinases for high-fidelity kilobase-scale human genome writing. INSTALL minimizes innate immune responses in primary human cells and in mice, improving recombinase-mediated integration efficiencies and supporting systemic in vivo non-viral DNA delivery by substantially increasing tolerability and broadening the dosing range compared with lipid nanoparticle-delivered dsDNA molecules. Together, INSTALL overcomes fundamental challenges for DNA delivery and integration methods by synergizing immune-stealth nucleic acids with recombinases to enable kilobase-scale integration strategies without viral vectors.},
}
@article {pmid41814120,
year = {2026},
author = {Simonneau, B and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {CRISPR-Cas9 genome editing in the parental iPSC line PCIi033-A to introduce the homozygous mutation p.F508del (c.1521_1523del) in the CFTR gene.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103947},
doi = {10.1016/j.scr.2026.103947},
pmid = {41814120},
issn = {1876-7753},
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Homozygote ; Cell Line ; *Mutation ; Cell Differentiation ; },
abstract = {Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. Patients carrying the most common mutation, p.F508del, benefit from the triple therapy Kaftrio®. We genome-edited the commercially available iPSC line PCIi033-A (wild-type CFTR) to generate the subclone PCIi033-A-5, which is homozygous for the mutation c.1521_1523del (p.F508del), using CRISPR-SpCas9 tools. PCIi033-A-5 has a normal karyotype and stem cell morphology, is pluripotent, and differentiates into the three germ layers. Introducing this mutation in a parental isogenic iPSC line is essential to demonstrate the feasibility of modeling CF disease after differentiation of the iPS cells into bronchial epithelium.},
}
@article {pmid41814945,
year = {2026},
author = {Lee, S and Kim, S and Chong, J and Kim, MG and Lim, K and Lee, NE and Koo, J and Kang, J and Lee, H},
title = {A Cryoprotectant-Compatible Nanoporous Platform for Stable and Scalable Delivery of Biopharmaceuticals.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {33},
pages = {e10532},
pmid = {41814945},
issn = {1521-4095},
support = {RS-2022-NR072331//National Research Foundation of Korea/ ; RS-2024-00403376//National Research Foundation of Korea/ ; RS-2023-KH135060//Korea Health Industry Development Institute, Ministry of Health &amp; Welfare, Republic of Korea/ ; RS-2024-00438476//Korea Health Industry Development Institute, Ministry of Health &amp; Welfare, Republic of Korea/ ; //KIST Institutional Program/ ; },
mesh = {*Cryoprotective Agents/chemistry ; Animals ; Humans ; Freeze Drying ; *Nanopores ; Gene Editing ; Ribonucleoproteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 ribonucleoproteins (RNPs) represent a promising class of biopharmaceuticals for treating genetic and complex diseases. However, their clinical translation is limited by instability during storage and delivery. Lyophilization offers a potential solution, though conventional approaches often compromise structural integrity and bioactivity under non-cryogenic conditions. Here, we have developed a nanostructured delivery platform, designated Nano Banker &amp; Blowball (NB[2]), which features a blowball-like architecture and tunable nanoscale pores. These pores are designed to protect RNPs and enable controlled release. The freeze-dried formulation (FNB[2]) integrates optimized cryoprotectants and a surface-engineered nanoparticle design, preserving morphology and function without excessive excipients. FNB[2] exhibits rapid rehydration and retains ∼70% of gene editing activity post-lyophilization, enabling robust functional gene editing in vitro and in vivo. It also maintains long-term stability and supports efficient cellular uptake, enabling administration via multiple routes. FNB[2] represents a scalable and robust platform for genetic therapeutics, vaccines, and biologics, particularly well-suited for resource-limited and emergency medical applications.},
}
@article {pmid41816357,
year = {2026},
author = {Pan, Z and Xu, L and Fan, Z and Cao, Y and Ren, F},
title = {CRISPR-based diagnostics for infectious diseases: mechanisms, advancements and clinical transformation prospects.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1769226},
pmid = {41816357},
issn = {2235-2988},
mesh = {Humans ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; *Communicable Diseases/diagnosis ; Gene Editing/methods ; Sensitivity and Specificity ; },
abstract = {Infectious diseases continue to pose significant global public health challenges, necessitating the development of rapid, sensitive, specific, and field-deployable diagnostic platforms. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) has revolutionized genome editing and concurrently enabled a new generation of molecular diagnostic tools. Leveraging the inherent trans-cleavage activities of Cas enzymes, platforms such as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) have emerged, combining target recognition precision with reporter systems to achieve ultra-sensitive detection of pathogen-specific nucleic acids. This review systematically examines the mechanistic foundations of CRISPR diagnostics, synthesizes recent advancements in infectious disease applications, evaluates their advantages in sensitivity, specificity, operational simplicity, and multiplexing capacity, and critically analyzes current implementation barriers and future translational pathways.},
}
@article {pmid41816845,
year = {2026},
author = {Chen, WD and Liu, L and Cheng, L},
title = {Nitroreductase-Responsive Oligomeric crRNAs for Enzyme-Triggered Regulation of CRISPR Activity.},
journal = {The Journal of organic chemistry},
volume = {91},
number = {12},
pages = {4494-4501},
doi = {10.1021/acs.joc.5c02287},
pmid = {41816845},
issn = {1520-6904},
mesh = {*Nitroreductases/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry ; },
abstract = {Hypoxic tumors overexpress nitroreductase (NTR), providing an endogenous trigger for selective biomolecular activation. Here, we describe the synthesis of NTR-responsive clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs via the site-specific incorporation of a p-nitrobenzyl (p-NB) phosphoramidite at the 5' terminus of crRNAs. Click-mediated oligomerization into trimeric and tetrameric constructs effectively suppressed Cas nuclease activity. Enzymatic reduction by NTR induced linker cleavage, releasing active crRNAs and restoring DNA cleavage in vitro, establishing a strategy for enzyme-regulated CRISPR control.},
}
@article {pmid41816914,
year = {2026},
author = {Zhao, H and Zhou, T and Zhang, M and Wang, C and Wang, R and Shu, X and Cheng, F and Xue, Q and Liu, C and Xu, J and Cao, X and Du, J and Wang, L and Liu, H and Li, M},
title = {Associate toxin-antitoxin with CRISPR-Cas to harness (ATTACH) engineered microbes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41816914},
issn = {1362-4962},
support = {2024YFA0918500//National Key Research and Development Project/ ; XDB0810000//Chinese Academy of Sciences/ ; 32370090//National Natural Science Foundation of China/ ; 32150020//National Natural Science Foundation of China/ ; 32400063//National Natural Science Foundation of China/ ; 32270092//National Natural Science Foundation of China/ ; 32200057//National Natural Science Foundation of China/ ; 32370120//National Natural Science Foundation of China/ ; 2020090//Youth Innovation Promotion Association of CAS/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Toxin-Antitoxin Systems/genetics ; Mice ; Plasmids/genetics ; Escherichia coli/genetics/metabolism ; Genetic Engineering/methods ; Lycopene ; },
abstract = {Robust biocontainment is essential for the safe use of engineered microbes, but existing strategies suffer from genetic instability and/or laborious construction. Here, we present ATTACH, a kill switch that associates toxin-antitoxin with CRISPR-Cas to harness engineered microbes. Our approach employs a CRISPR-repressed toxin-antitoxin (CreTA) module to make microbes addicted to the type I-F Cas effector proteins, and places both the Cas3 nuclease and the chromosome-targeting guide RNA under inducible promoters, thereby improving the genetic stability and stringency of the CRISPR-based suicidal program. Additionally, we have developed a single-plasmid, antibiotic-independent ATTACH device, which shows robust, stringent containment of a microbial chassis in murine gut, and negligible impacts on culture growth or lycopene production during batch fermentation. Our data highlight the potential of CreTA to stabilize CRISPR-based kill switches, advancing their development into more portable and reliable biocontainment tools for engineered microbes.},
}
@article {pmid41818620,
year = {2026},
author = {Huang, ZJ and Li, FM and Tu, YF and Feng, KK and Li, CL and Tian, SC and Hu, YS and Shao, JW and Liu, ZH},
title = {CRISPR/Cas9-Based Vanadium MXene-Free Radical Spatiotemporally Controlled Nanoreactor for Photothermal-Induced Multi-Effect Synergistic Antitumor Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {29},
pages = {e22535},
pmid = {41818620},
issn = {2198-3844},
mesh = {Animals ; *Photothermal Therapy/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Mice ; Free Radicals ; Tumor Microenvironment ; *Neoplasms/therapy ; Combined Modality Therapy ; Cell Line, Tumor ; Nitrites ; Transition Elements ; },
abstract = {Photothermal therapy (PTT), a non-invasive tumor treatment, shows promise but is limited in solid tumors by restricted tissue penetration, thermotolerance, anti-apoptotic and immunosuppressive effects. In this study, tumor microenvironment-responsive nanoplatform VARH was constructed based on MXene. Under NIR-II laser irradiation, VARH achieves a high photothermal conversion efficiency of 44.21%. Loaded 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride decomposes at high temperatures to generate alkyl radicals, synergizing with hydroxyl radicals from V[4+]-catalyzed endogenous H2O2 decomposition, enabling chemodynamic therapy (CDT) and thermal dynamic therapy to enhance tumor cell oxidative damage. Triggered by high glutathione, VARH releases ribonucleoprotein (RNP) complexes to knockout heat shock protein 90 (HSP90), attenuating cellular heat resistance and promoting apoptosis. It also enhances T cell-mediated anti-tumor immunity and, with free radicals, promotes tumor cell immunogenic cell death (ICD), achieving immunotherapeutic multi-effect synergy. Integrating nanotechnology with precise gene editing, this study develops a novel multimodal synergistic therapy system, providing new insights for multi-modal treatment R&amp;D and advancing PTT and free radical-based cancer therapies.},
}
@article {pmid41818662,
year = {2026},
author = {Wu, L and Chen, W and Huang, R and Zhou, F},
title = {EXAGO: An Argonaute-Based Primer-Free Exponential Amplification Strategy for Ultrasensitive Zero-Background Detection of Point Mutation.},
journal = {Analytical chemistry},
volume = {98},
number = {11},
pages = {8199-8208},
doi = {10.1021/acs.analchem.5c06961},
pmid = {41818662},
issn = {1520-6882},
mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; Humans ; *Point Mutation ; *Nucleic Acid Amplification Techniques/methods ; ErbB Receptors/genetics ; Limit of Detection ; },
abstract = {Ultrasensitive gene detection is crucial for precise molecular diagnostics. Conventional nucleic acid amplification methods frequently encounter nonspecific amplification triggered by exogenous primers, which limits their utility in ultrasensitive detection. Programmable gene editing tools, such as CRISPR/Cas and Argonaute (Ago), provide new avenues for developing next-generation detection technologies. Here, we develop an Ago-mediated exponential amplification strategy, EXAGO, for ultrasensitive detection of the epidermal growth factor receptor (EGFR) L858R mutation─a critical biomarker in nonsmall cell lung cancer. Leveraging the flexible programmability and single-base-resolution cleavage activity of Ago, EXAGO can specifically initiate DNA polymerase reactions at the target mutation site. The mechanism of dual-circuit operation allows the system to achieve an efficient exponential amplification under thermal cycling. By circumventing exogenous primers, it also prevents nonspecific amplification caused by primer misidentification at the source. Moreover, wild-type genes are entirely unable to trigger signal amplification, thereby underscoring their superior specificity and the potential for practical application. Experimental results demonstrated that EXAGO achieves femtomolar-level sensitivity and exhibits favorable recovery rates for detecting plasma-diluted samples. Moreover, the use of a thermostable enzyme allows direct compatibility with cell thermal lysis, enabling detection in cell lysates. In summary, EXAGO offers a robust and practical solution for accurate genetic mutation analysis in complex samples and promotes the application of Ago-based tools in molecular diagnostics.},
}
@article {pmid41819296,
year = {2026},
author = {Wang, C and Zhu, C and Liu, Q and Yang, L},
title = {Prokaryotic argonaute proteins: From ancient defense mechanisms to modern biosensing applications.},
journal = {Biotechnology advances},
volume = {89},
number = {},
pages = {108869},
doi = {10.1016/j.biotechadv.2026.108869},
pmid = {41819296},
issn = {1873-1899},
mesh = {*Biosensing Techniques ; *Argonaute Proteins/genetics/metabolism/chemistry ; *Prokaryotic Cells/metabolism ; },
abstract = {Prokaryotic Argonaute (pAgo) proteins constitute an evolutionarily ancient nuclease family that is rapidly maturing into a versatile molecular toolkit rivaling CRISPR-Cas. This review synthesizes recent advances in pAgo biology and biotechnology, tracing their phylogeny across thermophilic, mesophilic, and psychrotolerant lineages and highlighting temperature-adapted catalytic signatures that diverge from eukaryotic Agos. In vivo studies reveal pAgo roles in gDNA-guided host defense, transcriptional silencing and recombination, all executed through programmable DNA- or RNA-guided nuclease activity. We detail how guide length, 5' nucleotide identity, divalent cations and accessory factors modulate cleavage efficiency, enabling rational optimization. The review then maps the explosion of pAgo-based biosensing platforms, including selective nucleic acid enrichment platforms, ultrasensitive pathogen detection methods, programmable DNA cloning systems, and high-resolution imaging techniques. Their independence from protospacer adjacent motifs (PAMs), stable DNA guides, multi-turnover kinetics, and broad thermal tolerance position pAgos as ideal complements to CRISPR systems. Finally, we outline current limitations and future directions, including the discovery and engineering of novel variants, elucidation of guide-generation mechanisms, and development of next-generation gene-editing tools, aiming to accelerate translation of these versatile enzymes into practical biotechnological and therapeutic translation.},
}
@article {pmid41820207,
year = {2026},
author = {Porwal, S and Malviya, R and Belagodu Srighar, S and Shareef, J},
title = {Ribosome-targeted and Adjuvant Strategies to Combat Antibiotic Resistance.},
journal = {Infectious disorders drug targets},
volume = {},
number = {},
pages = {},
doi = {10.2174/0118715265396934250805055608},
pmid = {41820207},
issn = {2212-3989},
abstract = {INTRODUCTION: This study examines ribosome-targeted and adjuvant strategies to combat the growing threat of antimicrobial resistance (AMR), with a focus on novel therapeutic approaches, including phage therapy, monoclonal antibodies, CRISPR systems, and AI-driven drug discovery. The objective is to review current challenges and evaluate innovative strategies targeting bacterial ribosomes, a primary site for antibiotic action.<br><br>METHODS: A systematic literature review was conducted using databases, such as PubMed, ScienceDirect, Scopus, and Google Scholar.<br><br>RESULTS: The study indicates that bacteria evade ribosome-targeting antibiotics through various mechanisms, including porin modification, efflux pumps, ribosomal mutations, and enzymatic degradation. Innovative strategies, including AI-enabled virtual screening, phage-antibiotic synergy, ribosomal protein-targeted monoclonal antibodies and vaccines, and CRISPR-Cas systems, have shown potential in overcoming these mechanisms and restoring antibiotic efficacy.<br><br>DISCUSSION: These advanced strategies represent a significant shift from traditional approaches, as they directly target ribosomal functions or resistance genes. While promising, limitations such as phage specificity, challenges in CRISPR delivery, and regulatory concerns must be addressed to ensure clinical translation. The integration of AI with molecular techniques enhances therapeutic precision and development speed.<br><br>CONCLUSION: Ribosome-targeted therapies and adjunctive strategies, such as AI, phage therapy, monoclonal antibodies, and CRISPR, offer precise and innovative solutions to overcome antibiotic resistance.},
}
@article {pmid41820368,
year = {2026},
author = {Bertlin, JAC and Pauzaite, T and Liang, Q and Wit, N and Williamson, JC and Sia, JJ and Matheson, NJ and Ortmann, BM and Mitchell, TJ and Speak, AO and Zhang, Q and Nathan, JA},
title = {VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41820368},
issn = {2041-1723},
support = {R01 CA284591/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; STING Protein ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Membrane Proteins/metabolism/genetics ; Animals ; Cell Line, Tumor ; *Core Binding Factor beta Subunit/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Gene Expression Regulation, Neoplastic ; Interferon Type I/metabolism ; *Synthetic Lethal Mutations ; Mice ; cGAS-STING Signaling Pathway ; },
abstract = {Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.},
}
@article {pmid41820622,
year = {2026},
author = {Padureanu, T and Cocoș, R and Matache, IM and Bucur, O},
title = {Gene-sized editing for the therapy of genetic diseases.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {41820622},
issn = {1438-7948},
abstract = {Programmable genome editing technologies have reshaped the landscape of biomedical sciences, enabling the development of methods with great translational potential. CRISPR-Cas represents one of the most important and widely adopted genome editing tools, although its reliance on double-stranded DNA breaks implies inherent limitations on the precision and safety of genomic insertions. Thus, several research groups have focused on the development of new editing technologies, among which prime editing has emerged as a cutting-edge system. Ongoing advancements in prime editing, including protein engineering, have enhanced its efficiency and expanded its functionality. However, prime editing cannot achieve integration of large DNA sequences larger than 5 kilobases. To overcome this limitation, PASTE and PASSIGE methods were developed as novel genome editing methods that merge precise genome rewriting with efficient recombinase-mediated gene insertion. In this review, we investigate the mechanistic principles of these systems, compare their performance in cellular and animal models, and discuss the ongoing efforts to enhance system components and delivery. We extended our investigation to recent progress supporting their translational potential, assessing efficient delivery methods, genome site specificity, safety, and long-term efficacy, which are crucial for successful in vivo applications.},
}
@article {pmid41821244,
year = {2026},
author = {Elamin Eltom, A and Kareem, AK and Shaaban, Z and Sanaan Jabbar, H and Sharma, MK and Ghazi Al-Shawi, S and AlMohamadi, H and Abbas, Z and S Jabir, M and Ahmed AbdRabou, M},
title = {Progress of Magnetic Particles-Integrated CRISPR/Cas Biosensors for Pathogen Bacteria Detection: Design, Mechanism and Application.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10408347.2026.2636658},
pmid = {41821244},
issn = {1547-6510},
abstract = {Ensuring food safety through rapid, sensitive, and point-of-care (POC) detection of microbial pathogens is crucial for protecting public health and minimizing the socio-economic losses associated with foodborne diseases. Despite stringent regulatory measures, foodborne illnesses caused by microbial contamination continue to pose a significant global challenge. In this context, the emergence of CRISPR/Cas systems has significantly improved the performance of biosensors due to their programmability, high specificity, and precise recognition of target RNA and DNA sequences. Following target recognition, Cas proteins exhibit both cis- and trans-cleavage activities, enabling highly sensitive signal amplification. To achieve rapid analysis and low detection limits, recent studies have increasingly focused on integrating CRISPR/Cas system with magnetic particles (MPs). MPs offer key advantages, including superparamagnetism, biocompatibility, and facile surface functionalization, which enhance target enrichment, assay speed, and analytical sensitivity. Accordingly, substantial progress has been made in MP-conjugated CRISPR/Cas biosensors for the detection of diverse foodborne microbial pathogens. This review comprehensively summarizes recent advances in the integration strategies of magnetic particles with CRISPR/Cas-based biosensing platforms for the quantitative detection of microbial pathogens. Particular emphasis is placed on performance metrics, assay design, and the feasibility of these systems for POC applications, highlighting their potential to enhance food safety monitoring.},
}
@article {pmid41823840,
year = {2026},
author = {Loweree-Rivera, FD and Pérez-Álvarez, S and Castillo, AM and Vega Mares, JH and Leyva-Hernández, HA and Sánchez Chávez, E and Escobedo-Bonilla, CM and Uranga-Valencia, LP and Chávez Medina, JA},
title = {Pelecyphora chihuahuensis (Britton &amp; Rose) D. Aquino &amp; Dan. Sánchez: A Review on Its Taxonomy, Ecology and Conservation of an Endemic Mexican Cactus Species with Biotechnological Perspectives.},
journal = {Biology},
volume = {15},
number = {5},
pages = {},
pmid = {41823840},
issn = {2079-7737},
abstract = {The cactus Pelecyphora chihuahuensis is endemic to northern Mexico and represents an interesting subject on the integration of classical taxonomy with modern biotechnological tools to solve conservation issues. Because of its narrow ecological range and high ornamental value, the species is increasingly at risk from degradation of its habitats, climate change, and plant poaching. This review includes current knowledge on its taxonomic status, ecological distribution, and conservation needs, with a focus on biotechnological means to aid its preservation. Aspects such as molecular markers, next-generation sequencing, and previously reported GIS-based species distribution models provide valuable insights into its identity and ecological niche. Biotechnological tools for ex situ conservation include in vitro propagation and cryopreservation. Potential applications of CRISPR-Cas and synthetic biology in preserving rare cacti are also discussed. By uncovering gaps, this review opens a window of opportunity to urgently promote the sustainable management of P. chihuahuensis and related endangered cacti by merging biotechnology with ecology and taxonomy, the results presented here underscore the importance of integrating scientific findings into future research that supports the development and implementation of effective policies that prioritize the conservation and biocultural preservation of arid-land flora, ensuring that both ecological integrity and cultural values are maintained for these unique ecosystems.},
}
@article {pmid41824451,
year = {2026},
author = {Yuan, L and Liu, Q and Xiao, X and Xu, L and Liang, L and Guo, Y and Yao, Y and Wang, H and Feng, Y and Hua, X and Feng, Y},
title = {AlphaFold 3-powered discovery of phage proteins that inhibit bacterial transcription initiation.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117082},
doi = {10.1016/j.celrep.2026.117082},
pmid = {41824451},
issn = {2211-1247},
mesh = {*Viral Proteins/metabolism/chemistry ; DNA-Directed RNA Polymerases/metabolism ; *Bacteriophages/metabolism ; *Transcription Initiation, Genetic ; *Escherichia coli/genetics/metabolism/virology ; Transcription, Genetic ; Protein Binding ; Models, Molecular ; },
abstract = {Many phages encode proteins that specifically inhibit host RNA polymerase activity, thereby sabotaging and, in some cases, hijacking the host transcription machinery to serve their needs. Traditional methods for identifying new phage proteins that inhibit bacterial transcription are labor intensive and require access to live phages. To overcome these limitations, we develop a highly efficient pipeline for AlphaFold 3-guided discovery of phage proteins that inhibit bacterial transcription initiation. Using this pipeline, three phage proteins are identified and characterized. Structural and biochemical analyses demonstrate that these phage proteins bind to distinct sites on RNA polymerase and inhibit transcription initiation via different mechanisms. This study showcases the power of AlphaFold 3 in discovering novel binders of large protein complexes, and the pipeline developed here could be readily adapted to screen modulators of other large targets, such as the ribosome, proteasome, and CRISPR-Cas systems.},
}
@article {pmid41824934,
year = {2026},
author = {Arif, MA and Mubashir, F and Raffay, A and Talha, M and Zohaib, M and Khan, AR and Naseer, MU and Khan, MH and Idrees, M and Rafique, S and Afzal, S and Amin, I and Shahid, M},
title = {Recent Advances in CRISPR-Cas Systems for Dengue Virus Detection.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {36},
number = {1},
pages = {19-35},
doi = {10.1615/CritRevEukaryotGeneExpr.2025062420},
pmid = {41824934},
issn = {2162-6502},
mesh = {*Dengue Virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Dengue/diagnosis/virology/genetics ; RNA, Viral/genetics ; Nucleic Acid Amplification Techniques/methods ; Molecular Diagnostic Techniques/methods ; },
abstract = {Dengue virus (DENV) infections persist as a significant global health threat despite decades of surveillance and control efforts. The disease may progress to severe dengue, marked by hemorrhage, plasma leakage, and vital organ impairment, contributing to substantial worldwide morbidity and mortality. The rapidly escalating DENV burden demands rapid and innovative diagnostic approaches that move beyond conventional detection methods. CRISPR-Cas-based technologies have emerged as a revolutionary approach, offering next-generation solutions for DENV diagnostics. This review outlines the recent advances in the use of CRISPR based technologies for robust and more sensitive detection of dengue virus nucleic acids, critically evaluating their advantages over conventional diagnostics, current limitations, and future prospects. The roles of Cas12 and Cas13 in DENV-RNA detection are discussed in detail. Additional key areas mentioned include field-deployable and portable CRISPR-Cas technologies, serotype-specific detection, hybrid and isothermal amplification-based approaches, and a combination of CRISPR with electrochemical sensing techniques and nanotechnology. Collectively, these advances highlight the potential of CRISPR-based diagnostics in evolving future strategies for rapid and effective dengue virus detection and control of infections.},
}
@article {pmid41825104,
year = {2026},
author = {Li, P and Wang, L and Li, G and Li, R and Li, Y and Zhang, Z and Yang, S and Tang, H and Liu, Z},
title = {Transcriptomic and phenotypic analysis of maize with CRISPR/Cas9-mediated targeted mutagenesis of melatonin synthesis genes under drought stress.},
journal = {Plant physiology and biochemistry : PPB},
volume = {233},
number = {},
pages = {111189},
doi = {10.1016/j.plaphy.2026.111189},
pmid = {41825104},
issn = {1873-2690},
mesh = {*Zea mays/genetics/metabolism/physiology ; *Melatonin/biosynthesis/genetics ; *CRISPR-Cas Systems/genetics ; *Droughts ; Gene Expression Regulation, Plant ; Drought Resistance ; *Stress, Physiological/genetics ; *Transcriptome ; Abscisic Acid/metabolism ; Plant Proteins/genetics/metabolism ; *Mutagenesis ; Reactive Oxygen Species/metabolism ; Phenotype ; },
abstract = {Melatonin, a pleiotropic regulatory factor, plays a key role in mediating crop drought resistance. Herein, we conducted an integrated physiological and transcriptomic approach to elucidate the mitigating effect of endogenous melatonin in mitigating drought stress in maize. We generated a comt snat asmt maize mutant via CRISPR-Cas9-mediated simultaneous editing of ZmCOMT, ZmSNAT, and ZmASMT1-genes encoding rate-limiting enzymes in the endogenous melatonin biosynthesis pathway. Sequencing of the mutant lines revealed key amino acid substitutions (Gly168→Ala in ZmSNAT, Asp175→Glu in ZmCOMT, and Asp150→Glu in ZmASMT1) within critical protein domains, resulting from CRISPR-induced small insertions or deletions (indels), which led to subtle alterations in the tertiary conformation of corresponding proteins. These modifications resulted in an 86.70% increase in endogenous melatonin content. Under drought stress, the comt snat asmt maize exhibited enhanced antioxidant enzyme activities, leading to a significant reduction in reactive oxygen species (ROS) accumulation compared to the control. Furthermore, endogenous levels of melatonin, abscisic acid (ABA), cytokinin (CTK), and auxin (IAA) were markedly elevated, whereas gibberellin (GA) content was significantly reduced. Consistently, the activities of SNAT, ASMT, and COMT were also enhanced in the mutant. Transcriptomic profiling further revealed that endogenous melatonin regulates ABA, IAA, CTK, and GA signaling pathways to enhance drought tolerance. In particular, ZmCOMT, ZmSNAT, and ZmASMT1 apparently modulated the expression levels of key regulatory genes such as ZmIAA2, ZmIAA23, ZmIAA7, ZmSAUR24, ZmPYL8, and ZmPIF3.1, associated with these hormone pathways. Collectively, endogenous melatonin reinforces drought tolerance by reducing ROS accumulation and reprogramming phytohormone homeostasis through regulation of hormone-related gene expression. Our findings provide important insights into the regulatory mechanisms by which endogenous melatonin enhances drought resistance in crops.},
}
@article {pmid41825300,
year = {2026},
author = {Low, YC and McKnight, CL and Elliott, DA and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent embryonic stem cell TAFAZZIN hESC model (WAe009-A-3H) of Barth syndrome.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103948},
doi = {10.1016/j.scr.2026.103948},
pmid = {41825300},
issn = {1876-7753},
mesh = {Humans ; *Barth Syndrome/pathology/metabolism/genetics ; Female ; *Human Embryonic Stem Cells/metabolism/cytology ; Acyltransferases ; Cell Line ; Cell Differentiation ; *Transcription Factors/genetics/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; *Models, Biological ; },
abstract = {Barth syndrome is among the most common mitochondrial diseases presenting with cardiomyopathy. We have generated a human embryonic stem cell (hESC) model of Barth syndrome (TAFAZZIN[Δ3] C15) in a female background (H9 hESC) using CRISPR/Cas9 gene editing, with compound heterozygous variants in TAFAZZIN that result in exon 3 skipping in all stable transcripts. This cell line displayed characteristics consistent with pluripotent stem cells, including typical colony morphology, expression of pluripotency markers, trilineage potential, and a normal karyotype. This TAFAZZIN[Δ3] C15 line could be used for investigation of disease mechanisms in mitochondrial cardiomyopathy and preclinical therapeutic screening.},
}
@article {pmid41825380,
year = {2026},
author = {Wang, X and Zhang, X and Guo, Y and Zheng, Q and Hu, B and Liu, R and Cao, J},
title = {CRISPR/Cas12-driven portable paper-based electrochemical aptasensor based on 0D/2D Au@Ti3C2 MXene for AFB1 detection.},
journal = {Food chemistry},
volume = {511},
number = {},
pages = {148810},
doi = {10.1016/j.foodchem.2026.148810},
pmid = {41825380},
issn = {1873-7072},
mesh = {*Aflatoxin B1/analysis ; *Electrochemical Techniques/instrumentation/methods ; Gold/chemistry ; *Biosensing Techniques/instrumentation/methods ; Aptamers, Nucleotide/chemistry ; Food Contamination/analysis ; Paper ; CRISPR-Cas Systems ; Limit of Detection ; Titanium/chemistry ; Nitrites ; Transition Elements ; },
abstract = {Aflatoxin B1 (AFB1) is a widely distributed and potent carcinogenic small-molecule toxic contaminant, posing significant risks to food safety. Therefore, a portable, cost-effective, and field-deployable method is needed for sensitive detection of AFB1. This study developed a paper-based electrochemical aptasensor integrating CRISPR/Cas12a technology and 0D/2D Au@Ti3C2 MXene heterostructure for highly sensitive and rapid detection of AFB1. The aptasensor leveraged the Au@Ti3C2 MXene with excellent electron transfer property to amplify the electrochemical signals, while the CRISPR/Cas12a trans-cleavage activity, triggered by AFB1 specific aptamers, enabled high specificity. Utilizing a portable electrochemical workstation, the paper-based electrochemical sensing platform exhibited a linear detection range of 0.5 pg/mL-100 ng/mL with a limit of detection of 0.16 pg/mL and achieved recoveries of 93.0%-106.0% in spiked real samples, demonstrating promise as a rapid and portable tool for on-site analysis of mycotoxins in real samples.},
}
@article {pmid41825677,
year = {2026},
author = {Xu, Z and Haghighat, M and Shafiabady, N and Wu, R and Sang, R and Deng, W},
title = {Nuclear-targeted CRISPR/Cas delivery by using aptamer-modified polymer lipid nanoparticles.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {74},
number = {},
pages = {102928},
doi = {10.1016/j.nano.2026.102928},
pmid = {41825677},
issn = {1549-9642},
mesh = {*Nanoparticles/chemistry ; Humans ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Cell Nucleus/genetics/metabolism ; *Polymers/chemistry ; *Lipids/chemistry ; Oligodeoxyribonucleotides/chemistry ; Gene Editing ; Cell Line, Tumor ; Liposomes ; },
abstract = {Efficient nuclear delivery of CRISPR/Cas ribonucleoproteins (RNP) remains a significant hurdle for non-viral systems. To address this, we developed a polymer-lipid hybrid nanoparticle functionalized with the AS1411 aptamer, targeting nucleolin to facilitate nucleus-directed delivery. Confocal imaging confirmed the accumulation of these aptamer-modified nanoparticles within the cell nuclei. For precise quantification, we utilized an AI-assisted segmentation approach based on deep convolutional neural networks (CNN) to analyse nanoparticle and DAPI colocalization. We further evaluated in vitro gene knockout efficiency of Cas9/sgRNA by using this nucleus-targeted system. Aptamer-functionalised nanoparticles reduced GFP-positive cells to 30.0%, compared with 40.8% for untargeted nanoparticles. Further evaluation targeting the Lcn2 gene demonstrated higher knockout efficacy and a more potent inhibition of breast cancer cell proliferation. These findings indicate that aptamer-mediated nuclear targeting enhances CRISPR/Cas9 editing efficacy and may offer the potential to advance the performance of non-viral gene therapies.},
}
@article {pmid41826557,
year = {2026},
author = {Chao, K and Dietrich, ML and Covey, SC and Momoh, M and Gutt, EG and Sandi, JD and Kamara, MS and Fofanah, IU and Rogers, MM and Kallon, TMPS and Samuels, RJ and Grant, DS and Sabeti, PC and Garry, RF},
title = {Adaptable, quantitative CRISPR/Cas12a-based assay for cytomegalovirus DNA in infant saliva.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41826557},
issn = {2045-2322},
mesh = {Humans ; *Cytomegalovirus/genetics/isolation &amp; purification ; *DNA, Viral/genetics/analysis ; *Saliva/virology ; *Cytomegalovirus Infections/diagnosis/virology/genetics ; *CRISPR-Cas Systems ; Infant ; Sensitivity and Specificity ; Polymerase Chain Reaction/methods ; *Endodeoxyribonucleases/genetics ; *CRISPR-Associated Proteins/genetics ; Nucleic Acid Amplification Techniques/methods ; *Bacterial Proteins/genetics ; },
abstract = {Congenital cytomegalovirus (CMV) infection is the leading non-genetic cause of infant hearing loss worldwide, and a significant cause of neurodevelopmental disabilities. Reliance on polymerase chain reaction (PCR) for CMV DNA testing hampers diagnostic and research efforts in low-resource settings and universal screening implementation in high-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) detection and recombinase polymerase amplification (RPA) can be used together for low-cost viral detection. Here we describe an adaptable RPA-Cas12a assay for CMV DNA quantification based on the WHO international standard. Adequate quantification accuracy was achieved with contrived CMV samples but performance with Sierra Leonean infant saliva remains suboptimal. While improved quantification accuracy will require further optimization, our assay achieves screening test requirements, including > 80% sensitivity/specificity, quicker and more economically than PCR. This work highlights RPA-Cas12a-based assays for DNA quantification and suggests a path towards increased congenital CMV screening using PCR and RPA-Cas12a synergistically.},
}
@article {pmid41826615,
year = {2026},
author = {Fernandes, LGV and Nally, JE},
title = {CRISPR-based mutagenesis of lipopolysaccharide biosynthesis genes in Leptospira interrogans reveals gene essentiality and confirms the role of an O-antigen polymerase.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41826615},
issn = {2045-2322},
mesh = {*Leptospira interrogans/genetics/metabolism/pathogenicity/enzymology ; *Lipopolysaccharides/biosynthesis ; Animals ; *Mutagenesis ; Leptospirosis/microbiology/immunology ; CRISPR-Cas Systems ; *Genes, Essential ; O Antigens/genetics ; Cricetinae ; Mutation ; *Bacterial Proteins/genetics/metabolism ; Hexosyltransferases ; },
abstract = {Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira. Lipopolysaccharide (LPS) is an immunodominant and protective antigen for Leptospira, but its biosynthesis remains poorly understood. In this study, we employed CRISPR/Cas9-non-homologous end-joining and CRISPR-Prime Editing to mutate key genes within the rfb locus of L. interrogans, including those involved in core oligosaccharide assembly, and the biogenesis, polymerization, and ligation of O-antigen. Mutants were successfully generated in LIC11312 (waaF, heptosyltransferase II) and LIC12137 (wcaJ, undecaprenyl-phosphate glycosyltransferase) but yielded only in-frame deletions suggesting their essentiality. Mutants were also successfully generated in LIC12143, a putative O-antigen polymerase, which exhibited truncated LPS that failed to induce acute leptospirosis in hamsters but retained the ability to colonize kidneys. Mutation of LIC_RS09320, an O-antigen ligase, did not display a change in LPS phenotype. Bacterins prepared with either control wild-type or LIC12143 mutant cells conferred complete homologous protection with sterile immunity, though failed to protect against heterologous challenge. These findings confirm LIC12143 as a functional O-antigen polymerase and underscore the challenges in generating knockout mutants to understand LPS biosynthesis in leptospires.},
}
@article {pmid41826696,
year = {2026},
author = {Aird, EJ and Rabl, J and Knuesel, T and Groen, K and Awwad, SW and Korablev, B and Scherpe, L and Al-Herz, W and Hupfer, R and Recher, M and Jackson, SP and Hale, BG and Corn, JE},
title = {An SP110-SP100 axis is a critical regulator of promyelocytic leukaemia body dynamics and mitotic fidelity.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {684-695},
pmid = {41826696},
issn = {1476-4679},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 310030_188858//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 320030_232029//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; ALTF 144-2021//European Molecular Biology Organization (EMBO)/ ; Women's Postdoctoral Career Development Award in Science//Weizmann Institute of Science/ ; Outstanding Postdoctoral Women Fellowship//Council for Higher Education of Israel | Israeli Centers for Research Excellence (I-CORE)/ ; DRCPGM\100005//Cancer Research UK (CRUK)/ ; SEBINT-2024/100003//Cancer Research UK (CRUK)/ ; },
mesh = {Humans ; *Mitosis ; *Autoantigens/metabolism/genetics/chemistry ; *Promyelocytic Leukemia Protein/metabolism/genetics ; *Antigens, Nuclear/metabolism/genetics/chemistry ; *Leukemia, Promyelocytic, Acute/metabolism/genetics/pathology ; *Nuclear Proteins/metabolism/genetics ; DNA Damage ; CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {Stimulation of the innate immune system by foreign RNA elicits a potent interferon response and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Here, using genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we discover that promyelocytic leukaemia (PML) nuclear body-localized speckled protein 110 (SP110) is a potent inhibitor of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a toxic activity of SP100, a major constituent of PML bodies. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, leading to micronucleus formation, DNA damage and genotoxic cell death. A combination of cryo-electron microscopy, AlphaFold modelling and cellular biochemistry reveals that SP110 dissolves toxic SP100 oligomers via necessary and sufficient direct interactions between their caspase activation and recruitment domains. These data reveal the critical roles of SP100 and SP110 in governing the disassembly of PML bodies during mitosis, as well as the repercussions if this process is misregulated.},
}
@article {pmid41826699,
year = {2026},
author = {Skafar, V and de Souza, I and Ghosh, B and Ferreira Dos Santos, A and Porto Freitas, F and Chen, Z and Sun, S and Donate Castillo, M and Nepachalovich, P and Seufert, L and Bothe, S and Tschuck, J and Mathur, A and Nunes-Alves, A and Buhr, J and Aponte-Santamaría, C and Schmitz, W and Mack, M and Eilers, M and Bargou, R and Chaufan, M and Kaur, M and Palma, M and Ubellacker, JM and Elling, U and Augustin, HG and Hadian, K and Meierjohann, S and Proneth, B and Conrad, M and Fedorova, M and Alborzinia, H and Friedmann Angeli, JP},
title = {Riboflavin metabolism shapes FSP1-driven ferroptosis resistance.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {696-706},
pmid = {41826699},
issn = {1476-4679},
mesh = {*Ferroptosis/drug effects ; *Riboflavin/metabolism/pharmacology ; Humans ; Antioxidants/metabolism ; Lipid Peroxidation/drug effects ; *S100 Calcium-Binding Protein A4/metabolism/genetics ; Animals ; Cell Line, Tumor ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell's antioxidant capacity.},
}
@article {pmid41826749,
year = {2026},
author = {Valentino, LA and Hermans, C and Coffin, D and Miesbach, W and Mancuso, ME and Unzu, C and Jones, M and Gutstein, DE and McKeown, W and Kessler, CM},
title = {Building a gene editing lexicon: a model for rare and inherited disorders.},
journal = {Gene therapy},
volume = {33},
number = {3},
pages = {310-315},
pmid = {41826749},
issn = {1476-5462},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Hemophilia A/therapy/genetics ; CRISPR-Cas Systems ; *Rare Diseases/therapy/genetics ; },
abstract = {As more advanced cell and gene therapies, including gene editing technologies, progress through drug development, there is increased emphasis on the importance of stakeholders, including people living with disease, caregivers, and healthcare professionals, to communicate using clear, accurate, and consistent language. Lexicons explaining advanced gene therapies will support patients' and clinicians' understanding, enabling shared decision-making and informed consent for clinical trial participation and, in the future, healthcare choice. Early lexicon development is crucial for standardizing communication across clinical sites, geographies, clinicians, and patients. A lexicon for clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing for hemophilia was developed using comprehensive methodologies, gathering insight through qualitative research and in-depth interviews, language audits, and workshops, with input from lived experience experts, leading clinicians in hemophilia, gene therapy experts, and scientific and patient organizations. This lexicon serves as a gold standard template for future comprehensive patient lexicon development strategy and could be applied to other therapeutic areas where treatments are being developed and standardized, or where accessible vocabulary for patients, healthcare professionals, and the affected community is lacking. This communication highlights the need for lexicon development for advanced gene editing treatments across therapeutic areas to support standardized understanding and enhance communication.},
}
@article {pmid41826758,
year = {2026},
author = {Deol, KK and Harris, CA and Tomlinson, SJ and Delaney, CJ and Al-Farhan, A and Mathiowetz, AJ and Doubravsky, CE and Pratt, DA and Olzmann, JA},
title = {Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {3},
pages = {525-536},
pmid = {41826758},
issn = {1545-9985},
support = {R01 CA305423/CA/NCI NIH HHS/United States ; },
mesh = {*Ferroptosis ; Humans ; *Riboflavin/metabolism ; Protein Stability ; Flavin-Adenine Dinucleotide/metabolism ; CRISPR-Cas Systems ; Proteolysis ; HEK293 Cells ; },
abstract = {Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR-Cas9 screens using a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase and flavin adenine dinucleotide (FAD) synthase, enzymes that are essential for synthesizing FAD from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for both FSP1 activity and stability. FAD deficiency and mutations blocking FSP1-FAD binding triggered FSP1 degradation through a ubiquitin-proteasome pathway involving the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance, with implications for therapeutic strategies targeting FSP1 in cancer.},
}
@article {pmid41826830,
year = {2026},
author = {Zhang, H and Zhang, P and Bindels, E and Mulugeta, E},
title = {Insights from pooled CRISPRi single-cell screens in K562 cells reveal gene functions, regulatory networks, and highlight opportunities and limitations.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41826830},
issn = {1471-2164},
mesh = {Humans ; *Gene Regulatory Networks ; K562 Cells ; *Single-Cell Analysis/methods ; Transcription Factors/genetics ; Single-Cell Gene Expression Analysis ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Pooled CRISPR screening combined with single-cell RNA sequencing (scRNA-seq) has emerged as a powerful strategy for dissecting gene function and reconstructing gene regulatory networks (GRNs) in complex biological systems. This approach enables high-throughput, parallel perturbation of multiple genes while providing transcriptome-wide readouts at single-cell resolution, overcoming many limitations of traditional arrayed screens. However, its broader application remains limited by technical challenges, including variable perturbation efficiency and difficulties in accurately identifying perturbed cells.In this study, we adapted and applied a modified CRISPR droplet sequencing (CROP-seq) protocol using CRISPR interference (CRISPRi) in K562 cells to knockdown six transcription factors (TFs): LMO2, TCF3, LDB1, MYB, GATA2, and RUNX1. Our modified approach, which allows direct capture of sgRNAs from the cDNA library without a separate enrichment step, significantly improved sgRNA assignment per cell. We successfully achieved reproducible knockdown of three TFs (MYB, GATA2, and LMO2), captured the impact of these perturbations on the TF target genes, and enabled us to reconstruct their GRNs and identify key regulons and transcriptional targets. These networks revealed both previously established (such as LMO2 GATA2 interaction) and novel regulatory interactions, which we independently validated, providing new insights into hematopoietic transcriptional control. To assess the efficiency of CRISPRi based pooled perturbation, we additionally analyzed publicly available Perturb-seq CRISPRi datasets and found that only ~40-50% of targeted genes led to effective knockdown, underscoring the variability in perturbation efficiency across experiments.Together, our results demonstrate both the potential and the current technical limitations of pooled CRISPRi-based single-cell screens. While this integrated approach holds great promise for high-resolution functional genomics, further optimization and standardized benchmarking are essential to improve its reliability, scalability, and reproducibility.},
}
@article {pmid41827871,
year = {2026},
author = {Zheng, J and Wu, M and Wang, X and Zuo, Z and Zhou, C and Zuo, E and Lu, Y},
title = {Prime Editing Exhibits Limited Genome-Wide Off-Target Effects in Cellular and Embryonic Gene Editing.},
journal = {Cells},
volume = {15},
number = {5},
pages = {},
pmid = {41827871},
issn = {2073-4409},
support = {2023ZD0405302 and 2023ZD04074//the Biological Breeding-Major Projects/ ; 2021YFD1300100 and 2024YFC3406001//the National Key Research and Development Program of China/ ; 32371549 and W2533083//the National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Genome ; Polymorphism, Single Nucleotide/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *Embryo, Mammalian/metabolism ; },
abstract = {Prime editing (PE) is a precise genome-editing technology that avoids double-strand breaks, holding great promise for clinical and agricultural applications. However, its genome-wide off-target effects are not fully understood, raising safety concerns. Here, we systematically compared the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, and PE5max) using PEM-seq and RNA-seq. We further applied an ultra-sensitive method, Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI), to assess PE5max. Our results show that PE5max did not produce detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) in the GOTI assay and induced only limited large deletions and chromosomal translocations. Collectively, this side-by-side benchmarking under matched conditions demonstrates that PE5max achieves an improved specificity profile, with no detectable increase in genome-wide off-target SNVs, advancing its potential for safer therapeutic use.},
}
@article {pmid41827889,
year = {2026},
author = {Siles, L and Ruiz-Nogales, S and Méndez-Vendrell, P and Pomares, E},
title = {Precise CRISPR/Cas9 and Cas12 Correction Using Lipoplexes in Retinal Models Derived from Patients with Inherited Retinal Dystrophies.},
journal = {Cells},
volume = {15},
number = {5},
pages = {},
pmid = {41827889},
issn = {2073-4409},
support = {Fi-201401//Fundaci&#xf3; de Recerca de l'Institut de Microcirurgia Ocular/ ; //IMO Grupo Miranza/ ; },
mesh = {Humans ; *Retinal Dystrophies/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Retinal Pigment Epithelium/metabolism/pathology ; ATP-Binding Cassette Transporters/genetics ; *Retina/pathology/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Transfection ; Organoids/metabolism ; Bestrophins ; },
abstract = {Gene editing, particularly CRISPR/Cas technology, represents a promising approach for the treatment of rare genetic diseases, including inherited retinal dystrophies, for which effective therapies are largely unavailable. Despite extensive research investigating gene editing across a wide range of cell types, transient delivery of CRISPR/Cas components and efficient homology-directed repair (HDR) in differentiated cells remain challenging. In this study, we employed hiPSCs derived from patients with Stargardt disease or Best disease, carrying pathogenic variants in ABCA4 or BEST1, respectively, to explore gene editing in human models. CRISPR/Cas9 and Cas12 nucleases were delivered into hiPS-derived retinal pigment epithelium (RPE) and retinal organoids using lipoplexes and compared with electroporation. We evaluated transfection efficiency, sgRNA-mediated DNA cleavage, and HDR-based correction. Precise repair of the pathogenic BEST1 variant was successfully achieved in hiPS-derived RPE cells using both nucleases, with Cas12 yielding the highest efficiency, exceeding 10% of HDR correction. Edited RPE cells preserved normal morphology and expressed specific maturity markers. In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues. This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes. These findings underscore the therapeutic potential of CRISPR/Cas-based strategies for inherited retinal dystrophies and provide a proof of concept for future clinical approximations.},
}
@article {pmid41828461,
year = {2026},
author = {Ferrero, M and Acquadro, A and Moglia, A},
title = {From Lab to Field: CRISPRing Major Cultivated Solanaceae for Crop Improvement.},
journal = {International journal of molecular sciences},
volume = {27},
number = {5},
pages = {},
pmid = {41828461},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Solanaceae/genetics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant ; Plant Breeding/methods ; },
abstract = {The Solanaceae family includes some of the most economically and agronomically important crops, such as tomato, potato, pepper and eggplant. Recently, CRISPR/Cas-based genome editing has emerged as a powerful tool for functional genomics and crop improvement, enabling precise and efficient genetic modifications. This review provides an overview of CRISPR/Cas-mediated genome editing technologies and their applications in the major cultivated Solanaceae crops. The use of CRISPR/Cas9 systems for targeted gene knockout and knock-in approaches is described, together with advances in precision editing strategies such as base editing and prime editing, which allow precise nucleotide substitutions and small sequence changes. The expanding CRISPR toolbox is further explored through alternative Cas proteins, such as Cas12a and Cas13 with distinct targeting features and potential applications. Emerging delivery strategies, including ribonucleoprotein-mediated editing in protoplasts, virus-induced gene editing (VIGE), de novo induction of meristems and genome editing by grafting, represent promising approaches to generate transgene-free edited plants. In addition, the current status of field trials involving genome-edited Solanaceae crops in Europe is outlined, considering the regulatory landscape and legislative requirements for their release in the environment. Despite regulatory constraints, some genome-edited crops have reached the market, highlighting their potential to contribute to sustainable agriculture and crop improvement.},
}
@article {pmid41828635,
year = {2026},
author = {Dagdelen, B and Arikoglu, H and Erkoc-Kaya, D and Bozkurt, B},
title = {Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {5},
pages = {},
pmid = {41828635},
issn = {1422-0067},
support = {19102037//Sel&#xe7;uk University/ ; },
mesh = {Humans ; *Leukocytes, Mononuclear/metabolism ; *Corneal Dystrophies, Hereditary/genetics ; *Gene Editing/methods ; *Transforming Growth Factor beta/genetics ; *Mutation ; *CRISPR-Cas Systems ; *Extracellular Matrix Proteins/genetics ; betaIG-H3 Protein ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Over 70 mutations in the transforming growth factor beta-induced (TGFBI) gene are associated with corneal dystrophies that impair vision. The R555W hotspot mutation is a major cause of granular corneal dystrophy type 1 (GCD1). Here, we evaluated the technical feasibility of CRISPR/Cas9-mediated editing of the R555W mutation in peripheral blood mononuclear cells (PBMCs) obtained from a patient with GCD1. Three single guide RNAs (sgRNA1-3) and matched single-stranded oligodeoxynucleotide donors (ssODN1-3) were designed and co-transfected into PBMCs. Transfected cells were enriched by flow cytometric sorting, with GFP-positive cells representing approximately 2-4% of the total electroporated population. Editing outcomes were initially screened using high-resolution melting (HRM) analysis, and the sgRNA3-ssODN3 combination identified as the most promising candidate was subsequently validated by next-generation sequencing (NGS). Sequencing revealed a homology-directed repair efficiency of 98.2% among GFP-positive sorted cells, demonstrating efficient and precise genome editing within the enriched population. Because PBMCs are not disease-relevant corneal epithelial cells and only genomic endpoints were assessed, the clinical applicability of this study is limited and the work should be considered a technical proof-of-concept. This framework supports optimization of CRISPR-based strategies prior to studies in biologically relevant corneal models.},
}
@article {pmid41828895,
year = {2026},
author = {Gu, X and Zhou, Y},
title = {Unlocking the Potential of Macroalgae: Innovative Pretreatment Strategies for Efficient Biorefinery.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {5},
pages = {},
pmid = {41828895},
issn = {1420-3049},
support = {No. 31900088//the National Natural Science Foundation of China/ ; No. ZR2025MS493//the Natural Science Foundation of Shandong Province of China/ ; },
mesh = {*Seaweed/metabolism/chemistry/genetics ; Biomass ; Fermentation ; *Biofuels ; *Biotechnology/methods ; Gene Editing ; Cell Wall/chemistry/metabolism ; },
abstract = {Macroalgae represent a promising third-generation feedstock for biorefinery due to their high biomass productivity and non-reliance on arable land. However, their complex cell wall structure poses a significant barrier to efficient bioconversion. This review integrates current pretreatment methods, including physical, chemical, biological, and combined approaches, with a focus on their mechanisms, effectiveness, and limitations. Furthermore, it explores the conversion of pretreated macroalgal biomass into bioenergy and biochemicals, such as bioethanol, organic acid and polyhydroxyalkanoate, via microbial fermentation. The review also examines the application of genetic editing tools (e.g., CRISPR-Cas systems) for the targeted modification of macroalgae to improve their inherent characteristics for biorefinery, such as reducing biomass recalcitrance or increasing the content of target carbohydrates. Finally, future perspectives on technological innovations and integrated industrial chains of macroalgal biorefinery are discussed. This review serves as a systematic reference for deepening the understanding of macroalgal cell wall deconstruction processes and supports the development of efficient and environmentally benign pretreatment strategies to advance macroalgal biorefinery toward industrialization.},
}
@article {pmid41829816,
year = {2026},
author = {Al-Sawa'eer, AS and Al-Samydai, A and Odeh, L and Haj Ahmad, F and Obekh, R and Elqader, YMA and Khaleel, A and Al-Athamneh, AM and Gabriele, M and Di Simone, SC and Ferrante, C and Menghini, L and Ali Agha, ASA},
title = {Early Plant Development as a Systems-Level Trait: Integrating Omics, Artificial Intelligence, and Emerging Biotechnologies.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {5},
pages = {},
pmid = {41829816},
issn = {2223-7747},
abstract = {Seed germination and early seedling development are critical determinants of crop establishment, stress tolerance, and yield stability, yet these stages remain insufficiently integrated into contemporary crop improvement strategies. Recent advances across genome editing, microbiome-assisted seed treatments, nanotechnology-enabled priming, and artificial intelligence-guided phenotyping have generated substantial but fragmented insights into early developmental regulation. This review synthesizes recent advances across early plant development research. It demonstrates that seemingly diverse technologies converge on a limited set of regulatory control nodes, including abscisic acid-gibberellin balance, redox homeostasis, and root system architectural plasticity. By integrating evidence from molecular, microbial, physicochemical, and computational studies, early plant ontogeny is presented as a tunable regulatory state governed by quantitative thresholds rather than as a strictly predetermined genetic process. Advances in deep learning, reinforcement learning, and high-throughput phenotyping further enable the modeling and optimization of early developmental trajectories across genotype by environment contexts. Together, these insights establish early development as a programmable target for crop improvement and provide a mechanistic foundation for designing integrated interventions that enhance developmental uniformity, stress resilience, and yield stability across diverse agroecological systems.},
}
@article {pmid41830331,
year = {2026},
author = {Martínez-Alvarez, L and Peng, X},
title = {Diversity and evolution of archaeal immune strategies.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41830331},
issn = {1362-4962},
support = {DFF-0135-00402//Danish Council for Independent Research/ ; 10.46540/4264-00120B//Danish Council for Independent Research/ ; NNF17OC0031154//Novo Nordisk Foundation/ ; DeiC-KU-N1-2024089//Danish e-Infrastructure Consortium/ ; DFF-0135-00402//Natural Sciences/ ; 10.46540/4264-00120B//Natural Sciences/ ; },
mesh = {*Archaea/genetics/immunology/classification ; *Evolution, Molecular ; Phylogeny ; *Genome, Archaeal ; CRISPR-Cas Systems ; Archaeal Proteins/genetics/immunology ; Genetic Variation ; },
abstract = {Archaeal antiviral defense systems remain poorly characterized despite recent advances in understanding prokaryotic immunity. Here, we analyze 7747 archaeal genomes, the largest and most diverse dataset to date, revealing a striking disparity in defense system prevalence and diversity compared to Bacteria. Nearly one-third of archaeal genomes have no detected systems beyond CRISPR-Cas and restriction-modification (in contrast to only 2.2% bacterial genomes), and only 50-55% contain CRISPR-Cas systems, far below previous estimates. Many known defense systems appear restricted to Bacteria, while several single-gene putative candidate systems (PDCs) recently identified through a guilt-by-embedding approach are enriched in Archaea. Phylogenetic analyses suggest that PDC-S70 and PDC-M05 likely originated in Archaea, representing rare archaeal contributions to the prokaryotic immune repertoire. Consistent with earlier studies, our findings support the existence of deep evolutionary links between archaeal and eukaryotic systems for argonautes and viperins. These analyses highlight both the underexplored nature and the evolutionary significance of archaeal immunity, calling for expanded efforts to uncover archaeal-specific systems and improve our understanding of immune evolution across domains of life.},
}
@article {pmid41830765,
year = {2026},
author = {Leandro, K and Rufino-Ramos, D and Lopes, SM and Silva, FS and Rodrigues-Santos, P and Silva, AC and Fernandes, AR and Henriques, C and Pereira, D and Lobo, D and Gabriel, GL and Faro, R and Nobre, RJ and Perdigão, PRL and Kleinstiver, BP and de Almeida, LP},
title = {Extracellular vesicles-mediated delivery of SpCas9 RNPs for therapeutic gene editing in Spinocerebellar Ataxia Type 3.},
journal = {Biomaterials},
volume = {331},
number = {},
pages = {124119},
doi = {10.1016/j.biomaterials.2026.124119},
pmid = {41830765},
issn = {1878-5905},
support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Machado-Joseph Disease/therapy/genetics ; *Gene Editing/methods ; Ataxin-3/genetics ; *Extracellular Vesicles/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Ribonucleoproteins/genetics/administration &amp; dosage ; Mice ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Repressor Proteins ; },
abstract = {Spinocerebellar Ataxia Type 3 (SCA3) is a neurodegenerative dominantly-inherited disorder caused by an overexpansion of a CAG tract within the ATXN3 gene, conferring toxic properties to the ataxin-3 protein. Genome editing with CRISPR-Cas9 enzymes is a promising strategy to inactivate mutant ATXN3 alleles, however, in vivo delivery remains challenging. Extracellular vesicles (EVs) are promising delivery vehicles for Cas9 and single guide RNA (sgRNA) ribonucleoproteins that minimize genomic exposure to highly active endonucleases. In this study, we designed SpCas9 with a palmitoylation motif that enables SpCas9 and sgRNA enrichment into EVs. Introduction of a photocleavable linker - PhoCl - allowed the photo-inducible release of SpCas9 from the palmitoylation motif in EVs, increasing target engagement to ATXN3 in vitro. EVs loaded with SpCas9 ribonucleoproteins resulted in ATXN3 knockout in SCA3 patient-derived iPSCs and two SCA3 animal models. These findings highlight an innovative route for transient delivery of gene editing tools. This approach provides a promising therapeutic platform for the treatment of genetic diseases, including SCA3.},
}
@article {pmid41831412,
year = {2026},
author = {Liu, J and Luo, S and Chen, S and Chen, G and Zhu, Y and Lou, Y and Fan, R and Zhang, Y and Pan, J and Zhu, C and Xu, L and Li, L},
title = {Dual-function CRISPR/Cas12a assisted strand displacement reaction with RuHex-loaded DNA condensates for ultrasensitive electrochemical detection of hepatocellular carcinoma mRNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118610},
doi = {10.1016/j.bios.2026.118610},
pmid = {41831412},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; *Carcinoma, Hepatocellular/genetics/diagnosis/blood ; *RNA, Messenger/genetics/isolation &amp; purification/analysis ; *Liver Neoplasms/genetics/diagnosis/blood ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; DNA/chemistry ; CRISPR-Associated Proteins/chemistry ; Bacterial Proteins/chemistry ; Ruthenium Compounds ; },
abstract = {Hepatocellular carcinoma (HCC) typically develops in a clinically silent manner, and the suboptimal sensitivity and specificity of currently available diagnostic biomarkers remain significant obstacles to its accurate and early detection. To improve molecular diagnostic performance, we developed a dual-function CRISPR/Cas12a assisted strand displacement reaction (dCas12a-SDR) with hexaammine ruthenium(III) chloride (RuHex)-loaded DNA condensate for ultrasensitive and highly specific detection of HCC-associated mRNAs. Upon target recognition, the previously sequestered Cas12a activation site within the electrode-immobilized capture probe is exposed, thereby inducing hybridization between the accessible single-stranded domains retained in RuHex-loaded DNA condensates (RuDC) and the displacement strand (Ds), which ultimately leads to the release of electroactive Ds-RuDC condensates and the effective activation of Cas12a. Activated Cas12a then removes the activation site via cis-cleavage, releasing the target to enter subsequent reaction cycles; concurrently, the activated system initiates trans-cleavage of adjacent capture probes and Ds-RuDC assemblies on the electrode that harbor trans-cleavage motifs, thereby promoting the release of RuDC from the electrode interface. This cascade ultimately leads to a pronounced reduction in the electrochemical signal. Owing to this target-triggered dual cis- and trans-cleavage mechanism mediated by Cas12a, the biosensor achieves highly efficient signal amplification. The platform affords a limit of detection as low as 39.2 aM for PD-L1 mRNA and exhibits excellent specificity, stability, and reproducibility. Moreover, by jointly detecting a panel of HCC-associated mRNAs (PD-L1, GPC3, EpCAM, and FGA), the platform successfully discriminated healthy individuals from patients with early-stage hepatocellular carcinoma in clinical serum samples. Collectively, this platform provides a powerful tool for molecular diagnosis of hepatocellular carcinoma.},
}
@article {pmid41831437,
year = {2026},
author = {Yang, J and Wang, T and Liu, Z and Wu, W and Sun, Y and Zhan, Y and Zhang, S and Chen, H and Liu, B and Yue, C and Yin, Z and Shan, Z and Li, X and Li, Z and Yuan, Z and Yin, H and Zhang, H},
title = {Molecular basis for dual-spacer-guided target cleavage by the TIGR-TasH system.},
journal = {Molecular cell},
volume = {86},
number = {7},
pages = {1217-1229.e6},
doi = {10.1016/j.molcel.2026.02.017},
pmid = {41831437},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *Gene Editing/methods ; *Viral Proteins/genetics/metabolism/chemistry ; Nucleic Acid Conformation ; *Bacteriophages/genetics/enzymology ; Models, Molecular ; Protein Binding ; DNA/genetics/metabolism ; },
abstract = {The RNA-directed programmable nuclease systems, exemplified by the CRISPR-Cas system, have been widely used in genome editing. In contrast to the single-spacer configuration of CRISPR RNA (crRNA), the guide RNA (tigRNA) of the tandem interspaced guide RNA (TIGR) system features a dual-spacer arrangement, thereby directing the TIGR-associated (Tas) protein to engage both strands of the target double-stranded DNA (dsDNA). Here, we determine six cryo-electron microscopy structures of the Salicola phage TIGR-TasH complex. The central coiled-coil region of TasH mediates dimerization, while the C-terminal nucleolar protein (Nop) domain is able to autonomously process precursor tigRNA. Upon target binding, the dynamic N-terminal HNH nuclease domain is recruited for cleavage through a β-hairpin, which also determines the target preference. More interestingly, the conserved box C motif of tigRNA stabilizes this β-hairpin in an adenine-specific manner, enabling us to rationally design a guide RNA-defined nickase, distinct from conventional protein-based nickase strategies used in genome editing.},
}
@article {pmid41831575,
year = {2026},
author = {Park, E and Lee, S and Kim, D and Choi, Y and Choung, S and Kim, H and Kim, SG},
title = {Virus-induced genome editing enables functional genomics across diverse plant species.},
journal = {Molecules and cells},
volume = {49},
number = {5},
pages = {100348},
pmid = {41831575},
issn = {0219-1032},
mesh = {*Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; *Genome, Plant ; *Plants/genetics ; *Plant Viruses/genetics ; },
abstract = {Virus-induced genome editing (VIGE) is expanding plant functional genomics by enabling precise and heritable genome modification across diverse species. While classical model systems such as Arabidopsis thaliana have provided foundational genetic insights, many ecologically, agriculturally, and chemically important traits reside in species that remain difficult to manipulate genetically. By coupling CRISPR-Cas systems with plant viral vectors, VIGE bypasses key limitations of conventional transformation and enables rapid mutagenesis without repeated tissue culture and plant regeneration. This approach enables researchers to examine gene function in species selected for biological relevance rather than technical convenience. Here, we review the conceptual framework, technical considerations, and applications of VIGE, and discuss its current limitations and future prospects in ecological, evolutionary, and crop research.},
}
@article {pmid41832076,
year = {2026},
author = {Gundra, SR and Jiang, W and Aouida, M and Wang, Q and Kazlak, AM and Elbehery, AHA and Saleh, A and Masood, M and Ghouneimy, A and Mahfouz, M},
title = {Characterization and engineering of highly efficient Cas12j genome editors.},
journal = {Trends in biotechnology},
volume = {44},
number = {6},
pages = {1740-1765},
doi = {10.1016/j.tibtech.2026.02.001},
pmid = {41832076},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; Protein Engineering/methods ; },
abstract = {The large size of widely used CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) enzymes limits their delivery for therapeutic applications. Cas12j nucleases offer a hypercompact alternative but show modest editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologs from viral metagenomes, which in their native form exhibit low editing activity in mammalian cells. We therefore engineered T5 exonuclease-Cas12j fusions, resulting in substantially enhanced genome-editing activity across multiple mammalian cell types, reaching levels comparable to established compact CRISPR-Cas editors. Intriguingly, robust cellular editing occurred in the presence of a previously unrecognized trinucleotide sequence context within the target DNA. Furthermore, we developed Cas12j-based adenine base editors by coupling catalytically inactive Cas12j orthologs with adenine deaminase, enabling efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by establishing engineering principles that convert compact Cas12j nucleases into efficient and modular genome-editing platforms well suited for delivery-constrained therapeutic applications.},
}
@article {pmid41832226,
year = {2026},
author = {Lee, SY and Park, HH},
title = {AcrIIA7 hijacks tracrRNA to block CRISPR-Cas system.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41832226},
issn = {2041-1723},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Ribonucleoproteins/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Protein Binding ; },
abstract = {The CRISPR-Cas9 system provides adaptive immunity against invading genetic elements through a dual-RNA-guided DNA cleavage mechanism. This system relies on the precise assembly of a ribonucleoprotein (RNP) complex composed of the Cas9 endonuclease, a CRISPR-derived RNA (crRNA), and a trans-activating CRISPR RNA (tracrRNA). Around 100 anti-CRISPR proteins that inhibit CRISPR-Cas systems have been identified, and the mechanisms by which they act are increasingly being elucidated. However, the inhibitory mechanisms of many Acrs, including AcrIIA7, remain poorly understood. Here, we present the structure of AcrIIA7 and uncover a previously unrecognized mechanism by which it inhibits Cas9 function. Structural and biochemical analyses reveal that AcrIIA7 specifically binds to tracrRNA, preventing its association with crRNA and thereby blocking formation of the active Cas9 RNP complex. This tracrRNA hijacking mechanism represents a unique strategy for CRISPR inhibition, in which an anti-CRISPR protein targets an RNA scaffold essential for Cas9 activation rather than interacting directly with the Cas9 protein. Our findings provide the first structural insight into tracrRNA-targeted anti-CRISPR activity and highlight RNA-RNA interaction interfaces as vulnerable nodes in CRISPR-Cas immunity.},
}
@article {pmid41833124,
year = {2026},
author = {Park, JS and Kim, YM and Lee, HJ and Han, JY},
title = {Research note: Generation of ovalbumin-null chickens and characterization of altered protein compositions in their egg whites.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106715},
pmid = {41833124},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics/metabolism/physiology ; *Ovalbumin/genetics/metabolism ; Female ; *Egg Proteins/metabolism/genetics ; *Egg White/chemistry ; Chick Embryo ; CRISPR-Cas Systems ; Gene Knockout Techniques/veterinary ; Animals, Genetically Modified/genetics ; },
abstract = {Chickens are considered an efficient bioreactor platform for production of recombinant proteins due to their high egg laying rate and high capacity to produce proteins in egg white. Ovalbumin (OVAL) comprises 54% of egg white proteins, and targeted insertion of a recombinant protein construct into the OVAL locus leads to significant accumulation of the recombinant protein in egg white. However, it was reported that embryos could not develop or hatch from eggs laid by heterozygous OVAL-knockout hens in which OVAL gene was replaced by foreign protein coding sequences, a limitation that restricted the generation of homozygous OVAL-knockout chickens. In this study, we specifically targeted the OVAL locus using CRISPR/Cas9 and successfully generated OVAL-null chickens by mating heterozygous individuals. Both heterozygous and homozygous OVAL knockout embryos developed and hatched, notably, we found that embryos could develop and hatch from OVAL-deficient eggs, although the hatching rate was reduced by 52.30±14.42%. Furthermore, analysis revealed that concentrations of other major egg white proteins increased, indicating a compensatory accumulation of proteins in response to the removal of OVAL. Collectively, this study demonstrates that OVAL-null chicken lines can be established and that these chickens produce eggs with altered egg white protein compositions.},
}
@article {pmid41833778,
year = {2026},
author = {Quintana, AJ and García-Suárez, R and Prieto, A and Sánchez, J and Gómez, I and Verduzco-Rosas, LA and doNascimento, N and Lopez-Molina, S and Zhang, J and Soberón, M and Bravo, A and Pacheco, S},
title = {Genome editing of ABCB6 transporter confers resistance to cypermethrin in the major pest of corn, Spodoptera frugiperda.},
journal = {Insect biochemistry and molecular biology},
volume = {190},
number = {},
pages = {104537},
doi = {10.1016/j.ibmb.2026.104537},
pmid = {41833778},
issn = {1879-0240},
mesh = {Animals ; *Pyrethrins/pharmacology ; *Spodoptera/genetics/drug effects/metabolism/growth &amp; development ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; Gene Editing ; *Insect Proteins/genetics/metabolism ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Hemolysin Proteins ; Bacterial Proteins ; CRISPR-Cas Systems ; Larva/genetics/drug effects/growth &amp; development ; },
abstract = {Spodoptera frugiperda is a major global pest that affect multiple crops, mainly corn and rice. Unfortunately, this pest has evolved resistance to various chemical and biological pesticides. ATP-binding cassette (ABC) transporters, particularly members of the B subfamily, are associated with detoxification by exporting xenobiotics and plant-derived metabolites from the intoxicated insect cells. In addition, some are involved in the mode of action of Bacillus thuringiensis biopesticide Cry toxins, functioning as receptors for these proteins. In this study, we analyzed transcriptomic data from the midgut tissue of S. frugiperda and identified the ABCB6 as one of the most highly expressed transporters within the ABCB subfamily. To explore its functional role, we generated a CRISPR-Cas9 knockout (KO) mutation. Strikingly, loss of SfABCB6 conferred resistance to the chemical pyrethroid insecticide cypermethrin, while the susceptibility to B. thuringiensis Cry1Ab, Cry1Fa and Vip3Aa toxins remained unchanged. Consistently, the ABCB6 CRISPR-Cas9 KO in S. frugiperda derived Sf9 cells conferred resistance to cypermethrin, reiterating the observed larval phenotype. In contrast, the overexpressing of ABCB6 in Sf9 cells exhibited increased susceptibility to cypermethrin. However, SfABCB6 KO showed fitness costs in the insect, as this mutation drastically reduced fertility. Our results provide evidence that SfABCB6 transporter facilitates cypermethrin toxicity participating in insecticide resistance and pointing out its potential role as a novel target for pest management strategies.},
}
@article {pmid41833894,
year = {2026},
author = {Ma, Y and Liao, Y},
title = {CRISPR-mediated cancer therapies: Approaches to direct tumor targeting.},
journal = {Critical reviews in oncology/hematology},
volume = {222},
number = {},
pages = {105277},
doi = {10.1016/j.critrevonc.2026.105277},
pmid = {41833894},
issn = {1879-0461},
mesh = {Humans ; *Neoplasms/therapy/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Animals ; Tumor Microenvironment/genetics ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas9 technologies have opened new possibilities for precision cancer treatment, addressing limitations inherent in conventional therapies such as chemotherapy and radiation. This review examines CRISPR-based strategies for direct tumor targeting, including oncogene inactivation, tumor suppressor gene reactivation, and tumor microenvironment (TME) modification. Key advances include KRAS[G12D] inactivation via base editing, in which engineered deaminases introduce precise single-nucleotide changes without generating double-strand breaks; TP53 correction through homologous recombination, which uses a donor DNA template to repair mutant sequences at the targeted locus; and CDKN2A epigenetic remodeling using CRISPR-dCas9-TET1 demethylation, where catalytically inactive Cas9 guides the TET1 demethylase to hypermethylated promoters to restore gene expression. CRISPR screening has identified synthetic lethal interactions, such as PARP1 dependency in BRCA1[-/-] tumors. TME editing strategies, including modification of cancer-associated fibroblasts, demonstrate enhanced antitumor responses. Delivery challenges are being addressed through viral vectors, including adenovirus, AAV, and lentivirus. Non-viral approaches include lipid nanoparticles, gold nanoparticles, exosomes, and stimuli-responsive systems such as MMP-cleavable and hypoxia-responsive nanoparticles. Clinical trials with CRISPR-engineered T-cells (e.g., CTX130) have demonstrated remission rates in hematologic malignancies. However, significant challenges remain, including cytokine release syndrome, immunotoxicity, tumor heterogeneity, and limited delivery efficiency in solid tumors. Overcoming these barriers requires interdisciplinary innovation, ethical oversight, and technological refinement to support the safe and effective integration of CRISPR-based strategies into precision oncology.},
}
@article {pmid41834297,
year = {2026},
author = {Zhu, L and Xiong, W and Yang, S and Qi, Q and Liu, X and Zhou, X and Tian, T},
title = {Dynamic Control of RNA Structure and Function through Bioorthogonal Staudinger Chemistry.},
journal = {ACS chemical biology},
volume = {21},
number = {4},
pages = {835-843},
doi = {10.1021/acschembio.6c00067},
pmid = {41834297},
issn = {1554-8937},
mesh = {*RNA/chemistry/metabolism ; *Azides/chemistry ; Nucleic Acid Conformation ; Humans ; CRISPR-Cas Systems ; },
abstract = {Here, we report a reversible chemical strategy for regulating RNA function through a Staudinger reaction-mediated postsynthetic modification. We designed a bifunctional azide reagent, 1,3-diazidopropan-2-yl 1H-imidazol-1-carboxylate (DAPIC), which specifically modifies the 2'-hydroxyl of RNA, thereby disrupting RNA structure and function. Treatment with 2-diphenylphosphinoethylamine (DPPEA) reactivates the modified RNA through an efficient Staudinger reduction. This approach enables reversible modulation of RNA folding, hybridization, and protein-binding interactions, and can be applied to guide RNAs in the CRISPR-Cas9 system. DAPIC modification completely abrogates Cas9-mediated DNA cleavage, which is restored in a DPPEA concentration-dependent manner both in vitro and in living cells. Compared with monoazide derivatives, DAPIC exhibits enhanced reactivity and reduced reagent requirements. This Staudinger-based RNA regulation platform establishes a robust and generalizable chemical tool for conditional gene editing and studies of RNA function in complex biological environments.},
}
@article {pmid41834687,
year = {2026},
author = {Yang, J and Huang, Q},
title = {Cas13a/crRNA trans-cleavage triggered primer exchange reaction based self-priming chain extension for sensitive and label-free infantile pneumonia related microRNA analysis.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {12},
pages = {2524-2531},
doi = {10.1039/d6ay00090h},
pmid = {41834687},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; DNA Primers ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {Accurate and sensitive detection of microRNAs (miRNAs) is crucial for both pathophysiological studies and clinical diagnostics. Conventional amplification methods often face limitations such as dependence on thermal cycling, susceptibility to contamination, and insufficient specificity to discriminate among closely related miRNA family members. To address these challenges, we developed a label-free isothermal detection platform that integrates the precise RNA-targeting ability of the CRISPR/Cas13a system with a self-priming amplification cascade driven by the primer exchange reaction (PER). In this assay, target miRNA binding directly activates the trans-cleavage activity of the Cas13a/crRNA complex, which subsequently cleaves a uracil-rich toehold region on a stem-loop DNA primer (H1). Following dephosphorylation, the cleaved primer initiates a PER-mediated self-priming amplification process, generating long tandem double-stranded DNA products that can be sensitively detected using the fluorescent dye SYBR Green I. The proposed method demonstrates several key advantages: (i) high specificity enabled by the programmable Cas13a/crRNA complex, allowing clear distinction between the target miRNA and sequences with single-base mismatches or high homology; (ii) exceptional sensitivity, achieving a detection limit of 406 aM and a dynamic range spanning six orders of magnitude, through coupling Cas13a collateral cleavage with exponential isothermal amplification; (iii) excellent reproducibility, reflected by low relative standard deviations and a coefficient of variation of 3.65% in spiked serum samples; and (iv) strong concordance with the reference RT-qPCR method in mock clinical specimens, highlighting its reliability for potential clinical use. In summary, this CRISPR/Cas13a-coupled self-priming amplification strategy provides a robust, accurate, and highly sensitive means for miRNA quantification, offering a promising alternative for point-of-care molecular diagnostic applications.},
}
@article {pmid41834690,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO and Damayanti, D and Utari, NWA and Harmiansyah, and Karangan, A and Kusmali, M},
title = {Integration of CRISPR/Cas12a and Toehold-Mediated Strand Displacement for Alternative Conventional miRNA Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {6},
pages = {e202500932},
doi = {10.1002/cbic.202500932},
pmid = {41834690},
issn = {1439-7633},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression and serve as powerful biomarkers for cancer and other diseases. Conventional detection methods such as RT-qPCR, Northern blotting, microarrays, and next-generation sequencing provide robust analytical capabilities but remain limited by complexity, cost, and poor suitability for point-of-care diagnostics. CRISPR/Cas12a has emerged as a versatile nucleic acid detection platform with high specificity and sensitivity. However, its intrinsic preference for DNA substrates restricts direct application to miRNA sensing. Early CRISPR/Cas12a-based assays relied on enzymatic amplification, direct RNA-induced activation, or split-component designs, each offering proof-of-concept feasibility but facing trade-offs in sensitivity, workflow complexity, or robustness. Toehold-mediated strand displacement (TSD) provides a powerful alternative by converting miRNA inputs into DNA activators or crRNAs that efficiently trigger Cas12a. This integration enables enzyme-free amplification, programmable logic operations, and enhanced sensitivity, while reducing reliance on multienzyme cascades. This review critically evaluates conventional, enzymatic, direct, and split-based CRISPR/Cas12a strategies and emphasizes emerging TSD-assisted platforms as next-generation solutions for sensitive, specific, and portable miRNA detection.},
}
@article {pmid41834849,
year = {2026},
author = {Tenea, GN},
title = {Comprehensive genomic and metabolomic profiling of Weissella confusa UTNCys2-2 highlights bioactive potential.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1779198},
pmid = {41834849},
issn = {1664-302X},
abstract = {INTRODUCTION: The genus Weissella comprises a diverse group of lactic acid bacteria (LAB) widely distributed across plant- and food-associated ecosystems and recognized for their functional and technological versatility. Weissella confusa UTNCys2-2, a plant-derived strain isolated from Amazonian spiral ginger (Costus sp.), that produces exopolysaccharides (EPS) with documented antioxidant activity and promising probiotic properties.<br><br>METHODS: Whole-genome sequencing of UTNCys2-2 was performed to establish its taxonomic assignment, phylogenomic analysis, while genome mining was conducted to evaluate safety, metabolic potential, and biosynthetic capabilities. Carbohydrate-active enzymes (CAZymes), Kyoto Encyclopedia of Genes and Genomes (KEGG), and MetaCyc pathways were analyzed for functional insights. Moreover, the metabolite composition of the cell-free supernatant (CFS) was examined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with Sequential Windowed Acquisition of all Theoretical Fragment Ion Mass Spectra (SWATH-MS).<br><br>RESULTS: The genome consists of a 2.32&#x202f;Mb circular chromosome (44.59% GC) encoding 2,194 proteins, 76 tRNAs, and 10 rRNAs, with no plasmids. Phylogenomic analyses assigned the strain to the W. confusa clade, clustering closely with the reference strain DSM 20196. UTNCys2-2 harbors a complete Type II-A CRISPR-Cas system, intact prophages, and mobile elements, while lacking virulence determinants and transferable antimicrobial resistance genes. Functional annotation revealed 118 CAZymes supporting EPS biosynthesis, polysaccharide utilization, and carbohydrate metabolism. KEGG and MetaCyc pathways highlighted glycogen and riboflavin biosynthesis, stress tolerance, and metabolic versatility. Genome mining identified a Type III polyketide synthase (T3PKS) gene cluster with low similarity to known pathways, suggesting potential for novel secondary metabolites. Pangenome analysis showed extensive strain-specific genes linked to carbohydrate metabolism and EPS production. Metabolomic profiling of the CFS detected alkaloids, bioactive peptides, functional carbohydrates, and phenolics, supporting antimicrobial, probiotic, and host-interactive activities.<br><br>CONCLUSION: W. confusa UTNCys2-2 represents a biosafe and metabolically versatile strain with strong genomic capacity for EPS production, potential for novel secondary metabolite biosynthesis, and diverse bioactive properties, supporting its applicability in food fermentation, probiotic development, and microbial biotechnology.},
}
@article {pmid41834871,
year = {2026},
author = {Abdul Rehman, Y and Fayyaz, A and Alblooshi, AS and Muhammad, K and Mundra, S and Alam, MT},
title = {Molecular adaptations and engineering of extremophiles for synthetic biology and biotechnological applications.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1754802},
pmid = {41834871},
issn = {1664-302X},
abstract = {Extremophiles are microorganisms that thrive in environments previously thought to be uninhabitable, including extreme temperature, salinity, pH, pressure, and radiation. These organisms, found in Archaea, Bacteria, and Eukarya, exhibit distinct structural, metabolic, and genetic adaptations, such as enhanced enzyme stability, efficient DNA repair mechanisms, and robust stress-response systems that enable survival under extreme conditions. Understanding these adaptation mechanisms is key to engineering similar traits in mesophilic organisms. This review discusses the diversity of extremophiles and presents phylogenetic and comparative genomic insights which may provide insights into the origins and evolution of early life on Earth We highlight recent advances in CRISPR/Cas-based genome editing, genome-scale metabolic modeling (GEM), and synthetic biology that have expanded the use of extremophiles in sustainable industrial biotechnology. The exceptional stability and catalytic efficiency of extremozymes under harsh conditions underscore their potential in various biotechnological applications. Finally, we discuss the ecological significance of extremophiles in climate change mitigation and outline current challenges and future directions in extremophile research.},
}
@article {pmid41836275,
year = {2026},
author = {Otero, CP and Qi, LS},
title = {Rewriting the epigenome: CRISPR tools for biological discovery and therapeutics.},
journal = {Current opinion in biomedical engineering},
volume = {38},
number = {},
pages = {},
pmid = {41836275},
issn = {2468-4511},
support = {R21 AG077193/AG/NIA NIH HHS/United States ; },
abstract = {The eukaryotic epigenome plays a central role in regulating gene expression, cellular identity, and development through dynamic, multilayered biochemical modifications to DNA, histones, and chromatin architecture. Disruption of these regulatory mechanisms contributes to a wide range of human diseases, including cancer, neurodegenerative disorders, and immunological conditions. Targeted epigenome editing offers promising discovery and therapeutic strategies by enabling the correction of aberrant epigenetic states without the need for permanent changes to the DNA sequence. The catalytically inactive CRISPR-Cas (dCas) molecule fused to epigenetic effector domains has emerged as a versatile platform for programmable, locus-specific modulation of chromatin states. These CRISPR-based epigenetic editors can deposit or remove desired epigenetic marks and alter three-dimensional genome organization to fine-tune gene expression with high specificity. Recent developments have expanded the CRISPR epigenome editing toolbox by introducing new effector domains, improving multiplexing capabilities, and enabling large-scale genetic screening, leading to novel insights into the functional genomics across various cellular contexts. However, clinical translation remains challenged by inefficient delivery and suboptimal editing efficacy in vivo. This review highlights recent advances in CRISPR-based epigenetic editing, with a focus on applications in primary cells, new tool development, and the translational potential of epigenome modulation for safe, durable, and precise therapies.},
}
@article {pmid41836767,
year = {2026},
author = {Luo, L and Yang, Y and Zhang, Y and Mao, G},
title = {Advances in nanozyme-assisted CRISPR diagnostic technology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1796403},
pmid = {41836767},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system has significant potential in biological diagnostics because of its precise nucleic acid identification abilities. Traditional CRISPR diagnostics, however, have limitations such as insufficient signal output, dependence on exogenous enzymes, and high equipment demands. Nanozymes, as nanomaterials with enzyme-mimetic catalytic activity, integrate the catalytic efficiency of natural enzymes with the stability and modifiability of nanomaterials, providing a viable resolution to the limitations in CRISPR diagnostics. This article comprehensively evaluates the advancements in nanozyme-enhanced CRISPR diagnostic technologies. Furthermore, it delineates the fundamental attributes of the CRISPR diagnostic system and nanozymes, as well as the necessity of their integration. Moreover, the coupling mechanisms between the CRISPR/Cas system and nanozymes, including the regulation of nanozyme catalytic activity by Cas protein function and CRISPR signal amplification facilitated by nanozymes, were also comprehensively evaluated. The application of this technique in detecting nucleic acid and non-nucleic acid targets was assessed. Further, this study discusses the current limitations of this technology, such as complex separation of heterogeneous systems, laborious reaction protocols, and slow detection rates. The future advancements, such as the establishment of homogenous systems, the creation of integrated devices, and the utilization of single-atom nanozymes, have also been discussed in this review. The results of this study will provide references for the comprehensive integration of nanozymes and CRISPR technology, together with their diagnostic applications.},
}
@article {pmid41837829,
year = {2026},
author = {Bao, J and Ju, X and Zhang, H and Tang, D and Yan, J and Cui, C and Gao, SS},
title = {Synergistic CRISPR-Cas9 Host Engineering and Enzyme Evolution for Enantioselective Synthesis of a Vibegron Pyrrolidine Intermediate.},
journal = {Organic letters},
volume = {28},
number = {12},
pages = {3695-3700},
doi = {10.1021/acs.orglett.6c00065},
pmid = {41837829},
issn = {1523-7052},
mesh = {*Pyrrolidines/chemistry/chemical synthesis/metabolism ; Stereoisomerism ; Molecular Structure ; *CRISPR-Cas Systems ; Biocatalysis ; *Oxidoreductases/metabolism/chemistry ; Imines/chemistry ; Directed Molecular Evolution ; },
abstract = {The stereoselective synthesis of chiral pyrrolidine motifs is essential to vibegron production but remains challenging using conventional chemical routes. Here we report an imine reductase (IRED) catalyzed asymmetric imine reduction to access a key vibegron intermediate. Directed evolution afforded a highly efficient variant delivering 94% conversion and >99% d.e. Combined enzyme and host engineering enabled clean whole cell catalysis, establishing a robust and scalable biocatalytic platform.},
}
@article {pmid41837831,
year = {2026},
author = {Awinashe, M and Viswaja, K and Pathath, AW and Subair, FP and Varshney, A and Mishra, P and Mulla, M and Mulla, M},
title = {CRISPR-cas9-Mediated Gene Editing to Reverse Oncogenic Mutations in Oral Squamous Cell Carcinoma.},
journal = {Annals of African medicine},
volume = {25},
number = {4},
pages = {805-809},
doi = {10.4103/aam.aam_864_25},
pmid = {41837831},
issn = {0975-5764},
mesh = {Humans ; *Mouth Neoplasms/genetics/pathology/therapy ; *Carcinoma, Squamous Cell/genetics/therapy/pathology ; *Mutation ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing/methods ; Cell Proliferation/genetics ; Oncogenes ; },
abstract = {INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a genetically driven malignancy characterized by a high burden of oncogenic mutations that contribute to aggressive tumor behavior, therapeutic resistance, and poor survival outcomes. Conventional treatment modalities largely target downstream molecular pathways without correcting the underlying genetic aberrations, underscoring the need for precision-based therapeutic strategies. This study aimed to assess the feasibility and functional impact of CRISPR-Cas9-mediated gene editing in reversing oncogenic mutations associated with OSCC using an in vitro experimental model.<br><br>MATERIALS AND METHODS: A controlled in vitro experimental study was conducted using the established human OSCC cell lines harboring mutations in key oncogenic genes. Cells were divided into control, mock-transfected, and CRISPR-Cas9-edited groups. Target-specific single-guide RNAs were designed to correct oncogenic mutations using advanced CRISPR-based editing platforms. Gene-editing efficiency was validated by molecular assays, while functional outcomes were assessed using cell proliferation analysis. Statistical evaluation was done using the one-way ANOVA with significance set at P < 0.05.<br><br>RESULTS: CRISPR-Cas9-edited OSCC cells demonstrated a significant reduction in cell proliferation compared to control and mock-transfected groups (P < 0.001). No significant variation was found between control and mock-transfected cells, confirming minimal procedural influence.<br><br>CONCLUSION: CRISPR-Cas9-mediated correction of oncogenic mutations effectively suppresses malignant cellular proliferation in OSCC, highlighting its promising role as a precision therapeutic strategy in oral cancer management.},
}
@article {pmid41839994,
year = {2026},
author = {Fu, X and Zhao, F and Ge, J and Guan, H and Lv, P and Guo, F and Ma, H and Ainiwaer, M and Hu, S},
title = {Establishment of a rapid Brucella detection method based on MCDA-CRISPR dual signal amplification system for reducing transfusion-transmitted diseases.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839994},
issn = {2045-2322},
support = {CXCY2025024//the Xinjiang Medical University Graduate Innovation and Entrepreneurship Project/ ; KYQDJJ2024006//the Doctoral Scientific Research Startup Fund of Zhengzhou Central Hospital/ ; 2022-2-6042//the Capital's Funds for Health Improvement and Research/ ; 2025YLZDJH193//The Guidance Plan Project for Scientific and Technological Innovation in the Medical and Health Field of Zhengzhou City/ ; },
mesh = {*Brucella/genetics/isolation &amp; purification ; Humans ; *Brucellosis/diagnosis/transmission/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; Rapid Diagnostic Tests ; Blood Donors ; *Transfusion Reaction/prevention &amp; control ; },
abstract = {Brucellosis is a common zoonotic disease caused by Brucella and remains a globally concerning public health issue. Timely and effective detection methods are crucial for clinical diagnosis. We developed a novel Brucella detection platform (MCDA-CRISPR) by integrating multiple cross displacement amplification (MCDA) with a CRISPR-Cas12a-based biosensing system, and preliminarily applied it for the first time to screen for Brucella in voluntary blood donors from Xinjiang, China. This technology enables amplification under isothermal conditions at 64 °C using only a water bath, requires no specialized equipment, and completes detection within 60 min. Amplification products can be directly visualized under UV light without complex interpretation. Performance results demonstrated a minimum detection limit of 1 fg/μL for Brucella DNA, making the method 100 times more sensitive than conventional PCR. The assay showed 100% specificity for Brucella detection with no cross-reactivity to non-Brucella pathogens. The assay could also detect Brucella in blood donors samples and showed the same sensitivity and specificity as the culture method. The assay is a visual, sensitive, and highly specific detection technique. When applied to routine blood transfusion screening in areas with high prevalence of brucellosis, such as Xinjiang, can effectively reduce the risk of transfusion-transmitted brucellosis, and hold broad application prospects in resource-limited primary or field testing scenarios.},
}
@article {pmid41840308,
year = {2026},
author = {Madhi, M and Gholizadeh, P},
title = {CRISPR-Cas9: Genome Engineering and Future Vaccine Applications.},
journal = {Molecular biotechnology},
volume = {68},
number = {7},
pages = {3068-3090},
pmid = {41840308},
issn = {1559-0305},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Animals ; *Vaccines/genetics/immunology ; *Genetic Engineering/methods ; Vaccinology/methods ; Vaccine Development ; },
abstract = {The CRISPR-Cas9 system, a transformative genome engineering tool derived from prokaryotic adaptive immunity, is reshaping the landscape of biological research and therapeutic development. This review provides a critical synthesis of its rapidly evolving, yet underexplored, application in rational vaccine design. We analyze how CRISPR-Cas9 and its derivative platforms (including base editing, prime editing, and CRISPRi/a) are being repurposed from therapeutic gene editing to become indispensable assets in vaccinology. This transition is powered by the convergence of CRISPR-mediated precision with synthetic biology, enabling the rapid engineering of novel vaccine vectors and attenuated strains, the precise optimization of antigen sequences for enhanced breadth and potency, and the direct modulation of host immune responses. Notwithstanding this potential, significant technical and translational hurdles persist, including off-target editing risks, delivery inefficiencies in vivo, and unresolved regulatory pathways for genetically modified vaccines. We detail these mechanisms and evaluate the current preclinical and clinical landscape, while addressing persistent challenges in safety, delivery, and scalability. By delineating these advances and obstacles, this review outlines a forward-looking framework for leveraging CRISPR technology to create programmable, precision vaccines against emerging and re-emerging pathogens, moving the field beyond empirical methods toward a new paradigm of rational immunization.},
}
@article {pmid41840824,
year = {2026},
author = {Thalib, HI and Khan, S and Hanin Shaikh, A and Alawi, KM and Alabdrabalrasol, ZH and Mehveen, S and Haidar, S and ElSayed Hassan, F and Shaik, NA},
title = {CRISPR Gene Editing for Nucleotide Repeat Expansion Disorders: A Systematic Review of Preclinical and Clinical Evidence.},
journal = {Genetic testing and molecular biomarkers},
volume = {30},
number = {3},
pages = {71-80},
doi = {10.1177/19450265261434900},
pmid = {41840824},
issn = {1945-0257},
mesh = {Humans ; *Gene Editing/methods ; Myotonic Dystrophy/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Muscular Dystrophy, Duchenne/genetics/therapy ; Animals ; Genetic Therapy/methods ; Huntington Disease/genetics/therapy ; Trinucleotide Repeat Expansion/genetics ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {BACKGROUND: Incurable hereditary diseases such as Duchenne muscular dystrophy (DMD), Huntington's disease (HD), and myotonic dystrophy type 1 (DM1) fall into the nucleotide repeat expansion disorder (NRED) category. The discovery of CRISPR-Cas genome editing has paved the way toward hopeful strategies for accurate DNA-level repair. This systematic review presents preclinical data on the efficacy, molecular effects, and limitations of CRISPR-based treatments for NREDs.<br><br>METHODS: As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines, systematic PubMed, Scopus, and Embase searches up to June 2025 identified studies that evaluated CRISPR-Cas systems in human-derived in vitro models of NREDs. Methodological Index for Non-Randomized Studies tool was used to score eligible studies by methodological quality. CRISPR platforms, delivery systems, gene targets, molecular endpoints, and functional rescue data were extracted and synthesized descriptively.<br><br>RESULTS: Twenty-four out of 6510 records screened were included. They employed most of them to target specific DMD (n = 9), HD (n = 6), and DM1 (n = 3) with patient-derived induced pluripotent stem cells or differentiated myogenic/neuronal cells. Streptococcus pyogenes CRISPR-associated protein 9 as a nuclease was the most frequently used, although engineered Cas9 enzymes and dCas9 fusion proteins were also utilized to control transcription. Delivery was achieved through viral vectors (adeno-associated virus, lentivirus) and nonviral routes (plasmid, lipofection, electroporation). Uniform genomic editing, transcript rescue, and protein restoration were seen in CRISPR-mediated editing studies, and functional restoration was demonstrated for splicing correction and dystrophin restoration. Methodological flaws such as the absence of blinding, failure to follow up, and lack of full reporting of off-target effects limited robustness.<br><br>CONCLUSION: CRISPR-Cas systems exhibit reproducible molecular and functional correction in NRED models with their translational potential. Methodological strength, whole safety profiling, and in&#xa0;vivo verification remain a necessity, however, before clinical translation.},
}
@article {pmid41841492,
year = {2026},
author = {Li, Z and Kong, J and Wu, W and Duan, Y and Zhu, Z and Hua, C and Yan, P and Cao, C and Cao, X and Xiao, Y and Lu, M and Chen, M},
title = {Structural and functional insights into the adenosine deaminase of the type III-B CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41841492},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//National Key Research and Development Program of China/ ; 82473977//National Natural Science Foundation of China/ ; 32271330//National Natural Science Foundation of China/ ; 32471316//National Natural Science Foundation of China/ ; 82304614//National Natural Science Foundation of China/ ; 82373892//National Natural Science Foundation of China/ ; SBK2024010634//Basic Research Program of Jiangsu/ ; BK20250200//Basic Research Program of Jiangsu/ ; //State Key Laboratory of Natural Medicines/ ; SKLNMZZ2024JS31//China Pharmaceutical University/ ; 2632025TD02//Fundamental Research Funds for the Central Universities/ ; //Project Program of State Key Laboratory of Natural Medicines/ ; },
mesh = {*Adenosine Deaminase/chemistry/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Adenine Nucleotides/metabolism ; Adenosine Triphosphate/metabolism ; Models, Molecular ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Oligoribonucleotides/metabolism ; Ribonucleases/metabolism/chemistry/genetics ; },
abstract = {Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems confer antiviral immunity via cyclic oligoadenylate (cOA) signaling. Here, we elucidate a cooperative bacterial defense strategy involving two cOA-activated CRISPR-associated Rossmann fold (CARF)-containing effectors, adenosine deaminase CAAD and ribonuclease Csx1, in Thermoanaerobaculum aquaticum. Genomic analyses indicate widespread co-occurrence of CRISPR-associated adenosine deaminase (CAAD) with ancillary CARF-containing effectors in type III CRISPR systems, suggesting that multiple CARF-containing proteins may contribute to a coordinated cOA-dependent defense. Biochemical and structural studies reveal the intrinsic dynamics of CAAD hexamer, and demonstrate that cA4/cA6 binding stabilizes CAAD hexamers, triggering metal-ion-dependent conversion of ATP into inosine triphosphate. Concurrently, the downstream Csx1 is exclusively activated by cA4 to cleave single-stranded RNA. Strikingly, we found that both effectors are capable of degrading cA4, suggesting that this CAAD-Csx1 pair may be cross-regulated and achieve immunity through a dual-targeting mechanism: in response to infection, Csx1 degrades viral RNA while CAAD disrupts nucleotide metabolism via ATP deamination, which can be relieved via cA4 degradation when infection has been eliminated. This study proposes an enhanced defense mechanism through coordinated activation and regulation of multiple CRISPR effectors by a single signaling molecule, unveiling unprecedented complexity in CRISPR immunoregulation.},
}
@article {pmid41841497,
year = {2026},
author = {Nusawardhana, A and Hale, A and Straka, J and Nicolae, CM and Moldovan, GL},
title = {Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41841497},
issn = {1362-4962},
support = {R01ES026184/GF/NIH HHS/United States ; R01GM134681/GF/NIH HHS/United States ; R01CA244417/GF/NIH HHS/United States ; F31CA294862/GF/NIH HHS/United States ; 4D01_2024_1002//Four Diamonds Transformative Patient-Oriented Cancer Research/ ; /NH/NIH HHS/United States ; //Penn State University/ ; },
mesh = {*Exodeoxyribonucleases/genetics/metabolism ; *DNA Damage/genetics ; Cisplatin/pharmacology ; *R-Loop Structures/genetics ; DNA Repair/genetics ; CRISPR-Cas Systems ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae/genetics ; Genome, Fungal ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {DNA repair is critical for cellular homeostasis under both normal conditions as well as in response to genotoxic agents such as chemotherapeutics. EXO1 is a 5'-3' exonuclease with multiple roles in DNA biology. To better understand these roles, we employed CRISPR loss-of-function genome-wide screening to identify genes required for proliferation and cisplatin sensitivity in EXO1-deficient cells. We uncovered differential regulators of cisplatin sensitivity between wildtype (WT) and EXO1-deficient cells. By analyzing the genetic networks that these regulators belong to, we found that DNA repair was the main biological process suppressing cisplatin sensitivity in WT cells, but this was not the case in EXO1-deficient cells, indicating that EXO1 is critical for the repair of cisplatin-induced DNA damage. Moreover, synthetic lethality screens identified a genetic interaction between EXO1 and the histone chaperone CAF-1. Mechanistically, we show that EXO1 and CAF-1 are independently recruited to R-loops and participate in separate, synergistic pathway of R-loop suppression. Even in the absence of DNA damage treatment, concomitant loss of EXO1 and CAF-1 causes R-loop accumulation and increased R-loop-associated DNA damage. Our work sheds light on the critical roles of EXO1 in genomic stability.},
}
@article {pmid41841731,
year = {2026},
author = {Xiong, L and Yadav, V and Sun, S and Heitman, J},
title = {Dissecting the homeodomain MAT locus and engineering novel tripolar and bipolar mating systems in Cryptococcus amylolentus.},
journal = {mBio},
volume = {17},
number = {4},
pages = {e0005926},
pmid = {41841731},
issn = {2150-7511},
mesh = {*Genes, Mating Type, Fungal ; *Cryptococcus/genetics/physiology ; Gene Deletion ; Reproduction ; CRISPR-Cas Systems ; *Genes, Homeobox ; *Homeodomain Proteins/genetics ; },
abstract = {Sex in fungi is governed by the mating-type (MAT) locus, which exists as a bipolar, pseudobipolar, or tetrapolar system. The significance and impact of MAT on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal MAT loci shares features with the evolution of plant and animal sex chromosomes. Pathogenic Cryptococcus species harbor a bipolar system with a large contiguous MAT locus, whereas closely related species, such as the non-pathogen C. amylolentus, possess a tetrapolar system with unlinked P/R and HD loci. The P/R locus encodes pheromones and pheromone receptors that mediate partner recognition and cell-cell fusion, while the HD locus encodes homeobox domain-containing proteins (Sxi1 and Sxi2) that play important and evolutionarily conserved roles in sexual reproduction. Here, we explored the roles of HD genes in sexual reproduction and determined the implications of a tetrapolar to bipolar MAT transition. Using a CRISPR-Cas9 system we developed for C. amylolentus, we generated gene deletion mutants and demonstrated that a single compatible pair of heteroallelic Sxi1 and Sxi2 is both necessary and sufficient for mating. By relocating the HD genes to the P/R locus, we found that the artificially generated bipolar configuration led to defective sexual development, which could be partially alleviated through additional rounds of sexual reproduction. Transcriptomic profiling revealed the Sxi1-Sxi2 heterodimeric complex drives expression of genes required for DNA replication and ergosterol biosynthesis during sexual reproduction. These findings provide the first experimental demonstration of a tetrapolar-to-bipolar transition in a tetrapolar species, illuminating MAT locus evolution and homeodomain protein functions.IMPORTANCESexual reproduction is critical for fungal survival and adaptation, yet the mechanisms driving transitions between mating systems remain unclear. With Cryptococcus amylolentus, we provide the first experimental validation of a mating system transition from its original tetrapolar state, through an intermediate tripolar state, to a derived bipolar state in a tetrapolar species. We show that homeodomain (HD) protein heterodimers phenotypically govern dikaryotic filamentation and also transcriptionally modulate DNA replication. These findings establish a mechanistic basis for how MAT locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of MAT locus architecture and regulatory functions.},
}
@article {pmid41843435,
year = {2026},
author = {Wang, W and Lin, S and Luo, Y and Shao, C and Shi, Q and Shao, J and Chen, Y and Hu, H and Wan, S and Song, X and Jin, D and Jin, Y},
title = {Genomic and phenotypic insight into Clostridioides difficile RT027 isolates from China reveals diverse virulence associated with clinical symptoms.},
journal = {Emerging microbes &amp; infections},
volume = {15},
number = {1},
pages = {2637287},
pmid = {41843435},
issn = {2222-1751},
mesh = {*Clostridioides difficile/genetics/pathogenicity/isolation &amp; purification/classification/drug effects ; China/epidemiology ; Humans ; Virulence ; Genome, Bacterial ; *Clostridium Infections/microbiology/epidemiology ; Phenotype ; Phylogeny ; Ribotyping ; Bacterial Toxins/genetics ; Whole Genome Sequencing ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics/metabolism ; Drug Resistance, Bacterial ; Genomics ; },
abstract = {Clostridioides difficile (C. difficile) ribotype 027 (RT027) has caused severe outbreaks in North America and Europe over the past 20 years. However, RT027 infections are rare in Asia, particularly in China, with limited severe cases. To clarify its molecular and phenotypic features, we investigated 11 RT027 isolates collected from Shandong, China. Whole-genome sequencing, comparative transcriptomics, CRISPR-Cas analysis, and pan-genome profiling were combined with phenotypic assays of toxin production, sporulation, antimicrobial resistance, and motility. Evolutionary analysis demonstrated that isolates from China clearly diverged from the FQR2 lineage and were phylogenetically closer to FQR1, forming a distinct sublineage. All isolates from Shandong, China encoded a complete tcd and cdt locus and harboured rifamycin and aminoglycoside resistance genes. However, transcriptomic profiling demonstrated significantly reduced expression of binary toxin genes (cdtAB, cdtR), decreased spo0A transcription, and downregulation of flagellar pathways. Phenotypic assays confirmed impaired sporulation, motility and cytotoxicity, while TcdB production and adhesion was comparable to reference strains. CRISPR-Cas elements were conserved but showed reduced transcriptional activity, suggesting diminished host-pathogen interactions. The pan-genome revealed high genomic conservation, consistent with limited functional diversity. Together, these data indicate that RT027 isolates circulating in China possess unique evolutionary trajectories and attenuated virulence traits, helping to explain the rarity of severe RT027 infections in Asia. These findings provide important insights into the regional epidemiology of C. difficile and inform strategies for diagnosis, treatment, and prevention.},
}
@article {pmid41843552,
year = {2026},
author = {Leix, K and Serrano-Zayas, C and Vyas, HS and Graham, SE and Emmer, BT},
title = {Functional interrogation of candidate cis-regulatory elements at the LDLR locus.},
journal = {PLoS genetics},
volume = {22},
number = {3},
pages = {e1012082},
pmid = {41843552},
issn = {1553-7404},
support = {R01 HL167733/HL/NHLBI NIH HHS/United States ; R01 HL171013/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Receptors, LDL/genetics/metabolism ; Enhancer Elements, Genetic/genetics ; Promoter Regions, Genetic/genetics ; Genome-Wide Association Study ; Gene Expression Regulation ; *Regulatory Sequences, Nucleic Acid/genetics ; Introns/genetics ; Animals ; Cholesterol, LDL/genetics/blood ; Genetic Loci ; CRISPR-Cas Systems ; Atherosclerosis/genetics ; },
abstract = {Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. In this study, we developed and applied a high throughput CRISPR screen to test the functional importance of candidate LDLR cis-regulatory elements (CREs) in their native genomic context. In total, we found 25 discrete regions to exhibit a significant impact on LDLR expression. For one of these regions with particularly strong activity in the first intron, we validated the presence of an enhancer by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity within this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and clarify the functional landscape of noncoding sequences at the LDLR locus.},
}
@article {pmid41844662,
year = {2026},
author = {Zhang, J and Wang, Q and Cheng, Z and Liu, J and Liu, Q and Qi, S and Chen, Z and Duan, Y and Liu, Z and Jia, J and Li, C},
title = {Enhancer-mediated Etv4 activation stimulates osteogenic differentiation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41844662},
issn = {2041-1723},
support = {82072499, 32270610//National Natural Science Foundation of China (National Science Foundation of China)/ ; L242120//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {Animals ; *Osteogenesis/genetics ; *Cell Differentiation/genetics ; Mice ; *Osteoblasts/metabolism/cytology ; *Enhancer Elements, Genetic/genetics ; *Proto-Oncogene Proteins c-ets/genetics/metabolism ; STAT3 Transcription Factor/metabolism ; CRISPR-Cas Systems ; Mice, Knockout ; Humans ; },
abstract = {Enhancers, as cis-regulatory elements, play pivotal roles in transcriptional homeostasis. The abnormality in enhancers is highly associated with various diseases, including osteoporosis. However, the landscape of active enhancers underlying bone diseases remains incomplete. By conducting an integrative analysis of transcriptome and ChIP-seq data, we identify enh11 as an active enhancer during osteoblastogenesis. CRISPR/Cas9-mediated deletion of enh11 inhibits cell differentiation of pre-osteoblast MC3T3-E1 cells. The osteoblast-specific knockout of enh11 reduces bone formation and decreases bone mass in mice. In addition, Etv4 is identified as the downstream target of enh11. Functional experiments both in vitro and in vivo validate that Etv4 promotes osteogenesis and bone formation. Mechanistically, enh11 upregulates the expression of Etv4 to promote osteogenesis, probably via binding to the transcription factor Stat3. These findings not only deepen our comprehension of the molecular mechanisms of enh11 underlying bone formation but also highlight enh11 and Etv4 as promising therapeutic targets for osteoporosis.},
}
@article {pmid41846089,
year = {2026},
author = {Zhang, C and Wang, M and He, B and Yang, X and Li, XZ},
title = {High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106722},
pmid = {41846089},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Germ Cells ; *Gene Editing/veterinary/methods ; *DNA Damage ; Humans ; Male ; Female ; },
abstract = {CRISPR/Cas9 technology has transformed genome editing across species; however, its application in avian germ cells remains constrained-not only by editing efficiency, but also by limited evaluation of potential genotoxic effects. In this study, we systematically assessed the performance and genomic safety of CRISPR/Cas9 and CRISPR interference (CRISPRi) in chicken primordial germ cells (PGCs). While CRISPR/Cas9 achieved high editing efficiency, it simultaneously induced substantial DNA damage, apoptosis, and sex-specific cell cycle arrest, revealing the pronounced genotoxic sensitivity of PGCs. In contrast, CRISPRi was well tolerated but failed to achieve effective gene repression in chicken cells. Comparative experiments showed that CRISPRi functioned efficiently in human 293T cells but not in chicken PGCs or somatic DF-1 cells, suggesting species-dependent limitations of mammalian-optimized repression systems. Together, these findings reveal a fundamental trade-off-"efficient but toxic" versus "safe but ineffective"-when applying CRISPR tools to avian germ cells. Our results highlight the need for species-adapted, low-toxicity genome-editing platforms in poultry and provide a framework for evaluating editing strategies in developmentally sensitive cell types.},
}
@article {pmid41847198,
year = {2026},
author = {D, J and Mulavagili, S and Vijayasimha, M},
title = {Commentary: CRISPR-Cas systems against carbapenem resistance-from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1773181},
pmid = {41847198},
issn = {1664-302X},
}
@article {pmid41849360,
year = {2026},
author = {Lin, H and Wang, S and Xie, Y and Cheng, C and Jin, J and Song, X and Zhang, H},
title = {Single-tube two-step RPA-CRISPR/Cas12b platform for detection of Pseudomonas aeruginosa.},
journal = {PloS one},
volume = {21},
number = {3},
pages = {e0340856},
pmid = {41849360},
issn = {1932-6203},
mesh = {*Pseudomonas aeruginosa/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of significant clinical and public health concern, necessitating the development of rapid and reliable detection methods. Traditional diagnostic approaches, which rely on culture-dependent techniques and biochemical identification, are often labor-intensive, time-consuming, and technically demanding. This study describes a novel single-tube, two-step, rapid detection platform that integrates recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats-associated protein Cas12b technology. Through systematic experimental optimization, the study identified an optimal RPA primer pair (F2-R1) and single-guide ribonucleic acid 553 that targets the lasR gene of P. aeruginosa, with reaction conditions optimized at 42°C and a primer concentration of 10 μM. The RPA-clustered regularly interspaced short palindromic repeats/Cas12b fluorescence detection system (RPA-Cas12b-Fluo) demonstrated a sensitivity threshold of 10 copies of deoxyribonucleic acid (DNA) per reaction and a bacterial detection limit of 50 colony-forming units (CFU) per reaction. When coupled with a lateral flow strip (RPA-Cas12b-LFS), the sensitivity was slightly reduced but remained robust, achieving detection limits of 10[2] copies and 200 CFU per reaction. Specificity assays confirmed a high discriminatory capacity for P. aeruginosa with no cross-reactivity observed against P. fluorescens, P. putida, or six common foodborne pathogens, thereby validating the specificity profile of the platform. The applicability of the method was further validated by analyzing 20 water samples, which demonstrated 100% concordance with the national standard culture method. These findings have significant implications for improving outbreak surveillance and mitigating the risk of foodborne transmission associated with P. aeruginosa.},
}
@article {pmid41849610,
year = {2026},
author = {Dai, Y and Abudujielili, Z and Ding, Y and Huang, W and Yin, J and Ou, L and Hu, J and Zheng, S and Li, C},
title = {Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington's disease deficits in BAC226Q mice.},
journal = {Science advances},
volume = {12},
number = {12},
pages = {eaea8052},
pmid = {41849610},
issn = {2375-2548},
mesh = {Animals ; *Huntington Disease/genetics/therapy/pathology ; Mice ; *Huntingtin Protein/genetics ; *CRISPR-Cas Systems ; *Dependovirus/genetics ; Humans ; Disease Models, Animal ; *Gene Editing/methods ; Genetic Therapy ; Chromosomes, Artificial, Bacterial/genetics ; Mice, Transgenic ; },
abstract = {Huntington's disease (HD) is a monogenic autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HTT gene, yielding a gain-of-toxic-function mutant Huntingtin protein (mHTT). CRISPR-Cas9 is a potentially powerful therapeutic strategy for HD by eliminating mutant HTT (mHTT) gene. We developed a specific SaCas9 guide RNA to target human mHTT and a self-inactivating gene editing system that abolishes SaCas9 after a short transient expression for high gene editing efficiency and maximal safety to prevent off-target effects. Both conventional and the self-inactivating gene editing systems successfully eliminated mHTT gene, 60 to 90% mHTT protein and 90% of mHTT aggregation in BAC226Q mouse brains, which resulted in significant long-term rescue of neuropathology, motor deficits, weight loss, and shortened life span. These beneficial effects were observed when gene editing was applied before, at, and well after the onset of pathological and behavioral abnormalities. These proof-of-concept data demonstrate that gene editing can be a highly effective therapeutic approach for HD.},
}
@article {pmid41849748,
year = {2026},
author = {Wang, Q and Chen, D and Berr, A and Shen, WH},
title = {CRISPR gene editing of AtRING1 unravels a critical role of RAWUL domain in PRC1 repression of transcription.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70794},
pmid = {41849748},
issn = {1365-313X},
support = {ANR-12-BSV2-0013-02//Agence National de la Recherche/ ; //Centre National de la Recherche Scientifique/ ; },
mesh = {*Arabidopsis Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *Polycomb Repressive Complex 1/genetics/metabolism ; Gene Expression Regulation, Plant ; Gene Editing ; CRISPR-Cas Systems ; Histones/metabolism ; Protein Domains ; Transcription, Genetic ; RING Finger Domains ; Ubiquitin-Protein Ligases/genetics/metabolism ; Glucosyltransferases ; },
abstract = {Polycomb Group (PcG) proteins, including members of Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2), regulate many key developmental processes through transcriptional gene repression. While the molecular mechanisms of PRC2 and its histone methyltransferase involved in depositing histone 3 lysine 27 trimethylation (H3K27me3) are well understood, the components and E3 ubiquitin ligase functions of PRC1 in plants remain largely elusive. In Arabidopsis, AtRING1 is a key PRC1 component, containing an N-terminal RING-finger domain and a C-terminal RAWUL domain. Previous studies have relied on T-DNA insertion mutants in the investigation of AtRING1 function. By editing AtRING1A using CRISPR/Cas9 technology in the atring1b-1 background, here we have generated and characterized one N-terminal stop mutant atring1[ko] and two C-terminal deletion mutants atring1[▵C-terminal] lacking the RAWUL domain. We show evidence that atring1[ko] represents the strongest loss-of-function mutant, exhibiting embryonic callus-like structures, demonstrating the essential role of AtRING1 in cell differentiation. Remarkably, the atring1[▵C-terminal] mutants exhibit mild developmental defects, suggesting that the RING domain alone retains partial function, while the RAWUL domain fine-tunes PRC1 activity. Our molecular analyses support a model in which AtRING1/PRC1-mediated H2A monoubiquitination (H2Aub1) often precedes PRC2-mediated H3K27me3 deposition at some target loci. Strikingly, the RAWUL domain is required for efficient H2Aub1 enrichment and influences H3K27me3 deposition in a locus-specific manner. Taken together, our study provides new insights into the molecular mechanism underlying PRC1 E3 ligase activity, supporting that PRC1 function facilitates PRC2 activity in epigenetic gene silencing.},
}
@article {pmid41850054,
year = {2026},
author = {Jia, W and Wang, A and Wu, Z and Shi, L and Xie, J and Zhou, Q and Cheng, Y and Lei, X and Liu, L and Tian, L and Zhu, S},
title = {Construction and characterization of recombinant duck enteritis virus expressing duck hepatitis A virus 3 immunogenic genes.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106780},
pmid = {41850054},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Poultry Diseases/prevention &amp; control/virology/immunology ; *Viral Vaccines/immunology ; *Picornaviridae Infections/veterinary/prevention &amp; control/immunology/virology ; *Hepatitis Virus, Duck/immunology ; CRISPR-Cas Systems ; *Hepatitis, Viral, Animal/prevention &amp; control ; },
abstract = {Duck enteritis virus (DEV) is considered an ideal vector for waterfowl vaccine development due to its favorable safety profile and multiple genomic sites that accommodate foreign gene insertion. Duck hepatitis A virus (DHAV) causes acute hepatitis, neurological symptoms, and high mortality in young ducklings, and the predominant circulating serotype has shifted from type 1 to type 3, underscoring the urgent need for improved vaccines. In this study, we employed a CRISPR/Cas9 genome editing platform combined with dual single-guide RNAs (sgRNAs) and a homologous directed repair (HDR) strategy to construct two recombinant DEV strains (rDEV-DHAV) expressing the immunogenic VP0 or VP1 proteins of DHAV-3. The recombinants were purified by plaque selection and validated using PCR, Western blotting, indirect immunofluorescence, and animal experiments. Both recombinant viruses replicated efficiently in chicken embryo fibroblasts and exhibited growth kinetics comparable to the parental DEV vaccine strain. The inserted VP0 and VP1 genes remained genetically stable over at least 15 serial passages. Immunization trials in ducklings demonstrated that both recombinants elicited strong humoral responses against DEV and DHAV-3. Safety evaluation showed that neither recombinant virus induced clinical signs, pathological lesions, or abnormal viral shedding, and both displayed safety profiles equivalent to the parental vaccine strain. Overall, the two rDEV-DHAV strains generated in this study are genetically stable, safe, and exhibit good immunogenicity. The HDR-CRISPR/Cas9 strategy employing dual sgRNAs provides an efficient approach for the rapid construction of multivalent DEV vector vaccines, highlighting its substantial potential in poultry vaccine development.},
}
@article {pmid41850286,
year = {2026},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Gogo Liu, JJ and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {189},
number = {7},
pages = {2189-2193},
doi = {10.1016/j.cell.2026.03.014},
pmid = {41850286},
issn = {1097-4172},
}
@article {pmid41851456,
year = {2026},
author = {Nyberg, WA and Bernard, PL and Ngo, W and Wang, CH and Ark, J and Rothrock, A and Borgo, GM and Kimmerly, GR and Jung, JH and Allain, V and Hamilton, JR and Baldwin, A and Stickels, R and Wyman, S and Khan, SH and Lang, S and Marsh, D and Almudhfar, N and Novick, C and Mortazavi, Y and Zhang, S and AbdElwakil, MM and Sandoval, LR and Hwang, S and Chu, SN and Jung, H and Liu, C and Sharma, D and McCreary, T and Li, Z and Satpathy, AT and Carnevale, J and Rutishauser, RL and Cromer, MK and Roybal, KT and Dodgson, SE and Doudna, JA and Asokan, A and Eyquem, J},
title = {In vivo site-specific engineering to reprogram T cells.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {712-721},
pmid = {41851456},
issn = {1476-4687},
support = {P30 DK063720/DK/NIDDK NIH HHS/United States ; S10 1S10OD021822-0//NIH S10 Instrumentation grant/ ; },
mesh = {Animals ; Mice ; *T-Lymphocytes/metabolism/cytology/immunology ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; CRISPR-Cas Systems/genetics ; Humans ; Transgenes/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; Female ; *Cellular Reprogramming/genetics ; Genetic Vectors/genetics ; *Cell Engineering/methods ; Immunotherapy, Adoptive/methods ; Male ; },
abstract = {Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise[1]. However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR-Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.},
}
@article {pmid41851507,
year = {2026},
author = {Zheng, R and Lu, Z and Wei, R and Shin, YC and Du, J and Zhang, Q and Li, J and Wang, X and Wei, Y and Liu, B and Chen, Y and Ding, L and Zhang, H and Chen, H and Huang, J and Ma, L},
title = {Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {4},
pages = {590-602},
pmid = {41851507},
issn = {1545-9985},
mesh = {RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; Models, Molecular ; Mutation ; Endonucleases/genetics/chemistry/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; },
abstract = {Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.},
}
@article {pmid41851990,
year = {2026},
author = {Guo, Y and Li, J and Haque, A and Chen, J and Yang, X and Wang, Y and Yao, L and Zhuo, C and Wang, J and He, N and Lin, Y and Xiao, S and Liu, B and Zhuo, C},
title = {Subtypes of Type I-E CRISPR-Cas Systems Distribution in Human Escherichia coli Isolates from China.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {89-102},
doi = {10.1177/25731599261430828},
pmid = {41851990},
issn = {2573-1602},
mesh = {*Escherichia coli/genetics/isolation &amp; purification/classification/drug effects ; Humans ; China ; *CRISPR-Cas Systems/genetics ; Phylogeny ; Plasmids/genetics ; *Escherichia coli Infections/microbiology ; beta-Lactamases/genetics ; },
abstract = {The correlation between CRISPR-Cas systems and plasmid-mediated bacterial antibiotic resistance is increasingly growing attention. However, currently no reports exist on the relationship between the CRISPR-Cas systems and the carriage of blaNDM or plasmids in E. coli. Here, molecular characterization and phylogenetic analysis of 639 E. coli isolated from humans in China were carried out. Depending on similarity in sequence, the type I-E CRISPR-Cas systems in E. coli can be grouped into two distinct clades, which we refer to for descriptive purposes within this study as the type I-E-S1 and I-E-S2, whereas the type I-E-S2 CRISPR-Cas system is further divided into I-E-S2a and I-E-S2b systems based on the presence of cas8e and cas11. ST167 (phylogroup A) and ST410 (phylogroup C) E. coli were observed bearing the type I-E-S1 and I-E-S2b systems, respectively. Compared with strains carrying the I-E-S1 type CRISPR-Cas system, the blaNDM carrying rate, the positive rate of IncX3 plasmid, and the positive rate of IncF plasmid of strains with the I-E-S2a type CRISPR-Cas system were evidently lower (p < 0.05); the blaNDM carrying rate and the positive rate of IncF plasmid of strains with the I-E-S2b type CRISPR-Cas system were evidently higher (p < 0.05). The blaNDM positive rate and IncF plasmid positive rate of strains carrying the I-E-S2a type CRISPR-Cas system were significantly lower than those of strains carrying the I-E-S2b type CRISPR-Cas system (p < 0.001). It proves that the I-E-S1, I-E-S2a, and I-E-S2b type CRISPR-Cas systems are beneficial for spreading blaNDM and IncX3 plasmids. We found significant differences in the cas gene sequences of the I-E-S1 and I-E-S2 type CRISPR loci. The type I-E CRISPR-Cas systems in E. coli isolated from Chinese sources are classified further for the first time, revealing their high correlation with blaNDM, phylogenetic groups, and multilocus sequence typing. This work paves the way for a deeper understanding of the role that CRISPR-Cas systems play in the rise of resistant E. coli ST167 and ST410.},
}
@article {pmid41852146,
year = {2026},
author = {Mieritz, IK and Laustsen, C and Axelgaard, E and Dorset, SR and Bak, RO and Bertelsen, LB},
title = {Glycolytic alterations as biomarkers in polycystic kidney disease: A study using a PKD1 knockout model in NRK-52E rat kidney epithelial cells.},
journal = {Physiological reports},
volume = {14},
number = {6},
pages = {e70816},
pmid = {41852146},
issn = {2051-817X},
support = {R272-2027-4023//Lundbeckfonden (Lundbeck Foundation)/ ; R238-2016-534 3349//Lundbeckfonden (Lundbeck Foundation)/ ; 0077911//Novo Nordisk Fonden (NNF)/ ; R279-A16251//The Danish Cancer Society/ ; 500043//Karen Elise Jensens Fond (Karen Elise Jensen Foundation)/ ; 8056-00010B//Innovation Fund Denmark/ ; },
mesh = {Animals ; *Epithelial Cells/metabolism ; Rats ; *Glycolysis ; Monocarboxylic Acid Transporters/metabolism/genetics ; *Polycystic Kidney Diseases/metabolism/genetics ; *TRPP Cation Channels/genetics/metabolism ; Pyruvic Acid/metabolism ; Cell Line ; Biomarkers/metabolism ; *Kidney/metabolism ; Lactic Acid/metabolism ; Gene Knockout Techniques ; L-Lactate Dehydrogenase/metabolism/genetics ; },
abstract = {Polycystic kidney disease (PKD) is a genetic disorder characterized by the formation of fluid-filled cysts in the kidneys, often resulting in progressive renal impairment. Mutations in the PKD1 gene represent the predominant genetic cause of autosomal dominant PKD. Here, we investigated how PKD1 knockout affects glycolytic metabolism in NRK-52E kidney epithelial cells using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopy (MRS) with hyperpolarized [1-[13]C]pyruvate. PKD1 knockout NRK-52E kidney epithelial cells showed a significantly elevated pyruvate-to-lactate conversion as measured by hyperpolarized [1-[13]C]pyruvate (HP-MRS) and significantly increased lactate levels in culture medium, accompanied by upregulated lactate dehydrogenase (LDH) gene expression and enzymatic activity. Monocarboxylate transporter (MCT) expression was selectively altered (significant downregulation of MCT2 and MCT3; MCT1 not significantly changed). Pyruvate dehydrogenase (PDH) activity and transcript levels did not differ between groups. These results demonstrate glycolytic reprogramming associated with PKD1 deficiency and support hyperpolarized pyruvate MRS as a sensitive metabolic biomarker for detecting such alterations in real-time. These findings identify glycolytic remodeling as a robust metabolic consequence of PKD1 loss and demonstrate that HP-[1-[13]C]pyruvate MRS provides a flux-level biomarker suitable for real-time metabolic characterization.},
}
@article {pmid41854240,
year = {2026},
author = {Zhang, Y and Luo, X and Li, H and Jin, S and Zhang, X and Wang, M},
title = {Single-Cell CRISPR: An Efficient Strategy for Decoding Plant Cis-Regulatory Complexity.},
journal = {Plant biotechnology journal},
volume = {24},
number = {7},
pages = {4454-4467},
pmid = {41854240},
issn = {1467-7652},
support = {2023A01//Xinjiang Uyghur Autonomous Region/ ; //Tianchi Talent Introduction Plan of Xinjiang Uyghur Autonomous Region/ ; 2021YFF1000900//National Key Research and Development Program of China/ ; },
mesh = {*Single-Cell Analysis/methods ; *Plants/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Plant/genetics ; Single-Cell Gene Expression Analysis ; Gene Regulatory Networks/genetics ; *Regulatory Sequences, Nucleic Acid/genetics ; },
abstract = {The generation of complex traits involves the coordinated interplay of multiple gene networks. Elucidating the function of transcriptional cis-regulatory elements (CREs) in regulating gene expression is crucial for understanding complex regulatory pathways and improving our ability to modify macro-phenotypes. While traditional bulk sequencing approaches rely on tissue or cell population aggregates, single-cell transcriptomics provides a more precise perspective by capturing cell-type-specific information. The integration of single-cell technology with genome-wide genetic screening, particularly through the single-cell CRISPR (scCRISPR) system, enables the identification of critical regulatory elements and provides novel insights into gene-expression control mechanisms. Here, we summarise recent advances in diverse strategies for functional genome analysis using the scCRISPR system, with an emphasis on its potential to revolutionise single-cell genetic screening of CREs. We also explore the challenges and opportunities for applying these approaches in plant research.},
}
@article {pmid41854410,
year = {2026},
author = {Dias, RG and Freitas, FPM and de Almeida, ELM and Fietto, LG and Zsögön, A and Silveira, WBD},
title = {CRISPR/Cas9 Genome Engineering in Non-Conventional Oleaginous Yeasts: Applications, Challenges, and Prospects.},
journal = {Yeast (Chichester, England)},
volume = {43},
number = {3},
pages = {77-88},
pmid = {41854410},
issn = {1097-0061},
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Yeasts/genetics/metabolism ; *Genetic Engineering/methods ; Genome, Fungal ; Lipid Metabolism/genetics ; *Gene Editing/methods ; Fatty Acids/biosynthesis ; },
abstract = {Given the biotechnological potential of yeast-derived oils for oleochemical production, genes encoding lipid metabolism enzymes are key targets for metabolic engineering. Genetic engineering tools such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, Transcription Activator-Like Effector Nucleases (TALENs), Zinc-Finger Nucleases (ZFNs), RNA interference (RNAi), and integrative plasmids can be used to modulate fatty acid biosynthesis and optimize lipid production. Among them, the CRISPR/Cas9 system, recognized for its simplicity and efficiency, has been deployed as a tool to create oleaginous yeast strains with high lipid productivity and features suitable for application in biorefineries. Species such as Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Candida spp., and Yarrowia lipolytica have already been engineered using CRISPR/Cas9 to enhance the production of fatty acids and their derivatives. However, designing and constructing an efficient CRISPR/Cas9 platform for oleaginous yeasts faces several hurdles, including low transformation efficiency, difficulties in expressing Cas9 and sgRNAs efficiently and consistently, the lack of well-characterized promoters, limited availability of PAM sequences, and poorly understood DNA repair mechanisms. Here, we address the application of the CRISPR/Cas9 system in oleaginous yeasts, laying out the challenges to developing efficient platforms and highlighting key trends in the field. We compare and discuss alternative CRISPR-Cas9 expression strategies to provide an overview of the current landscape and support the development of new approaches.},
}
@article {pmid41854513,
year = {2026},
author = {Han, JH and Kang, YJ and Lee, SY and Jin, HB and Lee, CS and Park, HH},
title = {AcrVA3 Is a Double Strand DNA-Cleaving Anti-CRISPR That Indirectly Inhibits Cas12.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {6},
pages = {e71705},
pmid = {41854513},
issn = {1530-6860},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2026-25470081//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/antagonists &amp; inhibitors ; *DNA/metabolism/chemistry ; *Viral Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/metabolism/chemistry ; DNA Cleavage ; Models, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-Cas12 systems protect bacteria from foreign DNA, but are themselves targeted by anti-CRISPR (Acr) proteins evolved by phages. Among the eight known AcrV proteins that inhibit Cas12-based type V CRISPR-Cas systems, the mechanisms of all except AcrVA3 have been structurally and biochemically characterized. Here, we report the high-resolution structure of AcrVA3 and examine its inhibitory function in vitro. Unexpectedly, AcrVA3 does not directly work on Cas12. Instead, it exhibits double-stranded DNA (dsDNA) cleavage activity, suggesting an indirect mechanism of CRISPR inhibition through DNA degradation. This unique DNA-centric strategy contrasts with previously known Acr mechanisms and expands our understanding of how mobile genetic elements evade CRISPR immunity.},
}
@article {pmid41854526,
year = {2026},
author = {Gao, Z and Liu, G},
title = {Advancing Point-of-Care Testing for Helicobacter pylori toward CRISPR-Cas-Enabled Diagnostics.},
journal = {ACS sensors},
volume = {11},
number = {4},
pages = {2923-2939},
doi = {10.1021/acssensors.6c00089},
pmid = {41854526},
issn = {2379-3694},
mesh = {*Helicobacter pylori/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Helicobacter Infections/diagnosis/microbiology ; *Point-of-Care Testing ; Rapid Diagnostic Tests ; },
abstract = {Helicobacter pylori (H. pylori) chronically infects nearly half of the global population and is a major risk factor for gastric cancer. Timely and accurate diagnosis is critical to enable targeted eradication therapy and prevent disease progression. However, current gold-standard methods, such as invasive endoscopy and laboratory-based polymerase chain reaction, are costly, time-consuming, and logistically impractical for large-scale screening, particularly in resource-limited settings. Point-of-care testing (POCT) emerges as a transformative solution, offering rapid, user-friendly, and minimally invasive detection at the point of need. In this review, we systematically trace the evolution of H. pylori POCT, with a focus on revolutionary CRISPR-Cas-based diagnostic systems, cutting-edge advancements in substrate engineering (e.g., paper, polymer, hydrogels) and multi-modal signal transduction (e.g., optical, electrochemical). We further outline key design principles for next-generation POCT platforms that strictly align with the World Health Organization's ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Deliverable), aiming to accelerate early detection, reduce healthcare disparities, and improve global clinical management of H. pylori infection.},
}
@article {pmid41855944,
year = {2026},
author = {Miao, Y and Wang, C and Peng, Y and Sun, X and Zheng, Z and Zhang, Q and Cheng, W and Li, J},
title = {PAM-assembled CRISPR-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for detecting prostate cancer exosomes.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118635},
doi = {10.1016/j.bios.2026.118635},
pmid = {41855944},
issn = {1873-4235},
mesh = {Humans ; *Prostatic Neoplasms/diagnosis/genetics/blood ; Male ; *Biosensing Techniques/methods ; *Exosomes/chemistry/pathology ; Colorimetry/methods ; *CRISPR-Cas Systems/genetics ; *Glutamate Carboxypeptidase II ; *Antigens, Surface ; Aptamers, Nucleotide/chemistry ; Tetraspanin 30/chemistry ; },
abstract = {Prostate specific membrane antigen (PSMA)-positive exosomes hold significant potential for the diagnosis and risk assessment of prostate cancer. However, accurate detection is severely hindered by their low abundance in blood and interference from similarly sized particles. In this study, we have developed a protospacer adjacent motif (PAM)-assembled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for the highly sensitive detection of PSMA-positive exosomes. In this work, two split strands respectively containing CD63 and PSMA aptamers are utilized to bind CD63 and PSMA on the exosome surface, forming a template that induces the opening of a hairpin DNA (HP DNA). A PAM site forms via hairpin-to-double-stranded structure transition. CRISPR-Cas12a recognizes PAM, activates to cleave FAM-labeled probes for fluorescence, while cerium dioxide nanozyme (CeO2 NZ) (with phosphatase-mimicking activity) hydrolyzes cleavage products. Hydroxyl radicals from hydrolysis oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to induce colorimetry. CRISPR-Cas12a-nanozyme dual recognition significantly improves prostate cancer exosome detection selectivity and sensitivity. Under optimized conditions, the limits of detection for the fluorescence and colorimetric modes reach 49 particles/μL and 63 particles/μL, respectively. By mutually validating dual detection modes, this biosensing technology accurately distinguishes prostate cancer patients from healthy individuals, holding great promise for early diagnosis.},
}
@article {pmid41855972,
year = {2026},
author = {Bai, M and Li, Y and Hu, Q and Qing, M and Bai, L},
title = {Enzymatic crRNA stabilization strategy for enhanced CRISPR/Cas12a detection.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129674},
doi = {10.1016/j.talanta.2026.129674},
pmid = {41855972},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Humans ; Influenza A virus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *CRISPR-Associated Proteins/genetics ; Ribonucleases/antagonists &amp; inhibitors ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR/Cas12a system enables precise and rapid nucleic acid recognition through the programmable targeting capability of CRISPR RNA (crRNA). However, the intrinsic instability of crRNA limits the robustness and sensitivity of CRISPR-based molecular diagnosis in practical applications. Herein, we present a simple and general enhancement strategy that suppresses RNase-mediated crRNA degradation. This strategy, termed RNase inhibitor (RI)-assisted CRISPR (RI-CRISPR), leverages RI to specifically prevent crRNA degradation, thereby improving its stability and enhancing the detection performance and anti-interference capability of the CRISPR system. Using influenza A virus (IAV) and mycobacterium tuberculosis (MTB) as model targets, RI-CRISPR improves detection sensitivity by nearly twentyfold compared to conventional CRISPR/Cas12a. Clinical validation using 40 MTB samples, combined with recombinase polymerase amplification (RPA), achieves 100% specificity and 96% sensitivity compared with the GeneXpert assay. Overall, this work provides a practical strategy to enhance sensitivity and robustness of CRISPR-based diagnostics, and is expected to promote further biomedical applications of CRISPR technology.},
}
@article {pmid41855973,
year = {2026},
author = {Yang, M and Hu, J and Zhang, Z and Huang, L and Yu, Y and Qiu, D and Zu, Y and Liu, Y and Lin, Z},
title = {Homogeneous electrochemical sensor for sensitive detection of HBV DNA based on magnetic separation and CRISPR/Cas12a protein trans-cleavage.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129676},
doi = {10.1016/j.talanta.2026.129676},
pmid = {41855973},
issn = {1873-3573},
mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *DNA, Viral/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; Humans ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; Limit of Detection ; },
abstract = {Infection with hepatitis B virus (HBV) represents a major challenge to global health, especially in areas of inadequate healthcare infrastructure, rapid and on-site detection plays a crucial role in effective disease management and control. Consequently, it is necessary to develop some simple but sensitive HBV screening techniques. In this work, a homogeneous electrochemical biosensor was constructed for sensitive and specific HBV DNA detection through the integration of CRISPR/Cas12a target recognition and magnetic bead-based separation. The sensor exploits the collateral cleavage activity of CRISPR/Cas12a protein upon recognition of target double-stranded DNA, enabling the degradation of methylene blue-labeled single-stranded DNA captured on magnetic beads (MBs). Then the MBs are separated and redissolved in the solution and the electrochemical response of the system can be tested in homogeneous solution. The electrochemical signal has relationship with the concentration of HBV DNA within a range of 10 fM - 10 nM, and the LOD is 3.74 fM. The sensor demonstrates excellent selectivity and biological stability, which has potential application in clinical diagnosis, especially in resource-limited environments.},
}
@article {pmid41855975,
year = {2026},
author = {Yong, Q and Ou, X and Zhao, Y and Kang, X and Gao, H and Liu, H and Li, K and Guo, Y},
title = {CRISPR/Cas and isothermal amplification in Pathogen Detection: Applications and future perspectives.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129662},
doi = {10.1016/j.talanta.2026.129662},
pmid = {41855975},
issn = {1873-3573},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; },
abstract = {Conventional pathogen detection methods are often limited by prolonged turnaround times and laboratory dependency. The integration of CRISPR/Cas systems with isothermal amplification (IA) has emerged as a promising approach to enable rapid, accurate, and field-deployable molecular diagnostics. This review systematically outlines the principles, optimization strategies, and recent advances in CRISPR-Cas and IA-integrated platforms. It highlights how synergistic mechanisms enhance detection sensitivity and examines innovative integration strategies-such as physical compartmentalization, chemical regulation, and intelligent system design-that address key compatibility challenges. The role of nanomaterials in enhancing signal amplification and facilitating system integration is thoroughly discussed. Furthermore, the suitability of various readout modalities-including fluorescence, lateral flow assays, electrochemical sensing, and digital detection-is critically evaluated. While challenges remain in terms of stability, cost, and standardization, future advances in intelligent design, portable device development, and quantitative methodologies are expected to establish this technology as a versatile platform for public health control, food safety monitoring, and related fields. This review provides a comprehensive perspective and methodological reference for researchers engaged in point-of-care testing and diagnostic technology development.},
}
@article {pmid41856106,
year = {2026},
author = {Yuan, S and Zhu, H and Yu, M and Jia, H and Peng, S and Ma, Y},
title = {Discovery of human gut phage-encoded anti-CRISPR proteins unveils diverse mechanisms for phages to evade type II CRISPR immunity.},
journal = {Cell host &amp; microbe},
volume = {34},
number = {4},
pages = {708-719.e5},
doi = {10.1016/j.chom.2026.02.017},
pmid = {41856106},
issn = {1934-6069},
mesh = {Humans ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Viral Proteins/genetics/metabolism/chemistry ; *Gastrointestinal Tract/virology/microbiology ; *Bacteria/genetics/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; },
abstract = {Phages encode diverse anti-CRISPR (Acr) proteins to counteract bacterial CRISPR-Cas systems. However, gut phage Acrs remain poorly characterized. Using an integrated bioinformatics and high-throughput functional screening approach, we identify 651 phage-encoded positive Acr candidates that target type II CRISPR systems, which predominate in the human gut. Among these, a subset of Acrs is verified through plasmid interference assays, with plaque assays confirming CRISPR-Cas inhibitory activity for 36 Acr candidates. Mechanistic characterization of five Acrs, including the Acr against subtype II-B systems (AcrIIB-1), reveals distinct inhibition strategies. Remarkably, 213 positive Acr candidates, designated here as GutAcraca, exhibit structural convergence by adopting similar folds and exhibit dual functionality: transcription regulation to support their production and inhibition of CRISPR-Cas systems. These GutAcraca are widely distributed across microbial species (detected in 26% of species). Our work uncovers the extensive diversity of phage-encoded Acrs in the human gut and highlights their potential as biotechnology tools.},
}
@article {pmid41856683,
year = {2026},
author = {Wang, Y and Guo, Y and Lu, Q and Liu, X and Xu, H and Chen, J and Pi, R and Yuan, S and Yang, Z and Lu, R and Meng, FL and Gan, T and Hu, J},
title = {Restoring the potency of a neutralizing antibody via guided hypermutation with hyper-antibody editor HAE1.},
journal = {Genome research},
volume = {36},
number = {5},
pages = {1029-1039},
doi = {10.1101/gr.281396.125},
pmid = {41856683},
issn = {1549-5469},
mesh = {Humans ; HEK293 Cells ; *Antibodies, Neutralizing/genetics/immunology ; *Somatic Hypermutation, Immunoglobulin ; SARS-CoV-2/immunology ; CRISPR-Cas Systems ; Antibody Affinity ; *Antibodies, Viral/genetics/immunology ; Gene Editing ; },
abstract = {Somatic hypermutation (SHM) drives antibody affinity maturation in B cells. By mimicking this process, guided hypermutation (GHM) tools employing CRISPR systems and activation-induced cytidine deaminase (AID) have advanced antibody development. However, GHM-induced mutations in cultured cells exhibit mutation patterns distinct from those observed in natural antibody diversification following in vivo affinity selection. To address this, we engineer a hyper-antibody editor, HAE1, by integrating cytidine and adenine deaminases with a nicked, PAMless Cas9 variant, SpRY, to closely resemble the mutation spectrum of natural SHM. Moreover, to streamline mutation, selection, and validation within the same cells, we develop a dual-expression system in HEK293F cells that allows simultaneous expression of both transmembrane and secreted full-length antibodies. Using this system, we apply HAE1 to the SARS-CoV-2 neutralizing antibody CV07-209 and restore the antibody's binding affinity and neutralization potency against Omicron variants, specifically BA.1, including at least one mutation beyond the reach of current GHM tools. HAE1 thus provides a versatile, high-throughput strategy for expediting antibody evolution, presenting significant potential for therapeutic antibody development and protein engineering.},
}
@article {pmid41856855,
year = {2026},
author = {Tsai, FY and Sternberg, SH},
title = {Memory on demand: how RNA-free Cas9 recharges CRISPR immunity.},
journal = {Trends in biochemical sciences},
volume = {51},
number = {4},
pages = {310-312},
doi = {10.1016/j.tibs.2026.01.004},
pmid = {41856855},
issn = {0968-0004},
mesh = {*CRISPR-Cas Systems/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics/immunology ; Archaea/genetics/immunology ; Bacteria/genetics/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Bacteria and archaea acquire immune memories by integrating foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPR) arrays. Zhou et al. reveal that Cas9-thought to act only with guide RNAs-also functions in its RNA-free form, stimulating spacer acquisition. Rising CRISPR RNA levels shift the equilibrium toward the RNA-bound state, attenuating acquisition and minimizing autoimmunity.},
}
@article {pmid41856885,
year = {2026},
author = {Ahmar, S and Zhang, R and Pouramini, P and Janeczko, A and Shafique, MS and Rapacz, M and Reis, RS and Zhu, Q and Hensel, G and Pociecha, E},
title = {Precision harvest: path to genetically modified organism-free crops with CRISPR by 2035.},
journal = {Trends in plant science},
volume = {31},
number = {5},
pages = {719-730},
doi = {10.1016/j.tplants.2025.12.014},
pmid = {41856885},
issn = {1878-4372},
mesh = {*Crops, Agricultural/genetics ; *Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Gene Editing ; *CRISPR-Cas Systems ; },
abstract = {Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR) technology enable precise genetic modifications and produce genetically modified organism -free crops that match consumer preferences. By 2035, we will be able to consume CRISPR-edited crops, addressing food security issues and boosting economies for individual countries. This review highlights the progress of genetically modified crops and the regulatory challenges involved in bringing CRISPR-edited crops to market based on product- and process-based approaches across different regions. We also examine public preferences regarding these technologies and the current status of CRISPR-edited crops in terms of market availability. Furthermore, we stress the importance of establishing clear safety standards, effective patent management, and guidance on regulatory pathways for crop approval, as well as exploring future directions for integrating these technologies with artificial intelligence.},
}
@article {pmid41859774,
year = {2026},
author = {Fang, Z and Hao, Y and Zuo, X and Wang, S},
title = {Target-Gated Ratiometric pH Sensing via Tetrahedral DNA Framework-Based Dual-CRISPR System.},
journal = {Analytical chemistry},
volume = {98},
number = {12},
pages = {9208-9218},
doi = {10.1021/acs.analchem.5c07666},
pmid = {41859774},
issn = {1520-6882},
mesh = {Hydrogen-Ion Concentration ; DNA Nanostructures/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/chemistry ; Fluorescent Chemosensor Compounds ; Humans ; Fluorescent Dyes/chemistry ; },
abstract = {Extracellular vesicle (EV)-mediated communication is tightly regulated by local pH, which governs vesicle biogenesis, cargo release, and membrane fusion. Accurate and context-specific pH sensing is therefore crucial for elucidating EV function and disease-associated microenvironmental regulation. Here, we present a tetrahedral DNA framework (TDF)-orchestrated dual-CRISPR system that integrates orthogonal Cas12a and Cas13a nucleases for target-activated, ratiometric pH detection at lipid membranes. By exploiting the distinct pH-activity profiles of Cas12a (optimal pH ∼ 8.5) and Cas13a (optimal pH ∼ 7.2), combined with their complete substrate orthogonality, we constructed a self-calibrating nanosensor featuring equimolar coassembly of both nucleases and their corresponding fluorogenic reporters at the four vertices of a TDF. The well-defined tetrahedral geometry ensured reproducible molecular organization and stable fluorescence output, eliminating variability inherent to conventional single-fluorophore probes. The sensor exhibited quantitative assembly fidelity and robust pH responsiveness across physiological ranges. Importantly, the Cas module can be programmed for conditional activation, enabling pH sensing only upon recognition of disease-associated biomarkers. Using miR-146a, a regulatory microRNA enriched in EVs implicated in inflammation and cancer progression, as a model target, we demonstrated target-gated pH monitoring on cell-derived exosomes and during liposome fusion events. This work establishes a versatile and generalizable platform for programmable, ratiometric sensing at biomembrane interfaces, offering new opportunities to probe EV-mediated intercellular communication and dynamic microenvironmental regulation.},
}
@article {pmid41859925,
year = {2026},
author = {Tibebu, R and Gamo, ME and Ellison, EE and Myers, EA and Sahoo, R and Winecke, SR and Tan, GD and Fischer, JM and Leakey, ADB and Voytas, DF},
title = {Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70805},
pmid = {41859925},
issn = {1365-313X},
support = {SC0023160//U.S. Department of Energy/ ; SC0018277//U.S. Department of Energy/ ; },
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Plant Stomata/genetics ; *Plant Viruses/genetics ; Genomics/methods ; Plants, Genetically Modified ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; },
abstract = {Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.},
}
@article {pmid41860163,
year = {2026},
author = {Wang, B and Liu, W and Li, Y and Ouyang, Q and Wang, Y and Xu, X and Li, B and Xiu, R and Zhang, X and Liu, M},
title = {Consequences of CRISPR-Cas9-Mediated Stromelysin-1 Knockout in Pancreatic Islet Microvascular Endothelial Cells.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {6},
pages = {e71098},
pmid = {41860163},
issn = {1582-4934},
support = {7252093//Beijing Municipal Natural Science Foundation/ ; 81900747//National Natural Science Foundation of China/ ; },
mesh = {*Endothelial Cells/metabolism/drug effects ; Animals ; *Islets of Langerhans/blood supply/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Proliferation ; Glucose/toxicity ; Cell Movement/genetics ; *Microvessels/metabolism/cytology ; Gene Knockout Techniques ; Angiogenesis/genetics ; Mice ; *Matrix Metalloproteinase 10/genetics/metabolism ; Neovascularization, Physiologic ; Secretome/metabolism ; },
abstract = {The integrity of the pancreatic islet microvasculature is critical for endocrine function, yet it is progressively compromised by glucotoxicity in diabetes. While matrix metalloproteinases are implicated, the role of stromelysin-1 as a potential upstream driver of endothelial dysfunction remains poorly defined. The aim of our study was to elucidate the role of stromelysin-1 in mediating glucotoxic injury to islet microvascular endothelial cells (IMECs). To this end, we employed a CRISPR/Cas9-mediated knockout of stromelysin-1 in IMECs. Cellular functions, including proliferation, migration, and angiogenesis, were assessed using IncuCyte ZOOM live-cell imaging, while endothelial barrier integrity was quantified via a 40 kDa dextran flux assay. Additionally, the secretome was profiled using a cytokine antibody array. We found that genetic ablation of stromelysin-1 conferred protection against glucotoxicity. Stromelysin-1 KO IMECs exhibited significantly enhanced proliferation, migration, and angiogenic capacity compared to wild-type controls. Furthermore, stromelysin-1 deficiency restored endothelial monolayer integrity by attenuating high-glucose-induced hyperpermeability. These functional improvements were linked to a remodelling of the secretome, characterised by decreased secretion of the pro-degradative MMP-2 and increased secretion of the anti-inflammatory cytokine IL-10 and the endogenous inhibitor TIMP-2. Overall, our findings establish stromelysin-1 as a crucial mediator of glucotoxic injury in islet microvascular endothelial cells.},
}
@article {pmid41860589,
year = {2026},
author = {Walsh, DJ and Hynes, R and Guo, W and Surgenor, C and Prodöhl, PA and Parle-McDermott, A},
title = {Development and Laboratory Validation of a Field-Deployable CRISPR-Cas12a eDNA Assay for Phylogeographic Lineage Detection in Arctic Char (Salvelinus alpinus).},
journal = {Molecular ecology resources},
volume = {26},
number = {3},
pages = {e70125},
pmid = {41860589},
issn = {1755-0998},
support = {//Higher Education Authority/ ; },
mesh = {Animals ; Phylogeography/methods ; *CRISPR-Cas Systems ; *Trout/genetics/classification ; *DNA, Environmental/genetics ; Sensitivity and Specificity ; Ireland ; },
abstract = {Environmental DNA (eDNA) tools are increasingly used for biodiversity monitoring, with most existing assays targeting species-level identification. However, the use of eDNA to resolve intraspecific genetic variation remains rare and methodologically underdeveloped. This study presents the development and laboratory validation of a novel molecular assay capable of detecting specific phylogeographic lineages, advancing eDNA applications by enabling resolution below the species level. The assay combines Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a technologies with a lateral flow platform for field-ready, on-site detection. Irish Arctic char (Salvelinus alpinus) was selected as the model due to its conservation relevance and post-glacial lineage diversity in Ireland. Mitochondrial genome sequencing of known Irish lineages identified a Protospacer Adjacent Motif (PAM) site unique to the Atlantic Subclade 1 lineage, allowing clear discrimination from co-occurring lineages. Two assays were optimised: a species-specific assay detecting all Arctic char lineages and a lineage-specific assay targeting Lineage 1. Both showed high sensitivity and specificity under laboratory conditions, with LbCas12a outperforming AsCas12a at optimised buffer concentrations. The lateral flow adaptation, utilising a dual-labelled FAM-Biotin probe, enabled portable and rapid detection with minimal equipment. Field validation using eDNA from Irish lakes highlighted the need for improved sampling protocols, as lake-edge surface samples failed to yield detections. This assay represents the first reported example of a CRISPR-based eDNA tool for phylogeographic lineage detection in the field. It offers a novel, non-invasive, and scalable approach to fine-scale ecological monitoring and establishes a foundation for future conservation tools targeting intraspecific diversity.},
}
@article {pmid41860948,
year = {2026},
author = {Hong, Y and Si, X and Liu, W and Mai, X and Zhang, Y},
title = {Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41860948},
issn = {2050-084X},
support = {2021YFA1101002//National Key Research and Development Program of China/ ; 81773304//National Natural Science Foundation of China/ ; 81572795//National Natural Science Foundation of China/ ; 2019A39//the "Hundred, Thousand and Ten Thousand Talent Project" by Beijing municipal government/ ; },
mesh = {Animals ; Glycosylation ; Galactosyltransferases/metabolism/genetics ; Mice ; *Lymphocyte Activation ; *CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/immunology ; Humans ; Receptors, Antigen, T-Cell/metabolism ; },
abstract = {Cytotoxic CD8[+] T-cells play central roles in tumor immunotherapy. Understanding the mechanisms that regulate development, differentiation, and functions of cytotoxic CD8[+] T-cells leads to the development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8[+] T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1,4-galactosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8[+] T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8[+] T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8[+] T-cells. Finally, the expression levels of B4GALT1 normalized to tumor-infiltrated CD8[+] T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8[+] T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.},
}
@article {pmid41861112,
year = {2026},
author = {Fu, P and Zhang, X and Zhou, Y and Zheng, J and Sun, A and Zhuang, K and Bao, W and Gao, G},
title = {Embedded CRISPRi Enhances Gene-Silencing Efficiency in Drosophila.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {31},
pages = {e15849},
doi = {10.1002/advs.202515849},
pmid = {41861112},
issn = {2198-3844},
support = {32500494//National Natural Science Foundation of China/ ; 32370631//National Natural Science Foundation of China/ ; //priority academic program development of jiangsu higher education institutions/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Silencing/physiology ; Drosophila Proteins/genetics ; RNA Interference ; *Amyotrophic Lateral Sclerosis/genetics ; Disease Models, Animal ; },
abstract = {CRISPR interference (CRISPRi), leveraging catalytically inactive Cas9 (dCas9), has transformed transcriptional silencing. However, its application in Drosophila melanogaster has been constrained by inconsistent efficiency and limited repression amplitude. Here, we present embedded CRISPR interference (emCRISPRi), an advanced gene-silencing platform that integrates transcriptional repression domains (Mxi and TRD) into a structurally flexible region of dCas9. This design significantly enhances silencing efficiency, enabling robust repression of coding genes and cis-regulatory elements, particularly at transcription start site (TSS)-proximal regions. emCRISPRi demonstrates improved gene-silencing activity compared to RNA interference (RNAi) at several tested loci and facilitates strong phenotypic rescue via unmodified cDNA. Its versatility is demonstrated through the dissection of Hippo pathway interactions and the mitigation of TDP-43-induced neurotoxicity in an amyotrophic lateral sclerosis (ALS) model. These findings position emCRISPRi as a transformative tool for functional genomics, enhancer studies, and disease modeling in Drosophila, with significant potential for cross-species adaptation and therapeutic innovation.},
}
@article {pmid41861390,
year = {2026},
author = {Huang, Y and Yi, X and Yang, X and Li, C and Li, Y and Ye, Z and He, J},
title = {Characterization of CRISPR-Cas systems in the Haemophilus genus CRISPR-Cas in Haemophilus spp.},
journal = {Genetics and molecular biology},
volume = {49},
number = {1},
pages = {e20250166},
pmid = {41861390},
issn = {1415-4757},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system constitutes a crucial adaptive defense mechanism in prokaryotes against foreign genetic elements. Although CRISPR-Cas systems have been characterized in numerous bacteria, the architecture and function of these systems in the Haemophilus genus remain poorly understood. This study aims to analyze CRISPR-Cas systems in 138Haemophilusstrains and investigate their function, particularly in relation to virulence factors. Results revealed that CRISPR-Cas systems were identified in 31.88% of the Haemophilusstrains. Subtype I-C was the most prevalent, followed by subtypes II-C and III-A. Repeat sequences and thecas1gene were highly conserved within the same subtype. 29.62% of spacer sequences exhibited homology to plasmids or bacteriophages. phiMHaA1 was an important target of the CRISPR-Cas system in Haemophilusgenus. The protospacer adjacent motif sequences (PAM) were determined to be 5'-TTC-3' for subtype I-C and 5'-TTT-3' for subtype II-C. Comparative analysis of virulence genes showed that CRISPR-positive strains carried more ompP2 than CRISPR-negative strains, while the distribution of hmw2C and hmw1C exhibited an opposite trend. These findings provide novel insights into the diversity and function of CRISPR-Cas systems inHaemophilusgenus and propose potential strategies for attenuating the impact ofHaemophilusvirulence factors.},
}
@article {pmid41863808,
year = {2026},
author = {Louis, EM and Fu, L and Luu, N and Sachs, LM and Shi, YB},
title = {Protocol for streamlining genotyping of germline-transmissible mutants from genome editing by using a parallel qPCR-based index and R analysis.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104454},
doi = {10.1016/j.xpro.2026.104454},
pmid = {41863808},
issn = {2666-1667},
abstract = {Targeted genome editing using CRISPR-Cas, ZFNs, or TALENs enables precise gene function studies but often produces point mutations or insertions or deletions (indels) that are difficult to detect by conventional PCR. We developed a parallel qPCR assay with an iGenotype index for simple, reliable genotyping. iGenotype values (1, 0, -1) remained constant across allele-specific primers. qPCR data can be analyzed via an R program, enabling large-scale or automated genotyping. For complete details on the use and execution of this protocol, please refer to Fu et al.[1].},
}
@article {pmid41863928,
year = {2026},
author = {Chang, Z and Zhu, B and Wang, Y and Taylor, JA and Dong, H and Zhu, X and Hao, Y and Zhou, Y and Xu, M and Travas-Sejdic, J},
title = {Disposable laser-induced graphene-based biosensor strip for the detection of N-protein using CRISPR activation and ratiometric electrochemical readout.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129667},
doi = {10.1016/j.talanta.2026.129667},
pmid = {41863928},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Electrochemical Techniques/methods/instrumentation ; SARS-CoV-2 ; *Graphite/chemistry ; Humans ; Lasers ; COVID-19/diagnosis ; *Coronavirus Nucleocapsid Proteins/analysis ; *Phosphoproteins/analysis ; Ferrous Compounds/chemistry ; CRISPR-Cas Systems ; Metallocenes/chemistry ; Electrodes ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Betacoronavirus/isolation &amp; purification ; Methylene Blue/chemistry ; *Coronavirus Infections/diagnosis/virology ; Limit of Detection ; Pandemics ; },
abstract = {Pathogen detection is important for infectious diseases prevention and control. This study presents a novel positive-response, ratiometric electrochemical biosensor constructed on disposable laser-induced graphene (LIG) electrodes. The platform provides dual electrochemical signals for ultrasensitive detection of the SARS-CoV-2 N-protein by harnessing target-activated CRISPR-Cas12a trans-cleavage activity. The developed sensor incorporates a hairpin oligonucleotide (ON) functionalized on gold nanoparticles-deposited LIG electrode surface. Methylene blue (MB) molecules bind to guanine (G) bases of the hairpin ON through inherent affinity, generating the first electrochemical signal. Upon target-induced CRISPR-Cas12a activation, ferrocene (Fc)-labelled indicator ON hybridises with the hairpin ON, displacing the MB molecules and concurrently introduces the Fc as a secondary electrochemical signal reporter. Such displacement triggers a quantifiable decrease in MB electrochemical current and an increase in Fc electrochemical current, generating an Fc/MB ratiometric signal that enhances with increasing target levels and serves as the robust sensor response. The developed ratiometric biosensor achieves a linear response from 0.01 pM to 100 pM of N-protein with a detection limit of 1.3 × 10[-3] pM and with excellent selectivity. The clinical feasibility of the developed ratiometric electrochemical biosensor was confirmed by detecting N-protein in the inactivated cell-cultured SARS-CoV-2. This work demonstrates significant potential in rapid pathogen detection and point-of-care diagnostics.},
}
@article {pmid41863957,
year = {2026},
author = {Yang, Y and Huang, Z and Peng, X and Cui, F and Hu, X and Ren, H},
title = {Single-microsphere biosensors based on UiO-66 confinement-enhanced fluorescence emission for sensitive detection of antibiotics and pathogenic bacteria.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118632},
doi = {10.1016/j.bios.2026.118632},
pmid = {41863957},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Microspheres ; *Anti-Bacterial Agents/analysis/isolation &amp; purification ; *Staphylococcus aureus/isolation &amp; purification/pathogenicity ; Limit of Detection ; *Fluorescent Dyes/chemistry ; *Chloramphenicol/analysis/isolation &amp; purification ; Spectrometry, Fluorescence/methods ; Food Contamination/analysis ; CRISPR-Cas Systems ; Metal-Organic Frameworks/chemistry ; Phthalic Acids ; },
abstract = {To meet the urgent need for sensitive detection of foodborne pathogens and antibiotic residues-key to preventing outbreaks and curbing antimicrobial resistance-we developed a stable fluorescent probe, H4TCPE@UiO-66 (H@U). The probe retains strong fluorescence under harsh conditions (18.70% intensity change at pH 3, 11.36% at 80 °C) and exhibits a 6.90-fold signal amplification over H4TCPE. In addition, H@U is able to be a sensitive probe integrated with millimetre-scale polystyrene microspheres (mPS) and realize rapid detection of chloramphenicol within 20 min with a detection limit of 33.52 pg/mL, representing a 26.61-fold improvement in sensitivity over the enzyme-linked immunosorbent assay. Building on this, we harnessed the trans-cleavage activity of CRISPR/Cas12a to develop a CRISPR/Cas12a-enhanced H@U mPS (H@U-C mPS) biosensor for the quantitative detection of Staphylococcus aureus, achieving a linear range of 10-10[5] CFU/mL (two orders of magnitude lower than qPCR) and a detection limit of 6.97 CFU/mL. Furthermore, the practical applicability of H@U-C mPS biosensors was successfully demonstrated through validation using 30 real-world samples. Together, these platforms offer a robust, rapid, and highly sensitive strategy for monitoring chemical hazards and microbial in food safety and clinical diagnostics.},
}
@article {pmid41864147,
year = {2026},
author = {Zhang, L and Tang, J and Feng, M and Chen, S},
title = {Development of a plasmid-free Escherichia coli strain for high-yield production of ergothioneine.},
journal = {Enzyme and microbial technology},
volume = {197},
number = {},
pages = {110850},
doi = {10.1016/j.enzmictec.2026.110850},
pmid = {41864147},
issn = {1879-0909},
mesh = {*Ergothioneine/biosynthesis ; *Escherichia coli/genetics/metabolism ; Plasmids/genetics ; *Metabolic Engineering/methods ; Biosynthetic Pathways/genetics ; Transposases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Ergothioneine (ERG), a sulfur-containing amino acid derivative known for its antioxidant activity, has a wide range of applications in healthcare and nutrition. Escherichia coli has been extensively studied as a platform for ERG production due to its rapid growth and well-established genetic tools. However, most engineered strains rely on plasmid-based expression system, which are genetically unstable. Additionally, the requirement for antibiotics to maintain plasmid stability further limits the feasibility of plasmid-based systems for industrial-scale production. Here, we established a plasmid-free E. coli platform for ERG biosynthesis using a multi-copy chromosomal integration CRISPR-associated transposase (MUCICAT) system. We first integrated a three-gene ERG biosynthetic pathway into the E. coli genome at varying copy numbers, resulting in a five-copy strain (P5) that exhibited the highest ERG titer of 222.5 ± 5.0 mg/L. Subsequently, we reinforced the two key catalytic modules-histidine methylation and SAM biosynthesis-through iterative genomic integration of the corresponding genes, yielding a plasmid-free strain P18 that produced 370.0 ± 7.0 mg/L ERG. The engineered strain P18 exhibited excellent genetic stability, as confirmed by serial passaging. When scaled up in a 5-L bioreactor under fed-batch condition, an ERG titer of 10.1 g/L was achieved. This study demonstrates a plasmid-free ERG production strategy based on stable, multi-copy chromosomal integration of the ERG biosynthetic pathway in E. coli, highlighting its potential as an efficient platform for scalable ERG production.},
}
@article {pmid41865101,
year = {2026},
author = {Prakash, S and Mishra, C and Sinha, M and Kumari, H and Kumar, A},
title = {The evolution of next-generation lateral flow assays for bacterial and fungal diagnostics.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41865101},
issn = {1436-5073},
abstract = {Infectious diseases driven by increasingly resistant bacterial and fungal pathogens demand diagnostics that are faster and more accessible than conventional culture-based methods. This review traces the evolution of lateral flow assays (LFAs) from simple qualitative strips to sophisticated, molecularly enhanced diagnostic platforms. This review synthesizes literature mostly published between 2010 and 2025, identified through PubMed, Scopus, and Web of Science using search terms including 'lateral flow assay,' 'point-of-care diagnostics,' 'CRISPR diagnostics,' 'nanozyme biosensor,' 'antimicrobial resistance,' 'Candida auris,' and 'invasive aspergillosis. It highlights how advances in materials science (including quantum dots and nanozymes), isothermal amplification (RPA, LAMP), and CRISPR/Cas-based recognition have pushed LFAs toward laboratory-comparable sensitivity while preserving their simplicity. The clinical impact of these next-generation LFAs is illustrated using high-threat pathogens such as MRSA, Candida auris, and invasive Aspergillus, where rapid, point-of-care identification improves outcomes and supports antimicrobial stewardship. The review also examines the digital transformation of LFAs through smartphone-based readouts and artificial intelligence, which enable quantitative analysis and real-time epidemiological surveillance, even in remote settings. Despite ongoing challenges, including the hook effect, cross-reactivity, and regulatory fragmentation, the emerging technologies described here suggest that LFAs can help decouple high-quality infectious disease diagnostics from centralized laboratories, supporting a more equitable, global access to precision microbiological testing.},
}
@article {pmid41865126,
year = {2026},
author = {Marei, HE},
title = {Recent Advances in the Non-viral Delivery of Genes to Central Nervous System Disorders.},
journal = {Cellular and molecular neurobiology},
volume = {46},
number = {1},
pages = {},
pmid = {41865126},
issn = {1573-6830},
mesh = {Humans ; *Central Nervous System Diseases/therapy/genetics ; Animals ; *Gene Transfer Techniques/trends ; *Genetic Therapy/methods ; Nanoparticles/chemistry ; Blood-Brain Barrier/metabolism ; Gene Therapy Agents ; Genetic Vectors ; },
abstract = {Disorders of the central nervous system (CNS), neurological disorders, neurodegenerative disorders, genetic disorders) constitute a significant burden on global health, and current treatment options remain challenging. As treatment for CNS disorders is primarily palliative, the underlying causes of disease progression are not addressed through conventional pharmacologic therapies. Gene therapy has the potential to address these root causes of disease progression; however, many of the vectors used in gene therapy (e.g., adeno-associated viruses (AAVs)) have limitations such as immunogenicity, low cargo capacity, and crossing the blood-brain barrier (BBB). These limitations have led to significant progress in the development of non-viral gene delivery systems. Compared with viral vectors, non-viral platforms offer improved safety profiles, greater design flexibility, lower production costs, and superior suitability for repeated administration. This review reports recent advancements in the development of non-viral platforms for CNS gene delivery and focuses on lipid-based nanoparticles, polymeric nanoparticles, exosome-based techniques, and new hybrid technologies. Particular emphasis is placed on nanoparticle modification approaches to enhance BBB penetration and enable delivery of genome-editing technologies (CRISPR/Cas systems). The review provides explanations of clinical trials, regulatory considerations, and manufacturing issues that result from the recent developments noted above. It also explores the emerging role of artificial intelligence in supporting carrier design and enhancing delivery efficiency. Both artificial intelligence and non-viral platforms have the potential to facilitate the advancement of safe, effective, and repeatably administered gene therapies for patients with CNS disorders.},
}
@article {pmid41865271,
year = {2026},
author = {Kahr, J and Diaz-Peregrino, R and Sandalcioglu, IE and John, P and Mawrin, C},
title = {RagC and Map4K3 deficiency in high-grade gliomas drives proliferation and modulates mTORC1-dependent cellular functions.},
journal = {Journal of neuropathology and experimental neurology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jnen/nlag010},
pmid = {41865271},
issn = {1554-6578},
abstract = {Cellular growth and homeostasis via amino acid-responsive pathways are mediated by the mTOR signaling pathway. Rag GTPases and Map4K3 modify mTOR signaling as amino acid sensors. Altered mTOR signaling in relation to amino acid sensors might represent factors that modify proliferation and treatment responses in astrocytic tumors. To investigate this hypothesis, RagC and Map4K3 expression was studied in human gliomas, glioma cells (U87MG/U138MG), and nonglial cells (MCF-7, IOMM-Lee). RagC and Map4K3 knockout in glioma cells was generated using CRISPR-Cas and shRNA. High-grade astrocytomas had significantly reduced immunoreactivity for RagC and Map4K3 compared to low-grade astrocytomas. RagC- and Map4K3-deficient glioma cells had significantly increased proliferation and showed altered morphology and motility. Induced amino acid deficiency (leucine deprivation) reduced proliferation in Map4K3- but not in RagC-deficient cells. mTOR signaling in RagC- and Map4K3-deficient U87 cells was altered with increased phosphorylation of p70S6K and increased expression of RagD and transcription factor EB. In this context, uncoupled, exaggerated autophagy occurred in Map4K3-deficient U87 cells. In contrast, RagC-deficient U87 cells showed increased senescence but no autophagy induction. These data show that losses of RagC and Map4K3 in malignant gliomas have proliferation-inducing effects and differentially modulate key mTOR signaling-dependent cellular mechanisms.},
}
@article {pmid41865336,
year = {2026},
author = {An, H and Kim, H and Kim, DY and Yoon, HJ and Lee, JY and Eo, WK and Kim, MY and Kim, KH and Cha, HJ},
title = {Transcriptomic analysis of zonula occludens-1 (ZO-1) knockout in ovarian cancer cell lines.},
journal = {Genes &amp; genomics},
volume = {48},
number = {6},
pages = {911-921},
pmid = {41865336},
issn = {2092-9293},
mesh = {Humans ; Female ; *Zonula Occludens-1 Protein/genetics/metabolism ; *Ovarian Neoplasms/genetics/metabolism/pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; *Transcriptome/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {BACKGROUND: Zonula occludens-1 (ZO-1) is a crucial tight junction protein that regulates intercellular permeability and adhesion, thereby preserving the integrity of epithelial and endothelial barriers. ZO-1 is associated with tumorigenesis and the progression of epithelial-mesenchymal transition (EMT), invasion, and metastasis. In our previous study, knockout (KO) of ZO-1 using clustered regularly interspaced short palindromic repeats (CRISPR) reduced proliferation but increased migration and invasion, suggesting that ZO-1 may have a dual role. Therefore, this study aimed to elucidate the role of ZO-1 in ovarian cancer by analyzing transcriptomic changes associated with ZO-1.<br><br>OBJECTIVE: This study aims to elucidate the impact of ZO-1 KO on gene expression in ovarian cancer cells by performing comparative RNA sequencing (RNA-seq) analysis on two distinct ZO-1 KO ovarian cancer cell lines, SKOV3 and SNU119.<br><br>METHODS: ZO-1 was knocked out in SKOV3 and SNU119 cells using CRISPR-Cas9 technology. After identifying differentially expressed genes (DEGs) through RNA sequencing, Gene Ontology (GO) and pathway enrichment analyses were performed. The selected targets were subsequently validated using reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis to assess both transcript- and protein-level expression changes.<br><br>RESULTS: Transcriptomic analysis revealed over 400 DEGs in each cell line. Of these, 14 genes were consistently upregulated in both cell lines, while 24 genes were consistently downregulated. The common DEGs were visualized using a heatmap, and a subset of these genes was further validated by RT-qPCR and Western blot analyses. TGFB2 expression was consistently altered at both the mRNA and protein levels following ZO-1 KO in both cell lines. Similar expression patterns were observed for THBS1, VCAN, ITGB8, SEMA3A, and GAS6. The concordant changes observed in transcriptomic and protein analyses suggest a consistent association between ZO-1 KO and TGFB2 expression.<br><br>CONCLUSION: ZO-1 KO in ovarian cancer cells induces substantial transcriptional reprogramming, particularly affecting genes associated with extracellular matrix organization and signaling pathways. Multiple candidate genes showed consistent alterations at both the mRNA and protein levels, supporting the robustness of the observed transcriptional changes. These findings provide a framework for understanding ZO-1-associated regulatory networks in ovarian cancer.},
}
@article {pmid41865648,
year = {2026},
author = {Marková, K},
title = {Affinity-based nanostructured platforms for the selective pretreatment, enrichment and detection of miRNA biomarkers.},
journal = {Journal of chromatography. B, Analytical technologies in the biomedical and life sciences},
volume = {1276},
number = {},
pages = {125017},
doi = {10.1016/j.jchromb.2026.125017},
pmid = {41865648},
issn = {1873-376X},
mesh = {Humans ; *MicroRNAs/analysis/isolation &amp; purification/blood ; *Nanostructures/chemistry ; Biomarkers/analysis/blood ; Animals ; Spectrum Analysis, Raman ; },
abstract = {MicroRNAs (miRNAs) are clinically relevant liquid-biopsy biomarkers, yet their reliable quantification is still limited by low abundance, matrix complexity, and frequent association with protective carriers such as Argonaute-2 and exosomes. This review focuses on affinity-based nanostructured platforms as superior tools for the selective pretreatment and enrichment of miRNAs, bridging the gap between raw clinical samples and high-performance analysis. We summarize key nanomaterial architectures, including functional nanoparticles, electrospun nanofibers, 2D nanomaterial platforms, and hybrid nanocomposites, together with surface functionalization strategies that enable the liberation of miRNAs from protein complexes and their subsequent sequence-specific capture. Emphasis is placed on how these enrichment workflows address target accessibility in protein-rich biofluids and mitigate matrix-induced interference. Beyond sample preparation, we evaluate the integration of these platforms with advanced detection modalities, including Surface-Enhanced Raman Spectroscopy (SERS) using inverse molecular sentinel (iMS) nanoprobes, enzyme-free hybridization chain reactions (HCR) and CRISPR/Cas-based assays. These innovative strategies circumvent the limitations of enzymatic amplification, offering high sensitivity and specificity. Finally, we address challenges in automation and standardization, highlighting the need for integrated enrichment-to-detection workflows that accelerate the translation of nanomaterial innovation and next-generation, point-of-care miRNA diagnostics.},
}
@article {pmid41866453,
year = {2026},
author = {Wang, X and Xie, Y and Lin, Q and Xiong, Y and Liu, Y and Ge, S and Tan, Q and He, Z and Jiang, Y and Han, Q and Jin, S and Huang, P and Wang, Y and Guo, W and Ren, F and Gui, JF and Mei, J},
title = {Genetic deletion of miR-200a/200b increases growth and feed conversion efficiency in yellow catfish.},
journal = {Science China. Life sciences},
volume = {69},
number = {5},
pages = {1674-1687},
pmid = {41866453},
issn = {1869-1889},
mesh = {Animals ; *Catfishes/genetics/growth &amp; development/metabolism ; *MicroRNAs/genetics/metabolism ; *Gene Deletion ; Genome-Wide Association Study ; *Animal Feed ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; STAT5 Transcription Factor/genetics/metabolism ; Animals, Genetically Modified ; Energy Metabolism/genetics ; },
abstract = {The most effective approach for minimizing feed cost and maximizing animal production is the creation of breeding materials with simultaneous increases in growth and feed conversion efficiency (FCE). However, the key genes that regulate FCE are unknown. Here, we artificially selected specific strains of yellow catfish with simultaneous improvements in growth and FCE traits and then conducted a genome-wide association study to screen candidate SNPs and genes associated with these traits. A particular locus in the miR-200 cluster on chromosome 23 was identified, and the causal relationships between miR-200a/200b expression and growth/FCE were further validated. Genetic deletion of miR-200a/200b by CRISPR/Cas9 in yellow catfish significantly underpins phenotypic gains in growth and FCE by regulating genes involved in energy intake and energy metabolism without significantly affecting average feed intake or the expression of appetite-regulating genes. Several critical target genes of miR-200a/200b, such as stat5b and fasn, were identified via RNA-RNA pulldown and RNA-seq analyses, and stat5b-transgenic yellow catfish exhibited significantly increased growth and FCE. These findings highlight the pivotal role of the miR-200a/200b-stat5b signaling axis in controlling growth, metabolism, and FCE in yellow catfish, thus providing a strategy toward achieving more effective and sustainable animal agriculture by gene editing.},
}
@article {pmid41867107,
year = {2026},
author = {Zhuang, S and Li, J and Fang, Z and Zhou, H and Zhang, R and He, J and Zhu, L and Xu, Y and Xu, D and Gu, D and Wang, J},
title = {Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection.},
journal = {ACS infectious diseases},
volume = {12},
number = {4},
pages = {1423-1433},
doi = {10.1021/acsinfecdis.6c00013},
pmid = {41867107},
issn = {2373-8227},
mesh = {*Chikungunya virus/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Arboviruses/genetics/isolation &amp; purification ; *Chikungunya Fever/diagnosis/virology ; RNA, Viral/genetics ; *Phosphorothioate Oligonucleotides/chemistry ; Animals ; Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The ongoing arthropod-borne Chikungunya virus (CHIKV) highlights the requirements of rapid and accurate diagnostic methods to enhance the epidemic control. CRISPR diagnostic (CRISPR-Dx) technology holds promise, but the development of a highly efficient one-pot diagnostic system usually requires fine-tuning of the balance between isothermal amplification and Cas cleavage procedures. Here, we describe a simple method (psHOLMES) to create one-pot, two-step CRISPR-Dx systems, using photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity. Cas12a activity is first inactivated via binding of ppPS-DNA during the target sequence amplification procedure, which is then reactivated by ultraviolet (UV)-mediated photolysis of ppPS-DNA after amplification, triggering Cas12a trans-cleavage reactions. psHOLMES demonstrates attomolar sensitivity for CHIKV RNA detection and zero cross-reactivity against other related arboviruses. When applied to clinical samples, psHOLMES achieved 100% (50/50) accuracy and could detect CHIKV within 30 min. As traditional efforts for fine-tuning Cas cis-cleavage activity can be omitted, psHOLMES thus enables rapid development of one-pot CRISPR-Dx systems for clinical applications.},
}
@article {pmid41867779,
year = {2026},
author = {Wester, M and Lim, J and Van, AB and Koprowski, K and Valera, E and Bashir, R},
title = {Kinetic Modeling of Target-Amplification-Free CRISPR-Cas-Based Autocatalysis Reactions.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41867779},
issn = {2692-8205},
abstract = {CRISPR-Cas-based diagnostics utilize the Cas enzyme's trans-cleavage activity to generate signal and have become popular platforms for sensitive nucleic acid detection. Recently, autocatalytic systems have been demonstrated to improve the time to response and sensitivity in some cases. However, mechanistic description of these assays is limited and optimization relies on simple trial-and-error. In this work, we present the first comprehensive kinetic model that integrates all major biochemical processes involved in these assays, including cleavage reactions, nucleic acid equilibrium kinetics, inhibition of trans-cleavage by single-stranded DNA, and degradation of single-stranded reaction components. We discuss the biochemical foundations and implementation of the ordinary differential equation model, which is built for adaptation to different reaction schemes. We use the full model to investigate the role of nucleic acid stability in assay performance for a typical nucleic acid design and show that our model demonstrates inhibition effects consistent with experimental data. We describe the reaction behavior, derive a simplified analytical model and compare its performance to the full analytical model. Finally, we demonstrate tools developed for rapid in silico optimization to guide the rational design of future target-amplification-free CRISPR-Cas-based autocatalysis assays.},
}
@article {pmid41869310,
year = {2026},
author = {Petri, K and Ferrari, S and Cianciotti, BC},
title = {Editorial: Safety and efficacy of CRISPR/Cas-based genome editing tools: applications and considerations in cell and gene therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1809809},
doi = {10.3389/fimmu.2026.1809809},
pmid = {41869310},
issn = {1664-3224},
}
@article {pmid41869962,
year = {2026},
author = {Yang, L and Tan, H and Wang, Y and Zhang, J and Meng, X and Liu, X and Hou, T and Chen, W and Li, F},
title = {Fluidly Confined CRISPR-Magnetic Microbots Empowered Homogeneous Electrochemical Biosensor for Amplified Detection and Discrimination of Cancer-Derived Extracellular Vesicle Subtypes.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10103-10111},
doi = {10.1021/acs.analchem.6c00448},
pmid = {41869962},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/chemistry/metabolism ; Doxorubicin/chemistry ; *Neoplasms ; Biomarkers, Tumor/analysis ; },
abstract = {Accurate identification and profiling of multiple protein biomarkers on tumor-derived extracellular vesicles (tEVs) are crucial for noninvasive cancer subtyping diagnosis but remain technically challenging due to their high heterogeneity, low abundance in biofluids, and preisolation/purification processes. Herein, we developed a homogeneous electrochemical biosensor empowered by fluidly confined CRISPR-magnetic microbots for the amplified detection and sensitive discrimination of tEV subtypes. The CRISPR-magnetic microbots were constructed by engineering CRISPR/Cas12a and DNA icosahedra/doxorubicin (DNA-ICOS/DOX) on intracellularly gelated magnetic cells (IGMCs). Benefiting from the synergistic effects of spatial confinement and membrane fluidity to elevate the local concentration and collision efficiency, the activity of CRISPR/Cas12a was found to be greatly enhanced on IGMCs. For selective sorting of tEVs, a logic-gated aptamer system was used to orthogonally label tEV subpopulations, which further triggers the trans-cleavage activity of CRISPR/Cas12a, resulting in the release of massive DNA-ICOS/DOX into solution. After magnetic separation, the liberated DOX molecules generate a strong electrochemical signal. Particularly, the CRISPR-magnetic microbots could efficiently reduce the background signal, endowing a significantly improved signal-to-noise ratio. Therefore, by combining the CRISPR-magnetic microbots with the dual-target-guided orthogonal barcoding strategy in a homogeneous electrochemical biosensor, precise identification and sensitive detection of tEVs were successfully achieved. More significantly, this assay achieves accurate cancer subtyping in clinical samples, demonstrating its potential as a robust, noninvasive tool for high-accuracy disease screening, classification, and progression monitoring.},
}
@article {pmid41870078,
year = {2026},
author = {Bayurova, E and Kostyushev, D and Tikhonov, A and Chulanov, V and Gordeychuk, I},
title = {Broad-acting antivirals: the pursuit of pan-viral therapeutics in the era of pandemics.},
journal = {Journal of virology},
volume = {100},
number = {5},
pages = {e0007726},
pmid = {41870078},
issn = {1098-5514},
support = {25-65-00010//Russian Science Foundation/ ; },
mesh = {*Antiviral Agents/therapeutic use/pharmacology ; Humans ; *SARS-CoV-2/drug effects ; *COVID-19 Drug Treatment ; Host-Directed Therapy ; Pandemics ; Drug Repositioning ; Animals ; COVID-19/virology ; Drug Resistance, Viral ; },
abstract = {The ever-present threat of new viral epidemics makes the scientific community relentlessly work on the development of universal methods of antiviral therapy. The development of broad-spectrum antivirals (BSAs) focuses either on substances acting directly on viral proteins (direct-acting antivirals [DAA]) or on substances directed at the cell's own proteins (host-targeting antivirals [HTA]). Decades of development have led to the market entry of a number of DAAs with a wide range of antiviral activities; however, their clinical approval has been obtained for individual infections. HTAs have a number of advantages over DAAs, such as a wider range of antiviral activities and a high genetic barrier to viral resistance, which is undoubtedly important when preparing for a battle with an unknown pathogen. The COVID-19 pandemic has allowed for multiple clinical trials for repurposed HTAs, previously licensed for the treatment of other diseases, including cancer. Despite the enormous work done, the arsenal of BSAs capable of protecting against future pandemics caused by pathogen X is very limited. In this review, we described data on the most studied DAAs and HTAs, effective against at least two unrelated viral pathogens, focusing on those that have been studied in late preclinical and clinical trials. In the end, we highlighted alternative new approaches such as CRISPR-Cas therapy.},
}
@article {pmid41870471,
year = {2026},
author = {Zhao, X and Wang, Y and Wang, L and Liao, S and Gong, T and Xiong, M and Yu, B and Song, ZL},
title = {Direct RNA Triggering of Cas12a through the Native crRNA Architecture Enables Clinical Nucleic Acids Diagnostics.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9982-9992},
doi = {10.1021/acs.analchem.5c08220},
pmid = {41870471},
issn = {1520-6882},
mesh = {Humans ; Manganese/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; *RNA/chemistry/metabolism/genetics ; *MicroRNAs/blood/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; *Lung Neoplasms/diagnosis/blood ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for nucleic acid diagnostics, yet its activity is widely considered to be restricted to DNA targets, limiting its applicability for direct RNA detection. Here we report a manganese-ion (Mn[2+])-empowered Cas12a (MEC) platform that overcomes this constraint by allowing the robust RNA-mediated activation of Cas12a. Structural analyses reveal that Mn[2+] strengthens RNA engagement and reorganizes the catalytic center by coordinating RNA phosphates, resulting in an enhancement of trans-cleavage efficiency by 60-fold relative to the Mg[2+] conditions, without compromising sequence specificity. This Mn[2+]-dependent activation mechanism is conserved across multiple Cas12a orthologues (LbCas12a, AsCas12a, FnCas12a), permitting amplification-free detection of RNA with femtomolar sensitivity across diverse targets, particularly the ultrashort abortive transcripts (7 nt). Analysis of clinical serum samples further demonstrates that MEC quantitatively measures circulating miR-21 with performance concordant with reference clinical assays and effectively distinguishes lung cancer patients from healthy individuals. These results reveal an unrecognized role for Mn[2+] in Cas12a biochemistry and establish a simple, versatile, and highly sensitive framework for RNA diagnostics.},
}
@article {pmid41870705,
year = {2026},
author = {Khoshraftar, SH and Alirezae, P and Kiani Darabi, AH and Hadi, S and Gholami, A and Amirfiroozi, A and Pourseif, MM and Mansoori-Derakhshan, S},
title = {The role of circular RNAs as miRNA sponges in the mechanisms and therapeutic potential of triple negative breast cancer.},
journal = {Discover oncology},
volume = {17},
number = {1},
pages = {},
pmid = {41870705},
issn = {2730-6011},
support = {73169//Tabriz University of Medical Sciences/ ; },
abstract = {Triple-negative breast cancer (TNBC) is an aggressive subtype of breast carcinoma which lacks estrogen receptors, progesterone receptors and HER2 along with limited therapeutic options mainly based on chemotherapy. In this review, we outline the emerging function of circRNAs as key regulators in TNBC pathogenesis. CircRNAs are endogenous non-coding RNAs with a closed-loop structure, in contrast to the linear form. In TNBC cells, the underlying molecular mechanism mainly relies on their functions as a competitive sponge of miRNAs, which can absorb or bind to microRNAs (miRNAs) and hence regulate the expression of target genes. Such sponging can result in the activation of oncogenes or repression of tumor suppressor genes, which eventually affect cellular proliferation, apoptosis, and drug sensitivity. Crucial mechanisms include certain circRNAs, such as circEPSTI1, circRAD18, and hsacirc0000199 that enhance tumorigenesis and resistance to chemotherapy by targeting tumor-suppressor miRNAs and activation of oncogenic pathways (e.g., PI3K/Akt/mTOR pathway or Wnt/β-catenin). The potential clinical implications and dysregulation of circRNA-miRNA axes are highlighted, indicating that these may constitute promising diagnostic or prognostic markers by their stability in biofluids. Additionally, this review outlines the innovative treatment approaches regarding these interactions which have recently been addressed and described, novel methods include ASOs therapy, CRISPR/Cas system and nanoplatforms that may help to get over current therapeutic drawbacks in treating TNBC patients.},
}
@article {pmid41870756,
year = {2026},
author = {Gowtham, K and Shanmugaraj, B and Thangavel, LS and Srinivasan, A and Malla, A},
title = {Advancing the frontier of plant-based therapeutics: critical innovations in molecular farming and bioprocess Integration.},
journal = {Biotechnology letters},
volume = {48},
number = {2},
pages = {},
pmid = {41870756},
issn = {1573-6776},
support = {PSGCAS/IRSG/2024-2025/Biotechnology/004//PSG/ ; },
mesh = {*Plants, Genetically Modified/genetics/metabolism ; *Molecular Farming/methods ; Synthetic Biology ; *Plants/genetics/metabolism ; Polysaccharides ; CRISPR-Cas Systems ; *Biological Products/metabolism ; Biotechnology ; },
abstract = {Plant molecular farming (PMF) has emerged as a promising strategy for producing biopharmaceuticals and high-value biomolecules in plant systems. In this review, we present a comprehensive synthesis of current methodologies while introducing novel approaches to genetic transformation, protein expression, glycan engineering, and downstream processing. We offer in-depth analyses of recent advancements such as CRISPR/Cas9-mediated pathway editing, synthetic biology frameworks for optimizing protein yield and quality, and integrated bioprocessing solutions that enhance purification efficiency. Further, detailed case studies are discussed to illustrate actionable strategies, and future research directions are proposed to bridge current gaps. By focusing on transformative techniques and critical problem-solving perspectives, this review aims to guide researchers toward more effective and scalable PMF applications.},
}
@article {pmid41870761,
year = {2026},
author = {Dhaouadi, S and Titouche, Y and Dhaouadi, F and Akkou, M and Elandoulsi, RB},
title = {Mammaliicoccus sciuri as a sentinel for antimicrobial resistance and virulence: genomic epidemiology, transmission dynamics, and control in the one health era.},
journal = {Veterinary research communications},
volume = {50},
number = {3},
pages = {},
pmid = {41870761},
issn = {1573-7446},
abstract = {Mammaliicoccus sciuri (M. sciuri), a Gram-positive bacterium belonging to the group of coagulase-negative staphylococci (CoNS), has been taxonomically reclassified from the genus Staphylococcus to the newly established genus Mammaliicoccus. Its adaptability across diverse niches supports its relevance to One Health. Widely distributed in livestock and diverse environments, M. sciuri has been increasingly reported as an opportunistic pathogen and genetic reservoir for antimicrobial resistance (AMR) and virulence determinants of relevance to human and veterinary medicine. This review provides a synthesis of existing literature regarding the epidemiology, ecological niches, clinical relevance of the M. sciuri species, as well as the genomic and genetic features that underpin its contribution to the dissemination of resistance and virulence factors. Furthermore, it examined the global spread of methicillin-resistant M. sciuri (MR-M. sciuri) clones, characterized through phylogenetic analyses, sequence types (STs), and their complex transmission dynamics. The molecular basis of its resistance mechanisms is explored, with attention directed toward the genetic environments surrounding the mecA and mecC genes within the SCCmec cassettes, as well as the involvement of mobile genetic elements and heavy metal resistance systems. The genetic repertoire of M. sciuri, including the presence of CRISPR–Cas systems associated with adaptive immunity and genome plasticity, was also examined. Moreover, the review delves into the duality of M. sciuri as it can produce antimicrobials such as bacteriocins, which are active against bacteria and fungi. Finally, this review outlines control strategies, including genomic surveillance and stricter antimicrobial regulations, to address MDR M. sciuri strains in the human-animal-environment interface.},
}
@article {pmid41871065,
year = {2026},
author = {Huang, Y and Liang, W and Huang, M and Deng, Y and Huang, Z and Ai, C and Tan, W and Jiang, L},
title = {Application of CRISPR/Cas13a system on the rapid detection of Salmonella spp.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {3},
pages = {e0014150},
pmid = {41871065},
issn = {1935-2735},
mesh = {Humans ; *Salmonella/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Salmonella Infections/diagnosis/microbiology ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; Prospective Studies ; Diarrhea/microbiology/diagnosis ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Salmonella spp. infections can manifest in various clinical symptoms, from asymptomatic carriage to gastroenteritis, and even severe sepsis. Given the rapid progression of the disease and its potential to cause severe outcomes or trigger cluster outbreaks, making the detection of Salmonella spp. critically important. Although broth enrichment culture is considered the gold standard, it is time-consuming and involves multiple steps, making it difficult to meet urgent diagnostic needs. Hence, prompt and precise detection of Salmonella spp. is crucial not only for early diagnosis and effective treatment, but also for preventing transmission, controlling outbreaks, and screening asymptomatic Salmonella carrier.<br><br>METHODS: This study developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) -SE assay that integrated the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas13a system for detecting Salmonella spp. The clinical performance of CRISPR/Cas13a-SE assay was evaluated by a cohort of 94 inpatients with diarrhea and three prospective studies.<br><br>RESULTS: The CRISPR/Cas13a-SE assay can be completed within 60 minutes, and its limit of detection was 100 fg/&#x3bc;L. Compared to the broth enrichment culture, the CRISPR/Cas13a-SE assay demonstrated a sensitivity of 87.5% and a specificity of 98.8% in a cohort of 94 inpatients with diarrhea. In our prospective studies involved three distinct cohorts: 1,662 food handlers, 211 outpatients with diarrhea, and 154 inpatients with Gram-negative bacteremia. Compared with broth enrichment culture, CRISPR/Cas13a-SE assay had a high concordance rate of 98.79% (1,642/1,662), 99.52% (210/211), and 100.00% (154/154) respectively.<br><br>CONCLUSIONS: We demonstrated that the CRISPR/Cas13a-SE system showed excellent detection performance for infectious diarrhea caused by Salmonella spp. The combined use of CRISPR/Cas13a-SE with the blood culture method enhances the rapid diagnosis of invasive salmonellosis, which is crucial for early target-based therapy. Additionally, screening of asymptomatic Salmonella carrier will be benefit for disease prevention and control.},
}
@article {pmid41871234,
year = {2026},
author = {Liao, H and Xie, H and Ye, H and Liu, X and Chen, Y and Zhong, R and He, S and Xiao, X and Xie, Z and Shao, Z and Yu, L and Chen, Z},
title = {One-Pot CRISPR/Cas12a Assay Based on Ultrashort HDA for Ultrasensitive and Universal Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10004-10014},
doi = {10.1021/acs.analchem.5c08249},
pmid = {41871234},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Influenza A virus/genetics/isolation &amp; purification ; CRISPR-Associated Proteins ; *DNA Helicases/metabolism ; *Nucleic Acids/analysis ; },
abstract = {Isothermal amplification techniques, such as helicase-dependent amplification (HDA) combined with CRISPR, are cutting-edge approaches for nucleic acid detection. In this work, we developed a novel ultrashort mesophilic HDA (termed usHDA) for rapid, highly sensitive nucleic acid amplification at 37 °C and constructed a one-pot usHDA-CRISPR/Cas12 assay. The usHDA is specifically designed for rapid amplification of ultrashort sequences (about 40 nt) at 37 °C within 30 min. This usHDA-CRISPR/Cas12a detection can be completed within 1 h, achieving a limit of detection (LOD) of 5 aM. When tested on 58 clinical specimens from patients infected with respiratory pathogens, this assay identified 41 positive and 17 negative samples for influenza A virus. This assay achieved 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic curve (area under the curve value = 1.00; n = 58) compared with PCR analysis. Furthermore, 24 samples of Staphylococcus infection were detected using usHDA-CRISPR/Cas12a, and the same 100% sensitivity and specificity were achieved. These findings highlighted the strong applicability of our proposed assay for universal nucleic acid detection.},
}
@article {pmid41871879,
year = {2026},
author = {Lutz, S and Lawler, M and Amidon, S and Albert, FW},
title = {High-resolution genotype-free mapping of genetic variation with CRI-SPA-Map.},
journal = {Genome research},
volume = {36},
number = {5},
pages = {1016-1028},
doi = {10.1101/gr.281514.125},
pmid = {41871879},
issn = {1549-5469},
support = {R01 HG014395/HG/NHGRI NIH HHS/United States ; R35 GM124676/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *Chromosome Mapping/methods ; *Genetic Variation ; CRISPR-Cas Systems ; *Ploidies ; Phenotype ; Genotype ; Saccharomyces cerevisiae Proteins/genetics ; Genome, Fungal ; },
abstract = {Genetic variation within species shapes phenotypes, but identifying the specific genes and variants that cause phenotypic differences is costly and challenging. Here, we introduce CRI-SPA-Map, a genetic mapping strategy combining CRISPR-Cas9 genome engineering, selective ploidy ablation (SPA), and high-throughput phenotyping for precise genetic mapping with or without genotyping in the yeast Saccharomyces cerevisiae In CRI-SPA-Map, a donor strain carrying SPA machinery is mated to a genetically different recipient strain harboring a genome-integrated selectable cassette. In the resulting diploid, CRISPR-Cas9 cuts the cassette for replacement with DNA from the homologous donor chromosome. Donor chromosomes are then removed using SPA to yield haploid recombinant strains. To establish CRI-SPA-Map, we mate a W303 SPA strain to 92 strains from the BY4742 yeast knockout collection that carry gene deletion cassettes on the left arm of Chromosome XIV and create 1451 recombinant isolates. Whole-genome sequencing verifies that deletion cassette replacement introduces short donor DNA tracts of variable length, resulting in a finely recombined mapping population. Using only the known locations of the gene deletions, which mark where donor DNA is introduced, we identify a 6.5 kb region shaping yeast growth. We further dissect this region and identify two causal variants in two genes, MKT1 and SAL1 Engineering these variants alone and in combination reveals gene-by-environment interactions at both genes, as well as epistatic interactions between them that are dependent on the environment. CRI-SPA-Map is a cost-effective, meiosis-free strategy for creating high-resolution recombinant panels of yeast strains for identifying the genetic basis of phenotypic variation.},
}
@article {pmid41872214,
year = {2026},
author = {Khadake, RM and Shinde, K and Rode, AB},
title = {Engineering ligands for theophylline riboswitches expands its regulatory dynamic range in prokaryotic and eukaryotic systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41872214},
issn = {2041-1723},
support = {BT/ PR45158/DRUG/134/119/2022//Department of Biotechnology, Ministry of Science and Technology (DBT)/ ; CRG/ 2022/004570//DST | Science and Engineering Research Board (SERB)/ ; },
mesh = {*Riboswitch/genetics ; *Theophylline/metabolism/chemistry ; Ligands ; Aptamers, Nucleotide/metabolism/genetics/chemistry ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation ; },
abstract = {The theophylline riboswitch has been a foundational tool in synthetic biology for three decades, yet its regulatory performance remains constrained by the modest affinity of its native ligand. Enhancing the dynamic range of riboswitches is critical for precise gene regulation in biotechnological applications. Here, we show that synthetic 4-quinazolinone derivatives, designed through a structure-based approach, are significantly better than theophylline in both binding and functional activation across multiple biological systems. We demonstrate that these derivatives bind the theophylline aptamer with up to 30-fold higher affinity, thereby expanding regulatory performance. In the bacterial system, these ligands enhance "ON" gene expression by up to 380-fold, compared to 75-fold with theophylline. This superior control extends to diverse organisms; in mycobacteria, the activation ratio reached 20-fold, and in eukaryotes, expression increased 11-fold. Furthermore, in riboswitch-mediated conditional CRISPR-Cas9 applications, these ligands achieve 70% genome editing efficiency at 10-fold lower concentrations than theophylline. These results demonstrate that ligand optimization is a crucial driver for enhancing riboswitch performance for advanced biomedical engineering.},
}
@article {pmid41872289,
year = {2026},
author = {You, HJ and Kim, GY and Kang, MJ},
title = {CRISPR/Cas9-mediated targeted knock-in of human erythropoietin at the β-casein locus results in lactogenic hormone-responsive expression in HC11 mammary epithelial cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41872289},
issn = {2045-2322},
support = {RS-2024-00343478//National Research Foundation of Korea (NRF) grant awarded by the Korean government (MSIT)/ ; },
mesh = {*Erythropoietin/genetics/metabolism ; *CRISPR-Cas Systems ; Animals ; *Caseins/genetics/metabolism ; Humans ; *Epithelial Cells/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; *Mammary Glands, Animal/cytology/metabolism ; Female ; Cell Line ; Gene Expression Regulation ; },
abstract = {Precise genomic integration strategies are essential for achieving stable and regulated transgene expression. In this study, we established a CRISPR/Cas9-mediated targeted knock-in system to integrate the human erythropoietin (hEPO) gene into the endogenous regulatory region of the mouse β-casein locus in HC11 mammary epithelial cells. A donor vector carrying hEPO was designed for homology-directed repair and successfully introduced into the β-casein locus. A heterozygous single-cell knock-in clone was isolated and validated by genomic analysis. Upon stimulation with lactogenic hormones, the integrated hEPO transgene exhibited hormone-responsive expression, resulting in an approximately 20-fold increase in protein levels compared to non-induced conditions. To enable downstream purification and activity assessment, a GST-tagged hEPO construct was employed. The fusion protein was purified using glutathione affinity chromatography, followed by proteolytic cleavage to obtain recombinant hEPO. The purified protein displayed measurable biological activity with a specific activity of 53.4 mIU/μg. These findings demonstrate that targeted integration at the β-casein locus results in lactogenic hormone-responsive expression driven by endogenous regulatory elements in mammary epithelial cells. This approach provides a controlled gene expression platform that may be applicable to hormone-inducible expression systems for therapeutic protein studies.},
}
@article {pmid41872458,
year = {2026},
author = {Lkhagvadorj, K and Okamura, E and Taki, T and Suzuki, H and Kuno, A and Itoh, Y and Mizuno, S and Woltjen, K and Ema, M},
title = {Optimizing CRISPR precision in mouse embryos via microhomology-mediated end joining-dominant targeting.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41872458},
issn = {2399-3642},
support = {25K02195//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; 24K18045//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP223fa627008//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *DNA End-Joining Repair ; Female ; Mouse Embryonic Stem Cells/metabolism ; *Embryo, Mammalian/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblast Growth Factor 10/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 technology enables efficient gene editing in mice, but its reliance on non-homologous end joining often leads to unpredictable and mosaic mutations in founder (F0) animals. Here, we present a hybrid genome editing strategy that combines in silico prediction software with in vitro validation using mouse embryonic stem cells (mESCs). Although the software was trained on mESC datasets, actual editing outcomes in mESCs more accurately reflected mutation patterns observed in blastocysts and post-implantation embryos. Using this information to develop an integrated pipeline, we pre-selected guide RNAs (gRNAs) predicted to promote microhomology-mediated end joining (MMEJ)-dominant repair and validated them in mESCs prior to embryo injection. Applied to the Tyr and Fgf10 genes, this approach enabled efficient generation of F0 mice with highly uniform genotypes. Our strategy enhances the predictability and reproducibility of CRISPR-based genome editing in mice and may help reduce animal usage in gene editing studies.},
}
@article {pmid41872465,
year = {2026},
author = {Ahmed, MF and Sarkar, MMH and Mehzabin, K and Hossain, MI and Bhim, M and Chowdhury, SF and Naser, SR and Mumtaz, T and Faruk, MO},
title = {Genomic convergence of multidrug resistance, virulence-associated loci, and phage defense systems in Klebsiella pneumoniae from pharmaceutical wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41872465},
issn = {2045-2322},
mesh = {*Drug Resistance, Multiple, Bacterial/genetics ; Bangladesh ; *Klebsiella pneumoniae/genetics/pathogenicity/drug effects/isolation &amp; purification/virology ; Virulence/genetics ; *Wastewater/microbiology ; Phylogeny ; Genome, Bacterial ; *Bacteriophages/genetics ; Anti-Bacterial Agents/pharmacology ; Plasmids/genetics ; },
abstract = {Klebsiella pneumoniae strains that combine multidrug resistance and enhanced virulence pose a growing global public health threat. Understanding the genetic basis of these high-risk lineages is critical for surveillance and mitigation. We isolated K. pneumoniae JU-BAEC-01 from treated effluent of antibiotic-manufacturing pharmaceutical facilities in Bangladesh and performed whole-genome sequencing with comparative genomic analyses to characterize its phylogeny, resistome, virulence-associated loci, mobile genetic elements, and predicted antiviral defense systems. JU-BAEC-01 belongs to a phylogenetically distinct lineage, serotype O3b: KL150 with resistance to nearly all clinically relevant antibiotic classes except carbapenems and colistin, mediated by an extensive acquired resistome, including tmexCD3-toprJ3 (tigecycline), armA, aac(6')-Ib-cr, qnrB4, oqxAB, blaDHA-1, blaSHV-182, and blaTEM-1B, mostly carried on conjugative IncC, IncFIB, IncHI1B, and IncR plasmids. Classical hypervirulence markers are present: complete aerobactin (iucABCD-iutA) and salmochelin (iroBCDEN) clusters, rmpA2, type 1 and type 3 fimbriae, T6SS, and pgaABCD. Notably, the strain encodes one of the most elaborate anti-phage defense arsenals reported in Klebsiella to date, comprising functional Type I-E, III-A, and IV-A CRISPR-Cas systems, multiple restriction-modification systems, BREX Type I, abortive infection systems (AbiE, AbiU), and additional novel defenses that coexist with phage-derived anti-CRISPR (AcrIE9) and anti-restriction (ArdA) proteins. K. pneumoniae JU-BAEC-01 is a "perfect storm" pathogen that combines multi-drug resistance (MDR), hypervirulence, and a multilayered, highly developed defense against bacteriophages. Together, these findings highlight the environmental emergence of a genetically distinct, multidrug-resistant K. pneumoniae with substantial virulence potential and complex phage-host interaction capacity, underscoring the need for genomic surveillance of pharmaceutical wastewater systems.},
}
@article {pmid41873079,
year = {2026},
author = {Luan, M and Liu, S and Yang, Q and Zhang, Y and Wang, M and Liu, R and Niu, G},
title = {[Metabolic engineering of Streptomyces for production of hyaluronic acid].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {3},
pages = {1242-1260},
doi = {10.13345/j.cjb.250734},
pmid = {41873079},
issn = {1872-2075},
support = {2023YFD1700700//the National Key Research and Development Program of China/ ; },
mesh = {*Metabolic Engineering/methods ; *Streptomyces/genetics/metabolism ; *Hyaluronic Acid/biosynthesis ; Hyaluronan Synthases/genetics/metabolism ; },
abstract = {Hyaluronic acid (HA) is a glycosaminoglycan with significant biological activities, which render it widely applicable in the cosmetics and pharmaceutical industries. The development of safe and efficient chassis cells to enhance HA synthesis efficiency has thus emerged as a key factor in HA production. Our study aims to construct a high-performance HA biosynthesis system using Streptomyces as the chassis cell, thereby providing technical support for the efficient microbial production of HA. Thus, our study focused on the metabolic engineering of Streptomyces for strengthening the HA synthesis pathway and then optimized the culture conditions for efficient HA synthesis. First, the HA-synthesizing capabilities of four hyaluronate synthases from different sources were evaluated in two host strains: Streptomyces coelicolor M1146 and Streptomyces albus J1074. The results indicated that the hyaluronate synthases derived from Streptococcus pyogenes exhibited the strongest HA synthesis capability. Notably, the HA yield in S. albus J1074 was higher. Building on this finding, S. albus J1074 was selected as the starting strain to construct a chassis strain tailored for HA synthesis: key genes in the competitive metabolic pathway of HA synthesis were knocked out, while the expression levels of genes associated with the bypass pathway were down-regulated. Furthermore, different combinations of key genes involved in the HA precursor synthesis pathway were designed, and their expression levels were enhanced via a constitutive strong promoter. The recombinant strain obtained therefrom achieved a maximum HA yield of 2.62 g/L. Finally, the synthetic capacity of this high-yield engineered strain was further unleased through the optimization of culture conditions, leading to a final HA yield of 4.63 g/L. The recombinant strain constructed in this study not only lays a foundation for the development of engineered Streptomyces but also provides an excellent chassis strain for the microbial production of HA and other related bioproducts.},
}
@article {pmid41873757,
year = {2026},
author = {Yu, Z and Bao, Y and Alcalá-Lalinde, A and Andrade Dos Ramos, Z and Koroglu, M and Berkhout, B and Herrera-Carrillo, E},
title = {Elucidating the kinetics of CRISPR-SaCas9 action to obtain effective HIV DNA excision with two gRNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41873757},
issn = {1362-4962},
support = {1R01AI145045IH//NIH RO1/ ; DGP_EMEC_2023_00154//Junta de Andaluc&#xed;a/ ; PID2024-162385OB-I00//Spanish State Research Agency/ ; },
mesh = {*DNA, Viral/metabolism/genetics ; Kinetics ; *CRISPR-Cas Systems ; *HIV-1/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Proviruses/genetics ; },
abstract = {The persistence of integrated human immunodeficiency virus (HIV) proviral DNA poses a major barrier to viral eradication, as the viral reservoir enables rapid rebound upon treatment interruption, despite effective virus inhibition. CRISPR-Cas-based editing strategies, especially those using double-site cleavage, show promise in excising proviral DNA, yet the rate and determinants of excision efficiency remain poorly understood. In this study, we systematically evaluated both single- and dual-SaCas9/gRNA approaches for HIV-1 inactivation. Sequence analysis revealed that SaCas9 can eliminate all wild-type HIV-1 genomes with a single gRNA, unlike other CRISPR-Cas systems. Dual-gRNA strategies improved antiviral efficacy, with the Gag3 + Pol5 combination achieving 97% excision efficiency. Kinetic analysis showed that excision efficiency correlates with the kinetic compatibility of paired gRNAs. Pairs of gRNAs with fast and similar kinetics achieved the highest excision efficiency. In contrast, the Gag3 + Env4 pair exhibited discordant kinetic characteristics (fast and slow), resulting in the failure to induce excision as the cut DNA will be repaired before the second cut is realized. Consequently, no excision but regular editing occurred at the two target sites. These findings provide a mechanistic framework for optimizing CRISPR-Cas-mediated excision, highlighting the critical role of both antiviral activity and kinetic synergy in guiding gRNA selection.},
}
@article {pmid41873844,
year = {2026},
author = {Wang, Q and Sheng, M and Zheng, Y and Zhang, B and Jin, Z and Zhang, T and Li, Z and Huang, J and Yang, X},
title = {Electrochemiluminescence Biosensing Platform Based on CRISPR/Cas12a and DNA Nanotweezer-Mediated Catalytic Hairpin Assembly Amplification.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9832-9841},
doi = {10.1021/acs.analchem.5c07500},
pmid = {41873844},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; DNA Nanostructures ; Humans ; *DNA/chemistry ; Inverted Repeat Sequences ; },
abstract = {The detection of microRNAs (miRNAs) biomarkers has great potential in the early diagnosis of acute myocardial infarction (AMI). Herein, we constructed an electrochemiluminescence biosensing platform based on DNA nanotweezer (DNT)-mediated catalytic hairpin assembly (CHA) and CRISPR/Cas12a system for detecting potential AMI biomarker miRNA-133a. DNT, as a programmable molecular scaffold, can precisely organize molecules at the nanoscale and output high signal-to-background ratio detection signals, which is introduced into the construction of sensing platforms. When the target miRNA was presented, the hairpin in DNT was opened, which altered the DNT structure from a closed state to an open state and exposed the catalytic sequence for CHA. Subsequently, a large number of F/A-F duplexes were generated after the addition of fuel strands (F) and antifuel strands (A-F), which served as the target for activating the CRISPR/Cas12a system. The activated Cas12a collaterally cleaved the signal probe (H1) on the electrode surface, causing the labeled Ru(bpy)3[2+] to detach from the electrode surface, resulting in a weakened ECL signal. We found that compared with the general CHA reaction, the DNT-mediated CHA reaction significantly lowers the leakage of the circuit; thus, a high signal-to-background ratio and detection sensitivity can be obtained. Therefore, we developed a highly sensitive biosensing platform for detecting miRNA-133a with a detection limit of 0.12 fM. This sensing strategy provides a new approach for nucleic acid detection and disease diagnosis.},
}
@article {pmid41874403,
year = {2026},
author = {Zhang, H and Liu, L and Wang, D and Yang, X and Kang, Y and Huang, J and Ouyang, Y and Yu, H and Zhang, Y},
title = {CRISPR-Cas gene editing technology in biomanufacturing to enhance stress tolerance of microbial strains.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
doi = {10.1093/femsle/fnag030},
pmid = {41874403},
issn = {1574-6968},
support = {2023M742776//China Postdoctoral Science Foundation/ ; 2023BSHEDZZ12//Postdoctoral Research Program of Shaanxi/ ; GZB20240598//Postdoctoral Fellowship Program of CPSF/ ; 2025JC-YBQN-293//Natural Science Basis Research Plan in Shaanxi Province of China/ ; S202510698576//National Training Program of Innovation and Entrepreneurship for Undergraduates/ ; 202406280137//CSC/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Bacillus subtilis/genetics ; Corynebacterium glutamicum/genetics ; *Industrial Microbiology/methods ; Oxidative Stress ; Escherichia coli/genetics ; },
abstract = {In response to the loss of microbial efficiency caused by environmental stress in biomanufacturing, CRISPR-Cas gene editing technology has become a core tool for enhancing stress tolerance by accurately targeting genomic loci. This article systematically reviews the progress of its application. By optimizing engineered nucleases, gRNA design, and innovative delivery strategies, this technology successfully regulates key pathways in oxidative stress responses. It integrates functional genome screening with dynamic regulation to examine the networks of multi-gene collaborative tolerance. In the construction of high-stress-tolerant industrial chassis cells, the stress survival rate (>90% in Bacillus subtilis under thermal stress) and product synthesis ability (such as cellulose producing ethanol up to 4.5 g/l) of strains such as Escherichia coli and Corynebacterium glutamicum were significantly improved. Current challenges focus on delivery efficiency, off-target risks, and complex regulatory bottlenecks. In the future, the development of new editing tools and intelligent circuits will promote their industrial application in sustainable biomanufacturing.},
}
@article {pmid41874563,
year = {2026},
author = {Fang, J and Simon, JM and Wang, T and Gao, Y and Bi, X and Hu, L and Liao, C and Zhang, C and Adachi, Y and Zhou, J and Liu, H and Liang, Q and Nathan, JA and Mani, R and Brugarolas, J and Zhang, Q},
title = {Genome-wide CRISPR screen identifies a cytokine-enhancer circuit driving HIF-2α activation in renal cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {41874563},
issn = {1558-8238},
support = {P50 CA196516/CA/NCI NIH HHS/United States ; },
mesh = {*Basic Helix-Loop-Helix Proteins/genetics/metabolism ; Humans ; Endothelial PAS Domain-Containing Protein 1 ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; STAT3 Transcription Factor/metabolism/genetics ; Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; Animals ; Janus Kinase 1/metabolism/genetics ; Cell Line, Tumor ; Signal Transduction ; *Gene Expression Regulation, Neoplastic ; Mice ; *Neoplasm Proteins/genetics/metabolism ; *Enhancer Elements, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *CRISPR-Cas Systems ; *Cytokines/genetics/metabolism ; },
abstract = {Resistance to HIF-2α inhibitors such as belzutifan underscores the need to better understand how HIF-2α is transcriptionally regulated in clear cell renal cell carcinoma (ccRCC). Here, we uncover a cytokine-driven enhancer mechanism that sustains HIF-2α expression through the JAK1/STAT3 signaling pathway. Using a genome-wide CRISPR screen in von Hippel-Lindau-deficient (VHL-deficient) ccRCC cells, we identified SOCS3 as a key negative regulator of HIF-2α. Mechanistically, loss of SOCS3 activates JAK1/STAT3 signaling, leading to the recruitment of STAT3 to distal enhancers upstream of endothelial PAS domain-containing protein (EPAS1) that physically loop to its promoter to drive HIF-2α transcription. This cytokine-enhancer circuit was recapitulated in samples from patients with ccRCC and functionally validated using CRISPR interference (CRISPRi), which disrupted enhancer-promoter looping and reduced tumor growth in HIF-2α-dependent models. SOCS3 overexpression or pharmacologic inhibition of JAK1/STAT3 markedly suppressed HIF-2α expression and tumor progression both in vitro and in vivo. Unlike prior studies focusing on VHL/HIF occupancy-driven enhancer activation, this work defines a trans-acting cytokine-JAK1/STAT3 pathway that transcriptionally controls EPAS1. Together, these findings reveal a targetable enhancer mechanism that sustains HIF-2α expression and suggest that combined inhibition of JAK1/STAT3 and HIF-2α may overcome therapeutic resistance in kidney cancer.},
}
@article {pmid41875952,
year = {2026},
author = {Li, C and Zhang, L and Xu, Q},
title = {Extraction-free, rapid LAMP-CRISPR/Cas12a assay for detection of pseudorabies virus.},
journal = {Journal of virological methods},
volume = {343},
number = {},
pages = {115387},
doi = {10.1016/j.jviromet.2026.115387},
pmid = {41875952},
issn = {1879-0984},
mesh = {Animals ; *Herpesvirus 1, Suid/isolation &amp; purification/genetics ; Swine ; *Pseudorabies/diagnosis/virology ; Sensitivity and Specificity ; *Swine Diseases/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; DNA Primers/genetics ; Time Factors ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems ; },
abstract = {This study developed a LAMP-CRISPR/Cas12a detection system for rapid and visual identification of porcine pseudorabies virus (PRV). Optimal sgRNA and LAMP-specific primers were designed based on the conserved sequences of the viral pathogenic gene gG. The combined detection system demonstrated superior sensitivity compared to PCR-CRISPR/Cas12a and qPCR methods, achieving a detection limit of 1.0 × 10[-4] copies/μL for the target plasmid DNA. Specificity testing confirmed the selective identification of PRV without cross-reactivity to other porcine pathogens. Parallel comparison of 26 serum samples between LAMP-CRISPR/Cas12a and PCR-CRISPR/Cas12a systems showed 100% concordance for positive results, with both detecting 12 positive samples. The method eliminates the need for viral nucleic acid extraction and requires only a constant temperature device and/or basic fluorescence detection equipment. Results are obtainable within one hour and are readable by the naked eye. This simple, sensitive, and equipment-independent approach is ideal for on-site rapid diagnosis of pseudorabies in pigs, offering significant applications in clinical diagnosis, epidemiological surveillance, and field testing.},
}
@article {pmid41876528,
year = {2026},
author = {Lin, YH and Kompa, J and Sun, DE and Mao, R and Koch, B and Hinnah, K and Wilhelm, J and Franz, N and Kühn, S and Menche, T and Adow, A and Breuer, P and Hiblot, J and Johnsson, K},
title = {A high-affinity split-HaloTag for live-cell protein labeling.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41876528},
issn = {2041-1723},
mesh = {Microscopy, Fluorescence/methods ; Humans ; Fluorescent Dyes/chemistry ; *Staining and Labeling/methods ; CRISPR-Cas Systems ; Ligands ; Animals ; Peptides/metabolism/chemistry ; HEK293 Cells ; },
abstract = {We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHaloΔ3). Hpep binds to cpHaloΔ3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHaloΔ3 and in fixed cells through incubation with cpHaloΔ3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.},
}
@article {pmid41876645,
year = {2026},
author = {Ren, F and Liu, D and Ren, H and Zhang, H and Zhang, P and Lin, H and Li, C and Dong, J and An, S and Ge, X and Cheng, L and Yang, F and Liu, J and Fu, J and Tang, F and Wang, F and Liu, T and Pan, H and Rong, S and Ma, H and Zou, L},
title = {Synergistic integration of CRISPR/Cas and nanozymes in next-generation biosensors for ultrasensitive bacterial detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41876645},
issn = {1436-5073},
support = {82574099//National Natural Science Foundation of China/ ; LH2023H054//Natural Science Foundation of Heilongjiang Province/ ; 2022-MYHJ-014//Foundation for Huoju Plan Research of Mudanjiang Medical University/ ; YJSZX2022137//Foundation for Special Program of Supervisor Scientific Research of Mudanjiang Medical University/ ; 2024-KYYWFMY-0455//The Fundamental Research Funds for the Universities of Heilongjiang Province/ ; CYQN24028//Youth the Science and Technology Talents Team Project of Chunyan Plan of Heilongjiang Province/ ; },
abstract = {The rapid and accurate detection of pathogenic bacteria is paramount for global public health, food safety, and clinical diagnosis. While conventional methods face limitations in speed, sensitivity, and field-deployability, biosensors incorporating CRISPR/Cas systems and nanozymes have emerged as a transformative solution. This review provides a comprehensive analysis of this cutting-edge synergy. We first elucidate the fundamental mechanisms, highlighting the unparalleled programmability and specific nucleic acid cleavage activity of Class II CRISPR/Cas systems (e.g., Cas12, Cas13), and the robust, cost-effective, and tunable catalytic properties of various nanozymes (e.g., metal-based, MOF-derived). The core innovation lies in their integration: the CRISPR/Cas system acts as a highly specific molecular recognition unit, whose activation triggers the signal amplification function of nanozymes. We critically examine recent advancements in biosensing platforms that leverage this combination for the detection of diverse bacteria (e.g., Salmonella, Legionella, drug-resistant strains) via multiple readouts (colorimetric, fluorescent, electrochemical). The results demonstrate the exceptional sensitivity (often reaching attomolar or single CFU levels) and versatility achieved by these platforms. Finally, the current challenges, such as signal stability and the demand for multiplex detection, are discussed. Future development directions are also prospected, including the development of extraction-free detection methods, the fabrication of renewable sensors to achieve true point-of-care testing, and the design of novel nanozymes with higher specificity. This review not only summarizes the state-of-the-art but also charts a course for the next generation of intelligent, rapid, and accessible diagnostic tools.},
}
@article {pmid41876887,
year = {2026},
author = {Xu, R and Cong, T and Yuan, J and Chen, X and Li, Y and Lan, X and Zhu, M},
title = {Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {41876887},
issn = {1750-2799},
abstract = {Tracking-seq is a highly sensitive method for genome-wide detection of off-target effects in cells edited with diverse genome editing modalities, including Cas9, cytosine base editors, adenine base editors and prime editors. Since most genome editors induce DNA repair pathways and generate single-stranded DNA (ssDNA) intermediates, Tracking-seq leverages this process by tracking replication protein A-a key protein that binds and protects ssDNA-to identify on-target and off-target events. Here we provide a detailed protocol for Tracking-seq, covering genome editing of cells, extraction of replication protein A-bound ssDNA, sequencing library construction and data analysis using our custom computational tool Offtracker. Tracking-seq is applicable to various genome editing scenarios with low cell input, delivering high-performance results. The entire workflow, from genome editing to data analysis, can be completed within 1-2 weeks, making it a rapid solution for assessing genome-wide off-target activity.},
}
@article {pmid41877120,
year = {2026},
author = {Wang, Y and Cao, M and Hu, M and Xu, H and Du, Y and Sun, C and Kong, L and Luo, Y and Liu, X and Yang, J and Liang, B},
title = {Systematic engineering of Escherichia coli for biosynthesis of 3-hydroxypropionic acid from glucose and malonate.},
journal = {BMC biotechnology},
volume = {26},
number = {1},
pages = {},
pmid = {41877120},
issn = {1472-6750},
support = {22278233//National Natural Science Foundation of China/ ; 22378222//National Natural Science Foundation of China/ ; ZR2025MS127//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Malonates/metabolism ; *Glucose/metabolism ; *Metabolic Engineering/methods ; *Lactic Acid/analogs &amp; derivatives/biosynthesis/metabolism ; Malonyl Coenzyme A/metabolism ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: 3-Hydroxypropionic acid (3-HP) is a promising C3 platform chemical with wide industrial applications. However, its microbial production remains limited by insufficient intracellular malonyl-CoA availability and metabolic imbalance.<br><br>RESULT: In this study, we systematically engineered Escherichia coli for enhanced 3-HP biosynthesis. The malonate assimilation genes (matB, smatPQM) and 3-HP biosynthesis gene (mcr) were chromosomally integrated using CRISPR/Cas9, resulting in a plasmid-free, antibiotic-free strain (WYY04) that produced 21.97 mM 3-HP, 0.51-fold higher than the plasmid-based system. Further improvement was achieved by CRISPRi-mediated repression of fatty acid biosynthesis genes (fabD, fabF), increasing 3-HP titer by 66%. Introduction of a malonyl-CoA-responsive FapR/fapO biosensor enabled dynamic regulation of mcr expression, enhancing 3-HP production by 59%. Through all these above engineering, the 3-HP production of the strain WYY19 increased by 2.29 times compared to that of the plasmid-expressing system. Under optimized fermentation conditions, the final engineered strain WYY19 produced 42.22&#xa0;g/L 3-HP with the specific productivity of 0.69&#xa0;g/g and 0.46&#xa0;g/L/h from glucose and malonate in fed-batch bioreactor.<br><br>CONCLUSIONS: This study demonstrates a robust, genetically stable, and scalable microbial platform for 3-HP biosynthesis.},
}
@article {pmid41877594,
year = {2026},
author = {Cucuy, A and Ben-Tov, D and Melamed-Bessudo, C and Honig, A and Cohen, BA and Levy, AA},
title = {Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70809},
pmid = {41877594},
issn = {1365-313X},
support = {//Israel Innovation Authority/ ; },
mesh = {*Solanum lycopersicum/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.},
}
@article {pmid41877893,
year = {2025},
author = {Srinivasa, MA and Escobar, M},
title = {CRISPR-based Transcriptional Regulation: Technologies, Applications, and Future Directions.},
journal = {DNA},
volume = {5},
number = {4},
pages = {57},
pmid = {41877893},
issn = {2673-8856},
support = {25TPA1463933/AHA/American Heart Association-American Stroke Association/United States ; },
abstract = {CRISPR-based transcriptional regulation technologies, including CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), offer precise and programmable control over gene expression, representing a major advance in gene and epigenetic therapy. CRISPRa uses nuclease-inactive Cas proteins fused to transcriptional activators to upregulate target genes, while CRISPRi employs repressor domains for gene silencing. Preclinical studies have demonstrated the efficacy of CRISPRa/i in models of metabolic, neurological, muscular, and oncological diseases. Notably, CRISPRi-based therapies have entered clinical trials for conditions like hepatitis B and muscular dystrophy, showing encouraging safety and efficacy profiles. Despite ongoing challenges related to delivery efficiency, immunogenicity, and off-target activity, innovations in protein engineering and guide RNA design are rapidly enhancing the precision and safety of these technologies. Overall, CRISPRa and CRISPRi are poised to transform the treatment of genetic and epigenetic disorders, with continued optimization expected to accelerate their clinical adoption and broaden their therapeutic impact.},
}
@article {pmid41879319,
year = {2026},
author = {Cheng, W and Li, J and Lei, L and Zhu, Y and Luo, S and Wang, X and Zhang, Q and Cao, M and Zheng, Y and Peng, W},
title = {Unlocking genome engineering in Alcaligenes faecalis by exploiting its native type I-F CRISPR-Cas.},
journal = {Microbiology spectrum},
volume = {14},
number = {5},
pages = {e0278625},
pmid = {41879319},
issn = {2165-0497},
abstract = {Alcaligenes faecalis is an environmentally significant bacterium for pollutant biodegradation and aerobic denitrification, yet its genetic engineering has been hindered by a lack of high-throughput tools. Conventional methods like homologous recombination are time-consuming and cannot achieve large genomic deletions, while technologies based on heterologous CRISPR-Cas systems failed due to cytotoxicity. This study resolves these limitations by developing a genome editing toolkit based on the endogenous type I-F CRISPR-Cas of A. faecalis J481. The toolkit enables efficient single-gene knockout and accomplishes the previously unattainable precise deletion of large genomic fragments. By engineering a PheS-mutant counterselection marker, we achieved rapid plasmid curing, allowing two rounds of large-fragment removal (~47 kb total) within 5 days. This breakthrough provides the first CRISPR-based platform for complex genome engineering in A. faecalis, overcoming intrinsic constraints of heterologous systems. The work establishes a scalable genetic toolbox to enhance A. faecalis' capabilities in bioremediation and eutrophication control. Moreover, the strategy of harnessing endogenous CRISPR-Cas systems offers a blueprint for developing advanced genome editing tools in other prokaryotes.IMPORTANCEThis study breaks through the longstanding genetic engineering bottleneck in an environmentally crucial bacterium, Alcaligenes faecalis, by creating a fast, efficient, and versatile toolkit using its native CRISPR-Cas system. This enables complex edits, such as large genomic deletions previously impossible, unlocking new potential for bioremediation and eutrophication control, providing a blueprint for other prokaryotes, and setting a precedent for genetic tool development in other hard-to-engineer microbes.},
}
@article {pmid41880574,
year = {2026},
author = {Katsumura, T and Sato, S and Yamashita, K and Oda, S and Gakuhari, T and Tanaka, S and Fujitani, K and Nishimaki, T and Imai, T and Yoshiura, Y and Takeshima, H and Hashiguchi, Y and Sekita, Y and Mitani, H and Ogawa, M and Takeuchi, H and Oota, H},
title = {DNA methylation site loss for plasticity-led novel trait genetic fixation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {13},
pages = {e2534817123},
pmid = {41880574},
issn = {1091-6490},
support = {JP16K21352//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP19K16201//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP19H05737//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24K02078//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17H01453//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17H03738//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16J07227//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {*DNA Methylation ; *Oryzias/anatomy &amp; histology/genetics/physiology ; Epigenesis, Genetic ; *Adaptation, Physiological/genetics ; Seasons ; CpG Islands/genetics ; Animals ; CRISPR-Cas Systems ; Fish Proteins/genetics ; *Evolution, Molecular ; Organ Size/genetics ; Gastrointestinal Tract/anatomy &amp; histology ; Phenotype ; Male ; Female ; },
abstract = {Phenotypic plasticity allows organisms to adapt traits in response to environmental changes, yet the molecular basis by which such plastic traits become genetically fixed remains unclear. Here, we investigated gut-length plasticity in medaka fish (Oryzias latipes) through genome-wide methylation profiling, CRISPR/Cas9-mediated deletion, and population genomic analyses. We found that seasonal methylation of CpG sites upstream of the Plxnb3 is correlated with gut-length plasticity, and deletion of this region abolishes plasticity. Additionally, standing variation in Ppp3r1 is associated with genetically fixed longer gut length in populations lacking plasticity. These results suggest that loss of epigenetic regulation via CpG site reduction triggers the genetic fixation of novel traits. Our findings provide molecular evidence linking epigenetic plasticity and genetic assimilation, advancing understanding of plasticity-led evolution in natural populations.},
}
@article {pmid41880974,
year = {2026},
author = {Cantarini, C and Lorrain, V and Koutala, E and Olivieri, C and Raymond, K and Lebrin, F},
title = {Generation of ENG p.Met1Val mutant LUMCi029-A-2 for modeling Hereditary Hemorrhagic Telangiectasia type 1.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103973},
doi = {10.1016/j.scr.2026.103973},
pmid = {41880974},
issn = {1876-7753},
mesh = {Humans ; *Telangiectasia, Hereditary Hemorrhagic/genetics/pathology/metabolism ; *Endoglin/genetics/metabolism ; Mutation ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Hereditary Hemorrhagic Telangiectasia type I (HHT1) is an autosomal dominant vascular disease caused by pathogenic variants in endoglin (ENG) gene. It is located on chromosome 9 and encodes for the Endoglin protein, which is involved in the TGFb/BMP signalling pathway. Using CRISPR/Cas9-mediated gene editing, the ENG c.1A > G mutation was introduced in homozygous form in the well-characterized LUMCi029-A line. The resulting hiPSC line, LUMCi029-A-2, showed typical morphology, expressed pluripotency markers, was able to differentiate into the three germ layers in vitro and displayed a normal karyotype. The line represents a valuable HHT1 disease-model and an important tool for drug testing.},
}
@article {pmid41881766,
year = {2026},
author = {Li, S and Tang, S and Xu, W and Zhou, J and Li, X and Wang, J},
title = {SIMPLE-CRISPR: A Sample-to-Result Platform for Point-of-Care Detection of Nucleic Acids via a Functionalized Magnetic-Bead-Based CRISPR Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9964-9971},
doi = {10.1021/acs.analchem.5c08178},
pmid = {41881766},
issn = {1520-6882},
mesh = {*Point-of-Care Systems ; *CRISPR-Cas Systems/genetics ; Humans ; Smartphone ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/analysis/genetics ; Rapid Diagnostic Tests ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) systems show great promise for next-generation molecular diagnostics due to their programmability and specificity. Amplification-free CRISPR detection has a strong potential for point-of-care (POC) testing, and the digital format of the assay naturally improves the sensitivity of amplification-free CRISPR detection. However, integrating sample preparation, reaction implementation, and signal readout into a streamlined and user-friendly POC workflow remains a major technical challenge. We address this by developing SIMPLE-CRISPR (Sample-to-Result Integrated platform with Magnetic-bead extraction, Polydisperse droplets, Low-complexity operation, and Engineered smartphone readout), an amplification-free CRISPR-Cas12a system for POC diagnostics. Our innovation features functionalized magnetic-bead extraction for the efficient enrichment of low-abundance nucleic acid targets from large-volume samples with minimal loss, vortex-driven polydisperse emulsification for facile digital droplet generation, and smartphone-integrated fluorescence imaging for on-site portable signal detection. This integrated approach significantly improves sensitivity compared to bulk CRISPR, removes the need for amplification and complex microfluidics, and provides sample-to-result functionality for POC environments. Clinical validation for human papillomavirus type 18 (HPV18) detection confirmed that the assay achieved diagnostic performance consistent with quantitative real-time polymerase chain reaction (qRT-PCR), demonstrating its great potential for accessible POC nucleic acid diagnostics.},
}
@article {pmid41881989,
year = {2026},
author = {Ren, X and Zheng, L and Liu, Y and Maliskova, L and Tam, TW and Sun, Y and Liu, H and Cui, X and Lee, J and Takagi, MA and Li, B and Ren, B and Wang, W and Shen, Y},
title = {CRISPR tiling deletion screens reveal functional enhancers and allelic compensation effects (ACE) on SIN3A transcription.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41881989},
issn = {2041-1723},
support = {UM1HG009402//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30DK063720//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; S101S10OD021822-01//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; 1S10OD028511-01//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Humans ; *Enhancer Elements, Genetic/genetics ; *Transcription, Genetic ; *Repressor Proteins/genetics/metabolism ; Alleles ; Sin3 Histone Deacetylase and Corepressor Complex ; Induced Pluripotent Stem Cells/metabolism/cytology ; Promoter Regions, Genetic ; Neurons/metabolism/cytology ; Gene Expression Regulation ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Differentiation/genetics ; Methyl-CpG-Binding Protein 2/genetics ; },
abstract = {Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identify a total of 39 functional enhancers for four dosage-sensitive genes, APP, FMR1, MECP2, and SIN3A, using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. More importantly, we discover that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription are stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A, and potentially other dosage-sensitive genes.},
}
@article {pmid41882346,
year = {2026},
author = {Wang, F and Guo, R and Zhang, S and Cui, Y and Wang, J and Hu, T and Liu, K and Wang, Q and Liu, Y and Nam, KH and Zhao, ZW and Ji, Q and Xu, X and Wang, E and Zhu, Y and Yang, Y and Luo, M and Ma, P and Ma, S and Xu, C and Hu, C},
title = {Structural insight into IscB's RNA-lid-based inactivation mechanism.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {4},
pages = {603-614},
pmid = {41882346},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; Nucleic Acid Conformation ; Models, Molecular ; *RNA, Bacterial/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; Escherichia coli/genetics/metabolism ; },
abstract = {IscB, a compact Cas9 ancestor from the obligate mobile element guided activity system, has attracted growing interest as a programmable genome editor because of its small size and therapeutic delivery potential. Despite its promise, structural insights into IscB's regulation remain limited, with only a target-bound R-loop structure previously reported. Here, we present the structural trajectory of an engineered IscB, capturing its transition from a resting state to activation. Using cryo-electron microscopy, we resolve four high-resolution structures: the apo resting state, two intermediate complexes with 6-nt and 10-nt guide-target pairing and a fully paired 16-nt primed cleavage state. These structures uncover a dual inactivation mechanism mediated by RNA lids; the ωRNA lid blocks HNH domain access, while the guide RNA lid occludes the RuvC active site. As guide-target pairing progresses, the guide RNA undergoes a stepwise displacement, mimicking a 'car pedal' motion that triggers activation at 11-nt pairing. The HNH domain also contributes to R-loop stabilization through a positively charged R-wedge motif and undergoes a ~90° activation-driven rotation mediated by two hinge regions. In variants IscBHig1 and IscBHig2, engineering these hinge motifs to enhance conformational flexibility notably improved genome-editing efficiency in cells. In summary, our study reveals the molecular basis underlying IscB autoinhibition and activation, identifies previously uncharacterized regulatory features and establishes hinge elements as a target region for engineering compact, efficient genome editors.},
}
@article {pmid41882347,
year = {2026},
author = {Akdoğan, E and Lundgren, SM and Kamber, RA and Bassik, MC and Collins, SR},
title = {Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41882347},
issn = {2399-3642},
support = {P30CA093373//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; DP2HD094656//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01 GM148769/GM/NIGMS NIH HHS/United States ; S10OD018223//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; DP2 HD094656/HD/NICHD NIH HHS/United States ; R01GM148769//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CA259218//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Endocytosis ; *Receptors, Formyl Peptide/metabolism/genetics ; ADP-Ribosylation Factor 6 ; ADP-Ribosylation Factors/metabolism/genetics ; Signal Transduction ; G-Protein-Coupled Receptor Kinases/metabolism/genetics ; *Cell Membrane/metabolism ; *CRISPR-Cas Systems ; HEK293 Cells ; G-Protein-Coupled Receptor Kinase 2/metabolism/genetics ; },
abstract = {Chemoattractants generate strong chemotactic and cytotoxic responses in immune cells by activating cognate receptors. Cell surface receptor levels control sensitivity, which is critical for achieving effective responses without excessive inflammation. The surface levels of the attractant receptor FPR1 are controlled through a balance of delivery and removal, which responds to receptor activation and other stimuli. While this regulation is critical for appropriate responses, the underlying mechanisms remain unclear, including the roles of classic endocytosis regulators. We address these questions using both focused and genome-scale approaches. We find that the receptor kinase GRK6 acts in parallel with GRK2 and GRK3 to trigger internalization, and that internalization uses a β-arrestin-independent pathway, as well as pathways involving β-arrestin1 and 2. Moreover, we use an integrated analysis of two parallel CRISPR/Cas9 screens to classify regulators of FPR1 biogenesis, surface expression, recycling, and endocytosis. We identify the formin mDia1 and the small GTPase ARF6 as specific regulators of FPR1 internalization, which we confirm using chemical inhibitors in primary human neutrophils. Finally, we find that ARF6 contributes to the β-arrestin-independent pathway. Together, our results provide a systems overview of the control of FPR1 surface levels and offer insights into alternative endocytosis mechanisms used by chemoattractant receptors.},
}
@article {pmid41882360,
year = {2026},
author = {Smith, QM and Whittle, S and Aramayo, RJ and Rollins, DE and Jalal, ASB and Egharevba, DI and Morris, KL and Pyne, ALB and Rueda, DS},
title = {Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity.},
journal = {Nature},
volume = {653},
number = {8114},
pages = {627-635},
pmid = {41882360},
issn = {1476-4687},
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure ; *CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA, Circular/chemistry/ultrastructure/genetics/metabolism ; *DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics ; *Gene Editing/methods ; Models, Molecular ; Protein Domains ; RNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing tool[1], but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (-)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA-RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (-)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.},
}
@article {pmid41882696,
year = {2026},
author = {Ren, W and Yang, M and Zhou, Y and Yang, Y and Li, H and Chen, Y and Li, S and Pang, Y},
title = {An ultra-sensitive cell-free DNA-based diagnostic assay for Tuberculous pleurisy utilizing the CRISPR-Cas13a system.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {25},
number = {1},
pages = {},
pmid = {41882696},
issn = {1476-0711},
support = {S2023050//Popularization and application of appropriate medical and health technology in Guangxi Province/ ; 20230484295//Beijing Nova Program/ ; KJ2024CX028//Beijing Tongzhou District Science and Technology Commission/ ; 2024-4-1042//Health Improvement and Research/ ; },
mesh = {Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; Cell-Free Nucleic Acids/analysis ; CRISPR-Cas Systems ; *DNA, Bacterial/analysis ; *Molecular Diagnostic Techniques/methods/statistics &amp; numerical data ; *Mycobacterium tuberculosis/isolation &amp; purification ; Pleural Effusion/microbiology ; Prospective Studies ; Sensitivity and Specificity ; *Tuberculosis, Pleural/diagnosis ; },
abstract = {BACKGROUND: Tuberculous pleurisy (TP), a predominant form of extrapulmonary tuberculosis, presents significant diagnostic challenges attributable to the paucibacillary nature of pleural effusion (PE) specimens. Cell-free Mycobacterium tuberculosis (MTB) DNA in PE represents a promising biomarker for TP diagnosis. This study aimed to develop and assess a novel cell-free DNA (cfDNA)-CRISPR assay targeting MTB DNA in PE supernatants.<br><br>METHODS: Patients with suspected TP were prospectively enrolled at Beijing Chest Hospital. PE samples underwent centrifugation, with sediments tested by MTB/RIF Xpert (Xpert) testing and mycobacterial culture, while supernatants were analyzed using the cfDNA-CRISPR assay. Diagnostic performance was evaluated using a composite reference standard (CRS).<br><br>RESULTS: Of 276 participants, 237 (85.9%) were included in the final analysis. Based on the CRS, cases were stratified as follows: 63 definite TP, 70probable TP, and 104 non-TP controls. The cfDNA-CRISPR assay in definite TP demonstrated superior sensitivity (81.0%) compared to mycobacterial culture (33.3%, P&#x2009;<&#x2009;0.001) and Xpert (42.9%, P&#x2009;<&#x2009;0.001). In probable TP, where both Culture and Xpert were negative, cfDNA-CRISPR maintained high sensitivity (80.0%), exceeding that of ADA testing (64.3%, P&#x2009;<&#x2009;0.05). Overall sensitivity of cfDNA-CRISPR for TP was 80.5%, markedly higher than Culture (15.8%) and Xpert (20.3%) (both P&#x2009;<&#x2009;0.001). The cfDNA-CRISPR assay exhibited a specificity of 94.2%, while both Culture and Xpert achieved 100% specificity.<br><br>CONCLUSIONS: The cfDNA-CRISPR assay based on the CRISPR-Cas13a system offers significantly improved sensitivity over conventional methods for detecting MTB in PE. It represents a promising, non-invasive diagnostic tool for enhancing TP detection in clinical practice.},
}
@article {pmid41882914,
year = {2026},
author = {Kambakam, S and Thomas, J and Robbe-Austerman, S and Shanmuganatham, K and Palinski, R},
title = {Rapid identification of African swine fever virus in diagnostic samples using CRISPR-Cas.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {},
number = {},
pages = {10406387261432985},
pmid = {41882914},
issn = {1943-4936},
abstract = {African swine fever virus (ASFV) is a highly transmissible pathogen affecting swine, causing a devastating disease with high mortality rates in naive populations. Given the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten the U.S. swine industry, given their proximity and involvement in movement to and from North America. Although surveillance programs are ongoing, limited point-of-care (POC) tests are available during outbreaks with the sensitivity and specificity standards of laboratory testing (e.g., real-time PCR [rtPCR]). However, the recently developed CRISPR-Cas-based testing systems may offer comparable high-quality results. We sought to develop a low-cost visual detection method for ASFV by employing a recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a POC assay. Our CRISPR-Cas12a assay had comparable sensitivity and specificity to rtPCR, both visually and when quantified using a fluorescence reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, our CRISPR assay achieved a sensitivity of 98.3% (10[2] DNA copies) and a specificity of 100%. Test results of our RPA-CRISPR assay can be visualized in as few as 7 min, with peak fluorescence at 40 min (RPA and CRISPR steps). Our results lay the groundwork for a large-scale POC assay assessment for ASFV detection and offer a robust workflow that works with commonly submitted diagnostic samples.},
}
@article {pmid41883584,
year = {2026},
author = {Kobel, L and Van de Venn, L and Schröder, M and Bechter, LV and Huang, D and Abdolazimi, Y and Pertel, T and Gopalakrishnan, S and Corn, JE and Kontarakis, Z},
title = {DisTAL-Seq: A TALEN-specific adaptation of DISCOVER-Seq for off-target profiling.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {2},
pages = {102883},
pmid = {41883584},
issn = {2162-2531},
abstract = {Programmable guided nucleases have revolutionized genome editing and biomedical research, with transformative potential for gene and cell therapy. Although the widespread adoption of the CRISPR-Cas system has provided deep insights into target recognition and specificity, the behavior of clinically relevant tools like transcription activator-like effector nucleases (TALENs) remains poorly characterized in human cells. To address this gap, we implemented DisTAL-Seq, a TALEN-specific adaptation of the DISCOVER-Seq pipeline, which detects MRE11 recruitment to double-strand breaks (DSBs). Based on the DISCOVER-Seq principle, DisTAL-Seq incorporates alignment logic tailored to TALEN-binding properties, including variable RVD specificity, cleavage offset, and dimerization behavior. Using DisTAL-Seq, we identified and validated on- and off-target sites across diverse TALENs and T cell donors. This unbiased approach revealed key features of TALEN activity in human cells, including number of tolerated mismatches to a target site and relative location of the induced DSB. DisTAL-Seq thus extends DISCOVER-Seq to the TALEN family and provides a robust platform for assessing modifications in enzyme architecture and application contexts on a genome-wide scale, supporting the development of safer and more effective genome editing tools.},
}
@article {pmid41884948,
year = {2026},
author = {Ekstrand, F and Ruhrmann, S and Bacos, K and Bartel, S and Jellema, P and Rots, MG and Ling, C and Prinz, CN},
title = {Nanopore Electroporation: A New Delivery Method Within the Field of Epigenetic Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {28},
pages = {e13858},
pmid = {41884948},
issn = {1613-6829},
support = {682206//ERC CoG NanoPokers/ ; //Swedish Research Council (VR)/ ; //Novo Nordisk foundation/ ; //Region Sk&#xe5;ne/ ; Dnr 2009-1039//Strategic Research Area Exodiab/ ; //The Crafoord Foundation/ ; //NanoLund/ ; //The Swedish Diabetes Foundation/ ; ITM-17//The Swedish foundation for Strategic Research/ ; IRC15-0067//The Swedish foundation for Strategic Research/ ; },
mesh = {*Electroporation/methods ; Epigenome Editing ; *Nanopores ; Animals ; CRISPR-Cas Systems/genetics ; Insulin-Secreting Cells/metabolism ; *Gene Editing/methods ; Transfection ; Humans ; *Epigenesis, Genetic ; },
abstract = {Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR-dCas9-based tools offer this potential, yet β-cells are notoriously difficult to transfect, and efficient, non-viral delivery methods are lacking. Here, we developed nanopore-mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β-cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel-associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin-1 gene (Ins1) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult-to-transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR-dCas9-based epigenetic editors in pancreatic β-cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.},
}
@article {pmid41885207,
year = {2026},
author = {Yin, W and Jin, Z and Jiang, Q and Jin, S and Wang, X and He, R and Qiao, B and Qiao, J and Zhang, X and Liu, Y},
title = {Elimination of cis-cleavage in CRISPR diagnostics for one-pot rapid nucleic acid detection.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41885207},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Rapid Diagnostic Tests ; SARS-CoV-2/genetics/isolation &amp; purification ; Endodeoxyribonucleases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics/metabolism ; Sensitivity and Specificity ; },
abstract = {Current one-pot clustered regularly interspaced short palindromic repeats diagnostics are limited by the cis-cleavage activity of Cas nucleases, which leads to amplicon degradation during amplification. Here, we report a streamlined strategy that overcomes this limitation. By integrating a bipartite split-crRNA into Cas12a (SCas12a), we separate target recognition from PAM dependency and completely eliminate cis-cleavage while preserving robust trans-cleavage. This strategy is broadly applicable for one-pot testing, compatible with recombinase polymerase amplification, RT-RPA, and loop-mediated isothermal amplification, as well as multiple Cas12a orthologs, including As, Lb, and Ct Cas12a. Moreover, the SCas12a accelerates one-pot testing with 100-1000-fold improved sensitivity and achieves >10-fold reduction in time-to-signal, enabling detection of targets at attomolar levels within 30 min. Additionally, it provides single-base resolution with up to 91-fold selectivity. The system has been successfully applied to detect HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples. Together, we have developed a PAM-independent and cis-cleavage-free one-pot Cas12a assay, which holds strong potential for point-of-care diagnostics.},
}
@article {pmid41885416,
year = {2026},
author = {Ellis, AL and Stauss, M and Barros Tiburcio, P and Emmen, IE and Edlefsen, PT and Kosmider, E and Barlow, S and Goss, M and Temte, JL and Stachler, E and McMahon, K and Sabeti, P and O'Connor, DH and O'Connor, SL},
title = {Adaptation of the multiplexed CRISPR-Cas13 CARMEN RVP assay for longitudinal detection of respiratory pathogens from air samples.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {4},
pages = {e0211725},
pmid = {41885416},
issn = {1098-5336},
support = {5R01AI170737/NH/NIH HHS/United States ; 5U01CK000542-02-00//Centers for Disease Control and Prevention Foundation/ ; 6U01CK000630-01-01//Centers for Disease Control and Prevention Foundation/ ; },
mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; Wisconsin ; *Air Microbiology ; *CRISPR-Cas Systems ; *Influenza A virus/isolation &amp; purification/genetics ; COVID-19/diagnosis/virology ; Schools ; },
abstract = {UNLABELLED: Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the "gold standard" quantitative reverse transcription-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA), during the 2023-2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared the results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A positivity, were similar between qRT-PCR and RVP_air across the 2023-2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces.<br><br>IMPORTANCE: Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal coronaviruses, respiratory syncytial virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.},
}
@article {pmid41885428,
year = {2026},
author = {Carrington, E and Ballmer, D and Niederwieser, I and Thommen, BT and Brancucci, NMB and Voss, TS},
title = {A heterologous marker-free selection approach for CRISPR/Cas9-based gene editing in the malaria parasite Plasmodium falciparum.},
journal = {mSphere},
volume = {11},
number = {4},
pages = {e0088425},
pmid = {41885428},
issn = {2379-5042},
support = {310030_184785,310030_220001//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; 310030_200683//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; ALTF 920-2024//European Molecular Biology Organization/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Drug Resistance/genetics ; Pyrimethamine/pharmacology ; Thymidylate Synthase/genetics ; Antimalarials/pharmacology ; Tetrahydrofolate Dehydrogenase/genetics ; Protozoan Proteins/genetics ; Humans ; Multienzyme Complexes ; },
abstract = {CRISPR/Cas9-based gene editing of the malaria parasite Plasmodium falciparum has emerged as a transformative tool for advancing functional studies on parasite biology and identifying new therapeutic targets. Currently applied CRISPR/Cas9 methodologies depend on a limited set of heterologous drug resistance markers for the selection of transgenic parasites, which restricts the potential for iterative genetic modifications. Here, we developed a heterologous marker-free CRISPR/Cas9 gene editing strategy (CRISPR/Cas9[pyrR]) for P. falciparum based on the simultaneous editing of a gene of interest and introduction of pyrimethamine (PYR) resistance-conferring mutations into the dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) gene. By providing a pfdhfr[pyrR] donor sequence and the Cas9 expression cassette on separate plasmids, CRISPR/Cas9[pyrR] ensures that only parasites acquiring both plasmids survive under PYR pressure. As a proof of principle, we applied CRISPR/Cas9[pyrR] to generate two transgenic parasite lines expressing GFP-tagged versions of the putative nuclear envelope protein PfGEX1 and nuclear pore protein PfNUP116, respectively. We show that PfGEX1-GFP marks the nuclear envelope specifically in gametocytes, but not in asexual blood stage parasites. Similarly, and against previous reports, we find PfNUP116-GFP expression is undetectable in asexual parasites but instead localizes to a distinct perinuclear region in early gametocytes. These results suggest dynamic compositional changes of the nuclear periphery during sexual differentiation. We further demonstrate sequential genetic engineering of the PfNUP116-GFP-expressing line using the human dhfr drug resistance marker combined with WR99210-based selection by additionally tagging PfAP2-G, the master transcriptional regulator of sexual commitment, and the nuclear pore protein PfNUP313. Hence, CRISPR/Cas9[pyrR] provides a versatile and effective new method that enhances and complements the current genetic toolkit for malaria research.IMPORTANCEMalaria tropica, which is caused by the unicellular parasite Plasmodium falciparum, is one of the most devastating infectious diseases worldwide. The development of urgently needed effective vaccines and new antimalarial drugs with novel modes of action requires a profound understanding of parasite biology. CRISPR/Cas9-based genome engineering is beyond doubt the most important experimental approach to study the function and essentiality of parasite proteins and to identify and validate new vaccine and drug targets. In this study, we developed and successfully applied a modified CRISPR/Cas9 strategy, termed CRISPR/Cas9[pyrR], that avoids the use of a heterologous drug resistance marker for the selection of genetically modified parasites. CRISPR/Cas9[pyrR] thus complements the CRISPR/Cas9 toolbox available for gene editing in P. falciparum and overcomes some of the limitations of currently employed protocols.},
}
@article {pmid41885929,
year = {2026},
author = {Idrees, J and Shabbir, AQ and Alvi, IA and Sana, S and Rehman, SU and Asif, M},
title = {CRISPR-anti-CRISPR dynamics: evolutionary, ecological and biotechnological perspectives.},
journal = {Archives of microbiology},
volume = {208},
number = {6},
pages = {},
pmid = {41885929},
issn = {1432-072X},
mesh = {*Bacteriophages/genetics/physiology ; Biotechnology ; *Bacteria/genetics/virology ; *CRISPR-Cas Systems ; Biological Evolution ; Gene Editing ; Evolution, Molecular ; Ecosystem ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Viral Proteins/genetics/metabolism ; },
abstract = {Phages and bacteria are engaged in an evolutionary arms race. CRISPR-Cas systems allow bacteria to resist phage predation, contributing to competitive advantages for certain bacterial strains. However, phages generate a diverse anti-CRISPR (Acr) proteins to effectively neutralise the CRISPR-Cas system. Acr proteins restore phage infectivity, which may contribute in limiting the dominance of CRISPR-armed bacterial strains, potentially influencing the microbial diversity in certain environments. Acrs can influence microbial community dynamics which may indirectly affect ecosystem functions such as nutrient cycling in certain marine and soil ecosystems. The potential of these Acrs proteins in controlled and reversible genome editing, highlights their potential as regulatory components for genome editing systems. This review discusses the molecular mechanism of CRISPR and anti-CRISPRs, highlights the diversity and limitations of known inhibitory mechanisms, ecological role of anti-CRISPRs and highlights their expanding application in microbial evolution and biotechnology.},
}
@article {pmid41887170,
year = {2026},
author = {Liao, Y and Liang, L and Liang, R and Ding, J and Hu, D and Si, H and Song, X and Tang, X},
title = {Abnormal expression pattern of knock-in marker in Eimeria tenella using CRISPR/Cas9.},
journal = {Poultry science},
volume = {105},
number = {6},
pages = {106763},
pmid = {41887170},
issn = {1525-3171},
mesh = {*Eimeria tenella/genetics ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/veterinary ; *Gene Editing/methods ; Animals ; Chickens ; },
abstract = {Gene editing technology has been widely applied in the genetic manipulation of many organisms and is increasingly being utilized in eukaryotic pathogens. However, its efficiency often requires improvement. In our study using CRISPR/Cas9 to genetically manipulate Eimeria tenella, we aimed to insert a tag into a target gene locus via homologous recombination, but observed outcomes inconsistent with expectations. Whole-genome sequencing analysis of the integration sites revealed that the transgenic E. tenella did not exhibit correct targeted integration. These results indicate that creating double-strand breaks (DSB) at specific genomic sites to trigger homology-directed repair (HDR) for gene modification can lead to mislocalized expression. This study provides insights for utilizing CRISPR/Cas9 technology in genetic editing, particularly in E. tenella, and offers suggestions for improving strategies that employ the co-transfection of multiple plasmids, such as Cas9-gRNA and donor plasmids.},
}
@article {pmid41887225,
year = {2026},
author = {Drepanos, LM and Srikanth, S and Kaplan, EG and Shah, ST and Velasco, BE and Merzouk, S and Doench, JG},
title = {Balancing off-target and on-target considerations for optimized CRISPR-Cas9 knockout library design.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101190},
pmid = {41887225},
issn = {2666-979X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Library ; Animals ; Mice ; *Gene Knockout Techniques/methods ; Genome, Human ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations cause library deprecation over time, further motivating an updated genome-wide design effort. Additionally, while on-target efficacy and off-target avoidance are often optimized in isolation, we lack a robust framework for simultaneously weighing and balancing these competing priorities. Here, we present a selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides, allowing the inclusion of those with maximal on-target activity. We create, validate, and make available to the community the Jacquere library for knockout screens of the human genome, as well as its mouse counterpart, Julianna, to facilitate gene function discovery at scale.},
}
@article {pmid41887586,
year = {2026},
author = {Menestreau, M and Frostegård, &#xc5; and Kjos, M},
title = {Strain-specific challenges in applying CRISPR/Cas9-based genome editing in the novel genus Stutzerimonas.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107478},
doi = {10.1016/j.mimet.2026.107478},
pmid = {41887586},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems ; *Genome, Bacterial ; Denitrification/genetics ; Plasmids/genetics ; },
abstract = {Recently reclassified from the genus Pseudomonas, Stutzerimonas comprises metabolically versatile bacteria widely distributed across diverse environments and with a capacity to perform complete denitrification. Here, we evaluated the applicability of CRISPR/Cas9-based genome editing in Stutzerimonas species. Using a two-plasmid pCasPA/pACRISPR system, we achieved efficient deletion of the denitrification-associated narG and dnrE genes in Stutzerimonas decontaminans 19SMN4. On the other hand, Cas9-associated toxicity significantly limited transformation in Stutzerimonas perfectomarina ZoBell. These results highlight both the potential and the limitations of CRISPR/Cas9 editing in Stutzerimonas, emphasizing that genome editing efficiency and tolerance may vary even among closely related strains.},
}
@article {pmid41888353,
year = {2026},
author = {Wang, J and Bobrik, M and Pankaew, N and Gratacap, R and Digard, P and Bean, TP and Jin, Y and Robledo, D},
title = {Efficient genome editing in a Mozambique tilapia cell line using CAS ribonucleoprotein complexes.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41888353},
issn = {2045-2322},
support = {STG5443//Ministry of Science and Technology of Thailand/ ; BBS/E/RL/230002A/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; *Tilapia/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Cell Line ; *Ribonucleoproteins/genetics/metabolism ; },
abstract = {Genome editing using the CRISPR/Cas system makes it possible to rapidly characterise gene function in vitro and in vivo, and provides a powerful platform through which the genetics of farmed fish can be altered to improve traits such as resistance to important pathogens. Tilapia is one of the most important farmed fish globally; however, its farming is heavily impacted by Tilapia lake virus (TiLV). The Mozambique tilapia (Oreochromis mossambicus) brain (OmB) cell line is susceptible to TiLV, making it an ideal in vitro model for studying host-pathogen interactions and mechanisms of disease resistance. To establish OmB cells as a model for gene editing in Tilapia, it is essential to optimise genome editing protocols. In this study, we optimized a CRISPR/Cas9-based genome editing system for OmB cells using ribonucleoprotein complexes. With the optimized protocol, we successfully edited two endogenous genes with efficiencies ranging from 67% to 70%. In conclusion, we establish a highly efficient CRISPR/Cas9 RNP based gene editing workflow optimized for OmB tilapia cell lines. This optimized platform will facilitate future functional genomic studies in tilapia and support the development of TiLV-resistant tilapia.},
}
@article {pmid41889087,
year = {2026},
author = {Yang, L and Ma, W},
title = {CRISPR/Cas12a: A Comprehensive Review from Structural Foundations to Applications in Nucleic Acid Precision Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {13},
pages = {10689-10708},
doi = {10.1021/acs.jafc.5c16528},
pmid = {41889087},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acids/genetics/chemistry ; Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Humans ; *Bacterial Proteins/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; },
abstract = {CRISPR/Cas12a technology, characterized by its distinctive trans-cleavage activity, has evolved beyond its gene-editing function to emerge as a powerful tool for molecular detection. This review systematically delineates its structural foundation and molecular mechanism, with a focus on how the technology converts specific nucleic acid recognition into cascade signal amplification. Its applications span pathogen diagnosis, species identification, food safety, and authentication of traditional Chinese medicines. Through integration with isothermal amplification and multimodal detection platforms, Cas12a has driven molecular diagnostics toward portability, visualization, and quantification. The review further discusses challenges related to sensitivity, quantitative accuracy, crRNA design, and standardization, while outlining future directions through convergence with cutting-edge technologies such as microfluidics and artificial intelligence, offering a forward-looking perspective for the development of next-generation precision biosensing platforms.},
}
@article {pmid41889958,
year = {2026},
author = {Ocampo, RF and Orosco, C and Huang, B and West, MS and Jain, PK and Taylor, DW},
title = {Architecture of a DNA-guided Cas12a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.03.19.712971},
pmid = {41889958},
issn = {2692-8205},
support = {R35 GM147788/GM/NIGMS NIH HHS/United States ; R61 AI181016/AI/NIAID NIH HHS/United States ; },
abstract = {CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The ΨDNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering.},
}
@article {pmid41891780,
year = {2026},
author = {Gu, C and Gu, J and Li, J and Zou, D and Qi, Y and Xia, R and Zhou, Z and Li, M},
title = {Insights into the Arms Race between Prokaryotic Hosts and Their Viruses in Mangrove Ecosystem.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {14},
pages = {10912-10927},
doi = {10.1021/acs.est.5c14802},
pmid = {41891780},
issn = {1520-5851},
mesh = {*Wetlands ; Viruses ; Ecosystem ; *Prokaryotic Cells ; },
abstract = {The coevolutionary arms race between prokaryotes and viruses has driven the diversification of various microbial immune mechanisms including restriction-modification (RM) and clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems. While recent efforts have expanded the catalog of antiviral systems, their ecological dynamics within complex microbial communities remain underexplored. Here, we analyzed prokaryotic communities, viruses, and defense systems in mangrove habitats from Futian, China, by integrating DNA and RNA sequencing of sediment cores collected across multiple depths and seasons at two sites. Prokaryotic genomes harbored 65 distinct defense system types, representing ∼43% of known systems, with transcriptional activity dominated by Pseudomonadota, Planctomycetota, and Chloroflexiota. The key systems, including abortive infection (AbiD, AbiE, AbiU), Eleos, CRISPR-Cas, MazEF, Retron, and Wadjet, exhibited high transcriptional activity across samples. Strikingly, viruses encoded highly expressed defense systems such as AbiE and RM, despite their co-occurring prokaryotic hosts lacking detectable antiviral systems. The abundance ratio of prokaryotes and viruses with defense systems shows an opposite trend as the depth variation. Heterologous validation confirmed the antiviral efficacy of the selected systems. Our findings suggest that viral-encoded defense systems may functionally augment host immunity in mangrove habitats, revealing a nuanced coevolutionary interplay within these ecosystems. This study advances our understanding of host-virus interactions at the community level in mangrove wetland microbiomes.},
}
@article {pmid41891877,
year = {2026},
author = {Wan, Y and Zhao, X and Lin, X and Wang, L and Ai, X and Jiang, J and Han, L and Huang, D and Du, H and Huang, L},
title = {Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41891877},
issn = {1362-4962},
support = {2024ZDZ07//Guangdong Province Drug Administration Science and Technology Innovation/ ; 202206010073//Science and Technology Program of Guangzhou/ ; 2023A03J0542//Science and Technology Program of Guangzhou/ ; 2025A1515010459//Natural Science Foundation of Guangdong Provinc/ ; GZYZH2023001//Open Project of the China NMPA Key Laboratory for Animal Alternative Testing Technology of Cosmetics/ ; 2022A1515011733//Guangdong Basic and Applied Basic Research Foundation/ ; 2024ZDZ07//Guangdong Province Drug Administration Science and Technology Innovation Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; *INDEL Mutation ; Humans ; *Gene Editing/methods ; DNA End-Joining Repair/genetics ; Gene Regulatory Networks ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; },
abstract = {CRISPR/Cas9-induced DNA double-strand breaks (DSBs) trigger diverse repair outcomes, yet the dynamic regulatory networks governing these outcomes remain incompletely understood. Here, we develop indel pattern-guided repair mapping, an integrative framework that deciphers DSB repair mechanisms by integrating repair outcome spectra, kinetic dynamics, and functional gene regulation. Our analysis categorizes Cas9-mediated repair outcomes into seven distinct patterns based on their frequency and sequence characteristics, revealing differential repair kinetics among these subtypes. Functional clustering identifies three regulatory pillars: (i) microhomology-mediated end joining (MMEJ)-driven MH deletions form a cohesive module defined by a shared regulatory network of protein-coding genes and miRNAs, rather than by the core repair enzymes themselves; (ii) non-homologous end joining coordinates 1 bp insertions and non-MH deletions, with RFC4/5 stabilizing repair templates to suppress large deletions; (iii) Atypical repair outcomes show distinct genetic signatures: large insertions are associated with polymerase-related regulators, whereas mutations are associated with a signature enriched for chromatin-associated regulators. Strikingly, S100A8 emerges as a potent MMEJ suppressor via direct interaction with PARP1, revealing unappreciated cross-talk between inflammatory signaling and DSB repair pathway choice. By linking repair outcome patterns to molecular determinants, our work provides a transformative platform to interrogate DNA repair mechanisms for precise genome editing optimization and therapeutic genome stabilization.},
}
@article {pmid41892062,
year = {2026},
author = {Tantai, W and Xu, Q and Zhang, W and Li, Y and Liu, H},
title = {A One-Pot CRISPR/Cas12a-Based Platform for Contamination-Free Nucleic Acid Amplification Detection.},
journal = {Biosensors},
volume = {16},
number = {3},
pages = {},
pmid = {41892062},
issn = {2079-6374},
support = {22074085//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques ; Polymerase Chain Reaction ; *Biosensing Techniques ; DNA ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 10[6] copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples.},
}
@article {pmid41892280,
year = {2026},
author = {Woronkowicz, M and Thomas, MN and Saram, SJ and Carr, AF and Alonso-Carriazo Fernandez, A and Butt, Z and Skopiński, P and Ramsden, CM},
title = {CRISPR and Beyond: Genome-Editing Strategies in Retinal Stem Cell Research.},
journal = {Cells},
volume = {15},
number = {6},
pages = {},
pmid = {41892280},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Retina/cytology/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Stem Cell Research ; Embryonic Stem Cells/metabolism ; },
abstract = {Genome editing has emerged as a transformative approach for understanding and treating retinal degenerative diseases. Combining this technology with pluripotent stem cells provides an ideal platform for modeling human development and disease, and investigating emerging therapeutic strategies ultimately aimed towards in vivo correction. This approach enables both functional studies to understand retinal degeneration and the early development of targeted therapies for inherited disease. This review offers a comprehensive overview of genome-editing techniques and the ability to create new clinically relevant models to understand human disease in retinal research, focusing on the use of the CRISPR-Cas9 system in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), as well as highlighting recent advancements in base and prime editing. Gene editing in various retinal diseases is discussed in context of studies focusing on disease modeling or developing therapeutic strategies. Continued refinement of these techniques will be essential for advancing translational applications in retinal disease treatment.},
}
@article {pmid41892424,
year = {2026},
author = {Gomes, E and Mesquita, TG and Serra, P and Araújo, D and Almeida, C and Machado, A and Oliveira, R and Castro, J},
title = {Antimicrobial Resistance in the Food Chain: Bridging Knowledge Gaps for Effective Detection and Control.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41892424},
issn = {2079-6382},
support = {https://doi.org/10.54499/2024.13640.PEX//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; https://doi.org/10.54499/2022.07654.PTDC//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; APTA4shiga (number 14840)//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; },
abstract = {Antimicrobial resistance (AMR) poses a critical global public health threat, with the food chain serving as a significant transmission route connecting animals, environment, and humans. This review adopts a One Health perspective to analyze the key drivers of AMR dissemination across animal agriculture, aquaculture and food processing. We evaluate detection methodologies, contrasting the regulatory gold standard of culture-based phenotypic testing with rapid molecular advancements, including Whole Genome Sequencing (WGS), metagenomics, and emerging CRISPR-Cas diagnostics. While molecular tools offer unprecedented speed and resolution, challenges such as matrix interference, the viable but non-culturable (VBNC) state, and the genotype-phenotype disconnect remain. Finally, integrated mitigation strategies are also described, ranging from on-farm antimicrobial stewardship and innovative biofilm control to consumer hygiene practices. It is essential to bridge the technical and regulatory gaps in AMR surveillance in order to develop effective interventions and ensure a safer food system.},
}
@article {pmid41893903,
year = {2026},
author = {Baliyan, N and Upadhyay, P and Murugan, T and Srivastava, A and Singh, S and Tomar, BS and Mangal, M},
title = {Recent advances in generation of doubled haploid plants for genetic improvement in solanaceous vegetable crops.},
journal = {Planta},
volume = {263},
number = {5},
pages = {},
pmid = {41893903},
issn = {1432-2048},
support = {SR/WOS-A/LS-146/2019//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*Haploidy ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {Genotype-specific protocols and advancements focusing on CRISPR/Cas9-based haploid induction for doubled haploid (DH) production are poised to revolutionize plant breeding for faster genetic improvement in solanaceous crops. The need for swift development of improved cultivars offering greater resilience to biotic and abiotic stresses stems from the emergence of climate change risks. While conventional methods are effective, novel methods for precise crop genome manipulation are required. The in vivo and in vitro protocols leading to fixation of homozygosity and rapid attainment of homozygous DH (doubled haploids) lines have led to a resurgence in research on haploids and DH. The efforts for haploid production have been primarily concentrated on in vitro androgenesis and gynogenesis. The success of these in vitro techniques depends on various parameters, including culture conditions, developmental stage of the microspore, pretreatment, culture medium, growth regulators, and other media additives. Breakthroughs in genome-editing technologies, such as the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system, have provided a new avenue for exploring in vivo haploid embryogenesis for haploid induction by means of haploid inducer factors, such as DMP (DMP domain of unknown function 679 membrane protein), ECS (egg cell-specific), MTL (matrilineal), and BBM (Baby boom). A successful haploid induction system in vegetable crops will be possible through the development of efficient in vivo and in vitro androgenesis and gynogenesis protocols. For genome-editing to be feasible, protocols must be optimized for commercially significant solanaceous crops like pepper, potatoes, eggplant, and tomatoes. The existing protocols for genome doubling need to be further improved for solanaceous crops. These developments are pivotal for the advancement and harnessing of haploid technology to its full potential in crop breeding.},
}
@article {pmid41893914,
year = {2026},
author = {Pistone, D and Bevivino, G and Dipaola, MG and Bandi, C and Lombardo, F},
title = {Current and emerging molecular diagnostic approaches in the detection of human parasites.},
journal = {Parasitology research},
volume = {125},
number = {1},
pages = {},
pmid = {41893914},
issn = {1432-1955},
abstract = {Microscopy and morphological identification remain the gold standard for diagnosing most parasitic infections, yet their limited sensitivity in asymptomatic or low-burden cases, along with technical constraints, has accelerated the adoption of molecular diagnostics. Over the past three decades, advances in nucleic acid amplification and sequencing technologies have transformed parasite detection by improving sensitivity, specificity, and reproducibility, enabling earlier intervention and stronger surveillance. PCR remains the foundation of molecular diagnostics, with real-time PCR and digital PCR improving analytical performance and quantification. Multiplex qPCR supports simultaneous detection of multiple pathogens, while dPCR enables absolute quantification and rare variant detection, although broader implementation is limited by instrument cost. Isothermal amplification methods such as tHDA, NASBA, LAMP, and RPA offer rapid, low-cost amplification at constant temperature and are well suited for field diagnostics in resource-limited settings. Next-Generation Sequencing has advanced genotyping and epidemiological surveillance by resolving cryptic species, resistance mutations, and mixed infections through targeted panels, whole-genome sequencing, and metagenomics. CRISPR/Cas-based assays provide rapid and sensitive nucleic acid detection with strong potential for point-of-care deployment due to their simplicity and adaptability. Emerging biomarkers, including circulating cell-free DNA, non-coding RNAs, and microRNAs in extracellular vesicles, offer promising non-invasive diagnostic strategies, though further validation is required. This review offers a concise overview of these molecular approaches, emphasizing recent innovations such as dPCR, NGS, CRISPR/Cas systems, and biomarker-based detection. For each method, core technical principles, representative applications, and comparative strengths and limitations are presented to illustrate their diagnostic potential.},
}
@article {pmid41895443,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {5},
pages = {111401},
pmid = {41895443},
issn = {1083-351X},
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Mutation ; },
abstract = {Type I-E CRISPR-Cas (CRISPR associated) systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the protospacer adjacent motif and protospacer adjacent motif-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four Escherichia coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 22, and four. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cascade conformation and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}
@article {pmid41896556,
year = {2026},
author = {Walsh, LH and Soni, V and Ancla, J and Somerville, V and Segata, N and Joyce, S and Sinderen, DV and Mahony, J and Shkoporov, AN and Kenny, JG and Cotter, PD and O'Sullivan, O},
title = {Mining of food metagenomes reveals an unexplored diversity of dsDNA bacteriophages.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {},
pmid = {41896556},
issn = {2055-5008},
support = {DOMINO-101060218//European Union's Horizon Europe programme/ ; },
mesh = {*Bacteriophages/genetics/classification/isolation &amp; purification ; *Metagenome ; Metagenomics/methods ; *Bacteria/virology/genetics/classification ; *Food Microbiology ; Genome, Viral ; Phylogeny ; Biodiversity ; },
abstract = {Bacteriophages are key drivers of microbial ecology, co-existing and co-evolving with bacteria across diverse environments. Limitations in culturing, alongside advances in sequencing and bioinformatics, have driven the use of metagenomics to explore viral diversity. Viral-specific analysis of >3000 food metagenomes from cFMD produced the FVGC, comprising ~3400 metagenome-assembled viruses, most of which belong to novel Caudoviricetes lineages (n = 91), with only ~15% represented in IMG/VR v4. Together, these findings reveal extensive uncharacterized viral diversity in food systems. Beyond serving as a reference, the FVGC facilitates detailed investigation of virus-host interactions. Viral sequences were pervasive across microbial genomes, with several bacterial families exhibiting near-universal associations with viral elements. Bacterial antiviral defence systems were abundant and taxonomically diverse, dominated by restriction-modification systems, while CRISPR-Cas systems showed pronounced lineage-specific distributions; in contrast, viral anti-defence genes were detected at low frequency (<10% of MAVs). Host prediction linked MAVs to clinically relevant taxa, including expanded ESKAPE pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Enterobacter spp., highlighting the ecological connectivity between food-associated viruses and clinically important bacteria. Antimicrobial resistance signals were scarce, suggesting minimal phage-mediated AMR dissemination in food environments. This new publicly available viral database represents a valuable resource for further exploration of viral diversity.},
}
@article {pmid41896732,
year = {2026},
author = {Herrera-Cardoso, ED and Tapia-Cervantes, KA and Cepeda-Negrete, J and Gutiérrez-Vargas, S and León-Galván, MF},
title = {Genome sequencing analysis reveals probiotic potential of Lactiplantibacillus plantarum IGMA4EH isolated from the gut of the white maguey worm (Aegiale hesperiaris).},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41896732},
issn = {1471-2164},
support = {CIIC 147/2023//Direcci&#xf3;n de Apoyo a la Investigaci&#xf3;n y al Posgrado of the Universidad de Guanajuato (DAIP UG)/ ; },
abstract = {BACKGROUND: The use of probiotics is increasingly popular for health applications, and Lactiplantibacillus plantarum strains are among the most widely studied for their potential in promoting gut health. In this study, we present the first genomic characterization of L. plantarum IGMA4EH, isolated from the gut of the maguey worm (Aegiale hesperiaris), an edible insect with traditional significance in Mexico.<br><br>METHODOLOGY: Whole-genome sequencing was performed on the L. plantarum IGMA4EH strain. Bioinformatics analyses were conducted to identify probiotic-related genes, antimicrobial potential, and safety features. Genes associated with resistance to environmental stressors, adhesion, and immunomodulation were screened, and safety assessment included the detection of antibiotic resistance genes, virulence factors, and plasmids.<br><br>RESULTS: The genomic analysis revealed the presence of multiple probiotic-related genes, including those associated with resistance to temperature, acidity, oxidative stress, and bile. Additionally, gene clusters related to bacteriocins, and secondary metabolites were identified, suggesting strong antimicrobial potential. Safety evaluations showed no evidence of acquired antibiotic resistance genes, virulence factors, or plasmids. Furthermore, elements related to genetic stability, such as CRISPR-Cas systems and prophage regions, were also detected.<br><br>CONCLUSIONS: The findings highlight the potential of L. plantarum IGMA4EH as a novel probiotic strain with promising applications.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-026-12798-5.},
}
@article {pmid41897294,
year = {2026},
author = {Zhou, Z and Zhu, L},
title = {A Computational Model for Nme1Cas9 HNH Activation Driven by Dynamic Interface Engineering at Residues S593 and W596.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897294},
issn = {2218-273X},
support = {32471296//National Natural Science Foundation of China Projects/ ; },
mesh = {*Molecular Dynamics Simulation ; Thermodynamics ; *Endonucleases/chemistry/metabolism/genetics ; },
abstract = {Nme1Cas9 is an encouraging genome-editing tool with high fidelity and compactness, but its applications are limited by poor catalytic efficiency compared with SpyCas9. Understanding the dynamic activation mechanism of the HNH nuclease domain is the key to breaking the kinetic bottleneck. Here, we integrated Steered Molecular Dynamics (SMD) with the Traveling-Salesman-based automated Path Searching (TAPS) algorithm to reconstruct the atomic-level activation landscape of the L1-HNH module. The simulations suggest a complex "Lifting-Rearrangement-Sliding" pathway, revealing the critical role of a "Backbone Sliding" conformation; in this step, the HNH domain rotates across the R-loop surface. A thermodynamic analysis using free energy decomposition by MM/PBSA indicates that the intrinsic instability of the wild-type HNH/R-loop interface constitutes the predominant energetic barrier. Hyperactive variants (S593Q/W596K and S593Q/W596R) can overcome this barrier by substantially increasing binding affinity to the R-loop through a "Geometry-Electrostatics Synergism": S593Q improves interfacial proximity, whereas W596K/R acts as an "Electrostatic Anchor." The results of unbiased MD simulations demonstrate that strengthened interfacial interactions effectively promote spontaneous conformational drift toward the activated state. This computational study proposes a novel in silico model for "Dynamic Interface Engineering" in which reinforcing transient interfacial contacts during conformational sliding can be an effective strategy in developing high-efficiency CRISPR-Cas effectors.},
}
@article {pmid41897386,
year = {2026},
author = {Richter, PR and Graf, J and Haag, FWM and Scudlo, V and Wiesmeth, S and Hauslage, J and Richter, M and Geißler, D and Lebert, M},
title = {Acceleration and Light-Induced Changes in Cytosolic cAMP Concentration in Euglena gracilis.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897386},
issn = {2218-273X},
support = {50WB1128 and 50WB2218)//Deutsches Zentrum f&#xfc;r Luft- und Raumfahrt (DLR) BMWi project/ ; },
mesh = {*Euglena gracilis/metabolism/radiation effects ; *Cyclic AMP/metabolism ; *Light ; Adenylyl Cyclases/metabolism/genetics ; *Cytosol/metabolism/radiation effects ; Phototaxis ; },
abstract = {The second messenger cyclic AMP (cAMP) is very likely involved in phototactic as well as gravitactic behavior of the unicellular flagellate Euglena gracilis. A slight but significant increase in cAMP was observed when cells encountered sub-threshold acceleration (0.16 × g) force after microgravity [µg]. No differences in cAMP levels were found between cells on a clinostat and 1x-controls. This observation is consistent with the ones of earlier studies. Illumination of cells resulted in a significant increase in cellular cAMP levels. After RNAi-mediated knockdown or CRISPR-Cas9 knockout of the photoactivated adenylyl cyclases PACα and/or PACβ in the photoreceptor, light-induced changes in cAMP levels were no longer observed. In parallel, phototactic behavior was abolished, supporting the essential role of photoactivated adenylyl cyclases in phototaxis. Cells spin around their length axis during locomotion (1-2 Hz). In order to generate a signal in the light direction, the cells should be capable of synthesizing and degrading cAMP within 0.5-1 s. The rapid fixation of cells upon transition from dark to light or light to dark revealed that detectable changes in cAMP-levels (increase or decrease) occur within a 100-200 ms time window, which is sufficiently fast to account for the proposed theoretical kinetics of cAMP oscillations.},
}
@article {pmid41897402,
year = {2026},
author = {Shahannaz, DC and Sugiura, T},
title = {Next-Generation Metabolic Reprogramming in iPSC-Derived Cardiomyocytes: CRISPR-EV Synergy for Precision Cardiac Regeneration.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897402},
issn = {2218-273X},
mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; Metabolic Reprogramming ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Regeneration ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; Animals ; Oxidative Phosphorylation ; },
abstract = {Cardiovascular disease remains the leading global cause of mortality, largely due to the limited regenerative capacity of adult human myocardium. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a scalable platform for cardiac repair and disease modeling; however, their persistent metabolic immaturity-characterized by reliance on glycolysis, reduced oxidative phosphorylation (OXPHOS), and structurally underdeveloped mitochondria-limits functional integration and long-term therapeutic efficacy. Recent advances indicate that targeted metabolic reprogramming can enhance mitochondrial biogenesis, increase ATP production, and improve stress resilience in iPSC-CMs. This review examines the complementary integration of CRISPR-based metabolic engineering and extracellular vesicle (EV)-mediated metabolic modulation as a systems-level strategy for cardiac maturation. We discuss CRISPR activation, interference, and epigenome-editing approaches targeting regulators such as PGC-1α, TFAM, and PPARs to promote stable enhancement of mitochondrial networks and respiratory capacity. In parallel, engineered EVs delivering miRNAs, metabolic enzymes, and redox modulators provide non-genomic mechanisms to optimize bioenergetic function and mitigate oxidative stress. By synthesizing mechanistic insights, quantitative bioenergetic metrics, and translational considerations, we propose CRISPR-EV synergy as a precision framework for durable metabolic maturation of iPSC-CMs, with implications for regenerative therapy, pharmacologic screening, and myocardial repair.},
}
@article {pmid41898355,
year = {2026},
author = {Ye, Y and Huang, L and Fu, H and Wang, J and Jin, Y},
title = {Advances in SRNS Gene Research: From Precision Classification to Precision Diagnosis and Treatment.},
journal = {Biomedicines},
volume = {14},
number = {3},
pages = {},
pmid = {41898355},
issn = {2227-9059},
support = {2024C03211 to Jingjing Wang//Key Research and Development Program of Zhejiang Province/ ; GZY-KJS-ZJ-2026-071//Joint TCM Science &amp;Technology Projects of National Demonstration Zones for Comprehensive TCM Reform/ ; LTGD24H050002//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {To clarify the genetic classification, diagnostic strategies, and precision treatment pathways of steroid-resistant nephrotic syndrome (SRNS), this review systematically reviews the genetic stratification system of SRNS by integrating recent advances in genetic testing technologies and pathogenesis research. It contains the pathogenic mechanisms, diagnostic protocols, and therapeutic correlations of different genetic subtypes, while summarizing current progress and clinical challenges in gene therapy. Results indicate SRNS can be categorized into genetic (38-58%) and non-genetic/immune-mediated (40-60%). A stepwise diagnostic system comprising core proteinuria gene panel testing, whole-genome sequencing (WGS), whole-exome sequencing (WES), and supplementary multi-omics/long-range sequencing is proposed, suited for populations with "typical phenotypes and moderate genetic risk", "atypical phenotypes and high genetic suspicion", and "complex structural/non-coding region variants" respectively. Pathogenic mechanisms directly determine therapeutic strategies: COQ2/PDSS2 mutations respond to coenzyme Q10 suplementation, while NPHS1 mutations necessitate early renal transplantation. Adeno-associated virus (AAV)-mediated gene therapy and CRISPR-Cas editing show preclinical promise but face challenges including incomplete detection coverage and clinical translation difficulties. Genetic technologies are driving SRNS management transformation from "empirical treatment" to "mechanism-oriented precision diagnosis and therapy". Future efforts should focus on overcoming genetic testing limitations and gene therapy translation bottlenecks to enhance diagnostic and therapeutic efficacy.},
}
@article {pmid41898703,
year = {2026},
author = {Seh, BA and Rafiq, K and Legradi, A and Mir, MY},
title = {Targeted Gene and Genome-Editing Strategies for Epilepsy: Experimental Advances and Translational Challenges.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898703},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; *Epilepsy/genetics/therapy ; *Genetic Therapy/methods ; Animals ; Translational Research, Biomedical ; CRISPR-Cas Systems ; },
abstract = {Epilepsy affects more than 50 million individuals worldwide, and approximately one-third of patients remain refractory to existing antiseizure medications. Advances in gene therapy and genome editing have opened new possibilities for disease-modifying interventions that directly target the molecular and circuit-level mechanisms underlying epileptogenesis. Recent progress in central nervous system tropic viral vectors, non-viral delivery systems, and programmable genome-editing technologies has enabled precise manipulation of neuronal and glial function in preclinical epilepsy models. Strategies range from restoration of haploinsufficient genes implicated in monogenic epilepsies, such as SCN1A in Dravet syndrome, to modulation of neuronal excitability through engineered ion channels, neuropeptides, and astrocyte-based approaches. In parallel, CRISPR-derived platforms, including transcriptional activation and repression systems, base editing, and prime editing, offer new avenues for regulating gene expression in post-mitotic neurons without introducing double-strand DNA breaks. Despite these advances, significant translational challenges remain, including efficient and cell-type-specific delivery, long-term safety, and the risk of network-level side effects in the epileptic brain. This review critically examines recent gene therapy and genome-editing approaches for epilepsy, highlights key technological and biological barriers to clinical translation, and discusses emerging strategies that may enable durable and targeted treatments for drug-resistant epilepsies.},
}
@article {pmid41898716,
year = {2026},
author = {Calbay, O and Hsieh, CL and Lu, C and Ghosh, S and Vijaykumar, V and Watts, I and Sweigard, H and Gandhi, J and den Hollander, AI},
title = {Mapping the Hypoxic Fitness Landscape of Retinal Pigment Epithelial Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898716},
issn = {1422-0067},
mesh = {Humans ; *Retinal Pigment Epithelium/metabolism/cytology ; Cell Hypoxia/genetics ; Cell Line ; CRISPR-Cas Systems ; Gene Expression Profiling ; Oxygen/metabolism ; *Epithelial Cells/metabolism ; Macular Degeneration/genetics/metabolism/pathology ; Transcriptome ; *Hypoxia/genetics/metabolism ; },
abstract = {Chronic hypoxia is a hallmark of aging and retinal diseases such as age-related macular degeneration (AMD), yet the molecular mechanisms that enable retinal pigment epithelium (RPE) cells to survive under sustained low-oxygen conditions remain poorly understood. To address this, we conducted transcriptomic profiling and a genome-wide CRISPR-Cas9 loss-of-function screen in ARPE-19 cells exposed to chronic hypoxia (1% and 5% O2), mimicking the retinal disease environment. The CRISPR screen identified genes whose loss compromises RPE viability or fitness under hypoxia, while transcriptomic profiling revealed oxygen-dependent shifts in key functional modules. These findings converged on pathways related to mitochondrial function, extracellular matrix remodeling, vascular signaling, and cell cycle regulation, identifying unique functional nodes specific to RPE cells. These core processes are also implicated in retinal diseases, such as AMD. Together, these complementary approaches provide an integrated view of the molecular networks driving RPE adaptation to hypoxic stress and highlight novel gene candidates that may serve as therapeutic targets in retinal disease.},
}
@article {pmid41898816,
year = {2026},
author = {Abdalla Elsayed, MEA and MacLaren, RE},
title = {Precision Is Not Enough: When Tools Outpace Translation in Ocular Gene Therapy.},
journal = {Genes},
volume = {17},
number = {3},
pages = {},
pmid = {41898816},
issn = {2073-4425},
support = {//Foundation Fighting Blindness Clinical Research Fellowship/ ; //Oxford NIHR Biomedical Research Centre, UK Department of Health/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Eye Diseases/therapy/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; },
abstract = {Advances in molecular biology have positioned the eye as a leading platform for gene therapy, owing to its surgical accessibility, relative immune privilege, and the ability of the contralateral eye to serve as an anatomical control. We trace the historical evolution of gene discovery, synthesize current gene therapy strategies for inherited and acquired ocular disorders, critically evaluating the limitations of CRISPR and related genome-editing technologies, and examine the key scientific and translational challenges that must be addressed for genetic therapies to be integrated into routine ophthalmic practice.},
}
@article {pmid41899394,
year = {2026},
author = {Aliev, T and Imatdinov, A and Prudnikova, E and Taranov, O and Emtsova, K and Imatdinov, I and Agafonov, A},
title = {The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles.},
journal = {Current issues in molecular biology},
volume = {48},
number = {3},
pages = {},
pmid = {41899394},
issn = {1467-3045},
support = {The Federal Scientific-technical programme for genetic technologies development for 2019-2030, agreement &#x2116; 075-15-2025-526//The Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m-TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m-TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.},
}
@article {pmid41900389,
year = {2026},
author = {Shuang, W and Zeng, X and Li, T and Li, J and Sun, Q and Chen, L},
title = {Screening, Safety Assessment, and Process Optimization of Lactic Acid Bacteria from Traditional Yak Yogurt as Adjunct Cultures.},
journal = {Microorganisms},
volume = {14},
number = {3},
pages = {},
pmid = {41900389},
issn = {2076-2607},
support = {Southwest Minzu University (Grant No. ZYN2025240)//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Cheese ripening is slow and costly, driving interest in accelerating maturation. This study aimed to isolate a safe, efficient adjunct starter from traditional Sichuan yak yoghurt, a niche rich in stress-adapted lactic acid bacteria. From 295 isolates, 15 strains tolerant to high salt, low pH, and low temperature were selected. Using acidification, autolysis, proteolysis, and peptidase activity as indices, principal component analysis identified Limosilactobacillus fermentum 270 as the best candidate. Phenotypic assays showed no haemolysis, gelatin liquefaction, indole production, or amino acid decarboxylase activity. Whole-genome sequencing confirmed species identity and revealed 52 protease/peptidase genes, complete pathways for diacetyl/acetoin biosynthesis and branched-chain amino acid conversion, and no functional biogenic amine synthesis genes. Stress-related genes (F-ATPase, glycine-betaine transport, cold-shock proteins) support cheese adaptability. Antibiotic resistance gene homologs were mainly chromosomal and unlinked to mobile genetic elements; a functional CRISPR-Cas system lowers horizontal transfer risk. The strain was developed as a freeze-dried direct-vat starter (97.3% viability). Orthogonal optimisation of yak Gouda cheese-making defined best conditions: 0.018% adjunct, 45 min acidification, pH 5.8, and 30% curd washing. L. fermentum 270 thus combines proteolytic, flavour-enhancing, genetic safety, and processing traits, offering a promising adjunct for accelerated cheese ripening.},
}
@article {pmid41901428,
year = {2026},
author = {Hou, J and Li, H and Zhang, F and Yang, D and Xiong, Y and Zhu, X and Wen, M},
title = {From Gene Knockouts to Genome Remodeling: Large DNA Fragment Deletion Technologies in Plants.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {41901428},
issn = {2223-7747},
support = {2023YFA0913500//National Key Research and Development Program of China/ ; 25HHWCSS0007//Haihe Laboratory of Sustainable Chemical Transformations/ ; 32230012//the National Natural Science Foundation of China/ ; 32470363//the National Natural Science Foundation of China/ ; },
abstract = {Large DNA fragment deletion (LDFD) provides a powerful means to reconfigure plant genomes at the kilobase to megabase scale, enabling the dissection of genome function, elucidation of non-coding regulatory elements, modulation of gene dosage, reorganization of chromosomal architecture, and implementation of synthetic biology designs. In this review, we systematically compare the mechanisms, efficiencies, advantages, and limitations of the major LDFD technologies that have been applied in plants, including ZFNs, TALENs, CRISPR/Cas systems (Cas9, Cas12a, Cas3), site-specific recombinases, transposon-based systems, and prime editing-derived strategies. We highlight how plant-specific features of chromatin organization and DNA repair constrain large deletions, and discuss the current bottlenecks in achieving efficient, precise, and predictable LDFD across diverse crop genomes. Finally, we outline future directions for plant LDFD, emphasizing AI-assisted design of nucleases and recombinases, protein-directed evolution, and improved DNA- and RNP-based delivery systems. Together, these advances are expected to transform LDFD from a specialized tool into a broadly accessible platform for functional genomics, trait engineering and rational genome design in plants.},
}
@article {pmid41902223,
year = {2026},
author = {Zhang, J and Dang, TT and Lin, TY and Yu, X and Pellin, D and Tian, J and Simmons, O and Kou, E and Cornetta, K and Xiao, W},
title = {Development of a Novel Method to Detect AAV Vector Integration.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902223},
issn = {1999-4915},
support = {P01HL160472//National Institute of Health/ ; 75N92019D00018/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Dependovirus/genetics ; *Genetic Vectors/genetics ; *Virus Integration ; CRISPR-Cas Systems ; HeLa Cells ; Nanopore Sequencing ; Polymerase Chain Reaction ; },
abstract = {AAV integration has become an important safety consideration in gene therapy. However, accurately determining integration sites remains challenging due to biases introduced by library preparation methods, sequencing technologies, and bioinformatic pipelines. In this study, we developed a PCR-free amplification based on a CRISPR-Cas9 cleavage strategy for AAV DNA that overcomes the limitations of PCR amplification imposed by the ITR structure. When combined with long-read nanopore sequencing, this CRISPR-Cas9-based workflow preserves native AAV integration states and enables unbiased detection of integration junctions. We used AAV-transduced HeLa single-cell clones to evaluate the performance of this approach. To confirm integration site identification, AAV integration junctions were also detected using a probe hybridization capture strategy followed by Illumina short-read sequencing. Integration junctions identified by both methods were further confirmed by PCR. The results showed strong consistency between the two approaches in accurately identifying AAV integration sites in each clone. Overall, these findings demonstrate that the CRISPR-Cas9-enabled, PCR-free long-read sequencing workflow provides a promising tool for characterizing AAV integration events.},
}
@article {pmid41902231,
year = {2026},
author = {Qi, M and Liu, X and Wang, W and Lu, M and Zeng, Q and Li, N and Han, Y and Fan, S and Lu, C and Dai, J},
title = {Tree Shrew Genome-Wide CRISPR Screen Identifies RNF6 as a Proviral Host Factor for Zika Virus Replication in Brain Microvascular Endothelial Cells.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902231},
issn = {1999-4915},
support = {2019-1-R-24483//the Bureau of Science and Technology of Kunming/ ; 2017HC019//the Yunnan Science and Technology Talent and Platform Program/ ; D-2024006//the Training Program for High-level Health and Medical Technology Talents in Yunnan Province/ ; },
mesh = {Animals ; *Virus Replication ; *Endothelial Cells/virology ; Humans ; *Brain/virology/blood supply ; *Zika Virus/physiology/genetics ; *Zika Virus Infection/virology ; Viral Nonstructural Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; },
abstract = {Zika virus (ZIKV), a unique flavivirus with neurotropic and teratogenic potential, can cross the blood-brain barrier and persist in human brain microvascular endothelial cells (BMECs); however, no approved vaccines or specific antivirals exist, and its barrier-crossing and neuroinvasive mechanisms remain elusive. Innovative strategies to identify additional host factors mediating ZIKV infection could yield key insights and help address these challenges. To uncover novel host factors, we established the first tree shrew (Tupaia belangeri) genome-wide CRISPR/Cas9 knockout (GeCKO) library and performed a screen in BMECs, identifying ring finger protein 6 (RNF6) as a novel proviral factor for ZIKV. ZIKV infection in BMECs was significantly reduced following RNF6 knockout or knockdown but enhanced upon RNF6 overexpression or rescue. Mechanistically, RNF6 interacts with the ZIKV NS5 protein and acts as a potential negative regulator of the type I interferon and MAPK signaling pathways. Evolutionary and structural analyses revealed that RNF6 is highly conserved between humans and tree shrews; molecular docking further identified shared NS5-binding residues (Gln-59, Arg-140), supporting the conserved proviral role of human RNF6 in ZIKV infection. Our findings highlight tree shrew GeCKO screening as an efficient approach for identifying novel host factors and establish RNF6 as a critical proviral factor for ZIKV replication in BMECs, providing new insights into ZIKV neurotropic pathogenesis and informing potential antiviral strategies.},
}
@article {pmid41902262,
year = {2026},
author = {Jones, JE and Gunderson, CE and Wigdahl, B and Nonnemacher, MR},
title = {Breaking into HIV-1's Epigenetic Vault: Cure Strategies to Eliminate the Viral Reservoir.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902262},
issn = {1999-4915},
support = {MH110360/MH/NIMH NIH HHS/United States ; MH092177/MH/NIMH NIH HHS/United States ; MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *HIV-1/genetics/drug effects/physiology ; *Epigenesis, Genetic/drug effects ; *Virus Latency/drug effects ; *HIV Infections/virology/drug therapy/therapy ; Proviruses/genetics/drug effects ; Chromatin ; Gene Expression Regulation, Viral ; CRISPR-Cas Systems ; *Anti-HIV Agents/pharmacology/therapeutic use ; Virus Integration ; },
abstract = {Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that integrates into the host cell's DNA as a provirus. Transcription from the provirus is regulated in large part by cellular proteins and epigenetic factors. These may be repressive or permissive to productive infection. The host factors that regulate this balance are therefore attractive targets for HIV-1 therapeutics. Indeed, proviral chromatin is the focus of two of the current HIV-1 cure strategies. "Shock and Kill" uses latency reversal agents to open the provirus's chromatin, promoting high levels of gene expression that induce the killing of infected cells. "Block and Lock" uses latency promoting agents to induce heterochromatin, blocking transcription and forcing HIV-1 into a state of deep latency. Here, the compounds investigated in both strategies are reviewed, including their chemical structures, mechanisms of action, and clinical results. Finally, the use of CRISPR-Cas therapeutics and the impact of chromatin architecture on its efficacy are discussed.},
}
@article {pmid41903969,
year = {2026},
author = {Chen, F and Huang, M and Zeng, S and Wan, Y and Qin, A and Yang, X and Fu, D and Negahdary, M and Zhang, C},
title = {A CRISPR/dCas9 mediated electrochemical impedimetric biosensor for sensitive mtDNA detection.},
journal = {Analytica chimica acta},
volume = {1401},
number = {},
pages = {345323},
doi = {10.1016/j.aca.2026.345323},
pmid = {41903969},
issn = {1873-4324},
mesh = {*DNA, Mitochondrial/genetics/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; Limit of Detection ; Polymorphism, Single Nucleotide ; Electrodes ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Tin Compounds/chemistry ; Polyethyleneimine/chemistry ; },
abstract = {BACKGROUND: The accumulation of mitochondrial DNA (mtDNA) mutations in cells is closely linked to various human diseases. Detection of single-nucleotide variation (SNV) in mtDNA plays a crucial role in understanding the heteroplasmy of mtDNAs that contain pathogenic changes. While conventional nucleic acid sequencing-based methods are instrumental and complex, which hampered their capability in revealing the extensive diversity of mtDNA. In order to realize trace DNA analysis, recent CRISPR/Cas-based detection methods facilitated with target pre-amplification, while raised the risks of non-specific amplification and cross-carryover contamination. Thus, it is imperative to develop new methods for sensitive and precise SNV detection in mtDNA.<br><br>RESULTS: This study developed a CRISPR/dCas9 (deactivated Cas9) mediated electrochemical impedimetric biosensor without target pre-amplification for sensitive and specific detection of SNVs in mtDNA. dCas9/sgRNA complexes were immobilized on the surface of indium tin oxide (ITO) electrode to specifically recognize target mtDNA sequences and initiate hybridization chain reaction (HCR) for signal amplification. Subsequently, positively charged polyethylenimine-coated silver nanoparticles (PEI-Ag NPs) were electrostatically deposited onto the HCR-generated long double-stranded DNA (dsDNA) products, leading to a marked decrease in electrochemical impedance due to the high conductivity of the nanoparticles. The concentration of mtDNA was thus quantified by monitoring the impedance change via electrochemical impedance spectroscopy (EIS). The method could distinguish single- and multi-base mismatches with a low detection limit of 67&#xa0;fM without pre-amplification. It exhibits excellent anti-interference ability and excellent recovery rates of 90.0% to 108.0% in complex matrices (10% human plasma), and enables accurate mtDNA detection in cell lysates.<br><br>SIGNIFICANCE: This free of pre-amplification strategy offers a highly sensitive analytical tool that enables the successfully detection of mtDNA mutation in diverse cells types, and exhibited excellent anti-interference ability in complicated biological specimen. The work presents a viable and promising strategy for the electrochemical detection of cancer-related biomarkers, indicating substantial potential in early clinical diagnosis.},
}
@article {pmid41905261,
year = {2026},
author = {Li, J and Liu, Y and Gao, T and Zhang, Y and Wang, Y and Wu, B},
title = {Centromere protein CEP170 is dispensable for mouse spermatogenesis and male fertility.},
journal = {Theriogenology},
volume = {259},
number = {},
pages = {117912},
doi = {10.1016/j.theriogenology.2026.117912},
pmid = {41905261},
issn = {1879-3231},
mesh = {Animals ; Male ; *Spermatogenesis/physiology/genetics ; Mice ; *Fertility/genetics/physiology ; Mice, Knockout ; Testis ; *Centrosomal Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Microtubule-Associated Proteins/genetics/metabolism ; Gene Expression Regulation/physiology ; },
abstract = {Spermatogenesis is crucial for male fertility and involves highly coordinated cellular differentiation, which depends on mitosis, meiosis, and cellular transformation processes that all require dynamic remodeling of the microtubule cytoskeleton. Microtubule anchoring, spindle assembly, and ciliary control all depend on centromere protein 170 (CEP170), which is found in the subdistal pole of the centrosome. CEP170 is regarded as a promising option for controlling male fertility because of its high expression in the testes and interactions with meiotic proteins. Given the embryonic lethality associated with complete CEP170 knockout, we generated germline-specific Cep170 knockout (KO) mice using CRISPR/Cas9 technology to investigate its role in male fertility. Surprisingly, our results show that Cep170 KO males remain fertile, producing litter sizes comparable to those of wild-type (WT) controls. Testis weight and histological architecture were unaffected. Computer-assisted sperm analysis (CASA) revealed no significant differences in sperm motility, concentration, or morphology between KO and WT mice. Moreover, detailed analysis of testicular sections using stage-specific markers for spermatogonia, spermatocytes, and spermatids showed no disruptions in germ cell proliferation, meiotic progression, or spermatogenesis. These findings indicate that, despite its high expression in the testis and established cellular functions, CEP170 is not essential for spermatogenesis or male fertility in mice. This study further refines the molecular network governing male fertility, particularly with respect to centrosome-associated proteins.},
}
@article {pmid41905426,
year = {2026},
author = {Jerin, C and Seo, W and Nishikawa, H},
title = {Genetic disruption of Pdcd-1 upstream enhancer boosts T cell function and antitumor responses.},
journal = {Immunology letters},
volume = {280},
number = {},
pages = {107171},
doi = {10.1016/j.imlet.2026.107171},
pmid = {41905426},
issn = {1879-0542},
mesh = {Animals ; *Programmed Cell Death 1 Receptor/genetics/metabolism ; Mice ; *Enhancer Elements, Genetic/genetics ; Mice, Knockout ; CRISPR-Cas Systems ; *T-Lymphocyte Subsets/immunology/metabolism ; Humans ; T-Cell Exhaustion ; *Neoplasms/immunology/genetics ; },
abstract = {Programmed cell death 1 (PD-1) is an inhibitory receptor that drives T cell exhaustion in tumors, limiting antitumor immunity. Current PD-1 blockade therapies have shown limited success. To uncover new strategies for modulating PD-1, we investigated an upstream enhancer (UpEnh) of the Pdcd-1 gene using a CRISPR-Cas9 knockout mouse model. Deletion of the UpEnh reduced PD-1 expression across various T cell subsets. In a tumor setting, this deletion lowered PD-1 levels on intratumoral exhausted CD8[+], conventional CD4[+], Treg, and γδ T cells. This resulted in improved CD8[+] and γδ T cell function and promoted stronger antitumor immunity. Our findings establish UpEnh as a critical regulator of PD-1, presenting a potential therapeutic target.},
}
@article {pmid41905499,
year = {2026},
author = {Fu, T and Wang, F and Ren, J and Chen, S and Peng, N and Zhang, L and Pu, K and Li, Q},
title = {An extraction-free HLPCR-Cas12a assay for ultra-sensitive and rapid detection of Acinetobacter baumannii and Klebsiella pneumoniae following craniotomy.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107480},
doi = {10.1016/j.mimet.2026.107480},
pmid = {41905499},
issn = {1872-8359},
mesh = {*Acinetobacter baumannii/isolation &amp; purification/genetics ; *Klebsiella pneumoniae/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Polymerase Chain Reaction/methods ; Humans ; *Craniotomy/adverse effects ; Bacterial Proteins/genetics ; CRISPR-Cas Systems ; *Acinetobacter Infections/diagnosis/microbiology ; *Klebsiella Infections/diagnosis/microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Postoperative intracranial infection represents a significant complication in neurosurgical procedures, with Acinetobacter baumannii (Ab) and Klebsiella pneumoniae (Kp) frequently identified as common drug-resistant pathogens. In response to this challenge, we have developed an efficient, extraction-free detection method that facilitates the rapid and highly sensitive identification of Ab and Kp. This method employs a single-tube format that integrates heat lysis with PCR, allowing for the concurrent bacterial lysis and target gene amplification. Detection of the amplified products is achieved through the Cas12a/crRNA complex, which, upon activation, exhibits trans-cleavage activity that cleaves reporters to produce a signal detectable by microplate readers or lateral flow test strips (LFTS). The Heat Lysis-PCR-CRISPR/Cas12a (HLPCR-CRISPR/Cas12a) detection platform unifies lysis and amplification in one step, streamlining the process. It exhibits high specificity (no cross-reactivity) and achieves detection limits of 10[2] CFU/μL (fluorescence) and 10[3] CFU/μL (LFTS). When validated on clinical CSF samples, it delivered results in 90 min with perfect concordance with quantitative PCR (qPCR). Combining speed, high sensitivity, specificity, and operational simplicity, this platform provides a powerful diagnostic tool for early detection of Ab and Kp in postoperative intracranial infections.},
}
@article {pmid41905621,
year = {2026},
author = {Singh, RP and Maity, P and Jaiswal, C},
title = {Genome editing in Parkinson's disease: Unlocking therapeutic avenues through CRISPR-Cas systems.},
journal = {Neurochemistry international},
volume = {196},
number = {},
pages = {106149},
doi = {10.1016/j.neuint.2026.106149},
pmid = {41905621},
issn = {1872-9754},
mesh = {Humans ; *Parkinson Disease/genetics/therapy ; *CRISPR-Cas Systems/genetics/physiology ; Animals ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; },
abstract = {Parkinson's disease (PD) is an illness that causes both motor and non-motor symptoms in the patient which occurs as a result of a progressive loss of dopamine-producing neurons in the substantia nigra. Even though the success of symptomatic treatments is promising, at the same time there is currently no effective therapy that can halt or reverse disease progression. Key genes such as SNCA, LRRK2, and PINK1 are considered as the main hopefuls aspect for the treatment of Parkinson's because mutations of these genes are the reason for the appearance of the familial and sporadic kinds of the disease, respectively. The CRISPR-Cas system, a breakthrough genome-editing technology which enables precise and targeted genetic modifications, renders the possibilities of both PD research and therapy. Examining the mechanics of prime editing, base editing, and CRISPR-Cas9 highlights how effective and precise these methods are for modifying genes. An overview of recent developments in the use of CRISPR to create PD models is also included in the current review, with a focus on the roles these models play in clarifying disease pathways and locating new treatment targets. These models include isogenic cell lines, transgenic animals, and induced pluripotent stem cells (iPSCs). This review highlights the potential of CRISPR-based strategies to correct PD-associated mutations, modulate pathogenic gene expression, and develop neuroprotective interventions targeting key processes such as mitochondrial dysfunction. Furthermore, it critically evaluates the role of CRISPR-based technologies as transformative tools in PD research and therapy while highlighting key challenges for their clinical translation.},
}
@article {pmid41908308,
year = {2026},
author = {Xu, G and Li, S and Li, H and Ren, X and Ding, Y and Pang, X and Liu, X and Tang, Q and Tu, T and Wang, Y and Luo, H and Yao, B and Tian, J and Chen, R and Guan, F},
title = {CasMiner: a deep-learning tool for high-throughput mining and rational design of efficient Cas9.},
journal = {National science review},
volume = {13},
number = {6},
pages = {nwag090},
pmid = {41908308},
issn = {2053-714X},
abstract = {Since its inception, the CRISPR-Cas system, particularly Cas9, has demonstrated immense potential for life science applications, but expansion of the Cas9 toolkit is constrained by sequence-alignment-based strategies for mining and optimization. Here, we developed CasMiner-a deep-learning model for discovering and engineering novel Cas9 proteins. CasMiner achieved 99.63% accuracy in predicting Cas9s and identified VpCas9 from public databases. Experimental validation showed that VpCas9 exhibits robust double-strand cleavage activity. Combining CasMiner and evolutionary analysis, we engineered three mutants with markedly increased structural rigidity and positive charge. In vivo cleavage assays revealed that the mutant VPM2-3 achieved a higher average editing efficiency in rice callus and maize protoplasts than the wild-type VpCas9, the editing efficiency of which rivals that of SpCas9. This study thus establishes a comprehensive platform for mining and engineering Cas9 proteins, and provides VpCas9 and derivative nucleases as powerful tools that greatly broaden the horizon for genome-editing applications.},
}
@article {pmid41909927,
year = {2026},
author = {Liu, H and Farmer, R and Payyavula, R and Kudithipudi, C and Timko, MP},
title = {Functional Characterisation of HAIRPLUS (NtHAP) Genes Involved in Trichome Development and Specialised Metabolism of Tobacco.},
journal = {Plant biotechnology journal},
volume = {24},
number = {7},
pages = {4640-4655},
pmid = {41909927},
issn = {1467-7652},
support = {GI15099//Altria Client Services, LLC/ ; },
mesh = {*Trichomes/metabolism/genetics/growth &amp; development ; *Nicotiana/genetics/metabolism/growth &amp; development ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *Genes, Plant ; RNA Interference ; Nicotine/metabolism ; Diterpenes/metabolism ; CRISPR-Cas Systems ; Phylogeny ; },
abstract = {Plant glandular trichomes are specialised epidermal structures capable of synthesising, storing and secreting numerous varieties of secondary metabolites in different classes and are central to plant defence and the biosynthesis of high-value metabolites. In this study, we characterised the HAIRPLUS (HAP) gene family and uncovered its role as a conserved regulator of trichome development and metabolism in tobacco. Four homologues, NtHAP1a, NtHAP1b, NtHAP2a and NtHAP2b, were identified and functional studies using RNAi and CRISPR-Cas9 revealed that NtHAPs act as negative regulators of glandular trichome development. Suppression of NtHAPs resulted in increased trichome density and enlarged glandular heads, as well as enhanced accumulation of diterpenoids (e.g., neophytadiene) and increased nicotine levels. Additionally, NtHAP1 appeared to have a stronger effect on trichome density. This study establishes the NtHAP genes as key negative regulators of glandular trichome development in tobacco, expanding their functional scope from trichome morphogenesis to metabolic regulation and highlighting their evolutionary conservation across Solanaceae. These findings pave the way for both fundamental research into trichome biology and practical applications in metabolic engineering and crop improvement, such as pest resistance.},
}
@article {pmid41909946,
year = {2026},
author = {Zhong, H and Zhou, J and Qin, F and Zhang, XE and Chen, M},
title = {Programmable, target-induced fluorogenic CRISPR-tDeg platform for live-cell RNA visualization.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909946},
issn = {1362-4962},
support = {2023YFA0915603//National Key R&amp;D Program of China/ ; 32101210//National Natural Science Foundation of China/ ; //Young Elite Scientist Sponsorship Program by CAST/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Viral/metabolism/genetics ; SARS-CoV-2/genetics ; Fluorescent Dyes/chemistry ; *RNA/metabolism ; Degrons ; HEK293 Cells ; },
abstract = {RNA molecules display remarkable heterogeneity in structure, dynamics, and function, yet methods for their precise visualization in living cells remain limited. While CRISPR-based RNA imaging holds great potential, existing systems often suffer from high background fluorescence due to constitutive signal emission or non-specific binding. To overcome these challenges, we developed CtDeg (CRISPR-dCas13-tDeg), a modular RNA imaging platform that links fluorescence activation directly to target RNA recognition while leveraging degron-mediated degradation to suppress background signals. By engineering the crRNA scaffold to embed the Pepper RNA motif, CtDeg ensures that fluorescence is present only upon binding to the native RNA target. We systematically optimized C-terminal tDeg variants to maximize the signal-to-noise ratio and demonstrated that CtDeg achieves substantially lower background and higher specificity than conventional fluorescent protein-CRISPR-based RNA imaging approaches. Using CtDeg, we captured real-time paraspeckle assembly dynamics and visualized early-stage SARS-CoV-2 genomic RNA transport. Remarkably, CtDeg provided the first direct imaging evidence of virus-induced NEAT1_2 lncRNA accumulation, revealing a host-virus regulatory interaction. Beyond these applications, CtDeg is compatible with multiple Cas13 orthologs and fluorescent proteins, establishing a versatile, target-induced platform for probing RNA localization, dynamics, and function in living cells, with broad applications in synthetic biology and RNA biology.},
}
@article {pmid41909947,
year = {2026},
author = {Hille, F and Wang, C and Finstermeier, K and Beerens, D and Schmidt, K and Skibbe, M and Charpentier, E},
title = {Insights into spacer acquisition of the type V-A CRISPR-Cas system of Francisella novicida U112.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909947},
issn = {1362-4962},
support = {//Alexander von Humboldt Foundation/ ; //German Research Foundation/ ; //Max Planck Society/ ; },
mesh = {*Francisella/genetics/enzymology ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Integrases/metabolism/genetics ; Escherichia coli/genetics ; DNA, Single-Stranded/metabolism ; },
abstract = {CRISPR-Cas systems immunize prokaryotic cells through a CRISPR adaptation process, in which short DNA fragments from foreign elements are acquired and integrated into the CRISPR array. Here, we investigated the spacer acquisition mechanism of the type V-A CRISPR-Cas system from Francisella novicida U112. We characterized the Cas1-Cas2 integrase in vitro and elucidated the sequence requirements of the pre-spacer and the CRISPR array for optimal spacer incorporation. We demonstrated that Cas2 coordinates metals at its active site to facilitate full-site spacer integration. Furthermore, we introduced this spacer acquisition system into Escherichia coli cells and observed that, in vivo, all Cas proteins are required for efficient type V-A adaptation, with Cas12 significantly improving adaptation efficiency. We showed that spacers were acquired preferentially from plasmids encoding cas genes, and from genomic regions of prophages and origins of replication. In addition, we found that Cas4 possesses a 3'-5' exonuclease activity against single-stranded DNA and an ATP-independent unwinding activity towards double-stranded DNA. Cas4 interacts with the Cas1-Cas2 complex and processes pre-spacers in a PAM-dependent manner. The presence of Cas4 in vivo ensures that new spacers are derived from DNA immediately downstream of a 5'-TTTN-3' PAM, which is critical for targeting invaders.},
}
@article {pmid41909950,
year = {2026},
author = {Deng, J and Zhou, J and Xiang, H and Li, X and Han, X and Weng, Z and Jia, J and Shao, Y and Sima, Y and Niu, M and Li, D and Ouyang, H and Xu, B and Pang, D and Yang, L and Yuan, H},
title = {Regulated transformation system (RTS): sddi-mediated programmable shut-off and mode switching of base editors.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909950},
issn = {1362-4962},
support = {20260102218JC//Jilin Provincial Science and Technology Development Plan Project/ ; 2025YFF1000800//National Key Research and Development Program of China/ ; 32202754//National Natural Science Foundation of China/ ; 32372962//National Natural Science Foundation of China/ ; 20230205117RC//Jilin Province science and technology development plan project/ ; },
mesh = {Humans ; *Gene Editing/methods ; Cytosine/metabolism ; Adenine/metabolism ; CRISPR-Cas Systems ; Animals ; },
abstract = {Orthogonal and externally controllable base editors are critical for safe multiplexed single-nucleotide manipulation in vivo. Here, we identify ~140-aa miniature deaminase inhibitors (Sddis) that bind cognate single-stranded DNA deaminases (Sdds) with high affinity and specificity, occluding their DNA-binding surfaces to completely inhibit C-to-T activity. Based on these inhibitors, we engineer an adenine and cytosine base editing-regulated transformation system (ACBE-RTS). This platform features two inactive dSdds fused to nCas9 as docking arms, with effector modules provided by doxycycline-inducible SviSddi-SflSdd (CBE) and cumate-inducible Air1Sddi-ABE8e (ABE) fusions. Small-molecule regulation enables switching among four modes (OFF, CBE, ABE, ACBE), achieving up to 43.4% C-to-T or 42.9% A-to-G editing at four endogenous human sites. Using a 4000-member sgRNA library in MARC-145 cells stably expressing ACBE-RTS, a three-round screening identified four key amino acids in monkey CD163 that reduced replication of highly pathogenic PRRSV by >100-fold and eliminated detectable viral-antigen staining. Compact and multi-mode switchable on a single Cas9 scaffold, ACBE-RTS establishes a versatile framework for precision therapeutics and genetic interrogation. Its modular Sddi-Sdd interface could in principle be readily extended to other base editors, such as thymine and guanine base editors (TBE and GBE).},
}
@article {pmid41910274,
year = {2026},
author = {Qiao, W},
title = {From CRISPR functional genomics to synthetic interventions: engineering antiviral strategies.},
journal = {Journal of virology},
volume = {100},
number = {4},
pages = {e0005726},
pmid = {41910274},
issn = {1098-5514},
support = {C1032633001//Shenzhen Bay Laboratory/ ; },
mesh = {Humans ; *Antiviral Agents/pharmacology ; *Genomics/methods ; *CRISPR-Cas Systems ; Animals ; *Synthetic Biology/methods ; Virus Replication/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Viruses/genetics/drug effects ; Host-Pathogen Interactions/genetics ; *Virus Diseases/virology/genetics ; },
abstract = {Virus-host interactions govern infection outcomes and viral evolution, but host determinants that enable or restrict viral replication have been difficult to map comprehensively and in the right cellular contexts. Pooled CRISPR perturbation screens now provide scalable, mechanistic entry points into host dependency and restriction landscapes across diverse viruses. Recent extensions, including single-cell readouts, imaging and spatial phenotyping, organoid models, and in vivo selection, are shifting the field from static hit lists toward contextual maps that explain how host pathways and cell states shape permissiveness. In parallel, synthetic biology is translating these maps into programmable intervention classes, including receptor decoys and binders that intercept entry, conditional protein depletion systems that modulate host factors with temporal control, gene circuits that couple infection sensing to tailored responses, and engineered immune cells with tunable antiviral functions. This review highlights conceptual and technical advances that connect CRISPR functional genomics to synthetic antiviral design, summarizes emerging principles that generalize across viral families, and discusses constraints that will determine whether screen-nominated mechanisms can be engineered into effective and safe antiviral strategies.},
}
@article {pmid41911023,
year = {2026},
author = {Ye, J and Tang, X and Chen, Y and Guo, R and Zhang, W and Wang, M and Yang, X and Deng, W and Zhang, Y and Feng, X and Hu, N and Zhang, D},
title = {Conformationally Regulated CRISPR/Cas12a Activation Enabled by a Programmable DNA Dumbbell for Electrochemical SNP Genotyping.},
journal = {Analytical chemistry},
volume = {98},
number = {14},
pages = {10814-10823},
doi = {10.1021/acs.analchem.6c00333},
pmid = {41911023},
issn = {1520-6882},
mesh = {*Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; *Electrochemical Techniques/methods ; *Genotyping Techniques/methods ; Genotype ; Biosensing Techniques/methods ; Nucleic Acid Conformation ; },
abstract = {Single-nucleotide polymorphism (SNP) genotyping is crucial for genetic research and precision medicine, yet reliable discrimination of single-base variants in complex genomic backgrounds remains analytically challenging. Although CRISPR/Cas12a-based biosensing offers high sequence specificity, its intrinsic mismatch tolerance often leads to nonspecific activation by wild-type sequences, thereby compromising SNP fidelity. Herein, we report a highly sensitive electrochemical sensing platform based on a programmable DNA dumbbell (Dum) probe that functions as a conformational energy-barrier regulator of CRISPR/Cas12a activation. The closed-loop dumbbell architecture sterically shields the crRNA-activating sequence, establishing a high activation threshold that suppresses nonspecific Cas12a triggering. Only precise SNP hybridization induces a thermodynamically favorable conformational rearrangement, releasing the mediator and transitioning the CRISPR system from an inactive to an active state. To further enhance analytical sensitivity and reliability, nucleic acid-functionalized FeCo nanozymes were incorporated as catalytic signal transducers, enabling a self-validating dual-mode signal electrochemical readout through intrinsic metal redox and H2O2 electrocatalysis. The resulting platform achieved reliably discriminates mutation abundances down to 0.1%. Validation using soybean genomic DNA samples demonstrates the robustness and practical applicability of the proposed strategy. This work establishes a conformational energy-barrier-regulated CRISPR activation paradigm, providing a generalizable analytical framework for high-fidelity SNP genotyping in molecular breeding and clinical diagnostics.},
}
@article {pmid41911152,
year = {2026},
author = {Wu, Y and Qi, Y and Chen, Y and Liu, D and Liu, Q and Shang, X},
title = {CrisprPr: a hybrid-driven framework for CRISPR/Cas9 off-target prediction with analysis of prior-information updates.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {2},
pages = {},
pmid = {41911152},
issn = {1477-4054},
support = {62433016//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; Prediction Algorithms ; Deep Learning ; *Gene Editing/methods ; Humans ; },
abstract = {CRISPR/Cas9 specificity is critically affected by off-target effects. However, the complex patterns of mismatches and their combinations at off-target sites remain difficult to capture, and existing approaches show limited capacity to identify informative features. Here, we present CrisprPr, a hybrid-driven off-target prediction framework that integrates both prior information and data-driven modeling to improve the characterization of off-target activity. CrisprPr employs a synchronous updating strategy that jointly optimizes prior-knowledge and deep-learning modules, together with multi-source integration, to deliver accurate and stable off-target predictions. Evaluations on independent test sets indicate that CrisprPr achieves competitive predictive performance and generalization compared with existing deep learning methods, with statistically significant improvements observed on several datasets. Beyond predictive performance, its analysis module examines the patterns of prior embedding-space updates to reveal distinctive target-site features supported by literature evidence. Overall, CrisprPr proposes a novel framework that demonstrates competitive predictive performance while offering new insights into the characteristics of off-target effects.},
}
@article {pmid41911342,
year = {2026},
author = {Chi, H and McMahon, SA and Graham, S and White, MF},
title = {The CRISPR ring nuclease Csx15 oligomerises on cyclic nucleotide binding to regulate antiviral defence.},
journal = {The Biochemical journal},
volume = {483},
number = {5},
pages = {699-712},
pmid = {41911342},
issn = {1470-8728},
support = {101018608//EC | European Research Council (ERC)/ ; },
mesh = {*Nucleotides, Cyclic/metabolism/chemistry ; Protein Multimerization ; *CRISPR-Cas Systems ; Protein Binding ; Binding Sites ; },
abstract = {Prokaryotic type III CRISPR systems signal infection by generating cyclic oligoadenylate (cOA) second messengers, which activate defence proteins allosterically, providing immunity. cOA molecules are typically degraded by extrinsic, stand-alone ring nuclease (RN) enzymes with phosphodiesterase activity or by the intrinsic RN activity of the effectors themselves. Viruses and plasmids also encode RNs, which can function as anti-CRISPRs. Eight different families of extrinsic RNs are currently known. Here, we report the structural and biochemical analysis of one of these families: Csx15. We show that Csx15 is a dimeric protein of the CRISPR-associated Rossmann fold (CARF) superfamily with the ability to bind cyclic tetra-adenylate (cA4) molecules in a shared binding site formed by the head-to-tail stacking of dimers in a filament conformation. Some family members are non-enzymatic, relying on the sequestration (sponging) of cA4 to regulate the host immune response, while others act as canonical RNs, slowly degrading cA4.},
}
@article {pmid41911457,
year = {2026},
author = {Waild, NC and Ciesla, JH and Schafer, XL and Munger, J},
title = {Cellular antibody affinity-based CRISPR screening identifies JUNB as a broadly acting antiviral factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {14},
pages = {e2534154123},
pmid = {41911457},
issn = {1091-6490},
support = {AI150698//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI118689//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI184380/AI/NIAID NIH HHS/United States ; GM068411//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 AI150698/AI/NIAID NIH HHS/United States ; R01 AI181865/AI/NIAID NIH HHS/United States ; AI181865//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Cytomegalovirus/genetics/immunology ; *Transcription Factors/genetics/metabolism ; Virus Replication ; *Cytomegalovirus Infections/genetics/virology/immunology ; CRISPR-Cas Systems ; Viral Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR screening is a powerful approach to identify genetic perturbations that impact viral infection. However, most virus-focused CRISPR screens utilize selection strategies that limit the ability to identify genes important for infection. Here, we developed a CRISPR screening pipeline to identify cellular determinants of human cytomegalovirus (HCMV) infection based on virally induced remodeling of cellular antibody affinity (VIRCAA), which is scalable for large libraries and can identify cellular genes that impact HCMV infection at different life cycle stages. We utilized this pipeline to interrogate proteomic and transcriptomic datasets associated with the HCMV UL26 protein, which blocks antiviral signaling during infection. We find that JUNB drives antiviral gene expression, induces protein ISGylation, and suppresses diverse viral infections. Further, UL26 interacts with JUNB and suppresses JUNB-mediated condensation of viral DNA replication compartments. These results highlight the VIRCAA pipeline's utility for identifying important determinants of viral infection.},
}
@article {pmid41911458,
year = {2026},
author = {Richards, L and Lee, D and Wiktor, J and Truedson, A and Cederblad, J and Jones, D},
title = {Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {14},
pages = {e2525424123},
pmid = {41911458},
issn = {1091-6490},
support = {2020-05137//Swedish Research Council/ ; CTS 21:1334//Carl Tryggers Foundation/ ; },
mesh = {*Plasmids/genetics ; Kinetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Conjugation, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Understanding and manipulating the spread of mobile genetic elements represents a great challenge with potential benefits across synthetic biology, agriculture, and medicine. A key part of this challenge is the multiple scales in play, from the molecular kinetics of defense systems such as CRISPR-Cas, to single-cell variability in immunity levels, to spatial structure in bacterial populations. In this work, we use a time-lapse, imaging-based approach to characterize conjugative plasmid dynamics at the molecular, single-cell, and population levels. By fluorescently tagging the conjugative plasmid RP4 and CRISPR-Cascade complexes, we quantify population dynamics as a function of spacer target number, Cascade expression level, and the presence of plasmid addiction modules. Using single-cell tracking, we report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and quantify the effect of Cascade expression variability on plasmid clearance kinetics. Finally, using a spatially resolved, agent-based model, we show that plasmid population dynamics can be successfully predicted using these single-cell biophysical parameters as inputs. This synthesis of population and single-cell measurements suggests that plasmids are the subject of a dynamic tug-of-war between defense expression, spacer distribution, neighboring cell identity, and plasmid cost-benefit tradeoffs. The imaging and analysis techniques used here will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.},
}
@article {pmid41912937,
year = {2026},
author = {Zhang, D and Wang, H},
title = {Liquid Biopsy in Uterine Leiomyosarcoma: Current Biomarkers, Emerging Technologies, and Future Perspectives.},
journal = {Current oncology reports},
volume = {28},
number = {1},
pages = {},
pmid = {41912937},
issn = {1534-6269},
mesh = {Humans ; Female ; *Leiomyosarcoma/diagnosis/pathology/genetics/blood ; Liquid Biopsy/methods ; *Biomarkers, Tumor/blood/genetics ; *Uterine Neoplasms/pathology/diagnosis/genetics/blood ; Neoplastic Cells, Circulating/pathology ; Circulating Tumor DNA/blood ; },
abstract = {PURPOSE OF REVIEW: Uterine leiomyosarcoma (uLMS) is a rare but aggressive malignant mesenchymal tumor, accounting for 2-5% of uterine malignancies. Because its symptoms and imaging features often resemble those of benign uterine leiomyoma (LM), accurate preoperative diagnosis remain difficult. This review summarizes recent advances in liquid biopsy for uLMS and explores its potential for early detection, molecular characterization, and treatment monitoring.<br><br>RECENT FINDINGS: Liquid biopsy enables minimally invasive detection of tumor-derived components such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), non-coding RNAs, and extracellular vesicles (EVs). Recurrent mutations in TP53, RB1, and ATRX have been identified through ctDNA analysis, while CTCs, ncRNAs, and EVs provide complementary information for monitoring tumor dynamics and therapeutic response. Emerging technologies including CRISPR-Cas systems, nanotechnology, electrochemical biosensors, and multi-omics integration enhance detection sensitivity and specificity. Liquid biopsy holds promise for improving uLMS diagnosis and management. However, standardization and biomarker validation remain essential to achieve reliable clinical translation and enable earlier, more precise treatment strategies.},
}
@article {pmid41913088,
year = {2026},
author = {Zhang, S and Sun, W and Xiao, T and Wang, Y and Wu, X and Chen, H and Chen, M and Zhang, J},
title = {TopCas: Topology-Gated Cas12a via DNA-RNA Chimeric Circular crRNA for Amplification-Free Nucleic Acid Detection and Conditional Gene Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {33},
pages = {e75046},
pmid = {41913088},
issn = {2198-3844},
support = {2025ZD0551200//National Science and Technology Major Project on the Prevention and Treatment of Cancers, Cardiovascular and Cerebrovascular Diseases, Respiratory and Metabolic Diseases/ ; 2022YFC2603800//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; RNA/genetics ; *DNA/genetics ; *Bacterial Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {Controlled activation of CRISPR-Cas12a is critical for achieving conditional gene editing and molecular diagnostics. As an indispensable component for forming an active complex, CRISPR RNA (crRNA) represents a key route to regulate Cas12a activity. Here, we establish TopCas (Topology-gated Cas12a via DNA-RNA Chimeric Circular crRNA) as a platform for preamplification-free nucleic acid detection and conditional gene editing. Within TopCas, the circular crRNA sterically constrains Cas12a's nuclease activity until target-activated complexes trans-cleave the DNA segment of the chimeric crRNA, converting the circular guide into its linear form and initiating an autocatalytic cascade that culminates in fluorophore release and signal amplification. By the same mechanism, the system conditionally activates Cas12a's gene-editing function (cis-cleavage) exclusively in the presence of specific nucleic acid targets (e.g., viral DNA or RNA). We demonstrate that TopCas affords high specificity and sensitivity in nucleic acid detection, supports accurate detection in clinical viral nucleic acid samples, and shows potential for in vivo real-time molecular imaging, while also demonstrating the feasibility of conditional gene editing. This innovative chimeric circular crRNA-Cas12a system not only provides a new tool for precise disease diagnostics but also offers a promising strategy for personalized therapeutic intervention.},
}
@article {pmid41913646,
year = {2026},
author = {Streiber, M and Liu, N and Simon, L and Adermann, F and Bachmann, V and Gath, L and Hoeppener, S and Schubert, S and Werz, O and Lapinte, V and Morille, M and Bauer, M and Press, AT and Schubert, US and Traeger, A},
title = {Extrahepatic Gene Editing In Vivo Using Organic Solvent-Free Lipid Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {27},
pages = {e11489},
pmid = {41913646},
issn = {1613-6829},
support = {13XP5034A//Bundesministerium f&#xfc;r Bildung und Forschung/ ; 03RU2U071H//Bundesministerium f&#xfc;r Bildung und Forschung/ ; 2021 FGI 0005//Th&#xfc;ringer Aufbaubank/ ; 2023FGR0077//Th&#xfc;ringer Aufbaubank/ ; //Joachim Herz Stiftung/ ; P2024-02-016//Carl-Zeiss-Stiftung/ ; 57604510//German Academic Exchange Service/ ; 2018FGI0025//European Regional Development Fund/ ; ANR- 20-CE09-0011-01//Agence Nationale de la Recherche/ ; 514006196//Deutsche Forschungsgemeinschaft/ ; 316213987//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Nanoparticles/chemistry ; *Lipids/chemistry ; Humans ; Animals ; *Gene Editing/methods ; Polyethylene Glycols/chemistry ; *Solvents/chemistry ; CRISPR-Cas Systems/genetics ; },
abstract = {Targeted therapy, which modifies genes and their expression, holds great promise for treating a variety of diseases, including cancer, inborn errors of metabolism, and acute and chronic inflammatory and infectious conditions. However, it also presents challenges related to RNA delivery, immune responses, side effects of delivery vectors, and the need for individualized formulations. To overcome these limitations, the choice of lipids and formulation processes might be re-evaluated, with a focus on eliminating critical components, such as poly(ethylene glycol) (PEG) and ethanol. Thus, a purely water-based formulation for lipid nanoparticles was developed, offering a material-efficient, time-saving process with high reproducibility. Initially, a stealth lipid containing poly(2-methyl-2-oxazoline) (PMeOx) was used, and the formulation was later expanded to include approved lipids. These nanoparticles not only efficiently transfect primary human immune cells but also effectively deliver multiple nucleotides in CRISPR-Cas9 applications. Moreover, an in vivo comparison revealed that the nanoparticles exhibited preferential transfection in extrahepatic tissues. This distinguishes them from conventional cholesterol-rich lipid nanoparticles, which primarily target the liver regardless of the application route.},
}
@article {pmid41914300,
year = {2026},
author = {Wang, W and Li, L and Li, X and Zhang, Q and Liu, Y},
title = {Aptamer- and Ribozyme-Engineered sgRNAs for Conditional Control of CRISPR/Cas9 Function.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {31},
number = {3},
pages = {47300},
doi = {10.31083/FBL47300},
pmid = {41914300},
issn = {2768-6698},
support = {2024B02//Fund for Creative Research of The Second People's Hospital of Foshan/ ; 4SG24185G//Postdoctoral Initial Foundation of Guangdong Medical University/ ; },
mesh = {*RNA, Catalytic/genetics ; *CRISPR-Cas Systems/genetics ; *Aptamers, Nucleotide/genetics ; Genetic Engineering/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Synthetic Biology/methods ; },
abstract = {The clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as a versatile platform for genome editing, transcriptional regulation, and chromosomal imaging. Recent advances in synthetic biology have enabled the engineering of single guide RNA (sgRNA) to confer conditional responsiveness on the CRISPR/Cas9 system. By integrating functional nucleic acid elements, such as aptamers, ribozymes, and aptazymes, into specific structural regions of the sgRNA, researchers have developed systems that respond to a variety of molecular signals, including small molecules, proteins, and endogenous metabolites. These engineered sgRNAs enable spatiotemporal control of gene editing, activation, repression, and imaging in both prokaryotic and eukaryotic cells. This review summarizes the structural principles, design strategies, and applications of condition-responsive CRISPR/Cas9 systems, highlighting their potential in synthetic biology, disease modeling, and therapeutic development. Current challenges and future directions for improving the specificity, efficiency, and applicability of these systems are also discussed.},
}
@article {pmid41914367,
year = {2026},
author = {Lu, Q and Ye, C and Chen, R and Xing, Z and Liu, Z and Zeng, F and Gong, J and Gao, Y and Sun, X and Tang, S and Song, Y},
title = {High-Entropy Alloy Synergized with Gene Editing for Cocktail-Sensitized Radioimmunotherapy of Lung Metastases.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {25},
pages = {e22618},
doi = {10.1002/adma.202522618},
pmid = {41914367},
issn = {1521-4095},
support = {22477056//National Natural Science Foundation of China/ ; 82272138//National Natural Science Foundation of China/ ; 22375089//National Natural Science Foundation of China/ ; 82572370//National Natural Science Foundation of China/ ; 2024300315//Fundamental Research Funds for the Central Universities/ ; 2019YFA0709200//National Key R&amp;D Program/ ; BE2021373//Jiangsu Province Key R&amp;D Program/ ; 5431ZZXM2304//State Key Laboratory of Analytical Chemistry for Life Science/ ; },
mesh = {Cell Line, Tumor ; Animals ; *Lung Neoplasms/secondary/radiotherapy/pathology/therapy ; *Alloys/chemistry ; Humans ; *Radioimmunotherapy/methods ; *Gene Editing ; Mice ; Palladium/chemistry ; CRISPR-Cas Systems ; },
abstract = {Radiotherapy (RT) eliminates cancer cells either through direct DNA damage induced by ionizing radiation or indirectly by generating cytotoxic reactive oxygen species (ROS) via radiolysis. However, high-dose radiation often triggers DNA repair mechanisms, undermining therapeutic efficacy and causing damage to surrounding healthy tissues. Thus, enhancing anti-tumor effects at lower doses while minimizing normal tissue damage and improving safety remains a key challenge in advancing RT technologies. To tackle these issues, we developed an RT-sensitizing platform, referred to as HAEPRC, which integrates a novel high-entropy alloy (HEA) composed of gold (Au), bismuth (Bi), platinum (Pt), silver (Ag), and palladium (Pd), a CRISPR/Cas9 gene-editing system, and tumor cell membranes (CM) for enhanced home-targeting and biocompatibility. We demonstrated that HAEPRC exhibits exceptional dose enhancement factors (DEFs), significantly boosting RT sensitization and improving RT-induced immunotherapeutic outcomes. Furthermore, the gene-editing system modulates the cell cycle, transforming RT-resistant cancer cells into RT-sensitive ones and further amplifying RT efficacy. Additionally, Pd-mediated bioorthogonal catalysis activates immune adjuvant production, enhancing immune responses and reinforcing anti-tumor immunity. Collectively, these features synergistically promote an enhanced abscopal immune effect, inhibiting lung cancer growth and metastasis, and providing a promising strategy to improve the efficacy and safety of RT.},
}
@article {pmid41914500,
year = {2026},
author = {Liu, H and Jiao, K and Hao, A and Li, C and Hou, J and Lu, X and Liu, M and Qi, Q},
title = {Enhancement of single-stranded template annealing activity by Rad52 during repair of CRISPR-induced dsDNA breaks.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41914500},
issn = {1362-4962},
support = {U23A20268//National Natural Science Foundation of China/ ; ZR2025MS440//Shandong Provincial Natural Science Foundation/ ; SKLMTFCP-2023-03//SKLMT Frontiers and Challenges Project/ ; },
mesh = {*Rad52 DNA Repair and Recombination Protein/metabolism/genetics ; *DNA Breaks, Double-Stranded ; Yarrowia/genetics ; *DNA, Single-Stranded/metabolism/genetics ; DNA End-Joining Repair ; *CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Single-strand annealing protein (SSAP)-mediated recombination engineering has become a powerful tool for bacterial genome editing. However, in most eukaryotes, its efficiency is constrained by the dominant non-homologous end joining (NHEJ) repair pathway and the limited activity of exogenous SSAPs. Here, in the typically NHEJ-dominated yeast Yarrowia lipolytica, we found that 18.7% of Cas9-induced double-strand breaks (DSBs) were precisely repaired upon provision of single-stranded oligonucleotide templates, even in the absence of recombinase overexpression, indicating the presence of an endogenous eukaryotic SSAP-mediated recombination activity. Overexpression of recombination-related proteins revealed that Rad52 plays a key role in single-strand annealing. Structural truncation of Rad52(1-300) boosted genome-editing efficiency to 96.3%, comparable to that achieved by disrupting NHEJ via Ku70 deletion. Our ESTAR platform (enhancement of single-stranded template annealing activity by Rad52) enables precise and efficient genome editing, including small-fragment insertions, deletions, and replacements, as well as large-fragment deletions exceeding 20 kb. This gene-editing technology was further validated in Saccharomyces cerevisiae and other non-conventional yeast, offering new insights into the single-stranded DNA annealing step during the repair of Cas9-induced DSBs.},
}
@article {pmid41914542,
year = {2026},
author = {He, S and Connerty, P and de Weck, A},
title = {CRISPR-Cas9 Genome-Wide Screening in Paediatric Cancer: Functional Genomics for Target Discovery and the Improvement of Existing Therapies.},
journal = {Medicinal research reviews},
volume = {46},
number = {4},
pages = {989-1004},
doi = {10.1002/med.70046},
pmid = {41914542},
issn = {1098-1128},
support = {RG23-08//Cancer Council NSW/ ; RG212077//Cancer Institute NSW/ ; 2021/CBG003//Cancer Institute NSW Research Capacity Building/ ; //Sydney Partnership for Health, Education, Research and Enterprise/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Genomics/methods ; Drug Discovery ; Child ; Animals ; *Genome-Wide Association Study ; },
abstract = {CRISPR-Cas9 genome-wide screening has been instrumental towards identifying novel targets for drug discovery in cancer research. However, much of this research has centred specifically on adult cancers, with paediatric cancers being underserviced by current research and screening. With contemporary evidence increasingly highlighting the differences in biology between adult and children's cancers, more research has gradually occurred investigating dependencies and mechanisms of resistance in paediatric cancers through the use of CRISPR-Cas9 genome-wide screens. This review collates and summarises the experimental genome-wide screens that have been performed specifically in paediatric cancer models, and highlights the versatility of this technology and the knowledge gained through this research.},
}
@article {pmid41914596,
year = {2026},
author = {Ma, B and Wang, X and Cai, X and He, M and Lai, S and Zhang, P and Xu, J},
title = {Isothermal amplification and CRISPR/Cas one-pot detection systems: strategies and prospects.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {14},
pages = {2824-2837},
doi = {10.1039/d5ay01976a},
pmid = {41914596},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Rapid Diagnostic Tests ; Point-of-Care Systems ; },
abstract = {Molecular diagnostic techniques, known for their high sensitivity and specificity, are now the gold standard for detecting pathogen nucleic acids and are crucial in precision medicine. Traditional methodologies, including polymerase chain reaction (PCR) and gene sequencing, offer exceptional analytical precision and reliability. Nevertheless, their applicability for point-of-care testing (POCT) is limited due to the high cost of equipment, prolonged detection durations, and reliance on controlled laboratory settings. The CRISPR/Cas system is considered as the next-generation of nucleic acid-based molecular diagnostic techniques, attributed to its highly specific recognition of target nucleic acids, programmability, high sensitivity, and suitability for POCT. The one-pot CRISPR assay integrates the pre-amplification of DNA or RNA with CRISPR/Cas detection into a single reaction system. This consolidation streamlines experimental workflows, enhances the practicality of POCT, and minimizes the risk of aerosol contamination. Consequently, this method has received tremendous attention from researchers. However, challenges remain due to incompatibilities between isothermal amplification and the CRISPR/Cas system, including mutual interference, temperature mismatches, and buffer incompatibility. This paper reviews recent advancements in one-pot CRISPR/Cas detection strategies, highlighting potential solutions such as physical isolation, microfluidic integration, optimization of the reaction systems, modification of crRNA and PAM sequences and engineering of nucleases. Additionally, it explores the potential applications and challenges associated with these systems in the development of high-performance POCT platforms.},
}
@article {pmid41915142,
year = {2026},
author = {Gottipamula, S and Seetharam, RN and Nk, V},
title = {"Primed for Repair: Harnessing Hypoxia, Mechanobiology, and Gene Editing to Enhance MSC Potency and Clinical Translation".},
journal = {Stem cell reviews and reports},
volume = {22},
number = {4},
pages = {1782-1799},
pmid = {41915142},
issn = {2629-3277},
}
@article {pmid41915438,
year = {2026},
author = {Ma, Z and Jia, W and Zhou, X and Liu, J and Li, Q and Chang, R and Shiqi, G and Yuan, N and Chen, Z and Lan, P},
title = {In vivo CRISPR screens identify CBX4 as an epigenetic regulator for cancer immunotherapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {41915438},
issn = {1558-8238},
mesh = {Animals ; Mice ; Humans ; *Epigenesis, Genetic/immunology ; *Immunotherapy ; *CRISPR-Cas Systems ; Tumor Microenvironment/immunology/genetics ; *Polycomb-Group Proteins/genetics/immunology ; *Neoplasm Proteins/genetics/immunology ; Female ; },
abstract = {Epigenetic dysregulation is associated with immune evasion and immune checkpoint blockade (ICB) resistance. Here, using in vivo CRISPR/Cas9 screens targeting epigenetics-related factors in mouse tumor models treated with ICB, we identified chromobox 4 (CBX4) as a key negative regulator of the immune tumor microenvironment (TME). Single-cell RNA-seq and spatial transcriptomics analyses of patients receiving neoadjuvant anti-programmed cell death protein 1 (anti-PD-1) therapy revealed high CBX4 expression in both tumor cells and immunosuppressive tumor-associated macrophage subpopulations, with preferential accumulation in nonresponders. Deficiency of CBX4 in macrophages or tumor cells induced robust antitumor immunity and increased infiltration and the cytotoxic activity of CD8+ T cells and NK cells, thereby heightening the sensitivity of ICB treatment. Mechanistically, CBX4 targeted H3K9me3- and H3K27me3-marked endogenous retroelements such as RLTR4-Mm-int. Loss of CBX4 derepressed retrotransposons, activating cytosolic RNA-sensing pathways and triggering the type I IFN response, ultimately leading to a robustly inflamed TME. Moreover, we uncovered a negative correlation between CBX4 expression, immune responses, and retrotransposon levels, and were able to determine the prognosis of patients with hepatocellular carcinoma (HCC) undergoing ICB therapy. Our study establishes CBX4 as an epigenetic immune checkpoint through the epigenetic silencing of retrotransposons, remodeling the immune TME and thus providing a promising therapeutic target to enhance tumor immunogenicity and overcome immunotherapy resistance.},
}
@article {pmid41915857,
year = {2026},
author = {Ahmad, Z and Ramakrishnan, M and Varshney, RK and Shahzad, A and Rehman, S and Pant, B and Wei, Q},
title = {WUSCHEL Transcription Factor: From Stem Cell Maintenance to Crop Improvement.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {24},
pages = {e19705},
pmid = {41915857},
issn = {2198-3844},
support = {32471977//National Natural Science Foundation of China/ ; 32071848//National Natural Science Foundation of China/ ; BK20231289//Natural Science Foundation of Jiangsu Province/ ; Y20240114//Young Foreign Talent Program/ ; QN2022014012L//Young Foreign Talent Program/ ; 163100036//Metasequoia Faculty Research Start-Up Funding/ ; 163100028//Metasequoia Faculty Research Start-Up Funding/ ; JC2019004//Natural Science Foundation For Distinguished Young Scholars of Nanjing Forestry University/ ; 202211//Project for Ground breaking Achievements of Nanjing Forestry University/ ; },
mesh = {*Homeodomain Proteins/genetics/metabolism ; *Crops, Agricultural/genetics/growth &amp; development ; *Stem Cells/metabolism/physiology ; *Arabidopsis Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant/genetics ; Arabidopsis/genetics ; },
abstract = {The WUSCHEL (WUS) transcription factor, long recognized as a master regulator of stem cell maintenance in the shoot apical meristem (SAM), has expanded in significance as a multifaceted tool in plant biotechnology. With an emphasis on its new uses in crop regeneration, somatic embryogenesis (SE), stress tolerance, and developmental regulation in cereals, legumes, and other plant species, this review summarizes recent developments on WUS function outside of Arabidopsis. We emphasize how insights from WUS biology can be translated into practical strategies to improve yield, adaptability, and resilience, while also enhancing in vitro tissue culture systems. The objective of this review is to establish WUS as a crucial molecular target for future crop genetic improvement and sustainable farming methods by highlighting the current knowledge gaps and suggesting future directions.},
}
@article {pmid41916134,
year = {2026},
author = {Buakaew, T and Thaiwong, R and Inthanachai, T and Palaga, T and Weissman, D and Suppipat, K and Ausavarungnirun, C and Tawinwung, S},
title = {Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {198},
number = {},
pages = {119300},
doi = {10.1016/j.biopha.2026.119300},
pmid = {41916134},
issn = {1950-6007},
mesh = {Humans ; *RNA, Messenger/genetics ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes/immunology/metabolism ; Cryopreservation ; *Receptors, Chimeric Antigen/genetics/immunology ; *Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell/genetics/immunology ; Cell Survival ; *Gene Knockout Techniques ; Transfection ; },
abstract = {BACKGROUND: Autologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in hematologic malignancies but remain limited by complex manufacturing processes. Allogeneic, off-the-shelf CAR T cells derived from healthy donors represent a promising alternative; however, safe implementation requires elimination of endogenous T-cell receptor (TCR) expression and flexible CAR expression strategies.<br><br>OBJECTIVE: This study aimed to develop an optimized manufacturing workflow for allogeneic CAR T cells by combining CRISPR/Cas9-mediated TCR knockout with mRNA-based CAR expression, and to evaluate cryopreservation strategies enabling on-demand CAR T-cell generation.<br><br>METHODS: Healthy donor T cells were edited at the TRAC locus using CRISPR/Cas9 to generate TCR-deficient T cells. These cells were cryopreserved and subsequently transfected with mRNA encoding CD117, BCMA, or CD19 CARs. CAR expression, cell viability, immunophenotype, cytokine secretion, and antigen-specific cytotoxicity were assessed under different cryopreservation-transfection conditions.<br><br>RESULTS: TCR knockout T cells exhibited efficient TCR disruption with reduced alloreactive proliferation. CD117 mRNA CAR T cells derived from TCR-deficient T cells demonstrated CAR expression kinetics, immunophenotypic profiles, and antigen-specific cytotoxicity comparable to wild-type CAR T cells. Evaluation of two cryopreservation strategies revealed that cryopreservation prior to mRNA electroporation preserved cell viability, phenotype, and cytotoxic function, whereas cryopreservation after mRNA transfection was associated with reduced functional activity. The optimized protocol was successfully extended to CD19- and BCMA-targeting CAR mRNAs.<br><br>CONCLUSION: Collectively, these findings establish a modular platform for producing allogeneic CAR T cells using mRNA technology, offering a practical approach for rapid, on-demand CAR T-cell therapy.},
}
@article {pmid41916193,
year = {2026},
author = {Luo, Y and Vallone, VF and Blanc, E and Miller, DC and He, J and Stachelscheid, H and Beule, D and Scheffel, J and Siebenhaar, F},
title = {CRISPR/Cas9 engineered and whole-genome characterized KIT D816V-mutant human iPSC lines.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103975},
doi = {10.1016/j.scr.2026.103975},
pmid = {41916193},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Proto-Oncogene Proteins c-kit/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mutation ; *Genome, Human ; Cell Line ; Whole Genome Sequencing ; },
abstract = {We report on the generation of human induced pluripotent stem cell (iPSC) lines, BIHi005-A-86 and BIHi005-A-87, carrying the KIT D816V mutation associated with Indolent Systemic Mastocytosis (ISM). To overcome the confounding genetic backgrounds of existing leukemic models, we introduced this gain-of-function mutation into the healthy BIHi005-A line using CRISPR/Cas9 editing. The resulting clones were validated via whole-genome sequencing (WGS) to confirm specific on-target editing and lack of predicted or disease-relevant off-target effects, while maintaining genomic stability. Together with the parental line, this resource provides an isogenic controlled platform for investigating KIT D816V-driven pathogenesis.},
}
@article {pmid41917016,
year = {2026},
author = {Gong, X and Pożoga, M and Boyer, JB and Xue, Y and Meinnel, T and Bange, T and Giglione, C and Hell, R and Wirtz, M},
title = {The ribosome-associated N-terminal acetyltransferase B coordinates global proteostasis and autophagy in plants by creating Ac/N-degrons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41917016},
issn = {2041-1723},
support = {496871662//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 544882710//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 5041140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ANR-17-CAPS-0001-01//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-20-CE92-0040//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*Autophagy/genetics ; *Proteostasis ; Acetylation ; *Arabidopsis Proteins/metabolism/genetics ; Degrons ; *Arabidopsis/genetics/metabolism/enzymology ; *N-Terminal Acetyltransferase B/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; *Ribosomes/metabolism ; Ubiquitin/metabolism ; Proteome/metabolism ; Mutation ; Proteomics ; CRISPR-Cas Systems ; },
abstract = {The N-terminal acetyltransferase B (NatB) acetylates ~20% of the eukaryotic proteome. However, the role of NatB-mediated N-terminal acetylation (NTA) for the regulation of the proteome fate remains unclear in eukaryotes. In this study, we demonstrate that CRISPR-Cas9-mediated deletion of NatB activity in plants results in significantly lowered global protein turnover due to decreased ubiquitin-proteasome system (UPS) activity and protein translation. Quantitative proteomics uncovers that NatB substrates are significantly enriched in the fraction of stabilized proteins in natb mutants. We provide direct evidence that the absent NTA of KIN11, a subunit of the autophagy-controlling energy sensor SnRK1, protects it from UPS-mediated destruction. The resulting accumulation of KIN11 is responsible for the increased resistance of natb mutants to energy limitation induced by prolonged darkness. Our findings establish NatB as a central regulator of UPS-autophagy interplay and highlight its role in maintaining proteome stability and enabling dynamic stress responses in plants.},
}
@article {pmid41917051,
year = {2026},
author = {Lu, J and Lai, J and Cheng, L and Zhan, H and Jie, K and Liu, C and Huang, L and Cen, M and Liu, S and Chen, Z and Zhang, Q and Zhang, J and Wu, J and Pan, B and Chen, S and Zhong, J and He, B and Li, H and Chen, X and Lin, T},
title = {Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41917051},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Immunotherapy/methods ; CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; Dependovirus/genetics ; *Neoplasms/therapy/immunology/genetics ; Gene Editing/methods ; T-Lymphocytes/immunology ; },
abstract = {CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.},
}
@article {pmid41917262,
year = {2026},
author = {Shi, R and Yang, M and Liu, Y and Gao, H and Lin, Z},
title = {Mechanistic basis for selective Csm6-2 activation by cyclic penta-adenylate in a type III CRISPR-Cas system.},
journal = {The EMBO journal},
volume = {45},
number = {10},
pages = {3416-3429},
pmid = {41917262},
issn = {1460-2075},
support = {32471255//MOST | National Natural Science Foundation of China (NSFC)/ ; 32271258//MOST | National Natural Science Foundation of China (NSFC)/ ; 2024J02006//| Natural Science Foundation of Fujian Province (Fujian Natural Science Foundation)/ ; },
mesh = {*CRISPR-Cas Systems ; *Adenine Nucleotides/metabolism ; *Oligoribonucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Cryoelectron Microscopy ; *Ribonucleases/metabolism/chemistry/genetics ; },
abstract = {Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the effectors specific to cA5-one of the most abundant cOA species produced during phage infection-have remained unexplored. Here, we report that the CRISPR ribonuclease Csm6 (Csm6-2) from Actinomyces procaprae selectively employs cA5 as its activator. Csm6-2 utilizes its HEPN domain, rather than the CARF domain, to mediate self-limiting cleavage of cOA activators. Cryo-EM structural analyses reveal that Csm6-2 functions as a homotetramer, and disruption of tetramer formation significantly reduces its ribonuclease activity. Although cA6 and cA5 bind Csm6-2 with comparable affinity, only cA5 induces CARF domain closure, stabilizes the tetramer, and remodels the active site in the HEPN domain. In contrast, the sixth AMP of cA6 imposes significant steric hindrance on CARF domain movement, preventing its closure and subsequent allosteric activation. These findings expand our understanding of the cOA signaling diversity and specific cOA recognition mechanisms in type III CRISPR immunity.},
}
@article {pmid41917354,
year = {2026},
author = {Chen, Z and Huang, X and Pi, Y and Jiang, Y},
title = {Method for Generation of adamtsl4 Knock-Out Zebrafish Lines Using CRRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {73-82},
pmid = {41917354},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *ADAMTS Proteins/genetics ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Phenotype ; Mutation ; Disease Models, Animal ; *Ectopia Lentis/genetics ; Humans ; *Zebrafish Proteins/genetics ; },
abstract = {Congenital ectopia lentis (CEL) is a rare pediatric ocular disorder characterized by zonular fiber defects leading to lens dislocation and is genetically heterogeneous. Among known causes, biallelic mutations in ADAMTSL-4 represent the second most common genetic contributor, frequently associated with ectopia pupillae (EP)-a distinct and clinically significant feature. However, the mechanisms by which ADAMTSL-4 mutations lead to these ocular abnormalities remain poorly understood, partly due to the lack of effective animal models. In this study, we generated adamtsl-4 knock-out zebrafish lines using the CRISPR/Cas9 system. Through microinjection of sgRNA/Cas9 complexes and multigenerational screening, we established stable homozygous mutant lines. These mutants exhibited consistent phenotypes, including lens dislocation into the vitreous body and marked pupillary displacement, faithfully recapitulating human ADAMTSL-4-related EL and EP. This method provides a practical and scalable strategy for generating loss-of-function zebrafish models, with demonstrated utility in recapitulating phenotypes associated with ADAMTSL-4 mutations. Our approach offers a valuable tool for investigating the molecular mechanisms underlying CEL and EP and may support drug screening and therapeutic discovery in the future.},
}
@article {pmid41917356,
year = {2026},
author = {Zheng, L and Wu, Z and Zheng, XL},
title = {Generating adamts13[-/-] Zebrafish via CRISPR/Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {93-111},
pmid = {41917356},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *ADAMTS13 Protein/genetics ; Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; },
abstract = {The zebrafish (Danio rerio) is a powerful vertebrate model for studying hematologic and thrombotic diseases due to its genetic tractability and conservation of hemostatic pathways with humans. In this chapter, we describe a detailed workflow for generating adamts13 knockout zebrafish using CRISPR/Cas9-mediated genome editing. Methods include the design and preparation of guide RNAs, Cas9 mRNA synthesis, and embryo microinjection at the one-cell stage. Alternative strategies for gRNA generation, including synthetic crRNA:tracrRNA duplexes, are also outlined. We provide protocols for screening founder fish, genotyping, and establishing stable mutant lines through outcrossing, thereby minimizing potential off-target effects. Practical notes on embryo handling, RNA stability, and contamination prevention are highlighted to ensure reproducibility. Together, these procedures establish a robust framework for creating zebrafish loss-of-function models, enabling mechanistic studies of ADAMTS13 function in vivo and advancing the exploration of thrombotic disease pathophysiology. This approach can be readily adapted to knock out other genes or introduce specific mutations in zebrafish simply by altering the gRNA sequence.},
}
@article {pmid41919328,
year = {2026},
author = {Singh, P and Sharma, K and Tamrakar, VK and Khare, R and Bhargava, A and Negi, SS},
title = {CRISPR-Cas system: recent advancements in prompt diagnosis of high-risk HPV genotypes in cervical cancer.},
journal = {Expert review of molecular diagnostics},
volume = {26},
number = {3},
pages = {241-256},
doi = {10.1080/14737159.2026.2654503},
pmid = {41919328},
issn = {1744-8352},
mesh = {Humans ; *CRISPR-Cas Systems ; Female ; *Uterine Cervical Neoplasms/diagnosis/virology/etiology ; *Human Papillomavirus Viruses/genetics ; *Papillomavirus Infections/diagnosis/virology/complications/genetics ; Genotype ; *Molecular Diagnostic Techniques/methods ; *Papillomaviridae/genetics ; },
abstract = {INTRODUCTION: The CRISPR/Cas system has emerged as a highly versatile platform for diagnosing infectious diseases, particularly viral pathogens. Human papillomavirus (HPV) comprises of more than 200 types, with persistent infection by 14 high-risk genotypes recognized as the primary cause of cervical cancer worldwide. Early and accurate detection of these High-Risk HPV (HR-HPV) types is essential for effective clinical management and prevention of disease progression.<br><br>AREAS COVERED: This narrative review was based on literature searches in PubMed, Scopus, and Google Scholar covering studies published between 2015 and 2024. This review summarizes recent advances in CRISPR/Cas based diagnostics for HR-HPV, including both pre-amplification and amplification-free strategies. Integration of CRISPR systems with diverse readout modalities such as colorimetric, fluorescent, electrochemical, and lateral-flow biosensors has enabled rapid, sensitive, and user-friendly detection suitable for point-of-care testing (POCT), particularly in low-resource settings.<br><br>EXPERT OPINION: CRISPR/Cas assays demonstrate high sensitivity, specificity, and speed, offering a promising alternative to conventional molecular techniques for HR-HPV detection and genotyping. The convergence of CRISPR diagnostics with artificial intelligence, microfluidics, and affordable biosensors holds significant potential to transform community-level HPV screening. With continued innovation and regulatory support, CRISPR/Cas systems are poised to become indispensable tools for early HR-HPV detection and cervical cancer prevention.},
}
@article {pmid41919983,
year = {2026},
author = {Kaur, N and Raffan, S and Clark, SJ and Musa, S and Scherf, K and Elmore, JS and Curtis, TY and Honan, E and Halford, NG},
title = {Field Trials and Baking Studies of Ultra-Low Asparagine, Genome Edited (CRISPR/Cas9) and Mutant (TILLING) Wheat.},
journal = {Plant biotechnology journal},
volume = {24},
number = {7},
pages = {4704-4715},
pmid = {41919983},
issn = {1467-7652},
support = {BB/T017007/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/W007134/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Triticum/genetics/metabolism ; *Asparagine/metabolism/analysis ; Acrylamide/analysis/metabolism ; *CRISPR-Cas Systems/genetics ; Bread/analysis ; Plants, Genetically Modified ; Mutation ; Aspartate-Ammonia Ligase/genetics/metabolism ; Gene Knockout Techniques ; Mutagenesis ; Cooking ; Genome, Plant/genetics ; },
abstract = {Field trials were conducted of wheat (Triticum aestivum) cv. Cadenza in which asparagine synthetase gene, TaASN2, had been knocked out, either on its own or together with a partial knockout of the related gene, TaASN1, using CRISPR/Cas9. Chemical mutagenesis (TILLING) TaASN2 nulls in the Claire background were also included. The main aim was to assess the free asparagine content of the grain and the conversion of free asparagine to acrylamide, a toxic contaminant, in bread, toast and biscuits. Over 2 years of trials combined, the TaASN2 and TaASN1/2 CRISPR knockouts resulted in a reduction of free asparagine in the grain of 59% and 93%, respectively, compared with Cadenza. The reduction in the TaASN2 total knockout TILLING line compared with Claire was 50%. Yield was not affected in the edited lines but was reduced in the TILLING lines. Acrylamide in bread made from a TaASN1/2 CRISPR line was below detection levels, while in a TaASN2 CRISPR line it was 14% of the Cadenza control. Even after 4 min of toasting, acrylamide levels remained at 8% and 23%, respectively, of the control. The concentration in bread made from the TILLING TaASN2 knockout was 21% that for the Claire control, rising to 46% after 4 min of toasting. Acrylamide in biscuits made from a TaASN1/2 CRISPR line was reduced by 93% compared with the control. The relationship between acrylamide and colour was altered in the edited and mutant lines compared with the controls, with less acrylamide forming for the same degree of colour.},
}
@article {pmid41920952,
year = {2026},
author = {Nicolia, A and Cuccurullo, A and Tamada, K and Yoneyama, K and Rambla, JL and Granell, A and Camerlengo, F and Festa, G and Francese, G and Contaldi, F and D'Alessandro, A and Rigano, MM and Principio, L and D'Agostino, N and Cardi, T},
title = {Editing strigolactone biosynthesis genes in tomato reveals novel phenotypic effects and highlights D27 as a breeding target for parasitic weed resistance.},
journal = {Plant &amp; cell physiology},
volume = {67},
number = {4},
pages = {667-684},
doi = {10.1093/pcp/pcag042},
pmid = {41920952},
issn = {1471-9053},
support = {DM 15924//Italian Ministry of Agriculture, Food Sovereignty and Forests/ ; 18-05-2018//Italian Ministry of Agriculture, Food Sovereignty and Forests/ ; IJC2020-045612-I//Spanish Ministry of Science and Innovation/ ; PID2022-141438OB-I00//Spanish Ministry/ ; 101000716//EU for Harnesstom/ ; JPMJFR220F//Japan Science and Technology Agency/ ; },
mesh = {*Solanum lycopersicum/genetics/parasitology/metabolism ; *Lactones/metabolism ; Phenotype ; *Gene Editing ; *Plant Weeds/physiology ; Plant Proteins/genetics/metabolism ; Germination ; Orobanche ; CRISPR-Cas Systems ; *Genes, Plant ; Fruit/genetics ; Seeds ; },
abstract = {Parasitic weed infestations represent an increasing threat to agriculture worldwide, especially in the Mediterranean region. Phelipanche ramosa (L.) and Phelipanche aegyptiaca (Pers.) (broomrapes) cause severe yield losses in field-grown tomato (Solanum lycopersicum L.). Strigolactones (SLs) are apocarotenoid phytohormones that not only play a critical role in plant physiology and development but also act as the primary germination signals for parasitic weed seeds. In this study, we generated CRISPR/Cas9 tomato knock-out (KO) lines targeting the SlD27 gene and three other key genes involved in SL biosynthesis (SlCCD7, SlCCD8, and SlMAX1), all in the same genetic background. All the edited lines exhibited undetectable SL levels in root exudates, leading to a strong reduction in the in vitro germination of Phelipanche spp. seeds. Consistently, reduced parasitism was also observed in vivo when Sld27 lines were tested. A comprehensive evaluation of morphological, reproductive, and fruit-related traits revealed gene-specific phenotypic effects, including changes in vegetative growth, fruit set, fruit development, and volatilome. Specifically, KO of two carotenoid cleavage dioxygenases and SlMAX1 affected shoot architecture, fruit development, and the production of volatile organic compounds during fruit ripening. In contrast, the newly developed Sld27 lines in this study displayed a mild phenotype generally comparable to nonedited control plants and likely due to the expression of SlD27 paralogues. Overall, our results indicate that SlD27 represents a promising breeding target for enhancing resistance to parasitic weeds in tomato while minimizing negative impacts on plant development and fruit quality.},
}
@article {pmid41921842,
year = {2026},
author = {Liao, YJ and Li, YG and Li, YL},
title = {CRISPR/Cas9-mediated lgp2 knockout and cross-species rescue reveal the immunoregulatory role of LGP2 in zebrafish.},
journal = {Fish &amp; shellfish immunology},
volume = {173},
number = {},
pages = {111308},
doi = {10.1016/j.fsi.2026.111308},
pmid = {41921842},
issn = {1095-9947},
mesh = {Animals ; *Zebrafish/immunology/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques/veterinary ; *Zebrafish Proteins/genetics/immunology ; *Immunity, Innate/genetics ; Gene Expression Profiling/veterinary ; *Gene Expression Regulation/immunology ; *RNA Helicases/genetics/immunology ; Fish Diseases/immunology ; },
abstract = {LGP2 is a key regulator within the RIG-I-like receptor (RLR) pathway with dual roles in antiviral immunity, but its function under basal conditions and its evolutionary variation among fish species require further investigation. In this study, we constructed a zebrafish lgp2 knockout model using CRISPR/Cas9. Knockout lgp2 led to upregulation of key RLR signaling components (mda5, mavs), type I interferons (ifnphi1, ifnphi3, ifnphi4), and the interferon-stimulated gene mxa. Transcriptomic profiling of 3 dpf embryos identified 3186 differentially expressed genes (DEGs), including significantly elevated expression of five claudin-family genes. Protein-protein interaction (PPI) analysis of DEGs indicated that irf7 and claudin b exhibit an interaction at the protein level. Gene ontology analysis showed that LGP2 was enriched in terms such as helicase activity and ATPase activity, and was also significantly enriched in extracellular structure terms. Comparative sequence analysis revealed conserved DEXDc, HELICc and CTD domains in LGP2 from zebrafish, grass carp and barbel chub, with notable species-specific sequence variations. Functional rescue experiments demonstrated that all three LGP2 orthologs could restore lgp2 expression and modulate downstream interferon responses in lgp2[-/-] embryos; however, induction of the transcription factor irf7 was specifically mediated only by zebrafish-derived LGP2. These results provide insights into the functional diversity of LGP2 in teleost immunity and inform strategies for selective breeding aimed at disease resistance.},
}
@article {pmid41922342,
year = {2026},
author = {Carey, CM and Parvez, S and Brandt, ZJ and Bisgrove, BW and Yates, CJ and Peterson, RT and Gagnon, JA},
title = {MIC-Drop-seq: scalable single-cell phenotyping of mutant vertebrate embryos.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41922342},
issn = {2041-1723},
support = {F32HL156644//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 GM134069/GM/NIGMS NIH HHS/United States ; R00HG012593//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01GM134069//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R24 OD035409/OD/NIH HHS/United States ; K01HG013682//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R35GM142950//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R24OD035409//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Single-Cell Analysis/methods ; Phenotype ; Gene Expression Regulation, Developmental ; *Embryo, Nonmammalian/metabolism/cytology ; Transcription Factors/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Gene Regulatory Networks ; Mutation ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics/metabolism ; Sequence Analysis, RNA/methods ; Mesoderm/metabolism/embryology ; },
abstract = {Pooled perturbation screens can reveal cellular regulatory networks, yet scaling these techniques for large-scale screens in animals remains challenging. Here we present MIC-Drop-seq, a technique that addresses these challenges by combining high-throughput CRISPR gene disruption in zebrafish embryos with phenotyping by multiplexed single-cell RNAseq. In one MIC-Drop-seq experiment, we simultaneously identified changes in gene expression and cell abundance across 74 cell types resulting from loss of function of 50 transcription factors. These observations recapitulate many known phenotypes, while also uncovering previously uncharacterized roles for transcription factors in brain and mesoderm development. A key advantage of whole-animal screens is that they reveal how changes in one cell type affect the development of other cell types. Surprisingly, such cell-extrinsic phenotypes are abundant, indicating that transcription factors frequently exert effects beyond the cells where they are expressed to adjacent cells. We propose that MIC-Drop-seq will facilitate efforts to dissect the complete gene regulatory networks that guide animal development.},
}
@article {pmid41922875,
year = {2026},
author = {Burr, SP and Auckland, K and Glynos, A and Dhawanjewar, A and Ryall, C and Wei, W and Hynes-Allen, A and Prater, M and Sczaniecka-Clift, M and Prudent, J and Chinnery, PF and van den Ameele, J},
title = {MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {4},
pages = {711-723},
pmid = {41922875},
issn = {1545-9985},
mesh = {Animals ; *DNA, Mitochondrial/genetics ; Mice ; DNA-Binding Proteins/genetics ; *Heteroplasmy/genetics ; DNA Polymerase gamma/genetics ; Mitochondrial Proteins/genetics/metabolism ; *Mitochondria/genetics/metabolism ; *Single-Cell Analysis/methods ; Transcription Factors/genetics ; CRISPR-Cas Systems ; GTP Phosphohydrolases/genetics ; Activating Transcription Factor 4/metabolism/genetics ; High Mobility Group Proteins/genetics ; Mutation ; DNA-Directed DNA Polymerase/genetics ; Cell Proliferation ; Single-Cell Gene Expression Analysis ; },
abstract = {Mitochondria contain their own genome, mitochondrial DNA (mtDNA), which is under strict control by the cell nucleus. mtDNA occurs in many copies per cell and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the tissue-specific impact of mtDNA mutations, eventually giving rise to both rare mitochondrial and common neurodegenerative diseases. Here, we use MitoPerturb-Seq for CRISPR-Cas9-based, high-throughput single-cell interrogation of the nuclear genes and pathways that sense and control mtDNA copy number and heteroplasmy. We screened a panel of mtDNA maintenance genes in mouse cells with a heteroplasmic mtDNA mt-Ta mutation. This revealed both common and perturbation-specific aspects of the integrated stress response to mtDNA depletion caused by Tfam, Opa1 and Polg knockout. These responses are only partially mediated by ATF4 and cause cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq, thus, provides experimental insight into disease-relevant mitochondrial-nuclear interactions and may inform development of therapies targeting cell-type- and tissue-specific vulnerabilities to mitochondrial dysfunction.},
}
@article {pmid41923586,
year = {2026},
author = {Oh, Y and Lee, H and Jang, S},
title = {Emerging synthetic biology-assisted technologies for overcoming antibiotic resistance: CRISPR-Cas, bacteriophage, microbiome, and metabolic engineering-based solutions.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {64},
number = {3},
pages = {e2512002},
doi = {10.71150/jm.2512002},
pmid = {41923586},
issn = {1976-3794},
support = {//National Research Foundation of Korea/ ; RS-2025-02214910//Ministry of Science and ICT/ ; //Incheon National University/ ; },
mesh = {*Metabolic Engineering/methods ; *Synthetic Biology/methods ; *Bacteriophages/genetics ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; *Microbiota ; *Drug Resistance, Bacterial/genetics ; *Drug Resistance, Microbial ; Humans ; },
abstract = {Antibiotic resistance has become a critical global health challenge due to the decreased efficacy of existing antibiotics and the emergence of multidrug-resistant pathogens. In particular, the rapid horizontal transfer of resistance genes and the diverse mechanisms by which bacteria acquire resistance have significantly undermined the effectiveness of conventional therapeutic strategies, revealing fundamental limitations in current infectious disease management. In this context, synthetic biology provides a promising framework to overcome the limitations of conventional antibiotics by integrating engineering principles with bioengineering approaches, thereby enabling precise and programmable control of biological processes. These synthetic biology-based approaches offer substantial potential for developing sustainable and highly specific antimicrobial strategies. This review comprehensively examines recent advances in synthetic biology-assisted antimicrobial strategies, including CRISPR-Cas systems, bacteriophage engineering, microbiome engineering, and metabolic engineering-driven antibiotic discovery. Collectively, these approaches represent a precision antimicrobial paradigm that enables selective targeting of resistant bacteria while preserving microbiome homeostasis. These strategies also provide new directions for limiting resistance dissemination and guiding the development of next-generation therapeutics.},
}
@article {pmid41923628,
year = {2026},
author = {Ou, C and Huang, X and Huang, D and Luo, R and Chen, R and Li, X and Wu, X and Wu, Q and Gong, C},
title = {A harmless-to-harmful switchable and spatiotemporally activated nano-CRISPR hierarchically amplifies ferroptosis in melanoma.},
journal = {Cell reports. Medicine},
volume = {7},
number = {4},
pages = {102718},
pmid = {41923628},
issn = {2666-3791},
mesh = {*Ferroptosis/genetics ; Humans ; Reactive Oxygen Species/metabolism ; Animals ; *Melanoma/pathology/genetics/therapy/metabolism ; Cell Line, Tumor ; Iron/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Lipid Peroxidation ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; Lipid Peroxides/metabolism ; },
abstract = {Ferroptosis therapy faces challenges due to low lipid peroxide (LPO) levels. Herein, we develop a harmless-to-harmful switchable and spatiotemporally activated nano-CRISPR system (termed ARCHER) that sequentially amplifies ferroptosis sensitivity, iron ion levels, and reactive oxygen species (ROS) to amplify ferroptosis therapy efficiency. ARCHER targets cancer cells and releases CRISPR-Cas9 and Ce6Fe(III)Cl in response to hyaluronidase and tumor acidity. CRISPR-Cas9 sustains effective suppression of LPO-reducing protein GPX4. By persistently enhancing LPO accumulation through GPX4 downregulation, ARCHER primes cancer cells for ferroptosis and sensitizes them to subsequent interventions. Upon laser irradiation under acidic conditions, Ce6Fe(III)Cl undergoes spatiotemporal activation, transforming from inert form into cytotoxic Fe(III) ions and Ce6-generated ROS. Liberated Fe(III) and ROS synergistically amplify lipid peroxidation, driving LPO accumulation to trigger ferroptosis storm in sensitized cancer cells. In vivo studies demonstrated that ARCHER achieves 60% (3/5) tumor ablation with minimal off-target effects, validating its high therapeutic efficacy in ferroptosis-driven cancer treatment.},
}
@article {pmid41925118,
year = {2026},
author = {El-Sokkary, MMA and Ali, IEH},
title = {Association of CRISPR/Cas system with integrons and antibiotic resistance in Kliebsiella pneumoniae.},
journal = {Future microbiology},
volume = {21},
number = {3},
pages = {299-306},
pmid = {41925118},
issn = {1746-0921},
mesh = {*Integrons/genetics ; *Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; Klebsiella Infections/microbiology ; *Drug Resistance, Multiple, Bacterial/genetics ; },
abstract = {AIMS: The interaction between the CRISPR/Cas system and drug resistance was investigated in this study.<br><br>METHODS: In this study, 24 isolates, identified as Klebsiella pneumoniae, were subjected to antimicrobial sensitivity screening and detecting the presence of cas, integrons, and antibiotic resistance genes.<br><br>RESULTS: The highest resistance level could be detected in ceftriaxone and amoxicillin by 79% and 70.8%, respectively, while the lowest was 16.7% for chloramphenicol. Additionally, 50% of all isolates exhibited multidrug resistance (MDR); however, extensive drug resistance (XDR) was present in 12.5%. Class 1 integrons with different sizes could be identified. sul1 and sul2 were the most resistance genes identified, followed by aacA4 and aac(3)-II and qnrS, with 58%, 50%, 41.7%, 33%, and 33%, respectively. Concerning CRISPR genes, cas3 could be identified in nine different isolates; however, cas1 could be detected in 11 different isolated strains. In case of CRISPR1, it was found in seven isolates (29.3%), while CRISPR2 could be identified in three different isolates (12.5%).<br><br>CONCLUSIONS: Antibiotic resistance genes were mostly correlated with CRISPR genes. Interestingly, BOX-PCR group A, with a lower number of CRISPR positive isolates (p&#x2009;=&#x2009;0.046) had a limited number of detected genes, compared with other groups indicating a significant correlation between antibiotic resistance genes and cas genes (p&#x2009;=&#x2009;0.0023).},
}
@article {pmid41925899,
year = {2026},
author = {Wang, B and Liu, M and Wang, H and Wang, C and Zhang, W and Yan, J},
title = {Research progress on nucleic acid amplification-based detection technologies for phytopathogenic fungi.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41925899},
issn = {1432-0614},
support = {CARS-29//China Agriculture Research System of MOF and MARA/ ; JKTD2025002//the Outstanding Scientist Project of Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Fungi/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; CRISPR-Cas Systems ; },
abstract = {Phytopathogenic fungi are highly diverse and globally distributed, posing a major threat to agricultural production worldwide. The annual losses caused by plant diseases can reach up to 30% of global crop yields, with over 80% of infections caused by fungal pathogens. The accurate identification of pathogenic fungal species is crucial for effective disease prevention and control. Thus, establishing accurate and rapid detection technologies for phytopathogenic fungi is crucial for implementing targeted control strategies and reducing agricultural losses. Molecular detection technologies based on nucleic acid amplification have recently become indispensable tools for pathogen detection. This review examines the principles and advancements of nucleic acid-based detection techniques, including thermal cycling-based methods (e.g., conventional PCR, real-time quantitative PCR, and droplet digital PCR) and isothermal amplification platforms (e.g., loop-mediated isothermal amplification and recombinase polymerase amplification), as well as CRISPR/Cas-assisted assays coupled with isothermal amplification (e.g., RPA-CRISPR and LAMP-CRISPR), with the aim of evaluating their strengths, limitations, and practical applicability in the rapid diagnosis and precision management of phytopathogenic fungal diseases. KEY POINTS: Nucleic acid amplification technologies enable rapid and sensitive detection of phytopathogenic fungi. Isothermal amplification and CRISPR/Cas-assisted platforms facilitate field-deployable and low-instrumentation diagnostics. Integrated workflows support early diagnosis and precision management of phytopathogenic fungal diseases.},
}
@article {pmid41926741,
year = {2026},
author = {Driscoll, CS and Kim, J and Knott, JG},
title = {CRISPR-mediated editing of cis-regulatory elements in early mouse embryos: a tool for studying pluripotency gene regulation.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {5},
pages = {},
doi = {10.1093/reprod/xaag042},
pmid = {41926741},
issn = {1741-7899},
support = {HD095371//National Institutes of Child Health and Development/ ; //Michigan State University AgBioResearch/ ; HD087166//T32 doctoral fellowship from the NICHD/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Transcription Factor AP-2/genetics/metabolism ; *Gene Expression Regulation, Developmental ; *Gene Editing/methods ; *Blastocyst/metabolism/cytology ; Female ; *Embryonic Development/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; *Regulatory Sequences, Nucleic Acid ; Octamer Transcription Factor-3/genetics/metabolism ; },
abstract = {In brief Cis-regulatory elements and transcription factor binding motifs play crucial roles in regulating the spatial and temporal patterns of gene expression during development. This study tested the utility of CRISPR/Cas9 as a tool to interrogate the function of transcription factor AP2 gamma motifs in pluripotency gene expression during mouse preimplantation embryo development. Abstract Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) is a highly efficient tool that enables the generation of gene knockouts, knock-ins, and single base substitutions in a variety of organisms. Recently, we used CRISPR to examine the activity of cis-regulatory elements (CREs) in mouse preimplantation embryos. However, there is limited information on the feasibility of using CRISPR in preimplantation embryos to interrogate the function of select transcription factor (TF) binding motifs located within critical CREs in pluripotency genes. In the current study we employed CRISPR to disrupt TF AP2 gamma (TFAP2C) binding motifs located within key CREs involved in the regulation of Pou5f1 and Sox2 expression in early embryos. Microinjection of ribonucleoprotein complexes containing Cas9 and single guide RNAs (sgRNAs) targeting TFAP2C motifs located within a distal enhancer and proximal promoter substantially impaired Pou5f1 and Sox2 expression, respectively. Quantification of the editing efficiencies at each targeted CRE revealed that the targeting sgRNA sequences and the number of sgRNAs injected influenced the overall editing rates. Lastly, we investigated whether TFAP2C-induced activation of Sox2 expression in 2-cell embryos required TFAP2C motifs located within the Sox2 proximal promoter. CRISPR-mediated editing of these motifs diminished the activation of Sox2 expression. In summary, these findings indicate that CRISPR/Cas9 is a feasible approach for editing TF motifs in preimplantation embryos and provide evidence that TFAP2C directly contributes to Pou5f1 and Sox2 expression in preimplantation embryos.},
}
@article {pmid41927370,
year = {2026},
author = {Zheng, J and Wang, X and Wu, M and Liu, J and Feng, H and Yang, H and Li, D and Wang, H and Hu, J and Zuo, E},
title = {Large-scale parallel characterization of RNA-guided nuclease activity and specificity.},
journal = {Science bulletin},
volume = {71},
number = {8},
pages = {2044-2054},
doi = {10.1016/j.scib.2026.03.047},
pmid = {41927370},
issn = {2095-9281},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *Endonucleases/genetics/metabolism ; },
abstract = {As systematic comparisons of editing efficiency and specificity seldom keep pace with rapid developments in RNA-guided nucleases (RGNs), the current study examined 50 such editing systems and characterized the off-target effects and genomic structural impacts of a subset of high-efficiency RGNs. Among them, AsCas12a-Ultra, LbCpf1, and AsCas12a-Plus demonstrated similar or higher efficiency compared to SpCas9, while the relatively high efficiency and small size of enOsCas12f1 together support its suitability for in vivo delivery. AsCpf1-YH and FnCpf1 exhibited the lowest single-guide RNA-dependent (sgRNA-dependent) off-target risks, whereas DpFNuc showed the highest. Genomic structural analysis revealed that enCas12f-HKRA frequently introduces chromosomal translocations, while Cas12j-SF05 poses a lower risk of such mutations. Notably, the high-efficiency RGNs were associated with translocation hotspots. Additionally, enRhCas12f1 and SpaCas12f1 had the lowest cytotoxicity, while enAsCpf1-HF strongly inhibited cell proliferation. This study establishes the first multidimensional performance evaluation framework for RGNs, providing a data-driven tool to support precise genome editing.},
}
@article {pmid41927937,
year = {2026},
author = {Zhuravlev, IY and Lyakhovets, AA and Matveenko, AG and Lebedeva, MA and Zhernakov, AI and Simonova, VY and Sulima, AS and Tikhonovich, IA and Zhukov, VA},
title = {CRISPR/Cas9-mediated knockout of PsLykX gene of pea (Pisum sativum L.) leads to loss of symbiotic nodules.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {41927937},
issn = {1573-9368},
mesh = {*Pisum sativum/genetics/microbiology/growth &amp; development ; *Symbiosis/genetics ; *Root Nodules, Plant/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics/growth &amp; development ; *Plant Proteins/genetics ; Nitrogen Fixation/genetics ; Gene Knockout Techniques ; Rhizobium/genetics ; Plant Roots/genetics/microbiology/growth &amp; development ; },
abstract = {Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Caméor using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Caméor and Rhizobium ruizarguesonis RCAM1026.},
}
@article {pmid41928005,
year = {2026},
author = {Migur, A and Feussner, M and Liao, C and Alkhnbashi, OS and Chauvier, A and Walter, NG and Backofen, R and Weinberg, Z and Beisel, CL},
title = {A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems.},
journal = {The EMBO journal},
volume = {45},
number = {10},
pages = {3396-3415},
pmid = {41928005},
issn = {1460-2075},
support = {468749960//Deutsche Forschungsgemeinschaft (DFG)/ ; BA 2168/23-2//Deutsche Forschungsgemeinschaft (DFG)/ ; 865973//EC | Horizon Europe | Excellent Science | HORIZON EUROPE European Research Council (ERC)/ ; MCB 2140320//National Science Foundation (NSF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Bacterial/genetics/metabolism/chemistry ; *Porphyromonas gingivalis/genetics/virology ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Conformation ; CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {CRISPR RNAs (crRNAs) guide recognition and targeting of intracellular invaders as part of adaptive immunity by CRISPR-Cas systems. crRNAs are transcribed from CRISPR arrays of conserved repeats interlaced with invader-derived spacers. While crRNA production is essential for immunity, its optimization for defense remains poorly understood. Here, we show that, in diverse RNA-targeting type VI CRISPR-Cas systems, the leader RNA encoded upstream of the CRISPR array prevents formation of an invader-independent extraneous crRNA (ecrRNA) by blocking processing of the first repeat. Using the VI-B2 system from Porphyromonas gingivalis as a model, we demonstrate that the leader RNA and first repeat form a conserved inhibitory hairpin that precludes binding and processing by the system's Cas13b nuclease. Disrupting this hairpin enables ecrRNA production, which in turn can deplete invader-derived crRNAs and reduce Cas13b-mediated phage defense. Structure prediction indicates that these leader-repeat hairpins are widespread across diverse type VI subtypes, highlighting a conserved regulatory mechanism. Our findings reveal how a prevalent branch of CRISPR-Cas systems suppresses ecrRNA formation to promote RNA-guided immunity.},
}
@article {pmid41928521,
year = {2026},
author = {Diao, K and Duff, SMG and Li, H},
title = {An Idea to Explore: Enhancing the Teaching of Genome Editing Through 3D Printed Models of CRISPR-Cas9 Technology.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {54},
number = {3},
pages = {292-296},
doi = {10.1002/bmb.70047},
pmid = {41928521},
issn = {1539-3429},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Printing, Three-Dimensional ; Humans ; Teaching ; },
abstract = {Innovative biological discoveries are crucial for addressing global challenges, yet teaching these complex concepts poses significant difficulties due to the complexity of the subject matter as well as limited educational resources and methodologies. Genome editing, specifically with CRISPR-Cas9, represents a convergence of technology, molecular biology, and engineering, enabling precise manipulation of DNA sequences in various organisms. It leverages advanced tools and a deep understanding of molecular biology to target specific genes while applying engineering principles to improve editing mechanisms. However, the complexity of this field poses educational challenges due to a scarcity of accessible resources. To enhance the accessibility of genome editing to scientists and students, we propose utilizing 3D modeling and printing to create tangible models of key components in the genome editing process. By visualizing these molecular structures, our goal is to simplify and enrich the educational experience, making the intricate principles of genome editing more comprehensible and engaging for students.},
}
@article {pmid41928564,
year = {2026},
author = {Topfer, SK and Rutherford, M and Zewe, F and Strive, T and Oh, KP},
title = {Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {59-70},
doi = {10.1177/25731599261424870},
pmid = {41928564},
issn = {2573-1602},
mesh = {Animals ; Male ; *Spermatozoa/metabolism ; Rabbits ; *Transfection/methods ; *CRISPR-Cas Systems/genetics ; Electroporation/methods ; *Gene Editing/methods ; Cell Survival ; Sperm Motility ; Lipids ; },
abstract = {Modern genome editing tools such as CRISPR-Cas9 have revolutionized mammalian genome engineering, yet translation to in vivo applications remains limited by low efficiency and frequent occurrence of mosaicism. Sperm-mediated delivery of editing reagents is one proposed alternative that may mitigate these issues. This method depends on efficient transfection of genome editing materials into viable spermatozoa, a critical yet frequently overlooked parameter. Using FACS, we compared electroporation (Neon NxT) and lipofection (CRISPRMAX) for introducing CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa. Electroporation, shown to enable Cas9 and plasmid transfection in spermatozoa from other species, performed poorly once dead spermatozoa were excluded. In contrast, Lipofectamine CRISPRMAX improved transfection efficiency with minimal effects on spermatozoa viability and motility. These findings emphasize the importance of distinguishing true transfection (transfection of viable spermatozoa) from total transfection and highlight lipofection as a promising alternative to electroporation for sperm-based genome editing, with potential applications in rabbit genome engineering.},
}
@article {pmid41928587,
year = {2026},
author = {Gibson, JR and Dhungana, A and Pokhrel, M and Arthur, BB and Adebayo, O and Hossack, D and Cottle, RN},
title = {Toward Next-Gen Cell Therapy for Pediatric Patients: Neonatal Hepatocytes Tolerate Electroporation-Mediated Gene Editing and Engraft in the Liver.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {103-114},
doi = {10.1177/25731599261430830},
pmid = {41928587},
issn = {2573-1602},
support = {R01 HL168093/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Hepatocytes/transplantation/metabolism/cytology ; Mice ; *Gene Editing/methods ; Liver/metabolism/cytology ; Humans ; Tyrosinemias/therapy/genetics ; *Electroporation/methods ; Animals, Newborn ; CRISPR-Cas Systems ; Mice, Knockout ; *Cell- and Tissue-Based Therapy/methods ; Disease Models, Animal ; Hydrolases ; },
abstract = {Hepatocyte transplantation (HTx) offers a safer, less invasive alternative to orthotopic liver transplantation for inherited metabolic liver diseases, especially in high-risk pediatric patients. Combining HTx with ex vivo gene editing is a promising autologous therapeutic strategy using the patient's cells. We investigated the feasibility of this approach by applying CRISPR-Cas9 gene knock-out to neonatal mouse hepatocytes and comparing their engraftment potential with that of mature adult cells in the Fah[-/-] mouse model of hereditary tyrosinemia type I (HT1). Electroporation-mediated gene editing did not significantly impair the ability of neonatal hepatocytes to engraft in vivo. Quantitative histological analysis revealed comparable liver repopulation levels between recipients of gene-edited neonatal cells and adult cells after hepatoxicity-mediated selection, providing a benchmark for electroporation-mediated gene editing in neonatal hepatocytes, and supporting the development of genetically corrected neonatal hepatocyte products as a crucial long-term or bridge-to-transplant therapeutic strategy for pediatric liver disease.},
}
@article {pmid41928679,
year = {2026},
author = {Li, D and Feng, J and Wang, X and Zhao, E and Liang, Y and Li, K and Li, Y},
title = {An Anaerobic Fluorescent Reporter System and CRISPR-Cas12a Enable High-Throughput Metabolic Engineering of Clostridium butyricum.},
journal = {ACS synthetic biology},
volume = {15},
number = {4},
pages = {1669-1680},
doi = {10.1021/acssynbio.6c00158},
pmid = {41928679},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; *Clostridium butyricum/genetics/metabolism ; Anaerobiosis ; Genes, Reporter/genetics ; Promoter Regions, Genetic/genetics ; Gene Editing/methods ; Aldehyde Dehydrogenase/genetics/metabolism ; Plasmids/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clostridium butyricum is an important probiotic and industrial organism with significant potential for anaerobic bioproduction. However, the lack of efficient genetic tools, particularly for high-throughput screening under strict anaerobic conditions, has hindered its metabolic engineering. To address this, we first established a highly efficient conjugation method, significantly improving exogenous DNA transformation efficiency. Leveraging time-resolved transcriptomic data, we then mapped the dynamic activity of native promoters and developed a robust anaerobic fluorescent protein reporter system. This system overcomes the oxygen-dependent limitation of traditional reporters like GFP, enabling high-throughput and quantitative screening of promoter strength in live anaerobes. Furthermore, we constructed a CRISPR-Cas12a-based genome editing platform for scarless gene manipulation and a two-plasmid curing strategy to generate markerless and genetically stable engineered strains. The power of this integrated toolkit was demonstrated by engineering the acetaldehyde metabolic pathway. Overexpression of aldehyde dehydrogenase (ALDH) resulted in a 79.29% increase in enzyme activity, indicating an enhanced catalytic capacity for acetaldehyde oxidation. This proof-of-concept module, together with the anaerobic fluorescent reporter and CRISPR-Cas12a platform, supports a streamlined workflow for genetic part characterization and metabolic engineering in C. butyricum under strict anaerobic conditions.},
}
@article {pmid41930610,
year = {2026},
author = {Xing, B and Zhang, X and Shen, L and Zhang, X},
title = {CRISPR-mediated MLH1 disruption suppresses endometrial cancer growth via genomic instability induction and Wnt/β-catenin pathway inhibition.},
journal = {Folia histochemica et cytobiologica},
volume = {64},
number = {1},
pages = {75-87},
doi = {10.5603/fhc.111186},
pmid = {41930610},
issn = {1897-5631},
mesh = {Female ; *MutL Protein Homolog 1/genetics/metabolism ; Humans ; *Endometrial Neoplasms/genetics/pathology/metabolism ; *Wnt Signaling Pathway/genetics ; *Genomic Instability/genetics ; Animals ; Cell Line, Tumor ; Cell Proliferation/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Nude ; Cell Movement ; Apoptosis/genetics ; },
abstract = {INTRODUCTION: MutL homolog 1 (MLH1) loss is a defining molecular feature of endometrial cancer (EC) and a principal driver of microsatellite instability (MSI). Ishikawa cells harbor intrinsic MLH1 promoter hypermethylation, resulting in reduced but not abolished MLH1 expression and placing these cells in a vulnerable, partially compromised mismatch repair state. This study explores the effects of MLH1 knockdown (MLH1-KD) on MSI, cellular functions, signaling pathways, and tumor growth in Ishikawa EC cells.<br><br>MATERIAL AND METHODS: Using CRISPR/Cas9, we created an MLH1-KD Ishikawa EC cell line, validated through Sanger sequencing, qRT-PCR, western blotting, comet assays, and &#x3b3;-H2AX analysis. Functional assays assessed proliferation, migration, and cell cycle progression and apoptosis. RNA sequencing identified global transcriptomic changes, and Wnt/&#x3b2;-catenin pathway activity was measured by a dual-luciferase reporter assay. A xenograft model evaluated tumor growth in vivo.<br><br>RESULTS: MLH1-KD cells showed MSI-H characteristics, increased DNA damage, and downregulation of key EC-related genes. Functionally, MLH1-KD led to significant reductions in cell proliferation and migration, which was accompanied by cell cycle arrest and a marked increase in apoptosis. RNA sequencing revealed profound alterations in the Wnt signaling pathway. Crucially, this was confirmed by a dual-luciferase reporter assay, which showed a significant inhibition of Wnt/&#x3b2;-catenin signaling activity. In vivo, MLH1-KD significantly decreased tumor weight and size in nude mice.<br><br>CONCLUSIONS: In EC cells with pre-existing MLH1 promoter methylation, MLH1-KD leads to MSI-H, enhances genomic instability, disrupts Wnt signaling, impairs cellular functions, and inhibits tumor growth, highlighting Wnt signaling and MSI-H as potential therapeutic targets in EC.},
}
@article {pmid41930821,
year = {2026},
author = {Zheng, P and Zheng, S},
title = {Xenotransplantation: Promise, progress, and risks.},
journal = {Veterinary immunology and immunopathology},
volume = {296},
number = {},
pages = {111110},
doi = {10.1016/j.vetimm.2026.111110},
pmid = {41930821},
issn = {1873-2534},
mesh = {*Transplantation, Heterologous/trends/adverse effects/ethics/veterinary ; Animals ; Humans ; Graft Rejection/prevention &amp; control ; Gene Editing ; CRISPR-Cas Systems ; Heart Transplantation ; Kidney Transplantation ; },
abstract = {Xenotransplantation has seen remarkable progress in recent years, largely driven by the advent of CRISPR-Cas9 gene editing technology. Several patients have received genetically modified kidney and heart transplants, achieving graft and patient survival ranging from days to months. This review offers a brief history of xenotransplantation and highlights advancements in gene editing that represent breakthroughs in minimizing rejection. It also presents recent cases of xenotransplantation and their clinical outcomes. Finally, the review addresses the ethical considerations and risks associated with this emerging field.},
}
@article {pmid41930845,
year = {2026},
author = {Qin, Y and Zhang, S and Zhang, H and Jiang, D and Wen, P and Li, S and Wang, G and Qu, F and Zhang, N and Wang, L and He, Y and Shao, Q and Zhou, X and Zeng, X},
title = {Dual-lock gated DNAzyme-CRISPR cascade for amplification-free ultrasensitive profiling of FTO demethylase activity.},
journal = {Talanta},
volume = {306},
number = {},
pages = {129714},
doi = {10.1016/j.talanta.2026.129714},
pmid = {41930845},
issn = {1873-3573},
mesh = {*Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics/metabolism ; Humans ; *DNA, Catalytic/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Female ; *Breast Neoplasms/genetics ; Adenosine/analogs &amp; derivatives/metabolism ; Limit of Detection ; },
abstract = {N6-methyladenosine, the most prevalent internal mRNA modification in eukaryotes, dynamically regulates transcriptomic homeostasis via FTO-mediated demethylation. CRISPR-based FTO detection technologies represent a significant research direction. However, most current strategies depend critically on nucleic acid pre-amplification steps, which introduce substantial risks of amplification leakage, thereby compromising their ability to achieve stable, high-sensitivity FTO detection. To address this, we designed a dual-lock gated DNAzyme-CRISPR cascading platform that integrates a double-locked DNAzyme with a redundant structure regulated CRISPR-Cas12a trans-cleavage signal amplification system. This architecture achieves an unprecedented limit of detection of 0.083 pM, which is 12 times more sensitive than commercial ELISA kits. Clinical validation using 20 breast cancer patient tissues demonstrated robust cancer/normal discrimination (AUC = 0.9853) and significant FTO upregulation in triple-negative subtypes (p < 0.001). The modular design establishes a universal framework for epigenetic enzyme detection while enabling dynamic monitoring of tumor epigenetic remodeling.},
}
@article {pmid41931047,
year = {2026},
author = {Hanna, R and Frangoul, H and Pineiro, L and McKinney, C and Mapara, M and Dalal, J and Rangarajan, HG and Atkins, H and Sharma, A and Chang, KH and Jaskolka, MC and Kim, K and Yu, Q and Mei, B and Afonja, O and Walters, MC and , },
title = {CRISPR-Cas12a Gene Editing of HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.},
journal = {The New England journal of medicine},
volume = {394},
number = {13},
pages = {1281-1291},
doi = {10.1056/NEJMoa2415550},
pmid = {41931047},
issn = {1533-4406},
mesh = {Adolescent ; Adult ; Female ; Humans ; Male ; Young Adult ; *Anemia, Sickle Cell/blood/diagnosis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Fetal Hemoglobin/analysis/genetics ; *gamma-Globins/genetics ; Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Promoter Regions, Genetic/genetics ; Severity of Illness Index ; Follow-Up Studies ; Transplantation, Autologous/adverse effects/methods ; Transplantation Conditioning/adverse effects/methods ; Repressor Proteins/metabolism ; Busulfan/administration &amp; dosage/adverse effects ; Myeloablative Agonists/administration &amp; dosage/adverse effects ; *Vaso-Occlusive Crises/epidemiology/genetics/prevention &amp; control/therapy ; Treatment Outcome ; },
abstract = {BACKGROUND: Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of sickle cell disease.<br><br>METHODS: We conducted a phase 1-2, multicenter, open-label, single-group study involving patients with severe sickle cell disease who were 12 to 50 years of age and had had at least two severe vaso-occlusive events per year in the previous 2 years. The patients received a single infusion of reni-cel after myeloablative conditioning with busulfan. The patients were monitored for engraftment, hemoglobin-related measures, allelic editing levels, vaso-occlusive events, and adverse events over a 24-month period. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported.<br><br>RESULTS: As of October 29, 2024, a total of 28 patients with severe sickle cell disease had been treated with reni-cel. The median duration of follow-up was 9.5 months (range, 0.7 to 25.2). Among 27 patients who had neutrophil and platelet engraftment by the data-cutoff date, neutrophil engraftment occurred after a median of 23 days (range, 14 to 29), and platelet engraftment occurred after a median of 25 days (range, 17 to 51). At month 6, among 18 patients with at least 6 months of available data, the mean (&#xb1;SD) total hemoglobin level (9.8&#xb1;1.7 g per deciliter at baseline) had increased to 13.8&#xb1;1.9 g per deciliter, and the mean percentage of fetal hemoglobin (2.5&#xb1;2.5% at baseline) had increased to 48.1&#xb1;3.2%; both measures were maintained at or above these values thereafter. One patient had two severe vaso-occlusive events after infusion. Adverse events were consistent with those that occur after myeloablative busulfan-based conditioning and autologous hematopoietic stem-cell transplantation.<br><br>CONCLUSIONS: Treatment with reni-cel led to normalization of the total hemoglobin level and an increase in the percentage of fetal hemoglobin, with no vaso-occlusive events occurring in 27 of 28 patients after infusion. These results support further investigation of this gene-editing approach in the treatment of severe sickle cell disease. (Funded by Editas Medicine; RUBY ClinicalTrials.gov number, NCT04853576.).},
}
@article {pmid41931048,
year = {2026},
author = {Frangoul, H and Hanna, R and Walters, MC and Kao, RL and Carroll, C and McManus, M and Chang, KH and Jaskolka, MC and Kim, K and Yu, Q and Badamosi, N and Mei, B and Afonja, O and Thompson, A and , },
title = {CRISPR-Cas12a Gene Editing of HBG1 and HBG2 Promoters to Treat β-Thalassemia.},
journal = {The New England journal of medicine},
volume = {394},
number = {13},
pages = {1292-1301},
doi = {10.1056/NEJMoa2501277},
pmid = {41931048},
issn = {1533-4406},
mesh = {Adolescent ; Adult ; Female ; Humans ; Male ; Young Adult ; *beta-Thalassemia/therapy/genetics/blood ; *CRISPR-Cas Systems/genetics ; *Fetal Hemoglobin/analysis/genetics ; *gamma-Globins/genetics ; Gene Editing/methods ; *Genetic Therapy/adverse effects/methods ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Neutrophils ; Promoter Regions, Genetic ; Repressor Proteins/metabolism ; Busulfan/administration &amp; dosage/adverse effects ; Myeloablative Agonists/administration &amp; dosage/adverse effects ; Transplantation Conditioning/adverse effects/methods ; *Erythrocyte Transfusion/statistics &amp; numerical data ; Treatment Outcome ; },
abstract = {BACKGROUND: Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of transfusion-dependent β-thalassemia.<br><br>METHODS: We conducted a phase 1-2, multicenter, open-label, single-group study of reni-cel in participants 18 to 35 years of age with transfusion-dependent &#x3b2;-thalassemia. The participants received myeloablative conditioning with busulfan before reni-cel infusion. The primary end points were neutrophil engraftment by 42 days after infusion and frequency and severity of adverse events. Participants were monitored for hemoglobin-related measures and transfusion independence. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported.<br><br>RESULTS: Nine participants with transfusion-dependent &#x3b2;-thalassemia (four &#x3b2;[0]/&#x3b2;[0] or &#x3b2;[0]/&#x3b2;[0]-like and five non-&#x3b2;[0]/&#x3b2;[0] genotypes) received reni-cel and were included in the analysis. The median duration of postinfusion follow-up was 17.5 months (range, 3.8 to 23.4), and six participants could be evaluated for transfusion independence at 12 months or more. All the participants had neutrophil and platelet engraftment by 42 days after infusion. Rapid increases in total and fetal hemoglobin levels resulted in each of the nine participants being transfusion-free at their last follow-up visit. The six participants who could be evaluated at 12 months or later were transfusion-independent. The mean total and fetal hemoglobin levels were greater than 12 g per deciliter and greater than 11 g per deciliter, respectively, between months 6 and 18. A total of 69 grade 3 or 4 adverse events with onset or worsening during or after reni-cel infusion were reported in the nine participants. Six serious adverse events (infections, pyrexia, or pneumonitis) were reported in four participants. Adverse events were generally consistent with myeloablative conditioning. One patient had decreased lymphocyte counts attributed to reni-cel.<br><br>CONCLUSIONS: Treatment with reni-cel resulted in rapid neutrophil engraftment, an increase in fetal hemoglobin expression, and transfusion independence. These data support further investigation of Cas12a gene editing of the promoters of HBG1 and HBG2 in the treatment of transfusion-dependent &#x3b2;-thalassemia. (Funded by Editas Medicine; EdiThal ClinicalTrials.gov number, NCT05444894.).},
}
@article {pmid41932456,
year = {2026},
author = {Silva, CS and Nascimento, GR and Cruz, PEO and Arancibia, RH and Andrade Belitardo, EMM and Castro, TLP and Villar, LM and Pacheco, LGC},
title = {Design principles for LAMP-CRISPR molecular diagnostics.},
journal = {Methods (San Diego, Calif.)},
volume = {251},
number = {},
pages = {1-22},
doi = {10.1016/j.ymeth.2026.03.014},
pmid = {41932456},
issn = {1095-9130},
mesh = {*Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Humans ; Point-of-Care Systems ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to develop CRISPR-based diagnostic assays for a broad range of pathogens. Coupling these systems with portable result-readout platforms such as lateral flow devices, microfluidics, and smartphones offers a promising pathway for deploying LAMP-CRISPR diagnostics at the point-of-care (PoC), especially in settings where conventional, resource-intensive methods like real-time PCR are not feasible. However, the development of LAMP-CRISPR assays presents unique challenges not typically encountered in real-time PCR workflows. These include the need for a larger number of oligonucleotides, the complexity of integrating multiple biochemical conditions, and a heightened risk of false-positive results. Despite the growing number of bioinformatics tools designed to aid assay development, establishing a robust and reproducible workflow for LAMP-CRISPR remains a significant hurdle. In this review, we critically examine current strategies for designing LAMP-CRISPR assays and offer a detailed, step-by-step guide to achieving high-performance diagnostic tools using this approach. We cover key aspects of target sequence selection, oligonucleotide and CRISPR system design, and the strategic choice of readout methods. We further discuss available tools for LAMP primer and CRISPR guide RNA design, providing practical recommendations for optimizing sequence selection. Various probe formats for Cas-mediated trans-cleavage detection are summarized, and we present best practices for assay standardization and minimizing false-positive signals. Finally, we highlight the current limitations and outline future directions for LAMP-CRISPR diagnostics in decentralized and PoC testing environments.},
}
@article {pmid41934379,
year = {2026},
author = {Lin, WW and Su, J and Li, JX and Chen, YW and Chen, JY and Wu, B and Cai, NQ and Gan, LJ and Liu, ZJ},
title = {Enhanced SNV Detection of HLA-B*15:02TA by Integrating Blocking RPA and Inhibiting CRISPR-Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {15},
pages = {11234-11245},
doi = {10.1021/acs.analchem.5c08035},
pmid = {41934379},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide/genetics ; *Replication Protein A/metabolism ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {With the introduction of the RPA-based CRISPR-Cas12a method, crRNA allostery and Cas12a protein engineering have been applied to the identification of single-nucleotide variants (SNVs). However, the complicated testing procedure often falls short of the intended simple and rapid objectives of CRISPR-based diagnostics (CRISPR-Dx), and the discrimination factor (DF) for SNV distinction remains suboptimal (DF = 2-5). In this study, we proposed a one-tube "Blocking RPA-coupled Inhibiting CRISPR-Cas12a" (BRIC) cascade strategy that features a dual recognition of SNVs. In the BRIC cascade, a reduction of RPA amplification products of wild-type target (WT) and WT-activated Cas12a cleavage lowered the WT detection signal to near-background levels, without compromising the efficiency of RPA or CRISPR-Cas12a. With HLA-B*15:02TA (rs3909184, G > C), a gene associated with drug-induced cutaneous rash, as the SNV target, the proposed one-tube BRIC strategy achieved a DF of 32.51, with the WT signal approximating the background level, thereby greatly enhancing SNV detection specificity. On the other hand, MT can be detected in a large number of WT (with a resolution of 0.1%). Within the HLA-B*15:02TA concentration (CMT) range of 5.0 × 10[-7] to 1.0 × 10[-3] nM (500 aM to 1 pM), a linear relationship was evident between the fluorescence signal (FMT) and lgCMT (FMT = 4146.55 + 578.09lgCMT; R[2] = 0.9904), with a detection limit of 67.6 aM (>3σ). Combined with a 3D-printed portable fluorimeter, the proposed method reported high consistency with next-generation sequencing in detecting HLA-B*15:02TA in 22 clinical plasma samples, validating the reliability of the one-tube BRIC cascade assay. These improvements in assay design, process, and DF highlight the promising potentials of the BRIC strategy in clinical settings.},
}
@article {pmid41934615,
year = {2026},
author = {Wang, Z and Tamura, Y and Hashimoto, M and Nakazato, I and Yamaguchi-Nishimura, A and Arimura, SI},
title = {Using ngTALEN to improve genome editing efficiency on targets containing 5-methylcytosines.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {1},
pages = {e70826},
pmid = {41934615},
issn = {1365-313X},
support = {24H02271//Japan Society for the Promotion of Science/ ; JPJSCCA20230008//Japan Society for the Promotion of Science/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *5-Methylcytosine/metabolism ; *Gene Editing/methods ; DNA Methylation/genetics ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Genome, Plant/genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {We recently discovered distinct methylation patterns between the mitochondrial genome and the nuclear-encoded mitochondrial DNA sequences (NUMTs), with the mitochondrial genome being hypomethylated and NUMTs being hypermethylated. Given that genome editing using mitochondrial targeted transcription activator-like effector nucleases (TALEN) is highly efficient, while editing at NUMT is difficult, we hypothesized that the methylation status might affect editing outcomes. To test this, we attempted to use ngTALEN [employing RVD-NG to recognize 5-methylcytosine (5mC)] to target the Flowering Wageningen (FWA) locus of Arabidopsis thaliana, specifically the promoter and gene body regions with varying levels of cytosine methylation. Comparative analysis using the active epimutant allele fwa-d and wild-type Columbia-0 (Col-0) carrying a naturally silenced allele of FWA revealed that editing was impeded by 5mC at both the promoter and gene body of FWA for both CRISPR/Cas9 and TALEN. Overall, TALEN editing is robust and comparable to that of CRISPR/Cas9 at multiple sites, while ngTALEN showed improved editing at the CG-hypermethylated promoter of FWA compared with TALEN. Additionally, when targeting multiple genomic loci with identical sequences that differ in methylation levels and chromatin states, ngTALEN was less effective to induce edits. Therefore, this study represents the first systematic comparison of editing efficiency between CRISPR/Cas9 and TALEN in dealing with methylated or unmethylated DNA in plants. Furthermore, we have developed ngTALEN as a specific and robust tool for enhancing editing at sites with various levels of CG methylation.},
}
@article {pmid41935002,
year = {2026},
author = {Zhang, H and Zhu, H and Gao, H and Zhou, Y and Que, L and Rong, S and Ma, H and Chang, D and Pan, H},
title = {A colorimetric/fluorescent/electrochemical tri-modal biosensor based on dual CRISPR/Cas12a system for detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1402},
number = {},
pages = {345393},
doi = {10.1016/j.aca.2026.345393},
pmid = {41935002},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Colorimetry/methods ; *Electrochemical Techniques/methods ; Fluorescence ; },
abstract = {BACKGROUND: The precise detection of tumor markers is crucial for early cancer diagnosis and monitoring. Existing unimodal detection methods are susceptible to interference in complex biological samples, making it difficult to simultaneously achieve high sensitivity and reliability. MicroRNAs (miRNAs), as a key class of cancer-related biomarkers, necessitate novel detection methods capable of multi-layered verification. This study aims to develop an biosensing platform with multi-signal outputs to address the critical challenge of balancing sensitivity, specificity, and result credibility in current tumor marker detection.<br><br>RESULTS: We successfully constructed a tri-modal biosensor based on a dual CRISPR/Cas12a system for the highly sensitive and specific detection of miRNA let-7a. The sensor generates a trigger strand via an exponential amplification reaction (EXPAR), which concurrently regulates three independent signaling pathways: &#x2460; It initiates the first CRISPR/Cas12a to suppress G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme) formation, turning off the ABTS colorimetric signal. &#x2461; It also activates duplex-specific nuclease (DSN) to inhibit the hybridization chain reaction (HCR), thereby blocking FAM fluorescence coupling to streptavidin-coated magnetic nanoparticles (SMBs) and turning off the fluorescence signal. &#x2462; The absence of HCR products on the SMBs inhibits the second CRISPR/Cas12a system, thereby preserving the electrode's P1 probe for binding with P2-3D-CdCo-ONSs@AuNPs and maintaining a high "turn-on" electrochemical signal from the nanocomposite. Thus, the concentration of miRNA let-7a, ranging from 50&#xa0;fM to 1 pM, can be precisely quantified and validated through colorimetric, fluorescent, and electrochemical signals.<br><br>SIGNIFICANCE: This study integrates a dual CRISPR/Cas12a system with a tri-modal output strategy encompassing colorimetric, fluorescent, and electrochemical detection, thereby constructing a detection platform featuring a cross-verification mechanism. This design not only significantly enhances detection accuracy and anti-interference capability but also lays a solid foundation for developing next-generation, highly reliable molecular diagnostic tools. It holds considerable application potential in the fields of early cancer screening and precision medicine.},
}
@article {pmid41935011,
year = {2026},
author = {Zhang, Y and Liu, W and Guo, R and Qi, Y and Bai, B and Zhang, J and Hu, N and Gu, Y and Yang, Y and Wang, S},
title = {CRISPR/Cas12a-mediated electrochemiluminescent biosensor integrating Ag modified Co-doped metal-organic frameworks for dual detection of malathion and phorate.},
journal = {Analytica chimica acta},
volume = {1402},
number = {},
pages = {345402},
doi = {10.1016/j.aca.2026.345402},
pmid = {41935011},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Metal-Organic Frameworks/chemistry ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Luminescent Measurements/methods ; *Malathion/analysis ; Silver/chemistry ; Cobalt/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; },
abstract = {BACKGROUND: With growing concerns about food safety, the detection of pesticide residues in food has become increasingly important. The mixed organophosphorus pesticides (OPs) formulations are commonly employed to maximize crop production; however, the excessive application of OPs has posed severe threats to food safety and human health. Therefore, there is an urgent need to develop rapid and sensitive analytical methods that could simultaneously detect the multiple OPs in food samples. Currently, studies on high-performance electrochemiluminescence (ECL) sensors for two or more target sensing have been rarely reported.<br><br>RESULTS: This work reports a CRISPR/Cas12a-mediated ECL biosensor for the dual detection of two organophosphorus pesticides, i.e., malathion and phorate. The composite of metal-organic framework material (Co-PTC) loaded with silver nanoparticles (AgNPs@Co-PTC) serves as the single-signal probe, in which Co-PTC was the ECL emitter and AgNPs function as co-reaction accelerators to amplify ECL signals effectively. The method obtains an off-ECL signal by incubating the biosensor with malathion and DNA labelled with black hole quencher 1 (BHQ1-DNA); subsequently, after converting phorate to activator DNA, the sideloading activity of CRISPR/Cas12a was activated, which resulted in an on-ECL signal. By integrating spherical nucleic acid switching strategy with CRISPR/Cas12a for signal amplification, the method achieved the detection limits of 0.108 pM for malathion and 1.01 pM for phorate (S/N&#xa0;=&#xa0;3), with satisfactory recovery rates of 95.7%-106.4% in food samples (cabbage and lettuce).<br><br>SIGNIFICANCE: The established dual-detection mode eliminates the need for multiple signal reporters, simplifying the detection procedure and effectively avoiding cross-interference. Accordingly, the CRISPR/Cas12a-based ECL biosensors featured high selectivity and stability and offered a novel analytical strategy for food safety monitoring.},
}
@article {pmid41935495,
year = {2026},
author = {Zhang, J and Zhang, W and Yan, J and Zhang, Y and Zheng, J and Liu, Y and Gu, N and Han, K},
title = {A proximity induced strand displacement amplification and CRISPR/Cas12a-based SERS assay for ultrasensitive detection of dengue virus.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118651},
doi = {10.1016/j.bios.2026.118651},
pmid = {41935495},
issn = {1873-4235},
mesh = {*Dengue Virus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; *Dengue/diagnosis/virology ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Dengue virus (DENV) is a mosquito-borne single-stranded RNA virus that causes dengue fever and can progress to severe disease, posing a significant public-health threat. Rapid and highly sensitive detection is crucial for the early diagnosis of DENV, which, however, suffers from difficult discrimination from similar symptoms of other virus infection, such as Chikungunya or Zika virus, as well as insufficient specificity and time-consuming procedures. Here, we present a surface-enhanced Raman scattering (SERS) biosensor that integrates strand displacement amplification (SDA) with CRISPR/Cas12a for ultrasensitive detection of DENV. In the absence of DENV, SDA and Cas12a stay inactive and the SERS probes retain an aggregation state due to the crosslinking of single-stranded Linker, exhibiting strong SERS signals. In the presence of target sequences, SDA generates abundant single-stranded Trigger-strands, activating Cas12a through crRNA recognition, cleaving the Linker required to enable the crosslinking and leaving the SERS nanoprobes in a monodispersed state, demonstrating weak SERS signals. This method shows exceptional sensitivity for Dengue virus type 1 (DENV-1), with a limit of detection as low as 4.68 fM. It also provides excellent specificity by accurately distinguishing DENV-1 from other pathogens, while maintaining reliable performance. The modular design of this strategy endows the system capability to be adapted for different targets, making it not only a promising tool for the highly sensitive diagnosis of DENV-1, but also a versatile platform for the precise recognition of other pathogens.},
}
@article {pmid41935496,
year = {2026},
author = {Wu, Y and Lv, B and Zhou, R and Zhang, H and Li, D},
title = {Highly sensitive closed-tube detection of alkaline phosphatase based on phosphate-mediated CRISPR/Cas12a regulation.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118659},
doi = {10.1016/j.bios.2026.118659},
pmid = {41935496},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Alkaline Phosphatase/blood/isolation &amp; purification ; Humans ; *Phosphates/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism ; *Bacterial Proteins/chemistry/metabolism/genetics ; Endodeoxyribonucleases ; },
abstract = {Recently, we reported that a 5'-terminal phosphate at the junction of split activators inhibits the trans-cleavage activity of Cas12a. In this work, we discovered that phosphate-mediated Cas12a inhibition has two key features: strong position dependence and notable dual-phosphate enhancement. AlphaFold 3 (AF3) structural predictions indicate that the inhibition is mediated by interactions between the phosphate and specific local residues on Cas12a. On the basis of these findings, we developed a highly sensitive alkaline phosphatase (ALP) biosensor by harnessing the principle that ALP controls Cas12a activation through dephosphorylating activators. Without the need for upstream signal amplification, the biosensor achieves a high sensitivity of 6.07 × 10[-6] U/L for ALP across a broad dynamic range of 7.5 × 10[-6] to 2.5 × 10[-2] U/L. Furthermore, the assay is readily extendable to the evaluation of ALP inhibitors. Notably, a closed-tube assay system with lyospheres (CASL) was developed. Our biosensor equipped with the CASL enables closed-tube ALP detection in human serum, effectively preventing environmental contamination and interference. Owing to its simple closed-tube workflow and excellent performance, this biosensor holds great potential for point-of-care clinical diagnostics. Beyond its application, the phosphate-mediated CRISPR/Cas12a regulation strategy elucidated herein offers deeper mechanistic insight and a versatile blueprint for future biosensor design.},
}
@article {pmid41935970,
year = {2026},
author = {Chen, YW and Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC},
title = {Mismatch-Driven CRISPR/Cas12a Biosensing of UV-Induced DNA Lesions for Environmental Solar Exposure Surveillance.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {22},
pages = {15930-15939},
pmid = {41935970},
issn = {1520-5851},
mesh = {*Ultraviolet Rays ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *DNA Damage ; Sunlight ; },
abstract = {Monitoring environmentally relevant ultraviolet (UV) radiation is critical for understanding its biological impacts on ecosystems and human health. However, conventional UV dosimeters lack the molecular sensitivity to detect DNA-level damage that initiates such effects. Here, we present a CRISPR/Cas12a-based biosensing platform capable of quantifying solar UV exposure through the detection of UV-induced thymine dimers in DNA activators. This system harnesses mismatch-driven suppression of Cas12a activity, enabling a reduction in the fluorescence signal in response to UV-induced molecular lesions. The impact of thymine arrangement and the dimerization position of the activators on sensitivity were investigated. UV-induced diminution in Cas12a's trans-cleavage efficiency (kcat/Km) was also characterized, revealing a 1.67-fold decrease as the UVB dose increased from 0 to 2 J/cm[2]. Under optimized conditions, the sensor achieved a detection limit of 0.029 J/cm[2] for UVB and demonstrated high sensitivity to UVC. Field validation under natural sunlight showed a strong correlation with reference radiometric measurements, validating the biosensor's accuracy and environmental relevance. The system's sensitivity to low lesion densities, straightforward mechanism, and simple operation highlights its potential for environmental surveillance, human health risk assessment, and ecological monitoring in response to solar UV radiation.},
}
@article {pmid41937163,
year = {2026},
author = {Zhou, HR and Doan, DTH and Hartwig, T and Turck, F},
title = {Cis-regulatory architecture downstream of FLOWERING LOCUS T underlies quantitative control of flowering in Arabidopsis thaliana.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41937163},
issn = {1474-760X},
support = {EXC 2048/1 Project ID: 390686111//Cluster of Excellence on Plant Sciences/ ; },
mesh = {*Arabidopsis/genetics/growth &amp; development ; *Flowers/genetics/growth &amp; development ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Enhancer Elements, Genetic ; Chromatin/metabolism ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid ; Transcription Factors/metabolism ; },
abstract = {BACKGROUND: The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with its precise expression controlled by complex transcriptional networks. While upstream regulatory regions are well-studied, the role of downstream cis-regulatory elements in modulating FT expression remains poorly characterized.<br><br>RESULTS: Systematic dissection of the FT downstream region in its native chromosomal context using CRISPR/Cas9-mediated genome editing provides genetic evidence that a 2.3-kb sequence, encompassing the Block E enhancer immediately adjacent to the FT coding sequence, is essential for proper FT expression and timely flowering. Fine-scale deletions within Block E reveal that a 63-bp sequence containing one CCAAT-box and one G-box, both closely spaced, forms a core functional module, whereas other conserved motifs contribute modestly in a context-dependent manner. Strikingly, a cryptic CCAAT-box module downstream of Block E that becomes active when repositioned. This coincides with increased transcription factor occupancy and local chromatin accessibility.<br><br>CONCLUSIONS: Our work reveals that quantitative FT expression and flowering time are governed by the spatial organization and chromatin context of downstream cis-regulatory elements. The positional sensitivity and modular logic of these elements provide framework for understanding and engineering quantitative gene regulation through targeted cis-regulatory design, a concept broadly applicable across diverse developmental systems.},
}
@article {pmid41937727,
year = {2026},
author = {Kriete, A and Basika, T and Novas, R and Mathieson, OL and Belikoff, EJ and Kleiner, M and Scott, MJ},
title = {Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {71-88},
doi = {10.1177/25731599261436373},
pmid = {41937727},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Female ; *Lucilia cuprina/genetics ; *Gene Editing/methods ; Male ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sex Determination Processes/genetics ; },
abstract = {Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene transformer with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing transformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of transformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in L. cuprina and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.},
}
@article {pmid41937789,
year = {2026},
author = {Lv, J and He, X and Wu, Q and Sun, Y and Pu, W and Dai, C},
title = {Research progress and applications of gene activation editing technology in crops.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1787461},
pmid = {41937789},
issn = {1664-462X},
abstract = {In recent years, CRISPR/Cas gene editing technology has become a fundamental method in biological breeding. As a vital tool for overcoming technological obstacles, it is currently widely used in functional gene research and genetic enhancement across a variety of organisms. Currently, CRISPR activation (CRISPRa) technology based on dCas9 fusion transcription activation domains has emerged as a powerful tool for expanding the application of CRISPR/Cas systems in improving traits in plants, animals, and microorganisms. This overview starts by going over the underlying principles and components of gene activation editing technology, as well as the phases of development of its three generations. It summarises the present difficulties and potential directions in this field while concentrating on the use of gene activation editing in important crop traits including growth and development regulation, stress resistance, and quality regulation. The objective is to offer valuable insights for the research and development of crop breeding.},
}
@article {pmid41938062,
year = {2026},
author = {Wang, S and Thach, T and De Vita, A},
title = {Editorial: The role of nano-therapeutics in precision cancer medicine.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1818253},
pmid = {41938062},
issn = {2296-4185},
}
@article {pmid41938130,
year = {2026},
author = {Yin, X and Fan, Z and Tong, Z},
title = {CRISPR-based electrochemiluminescence biosensors: Principles, optimization strategies, and translational challenges - A review of recent progress.},
journal = {Food chemistry. Molecular sciences},
volume = {12},
number = {},
pages = {100392},
pmid = {41938130},
issn = {2666-5662},
abstract = {The integration of CRISPR/Cas systems with electrochemiluminescence (ECL) has emerged as a promising strategy for constructing high-performance biosensing platforms. CRISPR systems, particularly Cas12a and Cas13a, offer programmable recognition of nucleic acid targets and activatable trans-cleavage activity. ECL provides sensitive signal readout with low background and wide dynamic range. As a narrative review, this article provides a comprehensive overview of recent advances in CRISPR-ECL biosensors, with an emphasis on optimization strategies and practical applications. We first discuss the working principles of Cas12a and Cas13a relevant to biosensing, highlighting their distinct kinetic properties, crRNA design considerations, and reaction condition requirements. We then examine optimization approaches at three interconnected levels: nucleic acid probe design (signal-on, signal-off, and auxiliary probes), sensing interface engineering (probe structures, luminophores, electrode materials, and magnetic nanomaterials), and cascade signal amplification (PCR, CHA, RCA, SDA, EDA, and RPA). Through cross-study comparison, we evaluate the strengths and limitations of different approaches and identify critical knowledge gaps. Their applications in detecting disease biomarkers, pathogen nucleic acids, environmental contaminants, and enzyme activities are summarized. Despite remarkable sensitivity achieved, challenges remain in assay time, reproducibility in complex matrices, and clinical validation. From industrialization and global health perspectives, regulatory approval, manufacturing scalability, cost control, and deployment in low-resource settings are also discussed. Finally, future directions toward simplified workflows, enhanced matrix robustness, standardized validation, multiplexed detection, and point-of-care compatible platforms are proposed. This review provides a structured reference and critical perspective for researchers working on CRISPR-ECL biosensing and related fields.},
}
@article {pmid41938355,
year = {2026},
author = {Ma, G and Xu, H and Zhang, S and Li, X and Liu, J and Xie, J and Yan, F and Zhou, H},
title = {Enhancing the efficiency of nuclease-based prime editing in rice with the Tf1 reverse transcriptase.},
journal = {aBIOTECH},
volume = {7},
number = {2},
pages = {100026},
pmid = {41938355},
issn = {2662-1738},
abstract = {The development of efficient and precise genome-editing tools is crucial for advancing functional genomics and improving crops. Our previously established nuclease-mediated prime editing (NM-PE) system, which combines the SpCas9 nuclease with prime editing based on microhomology-mediated end joining, enables the seamless insertion of small DNA fragments into plant genomes to add tags to genes of interest. However, the efficiency of 3 × FLAG sequence insertion via NM-PE requires further improvement. Here, we report a significant optimization of this system by replacing the M-MLV reverse transcriptase (RT) with evolved variants of the retrotransposon RT Tf1 derived from the mammalian PE6 system. Through codon optimization, we generated the evoTf1M4 variant, which substantially enhanced the efficiency of NM-PE. The optimized construct rPE20aV3 achieved up to 18.75% precise insertion of a 66-bp 3 × FLAG sequence at endogenous loci, representing a three-fold improvement over the original NM-PE system. Our results demonstrate that Tf1-aided optimization of NM-PE serves as an efficient platform for seamless insertion of a 3 × FLAG sequence in rice, offering broad potential for advanced genome engineering in plants.},
}
@article {pmid41939725,
year = {2026},
author = {Xu, H and Liu, R and Zhou, H and Kong, B and Shen, K and Zhao, T and Du, X and Zhang, H and Song, H and Guo, D and Gu, X and Wang, Q and Lee, CW and Yin, G and Zhang, Y and Chen, W},
title = {Engineered small extracellular vesicles as bioactive materials: Integrating engineering strategies for cargo loading and targeted delivery systems.},
journal = {Bioactive materials},
volume = {59},
number = {},
pages = {96-134},
pmid = {41939725},
issn = {2452-199X},
abstract = {Small extracellular vesicles (sEVs) are increasingly regarded as a unique class of bioactive materials whose intrinsic membrane composition and nanoscale architecture provide a versatile platform for therapeutic engineering. Rather than passive carriers, sEVs can be actively programmed through diverse strategies to achieve efficient loading, precise targeting, and functional integration with synthetic systems. Endogenous modulation of donor cells-via genetic editing, priming with bioactive glass, cytokine stimulation, or hypoxic cues-enables selective packaging of nucleic acids, proteins, and metabolites into secreted vesicles. Exogenous techniques, including electroporation, sonication, and extrusion, allow controlled incorporation of therapeutic drugs or genome-editing complexes such as CRISPR/Cas. In parallel, surface modifications based on Lamp2b-fusion scaffolds, aptamers, antibodies, and click chemistry confer tissue tropism and extend circulation time. Integration with nanomaterials, scaffolds, and microfluidic platforms further enhances stability, scalability, and reproducibility, positioning sEVs at the intersection of biology and materials science. This review highlights recent advances in engineering sEVs as programmable bioactive materials and discusses their potential to transform regenerative medicine, oncology, and precision therapeutics.},
}
@article {pmid41940952,
year = {2026},
author = {Wang, N and Tang, J and Hou, X and Quan, C and Mai, J and He, C and Chen, R and Tao, B and Gu, Y and Jia, D and Fu, T and Zou, J and Zhao, L and Wen, J and Shen, J},
title = {BnaCIPK9 homoeologs mediate the dosage-dependent regulation of seed oil in allotetraploid Brassica napus L.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {4},
pages = {},
pmid = {41940952},
issn = {1432-2242},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022YFD1200804//National Key Research and Development Program of China/ ; 2021HSZD004//Hubei Hongshan Laboratory research funding/ ; 2662023PY004//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Brassica napus/genetics/metabolism ; *Seeds/chemistry/metabolism ; *Plant Oils/metabolism ; Phylogeny ; Gene Expression Regulation, Plant ; Tetraploidy ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Gene Duplication ; Gene Dosage ; Haplotypes ; Genes, Plant ; Plants, Genetically Modified ; },
abstract = {CRISPR/Cas9 mutagenesis and overexpression lines analyses revealed CIPK9 homoeologs function in oil regulation of allotetraploid Brassica napus, cooperative multicopy interactions, and a superior haplotype on chromosome A10. Rapeseed (Brassica napus, B. napus), a globally significant allopolyploid oilseed crop, fulfills substantial annual vegetable oil demand. Evolutionary adaptation in this species is underpinned by gene duplication and homoeolog retention, enhancing plasticity under dynamic environmental stresses. This study focuses on BnaCIPK9, a regulator of seed oil content. Phylogenomic and structural analyses demonstrate that BnaCIPK9 homoeologs underwent duplication followed by consistent evolutionary retention within the Brassica lineage, exhibiting remarkable sequence and structural conservation. Expression profiling revealed tissue-partitioned functional specialization among homoeologs, with BnaA10.CIPK9 and BnaC05.CIPK9 showing seed-preferential expression. CRISPR/Cas9 knockout in B. napus and heterologous overexpression in Arabidopsis demonstrate these homoeologs act as dosage-dependent regulators of oil accumulation, dependent on their distinct expression patterns. They further exhibit expression-driven functional diversification in abiotic stress responses during seedling development. Population genomics reveal differential evolutionary trajectories among duplicates, with intensified selection on chromosome A10 driving adaptive divergence. Crucially, haplotype-trait association identifies hap.qCIPK9.A10.0 as a major haplotype linked to elevated oil content. This work elucidates how homoeolog subfunctionalization fine-tunes critical agronomic traits, oil biosynthesis, and stress resilience, in polyploid crops, establishing haplotype-assisted breeding as imperative for developing crop cultivars. Favorable haplotypes, exemplified by hap.qCIPK9.A10.0, offer precise targets for high-oil crop breeding improvement.},
}
@article {pmid41941439,
year = {2026},
author = {He, Y and Shen, Y and Duan, M and Shen, J and Zhang, X and Chen, J and Liu, Z and Jia, F},
title = {An Environmentally Resilient, Metal-Organic Framework-Armored CRISPR/Cas12a Sensing Reactor (ENCASE) for the Ultra-Stable and On-Site Detection of Salmonella typhimurium in the Food Supply Chain.},
journal = {ACS sensors},
volume = {11},
number = {4},
pages = {3051-3062},
doi = {10.1021/acssensors.5c03704},
pmid = {41941439},
issn = {2379-3694},
mesh = {*Salmonella typhimurium/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Metal-Organic Frameworks/chemistry ; *Food Microbiology/methods ; Temperature ; },
abstract = {The development of reliable and field-deployable detection technologies of Salmonella typhimurium (S. typhimurium) throughout the food supply chain is crucial for the early warning and effective control of salmonellosis outbreaks. CRISPR-based biosensors offer excellent specificity, high sensitivity, and portability; however, their practical applications are significantly limited by the poor environmental stability of Cas enzymes, which are highly susceptible to temperature fluctuations and organic solvent interference. Here, a metal-organic framework (MOF) material, named ZIF-L, was employed as a sensing reactor to encapsulate the whole CRISPR sensing system, effectively enhancing its stability against variable external conditions such as temperature fluctuations and organic solvents encountered along the food supply chain. The feasibility of the conventional and encapsulated anti-S. typhimurium CRISPR sensor was confirmed through CLSM imaging, PAGE testing, and fluorescent verification. Importantly, the protective ability of the fabricated sensing reactor was precisely regulated by optimizing the pore size, ligand ratio, and dimensions of the MOFs and then evaluated under extreme detection conditions: (i) different external temperatures (4, 37, 50, 60, and 70 °C) and (ii) different organic solvents (methanol, acetone, and isopropyl alcohol). An impressive sensing performance of over 75% of its bioactivity, with a detection limit of 33 CFU/mL for S. typhimurium, was retained, confirming its detection ability under variable detection conditions. Moreover, recovery rates of 93.2-105.7% in spiked food samples, even when subjected to typical environmental interferences, including low temperatures (4 °C), high temperatures (60 °C), and organic solvent (methanol) exposure were obtained, showcasing its potential for the ultra-stable, on-site detection of S. typhimurium, particularly under variable external conditions.},
}
@article {pmid41942435,
year = {2026},
author = {Kaufmann, C and Sting, S and Dai, C and Wutz, A},
title = {Comprehensive CRISPR/Cas9-based mutagenesis identifies single-amino acid substitutions that abrogate SPEN function in X inactivation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41942435},
issn = {2041-1723},
support = {31003A_152814/1//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Mutagenesis ; *Amino Acid Substitution ; *X Chromosome Inactivation/genetics ; RNA, Long Noncoding/genetics/metabolism ; Hypoxanthine Phosphoribosyltransferase/genetics/metabolism ; MutS Homolog 2 Protein/genetics/metabolism ; Haploidy ; Mouse Embryonic Stem Cells/metabolism ; DNA-Binding Proteins/genetics ; Histones/metabolism ; },
abstract = {While genetic screens have facilitated the dissection of protein function in animal development, advances in systematic point mutagenesis open new opportunities for forward genetics in mammalian cells. Here, we develop a CRISPR/Cas9-mediated base editing screen that allows functional screening of extensive collections of single amino acid substitutions of endogenous proteins. We demonstrate the application on the X-chromosomal Hprt and the autosomal Msh2 gene in diploid male and haploid mouse embryonic stem cells, respectively. Finally, we use this methodology to generate a sequence-function map of the transcriptional co-repressor SPEN in X chromosome inactivation. We demonstrate that the substitution of the SPEN RRM4-residue W522 abrogates X-linked gene repression by Xist RNA and impairs the establishment of H3K27me3 deposition. Our results demonstrate that screening in haploid cells allows efficient identification of mutations that would be recessive in diploid cells, suggesting applications across a wide range of areas.},
}
@article {pmid41942889,
year = {2026},
author = {Qiu, X and Yuan, L and Liu, X and Li, Y and Ma, X and Du, B and Yuan, M and Li, Z},
title = {A one-pot CRISPR/Cas12b assay for extraction-free and visual detection of Haemophilus influenzae.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41942889},
issn = {1471-2180},
support = {2024A102//Chinese Center for Disease Control and Prevention/ ; 2021YFC2301105//National Key Research and Development Program of China/ ; },
mesh = {*Haemophilus influenzae/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics ; *Haemophilus Infections/diagnosis/microbiology ; Sputum/microbiology ; Rapid Diagnostic Tests ; },
abstract = {Haemophilus influenzae is a major respiratory pathogen, particularly in pediatric populations, and its rapid and accurate detection is critical for early diagnosis and targeted treatment. Traditional diagnostic methods, such as bacterial culture and PCR, are often time-consuming and require specialized equipment. In this study, we developed the Hi-ExCad assay, a one-pot, extraction-free CRISPR/Cas12b-based system for the rapid, simple, and accurate detection of H. influenzae. The assay integrates loop-mediated isothermal amplification and CRISPR/Cas12b detection in a single reaction, enabling direct detection from clinical sputum samples without the need for nucleic acid extraction. The Hi-ExCad assay demonstrated high specificity, correctly identifying H. influenzae in clinical samples, with a limit of detection of 1 pg of genomic DNA. The assay also exhibited excellent performance in real-time detection and visual result interpretation under blue light, making it highly suitable for point-of-care testing and resource-limited settings. These findings suggest that the Hi-ExCad assay provides a rapid, reliable, and user-friendly method for the detection of H. influenzae, with significant potential for clinical application.},
}
@article {pmid41942919,
year = {2026},
author = {Behera, L and Samal, KC and C, P and Agrawal, PK and Achary, VMM and Dash, M and Mishra, A and Rani, M and Masika, FB and Goud, GSD and Kesawat, MS and Samantaray, S},
title = {An improved Agrobacterium-mediated transformation method for genome editing using CRISPR/Cas9 in elite indica rice (Oryza sativa L.).},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41942919},
issn = {1471-2164},
abstract = {UNLABELLED: Rice feeds nearly half of the world’s population and underpins global food security. Climate change now poses a major threat to rice productivity worldwide. Genome editing has reshaped crop improvement strategies. Among these tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) system stands out for its precision, efficiency, and scalability. However, Agrobacterium-mediated transformation efficiency is often low, particularly in indica rice varieties. Here, we optimized an Agrobacterium-mediated transformation protocol for indica rice cultivars. The method was established in Lalat and MTU-1010. Seed-derived embryogenic calli were used to introduce the thermosensitive genic male sterile (OsTMS5) gene. A CRISPR/Cas9 vector carrying a gRNA and the selectable marker hptII was used for transformation. Callus induction reached 96.87% in MTU-1010 and 93.30% in Lalat MS medium supplemented with 3 mg/L 2,4-D and 0.5 mg/L BAP. In contrast, regeneration efficiency was higher in Lalat (90.28%) than in MTU-1010 (87.51%) on MS medium supplemented with 0.25 mg/L NAA, 0.5 mg/L kinetin, and 2 mg/L BAP. In addition, PCR analysis further verifies the integration of the transgene. Subsequently, the transformation efficiency was 37.20% in Lalat and 29.62% in MTU-1010. Therefore, this protocol provides a robust platform for gene function analysis and trait editing in rice. Its application may accelerate yield improvement and enhance stress tolerance under changing climatic conditions.<br><br>GRAPHICAL ABSTRACT: [Image: see text]<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-026-12800-0.},
}
@article {pmid41943368,
year = {2026},
author = {Sangeetha, VJ and Pawase, PA and Vasanthkumar, SS and Patrimath, SS and Bashir, O},
title = {Precision-engineered starch: Integrating metabolic engineering and cell-free synthetic biology for sustainable bioplastics and functional foods.},
journal = {Carbohydrate polymers},
volume = {381},
number = {},
pages = {125146},
doi = {10.1016/j.carbpol.2026.125146},
pmid = {41943368},
issn = {1879-1344},
mesh = {*Starch/chemistry/biosynthesis/metabolism/genetics ; *Metabolic Engineering/methods ; *Synthetic Biology/methods ; *Functional Food ; Amylose/chemistry ; CRISPR-Cas Systems ; },
abstract = {This review consolidates recent advances that reposition starch development from downstream modification towards deliberate biosynthetic engineering. It critically examines the starch biosynthetic pathway as a foundation for rational design, highlighting the coordinated roles of granule-bound starch synthase, soluble starch synthases, starch branching enzymes, and debranching enzymes in defining granule structure. Clear structure-function-nutrition relationships are delineated, demonstrating how amylose-amylopectin ratio, chain-length distribution, and phosphate esterification govern physicochemical behaviour, digestibility, glycemic response, and resistant starch formation. Insights derived from naturally occurring starch mutants are integrated to elucidate genotype-phenotype-function linkages. It further evaluates the recent progress in planta metabolic engineering, with particular emphasis on CRISPR-Cas-based genome editing as a precise strategy for generating food-relevant starch phenotypes without foreign DNA insertion. Representative applications include high-amylose, low-glycemic staple crops and starches with engineered branching patterns to enhance processing performance. In parallel, emerging cell-free synthetic biology platforms are presented as complementary systems for producing structurally defined glucans with high purity and reproducibility. Collectively, these developments establish engineered starch as a next-generation bio-based material aligned with nutritional quality, technological functionality and sustainability objectives.},
}
@article {pmid41943836,
year = {2026},
author = {Xu, J and Cheng, L and Ma, S and Gan, C and Chai, J and Zheng, X and Hu, L and Ling, M and Zhang, M and Zhao, B and Cheng, H},
title = {In vivo CRISPR/Cas9 Screening Reveals that UBE2L3 Modulates Autophagic Flux through TSC2 Ubiquitination and Potentiates PD-1 Blockade in Triple-Negative Breast Cancer.},
journal = {International journal of biological sciences},
volume = {22},
number = {6},
pages = {2950-2969},
pmid = {41943836},
issn = {1449-2288},
mesh = {Humans ; Autophagy/genetics/physiology ; *Ubiquitin-Conjugating Enzymes/metabolism/genetics ; *Tuberous Sclerosis Complex 2 Protein/metabolism/genetics ; Animals ; Female ; CRISPR-Cas Systems/genetics ; Ubiquitination/genetics ; Cell Line, Tumor ; *Triple Negative Breast Neoplasms/metabolism/genetics ; Mice ; },
abstract = {Triple-negative breast cancer (TNBC), a distinct breast cancer subtype, poses significant challenges to conventional therapeutic approaches, and effective targeted therapies are limited. CRISPR/Cas9 library screening has demonstrated unprecedented efficiency and revolutionary potential in the identification of therapeutic targets. In this study, we performed In vivo CRISPR/Cas9 library screening and identified the E2 ubiquitin-conjugating enzyme UBE2L3 as a critical regulatory factor in the progression of TNBC. Loss of UBE2L3 restricted tumor cell growth by modulating autophagy in TNBC cells. Mechanistically, UBE2L3 downregulation led to increased tuberous sclerosis complex 2 (TSC2) expression, suppressing mTOR activity and altering autophagic processes in tumor cells. This regulation was mediated through the interaction between UBE2L3 and the E3 ubiquitin ligase SMURF2, which together control TSC2 protein ubiquitination and degradation. Autophagy and the tumor microenvironment are closely associated, and we observed that UBE2L3 knockdown in TNBC tumors significantly increased CD8+ T lymphocyte infiltration and enhanced tumor sensitivity to anti-PD-1 therapy. Collectively, our findings provide a theoretical foundation for considering UBE2L3 as a potential therapeutic target in TNBC.},
}
@article {pmid41945389,
year = {2026},
author = {Li, M and Gao, G and Jiao, Y},
title = {A Hierarchical Screening Strategy for Genome-Edited Events in Polyploid Species: A Case Study on Hexaploid Common Wheat.},
journal = {Current protocols},
volume = {6},
number = {4},
pages = {e70354},
doi = {10.1002/cpz1.70354},
pmid = {41945389},
issn = {2691-1299},
support = {2024YFF1000301//National Key R&amp;D Program of China/ ; tsqn202312306//Taishan Scholars Program/ ; 2025HWYQ-071//Shandong Provincial Natural Science Fund for Excellent Young Scientists Fund Program (Overseas)/ ; SYS202206//Shandong Provincial Natural Science Foundation/ ; ZR2022ZD22//Shandong Provincial Natural Science Foundation/ ; ZR2021ZD30//Shandong Provincial Natural Science Foundation/ ; },
mesh = {*Triticum/genetics ; *Polyploidy ; *Genome, Plant ; *Gene Editing/methods ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Genome editing via CRISPR/Cas9 has been widely adopted in cereal crops. In diploid species such as rice and barley, the generation of knockout mutants is relatively straightforward for functional characterization of the genes of interest due to their single-copy nature in the genome. In contrast, common wheat (Triticum aestivum L.) is a hexaploid species comprising three subgenomes (AABBDD); consequently, most genes are present as three homoeoalleles that retained substantial function redundancy during evolution. The generation of a complete set of single, double, and triple mutants is therefore essential for elucidating homoeoallele-specific functions and dissecting their contributions to the developmental and agronomic traits. Moreover, ensuring germplasm purity through the elimination of residual T-DNA is critical for maintaining stable mutation, particularly in single- and double-mutant lines. Here, we describe a hierarchical screening strategy for efficient identification of a comprehensive series of CRISPR/Cas9-induced mutants. This approach integrates high-throughput DNA isolation, selection of T-DNA-free mutants, maintenance of a uniform genetic background via backcrossing, systematic screening of all mutant combinations, and molecular confirmation of genome edits. This screening pipeline has proven effective in hexaploid common wheat and is readily adaptable to other polyploid species that are amenable to crossing. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Wheat cultivation and leaf sample preparation Basic Protocol 2: High-throughput DNA isolation Basic Protocol 3: Genotyping using an optimized, cost-efficient T7E1 assay Alternate Protocol 1: Genotyping using the KASP assay Support Protocol 1: Screening of T-DNA-free triple mutants.},
}
@article {pmid41945404,
year = {2026},
author = {Yu, F and Zhang, D and Peng, C and Qin, H and Xu, F and Zhang, Y and Pan, Z and Xiao, P and Li, N},
title = {Development of a rapid and sensitive single-tube RAA-CRISPR/Cas12a assay for monkeypox virus detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {16},
pages = {3312-3320},
doi = {10.1039/d6ay00065g},
pmid = {41945404},
issn = {1759-9679},
mesh = {*Monkeypox virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Mpox, Monkeypox/diagnosis/virology ; Rapid Diagnostic Tests ; Animals ; Recombinases/metabolism ; Sensitivity and Specificity ; },
abstract = {Monkeypox (mpox) is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks historically concentrated in West and Central Africa. The current global outbreak of MPXV highlights the urgent need for an efficient detection system. This study presents a novel single-tube recombinase-aided amplification (RAA)-CRISPR/Cas12a assay for the rapid, sensitive, and specific detection of MPXV. Optimized to achieve a fluorescence-based sensitivity as low as 0.5 copies per µL within 35 min at 37 °C, the system demonstrates excellent specificity, accurately distinguishing MPXV from other viruses, including vaccinia virus, with no cross-reactivity observed. Additionally, a lateral flow assay (LFA) format was developed, enabling visual detection of concentrations as low as 5 copies per µL within 40 min. Validated with simulated clinical samples, the assay achieved 100% accuracy in distinguishing positives from negatives. This integrated single-tube approach eliminates the need for costly thermal cyclers, simplifying the detection process, reducing contamination risks, and delivering reliable results in a short time, making it ideal for point-of-care testing (POCT) in resource-limited settings. Furthermore, the RAA-CRISPR/Cas12a platform offers significant cost savings by requiring fewer reagents for virus detection. The extremely low template volume requirement of just 1 µL maximizes detection efficiency. This configuration allows for repeated testing without compromising result integrity, further enhancing the utility of this approach for surveillance and outbreak control, particularly in low-resource environments.},
}
@article {pmid41946009,
year = {2026},
author = {Sutaoney, P and Singh, P and Malakar, S and Arsi, L and Ghosh, P},
title = {Microbial lipases: Catalyzing sustainable solutions for industrial innovations.},
journal = {Enzyme and microbial technology},
volume = {198},
number = {},
pages = {110869},
doi = {10.1016/j.enzmictec.2026.110869},
pmid = {41946009},
issn = {1879-0909},
mesh = {*Lipase/metabolism/chemistry/genetics ; *Bacteria/enzymology/genetics ; *Fungi/enzymology ; Biocatalysis ; Substrate Specificity ; Protein Engineering ; Biotechnology ; *Bacterial Proteins/metabolism/chemistry/genetics ; Industrial Microbiology ; Enzyme Stability ; },
abstract = {Microbial lipases are multifaceted biological catalyst that have surfaced as a key driver in various industries and are both eco-friendly and cost efficient.In large scale applications, lipases produced from bacteria, fungi and yeasts function better than their equivalents generated from plants and animals due to their wide substrate specificity, catalytic efficacy and stability under physicochemical circumstances. Recent developments in microbial lipase research, including sources, screening techniques, assay procedures, production methods, purification tactics, and biochemical characterisation, are critically examined in this review.The structural and mechanistic elements that control lipase function-such as lid domains, interfacial activation, and catalytic triads-are given special attention since they all have an impact on the stability, specificity, and industrial performance of the enzyme.Large-scale screening is done to check for the production of lipase in Bacillus sp., Achromobacter sp., Alcaligenes sp., Arthrobacter sp., Pseudomonas sp., and Penicillium sp. Additionally, the combination of synthetic biology, metagenomics, CRISPR-Cas technologies, enzyme engineering, and AI-assisted modelling is emphasized as a revolutionary strategy for identifying and customizing lipases with desired characteristics, including extreme environment microbes and application-specific variants.The review also highlights the growing industrial uses of microbial lipases in the bio-fuel, food and beverage, detergent, textile, leather, pharmaceutical, and medical industries, highlighting their contribution to the development of economically feasible and ecologically safe bioprocesses. All things considered, microbial lipases are an important biotechnological tool for developing sustainable industrial innovation and green chemistry.},
}
@article {pmid41946722,
year = {2026},
author = {Malone, HA and Myers, JA and Gruss, EG and Morgan, MA and Friske, JD and McCarty, TC and Navarro, JJ and Robinson, S and Halliburton, RL and Kietlinska, SJ and De Luna Vitorino, FN and Hansen, BS and Pruett-Miller, SM and Garcia, BA and Roussel, MF and Partridge, JF and Roberts, CWM},
title = {PHIP suppresses NuRD to enable the growth of SWI/SNF-mutant cancers.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41946722},
issn = {2041-1723},
support = {R01-CA-273455//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-172152//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-113794//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; F31-CA-278355//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P01-CA-96832//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-HD-106051//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P01-CA-196539//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism/genetics ; Cell Line, Tumor ; *Neoplasms/genetics/metabolism/pathology ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; Mutation ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Chromatin Assembly and Disassembly/genetics ; Promoter Regions, Genetic ; Transcriptional Activation ; Ubiquitination ; DNA-Binding Proteins/metabolism/genetics ; Animals ; CRISPR-Cas Systems ; Chromatin/metabolism ; },
abstract = {SWI/SNF chromatin remodeling complexes are perturbed in 20% of all cancers and in several developmental disorders, yet the mechanisms by which these mutations dysregulate transcription and drive disease are poorly understood. To both elucidate these mechanisms and identify vulnerabilities caused by these mutations, we leverage genome-wide CRISPR-Cas9 screening in hundreds of cancer cell lines and identify the chromatin reader protein PHIP as a specific dependency in cancers with broadly disrupted SWI/SNF function. Mechanistically, we reveal that PHIP cooperates with SWI/SNF to facilitate transcriptional activation by ubiquitinating and suppressing subunits of the repressive Nucleosome Remodeling and Deacetylase (NuRD) complex. We demonstrate that loss of SWI/SNF results in NuRD complexes accumulating at promoters where they would otherwise cause widespread transcriptional silencing if not antagonized by PHIP. Collectively, we identify PHIP as a regulator of the interplay between distinct chromatin regulators that function in development and disease and as a targetable vulnerability in cancers with broad SWI/SNF inactivation.},
}
@article {pmid41946842,
year = {2026},
author = {Akana, RV and Yoe, J and Laveroni, O and Sun, C and Kim, YM and Jerby, L},
title = {High-content CRISPR activation screens identify synthetically lethal RNA-based mechanisms to sensitize cancer cells to targeted T cell cytotoxicity.},
journal = {Nature genetics},
volume = {58},
number = {4},
pages = {841-853},
pmid = {41946842},
issn = {1546-1718},
support = {1019508.01//Burroughs Wellcome Fund (BWF)/ ; OPP1113682//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; },
mesh = {Humans ; Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Hyaluronan Receptors/genetics ; *T-Lymphocytes/immunology ; *Melanoma/genetics/immunology ; Single-Cell Gene Expression Analysis ; *RNA/genetics ; *Cytotoxicity, Immunologic/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *T-Lymphocytes, Cytotoxic/immunology ; },
abstract = {T cells recognize their target cells through the T cell receptor (TCR). Combining gain-of-function, single-cell and optical high-content screens, we identified RNA-based mechanisms that selectively sensitize target cells to TCR-specific T cell cytotoxicity. First, CRISPR activation screens in melanoma cells identify functionally diverse regulators of TCR-specific cytotoxicity, including SAFB, KHDRBS1, MYC, CD44, WNT3A, WNT1 and others. Expressing sensitizing hits in cancer and virally infected cells restores TCR-specific cytotoxicity. Next, we developed in situ Perturb-seq for optical pooled genetic screens with in situ detection of perturbations and spatial transcriptomic readouts. Perturb-seq and in vivo-in situ Perturb-seq show that the hits converge on shared cell-autonomous and intercellular mechanisms, map gene-environment interactions and reveal that Wnt ligands activate T cells. Introducing a scalable approach to decode gene function at the cell and tissue level, the study uncovered context-specific gene functions to restore targeted T cell-based elimination of dysfunctional cells via synthetically lethal, RNA-based interventions.},
}
@article {pmid41946927,
year = {2026},
author = {Fang, GQ and Deng, Y and Lyu, XY and Yin, CQ and Song, J and Zhang, Y and Zhong, J and Shen, EZ and Song, CQ},
title = {Boosting prime editing with engineered non-canonical pegRNAs.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41946927},
issn = {2157-846X},
support = {32471552//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNA (pegRNA), with the reverse transcription template and primer binding site (RTT-PBS) attached to the 3' ends of CRISPR-Cas guide RNAs. Although PE ribonucleoprotein (RNP) delivery holds great therapeutic potential, its weak genomic editing capability limits therapeutic applications. Here we present structure-guided engineering of the PE complex using non-canonical pegRNAs (npegRNAs), with the RTT-PBS integrated within the single guide RNA loops, to improve PE efficiency. This approach demonstrates enhanced precise editing rates across various genomic sites and cell types, and improves therapeutic gene correction in a tyrosinaemia mouse model. Cas9-associated npegRNAs are more resistant to exonuclease degradation, probably enhancing the PE complex's targeting efficiency in living cells. Using PE RNP delivery, npegRNAs achieve increased average editing yields of 26.8-fold over canonical pegRNAs and 5.9-fold over engineered pegRNAs (epegRNAs). Furthermore, npegRNA-mediated RNPs increased the efficiency of installing disease-relevant mutations up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, our findings demonstrate a robust PE strategy and highlight the potential of npegRNAs for therapeutic PE applications.},
}
@article {pmid41949766,
year = {2026},
author = {Mostafa, K and Scarano, A and Abdulla, MF and Hacıkamiloğlu, S and Kurt, O and Santino, A and Kavas, M},
title = {A rapid Agrobacterium rhizogenes-mediated transient expression for assessing sgRNA efficiency in CRISPR-Act3.0 in tomato.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {41949766},
issn = {1432-203X},
support = {121O463//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;t&#x131;rma Kurumu/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Agrobacterium/genetics ; Plants, Genetically Modified ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Fatty Acids/metabolism ; Gene Expression Regulation, Plant ; Transformation, Genetic ; *CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {CRISPR-Act3.0 is a robust tool for modulating fatty acid profiles in plants. We demonstrate that Agrobacterium rhizogenes-mediated transformation provides a rapid, cost-effective, and equipment-independent platform for validating sgRNA efficiency and metabolic outcomes within a short time. The CRISPR-Act3.0 system offers a powerful strategy for activating endogenous gene expression in plants. However, the labor-intensive and time-consuming nature of stable transformation often hinders the rapid validation of multiple sgRNAs. In this study, we optimized a rapid Agrobacterium rhizogenes-mediated transient expression system in tomato to evaluate sgRNA efficiency within the CRISPR-Act3.0 framework. As a proof-of-concept, we targeted four genes involved in fatty acid biosynthesis: SlFATA, SlFATB-01, SlFATB-02, and SlFATB-03. To ensure precise control, we utilized the root-specific pSMB promoter to drive the CRISPRa components. Our results demonstrate that this system can successfully induce significant transcriptional activation and alter fatty acid compositions specifically increasing palmitic acid levels by up to 45%-within approximately 30 days. This approach bypasses the requirement for whole-plant stable transformation during the initial screening phase and prevents potential pleiotropic effects by restricting activation to root tissues. Overall, this study provides a highly efficient diagnostic pipeline for functional genomics and metabolic engineering in tomato, offering a significant advantage for rapid trait evaluation before committing to stable transgenic line production.},
}
@article {pmid41950725,
year = {2026},
author = {Clémençon, M and Brogard, J and Rozen, M and Hourton, C and García, RM and Goureau, O and Bigou, S and Reichman, S},
title = {Generation of human P347L RHO-associated retinitis pigmentosa iPSC lines by a mutation insertion in the RHODOPSIN gene carrying the RHO c.1040C &gt; T variant using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103968},
doi = {10.1016/j.scr.2026.103968},
pmid = {41950725},
issn = {1876-7753},
mesh = {Humans ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Rhodopsin/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; Mutation ; Cell Differentiation ; Base Sequence ; },
abstract = {The P347L RHODOPSIN-related retinal dystrophy leads an autosomal dominant Retinitis Pimentosa. Here we describe the generation of two isogenic human induced pluripotent stem cell (hiPSC) lines carrying the mutation c.1040C > T, p.Pro347Leu in the RHODOPSIN gene using CRISPR/Cas9 engineering from a control hiPSC clone. The two generated hiPSC lines can be differentiated in all the three germ layers, showed pluripotency makers expression and presented a normal karyotype. These hiPSCs will provide a new cell tool to better understand physiopathological mechanisms of retinitis pigmentosa and for the development of innovative treatment.},
}
@article {pmid41950758,
year = {2026},
author = {Han, L and Wang, SG and Teng, M and Zheng, LP and Ge, SY and Mao, Q and Han, F and Yang, L and Chai, SJ and Luo, Q and Yao, Y and Yu, ZH and Luo, J},
title = {Research note: A novel double-gene deleted vaccine against hypervirulent MDV variant generated by CRISPR/Cas9-based gene editing.},
journal = {Poultry science},
volume = {105},
number = {7},
pages = {106872},
pmid = {41950758},
issn = {1525-3171},
mesh = {Animals ; *Chickens ; CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Marek Disease/prevention &amp; control/virology/immunology ; *Marek Disease Vaccines/immunology/genetics ; *Poultry Diseases/prevention &amp; control/virology/immunology ; *Herpesvirus 2, Gallid/immunology/genetics ; Gene Deletion ; Virulence ; },
abstract = {Marek's disease (MD), an important avian immunosuppressive and neoplastic diseases, has resulted in huge economic losses for the poultry industry worldwide. Over the past 50 years, the long-term prevalence of this disease and the growing immune pressure of MD vaccination have triggered persistently increased virulence. The emerging hypervirulent variant of MDV (HV-MDV) overcomes the protection conferred by commercial vaccines, highlighting the urgent need to develop novel, highly efficient MD vaccines. To address this challenge, using CRISPR/Cas9-based gene editing, we generated a novel candidate vaccine, SQ01ΔmeqΔLAT, which features double deletions of both oncogene meq and LAT-clustered miRNAs. Comprehensive experiments confirmed the gene deletions, genetic stability and retention of the replication kinetics of the mutant, without revealing any adverse impact on the expression of essential viral genes. Further animal experiments showed no histopathological lesions or neoplastic changes in SQ01ΔmeqΔLAT-challenged birds, demonstrating favourable safety to chicken hosts. More importantly, the SQ01ΔmeqΔLAT vaccine achieved a high protection index (PI) of 88.9% against HV-MDV strain HNSQ01; this was an improvement compared to CVI988, which provided a PI value of only 70.6%. Our data provide an important basis for the development of highly efficient MD vaccines and shed new light on the design of herpesvirus vaccines, simultaneously targeting both viral protein-coding genes and non-coding RNAs.},
}
@article {pmid41950922,
year = {2026},
author = {Kang, T and Bleris, L},
title = {Cellular-state control using ribozyme-scaffolded miRNA-sensing and CRISPR-mediated actuation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101379},
pmid = {41950922},
issn = {2667-2375},
mesh = {*MicroRNAs/metabolism/genetics ; *RNA, Catalytic/metabolism/genetics ; Humans ; Epithelial-Mesenchymal Transition/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; },
abstract = {Cellular transitions between states are fundamental to development, adaptation, and pathological processes, but monitoring and guiding these transitions using endogenous signals remain challenging. MicroRNAs (miRNAs) represent a powerful modality, as distinct cell states are characterized by unique miRNA expression signatures. Here, we introduce a state-specific miRNA-directed CRISPR system for detecting and responding to epithelial-to-mesenchymal transition (EMT), a critical process in development, wound healing, and cancer metastasis. This system leverages EMT-specific miRNAs to regulate activation of type II polymerase-driven ribozyme-single-guide RNA (sgRNA) constructs, which direct CRISPR-based effectors to modulate gene expression. Using this approach, we demonstrate selective elimination of cells that have undergone mesenchymal transition and dynamic filtering of cell populations. This system provides a versatile platform for precise activation of CRISPR-Cas9 effectors using endogenous, state-specific cues. Integrating miRNA signatures with CRISPR technology to monitor, modulate, and reprogram cell-state transitions paves the way for applications in regenerative medicine, cancer therapy, and beyond.},
}
@article {pmid41951520,
year = {2026},
author = {He, Y and Ma, Y and Wu, Y and Tang, X and Liu, S and Yin, D and Zheng, X and Qi, Y and Zhang, Y and Zhang, T},
title = {Harnessing diverse tRNAs and AI-guided mining for compact and efficient plant multiplex genome editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.02.016},
pmid = {41951520},
issn = {1879-3096},
abstract = {The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.},
}
@article {pmid41951614,
year = {2026},
author = {Kudo, K and Hashimoto, T and Awakawa, T and Zhang, L and Nishimura, T and Hashimoto, J and Kozone, I and Kagaya, N and Suenaga, H and Keatinge-Clay, AT and Abe, I and Shin-Ya, K},
title = {Skeletal editing via multi-step engineering of a modular polyketide synthase.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41951614},
issn = {2041-1723},
support = {JP19ae0101045//Japan Agency for Medical Research and Development (AMED)/ ; JP23H04569//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; GM145992//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM145992/GM/NIGMS NIH HHS/United States ; JP23H05474//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {*Polyketide Synthases/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Protein Engineering/methods ; Streptomyces/genetics/metabolism/enzymology ; Acyltransferases/metabolism/genetics ; Macrolides/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Assembly line biosynthesis creates numerous structurally diverse natural products using a common modular synthetic strategy. The collinearity between the architectures of modular polyketide synthases (PKS) and the structures of their polyketide products would seem to render these biosynthetic machineries excellent platforms for designer biosynthesis, yet reliable strategies to reprogram these assembly lines without diminishing their activities have not been identified. Here, as a best practice for PKS engineering, we demonstrate the reprogramming of the mediomycin PKS without significant loss of productivity. Using in vitro CRISPR/Cas9 gene editing followed by heterologous expression, we reconstruct an inaccessible drug lead of the fibrinogen receptor, tetrafibricin, at 82 ± 3 mg/L yield, retaining 26% productivity after five-step module editing using an evolution-supported cut site, downstream of the acyltransferase domain. A macrocyclic aminopolyol is also accessed through thioesterase swapping. These results pave the way toward the rational reprogramming of PKSs to access desired complex organic molecules.},
}
@article {pmid41951658,
year = {2026},
author = {Lam, JKC and Leung, SSK and Li, JYK and Cheng, ECK and Kwon, SC},
title = {Molecular basis of target RNA cleavage by Cas13.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41951658},
issn = {2041-1723},
support = {17110223//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 17118324//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 17118724//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA Editing ; *RNA/metabolism/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Base Sequence ; },
abstract = {RNA-targeting CRISPR-Cas13 enzymes are robust RNA knockdown tools with both on-target and collateral cleavage activities. However, to date, the in vivo RNA cleavage mechanisms remain poorly understood. Here, we combine in vitro and in vivo methods to elucidate the exact cleavage sites of Cas13. We reveal that some subtypes of Cas13, including Cas13b and Cas13bt, cleave the target RNA at predominant positions, and rational engineering of Cas13 further improves precision. Building on these findings, we develop RNA segment editing (RSE), a targeted RNA cleavage and repair method, to restore dysfunctional RNA in cells. We anticipate that RSE will enable precision RNA engineering for therapeutics and basic research.},
}
@article {pmid41951661,
year = {2026},
author = {Li, Y and Feng, N and Wu, X and Zhu, B and Chen, Y},
title = {Activation mechanism and integrated one-pot assay of chiral-like cRNA-enhanced CRISPR/Cas12a systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41951661},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Gene Editing/methods ; Nucleic Acid Conformation ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {The 5'-repeat fragments released during pre-crRNA maturation are critical yet understudied components of the CRISPR/Cas12a system. Here, we demonstrate that engineered 5'-repeat fragments can potently activate Cas12a cleavage, with efficiency strongly dependent on the length of the 3'-spacer. Strikingly, complete truncation of the 3'-spacer generates a "chiral-like crRNA" conformation that induces a delayed-switch mode of Cas12a activation, fundamentally distinct from conventional mature crRNA. Leveraging this characteristic, we develop the delayed cleavage feature-mediated one-pot sensing strategy that resolves the long-standing challenge of incompatibility between Cas12a-based cleavage reaction and nucleic acid amplification, achieving a 1000-fold improvement in sensitivity over that of the conventional mature crRNA-mediated one-pot method. Furthermore, we integrate a cleavage-based one-pot assay with a portable temperature-controlled fluorescence imaging device to create an on-site diagnostic platform for high-throughput screening. Our study further advances the understanding of the crRNA-guided mechanism and facilitates the expansion of its applications in genome editing and molecular diagnostics.},
}
@article {pmid41951696,
year = {2026},
author = {Hu, C and Zhang, W and Ge, H and Wang, Y and Chao, C and Shi, X and Zhou, X and Wang, C},
title = {Effects of CRISPR-Cas9-mediated CEP55 gene knockout on immune evasion mechanisms of liver cancer cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41951696},
issn = {2045-2322},
mesh = {Humans ; *Liver Neoplasms/immunology/genetics/pathology ; *CRISPR-Cas Systems ; STAT1 Transcription Factor/metabolism ; *Cell Cycle Proteins/genetics ; Interferon-gamma/metabolism ; *Centrosomal Associated Proteins/genetics ; Cell Line, Tumor ; Gene Knockout Techniques ; B7-H1 Antigen/metabolism/genetics ; *Tumor Escape/genetics ; Reactive Oxygen Species/metabolism ; Tumor Microenvironment/immunology ; Signal Transduction ; *Carcinoma, Hepatocellular/immunology/genetics/pathology ; Apoptosis/genetics ; Hep G2 Cells ; Histocompatibility Antigens Class I/metabolism ; },
abstract = {This study investigates the role of centrosomal protein CEP55 in immune evasion by liver cancer cells and evaluates the effects of its knockout using CRISPR-Cas9 technology. CEP55-knockout models were established in human hepatocellular carcinoma cell lines Huh7 and HepG2, and alterations in immune-related molecules, tumor cell behavior, and antitumor immune responses were systematically assessed. CEP55 knockout significantly reduced PD-L1 expression while upregulating MHC class I levels, thereby enhancing tumor immunogenicity. Mechanistically, CEP55 deletion attenuated STAT1 activation, particularly under interferon-γ (IFN-γ) stimulation, suggesting involvement of the IFN-γ-STAT1 signaling axis in CEP55-mediated immune regulation. In parallel, CEP55 knockout markedly decreased intracellular reactive oxygen species (ROS) levels and suppressed the secretion of immunosuppressive cytokines IL-10 and TGF-β, indicating remodeling of the immunosuppressive tumor microenvironment. Functional assays demonstrated that CEP55 deficiency inhibited tumor cell migration and invasion and promoted apoptosis. Importantly, co-culture experiments revealed that CEP55 knockout enhanced T cell effector function, as evidenced by increased secretion of IFN-γ and Granzyme B and restored T cell-mediated cytotoxicity, even in the presence of IFN-γ stimulation. Collectively, these findings indicate that CEP55 promotes liver cancer immune escape and malignant progression through modulation of STAT1-dependent PD-L1/MHC-I expression, oxidative stress, and immunosuppressive signaling. Targeting CEP55 may therefore represent a potential strategy to improve antitumor immune recognition in liver cancer.},
}
@article {pmid41951915,
year = {2026},
author = {Zhu, Y and Moerner, WE and Qi, LS},
title = {CRISPR-Cas-based live cell imaging of genome dynamics.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {41951915},
issn = {1471-0064},
abstract = {The 3D architecture and dynamics of the genome are crucial for regulation of genome stability, transcription and cellular function. CRISPR-based live imaging technologies have enabled real-time visualization of specific genomic loci and transcripts in living cells. These tools harness customized guide RNAs and nuclease-deactivated Cas effectors to achieve precise genomic targeting, and recent methodological advances provide the 3D spatiotemporal resolution required to decipher real-time chromatin communication. These methods are elucidating the biophysical properties of chromatin, linking dynamic enhancer-promoter interactions directly to transcription, and revealing the role of 3D genome dynamics in basic cellular processes and disease. Here, we summarize the development of CRISPR-based live-cell imaging techniques, highlight the complementary 3D microscopy and analysis methods compatible with these methods, and offer perspectives on their applications to uncover fundamental principles that govern genome dynamics and function.},
}
@article {pmid41952056,
year = {2026},
author = {Salum, YM and Chen, J and Dang, J and Qiao, Q and Chen, T and Lin, T and Wei, H and He, W},
title = {Leveraging RNAi and CRISPR/Cas9-based strategies for target gene characterization and control of western flower thrips (Frankliniella occidentalis): Advances and perspectives.},
journal = {Pest management science},
volume = {82},
number = {7},
pages = {6024-6039},
doi = {10.1002/ps.70782},
pmid = {41952056},
issn = {1526-4998},
support = {//Major Project of Science and Technology of Fujian Province (2024NZ029029)/ ; //Fujian Provincial Science and Technology Project (2023L3021 and 2025N0081)/ ; },
mesh = {Animals ; *Thysanoptera/genetics ; *RNA Interference ; *CRISPR-Cas Systems ; *Insect Control/methods ; *Gene Editing/methods ; },
abstract = {Frankliniella occidentalis, the western flower thrips, is among the most destructive agricultural pests worldwide and a major vector of orthotospoviruses. Escalating insecticide resistance has intensified efforts to develop molecularly informed control strategies. RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing have emerged as principal tools for functional genomics in this species, enabling targeted analysis of genes involved in development, metabolism, and insecticide resistance. This review synthesizes recent advances in RNAi and CRISPR applications in F. occidentalis, with particular emphasis on delivery strategies, editing efficiencies, and resulting phenotypes. RNAi studies demonstrate growing technical feasibility through injection, feeding-based approaches, and plant-mediated systems, and emerging CRISPR/Cas investigations confirm the possibility of targeted gene knockouts affecting resistance traits and visible genetic markers. Despite these advances, genetic manipulation of F. occidentalis remains technically challenging, primarily due to biological and technical constraints, including inefficient delivery of ribonucleoprotein to the germline, the extremely small and fragile nature of eggs, and narrow developmental windows for manipulation. We discuss these barriers and highlight methodological innovations required to improve RNAi stability, embryo manipulation, and genome editing efficiency. Finally, we propose how these molecular and genetic tools may be incorporated into existing integrated pest management, emphasizing their potential as complementary, species-specific strategies rather than standalone control solutions toward sustainable F. occidentalis management. © 2026 Society of Chemical Industry.},
}
@article {pmid41952066,
year = {2026},
author = {Chatla, K and Ayalew, L and Yim, M and Ko, P and Lippold, S and Hernandez, G and Chua, BA and Patil, DP and Camperi, J},
title = {Analytical Assessment of sgRNA Impurities and Their Impact on Functional Performance.},
journal = {Analytical chemistry},
volume = {98},
number = {15},
pages = {11438-11447},
doi = {10.1021/acs.analchem.6c00747},
pmid = {41952066},
issn = {1520-6882},
mesh = {*RNA, Guide, CRISPR-Cas Systems/analysis/genetics ; Chromatography, Reverse-Phase ; Chromatography, Gel ; CRISPR-Cas Systems ; Humans ; },
abstract = {Single guide RNA (sgRNA) is a critical component of the clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system, guiding Cas9 to specific genomic loci for precise DNA modification. With its growing clinical potential, sgRNAs have emerged as a promising modality for gene editing-based therapeutics, underscoring the need for robust analytical characterization to ensure quality, safety, efficacy, and regulatory compliance. Here, we present an integrated strategy for deep profiling of sgRNA impurities, combining high-resolution ion-pairing reversed-phase liquid chromatography (IP-RPLC) and size-exclusion chromatography (SEC) with advanced technologies, namely, native mass spectrometry and nanopore direct RNA sequencing. The isolation and characterization of isolated chromatographic peaks revealed truncated, chemically modified, and deletion-prone species. Notably, early eluting fractions in IP-RPLC exhibited elevated deletion frequencies in the target-specific region, correlating with reduced gene-editing efficiency and increased variability in T cells when tested using the Cas-CLOVER system. In contrast, late-eluting fractions in IP-RPLC revealed polyphosphorylated variants with minimal functional impact, while off-target analyses suggested that early eluting impurities may paradoxically reduce off-target editing. Lastly, even at high levels, sgRNA aggregates showed only a limited impact on activity: fully aggregated preparations displayed ∼10% lower knockout efficiency; however, an increase in cumulative off-target frequencies was observed. Overall, this study highlights the value of combining advanced analytical tools to achieve deep profiling of sgRNA impurities. By linking specific impurity profiles to functional outcomes, these findings provide actionable insights for improving sgRNA quality control and advancing the development of safe and effective gene editing-based therapeutics.},
}
@article {pmid41952275,
year = {2026},
author = {Das, U and Prasad, SS and Sahoo, T and Paramanik, S and Halder, A and S, P},
title = {Unveiling the potential of banana (Musa spp.) improvement through genetic manipulation: current trends and future implications.},
journal = {Plant signaling &amp; behavior},
volume = {21},
number = {1},
pages = {2656013},
pmid = {41952275},
issn = {1559-2324},
mesh = {*Musa/genetics/microbiology ; Gene Editing ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Plant Breeding ; Plant Diseases/microbiology/genetics ; CRISPR-Cas Systems/genetics ; Disease Resistance/genetics ; },
abstract = {Banana (Musa spp.) is a globally important fruit crop and a staple food for millions of people. However, its narrow genetic diversity and clonal propagation make it highly vulnerable to pests, diseases, and abiotic stresses. Genetic improvement is limited by sterility, and triploid plants are reproduced clonally in most cultivated varieties, preventing traditional breeding based on genetic advancements. Transgenesis and gene editing are among the genetic engineering techniques used to increase yield, improve quality, and enhance resilience. Advances in Agrobacterium-mediated transformation and CRISPR/Cas tools have enabled the development of bananas with enhanced resistance to Fusarium wilt tropical race 4 (TR4), Black Sigatoka, and bacterial wilt, along with increased provitamin A content, longer shelf life, and reduced postharvest losses. DNA-free genome editing provides a promising approach to overcoming certain regulatory barriers and enhancing public acceptance. While challenges such as genotype-specific transformation efficiency, regulatory hurdles, and public perception persist, genetic manipulation holds the potential to both preserve and improve global banana production. This review synthesizes recent progress, key targets, and future prospects for the genetic improvement of banana.},
}
@article {pmid41952451,
year = {2026},
author = {Mengistu, G},
title = {CRISPR-Cas Systems in Human Disease Therapy: Advances, Clinical Applications, Limitations, and Future Directions.},
journal = {The journal of gene medicine},
volume = {28},
number = {4},
pages = {e70091},
doi = {10.1002/jgm.70091},
pmid = {41952451},
issn = {1521-2254},
mesh = {Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods/trends ; *Gene Editing/methods ; Animals ; Precision Medicine/methods ; Epigenome Editing ; },
abstract = {CRISPR-Cas systems have emerged as versatile platforms for targeted genome and transcriptome engineering, enabling precise manipulation of disease-associated genetic pathways. Continued advances in CRISPR technologies including base editing, prime editing, and epigenome modulation have expanded therapeutic possibilities beyond nuclease-mediated DNA cleavage, allowing programmable gene correction and regulation. Early clinical studies demonstrate sustained therapeutic benefit in selected monogenic disorders and highlight the feasibility of both ex vivo and in vivo editing strategies. However, clinical translation remains constrained by challenges such as off-target activity, delivery inefficiency, immune responses to Cas proteins, editing heterogeneity, and uncertainties regarding long-term safety. This review critically synthesizes recent advances in CRISPR-Cas systems for human disease therapy, integrating molecular innovations, delivery strategies, clinical progress, and ethical considerations. By evaluating both technological achievements and unresolved limitations, this article outlines key priorities for advancing CRISPR-based therapeutics toward safe, effective, and equitable precision medicine.},
}
@article {pmid41952472,
year = {2026},
author = {Martinkienė, J and Cui, T and Ciotta, G and Kupčinskas, J and Pažėraitis, D},
title = {RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.},
journal = {BioTechniques},
volume = {78},
number = {1-12},
pages = {149-157},
doi = {10.1080/07366205.2026.2649857},
pmid = {41952472},
issn = {1940-9818},
mesh = {*DNA, Single-Stranded/isolation &amp; purification/genetics/chemistry ; Humans ; Chromatography, High Pressure Liquid/methods ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Chromatography, Reverse-Phase/methods ; CD8-Positive T-Lymphocytes/metabolism ; },
abstract = {BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8[+] T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.},
}
@article {pmid41954204,
year = {2026},
author = {Zheng, S and Wang, C and Huang, Y and Meng, X and Zhao, D and Guan, E and Ma, F and Chen, A and Zheng, L},
title = {Dual-Mode CRISPR/Cas13a Assay for the Detection of Human Metapneumovirus in Clinical Respiratory Samples.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70921},
doi = {10.1002/jmv.70921},
pmid = {41954204},
issn = {1096-9071},
support = {2025YFC2311704//National Key R&amp;D Program of China/ ; },
mesh = {Humans ; *Metapneumovirus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Paramyxoviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/virology ; Reproducibility of Results ; *Molecular Diagnostic Techniques/methods ; RNA, Viral/genetics ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Human metapneumovirus (HMPV) is a significant pathogen, causing widespread acute respiratory infections (ARIs). Rapid and accurate detection is crucial for timely diagnosis and outbreak control. To address the limitations of real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based HMPV detection, we developed a novel method integrating recombinase-aided amplification (RAA) with CRISPR-Cas13a technology. Based on the alignment of 335 HMPV whole-genome sequences, a conserved region of the nucleocapsid (N) gene was selected for RAA primer and CRISPR RNA (crRNA) design. The concentrations of Cas protein, crRNA, and probe were optimized for specificity, sensitivity, and repeatability. A dual-mode platform was established, combining a fluorescence assay for quantitative detection and a lateral flow assay (LFA) for visual, on-site readout. Both methods were validated using 106 clinical samples and compared with RT-qPCR. Both methods demonstrated high specificity (no cross-reactivity with other respiratory viruses), a detection limit of 1 copy/μL, and excellent repeatability. Clinical validation showed complete concordance (κ = 1, p < 0.001) between the fluorescence assay and RT-qPCR, and high agreement for LFA (κ = 0.924, p < 0.001), with a sensitivity of 94.23%, specificity of 98.15%, positive predictive value (PPV) of 98%, and negative predictive value (NPV) of 94.6%. This study presents an RAA-CRISPR/Cas13a dual-mode platform for HMPV detection, providing a robust molecular diagnostic framework that paves the way for the future development of rapid testing assays.},
}
@article {pmid41954981,
year = {2026},
author = {Sentmanat, MF and Wang, ZT and Kouranova, E and Peters, ST and Chan, WC and Lin, J and Miao, Y and White, JM and Wallace, M and Cui, X},
title = {Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954981},
issn = {1362-4962},
support = {//Alvin J. Siteman Cancer Center/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/methods ; *Dependovirus/genetics ; Cell Line ; *Gene Editing/methods ; Genome ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles, imprecision in the insert and donor concatenation, genotypes of non-KI alleles, and more importantly, uniquely identifies and localizes random integration of the full or partial donor(s). Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models.},
}
@article {pmid41954985,
year = {2026},
author = {Zhang, S and Liu, Y and Wu, W and Liu, Z and He, Q and Wang, T and Yang, J and Yin, H and Yuan, Z and Zhang, H},
title = {Allosteric activation mechanism of the type VII CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954985},
issn = {1362-4962},
support = {32571682//National Natural Science Foundation of China/ ; 25JCZDJC00740//Natural Science Foundation of Tianjin Municipal Science and Technology Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; Models, Molecular ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Catalytic Domain ; RNA/chemistry/metabolism/genetics ; },
abstract = {Type VII CRISPR-Cas system, evolutionarily associated with type III systems, utilizes a Cascade complex formed by Cas5 and catalytically inactive Cas7 copies for target RNA binding, but instead incorporates a specialized Cas14 ribonuclease for target cleavage. Here, we report a high-quality cryo-EM structure at the target engagement state with a shortened crRNA and elucidate how the recruited Cas14 captures the target RNA and undergoes target-mediated activation. The signature Cas14 is homologous to eukaryotic CPSF73 and prokaryotic RNase J, comprising two conserved subdomains, MβL and β-CASP. Different from canonical type III systems, 5'-end target RNA, rather than 3'-end, is bent into the positively charged binding channel formed by the two subdomains to access the conserved catalytic pocket on Cas14. Two special structural features, α1 helix from Cas7 and α10 helix from Cas14, promote the bent target RNA docking into the catalytic pocket of Cas14 nuclease in concert. A dual-functional loop, displaced by the entering target RNA, induces a closed-to-open transition between the two subdomains for nuclease activation. More importantly, the flipped dual-functional loop also maintains the stabilization of incoming target RNA. Altogether, our work provides a more comprehensive understanding of type VII system mechanism, laying a mechanistic foundation for RNA-targeting tool development.},
}
@article {pmid41955489,
year = {2026},
author = {Jiang, B and Zhang, T and Lu, Y and Zhou, S and Xiao, W and Pan, S and Shi, N and Sheng, Y and Hu, J},
title = {CRISPR/Cas13a: Compensatory Target Activation Mechanism.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {32},
pages = {e24156},
pmid = {41955489},
issn = {2198-3844},
support = {22576127//National Natural Science Foundation of China/ ; 2023YFC2417200//National Key Research and Development Program of China/ ; 25ZR1401116//Sci-Tech Innovation Initiative Science and Technology Commission of Shanghai Municipality/ ; 24ZR1423000//Sci-Tech Innovation Initiative Science and Technology Commission of Shanghai Municipality/ ; 2025-YJRC-03//Putuo Hospital, Shanghai University of Traditional Chinese Medicine Start-up Grant/ ; 2019QN01Y725//Guangdong Provincial Pearl River Talents Program/ ; 202206010108//Science and Technology Program of Guangzhou/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; MicroRNAs/genetics ; Humans ; },
abstract = {CRISPR/Cas13a is a powerful RNA-targeting platform for molecular diagnostics, but conventional single-effector systems typically require contiguous RNA targets longer than ∼20-28 nt, limiting sensitivity and target flexibility. CRISPR/Cas13a-CTAM is presented as a compensatory target activation mechanism that facilitates synergistic Cas13a activation through two independently programmable short RNA effectors. By functionally decoupling allosteric activation and binding stabilization, CRISPR/Cas13a-CTAM supports robust activation by ultra-short RNA targets as short as 13 nt, substantially expanding the detectable target range. Compared with traditional single-effector Cas13a assays, CRISPR/Cas13a-CTAM achieves a detection limit of 1 fM for a 13-nt RNA target, representing an approximately tenfold sensitivity improvement. Notably, a single-nucleotide mismatch within the 13-nt target induces up to a 35-fold reduction in apparent cleavage rate, corresponding to a sevenfold enhancement in mismatch discrimination. The dual-effector architecture further enables simultaneous dual-target detection, demonstrated by dual miRNA profiling related to COVID-19 and combined detection of exosome membrane proteins. Moreover, the weakly activating effector was utilized as an anchoring module to achieve the first functional immobilization of Cas13a on a sensing surface, enabling in situ electrochemical miRNA detection. By overcoming the reliance on long RNA targets, CRISPR/Cas13a-CTAM provides a sensitive, programmable platform for RNA diagnostics and integrated biosensor development.},
}
@article {pmid41955743,
year = {2026},
author = {An, X and Tian, W and Wang, Y and Ren, Y and Xu, C and Chai, Q and Ai, P},
title = {Enhancing tomato fruit sweetness by CRISPR/Cas9-mediated SlVIF gene editing.},
journal = {Plant physiology and biochemistry : PPB},
volume = {233},
number = {},
pages = {111270},
doi = {10.1016/j.plaphy.2026.111270},
pmid = {41955743},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Fruit/genetics/metabolism ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Sucrose/metabolism ; Plants, Genetically Modified ; },
abstract = {In tomato plants, vacuolar invertase inhibitor (VIF) is a negative regulator of sucrose (SUC) degradation into fructose (FRU) and glucose (GLU), suggesting knockout of SlVIF promotes the accumulation of FRU and GLU. A dual-target vector of pKSE402-SlVIF was constructed and introduced into the tomato breeding parent '1912' via Agrobacterium-mediated transformation. The editing efficiency was as high as 47%. Two Cas9-free T1 lines exhibited delayed fruit ripening, significantly increased FRU and GLU levels, and reduced SUC content at the red ripe stage compared to wild-type (WT) plants, while fruit size and yield were unchanged. Additionally, in the SlVIF knockout mutants, the VIF protein structure was significantly altered, and VIN enzyme activity was markedly increased, which may underlie the enhanced degradation of sucrose in tomato fruits. The findings indicate that the sweetness of tomato fruit can be improved by CRISPR/Cas9-mediated SlVIF gene editing, which provides valuable resources for high-quality breeding in tomato.},
}
@article {pmid41955813,
year = {2026},
author = {Ji, Z and Zhao, Y and Huang, J and Shan, Y and Li, J and Huang, Q and Wu, M and Liu, F},
title = {Portable amplification-free digital droplet CRISPR/Cas12a platform for one-pot multiplexed viruses detection with attomolar sensitivity.},
journal = {Biosensors &amp; bioelectronics},
volume = {305},
number = {},
pages = {118668},
doi = {10.1016/j.bios.2026.118668},
pmid = {41955813},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; Animals ; *Biosensing Techniques/instrumentation ; Swine ; *Circovirus/isolation &amp; purification/genetics ; *Porcine epidemic diarrhea virus/isolation &amp; purification/genetics ; *Porcine respiratory and reproductive syndrome virus/isolation &amp; purification/genetics ; Limit of Detection ; Equipment Design ; Rapid Diagnostic Tests ; DNA, Viral/genetics ; },
abstract = {High-throughput, sensitive, and field-deployable nucleic acid detection is essential for timely pathogen surveillance. Here, we developed a novel amplification-free and portable platform based on an instrument-free polydisperse digital droplet CRISPR/Cas12a (DD-Cas12a) assay, enabling the simultaneous detection of three synthetic nucleic acid targets in a single reaction. The platform achieved attomolar-level sensitivity (0.15 fM) without pre-amplification, generated detection results within 40 min, and maintained high specificity even in the presence of a 10-fold excess of non-target nucleic acids. The DD-Cas12a was further applied to detect one DNA virus, porcine circovirus type 2 (PCV2), and two RNA viruses, porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection limits for PCV2, PEDV, and PRRSV were 126.0, 673.5, and 584.5 fg/μL, respectively, corresponding to sensitivity comparable to qPCR for PCV2 and 10-fold higher sensitivity for PEDV and PRRSV. To improve analytical throughput and objectivity, we established an Algorithm-Driven DD-Cas12a (ADC) platform for automated image analysis and result decoding. The custom-designed miniaturized portable instrument was developed for signal acquisition and field deployment. By combining the DD-Cas12a assay with the ADC platform and the portable instrument, we established a portable, digital, amplification-free, and ultrasensitive system for multiplex pathogen detection, providing a powerful tool for diagnostics, biosurveillance, and environmental monitoring.},
}
@article {pmid41956025,
year = {2026},
author = {Yang, M and Li, D and Wang, A and Zhang, Y and Ouyang, J and Wang, X and Hong, L and Tong, D and Wang, M and Lin, J and Luo, Q and Wang, G},
title = {CRISPR/Cas12a and Au@UiO-66 nanozyme synergistic dual amplification-based electrochemical biosensor for ultrasensitive ctDNA detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {171},
number = {},
pages = {109300},
doi = {10.1016/j.bioelechem.2026.109300},
pmid = {41956025},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; Humans ; *Gold/chemistry ; *Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/analysis/genetics ; *CRISPR-Cas Systems ; Breast Neoplasms/blood/diagnosis/genetics ; Hydrogen Peroxide/chemistry ; Female ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Breast cancer, a globally prevalent malignancy in women, requires early diagnosis to improve patient outcomes, where ctDNA serves as a key biomarker. In the present study, an electrochemical biosensor based on CRISPR/Cas12a and Au@UiO-66 nanozyme synergistic dual amplification was developed to achieve ultrasensitive detection of breast cancer marker ctDNA. Au@UiO-66-modified ssDNA probes were anchored to the gold electrode via thiol groups. The intact ssDNA probe anchors Au@UiO-66 nanozyme to the electrode, where its peroxidase-like activity efficiently catalyzes H2O2 reduction to generate amplified reduction peak currents. Target ctDNA activates Cas12a's trans-cleavage capacity by binding specifically to crRNA. Under the presence of the target, Au@UiO-66 nanozymes are released, resulting in a discernible drop in the peak current linked to H2O2 reduction. Benefiting from the optimized experimental conditions, the biosensor demonstrates a broad linear detection range (from 10 fM to 10 nM) for the target ctDNA, along with an exceptionally low detection limit of 6.14 fM. Successful detection in human serum samples demonstrates its practicality. The platform's high specificity is attributed to the programmable crRNA design, enabling detection of specified DNA sequences and showcasing significant adaptability for diagnosing multiple genetic targets, highlighting its potential in clinical cancer diagnosis.},
}
@article {pmid41956064,
year = {2026},
author = {Wang, M and Sternberg, SH},
title = {'Insane in the membrane': 2',3'-cGAMP triggers filamentous phage defense.},
journal = {Cell host &amp; microbe},
volume = {34},
number = {4},
pages = {556-558},
doi = {10.1016/j.chom.2026.03.009},
pmid = {41956064},
issn = {1934-6069},
mesh = {*Nucleotides, Cyclic/metabolism ; *Inovirus/physiology ; cGAS-STING Signaling Pathway ; Signal Transduction ; Animals ; Cell Membrane/metabolism ; Virus Replication ; *Bacteria/metabolism/virology ; },
abstract = {In this issue, Tak et al. report that bacteria produce 2',3'-cGAMP, a signaling molecule once considered unique to metazoans. This cyclic dinucleotide activates a SAVED-domain effector that polymerizes into membrane-disrupting filaments, inducing abortive infection and preventing phage replication, broadening the evolutionary scope of cGAS-STING-like signaling.},
}
@article {pmid41956072,
year = {2026},
author = {Decloquement, M and Macauley, MS},
title = {Revealing cancer glycome drivers using CRISPR activation screens.},
journal = {Cell genomics},
volume = {6},
number = {4},
pages = {101215},
pmid = {41956072},
issn = {2666-979X},
mesh = {Humans ; *Neoplasms/genetics/metabolism ; Glycosylation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; *Glycomics/methods ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics ; },
abstract = {In this study, Wisnovsky and colleagues[1] investigate the genetic determinants underlying cancer-associated glycome remodeling. Using genome-wide CRISPR-based approaches, they identified regulators of cell surface glycosylation. Specifically, genetic networks are discovered that upregulate cancer-related ligands of the sialic acid-binding immunomodulatory Siglecs.},
}
@article {pmid41956306,
year = {2026},
author = {Ding, Y and Lv, Z and Li, N and Ding, J and Zhao, GR and Wang, M and Zhang, Y},
title = {Pooled CRISPR screening enables genotype-phenotype association in industrial microorganisms.},
journal = {Biotechnology advances},
volume = {90},
number = {},
pages = {108893},
doi = {10.1016/j.biotechadv.2026.108893},
pmid = {41956306},
issn = {1873-1899},
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Industrial Microbiology ; *Genetic Association Studies ; Phenotype ; *CRISPR-Cas Systems ; Artificial Intelligence ; Genotype ; },
abstract = {Industrial microorganisms combine genomic robustness with process resilience to achieve high-level bioproduction, and these traits are usually governed by complex, multi-gene interactions. Pooled CRISPR screening-assisted genotype-phenotype association (GPA), an emerging approach attracting increasing attention, has recently evolved into a powerful platform for systematically interrogating gene function in industrially relevant strains and for rapidly identifying genotypes that drive desired phenotypes. In this review, we frame the GPA workflow as "Design-Build-Screen-Apply" and focus on the middle two steps. We compare state-of-the-art library-building technologies and catalogue positive hits obtained with diverse enrichment and screening strategies, evaluating their respective strengths, limitations, and applicability. Relevant applications from the past five years are then summarized to illustrate how GPA deciphers industrially relevant traits and accelerates the construction of high-performance microbial cell factories. Finally, we explore how artificial intelligence (AI) can streamline pooled CRISPR GPA workflows and outline remaining challenges.},
}
@article {pmid41957993,
year = {2026},
author = {Duhalde, MA and Martino, RA and Smania, A and Alvarez, HM and Hernández, MA},
title = {CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous Rhodococcus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1926-1942},
doi = {10.1021/acssynbio.6c00019},
pmid = {41957993},
issn = {2161-5063},
mesh = {*Rhodococcus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cytidine/genetics/metabolism ; Plasmids/genetics ; Genome, Bacterial/genetics ; DNA End-Joining Repair/genetics ; },
abstract = {Oleaginous Rhodococcus strains can degrade diverse compounds and synthesize and accumulate large amounts of intracellular lipids, making them attractive platforms for biotechnological applications. However, efficient genome editing in Rhodococcus remains challenging, and new molecular tools are needed to advance the understanding of its metabolism, stress responses, and cellular physiology. Here, we describe the nonhomologous end joining (NHEJ) system in rhodococci and implement an efficient genome-editing system based on a CRISPR/Cas9 nuclease approach that utilizes this repair mechanism, eliminating the need for donor DNA templates. In addition, we report the first implementation of a cytidine base-editing (CBE) system, enabling precise single-nucleotide substitutions (C•G → T•A) in oleaginous Rhodococcus strains. Both strategies rely on a dual-plasmid CRISPR platform, resulting in two plasmid sets: pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA. These systems enabled high rates of INDEL formation and C•G → T•A base conversions, with efficiencies of 70-80% and 75-85% at native genomic targets, respectively. Finally, for the CBE system, we implemented a Csy4-mediated sgRNA-processing module to support multiplex genome editing, enabling the simultaneous modification of multiple loci. Together, these tools outperform recombination-based approaches and will facilitate the study of complex metabolic pathways and the development of genetic strategies for biotechnological applications in Rhodococcus, while also being transferable to other actinobacteria.},
}
@article {pmid41958313,
year = {2026},
author = {Wong, E and Souza-Fonseca-Guimaraes, F},
title = {Gain-of-function enhancers optimize CAR-NK cell-based anti-cancer immunotherapy.},
journal = {Immunology and cell biology},
volume = {104},
number = {5},
pages = {494-498},
pmid = {41958313},
issn = {1440-1711},
mesh = {Humans ; Animals ; *Killer Cells, Natural/immunology/transplantation/metabolism ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; Mice ; *Receptors, Chimeric Antigen/metabolism/immunology/genetics ; *Neoplasms/therapy/immunology ; },
abstract = {Schematic overview of the two-stage screening approach used to identify NK cell fitness genes. (A) CRISPRa mechanism, showing dCas9-VP64-mediated upregulation of target genes. (B) Whole-genome CRISPRa screening in HER2-CAR-NK92 cells transduced with a CRISPR sgRNA library and transferred into mice bearing HT29 tumours, followed by tumour collection and next-generation sequencing (NGS). (C) Barcoded ORF mini-screen in primary peripheral blood NK (PBNK) cells transduced with HER2-CAR and an ORF library, transferred into HT29 tumour-bearing mice, with subsequent tumour collection and NGS analysis.},
}
@article {pmid41958681,
year = {2026},
author = {Zhang, R and Wu, Y and Sun, T and Luan, T and Ren, Y and Zhang, Y and Lin, L and Meng, T and Wang, J and Qiu, Y},
title = {Evolutionary insights and structural characterization guide the development of RAG1/RAG2-deficient swine models for immunological research.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1757508},
pmid = {41958681},
issn = {1664-3224},
mesh = {Animals ; *Homeodomain Proteins/genetics/chemistry/immunology ; Swine ; *DNA-Binding Proteins/genetics/chemistry/immunology ; *Evolution, Molecular ; V(D)J Recombination ; Humans ; *Severe Combined Immunodeficiency/genetics/immunology ; Disease Models, Animal ; CRISPR-Cas Systems ; Molecular Docking Simulation ; },
abstract = {INTRODUCTION: Defects in RAG1/RAG2-mediated V(D)J recombination cause severe combined immunodeficiency (SCID), a disorder characterized by the absence of mature T and B lymphocytes resulting from impaired antigen receptor gene rearrangement. Although evolutionary analyses demonstrate strong conservation of the catalytic core domains of RAG1/RAG2 across jawed vertebrates, structural divergence among species highlights the need for physiologically relevant large-animal models to evaluate human disease mechanisms. Pigs represent an advantageous model for human pathologies because of their close immunological and physiological similarities to humans.<br><br>METHODS: In this study, we performed evolutionary profiling, conservation analysis, tertiary structural modeling, and protein docking to characterize pig RAG1 and RAG2 and predict their functional roles in V(D)J recombination. These predictions were experimentally validated through the generation of RAG1 and RAG2 knockout pigs using CRISPR/Cas9-mediated genome editing.<br><br>RESULTS: Our findings define key structural features of pig RAG1 and RAG2 that are essential for V(D)J recombination and establish an integrated framework that combines comprehensive computational analyses with in vivo experimental validation.<br><br>DISCUSSION: This approach supports the development of pigs as physiologically relevant models of RAG deficiency and provides a foundation for future studies in pig immunology, genome editing, and translational research. Furthermore, these immunodeficient pigs permit long-term engraftment of human tissue xenografts and stem cell-derived teratomas, thereby enabling investigations of human immune reconstitution, cancer biology, and regenerative medicine. Collectively, this model offers a robust platform for immunotherapy development and for advancing studies in evolutionary and comparative immunology research.},
}
@article {pmid41959582,
year = {2026},
author = {Carrasco, E and Gutierrez-Marcos, J},
title = {Novel genome editing approaches to manipulate apical meristem activity for crop yield.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1743528},
pmid = {41959582},
issn = {1664-462X},
abstract = {Meristem function underlies organogenesis and yield potential in crop species, and its regulation depends on the crosstalk of genetic and hormonal networks that balance stem-cell niche maintenance and differentiation. During the shoot apical meristem (SAM) transition, developmental reprogramming shifts the meristem from a vegetative to a reproductive state, referred to as inflorescence meristem (IM). Major regulatory events in this transition include the cytokinin-gibberellin crosstalk, that regulate the expression of the CLAVATA/WUSCHEL (CLV/WUS) negative feedback loop and key transcription factor families like KNOTTED-LIKE HOMEOBOX (KNOX) and SHOOT MERISTEMLESS (STM). Despite the basic principles of apical meristem differentiation are well-described nowadays, major phenotypic bottlenecks were reached in major staple crops during the artificial selection process, known as domestication, leading to a final reduction in total crop yield. This review aims to describe the key processes and genes that play a role in this transition and how they can be artificially targeted to overcome these limitations. Major bioengineering approaches are covered, ranging from classical random mutagenesis with chemicals like ethyl methanesulfonate (EMS) to targeted genome editing using diverse Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR/Cas) systems. Finally, emerging strategies such as epibreeding are considered as promising tools to achieve precise, reversible modulation of meristem activity and to unlock new routes for crop yield enhancement.},
}
@article {pmid41960606,
year = {2026},
author = {Zhang, X and Nie, X and Yu, W and Du, G and Liu, S and Song, Y},
title = {Catalytic hairpin assembly-assisted split-T7 promoter-regulated CRISPR/Cas12a system for the sensitive analysis of microRNAs associated with coronary heart disease.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {16},
pages = {3303-3311},
doi = {10.1039/d6ay00524a},
pmid = {41960606},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *Coronary Disease/genetics/diagnosis ; Bacteriophage T7/genetics ; *Biosensing Techniques/methods ; },
abstract = {MicroRNAs (miRNAs) have emerged as promising non-invasive biomarkers for coronary heart disease (CHD); however, their accurate quantification remains challenging due to their short length, low abundance, and high sequence homology. Herein, we report a highly sensitive and specific biosensing platform by integrating catalytic hairpin assembly (CHA) with a split-T7 promoter-regulated CRISPR/Cas12a system. The key innovation lies in the split-T7 promoter architecture, which enables target-responsive reconstitution of a functional T7 promoter, driving robust in vitro transcription to generate abundant crRNA transcripts eliminating the need for pre-synthesized exogenous crRNA. This strategy achieves triple cascade amplification through three synergistic modules: CHA-mediated target recycling ensures efficient signal initiation; split-T7 promoter-driven transcription provides substantial signal enrichment; and CRISPR/Cas12a-based trans-cleavage delivers highly specific fluorescence readout. By eliminating pre-synthesized crRNA, the platform significantly reduces assay cost and complexity while maintaining excellent amplification efficiency. The method achieves a limit of detection as low as 38.9 aM for miRNA-155 within 90 min, exhibits single-base mismatch discrimination capability, and performs reliably in clinical specimens from CHD patients. With its modular design and robust performance, this cascade amplification platform offers a versatile and cost-effective tool for miRNA analysis, holding great promise for cardiovascular disease diagnosis and broader molecular diagnostics.},
}
@article {pmid41962435,
year = {2026},
author = {Jia, M and Ru, X and Bing, X and Zhai, F and Huang, M and Du, X},
title = {CRISPR/Cas12a-mediated aggregation of unmodified AuNPs via microwave-assisted heating-dry for label-free detection of Escherichia coli O157:H7.},
journal = {Biosensors &amp; bioelectronics},
volume = {305},
number = {},
pages = {118676},
doi = {10.1016/j.bios.2026.118676},
pmid = {41962435},
issn = {1873-4235},
mesh = {*Escherichia coli O157/isolation &amp; purification/genetics/pathogenicity ; Gold/chemistry ; *Biosensing Techniques/methods ; Microwaves ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Colorimetry ; Limit of Detection ; Escherichia coli Infections/microbiology/diagnosis ; Humans ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for developing next-generation biosensors, yet achieving rapid, instrument-free visual detection remains a challenging. Conventional CRISPR-AuNP assays rely on pre-functionalized, thiol-modified gold nanoparticles (AuNPs), which introduce steric hindrance that slows Cas12a kinetics and increases cost and complexity. Here, we developed a rapid, label-free colorimetric platform that integrates a mismatched catalytic hairpin assembly (MCHA) for low-background amplification with CRISPR/Cas12a trans-cleavage, coupled to a microwave-assisted dry-heating method for in-situ probe conjugation. Unlike conventional approaches where CRISPR acts on pre-conjugated probes, our method allows Cas12a to cleave free, non-thiolated ssDNA probes. Intact probes rapidly conjugate to unmodified AuNPs under microwave irradiation via their high-affinity domain, forming a protective corona that prevents salt-induced aggregation. Conversely, probes that have been cleaved by target-activated CRISPR/Cas12a lose this conjugation ability, rendering AuNPs susceptible to aggregation and producing a visible color change. To address the conflicting ionic requirements between the CRISPR reaction and AuNP stability, a Ba(OH)2-mediated ionic reset step is introduced to remove excess Mg[2+] prior to probe conjugation. This platform enables the sensitive detection of Escherichia coli O157:H7 within 2 h a limit of detection of 5 CFU/mL and robust performance in complex food and clinical matrices. Combined with smartphone-based RGB analysis, the instrument-minimized strategy eliminates the need for chemical modification of AuNPs and specialized operators, offering a versatile, low-cost, and user-friendly platform well-suited for pathogen detection in resource-limited settings.},
}
@article {pmid41962729,
year = {2026},
author = {Liu, Y and Chen, S and Zhang, C and Xue, H and Abubakar, MU and Yin, S and Yang, X and Fan, B and Tai, P and Xiong, M and Li, J and He, B},
title = {Asy-RPA/PCR combined with One-crRNA-CRISPR/Cas12a for simultaneous detection of multiple Clarithromycin resistance mutations in Helicobacter pylori.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {74},
number = {},
pages = {102942},
doi = {10.1016/j.nano.2026.102942},
pmid = {41962729},
issn = {1549-9642},
mesh = {*Helicobacter pylori/genetics/drug effects ; *Clarithromycin/pharmacology ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Drug Resistance, Bacterial/genetics ; *Polymerase Chain Reaction/methods ; Humans ; Anti-Bacterial Agents/pharmacology ; Polymorphism, Single Nucleotide ; Rapid Diagnostic Tests ; Helicobacter Infections/microbiology/drug therapy/genetics ; },
abstract = {METHODS: Genetic testing for Clarithromycin resistance-associated single-nucleotide variations (SNVs) in H. pylori could be applied for formulating individual eradication plan. In this study, we integrated asymmetric recombinase polymerase amplification (Asy-RPA) with a single crRNA for CRISPR/Cas12a-designated the ARoRC system-to circumvent protospacer adjacent motif (PAM) dependency.<br><br>RESULTS: The ARoRC platform detected all targeted mutations with 100% agreement compared to Sanger sequencing. Assay sensitivity was determined as follows: A2143G (10[-2]&#xa0;ng/&#x3bc;L), A2142C (2.58&#xa0;&#xd7;&#xa0;10[-3]&#xa0;ng/&#x3bc;L), A2142G (2.49&#xa0;&#xd7;&#xa0;10[-3]&#xa0;ng/&#x3bc;L), and A2142G&#xa0;+&#xa0;A2143G (2.39&#xa0;&#xd7;&#xa0;10[-3]&#xa0;ng/&#x3bc;L), enabling Asy-PCR-CRISPR/Cas12a detection suitable for fecal samples. The assay achieved visual results within 1&#xa0;h using lateral flow strips, with no cross-reactivity to WT or non-target sequences.<br><br>DISCUSSION: We developed a rapid, ultrasensitive, and portable assay for detecting Clarithromycin resistance-associated mutations in H. pylori. The robustness of the platform in complex matrices such as feces, along with its dual readout capability (fluorescence and lateral flow), supports its potential for point-of-care (POC) application.},
}
@article {pmid41963468,
year = {2026},
author = {Park, SB and Kim, JS and Ha, Y and Kim, MS and Kim, TW},
title = {Human pluripotent stem cell engineering with CRISPR-Cas9 for Parkinson's disease.},
journal = {Experimental &amp; molecular medicine},
volume = {58},
number = {4},
pages = {993-1009},
pmid = {41963468},
issn = {2092-6413},
support = {RS-2024-00351442//National Research Foundation of Korea (NRF)/ ; RS-2025-25437095//National Research Foundation of Korea (NRF)/ ; },
mesh = {Humans ; *Parkinson Disease/therapy/genetics/metabolism ; *CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism/cytology ; Dopaminergic Neurons/metabolism ; Animals ; *Gene Editing/methods ; *Cell Engineering/methods ; },
abstract = {Parkinson's disease (PD) entails loss of substantia nigra dopamine (DA) neurons and α-synuclein pathology. Currently, no effective disease-modifying therapies have been developed. Human pluripotent stem cells (hPS cells) can generate DA neurons on scale, enabling human genetic PD modeling of mitochondrial, lysosomal and synaptic connection failure that leads to DA neuron degeneration. Clustered regularly interspaced short palindromic repeats (CRISPR) extends this human model by providing causal, isogenic interrogation and transcriptional regulation of PD genes and reporter knock-ins that support purification and high-content screening. hPS cell-based DA cell grafts can restore motor function yet face >90% acute cell death and product heterogeneity in vivo post implantation. CRISPR enabled not only an in vivo cell survival screen to identify the cell death regulators but also a reporter-guided enrichment of DA neurons and chemogenetic control of grafted DA cell function in vivo. Here we summarize this progress and outline a practical road map to accelerate the development of precise human models and advanced hPS cell-based cell therapies for PD.},
}
@article {pmid41963733,
year = {2026},
author = {Le Phan, TH and Buchard, A and Brakebusch, C},
title = {Dispensable players: N-WASP and WASP are not crucial for homology-directed DNA repair.},
journal = {EMBO reports},
volume = {27},
number = {10},
pages = {2798-2822},
pmid = {41963733},
issn = {1469-3178},
support = {R302-A17455//Danish Cancer Society/ ; 101034291//EC | Horizon 2020 Framework Programme (H2020)/ ; },
mesh = {*Wiskott-Aldrich Syndrome Protein, Neuronal/genetics/metabolism ; Humans ; DNA Breaks, Double-Stranded ; *Wiskott-Aldrich Syndrome Protein/genetics/metabolism ; Actin-Related Protein 2-3 Complex/metabolism/antagonists &amp; inhibitors/genetics ; *Recombinational DNA Repair ; Cell Line, Tumor ; DNA End-Joining Repair ; RNA, Small Interfering/genetics ; *DNA Repair ; CRISPR-Cas Systems ; Indoles ; },
abstract = {N-WASP and WASP can induce actin polymerization via Arp2/3 and were reported to be crucial for homology-directed repair (HDR) of DNA double-strand breaks (DSB). The underlying mechanism was suggested to involve nuclear actin polymerization, but the mechanistic details were debated. Unexpectedly, we show now that neither WASP nor N-WASP is required for HDR during CRISPR-mediated genome editing. Using knock-out and overexpression of N-WASP and WASP in U2OS cells, we did not detect alterations in total gene editing, HDR, or the ratio of HDR to non-homologous end joining (NHEJ) as assessed by different methods. Furthermore, we could not observe colocalization of HA-tagged WASP or N-WASP with DSBs. Finally, while the Arp2/3 inhibitor CK-666 and ARPC4 knockdown by siRNA reduced HDR efficiency in U2OS cells, this corresponded with a decreased transfection efficiency and a reduction of the HDR-proficient cell cycle phases S and G2/M. In summary, contrary to expectations, these data do not support a crucial role for N-WASP and WASP in DSB repair.},
}
@article {pmid41964260,
year = {2026},
author = {Muthusamy, B and Nizan, S and Bar-Ziv, A and Perl-Treves, R},
title = {Functional Validation of the Melon Fom-1 Gene, Controlling Resistance to Fusarium oxysporum Races 0 and 2, by CRISPR/Cas9 Mutagenesis.},
journal = {Molecular plant pathology},
volume = {27},
number = {4},
pages = {e70258},
pmid = {41964260},
issn = {1364-3703},
support = {1137/16//Israel Science Foundation/ ; 873-0074-11//Chief Scientist grant for Biotechnology, Ministry of Agriculture, Israel/ ; },
mesh = {*Fusarium/physiology/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Genes, Plant ; *Mutagenesis/genetics ; Base Sequence ; *Plant Proteins/genetics/metabolism ; *Cucurbitaceae/genetics/microbiology ; *Cucumis melo/genetics/microbiology ; },
abstract = {Functional validation of NLR genes is critical for confirming their specific roles and developing durable disease-resistant crops. The Fom-1 gene of Cucumis melo, controlling resistance to races 0 and 2 of F. oxysporum f. sp. melonis (FOM), had been identified by map-based cloning as MELO3C022146, which encodes a TIR-NBS-LRR (TNL) protein. It resides in a head-to-head orientation adjacent to another TNL gene, Prv, controlling resistance to papaya ring spot virus (PRSV). In this study we validated the function of Fom-1 in mediating FOM resistance by applying CRISPR/Cas9 editing to the resistant cultivar, Védrantais. Two gRNAs were designed to target exons 1 and 2, respectively, and mutations were introduced at both target sites, resulting in truncated open reading frames in both alleles. Inoculation assays of T1 plants with FOM races 2 and 0 revealed a breakdown of resistance, manifested by leaf necrosis and wilting, and susceptibility was stably inherited in the T2 generation. This proved that MELO3C022146, the candidate gene for Fom-1, is responsible for resistance in melon cultivar Védrantais. Future studies will address the molecular functions of this gene as well as possible interactions between Fom-1 and its neighbour R-gene, Prv.},
}
@article {pmid41964558,
year = {2026},
author = {Guo, A and Guo, W and Guo, Y and Zhang, Y and Zhang, Z and Zou, X and Sun, Z},
title = {Clustered Regularly Interspaced Short Palindromic Repeat-Based Colorimetric Aptasensor Combined with Smartphone Imaging and Deep Learning Enables Selective Recycling and Visual Prediction of Microplastics in the Environment.},
journal = {Analytical chemistry},
volume = {98},
number = {16},
pages = {11886-11898},
doi = {10.1021/acs.analchem.5c08138},
pmid = {41964558},
issn = {1520-6882},
mesh = {*Colorimetry/methods ; *Smartphone ; *Microplastics/analysis ; *Aptamers, Nucleotide/chemistry ; *Deep Learning ; Polyvinyl Chloride/analysis ; *Water Pollutants, Chemical/analysis ; Polystyrenes/analysis ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Microplastics present significant risks to human health and ecosystem stability, creating an urgent need for analytical methods that are simple, rapid, sensitive, and field-deployable. Herein, we report a clustered regularly interspaced short palindromic repeat (CRISPR)-based colorimetric aptasensor for the detection of poly(vinyl chloride) (PVC) and polystyrene (PS) microplastics. This platform leverages the high specificity of PVC and PS aptamers integrated into a Fe3O4@Au-DNA magnetic complex, which facilitates capture, separation, and detection. Upon microplastic binding, a competitive reaction releases an activator DNA, initiating a dual CRISPR-Cas12a system for signal amplification. The activated Cas12a trans-cleavage activity is then linked to a hemin-aptamer DNAzyme colorimetric reaction, converting the signal into a visible color change. This colorimetric output is captured by smartphone imaging and processed in real time. Furthermore, a deep-learning-based regression model was developed to enable the quantitative prediction of PVC and PS micro/nanoplastics in diverse environmental matrices. The method exhibited high selectivity and a broad dynamic range from 10[-2] to 10[3] μg/mL. In smartphone detection mode, the limits of detection for PVC and PS reached 3.1 ng/mL and 3.7 ng/mL, respectively. This approach significantly enhances detection performance and stability, enabling visual monitoring of microplastics in complex real samples. Collectively, this work provides a rapid and effective strategy for the extraction and real-time quantification of small molecules.},
}
@article {pmid41965285,
year = {2026},
author = {Yang, X and Cai, J and Wang, J and Meng, Y and Shi, Y and Cai, H},
title = {CRISPR-Cas9 knockout screens to identify drug resistance genes in acute myeloid leukemia.},
journal = {Methods in cell biology},
volume = {205},
number = {},
pages = {199-216},
doi = {10.1016/bs.mcb.2026.01.006},
pmid = {41965285},
issn = {0091-679X},
mesh = {*Leukemia, Myeloid, Acute/genetics/drug therapy ; Humans ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Neoplasm/genetics ; *Gene Knockout Techniques/methods ; Cytarabine/pharmacology ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology ; Cell Line, Tumor ; Sulfonamides/pharmacology ; Antineoplastic Agents/pharmacology ; },
abstract = {Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.},
}
@article {pmid41965303,
year = {2026},
author = {Zhai, X and Yang, B and Deng, F and Gulati, S and Mckinnirey, F and Wu, X and Li, Y and Goldys, EM},
title = {CbAgo-enriched Cas12a biosensor for cancer mutations screening.},
journal = {Analytica chimica acta},
volume = {1403},
number = {},
pages = {345420},
doi = {10.1016/j.aca.2026.345420},
pmid = {41965303},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; Humans ; *Mutation ; CRISPR-Cas Systems/genetics ; Circulating Tumor DNA/genetics/blood ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Proto-Oncogene Proteins p21(ras)/genetics ; *Pancreatic Neoplasms/genetics/diagnosis ; },
abstract = {BACKGROUND: Accurate detection of low-frequency DNA mutations in body fluids is essential for cancer monitoring and treatment evaluation. However, the high abundance of wild-type DNA often masks rare mutant signals, making sensitive detection particularly challenging.<br><br>RESULTS: We developed a screening strategy termed the CbAgo-enriched Cas12a mutation screening system (CECMS). By integrating the single-nucleotide resolution of CbAgo with the trans-cleavage activity of CRISPR-Cas12a, this system selectively eliminates wild-type DNA while enriching targeted mutant alleles. CECMS achieves up to 100-fold higher sensitivity at 37&#xa0;&#xb0;C compared with conventional Cas12a biosensors, enabling reliable detection of variant allele frequencies (VAFs) as low as 0.01%. In undiluted serum spiked samples for circulating tumor DNA (ctDNA) detection, the method successfully detected pancreatic cancer-associated KRAS G12D mutations at a VAF of 0.1%.<br><br>SIGNIFICANCE: By leveraging CbAgo-mediated enrichment, the capability of exposing rare SNV for downstream detection is markedly improved. With its high efficiency and ease of use, CECMS holds strong potential as a convenient tool for clinical cancer diagnostics and monitoring.},
}
@article {pmid41965876,
year = {2026},
author = {Seibert, M and Kurrle, N and Kaleab, S and Wempe, F and von Metzler, I and Serve, H and Schnütgen, F},
title = {Endogenous protein tagging coupled with a CRISPR screening approach identifies UBE3C as a potential MYC oncogene regulator.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41965876},
issn = {2045-2322},
mesh = {*Proto-Oncogene Proteins c-myc/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Cell Line, Tumor ; *Multiple Myeloma/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Interferon Regulatory Factor-4 ; Green Fluorescent Proteins/genetics/metabolism ; F-Box-WD Repeat-Containing Protein 7/genetics/metabolism ; },
abstract = {The transcription factor MYC is a key regulator of cellular proliferation and metabolism and is frequently dysregulated in malignancies such as multiple myeloma (MM). Despite its clinical relevance, direct therapeutic targeting of MYC remains limited, emphasizing the need to identify upstream regulators that control endogenous MYC expression. To systematically uncover such regulators, we developed a genome-wide CRISPR-Cas9 loss-of-function screening approach, employing a custom-engineered MM reporter cell line (RPMI8226-F11), in which oncogenic MYC protein was endogenously tagged with EGFP (referred to as GFP). This fluorescent readout enabled a direct, quantitative assessment of endogenous MYC expression levels. A pooled genome-wide sgRNA library was introduced, and cells were sorted based on GFP fluorescent intensity to reflect varying MYC levels. Next-generation sequencing of sgRNA distributions across sorted populations enabled the identification of candidate MYC regulators. Validation of screen hits, including the established MYC activator IRF4 and repressor FBXW7, confirmed the reliability of our system. To further dissect regulatory networks, we performed an overrepresentation analysis of target genes, which revealed the enrichment of Mediator complex subunits among MYC activators and ubiquitin-proteasome pathway components among MYC repressors. Functional validation of prioritized hits-MED30 (Mediator complex) and UBE3C (E3 ubiquitin ligase)-demonstrated a strong impact on endogenous MYC levels. Notably, the knockout of UBE3C markedly increased MYC expression, whereas its paralogs, UBE3A and UBE3B, showed no measurable effect, suggesting a specific regulatory role for UBE3C in MM cells. Together, our study provides a comprehensive CRISPR screen-based resource for the discovery of MYC regulators and highlights UBE3C as a potential therapeutic node for modulating MYC expression in MM.},
}
@article {pmid41966744,
year = {2026},
author = {Tang, Q and Song, XH and Guo, AN and Chen, Y and Shao, JW},
title = {CRISPR-mediated regulation of apoptosis in cancer: Molecular targets, mechanisms, and translational challenges.},
journal = {Biochemical and biophysical research communications},
volume = {817},
number = {},
pages = {153719},
doi = {10.1016/j.bbrc.2026.153719},
pmid = {41966744},
issn = {1090-2104},
mesh = {Humans ; *Apoptosis/genetics ; *Neoplasms/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; Animals ; Gene Editing/methods ; Signal Transduction ; Translational Research, Biomedical ; },
abstract = {The CRISPR/Cas system, owing to its high gene-editing efficiency and relatively low off-target effects, has emerged as a pivotal technological platform in cancer research. By precisely modulating oncogenic signaling pathways and apoptosis-related molecules, CRISPR provides a critical tool for elucidating and reprogramming the regulatory mechanisms of apoptosis in tumor cells. However, despite substantial progress in preclinical studies, achieving efficient and selective induction of apoptosis in tumor cells remains a major translational challenge. Increasing evidence indicates that CRISPR-based strategies can achieve more effective antitumor outcomes by reprogramming tumor cell sensitivity to apoptosis, rather than relying solely on single-gene editing. This review systematically summarizes the molecular targets and underlying mechanisms of CRISPR-mediated regulation of tumor cell apoptosis, with particular emphasis on key apoptotic signaling pathways and representative research advances. It highlights the pivotal role of targeting survival-associated genes in suppressing tumor progression and promoting apoptotic cell death. Furthermore, this review discusses the potential synergistic mechanisms of CRISPR in combination with chemotherapy or immunotherapy, as well as the value of CRISPR-based functional screening in identifying apoptosis-regulatory targets and drug resistance-associated mechanisms. Finally, we analyze the key challenges facing the clinical translation of CRISPR-mediated apoptosis regulation and propose future research directions and conceptual frameworks to optimize CRISPR-based anticancer strategies and facilitate their clinical application.},
}
@article {pmid41967792,
year = {2026},
author = {Yuan, W and Jiang, Z and Li, F and Chen, H and Zhang, X and Fan, X},
title = {THOC6 deficiency leads to cardiomyopathy by reducing myocardial contractile proteins in cardiomyocytes.},
journal = {Experimental cell research},
volume = {459},
number = {2},
pages = {115025},
doi = {10.1016/j.yexcr.2026.115025},
pmid = {41967792},
issn = {1090-2422},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; Animals ; Humans ; *Cardiomyopathies/metabolism/pathology/genetics ; Rats ; Cell Proliferation/genetics ; Apoptosis/genetics ; Cell Line ; *Contractile Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism ; *Nuclear Proteins/genetics/deficiency ; },
abstract = {BACKGROUND: The THOC6 protein is an essential part of the THO complex. Biallelic loss-of-function variants in the THOC6 gene are linked to Beaulieu-Boycott-Innes syndrome (BBIS; OMIM #613680). Although research predominantly focuses on THOC6's involvement in neurodevelopmental disorders, approximately 80% of BBIS patients present with cardiac anomalies, including structural heart disease, cardiomyopathy, and arrhythmia. Despite this, the connection between THOC6 expression and cardiac development remains underexplored. This study firstly investigates THOC6's role in heart development.<br><br>METHODS AND RESULTS: This study we firstly utilized CRISPR/Cas9 to knock out THOC6 in H9C2 cardiomyocytes, revealing a reduction in cell proliferation and an increase in apoptosis. With RNA sequencing (RNA-seq) analysis we found abundant gene changes after THOC6 knockout (KO) in H9C2, which associated with hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and dilated cardiomyopathy. Protein-protein interaction analysis and experimental validation indicated that THOC6 regulates the expression of type I collagen (COL1A1, COL1A2) and cytoskeletal protein (Cardiac &#x3b1; actin 1) in cardiomyocytes. Subsequently, we generated a THOC6 knockout cell lines in human induced pluripotent stem cells (hiPSCs) derived from a healthy individual using CRISPR/Cas9 technology. THOC6 knockout (KO) in hiPSCs-derived cardiomyocytes (hiPSC-CMs) led to the early manifestation of hypertrophic cardiomyopathy and dilated cardiomyopathy phenotypic characteristics, including disrupted sarcomeric organization. Notably, THOC6 KO hiPSC-CMs demonstrated a significant decreased in COL1A2 and &#x3b2;-tubulin expression levels.<br><br>CONCLUSION: THOC6 may influence cardiac development by regulating myocardial contractile proteins, primarily type I collagen, cardiac &#x3b1; actin 1 and &#x3b2;-tubulin.},
}
@article {pmid41968146,
year = {2026},
author = {Nguyen, LT and Rakestraw, NR and Pizzano, BLM and Iyyappan, R and Young, CB and Huang, Y and Beerensson, KT and Fang, A and Antal, SG and Anamisis, KV and Peggs, CMD and Yan, J and Jing, Y and Lewis, JG and Burdine, RD and Adamson, B and Jiang, Z and Toettcher, JE and Myhrvold, C and Jain, PK},
title = {Efficient genome editing with chimeric oligonucleotide-directed editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41968146},
issn = {2041-1723},
support = {R35 GM147788/GM/NIGMS NIH HHS/United States ; U01 DK127429/DK/NIDDK NIH HHS/United States ; U01DK127429//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35GM147788//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01HD113698//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R61 AI181016/AI/NIAID NIH HHS/United States ; T32GM148739//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01HD102533//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; RM1HG009490//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; T32GM136583//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32 GM148739/GM/NIGMS NIH HHS/United States ; R61AI181016//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 75D30122C15113//U.S. Department of Health &amp; Human Services | Centers for Disease Control and Prevention (CDC)/ ; },
mesh = {*Gene Editing/methods ; Humans ; Animals ; HEK293 Cells ; *Oligonucleotides/genetics ; CRISPR-Cas Systems/genetics ; Mice ; Cattle ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report several nCas9-DNA polymerase fusion proteins and their engineered versions to create a simple and efficient two-component chimeric oligonucleotide-directed editing (CODE) system. CODE contains a derivative of Bst DNA polymerase engineered for increased thermostability and processivity as well as a chimeric pegRNA (cpegRNA) for programmable search and replace genome editing. Additionally, CODEMax(exo+) features a 5' to 3' exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate that CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci in HEK293T cells with plasmid- and RNP-based delivery. We also show that CODEMax can successfully modify mouse and bovine embryos with up to 9.3% precise editing. Further optimization of CODEMax systems may enhance editing outcomes in embryos and other challenging contexts. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.},
}
@article {pmid41968241,
year = {2026},
author = {Martin-Vicente, A and Fortwendel, JR},
title = {Cas9 Ribonucleoproteins (RNPs) for Gene Deletion.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {3-12},
pmid = {41968241},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Deletion ; *Ribonucleoproteins/genetics/metabolism ; *Aspergillus fumigatus/genetics ; Gene Targeting/methods ; *Gene Editing/methods ; Homologous Recombination ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Genetic manipulations in pathogenic microorganisms, like Aspergillus fumigatus, allow us to study the function of genes in pathogenicity or other important traits. Classic genetic engineering tools in filamentous fungi rely on DNA fragment sub-cloning or fusion PCR to build a gene deletion cassette containing extended flanking regions of homology to promote efficient recombination. However, the introduction of CRISPR technology into fungal genetics has greatly simplified and accelerated the genome editing process. Here, we describe a simple and universal, one-step CRISPR-Cas9-mediated genetic tool employing repair templates containing microhomology regions. This gene targeting system displays high homologous recombination efficiency rates and can be easily implemented across different genetic backgrounds.},
}
@article {pmid41968242,
year = {2026},
author = {Handelman, M and Werner, H and Osherov, N},
title = {Generation of Seamless Point Mutations with Cas9 RNP and pTel-hygR Plasmid in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {13-21},
pmid = {41968242},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/drug effects ; *CRISPR-Cas Systems ; Drug Resistance, Fungal/genetics ; *Point Mutation ; *Plasmids/genetics ; Antifungal Agents/pharmacology ; Triazoles/pharmacology ; Fungal Proteins/genetics ; Cytochrome P-450 Enzyme System ; },
abstract = {The environmental mold Aspergillus fumigatus is a common human fungal pathogen that causes a wide range of diseases. The antifungal triazoles that inhibit the Cyp51 enzyme involved in ergosterol biosynthesis are used to treat A. fumigatus infections. However, triazole resistance is an increasing concern due to mutations in the genes cyp51A, hmg1, and others, and efflux pumps overexpression. The process of verifying mutations is time-consuming, even with CRISPR-Cas9 methods, as it still requires constructing repair templates with selectable markers. This study presents a faster and more efficient method to introduce mutations conferring triazole resistance in A. fumigatus by using in vitro assembled CRISPR-Cas9, along with a recyclable selectable marker. With this approach, we successfully introduced triazole resistance-conferring mutations in A. fumigatus genes (cyp51A, cyp51B, and hmg1), both individually and in combination. The technique has the potential to introduce mutations for resistance to other antifungals, toxic metals, and environmental stressors, thus enhancing the ability to generate dominant mutations in A. fumigatus.},
}
@article {pmid41968243,
year = {2026},
author = {Valero, C and Tindale, J and Bromley, MJ and van Rhijn, N},
title = {Marker-Free CRISPR/Cas9 RNP Transformation for Gene Disruption in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {23-30},
pmid = {41968243},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics ; *CRISPR-Cas Systems ; *Transformation, Genetic ; *Ribonucleoproteins/genetics ; Genetic Engineering/methods ; Protoplasts/metabolism ; },
abstract = {CRISPR-based genetic engineering has resulted in the possibility of far more efficient and challenging genetic engineering in filamentous fungal pathogens. In this respect, selection marker-free CRISPR-Cas9 transformation has been possible in fungi such as Aspergillus fumigatus, one of the most prevalent fungal pathogens of humans. Here, we describe a protocol to perform this technique, which can be widely adapted to use multiple strains and other species.},
}
@article {pmid41968244,
year = {2026},
author = {Guo, Y and Scharf, DH},
title = {CRISPR-Cas9 and Microhomology-Mediated End-Joining for Biosynthetic Pathway Reconstruction in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {31-45},
pmid = {41968244},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/metabolism ; *CRISPR-Cas Systems ; *Biosynthetic Pathways/genetics ; Gliotoxin/biosynthesis ; Plasmids/genetics ; Piperazines/metabolism ; Genetic Vectors/genetics ; },
abstract = {We have recently engineered Aspergillus fumigatus using an AMA1-based episomal expression CRISPR vector and a microhomology-mediated end-joining repair system. We adopted A. fumigatus as a heterologous expression host to investigate the biosynthetic pathways of epipolythiodioxopiperazine-type compounds. In the present chapter, we describe the assembly of the CRISPR-Cas9 vector and microhomology template plasmid, their transformation into A. fumigatus, and metabolite detection.},
}
@article {pmid41968255,
year = {2026},
author = {Campanella, JEM and Arentshorst, M and Ram, AFJ and van den Hondel, CAMJJ and Malavazi, I},
title = {Molecular Biosensing of the Aspergillus fumigatus Cell Wall Integrity Pathway Using the (p)agsA::luc (Luciferase) Reporter System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {199-223},
pmid = {41968255},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/metabolism ; *Cell Wall/metabolism/genetics ; *Biosensing Techniques/methods ; *Genes, Reporter ; *Luciferases/genetics/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; },
abstract = {In this protocol, we describe a robust luciferase-based biosensor assay to monitor the activity of the cell wall integrity (CWI) pathway in Aspergillus fumigatus in real time. The method relies on the stable integration of a markerless, synthetic reporter cassette (p)agsA::luc at the aft4 Safe Haven (Sh) genomic locus using CRISPR-Cas9. This cassette comprises a modified A. niger agsA promoter containing three tandem RlmA-binding sites, which drives the expression of the luciferase gene. Upon exposure to cell wall stress, the endogenous transcription factor RlmA activates the reporter, generating a luminescent signal proportional to promoter activity. The protocol includes the construction of aft4 locus-specific CRISPR-Cas9 plasmids, A. fumigatus transformation and candidate selection, as well as the setup of the luminescence bioassay in white 96-well microplates. This system enables highly sensitive, nondestructive, and time-resolved quantification of CWI pathway activation during early fungal growth or biofilm under various genetic or chemical perturbations. Moreover, it supports comparative studies across wild-type and mutant strains, offering a powerful platform for dissecting stress response signaling and identifying antifungal compounds that target the CWI pathway.},
}
@article {pmid41972732,
year = {2026},
author = {Rutkowska, A and Strózik, T and Wasiak, T and Ciunowicz, D and Kapelan, N and Szczepaniak, N and Sosnowski, J and Goślińska, W and Bartkowiak, J and Budny-Lewandowska, A and Antończyk, P and Markiewicz, M and Gustaw, P and Filiks, K and Jaskólska, M and Stoczyńska-Fidelus, E},
title = {CRISPR-Cas9 Therapeutics in Early Clinical Development: Delivery and Molecular Diagnostics.},
journal = {Cells},
volume = {15},
number = {7},
pages = {},
pmid = {41972732},
issn = {2073-4409},
support = {2024/ABM/05/KPO/KPOD.07.07-IW.07-0242/24-00//LEK-AM Pharmaceutical Company Ltd/ ; 503/0-154-01/503-01-001//Medical University of Lodz/ ; },
mesh = {*CRISPR-Associated Protein 9/genetics/therapeutic use ; CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing ; *Genetic Therapy/methods ; Animals ; Mice ; },
abstract = {CRISPR-Cas9 has progressed from an experimental tool to a therapeutic modality, marked by the first regulatory approvals of an ex vivo-edited autologous CD34+ hematopoietic stem cell product that induces fetal hemoglobin (CASGEVY/exa-cel). In this narrative review, we synthesize modality-specific molecular diagnostic strategies used across early CRISPR clinical translation. In parallel, early clinical experience has begun to demonstrate the feasibility of in vivo editing, including subretinal delivery for CEP290-associated inherited retinal degeneration (EDIT-101 programme) and hepatocyte-targeted lipid nanoparticles (LNPs) for liver-derived targets such as transthyretin and plasma prekallikrein (KLKB1). As translation expands across hematologic, metabolic, ocular and oncology indications, development is increasingly constrained by the predictability and safety of editing outcomes, delivery-determined biodistribution and exposure time, and immune recognition of bacterial Cas9 orthologs and delivery components. We summarize diagnostic readouts for confirming patient genotype, quantifying on-target editing and expression changes, assessing off-target and structural outcomes using orthogonal assays, and monitoring clonal dynamics and immune responses during long-term follow-up. We also discuss how these readouts interface with CMC controls and regulatory expectations for advanced therapy medicinal products (ATMPs), highlighting the need for fit-for-purpose, standardized testing frameworks in early trials.},
}
@article {pmid41972830,
year = {2026},
author = {Chokwassanasakulkit, T and Ranasinghe, V and Woods, E and Nguyen, LQ and McMillan, NAJ},
title = {What If Trojan Horse Nanoparticles Could Change the Game for HPV Gene-Targeted Therapies?.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70916},
pmid = {41972830},
issn = {1096-9071},
support = {2027649//National Health and Medical Research Council (NHMRC)/ ; 2027569//National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Nanoparticles/administration &amp; dosage ; *Papillomavirus Infections/therapy/virology ; *Genetic Therapy/methods ; Uterine Cervical Neoplasms/therapy/virology ; Female ; RNA, Small Interfering/administration &amp; dosage/genetics ; *Papillomaviridae/genetics ; Animals ; CRISPR-Cas Systems ; },
abstract = {Human papillomavirus (HPV) is a common sexually transmitted infection linked to various cancers, particularly cervical cancer, primarily driven by high-risk strains like HPV16 and HPV18. While vaccines are effective in preventing new infections, they do not address existing cases, highlighting the need for innovative therapies. Gene-targeted approaches, such as CRISPR/Cas and siRNA, show promise in inhibiting HPV oncogenes. Recent advancements in Trojan horse nanoparticles (NPs) offer a strategy for delivering these therapies directly to HPV-infected cells. These NPs improve stability and targeted delivery, enhancing the biodistribution of CRISPR/Cas systems and siRNAs while protecting them from degradation. However, challenges like immune responses and regulatory hurdles persist. Therefore, this review emphasizes the potential of Trojan horse NPs in treating HPV-related cancers, identifies critical areas for future research, and provides updates on gene-targeted therapy encapsulated NPs in preclinical and clinical trials.},
}
@article {pmid41973392,
year = {2026},
author = {Shojaei Baghini, S and Esfahani, K and Rad, N and Arezoumandi, M and Taghipour, E and Salmanian, AH},
title = {Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {41973392},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Araceae/genetics ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics/growth &amp; development ; Fucosyltransferases/genetics ; Pentosyltransferases/genetics ; },
abstract = {Lemna minor (commonly known as duckweed) is a fast-growing aquatic plant recognized as a promising green bioreactor for recombinant protein production. Its rapid proliferation, high protein yield, environmental adaptability, and edibility make it highly attractive for biotechnological applications. It is essential to develop and expand genetic tools tailored to this species to maximize these advantages and further unlock its biotechnological potential. A key strategy for achieving this goal is the implementation of advanced genome editing technologies, such as the CRISPR/Cas9 system. Although multiplex CRISPR/Cas9 gene editing has previously been successfully applied in Lemna aequinoctialis, the capability of the endogenous plant tRNA processing system for multiplex editing in L. minor using the polycistronic tRNA-sgRNA (PTG)/Cas9 system has not yet been explored. In this study, a PTG construct was engineered to include four sgRNAs designed to simultaneously target two plant-specific glycosyltransferase genes: α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT). As anticipated, the PTG-Cas9 system successfully induced frameshift mutations, characterized by insertions and deletions (indels), in regenerated L. minor plants derived from transformed calli. Validation via PCR and RT-PCR analysis, followed by sequencing of the target loci, confirmed the presence of indels at the target sites. Furthermore, western blot analyses utilizing antibodies specific to XylT and FucT in two homozygous lines (lines 44 and 217) revealed truncated XylT proteins in both lines. Moreover, an in-frame FucT protein was detected in line 217, whereas FucT expression was absent in line 44. This study marked the first successful demonstration of PTG-Cas9 system for multiplex genome editing in L. minor, paving the way for advanced genetic engineering in this species.},
}
@article {pmid41973928,
year = {2026},
author = {Lian, G and Khabazeh, A and Sheen, V},
title = {A modified CRISPR/Cas9 approach in silencing the triplication in Down syndrome: A treatment path XISTs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {16},
pages = {e2517953123},
pmid = {41973928},
issn = {1091-6490},
support = {1R01HD109794-01//Foundation for the NIH (FNIH)/ ; 5R21NS115593-02//Foundation for the NIH (FNIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Long Noncoding/genetics ; *Down Syndrome/genetics/therapy ; Gene Silencing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {Down syndrome (DS) is one of the most common developmental human genetic disorders and is due to triplication of chromosome 21 (HSA21). Although previous studies using epigenetic suppression of HSA21 by the long noncoding RNA XIST showed a potential for DS treatment, integration efficiency of XIST by conventional zinc finger nucleases is too low to allow for practical implementation. Here, we report a modified CRISPR/Cas9 approach, which enhances the efficiency of XIST gene integration. First, a codon-optimized λ-phage exonuclease (exo) was fused with Cas9 to create 5'- and 3'-end overhangs at cutting sites of donor DNA and acceptor chromosome DNA. Second, four sgRNAs, two of which selectively targeted each the acceptor or donor DNA, were assembled tandemly into one Cas9 plasmid (PX459) to increase the Cas9-cutting efficiency and promote donor DNA integration. Third, sgRNAs were designed by searching for unique single nucleotide polymorphism nucleotides distinct between the three HSA21 copies, as a protospacer adjacent motif site to specifically target one HSA21 copy. Fourth, donor DNA plasmid containing XIST was modified to disable replication and inhibit transcription function and allow for inducible expression. Our modified CRISPR method significantly enhanced the integration efficiency (20 to 40%) of long XIST gene (14 kb) into an extra chromosome 21 (HSA21), as was identified with PCR, cell cloning, immunostaining, and FISH. RNA sequencing results showed that imbalance of gene transcription across extra HSA21 can be partially corrected by XIST gene integration. The modified CRISPR method with XIST paves a road for therapeutic treatment for DS.},
}
@article {pmid41974167,
year = {2026},
author = {Peng, Z and Liu, Z and Wei, S and Zhu, Y and Jia, X and Xiao, W and Dong, Z and Hu, H and Lu, C and Pan, M and Chen, P},
title = {Genome-Wide CRISPR/Cas9 Screening Reveals BmM-ALP Orchestrates the Antioxidant Response and Metabolic Adaptations for Heat Resistance in Bombyx mori.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1968-1979},
doi = {10.1021/acssynbio.6c00053},
pmid = {41974167},
issn = {2161-5063},
mesh = {Animals ; *Bombyx/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; *Antioxidants/metabolism ; Reactive Oxygen Species/metabolism ; *Thermotolerance/genetics ; Heat-Shock Response/genetics ; Adaptation, Physiological/genetics ; *Insect Proteins/genetics/metabolism ; Hot Temperature ; Animals, Genetically Modified ; Oxidative Stress ; },
abstract = {Climate change-induced heat stress represents an increasing threat to biodiversity, engendering the need to understand evolutionary adaptations to thermal extremes. Using Bombyx mori as an economically important insect and research model, we employed genome-wide CRISPR/Cas9 screening integrated with high-throughput sequencing to systematically identify adaptations for heat resistance. The analysis identified the Bombyx mori membrane-bound alkaline phosphatase-like gene (BmM-ALP) as a vital thermoregulator. BmM-ALP triggered a pleiotropic protective cascade by significantly decreasing reactive oxygen species (ROS), suppressing apoptosis, and reprogramming mitochondrial metabolism via the phosphorylation of Vitamin B1 (VB1), suggesting a potential role in sustaining oxidative phosphorylation and enhancing energy metabolism under stress. Transgenic validation experiments confirmed the consistency of this strategy, and the BmM-ALP-OE strain displayed significantly enhanced thermal tolerance and prolonged survival under high-temperature stress. Beyond establishing BmM-ALP as a key gene of heat resistance in silkworms, this study uncovered a regulatory axis linking BmM-ALP to Vitamin B1 metabolism, illustrating a novel connection between energy homeostasis and thermal adaptation. The findings provide new insights that can be applied to breeding resistant strains in agriculture and biodiversity conservation in the context of global warming.},
}
@article {pmid41974708,
year = {2026},
author = {Lai, S and Keller, MP and Zhang, J and Fang, Z and Xie, Y and Weng, C and Zhang, S and Zhang, S and Gao, P and Ke, L and Wang, Y and Mitok, KA and Clark, L and Schueler, KL and Liu, H and Hatipoglu, B and Hatzoglou, M and Chen, Y and Shalev, A and Jin, F and Attie, AD and Li, Y},
title = {Proinsulin regulators identified with CRISPR screen and in vivo mouse QTL mapping.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41974708},
issn = {2041-1723},
support = {R01 DK131437/DK/NIDDK NIH HHS/United States ; R01HG012384//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01 HG012384/HG/NHGRI NIH HHS/United States ; R01DK131437//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; P30 AG092586/AG/NIA NIH HHS/United States ; R01CA267872//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01HG009658//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01DK113185//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; R01 DK113185/DK/NIDDK NIH HHS/United States ; UG3NS132061//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *Quantitative Trait Loci/genetics ; *Proinsulin/metabolism/blood/genetics ; Mice ; Golgi Apparatus/metabolism ; Humans ; Protein Disulfide-Isomerases/genetics/metabolism ; Insulin-Secreting Cells/metabolism ; Insulin/metabolism ; Chromosome Mapping ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exocytosis ; Cell Line ; Secretory Vesicles/metabolism ; },
abstract = {Altered proinsulin levels in β-cells and bloodstream are hallmarks of diabetes and other diseases, but our knowledge about the proinsulin regulators remains limited. Here we perform a genome-wide CRISPR screen to identify 84 proinsulin regulators that alter intracellular proinsulin/insulin ratio in a mouse β-cell line. The proinsulin regulators are distinct from the insulin regulators from a previous orthogonal CRISPR screen. Functional annotation of the proinsulin regulators highlights Golgi as the primary organelle for proinsulin storage and regulation. Trafficking towards the Golgi increases the intra-cellular proinsulin/insulin ratio, while trafficking away from the Golgi, including exocytosis and Golgi-to-ER retrograde transport, decreases the intracellular proinsulin levels. We also map mouse quantitative trait loci (QTLs) associated with plasma proinsulin levels and use the CRISPR screen results to pinpoint the causal genes within the QTL loci. Interestingly, protein disulfide isomerase Pdia6 is the strongest hit from both CRISPR screen and the in vivo QTL mapping. Knocking down Pdia6 significantly reduce proinsulin accumulation in Golgi and secretory granules. Intriguingly, Pdia6-depletion in both human and mouse β-cells does not affect the folding status of proinsulin but causes significantly impaired proinsulin production through a UPR-independent mechanism. Taken together, our genetic profiles provide mechanistic insights into the regulation of proinsulin/insulin homeostasis.},
}
@article {pmid41974871,
year = {2026},
author = {Vitiello, A and Boccellino, M and Zovi, A and Bassetti, M},
title = {Antimicrobial resistance and gene therapy: emerging molecular strategies for a global health threat.},
journal = {Gene therapy},
volume = {33},
number = {3},
pages = {239-241},
pmid = {41974871},
issn = {1476-5462},
mesh = {Humans ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; Global Health ; Anti-Bacterial Agents/therapeutic use ; *Drug Resistance, Microbial/genetics ; Animals ; },
abstract = {Antimicrobial resistance (AMR) is one of the most serious and pressing health challenges facing modern medicine. Despite advances in antimicrobial stewardship, diagnostics and infection prevention, the rapid emergence and spread of resistant pathogens continues to limit treatment options and increase morbidity, mortality and healthcare costs. The discovery of new innovative antimicrobial therapies remains of paramount importance. In this context, gene therapy is gaining attention as a complementary strategy that can directly target the molecular genetic determinants of antimicrobial resistance. Recent advances in RNA-based technologies and CRISPR-Cas systems have enabled increasingly precise manipulation of microbial genomes, opening up the possibility of restoring antimicrobial susceptibility, reducing virulence and limiting the spread of resistance genes.},
}
@article {pmid41975095,
year = {2026},
author = {Guan, K and Ocampo, RF and Matheus Carnevali, PB and Castelle, CJ and Gonzalez-Osorio, L and Castanzo, DT and Thomas, NC and Brothers, M and Dangerfield, TL and Hooper, MM and West, MS and Appleby, NM and Krudop, I and Lamothe, RC and Aliaga Goltsman, DS and Alexander, LM and Butterfield, CN and Johnson, KA and Brown, CT and Taylor, DW},
title = {Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {5},
pages = {756-767},
pmid = {41975095},
issn = {1545-9985},
support = {R35 GM138348/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Models, Molecular ; R-Loop Structures ; HEK293 Cells ; },
abstract = {Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing because of their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here we identify and characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Alistipes sp. Cas12f (Al3Cas12f), which supports robust genome editing in human cells. Through structural, biochemical and kinetic analyses, we compare Al3Cas12f to two recently described orthologs, Oscillibacter sp. Cas12f and Ruminiclostridium herbifermentans Cas12f. These orthologs present divergent architectures and regulatory features governing protospacer-adjacent motif recognition, guide RNA (gRNA) binding, dimerization and DNA cleavage. Notably, Al3Cas12f achieves efficient R-loop formation through a stable dimer interface and a naturally optimized gRNA. Leveraging these structural insights, we generate an engineered Al3Cas12f variant (RKK) that increases editing and improves activity across several tested genomic loci. By overcoming locus-dependent variability and an apparent potency threshold, this engineered compact editor seems to expand the feasibility of low-dose, AAV-compatible therapeutic genome editing. Our results elucidate mechanistic determinants of Cas12f activity and offer a framework for engineering compact genome editors that may bear therapeutic potential.},
}
@article {pmid41975467,
year = {2026},
author = {Song, HY and Cao, H and Huang, SB and Elsheikha, HM and Zheng, Z and Lu, XS and Tian, X and Zheng, XN and Zhu, XQ},
title = {Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41975467},
issn = {1756-3305},
support = {2022Y309//the Graduate Innovation Project of Shanxi Province/ ; U2202201//the NSFC-Yunnan Joint Fund/ ; 2021YFC2300800 and 2021YFC2300802//National Key Research and Development Program of China/ ; 2021XG001//the Special Research Fund of Shanxi Agricultural University for High-level Talents/ ; },
mesh = {*Toxoplasma/genetics/pathogenicity/metabolism ; *Protozoan Proteins/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Mice ; Virulence ; Gene Knockout Techniques ; Female ; Toxoplasmosis, Animal/parasitology ; Gene Expression Profiling ; },
abstract = {BACKGROUND: Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence.<br><br>METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RH&#x394;ku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RH&#x394;rop64 and RH&#x394;rop65 knockout strains.<br><br>RESULTS: All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5, ROP39, TgIST, and PLP1. In addition, RH&#x394;rop64 exhibited broader suppression of ROPs than RH&#x394;rop65, suggesting it has a more pronounced role in immune modulation.<br><br>CONCLUSIONS: ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RH&#x394;rop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis.},
}
@article {pmid41977119,
year = {2026},
author = {Abubakar, S and Abdulsalam, L and Fatty, L and Kanwal, R and Naeem, M and Ahmad, I},
title = {Emerging CRISPR Approaches for Countering Immune Evasion: Insight from Recent Studies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977119},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology/genetics ; Gene Editing/methods ; Immunotherapy/methods ; *Tumor Escape/genetics ; Animals ; *Immune Evasion/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {Cancer immunotherapy has recently become an essential approach for treating cancer, showing considerable promise as a substitute for surgery, radiation therapy, and conventional chemotherapy. It primarily aims to boost the host's natural defense system to combat cancer malignancies by utilizing components of immune checkpoint blockades (ICBs), mainly programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), along with elements of adoptive cellular therapies (ACTs) like Chimeric Antigen Receptor (CAR) therapy, T Cell Receptor (TCR) therapy and Tumor-Infiltrating Lymphocyte (TIL) therapy. However, cancer cells tend to undermine the effectiveness of cancer immunotherapeutic strategies by employing one or more immune evasion mechanisms. This review briefly highlights how key mechanisms of cancer immune evasion confer resistance to immunotherapy and how the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR)/Cas9 systems, as gene-editing tools, are poised to enhance cancer immunotherapy for treating challenging cancers. We emphasize that (CRISPR/Cas9) systems can be used to explore and positively alter the genes of the immune system, boosting the effectiveness of cancer immunotherapy by editing immune checkpoints, TILs, and CAR-T cells, and disrupting genes, facilitating tumors' evasion of the immune system. Furthermore, we highlight the growing interest in emerging base editor technology to engineer natural killer (NK) cells to overcome NK-cell-based immunotherapy challenges, particularly human leukocyte antigens (HLA)-mediated limitations, and to engineer CAR-T cells for improved immunotherapy outcomes.},
}
@article {pmid41977403,
year = {2026},
author = {Ma, S and Li, Y and Fei, T},
title = {Functional CRISPR Screens Define Genetic Drivers for Cancer Transformation and Progression from Non-Cancerous Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977403},
issn = {1422-0067},
support = {2023A1515140084//Guangdong Basic and Applied Basic Research Foundation/ ; 32470673//National Natural Science Foundation of China/ ; B16009//the 111 Project/ ; 2022JH13/10200026//the Construction Project of Liaoning Provincial Key Laboratory, China/ ; },
mesh = {Humans ; Animals ; Mice ; *Cell Transformation, Neoplastic/genetics ; *CRISPR-Cas Systems ; *Liver Neoplasms/genetics/pathology ; Female ; *Carcinoma, Hepatocellular/genetics/pathology ; Disease Progression ; Gene Expression Regulation, Neoplastic ; *Breast Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution.},
}
@article {pmid41977439,
year = {2026},
author = {Bougea, A},
title = {Targeting Non-Coding RNAs as a Potential Therapeutic and Delivery Strategy Against Neurodegenerative Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977439},
issn = {1422-0067},
mesh = {Humans ; *Neurodegenerative Diseases/genetics/therapy ; Animals ; MicroRNAs/genetics ; *RNA, Untranslated/genetics ; Exosomes/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Long Noncoding/genetics ; },
abstract = {Neurodegenerative diseases (NDs), including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), represent a growing global health challenge characterized by progressive neuronal loss and a lack of definitive disease-modifying treatments. This review explores the emerging potential of targeting non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and exosomal RNAs, to modulate pathogenic molecular pathways and address the underlying molecular origins of neurodegeneration. We evaluate the integration of advanced computational techniques for RNA structure prediction and gene regulatory network analysis, alongside chemical engineering strategies-such as Locked Nucleic Acids (LNAs) and phosphorothioate modifications-aimed at enhancing the stability and specificity of RNA-based molecules. Furthermore, we analyze cutting-edge delivery and editing technologies, including nanotechnology-driven solutions for precise neuronal targeting and the CRISPR/Cas13 system for direct ncRNA manipulation.The findings indicate that while challenges in delivery efficiency and long-term efficacy persist, the synergy of chemical engineering and computational modeling significantly improves the therapeutic profile of ncRNAs, with exosomal pathways offering a novel route for intercellular signaling modulation and biomarker discovery. Therapeutic interventions directed at specific clinical targets, such as miR-34a and BACE1-AS, demonstrate the capacity to influence protein aggregation and neuroinflammatory cascades. Although ncRNA-based therapies are currently in nascent stages, ongoing technological advancements in RNA editing and nanotechnology offer a transformative framework that could redefine the future of ND treatment and successfully halt disease progression rather than merely managing symptoms.},
}
@article {pmid41979903,
year = {2026},
author = {Stuckless, EE and Gai, LS and Slattery, SS and Dempsey, KH and Browne, TS and Gloor, GB and Edgell, DR},
title = {PHYCUT: Scalable Multiplex CRISPR/Cas9 Editing for Genome Engineering in the Diatom Phaeodactylum tricornutum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1850-1865},
pmid = {41979903},
issn = {2161-5063},
mesh = {*Diatoms/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Glycosylation ; Plasmids/genetics ; Fucose/metabolism ; },
abstract = {Diatoms are globally significant microalgae that contribute ∼20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom Phaeodactylum tricornutum has emerged as a promising synthetic biology platform for the bioproduction of recombinant proteins, supported by a human-like N-linked glycosylation pathway. However, its α(1,3)-linked core fucose is potentially immunogenic in humans and thus limits its biopharmaceutical applications. One hurdle to efficient genome engineering in P. tricornutum is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (Phaeodactylum tricornutum Csy4-Cas9 multiplex tool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multiguide RNA arrays. To highlight PHYCUT applications, we demonstrate multiplex editing of all three FucT genes responsible for α(1,3) fucosylation in P. tricornutum, yielding strains with reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the P. tricornutum glycosylation pathway to support next-generation algal biotechnology.},
}
@article {pmid41980379,
year = {2026},
author = {Liu, X and Wang, C and Wang, W and Chen, Z and Luo, M and Yang, R and Deng, H},
title = {In situ thermosensitive mRNA-loaded hydrogel modulates post-surgery tumor immune microenvironment to prevent recurrence and metastasis.},
journal = {Biomaterials},
volume = {333},
number = {},
pages = {124188},
doi = {10.1016/j.biomaterials.2026.124188},
pmid = {41980379},
issn = {1878-5905},
mesh = {*Tumor Microenvironment/immunology/drug effects ; *Hydrogels/chemistry ; Animals ; Mice ; Nanoparticles/chemistry ; Macrophages/immunology ; Reactive Oxygen Species/metabolism ; *RNA, Messenger/administration &amp; dosage/chemistry ; Cell Line, Tumor ; Female ; *Neoplasm Recurrence, Local/prevention &amp; control/immunology ; Humans ; Neoplasm Metastasis/prevention &amp; control ; Temperature ; CRISPR-Cas Systems ; },
abstract = {Surgery remains the primary cancer treatment, but postoperative trauma disrupts the local immune microenvironment by altering the critical balance between M1 and M2 macrophages while simultaneously elevating reactive oxygen species (ROS) levels at the surgical site. The critical clinical dilemma in postoperative tumor management lies in achieving re-balancing over macrophage polarization within the post-operative niche - specifically maintaining tumor-suppressing M1 phenotype while permitting necessary wound-healing M2 function. Here, we developed an in situ thermosensitive hydrogel platform capable of co-delivering two nanoparticle systems (BC12D NPs and PPS NPs) to alleviate the immunosuppressive microenvironment. Specifically, CRISPR/Cas9-loaded nanoparticles (BC12D@CRISPR NPs) were incorporated into the hydrogel for addressing the high proportion of M2-type macrophages at the resection site, reprogramming the macrophages with an effective M1/M2 ratio to exert potent antitumor functions. Meanwhile, the PPS nanoparticles were employed for the clearance of ROS at the surgical site, thereby ensuring that the normal wound healing process remained unimpeded. Using an in situ tumor resection model, the synergistic effects of ROS clearance and macrophage repolarization at the postoperative site were leveraged to achieve efficient immune microenvironment modulation.},
}
@article {pmid41981893,
year = {2026},
author = {Jia, M and Xie, Y and Wei, C and Wang, H and Xue, L and Zou, C and Zhu, JK and Wang, M},
title = {Base editing in rice using nuclease-deactivated CRISPR/Cas-SF01.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70265},
pmid = {41981893},
issn = {1744-7909},
support = {YBXM2514//Nanfan special project of CAAS/ ; 32188102//National Natural Science Foundation of China/ ; KQTD20240729102038044//Shenzhen Science and Technology Program/ ; //Agriculture Science and Technology Major Project/ ; 2024B1111130001//Guangdong S&amp;T Program/ ; },
abstract = {Adenine and cytosine base editing using dCas-SF01 and the 35S-CmYLCV-U6 composite promoter successfully introduced targeted base substitutions at multiple loci in rice, with average editing efficiency of 33.3%. Using the protospacer adjacent motif (PAM)-relaxed SF01-IKRR variant enabled base editing using 5'-NTN PAMs in rice.},
}
@article {pmid41982105,
year = {2026},
author = {Cong, X and Zhang, X and Gu, F and Tan, N and Huang, S and Jia, P and Su, J and Sun, C and Tan, Q and Fang, L and Wang, J and Yan, J and Yu, C and Li, B and Huang, J},
title = {Development of a Reverse Transcription Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Visual and Highly Specific Identification of Zika Virus.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70917},
pmid = {41982105},
issn = {1096-9071},
support = {2026ZD01909700//Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project/ ; 2024A1515010148//Natural Science Foundation of Guangdong Province/ ; 0720240122//Guangdong Provincial Center for Disease Control and Prevention Supports Talent Projects/ ; 2023B1212010010//Guangdong Provincial Key Laboratory of Pathogen Detection for Emerging Infectious Disease Response/ ; A2024665//Medical Scientific Research Foundation of Guangdong Province, China/ ; 2024D343//Talent Support Program of Guangdong Provincial Center for Disease Control and Prevention/ ; },
mesh = {Humans ; *Zika Virus/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Zika Virus Infection/diagnosis/virology ; Reproducibility of Results ; Rapid Diagnostic Tests ; Recombinases ; RNA, Viral/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; DNA Primers/genetics ; Point-of-Care Testing ; Reverse Transcription ; },
abstract = {Zika virus (ZIKV), a single-stranded positive-sense RNA virus of the Flaviviridae family (Flavivirus genus), causes acute febrile illness and severe congenital anomalies. Serological cross-reactivity with Dengue virus (DENV) and Chikungunya virus (CHIKV) complicates diagnosis, underscoring the urgency of developing specific point-of-care tests for early detection, outbreak mitigation, and reduced misdiagnosis risks. This study established and optimized an RPA-CRISPR/Cas12a assay for the rapid, visual, and highly specific detection of ZIKV. Primers and crRNAs targeting the highly conserved capsid (C) gene were designed, and the assay was systematically evaluated for its specificity, sensitivity, reproducibility, and clinical applicability. The RPA-CRISPR/Cas12a assay enabled naked-eye detection under UV light within 35 min. It demonstrated single-copy sensitivity (1 copy/μL), no cross-reactivity with DENV 1-4, CHIKV, or Japanese encephalitis virus (JEV), and 100% concordance with RT-qPCR in clinical validation. Repeatability tests showed low variability (coefficient of variation (C.V.) < 15%), confirming robust reproducibility. This instrument-free platform integrates rapid visual detection, single-copy sensitivity, high specificity, and field-deployable features, making it particularly suitable for point-of-care testing (POCT) in resource-limited settings. The developed assay provides critical support for early outbreak containment and prenatal screening in ZIKV-endemic regions.},
}
@article {pmid41982408,
year = {2025},
author = {Ni, D},
title = {The Kongming defense: Host-pathogen battles take a new face.},
journal = {Engineering microbiology},
volume = {5},
number = {2},
pages = {100209},
pmid = {41982408},
issn = {2667-3703},
abstract = {Bacteria employ diverse immune systems, such as CRISPR-Cas, to fend off phage infections. A recent study uncovered the unprecedented mechanistic features of the Kongming bacterial defense system, which uniquely exploits phage-derived enzymes to synthesize deoxyinosine triphosphate (dITP), thereby triggering host immunity through NAD+ depletion. In response, some phages have evolved countermeasures to disrupt dITP synthesis, highlighting the ongoing evolutionary arms race between hosts and pathogens. This discovery not only deepens our understanding of bacterial defense strategies but also paves the way for new insights in biomedical research and synthetic biology.},
}
@article {pmid41985328,
year = {2026},
author = {Jigheh, MP and Ravanlo, ZZ and Shahrak, MZ and Mohabbat, A and Baghi, HB},
title = {Targeting human oncogenic viruses with CRISPR/Cas: New therapeutic opportunities and challenges.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {198},
number = {},
pages = {119284},
doi = {10.1016/j.biopha.2026.119284},
pmid = {41985328},
issn = {1950-6007},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Oncogenic Viruses/genetics ; Animals ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Tumor Virus Infections/therapy/genetics/virology ; Neoplasms/virology/therapy/genetics ; },
abstract = {CRISPR/Cas systems, initially characterized as bacterial adaptive immune mechanisms, have rapidly emerged as precise and versatile genome-editing tools with significant potential for antiviral research and therapeutic development. This review highlights the role of CRISPR/Cas systems in targeting persistent and human oncogenic viruses, including HPV, HBV, HCV, EBV, KSHV, HTLV-1, and MCPyV, as well as HIV, which may indirectly contribute to cancer through immune dysregulation. Many of these viruses can integrate into the host genome or persist as chronic or latent infections, contributing to cancers for which curative options are limited. CRISPR-based strategies enable the excision of integrated proviral DNA, disruption of viral replication, targeted silencing of viral transcripts, and modulation of host tumor-suppressor pathways. Cas9 efficiently targets DNA viruses, such as HBV and HPV, whereas RNA-targeting Cas13 allows precise silencing of RNA viruses, like HCV. Editing T-cell receptors, including CCR5 and CXCR4, offers the potential for long-term resistance to HIV. CRISPR-based preclinical studies indicate the potential to disrupt HBV cccDNA, suppress EBV and KSHV latency gene expression, and inactivate HTLV-1 oncogenes, thereby potentially reducing viral persistence and oncogenic progression. Despite these advances, challenges remain regarding off-target effects, delivery efficiency, immune responses, and ethical considerations. Innovations such as high-fidelity Cas variants, base and prime editing, and non-viral delivery systems are expected to enhance both safety and therapeutic precision. This review provides an overview of viral life cycles, oncogenic pathways, and therapeutic vulnerabilities of human oncogenic viruses and CRISPR-based genome-editing approaches under investigation for viral elimination and cancer therapy.},
}
@article {pmid41985457,
year = {2026},
author = {Kim, J and Hwang, Y and Kim, S and Kwon, D and Park, J and Cho, B and An, S and Kang, S and Kim, Y and Kim, S and Lengner, CJ and Kim, S and Kwon, Y and Sung, JS and Kim, J},
title = {Electromagnetic field-inducible in vivo gene switch for remote spatiotemporal control of gene expression.},
journal = {Cell},
volume = {189},
number = {11},
pages = {3465-3480.e23},
doi = {10.1016/j.cell.2026.03.029},
pmid = {41985457},
issn = {1097-4172},
mesh = {Animals ; Kruppel-Like Factor 4 ; Humans ; Mice ; *Electromagnetic Fields ; Alzheimer Disease/genetics/pathology/metabolism ; Amyloid beta-Protein Precursor/genetics/metabolism ; *Gene Expression Regulation/radiation effects ; *Genes, Switch ; Tryptophan Hydroxylase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Gaining precise control of gene expression is crucial in biomedical applications. However, spatiotemporal precision remains challenging. Here, we present a remotely controlled in vivo gene switch responsive to electromagnetic fields (EMFs) that enables precise spatiotemporal activation of target genes. We uncovered the EMF-inducible gene switch activation mechanism via a CRISPR-Cas9 screen, identifying cytochrome b5 type B (Cyb5b) as an essential mediator likely acting as an EMF sensor. The EMF-inducible gene switch was activated by rhythmic oscillatory calcium dynamics rather than generic calcium influx, defining a precisely tuned and bio-orthogonal induction mechanism. Functionally, EMF activation of the Oct4-Sox2-Klf4 (OSK) cassette induced in vivo partial reprogramming in aged mice, conditional expression of human mutant amyloid precursor protein (APP) for Alzheimer's disease (AD) modeling recapitulated pathological features, and EMF-mediated Tph2 expression restored serotonergic activity and ameliorated depressive-like behaviors in Tph2-mutant depression mice. Overall, a remotely controlled EMF-inducible gene switch represents a versatile and effective biomedical platform.},
}
@article {pmid41985719,
year = {2026},
author = {Liang, G and Li, G and Liang, T and Zhen, Z and Teng, C and Xiong, J and Guangqiang, J and Zheng, M and Pan, Y and Zhong, S and Wu, C and Li, J and Huang, W and Wei, Z},
title = {Construction of an RAA-CRISPR detection platform for differentiation of Brucella abortus A19-∆VirB12 vaccine strain from wild-type strains.},
journal = {Journal of microbiological methods},
volume = {245},
number = {},
pages = {107512},
doi = {10.1016/j.mimet.2026.107512},
pmid = {41985719},
issn = {1872-8359},
mesh = {*Brucella abortus/genetics/isolation &amp; purification/classification/immunology ; Animals ; Recombinases/metabolism ; Sensitivity and Specificity ; *Brucella Vaccine/genetics ; Cattle ; *CRISPR-Cas Systems ; *Brucellosis, Bovine/microbiology/diagnosis ; Bacterial Proteins/genetics ; *Nucleic Acid Amplification Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella abortus is a primary etiological agent of bovine brucellosis, a zoonosis posing significant threats to livestock industries and public health. The recently developed A19-∆VirB12 vaccine strain, which carries a deletion of the VirB12 gene, complicates serological differentiation from wild-type infections. This study aimed to establish a rapid, accurate, and economical detection strategy targeting the VirB12 gene to distinguish the A19-∆VirB12 vaccine strain from wild-type B. abortus. We developed a recombinase-aided amplification (RAA) coupled with CRISPR/Cas12a-based detection method. Following optimization, primer pair C and crRNA1 were selected as optimal components, with 150 nM each of Cas12a protein and crRNA identified as the ideal concentrations in a 50 μL reaction. The assay demonstrated high analytical specificity, showing no cross-reactivity with six non-target bacterial pathogens. Sensitivity analysis established a limit of detection of 10[2] copies per reaction. When evaluated on 52 clinical samples, the RAA-CRISPR assay detected two positive samples, outperforming conventional PCR which detected only one. Crucially, the method yielded no positive signal when challenged with the A19-∆VirB12 gene-deficient vaccine strain, while successfully detecting wild-type strains A19 and S2, confirming its discriminatory capability. The entire workflow, comprising RAA amplification (30 min) and CRISPR-mediated cleavage (20 min), can be completed within one hour, with results visualized via fluorescence or lateral flow strips. This study successfully establishes a rapid, sensitive, and specific diagnostic method for distinguishing the A19-∆VirB12 vaccine strain from wild-type B. abortus, offering a practical tool for field surveillance and eradication programs.},
}
@article {pmid41985988,
year = {2026},
author = {Yeo, JH and Kim, HH and Oh, SH and Lee, J},
title = {Highly efficient and scarless genome editing via essential-gene-coupled homology-directed repair.},
journal = {Genome research},
volume = {36},
number = {6},
pages = {1187-1198},
doi = {10.1101/gr.281194.125},
pmid = {41985988},
issn = {1549-5469},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; *Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {Homology-directed repair (HDR) enables precise genome editing; however, its application in mammalian cells is limited by low efficiency owing to competition from error-prone repair pathways and intrinsically restricted HDR activity. Existing HDR-enhancement strategies, including small-molecule treatments and marker-based selection, are constrained by cytotoxicity, genomic scarring, and inconsistent performance. Here, we present essential-gene-supported scarless HDR (ESS-HDR), a robust, drug- and marker-free platform that selectively enriches HDR-proficient cells. By leveraging essential-gene coediting, ESS-HDR enables precise and scarless genome modification with enhanced efficiency. CRISPR-Cas9 induces double-strand breaks at both the target locus and an essential gene, accompanied by two donor templates: one introducing the desired edit and the other restoring essential-gene function. Only cells that undergo accurate HDR at the essential locus survive, providing endogenous selection without exogenous markers. Single-cell clone analysis confirms that enrichment of HDR-proficient cells enhances editing at the target locus. Using ssODN donors carrying a 1 nucleotide substitution or a 10 nucleotide insertion, ESS-HDR increases HDR efficiencies by sevenfold to 16-fold in HEK293 cells and 41-fold in primary epidermal keratinocytes compared with conventional single-site HDR. With plasmid donors targeting TUBA1B, LMNB1, or ACTB, ESS-HDR improves knock-in efficiencies by sixfold to 34-fold across HEK293, U2OS, and HeLa cells. ESS-HDR also outperforms chemical enhancers including RS-1, SCR7, nocodazole, and AZD7648. Together, these findings establish ESS-HDR as a broadly applicable strategy for efficient, scarless genome editing without external selection markers.},
}
@article {pmid41986708,
year = {2026},
author = {Roth, MO and Shu, Y and Zhao, Y and Trasanidou, D and Hoffman, RD and Südfeld, C and Bouzetos, E and Trasanidis, N and Zawrotny, M and Gelasco, MK and Medina, ML and Das, A and Rai, J and Goswami, HN and Wang, B and van der Oost, J and Li, H},
title = {Molecular basis for methylation-sensitive editing by Cas9.},
journal = {Nature},
volume = {653},
number = {8116},
pages = {1229-1239},
pmid = {41986708},
issn = {1476-4687},
mesh = {Humans ; *DNA Methylation ; Cryoelectron Microscopy ; Models, Molecular ; *CRISPR-Cas Systems/genetics ; Cytosine/metabolism ; Genome, Human/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/chemistry/ultrastructure ; Nucleotide Motifs/genetics ; DNA/metabolism/chemistry/genetics ; CRISPR-Associated Protein 9/metabolism/chemistry ; Female ; *Gene Editing ; },
abstract = {The bacterial CRISPR-Cas9 (Cas9) nuclease has become a powerful genome manipulation tool for a wide range of organisms[1-3]. However, it has yet to fully leverage the pervasive presence of DNA methylation in genomes[4-10]. Here, to fill this gap, we report biochemical, structural and human genome-editing characterizations of a methylation-sensitive Cas9 (ThermoCas9). ThermoCas9 efficiently binds to and cleaves DNA upstream of its protospacer adjacent motif (PAM) 5'-NNNNCGA-3' or 5'-NNNNCCA-3' in vitro. Methylation of the fifth cytosine in either PAM sequence ([5m]CpG or [5m]CpC), however, significantly inhibits ThermoCas9 activity. Cryo-electron microscopy structures of ThermoCas9 in pre-cleavage and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provide guidance for further enzyme engineering. We demonstrate methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrate that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in patients with breast cancer. Owing to its sensitivity to DNA methylation, ThermoCas9 can specifically target cells with disease-related hypomethylation, which adds another layer of precision to genome-editing technologies.},
}
@article {pmid41986722,
year = {2026},
author = {Xu, Z and Lu, Z and Ugurbil, A and Abdulraouf, A and Liao, A and Zhang, J and Zhou, W and Cao, J},
title = {Mapping convergent regulators of melanoma drug resistance by PerturbFate.},
journal = {Nature},
volume = {654},
number = {8117},
pages = {261-271},
pmid = {41986722},
issn = {1476-4687},
mesh = {Humans ; Cell Dedifferentiation/genetics/drug effects ; Cell Line, Tumor ; Chromatin Assembly and Disassembly/genetics ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; Gene Expression Regulation, Neoplastic/drug effects/genetics ; Mediator Complex/metabolism/genetics ; *Melanoma/genetics/drug therapy/pathology ; Phenotype ; *Single-Cell Gene Expression Analysis/methods ; Transcription Factors/metabolism ; Transcriptional Activation ; *Vemurafenib/pharmacology ; },
abstract = {High-throughput genomic studies have uncovered associations between diverse genetic alterations and disease phenotypes. However, elucidating how perturbations in functionally disparate genes give rise to convergent cellular states remains challenging. Here we present PerturbFate, a high-throughput, cost-effective, combinatorial-indexing single-cell platform that enables systematic interrogation of massively parallel CRISPR interference[1] perturbations across the full spectrum of gene regulation, from chromatin remodelling and nascent transcription to steady-state transcriptomic phenotypes. Using PerturbFate, we profiled more than 300,000 cultured melanoma cells to characterize multimodal phenotypic and gene regulatory responses to perturbations in more than 140 vemurafenib resistance-associated genes. We uncovered a shared dedifferentiated cell state marked by convergent cooperative transcription factor activities across diverse genetic perturbations. We further dissected phenotypic responses to perturbations in Mediator complex components, linking module-specific biochemical properties to convergent transcriptional activations. We identified common regulatory nodes that drive similar phenotypic outcomes across distinct genetic perturbations. We also delineated how perturbations in functionally unrelated genes reshape cell state. Thus, PerturbFate establishes a versatile platform for identifying key molecular regulators by anchoring multimodal regulatory dynamics to disease-relevant phenotypes.},
}
@article {pmid41986860,
year = {2026},
author = {Armenta-Medina, A and Mora-Macias, J and Massange-Sánchez, JA and López-Valenzuela, BE and Rangel-Chavez, CP},
title = {Perspectives of Gene Editing for the Conservation of Plant Genetic Resources.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3011},
number = {},
pages = {365-381},
pmid = {41986860},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Conservation of Natural Resources/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Plants/genetics ; Crops, Agricultural/genetics ; Plant Diseases/genetics ; },
abstract = {The rapid development of genome editing tools, particularly CRISPR-Cas9 and virus-induced genome editing (VIGE), offers transformative opportunities for the conservation of plant genetic resources. These technologies enable precise modifications that can enhance genetic variation, restore endangered populations, and strengthen resilience against climate change and invasive species. This chapter reviews methodological frameworks, including guide design, delivery systems, regeneration protocols, and verification of off-target effects, as well as ecological and ethical considerations. Case studies are highlighted in both wild and cultivated species: American chestnut, ash, elm, Pacific yew, hemlock, and whitebark pine, alongside crops of global significance such as coffee, cacao, wild apple, and banana. Applications include resistance to blight, borers, Dutch elm disease, woolly adelgid, rust, rots, and endogenous viral elements. Finally, the chapter outlines future directions emphasizing biosafety, ecological validation, and equitable governance, underscoring the potential of CRISPR-based strategies to integrate conservation science with sustainable agricultural practices.},
}
@article {pmid41987334,
year = {2026},
author = {Kuo, CY and Harrington, S and Campo-Fernandez, B and Wyman, SK and Wu, X and Zhang, R and Espinoza, A and de Andrade Silva, BJ and Sanchez, JM and Fitz-Gibbon, S and Tseng, CH and Pellegrini, M and Bonner, M and Romero, Z},
title = {Comparative analysis of NSG and NBSGW mice for preclinical evaluation of gene-modified human hematopoietic stem and progenitor cells.},
journal = {Stem cell research &amp; therapy},
volume = {17},
number = {1},
pages = {},
pmid = {41987334},
issn = {1757-6512},
mesh = {Animals ; Humans ; *Hematopoietic Stem Cells/metabolism/cytology ; Mice ; *Hematopoietic Stem Cell Transplantation ; CRISPR-Cas Systems/genetics ; Gene Editing ; Lentivirus/genetics ; Mice, Inbred NOD ; },
abstract = {Humanized mouse models are essential for evaluating the engraftment capacity and genetic integrity of gene-modified hematopoietic stem and progenitor cells (HSPCs). Here, we compared two widely used xenotransplantation platforms, NSG and NBSGW mice, in the context of lentiviral vector (LVV) transduction and CRISPR/Cas9-mediated gene correction. HSPCs harboring high LVV copy numbers exhibited engraftment deficits in NSG mice that were not observed in NBSGW mice. This discrepancy highlights the potential for the NBSGW model to mask safety liabilities of LVV-modified products due to its higher overall levels of human chimerism. In contrast, CRISPR/Cas9 editing with a single-stranded oligodeoxynucleotide donor yielded comparable correction rates in both models, even across decreasing input cell doses, demonstrating that long-term repopulating hematopoietic stem cells (HSCs) retain equivalent engraftment capacity in each strain. Single-cell RNA-sequencing revealed distinct progenitor populations that were markedly under-represented in the NSG model but preserved in NBSGW recipients, emphasizing the greater capacity of NBSGW mice to better support multilineage human hematopoiesis. Together, these findings establish that both NSG and NBSGW mice are suitable for assessing long-term engraftment and gene modification outcomes in human HSPCs. However, the significantly higher percentage of human cell chimerism in the NBSGW model may obscure cell populations with engraftment deficits. Careful selection of in vivo models is therefore critical for rigorous preclinical evaluation of gene therapy products prior to clinical translation.},
}
@article {pmid41987615,
year = {2026},
author = {Eliwa, AI and Eldahshan, MM},
title = {CRISPR-Cas at a crossroads: from microbial immunity to precision biotechnology.},
journal = {Journal of immunoassay &amp; immunochemistry},
volume = {},
number = {},
pages = {1-24},
doi = {10.1080/15321819.2026.2658465},
pmid = {41987615},
issn = {1532-4230},
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) form RNA-guided adaptive immune systems in bacteria and archaea that mediate sequence-specific defense against invading genetic elements. Beyond their ecological role in restricting bacteriophage infection and horizontal gene transfer (HGT), CRISPR-Cas systems have been repurposed as programmable nucleases, enabling rapid, scalable, and precise genome engineering. Over the past decade, CRISPR platforms, most prominently Cas9, have transformed functional genomics, accelerated target discovery and drug development, and progressed from experimental tools to clinically evaluated gene and cell therapies. In parallel, growing attention has focused on both native and engineered roles of CRISPR-Cas in shaping HGT, plasmid ecology, and antimicrobial resistance (AMR), as AMR continues to expand globally. In this Review, we integrate advances spanning eukaryotic genome editing and prokaryotic antimicrobial applications. We summarize CRISPR-Cas classification and molecular mechanisms, highlighting spacer acquisition, guide RNA biogenesis, target recognition, and nucleic acid cleavage. We then examine how cellular DNA repair pathways influence editing outcomes and discuss strategies to enhance precision. We further review delivery strategies, such as conjugative plasmids, bacteriophages and phagemids, extracellular vesicles, and nanoparticles, together with evolutionary countermeasures encoded by mobile genetic elements, including anti-CRISPR proteins. Finally, we outline current limitations.},
}
@article {pmid41988391,
year = {2026},
author = {Tang, W and Yang, N and Shi, M},
title = {Applications and Challenges of CRISPR-Cas Technology for the Detection of Antimicrobial Resistance Genes.},
journal = {Infection and drug resistance},
volume = {19},
number = {},
pages = {578705},
pmid = {41988391},
issn = {1178-6973},
abstract = {The global rise of antimicrobial resistance (AMR) demands rapid and precise diagnostic tools capable of providing actionable results at the point of care. Traditional methods, including bacterial culture and PCR, face critical limitations: culture-based approaches require days to weeks for results, potentially delaying life-saving treatment decisions, while PCR-based methods, though faster, require expensive instrumentation, trained personnel, and are typically confined to centralized laboratory settings. CRISPR-Cas systems offer a promising approach for detecting bacterial drug resistance genes with high specificity, speed, and sensitivity. By utilizing Cas enzymes (eg, Cas12 and Cas13) and guide RNAs, CRISPR enables targeted recognition of resistance markers, typically requiring upstream nucleic acid amplification (eg, recombinase polymerase amplification or loop-mediated isothermal amplification) to achieve clinically relevant sensitivity. Platforms such as SHERLOCK and DETECTR facilitate rapid identification of markers associated with pathogens like MRSA and CRE directly from clinical samples, supporting point-of-care use. Integration with microfluidics and AI further expands its potential for personalized therapy and AMR surveillance. Despite ethical and regulatory challenges, CRISPR-Cas technology holds significant promise for advancing antimicrobial stewardship and public health responses to AMR.},
}
@article {pmid41989832,
year = {2026},
author = {Ceasar, SA and Pandey, H and Misra, V and Sharma, A and Kumar, R and Mall, AK},
title = {Biotechnological Improvement of Fiber Crops: Role of In Vitro Culture, Genetic Transformation, and Genome Editing.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {16},
pages = {12590-12610},
doi = {10.1021/acs.jafc.5c14248},
pmid = {41989832},
issn = {1520-5118},
mesh = {Gene Editing ; *Crops, Agricultural/genetics/growth &amp; development/metabolism/chemistry ; *Plants, Genetically Modified/genetics/metabolism/growth &amp; development/chemistry ; Transformation, Genetic ; Biotechnology/methods ; Plant Breeding ; *Dietary Fiber/analysis/metabolism ; Tissue Culture Techniques ; },
abstract = {Fiber crops face major challenges from climate instability, pests, and suboptimal fiber or oil quality. These challenges can be addressed using plant tissue culture and molecular breeding tools, including genetic transformation and CRISPR/Cas-mediated genome editing. Advances in in vitro regeneration have enabled efficient plant recovery in crops such as cotton, jute, mesta, flax, sunn hemp, and industrial hemp. Techniques including anther culture have facilitated the development of doubled haploid lines with improved fiber quality and uniformity. Genetic transformation and emerging genome-editing applications provide new opportunities for targeted trait improvement in fiber crops. This review covers in detail the application of in vitro regeneration, genetic transformation, and genome editing studies in fiber crops. This review also includes several insights for improving fiber crops by applying these tools. This review will be a rich resource for the details of in vitro regeneration, genetic transformation, and gene editing studies in fiber crops.},
}
@article {pmid41990079,
year = {2026},
author = {Schenstnyi, K and Zhang, Z and Liu, B and Nakamura, M and Schepler-Luu, V and Loo, EPI and Yang, B and Frommer, WB},
title = {Methylviologen resistance in loss-of-function mutants of the polyamine transporter gene OsLAT5.},
journal = {PloS one},
volume = {21},
number = {4},
pages = {e0346828},
pmid = {41990079},
issn = {1932-6203},
mesh = {*Oryza/genetics/drug effects ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Loss of Function Mutation ; *Membrane Transport Proteins/genetics ; Gene Editing ; Plants, Genetically Modified ; *Herbicide Resistance/genetics ; Herbicides/pharmacology ; },
abstract = {TALENs and CRISPR/Cas have become routine tools for genome editing. During stable plant transformation, genes coding for editing enzymes, e.g., Cas9, guide RNAs (gRNA), and selectable or screenable markers are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to herbicides or antibiotics. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used. Marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to methylviologen (MV) in rice. As proof of concept, CRISPR/Cas9 constructs with gRNAs were generated to target three close homologs, namely OsLAT1, OsLAT5, and OsLAT7. Loss of OsLAT5 (also known as OsPUT3 or OsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli. Loss-of-function alleles generated by editing of LAT5 can serve as a selectable marker at the seed germination stage. We discuss the potential utility of rice lat5 loss of function variants as selectable markers for genome editing.},
}
@article {pmid41990179,
year = {2026},
author = {Hartweger, H and Ruprecht, C and Yao, KH and Laffont, P and Lima Dos Reis, G and Zhou, P and Hägglöf, T and Binet, L and Loewe, M and Hong, JP and Xiao, T and Sefik, E and Hernandez, B and Gazumyan, A and Jankovic, M and Seaman, MS and Costa, G and Nelson, SA and Clark, J and Kanatani, S and Wilson, PC and Krammer, F and Levashina, EA and Julien, JP and Wardemann, H and Sinnis, P and Stamatatos, L and Flavell, RA and Nussenzweig, MC},
title = {B lymphocyte protein factories produced by hematopoietic stem cell gene editing.},
journal = {Science (New York, N.Y.)},
volume = {392},
number = {6795},
pages = {eadz8994},
doi = {10.1126/science.adz8994},
pmid = {41990179},
issn = {1095-9203},
mesh = {Animals ; Mice ; *B-Lymphocytes/immunology ; Broadly Neutralizing Antibodies/immunology ; CRISPR-Cas Systems ; *Gene Editing/methods ; Hematopoietic Stem Cell Transplantation ; *Hematopoietic Stem Cells/immunology ; HIV Antibodies/blood/immunology/biosynthesis ; HIV-1/immunology ; Malaria/prevention &amp; control/immunology/therapy ; Mice, Inbred C57BL ; Orthomyxoviridae Infections/prevention &amp; control/immunology ; Plasma Cells/immunology ; Precursor Cells, B-Lymphoid/immunology ; Antibodies, Protozoan/biosynthesis/blood/immunology ; },
abstract = {Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. In this study, we report on an alternative gene editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. In mice, edited B lymphocytes derived from transplanted HSPCs were activated by cognate antigen, underwent clonal expansion, and developed into specific antibody-synthesizing or cargo protein-synthesizing plasma cells. These cells produced long-lasting, therapeutic levels of serum antibody against HIV-1, malaria, or an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.},
}
@article {pmid41990423,
year = {2026},
author = {Wang, Q and Zhu, Y and Chen, B and Zhai, C and Xu, J and Xia, J},
title = {Asymmetric RPA-primed hybridization chain reaction enabling one-input-multiple-Cas12a activation for ultrasensitive Salmonella detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {305},
number = {},
pages = {118689},
doi = {10.1016/j.bios.2026.118689},
pmid = {41990423},
issn = {1873-4235},
mesh = {*Salmonella/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/methods ; Limit of Detection ; Food Microbiology ; Nucleic Acid Hybridization ; Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Humans ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics/chemistry ; },
abstract = {Ensuring food safety requires accurate, simple detection of pathogens such as Salmonella. However, conventional methods suffer from long processing times, complicated procedures, and inadequate accuracy. To address this, we developed an integrated platform combining asymmetric recombinase polymerase amplification (aRPA), programmable hybridization chain reaction (HCR), and CRISPR/Cas12a readout in a simplified single-tube integrated format. Unlike conventional CRISPR assays, in which a single target activates a single Cas12a complex in a PAM-dependent manner, our design uses aRPA-generated single-stranded DNA to trigger HCR, converting each target into a long dsDNA molecule with multiple PAM sites. This architecture enables one-input, multiple-Cas12a activation, representing a shift from linear to cooperative amplification. Under optimized conditions, the assay achieves a detection limit of 1.8 × 10[2] colony-forming units/mL (CFU/mL) in direct screening, and reaches as low as 5 CFU/mL after a 6 h enrichment step. The method shows high specificity toward common foodborne pathogens and reliable performance in spiked food samples. Furthermore, a DNA releaser simplifies sample preparation and enhances convenience. In summary, this integrated strategy offers a reliable and practical tool for food safety monitoring.},
}
@article {pmid41991098,
year = {2026},
author = {Lu, P and Guo, J and Jia, R},
title = {Alternative splicing of immune checkpoints: Classifications, mechanisms, and therapeutic implications for overcoming immune checkpoint blockade resistance.},
journal = {Critical reviews in oncology/hematology},
volume = {223},
number = {},
pages = {105339},
doi = {10.1016/j.critrevonc.2026.105339},
pmid = {41991098},
issn = {1879-0461},
mesh = {Humans ; *Alternative Splicing ; *Drug Resistance, Neoplasm/genetics/immunology ; *Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; *Neoplasms/genetics/immunology/drug therapy/therapy ; Animals ; *Immune Checkpoint Proteins/genetics ; Tumor Microenvironment/immunology/genetics ; Immunotherapy/methods ; },
abstract = {Immune checkpoint blockade (ICB) therapy has revolutionized oncology, yet its clinical efficacy remains limited due to primary and acquired resistance. Alternative splicing (AS), a fundamental post-transcriptional regulatory mechanism in eukaryotic gene expression, has been shown to profoundly remodel immune checkpoint molecules, driving immune evasion and ICB resistance. In this review, we systematically categorize immune checkpoint splicing based on splicing events, evolutionary conservation, altered domains, and functional impacts. We propose a new feature of immune checkpoint splicing, transmembrane exon splicing strategy. Mechanistically, we explain how dysregulated spliceosomes, cis-acting elements, and trans-acting factors within the tumor microenvironment orchestrate these splicing events, impacting ligand/receptor interactions and downstream immune signaling. Therapeutically, soluble isoforms serve as diagnostic/prognostic biomarkers. Engineered oncolytic viruses expressing soluble decoys offer novel combination strategies. Emerging therapeutic approaches, including antisense oligonucleotides, splicing modulators, RNA interference, and CRISPR/Cas systems, show promise for directly targeting aberrant splicing to overcome ICB resistance.},
}
@article {pmid41991300,
year = {2026},
author = {Ji, T and Wang, T and Yu, K and Gao, YZ and Gao, XZ},
title = {[Research progress in the application of RPA-CRISPR/Cas13a technology in the detection of pathogenic microorganisms].},
journal = {Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]},
volume = {60},
number = {4},
pages = {622-630},
doi = {10.3760/cma.j.cn112150-20250430-00378},
pmid = {41991300},
issn = {0253-9624},
support = {M2024086//2024 Research Project of Jiangsu Provincial Health Commission/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Bacteria/isolation &amp; purification/genetics ; },
abstract = {Recombinase polymerase amplification (RPA) is an emerging method for nucleic acid amplification. It can be performed under low-temperature conditions, making it suitable for point-of-care testing. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 13 (CRISPR/Cas13a), a type Ⅵ CRISPR-Cas system, employs a single-subunit RNA-guided Cas endonuclease to target exogenous RNA and exhibits distinct trans-cleavage activity. The integration of CRISPR/Cas13a with RPA technology significantly enhances detection sensitivity and accuracy, demonstrating considerable potential for molecular diagnostics. The RPA-CRISPR/Cas13a platform has achieved notable success in areas such as food safety, environmental monitoring, and clinical diagnosis, and has become an important tool in modern biomedical research. This article provides an in-depth analysis of the fundamental principles of RPA-CRISPR/Cas13a technology and comprehensively reviews its applications in pathogenic microorganism detection, aiming to offer new insights for accurate pathogen diagnosis.},
}
@article {pmid41991526,
year = {2026},
author = {Omura, SN and Nakagawa, R and Kajimoto, S and Okazaki, S and Ishiguro, S and Mori, H and Onishi, K and Kashiwakura, Y and Hiramoto, T and Horinaka, K and Tanaka, M and Hirano, H and Jividen, K and Yamashita, K and Tsai, SQ and Yachie, N and Ohmori, T and Nishimasu, H and Nureki, O},
title = {Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41991526},
issn = {2041-1723},
support = {JP25ama121012//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*Staphylococcus aureus/genetics/enzymology ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; Protein Engineering ; Streptococcus pyogenes ; Cryoelectron Microscopy ; },
abstract = {Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Although PAM-relaxed Cas9 variants have been developed, expanded targeting is often accompanied by compromised target specificity. Here, we report the rational engineering of eSaCas9-NNG, a SaCas9 variant that recognizes relaxed NNG PAMs while maintaining high target fidelity, thereby overcoming a fundamental trade-off in Cas9-based genome editing. eSaCas9-NNG efficiently induces indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice, with editing efficiencies comparable to those of other PAM-relaxed nucleases, including SpRY, SpG, and iGeoCas9, but with reduced off-target activity. We further determine the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for its relaxed PAM recognition, improved target specificity, and nuclease activation. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.},
}
@article {pmid41992303,
year = {2026},
author = {Wang, Y and Guo, Y and Xiong, Y and Ren, X and Zhao, Y and Song, L and He, L},
title = {The stimuli-responsive CRISPR-Cas12a system for modulating the selective aggregation of cell membrane receptors.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {41992303},
issn = {1477-3155},
support = {82373630//National Natural Science Foundation of China/ ; 2023GGJS009//the training grant of Henan Province for Young Teachers/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Vascular Endothelial Growth Factor A/metabolism ; A549 Cells ; *Receptors, Transferrin/metabolism/genetics ; Proto-Oncogene Proteins c-met/metabolism/genetics ; *Receptors, Cell Surface/metabolism ; },
abstract = {While CRISPR systems exhibit remarkable programmability in the field of nucleic acid editing, their extension to protein engineering faces a fundamental challenge, namely the traditional CRISPR tools lack the design to efficiently convert stimulus signals into the selective clustering of membrane receptors. This study develops a stimulus-responsive membrane-confined CRISPR-Cas12a platform that enhances selective clustering of membrane receptors for functional regulation. Specifically, a membrane-anchored DNA tetrahedral framework (TD-apt) was designed, which leverages vascular endothelial growth factor (VEGF) to activate Cas12a. Compared with unconfined CRISPR-Cas12a, membrane-confined CRISPR-Cas12a exhibits stronger cleavage activity, the interaction between the cellular-mesenchymal epithelial transition factor (c-Met) receptor and transferrin receptor (TfR) on A549 cells was efficiently modulated by nucleic acid assembly. This manipulation selectively inhibited c-Met function through spatial steric hindrance of TfR, modulating cellular behavior. Notably, the system's generality was validated by engineering of c-Met homodimerization for activation. This cascading regulatory paradigm of environmental sensing (VEGF response)-nucleic acid computation (CRISPR-based nucleic acid molecular computation)-protein assembly (receptor topological remodeling) effectively extends CRISPR's application boundaries to the field of non-genetic regulation protein-protein interaction (PPI) and establishes a versatile toolkit for dynamic and precise functional regulation.},
}
@article {pmid41992329,
year = {2026},
author = {Cho, E and Lee, J and Kim, J and Choi, J and Kang, M and Song, J},
title = {Application of extracellular vesicles in the CRISPR-based diagnosis and treatment: possibilities and challenges.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {},
pmid = {41992329},
issn = {1754-1611},
abstract = {The CRISPR–Cas system has revolutionized molecular diagnostics and gene editing, yet its clinical translation is hindered by delivery barriers, off-target activity, immunogenicity, and manufacturing challenges. Compared with viral vectors and synthetic non-viral carriers such as lipid nanoparticles, extracellular vesicles (EVs) offer a biologically derived delivery platform with superior biocompatibility, reduced immunogenicity, intrinsic cargo protection, and natural barrier-crossing capability. Engineered EVs can further achieve cell- or tissue-specific targeting. In diagnostics, endogenous EV proteins and nucleic acids provide disease-informative signatures that can interface with CRISPR nuclease readouts for highly sensitive detection. This review summarizes the therapeutic and diagnostic potential of EV-CRISPR platforms, covering strategies for loading CRISPR cargos (producer-cell engineering, post-isolation methods), cargo formats, and surface targeting approaches. We evaluate preclinical performance with attention to biodistribution, safety, innate and adaptive immune responses, and genomic integrity, as well as analytical assays and scalable manufacturing considerations essential for clinical translation. Finally, we discuss emerging opportunities, including AI-guided optimization of EV-CRISPR design and integrated EV platforms that combine disease detection with therapeutic intervention, highlighting their promise for advancing precision medicine.},
}
@article {pmid41992430,
year = {2026},
author = {Pinos, D and García-Marín, E and Ramírez-Serrano, B and Benavent-Albarracín, L and Gamir, J and Crava, CM},
title = {Maintenance of Gut Microbial Balance via the Kynurenine Pathway Improves Larval Performance and Resistance to Bacillus thuringiensis in Spodoptera exigua.},
journal = {MicrobiologyOpen},
volume = {15},
number = {2},
pages = {e70289},
pmid = {41992430},
issn = {2045-8827},
support = {PID2020-118787RA-I00//Agencia Estatal de Investigaci&#xf3;n/ ; MRR/RYC2021-033098-I//Agencia Estatal de Investigaci&#xf3;n/ ; PID2024-162058OB-C32//Agencia Estatal de Investigaci&#xf3;n/ ; RGY0052/2022//Human Frontier Science Program/ ; },
mesh = {Animals ; *Gastrointestinal Microbiome ; Larva/microbiology/growth &amp; development ; *Spodoptera/microbiology/growth &amp; development ; *Kynurenine/metabolism ; *Bacillus thuringiensis/physiology ; Kynurenine 3-Monooxygenase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The gut microbiota is a key determinant of insect physiology, influencing nutrition, immunity, and interactions with plants and pathogens. In Lepidoptera, larval gut communities are dynamic, but a core microbiota, often dominated by Enterococcus species, persists across instars. In Spodoptera littoralis, the enzyme kynurenine 3-monooxygenase (KMO) regulates gut bacterial composition via 8-hydroxyquinoline-2-carboxylic acid (8-HQA), a secreted iron-chelating compound. To investigate whether this mechanism is conserved in Noctuidae, we generated Spodoptera exigua kmo[-/-] mutants using CRISPR/Cas9 and analyzed bacterial communities in foregut, midgut, hindgut, and oral secretions by 16S metabarcoding, using RNA-derived cDNA for gut samples and DNA for oral secretions due to lower microbial biomass. The kmo deletion abolished 8-HQA production, reduced bacterial diversity, and collapsed compartment-specific bacterial communities in the gut, while also being associated with Enterococcus dominance in oral secretions. Fitness assays revealed that kmo[-/-] larvae exhibited reduced weight gain on artificial diet, and higher mortality and delayed growth when fed on pepper leaves. Moreover, kmo[-/-] larvae were threefold more susceptible to Bacillus thuringiensis, consistent with an interaction between host physiological state, gut microbial homeostasis, and pathogen susceptibility. Dietary supplementation with 8-HQA partially mitigated, but did not fully rescue, growth deficits. Our results demonstrate that the kynurenine pathway and 8-HQA production are crucial for maintaining gut microbial homeostasis, particularly within Enterococcus, thereby supporting larval development, dietary adaptation, and pathogen resilience. These findings reveal a conserved mechanism in noctuid moths linking host metabolism, microbiota regulation, and ecological performance, emphasizing the interplay between host genetics, microbiota composition, and environmental stressors.},
}
@article {pmid41992908,
year = {2026},
author = {Cleanclay, WD and Adedoyin, ED and Zakari, S and Ogunlana, OO and Iweala, EEJ and Chinedu, SN},
title = {Advancing Malaria Vector Control: Insights Into Mosquito Immunity and Genetic Strategies.},
journal = {TheScientificWorldJournal},
volume = {2026},
number = {1},
pages = {e7634044},
pmid = {41992908},
issn = {1537-744X},
mesh = {Animals ; *Mosquito Vectors/immunology/genetics/parasitology ; *Malaria/prevention &amp; control/transmission/immunology ; *Anopheles/immunology/genetics/parasitology ; *Mosquito Control/methods ; Plasmodium ; RNA Interference ; Humans ; Wolbachia ; Immunity, Innate ; CRISPR-Cas Systems ; },
abstract = {Malaria remains a major global health challenge, particularly in sub-Saharan Africa where Anopheles mosquitoes transmit the Plasmodium parasites. Resistance to insecticides remains an obstacle in spite of the efforts to control malaria vector. The interaction between Plasmodium parasites and mosquito vectors, with a focus on the immunity of mosquitoes and approaches to combat malaria, is examined in this review. This review explores the potential of genetic approaches including CRISPR-Cas9, Wolbachia, RNA interference (RNAi), and symbiont-based strategies for the control of malaria vector. The innate immune system of Anopheles mosquitoes that identify, recognize, and limit Plasmodium infection through pathogen recognition receptors, signaling pathways, and effector mechanisms like antimicrobial peptides and melanization is well developed. However, Plasmodium has developed several evasion mechanisms to establish infection. This led to various genetic modification techniques being designed to reduce vector population and transmission. Gene drive such as CRISPR-Cas9 can introduce genetic alterations to interfere with the transmission of malaria; Wolbachia interferes with vector competence, RNAi-mediated gene to target relevant genes involved in reproduction and survival. Self-limiting strategies such as RIDL and pgSIT genetically modified insect releasement to the environment. mosGILT is an emerging immune regulator which has shown relevance in blocking transmission. This review explores the potential of these genetic approaches in malaria vector control efforts, highlighting their advantages and imitations. Further research should explore mosquito immune genes and pathways in developing innovative and acceptable genetic vector control approaches.},
}
@article {pmid41993032,
year = {2026},
author = {Zhao, J and Sui, Z and Zhou, Y and Peng, Y and Xu, J},
title = {Rigidity-Responsive Fluorescence Polarization Detection of Aflatoxin B1 via Programmable RCA-Coupled CRISPR/Cas12a and a Conformation-Restricted Depolarization Reporter.},
journal = {Analytical chemistry},
volume = {98},
number = {16},
pages = {12023-12034},
doi = {10.1021/acs.analchem.6c00481},
pmid = {41993032},
issn = {1520-6882},
mesh = {*Aflatoxin B1/analysis ; *Fluorescence Polarization/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry/genetics ; *Nucleic Acid Amplification Techniques ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Aflatoxin B1 (AFB1) is one of the most toxic and carcinogenic mycotoxins, and its trace-level determination in complex food matrices remains a major analytical challenge. Conventional chromatographic methods, while highly accurate, rely on expensive instrumentation and labor-intensive sample pretreatment, whereas most CRISPR/Cas-based biosensors depend on fluorescence intensity turn-on readouts that are vulnerable to matrix autofluorescence, photobleaching, and signal instability, limiting their reliability in real samples. Herein, we propose a rigidity-responsive fluorescence polarization (FP) biosensing strategy that integrates aptamer-based molecular recognition, rolling circle amplification (RCA), and CRISPR/Cas12a trans-cleavage for robust and matrix-tolerant AFB1 detection. In this system, target binding induces the release of a complementary DNA strand from an immobilized aptamer duplex, initiating padlock probe circularization and RCA to generate abundant Cas12a-activating amplicons. A rationally engineered conformation-restricted depolarization reporter (CRD-Reporter), in which the fluorophore is confined within a rigid duplex framework, provides an intrinsically high FP signal. Upon Cas12a activation, collateral cleavage disrupts the rigid architecture, releasing freely rotating fragments and producing a pronounced FP decrease. Unlike intensity-based CRISPR assays, the FP readout effectively suppresses background interference and signal fluctuations. The proposed assay exhibits a wide linear range from 0.003 to 300 ng/mL with a low detection limit of 0.00113 ng/mL, high specificity, and excellent accuracy in grains, peanuts, and tea samples. This work establishes a robust FP-based CRISPR sensing paradigm for reliable mycotoxin monitoring in complex food systems.},
}
@article {pmid41993526,
year = {2026},
author = {Knight, AL and Belato, HB and Dresser, CS and Pindi, C and Mercado, BJ and Lasekan, P and Luo, J and Arantes, PR and Jogl, G and Palermo, G and Lisi, GP},
title = {Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on Geo Cas9 from integrated structural biophysics.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.04.08.717222},
pmid = {41993526},
issn = {2692-8205},
abstract = {Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus (Geo Cas9) via its HNH nuclease domain. An X-ray crystal structure of the Geo HNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of Geo Cas9. AcrIIC1 binding also rewires the intrinsic dynamics of the Geo HNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of Geo Cas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on Geo Cas9.},
}
@article {pmid41995110,
year = {2026},
author = {Dong, J and Liu, J and Li, Y and Sohail, H and Ji, Z and Chen, X and Xu, J},
title = {CsPNRC1 regulated the resistance of Fusarium wilt by interacting with Csfla15 to influence the cell wall structure of cucumber (Cucumis sativus L.).},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {1},
pages = {e70871},
doi = {10.1111/tpj.70871},
pmid = {41995110},
issn = {1365-313X},
support = {32372690//National Natural Science Foundation of China/ ; JBGS [2021]018//"JBGS" Project of Seed Industry Revitalization in Jiangsu Province/ ; },
mesh = {*Cucumis sativus/microbiology/genetics/metabolism ; *Fusarium/physiology ; *Plant Proteins/metabolism/genetics ; *Cell Wall/metabolism ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/immunology/genetics ; Gene Expression Regulation, Plant ; *Mucoproteins/metabolism/genetics ; },
abstract = {Cucumber (Cucumis sativus L.) Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. cucumerinum (Foc), is a devastating disease that significantly impacts cucumber production worldwide. The lack of natural resistant cultivars to FW has hindered the development of resistant cucumber varieties through conventional breeding methods. Interestingly, the disruption of susceptibility (S) genes has emerged as an effective alternative approach for enhancing crop resistance. Here, we identified a proline-rich nuclear receptor coactivator CsPNRC1 through transcriptomic analysis, and function as a susceptibility gene of cucumber FW using virus-induced gene silencing (VIGS), CRISPR/Cas- and overexpression-based methods. Seedlings overexpressing CsPNRC1 exhibited browning at the shoot bases 7 days after inoculation with Foc pathogens, while knockout seedlings demonstrated significantly greater resistance compared with wild-type (WT) plants. We identified CsPNRC1 interacting with a fasciclin-like arabinogalactan protein Csfla15 through yeast two-hybrid (Y2H) assays, bimolecular fluorescence complementation (BiFC), Co-immunoprecipitation (Co-IP), and luciferase complementation imaging (LCI) assay, and found that Csfla15 was regulated by CsPNRC1. Also, CsPNRC1 could influence the content of cell wall components pectin and cellulose in cucumber roots. Meanwhile, these findings provide valuable insights into the role of CsPNRC1 in cucumber's susceptibility to FW and highlight its potential use for resistance breeding.},
}
@article {pmid41996462,
year = {2026},
author = {Gu, Y and Hart, T and Leon-Novelo, L and Shen, JP},
title = {Double-CRISPR Knockout Simulation (DKOsim): A Monte-Carlo randomization system to model cell growth behavior and infer the optimal library design for growth-based double knockout screens.},
journal = {PLoS computational biology},
volume = {22},
number = {4},
pages = {e1013510},
pmid = {41996462},
issn = {1553-7358},
mesh = {Monte Carlo Method ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems/genetics ; Computer Simulation ; Humans ; Computational Biology ; Gene Library ; *Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Models, Genetic ; },
abstract = {Advances in functional genomic technology, notably CRISPR using Cas9 or Cas12, now allow for large-scale double perturbation screens in which pairs of genes are inactivated, allowing for the experimental detection of genetic interactions (GIs). However, as it is not possible to validate GIs in high-throughput, there is no gold standard dataset where true interactions are known. Hence, we constructed a Double-CRISPR Knockout Simulation (DKOsim), which allows users to reproducibly generate synthetic simulation data where the single gene fitness effect of each gene and the interaction of each gene pair can be specified by the investigator. We adapted Monte-Carlo randomization methods to extend single knockout simulation methods to double knockout designs, which simulate the gene-gene interactions between all possible combinations of the input genes. Using DKOsim, we generated simulated datasets that closely resemble real double knockout CRISPR datasets in terms of Log Fold Change (LFC), GI distribution, and replicate correlation. We further inferred optimal CRISPR library designs by systematically investigating critical experimental parameters including depth of coverage, guide efficiency, and the variance of initial guide distribution. This simulation scheme will help to identify optimal computational methods for GI detection and aid in the design of future dual knockout CRISPR screens.},
}
@article {pmid41996722,
year = {2026},
author = {Han, K and Qin, Z and Hu, S and Wu, H and Lei, W},
title = {CRISPR/Cas9-mediated generation of a REPS2 knockout human embryonic stem cell line.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103986},
doi = {10.1016/j.scr.2026.103986},
pmid = {41996722},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), an Xchromosome-linked gene, is broadly expressed in brain, adrenal gland, and other tissues. REPS2 encodes a protein that forms part of a complex involved in the regulation of growth factor receptor endocytosis, and has been associated with a variety of diseases. We generated a REPS2 knockout human embryonic stem cell line using CRISPR/Cas9 genome editing system, which remained typical stem cell morphology, a normal karyotype, and pluripotency, and demonstrated the capacity to differentiate into all three germ layers. The REPS2 knockout hESC line provides a valuable tool for modeling REPS2-associated pathological process.},
}
@article {pmid41996895,
year = {2026},
author = {Pindi, C and Palermo, G},
title = {Deep learning and cryogenic electron microscopy modeling for gene editing dynamics.},
journal = {Current opinion in structural biology},
volume = {98},
number = {},
pages = {103270},
doi = {10.1016/j.sbi.2026.103270},
pmid = {41996895},
issn = {1879-033X},
mesh = {*Deep Learning ; *Cryoelectron Microscopy/methods ; *Gene Editing/methods ; Molecular Dynamics Simulation ; Graph Neural Networks ; },
abstract = {Advances in cryogenic electron microscopy (cryo-EM) data modeling and deep learning are reshaping our ability to interrogate and engineer genome-editing systems. Their synergistic integration enables high-resolution structural interpretation, quantitative mapping of conformational landscapes, and rational design across diverse CRISPR-Cas architectures. By coupling molecular dynamics with cryo-EM refinement, we uncover functionally relevant dynamic ensembles, while quantum mechanical methods resolve ambiguous features in low-resolution density maps. Emerging deep-learning frameworks including graph neural networks, extract interpretable communication pathways from large-scale simulations and provide methods that are broadly transferable across biomolecular systems. These advances propel the field beyond static structural snapshots toward a dynamic, predictive, and data-driven approach for understanding and designing genome-editing systems.},
}
@article {pmid41997156,
year = {2026},
author = {Peng, J and Chan, DCT and Chu, HY and Fong, JHC and Lam, YK and Cheung, MSH and Leung, W and Choi, GCG and Wong, ASL},
title = {Rapid customization of base editors via machine learning-powered combinatorial mutagenesis.},
journal = {Molecular cell},
volume = {86},
number = {10},
pages = {1839-1855.e10},
doi = {10.1016/j.molcel.2026.03.030},
pmid = {41997156},
issn = {1097-4164},
mesh = {Humans ; *Mutagenesis ; *Gene Editing/methods ; *Machine Learning ; *CRISPR-Cas Systems ; *DNA/genetics ; Mutation ; Nucleotide Motifs ; HEK293 Cells ; Predictive Learning Models ; },
abstract = {Being able to control the complementarity and hindrance between target DNA and base editor proteins enables precise, bystander-free editing. Here, we combined combinatorial mutagenesis with machine learning to analyze and engineer these interactions at scale. By profiling DNA motif preferences across 160,000 evoAPOBEC1 and 64 million TadA variants in human cells, we used as little as 0.004% of the mutational landscape to make predictions. This identified variants with motif-specific activity and eliminated residual adenine editing in cytosine base editors. In correcting >800 disease-associated mutations, our variants outperformed previous versions in precluding unintended edits at purine motifs, achieving undetectable bystander edits in 50% of cases. Additionally, a pre-trained, structure-based deep learning model predicted functional TadA variants with 63% success across 20[26] variants spanning 26 amino acid sites, without experimental data and in a single prediction round. These approaches streamline the re-engineering of base editors for enhanced precision tailored to specific targets.},
}
@article {pmid41997283,
year = {2026},
author = {Zhang, Y and Yu, M and Huang, J},
title = {Arginase 2 regulates cholesterol biosynthesis in endothelial cells.},
journal = {Experimental cell research},
volume = {459},
number = {2},
pages = {115029},
doi = {10.1016/j.yexcr.2026.115029},
pmid = {41997283},
issn = {1090-2422},
mesh = {Humans ; *Arginase/metabolism/genetics ; *Cholesterol/biosynthesis ; *Human Umbilical Vein Endothelial Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Arginase 2 (Arg 2) is a mitochondrial enzyme that hydrolyzes L-arginine to L-ornithine and urea, influencing endothelial nitric oxide (NO) bioavailability and vascular function. Although Arg 2 is implicated in endothelial dysfunction under hypercholesterolemic and oxidative stress conditions, its potential role in endothelial cholesterol metabolism remains unknown.<br><br>METHODS: Arg 2 was deleted in immortalized human umbilical vein endothelial cells (HUVECs) using CRISPR/Cas9, followed by transcriptomic analyses. Differential gene expression was validated by quantitative RT-PCR and immunoblotting. Overexpression of wild-type Arg 2 and the catalytically inactive Arg 2 (H160F) mutant was achieved using recombinant lentiviral transduction. Arginase activity was quantified by measuring urea production using a colorimetric assay. Cholesterol intermediates were quantified by LC-MS.<br><br>RESULTS: RNA sequencing revealed that Arg 2 deletion markedly downregulated genes involved in the mevalonate and steroid biosynthesis pathways, including HMGCS1, FDFT1, FDPS, SQLE, and DHCR7. These transcriptional changes were accompanied by reduced protein levels of key cholesterol biosynthetic enzymes and decreased cellular concentrations of sterols, lanosterol, desmosterol, and cholesterol. Conversely, either overexpression of wild-type Arg 2 or the catalytically inactive Arg 2 (H160F) mutant enhanced the expression of these enzymes.<br><br>CONCLUSIONS: These findings identify a previously unrecognized role of Arg 2 in promoting endothelial cholesterol biosynthesis. Beyond competing with endothelial NO synthase for L-arginine, Arg 2 may regulate vascular homeostasis through modulation the mevalonate pathway, independent of its enzymatic activity. This dual function may link amino acid and lipid metabolism in the endothelium and suggests new mechanisms by which Arg 2 contributes to endothelial dysfunction and atherosclerotic progression.},
}
@article {pmid41997380,
year = {2026},
author = {Sahu, VK and Das, P and Choudhury, SR and Karmakar, S},
title = {Emerging advantages of nano delivery systems in enhancing CAR-T/CRISPR-Cas9 mediated cancer therapeutics.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1881},
number = {3},
pages = {189592},
doi = {10.1016/j.bbcan.2026.189592},
pmid = {41997380},
issn = {1879-2561},
mesh = {Humans ; *Neoplasms/therapy/immunology/genetics ; Animals ; *Nanoparticles/chemistry ; *Receptors, Chimeric Antigen/genetics/immunology ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; Cancer Vaccines/immunology/administration &amp; dosage ; *Drug Delivery Systems/methods ; *Nanoparticle Drug Delivery System ; Tumor Microenvironment/immunology ; },
abstract = {Viral vectors have long been central to cancer immunotherapy, particularly for ex vivo chimeric antigen receptor (CAR)-T cell engineering and cancer vaccine development. Despite their success, clinical translation remains limited by immunogenicity, insertional mutagenesis, restricted cargo capacity, and high production costs. These drawbacks not only compromise safety but also hinder scalability and repeated dosing, both of which are critical for durable cancer control. To overcome these barriers, non-viral nanocarrier systems have emerged as versatile and safer alternatives. Lipid nanoparticles, polymeric platforms, biomimetic exosome-like vesicles, and hydrogel-based systems enable targeted and controlled delivery of nucleic acids, immunomodulators, and chemotherapeutics with enhanced stability, reduced systemic toxicity, and improved biocompatibility. Beyond passive delivery, these smart nanocarriers can be engineered with tumor-targeting ligands, immune checkpoint modulators, or stimulus-responsive release mechanisms to reprogram the tumor microenvironment and potentiate T-cell and dendritic cell activation. Furthermore, the modularity of nanotechnology facilitates co-delivery of multiple therapeutic agents, including antigens, adjuvants, and checkpoint inhibitors, allowing synergistic immunotherapeutic outcomes. Recent advances in large-scale manufacturing and clinical translation of lipid nanoparticle-based mRNA vaccines underscore the feasibility of these systems for oncology applications. As cancer immunotherapy evolves toward personalization and combination regimens, nanobiotechnology offers a transformative platform to replace conventional viral vectors, advancing safer, more effective, and clinically scalable treatments.},
}
@article {pmid41997664,
year = {2026},
author = {Karan, R and Prasannakumar, MK and Kour Khera, H and Harish, J and Patil, SS and Devanna, P and Manjunatha, C and Mishra, RK},
title = {Unified Primer Enabled Detection (UPED) system for Magnaporthe oryzae infecting rice: A comparative study from conventional PCR to CRISPR Cas12a based detection systems.},
journal = {Analytica chimica acta},
volume = {1404},
number = {},
pages = {345418},
doi = {10.1016/j.aca.2026.345418},
pmid = {41997664},
issn = {1873-4324},
mesh = {*Oryza/microbiology ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques ; *DNA Primers/genetics ; *Plant Diseases/microbiology ; Molecular Diagnostic Techniques/methods ; *Magnaporthe/genetics/isolation &amp; purification ; *Ascomycota/genetics/isolation &amp; purification ; Real-Time Polymerase Chain Reaction ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is one of the most devastating fungal pathogens of rice. Early, rapid and accurate detection is critical for effective disease management and the prevention of outbreaks. Conventional and isothermal molecular methods vary widely in sensitivity and field applicability, making it difficult to understand the reliable detection. This study aimed to perform the first unified, side-by-side comparison of six molecular detection techniques using a common primer set to identify the most sensitive, specific and field deployable approach for M. oryzae detection.<br><br>RESULTS: In this study, we have evaluated six diagnostic methods such as PCR, qPCR, LAMP, RPA, and CRISPR-Cas12a integrated with LAMP or RPA targeting the multi copy Pot2 transposon in M. oryzae. Sensitivity assays using 10-fold genomic DNA dilutions (10[9] to 10[-1] copies/reaction) revealed that LAMP-CRISPR and RPA-CRISPR were most sensitive, detecting down to 1 copy/reaction. LAMP and qPCR detected down to 10[2] and 10[3] copies/reaction, while RPA and PCR were limited to 10[5] and 10[6] copies/reaction, respectively. All assays showed high specificity with no cross-reactivity to 10 non-target rice fungal pathogens. Field validation on 17 symptomatic samples confirmed that CRISPR-based methods outperformed traditional techniques, detecting positives missed by other platforms. This is the first systematic comparison applying the same primer set across multiple diagnostic methods for M. oryzae.<br><br>SIGNIFICANCE: Our findings demonstrate that CRISPR-Cas12a-based platforms combined with isothermal amplification offers high sensitivity and specificity. CRISPR based detection allows strong field applicability for M. oryzae detection. The use of a unified primer set allowed for a direct performance comparison. These results highlight the potential of CRISPR-based diagnostics to enhance early pathogen detection, enabling rapid interventions and improved management of rice blast disease.},
}
@article {pmid41997865,
year = {2026},
author = {George, G and Harding, HP and Kay, R and Ron, D and Ordoñez, A},
title = {Metabolite import by SLC33A1 is required for ATF6 activation during endoplasmic reticulum stress.},
journal = {Life science alliance},
volume = {9},
number = {6},
pages = {},
pmid = {41997865},
issn = {2575-1077},
support = {224407/Z/21Z//Wellcome Trust Principal Research Fellowship/ ; },
mesh = {Animals ; *Activating Transcription Factor 6/metabolism/genetics ; Unfolded Protein Response ; CHO Cells ; Cricetulus ; Endoplasmic Reticulum/metabolism ; *Endoplasmic Reticulum Stress/physiology/genetics ; Golgi Apparatus/metabolism ; Humans ; *Membrane Transport Proteins/metabolism/genetics ; Signal Transduction ; Protein Serine-Threonine Kinases/metabolism ; Protein Transport ; Cricetinae ; CRISPR-Cas Systems ; GABA Plasma Membrane Transport Proteins ; },
abstract = {The transcription factor ATF6α has a central role in adapting mammalian cells to ER stress via the unfolded protein response (UPR), prompting efforts to identify ATF6α modulators. Here, an unbiased genome-wide CRISPR-Cas9 screen performed in Chinese Hamster Ovary cells revealed that proteolytic processing of the ATF6α precursor to its active form was impaired in cells lacking the ER-resident solute carrier SLC33A1, a transporter previously implicated in acetyl-CoA import, sialylation, and Nε-lysine protein acetylation. Cells lacking SLC33A1 constitutively trafficked the ATF6α to the Golgi but exhibited impaired Golgi processing and activating proteolysis. IRE1α signalling was derepressed by SLC33A1 deficiency consistent with selective loss of ATF6α-mediated negative feedback in the UPR. Slc33a1-deleted cells accumulated unmodified sialylated N-glycans, precursors to acetylated glycans, likely reflecting impaired glycan processing. Deletion of ER-localised acetyltransferases NAT8 and NAT8B, which catalyse protein Nε-lysine acetylation in the secretory pathway, did not replicate the ATF6α processing defects observed in Slc33a1-deficient cells. Together, our findings highlight a role of SLC33A1-mediated metabolite transport in the post-ER ATF6α maturation, linking small-molecule metabolism to branch-specific signalling in the UPR.},
}
@article {pmid41999294,
year = {2026},
author = {Liu, S and Wang, L and Zhang, H and Jiang, F and Tang, F and Han, R and Guo, W and Gu, S and Chen, G and Zhang, D and Zhan, X},
title = {A novel rapid detection approach based on CRISPR-Cas13a for Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acariformes: Pyroglyphidae).},
journal = {Journal of insect science (Online)},
volume = {26},
number = {2},
pages = {},
pmid = {41999294},
issn = {1536-2442},
support = {H202112//Commissioned Research Project and the Anhui Provincial Graduate Innovation and Entrepreneurship Practice Project/ ; 2022cxcysj180//Commissioned Research Project and the Anhui Provincial Graduate Innovation and Entrepreneurship Practice Project/ ; },
mesh = {Animals ; *Dermatophagoides pteronyssinus/genetics ; *CRISPR-Cas Systems ; *Dermatophagoides farinae/genetics ; },
abstract = {Dermatophagoides pteronyssinus (Trouessart) and Dermatophagoides farinae (Hughes) (Acariformes: Pyroglyphidae) are the prevalent kinds of house dust mites (HDMs). HDM is a common indoor pest, which mainly breeds in indoor dust and is an important allergen source causing a variety of allergic diseases. Effective detection of these HDMs is crucial in preventing the allergic diseases they cause. The objective of this study was to develop an innovative method for the rapid visualization of HDMs (D. pteronyssinus and D. farinae) using recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) in combination with CRISPR-Cas13a (RPA-Cas13a-LFD). To achieve heightened sensitivity in the detection of HDMs, Cas13a was incorporated into the RPA process and coupled with T7 transcripts. Based on this approach, a total of 2.23-102 copies/μl of HDM were detected within 35 min (detection limit of 2.23 copies/μl for D. farinae and 39.7 copies/μl for D. pteronyssinus). No cross-reactivity occurred with Aleuroglyphus ovatus (Troupeau) (Acariformes: Acaridae), Tyrophagus putrescentiae (Schrank) (Acariformes: Acaridae), Blomia tropicalis (van Bronswijk, de Cock &amp; Oshima) (Acariformes: Acaridae), Suidasia nesbitti (Hughes) (Acariformes: Acaridae), and Carpoglyphus lactis(Linnaeus) (Acariformes: Acaridae). The RPA-Cas13a-LFD methodology demonstrated high specificity and sensitivity in detecting HDM. Given its advantages, such as ease of operation, rapid detection, and time efficiency, it is well-suited for rapid field-based detection of HDMs, providing a new technical tool for detecting D. farinae and D. pteronyssinus.},
}
@article {pmid41999750,
year = {2026},
author = {Thege, FI and Hoskins, A and Kramer, A and Salim, I and Seetharaman, A and Fowlkes, N and Rajapakshe, KI and Maitra, A and Wörmann, SM},
title = {An autochthonous CRISPR activation screening platform for characterizing tissue-specific oncogene selection.},
journal = {Cell reports. Medicine},
volume = {7},
number = {5},
pages = {102759},
pmid = {41999750},
issn = {2666-3791},
mesh = {Animals ; *Oncogenes/genetics ; Humans ; Mice ; *Pancreatic Neoplasms/genetics/pathology ; *Lung Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Organ Specificity/genetics ; *CRISPR-Cas Systems/genetics ; SOXB1 Transcription Factors/genetics/metabolism ; Mutation/genetics ; },
abstract = {Human adenocarcinomas exhibit tissue-specific mutation and copy-number patterns that suggest diverse selective pressures and distinct oncogene dependencies. Here, we use our FiCASCan platform to test whether in vivo CRISPR activation screening can recapitulate oncogene selection during tumor initiation. Using CRISPRa-competent PPKS mice and intranasal or intraductal delivery of pooled lentivirus, we screen frequently amplified and mutated genes in autochthonous lung and pancreas cancer models. We observe strong selection for Egfr, Myc, Sox2, and Pik3cb activation in lung tumors and near-complete dominance of Myc in pancreatic tumors, revealing striking tissue-specific differences. In our model, Sox2 activation suppresses Nkx2-1 signaling and drives aggressive mucinous lung adenocarcinoma. MYC activation in the pancreas mirrors MYC amplification in human PDAC, including the emergence of an immune-cold microenvironment. Overall, our findings show that in vivo CRISPR activation screening faithfully captures oncogene selection and provides a powerful approach for studying tumor initiation and progression.},
}
@article {pmid41999797,
year = {2026},
author = {Wang, Z and Zhou, L and Ming, L},
title = {From "Simulation" to "Mirror": Gene editing and humanization redefines the next-generation precision oncology animal model.},
journal = {Gene},
volume = {998},
number = {},
pages = {150161},
doi = {10.1016/j.gene.2026.150161},
pmid = {41999797},
issn = {1879-0038},
mesh = {Animals ; Humans ; *Gene Editing/methods ; *Precision Medicine/methods ; Tumor Microenvironment/genetics ; *Disease Models, Animal ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems ; Multiomics ; Mice ; },
abstract = {Patient-derived xenograft (PDX) models, although conventionally used in oncology, exhibit critical limitations: they frequently lose patient-specific genetic mutations and lack the human leukocyte antigen (HLA) diversity essential for immune recognition, and fail to recapitulate the human tumor microenvironment (TME). These deficiencies contribute to immunotherapy prediction failure rates exceeding 80% in clinical translation. To address these gaps, we propose a Tumor Model 2.0 framework. This framework integrates multi-omics data (whole-genome, transcriptomic, and proteomic) with precision genome editing technologies (CRISPR-Cas9 and Prime Editing) to reconstruct patient-specific mutations across multiple biological layers. Employing an organoid-animal coupling platform with stepwise immune system construction and microenvironment remodeling-subsequently validated in large animals-the framework enables the creation of programmable, patient-specific digital twins. These high-fidelity models support personalized N-of-1 clinical trials, bridging the gap between preclinical research and clinical precision oncology.},
}
@article {pmid42000058,
year = {2026},
author = {Carvalho, C and Hérivaux, A and Mello, T and Bastide, F and Thomas, O and Guillemette, T and Saulnier, P and Souza Dos Santos, AL and Papon, N and Bouchara, JP},
title = {TRXR2, a thioredoxin reductase-encoding gene, contributes to protection against the oxidative stress and virulence in Scedosporium apiospermum.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108498},
doi = {10.1016/j.micpath.2026.108498},
pmid = {42000058},
issn = {1096-1208},
mesh = {*Oxidative Stress ; *Scedosporium/pathogenicity/genetics/enzymology/drug effects ; Virulence/genetics ; *Thioredoxin-Disulfide Reductase/genetics/metabolism ; Animals ; Hyphae/genetics/ultrastructure ; Reactive Oxygen Species/metabolism ; Spores, Fungal ; Cell Wall/ultrastructure/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Humans ; Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; Benzene Derivatives ; },
abstract = {Scedosporium apiospermum has received an increased attention over the past decades, especially because of its low susceptibility to current antifungals and its clinical relevance in cystic fibrosis. Nevertheless, little is still known about its pathogenic mechanisms. During the inflammatory reaction, macrophages and neutrophils release antimicrobial compounds, especially reactive oxygen species (ROS). To cope with ROS, pathogens have developed various strategies, including synthesis of some immunoprotective secondary metabolites, and enzymatic mechanisms relying on antioxidant enzymes. A recent transcriptomic study showed that the TRXR2 gene, encoding one of its two thioredoxin reductases (TrxRs), is overexpressed upon exposure to oxidative stress. To investigate the role of TrxR2, the encoding gene was disrupted using the CRISPR-Cas9 technology. An ultrastructural study revealed that TRXR2 gene knock-out induced the loss of the outer cell wall layer of hyphae, thereby increasing their surface hydrophobicity. The effects on the tolerance of the fungus to chemically-induced oxidative stresses were also investigated, revealing a higher susceptibility to cumene hydroperoxide. Additionally, an overexpression of BoyT gene (encoding the other TrxR) was seen as a compensatory mechanism, but bioinformatic analysis also suggested distinct roles for the two TrxRs in relation with distinct subcellular localizations. Finally, conidia exhibited a reduced capacity to adhere to epithelial cells and a diminished virulence in the Galleria mellonella model. In conclusion, these results suggest that TrxR2 plays a contributory role in the pathogenicity of S. apiospermum, but is not a major determinant of its virulence, which justifies future studies on the other components of the thioredoxin system.},
}
@article {pmid42000334,
year = {2026},
author = {Sarsaiya, S and Jain, A and Chen, J and Gong, Q},
title = {Unlocking non-model organisms with CRISPR-Cas: A roadmap for sustainable biotechnology.},
journal = {Biotechnology advances},
volume = {90},
number = {},
pages = {108890},
doi = {10.1016/j.biotechadv.2026.108890},
pmid = {42000334},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems ; *Biotechnology ; *Gene Editing ; },
abstract = {The reliance on model organisms in biotechnology has advanced our understanding of fundamental biology but has failed to capture the complexity of real-world ecosystems, limiting applications in agriculture, biomanufacturing, and environmental remediation. This review critically evaluates the challenges and opportunities of applying CRISPR-Cas genome editing to non-model organisms, structured around a framework that systematically addresses host-specific barriers, enabling technical solutions, and real-world applications. Key obstacles are first delineated, such as restrictive genetic tools, inefficient DNA repair pathways (including NHEJ-dominance, HDR-deficiency, and polyploidy), and delivery limitations. Subsequently, innovative solutions are explored, including the engineering of Cas variants with expanded PAM flexibility and reduced toxicity, the development of host-adapted delivery systems such as phage-based vectors and conjugative plasmids, and the integration of synthetic biology tools and machine learning for optimization. Alternative, DSB-free modalities-comprising base editing, prime editing, CRISPR-associated transposases (CAST), and recombinase-assisted engineering-are further expanded upon, offering enhanced precision and expanded capabilities for complex genetic modifications. Major findings indicate that these approaches can unlock the potential of non-conventional hosts to address global challenges, such as low-energy biomanufacturing, environmental bioremediation, and carbon capture. It is concluded that bridging the gap between foundational CRISPR research and its real-world applications is imperative. Future efforts should focus on democratizing tools via open-source platforms, advancing delivery systems, establishing ethical governance-with detailed considerations for environmental release, horizontal gene transfer, regional regulatory frameworks, and biosafety in extremophile engineering-and fostering sustainable innovation through synthetic biology integration to fully realize the transformative potential of genome editing in organisms beyond model organisms.},
}
@article {pmid42000542,
year = {2026},
author = {Kerkhof, LMC and Pepers, BA and van der Graaf, LM and Santiago-Aranda, A and Voesenek, BJB and Reits, EAJ and Buijsen, RAM and van Roon-Mom, WMC},
title = {Generation of an isogenic human induced pluripotent stem cell line for spinocerebellar ataxia type 1.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103987},
doi = {10.1016/j.scr.2026.103987},
pmid = {42000542},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Spinocerebellar Ataxias/pathology/metabolism/genetics ; Cell Line ; Ataxin-1/genetics/metabolism ; Cell Differentiation ; CRISPR-Cas Systems/genetics ; },
abstract = {Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in exon 8 of the ATXN1 gene. In this study, an isogenic human induced pluripotent stem (hiPS) cell SCA1 line was generated using CRISPR/Cas9 genome editing. Characterization revealed an expanded repeat containing 54 CAG repeats in one allele and an unmodified second allele. The isogenic hiPS cell line showed a typical hiPS cell morphology, expressed pluripotency markers and was able to differentiate into all three germ layers.},
}
@article {pmid42001720,
year = {2026},
author = {Li, X and Lu, H and Li, S and Chitrakar, B and Gu, X},
title = {Rapid and simple detection of Pediococcus using ARMS-CRISPR/Cas12a method.},
journal = {Talanta},
volume = {308},
number = {},
pages = {129806},
doi = {10.1016/j.talanta.2026.129806},
pmid = {42001720},
issn = {1873-3573},
mesh = {*Pediococcus/genetics/isolation &amp; purification ; *RNA, Ribosomal, 16S/genetics ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; Polymorphism, Single Nucleotide ; },
abstract = {Pediococcus spp. are lactic acid bacteria, which are prevalent in various environments, including plants and animals. Notably, they constitute a significant component of the intestinal microbiota in both humans and animals. Despite this, numerous obstacles remain in developing tools that are both highly sensitive and specific for distinguishing this genus. This study established a fluorescent detection system using an amplification-resistant mutation system-based polymerase chain reaction (ARMS-CRISPR/Cas12a) for identifying 16S rRNA gene containing single nucleotide polymorphism (SNP) in Pediococcus spp. By aligning the sequences of Pediococcus spp. with those of other genera, we performed a comprehensive statistical analysis of SNP sites within Pediococcus spp. and designed specific primers using the 16S rRNA gene sequence of Pediococcus pentosaceus STS-6. The results demonstrated that, under optimised conditions (a Cas12a:crRNA ratio of 1:1 at 37 °C), the dual recognition process combining ARMS-PCR with CRISPR/Cas12a achieved high specificity and sensitivity in the detection of Pediococcus spp. The detection limit for genomic DNA was 8.15 × 10[-5] ng/μL, demonstrating significantly higher sensitivity than gel electrophoresis. The entire detection process took approximately 1.5 h. In summary, the ARMS-CRISPR/Cas12a detection system established in this study provided a rapid and effective method to detect the 16S rRNA gene of clinically relevant Pediococcus spp. probiotics, meeting the requirements for food production detection.},
}
@article {pmid42001795,
year = {2026},
author = {Radszuweit, P and Fitzel, R and Bruestl, S and Hentrich, T and Korkmaz, F and Mankel, B and González-Menéndez, I and Rudat, S and Marschalek, R and Erkner, E and Keppeler, H and Schairer, R and Luib, L and Mezger, M and Quintanilla-Martinez, L and Schulze-Hentrich, J and Lengerke, C and Schneidawind, D and Schneidawind, C},
title = {Characterizing the impact of MLL fusion variants and fusion partners on leukemia plasticity using a human CRISPR-engineered MLL-rearranged leukemia model.},
journal = {Neoplasia (New York, N.Y.)},
volume = {77},
number = {},
pages = {101308},
pmid = {42001795},
issn = {1476-5586},
mesh = {Humans ; *Myeloid-Lymphoid Leukemia Protein/genetics ; Animals ; *Oncogene Proteins, Fusion/genetics ; *Histone-Lysine N-Methyltransferase/genetics ; Mice ; *Gene Rearrangement ; Disease Models, Animal ; *Leukemia/genetics/pathology/metabolism ; CRISPR-Cas Systems ; },
abstract = {Acute leukemias involving KMT2A (MLL) rearrangements are aggressive hematologic malignancies associated with a poor prognosis, especially in infants. The majority of MLL breakpoints are located within the breakpoint cluster region spanning exons 8-14, with AFF1 (AF4) and MLLT3 (AF9) being the most frequent fusion partners. To study the contribution of different fusion partners and breakpoint locations to leukemogenesis, we created a human CRISPR/Cas9-based model. We introduced MLL::AF4 or MLL::AF9 fusions with MLL breakpoints in intron 9 or 11, respectively, into human cord blood-derived CD34[+] cells from the same donor. Compared to healthy control cells, all MLL-rearranged cells showed increased proliferation and stemness, as well as an altered immunophenotype characterized by the upregulation of leukemic markers. Transcriptomic profiling revealed breakpoint- and partner-specific gene expression patterns that influence the characteristics of the disease. Notably, even after prolonged in vitro culture MLL(intron 9)::AF9 cells displayed robust colony formation in semisolid media and engrafted robustly in NOD scid gamma mice. The cells still exhibited high lineage plasticity, switching from a myeloid to a B-lymphoid identity in vivo. In conclusion, this model enables the mechanistic dissection of MLL fusion variants in vitro and in vivo, providing a foundation for developing targeted therapies for MLL-rearranged leukemias.},
}
@article {pmid42001810,
year = {2026},
author = {Saleem, MS and Khan, SH and Rana, IA and Ahmad, A},
title = {CRISPR/Cas9-mediated editing of the GhJAZ2 gene improves fiber length and lint percentage in Gossypium hirsutum L.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2660546},
pmid = {42001810},
issn = {2164-5701},
mesh = {*Gossypium/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Cotton Fiber ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; },
abstract = {Cotton is regarded as a strategic agricultural commodity owing to its renewable and naturally derived fiber. With the escalating global demand for high-quality fiber, genetic improvement of fiber traits is a critical focus for sustaining and advancing the textile industry standards. The cotton GhJAZ2 gene encodes the Jasmonate ZIM-domain 2 protein, a known repressor in the jasmonic acid signaling pathway and negatively regulates fiber initiation. In this study, we designed a gRNA that simultaneously targets GhJAZ2 homologs and assembled it into the CRISPR vector (pHSE401). Subsequently, the construct (pHSE401-gRNA) was transformed into cotton (Gossypium hirsutum L.) using an Agrobacterium-mediated in planta transformation strategy, targeting the shoot apical meristem as the primary site of transformation. Sanger sequencing analysis revealed consistent single-base pair indels at the targeted site across both A and D sub-genomes, with edited T1 progenies showing both inherited and newly introduced indels at the targeted loci. Fiber analysis of edited lines compared to the control revealed a significant (p < .05) enhancement in lint percentage (≤13.74%) and fiber length (≤16.91%). This study demonstrated the effective application of CRISPR/Cas9 for targeted trait improvement in cotton, offering GhJAZ2-edited lines that can be advanced to develop transgene-free cultivars with improved fiber traits.},
}
@article {pmid42001897,
year = {2026},
author = {Li, Q and Cao, J and Deng, S and Yu, K},
title = {Precise Multi-Gene Editing Strategies for Xenotransplantation Donor Pigs: Overcoming Immune and Coagulation Barriers.},
journal = {Xenotransplantation},
volume = {33},
number = {2},
pages = {e70128},
doi = {10.1111/xen.70128},
pmid = {42001897},
issn = {1399-3089},
support = {32072722//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine ; Humans ; Graft Rejection/immunology ; CRISPR-Cas Systems/genetics ; *Blood Coagulation/genetics/immunology ; *Heterografts/immunology ; Tissue Donors ; Organ Transplantation ; },
abstract = {Organ transplantation is the preferred treatment for end-stage organ failure, but the severe shortage of donors severely restricts its clinical application. Xenotransplantation, especially using pigs as donors, is considered an ideal source of alternative donors due to the high similarity between their organ structures and those of humans. However, significant differences in immune recognition and coagulation regulation between species can easily induce a series of rejection reactions, including hyperacute rejection, acute humoral rejection, T-cell-mediated rejection, and chronic vascular complications. It also carries risks such as physiological metabolic incompatibility and potential viral transmission. In recent years, with the development of tools such as CRISPR/Cas, precise multi-gene editing technology has become possible, enabling the simultaneous knockout of multiple xenoantigen genes (such as GGTA1, CMAH, and B4GALNT2) and the introduction of human genes regulating complement, coagulation, and immune responses (such as hCD55, hTBM (THBD), and hCD47), significantly improving the immune tolerance and physiological compatibility of donor organs. This article systematically reviews the immune and coagulation barriers in xenotransplantation, focusing on precise multi-gene editing strategies for pigs used in xenotransplantation. It highlights editing pathways such as tandem knock-in at the same site, simultaneous multi-site editing, stepwise modular editing, and homology-directed repair (HDR) enrichment. Combined with representative organ-specific examples (heart, kidney, liver, and lung), including key non-human primate studies and early human exploratory cases where available, it explores the application prospects of these strategies in creating safe clinical-grade donor pigs and promoting the clinical translation of xenotransplantation.},
}
@article {pmid42002179,
year = {2026},
author = {Guha, S and Tharmatt, A and Yadav, S and Siwakoti, P and Kumeria, T and Mittal, A and Chitkara, D},
title = {Lipid-polymer hybrid nanoplex loaded microneedle patches as a corneal delivery platform for CRISPR/Cas expressing plasmid.},
journal = {International journal of biological macromolecules},
volume = {368},
number = {},
pages = {152092},
doi = {10.1016/j.ijbiomac.2026.152092},
pmid = {42002179},
issn = {1879-0003},
mesh = {Humans ; *Plasmids/genetics ; HEK293 Cells ; *Lipids/chemistry ; *Polymers/chemistry ; *CRISPR-Cas Systems/genetics ; Transfection ; *Cornea/metabolism ; Cell Survival ; Particle Size ; Microneedle Drug Delivery ; *Gene Transfer Techniques ; },
abstract = {Lipid-polymer hybrid (LPH) systems have evolved into a promising vehicle for delivering therapeutic agents. This study demonstrates cationic LPH nanoplexes composed of a biodegradable cationic mPEG-polycarbonate based cationic copolymer and cholesterol to enhance delivery efficiency of genetic materials. The optimized blank formulation, BNPX-3, exhibited a particle size of 93.60 nm, polydispersity index (PDI) of 0.116, and a zeta potential (ZP) of 21.9 mV. After complexation of pcDNA3-EGFP plasmid and CRISPRi plasmid at N/P ratios of 10 and 20, respectively, the nanoplexes exhibited particle sizes of 123.4 nm and 131.6 nm, with corresponding PDI values of 0.128 and 0.142 and zeta potentials of 12.2 mV and 10.5 mV, respectively. Cytocompatibility studies demonstrated >80% cell viability in HEK293 and SIRC cells over a wide concentration range. Transfection efficiencies using LPH nanoplexes for pcDNA3-EGFP (6159 bp) and CRISPRi plasmids (11,266 bp) were found to 60.09% and 48.02%, respectively, in HEK293 cells, and 56.04% and 40.34%, respectively, in SIRC cells. These efficiencies were comparable to Lipofectamine 3000 and superior to formulations prepared with cationic polymers alone. To enable efficient delivery of the nanoplexes, the developed nanoplexes were incorporated into dissolvable microneedle (MN) patches. The microneedle patch demonstrated successful loading of pDNA-complexed nanoplexes and efficient delivery ex-vivo.},
}
@article {pmid42002396,
year = {2026},
author = {Chen, L and Luo, J and Zhang, H and Zhao, P},
title = {RPA Combined With CRISPR/Cas12a for Rapid and Ultrasensitive Detection Dual-Gene of Methicillin-Resistant Staphylococcus aureus (MRSA).},
journal = {Journal of molecular recognition : JMR},
volume = {39},
number = {3},
pages = {e70035},
pmid = {42002396},
issn = {1099-1352},
support = {2025A1515010579//Natural Science Foundation of Guangdong Province, China/ ; 211102114530659//Shaoguan Municipal Science and Technology Program, China/ ; 220610154531525//Shaoguan Municipal Science and Technology Program, China/ ; 220525096180441//Shaoguan Engineering Research Center for Research and Development of Molecular and Cellular Technology in Rapid Diagnosis of Infectious Diseases and Cancer Program, China/ ; KEYANSHEN (2023) 01//Research Fund for Joint Laboratory for Digital and Precise Detection of Clinical Pathogens, Yuebei People's Hospital Affiliated to Shantou University Medical College, China/ ; RS202001//Research Project for Outstanding Scholar of Yuebei People's Hospital Affiliated to Shantou University Medical College, China/ ; 2023B110008//Research Project for Guangdong&#x2009;Provincial&#x2009;Clinical&#x2009;Research&#x2009;Center&#x2009;for&#x2009;Laboratory&#x2009;Medicine, China/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics ; Humans ; Rapid Diagnostic Tests ; Penicillin-Binding Proteins/genetics ; Sensitivity and Specificity ; *Staphylococcal Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The increasing issue of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) necessitates rapid and reliable diagnostic methods. While existing RPA-CRISPR/Cas12a platforms have demonstrated potential for MRSA detection, most rely on single-gene targets or require multiple Cas enzymes. Here, we have developed a novel dual gene detection strategy that simultaneously detects the S. aureus specific femA gene and the methicillin-resistant mecA gene in a single RPA-CRISPR/Cas12a reaction. This integrated approach enables clear discrimination between MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) in just 30 min, with results visualized via both fluorescence and lateral flow strips. The assay exhibited high specificity (no cross-reactivity with common pathogens) and a sensitivity of 10 copies/μL, comparable to qPCR. Validation with 39 clinical samples showed 100% concordance with antimicrobial susceptibility testing. Our dual-gene RPA-CRISPR/Cas12a platform represents a significant advancement in point-of-care MRSA diagnostics, offering enhanced accuracy and operational simplicity.},
}
@article {pmid42002643,
year = {2026},
author = {Marnet, K and Subramanian, H and Wiesler, M and Borst, A and Liedtke, D and Pattappa, G and Docheva, D and Nikolaev, VO and Stellzig-Eisenhauer, A and Eigenthaler, M and Herrmann, M},
title = {Live-cell imaging reveals decreased cAMP in a PFE-associated c.1050-3C&gt;G PTH1R cell model.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {104},
number = {1},
pages = {},
pmid = {42002643},
issn = {1432-1440},
support = {453600987//Deutsche Forschungsgemeinschaft/ ; D-361//Interdisziplin&#xe4;res Zentrum f&#xfc;r Klinische Forschung, Universit&#xe4;tsklinikum W&#xfc;rzburg/ ; CA22170//European Cooperation in Science and Technology/ ; },
mesh = {*Receptor, Parathyroid Hormone, Type 1/genetics/metabolism ; Humans ; *Cyclic AMP/metabolism ; Mutation ; Signal Transduction ; Fluorescence Resonance Energy Transfer ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Cell Line ; Periodontal Ligament/metabolism ; CRISPR-Cas Systems ; Phosphorylation ; },
abstract = {Primary failure of eruption (PFE) is a rare autosomal disorder provoked by heterozygous mutations in the parathyroid hormone receptor 1 (PTH1R) gene. PTH1R is a G protein-coupled receptor (GPCR) which regulates intracellular signaling molecules like cAMP. By using CRISPR/Cas9, the pathogenic PTH1R variant, c.1050-3C>G, was introduced into the periodontal ligament (PDL-hTERT) cell line to investigate molecular mechanisms in a PFE in vitro model. The PDL-hTERT immortal cell line, derived from human primary PDL cells, is a well-established model for dental diseases and expresses PTH1R. We performed different functional assays to compare the behavior of the PDL-hTERT WT versus PTH1R-mutated cells. cAMP synthesis and PKA activation were compared between different cell lines by live-cell imaging using Förster Resonance Energy Transfer (FRET)-based biosensors. Phosphorylation of VASP was measured to validate and compare the PKA activation between the cell lines. In summary, our experiments show that the mutated cell line has no major phenotypic changes, but the PTH1R downstream signaling cascade is impaired. KEY MESSAGES: A rare autosomal disorder linked to mutations in the PTH1R gene, which encodes a G protein-coupled receptor regulating intracellular signaling, including cAMP production. The pathogenic PTH1R mutation (c.1050-3C>G) was introduced into a periodontal ligament (PDL hTERT) cell line to model PFE and study molecular mechanisms in vitro. Functional assay revealed that while the mutated cell line displayed no major phenotypic changes, the PTH1R downstream signaling cascade, including cAMP synthesis and PKA activation, was disrupted. Techniques like FRET-based biosensors and VASP phosphorylation assays highlighted specific impairments in signaling pathways in cells with the PTH1R mutations.},
}
@article {pmid42002657,
year = {2026},
author = {Sichani, AS and Hassani, M and Gila, F and Shafieipour, N and Dabbaghipour, R and Heidari, Z and Sisakht, M and Hassani, M and Fallahi, J},
title = {A Comprehensive Review on CRISPR-Based Screening and Its Applications.},
journal = {Molecular biotechnology},
volume = {68},
number = {7},
pages = {3051-3067},
pmid = {42002657},
issn = {1559-0305},
support = {43016329//Shahid Beheshti University of Medical Sciences/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-based tools have quickly moved from specialist techniques to routine instruments in biology and medicine, and they are now central to large-scale loss-of-function and perturbation screens. In this review, we focus on how pooled CRISPR screens are used to interrogate gene function in living cells, most often through cell fitness or simple selectable markers, and contrast this with arrayed formats that trade throughput for richer molecular readouts, such as transcriptome-wide changes. We bring together current strategies for library design, delivery, and selection and show how different Cas nucleases, including Cas9, Cas12, and Cas13, broaden the range of genome and transcriptome perturbations that can be assayed. We then discuss recent applications in drug response, viral infection, and cancer biology and consider how improvements in high-content technologies, data analysis, and emerging diagnostic uses are likely to shape the next generation of CRISPR-based screening studies.},
}
@article {pmid42003550,
year = {2026},
author = {Marsic, T and Gundra, SR and Aouida, M and Masood, M and Salibi, A and Schmidt, F and Alquwayzani, R and Mahfouz, MM},
title = {Precise, specific gene editing via a compact GoCas12m-FokI chimeric nuclease.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
pmid = {42003550},
issn = {1362-4962},
support = {1/1035-01-01//BAS/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Deoxyribonucleases, Type II Site-Specific/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; },
abstract = {CRISPR gene editing technologies have transformed functional genomics and biotechnology. Despite these advances, challenges such as limited delivery capacity and off-target activity continue to hinder their therapeutic translation. We developed a chimeric gene editing platform by fusing the compact, catalytically inactive Cas12m guiding module (GoCas12m) with the FokI nuclease domain. GoCas12m-FokI system integrates the programmable DNA-binding capability of Cas12m with the dimerization-dependent cleavage mechanism of FokI, enabling precise genome editing. Our engineered XTEN-fused GoCas12m-FokI editor exhibits robust activity on both surrogate reporters and endogenous human loci, achieving high-efficiency editing at clinically relevant targets-including CLTA1, HBB, AIFM1, and ABL with no detectable off-target activity at in silico-predicted sites, as confirmed by targeted deep sequencing. Notably, GoCas12m-FokI is nearly half the size of conventional Cas9- or Cas12a-based editors, facilitating delivery via adeno-associated virus and other cargo-limited vectors. This dual-guided editor showed comparable editing efficiency to previously reported FokI-dCas9 systems on endogenous loci, while possessing a different PAM requirement and domain orientation. By combining compact architecture, high specificity, and modular programmability, the GoCas12m-FokI editor offers a powerful alternative for therapeutic genome editing and a promising tool for in vivo gene therapy applications.},
}
@article {pmid42003551,
year = {2026},
author = {Otten, C and Kutnjak, M and Supina-Pavic, C and Pranjic, M and Anticevic, I and Medved, V and Popovic, M},
title = {ACRC/GCNA is an essential protease that repairs DNA-protein crosslinks during vertebrate development.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
pmid = {42003551},
issn = {1362-4962},
support = {UIP-2017-05-5258//Croatian Science Foundation Installation Grant/ ; IPS-2020-01-4225//Slovenian-Croatian Bilateral Research Project grant/ ; KK.01.1.1.01.0003//European Structural and Investment Funds STIM - REI project/ ; HRZZ-IP-2024-05-9425//Croatian Science Foundation/ ; //NextGenerationEU/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *DNA Repair ; DNA/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; DNA Damage ; },
abstract = {DNA-protein crosslinks (DPCs) are toxic DNA lesions that block all DNA transactions including replication and transcription, and the consequences of impaired DNA-protein crosslink repair (DPCR) are severe. At the cellular level, impaired DPCR leads to the formation of double strand breaks, genomic instability, and cell death, while at the organismal level, it is associated with cancer, aging, and neurodegeneration. Despite its importance, the mechanisms of DPCR at the organismal level are largely unknown. Proteases play a central role in DPCR, as they remove proteinaceous part of the DPCs, while the peptide remnant crosslinked to DNA is subsequently removed by other repair factors. We characterized the role of putative protease ACRC/GCNA (ACidic Repeat Containing/Germ Cell Nuclear Antigen) in DPCR at the organismal level. For this purpose, we have created new animal models with CRISPR/Cas system: two zebrafish lines with inactive Acrc. We were able to overcome the early embryonic lethality caused by Acrc inactivation by injecting Acrc-WT messenger RNA and have created a viable animal model to study the role of Acrc in adult tissues. We identified histone H3, topoisomerases 1 and 2, Dnmt1, Parp1, Polr3a, and Mcm2 as putative DPC substrates of Acrc. We have shown that Acrc is essential for vertebrate development, and that the mechanism behind it is DPC removal.},
}
@article {pmid42003552,
year = {2026},
author = {White, N and Hu, YT and Chalk, JA and Kurgan, G and Naseem, A and Schmaljohn, E and Sturgeon, M and Cavazza, A and Thrasher, AJ and Turchiano, G},
title = {DNA-PKcs inhibitor AZD7648 reveals sgRNA cross-contaminants and enhanced sensitivity of genome engineering off-target activity in HSPCs.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
pmid = {42003552},
issn = {1362-4962},
support = {217112/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; 1160024//Great Ormond Street Hospital Children's Charity/ ; //Curing Rare Inherited Diseases Using Innovative Gene Therapies/ ; //European Union's Horizon 2020/ ; },
mesh = {Humans ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Pyrazoles/pharmacology ; DNA End-Joining Repair/drug effects ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Therapeutic gene editing with designer nucleases can be compromised by undesired repair outcomes. DNA repair inhibitors are used to bias DSB repair toward HDR, but their impact on larger structural rearrangements, including large deletions and translocations, remains unclear. We quantify the mutational burden associated with end-joining inhibitor compounds. With a highly precise Cas9 nuclease, repair inhibition yields modest increases in aberrations, whereas promiscuous single guide RNAs (sgRNAs) amplify aberrant outcomes by orders of magnitude. Donor templates mitigate mutational burden at on-target sites, and in rare cases donor sequences bridge translocations between on- and off-target loci. Because DNA-PKcs inhibition does not itself induce instability over short intervals but increases the likelihood of capturing chromosomal aberrations postediting, we leveraged this to enhance assay performance. Compared to CAST-Seq, high-resolution CAST-Seq achieved a median ~12-fold increase in detected aberrations and, in this higher-sensitivity context, revealed unintended, target-specific sgRNA contaminants in GMP-like batches, underscoring direct genotoxicity risk and the need for stricter guide purity controls. A modified, translocation-quantitative rhAmpSeq reports all translocation combinations between two loci, enabling robust off-target validation beyond indel-only readouts. Finally, we evaluate AZD7648, finding limited aberration increases with precise nucleases and reconciling reports of extensive large deletions by quantifying assay- and design-dependent biases.},
}
@article {pmid42003616,
year = {2026},
author = {Theriault, ME and Wong, AI and DeJesus, MA and Pisu, D and Nae Rin Lee, B and Kirukubar, G and Li, S and Wallach, JB and Schnappinger, D and Lê-Bury, G and Russell, DG and Rock, JM},
title = {Utilization of a CRISPRi-based ex vivo challenge model to reveal temporally dependent gene essentiality in intracellular Mycobacterium tuberculosis.},
journal = {mBio},
volume = {17},
number = {5},
pages = {e0061026},
pmid = {42003616},
issn = {2150-7511},
support = {R01 AI155319/AI/NIAID NIH HHS/United States ; AI155319//National Institute of Allergy and Infectious Diseases/ ; AI162598//National Institute of Allergy and Infectious Diseases/ ; INV-055894//Bill and Melinda Gates Foundation/ ; //Mueller Health Foundation/ ; //Rita Allen Foundation/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/growth &amp; development ; Animals ; Mice ; Macrophages/microbiology ; *Genes, Essential ; *CRISPR-Cas Systems ; *Tuberculosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Inbred C57BL ; Lung/microbiology ; Disease Models, Animal ; },
abstract = {UNLABELLED: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-embB, fadE29, and mbtI-confirmed their temporal significance in vivo. This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality in vivo.<br><br>IMPORTANCE: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. This novel approach enabled us to identify how different bacterial metabolic pathways were of greater importance to the bacterium at different time points post-infection. The results provide insights into how the evolving immune response to infection shapes the metabolic and replicative status of the bacterium. This information has significance in the design of therapeutic strategies toward cure.},
}
@article {pmid42003707,
year = {2026},
author = {Wu, Z and Jin, F and Zhu, W and Zhong, W and Qi, T and Luo, S and Liu, Q and Cai, Z and Dai, C and Chai, Z and He, Y and Rui, Y and Miao, Y and Zheng, L and Fu, Q},
title = {Photoactivated Digital Recombinase Polymerase Amplification/CRISPR-Cas12a Assay for Point-of-Care of BK Polyomavirus Quantification.},
journal = {ACS nano},
volume = {20},
number = {17},
pages = {13301-13313},
doi = {10.1021/acsnano.6c02651},
pmid = {42003707},
issn = {1936-086X},
mesh = {*BK Virus/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; DNA, Viral/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BK polyomavirus (BKV) serves as a critical biomarker for optimizing immunosuppressive therapy and preventing graft failure in kidney transplant recipients. Quantitative PCR (qPCR), the current gold standard for BKV load quantification, relies on batch-specific standard curves. For kidney transplant recipients, this not only elevates the risk of cross-contamination but also entails considerable economic burdens. Therefore, developing BKV quantification technologies independent of batch-specific standard curves is of great clinical significance for this patient population. The combination of CRISPR-Cas12a with recombinase polymerase amplification (RPA), termed DETECTR, offers notable advantages for digital nucleic acid analysis. However, due to the high viscosity of RPA reagents, the generation of high-throughput, uniform RPA microdroplets remains a significant technical challenge. In the present study, we developed a centrifugal RPA microdroplet generation method based on commercial capillaries, facilitating the production of high-throughput, uniform RPA microdroplets (23.1 μm in diameter) via simple centrifugation. Furthermore, by integrating a light-controlled RPA-CRISPR-Cas12a system, we established photoactivated digital DETECTR (pd-DETECTR) for precise, point-of-care, and cost-effective BKV quantification. When combined with a smartphone-based reader, the pd-DETECTR assay can be completed within 42 min. Clinical validation demonstrated a strong correlation (R[2] = 0.9801) with qPCR results, exhibiting high sensitivity (100.0%), specificity (98.0%), and accuracy (99.0%). The pd-DETECTR provides a rapid, convenient, and cost-effective tool for BKV load analysis, which can significantly reduce the economic burden and risk of opportunistic infections in kidney transplant recipients, thus holding significant clinical value.},
}
@article {pmid42007994,
year = {2026},
author = {Tiwari, P and Rathinasabapathi, P},
title = {Programmable CRISPR-Cas diagnostic platforms for rapid detection of uropathogens and antimicrobial resistance.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42007994},
issn = {1432-072X},
abstract = {Urinary tract infections (UTIs) represent a global burden, impacting more than 150 million individuals annually, and are a major contributor to the antimicrobial resistance (AMR) crisis because of the rampant empirical use of antibiotics. Although microbial culture remains the gold standard of diagnosis, its long turnaround time (48–72 h) limits timely, actionable decision-making for effective antimicrobial stewardship. This review is a critical analysis of the development of CRISPR-Cas biosensing as a better analytical platform to detect uropathogens in a rapid and point-of-care (POC) format. The diagnostic mechanisms of Class II Cas effectors (Cas12, Cas13, and Cas14) are discussed in detail, including programmable collateral trans-cleavage activity that allows programmable attomolar sensitivity and single-nucleotide specificity in the identification of specific resistance markers. New engineering advancements that have brought these assays from the bench to the bedside are heavily emphasized, particularly the integration of isothermal pre-amplification techniques (LAMP/RPA) into streamlined one-pot systems that lower the risk of contamination. Additionally, the review assesses the advancement of instrument-free formats, e.g., lyophilized reagents, paper-based microfluidics, and smartphone-based colorimetric readouts that would be suitable in resource-constrained environments. Lastly, the manuscript discusses important analytical issues, including sample preparation in complicated urine samples and multiplexing. It concludes that next-generation CRISPR diagnostics, when satisfying the requirements of the World Health Organization in its category of REASSURED, can fill the gap between molecular accuracy and decentralized patient care, and this will provide a powerful answer to the global AMR threat.},
}
@article {pmid42007998,
year = {2026},
author = {Sharma, D and Khan, M and Khan, JA},
title = {Genome editing‑based strategies to combat geminiviruses: CRISPR/Cas9 and emerging high‑fidelity tools.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42007998},
issn = {1432-072X},
abstract = {Plant virus diseases constitute a major constraint to agriculture and have adversely affected crop productivity worldwide. Over the past few decades, geminiviruses of the family Geminiviridae have emerged as some of the most destructive plant pathogens. Conventional. approaches for virus management have demonstrated varying degrees of success, their deployment is often hindered by prolonged timelines, limited availability of resistant sources, and reduced durability against rapidly evolving geminiviral pathogens. With the rapid evolution of viruses and the extensive damage they cause, it is imperative to develop rapid, cost-effective, and efficient antivirus resistance strategies. In recent years, CRISPR/Cas-based genome editing has made it possible to precisely target viral genomes, offering new avenues for developing antiviral resistance, although long-term durability remains under investigation. Derived from prokaryotic adaptive immune systems, the CRISPR/Cas platform confers resistance either by expressing genome-editing constructs that directly cleave viral genomes or by modifying host susceptibility (S) genes, representing mechanistically distinct strategies with different implications for durability of resistance. Recent advances have further expanded this toolkit with Cas-CLOVER, a dual guide RNA-dependent nuclease system designed to mitigate off-target effects associated with conventional Cas9. This review summarizes the principles of CRISPR/Cas-mediated antiviral resistance, critically evaluates viral escape, resistance durability, compares Cas9 with emerging platforms, and outlines conceptual frameworks for developing durable and field-ready genome-editing strategies.},
}
@article {pmid42008182,
year = {2026},
author = {Azhar, F and Mazhari, BBZ and Ibrahim, MN and Alanazi, A and Islam, F},
title = {CRISPR-based tools in food safety and microbiology: applications for pathogen detection and control.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42008182},
issn = {1432-072X},
abstract = {Foodborne diseases have continued to pose serious health and economic challenges worldwide, especially in low- and middle-income countries with weak food safety systems. The traditional forms of detection are effective but time-consuming and laborious, and may not be suitable for quick on-site use. In this regard, CRISPR-Cas technology has become a revolutionary technology in food safety because it is highly specific, sensitive and programmable. This is a review of CRISPR-based applications in food microbiology, with an emphasis on rapid pathogen detection methods and targeted microbial control. Recent diagnostic advances in CRISPR-based diagnostics, such as the addition of isothermal amplification, lateral flow assays, and biosensing platforms, have demonstrated fast, portable, and cost-effective methods for detecting foodborne pathogens. Also, CRISPR-mediated antimicrobial interventions, including programmed bacteriophages and gene targeting, provide effective control of pathogens and antimicrobial resistance. Notably, this review also discusses the comparative advantages of various Cas systems (Cas9, Cas12, Cas13), their industrial potential, and future opportunities to integrate them with smart technologies (artificial intelligence and IoT-based monitoring systems). In summary, CRISPR technologies are a next-generation approach to real-time, accurate, and sustainable food quality management. They are well-positioned to operate at an industrial scale, despite current regulatory and standardisation issues.},
}
@article {pmid42008698,
year = {2026},
author = {Wang, H and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {CRISPR/dCas9-Assisted On-Bead Multiplex Detection (BeadPlex2) for Genetically Modified Crops.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12586-12595},
doi = {10.1021/acs.analchem.5c08192},
pmid = {42008698},
issn = {1520-6882},
mesh = {*Plants, Genetically Modified/genetics ; Spectrum Analysis, Raman ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; DNA/genetics/analysis ; Glycine max/genetics ; Zea mays/genetics ; },
abstract = {This study leverages the precise recognition ability of CRISPR/dCas9 and the Raman coding feature of the gap-enhanced Raman tag-encoded magnetic beads (MagGERTs) to create a unique on-bead nucleic acid detection platform (BeadPlex2) for accurate and multiplex nucleic acid detection, which was proven to be applicable for the identification of diverse genetically modified (GM) events. Five distinct MagGERTs (MB@Au[Ra]) encoded with different Raman reporters (Ras) were constructed, followed by the conjugation of dCas9/single guide RNA (sgRNA) complexes in which the sgRNAs were explicitly designed for different target genes of GM events. These coding units could recognize and capture target double-stranded nucleic acid (dsDNA) sequences by the dCas9/sgRNA complexes. Then, SYBR Green I was applied to highlight positive beads by binding to target dsDNA due to its fluorescent emission under an imaging system. Decoding Raman signals from the Ras of the MagGERTs achieved the high-specific identification of GM events. Our BeadPlex2 platform has been demonstrated to be applicable for detecting GM maize and soybean seeds with high accuracy comparable to qPCR. This platform opens a new way to detect multiple target nucleic acids simultaneously and offers a powerful strategy for identifying genetically modified organisms.},
}
@article {pmid42009546,
year = {2026},
author = {Xu, T and Zhao, L and Dai, Y and Duan, J and Wang, Y and Shao, L and Chen, D and Zhu, L and Xu, Z},
title = {[A rapid visual detection method for porcine circovirus type 4 based on enzymatic recombinase amplification and CRISPR/EsCas13d].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {4},
pages = {1755-1768},
doi = {10.13345/j.cjb.250568},
pmid = {42009546},
issn = {1872-2075},
support = {2024YFD1800500 and 2024YFD1800102//the National Key Research and Development Program of China/ ; sccxtd-2024-08 and sccxtd-2024-18//the National Modern Agricultural Industrial Technology System/ ; },
mesh = {*Circovirus/isolation &amp; purification/genetics ; Animals ; Swine ; *Recombinases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Circoviridae Infections/diagnosis/veterinary/virology ; *Swine Diseases/virology/diagnosis ; },
abstract = {The emerging outbreaks of infectious diseases in humans and animals worldwide pose serious threats to public health and cause substantial economic losses. This has raised the demand for more efficient and sensitive diagnostic methods to strengthen disease surveillance and early warning. In this study, we developed a portable visual platform based on ERA-CRISPR/EsCas13d for the rapid detection of porcine circovirus type 4 under resource-limited conditions. We optimized the platform by integrating enzymatic recombinase amplification (ERA), T7 transcription, and CRISPR/EsCas13d cleavage in a single-tube reaction, thereby simplifying the workflow and shortening the total detection time to 30 min. In addition, a rapid nucleic acid release method was employed, eliminating the need for laboratory-based extraction and complex heating steps, which further improved the simplicity and usability. In addition, lyophilized reagents were modified to enhance stability, thus reducing cold-chain and storage requirements and enabling cost-effective transport and field deployment. The method achieved a visual limit of detection of 50 cp/μL for porcine circovirus type 4 and demonstrated no cross-reactivity with six other common swine pathogens. Moreover, the platform supported two visual readout formats-UV light (470 nm) and lateral flow assay (LFA)-providing flexible options for interpretation in different application scenarios. Validation with 60 clinical samples showed 100% concordance with quantitative PCR results, highlighting the practical utility and application potential of the ERA-CRISPR/EsCas13d-based portable visual platform for rapid on-site diagnostics.},
}
@article {pmid42009554,
year = {2026},
author = {Li, Y and Wen, D and Zhou, Z and Liao, C and Zhao, Q and Shuai, J and Zhang, X and Yu, X and Huang, J},
title = {[A CRISPR-Cas13a-based amplification-free electrochemical biosensor for rapid detection of bovine viral diarrhea virus].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {4},
pages = {1868-1880},
doi = {10.13345/j.cjb.250329},
pmid = {42009554},
issn = {1872-2075},
support = {2021YFF0600805 and 2021YFF0602801//the National Key Research and Development Program of China/ ; 2024SNJF044//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 20241203A23//the Key Research Program in the Field of Agriculture and Social Development of Hangzhou/ ; },
mesh = {*Biosensing Techniques/methods ; Cattle ; Animals ; *CRISPR-Cas Systems/genetics ; *Diarrhea Viruses, Bovine Viral/isolation &amp; purification/genetics ; *Electrochemical Techniques/methods ; Nucleic Acid Amplification Techniques ; Bovine Virus Diarrhea-Mucosal Disease/diagnosis/virology ; },
abstract = {Bovine viral diarrhea virus (BVDV), a major pathogen in the global bovine industry, causes diarrhea, fever, and reproductive disorders, leading to substantial economic losses. Developing the methods for rapid and accurate detection of BVDV is crucial for epidemic control. Current detection methods have notable limitations. PCR-based nucleic acid amplification techniques rely on sophisticated instruments and complex procedures. CRISPR-Cas13a systems, despite their high specificity, still require nucleic acid pre-amplification, which results in cumbersome workflows and contamination risks. To establish a simpler and more efficient on-site detection method for BVDV, this study integrated the CRISPR-Cas13a system characterized by specific recognition with electrochemical sensing praised for efficient signal transduction to establish a novel nucleic acid amplification-free method for the detection of BVDV. Through optimization of key parameters, including CRISPR RNA (crRNA) combination, buffer components, and Cas13a/crRNA concentration ratio, the biosensor achieved a detection limit of 3 090 copies/μL-representing a 4-5 order of magnitude improvement in sensitivity compared with conventional Cas13a fluorescence-based detection-and completed the entire process from sample loading to result output within 35 min. Specificity tests demonstrated that the sensor exclusively detected BVDV without cross-reactivity to other common bovine viruses (bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bluetongue virus, and foot-and-mouth disease virus). Clinical validation with 22 samples demonstrated 100% specificity and sensitivity. The developed CRISPR-Cas13a-based electrochemical biosensor offers the advantages of being nucleic acid amplification-free and operationally simple, serving as a powerful new tool for rapid on-site BVDV detection with significant potential for veterinary diagnostics and epidemic prevention and control.},
}
@article {pmid42009663,
year = {2026},
author = {Batty, P and Beneder, H and Schätz, C and Onea, G and Zaczek, M and Kutschat, AP and Abele, M and Müller, S and Superti-Furga, G and Winter, GE and Seruggia, D},
title = {Disruption of the SAGA CORE triggers collateral degradation of KAT2A.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42009663},
issn = {2041-1723},
support = {947803//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 10.55776/P36302//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; },
mesh = {*Histone Acetyltransferases/metabolism/genetics ; Humans ; Proteolysis ; Proteasome Endopeptidase Complex/metabolism ; Acetylation ; Histones/metabolism ; Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Proteomics ; *Trans-Activators/metabolism/genetics ; CRISPR-Cas Systems ; TATA-Binding Protein Associated Factors/metabolism/genetics ; p300-CBP-Associated Factor ; },
abstract = {The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex regulates gene expression through histone acetylation at promoters, mediated by its histone acetyl transferase (HAT), KAT2A. While SAGA structure and function are well characterised, mechanisms controlling the stability of individual subunits, including KAT2A, remain unclear. Here, using a fluorescence-based KAT2A stability reporter, we systematically dissect the molecular dependencies controlling KAT2A protein abundance, and identify the non-enzymatic SAGA CORE module subunits-TADA1, TAF5L, and TAF6L- as necessary for KAT2A stability. Loss of these subunits disrupts SAGA complex integrity, leading to non-chromatin-bound KAT2A that is degraded by the proteasome and consequent reduced H3K9 acetylation. Proteomic profiling reveals progressive loss of components from the CORE and HAT modules upon acute SAGA CORE disruption, indicating that an intact CORE is required for the stability of numerous SAGA components. Finally, a focused CRISPR screen of ubiquitin-proteasome system genes identifies the E3 ligase UBR5, a known regulator of orphan protein degradation, and the deubiquitinase OTUD5, as regulators of KAT2A degradation when the SAGA CORE is perturbed. Together, these findings reveal a dependency of KAT2A protein stability on SAGA CORE integrity and define an orphan quality control mechanism targeting unassembled KAT2A, revealing a potential vulnerability in SAGA-driven malignancies.},
}
@article {pmid42009664,
year = {2026},
author = {Corazzi, L and Ing, A and Benito, E and Cosenza, MR and Hasenfeld, P and Weber, T and Marx, AJM and Ionasz, VS and Trausch, N and Benedetto, S and Di Muzio, G and Ding, B and Berlanda, J and Giaisi, M and Claudino, N and Höfer, T and Korbel, JO and Wei, PC},
title = {Recurrent DNA break clusters drive replication-stress-induced copy number variants and genome diversification.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42009664},
issn = {2041-1723},
support = {949990//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 101098056//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; YIP-DKFZ//Helmholtz Association/ ; },
mesh = {*DNA Copy Number Variations/genetics ; Animals ; *DNA Replication/genetics ; Mice ; Neural Stem Cells/metabolism ; *DNA Breaks ; *Genome ; CRISPR-Cas Systems ; Whole Genome Sequencing ; Single-Cell Analysis ; DNA End-Joining Repair/genetics ; Humans ; DNA Repair ; },
abstract = {Copy number variants (CNVs) are strongly implicated in neurological and psychiatric disorders and brain cancer, yet the process by which replication stress generates CNVs-and why some recur while others remain rare-remains poorly understood. Here, we show that recurrent DNA-break clusters (RDCs) act as common initiating lesions that drive both recurrent and non-recurrent CNVs. In murine neural progenitor cells subjected to chemically induced replication stress, bulk whole-genome sequencing identifies recurrent CNVs enriched at late-replicating RDCs within actively transcribed genes. Single-cell genome sequencing further uncovers frequent, non-recurrent CNVs associated with RDCs that arise during the transition from early to late DNA replication. These CNVs represent stable, heritable structural variants with breakpoints consistently enriched at RDCs. CRISPR/Cas9-mediated transcriptional suppression abolishes both RDC formation and CNV generation, establishing RDC-associated breaks as a shared upstream source. Mechanistically, CNV formation depends on DNA repair context: CNVs are Pol θ-dependent in NHEJ-deficient cells but arise independently of Pol θ in NHEJ-proficient cells. Together, these findings define RDCs as central drivers of replication-stress-induced genome diversification.},
}
@article {pmid42010284,
year = {2026},
author = {Zhao, J and Zhang, J and Gao, M and Miao, Z and Zhang, Y and Guo, Y and Fan, Z and Tian, J and Yang, L and Jiang, N and Ma, J and Jiao, J and Pan, J and Ma, X},
title = {Photoactivatable CRISPR/Cas13d via upconversion nanoparticles for deep tissue RNA engineering and orthopedic therapy.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42010284},
issn = {2041-1723},
support = {82572860//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82102639//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302347//National Natural Science Foundation of China (National Science Foundation of China)/ ; 25JCYBJC01510, 22JCQNJC00850//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 25YDTPJC00330//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Animals ; Mice ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; *Tissue Engineering/methods ; Polyethyleneimine/chemistry ; *RNA/genetics ; Blue Light ; Bone and Bones ; Osteocytes/metabolism ; },
abstract = {Spatiotemporal control of RNA therapeutics remains a fundamental challenge limiting clinical translation. Here, we develop a photoactivatable CRISPR/Cas13d (paCas13d) system that enables non-invasive, light-controlled RNA manipulation in deep tissues. Through structure-guided engineering, we identify optimal split sites within RfxCas13d and create light-switchable fragments using CRY2PHR/CIBN optogenetic dimerization. To overcome the limited tissue penetration of blue light, we engineer polyethylenimine-functionalized upconversion nanoparticles (UCNPs-PEI) that serve dual roles as gene carriers and photon transducers, converting tissue-penetrating near-infrared (NIR) to blue light. The UCNPs-PEI@paCas13d system achieves precise spatiotemporal control of RNA targeting within bone tissue in vivo. In a murine steroid-associated osteonecrosis model, NIR-activated paCas13d achieves robust TET3 knockdown, disrupting the TET3-5hmC-PTEN axis that drives glucocorticoid-induced osteocyte apoptosis. This targeted intervention prevents bone deterioration, with treated mice showing preserved trabecular architecture, enhanced bone volume, and favorable shifts in bone turnover markers, while maintaining systemic glucocorticoid efficacy. Our platform combines the programmability of CRISPR/Cas13d with non-invasive optical control, offering a versatile approach for treating diseases requiring localized RNA modulation while minimizing systemic effects.},
}
@article {pmid42010707,
year = {2026},
author = {Milenkovic, A and Weber, BHF},
title = {Allele-specific suppression of pathogenic bestrophin-1 transcripts by CRISPR/Cas9-mediated genome editing.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42010707},
issn = {1756-994X},
mesh = {Humans ; *Bestrophins/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Alleles ; Induced Pluripotent Stem Cells/metabolism ; Retinal Pigment Epithelium/metabolism/cytology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; *Corneal Dystrophies, Hereditary/genetics/therapy ; },
abstract = {BACKGROUND: Treating autosomal dominant gene mutations remains challenging, particularly when mutations convey a gain-of-function or a dominant-negative effect, as standard gene supplementation strategies often fail to counteract the pathogenic allele.<br><br>METHODS: In this study, we employed human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) to investigate allele-specific CRISPR/Cas9 genome editing as a potential treatment for Best disease (BD), an autosomal dominant macular dystrophy caused by over 250 distinct mutations in the bestrophin-1 (BEST1) gene. We designed and evaluated single guide RNAs (sgRNA) targeting three known BEST1 mutations (p.(R218C), p.(A243V), and p.(I295del)), assessing their impact on BD-associated hiPSC-RPE phenotypes and BEST1 channel function. Computationally predicted sgRNAs were rigorously tested for on-target efficiency, allele specificity and genome-wide off-target activities.<br><br>RESULTS: We found that shortening sgRNA length improved specificity in some cases, while introducing an additional mismatch generally compromised editing efficiency. Notably, only one of the three mutations yielded an sgRNA with both high cleavage efficiency and undetectable off-target effects in hiPSC-RPE cells. We then explored the consequences of allele-specific editing on BEST1 expression and function in clonal BD hiPSC-RPE lines. Eliminating the mutant BEST1 transcript led to enhanced BEST1 localization, improved protein stability and restoration of anion transport function.<br><br>CONCLUSIONS: Taken together, our findings support allele-specific gene editing as a viable therapeutic strategy for selected BEST1 mutations, while underscoring the necessity for rigorous testing of computationally designed sgRNAs, given their mutation- and context-dependent variability.},
}
@article {pmid42011754,
year = {2026},
author = {Yang, G and Fang, Y and Liu, Y and Deng, Y and Nie, L and Li, Z and Li, S and Chen, Z and Su, E and Zai, Y and Umar Siddiqui, AM and He, N},
title = {Rapid and Specific Detection of Gastric Cancer EVs Using a Cas12a-Powered Aptasensor with a Novel Targeting Aptamer.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12529-12541},
doi = {10.1021/acs.analchem.5c08039},
pmid = {42011754},
issn = {1520-6882},
mesh = {*Stomach Neoplasms/diagnosis/pathology/metabolism ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Extracellular Vesicles/metabolism/chemistry ; SELEX Aptamer Technique ; *CRISPR-Associated Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; Cell Line, Tumor ; CRISPR-Cas Systems ; Biomarkers, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Gastric cancer remains a predominant contributor to global cancer-related mortality, characterized by a pronounced disparity in five-year survival rates between early stage (>90%) and advanced-stage (<30%) disease. This disparity underscores the urgent necessity for accessible early detection methods. Present diagnostic approaches, such as serum biomarkers and endoscopy, either lack adequate sensitivity or are invasive. Extracellular vesicles (EVs) represent promising biomarkers for liquid biopsy; however, a major limitation is the lack of probes that can specifically identify EVs derived from gastric cancer, as most existing markers are broad-spectrum and exhibit low specificity. To address this limitation, we isolated high-purity EVs from gastric cancer cells and utilized a combined immunomagnetic bead-based SELEX strategy to identify a novel aptamer, H-EV-4-1, which demonstrates high affinity (Kd = 13.32 ± 2.69 nM) and specificity for gastric cancer EVs. Subsequently, this aptamer was incorporated into a CRISPR-Cas12a-based biosensor. The aptamer was hybridized with a biotinylated oligonucleotide (H1-biotin) and immobilized on magnetic beads. Upon binding of the target EVs, the aptamer was displaced, thereby exposing H1-biotin to activate the Cas12a/crRNA complex. This activation induced the trans-cleavage of a fluorescent reporter, producing a quantifiable signal. This aptasensor facilitates the rapid, highly sensitive, and specific detection of gastric cancer EVs, presenting a promising platform for the development of noninvasive, point-of-care early diagnostic tools.},
}
@article {pmid42011779,
year = {2026},
author = {Biczók, Z and Krausz, SL and Simon, DA and Tóth, E and Varga, &#xc9; and Annus, T and Huba, F and Varga, M and Bakos, &#xc9; and Fodor, E and Welker, E},
title = {Disrupting pegRNA intramolecular complementarity via PBS and spacer sequence alterations can enhance prime editing efficiency.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
pmid = {42011779},
issn = {1362-4962},
support = {K134968//Hungarian Scientific Research Fund/ ; K142322//Hungarian Scientific Research Fund/ ; RRF-2.3.1-21-2022-00015//PharmaLab/ ; ELKH-PoC-2023//National Research, Development, and Innovation Office of Hungary/ ; //Hungarian Research Network/ ; //Ministry of National Economy/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; CRISPR-Cas Systems ; Binding Sites ; Plasmids/genetics ; Base Sequence ; Humans ; Base Pair Mismatch ; },
abstract = {The length and sequence of the primer binding site (PBS) are critical for efficient prime editing, and its intramolecular complementarity with the prime editing guide RNA (pegRNA) spacer is a major drawback. We investigated the effects of these factors by literature analyses and by testing over 300 modified pegRNAs with weakened PBS-spacer interactions. It has been suggested that the effective PBS length for plasmid-delivered pegRNAs without end protection is considerably longer than what efficient priming requires due to exonuclease digestion of the PBS ends; however, analysing literature data of over 3000 pegRNAs revealed no significant shift in the optimal PBS length for epegRNAs compared to conventional pegRNAs. We also found improvement in editing efficiency with up to seven-fold when mismatches were introduced in the spacer or PBS sequence disrupting complementarity, although this effect is more pronounced with non-optimal PBS lengths. A combination of spacer mismatches and PBS deletions led to further editing improvements, even compared to the optimal PBS, although finding the best combination requires extensive optimization. Here, we achieved near-optimal editing efficiency in the majority of cases without the need for prior pegRNA optimization by using SPELL (Streamlined Prime Editing with fixed-Length PBS Leverage), a prime editing approach that employs a 17-20 nucleotide-long PBS with a single nucleotide deletion.},
}
@article {pmid42013835,
year = {2026},
author = {Lontuo-Fogang, R and Bennuru, S and Nutman, TB},
title = {Development of Recombinase Polymerase Amplification and CRISPR-Cas12a-Enhanced Isothermal Amplification Assays for Strongyloides stercoralis DNA Detection: A Pilot Study.},
journal = {The American journal of tropical medicine and hygiene},
volume = {114},
number = {6},
pages = {1157-1164},
pmid = {42013835},
issn = {1476-1645},
mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA, Helminth/genetics/isolation &amp; purification ; Feces/parasitology ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Pilot Projects ; Rapid Diagnostic Tests ; Recombinases/metabolism ; Sensitivity and Specificity ; *Strongyloides stercoralis/genetics/isolation &amp; purification ; *Strongyloidiasis/diagnosis/parasitology ; },
abstract = {Soil-transmitted helminth (STH) infections are prevalent worldwide, but the true burden of strongyloidiasis is unclear due to lack of sensitive and field-friendly diagnostic tools. Diagnosis is often based on serological assays that are typically not point-of-care (POC). Although polymerase chain reaction (PCR) tests are sensitive and specific, the need for expensive equipment and highly skilled personnel limits their use in resource limited areas. Isothermal amplification assays are largely instrument-free, making them simpler to implement without loss of either sensitivity or specificity. We developed two recombinase polymerase amplification (RPA) assays to detect Strongyloides stercoralis (Ss) in human stool samples and a complementary CRISPR-Cas12a detection system with visual readouts. Primers, probes, and guide RNAs (crRNAs) for these assays were designed targeting the Ss-NIE sequence and Ss dispersed repetitive sequence (Ss-DRS). The assay's specificities and limits of detection (LOD) were assessed using gDNA from Ss L3 larvae or from other STH and filariae. The NIE RPA showed a LOD of 1 fg/µL, whereas the LOD for the Ss-DRS RPA was 1 pg/µL. The LOD was 500 fg/µL for the NIE RPA CRISPR-Cas12a assay. No cross-reactivity with any filarial parasite or other STH was observed. Because the NIE assays were more sensitive than the Ss-DRS assay, six patient samples positive for Ss by real-time PCR (qPCR) were tested using the NIE assays, of which four were positive. Though assay refinement and clinical validation are needed, this study establishes fast, highly sensitive and field-applicable POC diagnostic tools for Ss detection that are ideal for use in endemic areas with limited resources.},
}
@article {pmid42014015,
year = {2026},
author = {Li, R and Xie, L and Hu, J and Liu, B and Zhang, H and Qian, H},
title = {Genome-wide CRISPR knockout screening identifies novel disease-associated genes in retinal pigment epithelium cells.},
journal = {Experimental eye research},
volume = {268},
number = {},
pages = {111032},
doi = {10.1016/j.exer.2026.111032},
pmid = {42014015},
issn = {1096-0007},
mesh = {*Retinal Pigment Epithelium/metabolism/pathology ; Humans ; Gene Knockout Techniques ; *Retinal Degeneration/genetics/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome-Wide Association Study ; Cell Survival ; *CRISPR-Cas Systems ; *Gene Expression Regulation/physiology ; Cells, Cultured ; *Eye Proteins/genetics ; },
abstract = {Dysfunction and degeneration of retinal pigment epithelium (RPE) cells are common pathological features observed in various retinal degenerative diseases. It has been proposed to treat these diseases by either protecting RPE cells or replacing them with new RPE cells derived from stem cells. However, the development of effective therapeutic strategies is still limited due to the insufficient understanding of the pathogenic factors involved in retinal degeneration and their impact on the function and survival of RPE cells. In this study, we employed genome-scale CRISPR knockout (KO) screening in human RPE cells to identify genes critical for RPE cell survival. Over 300 genes were identified, including well-established housekeeping genes as well as several candidate genes previously linked to retinal degeneration, many of which still lack comprehensive investigation. Among these, we further validated PRPF38B, which was both enriched in our screening and highlighted in a prior family-based linkage study, as essential for RPE cell survival, thus confirming the effectiveness of our approach. As a component of the spliceosome, we found that PRPF38B is crucial for functions specific to RPE cells, offering new insights into its role in retinal degeneration. Our study presents a novel approach for investigating risk genes associated with retinal diseases and may inspire future research on RPE cells and vision disorders.},
}
@article {pmid42015509,
year = {2026},
author = {Lushington, C and Thomas, P and Adikusuma, F},
title = {A primer on prime: A prime editing update from advances to first-in-human trial.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {6},
pages = {3171-3191},
pmid = {42015509},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Animals ; Clinical Trials as Topic ; },
abstract = {The advent of CRISPR systems has transformed genome editing, offering unparalleled efficiency and versatility with wide therapeutic potential. However, conventional CRISPR systems face key limitations, including unpredictable and imprecise outcomes during repair of double-stranded breaks and reliance on specific protospacer adjacent motif sequences. In response, prime editing (PE) has emerged as a powerful alternative, enabling precise custom edits using a fusion of Cas9 nickase and an engineered reverse transcriptase (RT) together with a PE guide RNA (pegRNA) that encodes the desired repair template. PE enables edits to be installed at or downstream of the target site, expanding the range of targetable sequences. Since its inception, PE has undergone extensive optimization, including Cas variant selection, RT engineering, and pegRNA improvements. In parallel, advances in delivery, including nanoparticles and split viral systems, have accelerated translation across preclinical disease models. Notably, PE has now entered the clinic, with the first-in-human study reporting functional restoration with a promising safety profile to date. Here, we summarize recent mechanistic insights, architectural innovations, and therapeutic applications of PE and discuss the remaining challenges in efficiency, delivery, and safety that will shape broader clinical impact.},
}
@article {pmid42016307,
year = {2026},
author = {My, B and Lia, A and Rizzo, L and Maiorano, G and Galeone, A and Palamà, IE and Gigli, G},
title = {CRISPR-Cas9 engineering of CAR-T cells: Can non-viral nanoparticles unlock safer and scalable genome editing?.},
journal = {iScience},
volume = {29},
number = {5},
pages = {115422},
pmid = {42016307},
issn = {2589-0042},
abstract = {CAR-T cell therapy has revolutionized the treatment of hematologic malignancies. Still, durable activity in tumors remains limited by antigen heterogeneity and escape, immunosuppressive tumor microenvironment, and restricted persistence. Genome engineering with CRISPR-Cas systems offers a powerful route to reprogram CAR-T cells; however, translation increasingly depends on how editing payloads are delivered. Viral vectors remain a benchmark for efficient gene transfer, cargo constraints, insertional risk, immunogenicity, and manufacturing complexity motivate the development of safer, more scalable non-viral platforms. In this review, we provide an overview of CAR designs, clinical use, and current ex vivo manufacturing workflow; compare viral and non-viral delivery routes while distinguishing established ex vivo editing from emerging in vivo T cell programming; and outline genome-engineering strategies organized by therapeutic goals. We highlight feasibility trade-offs and discuss how nanoparticles could enable transient, non-viral delivery of genome editors, while noting that robust T cell targeting and standardized potency/safety assays remain key bottlenecks.},
}
@article {pmid42017790,
year = {2026},
author = {Gopinath, A and Shen, L and Ouellette, SP},
title = {CRISPRi and beyond: studying essential gene function in the obligate intracellular bacterium Chlamydia trachomatis.},
journal = {Journal of bacteriology},
volume = {208},
number = {5},
pages = {e0005926},
doi = {10.1128/jb.00059-26},
pmid = {42017790},
issn = {1098-5530},
support = {R35 GM151971/GM/NIGMS NIH HHS/United States ; R21AI175651/NH/NIH HHS/United States ; R35GM151971/NH/NIH HHS/United States ; },
mesh = {*Chlamydia trachomatis/genetics/metabolism ; *Genes, Essential ; Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Chlamydia Infections/microbiology ; },
abstract = {Chlamydia trachomatis is an obligate intracellular bacterium that is the leading cause of bacterial sexually transmitted infections (STIs) and preventable infectious blindness. Its unique biphasic developmental cycle comprises an infectious but non-dividing elementary body and a replicative but non-infectious reticulate body. C. trachomatis possesses a reduced genome where more than half of the open reading frames (ORFs) are predicted to code for essential genes, abrogation of which with traditional chromosomal disruption methods is expected to block bacterial growth and developmental cycle progression. However, understanding the function of such genes is critical to expand our knowledge of chlamydial biology and reveal new therapeutic targets. This review aims to compare and contrast four systems developed in the past 5 years for studying essential genes in Chlamydia. These include systems to conditionally knock down or knockout a target gene product using CRISPR interference (CRISPRi), inducible small RNAs (sRNA), fluorescence-reported allelic exchange mutagenesis (FRAEM) with inducible complementation of the target gene, and dependence on plasmid expression (DOPE).},
}
@article {pmid42018092,
year = {2026},
author = {Zhao, R and Wang, C and Li, J and Liao, Y and Huang, C and Hu, T and Zhang, H and Zhang, W},
title = {DNA and RNA editing for the therapy of human diseases: current status, challenges, and future prospects.},
journal = {Molecular biomedicine},
volume = {7},
number = {1},
pages = {},
pmid = {42018092},
issn = {2662-8651},
support = {82302421//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *RNA Editing ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; *DNA/genetics ; },
abstract = {The rapid development of DNA- and RNA-editing tools (collectively referred to as gene editing technologies) has caused a paradigm shift in the treatment of human diseases from symptomatic treatment to precision-based medicine. Both DNA-based and RNA-based editing systems, including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived technologies and newly developed RNA editing tools, have pushed technological frontiers in terms of editing precision, hierarchical control, and reversibility; they have accumulated a growing body of preclinical and clinical evidence across diverse diseases ranging from inherited disorders to cancer, infectious diseases, and neurodegenerative diseases (ND). This review systematically summarizes the core principles and representative advances of DNA-based genome editing and RNA-based transcriptome editing technologies, comprehensively compares the two categories of technical strategies in terms of therapeutic potential, durability of effects, and risk profiles, and further explores the key challenges for achieving long-term safe and efficient in vivo applications, covering core bottlenecks such as delivery efficiency, tissue specificity, genotoxicity, and immunogenicity. Safety assessment has broadened to include tracking genotoxicity and genomic structural variations, whereas delivery systems and tissue specificity are determinant factors for in vivo therapeutic applications. Through the employment of both permanent and reversible editing strategies with high cargo-writing capacity and low integration risk, combined with programmable delivery systems, the therapeutic potential of hard-to-transfect tissues and complex diseases is anticipated to be broadened, opening new paths for clinical translation.},
}
@article {pmid42018449,
year = {2026},
author = {Cheung, CY and Samuels, I and Klaus, HR and Cook, GM and McNeil, MB},
title = {Metabolic perturbation reduces antibiotic tolerance in Mycobacterium tuberculosis.},
journal = {Microbiology (Reading, England)},
volume = {172},
number = {4},
pages = {},
pmid = {42018449},
issn = {1465-2080},
mesh = {*Mycobacterium tuberculosis/drug effects/metabolism/genetics ; Humans ; Macrophages/microbiology ; THP-1 Cells ; *Antitubercular Agents/pharmacology ; Microbial Sensitivity Tests ; *Drug Tolerance ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Iron/metabolism ; Gene Knockdown Techniques ; },
abstract = {Mycobacterium tuberculosis is tolerant to many antibiotics, leading to impaired antibiotic killing. Using CRISPR interference (CRISPRi) transcriptional knockdowns, we generated a panel of metabolically compromised strains to identify tolerance pathways for pursuing in therapeutic development. Disrupting the regulation of intracellular iron storage, amino acid biosynthesis and redox defence mechanisms potentiated the lethality of multiple drugs and translated to infected THP-1 macrophages. This work reinforces the role of metabolism as a major contributor to drug tolerance in M. tuberculosis.},
}
@article {pmid42018671,
year = {2026},
author = {Umbach, A and Santini, A and Bulcaen, M and Guidone, D and Maule, G and Arosio, D and Carrozzo, I and Ciciani, M and Brugnara, E and Ramalho, A and Vermeulen, F and Galietta, LJV and Carlon, MS and Cereseto, A},
title = {Functional correction of the untreatable CFTR 1717-1G&gt;A mutation through mRNA- and sgRNA-optimized base editing.},
journal = {Science translational medicine},
volume = {18},
number = {846},
pages = {eadw8886},
doi = {10.1126/scitranslmed.adw8886},
pmid = {42018671},
issn = {1946-6242},
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Humans ; *Gene Editing/methods ; *RNA, Messenger/genetics/metabolism ; HEK293 Cells ; *Mutation/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cystic Fibrosis/genetics ; CRISPR-Cas Systems/genetics ; Organoids/metabolism ; Base Sequence ; },
abstract = {The 1717-1G>A is a prevalent splicing mutation causing cystic fibrosis (CF) for which no pharmacological treatments have been approved. This mutation disrupts a canonical 3' AG splice acceptor site in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to severe RNA missplicing, which prevents the correct synthesis of the encoded protein. In this study, we developed an adenine base editing (ABE) strategy to efficiently correct the 1717-1G>A mutation. By using the ABE9 base editor with the protospacer adjacent motif-relaxed Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) variant SpRY, we obtained up to 30% editing with limited bystander effects in a human embryonic kidney (HEK) 293-based cellular model. Through systematic optimizations of the ABE system, delivered by electroporation of base editor messenger RNA (mRNA) and single guide RNA (sgRNA), we demonstrated genetic repair of the 1717-1G>A mutation in airway epithelial cells and intestinal organoids derived from people with CF. Functional analysis was performed by measuring short-circuit current in air-liquid interface (ALI) culture and by assessing forskolin-induced swelling (FIS) in intestinal organoids, which revealed restoration of CFTR channel activity. These results highlight SpRY-ABE9 as a potential genome editing strategy to permanently correct the CFTR 1717-1G>A mutation and restore CFTR function.},
}
@article {pmid42019303,
year = {2026},
author = {Sun, X and Qian, L and Jin, D and Guo, B and Tao, S and Fang, J and Wang, S and Chen, H and Tian, T and Lei, H},
title = {Bifunctional DNA multivalent structure integrating stable capture of urothelial carcinoma cells with CRISPR/Cas12a signal amplification for bladder cancer detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {306},
number = {},
pages = {118710},
doi = {10.1016/j.bios.2026.118710},
pmid = {42019303},
issn = {1873-4235},
mesh = {Humans ; *Urinary Bladder Neoplasms/diagnosis/urine/genetics/pathology ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry/genetics ; *DNA/chemistry ; Nanostructures/chemistry ; Cell Line, Tumor ; Nucleic Acid Hybridization ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Noninvasive detection of bladder cancer remains challenging due to the limited analytical performance of current urine-based assays under realistic detection conditions. Here, we report a linear programmable DNA nanostructure that integrates multivalent aptamer recognition with CRISPR/Cas12a signal transduction for detecting tumor-derived urinary exfoliated cells. Assembled via hybridization chain reaction, the multivalent scaffold was shown to enhance ligand-cell binding stability under mechanically perturbed detection processes, thereby supporting mechanically stable biological recognition and yielding an approximately 14-fold increase in cellular binding affinity compared with monovalent aptamers. The optimized architecture (MAP12) enables detection with a LOD of 1.1 cells/mL in model systems and achieves high diagnostic performance in clinical urine samples (92% sensitivity, 88% specificity; AUC = 0.9424), supporting dual signal readouts via fluorescence and lateral flow devices (LFD). This work establishes a DNA nanostructural strategy for reliable, rapid, and noninvasive cancer cell detection under realistic operational conditions.},
}
@article {pmid42019459,
year = {2026},
author = {Yun, Q and Gu, M and Li, L and Xia, WH},
title = {Rapid and visual detection of Mycoplasma pneumoniae using a novel ERA-CRISPR/Cas12a-based lateral flow assay.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {1},
pages = {117416},
doi = {10.1016/j.diagmicrobio.2026.117416},
pmid = {42019459},
issn = {1879-0070},
mesh = {Humans ; *Mycoplasma pneumoniae/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Child ; Adhesins, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children. Conventional detection methods often lack the speed or accessibility required for point-of-care testing (POCT). This study aims to develop a rapid, highly sensitive, and instrument-free diagnostic platform integrating Enzymatic Rapid Amplification (ERA), CRISPR/Cas12a cleavage, and Lateral Flow Assay (LFA) for visual MP detection.<br><br>METHODS: Specific primers targeting the conserved P1 adhesin gene of MP were screened for ERA efficiency. A CRISPR/Cas12a system was designed to specifically recognize the ERA amplicons, triggering the trans-cleavage of a reporter probe. The results were visually interpreted using LFA strips. The assay's limit of detection (LoD), specificity against six common respiratory pathogens, and clinical performance on 80 throat swab samples were evaluated.<br><br>RESULTS: The optimized ERA-CRISPR/Cas12a-LFA assay can be completed within 40 minutes. The LoD was established at 200 copies/mL. Specificity testing showed no cross-reactivity with S. pneumoniae, H. influenzae, or other tested pathogens. In clinical validation (n = 80), the assay demonstrated a sensitivity of 96.23% and specificity of 100% compared to qPCR, with a Kappa value of 0.945.<br><br>CONCLUSION: The established ERA-CRISPR/Cas12a-LFA method offers a rapid, sensitive, and specific alternative for MP screening. Its minimal equipment requirements make it highly suitable for resource-limited settings and primary care clinics.},
}
@article {pmid42019502,
year = {2026},
author = {Vereecke, N and Behrmann, M and Khare, A and Dekker, JP},
title = {Improved Cas9-targeted nanopore sequencing facilitates ultra-deep analysis of genomic variation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101410},
pmid = {42019502},
issn = {2667-2375},
mesh = {*High-Throughput Nucleotide Sequencing/methods ; *Nanopore Sequencing/methods ; Staphylococcus aureus/genetics ; *CRISPR-Cas Systems/genetics ; Bacteroides fragilis/genetics ; *Nanopores ; *Genomics/methods ; *CRISPR-Associated Protein 9/metabolism ; Genome, Bacterial ; *Genetic Variation ; },
abstract = {We present nanopore adapter-enriched Cas9-targeted sequencing (nAECATS), a method permitting inexpensive, ultra-deep, selective long-read sequencing of targeted regions in native, unamplified DNA. This method modifies previous Cas9-targeted sequencing approaches through the inclusion of a bead-based capture step that exploits the poly(T)8 stretch within the R10.4.1 ligation adapter for additional purification. Testing on a 10 kb Bacteroides fragilis genomic region achieved 90% on-target yield with 51,000× coverage from a single Flongle flow cell (353-fold increase versus whole-genome sequencing). Applied to a variable-length (>20 kb) Staphylococcus aureus genomic target containing dynamic gene amplifications conferring antibiotic resistance, nAECATS achieved 46,000× coverage and 74% on-target yield, revealing 2-4 tandem amplifications at single-cell resolution. While efficiency decreased with longer fragments (up to 41 kb), substantial enrichment improvements were demonstrated. We anticipate that nAECATS ultra-deep sequencing will find broad application for a wide range of biological questions in pro- and eukaryotic (epi)genomics and microbiology.},
}
@article {pmid42019854,
year = {2026},
author = {Farheen, J and Iqbal, MZ and Mustaq, A and Kong, X},
title = {A synergistic CRISPR-nano-immunotherapeutic system for targeted Bcl-2 silencing in breast tumour.},
journal = {International journal of biological macromolecules},
volume = {363},
number = {},
pages = {152114},
doi = {10.1016/j.ijbiomac.2026.152114},
pmid = {42019854},
issn = {1879-0003},
mesh = {*Breast Neoplasms/genetics/therapy/pathology/immunology ; Humans ; Female ; Animals ; *Proto-Oncogene Proteins c-bcl-2/genetics ; Cell Line, Tumor ; Mice ; *Immunotherapy/methods ; *Gene Silencing ; Apoptosis/genetics ; *CRISPR-Cas Systems ; Nanoparticles/chemistry ; Metal Nanoparticles/chemistry ; Mice, Inbred BALB C ; Gold/chemistry ; },
abstract = {B-cell lymphoma-2 (Bcl-2) protein has an extensive role in anti-cell death regulation and immuno-response modulation. It seizes apoptosis when Bcl-2 interacts and binds to Bax via its BH domain. Breast tumour (BT) was found to have overexpression of Bcl-2 coupled with mitochondrial membrane deprivation and stumpy immune response. Here, we developed a phyto-nanomedicine (HRP-MET)-based immunotherapeutic system integrating gold di‑manganese tri-oxide nanoparticles (GMNPs) with a BT-directed gene knockdown strategy. The nanoformulation is designed to accumulate in tumour tissue through enhanced permeability and retention (EPR)-mediated passive targeting, followed by activation within the tumour microenvironment. Initially, the expression of the Bcl-2 gene was selectively silenced in BT cells using a one-step advanced GenCRISPR™ Ultra NLS-Cas9 ribonucleoprotein (RNP) system with four designed effective sgRNAs (gene-CRISPR). Subsequently, transfected BT cells were treated with GMNP@HRP-MET phyto-nanomedicine in vitro and in vivo. As a result, this combinatorial strategy significantly induced tumour cell death via a mitochondria-mediated apoptotic signalling cascade (mitochondria → Bh3-only → Bax/Bak → Cycs → Apoptosome → Casp-9/Casp-3 → apoptosis) in various BT cells. Notably, Bcl-2 gene expression was prominently blocked in BALB/c female mice, accompanied by enhanced T-cell activation and sustained immune responses at both proteomic and transcriptomic levels. Furthermore, the gene-CRISPR and phyto-nanomedicine combination significantly inhibited tumour growth, migration, and distant organ metastasis in xenograft and syngeneic mice models. Collectively, this study demonstrates a practical and durable therapeutic strategy based on gene-CRISPR-enhanced apoptosis integrated with microenvironment-responsive phyto-nanomedicine for BT treatment.},
}
@article {pmid42020604,
year = {2026},
author = {Calvo Fernández, E and Tomassoni, L and Zhang, X and Wang, J and Obradovic, A and Laise, P and Griffin, AT and Vlahos, L and Minns, HE and Morales, DV and Simmons, C and Gallitto, M and Wei, HJ and Martins, TJ and Becker, PS and Crawford, JR and Tzaridis, T and Wechsler-Reya, RJ and Garvin, J and Gartrell, RD and Szalontay, L and Zacharoulis, S and Wu, CC and Zhang, Z and Califano, A and Pavisic, J},
title = {Systematic design of combination therapy by targeting master regulators of coexisting diffuse midline glioma cell states.},
journal = {Nature genetics},
volume = {58},
number = {5},
pages = {1112-1125},
pmid = {42020604},
issn = {1546-1718},
support = {R35 CA197745/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Glioma/genetics/drug therapy/pathology ; Animals ; Mice ; Cell Line, Tumor ; Pyrazoles ; *Brain Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology ; Pyrimidines ; Gene Expression Regulation, Neoplastic/drug effects ; Nitriles ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Intratumor heterogeneity fundamentally challenges cancer treatment, as coexisting, molecularly distinct cell states with non-overlapping drug sensitivities can drive therapeutic resistance. We establish and validate a generalizable, network-based framework to systematically identify combination therapies targeting complementary tumor cell states. Applied to diffuse midline glioma (DMG)-a universally fatal pediatric malignancy-this approach identified master regulator protein dependencies in seven coexisting cell states, confirmed by pooled CRISPR-Cas9 assays. Perturbational transcriptional profiles for 372 clinically relevant drugs prioritized candidates predicted to invert state-specific master regulator activity. State-selective drug sensitivity was validated for eight out of nine (89%) drugs in vivo, including avapritinib, ruxolitinib and larotrectinib. Compared with monotherapy, co-administering drugs targeting complementary states significantly prolonged survival across virtually all combinations, with avapritinib plus ruxolitinib extending median survival nearly threefold versus vehicle and 1.5-fold versus avapritinib alone. These findings establish clinically actionable DMG combinations and a tumor-agnostic and mutation-agnostic framework for rational combination therapy design.},
}
@article {pmid42020735,
year = {2026},
author = {White, MT and Wang, K and Zhang, H and Eckhard, U and Hullahalli, K and Chen, J and Wu, S and Geis, AL and Zhang, J and Queen, J and Gomis-Ruth, FX and Waldor, MK and Dong, M and Sears, CL},
title = {A pro-carcinogenic bacterial toxin binds claudin-4 to cleave E-cadherin.},
journal = {Nature},
volume = {654},
number = {8118},
pages = {504-512},
pmid = {42020735},
issn = {1476-4687},
mesh = {*Cadherins/metabolism/chemistry ; *Claudin-4/metabolism/genetics ; Humans ; *Bacterial Toxins/metabolism ; Protein Binding ; Animals ; Bacteroides fragilis/enzymology/pathogenicity/metabolism ; *Proteolysis/drug effects ; *Metalloendopeptidases/metabolism ; CRISPR-Cas Systems/genetics ; Antigens, CD/metabolism ; Colorectal Neoplasms/microbiology/metabolism ; Epithelial Cells/metabolism ; },
abstract = {The human colon is colonized by trillions of bacteria that play substantial roles in human health and disease[1]. Epidemiological and experimental studies suggest that certain colonic bacteria can stimulate the development and progression of colorectal cancer[2]. One such bacterium, enterotoxigenic Bacteroides fragilis, drives colon tumour formation through the action of a single toxin, the B. fragilis toxin (BFT)[3,4]. BFT is a metalloprotease that binds to a colonic epithelial cell receptor and causes cleavage of the E-cadherin ectodomain, leading to epithelial barrier disruption, inflammation and increased cellular proliferation[4-6]. However, the identity of the BFT receptor is unknown and the molecular mechanism of BFT-initiated E-cadherin cleavage is not well understood. Here we identify claudin-4 as a BFT receptor through a genome-wide CRISPR screen and demonstrate that claudin-4 binding promotes BFT-mediated cleavage of cell surface E-cadherin. Our work both sheds light on BFT's mechanism of action and opens avenues for the development of anti-BFT therapies, which may prove useful for colorectal cancer prevention and treatment of acute enterotoxigenic B. fragilis infection.},
}
@article {pmid42020736,
year = {2026},
author = {Shi, YJ and Ding, ZY and Wu, Y and He, Z and Zhang, YZ and Zhang, YL and Zhang, YM and Huang, XR and Yin, H and Zhang, Y},
title = {Quadruple pegRNA enables programmable and efficient large genomic insertion.},
journal = {Nature},
volume = {654},
number = {8117},
pages = {272-281},
pmid = {42020736},
issn = {1476-4687},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Mutagenesis, Insertional/methods ; Animals ; *Gene Editing/methods ; Transposases/metabolism ; *Genomics ; DNA/genetics ; },
abstract = {Precise, site-specific insertion of large gene sequences holds great promise for the treatment of diverse genetic disorders. Although prime editing using paired guide RNAs (pegRNAs) can mediate targeted integration, insertion efficiency drops sharply for payloads exceeding 300 base pairs[1-3]. Here we present a rationally designed quadruple pegRNA strategy (QuadPE) for efficient and programmable insertion of large DNA fragments. Through screening different designs, we identified that combinations of two genome-targeting pegRNAs in a PAM-out or PAM-in orientation, when paired with two donor-targeting pegRNAs in linear or circular form, yield optimal efficiency. Using QuadPE, we achieved stable integration efficiency of DNA fragments ranging from 1.6 to 26 kb, with efficiencies of around 40% at multiple loci with minimal off-target insertion activity. QuadPE substantially outperformed recombinase-mediated (PASSIGE and PASTE)[4,5] and transposase-mediated (CAST)[6] insertion systems, particularly for larger payloads, showing a 11-fold, 61-fold and 12-fold improvement for a 9.5 kb insertion, respectively. Notably, QuadPE was effective in both dividing and non-dividing primary cells such as human primary T cells and post-mitotic neurons, establishing QuadPE as a powerful and precise platform for large-fragment gene insertion without the need for double-stranded breaks or recombinases.},
}
@article {pmid42020899,
year = {2026},
author = {Kumar, S and Zhao, D and Wong, VHY and Bui, BV and Liu, GS},
title = {Evaluation of CRISPR/CasRx-Mediated VEGF mRNA Knockdown in Mouse Retina.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3023},
number = {},
pages = {25-37},
pmid = {42020899},
issn = {1940-6029},
mesh = {Animals ; *Vascular Endothelial Growth Factor A/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics ; *Retina/metabolism ; *Gene Knockdown Techniques/methods ; Gene Editing/methods ; Humans ; },
abstract = {Neovascular eye diseases (NEDs) are a group of diseases caused by the abnormal overgrowth of blood vessels in the eye. Normal vasculature is maintained through a dynamic balance of the vascular endothelial growth factor (VEGF). In diseases such as advanced diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD), an overexpression of VEGF leads to the formation of structurally weak and leaky blood vessels, resulting in vision impairment and, without intervention, legal blindness. In the clinic, NEDs are presently managed through anti-VEGF agents that specifically bind and neutralize VEGF signaling. While effective, this approach requires invasive intravitreal injections monthly and places a heavy burden on patients and healthcare providers. A flexible, long-lasting therapeutic that can reduce or eliminate frequent anti-VEGF treatment will significantly advance NED management.RNA editing with clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) is an emerging strategy to achieve reversible gene editing. The CRISPR-Cas13 system exclusively targets single-stranded RNA and allows gene silencing in a safe manner through RNA knockdown, as the genome is left intact. In addition, the compact CasRx enzyme (930aa) allows RNA silencing to be achieved through the delivery of a single adeno-associated virus (AAV), ideal for gene therapy applications. Herein, we outline methods to target VEGFA mRNA using CRISPR/CasRx in a mammalian cell line and humanized VEGFA transgenic mice.},
}
@article {pmid42022672,
year = {2026},
author = {Edward, M and Owoicho, AW},
title = {Antimicrobial Resistance in Cancer Care: Challenge and Path Forward.},
journal = {Health science reports},
volume = {9},
number = {3},
pages = {e71976},
pmid = {42022672},
issn = {2398-8835},
abstract = {INTRODUCTION: Antimicrobial resistance (AMR) is a global health crisis that poses a devastating and specific threat to immunocompromised cancer patients, who are heavily reliant on antibiotics to navigate high-risk treatments. The rapid emergence of multidrug-resistant (MDR) pathogens compromises the efficacy of prophylaxis and empirical therapy, leading to increased morbidity, mortality, and economic burden. This article aims to critically analyze the unique challenges of AMR in oncology and evaluate the translational readiness of innovative therapeutic modalities as a sustainable path forward.<br><br>METHODS: We synthesize current literature to articulate the clinical and economic burden of AMR specifically within the oncology setting. The article goes beyond conventional antibiotic stewardship discussions to critically appraise non-traditional strategies, including the clinical and regulatory barriers facing bacteriophage therapy, antimicrobial peptides (AMPs), and CRISPR-Cas systems.<br><br>RESULTS: AMR threatens the safe delivery of systemic cancer therapies, as resistant infections necessitate dose modifications and treatment delays. Traditional antibiotic development is insufficient. The path forward requires a paradigm shift: coupling robust stewardship and rapid diagnostics with a critical investment in innovative therapies. While promising, these novel modalities are not "silver bullets" and face significant hurdles in standardization, manufacturing, and regulatory approval that must be addressed for clinical integration into cancer care.<br><br>CONCLUSION: The fight against AMR is inseparable from the fight against cancer. To safeguard the future of oncology, a concerted effort is required to advance non-traditional anti-infectives through the translational pipeline, while ensuring immediate, tailored infection management for this vulnerable population.},
}
@article {pmid42023266,
year = {2026},
author = {Merwaiss, F and García, A and Rogo, U and Querol-Martí, I and García-Sogo, B and de Paola, C and Rodriguez-Rodriguez, M and Pineda, B and Moreno, V and Vazquez-Vilar, M and Orzáez, D and Daròs, JA},
title = {Virus induced gene editing using potyviral vectors in Cas12a expressing plants.},
journal = {Horticulture research},
volume = {13},
number = {4},
pages = {uhag017},
pmid = {42023266},
issn = {2662-6810},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are revolutionizing precision genome editing and gene expression control in crop plants. While effective CRISPR-Cas applications traditionally rely on labor-intensive stable genetic transformation to deliver Cas nucleases and guide RNAs into plant cells, plant viruses have emerged as a faster and efficient alternative, a strategy known as virus-induced gene editing (VIGE). Cas12a, Class 2 Type V CRISPR nucleases, are an alternative to broadly used Cas9 for plant genome engineering. Both kind of nucleases offer precise editing, but some Cas12a unique features make them particularly well suited for VIGE. In this study, we first used a tobacco rattle virus vector to compare editing efficiency of various target sequences and CRISPR RNA (crRNA) architectures in Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a)-expressing Nicotiana benthamiana plants, evaluating results in infected tissues and seeds. Next, we developed a tobacco etch virus (genus Potyvirus)-derived vector efficiently delivering crRNAs throughout the plant. This approach enabled generation of plants with all four edited alleles in the allotetraploid N. benthamiana through in vitro regeneration from infected leaves, and to produce edited non-infected progeny, although at a very low frequency. We then demonstrated the successful application of the potyviral vector for VIGE in agronomically important crops, such as tomato or cultivated tobacco. Finally, we replicated this design using two other potyviral vectors, turnip mosaic virus, and lettuce mosaic virus. Given the conserved biological properties among potyviruses, we believe these findings are broadly applicable to the largest genus of plant RNA viruses, significantly expanding the host range of the VIGE technology.},
}
@article {pmid42023429,
year = {2026},
author = {Lin, TM and Chang, HF and Lin, TC and Lin, CH and Sun, YL and Lin, CS},
title = {Gene Therapy and Gene Editing in Type 1 Diabetes: CRISPR-Based β-Cell Replacement and Treg Immune Modulation Approaches.},
journal = {Diabetes, obesity &amp; metabolism},
volume = {28},
number = {7},
pages = {5476-5491},
doi = {10.1111/dom.70800},
pmid = {42023429},
issn = {1463-1326},
support = {//Ministry of Education (MOE), Taiwan/ ; NSTC 114-2321-B-A49-001//National Science and Technology Council (NSTC), Taiwan/ ; },
mesh = {*Diabetes Mellitus, Type 1/therapy/immunology/genetics ; Humans ; *Genetic Therapy/methods ; *Insulin-Secreting Cells/immunology/transplantation ; *Gene Editing/methods ; *T-Lymphocytes, Regulatory/immunology ; Animals ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease marked by the destruction of pancreatic β-cells, resulting in lifelong dependence on exogenous insulin. Despite advances in insulin delivery and glucose monitoring technologies, patients remain at risk for acute and long-term complications, underscoring the need for curative strategies. Gene therapy and gene-editing technologies are emerging as transformative approaches capable of restoring β-cell function, modulating immune responses and potentially achieving durable remission.<br><br>METHOD: This review synthesizes basic science foundations and clinical trial evidence, focusing on five key protocols (NCT03162237, NCT05210530, NCT05241444, NCT05565248 and NCT06938334).<br><br>RESULTS: Strategies include immune modulation (PD-L1, FOXP3), &#x3b2;-cell replacement (CRISPR-edited progenitors, xenotransplantation) and combination approaches. Early-phase clinical trials have demonstrated feasibility and safety; however, long-term efficacy, durability and scalability remain uncertain. Critical challenges include potential off-target effects in CRISPR editing, risks of insertional mutagenesis, safety concerns in xenotransplantation and achieving a balance between immune tolerance and protective immunity. Future directions emphasize combination therapies, personalized medicine and next-generation editing tools such as base and prime editing.<br><br>CONCLUSION: Together, these efforts represent a paradigm shift from symptomatic insulin replacement toward curative interventions, while highlighting the considerable translational hurdles that must be overcome before routine clinical application.},
}
@article {pmid42024193,
year = {2026},
author = {Mahmoud, LM and Killiny, N},
title = {Crop biofortification for global food security: advances in genetic engineering and biotechnological approaches.},
journal = {Plant molecular biology},
volume = {116},
number = {3},
pages = {},
pmid = {42024193},
issn = {1573-5028},
}
@article {pmid42024428,
year = {2026},
author = {Jia, H and Zhao, P and Pei, J and Tian, P},
title = {Rewiring the morphology and metabolism of Escherichia coli with an engineered CRISPRi system.},
journal = {Journal of applied microbiology},
volume = {137},
number = {5},
pages = {},
doi = {10.1093/jambio/lxag104},
pmid = {42024428},
issn = {1365-2672},
support = {22278022//National Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/metabolism/cytology ; *Metabolic Engineering/methods ; DNA Replication/genetics ; Escherichia coli Proteins/genetics ; *CRISPR-Cas Systems ; Lactic Acid/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Bacterial ; },
abstract = {AIMS: Microbial morphology is an increasingly leveraged target in metabolic engineering. To causally investigate the coupling between DNA replication, morphology, and metabolism, we constructed a CRISPR interference (CRISPRi) system in Escherichia coli BL21(DE3) to repress essential replication genes (dnaN, dnaG, polA, and ssb).<br><br>METHODS AND RESULTS: Transcriptional repression disrupted cell division and yielded distinct, quantifiable shape changes: knockdown of dnaG (encoding primase) and ssb (encoding single-stranded DNA-binding protein), in particular, induced significant cellular elongation and widening. This morphological reprogramming concurrently reallocated metabolic flux, as evidenced by a substantial increase in lactic acid titer from 0.89&#xa0;g L&#x207b;&#xb9; to 6.01&#xa0;g L&#x207b;&#xb9;. RNA-seq and subsequent analyses (differential expression, GO/KEGG enrichment, GSEA, and PPI) reveal that replication inhibition drives extensive metabolic reprogramming and cell envelope remodeling, with notable perturbations in peptidoglycan biosynthesis.<br><br>CONCLUSION: Our results suggest that targeted replication stress can coordinately reshape bacterial morphology and alter metabolic output, providing a controllable strategy for engineering microbial cell factories.},
}
@article {pmid42024433,
year = {2026},
author = {Sato, R and Maruyama, K and Ara, S and Shibata, M and Shida, Y and Ogasawara, W and Yamazaki, H and Takaku, H},
title = {A CRISPR/Cas9-based genome-editing platform enabling efficient and precise gene replacement in Lipomyces starkeyi.},
journal = {FEMS yeast research},
volume = {26},
number = {},
pages = {},
pmid = {42024433},
issn = {1567-1364},
support = {JPJS00420240017//JSPS/ ; 25K08907//JSPS/ ; 25K18164//JSPS/ ; JPMJPF2211//Japan Science and Technology Agency/ ; //New Energy and Industrial Technology Development Organization/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Lipomyces/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Lipomyces starkeyi is a promising oleaginous yeast with industrial potential. However, its genome engineering remains constrained by low gene-targeting efficiency and the requirement for long homologous regions. Herein, we established a CRISPR/Cas9 genome-editing platform for L. starkeyi by expressing codon-optimized Streptococcus pyogenes Cas9 fused to an SV40 nuclear localization signal. Furthermore, in vitro-transcribed single-guide RNAs (sgRNAs) were directly delivered into the host, eliminating the need for endogenous RNA polymerase III-dependent sgRNA expression. CRISPR/Cas9 activity was validated using a codon-optimized Aequorea coerulescens GFP reporter. Cas9-induced frameshift mutations caused GFP disruption, leading to fluorescence loss. Gene replacement at the LsURA3 locus was evaluated using donor constructs with homologous regions ranging from 50-3000 bp. In a Cas9-expressing wild-type background, precise gene replacement was dependent on homology arm length, increasing from 36% with 50-bp arms to 80% with 3000-bp arms. Notably, in a Cas9-expressing Δlslig4 strain with suppressed non-homologous end joining (NHEJ), precise gene replacement was achieved with 100% accuracy using 50-bp homology arms under CRISPR/Cas9-dependent conditions. Together, these results demonstrate that a Pol III-independent CRISPR/Cas9 system combined with NHEJ suppression enables precise genome editing in L. starkeyi, providing a foundation for functional genomics and metabolic engineering.},
}
@article {pmid42024482,
year = {2026},
author = {Liang, Q and Cao, Y and Zhang, X and Ye, R and Liu, M and Zhang, S and Wang, Y},
title = {A Rapid and Ultrasensitive Detection of Coxsackievirus A16 Using Reverse Transcription Multiple Cross Displacement Amplification Combined with the CRISPR-Cas12a-Based Biosensing System.},
journal = {ACS infectious diseases},
volume = {12},
number = {5},
pages = {1765-1775},
doi = {10.1021/acsinfecdis.6c00151},
pmid = {42024482},
issn = {2373-8227},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Enterovirus/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *Hand, Foot and Mouth Disease/diagnosis/virology ; Sensitivity and Specificity ; Reverse Transcription ; Limit of Detection ; RNA, Viral/genetics ; Child, Preschool ; },
abstract = {Coxsackievirus A16 (CVA16) is one of the primary viral etiological agents of hand, foot, and mouth disease (HFMD) in infants and children under five years of age. Prompt and reliable detection of CVA16 is crucial for guiding immediate therapeutic interventions and for implementing effective epidemic prevention and control strategies, particularly in settings with limited resources. Herein, a diagnostic platform for CVA16 (CVA16-RT-MCDA-CRISPR) was developed by combining reverse transcription multiple cross displacement amplification (RT-MCDA) with CRISPR-Cas12a-based detection. In this system, the CVA16 VP1 gene was preamplified using RT-MCDA technology. The resulting amplicons were then specifically recognized and cleaved by the CRISPR-Cas12a-based detection system. MCDA primers, an engineered CP1 primer, and a specific guide RNA (gRNA) were designed to target the VP1 gene of CVA16. The assay achieved a limit of detection of 2.8 × 10[-1] copies per microliter for CVA16 RNA standard templates and showed no cross-reactivity against non-CVA16 pathogens. Furthermore, the CVA16-RT-MCDA-CRISPR assay's feasibility was validated using 96 clinical samples. Taken together, these results demonstrate that the CVA16-RT-MCDA-CRISPR assay is a reliable diagnostic tool for rapidly and sensitively detecting CVA16.},
}
@article {pmid42024570,
year = {2026},
author = {Liu, X and Shi, F and Luo, G and Wang, Q and Deng, F and Luo, X and Huo, D and Hou, C},
title = {A Disposable CRISPR-Nanozyme Electrochemical Biosensor for Rapid and Sensitive Detection of Breast Cancer Circulating Tumor DNA.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12802-12810},
doi = {10.1021/acs.analchem.6c00173},
pmid = {42024570},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Circulating Tumor DNA/analysis/blood/genetics ; *Breast Neoplasms/blood/genetics/diagnosis ; *Electrochemical Techniques/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Female ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) serves as a promising next-generation biomarker for noninvasive cancer screening and monitoring. In this work, we report an electrochemical biosensor for ctDNA detection. The sensor is constructed based on the synergistic integration of CRISPR/Cas12a and PB-Au NPs. CRISPR/Cas12a provides precise target recognition and triggers trans-cleavage, while the nanozyme enables strong signal amplification through its catalytic activity. Using a disposable carbon-fiber paper as the biosensing interface, we developed this sensitive detection strategy. On this interface, a PB-AuNP-labeled single-stranded DNA reporter is immobilized. In the presence of target ctDNA, activated Cas12a cleaves the reporter, releasing the nanozyme and resulting in a quantifiable decrease in the TMB oxidation current. This dual-amplification strategy achieves a detection limit of 860 aM (S/N = 3) with a linear range from 1 fM to 1 nM. Overall, this approach provides a satisfactory demonstration toward the realization of a low-cost and highly sensitive biosensor for ctDNA detection.},
}
@article {pmid42025056,
year = {2026},
author = {Ren, Y and Du, W},
title = {Integrated single-tube detection of miRNAs in subpopulation-specific extracellular vesicles via spatially colocalized dual-module DNA scaffold.},
journal = {Biosensors &amp; bioelectronics},
volume = {306},
number = {},
pages = {118711},
doi = {10.1016/j.bios.2026.118711},
pmid = {42025056},
issn = {1873-4235},
mesh = {*MicroRNAs/isolation &amp; purification/genetics/analysis ; *Extracellular Vesicles/chemistry/genetics ; Humans ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; CRISPR-Cas Systems ; *DNA/chemistry ; Biomarkers, Tumor/genetics ; },
abstract = {Extracellular vesicles (EVs)-derived miRNAs are valuable non-invasive biomarkers for early cancer diagnosis. However, most current methods detect either total EVs-miRNAs or those from a single subpopulation, overlooking subpopulation heterogeneity. Moreover, EVs isolation and miRNA analysis are often conducted in separate workflows, increasing sample handling and compromising reproducibility. Herein, we presented an integrated, single-tube platform for subpopulation-specific miRNA profiling, in which multivalent aptamer-based EVs capture and CRISPR/Cas12a-mediated signal amplification were co-localized on a rationally designed DNA scaffold. The multivalent aptamer system achieved a capture efficiency of 84.3%, significantly outperforming monovalent aptamers (56.4%). Upon in situ lysis of captured EVs subpopulation, the released miRNAs preferentially encountered adjacent recognition probes due to the increased local concentration, thereby efficiently initiating catalytic hairpin assembly (CHA). The resulting abundant duplex subsequently activated Cas12a, achieving a limit of detection (LOD) as low as 1.42 × 10[4] particles/μL. Critically, the modularity of the platform allowed for straightforward reconfiguration to target distinct EVs subpopulations by exchanging the capture aptamer. We demonstrated this flexibility by profiling miR-21 and miR-155 across two specific subpopulations (CD63[+] and MUC1[+]). The results revealed both cell-line-specific expression patterns and marked heterogeneity across CD63/MUC1-captured EVs fractions. Meanwhile, MUC1[+] subpopulation outperformed CD63[+] in clinical diagnosis, with higher AUC values for both miR-21 (0.85 vs 0.75) and miR-155 (0.83 vs 0.73). Overall, our study highlighted the critical importance of EVs subpopulation heterogeneity in cancer diagnostics and provided more informative biomarker readouts.},
}
@article {pmid42026322,
year = {2026},
author = {Li, Y and Zhao, W and Peng, X and Liang, Y and Xue, R and Liu, J and Xu, Z and Cao, D and Liu, B},
title = {TaGα knockout in wheat causes early heading and short organ length, with dose-dependent effects through various pathways.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42026322},
issn = {1432-2242},
support = {2025-ZJ-947M//Qinghai Provincial Department of Science and Technology/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Leaves/growth &amp; development/genetics ; Phenotype ; Gene Knockout Techniques ; Mutation ; Glutathione Transferase/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {TaGα regulates wheat development in a dose-dependent manner: a single mutant of TaGα solely accelerates heading, while a double mutant not only accelerates heading but also shortens organ length. The Gα subunit plays a crucial role in plant organ development and heading time; however, the exact functions in wheat are unknown. In the present study, knockout mutations in functional TaGα-7A and TaGα-7D were generated in the spring wheat cultivar 'Fielder' using the CRISPR/Cas9 system. Both single and double mutants exhibited early heading time. Reduced plant height, leaf length, and grain length were observed exclusively in the double mutant Gα[Δaadd], whereas leaf and grain width were unaffected. Histological sections revealed that cell length did not vary between the leaf and stem among 'Fielder,' Gα[Δaa], Gα[Δdd], and Gα[Δaadd], which indicated that cell number caused the leaf and stem length differences. Transcriptome analysis revealed dysregulated expression of kinesin and tubulin genes in double-mutant leaves, whereas altered oxidoreductase activity and differential expression of flowering-related genes were detected in the leaves of both single and double mutants. Immunoprecipitation coupled with mass spectrometry revealed that glutathione S-transferase (GST) physically interacted with TaGα directly, which was confirmed by luciferase complementation imaging and yeast two-hybrid assay. Furthermore, GST expression and enzyme activity were suppressed in both single and double mutants, resulting in elevated glutathione (GSH) content. The peak GSH content in wheat leaves during development corresponded to the heading time, which implied that GSH may have participated in regulation of heading time. According to the results of the present study, TaGα exerted regulatory effects on plant height, leaf length, grain length, and heading time in a dose-dependent manner via various pathways.},
}
@article {pmid42026979,
year = {2026},
author = {Wu, Z and Chen, J and Huang, M and Hu, W and Liu, Y and Shao, H and Zhang, W},
title = {Strategies and Advances in Site-Specific Integration of Exogenous Large Genes.},
journal = {Human gene therapy},
volume = {37},
number = {13-14},
pages = {551-565},
doi = {10.1177/10430342261445050},
pmid = {42026979},
issn = {1557-7422},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; *Mutagenesis, Insertional ; Genetic Vectors/genetics ; DNA Transposable Elements ; Transgenes ; },
abstract = {Large genomic deletions (≥1 kb) are a recurrent class of disease-causing lesions in monogenic disorders, frequently leading to complete gene inactivation or the loss of critical cis-regulatory elements. Addressing these defects in a therapeutically relevant manner requires integration modalities capable of delivering and stably installing large exogenous DNA sequences at predefined genomic loci with an improved safety profile. By contrast, legacy approaches-including viral-vector delivery, recombinase-based strategies, and transposon-mediated insertion-typically achieve integration through random or semi-random mechanisms, which, despite their practicality and often favorable efficiencies, limit control over insertion site and copy number and may increase the risk of insertional mutagenesis and position-dependent variability in transgene expression. The past few years have witnessed rapid methodological diversification driven by genome editing, resulting in a growing repertoire of locus-specific strategies for large-fragment DNA insertion that are reshaping both disease-model construction and genetic therapeutics. In this Review, we synthesize the main classes of targeted large-fragment integration technologies reported to date. We begin with homology-directed repair (HDR)-dependent CRISPR-Cas9 knock-in strategies and discuss how donor architecture and local donor recruitment can be leveraged to improve integration outcomes for kilobase-scale payloads. We then examine approaches centered on prime editing, particularly those that couple prime editing with engineered serine/tyrosine recombinases to support programmable insertion of large DNA cargos. We close by surveying emerging HDR-independent systems based on CRISPR-guided transposition and retrotransposition, and we provide a comparative perspective on their performance envelopes, constraints, and trajectories toward broader biomedical applications.},
}
@article {pmid42027081,
year = {2026},
author = {Hou, Q and Ren, J and Wu, Y and Zhao, P and Yue, S and Bi, S},
title = {Nucleic Acid Nanotechnology-Empowered CRISPR-Cas12a Systems for Biosensing and Bioimaging Applications.},
journal = {Small methods},
volume = {},
number = {},
pages = {e70666},
doi = {10.1002/smtd.70666},
pmid = {42027081},
issn = {2366-9608},
support = {ZR2024QB071//Natural Science Foundation of Shandong Province/ ; 22474067//National Natural Science Foundation of China/ ; tstp20230623//Taishan Scholar Foundation of Shandong Province/ ; 2025SHFXTD001//Open Project of Shandong Provincial Key Laboratory for Tumor Imaging Equipment Development and Tumor Diagnosis &amp; Treatment Integration Technology/ ; X2025104521145//Innovation and Entrepreneurship Training Program for College Students/ ; },
abstract = {The development of highly sensitive and simple bioanalytical platforms is crucial for advancing disease diagnostics and biomedical research. In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have emerged as the "next-generation molecular technology" that profoundly impacts the field of genome editing and molecular diagnosis. In particular, CRISPR-Cas12a, an important CRISPR-Cas family member, has gained prominence as a robust tool in biosensing and bioimaging due to its easy design and high target specificity. Notably, the integration of nucleic acid nanotechnology, including nucleic acid amplification strategies and engineered functional nucleic acids, with CRISPR-Cas12a systems significantly improves detection sensitivity and specificity, enabling the analysis of low-abundance analytes. This review introduces the fundamentals of CRISPR-Cas12a and key nucleic acid-based toolboxes. Next, we systematically summarize the advantages of nucleic acid nanotechnology-empowered CRISPR-Cas12a platforms in detail and highlight recent advances in their applications in biosensing and bioimaging. Finally, current challenges and future perspectives of such nucleic acids-assisted CRISPR-Cas12a systems for disease diagnostics are discussed.},
}
@article {pmid42027134,
year = {2026},
author = {Xu, L and Zhao, X and Meng, X and Chen, J and Chen, P},
title = {Multiselective Recognition of Metal Ion-Nucleic Acid Complexes by CRISPR/Cas12a and Quantum Dots Enables the Profiling of Circulating Tumor DNA in Breast Cancer.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {13120-13133},
doi = {10.1021/acs.analchem.6c01333},
pmid = {42027134},
issn = {1520-6882},
mesh = {*Quantum Dots/chemistry ; *Breast Neoplasms/genetics/blood/diagnosis ; Humans ; *CRISPR-Cas Systems ; Female ; *Circulating Tumor DNA/blood/genetics ; *Silver/chemistry ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; },
abstract = {The rapid, noninvasive detection of circulating tumor DNA (ctDNA) is vital for the diagnosis and staging of breast cancer (BC). In this study, we developed a homogeneous CRISPR/Cas12a fluorescent platform using a hierarchical grape-cluster rolling circle amplification (GCRCA) nanomaterial to detect the PIK3CA E542K mutation. A pivotal discovery of this study is that activated Cas12a efficiently cleaves metal ion-mediated cytosine-Ag[+]-cytosine base pairs, which enables direct coupling between enzymatic activity and signal transduction. GCRCA, assembled by precise hybridization of long-chain RCA concatemers with auxiliary circular DNA, features Ag[+]-bridged dual-ring units that sequester both target sequences and Ag[+] reporters within a self-shielding framework. Upon target recognition, activated Cas12a dismantles the GCRCA architecture, initiating an autocatalytic feedback loop that releases caged Ag[+] to quench the quantum dot fluorescence. This label-free assay achieved attomolar sensitivity within 30 min without enzymatic preamplification or complex nucleic acid extraction. Importantly, the platform exhibits excellent sequence selectivity, enabling precise discrimination of single-base mutations against closely related sequences. Validation of 42 clinical plasma samples achieved 100% diagnostic specificity for BC. For staging, the platform yielded a sensitivity of 100%, a specificity of 92.3%, and an area under the curve of 0.978. With its exceptional sensitivity and operational simplicity, this platform offers a promising approach for precise ctDNA-based BC detection and staging, demonstrating significant translational clinical potential.},
}
@article {pmid42029722,
year = {2026},
author = {Gong, Z and Chen, M and Zhang, H and Mortimer, JC and Botella, JR},
title = {Evaluation of computational tools for the prediction of CRISPR/SpCas9 gRNA activity in plants.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {42029722},
issn = {1432-203X},
support = {CE230100015//P4S/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Nicotiana/genetics ; *Computational Biology/methods ; Genome, Plant/genetics ; Machine Learning ; *CRISPR-Associated Protein 9/genetics/metabolism ; Algorithms ; },
abstract = {CRISPR/Cas9 technologies are now routinely used in plant research, with guide RNA (gRNA) design being a critical determinant of genome editing success. However, rational design of highly active gRNAs is challenging due to complex sequence and biochemical factors affecting activity. While numerous computational prediction tools have been developed, they are predominantly trained on animal cell or microbial data and their performance in plants remains controversial or untested. In this study, using two independent Nicotiana benthamiana experimental datasets comprising a total of 52 gRNAs, we systematically evaluated over 20 freely accessible, Web-based in silico tools for predicting gRNA on-target efficiency. We identified several machine learning-based tools that showed strong correlation with experimental editing efficiency across both datasets. Importantly, gRNAs in the top quartile by prediction score produced significantly higher InDel frequencies than those in the lowest quartile for all tools tested. Furthermore, several algorithms available through CRISPOR, a platform containing a large number of non-model plant genomes, also showed good predictive performance. This may enable better integration of on-target and off-target predictions in gRNA design. Our findings provide practical guidance for improving gRNA design in plant genome editing applications.},
}
@article {pmid42029959,
year = {2026},
author = {Sarki, YN and Keot, AK and Marwein, R and Singha, DL and Gogoi, DJ and Velmurugan, N and Chikkaputtaiah, C},
title = {Genotype-specific optimization of in vitro regeneration and Agrobacterium-mediated transformation in indica rice with 35S:RUBY and CRISPR/LbCas12a system.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42029959},
issn = {1432-2048},
support = {MMP025301//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; CRG/2022/007073//Science and Engineering Research Board/ ; },
mesh = {*Oryza/genetics/physiology ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; Genotype ; Regeneration/genetics ; CRISPR-Cas Systems/genetics ; *Agrobacterium/genetics ; Seeds/genetics ; 2,4-Dichlorophenoxyacetic Acid/pharmacology ; },
abstract = {This study establishes a genotype-specific transformation system for indica rice cultivars Ranjit, Mahsuri, and Kon Joha using 35S:RUBY and CRISPR/LbCas12a constructs, enabling functional genomics studies and genetic improvement. The indica rice subspecies generally faces challenges in functional genomics and genetic improvement due to its recalcitrance to tissue culture and Agrobacterium-mediated transformation. Three indica rice varieties, Ranjit, Mahsuri, and Kon Joha, cultivated in Assam (India) were selected to optimize callus induction, regeneration, and genetic transformation. Ranjit and Mahsuri are high-yielding cultivars, whereas Kon Joha is an indigenous aromatic landrace of high commercial value. Initially, key steps, such as callus induction and regeneration, were optimized for Ranjit and Kon Joha using mature seed. Thereafter, for transformation, immature embryos were selected as explants because of their competence for agro-infection. The highest callus induction frequency of 70.33% and 90% was achieved with 3.0 and 3.5 mg L[-1] of 2,4-dichlorophenoxyacetic acid (2,4-D) in Ranjit and Kon Joha, respectively. The best regeneration, in Ranjit (77%) and Kon Joha (54.5%), was achieved with 4.0 mg L[-1] 6-benzylaminopurine (BAP), 1.0 mg L[-1] kinetin, and 0.5 mg L[-1] naphthalene acetic acid (NAA). Transformation efficiencies with CRISPR/LbCas12a (OD660 = 0.8) were 5.6% (Ranjit), 7.5% (Mahsuri), and 15.33% (Kon Joha). The optimal in vitro regeneration conditions previously identified for Ranjit were adopted for Mahsuri transformation. Furthermore, a visual non-invasive reporter, RUBY, was employed to investigate the transformation of immature embryos in Ranjit and Kon Joha. The vivid red coloration in the early callusing stage indicates successful transformation events. Kon Joha plants regenerated from red calli showed a distinct phenotype with pink leaves and red roots. These findings confirm RUBY's effectiveness as a non-invasive reporter for rapid monitoring of rice transformation and underscore the importance of immature embryos in indica rice transformation.},
}
@article {pmid42030398,
year = {2026},
author = {Henard, JM and Lee, SA and Yu, YC and Shao, D and Azad, RK and Henard, CA},
title = {CRISPRi-based functional genomic screening identifies genes essential for CH4-dependent growth in a methanotrophic bacterium.},
journal = {Science advances},
volume = {12},
number = {17},
pages = {eaed4234},
pmid = {42030398},
issn = {2375-2548},
mesh = {*Methane/metabolism ; *CRISPR-Cas Systems ; *Genomics/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genes, Essential ; *Methylococcus capsulatus/genetics/growth &amp; development/metabolism ; },
abstract = {Methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH4) and have potential to mitigate climate-active gases. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a CRISPR interference (CRISPRi) system to enable functional genomic screening in methanotrophic bacteria. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph, Methylococcus capsulatus, consisting of ~45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH4 identified genes associated with essential methanotrophic bacterial processes and previously unidentified essential gene candidates, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs. The CRISPRi screen also led to nitrate mineral salts medium optimization to improve methanotroph biomass productivity from CH4. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify genetic determinants of methanotrophy and isolation of improved methanotroph biocatalysts.},
}
@article {pmid42031218,
year = {2026},
author = {Wang, MR and Yi, J and Li, ZJ},
title = {Tailored base editing toolkits for functional genomics and metabolic engineering in the halophile Salinivibrio.},
journal = {Journal of biotechnology},
volume = {416},
number = {},
pages = {56-63},
doi = {10.1016/j.jbiotec.2026.04.012},
pmid = {42031218},
issn = {1873-4863},
mesh = {*Gene Editing/methods ; *Metabolic Engineering/methods ; *Genomics/methods ; *Vibrionaceae/genetics/metabolism ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Polyhydroxybutyrates ; Acetates/metabolism ; Polyesters/metabolism ; },
abstract = {The development of synthetic biology in non-model halophiles has been hindered by the lack of efficient genetic tools, limiting their industrial applications. In this study, we report the development of an efficient base editing platform for the industrially promising halophile Salinivibrio sp. TGB4. Both adenine and cytosine base editors (ABE and CBE) were engineered and validated, enabling single, dual, and triple-site substitutions at specific loci. ABE demonstrated high efficiency and broad PAM compatibility, while CBE enabled accurate C-to-T substitutions. Additionally, a novel fusion protein (PmCDA-TadA8e-nCas9-UGI) facilitated cooperative dual-base editing, allowing for simultaneous A→G and C→T edits, which expands the potential for genome engineering in halophiles. Beyond tool development, we applied this system to elucidate the acetate metabolism in Salinivibrio sp. TGB4, identifying acs1 as a predominant gene for acetate assimilation and poly(3-hydroxybutyrate) biosynthesis under the tested conditions. This work establishes a versatile base editing toolkit for halophilic bacteria and demonstrates its utility in metabolic pathway analysis, thereby enhancing the genetic engineering potential of Salinivibrio sp. TGB4 for the biotechnological conversion of low-cost carbon sources into valuable chemicals.},
}
@article {pmid42031246,
year = {2026},
author = {Han, T and Long, K and Hu, W and Liu, M and Guo, M and Huo, D and Hou, C},
title = {Dual-locked probes inhibit off-target circularization: Enhancing specificity in rolling circle transcription for nucleic acid detection.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152180},
doi = {10.1016/j.ijbiomac.2026.152180},
pmid = {42031246},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/genetics ; *Transcription, Genetic ; *Nucleic Acids/genetics/analysis ; Human papillomavirus 16/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; },
abstract = {Rolling Circle Replication (RCR) holds significant potential for detecting low-abundance nucleic acids. However, its practical application is often hindered by nonspecific ligation, which leads to high background signals and false-positive results. Herein, we designed a novel dual-locked circular template, termed no-bilateral-overhang dual-locked probe (ndRC). This probe employed steric hindrance from its dual hairpins lacking overhangs to create a "dual-locked" mechanism that specifically prevents off-target circularization. By integrating this template with exonuclease purification and a CRISPR/Cas12a module, we constructed the dual-locked RCT-CRISPR/Cas12a (DL-RCT-Cas12a) system, in which RCT generates long RNA transcripts containing crRNA repeats. These transcripts then activate Cas12a-mediated trans-cleavage, thereby amplifying the detection signal. The established assay achieved an ultralow detection limit of 40.31 aM, with a detection range from 100 aM to 1 μM. It demonstrated robust performance in complex matrices for the detection of partial HPV16 L1 gene sequences and exhibited a superior ability to discriminate high-concentration targets compared to qPCR. This work presents a versatile strategy to enhance the specificity of highly sensitive detection, improving performance in low-abundance nucleic acid analysis.},
}
@article {pmid42031757,
year = {2026},
author = {Ly, D and Jang, H and Goel, A and Singh, A and Raguram, A},
title = {Genome-wide screening reveals producer-cell modifications that improve virus-like particle production and delivery potency.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42031757},
issn = {2041-1723},
support = {DP5 OD037342/OD/NIH HHS/United States ; DP5OD037342//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; *Virion/genetics/metabolism ; HEK293 Cells ; *Gene Transfer Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of gene editing agents. While extensive particle engineering has yielded efficient eVLPs, it remains underexplored whether engineering the cells used to produce eVLPs could further improve eVLP properties. We report an unbiased genome-wide screening approach to systematically investigate how genetic perturbations in producer cells influence eVLP production. This approach generates eVLPs loaded with guide RNAs that identify the genetic perturbation in the cell that produced a particular particle; the abundance of each guide RNA in eVLPs therefore reflects how the corresponding genetic perturbation influences eVLP production or cargo loading. We apply this approach to identify several genes that regulate eVLP cargo expression and loading into particles during the production process. Leveraging these insights, we engineer producer cells that support increased eVLP cargo packaging and a 2- to 9-fold increase in eVLP delivery potency across several cargo, particle, and target-cell types in cultured cells and in mice. Our findings suggest the potential of producer-cell engineering as a useful strategy for improving the utility of eVLPs and related delivery methods.},
}
@article {pmid42033211,
year = {2026},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {8},
pages = {e71159},
pmid = {42033211},
issn = {1582-4934},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Genomics/methods ; Animals ; *Evolution, Molecular ; *Genetic Therapy/methods ; },
abstract = {CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&amp;C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.},
}
@article {pmid42033509,
year = {2026},
author = {Çelik, B and Kiraz, Y and Şahin, Y and Tezcanlı Kaymaz, B},
title = {Targeting STAT5A via CRISPR/Cas9 restores TKI sensitivity in resistant chronic myeloid leukemia cells.},
journal = {Medical oncology (Northwood, London, England)},
volume = {43},
number = {6},
pages = {},
pmid = {42033509},
issn = {1559-131X},
mesh = {Humans ; *STAT5 Transcription Factor/genetics/metabolism ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems ; *Protein Kinase Inhibitors/pharmacology ; K562 Cells ; Apoptosis/drug effects/genetics ; Gene Knockout Techniques ; Tumor Suppressor Proteins ; },
abstract = {Therapeutic resistance to tyrosine kinase inhibitors (TKIs) remains a major challenge in the clinical management of chronic myeloid leukemia (CML). The transcription factor STAT5A, a principal downstream effector of BCR::ABL1, has emerged as a key transcriptional regulator implicated in the development of TKI resistance. This study aims to functionally validate the role of STAT5A in TKI-resistant CML by employing CRISPR/Cas9-mediated gene knockout and assessing the downstream molecular and phenotypic alterations. We hypothesized that selective disruption of STAT5A would restore apoptotic sensitivity and TKI responsiveness in resistant CML models. Additionally, we sought to integrate bioinformatic transcriptional network analyses to confirm whether STAT5A directly regulates the genes modulated by its deletion, thus reinforcing its mechanistic relevance as a therapeutic target. STAT5A was knocked out using CRISPR/Cas9 in K562 cells and their TKI-resistant derivatives (K562/Ima-Res, K562/Pon-Res). Western blot analysis confirmed effective depletion of STAT5A protein following CRISPR/Cas9 editing, validating that the observed phenotypic and transcriptional changes were attributable to successful STAT5A knockout. Post-editing, XTT assays were performed to assess cell viability, followed by Annexin V/PI staining for apoptosis and PI-based flow cytometry for cell cycle analysis. RT-qPCR was used to quantify the expression of key genes involved in the JAK/STAT pathway (JAK2, STAT3, CISH) and apoptosis/DNA damage responses (TP53, ATM, CASP3, CASP8). In silico analyses were conducted using TRRUST and Harmonizome/ChEA3 to confirm whether the genes modulated by STAT5A deletion were direct transcriptional targets. For additional validation, expression matrices from GSE207627 and GSE208314 were reanalyzed to confirm STAT5A-centered pathway alterations in resistant CML datasets. STAT5A knockout significantly reduced cell viability and induced apoptosis across all CML cell models, accompanied by G0/G1 cell cycle arrest. RT-qPCR revealed altered expression of both JAK/STAT components (JAK2, STAT3, CISH) and apoptosis-related genes (TP53, ATM, CASP3, CASP8). Transcriptional target analysis confirmed that several of these genes-such as CDKN2B, BCL2L1, and CCND1-are direct STAT5A targets, reinforcing the functional consequences of STAT5A loss. Integration of these findings suggests that STAT5A knockout reprograms both intrinsic (CASP3, TP53, ATM) and extrinsic (CASP8, BCL2L1) apoptotic pathways, thereby restoring chemosensitivity. CISH dysregulation further suggested compensatory feedback within the signaling network. CRISPR/Cas9-mediated STAT5A disruption effectively reverses TKI resistance in CML cells by reprogramming apoptotic and proliferative signaling. These findings identify STAT5A as a mechanistically validated and clinically actionable target, supporting its potential for combination strategies with TKIs or STAT5 inhibitors such as pimozide. Integration of transcriptional network analysis supports the mechanistic basis of these effects. STAT5A emerges as a compelling therapeutic target, meriting further investigation in preclinical models and patient-derived samples to evaluate its translational potential. Future validation in patient-derived CD34[+] CML models may advance STAT5A-based therapeutic design.},
}
@article {pmid42033623,
year = {2026},
author = {Askri, SMH and Li, F and Wang, J and Shahwar, D and Zhang, X},
title = {Toward sustainable control of phyto-nematodes: integrating lessons from crops to advance genetic modification in tomato.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42033623},
issn = {1432-2242},
support = {ZR2024MC063//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Solanum lycopersicum/genetics/parasitology ; Animals ; *Plants, Genetically Modified/parasitology/genetics ; *Disease Resistance/genetics ; *Plant Diseases/parasitology/genetics/prevention &amp; control ; *Tylenchoidea/physiology ; Crops, Agricultural/genetics/parasitology ; Plant Breeding ; },
abstract = {Emerging molecular approaches offer promising avenues to enhance tomato defense, demonstrating potential to effectively suppress root-knot nematode infestation and improve crop resilience under diverse environmental conditions. Root-knot nematodes (RKN, Meloidogyne spp.) continue to challenge tomato production by disrupting root architecture, impairing nutrient uptake, and reducing yields. Recent advances in plant biotechnology provide multiple avenues to enhance nematode resistance beyond conventional resistance (R)-gene utilization. This review discusses molecular and transgenic strategies investigated for RKN resistance across diverse plant systems, including the transfer or engineering of R-genes, expression of anti-feedant proteins and nematotoxic peptides, host-induced gene silencing targeting essential nematode genes, and CRISPR/Cas-mediated modification of host susceptibility factors, with an emphasis on their relevance for future use in tomato. Each approach interferes with distinct stages of nematode infection-from host recognition and feeding-site establishment to reproduction-and has demonstrated measurable reductions in gall formation, egg production, or nematode fitness, primarily in controlled-environment studies and selected field evaluations conducted in non-tomato or model crop systems. The review further evaluates progress in translating these cross-species advances into field-relevant tomato production contexts, emphasizing considerations for tissue-specific expression, stacking of complementary traits, and regulatory compliance affecting practical implementation. By highlighting the mechanistic insights gained from these molecular interventions across multiple crops, this work identifies key opportunities for integrating complementary genetic strategies into tomato breeding programs. Collectively, these advances underscore the potential of combining precision genetics and biotechnology to develop durable, broad-spectrum nematode resistance, reducing dependence on chemical nematicides and supporting sustainable horticultural production.},
}
@article {pmid42033935,
year = {2026},
author = {Yao, F and Ziqing, Z and Jingxi, H and Yuxin, X and Tiancheng, Z and Qi, X and Guangjin, P},
title = {Generation of an IL2 knock-in human induced pluripotent stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103996},
doi = {10.1016/j.scr.2026.103996},
pmid = {42033935},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Interleukin-2/genetics/metabolism ; *Gene Knock-In Techniques ; Cell Differentiation ; Cell Line ; Animals ; },
abstract = {Interleukin-2 (IL2) is a critical cytokine that drives T-cell proliferation, activates NK cells, and holds significant therapeutic value. Its established clinical importance in cancer immunotherapy lies in sustaining the expansion, persistence, and effector function of adoptive cell therapies. Here, we established a stable IL2 knock-in human induced pluripotent stem cell (iPSC) line via CRISPR/Cas9 technology. This engineered line constitutively secretes IL2, maintains a normal karyotype, and retains typical pluripotent characteristics, including gene expression and in vivo differentiation potential. It thus provides a reliable model for studying IL2 signaling, immune crosstalk, and therapeutic screening.},
}
@article {pmid42035477,
year = {2026},
author = {Noronha, S and Liu, Y and Geneti, G and Li, H and Wu, X and Sun, D and Gujar, V and Furusawa, T and Lobanov, A and Cam, M and Pal, LR and Nair, NU and Day, CP and Ruppin, E and Ghosh, C and Hu, J and Ramamoorthy, B and Kumar, S and Andresson, T and Chan, K and O'Neill, M and Chari, R and Pommier, Y and Rivero, JD and Weyemi, U and Kebebew, E and Boufraqech, M},
title = {CRISPR-Based Gene Dependency Screens Reveal Mechanism of BRAF Inhibitor Resistance in Anaplastic Thyroid Cancer.},
journal = {Molecular carcinogenesis},
volume = {65},
number = {7},
pages = {874-887},
pmid = {42035477},
issn = {1098-2744},
mesh = {Humans ; *Proto-Oncogene Proteins B-raf/antagonists &amp; inhibitors/genetics ; *Thyroid Carcinoma, Anaplastic/genetics/drug therapy/pathology ; *Drug Resistance, Neoplasm/genetics ; *Thyroid Neoplasms/genetics/drug therapy/pathology ; Cell Line, Tumor ; *Protein Kinase Inhibitors/pharmacology ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; Imidazoles/pharmacology ; Oximes/pharmacology ; Unfolded Protein Response/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; },
abstract = {Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors. Using gene essentiality score across cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.},
}
@article {pmid42036673,
year = {2026},
author = {Jiang, CQ and Song, Z and Yan, ZC and Liu, Y and Zhang, JJ and Yue, B and Qiu, M and Hu, YJ},
title = {MXene-based CRISPR/Cas9 nanoplatform targeting FABP5 for ROS amplification and synergistic photothermal/photodynamic therapy of cervical cancer.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42036673},
issn = {1477-3155},
support = {62405299//the Young Scientists Fund of the National Natural Science Foundation of China/ ; ZR2024QE315//Youth Foundation of Shandong Natural Science Foundation/ ; 62375249//National Natural Science Foundation of China/ ; ZR2022JQ22//Natural Science Foundation of Shandong Province/ ; tsqn201909054//Taishan Scholar Project/ ; 202341006//Fundamental Research Funds for the Central Universities/ ; TJYXZDXK-3-029C//Tianjin Key Medicine Discipline (Specialty) Construction Project/ ; },
mesh = {Female ; *Uterine Cervical Neoplasms/therapy/metabolism/pathology ; Humans ; *Photochemotherapy/methods ; *Reactive Oxygen Species/metabolism ; Animals ; *CRISPR-Cas Systems/genetics ; *Fatty Acid-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Mice ; Gene Editing ; Apoptosis/drug effects ; Oxidative Stress/drug effects ; Photothermal Therapy/methods ; Nitrites ; Transition Elements ; },
abstract = {Cervical cancer remains a leading cause of cancer-related mortality among women worldwide, underscoring the need for more effective therapeutic strategies. Photodynamic therapy (PDT) has gained attention in tumor treatment owing to its high selectivity and minimal invasiveness. However, PDT is often compromised by the intrinsic antioxidant defense systems of cervical cancer cells. Herein, we developed a gene editing photonic nanoplatform, MXene@PEI-FABP5 (MPF), which integrates the photothermal/photodynamic properties of MXene with CRISPR/Cas9-mediated FABP5 gene editing to achieve synergistically enhanced antitumor effects. Fatty acid binding protein 5 (FABP5), highly expressed in cervical cancer, plays a pivotal role in regulating lipid peroxidation and oxidative stress tolerance. By delivering the CRISPR/Cas9 system using MXene into tumor cells, FABP5 expression was effectively silenced, thereby disrupting cellular antioxidant defenses at the genetic level. Meanwhile, under 808 nm laser irradiation, MXene generated robust hyperthermia and reactive oxygen species (ROS), jointly amplifying oxidative stress and inducing cell death predominantly through apoptosis. Both in vitro and in vivo results demonstrated that MPF achieved an impressive tumor inhibition rate of ~96% while maintaining excellent biosafety. This work presents a "gene editing and photothermal/photodynamic" hybrid therapeutic paradigm, offering a promising avenue to overcome the limitations of conventional PDT and improve cervical cancer treatment outcomes.},
}
@article {pmid42037564,
year = {2026},
author = {de Barros Rodrigues, DK and Leeuwerik, M and Brankovics, B and Fava, WS and Venturini, J and Meyer, W and Arai, T and Majima, H and Watanabe, A and de Souza Carvalho Melhem, M},
title = {Environmental Circulation of Aspergillus fumigatus With Reduced Susceptibility to Agricultural Triazole in Brazil: Clonal Dissemination of Potentially Resistant Genotypes.},
journal = {Mycoses},
volume = {69},
number = {5},
pages = {e70179},
pmid = {42037564},
issn = {1439-0507},
support = {201422/2024-7//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 443813/2023-0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 88887.658397/2021-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 88887.819536/2023-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; },
mesh = {*Aspergillus fumigatus/genetics/drug effects/isolation &amp; purification/classification ; *Triazoles/pharmacology ; *Drug Resistance, Fungal/genetics ; Genotype ; *Antifungal Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Brazil/epidemiology ; Aspergillosis/microbiology ; Fungal Proteins/genetics ; Whole Genome Sequencing ; Microsatellite Repeats ; Agriculture ; Cytochrome P-450 Enzyme System/genetics ; Tubulin/genetics ; Polymorphism, Single Nucleotide ; Air Microbiology ; Mutation ; },
abstract = {BACKGROUND: Aspergillus fumigatus resistance to triazole antifungals poses an increasing global health concern. Moreover, the cross-resistance between azole antifungal agents used in clinical settings and those applied in agriculture has become an important emerging issue.<br><br>OBJECTIVES: In this study, we investigated the five environmental A. fumigatus strains showing reduced susceptibility to tebuconazole.<br><br>METHODS: Fungal strains were recovered from air samples collected around the homes of two patients with suspected aspergillosis caused by resistant isolates. Species identification was performed by sequencing the &#x3b2;-tubulin gene, and minimum inhibitory concentrations were determined by broth microdilution. The cyp51A gene was sequenced to detect mutations, and CRISPR-Cas9 genome editing was employed to investigate their influence on susceptibility patterns. Microsatellite genotyping was performed to assess genetic variability, followed by whole genome sequencing and single nucleotide polymorphism analysis.<br><br>RESULTS: The environmental strains presented the same cyp51A genotype characterised by the M172V substitution and silent mutations. Microsatellite genotyping and whole genome sequencing confirmed that the strains were clonal. Functional validation demonstrated that the M172V and silent mutations partially contribute to reduced susceptibility to tebuconazole but are not the main mechanism of resistance involved. Analysis of polymorphisms in genes other than cyp51A revealed no resistance-conferring mutations.<br><br>CONCLUSIONS: The findings described herein suggest the possibility of local clonal dissemination of environmental strains under selective pressure from agricultural azoles in a major agribusiness region of the Midwest of Brazil. This study highlights the silent spread of potentially resistant genotypes in urban areas and reinforces the need for environmental surveillance and expanded genomic monitoring in South America.},
}
@article {pmid42040301,
year = {2026},
author = {Iqbal, Z and Awan, AZ and Atta, S and Hussain, K and Khurshid, M and Ahmad, F and Munir, M and Ghafoor, A and Hashedi, SAA and Ramadan, KMA and El-Gananiny, SM and AlSaleh, MA},
title = {Unveiling the landscape of plant virology in Saudi Arabia: seven decades of progress and future directions toward Vision 2030.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1758142},
pmid = {42040301},
issn = {1664-462X},
abstract = {Plant viruses pose a persistent and escalating threat to global agriculture and food security, inflicting over $30 billion in annual losses - a challenge acutely felt in Saudi Arabia as it strives for agricultural self-sufficiency under Vision 2030. This is the first comprehensive review which presents seven decades of plant virology research in the Kingdom, from early symptom-based diagnosis to advanced molecular, genomic, and bioinformatics advances. A total of ~81 plant viral species infecting 46 plant host species have been documented across the major agroecological regions, dominated by positive-sense single-stranded RNA viruses (~70%). Among these viruses, some are economically most destructive-including alfalfa mosaic virus, cucumber mosaic virus, soilborne cereal mosaic virus, tomato yellow leaf curl virus, zucchini yellow mosaic virus, watermelon chlorotic stunt virus, and barley mild mosaic virus-posing recurrent challenges to key crops such as alfalfa, cucurbits, and tomatoes. Network analysis of virus distribution revealed strong epidemiological linkages among central and western agricultural regions, possibly driven by intensive cultivation and vector ecology. The review highlights emerging management strategies including CRISPR-Cas diagnostics, RNA interference, AI-based detection, nanotechnology, and plant growth promoting rhizobacteria. Gaps persist in genomic surveillance, vector ecology, and biosecurity enforcement. The review concludes with future research priorities emphasizing innovation, interdisciplinary collaboration, and the development of a national plant virus genomic and surveillance framework to secure sustainable agriculture in line with Vision 2030.},
}
@article {pmid42041587,
year = {2026},
author = {Elias, A and Stern, S},
title = {Gene Editing Strategies for Neurological and Mental Disorders: Advances in Delivery, Methodology, and Clinical Translation.},
journal = {Cells},
volume = {15},
number = {8},
pages = {},
pmid = {42041587},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; *Nervous System Diseases/therapy/genetics ; *Mental Disorders/therapy/genetics ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; *Translational Research, Biomedical ; },
abstract = {Neurological and mental disorders are among the main causes of disability worldwide, affecting over three billion people and increasing the socioeconomic burden. Advances in molecular genetics and genome engineering have led to gene-targeted therapies that address root causes rather than just symptoms. This review covers current genome-editing tools, including CRISPR/Cas, base editing, and prime editing. The focus is on the benefits of gene editing in the central nervous system, where post-mitotic neurons allow lasting effects after a single treatment. It also discusses emerging delivery platforms such as viral vectors, nanoparticles, and exosome systems, as well as methods to bypass the blood-brain barrier. Recent clinical progress in spinal muscular atrophy, Parkinson's disease, Huntington's disease, and Alzheimer's disease is highlighted, with promising preclinical results for autism, bipolar disorder, epilepsy, and other neurogenetic conditions. The review concludes with regulatory issues, market trends, and ongoing clinical trials, underscoring the potential of gene therapies to transform disease management and provide long-term solutions.},
}
@article {pmid42042483,
year = {2026},
author = {Zhang, C and Wang, YX and Liu, XD and Iqbal, A and Wang, Q and Wang, Y},
title = {Integrated Pest Management Strategies for Controlling Phthorimaea (Tuta) absoluta: Advances in Biological, Pheromone, and Cultural Control Methods.},
journal = {Insects},
volume = {17},
number = {4},
pages = {},
pmid = {42042483},
issn = {2075-4450},
support = {No. XJ202411439006 and No. XJ202411439010.//Jilin Agricultural Science and Technology College Student Innovation and Entrepreneurship Training Program Project and the Jilin Agricultural Science and Technology University College Student Innovation and Entrepreneurship Training Program Project/ ; },
abstract = {The tomato leaf miner, Phthorimaea (Tuta) absoluta, Meyrick 1917 is recognized as a highly destructive pest, causing significant economic losses to crops in both greenhouse and open field environments across four continents: Asia, Africa, Europe, and South America. High genetic homogeneity among populations from various regions and countries indicates significant gene flow between P. absoluta populations, suggesting a lack of geographical barriers to dispersion. Furthermore, P. absoluta has developed resistance to insecticides due to target-site mutations or metabolic resistance, which enable the insect to withstand lethal doses of insecticides. To control this insect pest, the plant-mediated RNA interference (RNAi) is most promising host-induced gene silencing technique, utilized the plant's machinery to express double-stranded (dsRNA), which triggers the RNAi pathway in P. absoluta. Due to thermal tolerance, the P. absoluta has increased its area of invasion by 600 km per year over 9 years. Female P. absoluta releases pheromones that are recognized by males with a sophisticated olfactory circuit on their antenna. Pheromone binding proteins (PBPs) play a crucial role in mate recognition and attraction, and their expression peaks during courtship, specifically around 6:00 a.m. Given its potential to significantly alter the insect genome, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) offer a revolutionary strategy to control P. absoluta. Furthermore, this pest has developed remarkable adaptations to survive on unfavorable hosts by secreting specific proteins from its salivary glands that detoxify plant defenses. Insecticide resistance is likely the cause of field control failures of P. absoluta. Biological control, sex pheromone traps, and cultural control are the most promising approaches to address insecticide resistance resulting from these failures. Therefore, the implementation of integrated control programs and appropriate resistance management strategies is necessary to keep P. absoluta infestations under economic damage thresholds.},
}
@article {pmid42043550,
year = {2026},
author = {Yao, X and Yao, X and Luo, M and Luo, L and Zhou, L and Li, X},
title = {Establishment of a CRISPR-Cas12a based electrochemical detection method for Burkholderia gladioli and its subspecies cocovenenans in fresh noodles and tremella.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42043550},
issn = {1432-072X},
support = {2024CZ19//Science and Technology Plan Project of Administration for Market Regulation of Guangdong Province/ ; },
mesh = {*Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Burkholderia gladioli/isolation &amp; purification/genetics/classification ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Burkholderia gladioli pv. cocovenenans is a pathogenic bacterium of concern due to its potential threat to food safety. This study presents a rapid electrochemical detection method based on dual recombinase-aided amplification (RAA) coupled with CRISPR-Cas12a for the sensitive and specific identification of B. gladioli and its toxigenic subspecies B. gladioli pv. cocovenenans in food. The 16 S rDNA and bonA genes were selected as species- and subspecies-specific targets, respectively, and corresponding CRISPR-Cas12a reaction systems were established. An electrochemical biosensor incorporating a gold electrode functionalized with single-stranded DNA probes was constructed, and its specificity and sensitivity were evaluated using artificially contaminated fresh noodles and tremella samples. The method precisely distinguished B. gladioli and its toxigenic cocovenenans subspecies within 1 h, achieving a limit of detection limit of 10[2] CFU/g in both food matrices. This strategy provides a rapid and field-deployable approach for distinguishing non-toxigenic and toxigenic B. gladioli strains in food, supporting timely screening and food safety monitoring.},
}
@article {pmid42043584,
year = {2026},
author = {Ray, S and Vijayan, J and Vanchinathan, S and Dhakar, R and Nasrullah, N and Nagar, S and Dutta, TK and Chinnusamy, V},
title = {CRISP-PTG-Assembler Ver. 1.0: a primer design tool for polycistronic tRNA-gRNA (PTG) assembly for Cas9-based multiplex genome editing in plants.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42043584},
issn = {1432-2048},
support = {CRG/2021/002949//Science and Engineering Research Board (SERB), GoI/ ; Genome Editing Project//Indian Council of Agricultural Research/ ; Crop Science EFC-10//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *RNA, Transfer/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; DNA Primers/genetics ; },
abstract = {Multiplex genome editing using the CRISPR/Cas9 system allows simultaneous modifications at several genomic sites, offering great potential for crop improvement. Among various approaches, the polycistronic tRNA-gRNA (PTG) system is widely adopted due to its use of the host's native tRNA processing machinery, enabling the generation of multiple sgRNAs from a single transcript without the need for expressing any foreign RNA processing enzymes or ribozymes. However, designing the complete set of primers suitable for performing in vitro PTG assembly is complex and needs expertise, as a single mistake can lead to complete failure of the assembly process or subsequent editing. To overcome this challenge, we developed CRISP-PTG-Assembler Ver. 1.0, a user-friendly tool that takes only (i) 20-nucleotide sgRNA spacers and (ii) 4-nucleotide joiners as inputs; and produces colour-coded outputs in forms of (i) Primer-set required for complete PTG assembly, (ii) Primary PCR Amplicons, (iii) Overlap-Extension PCR Amplicons and (iv) Expected PTG assembly, for easy interpretation and construct making. Our novel assembly approach provides flexibility in sticky-end choice during golden gate ligation and ensures the fidelity of component sgRNAs in the PTG assembly by buffering against ligation errors (~ 1.5-40%) that may occur during the Golden Gate assembly process, thereby safeguarding the functionality of the in vivo-generated individual sgRNA molecules. We validated its effectiveness by editing two loci of the matrix metalloproteinase 1 gene in rice and demonstrated its applicability across various plant systems. With an intuitive interface and robust features, CRISP-PTG-Assembler empowers researchers of all levels to effectively implement PTG-based multiplex genome editing in plants.},
}
@article {pmid42044121,
year = {2026},
author = {Varderesian, HV and Utaegbulam, JN and Brown, HE and Ramirez, B and Velcani, M and Ryder, SP},
title = {The pos-1 3' untranslated region governs germline specification and proliferation to ensure reproductive robustness.},
journal = {PLoS genetics},
volume = {22},
number = {4},
pages = {e1012129},
pmid = {42044121},
issn = {1553-7404},
support = {R01 HD111505/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *3' Untranslated Regions/genetics ; Caenorhabditis elegans/genetics ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; Female ; Male ; Germ Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Proliferation/genetics ; Zygote/metabolism/growth &amp; development ; Reproduction/genetics ; Fertilization/genetics ; Oocytes/metabolism ; Fertility/genetics ; CRISPR-Cas Systems ; },
abstract = {During fertilization, haploid gametes combine to form a zygote. The male (sperm) and female (oocyte) gametes contribute a similar amount of DNA, but the oocyte contributes nearly all the cytoplasm. Oocytes are loaded with maternal mRNAs thought to be essential for embryonic patterning after fertilization. A conserved suite of RNA-binding proteins (RBPs) regulates the spatiotemporal translation and stability of maternal mRNAs. POS-1 is a CCCH-type tandem zinc finger RBP expressed in fertilized Caenorhabditis elegans zygotes from maternally supplied mRNA. POS-1 accumulates in the posterior of the embryo where it promotes posterior cell fate. Here, we show that the pos-1 3' untranslated region (UTR) is essential for POS-1 patterning and contributes to maximal reproductive fecundity. We engineered a pos-1 mutant where most of the endogenous pos-1 3'UTR was removed using CRISPR genome editing. Our results show that the 3'UTR represses POS-1 expression in the maternal germline but increases POS-1 protein levels in embryos after fertilization. In a wild-type background, POS-1 repression via the 3'UTR has little impact on fertility. In a sensitized background, the deletion mutant has a complex pleiotropic phenotype where most adult homozygous progeny lack either one or both gonad arms. Most phenotypes become more penetrant at elevated temperature. Together, our results support an emerging model where the 3'UTRs of maternal transcripts, rather than being essential, contribute to reproductive robustness during stress.},
}
@article {pmid42044361,
year = {2026},
author = {Huang, J and Zhang, C and Li, J and Ren, H and Yang, K and Zhang, Y},
title = {Comparative Evaluation of Engineered Bacteria and Yeast for Oral Delivery of CRISPR/Cas9 Systems in Colon Cancer Therapy.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1915-1925},
doi = {10.1021/acssynbio.6c00006},
pmid = {42044361},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Colonic Neoplasms/therapy/genetics ; Mice ; *Escherichia coli/genetics/metabolism ; Humans ; Administration, Oral ; Gene Editing/methods ; Cell Line, Tumor ; Female ; Genetic Therapy/methods ; Mice, Inbred BALB C ; },
abstract = {Colorectal cancer (CRC) poses a serious threat to human health. CRISPR/Cas9 technology offers new therapeutic strategies for the management of this disease, but its oral application is severely hindered by the limitations of suitable delivery systems. Herein, we develop and compare two separate orally delivered, genetically and chemically modified CRISPR/Cas9 delivery platforms based on E. coli BL21 and P. pastoris X33, which upon colonization in the intestine, secreted extracellular vesicles carrying the Cas9 protein and ART1-targeting sgRNA for tumor-specific gene disruption. Arginine ADP-ribosyltransferase 1 (ART1) plays a crucial role in the biological regulation of colon cancer, which was for the first time to the best of our knowledge, employed in vivo as a target gene in this study. Furthermore, we employed polydopamine (PDA) coating and gastrointestinal synthetic epithelial lining systems to facilitate microbial viability and intestinal retention, establishing on site cell factories for sustained CRISPR secretion. In subcutaneous tumor-bearing murine models, both delivery systems demonstrated comparable antitumor efficacy with significant tumor suppression. Taken together, the genetically modified microbial platform using bacterial and yeast strategies shows great potential and broad therapeutic versatility, offering a promising CRISPR-based solution for CRC treatment.},
}
@article {pmid42045369,
year = {2026},
author = {Ragucci, AE and Antine, SP and Leviss, EM and Mooney, SE and Garcia, JM and Shyrokova, L and Hauryliuk, V and Lee, ASY and Kranzusch, PJ},
title = {Nuclease-NTPase antiphage defence systems use conserved molecular features to control bacterial immunity.},
journal = {Nature microbiology},
volume = {11},
number = {5},
pages = {1424-1436},
pmid = {42045369},
issn = {2058-5276},
support = {1DP2GM146250-01//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {*Bacteriophages/physiology/genetics/immunology ; *Bacteria/virology/immunology/genetics/enzymology ; *Endonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Bacteria encode diverse defence systems, including restriction-modification and CRISPR-Cas, that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate that many recently identified antiphage defence operons comprise a nuclease and NTPase protein, suggesting that additional nucleic acid-targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyse 16 nuclease-NTPase systems and define molecular features that control antiphage defence. Purification, biochemical characterization and in vitro reconstitution of nucleic acid degradation demonstrates that protein-protein complex formation is a shared feature of multigene nuclease-NTPase systems. We show that PaAbpAB, BtHachiman and EcPD-T4-8 system nucleases use highly degenerate recognition site preferences to enable broad nucleic acid degradation, and the Azaca system exhibits specific phage targeting through the recognition of modified phage genomic DNA. Our results uncover principles of antiphage defence system function and highlight the mechanistic diversity of nuclease-NTPase systems in bacterial immunity.},
}
@article {pmid42045938,
year = {2026},
author = {Dinçer, C and Fussing, B and Garnett, MJ and Coelho, MA},
title = {BEstimate: a computational tool for the design and interpretation of CRISPR base editing experiments.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42045938},
issn = {1474-760X},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Software ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Computational Biology/methods ; },
abstract = {CRISPR base editors enable scalable targeted DNA mutagenesis and are a powerful tool for analysing the function of variants of uncertain significance and disease modelling. Existing guide RNA (gRNA) design tools lack comprehensive functional annotation of target sequences. Here we developed BEstimate, a flexible computational pipeline that systematically specifies base editor gRNA target sites, generates on-target activity and off-target predictions, and provides functional, structural and clinical annotations of installed variants. BEstimate supports custom gRNA design against variant alleles and reversion of disease variants. BEstimate is a freely available, versatile tool for designing gRNA libraries and analysing base editor screens.},
}
@article {pmid42045967,
year = {2026},
author = {Zhang, Y and Wang, Y and Ma, D and Xiao, G and Feng, L and Cai, J and Xu, Y and Wang, Y and Liu, X and Tian, J and Zuo, Z and Lan, J and Shen, B and Ding, S},
title = {Machine learning-advanced hydrogel-based transcription-coupled positive-feedback CRISPR/Cas13a analysis for novel microRNA signatures in differential diagnosis of non-small cell lung cancer.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42045967},
issn = {1477-3155},
support = {82502846//National Natural Science Foundation of China/ ; 2025ZNSFSC1562//Natural Science Foundation of Sichuan Province/ ; 25QNMP119//Health Commission of Sichuan Province Medical Science and Technology Program/ ; 25QNMP041//Health Commission of Sichuan Province Medical Science and Technology Program/ ; 2023CQBSHTB3028//Special Funding for Postdoctoral Research Projects in Chongqing/ ; },
mesh = {*Carcinoma, Non-Small-Cell Lung/diagnosis/genetics/blood ; Humans ; *Machine Learning ; *Lung Neoplasms/diagnosis/genetics ; *MicroRNAs/genetics/blood ; *Hydrogels/chemistry ; *CRISPR-Cas Systems/genetics ; Diagnosis, Differential ; Biomarkers, Tumor/blood/genetics ; Biosensing Techniques/methods ; },
abstract = {MicroRNAs (miRNAs) hold significant potential as biomarkers for the precise diagnosis of non-small cell lung cancer (NSCLC). However, miRNAs remain underused due to their low abundance, high heterogeneity, and complex matrix interference in liquid biopsies, as well as the requirement for specialized techniques. Herein, we devised a hydrogel-based transcription-coupled positive-feedback CRISPR/Cas13a (TCPFC) enhanced electrochemiluminescent (ECL) analyzer for advanced analysis of plasma miRNA signatures via machine learning (ML). Initially, three NSCLC-associated miRNA signatures (miR-203b, miR-450b, and miR-642a) were identified from plasma miRNA datasets and validated using RT-qPCR. An Au@ACZ-SA-PEG hydrogel emitter was engineered to deliver a robust ECL output on a glassy carbon electrode. Additionally, the TCPFC strategy utilized transcription-coupled positive-feedback CRISPR/Cas13a to achieve cascade signal amplification. Concurrently, collateral cleavage eliminated dopamine quenchers, thereby restoring the ECL signal ("OFF-ON") for readout and achieving attomolar-level detection. The integration of ML algorithms with the hydrogel-based TCPFC-ECL platform yielded differential diagnosis accuracies of 100.00% (train, n = 110) and 92.73% (test, n = 110), effectively distinguishing healthy controls (HC) from patients with stages I/II and III/IV NSCLC. Consequently, this biosensing platform demonstrates considerable promise as a practical tool for the precise diagnosis of NSCLC.},
}
@article {pmid42046057,
year = {2026},
author = {Hsu, CY and Abdelgawwad El-Sehrawy, AAM and Alshkarchy, SS and Abdul, AS and Ganesan, S and Gupta, PK and Sharma, R and Nayak, PP and Ebrahimpour, A and Khazaei, Y},
title = {Innovative approaches in the treatment of hematologic malignancies: the role of CRISPR-engineered microbiomes along the gut-immune axis in immunotherapy development.},
journal = {Cancer cell international},
volume = {26},
number = {1},
pages = {},
pmid = {42046057},
issn = {1475-2867},
abstract = {Hematologic malignancies encompass a diverse group of disorders characterized by the abnormal proliferation of blood-forming cells within the bone marrow, lymphatic system, and peripheral blood. These include leukemia, lymphoma, multiple myeloma, and other related conditions, which collectively pose significant health challenges worldwide. Despite advances in diagnosis and treatment, these malignancies often exhibit complex pathophysiology, heterogeneity, and resistance to conventional therapies, necessitating ongoing research to develop more effective and targeted interventions. Originally discovered as an adaptive immune mechanism in bacteria, CRISPR-Cas systems have been adapted for targeted gene editing in human cells. This technology offers unprecedented opportunities to correct genetic mutations, modulate gene expression, and engineer biological systems, including microbiomes. To address these challenges, we review recent strategies that harness CRISPR-engineered gut commensals as precision “living therapeutics” to modulate host immunity and directly target malignant clones. In this review, a living therapeutic is used operationally to mean a nonpathogenic live microorganism engineered with at least one therapeutic module and one control module, for example, a disease-responsive sensing circuit, a payload-production or secretion function, and basic biocontainment or stability safeguards. We organize our discussion around three mechanisms: (i) microbial secretion of immunomodulators (e.g., IL‑15, IFN‑γ, PD‑1/PD‑L1 blockers); (ii) delivery of tumor‑lytic payloads via phage‑ or nanoparticle‑mediated CRISPR systems; and (iii) production of anticancer metabolites (e.g., butyrate, indole derivatives). Preclinical models demonstrate that these engineered strains can reduce tumor burden by > 60%, restore CAR‑T cell function, and overcome drug resistance. We also analyze key technical barriers, strain stability, biocontainment, off‑target effects, and propose solutions including auxotrophic kill switches and AI‑guided strain optimization. Finally, we outline future directions, from in situ phage delivery to multi‑omics–driven patient stratification. CRISPR-microbiome editing represents a promising strategy for hematologic oncology, particularly because blood cancers arise in bone marrow and secondary lymphoid niches rather than in a compact solid mass. Although microbiome–immunotherapy interactions are also important in solid tumors, the strongest translational evidence currently available is concentrated in solid-tumor ICI response studies and in hematologic settings such as CAR-T and HSCT; therefore, this review is intentionally scoped to blood cancers.},
}
@article {pmid42046341,
year = {2026},
author = {Wang, Z and Chen, Y and Wang, Y and Li, W and Zhang, X and Zhang, S},
title = {One-Pot Ligation-Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a-Powered Trimode Lateral Flow Assay for Sensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13365-13376},
doi = {10.1021/acs.analchem.5c07208},
pmid = {42046341},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Gold/chemistry ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Platinum/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of microRNAs (miRNAs) holds significant importance for the early diagnosis of cancer. Since current sensitive nucleic acid detection methods like recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/Cas12a are not suitable for detecting short-stranded miRNAs, we introduced a T4 ligase-based ligation process to the RPA-CRISPR/Cas12a system and developed a novel miRNA detection method termed ligation-RPA-CRISPR/Cas12a (LRCC). This assay utilizes a glycerol-enhanced one-pot reaction strategy combined with a lateral flow assay (LFA) to streamline the operation, minimize aerosol contamination, and improve point-of-care testing performance. Kinetic studies have shown that the catalytic efficiency of the glycerol-enhanced one-pot reaction is 3.11 and 2.09 times higher than that of the direct one-pot and stepwise methods, respectively. By synthesizing "three-in-one" Au-Pt nanostars (Au@Pt NSs) as probes and stabilizing them via "click" chemistry modification, this work enabled a trimode detection approach (colorimetric, photothermal, and surface-enhanced Raman spectroscopy (SERS)) with improved accuracy. In the experiment, tetrahedron DNAs were immobilized on the test line of the strip to enhance the capture efficiency of probes, thereby improving the detection sensitivity. The entire detection process was completed in 70 min with detection limits of 23.6 fM for colorimetric (C-LFA), 2.19 fM for photothermal (P-LFA), and 72.29 aM for SERS (S-LFA). The results demonstrate the strong practical applicability of the LRCC strategy, which plays a crucial role in miRNA-based early disease diagnosis.},
}
@article {pmid42048304,
year = {2026},
author = {Mallon, J and Lenihan, CJ and Shridhar, S and Bailey, S},
title = {Target discrimination and PAM profiling of the Thermotoga maritima type I-B CRISPR system.},
journal = {The Biochemical journal},
volume = {483},
number = {6},
pages = {981-992},
pmid = {42048304},
issn = {1470-8728},
support = {GM097330 T32GM080189//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2T32CA009110//HHS | NIH | National Cancer Institute (NCI)/ ; N/A//Walder Foundation/ ; },
mesh = {*Thermotoga maritima/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; Escherichia coli/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Type I-B CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems represent the most abundant CRISPR subtype in nature and have emerged as powerful tools for endogenous genome editing in diverse prokaryotes. Here we reconstitute and characterize the type I-B1 system from the thermophile Thermotoga maritima (Tma) using purified components. We demonstrate that Tma Cascade requires standalone Cas11 expression, as the cryptic internal translation start site within cas8b1 is non-functional in Escherichia coli. The reconstituted system exhibits canonical type I function including RNA-guided DNA binding, protospacer adjacent motif (PAM)-dependent target discrimination, Cas3-mediated degradation, and seed region interrogation spanning seven PAM-proximal nucleotides. Using next-generation sequencing-based PAM library screens, we define a YYD consensus PAM (Y = C/T; D = G/A/T) with strong discrimination against the array repeat-adjacent sequence (AAC). Comprehensive PAM profiling reveals context-dependent tolerance for non-consensus sequences and identifies numerous intermediate-activity PAMs that may function in priming. Comparison with other characterized type I-B systems reveals a correlation between the Cas8b variant and position -3 specificity, conserved pyrimidine preference at position -2, and variability at position -1. This work establishes a thermostable type I-B platform for biotechnological applications and provides insights into evolutionary mechanisms balancing PAM promiscuity with self-discrimination in the most abundant CRISPR-Cas subtype.},
}
@article {pmid42048640,
year = {2026},
author = {Dominy, C},
title = {CRISPR Diagnostics, in Your Pocket.},
journal = {Journal of medical Internet research},
volume = {28},
number = {},
pages = {e98572},
pmid = {42048640},
issn = {1438-8871},
}
@article {pmid42048928,
year = {2026},
author = {Li, L and Ju, R and Yan, Y and Lou, Y and Chen, H and Wen, Q and Wang, M and Lei, R and Liu, J and Wang, X},
title = {Nanomaterial-enabled CRISPR-Cas biosensing for non-nucleic acid targets: Strategies, mechanisms, and applications.},
journal = {Talanta},
volume = {307},
number = {},
pages = {129831},
doi = {10.1016/j.talanta.2026.129831},
pmid = {42048928},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Nanostructures/chemistry ; Humans ; *Nucleic Acids/analysis ; },
abstract = {Nanomaterial-assisted CRISPR-Cas biosensing has transcended nucleic acid detection, emerging as a prominent paradigm for the ultrasensitive analysis of non-nucleic acid targets. This review systematically elucidates recent advances by proposing a novel three-dimensional framework encompassing transduction efficiency, signal output, and application adaptation. We first focus on molecular engineering approaches that convert non-nucleic acid recognition events into CRISPR activation signals, critically assessing the inherent efficiency limits and bottlenecks of key techniques such as aptamer switches, nuclease allosteric effects, substituent steric hindrance, Cas protein allosteric regulation, and Cas protein engineering. We then detail how various nanomaterials, including AuNPs, QDs, UCNPs, MOFs, nanozymes, synergistically enhance the efficiency of optical, electrochemical, and multimodal signal output. Utilizing this framework, the review analyzes representative applications in detecting proteins, small molecules, and heavy metal ions, clarifying the mechanisms behind performance enhancement and current limitations. Finally, we discuss persistent challenges and prospectively proposes disruptive future directions, such as constructing CRISPR sensing networks, developing intelligent nanocarriers, and creating closed-loop theranostic systems. This study provides a technical overview, a critical framework, and a design roadmap, facilitating the rational design of biosensors and enabling the realization of integrated bio-regulatory platforms.},
}
@article {pmid42049209,
year = {2026},
author = {Da Lage, JL and Bonneau, M and Moreno, C and Le Rouzic, A},
title = {Is the alpha-amylase paralogue Amyrel dispensable in Drosophila melanogaster?.},
journal = {Open biology},
volume = {16},
number = {4},
pages = {},
doi = {10.1098/rsob.250411},
pmid = {42049209},
issn = {2046-2441},
support = {//CNRS/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/metabolism/enzymology ; *Drosophila Proteins/genetics/metabolism ; Female ; *alpha-Amylases/genetics/metabolism ; Male ; CRISPR-Cas Systems ; },
abstract = {Divergent duplicated gene copies are considered to get new or variant function or regulation through sub- or neofunctionalization. In Drosophila and other flies (Muscomorpha), the alpha-amylase paralogue Amyrel is known to have peculiar enzymological properties compared with the classical enzyme Amy. Yet, its real function in fly biology is unclear. Here, we show that Amyrel and Amy share similar regulation patterns such as sugar downregulation and midgut-specific expression in Drosophila melanogaster. Most regulatory information lies within 500 bp of the upstream sequence, as enhanced green fluorescent protein expression under the Amyrel promoter mimics Amyrel expression quite well. To get an insight into Amyrel function, we knocked out the gene using CRISPR-Cas9. Setting a competition experiment between wild-type (wt) and null alleles over 40 generations, we estimated the selective advantage of the wt to be 2%. However, Amyrel-null mutant lines exhibited no clear defect in several life history traits. Interestingly, while Amyrel had very low expression in young adults, it was significantly upregulated in females aged two months; however, lifespan was not affected. Overall, we were able to document substantial functional and regulatory differences between the Amyrel copy and the regular amylase, and we showed that carrying an Amyrel gene conferred a competitive fitness advantage.},
}
@article {pmid42049231,
year = {2026},
author = {Zhou, J and Wang, X and Zhou, L and Zhang, H and Ye, S and Yuan, YJ},
title = {Automated linear DNA assembly of A. thaliana's chloroplast and mitochondrial genome.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42049231},
issn = {1362-4962},
support = {2021YFA0909300//National Key Research and Development Program of China/ ; //Ministry of Science and Technology of the People's Republic of China/ ; 22527901//National Natural Science Foundation of China/ ; //National Major Research Instrumentation Program/ ; JYB2025XDXM503//Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China/ ; 25ZXWCSY00330//Tianjin Municipal Science and Technology Major Program/ ; },
mesh = {*Arabidopsis/genetics ; *Genome, Mitochondrial ; *Genome, Chloroplast ; Escherichia coli/genetics ; Genome, Plant ; Synthetic Biology/methods ; CRISPR-Cas Systems ; },
abstract = {Synthetic genomics is advancing from microbial toward multicellular organisms. However, current manual methods for DNA and genome assembly remain inadequate for the efficient, large-scale production of long DNA constructs. Here, we present Programmed DNA Assembly via Cas9 and Conjugative Transfer (PACT), a method that integrates a linear vector system, bacterial conjugation, and programmable Cas9-mediated cleavage to achieve highly efficient, iterative assembly of large DNA fragments. PACT enhances assembly efficiency by ~30-fold compared to conventional circular vector strategies, enabling one-step assembly of DNA up to 80 kb. We engineered four single guide-RNA-Marker donor cassettes to support iterative assembly workflows. PACT can utilize low-recombination Escherichia coli strains as hosts to efficiently assemble Arabidopsis thaliana mitochondrial genome with high repeat units. Integrated with an automated robotic platform, we developed an unattended, high-throughput pipeline (aPACT) toward large-scale parallel DNA assembly. Using aPACT, we successfully assembled three large DNA constructs: a 210 kb digital DNA, the designed chloroplast (120 kb) and mitochondrial (350 kb) genome of A. thaliana. This automated system offers a powerful tool for scalable assembly of large DNA molecules, accelerating synthetic genomics research toward complex multicellular organisms.},
}
@article {pmid42051075,
year = {2026},
author = {Vats, S and Jadhav, H and Mahakalkar, B and Patil, G and Sonah, H and Sharma, TR and Deshmukh, R},
title = {Genome Editing of a Carotenogenic Gene for Lycopene Enhancement Increases Heavy Metal Stress Susceptibility in Tomato (Solanum lycopersicum L.).},
journal = {Physiologia plantarum},
volume = {178},
number = {3},
pages = {e70884},
doi = {10.1111/ppl.70884},
pmid = {42051075},
issn = {1399-3054},
support = {BT/PR38279/GET/119/351/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; //Anusandhan National Research Foundation, JC Bose Fellowship, Government of India./ ; HSCSIT/R&amp;D/2024/511//Haryana State Council for Science Innovation and Technology (HSCSIT)/ ; },
mesh = {*Solanum lycopersicum/genetics/drug effects/physiology/metabolism ; *Gene Editing/methods ; *Lycopene/metabolism ; Stress, Physiological/genetics/drug effects ; *Metals, Heavy/toxicity ; Fruit/genetics ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Intramolecular Lyases/genetics/metabolism ; Gene Expression Regulation, Plant ; Carotenoids/metabolism ; Plants, Genetically Modified ; },
abstract = {Improving the nutritional quality and abiotic stress tolerance of crop plants is essential for sustainable agriculture and global food security. Recent advances in genome editing, particularly the CRISPR/Cas9 system, have enabled precise modification of metabolic pathways to enhance valuable traits such as carotenoid accumulation. The present study aimed to enhance fruit lycopene content and assess associated stress responses in tomato (Solanum lycopersicum L.) through targeted genome editing of the lycopene beta cyclase (β-LCY, EC 5.5.1.19) gene, encoding for a key enzyme in the carotenoid biosynthetic pathway. A Csy4-based multiplex CRISPR/Cas9 approach was applied to edit β-LCY in three tomato genotypes, including two cultivated varieties and the wild species S. peruvianum L. Genotypic analysis revealed significant genotype-dependent differences in editing efficiency. The β-LCY knockout lines exhibited markedly increased lycopene accumulation in fruits, resulting in enhanced pigmentation. However, when subjected to cadmium stress, these lines showed greater susceptibility than wild-type plants, with pronounced wilting and stress symptoms. Physiological, biochemical, and metabolomic analyses confirmed disruption of stress-response mechanisms associated with carotenoid pathway modification. These findings demonstrate that while genome editing can successfully enhance desirable metabolic traits, it may also impair abiotic stress tolerance. This study provides new insight into the complex interplay between the carotenoid biosynthetic pathway and stress adaptation in tomato.},
}
@article {pmid42051315,
year = {2026},
author = {Nolan, M and Aryal, S and Ndayambaje, IS and Cao, M and Lee, P and Hovde, M and Yun, S and Wlaschin, J and Held, A and Beaussant, H and Wymann, B and Zong-Lee, C and Lim, SM and Jiang, X and Ramesh, N and Agra Almeida Quadros, AR and Boulos, A and Zinter, N and Salem, S and El-Tayar, L and Beccari, M and Presa, M and Jourdan Ferreras Reyes, C and Ruan, YY and Griesman, G and Aguilar, C and Hawrot, J and Wheeler, H and Melamed, Z and Kleinstiver, BP and Albers, M and Cleveland, DW and Tanzi, RE and Lutz, CM and Hubbard, RD and Kobayashi, D and Ward, M and R R Alves, C and Wainger, B and Pichon, CL and Lagier-Tourenne, C},
title = {Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42051315},
issn = {2692-8205},
abstract = {Loss of neuronal regenerative capacity is a common feature of neurodegenerative disease and axonal injury, yet the transcriptional programs governing this state remain poorly defined. Stathmin-2 (STMN2), a tubulin-binding protein essential for axon maintenance and repair, is profoundly depleted following loss of nuclear TDP-43 in neurodegenerative disease. Here, we identify statins as potent inducers of STMN2 expression. Pharmacological and genetic suppression of the mevalonate pathway, and subsequent prevention of protein geranylgeranylation, restored STMN2 levels in TDP-43 deficient cells and promoted neurite growth. STMN2 induction was abrogated when using a statin analogue unable to interact with HMG-CoA reductase, and through co-administration of mevalonate or geranylgeranyl diphosphate substrates. RNA-seq revealed that statins induce a coordinated pro-regenerative transcriptional response, including activation of the AP-1 transcription factor complex gene, ATF3. Loss of ATF3 attenuated STMN2 induction in vitro, and diminished injury-induced Stmn2 upregulation in spinal motor neurons in vivo. These results demonstrate statins as modulators of ATF3 and STMN2 expression and highlight their therapeutic potential in neurodegenerative disease.},
}
@article {pmid42052944,
year = {2026},
author = {Chen, Z and Hong, W and Wei, X and Li, Y and Feng, T and Ke, X and Li, X and Wang, Y and Hang, H and Tian, X and Chu, J},
title = {A Versatile tRNA-gRNA Array-Based CRISPR/Cas9 Platform Enabling Multiplex Genome Editing and Large-Fragment Engineering in Acremonium chrysogenum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1955-1967},
doi = {10.1021/acssynbio.6c00052},
pmid = {42052944},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Acremonium/genetics/metabolism ; *RNA, Transfer/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Cephalosporins/biosynthesis ; Metabolic Engineering/methods ; Gene Knockout Techniques ; },
abstract = {Cephalosporin C (CPC)-derived antibiotics have played a vital role in improving human health and extending life expectancy. Acremonium chrysogenum remains the only microorganism capable of industrial-scale CPC production to date. However, the lack of efficient multiplex genome-editing tools has limited studies on its gene function, high-yield mechanisms as well as metabolic engineering. To overcome this limitation, a rapid and efficient CRISPR/Cas9-based multiplex genome-editing system was developed, driven by endogenous tRNA promoters, enabling one-step multilocus knockout, large-fragment DNA deletion, and gene overexpression in A. chrysogenum. Given that many strains lack visible phenotypes associated with specific genes, we introduced a visually distinguishable red phenotype by expressing the heterologous protein mCherry under a strong promoter. In the wild-type strain, 20 endogenous tRNA promoters were evaluated and compared to the heterologous Aspergillus nidulans PgpdA and Aspergillus fumigatus U6 promoters. The endogenous tRNA[Val] promoter showed the highest knockout efficiency (95.5%). The tRNA-gRNA array-based CRISPR/Cas9 system enabled double- and triple-site knockouts without donor DNA in industrial strain, with efficiencies of 50.0-83.3% and 14.3%, respectively. This is the first demonstration of simultaneous triple-site knockout in A. chrysogenum, especially in industrial strain. Using this system, we successfully deleted a 50.7-kb DNA fragment containing the sorbicillinoids biosynthetic gene cluster with nearly 100% efficiency and achieved overexpression of the key gene pcbAB involved in CPC biosynthesis in high-yield strain, increasing CPC titer from 5.59 g/L to 6.92 g/L with an improvement of 23.8%. Overall, this tRNA-gRNA array-based CRISPR/Cas9 multiplex gene-editing system provides an efficient and versatile platform for functional genomics and industrial strain engineering in A. chrysogenum.},
}
@article {pmid42053263,
year = {2026},
author = {Turowski, P and Gatermann, SG and Pfennigwerth, N},
title = {Cefiderocol resistance mediated by mutation of the miniconductance mechanosensitive channel MscM in Klebsiella oxytoca.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {5},
pages = {},
doi = {10.1093/jac/dkag147},
pmid = {42053263},
issn = {1460-2091},
support = {//Robert Koch Institute/ ; 1369-402//German Ministry of Health/ ; },
mesh = {*Klebsiella oxytoca/drug effects/genetics/growth &amp; development/isolation &amp; purification ; *Anti-Bacterial Agents/pharmacology ; *Cephalosporins/pharmacology ; Microbial Sensitivity Tests ; Humans ; Klebsiella Infections/microbiology ; Cefiderocol/pharmacology ; *Mutation ; *Drug Resistance, Bacterial/genetics ; *Ion Channels/genetics ; *Bacterial Proteins/genetics ; Whole Genome Sequencing ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVE: To investigate the emergence of cefiderocol resistance in a clinical Klebsiella oxytoca isolate and to identify the underlying mechanism.<br><br>METHODS: A clinical isolate of K. oxytoca susceptible to cefiderocol was exposed to stepwise increasing cefiderocol concentrations via broth microdilution in iron-depleted CAMHB (ID-CAMHB) to select spontaneous mutants. WGS identified potential resistance-associated mutations. CRISPR-Cas9 genome editing was used to confirm causality. Growth curves in CAMHB and ID-CAMHB were performed to assess potential growth alterations.<br><br>RESULTS: A spontaneous mutant with elevated cefiderocol MIC (16&#x2005;mg/L) carried a Q1008L substitution in the miniconductance mechanosensitive channel MscM. CRISPR-edited strains reproduced this phenotype. Growth kinetics did not reveal an obvious growth defect under the tested in vitro conditions.<br><br>CONCLUSION: This is the first report linking cefiderocol resistance to a mutation in MscM in K. oxytoca. Although observed in a single isolate, the lack of an apparent growth defect under the tested conditions suggests that this resistance mechanism may persist in the absence of antibiotic pressure.},
}
@article {pmid42053665,
year = {2026},
author = {Lei, M and Bakhsh, MZM and Zhang, X and He, D and Dai, C and Ma, C and Tu, J and Shen, J and Wen, J and Fu, T and Yi, B},
title = {From haploid inducer to CMS donor: repurposing of CENH3 to create a CMS line in a single step in Brassica napus.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {42053665},
issn = {1432-203X},
support = {2024ZD04077//Biological Breeding-National Science and Technology/ ; },
mesh = {*Haploidy ; *Brassica napus/genetics ; *Plant Infertility/genetics ; *Plant Proteins/genetics/metabolism ; Plant Breeding/methods ; Cytoplasm/genetics ; CRISPR-Cas Systems/genetics ; *Histones/genetics/metabolism ; Mutation ; Centromere/genetics/metabolism ; },
abstract = {Mutation in centromere histone H3 (CENH3) protein could induce a paternal haploid with maternal cytoplasm in rapeseed. By paternal haploid induction, a cytoplasmic male sterile line can be created in any genetic background within one breeding cycle. Hybrid development in rapeseed relies primarily on the three-line system, which includes a cytoplasmic male sterile (CMS) line. Conventionally, these CMS lines are developed through backcrossing, a process that requires several breeding cycles to complete. More recently, the doubled haploid technique has been employed in various crops to generate homozygous lines within a single breeding cycle. In the present study, we utilized a haploid induction (HI) strategy to produce fertile homozygous lines and CMS lines via paternal haploid induction. We have created single homozygous and double heterozygous mutants of the BnaCENH3 gene in the rapeseed cultivar ganA (hau-CMS) using CRISPR/Cas9 technique. Upon hybridization of CMS-HI line with wild type can successfully induced paternal haploids with maternal sterile cytoplasm. This system offers the ability to introduce sterile cytoplasm into any genetic background within a single generation.},
}
@article {pmid42053710,
year = {2026},
author = {Panchuk, IO and Grigorieva, OV and Kurshakova, EV and Nagieva, SE and Shchagina, OA and Levchenko, OA and Pozhitnova, VO and Voronina, ES and Tabakov, VY and Smirnikhina, SA and Lavrov, AV},
title = {Generation of an Induced Pluripotent Stem Cell Line from a Duchenne Muscular Dystrophy Patient Carrying the DMD p.Ser429Ter (c.1286C&gt;G) Nonsense Mutation.},
journal = {Bulletin of experimental biology and medicine},
volume = {180},
number = {4},
pages = {548-554},
pmid = {42053710},
issn = {1573-8221},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Muscular Dystrophy, Duchenne/genetics/pathology/metabolism ; Kruppel-Like Factor 4 ; *Codon, Nonsense/genetics ; Cell Differentiation ; *Dystrophin/genetics/metabolism ; Cell Line ; Male ; Fibroblasts/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; CRISPR-Cas Systems ; Cellular Reprogramming ; Sendai virus/genetics ; },
abstract = {An induced pluripotent stem cell (iPSC) line was successfully generated from dermal fibroblasts of a patient with Duchenne muscular dystrophy (DMD) harboring the pathogenic nonsense variant c.1286C>G (p.Ser429Ter) in the DMD gene using non-integrating Sendai virus reprogramming. The iPSC clone exhibited typical pluripotent stem cell morphology, expressed key pluripotency markers (OCT4, SSEA4, NANOG, and TRA-1-60), and retained trilineage differentiation potential. The cell line had a normal karyotype, and elimination of reprogramming vectors (OCT3/4, SOX2, KLF4, and c-MYC) was confirmed. This isogenic cell model provides a valuable platform for investigating DMD pathogenesis associated with this specific mutation and for developing targeted therapeutic approaches, including CRISPR/Cas9-mediated gene correction.},
}
@article {pmid42053928,
year = {2026},
author = {Deres, D and Terefe, M},
title = {Beyond CRISPR/Cas9: emerging genome editing technologies for next-generation crop improvement.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42053928},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Genome, Plant/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Genome editing has changed plant biology and accelerated crop improvement. CRISPR/Cas9 allows precise and efficient genetic changes in many species. Still, Cas9 has limits like PAM restrictions, off-target effects, and varying editing success. This led to new systems. Editors like Cas12a, CasΦ, CasMINI, and CasX offer more targeting options, can edit RNA, and work better with hard to edit plant genomes. Precision tools such as base editors and prime editors make precise changes by swapping nucleotides or adding small pieces without cutting both DNA strands. This improves accuracy. Beyond single tools, combined and step by step editing methods can be used for handling complex traits controlled by many genes. Using several methods like CRISPR knockouts, base and prime editing, epigenome editing and recombinase systems-breeders can improve traits while reducing unwanted side effects. Stepwise editing helps to test changes, confirm their effects, and improve entire biological pathways. Combining these approaches with AI-driven analysis, target prediction, and design optimization makes it easier to pick the best genes and edits for desired traits. These advanced editing methods are used to boost stress tolerance, fight diseases, improve nutrition, increase yields, and enhance quality after harvest. Despite progress, problems remain with how efficient edits can be made, delivering tools into plants, reliance on specific genotypes, unclear regulations, and acceptance by society. Looking ahead, joying genome editing with AI, fast breeding techniques help develop stronger, high yielding crops and support global food security.},
}
@article {pmid42054542,
year = {2026},
author = {Gu, T and Xue, J and Zhang, Z and Cao, J and Song, J and Li, G and Ming, L and Zhu, Z and Wang, H},
title = {Mechanisms of Resistance to ALS Inhibitors and Bentazone in Fimbristylis littoralis and Rapid Identification of the ALS Trp-574-Leu Mutation Using LAMP-CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {18},
pages = {14311-14321},
doi = {10.1021/acs.jafc.5c17149},
pmid = {42054542},
issn = {1520-5118},
mesh = {*Acetolactate Synthase/genetics/antagonists &amp; inhibitors/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism/antagonists &amp; inhibitors/chemistry ; Herbicide Resistance ; Mutation ; CRISPR-Cas Systems ; *Enzyme Inhibitors/pharmacology ; },
abstract = {Fimbristylis littoralis Gaudich., a harmful sedge weed in Chinese rice paddy, impairs rice productivity and quality. In this study, we identified a resistant population (FL2) displaying multiple resistance to pyrazosulfuron-ethyl and bentazone, alongside cross-resistance to other acetolactate synthase (ALS)-inhibiting herbicides. The other population (FL6) showed exclusive resistance to bensulfuron-methyl. Sequencing demonstrated that FL2 carried a Trp-to-Leu mutation at codon 574 of ALS, whereas no mutations were detected in the psbA gene of bentazone-resistant FL2 or the ALS gene of bensulfuron-methyl-resistant FL6. Studies on nontarget-site resistance (NTSR) mechanisms indicated that FL2's resistance to pyrazosulfuron-ethyl was associated with neither PBO-inhibited P450s nor NBD-Cl-inhibited GSTs. In contrast, FL6's resistance to bensulfuron-methyl and FL2's resistance to bentazone were linked to P450 activity. A loop-mediated isothermal amplification (LAMP) coupled with CRISPR/FnCas12a assay was established for rapid detection of the Trp-574-Leu mutation, facilitating resistance management. These findings provide insights for managing resistant F. littoralis populations.},
}
@article {pmid42054950,
year = {2026},
author = {Wang, H and Yang, H and Zhong, K and Wang, R and Zhao, J and Zhao, Y and Zhang, G},
title = {Immune efficacy of two recombinant Turkey herpesviruses expressing the fusion protein of Newcastle disease virus genotype VII and hemagglutinin protein of H9N2 avian influenza virus generated by HDR/NHEJ-CRISPR/Cas9 systems.},
journal = {Veterinary microbiology},
volume = {318},
number = {},
pages = {111047},
doi = {10.1016/j.vetmic.2026.111047},
pmid = {42054950},
issn = {1873-2542},
mesh = {Animals ; *Influenza A Virus, H9N2 Subtype/immunology/genetics ; *Newcastle disease virus/genetics/immunology ; *Herpesvirus 1, Meleagrid/genetics/immunology ; *Influenza in Birds/prevention &amp; control/virology/immunology ; CRISPR-Cas Systems ; Chickens ; *Viral Fusion Proteins/immunology/genetics ; Newcastle Disease/prevention &amp; control/virology ; *Poultry Diseases/prevention &amp; control/virology ; Genotype ; *Viral Vaccines/immunology ; Virus Shedding ; *Hemagglutinin Glycoproteins, Influenza Virus/immunology/genetics ; Chick Embryo ; Vaccines, Synthetic/immunology ; Vaccines, Attenuated/immunology ; Gene Editing ; },
abstract = {Newcastle disease viruses (NDV) and H9N2 avian influenza viruses (AIV) are two major threats to poultry farming. Current vaccination programs against two diseases are complex, requiring considerable labor and resources, and repeated immunizations can induce stress in animals. Therefore, developing a simplified, single-dose strategy capable of providing protection against both infections is highly desirable. Recombinant turkey herpesviruses (rHVT)-based live vaccines provide an attractive and effective platform for controlling avian viral diseases. This study generated two rHVTs, rHVT-OHA-OF(U) and rHVT-OHA-OF(H), each co-expressing F protein of NDV strain aSG10 and HA protein of H9N2 strain G, using a homologous directed repair (HDR) and non-homologous end-joining (NHEJ)-dependent CRISPR/Cas9-based gene editing strategy. In vitro, the two rHVTs correctly expressed the F and HA protein. After 10 passages in primary chicken embryonic fibroblasts (CEF) cells, the exogenous proteins remained stable expressed. In vivo, although rHVT-OHA-OF(H) provided strong protection against H9N2 strain G shedding at 3 and 5 days post-challenge (dpc), it offered no significant protection against mortality following challenge with NDV strain SG10. In contrast, rHVT-OHA-OF(U) provided 100% protection against mortality and significantly suppressed viral shedding following challenge with SG10, as well as achieving a significantly reduction of viral shedding at 3 dpc after H9N2 strain G challenge, indicating a promising vaccine candidate against both viral diseases, which requires further optimization. This study provided a reference for developing rHVT-based live vaccines targeting NDV and/or H9N2, thereby establishing a foundation for the design of dual- or multi-insert rHVTs.},
}
@article {pmid42056528,
year = {2026},
author = {Marino, ND and Talaie, A and Gerovac, M and Rodriguez, JL and Schmidt, AD and Astmann, TJ and Carion, H and Taylor, AF and Liliedahl, J and Haniyur, S and Zoga, K and Johnson, MC and Buhlmann, L and Chen, KH and Silas, S and Yuping, L and Zhang, Y and Swaney, DL and Vogel, J and Bondy-Denomy, J},
title = {Translation-dependent degradation of cas12 mRNA triggered by an anti-CRISPR.},
journal = {Nature},
volume = {654},
number = {8119},
pages = {771-776},
pmid = {42056528},
issn = {1476-4687},
mesh = {*CRISPR-Associated Proteins/genetics/chemistry/metabolism/biosynthesis ; *Protein Biosynthesis ; *RNA, Messenger/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA Stability ; Ribosomes/metabolism ; Protein Domains ; *Viral Proteins/metabolism/chemistry/genetics ; Bacteriophages/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/biosynthesis/chemistry ; Conserved Sequence ; Bacterial Proteins ; },
abstract = {Bacteria encode diverse defence systems, including CRISPR-Cas, to recognize and cleave the DNA of bacteriophages (phages) and other mobile genetic elements[1]. In response, phages encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas activity by blocking DNA binding or cleavage[2]. Here we report an unexpected mechanism by which the anti-CRISPR AcrVA2 inhibits Cas12a biogenesis. AcrVA2 binds conserved and functionally important amino acid residues near the Cas12a N-terminus and triggers selective degradation of cas12a mRNA as it is translated. Additionally, conserved residues in the AcrVA2 C-terminal domain enable co-sedimentation with ribosomes and polysomes, which is required to achieve targeted co-translational mRNA degradation. The AcrVA2 C-terminal domain is broadly conserved in homologs encoded by diverse mobile genetic elements, typically in hosts that lack cas12a, suggesting that these homologues may recognize and downregulate alternative substrates in other bacteria. These findings reveal a novel mechanism for molecular conflict and gene regulation in bacteria.},
}
@article {pmid42057199,
year = {2026},
author = {Hatem, H and Mysara, M and Ramadan, R},
title = {Trends of nucleic acid - based point-of-care diagnostics for infectious diseases.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {},
pmid = {42057199},
issn = {1754-1611},
abstract = {The global emergence and spread of infectious diseases highlights the importance of having an easily accessible, decentralized testing modality that is adept at quickly identifying the nucleic acids present in a Point-of-Care (PoC) setting. PoC-based nucleic acid diagnostics encompass a broad range of platforms, such as lateral flow, biochips, and biosensors, whose primary goal has always been to offer sensitive, selective, and economically viable testing beyond conventional laboratory facilities. The current review illustrates a comprehensive overview of PoC-based nucleic acid diagnostics for infectious diseases, as it is divided into three major operational steps: nucleic acid extraction, amplification, and detection. Firstly, it describes strategies that could effectively work as a PoC, such as magnetic bead-based, paper-based, and integrated microfluidic approaches, while further focusing on their ability to remain simplified, rugged, and equipment-free. Secondly, it summarizes key amplification methods, such as Polymerase Chain Reaction (PCR), Nucleic Acid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), and Loop-Mediated Isothermal Amplification (LAMP), as they have all been modified to effectively work under more rapid, low-power, and portable conditions. Lastly, it introduces all presently known detection platforms, starting from simpler colorimetric and fluorescent assays present in lateral flow or device platforms, to innovative and advanced biosensors like those exploiting CRISPR/Cas systems and toehold switch principles, as these detectively offer highly sensitive and highly programmable nucleic acid recognition.},
}
@article {pmid42058176,
year = {2026},
author = {McNiven, C and Carnielli Trindade, JB and Geoghegan, V and Faria, JRC and Mottram, JC},
title = {CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1788564},
pmid = {42058176},
issn = {2235-2988},
mesh = {*Leishmania mexicana/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Kinetochores/metabolism ; *Protozoan Proteins/genetics/metabolism ; Phosphorylation ; *Codon/genetics ; },
abstract = {Leishmania mexicana, like other trypanosomatids, possess a unique kinetochore-the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160 bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.},
}
@article {pmid42059198,
year = {2026},
author = {Hellmer, H and Mayer, T and Bauersachs, L and Simmel, FC},
title = {Operating CRISPR/Cas12a in a complex nucleic acid sequence background.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42059198},
issn = {1362-4962},
support = {SI761/5-1//Deutsche Forschungsgemeinschaft/ ; 453249455//Deutsche Forschungsgemeinschaft/ ; CRC392 TP A5//Deutsche Forschungsgemeinschaft/ ; 521256690-TPA5//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA/metabolism/genetics/chemistry ; Kinetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Gene Editing/methods ; Base Composition ; Base Sequence ; Humans ; },
abstract = {Since their discovery, CRISPR-Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA (gRNA) design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a-a Class 2, Type V effector-and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR-Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the gRNA seed region significantly affect kinetic inhibition by background polynucleotides. gRNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. A gRNA with high uracil content in the seed region exhibited particularly strong inhibition in the presence of a dsDNA background. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.},
}
@article {pmid42059627,
year = {2026},
author = {Price, C and Lucas, JL and Davis, P and Smith, C and Jarvis, E and Fiore, J and Russell, JA and Winegar, R},
title = {CasCADE: Cas-CRISPR Automated Design and Evaluation for targeted gRNA detection assays.},
journal = {Microbiology spectrum},
volume = {14},
number = {6},
pages = {e0292025},
pmid = {42059627},
issn = {2165-0497},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Software ; Sensitivity and Specificity ; Humans ; },
abstract = {UNLABELLED: The adaptation of CRISPR technologies for molecular detection marks a significant advancement in the field of biosurveillance and infectious disease response. CRISPR-based detection systems offer superior specificity and sensitivity compared to traditional PCR methods by directly binding and cleaving target DNA or RNA sequences, thus signaling the presence of specific pathogens. These advantages include the elimination of non-specific amplification and the reduction of required genetic material, leading to faster time to results without the need for extensive amplification cycling. However, the efficacy of CRISPR technologies heavily depends on the design of specific guide RNA (gRNA) sequences tailored for each genomic target, a process that can be intricate and time-consuming. We present Cas-CRISPR Automated Design and Evaluation (CasCADE), a state-of-the-art gRNA design software platform with a high degree of flexibility and modularity. CasCADE incorporates k-mer set operations to reduce time to answer for large data inputs when compared to computationally costly multiple sequence alignment methodologies and uses an agnostic whole genome approach to maximize gRNA discovery. CasCADE can be scaled efficiently to problems of any input sequence size and can be used for design, candidate evaluation, or both, depending on user need.<br><br>IMPORTANCE: This work describes our software pipeline Cas-CRISPR Automated Design and Evaluation (CasCADE) that allows for in silico design of Cas-CRISPR detection assays. We demonstrate the viability of our design process in the lab and report 15 successful designs across nine diverse target organisms. The rapid time to answer afforded by CasCADE, combined with the superior specificity and sensitivity offered by emergent CRISPR detection assays compared to traditional PCR methods, makes this a timely contribution to pandemic preparedness and biosurveillance interests.},
}
@article {pmid42060320,
year = {2026},
author = {Zhong, N and Wang, M and Jiang, W and Li, G and Miao, J and Yin, H and Vanhnaseng, P and Gong, J and Yu, Z and Han, X},
title = {One-pot CRISPR/Cas12b-LAMP platform for dual-mode detection of Pasteurella multocida.},
journal = {Letters in applied microbiology},
volume = {79},
number = {5},
pages = {},
doi = {10.1093/lambio/ovag044},
pmid = {42060320},
issn = {1472-765X},
support = {2024-02-08-00-12-F00051//Shanghai Agriculture Applied Technology Development Program/ ; 2023YFD1800700//National Key Research and Development Program of China/ ; 2025I0030//External Cooperation Program of Fujian Science and Technology Plan Project/ ; },
mesh = {*Pasteurella multocida/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Animals ; *Molecular Diagnostic Techniques/methods ; Milk/microbiology ; *Pasteurella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; },
abstract = {Pasteurella multocida is a significant bacterial pathogen that poses a significant threat to public health and causes substantial economic losses. Existing detection methods for P. multocida have limitations, including time-consuming and technically complex methods. Here, we describe a simple and accurate detection platform that combines loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system. By using heparin sodium to modulate the cis-cleavage activity of Cas12b, we developed a one-pot LAMP-CRISPR/Cas12b assay in a single closed tube. The assay achieved a detection limit of 5.0 × 101 CFU ml-1 and showed no cross-reactivity with other bacterial species, indicating high sensitivity and specificity. Furthermore, we validated the clinical utility of the platform using milk samples artificially contaminated with P. multocida, which successfully detected P. multocida in the LAMP-CRISPR/Cas12b results. In summary, this study establishes a novel and robust detection system for P. multocida and highlights its potential for nucleic acid-based diagnostics in practical applications.},
}
@article {pmid42060714,
year = {2026},
author = {Jung, M and Wen, Z and Humbert, S and Lu, F and DeLeon, A and Marshall, L and Hastings, C and Cartwright, H and Thilges, K and Wang, N and Breckenridge, K and Wu, E and Ryan, L and Fengler, K and Simcox, K and Thatcher, S and Llaca, V and Woollums, G and Sander, J and Xu, D and Beatty, M and Brink, K and Fedorova, M and Jones, M and Ohlson, E and Suresh, LM and Beyene, Y and Olsen, M and Ogugo, V and Alakonya, A and Murithi, A and Mugo, S and Karanja, J and Boddupalli, P and Pixley, K and Albertsen, M and Jones, T and Meeley, R and Gutterson, N and Mazur, B and Dhugga, KS},
title = {Targeted knockout of a host peroxisomal peptidase confers field resistance to maize lethal necrosis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {18},
pages = {e2535202123},
pmid = {42060714},
issn = {1091-6490},
support = {OPP1193833//Bill and Melinda Gates Foundation (GF)/ ; INV-007833/GATES/Gates Foundation/United States ; },
mesh = {*Plant Diseases/virology/genetics ; *Zea mays/virology/genetics ; *Disease Resistance/genetics ; *Peroxisomes/enzymology/genetics ; Potyvirus/pathogenicity ; Gene Knockout Techniques ; *Peptide Hydrolases/genetics/metabolism ; Quantitative Trait Loci/genetics ; Tombusviridae/pathogenicity ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; },
abstract = {Maize lethal necrosis (MLN) is a severe disease caused by the combined infection of maize chlorotic mottle virus (MCMV) and a potyvirus, most often sugarcane mosaic virus (SCMV). This disease seriously threatens food security across sub-Saharan Africa (SSA). We investigated a major-effect quantitative trait locus for resistance on chromosome 6, named the maize lethal necrosis susceptibility locus 1 (qMLNS1), derived from the Thai line KS23-6. Fine mapping and CRISPR-Cas9 editing of the candidate genes within the narrowed 105 kb interval revealed a peroxisomal peptidase as the underlying cause of susceptibility. Confocal microscopy confirmed the localization of the MLNS1 protein within peroxisomes. Targeted knockout of the Mlns1 gene in the susceptible elite line CML536 from SSA conferred resistance comparable to KS23-6 in field trials conducted in Naivasha, Kenya. This knockout specifically blocked MCMV accumulation without affecting SCMV. The edited lines showed no yield penalty or agronomic defects under disease-free conditions. Our findings uncover a mechanistic link between a peroxisomal enzyme and viral susceptibility. They also establish a rapid, scalable gene editing strategy for incorporating MLN resistance into elite germplasm, offering a model for combating similar viral diseases in staple crops globally.},
}
@article {pmid42061344,
year = {2026},
author = {Han, X and Chen, H and Chang, Y and Zha, J and Lam, CYK and Yang, M and Wong, SHD and Yin, B},
title = {Surface-confined CRISPR-Cas12a biosensor with metal-enhanced fluorescence for rapid and ultrasensitive detection of SARS-CoV-2 nucleocapsid protein.},
journal = {Biosensors &amp; bioelectronics},
volume = {306},
number = {},
pages = {118649},
doi = {10.1016/j.bios.2026.118649},
pmid = {42061344},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *SARS-CoV-2 ; *Coronavirus Nucleocapsid Proteins/isolation &amp; purification ; Phosphoproteins/isolation &amp; purification ; Fluorescent Dyes ; Limit of Detection ; Signal-To-Noise Ratio ; Metal Nanoparticles ; Silicon Dioxide ; Gold ; },
abstract = {CRISPR-Cas12a integrated with nanomaterials has formulated powerful biosensors for viral protein detection, addressing the urgent need for point-of-care diagnostics. However, existing platforms are hindered by either multi-step separation procedures or insufficient signal amplification, limiting their sensitivity and practicality. Here, we report a one-pot "on-off" biosensor that combines metal-enhanced fluorescence (MEF) and nanoscale spatial confinement by co-localizing both reporter substrates and the CRISPR-Cas12a system on gold-silica core-shell nanoparticles (Au@SiO2 NPs), enabling rapid and ultrasensitive protein detection. Using SARS-CoV-2 nucleocapsid (N) protein as a model analyte, Au@SiO2 NPs are co-functionalized with (i) ssDNA activators blocked by N protein-specific aptamers, (ii) light-up hairpin DNA (DAP) complexed with auramine O (AO) as reporters, and (iii) short polyethylene glycol (PEG) spacers to mitigate steric hindrance. The nanoplatform displays an ultrabright "on-state" fluorescence, with an intensity >860-fold higher than that of free AO, enabled by the interaction with DAP and optimized fluorophore-metal spacing (∼20 nm). Upon target binding, aptamer displacement exposes the activator to locally initiate Cas12a trans-cleavage, disrupting proximal DAP structure and its interaction with AO, thereby producing a distinct "off-state" signal. Within the linear detection range, the system demonstrates up to ∼85% signal reduction relative to the initial signal and a signal-to-noise ratio (SNR) of 83.89, corresponding to a ∼2.5-fold higher SNR than the solution-phase system. The platform attains a limit of detection at 67.2 fg/mL within 30 min, with excellent sensitivity, selectivity, stability, and recovery in bronchoalveolar lavage fluid. By combining MEF-driven signal amplification with surface-confined CRISPR-Cas12a trans-cleavage, this platform establishes an efficient strategy for sensitive N protein detection.},
}
@article {pmid42061542,
year = {2026},
author = {Yuan, Y and Ni, B and Tian, X and Cui, J and Zhang, Y and Hu, Y and Zheng, D and Zou, Y and Yu, X and Liu, C and Liu, S and Ren, W and Chang, X and Wang, Y and Ge, S and Wei, R and Chen, Y and Wu, X and Li, J and Wang, Z and Huang, B},
title = {Development of a point-of-care diagnostic method for FMDV SAT2 using RT-RAA-CRISPR technology.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152271},
doi = {10.1016/j.ijbiomac.2026.152271},
pmid = {42061542},
issn = {1879-0003},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation &amp; purification ; Animals ; *Foot-and-Mouth Disease/diagnosis/virology ; *CRISPR-Cas Systems ; Cattle ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {In recent years, the foot-and-mouth disease virus (FMDV) serotype SAT2 has expanded beyond its traditionally recognized endemic areas on the African continent, leading to continuous dissemination in Middle Eastern countries and presenting a risk of further spread to Asia. In this study, the highly conserved sequence of the FMDV SAT2 genome was initially selected as the target, and five pairs of reverse transcription-recombinase-aided amplification (RT-RAA) primers were designed. By comparing fluorescence signal intensities, the CRISPR RNA (crRNA) with the highest sensitivity and specificity was identified and subsequently integrated with the CRISPR/Cas13a gene-editing system to establish a novel nucleic acid detection method. This method possesses a robust capacity for differential diagnosis and shows no cross-reaction with other serotypes of FMDV. Moreover, this method demonstrated high specificity and no cross-reactivity with the nucleic acid sequences of various common pathogens in porcine and bovine populations. The test results are readily interpretable and can be directly visualized using a fluorescence reader or lateral flow test strips (LFSs). In simulated clinical samples, this method achieved a concordance rate of 100% with the detection results of fluorescence quantitative RT-PCR. This study successfully developed a highly sensitive and specific FMDV SAT2 nucleic acid detection method based on RT-RAA-CRISPR/Cas13a technology. This method is straightforward to perform, does not require costly experimental equipment, and is suitable for rapid onsite detection, offering a convenient and efficient diagnostic tool for the early diagnosis, prevention, and control of FMDV SAT2 epidemics.},
}
@article {pmid42061934,
year = {2026},
author = {Gunasekaran, H and Najwa, KV and Nidarshan, NC and Porkodi, M and Singh, LS and Rasal, KD and Brahmane, MP and Goswami, M and Sonwane, AA},
title = {Characterization of Trachinotus blochii mstnb gene and construction of cognate gRNA vector.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152225},
doi = {10.1016/j.ijbiomac.2026.152225},
pmid = {42061934},
issn = {1879-0003},
mesh = {Animals ; *Myostatin/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Fishes/genetics ; Amino Acid Sequence ; Base Sequence ; *Fish Proteins/genetics ; },
abstract = {Skeletal muscle development is negatively regulated by the myostatin b (mstnb) gene in finfishes. CRISPR-Cas9-mediated knockout of mstnb has been used to develop fish strains with increased muscle mass. Trachinotus blochii (Silver Pompano), a high-value, moderately sized (~500 g), and cultivable marine finfish, is a promising candidate for mstnb knockout using CRISPR/Cas9. However, limited studies exist on the characterization of T. blochii mstnb and no CRISPR-based knockout studies have been reported in this species. This study aimed to partially characterize the mstnb gene of T. blochii and develop a CRISPR/Cas9-based guide RNA (gRNA) expression vector for its knockout. The T. blochii mstnb sequence available in NCBI was used as a reference to design exon- and intron-specific primers for PCR amplification and sequencing. The sequence revealed three exons and two introns. A predicted 1131 bp open reading frame encodes a 376 amino acid protein containing conserved domains typical of the TGF-β family which includes an N-terminal signal peptide, a propeptide region, a conserved RARR cleavage motif, and a C-terminal GF domain containing nine conserved cysteine residues. Secondary and 3D structure predictions confirmed the protein's functional integrity. Sequence analysis revealed novel putative polymorphisms, including SNPs and a (CA)n microsatellite. Phylogenetic analysis clustered T. blochii with related Trachinotus and other Carangiformes species. A gRNA targeting exon 1 was designed using CRISPOR and successfully cloned into expression vectors. This novel sequence information can aid population-level studies and genetic marker discovery. The constructed gRNA vectors can facilitate CRISPR/Cas9-mediated mstnb knockout in T. blochii to study gene function and develop a fleshy strain.},
}
@article {pmid42062574,
year = {2026},
author = {Xu, Y and Stubbendieck, RM and Viswanatha, R and Krč, A and Baik, LS and Suh, WS and Hu, Y and Wang, H and Yin, L and Mameli, E and van der Meij, A and Carlson, JR and Doxey, AC and Stenmark, P and Perrimon, N and Currie, CR and Dong, M},
title = {Streptomyces produce a diphtheria toxin-like exotoxin that targets insects.},
journal = {Nature microbiology},
volume = {11},
number = {5},
pages = {1271-1285},
pmid = {42062574},
issn = {2058-5276},
support = {R01AI170835//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI189789//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01NS080833//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Animals ; *Streptomyces/metabolism/genetics ; Drosophila melanogaster/drug effects ; Virulence Factors/metabolism/genetics ; *Diphtheria Toxin/genetics/metabolism/chemistry ; *Insecta/drug effects/microbiology ; Bacterial Proteins/genetics/metabolism/chemistry ; *Exotoxins/metabolism/genetics/chemistry/toxicity ; Phylogeny ; *Insecticides/metabolism ; Peptide Elongation Factor 2/metabolism ; CRISPR-Cas Systems ; Insect Proteins/metabolism ; },
abstract = {Streptomyces and insects engage in complex interactions shaped by millions of years of evolution. While many beneficial relationships are well recognized, it remains unknown whether Streptomyces produce virulence factors targeting insects specifically. Here, through bioinformatic analysis, we identified diphtheria toxin (DT) homologues, which we named Streptomyces antiquus insecticidal proteins (SAIP), within a monophyletic lineage of Streptomyces that emerged more than 100 million years ago. SAIP is cytotoxic to insect cells and lethal to Drosophila melanogaster, suppressing neuronal activity and immune responses in vivo. Structural and functional studies validated that SAIP is homologous to DT and acts by ADP ribosylation of eukaryotic elongation factor 2. CRISPR-Cas9 screening identified the insect protein Flower as the SAIP receptor across a range of insects. Toxigenic Streptomyces can consume dead insects and produce bioactive secondary metabolites while growing on insect carcasses. These findings establish an insecticidal toxin in Streptomyces and demonstrate that Streptomyces have evolved highly specific virulence factors against insects.},
}
@article {pmid42063341,
year = {2026},
author = {Nelson, C and Ambros, V},
title = {Multi-dimensional regulation of LIN-28 temporal expression dynamics in the C. elegans heterochronic gene cascade.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {10},
pages = {},
doi = {10.1242/dev.205391},
pmid = {42063341},
issn = {1477-9129},
support = {R35GM131741/NH/NIH HHS/United States ; R35GM131741/NH/NIH HHS/United States ; //University of Massachusetts Chan Medical School/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Caenorhabditis elegans Proteins/genetics/metabolism ; MicroRNAs/genetics/metabolism ; *Gene Expression Regulation, Developmental ; 3' Untranslated Regions/genetics ; *RNA-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Repressor Proteins ; },
abstract = {LIN-28 is an evolutionarily conserved RNA-binding protein that is crucial for regulating pluripotency and cell fate determination during animal development. In Caenorhabditis elegans, lin-28 is an integral component of the heterochronic (developmental timing) gene regulatory cascade. Loss-of-function mutations in lin-28 cause precocious cell fate determination during larval development. Previous studies indicate that proper progression of larval stage-specific cell fates relies on the downregulation of LIN-28, which is negatively regulated by the lin-4 microRNA through complementary sequences in the lin-28 3' untranslated region (UTR). This study employs CRISPR/Cas9 editing of the endogenous lin-28 locus to demonstrate that developmental downregulation of LIN-28 involves multiple inputs, including the action of the let-7 family and lin-4 microRNAs via adjacent complementary sites in the lin-28 3' UTR, along with post-translational inhibition of LIN-28 by the lep-5 long non-coding RNA, collectively accounting for nearly all LIN-28 repression. Additionally, systematic testing of truncations of the lin-28 3' UTR identifies three positive regulatory regions that enhance LIN-28 expression, counteracting the negative effects of the let-7 and lin-4 microRNAs and the lep-5 long non-coding RNA.},
}
@article {pmid42064640,
year = {2026},
author = {Pandey, H and Sharma, A and Misra, V and Mall, AK and Ceasar, SA},
title = {Pre-validation strategies for CRISPR/Cas-based genome editing in plants: a critical analysis of in vitro RNP cleavage assays.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {32},
number = {4},
pages = {677-691},
pmid = {42064640},
issn = {0971-5894},
abstract = {The advent of CRISPR/Cas-based genome editing has revolutionized crop improvement. However, the genome editing success rate remains a major challenge in many crops, especially those with challenging transformation protocols. We critically evaluate the integration of in vitro cleavage assays using naked target DNA and guide RNA-Cas9 nuclease (gRNA-Cas9) ribonucleoprotein (RNP) complexes as a pre-transformation validation step in genome editing workflows. We also compare other pre-validation methods with in vitro cleavage assays and present their advantages and limitations. In vitro assays can help directly confirm target cleavage and biochemically validate gRNA specificity. This strategy may facilitate the functional screening of gRNAs for plants with challenging and low transformation efficiency. In vitro assays can also reduce the unnecessary waste of resources and time associated with intensive transformation processes using non-specific gRNAs. Researchers can prioritize effective constructs based on the cleavage efficiency and specificity of the gRNAs. However, this assay may not guarantee simulation of the natural cellular environment for in vivo editing. We also present this pre-validation approach, which is particularly helpful for polyploid crops like wheat and cotton. In vitro cleavage assays offer a reliable pre-transformation screening step to identify highly active and specific gRNAs, thereby reducing resource-intensive transformation attempts. Future studies should integrate in vitro assays with advanced computational and in vivo validation tools to create a more predictive and efficient gRNA selection pipeline.},
}
@article {pmid42065027,
year = {2026},
author = {Ezeobiora, CE and Igbokwe, NH and Amin, DH and Mendie, UE},
title = {Rare endophytic actinobacteria from nigeria harbor unique biosynthetic gene clusters with novel antibiotic potential.},
journal = {3 Biotech},
volume = {16},
number = {5},
pages = {174},
pmid = {42065027},
issn = {2190-572X},
abstract = {UNLABELLED: Actinobacteria are prolific producers of specialized metabolites, including antibiotics; however, much of their biosynthetic potential remains unexplored, particularly within rare genera. This study presents the first genomic insights into the biosynthetic capacity of two rare endophytic actinobacteria, Saccharomonospora xinjiangensis strain PNSac2 and Saccharopolyspora cebuensis strain PGLac3, isolated from medicinal plants in Nigeria. The strains were characterized using morphological analysis, molecular sequencing, phylogenetic inference, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH). Whole-genome sequencing revealed that PNSac2 possesses a 4.7 Mb genome with 45 tRNA genes, 3 rRNA operons, and 4,541 coding sequences (CDSs), while PGLac3 harbors a 6.4 Mb genome comprising 48 tRNA genes, 4 rRNA operons, and 6,372 CDSs. Genome mining using antiSMASH identified 24 biosynthetic gene clusters (BGCs) in PNSac2 and 28 in PGLac3, including clusters encoding polyketides, nonribosomal peptides, siderophores, terpenes, and ribosomally synthesized and post-translationally modified peptides (RiPPs). Many BGCs showed low similarity to known clusters, indicating a strong potential for novel metabolite discovery. Notably, PNSac2 encoded BGCs related to bleomycin, oxalomycin, desertomycin, and ossamycin, while PGLac3 harbored predicted arylpolyene, lanthipeptide, and a unique lassopeptide cluster. Comparative genomics revealed conserved synteny with related species alongside strain-specific BGCs, and phylogenomic analysis confirmed their taxonomic placement. Overall, these findings highlight the untapped biosynthetic diversity of rare Nigerian endophytic actinobacteria and underscore their promise as sources of novel antimicrobial compounds. Targeted genome engineering approaches, including CRISPR-Cas-based strategies, may further enable the activation and exploitation of cryptic biosynthetic pathways in these strains.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-026-04781-4.},
}
@article {pmid42065492,
year = {2026},
author = {Han, R and Xiao, N and Wu, Z and Zhao, Y and Wang, X and Tang, X},
title = {Sensitive and Robust One-Pot RPA-CRISPR/Cas12a Assay with Elimination of cis-Cleavage.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13307-13318},
doi = {10.1021/acs.analchem.5c06674},
pmid = {42065492},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Limit of Detection ; DNA, Single-Stranded/genetics/chemistry ; *Replication Protein A/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection technology utilizing CRISPR/Cas12a is characterized by high sensitivity, portability, and efficiency, making it a prominent focus in the field of point-of-care testing (POCT). However, it still has limitations in one-pot detection systems. This study developed a one-pot assay based on CRISPR/Cas12a and RPA (11bp+9ss one-pot assay), which nearly eliminates the cis-cleavage activity of Cas12a while retaining its trans-cleavage activity. Specifically, cis-cleavage was abolished by shortening the complementary length between the crRNA and the target to maintain the double-stranded conformation at the cis-cleavage site, whereas trans-cleavage activity was preserved by using an ssDNA complementary to the remaining region. The trans-cleavage activity was applicable to targets within a 200 bp range and under suboptimal PAM conditions. Moreover, rational design of the ssDNA enables effective discrimination of single-base mutations. The 11bp+9ss one-pot assay achieved a limit of detection (LOD) of 1 × 10[0] copies/μL for various targets, demonstrating robust performance even in suboptimal RPA systems. Furthermore, the assay was successfully applied to the detection of Salmonella and Avian Leukosis Virus subgroup J (ALV-J) samples. Overall, the 11bp+9ss one-pot assay exhibits superior sensitivity and robustness, showing great potential for POCT of bacteria and viruses.},
}
@article {pmid42065511,
year = {2026},
author = {Chen, Y and Tang, D and Zhan, J and Wang, Y and Song, Z},
title = {Study on the detection of prostate cancer using MIRA-CRISPR/Cas12a technology.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3848-3857},
doi = {10.1039/d6ay00025h},
pmid = {42065511},
issn = {1759-9679},
mesh = {Humans ; Male ; *Prostatic Neoplasms/diagnosis/urine/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Antigens, Neoplasm/genetics/urine ; *Molecular Diagnostic Techniques/methods ; Biomarkers, Tumor/urine/genetics ; Sensitivity and Specificity ; },
abstract = {Background: Prostate cancer (PCa) is the most diagnosed cancer among men globally and a leading cause of cancer-related mortality. However, current conventional prostate diagnostic methods fail to meet the growing demands of clinical practice in terms of speed, simplicity, sensitivity, and specificity. To address these limitations, we established a molecular detection system based on MIRA-CRISPR/Cas12a technology. Using reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) to amplify minute PCA3-specific fragments in samples, we employ CRISPR/Cas12a to detect the fluorescence signal released by these fragments, enabling the detection of trace PCA3 molecules in urine. Methods: PCA3 standard strain cultivation and total RNA extraction; establishment and optimization of the MIRA amplification system using primers designed for the PCA3 molecular marker; design of crRNA targeting optimal sites within the detection sequence, combined with CRISPR/Cas12a technology to establish the detection system; preliminary validation of the technology's sensitivity and specificity. Results: the MIRA-CRISPR/Cas12a technology was successfully established for visual detection of PCA3 molecules in prostate cancer. Primer and crRNA sequences within the reaction system were determined. The detection sensitivity for PCA3 molecules in urine reached 1 × 10[0] copies per µL with excellent specificity. Conclusion: the MIRA-CRISPR/Cas12a technology enables specific detection of PCA3 molecules in urine. This technique features high sensitivity, high specificity, visual results, and simple operation. It does not require specialized laboratory UV imaging equipment; results are visible to the naked eye under LED blue light. Following further optimization, it offers a feasible technical solution for rapid molecular screening of prostate cancer.},
}
@article {pmid42067160,
year = {2026},
author = {Dai, S and Niu, L and Lv, Y and Zhang, H and Xu, L and Liao, Y and Hu, X and Xie, X and Yan, J and Yan, Y},
title = {Semi-artificial photobiocatalysis via genetically modified sulfur metabolism to in situ assembly of a solar-biohybrid for antibiotic degradation.},
journal = {Bioresource technology},
volume = {454},
number = {},
pages = {134750},
doi = {10.1016/j.biortech.2026.134750},
pmid = {42067160},
issn = {1873-2976},
mesh = {*Sulfur/metabolism ; *Anti-Bacterial Agents/metabolism ; Sulfides/metabolism ; Biodegradation, Environmental ; Nanoparticles/chemistry ; Gene Editing ; CRISPR-Cas Systems/genetics ; Solar Energy ; Tetracycline/metabolism ; },
abstract = {The solar-driven semi-artificial biohybrid system incorporates semiconductor materials with microbial metabolism, affording an innovative strategy for antibiotic degradation via photocatalysis. In this study, the sulfur metabolic pathway of biological cells was rationally engineered using CRISPR-Cas9 and Cre-loxP site-specific gene editing systems, successfully achieving intracellular accumulation of sulfide up to 552.84 ppm. Based on this capability, In(Ⅲ) was adsorbed in situ onto the cell surface, leading to the self-assembly of photosensitive In2S3 nanoparticles (NPs). The resulting inorganic-biological hybrid system of In2S3-cell exhibited a broad-spectrum light-harvesting capability with an ideal optical bandgap of 1.96 eV. Photoelectrochemical analysis confirmed the charge transfer process and the semiconductor biointerface based regeneration mechanism of redox cofactors in the cytosol. Photogenerated electrons directly form ROS for tetracycline oxidation, and are transferred to cells for enhance the regeneration of intracellular reducing cofactors. This light-driven photocatalytic biohybrid system enabled efficient tetracycline degradation of over 98% within 4 h and demonstrated excellent stability over consecutive cycles. Transcriptomic analysis identified key genes involved in solar energy capture, electron transport, and metabolic regulation, elucidating their functional roles in biomanufacturing processes and photocatalytic degradation. This study presents a bottom-up paradigm for the biotic-abiotic system from electronic and molecular perspectives to develop efficient and sustainable technologies for antibiotic remediation and solar energy conversion.},
}
@article {pmid42067223,
year = {2026},
author = {Andres-Lopez, Y and Santambrogio, A and Kafetzopoulos, I and Todd, CD and El Khouri-Gonzalez, C and Gonzalez-Alvarez, JE and Alda-Catalinas, C and Clark, SJ and Reik, W and Hernando-Herraez, I},
title = {Using CRISPR barcoding as a molecular clock to capture dynamic processes at single-cell resolution.},
journal = {Genome research},
volume = {36},
number = {5},
pages = {1005-1015},
doi = {10.1101/gr.280915.125},
pmid = {42067223},
issn = {1549-5469},
mesh = {Animals ; *Single-Cell Analysis/methods ; Mice ; *CRISPR-Cas Systems ; Mouse Embryonic Stem Cells/metabolism/cytology ; *DNA Barcoding, Taxonomic/methods ; Mutation ; Transcriptome ; },
abstract = {Biological processes are inherently dynamic, yet current methods for capturing temporal changes remain limited. Here, we present scDynaBar, a novel approach that combines CRISPR-Cas9 dynamic barcoding with single-cell sequencing. In this system, genetic barcodes gradually accumulate mutations over time; these barcodes are sequenced alongside the transcriptome of individual cells. We propose that the divergence of these barcodes from the original sequence can serve as a record of the timing of cellular events. To demonstrate the potential of this method, we track the transition from a pluripotent state to a two-cell (2C)-like state in mouse embryonic stem cells (mESCs), providing evidence for the transient nature of the 2C-like state. Additionally, our system shows consistent mutation rates across diverse cell types in a mouse gastruloid model, highlighting its applicability to other biological systems. This approach not only improves our ability to study single-cell dynamics but also opens up new possibilities for recording other temporal signals-in other words, using dynamic barcoding as a molecular clock in individual cells.},
}
@article {pmid42067284,
year = {2026},
author = {Zhang, X and Tian, C and Wang, M and Jia, H and Li, X and Tian, G},
title = {Performance enhancement of CRISPR-Cas system based on improved guide RNA: a review.},
journal = {Analytica chimica acta},
volume = {1406},
number = {},
pages = {345472},
doi = {10.1016/j.aca.2026.345472},
pmid = {42067284},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Biosensing Techniques ; },
abstract = {BACKGROUND: CRISPR-Cas technology has emerged as a transformative tool with widespread applications in gene editing and biosensing research; nevertheless, it is plagued by a suite of performance-related bottlenecks, including suboptimal targeting efficiency, undesirable off-target effects, insufficient sensitivity and recognition specificity, restricted target scope, limited multiplexing capacity, incompatible reaction systems, and compromised stability. As gRNA optimization has emerged as a core strategy to address these bottlenecks, there is an urgent need to consolidate recent breakthroughs in this rapidly advancing field. Existing literature lacks a comprehensive, focused synthesis of how gRNA optimization mitigates these key limitations, alongside an analysis of current challenges and future directions.<br><br>RESULTS: Herein, this review comprehensively summarizes recent breakthroughs in augmenting CRISPR-Cas system performance through guide RNA (gRNA) optimization, and further dissects the current challenges, future prospects, and promising research directions in this rapidly advancing field.<br><br>SIGNIFICANCE: It is timely to guide researchers in overcoming CRISPR-Cas performance barriers and accelerating its applications in gene editing and biosensing.},
}
@article {pmid42067668,
year = {2026},
author = {Wu, X and Lam, WH and Zhao, Z and Cao, Y and Lin, H and Feng, X and Zhai, Y and Hsing, IM},
title = {DNA-guided CRISPR-Cas12a effectors for programmable RNA recognition and cleavage.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42067668},
issn = {1546-1696},
support = {16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 6107-20G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C6053-25G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C7035-23GF//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 325014//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR-Cas12a and expanding the design space for programmable RNA manipulation.},
}
@article {pmid42068455,
year = {2026},
author = {Chakraborty, A and Yu, ASL},
title = {Miniaturization of CRISPRa plasmids for efficient delivery into renal epithelial cells and Pkd1 transactivation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42068455},
issn = {1573-4978},
support = {24PRE1194472//American Heart Association/ ; U54 DK126126/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *TRPP Cation Channels/genetics/metabolism ; *Plasmids/genetics ; Mice ; Epithelial Cells/metabolism ; Kidney/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Transcriptional Activation/genetics ; Polycystic Kidney, Autosomal Dominant/genetics/therapy ; Genetic Vectors/genetics ; Gene Editing/methods ; },
abstract = {BACKGROUND: Autosomal Dominant Polycystic Kidney Disease is caused by loss-of-function mutations in PKD1 or PKD2 genes, leading to reduced polycystin protein levels. Increasing PKD1 expression via CRISPR activation (CRISPRa) represents a promising therapeutic strategy; however, delivery of large CRISPRa plasmids into renal epithelial cells, and particularly primary cells, remains inefficient due to size-related barriers. We aimed to enable Pkd1 transactivation by miniaturizing CRISPRa plasmids into ~ 6 kb vectors using a one-pot method to enhance cellular uptake in mouse kidney epithelial cells.<br><br>METHODS AND RESULTS: Using type IIS restriction enzymes, we excised the mammalian expression cassette from full-length large 9-11 kB plasmids. The excised cassette was engineered to have complimentary overhangs. Thermocycling with T4 DNA ligase promoted circularization of the excised cassette (forming&#x2009;~&#x2009;6kB mini-CRISPRa vectors), and T5 exonuclease digestion removed residual backbone fragments. These mini vectors substantially enhanced nucleofection efficiency from 16.10% &#xb1; 0.53 to 54.17% &#xb1; 2.10 in Pkd1[RC/-] cells, and from 10.14% &#xb1; 1.40 to 31.27% &#xb1; 0.12 in primary Pkd1[RC/Cond]; Pkhd1[Cre+] cells. Functionally, the mini-CRISPRa plasmid (mdCas9-VPR) with Pkd1-targeting sgRNAs induced robust endogenous Pkd1 upregulation compared with non-targeting controls: a 4.1-fold increase in Pkd1[RC/-] cells (p&#x2009;<&#x2009;0.001) and a 2.9-fold increase in primary cells (p&#x2009;<&#x2009;0.001). Full-length plasmids produced no significant activation in either cell type.<br><br>CONCLUSIONS: Miniaturization of CRISPRa vectors with this one-pot approach overcomes delivery limitations in hard-to-transfect renal epithelial cells and enables efficient, functional Pkd1 activation, in vitro.},
}
@article {pmid42068556,
year = {2026},
author = {Ban, H and Rondthaler, SN and Lebovich, M and Lora, MA and Ugbesia, B and Andrews, LB},
title = {Cross-Strain Transferability of CRISPRi Systems and Design Rules from Laboratory to Clinical Escherichia coli Strains.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1993-2010},
pmid = {42068556},
issn = {2161-5063},
support = {T32 GM135096/GM/NIGMS NIH HHS/United States ; NSF CBET-1943695//NSF National Science Foundaion/ ; NSF EES-1824090//NSF UMass ADVANCE program National Science Foundation/ ; NSF DMR-1904901//NSF National Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to nonmodel strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different Escherichia coli strains, including a laboratory K-12 strain (MG1655) and three nonmodel strains that are clinical isolates (probiotic Nissle 1917, uropathogenic CFT073, and uropathogenic UMN026). We evaluated the repression in each strain using sets of guide RNAs (gRNAs) targeting along the gene sequence and assayed cytotoxicity of expressing each dCas protein. Growth toxicity from expression of the different dCas proteins notably differed and showed high variation between some host strains. We also observed variable repression among the strains and notably poorer repression in multiple clinical strains. Therefore, we developed a dual gRNA CRISPRi system for enhanced gene silencing among the strains, which achieved up to 824-fold repression in CFT073. The results demonstrate that strain-specific design considerations can arise when a CRISPRi genetic system is transferred to a closely related bacterial strain. These findings provide insight into the relationships between criteria used for CRISPRi genetic design and in vivo activity across nonmodel E. coli strains, providing guidelines for diverse applications of these tools.},
}
@article {pmid42068913,
year = {2026},
author = {Shen, M and Zhang, P and Ding, L and Yang, X and He, L and Wu, Y and Yu, S},
title = {Direct microRNA detection via topologically engineered CRISPR/Cas12a cascade amplification assay.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {360},
number = {},
pages = {128016},
doi = {10.1016/j.saa.2026.128016},
pmid = {42068913},
issn = {1873-3557},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Cell Line, Tumor ; CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Base Sequence ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its high specificity, sensitivity and programmability. However, direct RNA detection is hindered by its inherent DNA-targeting trans-cleavage activity, which typically necessitates a reverse transcription amplification to convert RNA into DNA. Herein, we report a one-pot assay that enables direct detection of microRNA-21 without target amplification. The method employs a molecular switch probe (MSP) that recognizes miRNA-21 and activates Cas12a activity, along with an amplifier probe (AMP) that establishes a self-driven cascade signal amplification. This biosensor achieves a detection limit of 2.88 pM, with high accuracy (recovery of 95.5%-108.6%) and good precision (RSD: 2.35%-7.18%), and exhibits excellent specificity against homologous miRNAs. Using this assay, we successfully quantified the elevated levels of miRNA-21 in lung squamous carcinoma (H520) cells compared to normal bronchial epithelial cells (BEAS-2B). Furthermore, a methodological comparison with RT-qPCR revealed a similar trend between the two methods. This study provides a simple and reliable strategy for direct RNA detection using CRISPR/Cas12a.},
}
@article {pmid42069941,
year = {2026},
author = {Singh, A and Bhattacharjee, S and Singh, Y and Kostova, I},
title = {Parabiotics as Next-Generation Microbiome Therapeutics: Insights into Mechanisms, Evidence, and Therapeutic Potential.},
journal = {Current microbiology},
volume = {83},
number = {6},
pages = {},
pmid = {42069941},
issn = {1432-0991},
mesh = {Humans ; *Prebiotics/administration &amp; dosage ; Animals ; *Gastrointestinal Microbiome/drug effects ; Probiotics ; *Microbiota ; },
abstract = {Parabiotics (also termed paraprobiotics) are defined as non-viable microbial cells or their components, including peptidoglycans, teichoic acids, surface proteins, that confer health benefits without requiring viability which distinguishes them from traditional probiotics. Their non-viable nature eliminates risks such as microbial translocation, bacteremia, and sepsis, making them suitable for vulnerable populations including immunocompromised, critically ill, paediatric and elderly individuals. In addition, parabiotic exhibit improved thermal stability, extended shelf life, and easier incorporation into functional foods, nutraceuticals, and pharmaceutical formulations without cold-chain requirements. Mechanistically, parabiotics retain immunomodulatory, anti-inflammatory and have barrier-enhancing activities through interactions with host pattern recognition receptors, including Toll-like receptors, modulation of cytokine responses, and reinforcement of gut epithelial integrity. Preclinical and clinical studies support their therapeutic potential such as in case of heat-killed Lactobacillus acidophilus LB (L. acidophilus) has shown efficiency in managing acute paediatric diarrhoea, while heat-inactivated Lacticaseibacillus paracasei PS23 (Lcb. paracasei) has demonstrated improvements in muscle strength and inflammatory markers, including reduced C-reactive protein and interleukin-6 and increased interlukin-10 in elderly individuals. Similarly, inactivated Lactiplantibacillus plantarum (Lpb. plantarum) and Bifidobacterium strains have been associated with benefits in irritable bowel syndrome, atopic dermatitis, respiratory infections, visceral fat reduction, and antibiotic-associated dysbiosis. Synergistic combinations with prebiotics, postbiotics and related bioactives further enhance therapeutic outcomes in inflammatory, metabolic and infectious conditions. Advances in metagenomics, next-generation sequencing, proteomics, metabolomics, CRISPR-Cas systems, and synthetic biology are accelerating strain characterization, functional evaluation, and scalable production. Despite ongoing challenges in standardization and regulated harmonization, parabiotics represent a safe and effective approach for microbiome-targeted interventions. This review synthesizes current evidence on their therapeutic applications, technological advancements, and translational potential, highlighting their role in precision health and next-generation functional nutrition.},
}
@article {pmid42070239,
year = {2026},
author = {Geerthana, S and Yogi, D and Kumar, A and Asokan, R and Suresh, K and Prabhakar, N and Rani, BU and Thangaraj, K and Paramasivam, M and Kaninika, V and Chiranth, RK and Shankarnarayan, AM and Pradeep, C and Manamohan, M},
title = {Towards CRISPR/Cas9 Genome Editing in Spodoptera frugiperda: A Proof-of-Concept Targeting Wing and Eye Color Genes.},
journal = {Archives of insect biochemistry and physiology},
volume = {122},
number = {1},
pages = {e70161},
doi = {10.1002/arch.70161},
pmid = {42070239},
issn = {1520-6327},
support = {//ICAR-IASRI for funding under the CABin programme/ ; },
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; *Spodoptera/genetics/growth &amp; development ; Wings, Animal ; *Eye Color/genetics ; Pigmentation/genetics ; Tryptophan Oxygenase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; },
abstract = {Fall armyworm (Spodoptera frugiperda), a globally significant destructive lepidopteran invasive pest, has recently invaded Africa and Asia, threatening food security. Conventional method of management, including chemical insecticides, are often ineffective due to various reasons compelling the need to explore alternative strategies. In this regard, CRISPR/Cas9 based genome editing has emerged as a powerful tool for functional genomics in insects, enabling to introduce site-specific mutations for various purposes. In this study, we applied multiplex CRISPR/Cas9 ribonucleoprotein (RNP) injections to disrupt two key genes in S. frugiperda: the spalt (Sfspalt), which regulates wing patterning, and tryptophan 2,3-dioxygenase (Sfto/vermillion) gene, involved in eye pigmentation. Microinjection of sgRNA/Cas9 ribonucleoprotein complex into freshly laid eggs resulted in distinct phenotypic alterations, including altered wing pigmentation and modified eyespot patterns, as well as golden-yellow eye color mutants. Genotyping and ICE analysis confirmed the presence of frameshift mutations in the target loci, supporting the phenotypic changes. Notably, while mutations were detected, only a single individual was confirmed to carry mutations in both genes simultaneously. These results demonstrate the technical feasibility of multiplex CRISPR/Cas9 editing in S. frugiperda, but also reveal a low frequency of confirmed events under the present experimental conditions. Therefore, this study is considered a proof of concept establishing a preliminary workflow in multiplex platform. The findings provide foundational insights for further optimization of genome editing strategies targeting sex related genes in this agriculturally important pest.},
}
@article {pmid42070443,
year = {2026},
author = {Awais, M and Chen, Y and Bibi, S and Wu, J and Zhang, W and Tlili, I and Hu, J},
title = {Breaking boundaries for PFAS surveillance in water reservoirs: CRISPR-electrochemical synergies from MXene to microfluidics- A review.},
journal = {Biosensors &amp; bioelectronics},
volume = {307},
number = {},
pages = {118756},
doi = {10.1016/j.bios.2026.118756},
pmid = {42070443},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; *Water Pollutants, Chemical/analysis/isolation &amp; purification ; *Fluorocarbons/analysis/isolation &amp; purification ; Lab-On-A-Chip Devices ; Environmental Monitoring/methods ; Electrochemical Techniques/methods ; Nitrites ; Transition Elements ; },
abstract = {The hydro-spherical contamination of water resources by per- and polyfluoroalkyl substances (PFAS) demands analytical technologies that transcend the limitations of current methods, which struggle to simultaneously achieve ultra-sensitivity, specificity, portability, and low cost. Therefore, this review advances next-generation PFAS surveillance by proposing a framework built on the synergistic convergence of molecularly engineered sulfonate-MXenes, CRISPR-Cas12a, and microfluidic automation. The manuscript critically analyzes how sulfonate-terminated Ti3C2Tx MXenes achieve picomolar affinity and rapid preconcentration of PFAS through biomimetic binding architectures. It also details the mechanism by which CRISPR-Cas12a, guided by PFAS-specific aptamers, enables single-molecule discrimination with attomolar sensitivity. Finally, this review paper demonstrates how microfluidic networks orchestrate this synergy, miniaturizing the entire assay into a portable, multiplexed platform that reduces analysis time from hours to minutes. Synergistically unifying breakthroughs in nanomaterials, synthetic biology, and lab-on-a-chip design, this work provides both a methodological blueprint for ultrasensitive PFAS sensors and a relevant roadmap for implementing proactive, decentralized water quality monitoring.},
}
@article {pmid42070526,
year = {2026},
author = {Jerred, C and Ramachandran, H and Hildebrandt, B and Zink, A and Ventura, N and Rossi, A and Prigione, A},
title = {Generation of an iPSC line IUFi004-A-13 with homozygous NDUFS1 mutation for the study of Leigh syndrome.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104002},
doi = {10.1016/j.scr.2026.104002},
pmid = {42070526},
issn = {1876-7753},
mesh = {Humans ; *Leigh Disease/genetics/pathology/metabolism ; *NADH Dehydrogenase/genetics/metabolism ; Homozygote ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Mutation ; *Electron Transport Complex I/genetics ; Cell Differentiation ; CRISPR-Cas Systems ; Mutation, Missense ; },
abstract = {NDUFS1 is a critical component of mitochondrial respiratory chain Complex I (CI). Pathogenic variants of NDUFS1 can cause Leigh syndrome (LS), a severe pediatric mitochondrial disorder. To model NDUFS1-linked LS, we generated an iPSC line with homozygous missense mutations in exon 8 using CRISPR/Cas9. The cell line demonstrated typical morphology, expression of iPSC markers, ability to differentiate into all three germ layers, and genomic integrity. This model will enable the study of LS caused by CI in an isogenic context.},
}
@article {pmid42072072,
year = {2026},
author = {Bartusik-Aebisher, D and Justin Raj, DR and Aebisher, D},
title = {Nanomaterial-Based Therapeutic Delivery: Integrating Redox Biology, Genetic Engineering, and Imaging-Guided Treatment.},
journal = {Antioxidants (Basel, Switzerland)},
volume = {15},
number = {4},
pages = {},
pmid = {42072072},
issn = {2076-3921},
abstract = {Nanomaterials are emerging versatile platforms for therapeutic delivery, as they offer precise control over drug, antioxidant, and genetic payload transport across biological barriers. Inorganic, organic, hybrid, and biomimetic systems are the major classes of nanomaterials, which all have different physicochemical properties such as size, surface charge, and surface functionalization. These properties collectively influence stability, biodistribution, cellular uptake, and release kinetics. Engineering strategies are increasingly using stimuli-responsive designs that are triggered by pH, reactive oxygen species (ROS), and intracellular redox gradients to perform spatially and temporally controlled delivery. Antioxidant and redox-modulating nanocarriers are of great importance as they overcome the limited bioavailability and nonspecific activity of conventional antioxidants by improving stability, targeting oxidative microenvironments, and allowing for regulated release. Improvements in lipid, polymeric, and inorganic nanoplatforms have also developed gene delivery applications, including siRNA, mRNA, and CRISPR/Cas systems, to provide better cytosolic release and precise therapeutics. When diagnostic imaging is integrated with therapy through theranostic nanoparticles, real-time monitoring and personalized intervention are possible. Safety, scalable manufacturing, and regulatory alignment are some challenges that show the need for standardization and translational procedures to utilize the potential of theranostic nanomedicine.},
}
@article {pmid42074014,
year = {2026},
author = {Li, Y and Ma, S and Fei, T},
title = {CRISPR Applications in Alzheimer's Disease: From High-Throughput Genetic Screening to Precision Editing and CNS Delivery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
pmid = {42074014},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; tau Proteins/genetics/metabolism ; *Genetic Testing/methods ; High-Throughput Screening Assays ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Alzheimer's disease is a devastating progressive neurodegenerative disorder characterized by extracellular amyloid-beta plaques and intracellular tau tangles. Despite recent advancements in amyloid-beta-targeting immunotherapies, achieving safe and definitive disease control remains a profound clinical challenge. The CRISPR/Cas9 system has emerged as a powerful technology for precision neurogenetics, offering significant potential to address the fundamental questions behind Alzheimer's disease. This comprehensive review delineates the trajectory of CRISPR applications in Alzheimer's disease research and therapeutics. First, we explore the integration of CRISPR in engineering high-fidelity in vitro models, such as isogenic induced pluripotent stem cells and three-dimensional cerebral organoids, alongside advanced in vivo mammalian models. Second, we examine how these platforms facilitate unbiased high-throughput genetic screening to uncover molecular underpinnings regulating tau, lipid metabolism, and neuroinflammation. Third, we critically evaluate precision editing strategies targeting core risk genes (APP, MAPT, APOE, and TREM2), explicitly highlighting the severe physiopathological trade-offs between therapeutic efficacy and loss-of-function toxicity. Finally, we address the ultimate translational bottlenecks impeding clinical application. By dissecting the packaging limits of adeno-associated viral vectors and the physical barricade of the blood-brain barrier, we underscore the necessity of transitioning toward next-generation base editors and non-viral lipid nanoparticles to realize safe and efficacious in vivo clinical gene therapies against Alzheimer's disease.},
}
@article {pmid42074029,
year = {2026},
author = {O'Hanlon Cohrt, K and O'Dea, S},
title = {Clinical Trial Landscape of Gene-Edited Autologous Hematopoietic Stem Cells for Hemoglobinopathies and Immunodeficiencies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
pmid = {42074029},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; *Hemoglobinopathies/therapy/genetics ; *Hematopoietic Stem Cell Transplantation/methods ; *Hematopoietic Stem Cells/metabolism ; Transplantation, Autologous ; Clinical Trials as Topic ; *Immunologic Deficiency Syndromes/therapy/genetics ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Allogeneic hematopoietic cell transplantation (HCT) has been used for decades to treat certain malignant and non-malignant hematological conditions, but challenges remain. Increased understanding of disease mechanisms and recent developments in genome editing have enabled alternative strategies utilizing gene-edited autologous HCT and many of these have progressed to the clinic. We present here a comprehensive review of clinical trials of gene-edited autologous hematopoietic stem cells for the treatment of hemoglobinopathies and immunodeficiencies. Searches of major international clinical trial registries were carried out using specific key words. In total, 44 interventional clinical trials investigating gene-edited autologous stem cell therapies were identified, with CASGEVY (exagamglogene autotemcel) being the only product approved to date. Hemoglobinopathies were the most common indication (n = 37) followed by immunodeficiencies (n = 4), with single trials in HIV-1 infection, pyruvate kinase deficiency and limb-girdle muscular dystrophy. Gene-editing strategies fall into three categories: disruption of the BCL11A erythroid enhancer, editing of the γ-globin promoter and direct correction or disruption of disease-relevant genes. CD34[+] hematopoietic stem and progenitor cells are the most common cell types edited, and CRISPR-Cas9 is the most widely used gene-editing modality. While results are encouraging, efficient intracellular delivery of gene-editing tools, editing efficiencies and off-target editing remain challenges for the field.},
}
@article {pmid42074108,
year = {2026},
author = {An, L and Xu, Z and Zhang, X},
title = {Self-Assembling Short Peptide Carriers for Gene Delivery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
pmid = {42074108},
issn = {1422-0067},
support = {12574240//National Natural Science Foundation of China/ ; 12174462//National Natural Science Foundation of China/ ; IS24073//Beijing Natural Science Foundation/ ; 2025XYCM83//Organized Research Program in Minzu University of China/ ; },
mesh = {Humans ; *Gene Transfer Techniques ; *Peptides/chemistry ; Animals ; Genetic Therapy/methods ; Gene Editing ; *Drug Carriers/chemistry ; CRISPR-Cas Systems ; },
abstract = {Gene therapy relies on safe and efficient delivery systems, yet traditional viral vectors and synthetic polymers often fail to meet these requirements due to immunogenicity and biocompatibility concerns. This review highlights self-assembling short peptides as a highly programmable and biocompatible non-viral platform uniquely positioned to overcome these translational bottlenecks. To provide a comprehensive overview of next-generation gene delivery, we systematically trace the trajectory from fundamental chemistry to clinical applications. First, we elucidate the supramolecular interactions and mechanisms driving peptide-nucleic acid co-assembly. Second, we outline concrete design strategies, detailing how sequence engineering and environmental responsiveness dictate the formation of optimized nanomorphologies. Third, we critically analyze how these nanocarriers navigate critical physiological and intracellular barriers, with a specific focus on cellular uptake, endosomal escape, and cargo release. Finally, we demonstrate the platform's versatility in emerging frontiers, particularly mRNA vaccines and CRISPR/Cas9 gene editing. We conclude by identifying current obstacles to clinical translation and proposing future directions centered on multifunctional integration and stimuli-responsive design.},
}
@article {pmid42074129,
year = {2026},
author = {Lan, Z and Tian, M and Liu, J and Shi, W and Chen, T and Ma, Q and Jin, B and Zhao, Y and Zhang, H and Lai, CJ and Cui, G},
title = {Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
pmid = {42074129},
issn = {1422-0067},
support = {CI2021A05051//China Academy of Chinese Medical Sciences/ ; },
mesh = {*Salvia miltiorrhiza/genetics/metabolism ; *CRISPR-Cas Systems ; *Abietanes/biosynthesis ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Phylogeny ; *Hydroxymethylglutaryl CoA Reductases/genetics/metabolism ; },
abstract = {3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) serves as a key rate-limiting enzyme in the mevalonate pathway and plays a central regulatory role in the biosynthesis of tanshinones. To date, four HMGR family members (SmHMGR1-4) have been identified in Salvia miltiorrhiza. Here, we cloned and identified a novel member, SmHMGR5, by integrating multiple genomic datasets. Genomically, SmHMGR5 formed an inverted repeat with SmHMGR3 (98.04% homology) and phylogenetically clustered with SmHMGR2. Based on the expression patterns of the five HMGR genes, we further generated SmHMGR2 and SmHMGR5 knockout mutants using CRISPR/Cas9 technology and compared their effects on the accumulation of 12 tanshinones and 4 phenolic acids via UPLC-MS-based metabolomic analysis. Knockout of SmHMGR2 significantly suppressed the accumulation of seven tanshinones, whereas SmHMGR5 knockout downregulated only three tanshinones, and neither mutation affected phenolic acids. Notably, the major compound tanshinone IIA remained stable across different mutants, but tanshinone IIB was markedly reduced upon SmHMGR2 knockout, suggesting complex regulatory mechanisms in tanshinone biosynthesis. These findings provide new insights into the biosynthetic network of tanshinones and establish a theoretical foundation for metabolic engineering strategies aimed at enhancing the production of bioactive constituents in S. miltiorrhiza.},
}
@article {pmid42074203,
year = {2026},
author = {Guo, B},
title = {CRISPR Interference to Inhibit Oncogenes for Cancer Therapy.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
pmid = {42074203},
issn = {1422-0067},
support = {1R01CA293945-02/NH/NIH HHS/United States ; },
mesh = {Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; *Oncogenes/genetics ; Animals ; Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {CRISPR interference (CRISPRi), a programmable transcriptional repression technology derived from nuclease-deficient CRISPR-Cas systems, has emerged as a powerful method for selectively inhibiting oncogene expression without altering the genomic DNA. This feature offers a major advantage over other oncogene targeting technologies such as CRISPR-mediated gene knockout, mRNA inhibition by siRNA or miRNA, or small-molecule inhibitors of the proteins encoded by the oncogenes, especially in cancers driven by transcriptional dysregulation or otherwise undruggable oncogenes. Here, I present a comprehensive review of CRISPRi mechanisms, delivery strategies, and preclinical applications in oncology (including advances in targeting core oncogenic drivers like MYC and KRAS). The advantages of CRISPRi as well as in vivo validation of CRISPRi-mediated tumor suppression are discussed. Finally, I outline translational challenges and future directions for incorporating CRISPRi into precision cancer therapies. The accumulated evidence suggests that CRISPRi could become a cornerstone for next-generation gene-regulatory therapeutics.},
}
@article {pmid42074279,
year = {2026},
author = {Sterckel, S and Chávez Martínez, IL and Schwach, V},
title = {CRISPR and the Future of Cardiac Disease Therapy: A New Genetic Frontier.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
pmid = {42074279},
issn = {1422-0067},
support = {10250042110011//ZonMw, The Dutch Organisation for knowledge and innovation in health, healthcare and well-being/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Heart Diseases/therapy/genetics ; *Genetic Therapy/methods ; Animals ; },
abstract = {CRISPR technologies are transforming cardiovascular therapy development by creating an increasingly seamless pipeline from potential target discovery to clinical translation. What began as a genome-editing tool has evolved into a versatile platform that enables researchers to precisely interrogate and modulate cardiac biology with tools such as base- and prime-editors, and CRISPR inhibition and activation. In this review, we follow the use of CRISPR across the stages of biomedical research through to bench-to-bedside application. This review begins by addressing how genome-wide and focused CRISPR screens discover developmental regulators, disease drivers, and drug-response pathways, making the first steps in identifying therapeutic targets. We then explore how CRISPR engineering creates progressively more relevant disease model systems to validate mechanisms of disease and test interventions, helping bridge the translational gaps between the lab and the clinic. Finally, we consider how CRISPR technologies are beginning to enter cardiovascular clinical trials, while highlighting the key challenges that still limit this translation. By linking the latest advances of modern CRISPR platforms to the stages of therapeutic development, this review highlights how CRISPR technology is reshaping the pipeline from molecular insight to clinical innovation in cardiac disease.},
}
@article {pmid42075136,
year = {2026},
author = {Zhang, E and Yan, J and Du, J and Chu, X and Chen, D},
title = {Application and Research Prospects of CRISPR/Cas Gene Editing Technology in Lactic Acid Bacteria.},
journal = {Microorganisms},
volume = {14},
number = {4},
pages = {},
pmid = {42075136},
issn = {2076-2607},
abstract = {Lactic acid bacteria (LAB) are pivotal microorganisms in the food industry. Current approaches for functional gene validation and trait improvement in LAB primarily rely on traditional gene editing and homologous recombination techniques. These methods are often cumbersome, inefficient, and time-consuming, hindering the rapid and precise customization of strains. This limitation has, to some extent, constrained the rapid selection and industrial application of functional LAB strains. The engineering of LAB through gene editing technologies has significantly advanced both fundamental and applied research. Among these, CRISPR/Cas gene editing has successfully achieved precise modification of multiple genes in various LAB species. Compared to conventional methods, it offers superior editing efficiency and lower operational costs, opening new avenues for functional gene identification and genetic improvement in LAB. However, the application of exogenous CRISPR/Cas systems in LAB faces technical challenges such as high off-target rates, chromosomal abnormalities, and cytotoxicity. The development of endogenous CRISPR/Cas-based editing tools for LAB provides novel pathways for precise regulation, rational design, and flexible application. This paper first outlines the structural components and mechanistic principles of CRISPR/Cas gene editing tools. It then explores the research progress and applications of both endogenous and exogenous CRISPR/Cas systems in LAB. Finally, it provides an outlook on the future application of CRISPR/Cas gene editing technology in LAB, offering a reference for its implementation in this field. The advent of gene editing technologies has significantly propelled functional gene validation and trait improvement in lactic acid bacteria (LAB), thereby advancing both fundamental research and industrial applications. Notably, the CRISPR/Cas system has emerged as a transformative tool enabling precise genetic modification in diverse LAB species, offering marked improvements in editing efficiency and cost reduction relative to conventional approaches. CRISPR/Cas-based editing strategies in LAB are broadly classified into exogenous and endogenous systems. Exogenous systems operate independently of the host's native immune repertoire, conferring the advantages of broad strain applicability and high editing efficiency. These systems have been successfully deployed for functional gene characterization, metabolic pathway engineering, such as augmenting antimicrobial production, and probiotic safety enhancement via virulence gene deletion. Conversely, endogenous systems leverage the intrinsic CRISPR/Cas machinery of LAB, offering superior biocompatibility and minimized off-target risks. Notable applications include precise gene knockout and integration using the native Type I-E system in Lacticaseibacillus paracasei. This review provides a concise overview of CRISPR/Cas system architecture and mechanisms, followed by a systematic synthesis of research progress and applications for both exogenous and endogenous systems in LAB. Finally, future directions are outlined to guide the continued development and application of CRISPR/Cas technologies in this field.},
}
@article {pmid42075335,
year = {2026},
author = {Liu, Q and Qiu, Z and Yao, M and Jiao, B and Zhou, Y and Li, C and Liu, H and Xin, L},
title = {Progress of Rapid Detection Technology for Aquatic Microorganisms: A Comprehensive Review.},
journal = {Microorganisms},
volume = {14},
number = {4},
pages = {},
pmid = {42075335},
issn = {2076-2607},
support = {No. 2023YFD2403000//National Key Research and Development Program of China/ ; 2024KJN029//the Ministry of Science and Technology of the People's Republic of China, Shandong Provincial University Youth Innovation Team Project/ ; },
abstract = {Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020-2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption-from days to less than one hour-while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements.},
}
@article {pmid42075363,
year = {2026},
author = {Di Pinto, A and Forte, V and D'Attilia, C and Possenti, M and Felici, B and Augelletti, F and Sessa, G and Carabelli, M and Morelli, G and Frugis, G and D'Orso, F},
title = {A Rapid Hairy Root-Based Platform for CRISPR/Cas Optimization and Guide RNA Validation in Lettuce.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
pmid = {42075363},
issn = {2223-7747},
support = {CN00000022//European Union Next-GenerationEU/ ; A0375E0166//Lazio Innova/ ; },
abstract = {Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, 'Saladin' and 'Osiride', identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing.},
}
@article {pmid42079440,
year = {2026},
author = {Yang, L and Luo, R and Zhou, W and Yin, P and Feng, Y and Zhang, Y},
title = {Recent advances in noncanonical inhibition mechanisms of anti-CRISPR proteins.},
journal = {mLife},
volume = {5},
number = {2},
pages = {133-147},
pmid = {42079440},
issn = {2770-100X},
abstract = {The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage-host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.},
}
@article {pmid42079647,
year = {2026},
author = {Requejo Cier, CJ and Valentini, N and Boudreau, G and Delisle, JS and Lamarche, C},
title = {Engineering human Tregs to resist tacrolimus via FKBP12 gene editing.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1756624},
pmid = {42079647},
issn = {1664-3224},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/drug effects/metabolism ; *Tacrolimus/pharmacology ; *Tacrolimus Binding Protein 1A/genetics/metabolism ; *Gene Editing ; Cell Proliferation/drug effects ; CRISPR-Cas Systems ; *Immunosuppressive Agents/pharmacology ; Interleukin-2 ; Lymphocyte Activation/drug effects ; Cells, Cultured ; },
abstract = {Regulatory T cells (Tregs) are essential for immune tolerance and are under active development as cell therapy in transplantation. However, the widespread use of the calcineurin inhibitor tacrolimus may inadvertently suppress Treg proliferation and activation, undermining their therapeutic potential. Tacrolimus binds to the FKBP12 protein in T cells, forming a complex that blocks calcineurin-NFAT signaling and suppresses IL-2 gene transcription, thereby inhibiting T cell activation. In this study, we investigated whether deleting FKBP12 in human Tregs could prevent tacrolimus-mediated suppression. Using CRISPR-Cas9 gene editing, FKBP12 was knocked out in ex vivo expanded human Tregs, which were then cultured for seven days with tacrolimus (10 ng/mL) or control, under varying IL-2 concentrations (100-500 IU/mL). We observed that tacrolimus significantly reduced the proliferation of control Tregs, even in conditions with 500 IU/mL IL-2, whereas FKBP12-knockout Tregs maintained robust proliferation comparable to untreated cells. We found no discernible changes in Treg phenotype or stability following FKBP12 deletion or tacrolimus exposure: edited Tregs retained normal expression of the lineage-defining marker FOXP3, displayed a global transcriptomic profile nearly indistinguishable from controls, and were similarly suppressive, indicating that they remained bona fide Tregs. These findings demonstrate that the antiproliferative effect of tacrolimus on Tregs is critically dependent on FKBP12, mirroring its mechanism in conventional T cells. By genetically uncoupling tacrolimus from its target in Tregs, this approach suggests a strategy to preserve Treg numbers during tacrolimus-based immunosuppression in transplant recipients, potentially enhancing Treg-based therapies for transplantation tolerance.},
}
@article {pmid42080108,
year = {2026},
author = {Yousefian, M and Baharmast, M},
title = {Artificial Intelligence-Assisted CRISPR Gene Editing: Current Advances, Clinical Challenges, and Future Directions in Precision Medicine.},
journal = {Avicenna journal of medical biotechnology},
volume = {18},
number = {1},
pages = {3-15},
pmid = {42080108},
issn = {2008-2835},
abstract = {Recent advances in Artificial Intelligence (AI) have profoundly transformed the field of genome editing, particularly through integration with the Clustered Regularly Inter-spaced Short Palindromic Repeats (CRISPR) technology. This review highlights how AI-driven computational models are reshaping guide RNA (gRNA) design, off-target prediction, and editing precision in CRISPR-Cas systems. A PRISMA-informed literature survey was conducted using PubMed, Scopus, EMBASE, and Google Scholar databases to identify studies exploring AI-assisted CRISPR applications in gene therapy and biomedical research. The results demonstrate that deep learning, machine learning, and reinforcement learning approaches significantly enhance prediction accuracy, algorithmic efficiency, and translational potential across genetic diseases such as β-thalassemia, muscular dystrophy, and cancer. Moreover, ethical challenges, algorithmic bias, and data security concerns remain critical barriers to clinical adoption. This review also discusses the emerging landscape of AI-assisted CRISPR research in Iran, emphasizing national progress, infrastructural constraints, and future opportunities. Overall, the convergence of AI and CRISPR technologies promises to advance precision medicine by accelerating the development of personalized, efficient, and ethically responsible genome-editing solutions.},
}
@article {pmid42080223,
year = {2026},
author = {Zhao, S and Huang, S and Li, N and Wang, N and Yu, Z and Liang, X and Yang, Z and Guo, H},
title = {Piperazine-Derived Diamine Lipid Nanoparticles Targeting to the Liver for Delivering Clustered Regularly Interspaced Short Palindromic Repeat Editing of PCSK9 to Durably Maintain Plasmatic Low-Density Lipoprotein Cholesterol in Low Levels.},
journal = {ACS applied bio materials},
volume = {9},
number = {10},
pages = {4465-4476},
doi = {10.1021/acsabm.6c00032},
pmid = {42080223},
issn = {2576-6422},
mesh = {*Proprotein Convertase 9/genetics/metabolism ; *Nanoparticles/chemistry ; Humans ; Animals ; Mice ; *Gene Editing ; *Liver/metabolism/drug effects ; *Cholesterol, LDL/blood ; *Piperazine/chemistry ; *Lipids/chemistry ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; Materials Testing ; Mice, Inbred C57BL ; Hep G2 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cardiovascular diseases remain a leading cause of morbidity and mortality worldwide, and durable suppression of low-density lipoprotein cholesterol (LDL-C) via genome editing represents a promising therapeutic strategy. Here, we report the rational design of a piperazine-derived bis-tertiary amine ionizable lipid (M10) and its optimized lipid nanoparticle formulation (M10-F4) for efficient and liver-targeted CRISPR/Cas9 delivery. Compared with benchmark lipids such as SM-102, M10 enables a reduced molar ratio of ionizable lipid while maintaining high nucleic acid encapsulation efficiency (>80%) and forming stable, spherical nanoparticles. The piperazine-based multi-cationic core confers an optimized apparent pKa of 6.56, facilitating endosomal escape through enhanced protonation under acidic conditions. Confocal microscopy in HepG2 and Huh-7 cells reveals efficient cellular uptake and enhanced cytosolic release of RNA cargo with minimal lysosomal entrapment. In vivo, M10-F4 exhibits strong liver tropism following systemic administration. A single intravenous dose mediates robust PCSK9 gene editing in C57BL/6 mice, resulting in sustained reductions of circulating PCSK9 and LDL-C levels under both normal and high-fat diet conditions for up to 48 days, accompanied by decreased hepatic PCSK9 expression. Importantly, acute safety evaluation in BALB/c mice showed no obvious signs of short-term systemic toxicity, including stable body weight, minimal induction of inflammatory cytokines (IL-6, TNF-α, and CXCL-10), no significant elevation of liver enzymes, and normal gross organ morphology. Collectively, this work establishes M10-F4 as a molecularly engineered, liver-targeted LNP platform in which ionizable lipid architecture and formulation composition enable effective in vivo genome editing with favorable tolerability, highlighting the importance of rational materials design at the materials-bio interface for cardiometabolic gene-editing applications.},
}
@article {pmid42080254,
year = {2026},
author = {Cho, SW and Kim, T and Yang, J and Byun, G and Seo, SW},
title = {Multiplexed CRISPR base editing enables pulse-activated irreversible biocontainment of engineered bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42080254},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2024-0035256//Ministry of Science and ICT/ ; RS-2025-02214910//Ministry of Science and ICT/ ; RS-2025-02309093//Ministry of Science and ICT/ ; RS-2024-00345885//Korean government/ ; SRFC-MA1901-11//Samsung Research Funding &amp; Incubation Center of Samsung Electronics/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Genes, Essential ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli Proteins/genetics ; },
abstract = {The environmental and therapeutic application of genetically engineered microorganisms necessitates the development of robust, irreversible biocontainment systems. In this study, we present an eEGM (editing-driven essential gene multiplex inactivation) module that utilizes CRISPR-mediated cytidine base editing to induce permanent self-killing via a single transient induction. By targeting the start codons of essential genes, we achieved an irreversible translational blockade that avoids the fitness costs associated with basal toxicity in nuclease-based systems. Multiplexed targeting of non-redundant essential loci (holA, ftsB, and dfp) yielded escape frequencies at or below the NIH guideline criterion (10-8) within 1 h of pulse induction. Furthermore, the eEGM system exhibited robust functional orthogonality and portability across laboratory, industrial, and therapeutic Escherichia coli strains, including MG1655, W3110, and Nissle 1917, without detectable interference with heterologous protein expression. This work establishes base editing as a cleavage-free CRISPR effector for pulse-activated, irreversible biocontainment and provides a practical framework for safer deployment of engineered microbes.},
}
@article {pmid42080258,
year = {2026},
author = {Yang, H and Shen, B and Wang, Y and Liu, J and Zhou, F and Liu, M and Li, J and Fan, J and Ding, S and Guo, J and Zhang, J and Li, X},
title = {Flexible regulation of CRISPR/Cas12a activity by spatial confinement effect.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42080258},
issn = {1362-4962},
support = {82202649//National Natural Science Foundation of China/ ; 82202649//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0007//Chongqing Natural Science Foundation Joint Fund for Innovation and Development/ ; CSTB2023NSCQ-MSX0897//Natural Science Foundation of Chongqing/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Single-Stranded/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics ; HIV-1/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise regulation of the trans-cleavage activity of CRISPR/Cas12a has substantially expanded its utility in molecular diagnostics. However, existing strategies rely predominantly on systems with freely diffusing components, necessitating intricate CRISPR RNA (crRNA) designs or specialized chemical modifications, which hinder their simplicity and broader applicability. Here, we demonstrate that the activity of spatially confined Cas12a on fluid membranes (CAS-FLIER) can be facilely modulated by simply adjusting the length of crRNA and the duplex-strand reporters. We reveal that fine-tuning the movement range of membrane-Cas12a and the accessibility of the reporter to Cas12a enables precise, scalable control over trans-cleavage activity. As a proof of concept, we show that the activity of confined Cas12a can be co-activated by single-stranded DNA (ssDNA) and RNA inputs, a capability that remains unattainable in conventional freely diffusing systems. Furthermore, by incorporating a DNA reverse-transcriptor into the CAS-FLIER system, we achieve one-pot, highly sensitive detection of HIV RNA, supporting accurate diagnosis of HIV infection. Notably, this assay is compatible with a lateral-flow format for direct visual readout, highlighting its potential as a point-of-care diagnostic tool for HIV. Collectively, our findings shed new light on modulating Cas12a activity, advancing its applications in molecular diagnostics.},
}
@article {pmid42080260,
year = {2026},
author = {Luo, W and Wu, Y and Ni, D and Zhang, L and Zhang, Y and Han, X and Zhang, Y and Pu, J and He, Y and Yin, N and Wang, W and Huang, R and Guo, Y and Sun, Y and Xie, G},
title = {A universal and orthogonal safety valve for CRISPR/Cas12a without chemical modification or external stimulation.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42080260},
issn = {1362-4962},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; 82501041//National Natural Science Foundation of China/ ; BJRC202410//Chongqing Medical University/ ; GZC20251421//National Postdoctoral Researcher Support Program/ ; CSTB2025NSCQ-GPX1184//Chongqing Natural Science Foundation General Project/ ; HBRC202404//Chongqing National Reserve Talent Program in Health and Wellness/ ; CSTB2025NSCQ-JQX0016//Chongqing Outstanding Youth Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/antagonists &amp; inhibitors/chemistry ; Humans ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism ; RNA/genetics/antagonists &amp; inhibitors/chemistry ; Proprotein Convertase 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {CRISPR/Cas-based gene editing technologies have achieved remarkable progress over the past decade, yet their broad practical applications remain limited by safety concerns. Although regulatory strategies applied before or during CRISPR/Cas activation have substantially improved sequence, temporal, and spatial specificity, persistent activity of already activated Cas nucleases may still increase the risk of uncontrolled editing. Therefore, an effective post-activation control strategy is urgently needed. Here, we report a modification- and stimulation-free RNA inhibitor (iRNA) that functions as a post-activation safety valve for CRISPR/Cas12a. By exploiting Cas12a's allosteric sensitivity and the thermodynamic and kinetic programmability of nucleic acid strand displacement, iRNA drives already activated Cas12a ribonucleoproteins back to an inactive state, enabling universal, sequence-programmable, and orthogonal post-activation inhibition within the validated Cas12a framework. Experiments and simulations elucidate the mechanistic basis of iRNA-mediated strand displacement and demonstrate its high inhibitory efficiency, reversible cyclic control, compatibility, expandability, orthogonality, and universality. Importantly, iRNA also acts as a programmable, autonomously operating safety valve in cells, suppressing uncontrolled editing while preserving PCSK9 gene knockout. With its simple design, excellent biocompatibility, and autonomous intracellular expression, iRNA provides a foundation for next-generation controllable CRISPR systems and holds broad potential for precision therapeutics, cell therapy, and molecular diagnostics.},
}
@article {pmid42080263,
year = {2026},
author = {Feng, W and Hu, J and Zhang, H and Le, XC},
title = {A kinetic approach for mapping seed regions of CRISPR ribonucleoprotein and improving specificity.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42080263},
issn = {1362-4962},
support = {TIPS CBRF2-2023-001//Canadian Biomedical Research Fund/ ; RGPIN-2024-05974//Natural Sciences and Engineering Research Council of Canada/ ; 22506171//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *Ribonucleoproteins/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Binding ; Binding Sites ; },
abstract = {The binding of an activator (target nucleic acid) to a crRNA-Cas ribonucleoprotein (RNP) in CRISPR systems is critical to the activation, kinetics, and specificity of the CRISPR technology. Key to this activation process is the interaction between the protospacer region of the activator and the spacer region of the crRNA in the RNP complex. However, how the nucleotides in the spacer region of the crRNA contribute to the kinetics of RNP binding is not well characterized. We report here profiling of the kinetically critical regions in the process of RNP binding to activators (RNA targets). We introduced the concept and strategy of kinetic manipulators, which enabled mapping of the seed regions (6-9 nucleotides within the spacer that is sensitive to mismatches) of the CRISPR-Cas13a system, including the LbuCas13a and LwaCas13a homologs. The characterization of the binding kinetics and the introduction of kinetic manipulators provided the foundation for a new kinetic approach to improve the specificity of CRISPR techniques without sacrificing the activity. Profiling the kinetically critical regions in the CRISPR system and designing corresponding manipulators maximized the kinetic differences, between the on-target and off-target, and increased discrimination of single-nucleotide mismatches.},
}
@article {pmid42080266,
year = {2026},
author = {Purcell, J and Liu, L and Calvert, RW and Hayes, BK and Huang, C and Davidovich, C and Knott, GJ and Rosenbluh, J},
title = {DUSP11 is an RNA triphosphatase that limits PspCas13b activity by destabilizing gRNA abundance in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42080266},
issn = {1362-4962},
support = {APP2011329//National Health and Medical Research Council/ ; //Australian Government Research Training Program Scholarship/ ; APP1175568//National Health and Medical Research Council/ ; SMRF2021-276//Snow Medical Research Foundation/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Dual-Specificity Phosphatases/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; HEK293 Cells ; *Mitogen-Activated Protein Kinase Phosphatases/genetics/metabolism ; RNA Stability ; Animals ; Gene Knockout Techniques ; RNA Polymerase III/metabolism/genetics ; },
abstract = {The CRISPR-Cas13 system enables programmable RNA targeting with potential applications in therapeutics and research. However, while PspCas13b mediates efficient RNA knockdown following transient transfection, stable lentiviral delivery results in minimal activity, limiting its utility. Here, we performed a genome-wide CRISPR-Cas9 knockout screen to identify mammalian factors that restrict PspCas13b activity. We discovered that DUSP11, an RNA triphosphatase, suppresses PspCas13b function by dephosphorylating the 5'-triphosphate of Pol III-transcribed guide RNAs (gRNAs), triggering their degradation. DUSP11 knockout increased gRNA levels 2.5-4-fold and enhanced PspCas13b-mediated knockdown across multiple cell lines. This enhancement was sustained for at least 27 days and enabled targeting of endogenous transcripts previously refractory to PspCas13b. Our findings reveal an unexpected host restriction of bacterial CRISPR systems and demonstrate that gRNA levels are a limiting factor. We provide a simple strategy to improve PspCas13b activity in mammalian cells. These results have implications for developing PspCas13b-based therapeutics and suggest that systematic identification of host factors regulating CRISPR components could enhance genome editing technologies.},
}
@article {pmid42080267,
year = {2026},
author = {Pan, L and Sang, R and Xue, R and Ma, Y and Goldys, E and Deng, F},
title = {AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
pmid = {42080267},
issn = {1362-4962},
support = {//Henan Province High-level Talent International Exchange Project/ ; 252300423880//Henan Provincial Natural Science Foundation/ ; //UNSW SHARP/ ; 2024/ECF1573//Cancer Institute NSW/ ; DP240103024//ARC/ ; 2030464//NHMRC/ ; 2024/ECF1573//Cancer Institute NSW/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *Ribonucleoproteins/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Gene Editing ; DNA, Single-Stranded/metabolism ; Models, Molecular ; Protein Multimerization ; },
abstract = {Although Cas12f (Cas14) is among the smallest Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effectors, it assembles into dimeric ribonucleoprotein (RNP) complexes with guide RNA, substantially increasing its functional size and limiting its suitability for gene editing and biosensing applications. To overcome this limitation, we systematically investigate the structural and functional roles of Cas12f dimerization using a combination of computational modeling and experimental validation. Structural analysis using Protein Data Bank data and AlphaFold-3 predictions revealed that the 5'-end sequence of tracrRNA is essential for dimer formation but dispensable for substrate cleavage. Based on this, we designed a truncated tracrRNA by removing 70 nucleotides from its 5'-end. This shortened tracrRNA successfully loaded into Cas12f to form a one guide RNA-one Cas12f monomer RNP. This functionally monomeric RNP demonstrated substantially enhanced trans-cleavage activity: 4.5-fold for ssDNA, 3.5-fold for dsDNA, and 2.5-fold for RNA, resulting in markedly improved detection sensitivity: 10-fold for ssDNA and dsDNA, and 4-fold for RNA. In addition, the functionally monomeric RNP exhibits cis-cleavage activity and gene editing efficiency comparable to that of the dimeric RNP, thereby restoring the advantage of Cas12f as a compact enzyme for in vivo gene editing. These results highlight that the functionally monomeric Cas12f RNP combines enhanced biosensing performance with retention of its uniquely compact size, benefiting gene editing applications.},
}
@article {pmid42080294,
year = {2026},
author = {Liu, Q and Guan, J and He, X and Xie, P and Zhao, Z and Liu, X and Lee, TY and Chiang, YC and Yao, L},
title = {EnAcrPred: A robust ensemble machine learning framework for identifying anti-CRISPR proteins.},
journal = {Protein science : a publication of the Protein Society},
volume = {35},
number = {6},
pages = {e70559},
pmid = {42080294},
issn = {1469-896X},
support = {JCYJ20230807114206014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2025A1515011753//Guangdong Province Basic and Applied Basic Research Fund/ ; 2025XAKJ0102017//Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory/ ; 20720250172//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Machine Learning ; *CRISPR-Cas Systems ; Gene Editing ; *Software ; },
abstract = {The identification of anti-CRISPR proteins (Acrs) is crucial for understanding the regulation of CRISPR-Cas systems and their application in gene editing. However, current experimental methods face challenges, particularly in detecting Acrs with low similarity to known protein sequences. To address these challenges, we propose EnAcrPred, an advanced prediction framework based on ensemble learning. The model combines features such as sequence composition, order correlation, and inferred structure and utilizes a stacking ensemble architecture to integrate multiple base models, which enhances both the accuracy and generalization ability of the predictions. Experimental results demonstrate that EnAcrPred achieves superior performance over existing methods across multiple evaluation metrics, further confirming its robustness. Additionally, SHapley Additive exPlanations (SHAP) value analysis identifies the key features influencing Acrs recognition. To facilitate broad adoption in practice, we developed an online platform where users can quickly obtain Acrs predictions by entering a protein sequence. EnAcrPred offers an effective solution for Acrs identification, contributing to the advancement of gene editing research and safety. The platform is accessible via the link at https://ycclab.cuhk.edu.cn/EnAcrPred/.},
}
@article {pmid42080370,
year = {2026},
author = {Wang, Z and Li, J and Yue, Z and He, B and Zhang, W and Fang, Z and Xia, Q and Liu, Y and Li, Y},
title = {A Modular and Programmable Cas13d Platform for RNA Single Nucleotide Variant Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {32},
pages = {e23680},
pmid = {42080370},
issn = {2198-3844},
support = {32471528//National Natural Science Foundation of China/ ; 82404929//National Natural Science Foundation of China/ ; zz-RCPY-23-25//State Key Laboratory of Systems Medicine for Cancer/ ; zz-94-25-22//State Key Laboratory of Systems Medicine for Cancer/ ; SB2023-13//State Key Laboratory of Systems Medicine for Cancer/ ; zz-GZR-25-09//State Key Laboratory of Systems Medicine for Cancer/ ; 23QC1400600 to Y.L//Shanghai Rising-Star Program/ ; DFYC-LYJ2022 to Y.L//Shanghai Oriental Talent Youth Program/ ; 10000015Z155080000004//National Clinical Key Specialty Construction Project/ ; YG2023QNA09//Shanghai Jiao Tong University Star Program Medical, Industrial Cross Research Fund/ ; QiankeherencaiXKBF[2025]025)//Guizhou Provincial Science and Technology Projects/ ; XK202401//Jiading district medical key discipline construction project/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/genetics ; },
abstract = {CRISPR-based nucleic acid diagnostics have shown broad potential, yet reliable single-nucleotide variant (SNV) discrimination remains limited by flanking sequence requirements that constrain targetability, and an inherent specificity-sensitivity trade-off where mismatch designs used to suppress wild type recognition often penalize enzymatic activity. Here we develop a scenario-guided Cas13d framework that supports pre-defined operating modes tailored to distinct analytical goals. Leveraging the minimal protospacer flanking site constraints of Cas13d, we first map mismatch-sensitive windows to derive rule-based crRNA designs that improve allelic discrimination. We then restore assay performance through structure-guided engineering of a miniaturized Cas13d scaffold by internally inserting auxiliary RNA binding domains (RBDs). Systematic benchmarking across representative oncology hotspots delineates two practical regimes comprising an ultra-sensitive, amplification-free mode in which a dual-RBD variant paired with optimized mismatched crRNAs achieves ∼0.6% variant allele fraction (VAF) detection, and a robust amplified mode incorporating optional loop-mediated isothermal amplification coupling that favors simpler architectures to balance performance and background across broader low-VAF ranges. In an evaluation of 45 clinical tumor RNA specimens spanning pancreatic, cholangiocarcinoma, and colorectal cancers, the assay correctly classified mutation status with full concordance for KRAS G12D, IDH1 R132C and BRAF V600E, with a subset of positive cases corroborated by orthogonal RT-ddPCR. A prospective IDH1 R132C clinical-matrix spike-in further supported sub-1% detection without pre-amplification. Collectively, this work establishes a configurable Cas13d toolkit and a rule-guided strategy for deploying CRISPR-based RNA SNV diagnostics with application-specific performance objectives.},
}
@article {pmid42080605,
year = {2026},
author = {Merwaiss, F and Aragonés, V and García, A and Daròs, JA},
title = {A Modified Cas9 Scaffold Allows Extension of the Virus-Induced Gene Editing Technology to the Large Potyvirus Genus.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70675},
pmid = {42080605},
issn = {1467-7652},
support = {PID2023-146418OB-I00//Spanish Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; FPU20/05477//Spanish Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CIPROM/2022/21//Generalitat Valenciana/ ; },
abstract = {Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprise the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompasses most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (Potyvirus nicotianainsculpentis)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed us to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings may be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.},
}
@article {pmid42081667,
year = {2026},
author = {Xie, H and Geng, L and Hu, Z and Chen, F and Tan, M and Wang, D and Huang, Z and Ye, W and Chen, P and Zhu, J},
title = {Genome editing generates high oleic soybean and eliminates beany flavors.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70282},
pmid = {42081667},
issn = {1744-7909},
abstract = {Soybeans serve as excellent sources of vegetable oil, protein, and other valuable nutrients for human consumption, materials for diverse industries, including the cosmetics and medical industries, and feedstocks for animals. Nevertheless, some people do not favor soy oil or other various food products derived from soybeans, due to inadequate levels of oleic acid in the oil and the presence of undesirable grassy and beany flavors associated with oxidation products of polyunsaturated fatty acids in the seeds. In this study, we developed soybean cultivars with very high levels of oleic acid in the seeds, and without grassy and beany flavors. We achieved this by using CRISPR-Cas-SF01 to edit genes in the elite cultivar Xudou 18 (XD18), targeting two microsomal Δ-12 fatty acid desaturase 2 (GmFAD2-1A and GmFAD2-1B) and three lipoxygenase (GmLOX1, GmLOX2, and GmLOX3) genes. Our findings showed that fad2-1a/b and fad2-1a/b/lox1/2/3 plants performed similarly to XD18 plants in the field, indicating no obvious growth penalties. Overall, this research has demonstrated that the development of soybean germplasms with high levels of oleic acid and without undesirable beany flavors through gene-editing of multiple relevant genes is effective, and this endeavor can contribute to the health of a broader global consumer population.},
}
@article {pmid42082082,
year = {2026},
author = {Saliani, N and Hejazi, MS and Zununi Vahed, S and Mehdizadeh Aghdam, E and Mori, Z and Hasheminejad, N and Daroon Parvar, M and Montazersaheb, S and Ebrahimi, V},
title = {Synthetic biology-driven innovations in triple-negative breast cancer: Integrating engineering design with targeted therapeutics.},
journal = {Journal of biotechnology},
volume = {416},
number = {},
pages = {125-137},
doi = {10.1016/j.jbiotec.2026.04.017},
pmid = {42082082},
issn = {1873-4863},
mesh = {Humans ; *Synthetic Biology/methods ; *Triple Negative Breast Neoplasms/therapy/genetics ; CRISPR-Cas Systems ; Female ; Gene Editing ; Animals ; Precision Medicine ; },
abstract = {Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited therapeutic options and poor clinical outcomes due to the absence of hormone-responsive receptors. The advent of synthetic biology, which integrates molecular biology with engineering design principles, has introduced new opportunities to develop precise and programmable therapeutic and diagnostic strategies for TNBC. Engineered immune cells, such as chimeric antigen receptor (CAR)-T constructs, can selectively recognize tumor-associated antigens and overcome immunosuppressive barriers. Synthetic gene circuits and engineered bacteria enable tumor-specific delivery of cytotoxic or immunomodulatory agents, while induced pluripotent stem cells (iPSCs) provide patient-specific platforms for disease modeling and drug screening. In parallel, CRISPR/Cas-based genome editing facilitates targeted modulation of oncogenic and tumor-suppressor networks, offering both mechanistic insights and therapeutic innovation. This review highlights current advances in synthetic biology-driven approaches for TNBC, encompassing cell-based, microbial, and nucleic acid-engineered systems. It also discusses their synergistic potential to mitigate tumor heterogeneity, enhance therapeutic specificity, and overcome drug resistance. Collectively, the intersection of synthetic biology, immuno-oncology, and precision medicine holds significant promise for next-generation, adaptive, and patient-tailored treatments for TNBC.},
}
@article {pmid42083602,
year = {2025},
author = {Taebi, S and Eskandari, F and Kohandani, M and Manoochehrabadi, T and Nasiri, H},
title = {Genetic Engineering in Hematopoietic Stem Cells for β-Hemoglobinopathies Treatment: Advances, Challenges, and Clinical Translation.},
journal = {International journal of hematology-oncology and stem cell research},
volume = {19},
number = {4},
pages = {399-423},
pmid = {42083602},
issn = {2008-3009},
abstract = {β-hemoglobinopathies rank among the most prevalent inherited blood disorders globally. Traditional management strategies are primarily palliative and often associated with significant challenges, including iron overload and limited long-term efficacy. Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for transfusion-dependent patients, but its broader applicability is constrained by factors that limit its use. Utilizing viral vectors and gene-editing tools, particularly CRISPR-Cas9 technology, researchers have developed therapies that target the root causes of these disorders. These innovative approaches have demonstrated substantial therapeutic potential, accompanied by favorable safety profiles, in clinical settings. Since the initial investigations, the genome editing tool has rapidly advanced for genetic abnormalities, particularly monogenic blood diseases, including β-hemoglobinopathies. This method suggests an approach with lower concerns in viral gene integration and insertional mutagenesis issues. This review comprehensively surveys the therapeutic strategies for β-thalassemia and sickle cell disease (SCD) currently in preclinical and clinical development, with a focus on the evolving treatment paradigm. Looking forward, critical research priorities include optimizing the efficiency and specificity of gene-editing platforms and pioneering novel delivery systems to guarantee both therapeutic efficacy and clinical safety.},
}
@article {pmid42083769,
year = {2026},
author = {Chen, Z and Mao, K and Meng, H and Gao, C and Lv, H and Li, X and Zheng, Q and Yang, Z and Hamza, IA and Tu, C and Zhang, H},
title = {String-Powered Microfluidic Chip Integrating Heparin-Mediated One-Pot RT-RPA/CRISPR-Cas12a for Multiplex Detection of HFMD Viruses.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {14315-14329},
doi = {10.1021/acs.analchem.6c00663},
pmid = {42083769},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Hand, Foot and Mouth Disease/virology/diagnosis ; *Heparin/chemistry ; Humans ; *Lab-On-A-Chip Devices ; Wastewater/virology ; Nucleic Acid Amplification Techniques ; },
abstract = {Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by enteroviruses, which exhibit distinct regional and seasonal epidemiological patterns. Wastewater-based epidemiology is a crucial tool for monitoring population infection dynamics and viral subtype distribution. However, the lack of effective on-site viral detection methods limits timely early warning and effective surveillance of infectious disease outbreaks. This study developed a one-pot RT-RPA/CRISPR-Cas12a assay-based, string-powered flywheel microfluidic chip for the multiplex detection of HFMD viruses in wastewater. First, by leveraging the regulatory effect of heparin sodium on CRISPR/Cas12a activity, a one-pot RT-RPA/CRISPR-Cas12a system was constructed to detect four major subtypes of HFMD virus (EV-A71, CV-A16, CV-A6, and CV-A10). Subsequently, this method was integrated into a pull-wire, flywheel-type, dual-axis centrifugal microfluidic chip, named the Heparin-Inhibited CRISPR-Associated System Chip (HICAS-Chip), enabling integrated enrichment, purification, elution, and multiplexed detection. The HICAS-Chip allowed visual detection of nucleic acids at 10 aM sensitivity within 1 h, corresponding to the sensitivity of the one-pot RT-RPA/CRISPR-Cas12a assay. During a year-long wastewater monitoring program in Guiyang City, China, the HICAS-Chip identified EV-A71 and CV-A10 as the predominant circulating subtypes, with incidence peaks observed in June, November, and December. The wastewater detection results obtained using HICAS-Chip showed high concordance (95.83%) with RT-qPCR assays. This platform provides an efficient portable device for the early detection and continuous monitoring of HFMD epidemic trends by wastewater-based epidemiology.},
}
@article {pmid42084031,
year = {2026},
author = {Ma, R and Xiao, Y and Yu, W and Huo, C and Meng, S and Zhao, Z and Guo, Z and Ren, X and Zhang, H and Li, B and Wang, Y and Liu, S and Huang, J},
title = {A novel high-sensitivity fluorescence detection technology for zearalenone based on the PER-triggered crRNA conformational change and CHA-coordinated energy supply.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3865-3873},
doi = {10.1039/d6ay00474a},
pmid = {42084031},
issn = {1759-9679},
mesh = {*Zearalenone/analysis ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Nucleic Acid Conformation ; Spectrometry, Fluorescence/methods ; Fluorescence ; },
abstract = {Zearalenone (ZEN), frequently encountered in corn, is a hazardous mycotoxin capable of impairing liver and kidney function, compromising immune responses, and potentially inducing carcinogenesis. Current detection methodologies are hampered by elevated costs, complex workflows, limited sensitivity, and poor specificity. There is a pressing need to develop simple, rapid, and ultrasensitive assays that combine high specificity with operational convenience, thereby facilitating precise biotoxin surveillance and control. This study developed a novel biosensing strategy for ultrasensitive detection of zearalenone (ZEN) by engineering a blocked Primer Exchange Reaction (PER) dumbbell-hairpin structure integrated with a Catalytic Hairpin Assembly (CHA)-based DNA machine. We constructed a highly specific and sensitive fluorescence biosensor for zearalenone (ZEN) by integrating a Primer Exchange Reaction (PER) with the trans-cleavage activity of CRISPR/Cas12a. This strategy significantly simplifies the operational procedure compared to conventional techniques. Furthermore, its modular design establishes a versatile and efficient platform adaptable for the detection of various trace analytes, offering a promising proof-of-concept for mycotoxin screening in agricultural products, although further extensive validation across diverse realistic matrices is warranted.},
}
@article {pmid42084552,
year = {2026},
author = {Huang, X and Liu, M and Chen, Y and Fang, L and Cao, Y},
title = {Genome-Scale CRISPRi Screening Identifies Gene Targets for Enhanced Octanoic Acid Tolerance and Production in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1771-1778},
doi = {10.1021/acssynbio.6c00105},
pmid = {42084552},
issn = {2161-5063},
mesh = {*Escherichia coli/genetics/metabolism/drug effects ; *Caprylates/metabolism/pharmacology ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; NADP/metabolism ; Genome, Bacterial ; },
abstract = {Medium-chain fatty acids (MCFAs) are valuable precursors for biofuels and other commodity chemicals; however, the microbial biosynthesis of these compounds is severely constrained by cytotoxic effects. Here, we employed a genome-scale CRISPR interference (CRISPRi) library to systematically identify gene targets whose repression enhanced the tolerance to octanoic acid (C8) in Escherichia coli. Among the identified targets, repression of ygaM, gluQ, gatY, and talA enabled a 1.1- to 1.7-fold increase in C8 production relative to the parental strain MS-1. Mechanistic analyses revealed that the enhanced tolerance was associated with improved membrane properties, reduced reactive oxygen species (ROS) levels, and a shorter cell morphology. Further metabolic engineering to optimize NADPH availability increased C8 production to 1083 mg/L, representing a 2.3-fold increase over that of the MS-1 strain. This study provides new insights into engineering robust E. coli strains for MCFAs production and highlights the utility of genome-scale CRISPRi screening for identifying genetic determinants of microbial stress tolerance.},
}
@article {pmid42084692,
year = {2026},
author = {Minami, A and Shimizu, M and Tamaki, S and Nishinarizki, V and Yosua, and Mochida, K},
title = {Affordable CRISPR RNP-Based Genome Editing in Euglena gracilis.},
journal = {Current protocols},
volume = {6},
number = {5},
pages = {e70357},
pmid = {42084692},
issn = {2691-1299},
support = {JPMJSA2204//This work was supported by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) through the Science and Technology Research Partnership for Sustainable Development (SATREPS) Program/ ; },
mesh = {*Gene Editing/methods/economics ; *Euglena gracilis/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing can enhance basic research and enable industrial applications of green algae. Here, we present an affordable, broadly applicable workflow for genome editing in the unicellular green alga Euglena gracilis using Cas9 nucleases. This method retains high editing efficiency while significantly lowering technical barriers. Unlike previous approaches that required specialized equipment, this protocol can be performed using a general-purpose laboratory electroporator and a simplified clonal isolation procedure without the need for specialized micromanipulation devices. This protocol is compatible with a range of editing outcomes, such as targeted deletions and precise base substitutions, enabling more widespread genome editing in Euglena. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of Euglena gracilis Basic Protocol 2: sgRNA synthesis Basic protocol 3: Transformation Basic protocol 4: Genotyping.},
}
@article {pmid42084860,
year = {2026},
author = {Tian, Y and Li, C and Zhao, E and Chen, Y and Shen, X and Xu, S and Yu, Y and Sun, L},
title = {Recent Advances in the Detection of Plant Diseases Based on the CRISPR-Cas System.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {13951-13966},
doi = {10.1021/acs.analchem.5c07871},
pmid = {42084860},
issn = {1520-6882},
}
@article {pmid42087437,
year = {2026},
author = {Yang, Y and Liu, Y and Xu, H and Zhou, F and Ji, X and He, Z},
title = {CRISPR/Cas13a-Driven Catalytic Hairpin Assembly of a Quantum Dot Nanobeacon for Viral RNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {14163-14172},
doi = {10.1021/acs.analchem.5c08146},
pmid = {42087437},
issn = {1520-6882},
mesh = {*Quantum Dots/chemistry ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Influenza A Virus, H1N1 Subtype/genetics/isolation &amp; purification ; SARS-CoV-2/genetics/isolation &amp; purification ; Limit of Detection ; Humans ; Nucleic Acid Amplification Techniques ; COVID-19/diagnosis ; },
abstract = {Influenza A (H1N1), which is a highly contagious respiratory pathogen, poses a serious public health threat. Its effective control necessitates rapid and accurate detection methods. Here, we developed a novel quantum dot (QDs) nanobeacon-based biosensor that synergistically integrates the precise target recognition and trans-cleavage activity of CRISPR/Cas13a with the isothermal, enzyme-free signal amplification power of catalytic hairpin assembly (CHA) for sensitive detection of H1N1 RNA. This system achieves dual signal amplification, enabling detection with a remarkably low limit of detection (LOD) of 95.2 aM within 40 min. Excellent specificity distinguishes H1N1 from other common influenza viruses. Crucially, the programmability of crRNA endows the method with broad versatility. Beyond influenza virus detection, it was successfully applied to SARS-CoV-2 RNA detection, achieving an LOD of 87.0 aM, demonstrating its potential for diverse pathogen diagnostics. These results collectively demonstrate the high performance and adaptability of this diagnostic platform, highlighting its significant promise for future applications in the rapid and sensitive detection of diverse viral pathogens.},
}
@article {pmid42087786,
year = {2026},
author = {Ramongolalaina, C and Pastor-Pareja, JC and Zhang, E and Jia, Y},
title = {Optimized optogenetic anti-CRISPR for endogenous gene regulation in Drosophila.},
journal = {Nucleic acids research},
volume = {54},
number = {9},
pages = {},
pmid = {42087786},
issn = {1362-4962},
support = {YB202212280503//Tsinghua-Peking Center of Life Sciences/ ; 100401473//Tsinghua-Peking Center of Life Sciences/ ; 32150710524//J. C. Pastor-Pareja/ ; PID2021-122119NB-I00//National Natural Science Foundation of China/ ; //Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CEX2021-001165-S//"Severo Ochoa" Program for Centers of Excellence/ ; 2025-I-ZD-004//"Severo Ochoa" Program for Centers of Excellence/ ; 2025-O-ZD-004//"Severo Ochoa" Program for Centers of Excellence/ ; //State Key Laboratory of Complex, Severe, and Rare Diseases/ ; 2025B-07-08//Changping Laboratory/ ; //SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; },
mesh = {Animals ; *Optogenetics/methods ; *Gene Expression Regulation/radiation effects ; Light ; *CRISPR-Cas Systems ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics ; *Drosophila/genetics ; },
abstract = {Optogenetic tools-light-responsive proteins that enable to regulate specific cellular activities, study biological processes, and develop new therapies-are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. Our first step in constructing an optogenetic system to regulate endogenous Drosophila gene expression was to identify inhibitory anti-CRISPR (Acr) proteins that block CRISPRa-mediated activation. Next, we inserted optogenetic protein LOV2 into these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. We therefore engineered an optimized Acr-LOV2 fusion module by refining length of intrinsically disordered and ordered regions (IDR and IOR) of Acrs. This optimization yielded a variant with significantly greater sensitivity to blue-light-induced endogenous gene upregulation than the prototypes, leading to new in vivo discoveries. In addition, this work provides insights for in vivo functional characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-controlled endogenous gene regulation in Drosophila.},
}
@article {pmid42088606,
year = {2026},
author = {Fujii, T and Sakoda, Y and Yoshimi, K and Takeshita, K and Watanabe, S and Iida, R and Obo, T and Yokoyama, K and Tamada, K and Mashimo, T},
title = {Efficient gene disruption with CRISPR-Cas3 in human T cells.},
journal = {NAR cancer},
volume = {8},
number = {2},
pages = {zcag009},
pmid = {42088606},
issn = {2632-8674},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes/immunology/metabolism ; *Gene Editing/methods ; *beta 2-Microglobulin/genetics ; Graft vs Host Disease/genetics/immunology ; Immunotherapy, Adoptive/methods ; Gene Deletion ; },
abstract = {The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.},
}
@article {pmid42089305,
year = {2026},
author = {Kooistra, T and Sarraf, TR and Chen, M and Gally, CK and Medoff, BD},
title = {Efficient CRISPR-Cas RNP-based gene targeting of human AT2 cells.},
journal = {American journal of respiratory cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1093/ajrcmb/aanag062},
pmid = {42089305},
issn = {1535-4989},
support = {U01 HL175384/HL/NHLBI NIH HHS/United States ; },
abstract = {Alveolar type 2 (AT2) cells play numerous roles in the alveolus related to stem cell, immunoregulatory, and secretory functions. Primary human AT2 cells can now be isolated and studied as organoids consisting of self-organizing epithelial tissues as pure populations without the need for stromal support cells. However, genetic manipulation of AT2 cells to investigate their biology has relied on expensive and time-consuming processes requiring the use of viral vectors or conducting gene editing with induced pluripotent stem cells (iPSCs)-derived AT2 cells. Here we describe a high-efficiency method of accomplishing highly effective gene editing in cultured primary human AT2 cells, which can be done rapidly and at significantly lower costs. Using an optimized CRISPR ribonucleoprotein (RNP) approach, we can achieve nearly complete genetic knockout while preserving AT2 identity and viability. Our results simplify the process of genetically manipulating human AT2 cells to better understand the role of the alveolar epithelium in human lung biology.},
}
@article {pmid42089665,
year = {2026},
author = {Liu, S and Bao, Y and Yilihaer, Y and Guo, W and Ma, C and Xue, C and Yang, Z and Chen, Y and Xie, Z},
title = {VEGFA-Targeted M3-F4 Ionizable Lipid Nanoparticles Improve Diabetic Retinopathy.},
journal = {Molecular pharmaceutics},
volume = {23},
number = {6},
pages = {3367-3382},
pmid = {42089665},
issn = {1543-8392},
mesh = {Animals ; *Diabetic Retinopathy/therapy/genetics/metabolism/drug therapy/pathology ; *Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists &amp; inhibitors ; Humans ; *Nanoparticles/chemistry ; Mice ; *Lipids/chemistry ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Endothelial Cells/metabolism/drug effects ; Diabetes Mellitus, Experimental ; Mice, Inbred C57BL ; Male ; Liposomes ; },
abstract = {Diabetic retinopathy (DR) is one of the leading causes of visual impairment and blindness worldwide. Current therapies for DR primarily focus on inhibiting vascular endothelial growth factor A (VEGFA); however, their efficacy remains limited due to drug resistance and the requirement for repeated intravitreal injections. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technology enables specific targeting and knockout of the VEGFA gene, offering a novel therapeutic approach for DR. In this study, we synthesized a novel ionizable lipid, M3, and assembled the optimal-performing M3-F4 into lipid nanoparticles (M3-F4 LNP) for codelivery of VEGFA-targeting Cas9 mRNA (mCas9) and single guide RNA (sgRNA). The optimized formulation, composed of M3:cholesterol:DSPC:DMG-PEG at a molar ratio of 45:42.5:10:2.5, exhibited a particle size below 100 nm, a PDI below 0.2, and an encapsulation efficiency above 80%. Sanger sequencing-based indel analysis confirmed VEGFA editing in HRMECs, with sgRNA1 achieving an indel frequency of approximately 28.7%. In high glucose-induced human retinal microvascular endothelial cells (HRMECs), the mCas9/sgVEGFA@M3-F4 LNP reduced cell proliferation, migration, invasion, and tube formation, while restoring endothelial barrier integrity and exerting anti-inflammatory effects. A single intravitreal injection of mCas9/sgVEGFA@M3-F4 LNP effectively inhibited pathological neovascularization and retinal leakage in both oxygen-induced retinopathy mice and streptozotocin-induced diabetic mice in vivo. Furthermore, it markedly attenuated VEGFA-induced inflammation while maintaining excellent biocompatibility. This study demonstrates M3-F4 LNP as a promising method for efficient CRISPR/Cas9 delivery and provides robust support for gene therapy strategies in DR treatment.},
}
@article {pmid42090038,
year = {2026},
author = {Jaffal, S and Jaffal, G},
title = {Genetic modulation of pain pathways: toward a new era in pain therapy- a systematic review.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42090038},
issn = {1573-4978},
mesh = {Humans ; *Pain Management/methods ; *Genetic Therapy/methods ; *Pain/genetics ; CRISPR-Cas Systems/genetics ; RNA Interference ; Animals ; Gene Transfer Techniques ; },
abstract = {Gene therapy represents a new strategy in pain management targeting the causes of pain rather than its symptoms. This review summarizes recent advances across RNA interference (RNAi), viral vector delivery systems, CRISPR/Cas9, gene replacement therapy, and endogenous opioid gene delivery, emphasizing efficacy, safety, and mechanisms of action in pain conditions. RNAi and gene replacement techniques remain powerful tools for reducing pain and improving the quality of life by modulating pain-associated genes. Long-term relief may also be achieved with CRISPR/Cas9 and site-directed delivery using nanoparticle systems. Safety profiles especially with CRISPR/Cas9 remains a concern. A systematic search of Web of Science, Medline, Scopus, and Google Scholar identified 512 records (January 2010-March 2023). Following PRISMA screening, 18 studies met inclusion criteria. These studies evaluated strategies of gene therapy in neuropathy, arthritis, fibromyalgia, and complex regional pain syndrome. The review outlines available options and highlights preclinical findings.},
}
@article {pmid42090053,
year = {2026},
author = {Dhara, C and Sah, H and Gantayat, S and Rajput, M and Mishra, S},
title = {CRISPR/Cas9 in perspective: evaluating efficacy, delivery methods, and ethical challenges in genome editing.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42090053},
issn = {1573-4978},
mesh = {*Gene Editing/ethics/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Transfer Techniques/ethics ; Genetic Engineering/methods/ethics ; },
abstract = {The latest developments in DNA sequencing techniques have revealed genes that play a role in determining our vulnerability to diseases and have given us a deeper understanding of our genetic composition. A groundbreaking milestone in genetic engineering has transformed the capabilities of the scientific community in altering the genetic material of different organisms. Among recent innovations, the clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) has emerged as a powerful and precise tool for genome editing across diverse organisms. Its applications span immunotherapy, agriculture, poultry science, and human therapeutics, marking a transformative shift in biomedical and biotechnological research. However, the rapid progress and clinical translation of CRISPR/Cas9 have raised significant concerns regarding off-target effects, delivery challenges, long-term safety, and ethical implications. This review critically evaluates the CRISPR/Cas9 system by examining its molecular mechanism, editing efficiency, gene delivery approaches, and potential for inducing unintended mutations. A comparative analysis with other gene-editing tools is presented, emphasizing the advantages of CRISPR/Cas9 in programmability and editing efficiency. Furthermore, we discuss current advances including base editing, prime editing, and high-fidelity Cas variants, along with the ethical and societal dimensions of genome editing. This synthesis provides an updated perspective on the potential and limitations of CRISPR/Cas9 technology and highlights key areas for future research and responsible application.},
}
@article {pmid42090299,
year = {2026},
author = {Xiong, Y and Tsai, LK and Zhou, J and Chen, S and Xia, X and Zhang, J and Chen, YE and Xu, J and Huang, X},
title = {Fully computational design of PAM-relaxed Staphylococcus aureus Cas9 with expanded targeting capability using UniDesign.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42090299},
issn = {2050-084X},
support = {GM149016/NH/NIH HHS/United States ; HL164205/NH/NIH HHS/United States ; },
mesh = {*Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 nucleases have transformed genome engineering, yet their application is often constrained by protospacer-adjacent motif (PAM) requirements. Staphylococcus aureus Cas9 (SaCas9) is particularly attractive for in vivo applications due to its compact size; however, its NNGRRT PAM limits targetable genomic sites. Here, we report KRH, a SaCas9 variant designed entirely from the wild-type enzyme through a fully computational point-mutation design workflow, UniDesign, without additional experimental optimization. As expected, KRH efficiently recognizes an expanded NNNRRT PAM and exhibits substantially enhanced editing efficiency at non-canonical PAM sites, with improvements of up to 116-fold over the wild type. KRH achieves genome- and base-editing efficiencies comparable to, or exceeding, those of the well-known evolution-derived KKH variant. Computational modeling by UniDesign provides a mechanistic explanation for the PAM relaxation observed in both KRH and KKH, with structural and energetic analyses revealing that KRH relaxes PAM specificity by fine-tuning the balance between sequence-specific interactions with PAM bases and nonspecific contacts with the DNA backbone. Beyond its practical utility, KRH demonstrates that computational design can identify a minimal set of mutations sufficient to remodel the PAM interface while preserving high nuclease activity. This approach recapitulates-and in some cases surpasses-the performance of evolution-derived variants, offering a scalable strategy for high-throughput Cas9 engineering. Overall, these results establish KRH as a blueprint for rationally engineered, PAM-relaxed nucleases and underscore the power of computational design to accelerate next-generation genome editing.},
}
@article {pmid42090813,
year = {2026},
author = {Hyeon, LS and Yang, JW and Bae, S and Yoo, JS and Kim, E and Lee, SS and Kang, S},
title = {Rapid and sensitive detection of blaKPC and blaNDM in carbapenemase-producing Enterobacterales using a one-pot recombinase polymerase amplification-CRISPR/Cas12a system with modified protospacer adjacent motifs.},
journal = {Biosensors &amp; bioelectronics},
volume = {307},
number = {},
pages = {118725},
doi = {10.1016/j.bios.2026.118725},
pmid = {42090813},
issn = {1873-4235},
mesh = {*beta-Lactamases/genetics/isolation &amp; purification ; *Bacterial Proteins/genetics/isolation &amp; purification ; Humans ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Enterobacteriaceae/genetics/enzymology/isolation &amp; purification ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Point-of-Care Systems ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification ; },
abstract = {Carbapenem-resistant Enterobacterales pose a critical global health threat because of their rapid transmission and resistance to last-line antibiotics. To address this threat, rapid and user-friendly point-of-care (POC) detection is essential. Quantitative polymerase chain reaction (qPCR), while providing high sensitivity, relies on expensive equipment and skilled personnel, hindering its practical use in resource-limited settings. Here, we aimed to develop an optimized one-pot RPA-CRISPR/Cas12a (RCCS) system with enhanced diagnostic performance to provide a reliable and user-friendly platform for rapid carbapenemase gene screening in both clinical and resource-limited settings. The developed assay was used for the detection of blaKPC and blaNDM genes and was integrated into a portable diagnostic device. By using suboptimal protospacer adjacent motif sequences, the assay provided a streamlined workflow and substantially enhanced detection sensitivity. This platform achieved a limit of detection of 10[-17] M for blaKPC and 10[-16] M for blaNDM within 30 min. Validation with 44 clinical samples demonstrated that the assay had 100% sensitivity and specificity, matching the effectiveness of qPCR. The one-pot RCCS platform offers a robust and highly sensitive POC solution for on-site testing.},
}
@article {pmid42090855,
year = {2026},
author = {Tian, Y and Chen, J and Chen, F and Song, J and Shi, K and Yi, Z and Wang, S},
title = {A novel fluorescent aptasensor for sensitive and label-free detection of Di(2-ethylhexyl) phthalate based on catalytic hairpin assembly-assisted CRISPR/Cas9 and G-triplex.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {360},
number = {},
pages = {128020},
doi = {10.1016/j.saa.2026.128020},
pmid = {42090855},
issn = {1873-3557},
mesh = {*Diethylhexyl Phthalate/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; Spectrometry, Fluorescence/methods ; Limit of Detection ; Fluorescent Dyes/chemistry ; Plasticizers/analysis ; DNA/chemistry ; },
abstract = {Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that poses significant risks to human health and ecosystems, making its sensitive, rapid, and reliable detection critically important for environmental monitoring and food safety assessment. CRISPR-Cas9, beyond its canonical site-specific endonuclease function, has recently been reported to exhibit trans-cleavage activity upon target recognition, enabling its application in nucleic-acid-triggered signal amplification and biosensing. However, the exploration of Cas9-based trans-cleavage strategies for small-molecule targets such as DEHP remains challenging. By effectively integrating catalytic hairpin assembly (CHA) with Cas9-mediated trans-cleavage, a sensing platform termed Cas9/CHA was constructed for DEHP detection. Within this platform, aptamer-specific recognition of DEHP triggers the CHA reaction, producing abundant double-stranded DNA products that activate Cas9's DNase activity. The activated Cas9 system subsequently cleaves the loop region of the G3-based hairpin probe, releasing the G3 sequence. Upon binding to thioflavin T (ThT), the liberated G3 produces a fluorescence signal through the formation of a G3/ThT complex. Quantitative analysis is achieved by establishing a linear relationship between fluorescence intensity and DEHP concentration over the range of 10 pM to 1 μM. Importantly, the Cas9/CHA platform allows selective detection of DEHP down to 3 pM and shows reliable performance in milk, bottled water and tap water samples. The Cas9/CHA system extends CRISPR/Cas9 applications beyond nucleic acid analysis and provides a versatile and robust framework for developing next-generation analytical tools for food safety surveillance and environmental monitoring.},
}
@article {pmid42091816,
year = {2026},
author = {Malapaka, A and Meunier, FA and Gormal, RS},
title = {Single-Molecule Imaging of Endogenous Proteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3034},
number = {},
pages = {211-237},
pmid = {42091816},
issn = {1940-6029},
mesh = {*Single Molecule Imaging/methods ; Microscopy, Fluorescence/methods ; Humans ; *Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Single-Domain Antibodies ; Plasmids/genetics ; },
abstract = {Single-molecule imaging is a technique of choice to investigate the dynamic nanoscale organization of proteins of interest, revealing a wealth of new information on how proteins perform their biological function. However, overexpression, which is often used in this context, can alter biological functions. It is therefore useful to implement tools to visualize and track endogenous proteins in living cells and observe their dynamic clustering and motion within their native environment. Herein, we describe two approaches: Fluorescent intrabody Localization Microscopy (FiLM) and gene-editing (CRISPR/Cas9) and provide a workflow including the design of plasmid backbones used to track endogenous proteins by either i) introducing nanobodies that target endogenous proteins to perform single-particle tracking or ii) gene-editing cells in culture to express tagged endogenous proteins at biologically relevant expression levels.},
}
@article {pmid42092764,
year = {2026},
author = {Liu, J and Jiang, W and Wang, X and Azoitei, A and Liu, H and Najjar, G and Liu, K and Melzer, MK and Stilgenbauer, S and Elati, M and Burkhalter, MD and Philipp, M and Wezel, F and Zengerling, F and Bolenz, C and Günes, C},
title = {CRISPR and compound screens in a novel ex vivo tissue model identify DDR1 and ETA as regulators of cancer cell invasion.},
journal = {Cellular &amp; molecular biology letters},
volume = {31},
number = {1},
pages = {},
pmid = {42092764},
issn = {1689-1392},
mesh = {Animals ; Humans ; Neoplasm Invasiveness ; *Urinary Bladder Neoplasms/pathology/genetics/metabolism ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Swine ; Cell Movement/drug effects ; Non-Muscle Invasive Bladder Neoplasms ; Small Molecule Libraries/pharmacology ; },
abstract = {BACKGROUND: Bladder cancer (BC) can be characterized clinically as either non-muscle-invasive (NMIBC) or muscle-invasive (MIBC). While NMIBC generally has a favorable prognosis, MIBC is characterized by high morbidity and mortality. Understanding the molecular determinants of tumor invasion is critical, yet research is hampered by the limitations of current experimental models. Standard assays such as the Boyden chamber lack physiological complexity, while porcine bladder models suffer from tissue contamination and genetic variability. There is an urgent need for reliable models that mimic the intact tissue architecture.<br><br>METHODS: We established a unique ex vivo tissue invasion model (EXTIM) to evaluate the invasive capacity of BC cells within a largely intact tissue context, using freshly prepared bladders from mice. The invasiveness of human BC cells (RT4, T24, UMUC3) and the immortal urothelial cell strain (Y235T) was comparably evaluated using EXTIM, the Boyden chamber, and porcine models. Gene knockdown or ectopic expression of GJB3 or ORP3 indicated the suitability of EXTIM to investigate the impact of specific factors on tumor cell invasion. To identify novel genetic regulators of cell invasion, we combined EXTIM with a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen. Additionally, we utilized the EXTIM to perform a pharmacological screen of a small molecule library comprising 90 substances to identify compounds capable of suppressing BC cell dissemination.<br><br>RESULTS: Importantly, by combining EXTIM with genomewide CRISPR-Cas9 screening, we identified several candidate genes involved in BC progression. Notably, discoidin domain receptor tyrosine kinase 1 (DDR1) was identified as a functional inhibitor of tumor cell invasion. Furthermore, the small-molecule screen revealed that PD-156707, a selective antagonist of the endothelin receptor A (ETA), significantly suppresses cancer cell invasion within the EXTIM environment.<br><br>CONCLUSIONS: EXTIM serves as a robust and physiologically relevant tool for assessing tumor cell invasion and migration under ex vivo conditions. EXTIM can be used to identify factors involved in the progression of invasive BC by high-throughput genetic screenings in an ex vivo organ culture system, by culturing cells after transmigration through the bladder tissue. Moreover, the impact of specific genetic factors in the process of tumor cell dissemination can be assessed by placing bladders from genetically modified mice into the EXTIM.},
}
@article {pmid42093418,
year = {2026},
author = {Cho, HM and Ryu, YC and Cho, MK and Hwang, BH},
title = {Functional peptide-assisted delivery of CRISPR/Cas9: recent progress on gene editing.},
journal = {Biomaterials science},
volume = {14},
number = {12},
pages = {3132-3156},
doi = {10.1039/d6bm00231e},
pmid = {42093418},
issn = {2047-4849},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Peptides/chemistry/metabolism ; Animals ; *Gene Transfer Techniques ; *Cell-Penetrating Peptides/chemistry ; },
abstract = {The CRISPR/Cas9 system has transformed the field of genome engineering by enabling precise and programmable modification of genomic sequences. Despite its remarkable potential, efficient and safe delivery of CRISPR/Cas9 components remains a significant bottleneck, particularly in translational and therapeutic applications. In the past decade (2015-2025), peptide-based delivery systems have gained increasing attention as versatile non-viral alternatives due to their modular design, inherent biocompatibility and potential for tissue-specific targeting. This review provides a critical synthesis of recent advances in functional peptide-based delivery strategies for CRISPR/Cas9. We focus on key categories of peptides-cell-penetrating peptides, cell-targeting peptides, endosomal escape peptides, and nuclear localization signals-and their multifunctional or hybrid combinations. By identifying key design principles and failure modes, this review discusses how these platforms can be optimized to support the development of next-generation CRISPR therapeutics, ultimately moving closer to safe and effective genome editing in future clinical applications.},
}
@article {pmid42094414,
year = {2026},
author = {Yoon, PH and Loi, K and Zhang, Z and Docter, TA and Lopez, SC and Langeberg, CJ and Moez Ur-Rehman, M and Vohra, K and Zhou, Z and Shi, H and Boger, R and Wang, PY and Adler, BA and Brohawn, SG and Doudna, JA},
title = {A Noncontiguous Code for RNA-Guided DNA Recognition Preceded CRISPR.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42094414},
issn = {2692-8205},
support = {K99 GM160778/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas systems use RNA-guided proteins for adaptive immunity through a mechanism whose origin is unknown. Here we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein more ancient than CRISPR-Cas and vrRNAs comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that base pair with nucleic acid targets using an uninterrupted sequence, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NNs of each repeat collectively specify a target that itself contains a gapped recognition sequence. Analysis of natural vrRNA targets suggests VIPR acts against competing phages. We demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that the roots of adaptive immunity lie in ancient warfare between viruses, and reveal a new logic for programmable genetic control.},
}
@article {pmid42094579,
year = {2026},
author = {Malaluan, RPP and Dy, RLV},
title = {Acquisition of novel arrays via horizontal gene transfer rewire CRISPR-mediated defense in Pseudomonas aeruginosa.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42094579},
issn = {2692-8205},
abstract = {CRISPR-Cas systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the CRISPR-Cas system of Pseudomonas aeruginosa ATCC 10145 (PA10145), which led us to discover the existence of an isolated CRISPR array, unique to this system. PA10145 possesses a type I-F CRISPR-Cas composed of a cas operon flanked by two divergently organized CRISPRs. The isolated CRISPR array, CRISPR3, is located ~1.3 million bp away from the cas loci. The cas and three CRISPR arrays are active. Plasmids with an engineered protospacer matching any of the three arrays were targeted and stimulated hyperactive adaptation in all CRISPR arrays of PA10145 if the plasmids possessed an intact protospacer adjacent motif (PAM), whereas minimal to no adaptation was observed when PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and the cas-adjacent CRISPR2 have nearly identical leader sequences with only 3 bp mismatches. From a survey of CRISPR loci in 1,198 P. aeruginosa genomes, isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Highly-transmissible mobile genetic elements (MGEs) associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these MGEs. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas-adjacent arrays revealed that CRISPR3 is laterally transferred across P. aeruginosa genomes. Taken together, these results implicate the role of horizontally-acquired isolated arrays in CRISPR-mediated pan-immunity as gateways to mobilize genetic memories.},
}
@article {pmid42096148,
year = {2026},
author = {Zhu, D and Wang, S and Sun, X and Britton, RA},
title = {CRISPR-AsCas12a and dAsCas12a-Mediated Gene Knockout and Knockdown in Clostridioides difficile.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3046},
number = {},
pages = {47-55},
pmid = {42096148},
issn = {1940-6029},
mesh = {*Clostridioides difficile/drug effects/genetics/pathogenicity ; *Gene Knockout Techniques ; *Gene Knockdown Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Acidaminococcus/enzymology ; Virulence/genetics ; *Drug Resistance, Bacterial/genetics ; *Host-Pathogen Interactions/genetics ; },
abstract = {Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and severe colitis, yet its genetic manipulation has long been constrained by low DNA transfer efficiency and limited recombination systems. Recent advances in CRISPR-based technologies have revolutionized the genetic toolkit for this pathogen, enabling precise genome editing and transcriptional regulation. Among CRISPR nucleases, Cas12a offers distinct advantages over Cas9 for bacterial applications, including a smaller size, T-rich PAM recognition, single-crRNA requirement, and reduced toxicity, which enhances conjugation efficiency in genetically recalcitrant organisms. AsCas12a-based platforms have enabled large fragment deletions, multiplex editing, and rapid generation of marker-free mutants in C. difficile. Complementing these nuclease-active systems, nuclease-deactivated variants (dCas9 or dAsCas12a) support CRISPR interference (CRISPRi)-a reversible, tunable approach for transcriptional repression without altering genomic sequences. Compared to traditional mutagenesis, CRISPRi greatly accelerates functional genomics by enabling high-throughput screening and drug target discovery. Together, our lab has independently developed CRISPR-AsCas12a-mediated genome editing and dAsCas12a-based CRISPRi tools, providing complementary strategies to overcome longstanding genetic barriers in C. difficile. These tools open new avenues for system-level interrogation of virulence, antibiotic resistance, and host-pathogen interactions.},
}
@article {pmid42096830,
year = {2026},
author = {Yin, Y and Wang, M and Sun, Y and Zhao, P and Ye, J},
title = {Sugarcane viral diseases: Epidemiology, detection, and advanced breeding methods for resistance.},
journal = {Virology},
volume = {621},
number = {},
pages = {110940},
doi = {10.1016/j.virol.2026.110940},
pmid = {42096830},
issn = {1096-0341},
mesh = {*Saccharum/virology/immunology/genetics ; *Plant Diseases/virology/prevention &amp; control ; *Disease Resistance ; *Plant Viruses/genetics/isolation &amp; purification/physiology ; *Plant Breeding/methods ; },
abstract = {Sugarcane viruses threaten yield and sucrose content, imposing substantial global economic losses. This review provides current knowledge on the major sugarcane pathogens, with a focus on virus biology, transmission, and virus-host interactions, and uses this foundation to frame practical control strategies. We first describe the epidemiology and economic impact of sugarcane viral diseases to establish the urgency of robust surveillance and management. Building on these insights, we then evaluate detection and surveillance technologies, ranging from traditional immunoassays and nucleic acid hybridization to molecular diagnostics, isothermal amplification, and CRISPR-based approaches, and discuss how high-throughput sequencing accelerates virus discovery. Next, we assess management strategies that include virus-free planting material, vector control, sanitation, and modern breeding technologies, like RNA interference (RNAi), CRISPR/Cas genome editing, marker-assisted selection (MAS), and genome wide association studies (GWAS) within genomic selection (GS) frameworks. We also consider supplementary strategies such as plant-derived antivirals and environmental management, and discuss how they complement core approaches. Crucially, we identify key knowledge gaps in sugarcane virus-host interactions and resistance breeding, and propose data-driven, multi-omics and AI-assisted approaches to precision breeding and integrated disease management. The review concludes with a practical roadmap for advancing sugarcane virus control and promoting sustainable production.},
}
@article {pmid42097051,
year = {2026},
author = {Liao, J and Rima, J and Sharma, A and Tsade, J and Jiang, F},
title = {Machine learning-enabled smartphone CRISPR-Cas12a lateral flow platform for sensitive detection of circulating HPV DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {307},
number = {},
pages = {118765},
doi = {10.1016/j.bios.2026.118765},
pmid = {42097051},
issn = {1873-4235},
mesh = {*Smartphone ; Humans ; *CRISPR-Cas Systems/genetics ; Machine Learning ; *Biosensing Techniques/instrumentation ; *Papillomavirus Infections/diagnosis/virology/blood ; *DNA, Viral/blood/genetics/isolation &amp; purification ; Female ; *Papillomaviridae/genetics/isolation &amp; purification ; Point-of-Care Systems ; Sensitivity and Specificity ; },
abstract = {Persistent infection with high-risk human papillomavirus (HPV) is a major cause of cervical cancer, and improved point-of-care (POC) detection is critical for early intervention. Although PCR-based assays are highly sensitive, their reliance on centralized laboratory infrastructure limits accessibility in decentralized settings. CRISPR-Cas diagnostics combined with lateral flow assays (LFA) offer a rapid alternative; however, visual interpretation of faint test bands remains subjective and variable. Here, we developed a smartphone-based CRISPR-Cas12a LFA platform integrated with an interpretable machine learning (ML) framework for quantitative detection of circulating HPV DNA in plasma. Standardized image acquisition was implemented using a light-controlled enclosure, and radiomics-inspired features were analyzed using a multivariable logistic regression model. The system was trained on 150 plasma samples and validated in an independent cohort of 60 samples. The optimized model achieved 96.7% sensitivity and 100% specificity, outperforming visual interpretation, particularly for low-signal samples. Performance remained stable across different smartphone models, lighting conditions, and operators, with rapid on-device inference enabling consistent and reliable operation. This integrated CRISPR-LFA platform demonstrates accurate and reproducible detection of circulating HPV DNA and supports feasibility for POC applications, pending further validation in broader clinical settings.},
}
@article {pmid42097949,
year = {2026},
author = {Fuller, MGA and Foley, M and Barrangou, R},
title = {CRISPR-based technologies for large DNA insertions.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.04.004},
pmid = {42097949},
issn = {1879-3096},
abstract = {While the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technologies has democratized the genesis of precise mutations, there is a need for more sophisticated tools to enable large-scale DNA manipulations, advancing genome editing across medicine, biotechnology, and agriculture. The success of Cas9 and Cas12 has hinged on the generation of precise DNA nicks and double-stranded breaks (DSBs), enabling local sequence mutagenesis, albeit of a limited size range. Emerging effectors combining Cas with other enzymatic functions, such as CRISPR-associated transposons and site-specific recombinases, enable larger integrations. Sophisticated combinations such as programmable addition via site-specific targeting element (PASTE), prime-editing-assisted site-specific integrase gene editing (PASSIGE), and prime-editing-mediated recombination of opportune target (PrimeRoot) expand payload options and DSB-free editing modalities, with translational potential for next-generation crop breeding in sustainable agriculture and the development of gene and cell therapies in personalized medicine.},
}
@article {pmid42098118,
year = {2026},
author = {Gierisch, ME and Barchi, E and Marogna, M and Wallnöfer, MH and Ankarcrona, M and Naia, L and Salomons, FA and Dantuma, NP},
title = {Mitochondria serve as a holdout compartment for aggregation-prone proteins hindering efficient degradation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42098118},
issn = {2041-1723},
support = {2021-02562//Vetenskapsr&#xe5;det (Swedish Research Council)/ ; 211653Pj//Cancerfonden (Swedish Cancer Society)/ ; FO2022-0271//Hj&#xe4;rnfonden (Swedish Brain Foundation)/ ; FO2023-0376//Hj&#xe4;rnfonden (Swedish Brain Foundation)/ ; KID grant//Karolinska Institutet (Karolinska Institute)/ ; GI-1329/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Mitochondria/metabolism ; Humans ; Huntingtin Protein/metabolism/genetics ; Proteolysis ; alpha-Synuclein/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; HEK293 Cells ; *Protein Aggregates ; Neurodegenerative Diseases/metabolism/genetics ; CRISPR-Cas Systems ; Protein Aggregation, Pathological/metabolism ; },
abstract = {The accumulation of protein aggregates has been causatively linked to the pathogenesis of neurodegenerative diseases. Here, we conduct a genome-wide CRISPR-Cas9 screen to identify cellular factors that regulate the degradation of an aggregation-prone reporter. Genes encoding proteins involved in mitochondrial homeostasis, including the translation factor eIF5A, are enriched among suppressors of the degradation of the reporter. Genetic or chemical inhibition of eIF5A leads to dissociation of the aggregation-prone substrate from mitochondria, which is accompanied by enhanced ubiquitin-dependent proteasomal degradation. The presence of an aggregation-prone, amphipathic helix that localizes the reporter to mitochondria is crucial for the stimulatory effect of eIF5A inhibition on proteasomal degradation. Additionally, inhibition of eIF5A also enhances degradation of mutant huntingtin and α-synuclein, two disease-associated proteins that contain amphipathic helices and mislocalize to mitochondria. We propose that mitochondria serve as a holdout compartment for aggregation-prone proteins. Therefore, preventing mitochondrial localization of aggregation-prone proteins may offer a viable therapeutic strategy for reducing disease-associated proteins in neurodegenerative disorders.},
}
@article {pmid42098301,
year = {2026},
author = {He, L and Na, H and Zhang, Y and Gong, P and Zhou, H and Wan, F},
title = {Ultrasensitive Detection and Its Potential Applications in the Diagnosis of Brucellosis.},
journal = {Current microbiology},
volume = {83},
number = {6},
pages = {},
pmid = {42098301},
issn = {1432-0991},
support = {2024LHMS08055//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2025MS08058//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2025---14//Hohhot Basic and Applied Basic Research Science and Technology Plan Project/ ; DC2000000754//the Prairie Talent Leadership Program/ ; BR22-14-02//the Basic Scientific Research Funds for Inner Mongolia Autonomous Region Government/ ; },
mesh = {*Brucellosis/diagnosis/microbiology ; Humans ; Animals ; *Brucella/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Biosensing Techniques/methods ; Zoonoses/diagnosis/microbiology ; Sensitivity and Specificity ; },
abstract = {Brucellosis is a major zoonotic disease caused by Brucella spp., notably B. melitensis, B. abortus, B. canis, and B. suis. It continues to impose a significant public health and economic burden in endemic regions, including parts of Asia, Africa, the Middle East, and Latin America. The clinical presentation of the disease is often nonspecific, and early, accurate diagnosis is further complicated by the susceptibility of conventional serological assays to cross-reactivity. This narrative review covers literature published between 2000 and 2025 regarding the detection of Brucella, indexed in PubMed, Web of Science, and China National Knowledge Infrastructure (CNKI). The review focuses on the limitations of traditional diagnostic methods and the performance characteristics and latest developments of emerging ultrasensitive detection technologies, including nucleic acid amplification (LAMP, RPA, SRCA), droplet digital PCR, CRISPR-Cas-based integrated systems, nanomaterial-enhanced assays, and protein/antigen-based biosensors. Their potential for integration into the One Health framework for zoonotic disease surveillance is also discussed.},
}
@article {pmid42100374,
year = {2026},
author = {Schrage, PR and Afonina, U and Wörtz, J and Marchfelder, A and Martens, KJA and Sáenz, JP and Endesfelder, U},
title = {A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization of altered Cas1 dynamics during UV-induced DNA damage response.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag014},
pmid = {42100374},
issn = {2633-6693},
abstract = {Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and cellular processes in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free H. volcanii strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel plasmid system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins-from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1-while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal Cas1 colocalizing with DNA-dense cellular regions and significant, dose-dependent changes in Cas1 mobility following UV-light-induced damage, providing evidence for its possible involvement in DNA damage response processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.},
}
@article {pmid42100688,
year = {2026},
author = {Yadav, J and Gehlot, P and Soni, P and Jain, T},
title = {Plant microbiome engineering: from inoculation to genome editing.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1781381},
pmid = {42100688},
issn = {1664-302X},
abstract = {Plant-associated microbiomes are central to crop productivity, nutrient efficiency, and stress resilience, yet conventional microbiome manipulation strategies, largely based on microbial inoculation and agronomic management, often suffer from inconsistent field performance and limited persistence. Although several recent reviews have discussed CRISPR-mediated plant-microbe engineering and synthetic microbial community (SynCom) design separately, few reviews integrate genome editing, ecological stability of microbiomes, and climate-resilient agricultural applications within a unified conceptual framework. Recent advances in molecular biotechnology are transforming this landscape by enabling precision engineering of plant-microbe interactions at genetic, metabolic, and community levels. In particular, synthetic biology tools including CRISPR/Cas genome editing, RNA interference, and synthetic microbial communities (SynComs), now allow targeted modification of plant traits governing microbial recruitment, microbial pathways underpinning nutrient cycling and stress tolerance, and community-level functional complementarity. This review integrates molecular genetics, microbial ecology, and systems-level microbiome design to frame the plant and its microbiome as an engineerable holobiont. We integrate insights from genome editing in plants and microbes, omics-guided SynCom design, climate-resilience mechanisms, and emerging AI-assisted decision frameworks, including machine learning and ecological modeling approaches used to analyze multi-omics datasets, and predict plant-microbiome interactions across experimental and field-based studies. Importantly, we critically assess limitations related to ecological stability, trait trade-offs, biosafety, and regulatory challenges that constrain large-scale deployment. By bridging genome-enabled microbiome manipulation with ecological design principles, this review proposes an integrative framework for climate-smart microbiome engineering and identifies key research priorities required to transition from empirical inoculation toward predictive, sustainable, and socially responsible agricultural biotechnology.},
}
@article {pmid42100873,
year = {2026},
author = {Kavanagh, EW and Joynt, AT and Pion, AR and Eastman, AC and Parr, AI and Starego, KL and Jain, M and Shannon, SR and Yoo, EJ and Newby, GA and Tzeng, SY and Sharma, N and Green, JJ and Cutting, GR},
title = {Base editing and nanoparticle transfection of airway cell types essential for treatment of cystic fibrosis.},
journal = {JCI insight},
volume = {11},
number = {9},
pages = {},
pmid = {42100873},
issn = {2379-3708},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Nanoparticles/administration &amp; dosage/chemistry ; *Gene Editing/methods ; *Transfection/methods ; Epithelial Cells/metabolism ; Genetic Therapy/methods ; Respiratory Mucosa/metabolism ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; },
abstract = {Cystic fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucus impairing the airway epithelia. Durable correction of these variants in airway cells remains a therapeutic challenge for about 10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant, 3120+1G>A, was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types resulting in notable enrichment of CFTR-expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited, but they decreased as a fraction of total cells and CFTR-expressing cells compared with unedited cells. CRISPR base editors delivered by polymeric nanoparticles (PNPs) facilitated functional rescue of CFTR to clinically meaningful levels in immortalized and primary airway cells. PNPs delivered GFP-encoding RNA to progenitor airway cells in fully differentiated airway cultures. Vitronectin was a major component of the PNP corona that formed in vivo, but preincubation with vitronectin did not enhance delivery. Together, these findings validate a scalable, nonviral platform with compelling translational promise for treating CF and other respiratory diseases involving respiratory epithelial cell dysfunction.},
}
@article {pmid42101028,
year = {2026},
author = {Madden, D and Trujillo, JD and Elango, S and Fitz, I and McDowell, CD and Assato, P and Lyoo, E and Kwon, T and Cool, K and Li, Y and Ferreyra, FM and Gaudreault, NN and Morozov, I and Lee, K and Driver, J and Richt, JA},
title = {CD1D knockout swine are permissive to African swine fever virus genotype II infection.},
journal = {Emerging microbes &amp; infections},
volume = {15},
number = {1},
pages = {2671519},
pmid = {42101028},
issn = {2222-1751},
mesh = {Animals ; *African Swine Fever Virus/genetics/pathogenicity/physiology ; *African Swine Fever/virology/genetics/immunology/pathology ; Swine ; Genotype ; *Antigens, CD1d/genetics/metabolism ; Virus Replication ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Virulence ; },
abstract = {African swine fever virus (ASFV) is an important pathogen of domestic and wild suids and the causative agent of African swine fever (ASF). ASFV displays a tropism for myeloid cells, predominately of the monocyte/macrophage lineage, which is critical for ASFV pathogenesis. However, the mechanisms that govern the cellular tropism of ASFV are complex and incompletely understood. ASFV can enter susceptible cells through several mechanisms. One way is by clathrin-mediated endocytosis (CME) via interaction with the host glycoprotein CD1d, encoded by the CD1D gene, and disruption of CD1D expression significantly reduces ASFV replication in cell culture. In order to evaluate the role of CD1d in the replication and pathogenesis of ASF in vivo, CD1D knockout (KO) pigs, generated using a CRISPR-Cas9 system, were challenged with the highly virulent genotype II ASFV isolate MNG19. Notably, CD1D KO pigs were highly permissive to ASFV infection and developed severe acute ASF similar to age-matched wildtype (WT) controls. No significant differences in disease severity or mortality between ASFV-infected WT and KO pigs were observed. ASFV DNA levels in blood or in the majority of visceral and lymphoid tissues were also not significantly different between ASFV-infected wild-type and KO pigs. In addition, pathological changes were similar between both groups and typical of acute ASF, such as fibrinous polyserositis, haemorrhagic lymphadenopathy, and systemic coagulopathy. This work demonstrates that porcine CD1d is not critical for genotype II ASFV replication and virulence in vivo, and that CD1D KO piglets possess no resistance to virulent genotype II ASFV infection.},
}
@article {pmid42101329,
year = {2026},
author = {Moss, O and Li, X and Kanagarajan, S and Wang, ES and Ivarson, E and Zhu, LH},
title = {Evaluation of Bna.SCT and Bna.REF1 as Target Genes to Reduce Sinapine in Rapeseed Using a Protoplast-Based CRISPR RNP Approach.},
journal = {Physiologia plantarum},
volume = {178},
number = {3},
pages = {e70905},
pmid = {42101329},
issn = {1399-3054},
support = {//SLU Grogrund- Centre for Breeding of Food Crops/ ; //Trees and Crops for the Future (TC4F)/ ; //The Royal Physiographic Society of Lund/ ; },
mesh = {Gene Editing/methods ; *Brassica napus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Protoplasts/metabolism ; *Brassica rapa/genetics/metabolism ; Mutation ; Choline/analogs &amp; derivatives ; },
abstract = {Rapeseed is a major oil crop worldwide, producing both oil and a high amount of protein. However, the use of its seed meal as a protein source for animal feed is limited by antinutritional factors, such as sinapine, which reduces nutrient absorption and affects the palatability. Efforts to reduce sinapine levels through conventional breeding have had limited success. Given the challenges of a changing climate and a growing global population, maximising crop utility, particularly the value of seed meal as a byproduct, is increasingly important. Genetic modification has been successfully used to reduce sinapine in rapeseed, but regulatory restrictions limit its commercial adoption in some regions. CRISPR-Cas gene editing, which is gaining broader global acceptance, offers a promising alternative to directly produce transgene-free mutants. In this study, we build on our previous work by generating transgene-free rapeseed mutants using protoplast-based CRISPR RNP gene editing. We successfully targeted the Bna.SCT and Bna.REF1 genes with editing efficiencies of 22%-63%, frequently achieving mutations in all four alleles of the target genes in T2 plants with a single sgRNA. Seed sinapine content was reduced by up to 38% in Bna.SCT mutants and 77% in Bna.REF1 mutants, with no observed effects on plant growth or development. These findings suggest that Bna.REF1 is the most effective target for sinapine reduction in transgene-free mutants among the genes tested in our lab.},
}
@article {pmid42101638,
year = {2026},
author = {Uc-Chuc, MA and Jiménez-Ramírez, IA and Guzmán-Marín, EDS and Chan-Pérez, JI and Acosta-Viana, KY},
title = {Beyond the catalysis of Trypanosoma cruzi trans-sialidases: structure, function, post-translational modifications, intrinsically disordered regions and use of CRISPR/Cas9.},
journal = {Archives of microbiology},
volume = {208},
number = {8},
pages = {},
pmid = {42101638},
issn = {1432-072X},
mesh = {*Trypanosoma cruzi/enzymology/genetics ; *Neuraminidase/metabolism/genetics/chemistry ; Protein Processing, Post-Translational ; *Glycoproteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism/chemistry ; Chagas Disease/parasitology ; Intrinsically Disordered Proteins/metabolism/genetics/chemistry ; Phylogeny ; Humans ; Gene Editing ; },
abstract = {Chagas disease, caused by the protozoan Trypanosoma cruzi, is a global health problem with limited treatment options. The parasite's trans-sialidase (TS) protein family has been widely described as a key component in its life cycle. In this work, we present a comprehensive review of the TS family, including its molecular structure, phylogenetic relationships, and known functions. Based on in silico analyses, we propose that intrinsically disordered regions (IDRs) present in the TS family may play a role in the spatial organization of these proteins. We also hypothesize that these IDRs could contribute to the formation of biomolecular condensates through liquid-liquid phase separation, providing a potential dynamic platform that is not fully explained by conventional structural models. In addition, we discuss recent advances in the application of the CRISPR/Cas9 gene-editing system to T. cruzi TS proteins. The available evidence indicates the multifunctional nature of these proteins, including enzymatic and non-enzymatic isoforms, as well as the presence of conserved motifs associated with host interactions and post-translational modifications. Taken together, this review integrates previous findings and proposes new hypotheses regarding additional functions of the TS family. The need for experimental studies to validate these proposals and clarify their relevance to the parasite's biology is emphasized. This approach could contribute to evaluating the potential of TS as a therapeutic target in Chagas disease.},
}
@article {pmid42102651,
year = {2026},
author = {Walus, M and Kida, E and Golabek, AA},
title = {Lysosomal storage, mitochondrial pathology, and autophagy in knockout of tripeptidyl peptidase 1 in human neuroblastoma cells in vitro.},
journal = {Molecular genetics and metabolism},
volume = {148},
number = {2},
pages = {110130},
doi = {10.1016/j.ymgme.2026.110130},
pmid = {42102651},
issn = {1096-7206},
mesh = {Humans ; Tripeptidyl-Peptidase 1 ; *Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics/deficiency/metabolism ; *Autophagy/genetics ; *Mitochondria/pathology/metabolism/genetics ; *Lysosomes/metabolism/pathology/genetics ; *Neuronal Ceroid-Lipofuscinoses/genetics/pathology/metabolism ; *Aminopeptidases/genetics/metabolism/deficiency ; *Serine Proteases/genetics/metabolism/deficiency ; Cell Line, Tumor ; Gene Knockout Techniques ; *Neuroblastoma/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {Deficiency of tripeptidyl-peptidase 1 (TPP1; EC 3.4.14.9), a lysosomal enzyme encoded by the CLN2 gene, is associated with the lysosomal storage disorder - classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease). The classic form of CLN2 disease leads to the accumulation of autofluorescent lysosomal storage and a massive loss of neurons with gliosis in the brain. The major component of the storage is subunit c of mitochondrial ATP synthase, a highly hydrophobic, 75-aminoacid polypeptide. We developed an in vitro model of CLN2 disease by knocking out CLN2 in the human neuroblastoma cells SH-SY5Y by using CRISPR/cas9 technology. We focused on defining the pattern of deposition of subunit c, factors contributing to subunit c accumulation, and subcellular morphometry to identify differences between the model cells knockout (KO) and controls. Implementation of acetone for cell fixation allowed us to: i. identify higher levels of subunit c in the mitochondria of KO cells than controls; ii. characterize in detail subunit c inclusions, also present in controls; iii. identify other mitochondrial proteins colocalizing with subunit c in inclusions; and iv. detect mitochondrial pathology in degenerating cells often accompanied by deposition of subunit c. Differentiation of cells with retinoic acid and brain-derived neurotrophic factor led to a substantial increase in the levels of subunit c and to significant differences in the levels of autophagy-related proteins between KO and control cells. Inhibition of induced autophagy by bafilomycin A1 (Baf.A1) decreased subunit c levels in controls but not in KO cells, whereas the levels of subunit c were unaffected by Baf.A1 treatment upon basal autophagy. Finally, subcellular morphometry showed differences in the number and size of vesicular structures immunostained for autophagy-related proteins between KO cells and controls upon both induced and basal autophagy, further supporting the association of TPP1 deficiency with autophagy.},
}
@article {pmid42103069,
year = {2026},
author = {Vikal, A and Maurya, R and Kumar, P and Verma, N and Mishra, AK},
title = {Next-generation gene editing strategies in cancer: Integrating CRISPR, PROTACs, and advanced molecular technologies.},
journal = {Life sciences},
volume = {397},
number = {},
pages = {124434},
doi = {10.1016/j.lfs.2026.124434},
pmid = {42103069},
issn = {1879-0631},
mesh = {Humans ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Proteolysis ; Genetic Therapy/methods ; Precision Medicine/methods ; Proteolysis Targeting Chimera ; },
abstract = {Cancer is a significant therapeutic problem as tumors are heterogeneous, multidrug-resistant, and oncogenic drivers are undruggable. Genome editing and targeted protein degradation are emerging approaches that are transforming precision oncology by allowing genetic and proteomic interventions. Such technologies as CRISPR-based systems and proteolysis-targeting chimeras (PROTACs) are alternative methods of correcting genes and the selective removal of a protein. Their combination provides them with new possibilities regarding long-lasting and specific cancer treatment and discovery of new therapeutic targets. Although this has good news, there are delivery, off-target effects and safety challenges. The continued advancements in nanotechnology, artificial intelligence (AI), and personalized medicine will be likely to improve clinical translation. Generally, the integration of these technologies is an inducing trend in the treatment of cancer in the next generation.},
}
@article {pmid42103732,
year = {2026},
author = {Barazas, M and van Schendel, R and Tijsterman, M},
title = {A mutational scar-based genome-wide map of DNA double-strand break repair.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103732},
issn = {2041-1723},
support = {2021-2/13905//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 2024-3/16583//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; DNA Polymerase theta ; *DNA Repair/genetics ; *Mutation ; CRISPR-Cas Systems ; DNA End-Joining Repair/genetics ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; Werner Syndrome Helicase/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Genome alterations arise from inaccurate DNA repair and accumulate as distinct mutational signatures. Here, we systematically interrogate the contribution of every protein-coding gene to double-strand break (DSB) repair by generating high-resolution outcome profiles following gene knockouts. Using a CRISPR/Cas9-based, massively parallel bulk screening approach, we establish a comprehensive catalogue of MUtational Scars of Induced DNA Cleavage (MUSIC) that maps the full landscape of DSB repair factors. Our analysis identifies and validates gene clusters - including nearly all known components and several previously unrecognised factors - associated with non-homologous end-joining, the 53BP1 pathway, homology-directed repair, and polymerase theta (POLQ)-mediated end-joining. By focusing on pathway-specific repair outcomes, we uncover an unexpected role for the WRN helicase in suppressing inverted templated insertions, a poorly understood POLQ-associated mutational signature. Finally, dissection of MUSIC features reveals unanticipated functional distinctions among genes within the same DSB pathway, providing mechanistic insight and enabling further investigation into chromosomal break repair.},
}
@article {pmid42105555,
year = {2026},
author = {Xiong, Y and Ye, S and Guo, Y and Li, L and Huang, X and Xiong, Y},
title = {Size-dependent AIENPs enhanced RPA-CRISPR/Cas12a mediated lateral flow assay for ultrasensitive detection of Staphylococcus aureus.},
journal = {Food chemistry},
volume = {517},
number = {},
pages = {149477},
doi = {10.1016/j.foodchem.2026.149477},
pmid = {42105555},
issn = {1873-7072},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics/chemistry ; Milk/microbiology ; Animals ; CRISPR-Cas Systems ; Particle Size ; Food Contamination/analysis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Bacterial Proteins/genetics ; *Biosensing Techniques/methods/instrumentation ; Rapid Diagnostic Tests ; Nanoparticles/chemistry ; },
abstract = {In this study, we developed a lateral flow assay (LFA) utilizing aggregation-induced emission nanoparticles (AIENPs) integrated with recombinase polymerase amplification (RPA) and CRISPR/Cas12a for the ultrasensitive detection of Staphylococcus aureus (S. aureus). We systematically evaluated the influence of AIENP particle size (250, 350, 450, and 600 nm) on the performance of LFA detection. Notably, the 450 nm AIENPs significantly enhanced the sensitivity of the strips due to their low background signal and size-dependent luminescent property. Under optimal conditions, the AIENPs-LFA demonstrated a 100-fold increase in sensitivity over conventional AuNPs-LFA. Combined with RPA, the assay achieved a remarkably low LOD of 9.95 CFU/mL for S. aureus, with a broad linear range (2.4-2.4 × 10[4] CFU/mL) and high specificity against other foodborne pathogens. Intra- and inter-assay validations indicated high accuracy (recoveries of 83.13%-106.93%) and precision (RSD < 14.42%) in S. aureus fortified milk samples.},
}
@article {pmid42105559,
year = {2026},
author = {Shi, T and Dai, T and Zhou, J and Li, J and Zhang, L and Cui, J and Ma, X and Dai, J and Chen, A and Wang, X},
title = {Rapid identification of binary and ternary adulteration in camellia oil by CRISPR/Cas12a assay.},
journal = {Food chemistry},
volume = {517},
number = {},
pages = {149501},
doi = {10.1016/j.foodchem.2026.149501},
pmid = {42105559},
issn = {1873-7072},
mesh = {*Food Contamination/analysis ; *Plant Oils/chemistry/analysis ; *Camellia/chemistry/genetics ; *CRISPR-Cas Systems ; },
abstract = {Camellia oil adulteration, bring about a serious potential threat to consumer health and food safety, yet current chromatography-based techniques require costly instruments and trained personnel, limiting their on-site detection. In this study, CRISPR/Cas12a was used for rapid identification of binary and ternary adulteration in camellia oil. On the basis of our screened RPA primer and crRNAs from corresponding rbcL genes, the CRISPR/Cas12a reaction, was completed within 40 min at a detection limit of 5% (w/w) in camellia oil adulterated with soybean oil, rapeseed oil, corn oil, and peanut oil, by visual naked-eye readout. Referring to the traditional gas chromatography technology, our constructed CRISPR/Cas12a method achieved 100% accuracy, highlighting its potential for routine market surveillance, especially for those oil samples at adulterated ratio ≤ 10%. This cost-effective and equipment-minimal assay, provide a promising tool for adulterated camellia oil on site detection, thus strengthening food quality control and consumer protection.},
}
@article {pmid42105578,
year = {2026},
author = {Wei, J and Wang, J and Wu, R and Zhang, J and Ren, H and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Aptakiss-guided CRISPR/Cas13a signal amplification for ultrasensitive FEN1 detection using CsPbBr3@PDA@AuNPs-based electrochemiluminescence platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {308},
number = {},
pages = {118775},
doi = {10.1016/j.bios.2026.118775},
pmid = {42105578},
issn = {1873-4235},
mesh = {*Flap Endonucleases/isolation &amp; purification/analysis ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; Humans ; Polymers/chemistry ; Indoles/chemistry ; Limit of Detection ; Titanium/chemistry ; },
abstract = {Sensitive and reliable detection of DNA repair enzymes is critical for early cancer diagnosis and therapeutic monitoring. Herein, we report a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Flap Endonuclease 1 (FEN1), integrating an aptakiss-assisted CRISPR/Cas13a signal amplification strategy with a perovskite-based nanocomposite sensing interface. Specifically, a hybrid nanomaterial composed of CsPbBr3 nanocrystals coated with polydopamine (PDA) and decorated with gold nanoparticles (AuNPs) was constructed to form a core-shell-satellite structure (CsPbBr3@PDA@AuNPs). The PDA coating enhanced the aqueous stability of CsPbBr3 and introduced functional groups for probe attachment, while the AuNPs facilitated electron transfer and signal amplification. Upon recognition and cleavage of a flap-structured DNA substrate by FEN1, a downstream transcription reaction was initiated to generate RNA triggers, which in turn activated the Cas13a system. Cas13a cleaved a surface-tethered RNA strand required for forming the aptakiss complex, thereby removing ferrocene-based ECL quenching and restoring strong luminescence. The proposed biosensor exhibited an excellent detection limit of 1.73 fM, with high selectivity against non-specific nucleases, and demonstrated remarkable reproducibility and long-term stability. This study not only presents a powerful biosensing platform for FEN1 but also highlights the potential of CsPbBr3@PDA@AuNPs as a versatile ECL-active material. The modular design and tunable functionality of the nanocomposite enable broad applicability in detecting various biomolecules, paving the way for the development of advanced perovskite-based ECL biosensors for clinical and environmental diagnostics.},
}
@article {pmid42106044,
year = {2026},
author = {Dutta, TK and Ray, S and Akhil, VS and Rupinikrishna, K and Chauhan, U and Dutta, A and Vijayan, J and Iquebal, MA and Chinnusamy, V},
title = {A synthetic guide RNA scaffold enhanced CRISPR/Cas9 editing efficiency in plants across multiple gene targets.},
journal = {International journal of biological macromolecules},
volume = {365},
number = {},
pages = {152456},
doi = {10.1016/j.ijbiomac.2026.152456},
pmid = {42106044},
issn = {1879-0003},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Arabidopsis/genetics ; Oryza/genetics ; *Plants/genetics ; },
abstract = {CRISPR/Cas9 mediated genome editing is a highly powerful and versatile tool for accelerating crop improvement. The editing efficiency of CRISPR/Cas9 system in planta has been highly variable owing to the variable binding affinity between native CRISPR RNA and Cas9 protein in vivo. In plant systems, systematic, large-scale engineering and benchmarking of guide RNA (gRNA) scaffold variants is still relatively limited compared with work in mammalian systems, despite several important studies demonstrating that scaffold and expression-cassette engineering can substantially improve CRISPR/Cas9 efficacy. Current study addresses the limitations of commonly used gRNA scaffold architecture by incorporating a stabilized stem-loop RAR (tetra loop) extension and a transcription-termination site mutation, resulting in improved RNA folding, increased Cas9 binding affinity, and enhanced in vivo editing outcomes. The synthesized scaffold boosted CRISPR/Cas9 efficiency in monocot or dicot plants across the 19 diverse target sites in Arabidopsis, rice and tomato. Furthermore, the synthetic scaffold is compatible with multiplex genome editing architectures, including polycistronic tRNA-gRNA (PTG) expression systems, enabling efficient simultaneous targeting of multiple genomic loci. The findings of this study have broad applications in precision plant breeding, functional genomics, and agricultural biotechnology, facilitating reliable gene modification across diverse plant species and transformation platforms.},
}
@article {pmid42106593,
year = {2026},
author = {Enciso-Rodríguez, F and Barrero, LS and Garzón-Martínez, GA and Kim, JH and Kumam, Y and Pagliai, FA and Jiang, T and Huo, H and Munoz, P},
title = {Overcoming breeding barriers with genome editing in autopolyploid crops.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08862-7},
pmid = {42106593},
issn = {1471-2229},
abstract = {Autopolyploid crops play a central role in global agriculture, yet their complex genomes pose significant barriers to genetic improvement. High allelic diversity, extensive redundancy, and polysomic inheritance impede both conventional breeding and the implementation of modern biotechnological tools. Genome editing offers a powerful alternative by enabling precise, multi-allelic modification of traits associated with yield, quality, and stress resilience. However, progress across autopolyploid crops remains uneven due to low transformation and regeneration efficiencies, limited genomic resources, and challenges in achieving complete allele disruption. This review focuses on recent advances in genome editing across four economically important autopolyploid crops-potato (Solanum tuberosum), alfalfa (Medicago sativa), sugarcane (Saccharum spp.), and blueberry (Vaccinium corymbosum). We highlight the diversity of traits targeted through CRISPR/Cas systems, including reporter and selectable marker validation, tuber and forage quality, biomass composition, stress tolerance, flowering modulation, and plant regeneration. We also describe technical constraints affecting genome editing in autopolyploids including genotype-dependent recalcitrance, low transformation and editing efficiency, multiallelic targeting and chimerism, outlining emerging solutions such as multiplexed designs, endogenous promoters, morphogenic regulators and virus-based approaches, among others. Together, these developments provide a path toward efficient and heritable genome editing in complex polyploid genomes, setting the stage for next-generation precision breeding in crops vital to food, forage, and bioenergy security.},
}
@article {pmid42106633,
year = {2026},
author = {Hannan, MN and Khan, S and Siddique, N and Hossain, MN and Islam, R and Nahar, K and Rahman, GKMM and Shozib, HB and Molla, AH and Hoque, MN and Haque, MM},
title = {Draft genome sequence of Priestia megaterium MHES4, a biofertilizer candidate isolated from tomato rhizosphere in Bangladesh.},
journal = {BMC genomic data},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12863-026-01431-9},
pmid = {42106633},
issn = {2730-6844},
support = {Project number: 002, Financial year: 2024 - 2027//This research was supported by Research Management Wing, Gazipur Agricultural University, Bangladesh./ ; },
abstract = {OBJECTIVE: The genus Priestia has recently gained attention for its plant growth-promoting potential. To examine the genomic traits and biosafety profile for potential field application as a native, climate-smart bioinoculant, we sequenced, assembled and annotated the genome of Priestia megaterium strain MHES4, isolated from the rhizosphere of tomato plant grown in drought-prone ecosystem of Rajshahi, Bangladesh.<br><br>DATA DESCRIPTION: Genome assembly data from the shotgun whole genome sequencing (WGS) of the P. megaterium MHES4 revealed 60 contigs with a total length of 5,267,048&#xa0;bp, an N50 of 446,003&#xa0;bp and 37.9% G&#x2009;+&#x2009;C content. The mean sequencing depth was 127.58&#xd7;, with 100% breadth of coverage. Genome completeness assessed was 97.43% with 3.5% contamination, confirming high assembly quality. In total, 5,484 protein-coding genes were annotated. Additionally, 5,445 protein-coding sequences, 28 tRNAs, and 5 rRNAs were identified. Functional analysis identified gene clusters involved in the synthesis of secondary metabolites, such as phytoene synthase and alpha-amylase, and a Type I CRISPR-Cas system. Biosafety assessment using in silico tools detected no virulence factors or transmissible antibiotic resistance genes, indicating its potential safe use in agriculture. Overall, this genomic resource provides valuable insights into the genetic potential of P. megaterium MHES4 for nutrient cycling and adaptation to the rhizosphere environment.},
}
@article {pmid42106677,
year = {2026},
author = {Peng, Z and Duan, W and Fan, Y and Yang, Q and Ye, Y and Xing, Y},
title = {PAM-flexible SpCas9 variants expand the targeting scope for porcine genome editing and cellular disease modeling.},
journal = {BMC biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12896-026-01164-8},
pmid = {42106677},
issn = {1472-6750},
support = {2023YFC3404302//National Key Reaearch and Development Program of China/ ; 32260825//National Natural Science Foundation of China/ ; },
abstract = {BACKGROUND: CRISPR-Cas-mediated gene editing has revolutionized life sciences, yet the targeting scope of the widely used SpCas9 is limited by its strict requirement for the NGG protospacer adjacent motif (PAM). To overcome this limitation, PAM-flexible SpCas9 variants have been developed and characterized in multiple species; however, their potential in pigs (an important biomedical model for humans) remains unexplored. Here, we systematically evaluated the editing performance of three PAM-flexible SpCas9 variants (SpRY, SpG, and SpCas9-NG) and their derived base editors in porcine fetal fibroblasts (PFFs).<br><br>RESULTS: Profiling across 228 target sites revealed that SpRY exhibits nearly PAM-less activity, with significantly higher editing efficiency at NRN (15.82%, R&#x2009;=&#x2009;A/G) than at NYN PAMs (5.75%, Y&#x2009;=&#x2009;C/T). SpG and SpCas9-NG preferentially targeted NGN PAMs, achieving mean efficiencies of 14.81% and 16.33%, respectively. PAM&#x2011;flexible cytosine base editors (CBEs) mediated efficient C:G&#x2011;to&#x2011;T:A conversion, with mean efficiencies of 12.01% for SpRY&#x2011;BE4max (NNN PAMs), 15.43% for SpG&#x2011;BE4max (NGN PAMs), and 18.39% for SpCas9&#x2011;NG&#x2011;BE4max (NGN PAMs). Similarly, PAM&#x2011;flexible adenine base editors (ABEs) mediated efficient A:T&#x2011;to&#x2011;G:C conversion, with mean efficiencies of 15.66% for SpRY&#x2011;ABE8e (NNN PAMs), 24.16% for SpG&#x2011;ABE8e (NGN PAMs), and 20.50% for SpCas9&#x2011;NG&#x2011;ABE8e (NGN PAMs). By exploiting this expanded targeting scope, we successfully introduced 16 pathogenic single&#x2011;nucleotide variants (SNVs) at NRN PAM sites in the porcine genome, with editing efficiencies reaching up to 40.68% for CBEs and 61.76% for ABEs.<br><br>CONCLUSIONS: PAM-flexible SpCas9 variants and their derived base editors greatly expand the targeting scope for porcine genome engineering, thereby substantially broadening the applicability potential of CRISPR-Cas-mediated genome editing tools in porcine genetic improvement and disease model generation.},
}
@article {pmid42107032,
year = {2026},
author = {Singh, V and Fetoh, MEA and Fetoh, IEA},
title = {Degradation dynamics: an insight into microbial interactions with explosive compounds.},
journal = {Biodegradation},
volume = {37},
number = {3},
pages = {},
pmid = {42107032},
issn = {1572-9729},
mesh = {Biodegradation, Environmental ; *Explosive Agents/metabolism ; *Bacteria/metabolism ; *Fungi/metabolism ; *Microbial Interactions ; },
abstract = {Degradation dynamics is an essential aspect in the field of environmental science and is crucial in understanding the interaction between microbes and explosive compounds. Explosive compounds and their residues, such as nitramines, nitro-substituted aromatics, picric acid, TETRYL, and HEXYL), and aliphatic, RDX, etc.are highly persistent in the environment. These compounds are toxic to many life forms at high concentrations, specific microbial species have evolved resistance and degradation capabilities, though their growth can still be inhibited beyond certain thresholds, The results of microbial biodegradation can range from complete mineralization to only the biotransformation into less toxic or more resistant metabolites. Research using pure cultures of bacteria and fungi has provided insight into the degradation pathways of certain nitro-organic compounds, and some key enzymes (laccases and lignin peroxidases) have been identified and studied. This review mainly aims to provide an overview of the current state of research on the degradation dynamics of explosive compounds Recent advancements have pivoted toward 'Bio-omics' and synthetic biology tools, such as CRISPR/Cas systems, to engineer high-activity microbial strains.},
}
@article {pmid42107569,
year = {2026},
author = {Cora, D and Seijas, A and Al-Soufi, W and Sánchez, L and Arana, &#xc1;J and Novo, M},
title = {Critical role of Cas9 aggregation on in vitro DNA cleavage.},
journal = {International journal of biological macromolecules},
volume = {366},
number = {},
pages = {152466},
doi = {10.1016/j.ijbiomac.2026.152466},
pmid = {42107569},
issn = {1879-0003},
mesh = {*DNA Cleavage ; *Protein Aggregates ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Osmolar Concentration ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {The CRISPR/Cas9 system is a powerful genome-editing tool widely used in molecular biology and gene therapy, whose efficiency strongly depends on the physicochemical properties of the Cas9 ribonucleoprotein complex. Optimizing Cas9 activity remains essential for reliable genome-editing applications, yet the factors limiting its in vitro cleavage efficiency are not fully understood. Among these, protein aggregation has been suggested to critically impair Cas9 functionality, although its role has not been systematically analysed. Here, we investigate Cas9 aggregation under different environmental conditions and evaluate its impact on in vitro DNA cleavage efficiency. Using fluorescently labelled Cas9 and single-molecule fluorescence techniques, we quantify aggregation as a function of buffer composition, ionic strength, salt concentration and sgRNA presence, and relate these properties to cleavage activity. Our results show that Cas9 aggregation significantly reduces DNA cleavage efficiency, with higher aggregation levels consistently correlating with lower activity. In contrast, buffers with higher ionic strength or stabilizing components reduce aggregation and enhance Cas9 performance. Overall, this study demonstrates that Cas9 aggregation plays a critical role in determining in vitro cleavage efficiency and highlights the importance of controlling protein aggregation to optimize CRISPR/Cas9-based genome-editing applications and delivery strategies.},
}
@article {pmid42107826,
year = {2026},
author = {Xu, T and Guo, Z and Li, Y and Lyu, H},
title = {Determining optimal sgRNA coverage and screening duration for pooled CRISPR screens: A quantitative framework.},
journal = {Methods (San Diego, Calif.)},
volume = {253},
number = {},
pages = {49-60},
doi = {10.1016/j.ymeth.2026.04.016},
pmid = {42107826},
issn = {1095-9130},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Reproducibility of Results ; Gene Library ; },
abstract = {CRISPR-based loss-of-function screening has emerged as a powerful tool for systematically characterizing gene functions. However, standardized quantification metrics for sgRNA coverage-a critical parameter determining genome-wide screening reliability and resource efficiency, remain undefined. In this study, we first conducted systematic sgRNA coverage tests in HeLa cells to determine the optimal coverage for CRISPR[iBAR] knockout libraries. Furthermore, we incorporated multiple timepoints to monitor sgRNA-mediated gene knockout dynamics. Here, balancing data quality, time efficiency, and cost, we identify 15 days and 800 × coverage as the optimal screening parameters for standard iBAR‑based CRISPR screens. Data from varying coverage levels can also serve as essential references for screening under different experimental conditions. Longitudinal analysis revealed that extending the screening period beyond 15 days maintains stable sgRNA distribution patterns within the cell population. This study establishes key parameter benchmarks to ensure CRISPR knockout screening efficacy and reproducibility, providing a solid foundation for downstream drug screening and target identification.},
}
@article {pmid42108387,
year = {2026},
author = {Pandya, K and Jaisinghani, LS and Tripathi, A and Kumar, D and Saraf, SK and Sahoo, J and Kumar, D},
title = {Rational Design and Optimisation of CRISPR-Cas9 Delivery Systems for Targeted Genomic Transformation.},
journal = {The journal of gene medicine},
volume = {28},
number = {5},
pages = {e70095},
doi = {10.1002/jgm.70095},
pmid = {42108387},
issn = {1521-2254},
support = {EMDR/SG/11/2023-5582//Indian Council of Medical Research/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; *Gene Transfer Techniques ; Blood-Brain Barrier/metabolism ; Dependovirus/genetics ; Alzheimer Disease/genetics/therapy ; },
abstract = {The CRISPR-Cas9 genome-editing technique offers a promising therapeutic strategy for genetic disorders, including neurodegenerative diseases like Alzheimer's disease (AD), characterised by inherited susceptibility and progressive cognitive decline, as well as other hallmarks such as amyloid beta (Aβ1-42) plaques and neurofibril tangles (NFTs). However, the blood-brain barrier (BBB) poses a significant challenge to the effectiveness of gene editing components in the affected brain region and impedes clinical translation. This comprehensive review compares various CRISPR-Cas9 delivery vectors, viral, nonviral and physical, with a focus on their efficacy in neurological diseases such as AD. Viral vectors viz., adeno-associated viruses (AAVs) and lentiviruses (LVs) demonstrate high transduction efficiency and BBB permeability. AAVs are preferred for their low immunogenicity, minimal toxicity, high neuronal tropism and episomal persistence, enabling sustained expression without insertional mutagenesis. LVs offer larger genetic payloads but raise concerns about genomic integration and potential oncogenesis, though integration-defective variants mitigate these risks. Nonviral vectors, including peptide and polymer-based nanoparticles, lipid nanoparticles (LNPs) and Inorganic carriers such as gold and silver nanoparticles, are less immunogenic and easier to handle but require further optimisation for in vivo BBB crossing and endosomal escape. Physical methods such as electroporation and microinjection are suitable for in vitro/ex vivo use, while novel CNS-targeted strategies, such as RVG-tagged particles, TfR-directed LNPs and engineered AAV variants, enhance brain penetration via receptor-mediated transcytosis. These preclinical studies show that these technologies can successfully edit genes and provide therapeutic benefits, including amyloid reduction and cognitive improvement in AD models. Yet off-target effects, immune responses and regulatory hurdles persist. Overall, continuous innovation in delivery vector design and safety profile-targeting strategies is crucial for advancing CRISPR-Cas9 towards clinical therapies for AD-based therapies and related neurological disorders.},
}
@article {pmid42108472,
year = {2026},
author = {Maxim, DS and Sostena, J and Johnson, NS and Wu, DW and Charu, V and Carter, JN and Anand, S and Church, GM and Bhalla, V},
title = {Designing genome editing experiments with EditABLE.},
journal = {Genome biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13059-026-04095-x},
pmid = {42108472},
issn = {1474-760X},
support = {1R41DK138689/DK/NIDDK NIH HHS/United States ; },
abstract = {While many computational tools exist for designing CRISPR-Cas experiments, there is a need for a centralized resource that combines individual tools to predict the most efficient genome editing strategy for a given application. To fill this gap, we develop EditABLE (EditABLE-app.stanford.edu), an online resource that provides optimal CRISPR editors and guide RNAs based on user provided sequence data with functionalities for base editing, prime editing, and integrase-mediated editing. We demonstrate the utility of EditABLE by applying it to one of the most common monogenic disorders, autosomal dominant polycystic kidney disease (ADPKD), identifying specific editing tools across the ADPKD mutation landscape.},
}
@article {pmid42109826,
year = {2026},
author = {Sun, A and Jin, SL and Liu, JG},
title = {A practical guide for characterization of novel CRISPR-Cas systems with Pro-CRISPR factors.},
journal = {Biophysics reports},
volume = {12},
number = {2},
pages = {85-99},
pmid = {42109826},
issn = {2364-3420},
abstract = {The emergence of advanced genome editing technologies has revolutionized research in life sciences, offering an unprecedented way to uncover unknown biological functions and innovative therapeutic strategies. Among all genome editing tools, CRISPR-Cas-based technologies play a pivotal role in this revolution, particularly Class 2 effectors such as Cas9 and Cas12, owing to their high efficacy and ease of programmability. With the advancements in genome sequencing and metagenomics, an increasing number of novel CRISPR-Cas systems have been discovered, including those found in extreme environments and viruses. Furthermore, recent studies have revealed an unexpected role of non-Cas accessory genes, such as the Tn7-like transposon and Pro-CRISPR factors (Pcr), in conferring additional functionalities to the CRISPR system, providing new insights into the understanding of CRISPR-mediated bacterial immunity and advancing the development of genome editing technologies. Therefore, it is essential to develop comprehensive methods for characterizing the Cas proteins and Pro-CRISPR factors with a growing diversity. In this protocol, we provide a method encompassing protein purification, biochemical characterization, validation of protein-protein interactions, and preliminary in vivo functional assays in bacteria for Cas nuclease and its associated Pro-CRISPR factor. We hope this protocol will not only assist in the characterization of the CRISPR-Cas system, but also provide valuable guidance for the characterization of other nucleases or nucleic acid modification systems.},
}
@article {pmid42112301,
year = {2026},
author = {Saedi, S and Nezhadi, J and Feizi, H and Memar, MY and Arefi, V and Kadkhoda, H},
title = {A comparative analysis of CRISPR systems, virulence factors, and antibiotic resistance genes in carbapenem-sensitive and carbapenem-resistant Klebsiella pneumoniae.},
journal = {Iranian journal of microbiology},
volume = {18},
number = {1},
pages = {1-13},
pmid = {42112301},
issn = {2008-3289},
abstract = {BACKGROUND AND OBJECTIVES: Klebsiella pneumoniae is a major cause of healthcare-associated infections, particularly in immunocompromised patients. This study compares the CRISPR systems, virulence factors, and antibiotic resistance genes in carbapenem-sensitive (CSKP) and carbapenem-resistant (CRKP) clinical isolates.<br><br>MATERIALS AND METHODS: Carbapenemase-producing isolates were identified by mCIM/eCIM. PCR and RT-qPCR detected key genes, including cas3, involved in CRISPR-Cas function. In silico analyses included STRING for protein interactions, CRISPRCasdb for CRISPR subtype distribution, and Phyre2/AlphaFold for cas3 structure prediction.<br><br>RESULTS: Among the isolates, 35.2% were resistant to carbapenems. Among CRKP strains, high prevalence of bla-NDM-1 (82%) and bla-OXA-48 (64%) was observed. The cas3 expression was significantly upregulated in resistant isolates (P = 0.002). CRISPR subtype I-E was identified in 16% of CRKP and 36% of CSKP isolates. Structural-functional analysis supported the integrity of Cas3 and revealed interactions with regulatory and iron acquisition proteins. Statistically significant differences in virulence and resistance gene profiles were found between CRKP and CSKP groups (P < 0.05).<br><br>CONCLUSION: This study highlights key differences between CRKP and CSKP isolates, particularly in CRISPR-Cas systems, resistance, and virulence. The findings suggest that cas3 plays a critical role in genomic adaptation and resistance mechanisms in K. pneumoniae, offering insights for future therapeutic strategies.},
}
@article {pmid42112626,
year = {2026},
author = {Chhabada, Y and Yadav, KS},
title = {Pulmonary delivery of gene-loaded lipid nanoparticles for targeting TGF-β signaling in idiopathic pulmonary fibrosis.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {11},
pages = {1671-1681},
pmid = {42112626},
issn = {1748-6963},
mesh = {Humans ; *Idiopathic Pulmonary Fibrosis/therapy/genetics/pathology/metabolism ; *Transforming Growth Factor beta/metabolism/genetics ; *Nanoparticles/chemistry ; Signal Transduction ; *Lipids/chemistry ; Animals ; RNA, Small Interfering/genetics/administration &amp; dosage ; Gene Transfer Techniques ; Genetic Therapy/methods ; Lung/metabolism/pathology ; MicroRNAs/genetics/administration &amp; dosage ; CRISPR-Cas Systems ; Liposomes/chemistry ; },
abstract = {Idiopathic Pulmonary Fibrosis (IPF) is chronic, progressive interstitial lung disease characterized by the accumulation of extracellular matrix (ECM) and remodeling of lung tissue. The existing antifibrotic therapies offer limited disease modification, underscoring the need for targeted and precise treatment strategies. Advances in LNP platforms, including solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and liposomes, are studied for their ability to encapsulate and deliver nucleic acid therapies such as Small interfering RNA (siRNA), microRNA (miRNA) and CRISPR/Cas9 directly to the lungs. This review addresses a key gap by critically evaluating inhalable lipid nanoparticle (LNP) platforms for gene modulation of transforming growth factor-β (TGF-β) signaling in IPF. Additionally, LNP-mediated delivery of CRISPR components enables durable gene editing with minimal systemic toxicity. Overall, inhalable gene-loaded LNPs represent a promising direction for disease-modifying therapy in IPF, provided that delivery efficiency, safety, and regulatory challenges are systematically addressed.},
}
@article {pmid42112836,
year = {2026},
author = {Hollomon, JM and Dahlstrom, KM},
title = {CRISPR-Cas9-mediated targeted gene deletion in Aspergillus calidoustus, a non-model environmental mold.},
journal = {Microbiology spectrum},
volume = {14},
number = {6},
pages = {e0389925},
pmid = {42112836},
issn = {2165-0497},
support = {5R35GM150797/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Deletion ; *Aspergillus/genetics ; Orotidine-5'-Phosphate Decarboxylase/genetics ; Fungal Proteins/genetics/metabolism ; },
abstract = {Reverse genetic approaches in molds are complicated by their recalcitrance to transformation, multicellular growth during much of the vegetative life cycle, and frequent off-target integration of deletion constructs. Thus, protocols for gene deletion in filamentous fungi are predominantly confined to established model organisms. Adapting an existing protocol developed for gene deletion in the related fungus Aspergillus fumigatus, we employed an expression-free CRISPR/Cas9 directed mutagenesis strategy to the non-model environmental mold Aspergillus calidoustus. As a test case, we have deleted A. calidoustus pyrG, encoding orotidine-5'-phosphate decarboxylase, using short regions of homology to guide on-target integration of a nourseothricin resistance cassette (NatR) to CRISPR/Cas9-induced double strand breaks. We genotypically and phenotypically validated two A. calidoustus ΔpyrG deletion strains generated using this methodology, with whole-genome sequencing revealing one ΔpyrG strain to have integrated the resistance cassette by homologous recombination, and another strain by non-homologous end joining. Thus, distinct modes of double-strand break repair were responsible for on-target integration of the homology-bearing NatR cassette at the pyrG locus in A. calidoustus.IMPORTANCEIn the environment, filamentous fungi play essential roles in soil health and agriculture, decomposition, and nutrient cycling. The study of these organisms is often limited by an inability to dissect the functions of their genes and the roles that they play by modifying the genomes of these organisms. Here, we adapt and validate a tool for genome modification to Aspergillus calidoustus, a soil fungus that forms a partnership with a bacterium (Paraburkholderia edwinii) to resist natural toxins found in the soil.},
}
@article {pmid42113695,
year = {2026},
author = {Yao, Y and Li, J and Duan, L and Chen, J and Li, G and Hu, Z},
title = {One-step Overlapping PCR for Rapid Synthesis of Single-guide RNA DNA Templates for the CRISPR System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {230},
pages = {},
doi = {10.3791/70369},
pmid = {42113695},
issn = {1940-087X},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/biosynthesis/chemical synthesis ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA/genetics/chemical synthesis ; Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; },
abstract = {The CRISPR-Cas system has revolutionized genome editing; however, conventional methods for generating single-guide RNA (sgRNA) often involve time-consuming cloning steps or expensive commercial synthesis kits. An optimized one-step overlapping PCR strategy is presented for the rapid, cost-effective synthesis of DNA templates for in vitro sgRNA transcription. Using four partially overlapping primers spanning the T7 promoter, target-specific guide sequence, and sgRNA scaffold, full-length templates are assembled in a single PCR reaction without cloning. Systematic experimental optimization established an optimal primer ratio (AF1:AF2:AF3:Tracr-R = 50:5:1:50), minimizing non-specific byproducts while maximizing full-length product yield, as confirmed by agarose gel electrophoresis. This approach was successfully extended to generate templates for Staphylococcus aureus Cas9 (saCas9) sgRNA, demonstrating cross-system applicability beyond Streptococcus pyogenes Cas9 (SpCas9). Although direct chemical synthesis of sgRNAs offers advantages such as high purity, chemical modifications to enhance stability, and reduced off-target effects, it remains prohibitively expensive for high-throughput applications or large-scale screens that require numerous sgRNAs. In vitro cleavage assays demonstrated that guide RNAs generated using this method achieve editing efficiencies comparable to those obtained via conventional plasmid-based cloning. Furthermore, ribonucleoprotein complexes assembled with these sgRNAs and delivered into HEK293T cells via electroporation resulted in detectable indel formation at the target locus, confirming functionality in vivo. Cost analysis indicates that this method substantially reduces template preparation costs compared to commercial synthesis kits while reducing turnaround time from days to hours, thereby providing an accessible and scalable approach for laboratories engaged in genetic research.},
}
@article {pmid42113764,
year = {2026},
author = {Iwata, S and Miura, Y and Iwamoto, T},
title = {Non-viral in vivo electroporation-based chromosomal engineering and repair assessment in the murine uterine epithelium.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0348797},
pmid = {42113764},
issn = {1932-6203},
mesh = {Animals ; Female ; *Electroporation/methods ; *Uterus/metabolism ; Mice ; CRISPR-Cas Systems ; Epithelium/metabolism ; *DNA Repair ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Translocation, Genetic ; },
abstract = {Chromosomal rearrangements generated by CRISPR/Cas systems are valuable for studying genomic architecture and repair mechanisms. However, most in vivo approaches rely on viral vectors, which require specialised production, prolonged nuclease expression, and elevated biosafety containment. Here, we applied Cas9 ribonucleoprotein (RNP) electroporation to the murine uterine epithelium as a simple, non-viral strategy for somatic chromosomal engineering. This method successfully induced defined interchromosomal translocations at multiple loci and enabled the molecular assessment of large-scale inversion repair (57.8 Mb) using paired gRNAs with an ssODN donor. While rearranged alleles were detected at low apparent frequencies in bulk uterine DNA-consistent with epithelial-restricted delivery and somatic mosaicism-high-depth whole-genome sequencing (WGS) and PCR provided nucleotide-resolution confirmation of precise junction formation. Our findings demonstrate that uterine electroporation of CRISPR RNPs is a feasible, rapid approach for evaluating engineered chromosomal rearrangements in vivo, providing a controlled platform for analyzing somatic DNA repair outcomes without viral confounds.},
}
@article {pmid42114484,
year = {2026},
author = {Wan, Y and Hu, Q and Zhu, J and Oung, QW and Lee, CH and Han, H and Lee, HL and Chen, X},
title = {PCR-free cascaded CRISPR-Cas13a colorimetric platform (CLAMP) for non-invasive miRNA-based allergen-specific subtyping of allergic rhinitis.},
journal = {Biosensors &amp; bioelectronics},
volume = {308},
number = {},
pages = {118773},
doi = {10.1016/j.bios.2026.118773},
pmid = {42114484},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/analysis/isolation &amp; purification ; *Colorimetry/methods ; CRISPR-Cas Systems/genetics ; Animals ; *Rhinitis, Allergic/diagnosis/genetics/immunology ; *Allergens/genetics/immunology ; *Biosensing Techniques ; Humans ; Mice ; Limit of Detection ; Nasal Lavage Fluid/chemistry ; },
abstract = {Accurate identification of causative allergens is critical for personalized management of allergic rhinitis (AR), yet current methods often fail to distinguish clinical relevance from sensitization, particularly in polysensitized patients. To address this, we developed CLAMP (Cascaded CRISPR-Cas13a LAMP Colorimetric Assay), a platform that leverages a single Cas13a/crRNA complex to initiate a simplified CRISPR cascade amplification, enabling one-pot two-step detection without tube opening after sample addition. The assay employs a colorimetric readout based on CIELAB ΔE, allowing quantitative biomarker analysis with minimal equipment, requiring only a simple heat source. CLAMP achieves a detection limit of 1.15 × 10[-15] M with excellent specificity and a turnaround time under 60 min. In preclinical validation using nasal lavage fluid from 200 allergen-specific AR mouse models, CLAMP enabled allergen-based AR classification via multiplex miRNA detection, achieving 91.5% accuracy in distinguishing major allergen subtypes. This low-cost, non-invasive platform holds strong potential for rapid allergen classification and future translation toward personalized immunotherapy in AR.},
}
@article {pmid42115498,
year = {2026},
author = {Valiñas, MA and Cerrudo, I and Marchetti, F and Villarreal, F and Pagnussat, G and Zabaleta, E},
title = {CRISPR/Cas9-mediated disruption of the gamma carbonic anhydrase 2 gene leads to reduced mitochondrial complex I and growth alterations in tomato.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42115498},
issn = {1432-2048},
support = {PICT 2020 00013//ANPCyT/ ; },
mesh = {*Solanum lycopersicum/genetics/growth &amp; development/enzymology/metabolism ; *CRISPR-Cas Systems/genetics ; *Electron Transport Complex I/metabolism/genetics ; Mitochondria/metabolism ; *Carbonic Anhydrases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Germination ; },
abstract = {Despite similar complex I reduction, γCA2 disruption in tomato, unlike in arabidopsis, triggers hormonal and developmental changes, challenging assumptions of conserved mitochondrial responses across plant species. NADH-ubiquinone oxidoreductase [complex I (CI)] is the main entry point of electrons to OXPHOS being essential for metabolism and redox balance. In most organisms, except animals and fungi, CI contains an additional domain composed of gamma carbonic anhydrase (γCA) subunits, termed the CA module. In Arabidopsis thaliana, this module includes AtɣCA1/3, AtɣCA2, and AtɣCAL1/2. AtɣCA2 is critical for CI biogenesis, yet its role in other species remains unclear. In tomato, the γCA family comprises SlɣCA1a, SlɣCA1b, SlɣCA2, and SlɣCAL. Here, we report the inactivation of the tomato SlɣCA2 using CRISPR/Cas9 technology. As in arabidopsis, SlɣCA2-KO tomato plants show comparable reduction in CI levels and activity and a similar decrease in oxygen consumption, yet display increased ATP levels in seeds. However, unlike arabidopsis, mutant tomato plants exhibit delayed seed germination and retarded growth and development. Our results further suggest that abscisic acid and gibberellin homeostasis is altered in SlɣCA2-KO plants. Together, these findings support a connection between mitochondrial respiration and hormonal regulation, by which plants adjust developmental processes to mitochondrial electron transport chain functionality, thereby preventing energy depletion during early growth stages.},
}
@article {pmid42115922,
year = {2026},
author = {Bhowmik, PK and Williams, JT and Polley, B and Chen, N and Kavuri, NR and Zang, W and Barakate, A and Yang, H and Narra, MK and Beattie, AD and Starker, C and Voytas, DF and Baysal, C},
title = {BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08866-3},
pmid = {42115922},
issn = {1471-2229},
abstract = {BACKGROUND: Plant RNA virus-mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta. However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms.<br><br>RESULTS: We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase (PDS) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated &#x3b3;b subgenomic promoter or when fused directly to the &#x3b3;b genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas &#x3b2;1- and &#x3b2;2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley (Hordeum vulgare) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission.<br><br>CONCLUSION: Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector-mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana, highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture-free, and transformation-independent genome editing in cereals and other recalcitrant monocots.},
}
@article {pmid42116436,
year = {2026},
author = {Sun, L and Yan, J and Xie, J and Wang, XY},
title = {From interactions to applications: the role of microbial communities in shaping the physicochemical, safety, and quality attributes of aquatic products.},
journal = {Food research international (Ottawa, Ont.)},
volume = {236},
number = {},
pages = {119126},
doi = {10.1016/j.foodres.2026.119126},
pmid = {42116436},
issn = {1873-7145},
mesh = {*Food Safety ; *Food Microbiology ; *Microbiota ; *Seafood/microbiology ; *Microbial Interactions ; Bacteria/growth &amp; development ; Animals ; Food Handling ; Food Quality ; Quality Control ; },
abstract = {Aquatic products are regarded as important dietary resources due to their abundant high-quality proteins, while their physicochemical properties, safety, and quality attributes during storage and processing are strongly influenced by microbial communities. Microbial interactions in aquatic products can exacerbate spoilage and pathogenic contamination, thereby intensifying food safety issues. However, previous studies primarily focused on identifying and characterizing individual microbial species in aquatic products, whereas current research increasingly emphasizes the critical role of multi-species interactions and applications in quality and safety evaluation of aquatic products. This review focuses on the interactions, characteristics, and growth models of spoilage and pathogenic microorganisms in aquatic products. The regulation mechanisms underlying microbial interactions and their neutral, negative, and positive effects on aquatic products' quality were summarized. Additionally, the paper analyzes targeted strategies for regulating microbial interactions in aquatic product preservation, providing theoretical and practical support for quality control. The microbiota primarily consists of spoilage and pathogenic bacteria, whose growth dynamics and interactions can be quantitatively modeled using models such as the Baranyi and P-model to predict their behavior. These microorganisms directly regulate the spoilage process and health risks via physical structures (e.g., extracellular polymeric substances, intercellular nanotubes) and chemical signaling networks (e.g., metabolic cross-feeding, quorum sensing). Conventional strategies (e.g., modified atmosphere packaging, ultra-high pressure processing) and precision approaches (e.g., digital twin technology and CRISPR/Cas system) have been applied to regulate the microbial interactions, thereby significantly enhancing the quality control in aquatic products.},
}
@article {pmid42117491,
year = {2026},
author = {Fan, X and Widodo, WS and Rozeboom, HJ and Fraaije, MW},
title = {Engineered Escherichia coli Strains for Flavoprotein Research: From Production of Apoproteins to Incorporation of Flavin Derivatives.},
journal = {ACS synthetic biology},
volume = {15},
number = {6},
pages = {2635-2648},
doi = {10.1021/acssynbio.6c00269},
pmid = {42117491},
issn = {2161-5063},
mesh = {*Escherichia coli/genetics/metabolism ; *Flavoproteins/metabolism/genetics/biosynthesis ; *Flavins/metabolism ; Flavin Mononucleotide/metabolism ; *Apoproteins/genetics/metabolism/biosynthesis ; Flavin-Adenine Dinucleotide/metabolism ; Riboflavin/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; },
abstract = {Flavoproteins are involved in a wide array of biological processes. These proteins contain one or more flavins as their cofactor, bound either noncovalently or covalently, and catalyze a wide breadth of redox reactions. In commonly used expression strains, flavoproteins are typically produced as holo (flavin-bound) flavoproteins. The ability to produce their apo form (flavoprotein devoid of flavins) will facilitate dedicated structural and mechanistic studies, while it also allows the incorporation of new-to-nature flavin-like cofactors. To facilitate this, we constructed, using the CRISPR/Cas9 system, an Escherichia coli strain that is impaired in producing the canonical FMN and FAD cofactors, due to a deletion in the ribB gene. This riboflavin auxotrophic strain is able to produce apoproteins of FMN- and FAD-dependent flavoproteins. We demonstrate that it can also be used for the incorporation of flavin derivatives by supplementing the medium with the respective riboflavin derivative. In parallel, we constructed, by genomic integration, a strain expressing an FAD synthetase from a T7 promoter and a flavin transferase from a lac promoter. This strain facilitates employing the newly developed methodology of flavin-tagging and flavin-fixing of target proteins, resulting in proteins carrying a covalently tethered FMN. It eliminates the need for two or more plasmids to generate covalently flavinylated flavoproteins. A third strain was prepared in which the features of riboflavin auxotrophy and flavin transferase activity were combined. This strain is perfectly suited for generating flavoproteins carrying a covalently anchored flavin derivative. These newly engineered strains, derivatives of E. coli BL21-AI, represent powerful tools for producing, investigating, and applying flavoproteins.},
}
@article {pmid42117889,
year = {2026},
author = {Wu, W and Jin, F and Xu, H and Liao, R and Fang, Z},
title = {Negative Regulators of Rice Agronomic Traits: Functional Insights and Applications in Genome Editing-Based Breeding.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70684},
pmid = {42117889},
issn = {1467-7652},
support = {32572249//National Natural Science Foundation of China/ ; qiankehepingtairencai-YQK (2023) 002//Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; qiankehejichu-ZD(2026)068//Science and Technology Program of Guizhou Province/ ; qiankehechengguo (2024) general 116//Science and Technology Program of Guizhou Province/ ; Qiankehepingtairencai-BQW (2024) 001//Science and Technology Program of Guizhou Province/ ; Qiankehepingtai ZSYS (2025) 037//Guizhou Key Laboratory of High Quality, High Efficiency, and Yield Enhancement in Grain and Oil Crops/ ; Qianjiaoji (2023) 007//Key Laboratory of Functional Agriculture of Guizhou Provincial Higher Education Institution/ ; GZSDCYJSTX-202602//Guizhou Provincial Modern Agricultural Industry Technology System Construction Special Program/ ; },
abstract = {Rice is the staple crop for more than half of the global population, and improving grain yield, grain quality, and stress resistance remain central goals of modern rice breeding. Among current precision breeding strategies, genome editing has created new opportunities for crop improvement, but its success depends heavily on the selection of effective target genes. In this context, negative regulators of agronomic traits are particularly valuable because their disruption or attenuation can relieve constraints on desirable phenotypes and generate beneficial variation. In this review, we summarize recent progress in the identification and functional characterization of negative regulatory genes associated with rice grain yield, grain quality and stress resistance. We further integrate the current knowledge of their molecular functions, regulatory mechanisms, and genetic networks and discuss their potential applications in genome editing-assisted breeding. This review provides a target-oriented framework for understanding negative regulation in rice and facilitating the development of improved varieties with increased productivity, quality and stress resistance.},
}
@article {pmid42119166,
year = {2026},
author = {Huang, D and Sun, D and Ou, C and Zhang, X and Luo, R and Kou, X and Li, X and Li, Y and Le, H and Li, W and You, Y and Gong, C},
title = {A hierarchical self-adjuvanted nanoCRISPR-based vaccine restores endogenous immune recognition and surveillance to amplify adaptive immune responses.},
journal = {Biomaterials},
volume = {334},
number = {},
pages = {124285},
doi = {10.1016/j.biomaterials.2026.124285},
pmid = {42119166},
issn = {1878-5905},
mesh = {Animals ; *Adaptive Immunity ; *Cancer Vaccines/immunology ; Humans ; *Nanoparticles/chemistry ; B7-H1 Antigen/genetics/immunology ; Nanovaccines ; Cell Line, Tumor ; CRISPR-Cas Systems ; Adjuvants, Immunologic ; Mice ; Female ; Mice, Inbred C57BL ; },
abstract = {Tumor vaccines are considered a promising approach in immunotherapy, designed to boost the immune system's capacity to identify tumor-associated antigens and subsequently trigger immune responses against tumors. However, the inherent genetic instability of tumor cells frequently results in decreased expression or loss of antigen and/or major histocompatibility complex (MHC) expression and upregulation of immune checkpoint molecule PD-L1, thus evading endogenous immune recognition and surveillance. Herein, we developed a hierarchical self-adjuvanted nanoCRISPR-based vaccine (HEDERA) loaded with LSD1/PD-L1 dual-editing CRISPR/Cas9 system, seeking to reinstate the endogenous immune detection and monitoring mechanisms to enhance adaptive immune reactions. Knockdown of LSD1 increases the presence of tumor-specific antigens and major histocompatibility complex class I molecules on the surface of cancer cells, thereby restoring immune recognition. Simultaneously, silencing PD-L1 alleviates the "exhaustion" of T cells and reactivates their cytotoxic activity. Moreover, LSD1 knockdown activates the type I interferon pathway to induce a self-adjuvant effect that enhances innate immune responses and thereby strengthens T cell-mediated adaptive immunity. This dual strategy achieves unprecedented efficacy, with 90% primary tumor inhibition, and demonstrates an 87.3% and 90.6% inhibition rate for post-surgical metastatic and recurrent tumors, respectively. Overall, HEDERA overcomes the single-action constraint of traditional tumor vaccines, and avoids combined medication-related poor patient compliance, delivering a more efficient, convenient integrated tumor immunotherapy solution.},
}
@article {pmid42119611,
year = {2026},
author = {Ghonse, K and Dandekar, S and Koratkar, S},
title = {Multidrug-resistant Acinetobacter baumannii: Molecular insights, clinical challenges, and therapeutic approaches.},
journal = {Journal of microbiological methods},
volume = {246},
number = {},
pages = {107544},
doi = {10.1016/j.mimet.2026.107544},
pmid = {42119611},
issn = {1872-8359},
mesh = {*Acinetobacter baumannii/drug effects/genetics/pathogenicity ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; *Acinetobacter Infections/microbiology/drug therapy/therapy/epidemiology ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Cross Infection/microbiology/drug therapy ; Biofilms ; Phage Therapy ; Virulence Factors ; },
abstract = {Acinetobacter baumannii has emerged as an agent of potentially life-threatening nosocomial infections, particularly among immunocompromised patients. Its ability to rapidly acquire resistance genes has made traditional antibiotic therapies progressively ineffective. The spread of bacterial contamination in hospital facilities increased due to lack of awareness among healthcare workers and improper management/handling of infectious samples. The persistence of pathogen in the hospital environment is increased with its ability to form biofilms, quorum sensing, and virulence factors. The infections caused by these agents are increasing in incidence and severity; necessitating efficient and timely management. This review highlights the epidemiological trends and molecular mechanisms involved in the pathogenesis and resistance of A. baumannii. The key resistance mechanisms that the organism possess include the activity of efflux pumps, beta-lactamase enzymes, and mobile genetic elements. This review discusses emerging treatment strategies - such as phage therapy - antimicrobial peptides, CRISPR-Cas-based technologies, and nanotechnology-enabled drug delivery- highlighting their respective benefits and limitations, with special emphasis on innovations like phage-antibiotic synergy and precision genome editing approaches. Despite promising advances, challenges remain, including the emergence of resistance pathogen, limited clinical scalability, and concern regarding the safety and toxicity of novel treatment options. Addressing these issues require focus on molecular insights of resistance mechanisms, the development of effective alternative therapies, and implementation of preventive strategies such as vaccines. Furthermore, execution of global antimicrobial stewardship program and robust surveillance systems are critical for effectively control and manage the threat posed by A. baumannii.},
}
@article {pmid42119627,
year = {2026},
author = {Guo, K and Yu, S and Yang, N and Wang, X and Liu, Q and Fu, Y and Liu, J},
title = {Serial passage is associated with virulence attenuation in Neospora caninum and transcriptomic remodeling of invasion- and chronic-stage-associated pathways.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108549},
doi = {10.1016/j.micpath.2026.108549},
pmid = {42119627},
issn = {1096-1208},
mesh = {*Neospora/pathogenicity/genetics/growth &amp; development ; Virulence/genetics ; Animals ; Gene Expression Profiling ; *Transcriptome ; Serial Passage ; Cattle ; Protozoan Proteins/genetics ; Host-Parasite Interactions/genetics ; Coccidiosis/parasitology/veterinary ; CRISPR-Cas Systems ; },
abstract = {N. caninum is an obligate intracellular apicomplexan parasite and a major cause of abortion in cattle worldwide. However, the molecular basis underlying virulence attenuation during long-term in vitro passage remains poorly understood. Here, we performed comparative transcriptomic profiling between the virulent Nc1-09 strain and its attenuated derivative Nc1 to investigate transcriptional changes associated with virulence reduction. A total of 487 differentially expressed genes (DEGs) were identified. Functional enrichment analyses revealed prominent changes in pathways related to protein modification, host-parasite interaction, motility, and metabolism. Notably, multiple members of the surface antigen glycoprotein (SRS) family displayed marked transcriptional shifts, consistent with altered host cell interaction and immune-related functions. CRISPR/Cas9-mediated disruption of three representative DEGs (NCLIV_016320, NCLIV_024950, and NCLIV_022830) did not reproduce the attenuated phenotype, suggesting that disruption of these individual genes is insufficient to account for the full attenuated phenotype under the conditions tested. Gene set enrichment analysis further demonstrated systematic downregulation of protein modification-associated pathways in the attenuated strain. Overall, the data indicate that virulence attenuation in Neospora caninum （N. caninum） is associated with coordinated transcriptional remodeling across multiple functional pathways rather than discrete gene loss.},
}
@article {pmid42119765,
year = {2026},
author = {Bakadlag, R and Li, S and Guilbert, C and Huard, C and Bertomeu, T and Coulombe-Huntington, J and Jackson, BP and Grant, MP and Iskandarani, L and Paganini, C and Rossi, A and Mwale, F and Tyers, M and Hales, BF and Mann, KK},
title = {CRISPR screen uncovers SLC26A2 as a modulator of tungsten toxicity in endochondral ossification.},
journal = {Environmental research},
volume = {303},
number = {Pt 1},
pages = {124634},
doi = {10.1016/j.envres.2026.124634},
pmid = {42119765},
issn = {1096-0953},
mesh = {Animals ; *Sulfate Transporters/genetics/metabolism ; *Osteogenesis/drug effects ; *Tungsten/toxicity ; Mice ; Humans ; Chondrogenesis/drug effects ; CRISPR-Cas Systems ; Cell Line ; *Environmental Pollutants/toxicity ; },
abstract = {Tungsten is an emerging environmental contaminant, highlighting the urgent need to elucidate its toxicological characteristics and assess long-term health risks. Our previous investigations show that tungsten deposition in the bone is associated with stalled pre-B lymphocyte differentiation, inhibition of osteogenesis, and increased intervertebral disc degeneration and fibrosis. To delineate the underlying molecular mechanisms, we employed CRISPR-based genomics on NALM-6 cells and identified Solute Carrier Family 26 Member 2 (SLC26A2), a sulfate/chloride antiporter, as a pivotal mediator of tungsten-induced toxicity. SLC26A2 deletion reduced tungsten-induced growth inhibition and intercellular tungsten levels. Functional impairment of SLC26A2 is associated with chondrodysplasias, thus, we hypothesized that tungsten would impair the development of cartilage and bone tissues. Indeed, tungstate exposure impaired chondrogenesis and osteogenesis in murine limb cultures, which was reversible by sulfate supplementation. Our study demonstrates that tungsten exploits SLC26A2 for cellular entry and correlates with bone development disruption through proteoglycan and collagen depletion.},
}
@article {pmid42120728,
year = {2026},
author = {DiPersio, JF and Koehne, G and Shah, NN and Bernard, L and Suh, HC and Koura, D and Tamari, R and Mushtaq, MU and Maakaron, J and Rimando, J and Kennedy, VE and Patel, SS and Hudson, C and Loken, MR and Slapak, CA and Lloyd, DM and Stanizzi, DA and Lee-Sundlov, MM and Thosar, S and Mundelboim, G and Guo, G and Ge, HG and Li, BE and Xavier-Ferrucio, J and Hyzy, SL and Lin, MI and Raffel, GD and Cooper, BW},
title = {CRISPR-Cas9 CD33-deleted allogeneic hematopoietic cell transplantation with gemtuzumab ozogamicin maintenance in AML: a phase 1/2 trial.},
journal = {Nature medicine},
volume = {32},
number = {5},
pages = {1763-1772},
pmid = {42120728},
issn = {1546-170X},
mesh = {Humans ; *Hematopoietic Stem Cell Transplantation/methods/adverse effects ; *Gemtuzumab/therapeutic use/administration &amp; dosage/adverse effects ; *Leukemia, Myeloid, Acute/therapy/genetics/drug therapy ; *Sialic Acid Binding Ig-like Lectin 3/genetics ; Middle Aged ; Female ; Male ; Adult ; Aged ; *CRISPR-Cas Systems/genetics ; Transplantation, Homologous ; Graft vs Host Disease ; Myelodysplastic Syndromes/therapy/genetics ; },
abstract = {Patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are likely to relapse despite allogenic hematopoietic cell transplantation (HCT). Post-HCT preventative maintenance can be limited by toxicity toward the normal donor cells. Tremtelectogene empogeditemcel (trem-cel) is a CRISPR-Cas9 gene-edited allogeneic HCT product lacking CD33, designed to shield the donor graft from cytotoxicity of subsequent CD33-targeted therapies such as gemtuzumab ozogamicin (GO). In this multicenter, phase 1/2a, open-label study, adult patients with AML/MDS with high relapse risk received trem-cel after myeloablative conditioning followed by GO maintenance (0.5-2.0 mg m[-][2] day 1 per 28-day cycles). Patients receiving trem-cel were assessed for the primary safety endpoint of neutrophil engraftment by day 28 and secondary endpoints including time to neutrophil engraftment, incidence of graft-versus-host disease and graft failure, transplant-related mortality, percentage of CD33-negative myeloid cells and survival. Patients receiving trem-cel and GO were assessed for the additional secondary endpoints of safety of maintenance GO with trem-cel and pharmacokinetics of GO after trem-cel transplant. All 30 patients receiving trem-cel achieved the primary safety endpoint of neutrophil engraftment by day 28 with a median engraftment time of 10 days (95% confidence interval: 9-10). Nineteen patients received GO maintenance in phase 1 dose escalation (n = 15) and in phase 2 dose expansion (n = 4). The trial was stopped early, and this is the final report on the trial including the completed phase 1 portion. GO treatment was safely tolerated up to the recommended phase 2 dose of 2 mg m[-][2], and no prolonged high-grade cytopenias were observed. The most common adverse events were cytopenias and infections. Three cases of transplant-related mortality were observed due to renal failure, sepsis and sinusoidal obstruction syndrome, respectively. In summary, trem-cel demonstrated safe, rapid, robust engraftment, and GO maintenance was administered without prolonged hematologic toxicity. ClinicalTrials.gov identifier: NCT04849910 .},
}
@article {pmid42121843,
year = {2026},
author = {Siddika, A and Husseiny, FE and Rousseau, J and Tremblay, JP},
title = {Successful In Vitro Modification of the Dmd Gene Using Prime Editing.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
pmid = {42121843},
issn = {2073-4409},
support = {53320215//Defeat Duchenne Foundation/ ; CIHR; Application No. 492510/CAPMC/CIHR/Canada ; },
mesh = {*Gene Editing/methods ; *Dystrophin/genetics ; Animals ; Mice ; *Muscular Dystrophy, Duchenne/genetics ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Base Sequence ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5'-TTCT-3' motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.},
}
@article {pmid42121869,
year = {2026},
author = {Siwak, JF and Connelly, JP and Pruett-Miller, SM},
title = {Essential HDRescue: A Co-Targeting Strategy to Enhance Precision Genome Editing by Co-Editing Essential Genes.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
pmid = {42121869},
issn = {2073-4409},
support = {N/A//American Lebanese Syrian Associated Charities/ ; NA//St. Jude Graduate School of Biomedical Sciences/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair/genetics ; *Genes, Essential/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Induced Pluripotent Stem Cells/metabolism ; Animals ; },
abstract = {Genome editing is widely used and conceptually simple, yet in practice, it is hindered by laborious workflows and high costs. These challenges stem from the difficulty of identifying and isolating cells that contain the desired user-defined modifications, a problem compounded by the wide variability in editing efficiencies across cell types. While homology-directed repair (HDR) provides a mechanism for precise genome modification following nuclease-induced double-strand breaks (DSBs), it is frequently outcompeted by the dominant mutagenic non-homologous end-joining (NHEJ) pathway in mammalian cells. Therefore, we developed a novel enrichment method, Essential HDRescue, to increase the frequency of HDR events at a target site by co-targeting an essential genomic locus. Using both intrinsic positive and negative selection at a common essential gene, we enabled enrichment of precise editing events at a second, unlinked target site. We demonstrated that co-targeting essential genes in cancer cell lines and iPSCs increased HDR rates without the need for an exogenous reporter or selective drug. Analysis of resulting clones revealed that Essential HDRescue produced up to a 6-fold increase in single-allele edits and an ~4-fold increase in homozygous edits relative to single-targeted controls. By harnessing the intrinsic cellular dependencies that arise from DSB repair at essential loci, Essential HDRescue offers a widely applicable method to improve precise genome editing outcomes in mammalian cells, leaving only a minimal, protein-silent scar at the essential gene.},
}
@article {pmid42122867,
year = {2026},
author = {Wen, Y and Li, Y and Bao, S and Cao, G and Li, M and Wang, J and Ding, B and Xie, X and Qiu, L},
title = {The Auxin Response Factor TaARF18-A Negatively Regulates Salt Tolerance in Common Wheat (Triticum aestivum L.).},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {9},
pages = {},
pmid = {42122867},
issn = {2223-7747},
support = {32301817//National Natural Science Foundation of China/ ; 22JCQNJC01470//Natural Science Foundation of Tianjin/ ; 26CXNE010//Gansu Provincial Science and Technology Program Project/ ; KLIBMC2507//Open Fund from the Tianjin Key Laboratory of Intelligent Breeding of Major Crops/ ; },
abstract = {Soil salinization is one of the major abiotic stresses that influences agricultural production and the environment. Auxin response factors (ARFs) are key components of the auxin signal transduction pathway, while their role in wheat salt stress responses remains unclear. In this study, we identified TaARF18 as a negative regulator of salt tolerance in wheat. The coding sequences of TaARF18-A, TaARF18-B, and TaARF18-D were 2106, 2088, and 2088 bp, respectively. TaARF18 is a hydrophilic protein featuring typical Auxin-resp and B3 DNA-binding domains and exhibits relatively high evolutionary conservation among Poaceae species. The expression of TaARF18 was upregulated under salt stress. TaARF18 predominantly accumulated in the nucleus. Silencing of TaARF18 via the BSMV-VIGS approach enhanced salt tolerance in wheat seedlings. In addition, haplotype analysis based on resequencing data from 355 wheat accessions identified 25, 31, and 16 haplotypes for TaARF18-A, TaARF18-B, and TaARF18-D, respectively. Fourteen wheat accessions carrying different haplotypes were evaluated under salt stress, and HapIII of TaARF18-A exhibited the highest level of salt tolerance, which can act as a strong selection locus in global wheat breeding. Our findings provide insight into the function of ARFs in salt stress responses and offer a potential target for CRISPR/Cas-mediated salt-tolerant wheat breeding programs.},
}
@article {pmid42123509,
year = {2026},
author = {Li, Y and Yao, Y and Xu, Z and Xiong, Y and Zhang, C and Yu, L and Gao, H and Fei, T},
title = {Genome-Wide CRISPR Screening Identifies Genetic Modulators of Amyloid Precursor Protein Processing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
pmid = {42123509},
issn = {1422-0067},
mesh = {Humans ; *Amyloid beta-Protein Precursor/metabolism/genetics ; *CRISPR-Cas Systems ; *Alzheimer Disease/genetics/metabolism ; Amyloid beta-Peptides/metabolism/genetics ; HEK293 Cells ; },
abstract = {The proteolytic processing of the amyloid precursor protein (APP) is a core pathological event in Alzheimer's disease (AD) pathogenesis, yet the global genetic regulatory networks modulating this process have not been fully characterized. To systematically identify novel regulators of APP cleavage, we performed a genome-wide CRISPR/Cas9 knockout screen utilizing an optimized UAS-GAL4-based cellular reporter, and identified genetic modulators governing amyloidogenic and non-amyloidogenic processing. The screen uncovered distinct functional gene clusters regulating the APP, prominently involving cellular metabolism, protein modification, and vesicular trafficking. Specifically, LDHB, PIAS2, CCDC53, and TRIM61 emerged as novel functional modulators. Biochemical validation confirmed that ablating these genes significantly alters the metabolic balance between sAPPα and amyloid-β (Aβ) production. Finally, integration with human AD transcriptomic datasets demonstrated that these identified modulators undergo significant dysregulation in clinics. Together, these findings establish a reporter-based functional screening framework for APP processing and identify candidate regulatory nodes linked to metabolism, protein modification, and vesicular trafficking. These candidates provide a resource for future mechanistic investigation and validation in more disease-relevant AD models.},
}
@article {pmid42123512,
year = {2026},
author = {Yuan, Y and Yuan, J and Deng, D and Wu, J and Zhou, X and Jiang, A and Wang, J and Wang, X and Li, M and Long, K and Zhao, L},
title = {CRISPR/Cas9-Mediated Knockout of CGNL1 Confers Resistance to Aflatoxin B1 in Porcine Intestinal Epithelial Cells via Suppressing ROS Generation.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
pmid = {42123512},
issn = {1422-0067},
support = {XZ202501ZY0147//Tibet Autonomous Region Science and Technology Agency/ ; sccxtd-2025-08-13//the Program for Pig Industry Technology System Innovation Team of Sichuan Province/ ; 32472884//National Natural Science Foundation of China/ ; 32573165//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Aflatoxin B1/toxicity ; *CRISPR-Cas Systems ; Swine ; *Epithelial Cells/metabolism/drug effects ; *Reactive Oxygen Species/metabolism ; Oxidative Stress/drug effects ; *Intestinal Mucosa/metabolism/drug effects ; Gene Knockout Techniques ; Cell Line ; Cell Survival/drug effects ; Signal Transduction ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent and highly toxic mycotoxin in the food and feed chain and can directly injure the intestinal epithelium. Yet, its upstream determinants linking epithelial stress to cytotoxicity remain insufficiently defined. Here, we used porcine intestinal epithelial IPEC-J2 cells to characterize AFB1-induced cytotoxic and transcriptomic responses and to determine the role of the tight-junction scaffold, Cingulin-like 1 (CGNL1), a candidate gene identified through genome-scale CRISPR knockout library screening. The results showed that AFB1 exposure reduced cell viability in a dose-dependent manner and induced oxidative stress. RNA-seq profiling analysis revealed broad transcriptional remodeling, with activation of inflammatory pathways (including NF-κB and JAK-STAT signaling). Based on our constructed CGNL1-knockout IPEC-J2 cell line (CGNL1-KO IPEC-J2) using CRISPR/Cas9, it was found that CGNL1 deficiency markedly alleviated AFB1-induced cytotoxicity and oxidative stress. Comparative transcriptomics analysis showed that CGNL1 knockout attenuated AFB1-triggered aberrant expression of some CGNL1-dependent AFB1-responsive genes related to immune response under AFB1 challenge. Together, these findings identify CGNL1 as a potential modulator of epithelial susceptibility to AFB1 and support its involvement in the regulation of toxin-induced oxidative response.},
}
@article {pmid42123673,
year = {2026},
author = {Jing, L and Roy, D and Kalischuk, M},
title = {Advances in CRISPR Plant Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
pmid = {42123673},
issn = {1422-0067},
support = {Alliance #44842//Natural Sciences and Engineering Research Council of Canada/ ; 45402//Solanum International Inc./ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; *Plants/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The ability to precisely edit genetic characteristics with a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immunity complex is a revolutionary advance in science. Originally discovered in bacteria as part of a natural defense mechanism against viruses, CRISPR/Cas provides a precise, efficient, and relatively simple method for editing genes in microbes, plants, animals, and humans. The process relies on the Cas protein, an enzyme that cleaves and unwinds DNA at targeted locations. This process is guided by RNA sequences complementary to the DNA or RNA sequence of interest, allowing for changes to the genome through innate non-homologous end joining (NHEJ) and homology-directed repair (HDR). The potential applications of CRISPR/Cas are immense and, in agriculture, is facilitating crop development with resistance to abiotic, biotic, and agronomic characteristics that improve yield, quality, and food security. Gene editing also facilitates the relatively rapid modification of regulatory and complex pathways that enable studies to advance our understanding of gene function. This review provides an update of the fast-evolving CRISPR/Cas modification of important crops to address emerging global population, as well as environmental and climate challenges.},
}
@article {pmid42124669,
year = {2026},
author = {Sanderson, EM and Peralta, J and Nouwens, S and Oriolt, L and Hayes, VM and Kaiser, BK and Meeske, AJ},
title = {Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.02.23.707548},
pmid = {42124669},
issn = {2692-8205},
abstract = {Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri . Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.},
}
@article {pmid42126187,
year = {2026},
author = {Huang, J and Ma, K and Ding, S and Wang, Y and Xiong, J and Yi, J and Zhang, J and He, Z and Huang, L and Ren, X and Zhou, J and Chen, X and Liu, L and Qi, W and Wang, S and Liao, M},
title = {A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol.},
journal = {Emerging microbes &amp; infections},
volume = {15},
number = {1},
pages = {2651464},
pmid = {42126187},
issn = {2222-1751},
mesh = {*Cholesterol/metabolism ; Animals ; Humans ; *Steroid Hydroxylases/genetics/metabolism ; *Influenza A Virus, H7N9 Subtype/physiology ; Virus Internalization/drug effects ; *Influenza, Human/virology ; Mice ; CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; Cell Membrane/metabolism ; Dogs ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Influenza A viruses (IAVs) cause severe outbreaks with high mortality in birds and humans. A deeper understanding of cell-intrinsic defense mechanisms against influenza viruses is therefore crucial for developing novel antiviral strategies. Herein, we perform a genome-wide CRISPR activation screen to systematically elucidate host restriction factors against influenza A (H7N9) virus. Among multiple candidates, cholesterol 25-hydroxylase (CH25H) is shown to be induced by influenza virus infection and inhibit viral membrane fusion. Notably, our previous work demonstrated that CH25H blocks the entry of plasma membrane-fusing viruses such as coronaviruses. This inhibition occurs by relocating accessible cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). Here, we extend this finding and show that the same mechanism works against endocytosis-dependent viruses such as influenza viruses. The exogenous supplementation of cholesterol can restore depleted accessible cholesterol and reverse the CH25H-mediated restriction. Additionally, we prove that acyl-CoA:cholesterol acyltransferase (ACAT) is required to recruit the accessible cholesterol in this process. However, how hydrophobic accessible cholesterol is transported remains unclear. Here, we demonstrate that GRAMD1/Aster-mediated non-vesicular cholesterol transport is utilized to mobilize accessible cholesterol upon stimulation of CH25H. 25-hydroxycholesterol (25HC), the catalytic product of CH25H, is a natural metabolite that potently inhibits influenza virus infection both in vitro and in vivo. These findings underscore the promising therapeutic potential of 25HC against influenza viruses.},
}
@article {pmid42126246,
year = {2026},
author = {Song, R and Yin, C and Chen, B and Qu, B and Qiao, W and Li, R and Gao, Y and Song, X},
title = {Machine Learning-Assisted Portable Ai BOX Based on RPA-CRISPR/Cas12a for Rapid On-Site Detection of Foodborne Pathogens.},
journal = {Analytical chemistry},
volume = {98},
number = {20},
pages = {14913-14927},
doi = {10.1021/acs.analchem.6c00303},
pmid = {42126246},
issn = {1520-6882},
mesh = {*Machine Learning ; *CRISPR-Cas Systems ; *Food Microbiology ; *Listeria monocytogenes/isolation &amp; purification/genetics ; },
abstract = {Foodborne pathogens present a major threat to global public health. However, conventional detection methods and equipment are often unsuitable for the on-site and timely monitoring of these pathogens. To overcome this critical limitation and establish a rapid detection workflow, we developed the portable smart Ai BOX (artificial intelligence BOX). This device is a compact, palm-sized, internet of things (IoT)-enabled instrument that utilizes isothermal fluorescence diagnostics and weighs only 180 g. The Ai BOX features an optimized minimalist industrial design, ultralow power consumption, and a high-sensitivity optical sensing system. The device performs real-time fluorescence detection, with results automatically interpreted and transmitted to a dedicated mobile application (APP) via an integrated smart camera, enabling comprehensive food monitoring. Furthermore, the incorporation of artificial intelligence and machine learning (ML) algorithms significantly enhances the processing capability of the RPA-CRISPR/Cas12a fluorescence signal, thereby ensuring superior detection accuracy. The Ai BOX is ideally suited for on-site point-of-care testing (POCT) of foodborne pathogens. By integrating the one-pot-RPA-CRISPR/Cas12a method, the device achieves an exceptionally low limit of detection (LOD) of 1 × 10[1] CFU/mL for Listeria monocytogenes. In tests using simulated samples, it demonstrated 100% sensitivity and specificity. Consequently, the Ai BOX exhibits promising application potential for diverse public and personal health scenarios, including the detection of meat adulteration, food contamination, and wastewater monitoring.},
}
@article {pmid42126429,
year = {2026},
author = {Huang, RS and Phung, SK and Sumstad, D and Weis, AJ and Kile, QM and Bendzick, L and Khaw, MJ and Vue, YY and McKenna, DH and Kennedy, PR and Miller, JS and Felices, M},
title = {Reprogramming endogenous NK circuits by highly efficient nonviral genome editing.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {7},
pages = {},
doi = {10.1084/jem.20260192},
pmid = {42126429},
issn = {1540-9538},
support = {R35 CA283892/NH/NIH HHS/United States ; P01 CA111412/NH/NIH HHS/United States ; P01 CA065493/NH/NIH HHS/United States ; P30 CA 77598/NH/NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; *Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Mice ; Gene Regulatory Networks ; Interleukin-12/genetics/metabolism ; Receptors, Chimeric Antigen/genetics/immunology ; },
abstract = {Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.},
}
@article {pmid42127676,
year = {2026},
author = {Cheng, M and Chen, X and Cheng, H and Gao, X and Ao, H and Bao, X and Song, X and Tai, Y and Jin, D and Zhang, L},
title = {An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for detection of toxigenic Clostridioides difficile.},
journal = {Biosensors &amp; bioelectronics},
volume = {308},
number = {},
pages = {118779},
doi = {10.1016/j.bios.2026.118779},
pmid = {42127676},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Clostridioides difficile/isolation &amp; purification/genetics/pathogenicity ; *Bacterial Toxins/genetics/isolation &amp; purification ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/isolation &amp; purification ; Humans ; *Clostridium Infections/microbiology/diagnosis ; Limit of Detection ; Rapid Diagnostic Tests ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Clostridioides difficile (C. difficile) infection (CDI) represents a formidable global healthcare challenge, necessitating the development of rapid, accurate, and cost-effective diagnostic platforms to mitigate nosocomial transmission and improve patient outcomes. Compared with the conventional methods, CRISPR/Cas systems featured by specific target reorganization by a single chain RNA, coupled with electrochemical technology enables highly sensitive detection of various biomarkers. However, their application to CDI has remained unexplored due to the lack of tailored crRNAs. Herein, we present the integration of CRISPR/Cas12a with electrochemical transduction for the direct detection of C. difficile. A novel crRNA was engineered to specifically recognize the toxin B gene (tcdB), activating the trans-cleavage activity of Cas12a upon target binding. This cascade triggers the cleavage of immobilized ssDNA reporters on the electrode surface, generating measurable amperometric signal changes. The developed biosensor demonstrates exceptional performance, achieving a detection limit of pM level for tcdB DNA within 40 min, while exhibiting high specificity against non-target pathogens and robust stability over 7 days. This work establishes a rapid and reliable CRISPR-electrochemical diagnostic platform, offering significant potential for point-of-care CDI management.},
}
@article {pmid42128117,
year = {2026},
author = {Zou, X and Gu, T and Li, X and Xia, X and Yang, M and Huo, D and Hou, C},
title = {One-tube, protospacer adjacent motif-free and AI-enhanced CRISPR/Cas12a platform for ultra-sensitive detection of human papillomavirus 16 double-stranded DNA.},
journal = {International journal of biological macromolecules},
volume = {366},
number = {},
pages = {152525},
doi = {10.1016/j.ijbiomac.2026.152525},
pmid = {42128117},
issn = {1879-0003},
mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics ; Humans ; Limit of Detection ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer, primarily caused by HPV16 infection, remains a major global health concern. While PCR is the gold standard for HPV DNA detection, its limitations include lengthy analysis and equipment dependency. To address these challenges, we developed an asymmetric semi-nested RPA combined with Lambda-assisted Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a CRISPR/Cas12a (ASN-RPA-LCRISPR) for protospacer adjacent motif (PAM) independent, ultrasensitive Human Papillomavirus 16 (HPV16) dsDNA detection. This method employs a three-primer asymmetric amplification strategy combined with Lambda nuclease and the CRISPR/Cas12a system to achieve signal amplification. This approach significantly increases single-stranded DNA yield while overcoming PAM restriction issues, achieving a detection limit of 11.8 aM within 35 min. A single-tube system designed in this study prevents aerosol contamination, while a DenseNet121-based image classifier enables 97% accurate fluorescence interpretation under ambient light, eliminating reliance on specialized equipment. This platform offers rapid, ultrasensitive, and portable HPV16 detection, advancing cervical cancer screening and PAM-free nucleic acid diagnostics.},
}
@article {pmid42128325,
year = {2026},
author = {Cai, Y and Yang, J and Hou, M and Su, W and Liang, F and Zhu, M and Wu, T},
title = {Multi-omics precision diagnosis of brucellosis: Advances in biomarker discovery and clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {590},
number = {},
pages = {121074},
doi = {10.1016/j.cca.2026.121074},
pmid = {42128325},
issn = {1873-3492},
abstract = {Brucellosis, a neglected zoonosis caused by intracellular Brucella bacteria, remains a formidable global public health challenge, especially in developing regions. The notorious ability of Brucella to evade host immunity and establish chronic focal infections limits the utility of traditional diagnostic methods like bacterial culture and serology for early detection, therapeutic monitoring, and disease staging. This review comprehensively synthesizes the ongoing paradigm shift from pathogen-centric detection toward multi-omics precision diagnosis. We critically evaluate advances in nucleic acid amplification technologies (NAATs), charting the progression from quantitative PCR to absolute quantification via droplet digital PCR (ddPCR) and examining the transformative potential of CRISPR-Cas biosensing for ultrasensitive, instrument-free detection. The discussion also encompasses the renaissance of serology through immunoproteomics, which has identified novel serodominant antigens and multi-epitope fusion proteins to address the persistent specificity problems arising from cross-reacting bacteria. Furthermore, we analyze the emerging landscape of host-response biomarkers, integrating transcriptomic, metabolomic, and single-cell RNA sequencing data to delineate distinct immune signatures of acute and chronic infection. Finally, we consider how artificial intelligence (AI) can integrate these multi-dimensional datasets to build predictive diagnostic models. This consolidated multi-omics framework charts a course for precision medicine in brucellosis, aiming to bridge the gap between biomarker discovery and point-of-care clinical application. SUMMARY: Multi-omics technologies (genomics, proteomics, metabolomics, transcriptomics) are advancing brucellosis diagnosis via sensitive detection and accurate biomarkers, and improving treatment through novel strategies like nano-delivery, vaccines, and AI integration.},
}
@article {pmid42128554,
year = {2026},
author = {Xu, W and Cheng, Y and Sun, K and Wu, Y and Xu, Y and Ye, J and Li, P and Wu, H},
title = {Inception-level signal amplification: Cascaded DNAzyme-Cas9 nickase achieves sub-nanomolar kanamycin tracking.},
journal = {Analytica chimica acta},
volume = {1408},
number = {},
pages = {345562},
doi = {10.1016/j.aca.2026.345562},
pmid = {42128554},
issn = {1873-4324},
mesh = {*Kanamycin/analysis/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *Biosensing Techniques/methods ; Milk/chemistry ; *Nucleic Acid Amplification Techniques ; *Deoxyribonuclease I/metabolism/chemistry ; Limit of Detection ; Animals ; *Anti-Bacterial Agents/analysis ; CRISPR-Cas Systems ; Water Pollutants, Chemical/analysis ; },
abstract = {BACKGROUND: Kanamycin's persistent contamination in agricultural products poses significant human health risks due to its nephrotoxicity and bioaccumulation via the food chain. Existing methods for on-site kanamycin monitoring lack sufficient sensitivity and portability, limiting their utility in field settings. The urgent need for rapid detection technologies remains unmet. This work addresses the critical gap in developing a field-deployable platform for ultrasensitive kanamycin residue screening.<br><br>RESULTS: We engineered a fluorescence biosensor integrating DNAzyme-assisted Cas9 nickase-based amplification reaction (Cas9nAR). Kanamycin binding induces aptamer conformational changes, triggering Cas9nAR-driven cascade amplification that continuously generates DNAzymes. These cleave reporter probes to enable quantitative detection. The system achieved a broad linear range (1&#x202f;nM - 5&#x202f;&#x3bc;M) with an ultralow detection limit (0.3&#x202f;nM), surpassing conventional methods. It exhibited high specificity against interfering antibiotics and delivered consistent recoveries (97% to 103%) in spiked water and milk samples. Following pretreatment, analysis can be completed within 120&#x202f;min, validating its operational simplicity and robustness for complex matrices.<br><br>SIGNIFICANCE AND NOVELTY: This work reports the first CRISPR-Cas9 nickase/DNAzyme cascade amplification platform for small-molecule detection, establishing a new paradigm that integrates programmable nucleic acid amplification with catalytic signal turnover. By using Cas9nAR-generated ssDNA as an in situ template for autonomous DNAzyme assembly, the biosensor achieves ultrasensitive, and homogeneous detection of kanamycin-addressing a critical gap in field-deployable antibiotic residue monitoring. The modular design offers a generalizable strategy for translating non-nucleic acid recognition events into amplified fluorescent outputs, with broad implications for point-of-need diagnostics in food safety and environmental analysis.},
}
@article {pmid42128971,
year = {2026},
author = {Xie, R and Zhu, C and Liang, X and Lu, K and Wu, J},
title = {Knockout of bsal/cel.2 results in growth retardation, reduced lipid digestion and altered energy metabolism in medaka larvae (oryzias latipes).},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42128971},
issn = {1438-7948},
support = {2024YFD2401502//the National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Oryzias/genetics/growth &amp; development/metabolism ; *Energy Metabolism/genetics ; *Lipid Metabolism/genetics ; *Fish Proteins/genetics/metabolism ; Larva/growth &amp; development/genetics/metabolism ; *Lipase/genetics/metabolism ; CRISPR-Cas Systems ; Lipolysis/genetics ; },
abstract = {To evaluate the significance of bile salt-activated lipase (BSAL) in lipid digestion and metabolism in fish, this study used CRISPR/Cas9 gene editing to generate medaka (Oryzias latipes) bsal mutant lines. Given that the bsal gene comprises three copies (bsal, cel.2, and bsal-like) in the medaka genome, bsal-like variants may lead to functional loss in lipid hydrolysis owing to amino acid residue alterations in the bile salt binding site. Consequently, two types of medaka mutants, bsal[-/-] and bsal[-/-]/cel.2[-/-], were generated for experimental exploration in this study. Compared to wild-type (WT) medaka, the bsal[-]/[-]/cel.2[-]/[-] group showed significant reductions in body length, expression of growth-related genes (gh and igf), total lipase and protease activities, and body composition (cholesterol, triglyceride, and protein levels). The bsal[-]/[-]/cel.2[-]/[-] group also exhibited upregulated expression of lipid synthesis genes (fas, acc1, scd) and downregulated expression of lipolysis-related genes (cpt1, acox1). Notably, key glycolysis genes (pk, gk) and gluconeogenesis-related genes (pck2) were significantly upregulated in the bsal[-]/[-]/cel.2[-]/[-] group. However, the bsal[-/-] group exhibited no significant differences from the WT group in all assays, except for notable reductions in protease activity and expression levels of the cpt1 and gk genes, as well as a significant increase in pck2 gene expression compared to the WT group. Remarkably, the expression level of the cel.2 gene was significantly elevated in the bsal[-/-] group compared to the WT group. In summary, this study demonstrates the pivotal role of the bsal gene in lipid digestion and metabolism in medaka. Furthermore, the presence of multiple copies of the bsal gene aids in fulfilling the demands of lipid digestion in medaka. This conclusion can provide insights into the research on lipid digestion and metabolism in other fish species.},
}
@article {pmid42128985,
year = {2026},
author = {Hou, M and Li, Y and Wu, X and Long, D and Sun, D and Chen, P and Huang, H},
title = {Recent Advances in the Development of CRISPR-Based Live-Cell Molecular Imaging and Sensing.},
journal = {Molecular imaging and biology},
volume = {},
number = {},
pages = {},
pmid = {42128985},
issn = {1860-2002},
support = {2026JJ90110//Natural Science Foundation of Hunan Province Joint Fund for Universities/ ; S202512034126//Hunan Province Undergraduate Innovation Training Program/ ; },
abstract = {Visualizing genome organization and transcriptional dynamics with spatial and temporal precision in living cells is essential for elucidating gene regulation and chromatin-associated disease mechanisms, yet conventional methods confront a fundamental tension between endogenous-sequence targeting and live-cell compatibility. Operator-repressor systems require prior insertion of repetitive arrays at engineered loci, whereas fluorescence in situ hybridization mandates cell fixation and thereby precludes temporal analysis. CRISPR-Cas technologies, originally developed for genome editing, have been re-engineered into a versatile molecular-imaging toolkit capable of interrogating native sequences in living cells. Here, we systematically review CRISPR-based live-cell imaging and sensing platforms, critically evaluating their design principles, mechanistic foundations, and performance limitations. We examine dCas9-based DNA labeling, dCas12a systems for non-repetitive loci, Cas13- and Csm-mediated RNA imaging, novel fluorescent reporters, engineered ribonucleoproteins, and delivery innovations including reagent-based Oligo-LiveFISH. To organize this diverse literature, we distinguish three operationally distinct modalities-live-cell imaging, intracellular sensing, and diagnostic biosensing-and assess each platform through three unifying design trade-offs: sensitivity versus cellular perturbation, multiplexing capacity versus system complexity, and detection threshold versus biological fidelity. Building on this framework, we evaluate the integration of CRISPR imaging with super-resolution microscopy, artificial-intelligence-driven computational analysis, and multimodal spatial omics. Collectively, this synthesis clarifies current capabilities, delineates unresolved constraints, and charts a coherent path toward clinically relevant applications of CRISPR-based live-cell molecular imaging.},
}
@article {pmid42129030,
year = {2026},
author = {Watanabe, K and Ishikawa, M and Ishibashi, K},
title = {Development of Tobamovirus-Resistant Tomato Plants by CRISPR-Cas9-Mediated Knockout of Susceptibility Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {1-10},
pmid = {42129030},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Tobamovirus/pathogenicity/physiology ; Gene Editing/methods ; *Gene Knockout Techniques/methods ; Host-Pathogen Interactions/genetics ; Plants, Genetically Modified/genetics/virology ; },
abstract = {The advent of genome editing technologies such as CRISPR-Cas9 has revolutionized the development of disease-resistant crops, offering precision and efficiency in targeting specific genetic loci responsible for susceptibility. In this protocol, we harness the CRISPR-Cas9 system to disrupt key susceptibility genes in tomato, aiming to fortify resistance against tobamoviruses, particularly the aggressive tomato brown rugose fruit virus (ToBRFV). By systematically knocking out four TOM1 homologs, genes essential for tobamoviral replication, tomato lines with robust and heritable resistance can be developed while minimizing adverse developmental effects. The approach not only underscores the significance of basic research on host-pathogen interactions in modern crop protection but also lays the groundwork for sustainable, gene-driven resistance strategies in commercial tomato breeding.},
}
@article {pmid42129032,
year = {2026},
author = {Yoshida, T and Ishibashi, K},
title = {Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana Using a Meristem-Invading Virus Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {21-27},
pmid = {42129032},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Meristem/genetics/virology ; CRISPR-Cas Systems ; *Potyvirus/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Gene editing can be achieved using sequence-specific nucleases. This protocol describes a plant gene editing method that eliminates the need for tissue culture by employing a virus-based delivery system. Tobacco ringspot virus (TRSV) can access meristematic tissues in infected plants, enabling the introduction of site-directed mutations into germline cells. This allows for heritable gene modification in the model plant Nicotiana benthamiana without tissue culture.},
}
@article {pmid42129103,
year = {2026},
author = {Ash, S and Attianese, GMPG and Kosti, P and Semilietof, A and Stefanidis, E and Triboulet, M and Irving, M},
title = {Generation and Characterization of CAR-T Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2998},
number = {},
pages = {439-504},
pmid = {42129103},
issn = {1940-6029},
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Neoplasms/therapy/immunology ; Lymphocyte Activation ; Lentivirus/genetics ; Retroviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.},
}
@article {pmid42129496,
year = {2026},
author = {Duchêne, C and Craig, RJ and Martinho, C and Luthringer, R and Agullo, F and Hipp, K and Escudeiro, P and Alva, V and Haas, FB and Coelho, SM},
title = {Latent endogenous giant viruses drive active infection and inheritance in a multicellular algal host.},
journal = {Nature microbiology},
volume = {11},
number = {6},
pages = {1547-1558},
pmid = {42129496},
issn = {2058-5276},
support = {101109906//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; },
mesh = {*Giant Viruses/genetics/physiology ; Virus Integration ; *Phycodnaviridae/genetics/physiology ; *Virus Latency ; Virus Replication ; *Haptophyta/virology/genetics ; CRISPR-Cas Systems ; Virus Activation ; Host-Pathogen Interactions ; Genome, Viral ; *DNA Viruses/genetics/physiology ; },
abstract = {Endogenous viral elements inserted in host genomes are often regarded as inert relics of past infections. Whether they can retain infective potential and contribute to active viral cycles has remained largely unresolved. Here we demonstrate that giant viral elements in the multicellular alga Ectocarpus can reactivate and drive productive viral infections. Using long-read sequencing and transcriptomics, we identify full-length, transcriptionally active phaeoviruses integrated within the host genome, and we use classical genetics and CRISPR-Cas to demonstrate that these elements are stably inherited through the germline, while their reactivation is precisely regulated by developmental and environmental cues including temperature. We resolve the genomic integration sites and propose a mechanism for phaeovirus integration and replication. Our work provides direct evidence and uncovers the mechanisms by which giant viral elements can reactivate, replicate and transmit both horizontally and vertically in a multicellular eukaryote, establishing a new model of latency, inheritance and evolutionary impact of giant dsDNA viruses.},
}
@article {pmid42129543,
year = {2026},
author = {Bilanovic, J and Bortolatto, J and Duan, S and Bjørnsdottir, V and Teetz, AK and Thoms, A and Fischbach, J and Schmidt, F and Nyberg, WA and Hartweger, H and Escolano, A and Thomas, PG and Victora, GD and Bilate, AM and Jacobsen, JT},
title = {One-step generation of T-cell receptor knock-in mice in the TCRβ locus.},
journal = {The EMBO journal},
volume = {45},
number = {12},
pages = {4321-4336},
pmid = {42129543},
issn = {1460-2075},
support = {101042650//EC | European Research Council (ERC)/ ; },
mesh = {Animals ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; Mice, Transgenic ; Dependovirus/genetics ; T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Transgenic mouse models expressing predefined T-cell receptors (TCRs) have been instrumental in advancing our understanding of T-cell biology. However, these traditional models rely on random genomic insertion of large constructs, require labor-intensive embryo manipulation, and frequently result in aberrant TCR expression and phenotypes. These limitations render traditional models insufficient to meet the mounting demands for rapid and precise model systems to evaluate TCR specificities. In this study, we developed a streamlined method that uses adeno-associated virus (AAV) and CRISPR/Cas9-mediated genome editing to precisely integrate pre-rearranged TCRα/β sequences into the mouse TCRβ (Trb) locus, enabling the rapid generation of TCR knock-in mice with physiological TCR expression and functional T-cell differentiation upon antigenic challenge. This approach bypasses the need for screening multiple founders for faithful TCR expression, enhancing the versatility and utility of monoclonal TCR mice in basic immunology and preclinical research, such as in the fields of cancer immunotherapy and vaccine development.},
}
@article {pmid42132069,
year = {2026},
author = {Liu, Z and Qi, J and Sun, L and Su, J and Li, W and Wu, L and Liang, Y and Wei, C and He, F and Han, Y and Sun, Y and Yan, H and Li, H and Xiao, R},
title = {Oxygen-Vacancy-Engineered WOX Nanowire-Based Surface-Enhanced Raman Scattering Biosensor with Lyophilized CRISPR/Cas13a Platform for CHIKV Detection.},
journal = {ACS sensors},
volume = {11},
number = {5},
pages = {4132-4144},
doi = {10.1021/acssensors.6c00934},
pmid = {42132069},
issn = {2379-3694},
mesh = {*Nanowires/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Spectrum Analysis, Raman/methods ; *Chikungunya virus/isolation &amp; purification ; Gold/chemistry ; Humans ; *Oxygen/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; *Chikungunya Fever/diagnosis/virology ; Immunoassay/methods ; },
abstract = {The outbreak of Chikungunya virus (CHIKV) has caused widespread acute morbidity with severe polyarthralgia or chronic arthritis, placing a great challenge to public health and socioeconomic development. Establishing a rapid and highly sensitive detection technology is crucial for achieving precise control and prevention. Here, we established a CRISPR/Cas13a-mediated SERS lateral flow immunoassay platform for rapid and highly sensitive detection of CHIKV. One-dimensional nanowires loading Au nanoparticles were used to prepare SERS tags, presenting excellent SERS-enhanced performances and superior applicability to directional flow on the test strip. For the best performance, the structure of WOX nanowires was regulated by adjusting the addition concentration of ascorbic acid during the synthesis process, resulting in a stronger LSPR effect derived from more Au NPs in situ grown on highly reducing WOX. Furthermore, the lyophilized CRISPR/Cas system greatly simplified the workflow. In the optimal conditions, the limit of detection reached 0.56 and 1.03 copies/μL for the CHIKV plasmid and inactivated viruses by this method, respectively. Furthermore, 34 clinical serum samples were accurately diagnosed by our proposed method, 100% consistent with qPCR. This platform with the advantages of simple operation and rapid response provides a reliable technical tool for the early precise identification and efficient monitoring of CHIKV.},
}
@article {pmid42132561,
year = {2026},
author = {Chen, H and Zhang, C},
title = {Simple yet sensitive MicroRNA detection using allosteric probe-initiated triple amplification and Cas13a/crRNA-based amplification reaction.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {20},
pages = {4154-4160},
doi = {10.1039/d6ay00578k},
pmid = {42132561},
issn = {1759-9679},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Allosteric Regulation ; },
abstract = {MicroRNAs (miRNAs) have emerged as promising biomarkers for early diagnosis and management of degenerative disc disease (DDD); however, their low abundance and high sequence homology pose significant challenges for clinical detection. Herein, we develop a novel, highly sensitive miRNA detection platform by integrating an allosteric probe-initiated triple-cycle amplification strategy with the CRISPR-Cas13a/crRNA system. The designed allosteric probe undergoes a conformational switch upon target miRNA binding, triggering successive enzymatic amplification steps, including polymerase-mediated extension, nicking enzyme-driven recycling, and T7 RNA transcription, to generate numerous single-stranded RNA activators. These activators specifically recruit the Cas13a/crRNA complex, unleashing its collateral cleavage activity to degrade reporter RNAs and produce amplified fluorescence signals. This method demonstrates a wide dynamic range from 1 fM to 100 pM and achieves an ultra-low detection limit of 548 aM. Notably, the approach exhibits excellent specificity, distinguishing target miRNA-155 from closely related variants and non-target miRNAs. Its operational simplicity, rapid turnaround (60 min), and robustness in serum samples highlight strong potential for clinical translation. By combining catalytic allosteric probing with CRISPR-based signal amplification, this work provides a versatile and powerful tool for miRNA quantification, paving the way for early, minimally invasive diagnosis of degenerative disc diseases and other miRNA-associated pathologies.},
}
@article {pmid42133116,
year = {2026},
author = {Rajan, A and Raveendran, M and Shanmugam, V and Arul, L and Kumar, KK and Subramanian, A and Mannu, J and Eswaran, K},
title = {Engineering crop determinacy: CRISPR/Cas based advances in self-pruning gene function and application.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42133116},
issn = {1573-4978},
support = {BT/INF/22/SP45584/2022//Department of Biotechnology, Government of India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/growth &amp; development ; Gene Editing/methods ; Flowers/genetics/growth &amp; development ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genetic Engineering/methods ; Plant Proteins/genetics ; },
abstract = {The transition from indeterminate to determinate growth represents a key achievement in crop improvement, as it enhances agricultural productivity by synchronizing flowering, facilitating uniform harvest, and improving overall efficiency. In tomato and other crops, this shift is largely governed by mutations in the SELF-PRUNING (SP) gene, a key member of the CENTRORADIALIS (CEN), TERMINAL FLOWER 1 (TFL1), and SELF-PRUNING (SP) (CETS) gene family that regulates the vegetative to reproductive phase transition and influences overall shoot architecture. With increasing labour constraints, climate variability and rising global food security challenges, the ability to engineer optimized plant architectures has become increasingly important. CRISPR-based genome editing provides a precise and efficient strategy to modify SP/TFL1 homologs, enabling targeted transition from indeterminate to compact, determinate growth forms that exhibit synchronized flowering and enhanced mechanical harvestability. These genome editing approaches have been successfully applied across diverse crop species, including tomato, legumes, cotton, cereals and horticultural crops. This review consolidates current understanding of the molecular mechanisms governing determinacy, with emphasis on the central role of SP/TFL1 genes and their interactions with hormonal pathways such as auxin and cytokinin. By integrating these insights with recent advances in CRISPR-based editing platforms, this review provides a practical framework for researchers and breeders aiming to leverage CRISPR technology for next-generation crop improvement. Such strategies hold significant potential for enhancing productivity, resilience and sustainability within modern agricultural systems.},
}
@article {pmid42133523,
year = {2026},
author = {Nammi, B and Madugula, SS and Jayasinghe-Arachchige, VM and Pham, T and Liu, J and Wang, S},
title = {Robust CRISPR-Cas Protein Identification using Max-Margin Regularized Transformer Models.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {PP},
number = {},
pages = {},
doi = {10.1109/TCBBIO.2026.3693528},
pmid = {42133523},
issn = {2998-4165},
abstract = {The discovery of CRISPR-Cas system has significantly advanced genome editing, offering vast applications in medical treatments and life sciences research. Despite their immense potential, the existing CRISPR-Cas systems still face challenges concerning size, delivery efficiency, and cleavage specificity. Addressing these challenges requires a deeper understanding of CRISPR-Cas proteins to advance the design and discovery of novel Cas proteins. Here, we study CRISPR-Cas proteins extensively using deep-learning techniques to build classification models that can differentiate between Cas and non-Cas proteins, as well as identify subfamilies Cas9 and Cas12. We developed two types of deep learning models: 1) a transformer encoder-based classification model, trained from scratch; and 2) a large protein language model fine-tuned on ProtBert, pre-trained on more than 200 million proteins. To boost learning efficiency for the model trained from scratch, we introduced a novel margin-based loss function to maximize inter-class separability and intra-class compactness in protein sequence embedding latent space of a transformer encoder. Our results show that the Fine-Tuned ProtBert-based (FTPB) classification model achieved accuracies of 99.06%, 94.42%, 96.80%, 97.57% for Cas9 vs. non-Cas, Cas12 vs.non-Cas, Cas9 vs. Cas12, and multi-class classification of Cas9 vs. Cas12 vs. non-Cas proteins, respectively. The Latent Space Regularized Max-Margin Transformer (LSRMT) model achieved classification accuracies of 99.81%, 99.81%, 99.06%, and 99.27% for the same tasks, respectively. These results demonstrate the effectiveness of the proposed Max-Margin-based latent space regularization in enhancing model robustness and generalization capabilities. Remarkably, the LSRMT model, even when trained on a significantly smaller dataset, outperformed the fine-tuned state-of-the-art large protein model. The high classification accuracies achieved by the LSRMT model demonstrate its proficiency in identifying discriminative features of CAS proteins, marking a significant step towards advancing our understanding of CAS protein structures in future research endeavors.},
}
@article {pmid42133534,
year = {2026},
author = {Liu, C and Feng, M and Yu, H and Zhang, X and Li, Y and Sui, G and Jing, W and Cheng, X},
title = {CRISPR-Cas12a2-Based Multiplexed Diagnostic for Rapid and Highly Sensitive Detection of Respiratory Viruses.},
journal = {Analytical chemistry},
volume = {98},
number = {20},
pages = {14775-14787},
pmid = {42133534},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Lab-On-A-Chip Devices ; *Respiratory Syncytial Viruses/isolation &amp; purification/genetics ; Molecular Diagnostic Techniques ; *Influenza A virus/isolation &amp; purification/genetics ; },
abstract = {Infectious diseases severely threaten global public health security, necessitating rapid and highly sensitive diagnosis. This study presents a novel multiplex diagnostic platform combining transcription-mediated amplification (TMA) with the CRISPR-Cas12a2 system for rapid and highly sensitive detection of respiratory viruses. The assay uses an integrated microfluidic chip, which can simultaneously identify influenza A/B and respiratory syncytial viruses (RSV-A/B) with optimized CRISPR RNAs and isothermal amplification, achieving detection limits as low as 10[2] copies/μL within 60 min. The detection system showed excellent specificity; nonspecific reactions were not observed in the presence of nucleic acids from other respiratory pathogens. Clinical validation using nasopharyngeal swabs demonstrated high concordance with real-time quantitative reverse transcription polymerase chain reaction, with most positive samples detected within 40 min. The system eliminates DNA amplification steps, reduces contamination risk, and simplifies the workflow. Using two-step reactions on a centrifugal microfluidic chip, the TMA-CRISPR-Cas12a2 platform offers a promising integrated platform for multiplex respiratory pathogen screening, thereby supporting timely diagnosis and outbreak management.},
}
@article {pmid42134322,
year = {2026},
author = {Lei, L and Kaufmann, MM and Lao, J and Thoulass, G and Ammann, S and Xiao, H and Rhiel, M and Dettmer-Monaco, V and Grünewald, J and Andrieux, G and Alzubi, J and Miller, BR and Weißert, K and Gräßel, L and Schell, C and Illert, AL and Joung, JK and Boerries, M and Cornu, TI and Ehl, S and Erlacher, M and Aichele, P and Cathomen, T},
title = {Genotoxicity profiling reveals distinct platform-and cell type-specific effects in therapeutic gene editing for genetic hyperinflammation.},
journal = {Cell stem cell},
volume = {33},
number = {6},
pages = {930-944.e5},
pmid = {42134322},
issn = {1875-9777},
support = {R35 GM118158/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; *Inflammation/genetics/therapy ; Mice ; CRISPR-Cas Systems/genetics ; Mutagenicity Tests ; Humans ; *Genetic Therapy ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Base editors enable precise correction of point mutations without requiring DNA double-strand breaks, yet platform- and cell type-specific genotoxicities remain incompletely characterized. Here, we applied cytosine base editing (CBE) to disrupt a cryptic splice-site mutation in the Unc13d locus of Jinx mice, a model of familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Efficient editing (62%-89%) in fibroblasts, T cells, and hematopoietic stem cells (HSCs) restored Unc13d splicing, reconstituted cytotoxic T cell function, and protected mice from virus-triggered hyperinflammation after transplantation of edited HSCs. Comparative genotoxicity profiling revealed distinct platform- and cell type-specific patterns: hyperactive CBE induced broader off-target activity and more structural variants than CRISPR-Cas9. Although off-target sequence edits persisted, the stability of CBE-induced chromosomal translocations differed between cell types. These findings establish base editing as a therapeutic strategy for a genetically predisposed hyperinflammatory syndrome and underscore the importance of context-specific safety profiling to guide the clinical translation of genome editors.},
}
@article {pmid42134990,
year = {2026},
author = {An, K and Prillo, S and Wu, W and Kristanto, I and Jones, MG and Song, YS and Yosef, N},
title = {Tree reconstruction guarantees from CRISPR-Cas9 lineage tracing data using Neighbor-Joining.},
journal = {Genome research},
volume = {36},
number = {6},
pages = {1199-1208},
pmid = {42134990},
issn = {1549-5469},
mesh = {*CRISPR-Cas Systems ; Animals ; *Algorithms ; Mice ; *Cell Lineage/genetics ; *Phylogeny ; Lung Neoplasms/genetics ; },
abstract = {CRISPR-Cas9-based lineage tracing technologies have enabled the reconstruction of single-cell phylogenies from transcriptional readouts. However, developing tree-reconstruction algorithms with theoretical guarantees in this setting is challenging. In this work, we derive a reconstruction algorithm with theoretical guarantees using Neighbor-Joining (NJ) on distances that are moment-matched to estimate the true tree distances. We develop a series of tools to analyze this algorithm and prove its theoretical guarantees. When the parameters of the data generating process are known and there is no missing data, our results align with established results from common evolutionary models, such as Cavender-Farris-Neyman and Jukes-Cantor. However, to account for the realistic case where the parameters of the data generating process are not known and there is missing data, we develop new theory that shows for the first time that it is still possible to obtain reconstruction guarantees in the CRISPR-Cas9 case and in other models of evolution. Empirically, we show on both simulated lineage tracing data and on real data from a mouse model of lung cancer the improved performance of our method as compared to the traditional use of NJ.},
}
@article {pmid42136289,
year = {2026},
author = {Sorourian, S and Behbahani, AB},
title = {CRISPR-Cas Systems and CHO in Biopharmaceuticals: Unlocking New Possibilities in Gene Editing.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010408149251016112221},
pmid = {42136289},
issn = {1873-4316},
abstract = {The CRISPR-Cas system has significantly advanced genome editing, offering superior efficiency, precision, and ease of use compared to traditional technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). CHO cells are a widely used mammalian cell line for large-scale therapeutic protein manufacturing due to their ability to produce human-like glycosylation patterns and grow in serum-free media. Recent CRISPR-based CHO cell engineering enables precise genetic modifications, improving productivity, stability, scalability, and reducing costs. This article highlights the transformative role of CRISPR technologies in addressing genetic disorders and expanding the frontiers of multiple scientific fields. It offers a comprehensive analysis of several CRISPR-Cas systems, including Cas9, Cas12, Cas13, and Cas14, emphasizing their unique structural features and functional capabilities. While Cas9 has dominated many genomeediting applications, the use of Cas13 in Chinese Hamster Ovary (CHO) cells has opened up promising RNA-targeting strategies. Moreover, the compact Cas14 system presents notable potential for applications requiring ultra-precise genome manipulation. With their critical role in therapeutic protein production, CHO cells have greatly benefited from CRISPR-enabled engineering, leading to measurable improvements in productivity, stability, and cost-efficiency. Key advancements in CRISPR delivery platforms, including both viral and nonviral vectors, are discussed alongside ongoing challenges such as off-target effects and regulatory considerations. Emerging trends such as base editing, prime editing, and the integration of artificial intelligence for system optimization are also explored. Altogether, the discussion underscores the pivotal contribution of CRISPR technologies to CHO cell engineering and their broader impact on the future of biopharmaceutical manufacturing.},
}
@article {pmid42137038,
year = {2026},
author = {Koshi, N and Kobayashi, M and Ezura, H and Miura, K},
title = {Enhancement of parthenocarpy and fruit set through genome editing in tomato variety for processing use.},
journal = {Plant biotechnology (Tokyo, Japan)},
volume = {43},
number = {1},
pages = {127-131},
pmid = {42137038},
issn = {1342-4580},
abstract = {Tomatoes are extremely important plants that are cultivated worldwide, with various varieties grown in different regions. The traits required can vary depending on the region and intended use. Parthenocarpy, a trait that confers numerous advantages, reduces the labor required for pollination and minimizes the incidence of poor fruit set owing to temperature fluctuations. Mutations in SlIAA9 induce parthenocarpy in tomatoes, and the introduction of this trait into processed varieties via genome editing suggests its potential to markedly shorten the breeding timeline. Genome editing has gained considerable attention as a breeding technique because it enables precise mutations in specific genes. However, only a few recent studies have reported examples of genome editing in Japanese tomato varieties for processing. In this study, we employed a genome-editing technique targeting SlIAA9 to induce parthenocarpy in the Japanese tomato variety Natsunokoma for processing purposes, thereby reducing the labor required for pollination. The null-segregant Sliaa9 mutant exhibited enhanced parthenocarpy and fruit set. These results suggest that improvements in fruit-bearing and parthenocarpic traits enhance the quality of tomato varieties that are mainly used for processing.},
}
@article {pmid42137600,
year = {2026},
author = {Schönberg, PY and Muñoz-Ovalle, &#xc1; and Saleh, HA and Crespo, E and Kuhnert, R and Michen, S and Loureiro, L and Temme, A and Feldmann, A and Buchholz, F},
title = {Epigenetic editing balances TCR suppression and persistence in CAR T cells.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201712},
pmid = {42137600},
issn = {3117-387X},
abstract = {Allogeneic chimeric antigen receptor (CAR) T cell therapies offer a scalable, off-the-shelf option for cancer treatment, but their clinical use is limited by the risk of graft-versus-host disease (GvHD), mediated by the endogenous T cell receptor (TCR). Conventional strategies to eliminate TCR expression rely on genome editing tools such as CRISPR-Cas9 or base editing, which introduce permanent DNA changes and pose safety concerns. Here, we present an epigenetic editing approach that enables efficient, specific, and reversible silencing of the CD3ε gene, a critical component of the TCR complex, without altering the genome. We systematically optimized the epigenetic editor and guide RNA in a cell line and achieved robust TCR silencing in primary T and CAR T cells while preserving CAR expression, activation, and effector function. Transcriptome analysis confirmed minimal off-target effects. In vivo observations suggest the epigenetically silenced T cells to prevent GvHD while persisting longer than TCR-knockout cells, supporting the notion that transient TCR suppression may help balance safety and long-term efficacy. Our findings establish epigenetic editing as a non-genotoxic alternative to genome editing, offering a flexible and safer route to generate next-generation allogeneic CAR T cells.},
}
@article {pmid42137606,
year = {2026},
author = {Campbell, JM and Korpela, DM and Han, H and Zhao, S and Webster, DA and Nguyen, YAH and Koes, N and Aune, RO and Dagan, H and Milliken, R and Watts, JK and Murthy, N and Carlson, DF},
title = {Swine reporter model for preclinical evaluation and characterization of gene delivery vectors.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201729},
pmid = {42137606},
issn = {3117-387X},
abstract = {Delivery of gene therapy vectors efficiently targeted to any somatic cell remains a key barrier for the development of genetic medicines. While rodent models provide insights into vector biodistribution and cellular tropism, their anatomical and physiological differences from humans limit their translational potential and studies in large animal models are often required. In this study, we developed a swine reporter model (SRM-1) to evaluate both viral and non-viral vector delivery in a large animal system. The SRM-1 model harbors a tdTomato reporter at the ROSA26 locus that can be activated by Cre recombinase or CRISPR-Cas reagents and allows for tracking of gene delivery vectors in vivo. To evaluate this model, we administered adeno-associated virus serotype 9 (AAV9) and lipid nanoparticles (LNPs) carrying messenger RNA (mRNA) systemically and found successful in vivo reporter activation across a variety of tissues. Intracerebroventricular (i.c.v.) administration of LNP-mRNA was also performed and demonstrated localized activation in cortical brain cells. In addition to systemic biodistribution studies, this model has utility for testing clinically relevant local administration methods, surgical and non-surgical, of delivery vectors. Our findings support the SRM-1 model as a valuable tool for advancing gene therapies from preclinical testing to clinical application.},
}
@article {pmid42137980,
year = {2026},
author = {Xiong, W and Zhou, E and Qi, Q and Liu, X and Zhang, K and Huang, S and Li, M and Wang, C and Zhou, X and Tian, T},
title = {Harnessing a single molecule for dual bioorthogonal regulation of RNA function and m6A methylation.},
journal = {Nucleic acids research},
volume = {54},
number = {9},
pages = {},
pmid = {42137980},
issn = {1362-4962},
support = {22377094//National Natural Science Foundation of China/ ; 22177089//National Natural Science Foundation of China/ ; 22037004//National Natural Science Foundation of China/ ; 91853119//National Natural Science Foundation of China/ ; 91753201//National Natural Science Foundation of China/ ; 22177088//National Natural Science Foundation of China/ ; 22377095//National Natural Science Foundation of China/ ; 22407104//National Natural Science Foundation of China/ ; 22407106//National Natural Science Foundation of China/ ; 22407107//National Natural Science Foundation of China/ ; 22377094//National Natural Science Foundation of China/ ; 2022YFA1502902//National Key Research and Development Program of China/ ; },
mesh = {Methylation ; Click Chemistry/methods ; *Adenosine/analogs &amp; derivatives/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Humans ; *RNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Azides/chemistry ; RNA Processing, Post-Transcriptional ; },
abstract = {This study presents a multifunctional RNA regulation strategy that enables RNA molecules to undergo both bioorthogonal ligation and cleavage reactions within the same system. Using guide RNA (gRNA) as an example, we demonstrate on-demand inactivation via click chemistry and subsequent reactivation by light exposure. Applied to CRISPR-mediated, site-specific RNA methylation, this technology enables continuous, multistep programmable control, overcoming the one-way limitation of traditional methods. Results show that gRNA can stably tolerate both azide and photodegradable groups, facilitating efficient targeting of M3M14-dCas9 and dCas13b-M3M14 methylation systems to introduce m6A at specific RNA sites. Small molecules can turn off methylation through in situ click chemistry, while 365-nm light exposure rapidly restores gRNA function, allowing precise control over RNA methylation. This strategy highlights the efficiency and flexibility of progressive bioorthogonal RNA modulation and paves the way for multisite, dynamic regulation in complex biological systems.},
}
@article {pmid42138076,
year = {2026},
author = {Hale, AT and Kundishora, AJ and Kalailingam, P and Barak, T and Duy, PQ and Ramundo, CM and Fan, B and Li, Q and Brastianos, PK and Shankar, GM and Alper, SL and Kleinstiver, BP and Musolino, PL and Kahle, KT},
title = {Towards precision medicine for brain arteriovenous malformations.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {42138076},
issn = {1558-8238},
mesh = {Humans ; *Precision Medicine ; *Intracranial Arteriovenous Malformations/genetics/therapy/metabolism/pathology ; Animals ; Mutation ; Proto-Oncogene Proteins B-raf/genetics/metabolism ; },
abstract = {Recent advances in cerebrovascular genomics, single-cell biology, pharmacology, and gene editing technology are transforming our understanding of brain arteriovenous malformations (bAVMs) - a leading cause of pediatric hemorrhagic stroke. Once considered static anatomical defects, bAVMs are now recognized as dynamic, genetically driven lesions resulting from somatic mutations in KRAS, BRAF, and pathways involved in arteriovenous specification, angiogenesis, and vascular remodeling. By integrating human genetics, animal models, and endovascular innovations, researchers have uncovered convergent mechanisms that link endothelial Ras/MAPK hyperactivation to abnormal vessel growth and higher rupture risk. These insights provide a foundation for precision medicine approaches that combine molecular diagnostics - such as liquid or endoluminal biopsies - with mutation-specific pharmacotherapies and emerging CRISPR-based gene editing strategies. We suggest that genotype-guided interventions, tailored by spatial and developmental cerebrovascular context, could ultimately reclassify bAVMs from surgically incurable malformations to treatable molecular conditions.},
}
@article {pmid42138081,
year = {2026},
author = {Chang, LC and Eyler, CE and Lee, CL},
title = {Chromosomal instability induced by CRISPR/Cas9: implications for pancreatic cancer therapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {42138081},
issn = {1558-8238},
mesh = {Humans ; *Pancreatic Neoplasms/genetics/therapy/pathology/metabolism ; *Chromosomal Instability ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; },
abstract = {Clinical management of pancreatic cancer (PC) remains severely limited, primarily due to the complex tumor microenvironment. Emerging DNA damage-targeted strategies have demonstrated considerable therapeutic potential in PC. In this issue of the JCI, Teh et al. employed cancer-specific multitarget sgRNAs to induce DNA double-strand breaks (DSBs), resulting in lethal effects in PC cells. Integrative bioinformatic and cytogenetic analyses revealed that CRISPR/Cas9-mediated DSBs provoked persistent chromosomal instability, ultimately leading to chromosome catastrophe and cell death. Compared with equivalent radiation-induced DSBs, these sgRNAs exhibited superior cytotoxicity and were able to eliminate cells resistant to a specific sgRNA via subsequent targeting at distinct genomic sites, highlighting a promising and innovative precision therapeutic approach for clinical treatment of PC.},
}
@article {pmid42138085,
year = {2026},
author = {Teh, SSK and Kotwal, A and Bennett, A and Halper-Stromberg, E and Morsberger, L and Zamani, S and Shi, Y and Skaist, A and Zhu, Q and Bowland, K and Liang, H and Hruban, RH and Hung, CF and Anders, RA and Roberts, NJ and Scharpf, RB and Goldstein, M and Zou, YS and Eshleman, JR},
title = {Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {42138085},
issn = {1558-8238},
mesh = {*Pancreatic Neoplasms/genetics/pathology/therapy/metabolism ; *CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded ; Cell Line, Tumor ; Animals ; Mice ; Gene Editing ; },
abstract = {While radiation is an effective oncologic therapy, killing cancer by inducing DNA double-strand breaks (DSBs), it lacks specificity for neoplastic cells. We have previously adapted the CRISPR/Cas9 gene-editing technology as a cancer-specific treatment modality targeting somatic mutations in pancreatic cancer (PC). However, its tumoricidal potential remains unclear, especially in comparison with therapeutic doses of radiation. Here, we demonstrate that CRISPR/Cas9-induced DSBs are more cytotoxic in PCs than a comparable number of radiation-induced DSBs. We observed more than 90% tumor growth inhibition by targeting 9 sites with cancer-specific sgRNAs. Through both bioinformatics and cytogenetics analyses, we found that CRISPR/Cas9-induced DSBs triggered ongoing chromosomal rearrangements, with 87% of structural variants not directly produced from the initial CRISPR/Cas9-induced DSBs, and chromosomal instability peaking before cell death. By comparing the cytotoxicity of CRISPR/Cas9- and radiation-induced DSBs, we demonstrated that the number of DSBs required to achieve equitoxic effects was approximately 3 times higher for radiation than CRISPR/Cas9. Finally, we showed that PC cells that had survived CRISPR/Cas9 targeting retained susceptibility to subsequent CRISPR/Cas9-induced DSBs at different genomic sites with more than 87% growth inhibition. Together, our data support the therapeutic potential of CRISPR/Cas9 as an anticancer strategy.},
}
@article {pmid42138212,
year = {2026},
author = {Xu, B and Tian, S and Yang, N and Cai, J and Yi, Q and Wang, Y and Ou, X and Jin, Y and Bai, S and Xue, J and Wang, J},
title = {Re-Engineering CRISPR-Cas12a into a Multimodal Biosensing Platform with Programmable Precursor crRNA.},
journal = {Analytical chemistry},
volume = {98},
number = {21},
pages = {15709-15719},
doi = {10.1021/acs.analchem.6c01251},
pmid = {42138212},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *RNA/analysis/genetics ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; },
abstract = {The CRISPR-Cas12a system has emerged as a powerful tool for molecular diagnostics due to its trans-cleavage activity. However, its utility in point-of-care testing is constrained by several inherent limitations: strict dependence on DNA for activation, compromised specificity against single-stranded DNA targets, and a basal catalytic rate often insufficient for direct detection. Here, we engineered a class of programmable precursor CRISPR RNAs (pcRNAs), which re-engineer Cas12a into a multimodal biosensing platform. Our platform enables Cas12a to respond to diverse programmable inputs, including direct RNA detection without reverse transcription, and features a built-in autocatalytic circuit for signal amplification. It demonstrates programmable high specificity, discriminating single-nucleotide variants in DNA with selectivity up to 908.7, and achieves high sensitivity by directly detecting synthetic circHER2 RNA, with a detection limit of 0.5 aM. The robust performance of the platform is validated through the quantitative assessment of circHER2 levels in breast cancer cell lines within complex cellular lysates. By employing a modular nucleic acid design strategy, this work breaks the intrinsic functional constraints of Cas12a and establishes a generalizable framework for the development of next-generation intelligent and programmable molecular diagnostic and sensing systems.},
}
@article {pmid42138264,
year = {2026},
author = {Gao, M and Jiang, T and Si, W and Rong, Y and Hu, Y and He, X and Liu, BF and Chen, P and Yang, Y and Deng, Y and Wang, FB},
title = {Harnessing CRISPR-Cas12 and Microfluidics Chips for Multiplex Respiratory Pathogens Diagnosis.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03226},
pmid = {42138264},
issn = {2379-3694},
abstract = {Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.},
}
@article {pmid42138716,
year = {2026},
author = {Lefrançois, G and Lavallée, E and Rowell, MC and Bourdeau, V and Mohebali, F and Bertomeu, T and Duman, AM and Nikolova, M and Tyers, M and Gravel, SP and Schmitzer, AR and Ferbeyre, G},
title = {The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {42138716},
issn = {2050-084X},
support = {TFRI Project #1123//Terry Fox Research Institute/ ; Operating Grant 2016//Cancer Research Society/ ; 935858//Cancer Research Society/ ; 840633//Cancer Research Society/ ; RGPIN-2021-03128//Natural Sciences and Engineering Research Council of Canada/ ; Ganotec/Marc-Andr&#xe9; Pigeon Fund//Cancer Research Society/ ; 1054571//Cancer Research Society/ ; },
mesh = {*Metformin/pharmacology/metabolism ; Humans ; Cell Line, Tumor ; *Mitochondrial Proton-Translocating ATPases/metabolism/genetics ; *Cell Proliferation/drug effects ; Oxidative Phosphorylation/drug effects ; *Antineoplastic Agents/pharmacology ; Mitochondria/drug effects/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Hypoglycemic Agents/pharmacology ; },
abstract = {Here, we identify the subunit e of F1F0-ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F1F0-ATP synthase dimers, which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro, and disabling its expression by CRISPR-Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide-treated cells, including mitochondrial morphology alterations, reduction of the NAD[+]/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers, and a compensatory increase in glycolysis. Notably, metformin disrupts F1F0-ATP synthase oligomerization, leading to the accumulation of vestigial assembly intermediates in pancreatic and osteosarcoma cancer cells, a phenotype also observed upon ATP5I inactivation in pancreatic cancer cells. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and antiproliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F1F0-ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.},
}
@article {pmid42139236,
year = {2026},
author = {Quiroz-Huanca, A and Vargas-Reyes, M and López, JD and Flores-Jimenez, K and Saldarriaga-Morán, S and Cifuentes, K and Alcántara, R},
title = {Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0337675},
pmid = {42139236},
issn = {1932-6203},
mesh = {*beta-Lactamases/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/drug effects/isolation &amp; purification/enzymology ; *Polymerase Chain Reaction/methods ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The β-lactams are critically important broad-spectrum antibiotics, widely used as first-line treatments; however, their effectiveness is increasingly compromised by β-lactamase enzymes. Among these, OXA-type enzymes have expanded to over 400 variants and are highly prevalent in Enterobacteriaceae. Current phenotypic and molecular detection tests have long turnaround times or require specialized equipment, respectively. In this study, we optimize a rapid molecular assay combining a PCR with modified thermal ramp rate (TRR) along with CRISPR-Cas12a fluorescence detection for blaOXA-1-harboring E. coli isolates. Using a commercial DNA Taq polymerase (TRR: 2.2 °C/s, annealing and extension hold time: 1 s), amplification time was reduced from 80 to 30 min, enabling detection within 50 min (PCR: 30 min; CRISPR: 20 min). With a locally produced enzyme (hold: 10 s), amplification time was 44 min. To demonstrate the practical application of the assay, we evaluated spiked poultry fecal samples achieving an analytical sensitivity of 8 CFU/reaction using commercial DNA Taq polymerase. The accelerated PCR:CRISPR workflow delivers results in less than one hour without compromising technical sensitivity (attomoles range), not requiring high technical expertise, and can be implemented in laboratories with basic molecular biology equipment.},
}
@article {pmid42139565,
year = {2026},
author = {Liu, Y and Wang, C and Tian, Y and Li, J and Wu, Z and Wang, P and Xiong, W},
title = {CRISPR/Cas9 Recombineering with Bacteriophage Recombinases Enables Efficient and Scarless Genome Editing in Cupriavidus necator H16.},
journal = {ACS synthetic biology},
volume = {15},
number = {6},
pages = {2390-2402},
doi = {10.1021/acssynbio.6c00055},
pmid = {42139565},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Cupriavidus necator/genetics/metabolism ; *Gene Editing/methods ; *Bacteriophages/enzymology/genetics ; *Recombinases/genetics/metabolism ; Plasmids/genetics ; Gene Knockout Techniques ; },
abstract = {Cupriavidus necator H16 is a promising chassis for one-carbon (C1) biomanufacturing due to its ability to assimilate CO2 with H2-derived reducing power, yet genome engineering in this bacterium remains constrained by inefficient DNA delivery and low homologous recombination efficiency. Here, we establish a modular two-plasmid CRISPR/Cas9 recombineering system that decouples Cas9 expression from sgRNA turnover, incorporates a self-splicing intron riboswitch to tightly control Cas9 counterselection, and introduces a pair of bacteriophage recombinases, Che9c60 and Che9c61, to markedly enhance homologous recombination efficiency. With Che9c60 and Che9c61, the gene knockout efficiency increased from undetectable levels to 58.3% and reached 100% at certain loci. Importantly, introducing these two recombinases enabled scarless chromosomal knock-in by boosting insertion efficiency from undetectable levels to nearly 40%, and supported targeted integration of DNA payloads up to 4.0 kb. To facilitate rapid multiround engineering, we integrated a tdk-based counterselection marker into the sgRNA plasmid, reducing plasmid-retaining cells to 1.1% within 24 h under 5-fluoro-2'-deoxyuridine (FUDR) selection. Overall, this streamlined toolkit enables efficient, iterative, and scarless genome editing in C. necator, accelerating C. necator strain development for C1 metabolic engineering and industrial applications.},
}
@article {pmid42141309,
year = {2026},
author = {Qin, L and Liu, D and Wang, Z and Sun, X and Zhao, X},
title = {Molecular biology of Pleurotus mushrooms: genomic resources, genetic manipulation, and regulation mechanisms.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {6},
pages = {},
pmid = {42141309},
issn = {1572-9699},
support = {32402651//National Natural Science Foundation of China/ ; 2024AFB274//Hubei&#x3000;Provincial&#x3000;Natural&#x3000;Science&#x3000;Foundation&#x3000;of&#x3000;China/ ; Q20231511//Youth Talent Project of Scientific Research Plan of Hubei Provincial Education Department of China/ ; K2023058//Scientific Research Fund of Wuhan Institute of Technology/ ; },
mesh = {*Pleurotus/genetics ; *Genomics/methods ; *Genome, Fungal ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; RNA Interference ; },
abstract = {As one of the most widely cultivated edible mushrooms in the world, Pleurotus mushrooms are popular among people for their delicious taste and rich nutritional value. Because of their great economic value, the research on the molecular biology of Pleurotus spp. has been deepening in recent years. The study first summarized the current situation of genomic resources available for this genus. The whole genome sequencing of 14 species, including Pleurotus tuoliensis and Pleurotus ostreatus, provides reference data for mining functional genes. Although the genomic data for Pleurotus mushrooms are continuously increasing, actual instances of successful genetic transformation remain restricted. Research on the regulatory mechanisms of key genes at different developmental stages and under various environmental stresses is insufficient. Then, the application of gene editing methods (CRISPR/Cas9, RNAi, and gene overexpression) in Pleurotus mushrooms was systematically described. RNAi and gene overexpression technologies have become well-established and are routinely used in most Pleurotus mushrooms. However, the application of CRISPR/Cas9 technology is still limited to P. eryngii and P. ostreatus. This limitation is attributed to the difficulties in establishing genetic transformation systems and the low efficiency of homologous recombination. Furthermore, this review explored the value of multi-omics technologies in elucidating the molecular mechanisms of morphogenesis and stress responses. To address the lack of specific antibodies for non-model organisms, we evaluated the application potential of DAP-seq technology in Pleurotus mushrooms and discussed its limitations, including the risk of false positives arising from the absence of a genuine environment in vivo. The purpose of this review is to evaluate the current molecular biology research on Pleurotus spp., and to provide systematic technical support and insights for functional genomics research and the analysis of molecular mechanisms of complex traits in Pleurotus mushrooms.},
}
@article {pmid42142405,
year = {2026},
author = {Zhang, X and Zhang, Z and Chen, K and Zhang, J and Wu, Z and Tang, B and Cheng, Y},
title = {CRISPR/Cas12a-powered tri-mode aptasensor for ultrasensitive and multiplexed detection of microcystin-LR.},
journal = {Biosensors &amp; bioelectronics},
volume = {308},
number = {},
pages = {118786},
doi = {10.1016/j.bios.2026.118786},
pmid = {42142405},
issn = {1873-4235},
mesh = {*Microcystins/analysis/isolation &amp; purification/chemistry ; Marine Toxins ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation &amp; purification ; Metal Nanoparticles/chemistry ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Exploiting CRISPR/Cas12a-powered multiple-mode sensing platforms is urgently needed to meet flexible and multi-scenario analytical requirements while improving detection accuracy for target pollutants. Herein, we developed a novel CRISPR/Cas12a-powered tri-mode aptasensor for ultrasensitive and multiplexed detection of microcystin-LR (MC-LR) in environmental water. First, target-specific recognition and the subsequent release of activator DNA were achieved through an aptamer-based competitive displacement reaction. Then, the trans-cleavage activity of Cas12a was activated in the presence of MC-LR, and a fluorescence-quenched ssDNA (FAM-ssDNA-BHQ1) and a dual -functionalized probe (MBs-ssDNA-AuNPs-4MBA) as signal reporters were employed for the tri-signal readouts. In the fluorescence mode, an ultrasensitive digital droplet fluorescence system was employed. The released activator DNA and Cas12a/crRNA complex were co-encapsulated into monodisperse nanoliter droplets, which not only accelerated the reaction but also amplified local fluorescence signals. This approach enabled rapid detection of MC-LR within 35 min, with a detection limit as low as 1.0 aM. For SERS and colorimetric modes, a novel dual-functionalized reporter was designed to provide both Raman and colorimetric readouts following magnetic separation. This extended the dynamic detection range from 1.0 × 10[-13] M to 1.0 × 10[-6] M. The proposed tri-mode aptasensor, using a single workflow, addresses both trace-level warning and high-concentration detection of MC-LR. It demonstrated good analytical performance in real samples, with recoveries ranging from 81.26% to 113.21% (n = 3). This CRISPR/Cas12a-driven strategy thus provides a versatile tool for reliable MC-LR monitoring across diverse water scenarios.},
}
@article {pmid42142541,
year = {2026},
author = {Jiang, H and Yang, S and Huang, Q and Miao, T and Ren, J and Yu, K and Yang, J and Yang, C and Liang, Y},
title = {CRISPR/Cas9-Mediated triple gene editing of Pi21, Bsr-d1, and Xa5 confers broad-spectrum disease resistance in elite early-season rice without yield compromise.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {370},
number = {},
pages = {113217},
doi = {10.1016/j.plantsci.2026.113217},
pmid = {42142541},
issn = {1873-2259},
mesh = {*Oryza/genetics/microbiology/immunology/growth &amp; development ; *Disease Resistance/genetics ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology/immunology/genetics ; *Plant Proteins/genetics/metabolism ; Xanthomonas/physiology ; *Gene Editing/methods ; Magnaporthe/physiology ; Plants, Genetically Modified ; Protein Serine-Threonine Kinases ; },
abstract = {Rice blast and bacterial blight are devastating diseases that severely threaten global rice production. To develop durable resistance without compromising yield, we utilized CRISPR/Cas9 gene editing to simultaneously knock out three negative immune regulators, Pi21, Bsr-d1, and Xa5, in two elite early-rice cultivars (ZZU53 and ZZU100). We successfully obtained transgene-free homozygous triple mutants that exhibited significantly enhanced resistance to both Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). This broad-spectrum resistance correlates with the constitutive upregulation of key defense genes in the salicylic acid (SA) pathway (OsPR1a, OsPR1b, and OsWRKY45) and jasmonic acid (JA) pathways (OsPR4). Comprehensive agronomic evaluation demonstrated that critical agricultural traits such as thousand-grain weight, panicle architecture, heading date, and tiller number remained comparable to wild-type plants, indicating no substantial yield penalty. This approach provides an efficient, transgene-free pathway for developing disease-resistant early-rice cultivars. Multiple environment field trials are now required to evaluate its long-term agricultural viability.},
}
@article {pmid42142549,
year = {2026},
author = {Ren, X and Zhou, Z and Kong, L and Guo, Y and Liu, Y and Li, J and Gu, X and Jiang, C and Wu, J},
title = {Re-balancing immunity with CRISPR-Cas9: Novel strategies for cancer and autoimmune disorders.},
journal = {Biotechnology advances},
volume = {90},
number = {},
pages = {108921},
doi = {10.1016/j.biotechadv.2026.108921},
pmid = {42142549},
issn = {1873-1899},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/immunology/genetics ; *Autoimmune Diseases/genetics/therapy/immunology ; *Gene Editing ; Animals ; Immunotherapy ; },
abstract = {Dysregulated immune activity is a cornerstone of numerous diseases, where excessive suppression enables cancer progression and uncontrolled activation drives autoimmunity. Immunotherapy aims to restore this balance, however, conventional approaches are often limited by suboptimal precision, efficacy, or long-term safety. The emergence of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) genome-editing system has opened transformative avenues for overcoming these hurdles. This review traces the development of the CRISPR-Cas9 toolkit, encompassing gene knock out (CRISPR-KO), knock in (CRISPR-KI), transcriptional activation (CRISPRa), interference (CRISPRi), base editing and prime editing-and examines their application in reprogramming immune responses. We focus on how these six strategies enhance anti-tumor immunity by engineering immune cells to bypass inhibitory checkpoints and the tumor microenvironment, and suppress autoimmunity by restoring immune tolerance through precise genomic and transcriptional interventions. Finally, we discuss translational challenges and future directions, including the adoption of next-generation editors and smart delivery systems, which are poised to maximize the therapeutic potential of CRISPR-based cellular interventions.},
}
@article {pmid42142632,
year = {2026},
author = {Yang, Y and Zhang, T and Pan, Y and Wang, D and Lü, P},
title = {Development of a CRISPR-Cas13a assay for mouse hepatitis virus detection.},
journal = {Journal of virological methods},
volume = {344},
number = {},
pages = {115407},
doi = {10.1016/j.jviromet.2026.115407},
pmid = {42142632},
issn = {1879-0984},
mesh = {Animals ; *Murine hepatitis virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Mice ; *Hepatitis, Viral, Animal/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {Mouse hepatitis virus (MHV) is a significant pathogen that undermines the health of laboratory animals and the reliability of research data. The development of rapid and sensitive detection methods for MHV is therefore crucial for ensuring laboratory animal quality and maintaining laboratory biosafety. Here, we established a CRISPR-Cas13a-based assay targeting the E, M, and N genes of MHV by constructing a recombinase-aided amplification (RAA)-coupled detection system. Results demonstrated that the N1 target exhibited the highest sensitivity, achieving a detection limit of 0.1 copy/μL for plasmid templates and 1 copy/μL in spiked serum samples. The system also exhibited robust specificity and a strong signal response in spiked serum samples. Furthermore, we developed a multiplex amplification-free CRISPR-Cas13a assay by combining Cas13a/crRNAs targeting different genes, which achieved a detection limit of 3.06 pM. In summary, this study presents two distinct CRISPR-Cas13a-based strategies for MHV detection, demonstrating the considerable application potential of this technology for pathogen detection in laboratory animal settings.},
}
@article {pmid42143446,
year = {2026},
author = {Martin, L and Rostami, S and Schuster, I and Qin, PZ and Rajan, R},
title = {Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.},
journal = {Biochemical and biophysical research communications},
volume = {823},
number = {},
pages = {153760},
pmid = {42143446},
issn = {1090-2104},
support = {R35 GM145341/GM/NIGMS NIH HHS/United States ; },
mesh = {*DNA Cleavage ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/chemistry/metabolism/genetics ; Gene Editing ; *DNA/metabolism/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Francisella/genetics/enzymology ; },
abstract = {CRISPR-Cas systems comprise a CRISPR RNA (crRNA)-guided CRISPR-associated (Cas) nuclease for providing immune protection. The complementary base pairing between crRNA and the invader genome leads to the formation of an "R-loop", which triggers the nuclease activity of the Cas protein, effectively neutralizing the invasion. This molecular mechanism has been repurposed for genome applications using Cas9 and Cas12a. Cas12a has several favorable features for applications including its smaller size, crRNA processing ability, and creation of staggered double-stranded DNA (dsDNA) cleavage. Gene editing with these Cas proteins, however, has some setbacks due to off-target and non-specific DNA cleavages. To increase the specificity in DNA cleavage, we introduced proline/alanine substitutions at different positions along a conserved arginine/lysine-rich "bridge helix" (BH) of Cas12a that plays an integral role in mediating conformational changes needed for DNA cleavage. Cleavage kinetics analyses reveal that enhanced helical integrity of the BH of Francisella novicida Cas12a provided by alanine substitutions increases DNA cleavage efficiency, while reducing the ability of the variants to discriminate DNA mismatches. Proline substitutions demonstrate an opposite effect by reducing the efficiency of cleaving on-target DNA, but almost completely abolishing linearization of a target with a mismatch in the middle of the R-loop. These results parallel those reported for Cas9 and show that balancing the helicity of BH through rational amino acid substitutions can finetune Cas12a's off-target profiles. This may provide a strategy for enhancing specificity of Cas12a in genome manipulation.},
}
@article {pmid42143634,
year = {2026},
author = {Mahendrarajan, V and Sankaranarayanan, GN and Muthukaliannan, GK and Easwaran, N},
title = {Triphala Modulates the Membrane Vesicle Transcriptome of Enterococcus durans VIT3 to Influence Antibiotic Response and Probiotic Functions.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42143634},
issn = {1559-0305},
support = {SG20210254//VIT University/ ; },
abstract = {Membrane vesicles (MVs) are critical mediators of bacterial communication, physiology, and host interactions. This study explores how triphala, a polyherbal formulation, modulates the RNA cargo of MVs in Enterococcus durans VIT3 isolate under varied stress conditions. The isolated MVs from all treatment conditions (triphala, antibiotics, or sequential combinations) were spherical, intact, with negative zeta potential and sizes ranging from 56.4 to 462.3 nm. Cytotoxicity assays indicated no toxicity toward CaCo2 cells under the tested conditions. Transcriptomic profiling suggested that triphala pretreatment enhanced relative expression of genes linked to oxidative stress defense (ohrB, sodA), envelope integrity (dlt operon, pbp, mprF), and CRISPR-Cas-associated genes (cas1). In contrast, antibiotic first regimens suppressed stress and defense-associated genes, underscoring treatment order effects. Notably, triphala antibiotic sequence condition showed coordinated expression patterns within ATP synthase, arginine deiminase, and CRISPR loci, suggesting operon-level transcriptional organization of metabolic and stress-response pathways. STRING network analysis further supported clustering of genes showing relatively higher expression into functionally related molecules. Collectively, these findings suggest a potential role for triphala in shaping MV RNA cargo, offering a conceptual framework for synbiotic strategies aimed at supporting microbial stress resilience. All transcriptomic observations are based on pooled MV samples, which represent exploratory expression trends rather than statistically inferred differential expression or functional outcomes.},
}
@article {pmid42143943,
year = {2026},
author = {Huang, Y and Lau, CH and Cai, W and Chen, X and Li, J and Xia, Q and Huang, T and Xiao, B and Zhu, H},
title = {Rapid, portable, one-pot CRISPR/Dx system for multiplex detection of human rhinovirus, adenovirus, and Mycoplasma pneumoniae.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {2},
pages = {117453},
doi = {10.1016/j.diagmicrobio.2026.117453},
pmid = {42143943},
issn = {1879-0070},
mesh = {Humans ; *Adenovirus Infections, Human/diagnosis/virology ; Adenoviruses, Human/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Mycoplasma pneumoniae/genetics/isolation &amp; purification ; *Picornaviridae Infections/diagnosis/virology ; *Pneumonia, Mycoplasma/diagnosis/virology ; *Rapid Diagnostic Tests ; Rhinovirus/isolation &amp; purification/genetics ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Human rhinovirus (HRV), human adenovirus (HAdV), and Mycoplasma pneumoniae (Mp) are the primary pathogens responsible for childhood pneumonia cases. These pathogens often co-infect the respiratory system, and their infections exhibit highly similar symptoms, making a differential diagnosis challenging. The inability to identify the specific causative pathogen often results in the administration of empirical antibiotics. Empirical antibiotic treatment may not be effective and can cause adverse patient outcomes and drive antibiotic resistance.<br><br>METHOD: We developed a rapid and one-pot RPA-CRISPR/Cas12a system (CRISPR/Dx) for multiplex detection of HRV, HAdV, and Mp. It is also equipped with our customized miniature device to enable portable diagnostics and visible signal readout.<br><br>RESULTS: Our CRISPR/Dx system is able to detect 5 copies/&#x3bc;L of these respiratory pathogens, with a detection limit of 0.57 copies/&#x3bc;L for HRV, 0.18 copies/&#x3bc;L for HAdV, and 2.6 copies/&#x3bc;L for Mp. It completes the reaction and detection within 35 minutes, excluding sample preparation time or pretreatment steps. It has high detection specificity and no cross-reactivity between these respiratory pathogens. This CRISPR/Dx system could be integrated with a portable fluorescent detector to realize point-of-care testing of these pathogens.<br><br>CONCLUSION: Our CRISPR/Dx system allows multiplex detection, intuitive results readout, and obviates the necessity for large instruments, thereby making it well-suited for field-deployable and point-of-care diagnostics of infectious diseases.},
}
@article {pmid42144412,
year = {2026},
author = {Kasapoglu, MZ and Acar, I and Gumustop, I and Erol, I and Kurt, IC and Ortakci, F},
title = {Pangenomics of Limosilactobacillus fermentum reveals genomic diversity and bacteriocin activity against Staphylococcus aureus and Escherichia coli.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-51219-1},
pmid = {42144412},
issn = {2045-2322},
support = {MGA-2024-45355//Bilimsel Ara&#x15f;t&#x131;rma Projeleri Birimi, &#x130;stanbul Teknik &#xdc;niversitesi/ ; },
abstract = {Limosilactobacillus fermentum is a versatile lactic acid bacterium with significant probiotic and biotechnological potential, yet the genomic determinants underlying its ecological adaptation and therapeutic applications remain underexplored. This study performed a comparative genomic analysis of 52 L. fermentum strains, with a focus on the novel strain ATT-06 isolated from traditional Turkish shalgam. We uncovered substantial genetic diversity, evidenced by an open pan-genome (18,647 genes) and a small core genome (718 genes). CRISPR-Cas systems were prevalent (46 strains), with Type-IE and Type-IIA being most common and mutually exclusive from Type-IC. Strain ATT-06, which uniquely produced gamma-aminobutyric acid at 17.74 µg/mL, harbored a Type-IIA CRISPR system and a single prophage. In silico molecular docking and dynamics simulations revealed that the bacteriocin Lactococcin, encoded by strain ATA-LTC-Lf170503, exhibited strong binding affinities (ΔG: -8.1 to -13.0 kcal/mol) against Rho proteins of Staphylococcus aureus and Escherichia coli, outperforming Acidocin A. These findings highlight the genomic plasticity and adaptive mechanisms of L. fermentum, and position strain ATT-06 as a promising probiotic candidate with potential neuroactive and antimicrobial applications.},
}
@article {pmid42144912,
year = {2026},
author = {Guo, X and Tian, T and Zhou, X},
title = {Split crRNAs Enhance Cas12a Diagnostic Performance.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70383},
doi = {10.1002/cbic.70383},
pmid = {42144912},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {CRISPR RNA (crRNA) is the key guide molecule in the CRISPR-Cas12a system, directing the Cas protein to recognize target sequences. It consists of a repeat-derived stem loop that binds Cas12a and helps stabilize the ribonucleoprotein complex, and a spacer region that base-pairs with the target and determines recognition specificity. Recently, multiple studies have shown that crRNAs can be split and reassembled in vitro in diverse ways. These split-and-reconfigured strategies have enabled detection schemes that are more flexible than full-length crRNAs, cover a broader range of targets, and achieve higher signal-to-background ratios. Here, we focus on split crRNA strategies for CRISPR-Cas12a and systematically summarize existing split crRNA-based detection platforms. We outline their design principles, reaction mechanisms, and performance features, and we synthesize how these approaches improve key metrics-including target scope, sensitivity, specificity, and controllability. Finally, we discuss the major advantages and current limitations of split crRNA strategies and highlight directions for further design optimization and translational applications. Schematic overview of split crRNA strategies for enhancing CRISPR-Dx performance. These advances are mainly reflected in four aspects: broadening the target range, thereby enabling Cas12a to be applied to the detection of short RNAs, structured RNAs, and certain non-nucleic acid targets; improving sensitivity by enhancing detection signals through reassembly-dependent activation, cascade amplification, or auxiliary activation strategies; increasing specificity by strengthening the discrimination of single-nucleotide differences through stepwise recognition and conditional assembly; and enhancing controllability by achieving on-demand activation of Cas12a activity via light, enzymes, small molecules, or proximity effects.},
}
@article {pmid42145154,
year = {2026},
author = {Dong, R and Zhang, Y and Yuan, G and Yue, X and Ding, Y and Zeng, X and Xiao, H},
title = {A Novel Strategy to Produce CAR-γδ T Cells via Site-Directed Gene Integration by a Combination of CRISPR/Cas9 and AAV.},
journal = {Cancer medicine},
volume = {15},
number = {5},
pages = {e71918},
pmid = {42145154},
issn = {2045-7634},
support = {82170141//National Natural Science Foundation of China/ ; 82470209//National Natural Science Foundation of China/ ; },
mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; Genetic Vectors/genetics ; Cell Line, Tumor ; },
abstract = {Chimeric antigen receptor (CAR)-αβ T cells are commonly employed in tumor therapy but hindered by some limitations. γδ T cells are promising substrates for CAR therapy for their major histocompatibility complex (MHC)-unrestricted recognition manner and innate immune function. Here, we established a novel method for generating CAR-γδ T cells. We utilized the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) method to interrupt the TCR delta chain constant region (TRDC) sequence, followed by the site-directed insertion of the CAR sequence into the TRDC locus via homologous complementation mediated by adeno-associated virus (AAV) gene delivery. We optimized electroporation parameters for Cas9/ribonucleoproteins (RNP) delivery and infection conditions for CAR-gene carrying AAV in γδ T cells. These optimizations facilitated efficient TCR knockout and site-directed CAR insertion, ultimately yielding functional CAR-γδ T cells. In vitro experiments demonstrated that these newly prepared CAR-γδ T cells could stimulate cytokine production, kill tumor cells as well as exhibit robust proliferative potential and memory-like phenotype. These state-of-the-art CAR-γδ T cells could reduce tumor burden and extend the survival period of tumor-bearing mice.},
}
@article {pmid42145193,
year = {2026},
author = {Yin, XY and Wang, SZ},
title = {High-throughput identification methods of genomic functional variation in post-GWAS era and their application in agricultural animals.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {5},
pages = {451-470},
doi = {10.16288/j.yczz.25-319},
pmid = {42145193},
issn = {0253-9772},
mesh = {Animals ; *Genome-Wide Association Study/methods ; *Genetic Variation ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-wide association study (GWAS) has identified a large number of genetic variations that are significantly associated with human diseases and animal and plant economic traits. However, the majority of these variants are located in non-coding regions of the genome, which makes it challenging to accurately pinpoint functional variants of biological significance from a vast number of candidate loci. In the post-GWAS era, high-throughput analytical approaches, such as high-throughput reporter gene analysis, CRISPR/Cas9-based gene editing technologies, and epigenetic analyses, have become powerful tools for systematically uncovering functional variants in the genome. These methods not only enable efficient identification of functional variants but also help elucidate the mechanisms through which they regulate gene expression, thereby clarifying the molecular basis underlying trait formation or disease pathogenesis. In this review, we systematically summarize current high-throughput strategies for identifying functional genomic variants, highlight their applications and recent advances in major agricultural animal species, and outline future research directions, with the aim of providing a reference for subsequent studies in related fields.},
}
@article {pmid42146559,
year = {2026},
author = {Butnaru, M and McKenna, W and Goswami, S and Wu-Chuang, A and Mameli, E and Wilcox, A and Quennesson, L and Kim, AR and Veal, A and Chen, W and Verzone, H and Lane, EA and Laukaitis-Yousey, HJ and Araneo, C and Singh, N and Pedra, JHF and Hu, Y and Viswanatha, R and Perrimon, N and Mohr, SE},
title = {Genome-wide CRISPR knockout cell screening platform for the disease vector tick species Ixodes scapularis.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42146559},
issn = {2692-8205},
abstract = {The black legged tick, Ixodes scapularis, is a vector of the bacterium that causes Lyme disease and several other illnesses, including anaplasmosis, babesiosis, and tick-borne encephalitis. Although high-quality genome annotations are available for I. scapularis, functional understanding of I. scapularis genes is limited. To address this, we developed a platform for genome-wide CRISPR-Cas9 knockout screening in I. scapularis cells. To evaluate the platform, we performed a screen to identify genes associated with cellular fitness, and screens for resistance to treatment with copper chloride, Antimycin A, or Destruxin A (DA), a cyclic hexadepsipeptide produced by the pathogenic fungus Metarhizium anisopliae. In each case, the screens implicate specific sets of conserved and non-conserved I. scapularis genes in relevant cellular functions, providing the first experimental evidence of function for a large set of I. scapularis genes. Altogether, in this first-of-its-kind effort for the arthropod subclass Acari, we present an unbiased genome-wide CRISPR-Cas9 knockout cell screening platform, related resources, and datasets that will be broadly useful to efficiently uncover cellular functions of I. scapularis genes.},
}
@article {pmid42148306,
year = {2026},
author = {Jezierski, A and Huang, J and Desbiens, L and Benabdallah, B and McComb, S and Beausejour, C},
title = {Induced pluripotent stem cells as platforms for engineering NK cell immunotherapies.},
journal = {Frontiers in cell and developmental biology},
volume = {14},
number = {},
pages = {1810206},
pmid = {42148306},
issn = {2296-634X},
abstract = {Human induced pluripotent stem cells (iPSCs) are transforming adoptive cell therapy by combining unlimited self-renewal, broad differentiation potential, and high amenability to genome engineering. These attributes make iPSCs a versatile source for the development of standardized immune effector cells at industrial scale, enabling a shift from patient- or donor-restricted cell products toward true off-the-shelf immunotherapies that can be improved through iterative genome engineering. iPSC-derived natural killer (iNK) cells are the most clinically advanced and exemplify the platform's advantages over conventional autologous or donor-sourced approaches. Unlike autologous therapies, which require labor-intensive and expensive personalized clinical-grade manufacturing, and are constrained by variable quality and genetic intractability of donor products, iPSC technology supports the creation of renewable, clonally defined master cell banks as uniform starting material for NK-cell therapy products. Advances in CRISPR/Cas-based editing now permit multiplex introduction of functional traits, enhanced cytokine signaling, antibody-dependent cytotoxicity, checkpoint resistance, optimized trafficking, safety switches, and increasing signal complexity, directly at the pluripotent or progenitor stages; ultimately allowing for fully-programmable iNK cells with customizable potency and persistence. Early clinical studies of iNK products validate the feasibility, safety, and therapeutic potential of this approach, but also underscore the need for continued refinement of differentiation protocols, manufacturing pipelines, and regulatory standards to ensure efficacy, genomic stability, phenotypic maturity, and long-term safety. This review outlines current breakthroughs and future directions of iNK cell therapies, emphasizing how programmable iPSC chassis platforms are enabling modular and off-the-shelf targeted immunotherapies.},
}
@article {pmid42148710,
year = {2026},
author = {Du, T and He, J and Wang, Y and Xie, X and Chen, J and Xu, Z and Zhou, C and Sun, W},
title = {An Evolutionarily Conserved Function of Grainyhead in Orchestrating Insect Wing Development.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {10},
pages = {e71927},
doi = {10.1096/fj.202600640R},
pmid = {42148710},
issn = {1530-6860},
support = {CSTB2025NSCQ-GPX0734//CSTC | Natural Science Foundation of Chongqing Municipality/ ; 32070499//MOST | National Natural Science Foundation of China (NSFC)/ ; 2025CDJ-IAISYB-022//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; },
mesh = {Animals ; *Wings, Animal/growth &amp; development/metabolism ; *Insect Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics/growth &amp; development/metabolism ; *Bombyx/genetics/growth &amp; development/metabolism ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; },
abstract = {The development of insect appendages is governed by deeply conserved genetic programs, even as developmental strategies diverge widely across taxa. In this study, we identify the conserved transcription factor Grainyhead (Grh) as a crucial regulator of insect wing development. In the silkworm Bombyx mori, CRISPR/Cas9-mediated knockout of BmGrh did not compromise larval viability but resulted in severe wing defects in adults, including crumpled and non-expandable wings. Similarly, tissue-specific knockdown of DmGrh in Drosophila melanogaster wing imaginal discs led to pupal lethality, a sex-biased eclosion rate, and adults exhibiting crumpled wings with disrupted wing hair polarity. Comparative transcriptomics revealed that loss of DmGrh function predominantly downregulates genes associated with cuticle structure and extracellular matrix organization in both species. In Drosophila, chromatin immunoprecipitation further confirmed that DmGrh directly binds to regulatory regions of these downregulated cuticle-related genes. Through functional screening, we identified the cuticle protein gene cpr65Ea as a key downstream effector, whose knockdown recapitulated the wing morphogenesis and eclosion defects observed in DmGrh RNA interference individuals. Moreover, extending our investigation to an agricultural pest, RNAi-mediated silencing of SfGrh in the white-backed planthopper (Sogatella furcifera) impaired wing expansion and completely abolished flight ability. Our findings establish Grh as an evolutionarily conserved regulator of insect wing development and highlight its potential as a novel genetic target for pest management by disrupting flight capacity and dispersal.},
}
@article {pmid42149441,
year = {2026},
author = {Klijnhout, JA and Senders, EAW and van den Bogaard, EH and Smits, JPH},
title = {Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding mRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3031},
number = {},
pages = {87-101},
pmid = {42149441},
issn = {1940-6029},
mesh = {Humans ; *Keratinocytes/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Messenger/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Cells, Cultured ; },
abstract = {CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.},
}
@article {pmid42149727,
year = {2026},
author = {Jong, RM and Ching, KL and Garelis, NE and Zilinskas, A and Wrynla, XH and Rawal, S and Hill, BC and Luckie, BA and Shallow, L and Cox, JS and Barton, GM and Stanley, SA},
title = {Cas9[+] conditionally immortalized neutrophil progenitors as a tool for genome-wide CRISPR screening for neutrophil differentiation and function.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42149727},
issn = {2050-084X},
support = {U19AI135990-01//NIH Office of the Director/ ; DGE-1752814//National Science Foundation Graduate Research Fellowship Program/ ; T32 GM 7232-40//NIH Office of the Director/ ; },
mesh = {*Neutrophils/physiology/cytology ; Animals ; *Cell Differentiation ; Mice ; *CRISPR-Cas Systems ; Homeodomain Proteins/genetics/metabolism ; Cell Line ; Gene Editing ; },
abstract = {Neutrophils are short-lived cells of the innate immune system that play numerous roles in defense against infection, regulation of immune responses, tissue damage and repair, autoimmunity, and other non-communicable diseases. Understanding neutrophil function at a mechanistic level has been hampered by the difficulty of working with primary neutrophils, which die rapidly upon isolation, and the relative paucity of neutrophil cell lines. Here, we report the creation of a Cas9 +ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils. By editing progenitors via transduction with sgRNAs and then withdrawing estrogen, Cas9-edited neutrophils are produced with high efficiency. Importantly, neutrophil differentiation of edited progenitors occurs both in vitro in cell culture and when transferred into murine recipients. To demonstrate the utility of Cas9 +ER-Hoxb8 progenitors for forward genetics, we performed a pooled CRISPR screen to identify factors required for survival during neutrophil differentiation. This screen identified hundreds of genes, including Cebpe, a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using this progenitor cell line, we confirmed that Cebpe is required for neutrophil differentiation in vivo, validating the utility of this line for studying in vivo phenotypes. The screen also identified all components of the WASH complex as being required for neutrophil differentiation, extending its known role in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, this resource enables the analysis of the role of neutrophils in numerous disease states using genetics for the first time.},
}
@article {pmid42149978,
year = {2026},
author = {Verzier, LH and Hesping, E and Doerflinger, M and Herold, MJ and Boddey, JA},
title = {Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions.},
journal = {PLoS genetics},
volume = {22},
number = {5},
pages = {e1012137},
pmid = {42149978},
issn = {1553-7404},
mesh = {*Plasmodium falciparum/genetics/pathogenicity ; Humans ; *Hepatocytes/parasitology/metabolism ; *Sporozoites/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Host-Parasite Interactions/genetics ; *Malaria, Falciparum/parasitology/genetics ; Cell Line ; Animals ; Erythrocytes/parasitology ; Liver/parasitology ; },
abstract = {Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are injected into the skin by infected mosquitoes and must reach the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that eventually enter the bloodstream and invade erythrocytes, causing malaria. The sporozoite's journey requires cell traversal, where sporozoites transiently enter and exit host cells, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish an exoerythrocytic form. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host factors. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity of HC-04.2B3 and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining. Optimized traversal and invasion assays led to a robust cytometric readout suitable for screening human genes involved in P. falciparum infection. Disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens confirmed utility of this platform. This study provides the basis for genome-wide CRISPR screens to uncover hepatocyte biology and host determinants of infection.},
}
@article {pmid42150000,
year = {2026},
author = {Hu, Q and Lin, J and Cui, H and Zhao, X and Wu, M and Wei, S and Deng, M and Wang, B and Liu, T and Gao, X and Huang, Q and Liang, Y and Liu, S and Yang, H and Lin, HB and Li, G},
title = {Cas13-Mediated RNA Base Editing for the Treatment of Hereditary Hypertrophic Cardiomyopathy.},
journal = {Circulation},
volume = {153},
number = {20},
pages = {1598-1602},
doi = {10.1161/CIRCULATIONAHA.125.076905},
pmid = {42150000},
issn = {1524-4539},
}
@article {pmid42152766,
year = {2026},
author = {Zhu, Y and Xu, W and Li, Y and Feng, W and Han, L and Zhong, Y and Li, C and Wan, Y and Hao, L and Gao, S and Huang, L},
title = {Dual Mechanisms of crRNA 3'-Extension-Mediated Cas12a Attenuation Enable Programmable One-Pot CRISPR Diagnostics.},
journal = {Analytical chemistry},
volume = {98},
number = {21},
pages = {15830-15842},
doi = {10.1021/acs.analchem.6c01964},
pmid = {42152766},
issn = {1520-6882},
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Precise control of Cas12a trans-cleavage activity is essential for developing robust one-pot CRISPR diagnostics, yet existing strategies remain largely empirical. In this study, we show that minimal 3'-RNA extensions of 7-13 nucleotides reliably attenuate Cas12a activity through a dual mechanism: competitive 3'extension cleavage and steric blockade of the RuvC catalytic pocket. Leveraging this mechanistic insight, we developed SMART-POT (Simple Modulation via Attenuating RNA Tail in One-POT detection), an integrated one-pot RPA-Cas12a platform that bypasses noncanonical PAM crRNA screening. Using a canonical PAM HPV18-targeting crRNA with a 7-nt extension, the assay achieved 10[-17] M sensitivity, perfect specificity against 13 other high-risk HPV genotypes, and 100% clinical concordance (20/20 samples) with qPCR. This work establishes a rational design rule for Cas12a regulation and provides a generalizable framework for field-ready one-pot CRISPR diagnostics.},
}
@article {pmid42153625,
year = {2026},
author = {Djouad, ME and Bendaha, MEA and Belaouni, HA and Bensalah, F},
title = {Comparative genomics of Lacticaseibacillus paracasei strain 69Bis reveals encoded probiotic traits and genomic specificities.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
doi = {10.1093/femsle/fnag060},
pmid = {42153625},
issn = {1574-6968},
support = {//Directorate General for Scientific Research and Technological Development/ ; //Ministry of Higher Education and Scientific Research/ ; },
mesh = {*Probiotics ; *Genome, Bacterial ; Animals ; Genomics ; *Lacticaseibacillus paracasei/genetics/isolation &amp; purification/classification/physiology ; Whole Genome Sequencing ; Goats ; Bacterial Adhesion ; Milk/microbiology ; Multigene Family ; Phylogeny ; },
abstract = {This study provides a comprehensive characterization of Lacticaseibacillus paracasei strain 69Bis, isolated from traditional fermented goat milk collected in Biskra (southeast Algeria). Phenotypic investigations revealed robust probiotic traits across all isolates, including NaCl tolerance at 10%, acid resistance at pH 2.0 for 3 h, bile tolerance at 0.3%, and survival in simulated gastrointestinal digestion. Adhesion properties included high auto-aggregation and hydrophobicity. The strain exhibited potent antimicrobial activity, particularly against Pseudomonas aeruginosa. Whole-genome sequencing yielded a genome of 2.9 Mbp, with 46.38% GC and 2969 protein-coding sequences, enriched in carbohydrate and amino acid metabolism. Functional annotation highlighted genomic features associated with probiotic functions, including adhesion factors (acm, fimA, lapA), stress response pathways, and exopolysaccharide synthesis (eps gene family). Three biosynthetic gene clusters were detected, encoding bacteriocins and terpene synthesis. The absence of known acquired antimicrobial resistance genes and the susceptibility to all tested antibiotics indicate a favourable safety profile. The presence of CRISPR-Cas systems was confirmed, which may contribute to genomic stability and protection against phages. Comparative genomics, through genome-based taxonomy, confirmed the assignment to L. paracasei. Pan-genome analysis of 122 genomes from the same species (including 69Bis) revealed an open pan-genome (6878 genes, 25.76% core), along with several unique and rare determinants linked to probiotic traits in 69Bis. Overall, these results position strain 69Bis as a promising candidate for use in functional foods, particularly in the context of nutrition in arid regions.},
}
@article {pmid42153634,
year = {2026},
author = {Shang, W and Lyu, Z and Chen, G},
title = {Harnessing Nature's Algorithm: From Test Tubes to Autonomous In Vivo Evolution.},
journal = {Biotechnology journal},
volume = {21},
number = {5},
pages = {e70237},
doi = {10.1002/biot.70237},
pmid = {42153634},
issn = {1860-7314},
support = {2024JJ4053//Hunan Provincial Natural Science Foundation/ ; 2025PT5021//Scientific Research Program of FuRong Laboratory/ ; },
mesh = {*Directed Molecular Evolution/methods ; *Algorithms ; Machine Learning ; Mutagenesis ; CRISPR-Cas Systems ; },
abstract = {Directed evolution (DE) enables the engineering of biomolecules without prior structural knowledge. However, traditional step-wise DE is constrained by limited screening throughput. To more efficiently navigate epistatic fitness landscapes, the field is increasingly adopting autonomous, continuous in vivo evolution systems. This review critically examines the molecular architectures and engineering principles driving this transition. We evaluate strategies for continuous genetic diversification-ranging from orthogonal replication systems (e.g., OrthoRep, T7-ORACLE) to CRISPR-guided mutagenesis (e.g., EvolvR)-with a focus on the fundamental trade-off between mutational load and host viability. Furthermore, we analyze the biophysical constraints of screening and the kinetic demands of coupling real-time selection with ultra-fast mutagenesis, as exemplified by phage-assisted continuous evolution (PACE). Crucially, we explore the functional integration of machine learning (ML), highlighting how active learning models and zero-shot predictions via protein language models (PLMs) can resolve epistatic complexities and mitigate the latency of next-generation sequencing. Finally, we discuss the multidimensional hardware and algorithmic bottlenecks currently impeding the realization of fully closed-loop biofoundries, and assess the strategic implications of these technologies for accelerating the engineering of complex therapeutics.},
}
@article {pmid42153784,
year = {2026},
author = {Estay-Ahumada, CE and Roux, M and Ciocca, D and El-Kholti, N and Birling, MC and Rossolillo, P and Felder-Schmittbuhl, MP and Hicks, D},
title = {Abca4 Knockdown in the Cone-Rich Rodent Psammomys Obesus Leads to Stargardt's Disease-Like Progressive Retinal Degeneration.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {67},
number = {5},
pages = {47},
pmid = {42153784},
issn = {1552-5783},
mesh = {Animals ; Gerbillinae ; *ATP-Binding Cassette Transporters/genetics ; Electroretinography ; Stargardt Disease/genetics ; Disease Models, Animal ; *Retinal Cone Photoreceptor Cells/metabolism/pathology ; Tomography, Optical Coherence ; *Macular Degeneration/genetics/congenital/metabolism ; *Retinal Degeneration/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Genetic Vectors ; Dependovirus/genetics ; },
abstract = {PURPOSE: Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts disease type 1 (STGD1), the most common form of inherited macular degeneration. We recently showed that injection of viral vectors expressing CRISPR/Cas9 tools directed against Abca4 into young Sand Rat (Psammomys obesus) eyes led to extensive structural and functional retinal degeneration resembling STGD1. Here we provide further evidence that this is highly likely due to specific knockdown of Abca4 and not off-target errors.<br><br>METHODS: We performed subretinal injections of Adeno-Associated Virus-CRISPR/Cas9-Abca4 constructs into postnatal (&#x223c;P15) Psammomys obesus. Eyes were examined by noninvasive exploration (ocular coherence tomography, fundus and electroretinography) at 15-60 days after injection. Additionally, subgroups were euthanized over the same time period, and ocular tissue was used for immunochemical analyses.<br><br>RESULTS: RNAscope analysis of injected eyes showed knockdown of Abca4, rhodopsin and cone transducin mRNA in transduced regions; neighbouring tissue that was not transduced showed robust expression of all three. Injection of control AAV, expressing CAS9 alone, induced only mild glial activation. Statistically significant decreases in visual responses to light flashes were only seen in eyes injected with the fully active CRISPR/Cas9-Abca4 probes.<br><br>CONCLUSIONS: Taken together, these data rule out off-target effects as responsible for the observed degeneration, and indicate that Psammomys obesus faithfully recapitulates many of the features seen in human STGD1, thus positioning it as an important research opportunity to further explore genotype-phenotype relationships and test putative therapeutic approaches.},
}
@article {pmid42154426,
year = {2026},
author = {Mayuri, K and Saravanan, KM and Somala, CS and Selvaraj, C and Anand, T and Vickram, S},
title = {CRISPR-Cas and Infectious Diseases: A Decade of Translational Advances in Molecular Biotechnology.},
journal = {Biochemical genetics},
volume = {},
number = {},
pages = {},
pmid = {42154426},
issn = {1573-4927},
abstract = {CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.},
}
@article {pmid42155174,
year = {2026},
author = {Choi, W and RuizdelRio, J and Embrione, V and Agarwal, D and Blecke, K and Gaffey, AC and Wahlin, KJ and Eliceiri, BP},
title = {Peptide display on small extracellular vesicles directs tissue-specific tropism and delivery of gene editing machinery.},
journal = {Biomaterials},
volume = {334},
number = {},
pages = {124312},
doi = {10.1016/j.biomaterials.2026.124312},
pmid = {42155174},
issn = {1878-5905},
support = {R35 GM149245/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Extracellular Vesicles/metabolism ; Mice ; *Peptides/chemistry/metabolism ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Peptide Library ; Organ Specificity ; *Gene Editing/methods ; *Gene Transfer Techniques ; },
abstract = {Small extracellular vesicles (EVs) deliver nucleic acid and protein therapeutics that promote tissue repair, however, there are few approaches to direct the tropism of EVs to specific tissues. Here, we address this challenge by engineering EVs to display a peptide library on the surface of EVs that is linked to barcoded guide RNA payloads (gRNAs). We show how these EVs can be administered systemically into mouse models and the cellular uptake of these gRNA-bearing EVs assessed by recovery of the small RNAs followed by PCR amplification and barcode sequencing. Since the design of the peptide library was linked to specific barcodes encoded on the same plasmid, subsequent sequencing of barcode sequences recovered from tissues revealed profiles of EV uptake associated with the display of specific peptide sequences on the surface of EVs. Therefore, candidate peptide motifs were cloned for validation using in vivo and in vitro readouts. In addition to the barcode-based tissue profiling, gRNA-laded EVs mediated functional gene editing in the Cre-loxP R26 LSL-tdTomato reporter mouse model in vivo. These studies demonstrated how EV display linked with barcoded gRNA payloads can address barriers to EV-based delivery of gene editing therapies.},
}
@article {pmid42155939,
year = {2026},
author = {Shi, Y and Gao, P and Wu, D and Wu, Y and Li, G},
title = {Enzyme-mediated "one to more" PAM-independent and amplification free CRISPR/Cas12a for one-pot rapid detection of food contaminated bacteria.},
journal = {Biosensors &amp; bioelectronics},
volume = {309},
number = {},
pages = {118815},
doi = {10.1016/j.bios.2026.118815},
pmid = {42155939},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Alicyclobacillus/isolation &amp; purification/genetics ; *Food Contamination/analysis ; *Biosensing Techniques/methods ; Food Microbiology ; DNA, Bacterial/genetics ; *Cronobacter/isolation &amp; purification/genetics ; Endodeoxyribonucleases/chemistry ; CRISPR-Associated Proteins/chemistry/genetics ; Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; },
abstract = {The requirement of protospacer-adjacent motif (PAM) and nucleic acid amplification have restricted the application of CRISPR/Cas12a system in bacteria detection. Herein, we proposed a restriction endonuclease mediated, "one to more", PAM-independent and amplification-free CRISPR/Cas12a strategy for bacteria detection. The bacterial genomic DNA can be cleaved by specific endonuclease, exposing a large number of sticky ends. The combination of these exposed sticky ends with a short ssDNA could effectively activate the trans-cleavage activity of Cas12a to break a FQ reporter probe, achieving the purpose of detection. This approach not only overcomes the dependence of PAM sequence but also enables one-pot detection of target bacteria within 60 min without the requirement of nucleic acid amplification. The detection sensitivity of two typical food contaminated bacteria, Alicyclobacillus acidoterrestris and Cronobacter sakazakii was 13.38 CFU/mL and 18.67 CFU/mL, respectively. In summary, this work provided a novel strategy and powerful tool for bacteria analysis and molecular diagnosis.},
}
@article {pmid42156407,
year = {2026},
author = {Sa, Y and Liu, C and Yang, L and Yue, L and Zhu, L and Guo, Y and Wang, R and Wang, Y and Feng, Y and Wang, Y and Zhang, Y and Wang, W and Xie, Y},
title = {Structural basis for dual mechanism of Cas2/3 nuclease inhibition by anti-CRISPR protein AcrIF19.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-73156-3},
pmid = {42156407},
issn = {2041-1723},
support = {32301058//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32371329, 32301055&#xff0c;32400565&#xff0c;32560225//National Natural Science Foundation of China (National Science Foundation of China)/ ; ZR202211190090//Natural Science Foundation of Shandong Province (Shandong Provincial Natural Science Foundation)/ ; tsqn202306146//Taishan Scholar Project of Shandong Province/ ; },
abstract = {CRISPR-Cas systems are prokaryotic immune mechanisms often targeted by phage-encoded anti-CRISPR (Acr) proteins. This study characterizes AcrIF19, a potent inhibitor of the type I-F system in Pectobacterium atrosepticum. The cryo-EM structure of the apo Cas2/3 and Cas2/3-AcrIF19 complex reveals a dual inhibitory mechanism. AcrIF19 employs a negatively charged β2-β3 loop to sterically occlude the non-target DNA strand entry channel, acting as a competitive inhibitor to disrupt Cas2/3 recruitment. Concurrently, this steric occlusion impedes ssDNA-mediated allosteric activation, which locks the critical helix-like loop motif in an inhibitory conformation and thereby abrogates DNA cleavage activity. AcrIF19 represents an anti-CRISPR protein inhibiting Cas2/3 via two different mechanisms, integrating a competitive ssDNA inhibitor with an allosteric blockade to suppress both target recruitment and DNA cleavage.},
}
@article {pmid42157911,
year = {2025},
author = {Waqar, Z and Sethi, P and Jain, D and Singh, K and Alsaidan, OA and Alzarea, SI and Gupta, JK and Saxena, S and Sharma, MC},
title = {Precision Medicine in Neurodegenerative Diseases: Genomic Approaches to Target Amyloid-β, Tau, and Alpha-Synuclein Pathways.},
journal = {Current genomics},
volume = {26},
number = {6},
pages = {469-494},
pmid = {42157911},
issn = {1389-2029},
abstract = {Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by the pathological aggregation of proteins such as amyloid-β, tau, and alpha-synuclein. These hallmark proteins play central roles in disease progression and represent promising targets for therapeutic intervention. Advances in precision medicine, driven by genomic technologies such as CRISPR-Cas systems, RNA-based therapies, and high-throughput sequencing, have enabled the development of tailored strategies to modulate these pathological pathways. This review examines the integration of genomic approaches in targeting amyloid-β, tau, and alpha-synuclein, emphasizing their potential to mitigate disease progression and improve patient outcomes. We highlight current progress in preclinical and clinical studies, discuss challenges associated with translating these therapies into clinical practice, and explore future directions for achieving therapeutic precision in neurodegenerative disorders. By examining the interplay of genetic, molecular, and therapeutic innovations, this review underscores the transformative potential of genomic medicine in addressing the unmet needs of neurodegenerative disease treatment.},
}
@article {pmid42158919,
year = {2026},
author = {Zhou, C and Arimura, SI},
title = {TALENs and Related Technologies for Editing Nuclear and Organellar Genomes in a Model Plant, Arabidopsis thaliana.},
journal = {Bio-protocol},
volume = {16},
number = {9},
pages = {e5668},
pmid = {42158919},
issn = {2331-8325},
abstract = {Plant genome editing is a powerful approach for modifying plant DNA to investigate gene function and to engineer desirable traits. Several genome-editing technologies have been developed, among which CRISPR/Cas systems and transcription activator-like effector nucleases (TALENs) are widely used to introduce targeted double-stranded DNA breaks. While CRISPR/Cas systems are highly efficient for nuclear genome editing, their application to plant organellar genomes remains limited, largely due to difficulties in guide RNA delivery into mitochondria and chloroplasts. Here, we present a detailed and reproducible protocol for constructing TALEN-based binary vectors for targeted genome editing in Arabidopsis thaliana. This protocol describes the assembly of TALE repeat arrays, the generation of nuclear-, mitochondrial-, and plastid-targeted TALEN expression vectors using MultiSite Gateway cloning, and subsequent Agrobacterium-mediated plant transformation and genotyping. The workflow enables the production of nTALENs, mitoTALENs, and ptpTALENs using a unified vector design strategy. In addition, the protocol briefly outlines the construction principles of TALE-based cytidine deaminases (TALECDs) for targeted C-to-T base editing in plant organellar genomes. The protocol provides a flexible and robust framework for plant nuclear and organellar genome editing and can be readily adapted to different target genes and experimental purposes. Its modular design and compatibility with standard molecular cloning techniques make it accessible to laboratories aiming to perform precise genome manipulation in plants. Key features • Requires experience in basic molecular cloning and Arabidopsis transformation; suitable for laboratories performing plant nuclear and organellar genome editing. • Enables construction of nuclear-, mitochondrial-, and plastid-targeted TALENs using a unified MultiSite Gateway-based vector system. • Provides a modular workflow for assembling large TALEN binary vectors compatible with Agrobacterium-mediated transformation in Arabidopsis thaliana. • Includes optional extension to TALE-based cytidine deaminases for targeted C-to-T base editing in plant mitochondrial and plastid genomes.},
}
@article {pmid42159153,
year = {2026},
author = {Gonçalves, LDS and Domingues, WB and Nunes, LS and Blödorn, EB and Dellagostin, EN and Martins, AWDS and Acosta, IB and Corcini, CD and Varela, AS and Fróes, CN and Campos, VF},
title = {First Report of CRISPR-Cas9 Ribonucleoprotein Delivery Into Teleost Spermatozoa With Preserved Membrane and Genomic Integrity.},
journal = {Molecular reproduction and development},
volume = {93},
number = {5},
pages = {e70114},
doi = {10.1002/mrd.70114},
pmid = {42159153},
issn = {1098-2795},
support = {23/2551-0000181-0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado do Rio Grande do Sul/ ; 22/2551-0001645-6//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado do Rio Grande do Sul/ ; 24/2551-0002158-2//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado do Rio Grande do Sul/ ; 440636/2022-1//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 407610/2024-3//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; AUXPE #2537/2018//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; },
mesh = {Animals ; Male ; *Spermatozoa/metabolism/cytology ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Cichlids/genetics ; *Cell Membrane/metabolism ; Electroporation/methods ; Sperm Motility ; DNA Fragmentation ; },
abstract = {Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR-Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%-13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR-RNP delivery in teleost reproductive biology.},
}
@article {pmid42159719,
year = {2026},
author = {Wu, B and Luo, H and Xie, H and Zhao, C and Xie, P and Yang, X and Shi, J and Huang, W and Fang, Z},
title = {Cis-regulatory editing of SD1 promoter enhances TCP19-mediated repression to optimize plant height in Kam sweet rice.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {6},
pages = {},
pmid = {42159719},
issn = {1432-2242},
mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing ; *Promoter Regions, Genetic ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Transcription Factors/genetics/metabolism ; Gibberellins/metabolism ; Phenotype ; Plants, Genetically Modified ; Plant Breeding ; },
abstract = {This study demonstrates that CRISPR-mediated cis-regulatory element editing (CRE editing) of the SD1 promoter effectively reduces plant height in Kam sweet rice, without compromising yield or grain quality, offering a precise strategy for crop improvement. Kam sweet rice, a unique aromatic variety, faces challenges with excessive plant height and suboptimal yield. This study explores a precision breeding approach by strengthening an endogenous TCP19-SD1 repression module through CRISPR-Cas9-mediated CRE editing to modulate the expression of the SD1 gene, a key regulator of gibberellin biosynthesis and stem elongation. By introducing an adenine insertion in the GGCCCCCC cis-regulatory element in the SD1 promoter, we enhanced the binding affinity of the transcription factor TCP19, resulting in down-regulated SD1 expression. This led to a reduction in gibberellin levels, shortening internodes, and reducing plant height. Phenotypic evaluations revealed that the edited lines exhibited significantly shorter plant height while maintaining grain yield and nitrogen utilization efficiency compared to wild-type plants. Microscopic analysis of the internodes confirmed that the reduced plant height correlated with decreased cell length. Transcriptomic studies indicated that CRE editing modulated a network of genes involved in both gibberellin and auxin signaling pathways, critical for plant growth. Importantly, the genetic modification did not adversely affect grain quality. This study demonstrates the potential of strengthening endogenous transcriptional repression via CRE editing as a precise alternative to conventional gene knockout techniques, offering a powerful strategy for optimizing complex agronomic traits in rice, with applications in modern crop breeding strategies.},
}
@article {pmid42161120,
year = {2026},
author = {Song, J and Kang, M and Cha, B and Lee, JC and Kim, S and Lim, EK and Jung, J and Lee, SW and Nam, HC and Castro, CM and Lee, H and Kang, T},
title = {Self-amplifying CRISPR-based one-pot ultrasensitive testing for rapid SARS-CoV-2 and its variant detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {309},
number = {},
pages = {118814},
doi = {10.1016/j.bios.2026.118814},
pmid = {42161120},
issn = {1873-4235},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *COVID-19/diagnosis/virology ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; RNA, Viral/genetics/analysis ; Rapid Diagnostic Tests ; Limit of Detection ; Clustered Regularly Interspaced Short Palindromic Repeats ; Spike Glycoprotein, Coronavirus/genetics ; *COVID-19 Nucleic Acid Testing/instrumentation ; Nucleic Acid Amplification Techniques/methods ; Smartphone ; },
abstract = {Rapid, accessible molecular tests that can resolve viral variants remain a critical unmet need. We report a one-tube clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) assay that couples target recognition to a T7-promoter-driven self-amplifying loop, thereby achieving exponential fluorescence amplification at a single temperature (37 °C) within 40 min. Without separate pre-amplification, the assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open-reading frame 1a (ORF1a), nucleocapsid (N), spike (S), and envelope (E) RNAs with limits of detection (LoDs) of 0.32-0.96 copies μL[-1], corresponding to attomolar level sensitivity. A compact 16-well reader and a smartphone application enable real-time quantification and field-deployable operation. The system discriminates S mutations (D614G, H69-70del, D80A, L452R, P26S, A67V, and A27S) and maintains specificity in mixed-variant samples. In a clinical study (n = 105; 75 positives and 30 negatives), assay calls are concordant with routine reverse transcription quantitative polymerase chain reaction (RT-qPCR). These results establish a minimal-handling, extraction-free workflow that quantitatively detects SARS-CoV-2 and resolves key mutations, suggesting a generalizable architecture for point-of-care (POC) nucleic-acid testing.},
}
@article {pmid42161262,
year = {2026},
author = {Li, Q and Yin, Y and Liu, Y and Ding, X and Nie, X and Zhao, Z and Zhang, R and Ma, H and Zhu, W and Xiang, S and Ouyang, H and Yang, F and Yang, Z and Li, Y and Gu, J and Yang, M and Wang, D and Zhu, B and Si, Y and Chen, H and Diamond, MS and Cao, S and Ye, J},
title = {A CRISPR activation screen identifies SPART as a pan-orthoflavivirus restriction factor.},
journal = {Cell host &amp; microbe},
volume = {34},
number = {6},
pages = {1082-1099.e12},
doi = {10.1016/j.chom.2026.04.018},
pmid = {42161262},
issn = {1934-6069},
mesh = {Animals ; Ubiquitin-Protein Ligases/metabolism/genetics ; Mice ; Virus Replication ; Mice, Knockout ; Humans ; Female ; Zika Virus/physiology ; Zika Virus Infection/virology ; CRISPR-Cas Systems ; Ubiquitination ; Capsid Proteins/metabolism ; Viral Load ; Host-Pathogen Interactions ; *Flavivirus ; },
abstract = {Orthoflaviviruses, including Zika (ZIKV), dengue, Japanese encephalitis, and West Nile viruses, cause diverse clinical syndromes and threaten human health. Identifying factors that inhibit orthoflavivirus infection could lead to antiviral countermeasures. Here, we conducted a genome-wide CRISPR activation screen and identified the host gene SPART (Spartin/SPG20) as a restriction factor against ZIKV and other orthoflaviviruses. SPART interacts with and disrupts the endosomal localization of Itchy E3-ubiquitin ligase (ITCH), which we determine ubiquitinates the ZIKV capsid, thereby triggering uncoating. Loss of SPART enhances ZIKV replication, an effect not observed in SPART-ITCH double knockout mutants. Maternal ZIKV infection of Spg20[-/-] mice results in heightened maternal and fetal viral loads and greater fetal abnormalities, whereas infection of Itch[-/-] mice yields opposite outcomes. Similar results were observed in these gene-edited mice upon infection with related orthoflaviviruses. Overall, this approach identified a broad orthoflavivirus restriction factor, providing a potential target against these emerging pathogenic viruses.},
}
@article {pmid42162502,
year = {2026},
author = {Makhijani, S and Alasiri, G and Quadri, MSA and Ingle, RG},
title = {From mechanism to medicine: CRISPR‒Cas9 delivery strategies, therapeutic applications and translation challenges.},
journal = {Discover nano},
volume = {21},
number = {1},
pages = {},
pmid = {42162502},
issn = {2731-9229},
abstract = {The advent of CRISPR‒Cas technology has revolutionized genome editing by providing unprecedented precision, efficiency, and versatility in genetic manipulation. This revolutionary system originated from bacterial adaptive immunity. It allows the programmed modification of DNA sequences in a highly specific way, as long as the desired modification site contains an enzyme-compatible protospacer adjacent motif (PAM) sequence. Recent innovations with CRISPR‒Cas systems have focused on optimizing delivery methods, addressing challenges and expanding their therapeutic applications. Delivery approaches, including viral vectors, lipid nanoparticles, and other nonviral vectors, have been improved to increase target specificity while minimizing off-target effects, ensuring safe and efficient In Vivo genome editing. The applications of CRISPR‒Cas technology include the correction of genetic disorders, engineering of immune cells for cancer therapy, and combating viral infections. However, significant challenges in the form of unintended edits, immune responses, and delivery to specific tissues or organs remain formidable barriers to clinical translation. New CRISPR variants, such as base editing, prime editing, and epigenome editing, provide solutions to improve accuracy and broaden intervention. This review covers recent progress in various CRISPR delivery methodologies, discusses broadening therapeutic applications, highlights ongoing challenges, and describes the future trajectory of this disruptive technology.},
}
@article {pmid42164567,
year = {2025},
author = {Li, X and Zhang, Z and Zhu, J and Zhang, Y and Guo, Y},
title = {Innovative Applications and Challenges of Isothermal Amplification Technology in miRNA Detection.},
journal = {Current genomics},
volume = {26},
number = {6},
pages = {581-598},
pmid = {42164567},
issn = {1389-2029},
abstract = {A class of endogenous non-coding RNAs with a length of roughly 18-25 nucleotides is known as microRNAs (miRNAs). They have been established as the best biomarkers for early cancer diagnosis, molecular subtyping, and prognostic evaluation. They also play important roles in important biological processes like cell proliferation, differentiation, apoptosis, and tumorigenesis through post-transcriptional regulatory networks. Traditional detection technologies (such as quantitative Polymerase Chain Reaction (qPCR), Northern blot, and microarrays) exhibit high sensitivity, yet they rely on expensive thermocycling equipment, involve cumbersome procedures, and are time-consuming, making it difficult to meet the demands of clinical point-of-care testing. In recent years, isothermal amplification technologies, represented by loop-mediated isothermal amplification (LAMP), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and Exponential Amplification Reaction (EXPAR), have emerged as a research frontier in the field of miRNA detection, by virtue of their advantages including elimination of thermal cycling requirements, fast reaction kinetics, high sensitivity, and simple operation. This study aims to systematically organize the core principles, latest advancements, and integrated innovation patterns of isothermal amplification-based miRNA detection technologies, and provide support for the development of miRNA detection platforms suitable for clinical point-of-care testing.},
}
@article {pmid42164670,
year = {2026},
author = {Islam, MS and Fan, J and Vabna, MA and Haque, N and Monir, SB and Li, Y and Nime, I and Acharjee, M and Pan, F},
title = {Phage enabled precision drug delivery: dual function platforms for therapeutics and genetic cargo transport.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1812871},
pmid = {42164670},
issn = {1664-302X},
abstract = {The rising concern of antimicrobial resistance, coupled with the continually challenging management of complicated diseases such as cancer, has provided momentum toward precision molecular medicine. This review provides an overview of bacteriophage enabled strategies encompassing both conventional antibacterial applications and advanced bioengineered delivery systems. Recent advances in phage therapy include the use of tailored phage formulations, phage immobilization approaches and phage antibiotic combinations to achieve targeted bacterial lysis particularly against multidrug-resistant pathogens and biofilm-associated infections. Beyond their intrinsic antibacterial activity, phages can be genetically and chemically engineered as nanoscale scaffolds. Phage display technologies enable the incorporation of targeting ligands for selective binding to specific tissues including tumor cells. Furthermore, phage capsids can be modified to encapsulate and deliver diverse therapeutic payloads such as small-molecule drugs, nucleic acids and gene-editing systems such as CRISPR-Cas, thereby expanding their utility beyond infectious diseases. The integration of phage biology with nanobiotechnology positions these viral platforms at the forefront of next generation therapeutics. Engineered phages have demonstrated potential as precision delivery vectors for cytotoxic agents, immunomodulators and genetic material with improved specificity and reduced off-target effects. Emerging strategies including phage antibiotic conjugates and enzyme functionalized phages further enhance therapeutic efficacy and facilitate penetration of physiological barriers. Collectively, phage-based platforms represent a versatile and transformative approach with significant implications for the treatment of infectious, oncologic and genetic disorders, supporting the advancement of targeted and personalized medicine.},
}
@article {pmid42164832,
year = {2026},
author = {Kumar, A and Krishan, B and Dhiman, S and Sharma, A and Thadiyan, V and Azmi, W},
title = {Beyond antibiotics: innovative and translational strategies to overcome antimicrobial resistance.},
journal = {3 Biotech},
volume = {16},
number = {6},
pages = {201},
pmid = {42164832},
issn = {2190-572X},
abstract = {The rapid rise of antimicrobial resistance demands therapeutic strategies that extend beyond conventional antibiotics. However, most existing reviews describe emerging alternatives without systematically linking their mechanistic advances to translational readiness and clinical implementation barriers. This review addresses this gap by integrating evidence across multiple beyond-antibiotic approaches, including antimicrobial peptides, bacteriophage therapy, CRISPR-based antimicrobials, nanotechnology-enabled delivery systems, anti-virulence agents, host-directed immunotherapies, microbiome modulation (engineered probiotics and fecal microbiota transplantation), and drug-repurposing or combination therapies. The principal contribution of this synthesis is a comparative framework that maps mechanisms of action, engineering innovations, and translational evidence across these diverse strategies. Advances such as peptidomimetics, engineered phages, and nanoparticle carriers that enhance stability, targeting, and therapeutic efficacy are highlighted, along with synergistic strategies including phage-antibiotic and CRISPR-nanocarrier combinations. The review further identifies major barriers limiting clinical translation, including delivery efficiency, toxicity and ecological concerns, large-scale production challenges, cost, inconsistent clinical outcomes, and regulatory fragmentation for biologics and live therapeutics. To facilitate clinical implementation, the study proposes a translational roadmap emphasizing standardized evaluation assays, physiologically relevant infection models, integrated rapid diagnostics, and regulatory frameworks tailored for emerging antimicrobial platforms, thereby supporting the development of sustainable therapies for the post-antibiotic era.},
}
@article {pmid42164989,
year = {2025},
author = {Taumar, D and Singh, AP and Sharma, H and Chaudhary, V},
title = {RNA Modifications as Drug Targets: Unlocking the Therapeutic Potential of the Epitranscriptome.},
journal = {Current genomics},
volume = {26},
number = {7},
pages = {802-818},
pmid = {42164989},
issn = {1389-2029},
abstract = {INTRODUCTION: The epitranscriptome covers reversible changes to RNA. These changes help control gene expression by making RNA more stable, easier to use, or more prone to degradation. They are increasingly implicated in disease development and offer a promising target for treatment. This review covers RNA alterations and their potential clinical applications.<br><br>METHODS: The systematic analysis of the peer-reviewed literature was performed, including experimental, clinical, and computational research. The biochemical properties and biological functions were used to classify RNA modifications. In addition, we evaluated current therapeutic options, including small molecules, CRISPR/Cas-based technologies, and RNA-targeted approaches.<br><br>RESULTS: RNA undergoes immense changes, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine. By these modifications, the genes are regulated by coordinated writers, erasers, and readers. Targeted therapeutics aim to regulate changes in cancer, neurodegenerative disease, and viral diseases. Several limitations remain, including overly general framing, suboptimal clarity in conveying the message, and inadequate response mechanisms.<br><br>DISCUSSION: The epitranscriptome provides an additional regulatory layer with significant therapeutic applications. New techniques appear to be successful. Researchers, in turn, should be more specific. These therapies require improved delivery vehicles and reduced side effects not associated with the treatment to be safe and effective for the patient.<br><br>CONCLUSION: Therapy targeting RNA-based modifications is a breakthrough in disease treatment. These chemical modifications affect gene function and expression patterns. The next step in the research requires overcoming current limitations to fully realize the therapeutic potential of epitranscriptomics-based interventions.},
}
@article {pmid42165133,
year = {2026},
author = {Liu, B and Klatt, D and Harris, C and McGuinness, M and Brendel, C and Williams, DA},
title = {Optimized lentivirus-derived virus-like particles for efficient delivery of Cas9-based genome editors.},
journal = {Nucleic acids research},
volume = {54},
number = {10},
pages = {},
pmid = {42165133},
issn = {1362-4962},
support = {INV-021791/GATES/Gates Foundation/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Proprotein Convertase 9/genetics ; Animals ; *Lentivirus/genetics ; HEK293 Cells ; Mice ; Genetic Vectors/genetics ; Ribonucleoproteins/genetics ; Virion/genetics ; },
abstract = {Implementation of therapeutic genome editing requires a potent, versatile, and transient delivery system to enable safe and effective in vivo applications. Here, we report on an optimized virus-like particle (VLP) platform for protein-based delivery of Cas9 ribonucleoproteins and Cas9-derived base editors and prime editors, termed LV-VLP-MA, that enables flexible editor deployment. By systematically engineering a panel of truncated Gag-Cas9 fusion variants, we identify a minimal MA-Cas9 configuration that maximizes editor packaging while effectively preserving efficient particle production and functional delivery. Systematic refinement of VLP production parameters enhances particle yield, supporting robust editing activity across diverse genomic targets. Importantly, systemic administration of LV-VLP-MA mediates efficient in vivo editing of the Pcsk9 locus with functional target suppression, establishing proof-of-concept for therapeutic application. Together, these results define a programmable, modular VLP-based platform that combines potency, flexibility, and transient delivery to expand the scope of in vivo genome engineering for therapeutic development.},
}
@article {pmid42165845,
year = {2026},
author = {Oxendine, J and Ibarra-Reyes, E and Ma, J and Li, C and Baron, S and Hwang, AE and Wang, R and Rodriguez-Leal, D},
title = {Optimization of Agrobacterium-mediated transformation of commercial heirloom tomato cultivars to develop novel traits via CRISPR/Cas9 genome editing.},
journal = {Planta},
volume = {264},
number = {1},
pages = {},
pmid = {42165845},
issn = {1432-2048},
support = {89365//California Tomato Research Institute/ ; MD-PSLA-243084//University of Maryland/ ; Startup funds//University of Maryland/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Solanum lycopersicum/genetics/growth &amp; development ; Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; Transformation, Genetic ; Plant Breeding/methods ; Genome, Plant ; },
abstract = {Trait development for commercial heirloom tomatoes can be advanced by optimization of tissue culture and transformation via Agrobacterium and CRISPR/Cas9 mutagenesis. Genetic improvement using new genome editing approaches relies on the efficient delivery of the CRISPR/Cas system in the vegetable crop tomato. Previous protocols for tomato transformation have primarily focused on a handful of cultivars (M82, Ailsa Craig, Microtom, Sweet-100) with very little commercial relevance, and it is not clear if these protocols can be implemented directly in other commercially relevant varieties. Heirloom tomatoes are sought for their deep and diverse flavor but have not been subjected to systematic crop improvement via conventional breeding or biotechnology approaches such as transgenesis or genome editing. Therefore, we tested the transformation and regeneration capacity of six different heirloom cultivars known for their superior taste and market relevance in the US. Subsequently, we optimized rooting conditions and used the GRF4-GIF1 chimeric developmental regulator to successfully recover transgenic plants. Finally, we evaluated the efficiency of targeted genetic modification using the CRISPR/Cas9 genome editing system in several of these cultivars. We demonstrate that our optimizations led to successful transformation of several heirloom varieties, including the generation of edited plants for target genes modifying plant architecture and flowering time. Our results set the foundation for a biotechnology platform to deliver improved traits to local and regional heirloom varieties using genome editing.},
}
@article {pmid42165978,
year = {2026},
author = {Zhu, B},
title = {Nanomaterial-nucleic acid probe synergy: accelerating rapid pathogen detection and antimicrobial susceptibility testing in bloodstream infections.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {42165978},
issn = {1874-9356},
abstract = {Bloodstream infections (BSIs) remain among the most lethal clinical syndromes, driven in large part by diagnostic delays that compel empiric, broad-spectrum antimicrobial therapy and expose patients to avoidable toxicity and resistance selection. Conventional blood culture-based workflows, although diagnostically definitive, are intrinsically slow, often requiring 24-72 h, and are therefore poorly matched to the time-critical demands of sepsis management, where each hour of delayed appropriate therapy measurably increases mortality. In this context, nano-enabled nucleic acid diagnostics represent a promising but largely preclinical strategy for improving analytical sensitivity and turnaround time. This critical translational review examines how engineered nanomaterials spanning plasmonic and magnetic nanoparticles, fluorescent quantum dots, upconversion nanoparticles, and two-dimensional materials synergize with programmable nucleic acid recognizers, including aptamers, CRISPR/Cas effectors, DNAzymes, and conformational probes, to enable rapid, ultrasensitive detection of pathogens and resistance determinants directly from whole blood. Rather than reviewing nanomaterials and nucleic acid probes as separate toolkits, this article focuses on how their co-design at the nano-bio interface enables clinically actionable whole-blood diagnostics. We elucidate how convergence engineering at the nano-bio interface governs signal amplification, background suppression, and assay robustness in complex biological matrices. Particular emphasis is placed on front-end enrichment strategies, optical and magnetic transduction mechanisms, and multiplexed readout architectures that together enable species-level identification and early antimicrobial susceptibility profiling within clinically relevant timeframes, typically ~ 1-6 h in research settings. Beyond analytical performance, we critically assess interconnected translational barriers including batch-to-batch reproducibility, standardization of bioconjugation protocols, antifouling strategies, and evolving regulatory frameworks, which collectively govern the trajectory from laboratory innovation to clinical adoption. At present, direct-from-blood phenotypic antimicrobial susceptibility testing remains technically challenging, and clinical adoption is limited by reproducibility, matrix tolerance, and workflow integration. By integrating mechanistic insight with clinical positioning, this review frames nano-probe diagnostics as promising candidates for next-generation BSI management that may support more timely and precise therapy once analytical robustness, standardization, and clinical validation are achieved.},
}
@article {pmid42166245,
year = {2026},
author = {Lim, J and Van, AB and Wester, M and Koprowski, K and Valera, E and Bashir, R},
title = {Amplification-free dual-blocking autocatalytic CRISPR-Cascade for attomolar DNA detection with low nonspecific signal.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {21},
pages = {e2537414123},
pmid = {42166245},
issn = {1091-6490},
support = {R01EB032725 A//HHS | NIH (NIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA, Bacterial/genetics ; Ribonucleoproteins/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Autocatalytic CRISPR architecture offers amplification-free nucleic acid detection by directly linking target recognition to self-reinforcing ribonucleoprotein (RNP) generation. However, spontaneous background activation remains a key barrier, because strand invasion or unwinding events can initiate unintended amplification and diminish assay specificity. Here, we introduce a dual-blocking CRISPR-Cascade design that independently cages both the guide RNA and trigger DNA, establishing an intrinsic AND gate to raise the effective kinetic barrier for unintended RNP formation. This strategy suppresses leakage by approximately 3- to 18-fold relative to single blocking configurations in full Cascade reactions, while preserving rapid detection (10 min), achieving single-copy sensitivity, and enabling quantitative detection. When paired with a competitive guide RNA decoy, the system further reduces background signals without affecting true target detection. Finally, we demonstrate robust Methicillin-resistant Staphylococcus aureus detection from whole blood in under 40 min including the sample purification and extraction. These results establish dual-blocking as a generalizable molecular gating framework for constructing leakage-resistant, amplification-free CRISPR systems suitable for rapid and decentralized diagnostics.},
}
@article {pmid42166810,
year = {2026},
author = {Huang, H and Yang, K and Wang, C and Bu, R and Chen, W},
title = {Heptameric transmembrane assembly of the CRISPR-Cas13b regulator Csx27.},
journal = {Biochemical and biophysical research communications},
volume = {824},
number = {},
pages = {153975},
doi = {10.1016/j.bbrc.2026.153975},
pmid = {42166810},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; Protein Multimerization ; *Cell Membrane/metabolism/chemistry ; Cryoelectron Microscopy ; *Bacterial Proteins/chemistry/metabolism ; Models, Molecular ; *Membrane Proteins/chemistry/metabolism ; },
abstract = {The type VI-B CRISPR-Cas13 system is an RNA-guided immune pathway in which the Cas13b effector is negatively regulated by the accessory protein Csx27. However, the structural and biochemical basis of Csx27 function remains poorly defined. Here, we characterize Bergeyella zoohelcum Csx27 as a membrane associated oligomeric protein. Bioinformatic prediction, detergent dependent purification, and membrane fractionation followed by western blotting support its membrane association. Native PAGE of Csx27 and BS3 cross-linking of Csx27 reconstituted in MSP2N2 nanodiscs further provide orthogonal evidence for oligomerization. Consistently, cryo-EM 2D class averages revealed ring-like particles with apparent sevenfold symmetry, and AlphaFold-assisted modeling supported a heptameric arrangement. Together, our results provide the first direct experimental evidence for the oligomeric, membrane-embedded assembly of Csx27, establishing a crucial structural foundation for elucidating its regulatory mechanism within the CRISPR-Cas13b immune response.},
}
@article {pmid42166840,
year = {2026},
author = {Simonneau, B and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {Generation of two iPSC lines carrying two cystic fibrosis rare intronic mutations c.1585-1G&gt;A and c.1680-886A&gt;G in the CFTR gene of the parental line PCIi033-A using CRISPR/Cas tools.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104016},
doi = {10.1016/j.scr.2026.104016},
pmid = {42166840},
issn = {1876-7753},
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Cystic Fibrosis/genetics/pathology ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Introns/genetics ; *Mutation/genetics ; Cell Line ; },
abstract = {CFTR gene mutations are responsible for Cystic Fibrosis. For half a decade, a triple therapy has been available for patients carrying the most frequent mutation: p.F508del. Among classified mutations, intronic mutations are rare, and no therapeutic strategies have yet been developed for such patients. We genome edited the parental iPSC line PCIi033-A to introduce two intronic mutations: A.s.Cas12a for c.1585-1G>A (PCIi033-A-8) and SpCas9 for c.1680-886A>G (PCIi033-A-9). Both cell lines have normal morphology and karyotype, conserved pluripotency, and differentiate into the three germ layers. Obtaining iPSC-derived mutant lung epithelia would be an efficient tool for testing new therapeutic strategies.},
}
@article {pmid42167149,
year = {2026},
author = {Buchholz, F},
title = {INSTALLing recombinase-driven genome writing: Engineered single-stranded donors bypass innate immunity to enable large DNA integration.},
journal = {Molecular cell},
volume = {86},
number = {10},
pages = {1836-1838},
doi = {10.1016/j.molcel.2026.04.018},
pmid = {42167149},
issn = {1097-4164},
mesh = {*Immunity, Innate/genetics ; *DNA, Single-Stranded/genetics/immunology ; Humans ; *Gene Editing/methods ; *Recombinases/genetics/metabolism ; CRISPR-Cas Systems ; *DNA, Circular/genetics/immunology ; },
abstract = {In a recent Nature article, Tou et al.[1] introduce INSTALL, a strategy that couples immune-evasive circular single-stranded DNA (cssDNA) technology with recombinase-based genome editing to overcome innate immune barriers and enable the integration of large DNA cargos.},
}
@article {pmid42167165,
year = {2026},
author = {Weerasinghe, PR and Tsugama, D},
title = {Diverse novel RNA polymerase III promoters and dual-activity promoters identified through motif and transcriptomic analyses across multiple plant species.},
journal = {Biochemical and biophysical research communications},
volume = {825},
number = {},
pages = {153936},
doi = {10.1016/j.bbrc.2026.153936},
pmid = {42167165},
issn = {1090-2104},
mesh = {*RNA Polymerase III/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; RNA, Untranslated ; CRISPR-Cas Systems ; *Plants/genetics ; *Promoter Regions, Genetic/genetics ; Transcriptome ; },
abstract = {RNA polymerase III (Pol III) promoters are essential tools for driving small RNAs such as guide RNAs and CRISPR RNAs (crRNAs) in CRISPR-Cas systems, yet their diversity and regulatory potential in major crops remain largely uncharacterized. In this study, we conducted a systematic, promoter motif-based scan for potential Pol III-transcribed genes across nine plant species including eight major crops. We identified 824 non-coding RNA (ncRNA) genes across 14 categories (consisting of 13 ncRNA families and 'Others') as Pol III-transcribed candidates; these included traditional Pol III-transcribed small nuclear RNA (snRNA) families and the families conventionally regarded as Pol II-transcribed. Small RNA sequencing revealed expression variability within families and across species, highlighting the importance of species-specific promoter selection. We also identified several rice ncRNA promoters with potential dual Pol II/Pol III activity and tested them for simultaneously expressing both Cas nucleases (AsCas12f-HKRA and Cas12j-8) and their crRNAs. While gene editing efficiencies in this single-promoter-driven architecture were low, the appearance of GFP signals in reporter assays confirms the functional feasibility of dual-polymerase-mediated transcription in plants. Our findings provide a roadmap for Pol III transcriptional diversity in crops and offer a simplified integrated promoter architecture for the development of compact genome-editing toolsets.},
}
@article {pmid42167468,
year = {2026},
author = {Yang, M and Gao, J and Han, Y and Ji, G and Liu, Z},
title = {In vivo evidence for the role of CRFB5 in zebrafish resistance to grass carp reovirus (GCRV) infection.},
journal = {Developmental and comparative immunology},
volume = {180},
number = {},
pages = {105631},
doi = {10.1016/j.dci.2026.105631},
pmid = {42167468},
issn = {1879-0089},
mesh = {Animals ; *Zebrafish/immunology/virology ; *Reoviridae Infections/immunology ; *Reoviridae/immunology/physiology ; Immunity, Innate/genetics ; *Fish Diseases/immunology/virology ; *Zebrafish Proteins/genetics/metabolism ; *Receptors, Interferon/genetics/metabolism ; *Carps/immunology/virology ; *Fish Proteins/genetics/metabolism ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Liver/immunology/metabolism ; Interferons/metabolism ; Animals, Genetically Modified ; },
abstract = {The interferon (IFN) system is central to vertebrate antiviral innate immunity, whose activation requires the binding of IFNs to specific cell membrane receptors. Teleost IFN receptors CRFB5 is a key component of this receptor complex. However, there is no solid in vivo genetic evidence to verify its physiological function in antiviral defense. Using a zebrafish model, we found that crfb5 expression was rapidly up-regulated in the liver at 4 h post-infection with grass carp reovirus (GCRV). We then constructed crfb5 homozygous knockout zebrafish (crfb5[-/-]) using CRISPR/Cas9 technology. Challenge with GCRV showed that crfb5[-/-] zebrafish had a significantly lower survival rate than wild-type (WT) zebrafish. Compared with WT, GCRV-infected crfb5[-/-] zebrafish showed suppressed mxa expression but elevated levels of ifnφ3 and il6. Collectively, our in vivo data demonstrate that CRFB5 plays a key role in zebrafish anti-GCRV immune response and the maintenance of inflammatory homeostasis by regulating the transcriptional balance of key immune factors.},
}
@article {pmid42167501,
year = {2026},
author = {Amezian, D and De Graeve, F and Mettumpurath Sasi, R and Vanhaecht, L and Mocchetti, A and Villacis Perez, E and Kant, MR and De Rouck, S and Van Leeuwen, T},
title = {A genome sequence and efficient CRISPR/Cas9 gene editing tools for the solanaceous specialist pest Tetranychus evansi.},
journal = {Insect biochemistry and molecular biology},
volume = {192},
number = {},
pages = {104588},
pmid = {42167501},
issn = {1879-0240},
mesh = {Animals ; *Tetranychidae/genetics ; *CRISPR-Cas Systems ; Pyrethrins/pharmacology ; Acaricides/pharmacology ; Female ; Genome ; },
abstract = {Tetranychus evansi is an invasive spider mite pest of solanaceous crops worldwide. Its rapid global spread and ability to develop acaricide resistance highlight the need for robust genomic resources and functional genetic tools for custom control strategies. Here, we present a genome assembly and establish efficient CRISPR/Cas9 editing in T. evansi to enable mechanistic studies of host adaptation and pesticide resistance. Using an inbred line and Oxford Nanopore Technologies long-read sequencing, we assembled an 89 Mb genome into 13 contigs (N50 = 18.6 Mb) and annotated 14,246 protein-coding genes. Manual curation of detoxification gene families (P450s, CCEs, GSTs, UGTs, ABC transporters and DOGs), revealed smaller repertoires than in the polyphagous relative Tetranychus urticae. To enable reverse genetics, we adapted SYNCAS for maternal delivery of CRISPR/Cas9 in T. evansi. Targeting the phytoene desaturase (PD) pigmentation marker produced reliable knockouts with visible lack of red pigmentation and editing efficiencies of ∼10-15%, allowing the efficient creation of stable mutant lines. We further applied precision gene editing to knock-in (KI) the M918T and M918L substitutions into the voltage-gated sodium channel (VGSC). While M918L was lethal in T. evansi, we generated multiple homozygous lines for M918T (KI efficiency ∼ 4.4%). Bioassays demonstrated that while the mutation caused extremely high levels of bifenthrin resistance (RR = 345-645), this was less so for β-cyfluthrin (RR = 113-127), deltamethrin (RR = 58.2-65.6) and tau-fluvalinate (RR = 87.7-98), revealing the specific role of M918T in pyrethroid resistance. Collectively, these resources establish T. evansi as a tractable system for reverse genetic analysis and provide a reference for future comparative and population genomics.},
}
@article {pmid42169298,
year = {2026},
author = {Zhao, G and Wei, J and Li, Y and Zhu, C and Tang, J and Gong, J and Huang, Y},
title = {Quantitative monitoring of Bacillus licheniformis during fermentation using PCR-CRISPR/Cas12a.},
journal = {Food research international (Ottawa, Ont.)},
volume = {237},
number = {},
pages = {119334},
doi = {10.1016/j.foodres.2026.119334},
pmid = {42169298},
issn = {1873-7145},
mesh = {*Fermentation ; *Bacillus licheniformis/genetics/isolation &amp; purification/metabolism ; *CRISPR-Cas Systems ; *Real-Time Polymerase Chain Reaction/methods ; *Food Microbiology/methods ; Limit of Detection ; DNA, Bacterial/genetics ; *Polymerase Chain Reaction/methods ; },
abstract = {Bacillus licheniformis is a key functional bacterium in Baijiu fermentation and plays an essential role in the formation of flavor compounds, making its quantitative detection crucial. This study developed a CRISPR/Cas12a-based microbial nucleic acid quantification (CRMNQ) method to quantify B. licheniformis in Baijiu fermentation. This method combines efficient PCR amplification with highly specific CRISPR/Cas12a recognition, enabling quantitative analysis through end-point fluorescence measurement without the need for a quantitative real-time PCR instrument. Under optimized conditions, a limit of detection (LOD) of 72.3 copies/μL was achieved with high specificity. The method was successfully applied to monitor the dynamics of B. licheniformis in both simulated and in situ samples of Baijiu fermentation, with higher robustness and anti-interference ability compared with the qPCR method. In summary, the method provides an effective and practical tool for highly sensitive and specific detection of B. licheniformis in Baijiu fermentation, facilitating process monitoring and quality control during fermentation.},
}
@article {pmid42169395,
year = {2026},
author = {van Roosmalen, RN and Murphy, CD and Sweeney, JB},
title = {A Stereospecific Lactic Acid Exclusion Biosensor for Grass Silage Fed Green Biorefinery.},
journal = {Microbial biotechnology},
volume = {19},
number = {5},
pages = {e70374},
pmid = {42169395},
issn = {1751-7915},
support = {//European Climate, Enviornment and Infrastructure Executive Agency (CINEA) LIFE Programme/ ; 860477//European Union's Horizon 2020 Research and Innovation Programme AgRefine/ ; LIFE18CCM/IE/001195//Government of Ireland Department of Climate, Energy and the Environment (DCEE)/ ; },
mesh = {*Silage/analysis/microbiology ; *Lactic Acid/analysis/metabolism ; *Biosensing Techniques/methods ; *Escherichia coli/genetics/metabolism ; *Poaceae/metabolism/chemistry ; Gene Knockout Techniques ; Gene Deletion ; Lactate Dehydrogenases ; },
abstract = {Grass silage fed green biorefineries require high quality grass silage leachates to produce high quality products. Real-time, cost-effective methods to monitor key leachate analytes are needed for small-scale decentralised systems. In this study, Escherichia coli mutants, designated JSP0090 and JSP0094, were created to quantify d- and l-lactic acid concentrations in grass leachate samples using an oxygen probe. The genes encoding either d- or l-lactate dehydrogenase were expressed in an exclusion biosensor strain (JSK0115). This strain required successive gene deletions to prevent interference from sugars, amino acids and organic acids present in grass silage leachates. This strain was incapable of catabolising d-lactic acid, l-lactic acid, acetic acid, propionic acid, formic acid, ethanol, glucose, fructose, l-alanine, VFAs, glycerol, mannitol and succinate. Gene knockouts were achieved using P1 phage lysates and CRISPR Cas 9 methods to target key steps in the catabolism of these compounds, with the exception of succinate. For this metabolite, the di-carboxylic acid transporters YaaH, DctA, YchM, DcuA and DcuB had to be deleted. The effectiveness of the biosensors for selectively measuring d- and l-lactic acid was assessed in Austrian and Irish grass silage leachate. The concentrations measured were comparable to those obtained using a commercial enzyme kit.},
}
@article {pmid42170179,
year = {2026},
author = {Karagyaur, M and Averina, O and Bozov, K and Dzhauari, S and Priymak, A and Khaybullina, R and Permyakov, O and Popov, V and Grigorieva, O and Illarionova, M and Shkarina, L and Gulyaev, M and Lebedev, D and Primak, A and Sergiev, P and Semina, E and Klimovich, P and Samokhodskaya, L and Malkov, P and Pirogov, Y and Tsygankov, B and Chaika, Y and Tkachuk, V and Neyfeld, E},
title = {Novel mouse line with D277N mutation in the Plau gene displays autism spectrum disorder-like traits.},
journal = {Frontiers in cell and developmental biology},
volume = {14},
number = {},
pages = {1762737},
pmid = {42170179},
issn = {2296-634X},
abstract = {INTRODUCTION: Genetic technologies provide an opportunity to study the molecular basis of a wide range of hereditary pathologies, including mental disorders. Reproducing of potentially pathogenic genomic variants in cellular and animal models allows establishing their functional significance and possible mechanisms of involvement in the pathogenesis of certain disorders.<br><br>METHODS: In this study, a genetic variant of urokinase type plasminogen activator (uPA, gene Plau) was modeled in mice using CRISPR/Cas genome editing tool, enabling a better understanding of the role of this molecule and its associated pathways in brain development. The protease uPA plays an important role in the directed migration of neural progenitors, glial, endothelial and immune cells, it participates in axon guidance and maturation of synaptic connections, activation of growth factors and degradation of the extracellular matrix. To study the contribution of the catalytic function of uPA to brain development, we have created for the first time a mouse line carrying the D277N (rs1243306395) mutation. We assessed social activity, anxiety, memory, problem-solving ability and stress resistance of these mice, as well as histological features of their brains.<br><br>RESULTS: Timely and correct functioning of the Plau gene ensures adequate positioning of crucial cellular components in the developing nervous system. According to bioinformatic calculations, the D277N (corresponds to the human single nucleotide variant rs1243306395) substitution that happens due to C-to-T mutation in the murine Plau gene may impair the catalytic activity of the uPA protein. While retaining their ability to find solutions in the escape test, this mouse line is characterized by high levels of anxiety, impaired social behavior, slowed learning dynamics (spatial memory), and impaired adaptation to stressors. This behavioral pattern can potentially be interpreted as autism spectrum disorder Histological analysis of the brain and cerebral cortex in Plau-D277N mice revealed brain volume enlargement and cortical thickening of approximately 10-15% compared to wild-type mice.<br><br>DISCUSSION: In this study, we draw attention for the first time to the genomic variant rs1243306395 in the Plau gene as a potential cause of autism spectrum disorder and propose the genetically modified Plau-D277N mouse line as a model object for studying the pathogenesis of this disorder. These models can also be used for the development and testing of promising therapeutic approaches and pharmacological agents.},
}
@article {pmid42171238,
year = {2026},
author = {Chu, Z and Yong, H and Li, Z and Wang, X and Li, B and Yan, C and Li, Q and Tao, Y and Zhou, D and Geng, S},
title = {Effective gene editing of melanoma by delivery of Cas9 mRNA with highly branched poly(β-amino ester).},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {23},
pages = {7142-7149},
doi = {10.1039/d5tb02556g},
pmid = {42171238},
issn = {2050-7518},
mesh = {Humans ; Animals ; *RNA, Messenger/genetics/chemistry/metabolism ; Mice ; *Gene Editing/methods ; *Melanoma/genetics/therapy/pathology ; *Polymers/chemistry ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; Cell Line, Tumor ; },
abstract = {Melanoma, the most aggressive form of skin cancer, still lacks clinically reliable treatments. CRISPR/Cas9 gene editing offers a promising approach for melanoma therapy; however, the efficient and safe delivery of CRISPR/Cas9 components to melanoma cells remains a formidable challenge. Here, we report the development of highly branched poly(β-amino ester) (HPAE) for the efficient delivery of Cas9 mRNA to melanoma cells and for transdermal application in vivo. HPAE is synthesized via a one-pot "A2 + B4 + C2" type Michael addition strategy and is capable of efficiently condensing mRNA to form nanosized polyplexes with a moderate positive charge. In human embryonic kidney cells (HEK293T), mouse melanoma cells (B16F10), and human melanoma cells (A2058 and A375), up to 76.3%, 74.6%, 16.5%, and 57.1% mRNA transfection efficiency was achieved without obvious cytotoxicity. Moreover, repeated dosing further enhanced mRNA transfection efficiency, resulting in sustained gene expression. Importantly, HPAE enables the effective co-delivery of Cas9 mRNA and sgRNA to melanoma cells, facilitating up to 25.5% gene knockout. HPAE also demonstrates strong transdermal capability, resulting in high in vivo mRNA expression following direct topical administration. This study demonstrates the feasibility of treating melanoma through the transdermal application of the CRISPR/Cas9 system, enabled by the efficient mRNA delivery provided by HPAE.},
}
@article {pmid42171807,
year = {2026},
author = {Li, Z and Guo, H and Yang, Y and Cheng, Q and Dao, L and Liu, J and Ding, P and Wu, P},
title = {An Fe single-atom nanozyme-sensitized RPA-CRISPR/Cas12a biosensor for the early detection of potential microcystins-producing cyanobacteria.},
journal = {Mikrochimica acta},
volume = {193},
number = {6},
pages = {},
pmid = {42171807},
issn = {1436-5073},
support = {2025JJ60624//Natural Science Foundation of Hunan Province/ ; 82504465//National Natural Science Foundation of China/ ; 502044013//Start-up Funds from Central South University/ ; },
mesh = {*Biosensing Techniques/methods ; *Microcystins/analysis/biosynthesis ; *Cyanobacteria/metabolism/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Limit of Detection ; *Iron/chemistry ; Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Lakes/microbiology ; },
abstract = {Harmful cyanobacteria are capable of producing hepatotoxic microcystins (MCs), which pose a significant risk to both aquatic ecosystems and public health. Since cyanobacterial strains that produce MCs harbor the mcy gene cluster, monitoring and analyzing the levels of the mcy gene is critical for identifying potential MCs-producing strains and assessing periods of elevated risk. In this study, Fe single-atom nanozymes (Fe SANs) with enhanced peroxidase-like (POD) activity were synthesized, which were then used to form a Fe SANs-DNA-magnetic bead complex (Fe SANs-DNA-MB). By integrating pre-amplification via recombinase polymerase amplification (RPA), trans-cleavage by CRISPR/Cas12a, and catalytic activity by Fe SANs, a novel triple signal amplification biosensor was developed for the detection of the microcystin synthase gene E (mcyE), a critical biomarker for MCs-producing cyanobacteria. The biosensor demonstrated significantly improved analytical performance, achieving a broad dynamic rangefor mcyE detection from 0.1 pM to 20 nM (R2 = 0.99), with a low limit of detection (LOD) of 0.05 pM. Furthermore, the biosensor provides high accuracy, as evidenced by spiked recoveries of mcyE in natural lake water samples ranging from 97.93% to 103.3%. It also enabled the selective identification of the mcyE gene in toxin-producing cyanobacterial strains (FACHB-905 and FACHB-979), thereby effectively distinguishing between toxin-producing and non-toxin-producing cyanobacteria. This innovative approach advances the field of molecular biosensing by providing a highly sensitive platform for the quantitative detection of the mcyE gene, facilitating accurate monitoring of MCs-producing cyanobacteria in environmental contexts. The method holds considerable promise for enhancing water quality management and protecting public health.},
}
@article {pmid42172144,
year = {2026},
author = {Aguilar, G and Sickmann, ME and Bieli, D and Born, G and Affolter, M and Müller, M},
title = {In situ mutational screening and CRISPR interference define apterous cis-regulatory inputs during compartment boundary formation.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {42172144},
issn = {2050-084X},
support = {310030_192659/SNSF_/Swiss National Science Foundation/Switzerland ; 310030B_176400/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; *Wings, Animal/embryology/growth &amp; development ; *Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics/embryology ; CRISPR-Cas Systems ; Homeodomain Proteins/genetics/metabolism ; Body Patterning/genetics ; Mutation ; },
abstract = {The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and dorsal fate by the selector gene apterous (ap). While the en expression domain is set up during embryogenesis, ap expression begins only during early wing development. Thus, the correct establishment of the ap expression pattern relative to en must be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the early ap enhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX-binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.},
}
@article {pmid42172311,
year = {2026},
author = {Guo, C and Zhang, S and Yerramsetti, R and Zhang, J and Guan, X and Yang, R and Hou, C and Pei, M and Schalper, KT and Liu, X and Li, Z and Perpetua, L and Gan, W and Ibrahim, O and Clark, RA and Liu, C},
title = {Single-nucleotide variant profiling in liquid biopsy with RECO-Cas.},
journal = {Science advances},
volume = {12},
number = {21},
pages = {eaed1757},
pmid = {42172311},
issn = {2375-2548},
mesh = {Humans ; Liquid Biopsy/methods ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; *Cell-Free Nucleic Acids/genetics/blood ; ErbB Receptors/genetics ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Neoplasms/genetics/diagnosis ; Mutation ; },
abstract = {Mutation detection of cell-free DNA (cfDNA) through liquid biopsy is essential for precision oncology, resistance profiling, and informed clinical decision-making. However, its clinical application has remained limited by the lack of simple, rapid, accurate, and cost-effective detection approaches. Here, we report a recombined DNA construct-activated Cas12a (RECO-Cas) assay for profiling cfDNA mutations. The RECO-Cas assay uses a recombined DNA construct generated from Argonaute-nicked mutant DNA and an artificial DNA activator to selectively trigger CRISPR-Cas12a, enabling 0.01% variant allele frequency sensitivity and single-nucleotide resolution. Using the assay, we detect KRAS, EGFR, and PIK3CA point mutations in cfDNA from clinical plasma samples, demonstrating high sensitivity (90.48%) and excellent specificity (100%). We also apply it to successfully classify and identify KRAS missense variants. RECO-Cas provides a simple, rapid, and affordable solution that is compatible with a compact, wirelessly powered point-of-care diagnostic platform incorporating smartphone-based fluorescence detection. This assay enables highly sensitive and specific detection of low-frequency mutations, facilitating early cancer diagnosis and supporting the development of personalized treatment strategies.},
}
@article {pmid42172545,
year = {2026},
author = {Jiang, Y and Wen, H and Xu, J and Peng, W and Zhou, J and Mao, H and Gu, Z and He, Y},
title = {Dual-Gene CRISPR Editing via Peptide Dendrimers Regulates Redox Balance for Diabetic Wound Repair.},
journal = {Biomacromolecules},
volume = {27},
number = {6},
pages = {3647-3661},
doi = {10.1021/acs.biomac.6c00110},
pmid = {42172545},
issn = {1526-4602},
mesh = {Animals ; *Wound Healing/genetics/drug effects ; Mice ; *Dendrimers/chemistry/pharmacology ; Oxidation-Reduction ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Oxidative Stress ; Kelch-Like ECH-Associated Protein 1/genetics/antagonists &amp; inhibitors ; NF-E2-Related Factor 2/genetics/metabolism ; *Peptides/chemistry ; *Diabetes Mellitus, Experimental/genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Nanoparticles/chemistry ; Genetic Therapy/methods ; Hypoxia-Inducible Factor-Proline Dioxygenases/genetics ; },
abstract = {The management of chronic diabetic wounds, plagued by persistent oxidative stress, remains a major clinical challenge. We devised a CRISPR/Cas9-based gene therapy to fundamentally reprogram this pathological microenvironment. A single system was engineered for the simultaneous knockdown of Keap1 and PHD2, key negative regulators of the Nrf2 and HIF-1α pathways, respectively. This payload was delivered by multifunctional peptide-modified lysine dendrimers (MsRNPs), which self-assembled into stable, positively charged nanoparticles that effectively complexed with DNA. The MsRNPs showed excellent biocompatibility and mediated efficient cellular uptake and gene editing in vitro, leading to reduced ROS levels. Consequently, a single topical application of the polyplexes in a diabetic mouse model robustly accelerated wound closure, enhanced collagen deposition, and promoted angiogenesis, driven by the synergistic activation of Nrf2 and HIF-1α. This study establishes a novel combinatorial gene-editing strategy and a versatile nanoplatform for treating oxidative stress-related pathologies.},
}
@article {pmid42173102,
year = {2026},
author = {Peng, S and Xie, W and Zhu, J and Bao, Z},
title = {Multiplex genome engineering: Methodologies and applications.},
journal = {Cell systems},
volume = {},
number = {},
pages = {101614},
doi = {10.1016/j.cels.2026.101614},
pmid = {42173102},
issn = {2405-4720},
abstract = {To facilitate the study of applied genetics and enable a rapid translation of genetic insights, it is highly desirable to concurrently modify many genomic loci in an organism of interest. While single-locus editing is well-established and technically straightforward, multiplex genome engineering (MGE) poses significant technological barriers. With the convergence of low-cost DNA synthesis, advanced genome editing techniques, and laboratory automation, a plethora of MGE methodologies were recently developed and applied in fields ranging from basic research to applied sectors. This review analyzes one-step and iterative MGE methodologies, with an emphasis on recombineering and CRISPR-Cas systems, and showcases emerging paradigm-shifting applications in biomanufacturing, agriculture, and therapeutics. We conclude by analyzing the limitations of existing technologies and discussing future directions for further optimizing MGE to solve system-level problems.},
}
@article {pmid42173386,
year = {2026},
author = {Mishra, S and Rehan, S and Barekzai, AM and Sharma, A and Raghav, A},
title = {Emerging frontiers in genome editing: From CRISPR to next-generation technologies.},
journal = {Methods (San Diego, Calif.)},
volume = {253},
number = {},
pages = {71-84},
doi = {10.1016/j.ymeth.2026.05.011},
pmid = {42173386},
issn = {1095-9130},
mesh = {*Gene Editing/methods/trends ; Humans ; *CRISPR-Cas Systems/genetics ; Epigenome Editing ; Zinc Finger Nucleases/genetics ; Animals ; },
abstract = {Genome editing has revolutionized molecular biology. It offers precise modification of genetic material across diverse organisms. This review outlines the evolution of genome editing technologies from homologous recombination to advanced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-9) based systems that now dominate the field. Early methods, such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), established the foundation for site-specific DNA cleavage. However, they were limited by complexity and cost. The advent of the CRISPR-Cas systems, particularly CRISPR-Cas9, transformed the landscape due to their simplicity, high efficiency, and adaptability. Variants such as CRISPR-Cas12a, base editors, and prime editors enhanced editing precision. They enable single-nucleotide modifications and targeted insertions without double-strand breaks. Emerging tools such as CRISPR-associated transposases, recombinase fusions, and RNA-targeting Cas13 enzymes expand the scope of manipulation beyond DNA to RNA. At the same time, epigenome editing, and gene drives present new therapeutic and ecological applications. Efficient delivery systems, both viral (Adeno-Associated Virus (AAV), lentivirus, adenovirus) and non-viral (lipid nanoparticles, gold nanoparticles, DNA nano clews), remain critical for clinical translation. Future directions emphasize artificial intelligence-guided design, retroelement-based integration, and novel biomimetic delivery vehicles to overcome current efficiency and safety barriers. These innovations can help overcome current efficiency and safety barriers. Together, they are propelling genome editing toward precise, programmable, and ethically responsible therapeutic applications. Despite unresolved challenges involving off-target effects, immunogenicity, and germline ethics, genome editing redefines biomedical research, drug development, and disease correction. CRISPR-derived technologies now stand at the forefront of next-generation genetic medicine.},
}
@article {pmid42173587,
year = {2026},
author = {Lu, X and Wang, Y and Che, Y and Li, Y and Nong, B and Ge, Y and Wang, X and Guo, Y and Li, R and Liu, J and Guo, J and Yao, Y and Geng, M},
title = {CRISPR/Cas9 mediated knockout of MeSSI enhances resistant starch content without compromising yield in cassava.},
journal = {Carbohydrate polymers},
volume = {385},
number = {},
pages = {125382},
doi = {10.1016/j.carbpol.2026.125382},
pmid = {42173587},
issn = {1879-1344},
mesh = {*Manihot/genetics/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Starch/chemistry/metabolism ; *Plant Proteins/genetics/metabolism ; Amylopectin/metabolism/chemistry ; Gene Editing ; Amylose/metabolism ; Gene Expression Regulation, Plant ; *Resistant Starch/metabolism ; },
abstract = {Enhancing resistant starch (RS) content in cassava is vital for developing nutritionally improved, functional food crops. In this study, targeted mutagenesis of the MeSSI gene via CRISPR/Cas9 was conducted to investigate its role in starch biosynthesis and RS accumulation. MeSSI knockout lines exhibited a 6.74-fold increase in RS content and a 16.42% elevation in amylose levels compared to the wild-type, without compromising total starch content or root yield. Starch structural analysis revealed an increased number of smaller granules per amyloplast and a shift in amylopectin chain-length distribution, characterized by reduced short chains (DP 6-12) and enrichment of intermediate and long chains, resulting in a lower branching degree. These modifications were associated with enhanced thermal stability and altered pasting behavior. Transcriptomic profiling indicated compensatory upregulation of AGPase subunits, and glycolytic genes, suggesting a reprogramming of carbon metabolism to sustain starch accumulation. This work identifies MeSSI as a key determinant of amylopectin fine structure and RS formation, providing a precise genome-editing strategy to improve the nutritional profile of cassava.},
}
@article {pmid42173700,
year = {2026},
author = {Ramachandran, H and Dobner, J and Nguyen, T and Binder, S and Tolle, I and Vykhlyantseva, I and Krutmann, J and Miccio, A and Staerk, C and Brusson, M and Kontarakis, Z and Prigione, A and Rossi, A},
title = {CleanFinder: a scalable framework for comprehensive genome editing analysis.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.04.024},
pmid = {42173700},
issn = {1879-3096},
abstract = {Genome editing often generates complex mixtures of alleles rather than single, predefined outcomes. Resolving these heterogeneous edits across diverse editing modalities, sequencing platforms, and multiplexed designs remains a persistent analytical challenge. To address this, we developed CleanFinder, a browser-native framework for genotyping genome editing outcomes using a constrained semi-global alignment strategy. Context-aware alignment modes support a broad spectrum of editing scenarios, including indels, base substitutions, and complex prime editing modifications across nuclear and mitochondrial targets. Additional modules include an optional turbo mode for high-throughput heuristic alignment in exploratory workflows and an allele-aware module that leverages heterozygous single-nucleotide polymorphisms to detect allelic dropout. To evaluate scalability and practical performance, we applied CleanFinder to a primary small-molecule screen of 1849 compounds in HEK293T cells. The software efficiently processed the dataset, enabling high-throughput comparison of editing outcomes and nomination of candidate compounds for follow-up analysis. Together, CleanFinder provides a flexible and scalable platform for genome editing analysis, enabling detailed genotyping and systematic comparison of editing outcomes across diverse edit types and genomic contexts.},
}
@article {pmid42176206,
year = {2026},
author = {Dias, RG and Freitas, FPM and Barbosa, SL and Assis, JVMG and Entringer, TL and Fonseca, JSC and Romanizio, ME and Pimentel, BBZ and Campos-Galvão, MEM and Vieira, NM and Fietto, LG and Zsögön, A and da Silveira, WB},
title = {10 years of CRISPR/CAS genomic engineering in Yarrowia lipolytica.},
journal = {Bioprocess and biosystems engineering},
volume = {},
number = {},
pages = {},
pmid = {42176206},
issn = {1615-7605},
abstract = {Yarrowia lipolytica is a versatile cell factory widely used in bioprocesses for producing lipids, organic acids, and other high-value compounds. Historically, its genetic engineering was constrained by low homologous recombination (HR) efficiency and the predominance of non-homologous end joining (NHEJ), limiting strain development and metabolic pathway optimization. The advent of CRISPR-Cas technologies has revolutionized genome editing in Y. lipolytica, enabling precise, efficient, and multiplexed modifications. Innovations such as pCAS1yl and pCRISPRyl plasmids, along with genomic Cas9 integration, have enhanced targeted editing efficiency. CRISPR applications now extend to metabolic engineering for lipids, itaconic acid, erythritol, and other compounds. Beyond canonical Cas9, alternative systems, including CRISPRa, Cas12a, base editors, and sgRNA libraries, provide increased flexibility and functional diversity. Central to these approaches is the rational design of guide RNAs (gRNAs), supported by bioinformatics platforms such as CHOPCHOP v3, CRISPOR, CCTop, and Cas-OFFinder, which assist in target site selection, off-target prediction, and editing optimization. This review summarizes the main CRISPR/Cas9 applications in Y. lipolytica, highlighting key engineered strains and emphasizing the critical role of bioinformatics in improving editing strategies. We also propose a pipeline for systematic gRNA design based on published evidence and discuss future perspectives, including the integration of machine learning, artificial intelligence, and emerging CRISPR variants to further advance yeast metabolic engineering.},
}
@article {pmid42178819,
year = {2026},
author = {Yadav, A and Saini, A and Dilbaghi, N and Yadav, N},
title = {Advances in Nano-Enabled Biosensing Technology for Gastric Cancer Diagnosis: Mechanistic Insights and Translational Perspectives.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-28},
doi = {10.1080/10408347.2026.2672560},
pmid = {42178819},
issn = {1547-6510},
abstract = {Gastric cancer (GC) remains a critical burden on healthcare, affecting millions of people annually. Helicobacter pylori infection is a critical contributor to GC. The diagnostic confirmation of GC is generally obtained at late stages owing to the delayed onset of symptoms. Early detection can significantly improve the disease outcomes. Several approaches, like endoscopy, MRI, and computed tomography, are conventionally employed. But they often have certain drawbacks, such as less accessibility, invasiveness, and potential false results. Delayed diagnosis and poor prognosis by conventional strategies have underlined the need for an efficient and precise solution. These obstacles can be mitigated by implementing advanced biosensing platforms. The amalgamation of nanotechnology, machine learning, and advanced computational intelligence has extensively evolved sensor technology. This review offers a holistic overview of GC pathogenicity and conventional diagnostics with special emphasis on recently fabricated biosensors. Advanced biosensing platforms, like CRISPR-Cas, smartphone-integrated, breath-based, and ingestible biosensors, are also explored. This review further highlights the translational perspectives along with the increasing role of AI and advanced algorithms. With a critical discussion on key challenges, this article provides a future roadmap for the detection of GC biomarkers. Significant innovations are needed to translate biosensors into a state-of-the-art technique in GC diagnostics.},
}
@article {pmid42178964,
year = {2026},
author = {Sha, T and Zhao, D and Zhao, X and Lu, Y and Wang, R and Liu, J and Li, Y and Li, S and Chen, M and Bi, C and Zhang, X},
title = {Mutant-Initiated Structure-Guided Refinement Enables Second-Generation Compact IscB Genome Editors.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70392},
doi = {10.1002/cbic.70392},
pmid = {42178964},
issn = {1439-7633},
support = {XDC0110200//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 32225031//National Science Fund for Distinguished Young Scholar/ ; 32488301//the Basic Science Center Project of the National Natural Science Foundation of China/ ; 32271483//National Natural Science Foundation of China/ ; 2022177//Youth Innovation Promotion Association CAS/ ; },
mesh = {*Gene Editing/methods ; Humans ; Mutation ; DNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Compact RNA-guided nucleases such as IscB represent an attractive foundation for next-generation genome editors, yet their application in mammalian cells has been constrained by suboptimal activity. Here, instead of re-engineering enzymes, we establish a mutant-initiated, structure-guided optimization strategy to generate second-generation high-activity IscB editors. Using AlphaFold3 to model the engineered IscB*-ωRNA-DNA complex, we reveal remodeling of the nucleic-acid-binding interface induced by activity-enhancing substitutions. Guided by this predicted structure, we perform a focused mutational scan and identify V367 as an activity hotspot. Saturation mutagenesis at this position yields a single substitution, V367Y (IscB*-Act), which increases mean editing efficiency by 34% and achieves up to 2.1-fold improvement across endogenous targets in mammalian cells. Importantly, the V367Y substitution is transferable to an IscB-based adenine base editor, elevating A-to-G conversion by 68% on average and up to 4.46-fold at individual loci without altering the intrinsic editing window. Targeted off-target profiling at loci suggests that V367Y does not substantially increase off-target indels or A-to-G conversion. Together, our work demonstrates a practical framework for second-generation refinement of compact genome editors, bridging deep-learning-enabled structural prediction with interpretable protein engineering, and expands the functional potential of miniature IscB systems for both nuclease and base editing applications.},
}
@article {pmid42179772,
year = {2026},
author = {Vats, P and Baweja, B and Saini, C and Kushwah, AS and Kumar, A and Srivastava, SK and Nema, R},
title = {An overview of CRISPR-artificial intelligence theranostics: Current and emerging applications.},
journal = {Biomaterials translational},
volume = {7},
number = {1},
pages = {79-120},
pmid = {42179772},
issn = {2096-112X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics are revolutionizing precision medicine by enabling highly sensitive detection of nucleic acid and protein biomarkers. Building on these capabilities, CRISPR-based theranostics now aim to unify real-time disease detection with targeted therapeutic interventions. However, traditional CRISPR diagnostics face several limitations, including restricted multiplexing, off-target effects, and challenges in delivery efficiency. To overcome these issues, artificial intelligence (AI) has significantly enhanced CRISPR platforms by enabling intelligent guide RNA (gRNA) design, interpretation of complex biosensor outputs, and facilitation of rapid clinical decision-making. Machine learning tools such as DeepCRISPR, Azimuth 2.0, DeepHF, and CRISPRpred support the development of highly specific gRNAs, reduce off-target events, and personalize genome-editing strategies based on individual genomic profiles. Recently, by combining CRISPR systems with nanomaterials, fluorescence-based detection, and electrochemical sensing, researchers have developed advanced biosensors capable of detecting a broad spectrum of disease biomarkers, from cancer-associated nucleic acids to viral and genetic disorders. These advances support both diagnostics and gene therapy, enabling accurate, low-cost testing at home, in point-of-care settings, and in resource-limited environments. Together, the integration of AI and CRISPR is accelerating biomarker discovery and the development of intelligent, adaptive therapeutic platforms. New point-of-care diagnostic tests (POCTs) based on CRISPR-AI are essential for early screening of high-mortality diseases, and CRISPR-based diagnostic assays have emerged as powerful, versatile alternatives to traditional nucleic acid tests, offering rapid, programmable, and portable diagnostic solutions. This review explores the evolution of CRISPR-AI theranostic systems, current and emerging POCT applications. It highlights the technological, clinical, and ethical challenges shaping their translation into next-generation precision diagnostics.},
}
@article {pmid42180316,
year = {2026},
author = {Chen, X and Zhang, M and Yang, L and Chen, Y and Chi, Y and Zhao, Y and Ma, Z and Li, Y and Wang, X},
title = {CRISPR spacer profiling and prophage mining reveal diverse bacteriophages associated with Streptococcus Mutans.},
journal = {Journal of oral microbiology},
volume = {18},
number = {1},
pages = {2674332},
pmid = {42180316},
issn = {2000-2297},
abstract = {BACKGROUND: Streptococcus mutans is a key cariogenic bacterium. Current antimicrobials lack species specificity, while phage-based approaches remain experimental and require more S. mutans phage isolates.<br><br>OBJECTIVE: To profile the diversity of S. mutans-associated phages and strain-level heterogeneity in phage exposure using genome-informed CRISPR spacer and prophage analyses.<br><br>MATERIALS AND METHODS: We compiled 944 publicly available S. mutans genomes and dereplicated them into 735 non-redundant strains. CRISPR-Cas systems, spacers, spacer targets, and putative prophages were identified, quality-assessed, and functionally annotated. Phylogenetic relationships of (pro)phages were evaluated using terminase large subunit proteins, and comparative genomics compared spacer-positive and spacer-negative strains.<br><br>RESULTS: CRISPR systems were detected in 548/735 strains, yielding 14,263 spacers, 1,864 phage-targeting spacers mapped to 110 viral genomes, including 41 cultured isolates, 51 metagenome-assembled phages, and 18 uncultured viral genomes. The most frequently targeted cultured phage was phiKSM96, whereas metagenome-assembled Caudoviricetes ctNo011 showed broader targeting. Prophage mining identified 186 regions in 130 strains, including 37 of &#x2265; medium quality and elements related to ctNo011 and phiKSM96. TerL phylogeny showed that most high-quality endogenous prophages clustered with phiKSM96 and ctNo011.<br><br>CONCLUSION: These findings reveal a vast, uncultivated phage repertoire targeting S. mutans, providing a critical genomic roadmap to guide the future isolation of novel phages for caries prevention.},
}
@article {pmid42180538,
year = {2026},
author = {Yang, J and Chen, Y and Chen, X and Ping, Y},
title = {Non-viral delivery of genome-editing tools for treatment of genetic disorders.},
journal = {Acta pharmaceutica Sinica. B},
volume = {16},
number = {5},
pages = {2903-2928},
pmid = {42180538},
issn = {2211-3835},
abstract = {Pathogenic mutations within protein-coding regions of genomic DNA can disrupt protein structure and lead to hereditary disorders. Genome-editing technologies, particularly those based on clustered, regularly interspaced, short palindromic repeats-associated protein (CRISPR-Cas), are promising therapeutic tools for correcting genetic abnormalities. To date, viral delivery vectors for genome-editing biomacromolecules have shown numerous promises in treating genetic disorders. However, safe viral delivery for genome-editing components remains challenging, largely due to the immunogenicity of viruses. As an alternative, non-viral delivery systems are emerging as a safer choice and may offer solutions to address the safety challenges. In this review, we first introduce CRISPR-Cas9-based genome editing tools and their delivery formats. Then, we outline the pathology of major genetic disorders and both preclinical and clinical approaches for these diseases by therapeutic genome editing, and provide an overview of current non-viral delivery strategies and their potential to overcome existing limitations. Finally, we discuss the current challenges and future outlooks of non-viral delivery of gene-editing components in treating genetic diseases.},
}
@article {pmid42182021,
year = {2026},
author = {Li, X and Huang, Y and Zhang, X and Du, L and Qiu, Y and Jiang, L},
title = {Recent advances in rapid multiplex detection of nucleic acid markers using RPA and CRISPR-Cas.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1810544},
pmid = {42182021},
issn = {1664-302X},
abstract = {The integration of recombinase polymerase amplification (RPA) with CRISPR-Cas systems has emerged as a powerful platform for rapid multiplex nucleic acid detection. Compared with quantitative polymerase chain reaction (qPCR) and Next-generation sequencing (NGS), RPA-CRISPR operates isothermally (37 °C-42 °C), requires minimal equipment, and achieves attomolar sensitivity in 20-90 min via collateral cleavage. Recent multiplex strategies, namely two-tube, spatial separation one-tube, and homogeneous one-pot, they have overcome crosstalk and enabled highly multiplexed detection in complex food matrices such as poultry, milk, and lettuce. These approaches are particularly suited for foodborne pathogen screening (e.g., Salmonella, Listeria), antimicrobial resistance profiling, and on-site surveillance, aligning with the scope of research at the frontier of food microbiology diagnostics. Despite advances, challenges persist in standardization, matrix inhibition, and regulatory approval. This mini-review summarizes recent advances (2020-2025) in RPA-CRISPR multiplex detection, outlines future directions for clinical implementation and food safety deployment, and provides guidance for subsequent research on its practical applications in these fields.},
}
@article {pmid42182524,
year = {2026},
author = {Huang, X and Sun, X and Dong, X and Tang, Y and Xu, S and Wu, Y and Hu, S and Ren, Y and Tu, Q and Zhang, Y},
title = {Prospects and challenges in using engineered lactic acid bacteria in aquaculture applications.},
journal = {Engineering microbiology},
volume = {6},
number = {2},
pages = {100275},
pmid = {42182524},
issn = {2667-3703},
abstract = {Despite the considerable potential of lactic acid bacteria (LAB) as probiotics, there is a fundamental gap between the functional limitations of wild-type strains and the complex demands of aquaculture. Modular and intelligent engineering strategies are the primary avenues for bridging this gap. This article systematically reviews the strategies and advances in the application of genetically engineered LAB. Technologies, including clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems, Red/ET recombination, and functional modifications have significantly enhanced the targeted delivery, environmental tolerance, and multiple probiotic functions of LAB, successfully yielding engineered strains such as oral vaccine strains expressing pathogen antigens, antimicrobial peptide-high-yielding antibacterial strains, and nitrite-degrading water-improving strains. These engineered strains have demonstrated superior performance in disease prevention, growth promotion, and environmental remediation compared to wild-type strains in the farming of tilapia, shrimp, and shellfish. However, challenges, such as plasmid instability, biosafety risks, and regulatory barriers, remain unresolved. Future research should focus on multi-omics-guided precision design, development of environmentally responsive genetic circuits, and full-cycle risk assessment, promoting engineered LAB as a core solution for sustainable aquaculture through collaboration across industries, academia, and research.},
}
@article {pmid42184324,
year = {2026},
author = {Aliev, TI and Imatdinov, AR and Prudnikova, EY and Imatdinov, IR},
title = {[Experimental Selection of Effective sgRNAs for MmCas12m Targeting the Region of the Start Codon of the HIV-1 gag Gene].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {1},
pages = {120-131},
doi = {10.7868/S3034555326010073},
pmid = {42184324},
issn = {0026-8984},
mesh = {*HIV-1/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Codon, Initiator/genetics ; *Gene Editing ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *gag Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; *HIV Infections/genetics/therapy ; },
abstract = {HIV-1 remains a threat to global health. There is no effective vaccine or drug for a complete cure of HIV infection. Work continues on the development of gene therapy drugs against HIV-1. The use of DNA base editors delivered to the editing site by CRISPR-Cas systems has shown some success in the field of HIV-1 gene therapy. The MmCas12m isoform obtained from Mycolicibacterium mucogenicum can become a promising platform for this task. MmCas12m has a compact size, the ability to bind strongly to the target DNA sequence, and an absence of nuclease activity. Thus, MmCas12m can act not only as a platform for the delivery of DNA base editors, but also as an inhibitor of transcription from HIV-1 proviral DNA. We experimentally selected in vitro the most effective sgRNAs for MmCas12m to target the start codon of the gag HIV-1, the product of which is important in virion assembly. Of the nine sgRNAs we selected, four showed statistically significant effectiveness in targeting the desired region of the HIV-1 genome. To test the effectiveness of each sgRNA, we have developed a system suitable for evaluating the binding of any Cas protein to the target site of HIV-1 genome editing.},
}
@article {pmid42184325,
year = {2026},
author = {Kolesnikova, OA and Svetlakova, AV and Furtak, ED and Zvereva, SD and Kukushkin, ID and Komedchikova, EN and Shipunova, VO},
title = {[Establishment and Validation of Cancer Cell Lines with HER2 Receptor Gene Deletion].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {1},
pages = {132-143},
doi = {10.7868/S3034555326010086},
pmid = {42184325},
issn = {0026-8984},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Gene Deletion ; Gene Editing ; Cell Proliferation ; },
abstract = {One of the clinically significant molecular targets in oncotheranostics is the receptor tyrosine kinase HER2 (HER2/neu, ERBB2), which is involved in the activation of various cellular programs. The overexpression of this receptor leads to uncontrolled cell proliferation, the initiation of oncogenesis, and is considered one of the most important oncogenic biomarkers. In vitro studies utilizing various HER2-positive cancer cell lines play a crucial role in the development of anti-HER2 targeted drug formulations. These cell lines differ in their structural and metabolic features, as well as in their sensitivity to hormones and other factors; therefore, the selection of an optimal cellular control is essential for the successful testing of HER2-specific agents. In the present study, a deletion in the ERBB2 gene was generated using CRISPR/Cas9 technology, with the success of the editing confirmed by sequencing of the target locus. A reduction in ERBB2 mRNA levels was demonstrated in three cancer cell lines with varying baseline HER2 receptor levels, alongside an alteration in the receptor's functional activity on the cell surface. Assessment of the binding efficiency of a fluorescently labeled HER2-specific antibody to the generated cell clones revealed a decrease in fluorescence intensity by 80.6-fold, 33.7-fold, and 2-fold in the SK-BR-3, SK-OV-3, and A549 cell lines, respectively. The generated cell lines with ERBB2 deletion represent a key tool for testing targeted therapeutics and can be utilized in the development of treatment modalities aimed at HER2-overexpressing malignant neoplasms.},
}
@article {pmid42184674,
year = {2026},
author = {Wang, H and Shi, Y and Feng, T and Ling, W and Huang, Y and Zhang, Y and Dai, Z and Yi, C},
title = {A portable Cas13a self-cascading cyclic amplification-integrated system enables multiple respiratory tract viruses analysis within minutes at point-of-needs.},
journal = {Biosensors &amp; bioelectronics},
volume = {309},
number = {},
pages = {118836},
doi = {10.1016/j.bios.2026.118836},
pmid = {42184674},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; *SARS-CoV-2/isolation &amp; purification/genetics ; RNA, Viral/genetics/analysis ; Humans ; *Biosensing Techniques/instrumentation ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; *COVID-19/diagnosis/virology ; Rapid Diagnostic Tests ; Respiratory Syncytial Viruses/isolation &amp; purification/genetics ; Influenza A virus/isolation &amp; purification/genetics ; Point-of-Care Systems ; Limit of Detection ; Nucleic Acid Hybridization ; Equipment Design ; },
abstract = {CRISPR-based molecular detection systems typically require pre-amplification for adequate sensitivity that prolongs reactions and increases risks of nonspecific amplification, primer interference, and aerosol contamination. In this work, we developed a LbuCas13a-based self-cascading cyclic amplification assay (SCC-Cas13a) for simultaneous detection of multiple respiratory tract viruses in a pre-amplification-free manner. It was achieved by designing CRISPR RNAs (crRNAs) that target specific viral RNA sequences and hairpin probes (HP) that contain multiple uracil structures. The sequence-specific hybridization of crRNA and target RNA activates trans-cleavage activity of LbuCas13a, which cleaves the HP at its poly-U-rich stem-loop region. This results in the release of single-stranded RNA activators that are identical to the target RNA from the HP. This triggers another round of sequence-specific hybridization with crRNA, and therefore a self-cascading cyclic amplification. In a proof-of-concept demonstration, respiratory syncytial virus (RSV), influenza A (Flu A), and SARS-CoV-2 were detected within 3 min, exhibiting limit of detections (LODs) of 230 aM (RSV), 310 aM (Flu A), and 420 aM (SARS-CoV-2), respectively. Notably, these LODs represent a 10[4]-fold enhancement over conventional CRISPR-based systems. The SCC-Cas13a eliminates reverse transcription and pre-amplification steps, streamlining workflow into a single-step reaction. Furthermore, a Radial microfluidic chip (R-chip) was engineered to integrate this SCC-Cas13a for simultaneous detection of RSV, Flu A, and SARS-CoV-2 in clinical nasopharyngeal swab samples. Assisted by a smartphone-based device which can stably excite and accurately collect fluorescence signals from R-chips, this research established an innovative solution for multiplexed pathogen identification and precise molecular diagnostics in resource-limited settings.},
}
@article {pmid42184675,
year = {2026},
author = {Yan, J and Xiao, M and Ho, KHW and Zhu, J and Zhang, Q and Xiao, S and Yin, B and Gu, B and Wong, SHD and Yang, M},
title = {A CRISPR-Cas12a system integrated with metal-enhanced light-up aptamer-fluorophore nanoreporter for ultrasensitive detection of prostate-specific antigen.},
journal = {Biosensors &amp; bioelectronics},
volume = {309},
number = {},
pages = {118834},
doi = {10.1016/j.bios.2026.118834},
pmid = {42184675},
issn = {1873-4235},
mesh = {*Prostate-Specific Antigen/blood/isolation &amp; purification ; *Biosensing Techniques/methods ; Humans ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; Gold/chemistry ; Silver/chemistry ; Limit of Detection ; *Nanotubes/chemistry/ultrastructure ; Fluorescent Dyes/chemistry ; Male ; Spectrometry, Fluorescence/methods ; },
abstract = {In this study, a CRISPR-Cas12a-based biosensing system integrated with metal-enhanced light-up aptamer fluorescence (MELAF) nanoreporters was developed for ultrasensitive detection of prostate-specific antigen (PSA). Here, the MELAF nanoreporters are constructed with a core-shell architecture consisting of a gold nanorod core, a silver inner shell, a mesoporous silica spacer, and surface linked light-up DNA aptamer-fluorogen complexes, enabling cascade fluorescence enhancement. This cascade fluorescence enhancement is accomplished through a two-stage process: (i) aptamer-fluorogen binding restricts intramolecular rotation, thereby activating fluorogen emission; and (ii) spectrally and spatially optimized Au@Ag core-shell structure provides plasmonic amplification, further boosting the fluorogen signal. In the presence of PSA, the PSA-specific aptamers preferentially bind the antigen, thereby blocking activation of the CRISPR-Cas12a system and preserving the "On" fluorescence state of the nanoreporter. In the absence of PSA, unbound PSA-specific aptamers activate the CRISPR-Cas12a system, inducing trans-cleavage of the MELAF nanoreporter and simultaneously abolishing both the light-up effect and plasmonic enhancement, which leads to a pronounced reduction in fluorescence. As a proof of concept, the platform enables rapid (approximately 75 min) and highly sensitive detection of PSA with a limit of detection of 0.36 pg/mL. The assay exhibits excellent specificity and robustness in complex biological matrices, and measurements in clinical specimens demonstrate high accuracy and diagnostic utility.},
}
@article {pmid42184676,
year = {2026},
author = {Li, P and Hu, Q and Li, Y and Li, S and Ruan, Z and Yin, K and Sun, G},
title = {Amplification-free light-activated CRISPR/Cas12a system with nano-amplifier for quantitative detection of non-nucleic acid targets.},
journal = {Biosensors &amp; bioelectronics},
volume = {309},
number = {},
pages = {118844},
doi = {10.1016/j.bios.2026.118844},
pmid = {42184676},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Troponin I/blood/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Humans ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Quantitative amplification-free CRISPR/Cas diagnosis to non-nucleic acid clinical biomarkers is limited by inefficient signal conversion and suboptimal sensitivity. Here, we present a light-activated CRISPR/Cas12a biosensing platform (LANA) to response this challenge. The LANA arms with a cascade signal enhancing strategy that integrates immunomagnetic enrichment, aptamer-functionalized gold nanoparticles carrying photocleavable activators, and UV-triggered release of activators to initiate Cas12a trans-cleavage. This light-controlled mechanism overcomes steric hindrance caused by surface confinement, provides precise temporal control of signal initiation, and effectively suppresses background activation. Using cardiac troponin I (cTnI) as a model analyte, LANA achieved a detection limit of 50 pg/mL, a wide dynamic range of 0.05-500 ng/mL, demonstrating high sensitivity and reliable quantitative performance with a simple fluorescence readout. Owing to its modular and amplification-free design, the platform can be readily adapted to other protein or small-molecule biomarkers, offering a generalizable and amplification-free framework for sensitive detection of non-nucleic acid targets.},
}
@article {pmid42184721,
year = {2026},
author = {da Silva, GE and Obst, S and Carvalho, P and Forner, J and Ruf, S and Saibo, NJM and Bock, R},
title = {Generation of a recipient line for Rubisco engineering by multiplex genome editing in tobacco.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {4},
pages = {e70930},
doi = {10.1111/tpj.70930},
pmid = {42184721},
issn = {1365-313X},
support = {//Max-Planck-Gesellschaft/ ; UIDB/04551/2020//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; PD/BD/138096/2018//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; },
mesh = {*Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Nicotiana/genetics/enzymology ; *Gene Editing/methods ; CRISPR-Cas Systems ; Photosynthesis/genetics ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics/metabolism ; Genetic Engineering/methods ; },
abstract = {Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) is the primary CO2-fixing enzyme on our planet. Its slow kinetics and poor discrimination between carbon dioxide and oxygen as substrates severely limit the efficiency of photosynthetic carbon fixation by plants. Attempts to improve Rubisco by genetic engineering have been hampered by the complexity of the Rubisco system, with a gene family for the enzyme's small subunit being encoded in the nuclear genome and the gene for the large subunit residing in the plastid (chloroplast) genome. Another nuclear gene family encodes the enzyme Rubisco activase, which is required to facilitate catalysis in the active site of the enzyme. The Rubisco subunits and the activase have co-evolved, making single-gene replacements largely fruitless and typically resulting in dysfunctional Rubiscos. Here, we have generated a dedicated plant line for Rubisco engineering that lacks nuclear genes for components of the Rubisco system. Using multiplex genome editing by CRISPR-Cas9 in the diploid tobacco species Nicotiana sylvestris, we have knocked out six loci encoding the small subunit of Rubisco and three loci encoding Rubisco activase. The generated mutants are incapable of autotrophic growth, and grafting experiments are underway to obtain transgene-free T1 progeny. The recipient line produced here provides a clean genetic background, in which heterologous Rubisco systems from other organisms can be readily implemented and systematically tested in comparative functional studies. It greatly simplifies Rubisco engineering and creates new opportunities for future efforts to enhance photosynthetic carbon assimilation and increase crop yields.},
}
@article {pmid42185214,
year = {2026},
author = {Zhao, G and Wang, S and Shen, G and Zhuo, L and Ruan, T and Wang, X and Jiang, C and Liu, Y and Jiang, X and Li, D and Shen, Y},
title = {Investigating the role of a testis-expressed gene Tex2 in spermatogenesis in mice.},
journal = {Molecular human reproduction},
volume = {32},
number = {2},
pages = {},
doi = {10.1093/molehr/gaag031},
pmid = {42185214},
issn = {1460-2407},
support = {20PJ085//the Sichuan Provincial Health and Wellness Committee/ ; },
mesh = {Animals ; Male ; *Spermatogenesis/genetics ; Mice, Knockout ; Mice ; *Testis/metabolism ; Sperm Motility/genetics ; CRISPR-Cas Systems ; Fertility/genetics ; Spermatids/metabolism ; Spermatozoa/metabolism ; },
abstract = {The testis-expressed (TEX/Tex) genes are highly expressed in the testes of various species. Several members of the TEX catalog have been suggested to play a role in spermatogenesis, but the functions of most members remain unknown. Here, we initially confirmed that TEX2 is significantly upregulated in spermatids and further successfully generated a Tex2 knockout (KO) mouse model using CRISPR/Cas9 technology. However, we found that the Tex2 KO male mice were fertile. Interestingly, Papanicolaou staining and scanning electron microscopy showed slight abnormalities in sperm morphology and reduced sperm motility as assessed by computer-assisted sperm analysis in Tex2 KO mice. Collectively, despite Tex2 having enriched expression in mouse testis, our genetic KO studies revealed that Tex2 is not essential for spermatogenesis or fertility in male mice, although its absence in sperm causes subtle abnormalities in morphology and motility.},
}
@article {pmid42185540,
year = {2026},
author = {Oberlin, S and Tay, NQ and Xue, A and Mosadeghi, R and Pimentel, H and McManus, MT},
title = {Multiplexed perturbation enables scalable pooled screens.},
journal = {Nature methods},
volume = {23},
number = {6},
pages = {1163-1173},
pmid = {42185540},
issn = {1548-7105},
support = {5U01CA272546-03//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 188001//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; T32HG002536//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Intercellular Adhesion Molecule-1/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; HEK293 Cells ; },
abstract = {CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.},
}
@article {pmid42185623,
year = {2026},
author = {Tsai, FY and Sternberg, SH},
title = {Flying under the radar: immune-evasive DNA for genome engineering.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
pmid = {42185623},
issn = {1748-7838},
support = {RM1HG009490//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01EB027793//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; CAREER 2239685//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
}
@article {pmid42186741,
year = {2026},
author = {Li, J and Ji, C and Yang, W and Han, Y and Zhao, P and Cai, X and Tian, S and Zhu, W and Zhang, J and Xu, J and Yang, W and Li, F and Liu, P},
title = {Engineered CRISPR/Cas12a2 Nanoprobe Imaging in Living Cells for Precise Tumor Diagnosis.},
journal = {Small methods},
volume = {10},
number = {11},
pages = {e70727},
pmid = {42186741},
issn = {2366-9608},
support = {U25C2029//National Natural Science Foundation of China/ ; 32371468//National Natural Science Foundation of China/ ; 22474077//National Natural Science Foundation of China/ ; 23ZR1461400//Shanghai Municipal Natural Science Foundation/ ; 22ZR1459600//Shanghai Municipal Natural Science Foundation/ ; YG2023ZD07//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; YG2024QNB09//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; 20234Y0201//Foundation of Shanghai Municipal Health Commission/ ; 2022JC002//Foundation of Shanghai Municipal Health Commission/ ; TMSK-2024-203//National Key Scientific Infrastructure for Translational Medicine (Shanghai)/ ; 10000015Z155080000004//2024 National Clinical Key Specialty Construction Project/ ; XK202401//Jiading District Medical Key Discipline Construction Project/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; RNA, Messenger/genetics/metabolism ; Mice ; Cell Line, Tumor ; Glutathione ; CRISPR-Associated Proteins/genetics ; Molecular Imaging/methods ; *Neoplasms/diagnosis/diagnostic imaging ; },
abstract = {Messenger RNA (mRNA) imaging in tumor cells plays a crucial role in monitoring the occurrence and development of tumors. However, achieving highly specific and sensitive mRNA imaging remains a significant challenge due to the complex intracellular environment and high background signal. Here, we engineered a CRISPR/Cas12a2 system with an RNA blocking strand that binds to CRISPR RNA (crRNA). After glutathione (GSH) stimulation, the RNA blocking strand is cleaved, allowing the release of crRNA and restoring the capability of CRISPR/Cas12a2 ribonucleoprotein (RNP). Furthermore, we developed a nanoprobe (termed eRNP-FHR) by converging engineered Cas12a2 RNP (eRNP) with framework-hotspot reporters (FHR). FHR features four vertices that modify the sgc8 aptamer to specifically target the protein tyrosine kinase 7 receptor on the surface of tumor cell membranes, link to the eRNP by hybridizing with crRNA, and incorporate fluorescence quenching groups. The eRNP-FHR precisely targets tumor cells through aptamer-mediated endocytosis, specifically recognizes mRNA upon GSH stimulation, and simultaneously cleaves FHR to release a significant fluorescent signal. Excitingly, eRNP-FHR successfully achieved imaging of baculoviral IAP repeat-containing 5 mRNA in pancreatic tumor cells, accurately distinguishing pancreatic tumor cells from normal cells. In a murine pancreatic tumor model, eRNP-FHR exhibited excellent mRNA imaging, highlighting significant potential for precise tumor diagnosis.},
}
@article {pmid42186844,
year = {2026},
author = {Zhao, Y and Sun, H and Wan, Y and Zhang, Z and Xiao, L and Han, T and Hao, Z and Li, N and Pang, C and Zhang, W and Wan, J},
title = {Research on a highly sensitive aptamer sensor for Vibrio alginolyticus based on CRISPR-Cas13a and T7 transcription cascade amplification.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {22},
pages = {4701-4711},
doi = {10.1039/d6ay00035e},
pmid = {42186844},
issn = {1759-9679},
mesh = {*Vibrio alginolyticus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {Vibrio alginolyticus is a prevalent aquatic pathogen that poses significant risks to public health. Thus, the development of rapid and highly sensitive detection methods is imperative. In this study, we developed a novel aptasensor leveraging the CRISPR/Cas13a system. By integrating a triple-amplification strategy comprising "aptamer competition recognition-T7 transcription amplification-Cas13a trans-cleavage", this platform enables efficient and specific detection of V. alginolyticus. The biosensor employs a V. alginolyticus-specific aptamer (Apt) as the recognition element. In the presence of the target bacterium, Apt binds to a surface membrane protein, resulting in the release of a blocking strand (Block). This triggers a conformational change in a hairpin probe (HP), thereby exposing the T7 promoter sequence. Subsequently, T7 RNA polymerase initiates an isothermal transcription reaction, producing abundant RNA products. These RNAs activate the trans-cleavage activity of Cas13a, which cleaves a fluorescent reporter probe to generate a quantifiable signal. This method eliminates the need for nucleic acid extraction and sophisticated instrumentation. It achieves a detection limit as low as 2 CFU mL[-1], and demonstrates high specificity by effectively distinguishing closely related species (e.g., Vibrio parahaemolyticus). When applied to simulated seawater and seafood samples, the recovery rates ranged from 94.61% to 106.56%, indicating robust anti-interference capacity and reproducibility. This work establishes a highly sensitive and specific biosensing technology for the on-site rapid detection of aquatic pathogens, offering promising applications in environmental monitoring and food safety.},
}
@article {pmid42187217,
year = {2026},
author = {Awotundun, TA and Samson, OJ and Olanbiwoninu, AA},
title = {Viruses that heal: harnessing bacteriophages in the era of antibiotic resistance.},
journal = {Voprosy virusologii},
volume = {71},
number = {2},
pages = {91-108},
doi = {10.36233/0507-4088-366},
pmid = {42187217},
issn = {2411-2097},
mesh = {*Bacteriophages/genetics/pathogenicity ; Humans ; *Phage Therapy/methods ; *Bacteria/virology/genetics ; *Bacterial Infections/therapy/genetics/microbiology/virology ; Anti-Bacterial Agents/therapeutic use ; Genetic Engineering ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Microbial/genetics ; },
abstract = {The global rise in antimicrobial resistance (AMR) poses an urgent threat to public health, and novel alternatives to traditional antibiotics are needed. One of the most promising options is bacteriophages, viruses that infect and destroy bacteria. Once overshadowed by the discovery of antibiotics, phage therapy is now regaining attention, driven by advances in genomics, synthetic biology, and targeted medicine. This review examines the biology, diversity, and therapeutic use of bacteriophages in treating bacterial infections, especially those caused by multidrug-resistant pathogens. It also discusses how phages act through natural mechanisms, such as lytic enzymes (holins, endolysins, and muralysins), and highlights new genetic engineering techniques, such as CRISPR-Cas systems, phage recombineering, and synthetic genome reboots. In addition to clinical applications, we evaluate phages as biocontrol agents for food safety, environmental sanitation, and biofilm management. Additionally, the article explores key issues in phage therapy, including regulatory frameworks, formulation stability, dynamics of phage-host resistance, and the importance of rapid diagnosis. When properly integrated into modern health and biotechnology practices, bacteriophages offer significant potential and a sustainable solution to the global challenge of antimicrobial resistance.},
}
@article {pmid42187489,
year = {2026},
author = {Liu, T and Guo, H and Yu, M and Peng, J and Guan, L and Xie, S and Hao, X and Yang, Y},
title = {A Magnetic-Assisted CRISPR-Cas12a Biosensor Incorporating a Y-DNA Probe for Sensitive Detection of Schistosoma japonicum Eggs.},
journal = {Biosensors},
volume = {16},
number = {5},
pages = {},
pmid = {42187489},
issn = {2079-6374},
support = {22277047//National Natural Science Foundation of China/ ; 82160631//National Natural Science Foundation of China/ ; 20252BAC250153//Jiangxi Provincial Natural Science Foundation/ ; },
mesh = {Animals ; *Biosensing Techniques ; *Schistosoma japonicum/isolation &amp; purification ; *CRISPR-Cas Systems ; DNA Probes ; Rabbits ; Ovum ; Magnetics ; Aptamers, Nucleotide ; },
abstract = {Schistosomiasis, caused by Schistosoma species, is notoriously difficult to accurately diagnose with conventional methods. In this study, we present an innovative biosensor that integrates CRISPR-Cas12a technology with nucleic acid aptamers for the highly sensitive detection of Schistosoma japonicum eggs. The biosensor leverages a Y-shaped DNA structure (Y-DNA) that incorporates an aptamer specific to S. japonicum eggs, along with an activator DNA and a segment for immobilization on magnetic nanomaterials. Upon target recognition, the Y-DNA releases the activator, which triggers the collateral cleavage activity of Cas12a, enabling the direct detection of eggs. This system demonstrates remarkable sensitivity, being capable of detecting individual eggs in infected rabbit serum and feces. Moreover, it effectively distinguishes the eggs of S. japonicum from those of other parasitic species. The simplicity, high sensitivity, and rapid detection of our biosensor offer significant potential for improving the diagnosis of schistosomiasis, providing a novel, reliable tool for early detection in clinical settings.},
}
@article {pmid42187493,
year = {2026},
author = {Bao, C and Zhang, H and Jiang, L and Liu, T and Liu, W and Qi, Q and Ren, X and Fu, H and Sun, M},
title = {Synthetic Biology-Enabled Biosensing Platforms for Point-of-Care In Vitro Diagnostics: Programmable Modules, Clinical Applications, and Translational Challenges.},
journal = {Biosensors},
volume = {16},
number = {5},
pages = {},
pmid = {42187493},
issn = {2079-6374},
support = {JYBS2025011LK//Jilin Medical University Institutional Research Project/ ; No. S202413706005, No. 202113706067, and No. 202513706018).//National Innovation and Entrepreneurship training Program for College Students/ ; 20250204071YY//the Science and Technology Department of Jilin Province/ ; },
mesh = {*Biosensing Techniques ; *Synthetic Biology ; Humans ; *Point-of-Care Systems ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; },
abstract = {Synthetic biology is reshaping in vitro diagnostics (IVD) by enabling programmable and modular biosensing elements that can be integrated into point-of-care testing (POCT) platforms. Compared with conventional assays that depend on fixed chemistries and centralized instrumentation, synthetic biology-based systems offer adaptable molecular recognition, tunable signal processing, and flexible readout formats for decentralized diagnostics. In this review, we present synthetic biology-enabled IVD as programmable biosensing platforms organized into four functional layers: molecular recognition, signal transduction and amplification, output generation, and system integration. We discuss four major enabling modules, including cell-free protein synthesis (CFPS) systems, aptamer and riboswitch sensors, CRISPR-Cas diagnostic platforms, and microfluidic integration technologies. We summarize representative clinical applications from 2021 to 2025 in infectious disease detection, cancer biomarker analysis, and drug metabolism/toxicity screening. In addition, we examine practical considerations beyond analytical sensitivity, including matrix tolerance, workflow complexity, manufacturability, quantitative capability, and regulatory readiness. Finally, we highlight future directions for programmable diagnostics, including AI-assisted biosensor design, multimodal readouts, interoperable platform architectures, and real-world clinical validation.},
}
@article {pmid42187704,
year = {2026},
author = {Jiang, C and Yang, D and Sun, C and Ren, X and Li, T and Wu, J and Tian, J and Feng, M and Yao, Y and Song, J and Weng, X and Mu, Y},
title = {An Episomal Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System for Transgene-Free Multiplex Gene Editing in Pig Cells.},
journal = {Biology},
volume = {15},
number = {10},
pages = {},
pmid = {42187704},
issn = {2079-7737},
support = {2022YFA1105402//Key Project of Natural Science Foundation of Heilongjiang Province of China/ ; 32272885//National Natural Science Foundation of China/ ; },
abstract = {Despite significant advancements in CRISPR/Cas-based genome editing technology over the past decade, achieving simultaneous homozygous gene editing at multiple targets in primary cells remains a major challenge. In this study, we developed and constructed a CRISPR multi-gene targeting system that integrates episomal vectors with tRNA-sgRNA array technology. This approach leverages scaffold/matrix attachment region (S/MAR) sequences to enable sustained episomal expression of both Cas9 and single-guide RNAs (sgRNAs) without genomic integration, thereby enhancing gene editing efficiency. For simultaneous editing of multiple loci, we used the tRNA-sgRNA architecture to process multiple sgRNAs from a single vector. Using this system in porcine fetal fibroblasts, we achieved concurrent editing of six genes, namely ANXA7, GSK3A, ENTPD6, SIRT3, CYP20A1, and SOCS2, in individual cells. These edited cells supported normal development following somatic cell nuclear transfer, yielding blastocysts with unaltered developmental competence. Collectively, our findings establish a framework for the application of CRISPR/Cas9 in gene-edited pigs, facilitating the generation of multi-gene-edited animals for biomedical and agricultural applications.},
}
@article {pmid42187710,
year = {2026},
author = {Singh, S and Tiwari, H and Singh, M and Gautam, V and Gautam, A and Gautam, HK},
title = {Expanding the Microbial Genomic Landscape and Biotechnological Applications of CRISPR-Cas Systems.},
journal = {Biology},
volume = {15},
number = {10},
pages = {},
pmid = {42187710},
issn = {2079-7737},
support = {(File No.: ANRF/IRG/2025/000135/LS)//Anusandhan National Research Foundation (ANRF)/ ; CST/D-1187//Council of Science and Technology, Uttar Pradesh, India (CST-UP)/ ; },
abstract = {The CRISPR-Cas systems, identified initially as adaptive immune mechanisms in bacteria and archaea against viral threats, have rapidly evolved into transformative tools in genetic engineering and biotechnology. These RNA-guided systems are broadly classified into Class 1, comprising multi-subunit complexes, and Class 2, characterized by compact single-effector protein, such as Cas9, Cas12, and Cas13. Their remarkable structural and functional diversity enables microorganisms to adapt to diverse ecological niches, offering a vast repertoire of genome-editing strategies. Beyond their natural role in maintaining genome integrity and defense, CRISPR-Cas systems have been extensively repurposed for precise genome modification, transcriptional regulation, epigenetic editing, and nucleic acid detection. Recent advances in computational mining of microbial genomes and metagenomes have uncovered a broad range of novel CRISPR effectors with unique properties, distinct protospacer adjacent motif (PAM) requirements, RNA-targeting capabilities, miniature architectures, and promiscuous cleavage activities that significantly expand the molecular biology toolkit. The development of CRISPR-based technologies such as base editing, prime editing, gene knock-in/out, and live-cell DNA/RNA imaging exemplifies the versatility of these systems. Despite the challenges associated with delivering complex Class 1 systems, both classes are now being actively harnessed across diverse microbial platforms. Concurrently, the CRISPR-Cas research, particularly for guide RNA (gRNA) design and activity prediction, has revolutionized target specificity and editing efficiency. This review presents a comprehensive overview of CRISPR-Cas system diversity, their genomic landscape in microorganisms, and their cutting-edge biotechnological applications. It also emphasizes the transformative potential of CRISPR in synthetic biology, therapeutics, diagnostics, environmental remediation, and agriculture, while also addressing the ethical and biosafety considerations surrounding its deployment. As CRISPR-Cas systems continue to evolve, they stand at the forefront of innovations that bridge natural microbial immunity with engineered precision tools for next-generation biotechnology.},
}
@article {pmid42189082,
year = {2026},
author = {Wu, Y and Jin, R and Lei, T and Liu, J and Chang, Y and Zhang, Z and Li, J and Liu, M},
title = {Aptamer-Coupled Droplet CRISPR/Cas12a Enables Ultrasensitive sPD-L1 Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {22},
pages = {16296-16305},
doi = {10.1021/acs.analchem.6c00779},
pmid = {42189082},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; *B7-H1 Antigen/blood ; Limit of Detection ; Lung Neoplasms/blood/diagnosis ; },
abstract = {Ultrasensitive detection of soluble programmed death-ligand 1 (sPD-L1) in peripheral blood is essential for early cancer diagnosis and immunotherapy monitoring. Conventional enzyme-linked immunosorbent assays lack the requisite sensitivity, whereas PCR quantifies nucleic-acid surrogates rather than the immunologically active protein. Here we report an aptamer-coupled droplet CRISPR/Cas12a (ADC) platform that integrates a structure-switching aptamer with picolitre droplet microfluidics to achieve femtomolar quantification of sPD-L1 within 70 min. Target binding with aptamer displaces a blocking sequence that activates Cas12a trans-cleavage, generating fluorescent droplets without preamplification. Confinement in picolitre droplets accelerates reaction kinetics through elevated local reagent concentrations and suppresses background fluorescence, collectively enhancing sensitivity. The assay exhibits a 0.5 pM limit of detection for sPD-L1, a dynamic range spanning 3 orders of magnitude, and 100% diagnostic accuracy in blinded plasma from lung cancer patients and healthy donors. The modular ADC architecture is readily adaptable to other protein biomarkers, offering a universal strategy for rapid, ultrasensitive liquid-biopsy analysis.},
}
@article {pmid42189993,
year = {2026},
author = {Sabol, AL and Mengiste, AA and Singh, P and Sreekanth, V and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, KE and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2536003123},
pmid = {42189993},
issn = {1091-6490},
support = {N66001-17-2-4055//DOD | ARPA | Defense Sciences Office, DARPA (DSO)/ ; R35GM136354//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM132825//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01DK132900//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; R01GM137606//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2330699//National Science Foundation (NSF)/ ; 587836-2024//Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; DRG-2539-24//Damon Runyon Cancer Research Foundation (DRCRF)/ ; P30-CA14051//HHS | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Directed Molecular Evolution/methods ; *CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics ; Mutation ; Adenoviridae/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Engineering CRISPR-Cas systems for improved or altered function is critical to both research and therapeutic applications. Unfortunately, most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1,000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns, the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}
@article {pmid42190245,
year = {2026},
author = {Choi, M and Byun, G and Kim, G and Yang, J and Seo, SW},
title = {Tunable Transcription-Level CRISPR Interference in Vibrio natriegens Using Engineered Single Guide RNAs.},
journal = {ACS synthetic biology},
volume = {15},
number = {6},
pages = {2416-2423},
doi = {10.1021/acssynbio.6c00057},
pmid = {42190245},
issn = {2161-5063},
mesh = {*Vibrio/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Lycopene/metabolism ; Metabolic Engineering/methods ; *Transcription, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Vibrio natriegens, a fast-growing bacterium, has emerged as a promising next-generation microbial platform for microbiology and biological engineering. While an expanding toolkit of genetic parts and genome engineering methods has been established, strategies for precise and predictable control of gene expression remain limited. Here, we report a dCas9-based tunable CRISPR interference (CRISPRi) system that enables multilevel transcriptional regulation in V. natriegens. By engineering the tetraloop and flanking regions of single-guide RNA (sgRNA), we constructed a synthetic sgRNA library that modulates the binding affinity between sgRNA and dCas9. The resulting sgRNA variants exhibited modular repression behavior across multiple protospacer targets. We further demonstrated the utility of this tunable CRISPRi system in metabolic engineering applications by redirecting intracellular carbon flux. Tunable repression of endogenous genes led to a 2.2-fold increase in 3-hydroxypropionic acid (3-HP) production and a 1.5-fold increase in lycopene production. Collectively, this work provides a simple and effective strategy for tunable gene regulation in V. natriegens and expands its potential as a versatile platform for the production of value-added chemicals.},
}
@article {pmid42190501,
year = {2026},
author = {Adams, BG and Wu, J},
title = {Imaging genome dynamics in real time with CRISPR-based technologies.},
journal = {Current opinion in chemical biology},
volume = {93},
number = {},
pages = {102700},
doi = {10.1016/j.cbpa.2026.102700},
pmid = {42190501},
issn = {1879-0402},
abstract = {Gene expression is a fundamental aspect of cellular function, driving diverse biological processes and disease. Dynamic interactions between genomic loci play an essential role in gene regulation. Therefore, visualizing the spatiotemporal dynamics of these interactions is vital to elucidating their function. CRISPR-Cas technology has enabled many powerful techniques for dynamic genome imaging. Recently, new methods for imaging single and multiple loci in live cells have been developed. This review describes the most recent advancements in CRISPR-based genome imaging, covering background reduction, signal amplification, and guide RNA tiling approaches. Fluorescence microscopy techniques complementing CRISPR-based imaging methods are also discussed.},
}
@article {pmid42190604,
year = {2026},
author = {Kim, JH and Cho, HJ and Lee, HM},
title = {Leveraging CRISPR/Cas9 for optimized adoptive T cell therapies: From molecular engineering to clinical manufacturing.},
journal = {Biochemical and biophysical research communications},
volume = {826},
number = {},
pages = {154003},
doi = {10.1016/j.bbrc.2026.154003},
pmid = {42190604},
issn = {1090-2104},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; *Gene Editing/methods ; *T-Lymphocytes/immunology/transplantation ; Animals ; *Neoplasms/therapy/immunology/genetics ; Receptors, Chimeric Antigen/genetics/immunology ; Genetic Engineering/methods ; },
abstract = {Cancer immunotherapy is rapidly evolving from pharmacologic immune modulation to adoptive cell therapy (ACT). In ACT, T cells are expanded and genetically engineered ex vivo to achieve long-lasting antitumor activity. The primary ACT platforms-tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR) T cells, and T-cell receptor (TCR) T cells-rely on T-cell effector function but differ in their mechanisms of antigen recognition, HLA dependence, and engineering requirements, leading to unique clinical strengths and limitations. CRISPR/Cas9 genome editing provides precise knock-out (KO) and knock-in (KI) strategies, allowing for multiplex editing and functional modulation across the genome. In the context of ACT manufacturing, CRISPR/Cas9 addresses critical challenges such as T-cell exhaustion, graft-versus-host disease (GvHD), and ensuring product consistency and quality. This article explores how CRISPR/Cas9 can be utilized to overcome the limitations of ACT and summarizes the current clinical landscape of CRISPR-engineered ACT products. Finally, we discuss the ongoing challenges associated with CRISPR-based genome editing and propose potential solutions.},
}
@article {pmid42190655,
year = {2026},
author = {Laub, S and Tulina, N and Hoffman, M and Faryean, JB and Ramachandran, S and Trang, K and Lewkiewicz, SM and Chesi, A},
title = {Integrative genomics and single-cell CRISPRi screening dissect Alzheimer GWAS non-coding variants regulating TSPAN14.},
journal = {American journal of human genetics},
volume = {113},
number = {6},
pages = {1253-1278},
pmid = {42190655},
issn = {1537-6605},
support = {R35 HG011959/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Alzheimer Disease/genetics ; *Genome-Wide Association Study ; *Tetraspanins/genetics ; *Genomics/methods ; Microglia/metabolism ; Polymorphism, Single Nucleotide/genetics ; Interleukin-8/genetics/metabolism ; Single-Cell Analysis/methods ; Cell Line ; Genetic Predisposition to Disease ; Interleukin-6/genetics/metabolism ; Astrocytes/metabolism ; Enhancer Elements, Genetic ; *CRISPR-Cas Systems/genetics ; Neurons/metabolism ; },
abstract = {Genome-wide association studies (GWASs) have uncovered many associations for human complex diseases, but functional dissection of the discovered loci has lagged behind. We present a variant-to-gene (V2G) mapping effort for Alzheimer disease (AD) leveraging the most recent AD GWAS meta-analyses. In this study, we integrated ten brain-relevant genomics datasets-including promoter Capture C, ATAC-seq, and RNA-seq from microglia, neurons, and astrocytes-to fine-map AD GWAS variants and identify effector genes. We then performed a single-cell CRISPRi Perturb-seq screen targeting 74 candidate regulatory regions in the human microglial cell line HMC3. Our V2G mapping effort identified 93 candidate causal variants and 94 effector genes (72 coding) for 35 AD loci. Our CRISPRi screen across ∼97,000 cells validated 21 variant-gene pairs. We showed that an intronic region at the TSPAN14 locus containing rs7080009, rs1870138, and rs1870137 is a microglial-specific enhancer activated by the AD-risk haplotype. CRISPR-mediated deletion of this region reduced TSPAN14 expression, disrupted cell-adhesion pathways, and lowered secretion of pro-inflammatory cytokines interleukin 6 (IL-6) and IL-8. Our study provides a systematic framework for mapping GWAS signals to effector genes in a cell-type-specific manner and identifies robust leads for in-depth functional investigations.},
}
@article {pmid42190658,
year = {2026},
author = {Hoshino, M and Nehlsen, M and Batista, RA and Raphalen, M and Wakimoto, T and Uwai, S and Kogame, K and Alva, V and Coelho, SM},
title = {PKN is a sex- and species-specific fertilization factor in brown algae.},
journal = {Current biology : CB},
volume = {36},
number = {11},
pages = {2866-2878.e5},
doi = {10.1016/j.cub.2026.04.065},
pmid = {42190658},
issn = {1879-0445},
mesh = {*Phaeophyceae/physiology/genetics ; *Fertilization/physiology ; Species Specificity ; *Algal Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Reproductive Isolation ; },
abstract = {Fertilization, the fusion of male and female gametes, is fundamental to sexual reproduction, yet the molecular mechanisms that mediate gamete recognition and enforce species specificity remain poorly understood, and only a handful of proteins are known to act as core fertilization factors across eukaryotes. Here, we identify PICKINESS-ASSOCIATED PROTEIN (PKN), a female gamete-specific transmembrane protein, as an essential determinant of fertilization in brown algae. CRISPR-Cas-mediated knockout of PKN abolishes successful male-female gamete interactions and prevents fertilization without affecting earlier mating behaviors, such as gamete attraction. Remarkably, PKN also enforces reproductive isolation by preventing interspecific fertilization, establishing it as a molecular gatekeeper of species specificity. Structural analyses reveal extracellular β-propeller and mucin-like domains enriched in predicted glycosylation sites and displaying rapid sequence evolution. Functional and comparative analyses suggest that PKN-dependent recognition mechanisms are conserved across diverse brown algal lineages. Because PKN originated within brown algae, its dual role in mediating both male-female gamete recognition and species-specific fertilization reveals a striking conceptual parallel with fertilization factors described in animals, suggesting that evolution repeatedly converges on lineage-specific gamete-expressed membrane proteins as key arbiters of reproductive recognition.},
}
@article {pmid42190792,
year = {2026},
author = {Zhang, YH and Yuan, Y and Chen, BT and Yang, N and Chen, TJ and Lin, YT and Na, XM and Wang, SH and Xiong, YN and Zhu, MX and Chen, LZ and Mokwatlo, SC and Ouyang, P and Ling, C},
title = {Engineering complex phenotypes in Halomonas bluephagenesis TD01 via large-fragment manipulation and multiplex base editing.},
journal = {Metabolic engineering},
volume = {96},
number = {},
pages = {405-419},
doi = {10.1016/j.ymben.2026.05.008},
pmid = {42190792},
issn = {1096-7184},
mesh = {*Halomonas/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Polyhydroxyalkanoates/biosynthesis/genetics ; },
abstract = {Halomonas bluephagenesis is a representative platform strain of next generation industrial biotechnology (NGIB), enabling contamination-resistant open fermentation due to inherent tolerance to high salinity and alkalinity. However, progress in strain development has been constrained by limited genome engineering tools, particularly for large-fragment manipulation and multiplex base editing. Herein, we developed a counterselection marker-based single-plasmid system (pHaloFM) that leverages native homologous recombination to enable sequential insertion of fragments up to 8 kb and deletion of regions up to 50 kb. Additionally, we re-engineered a CRISPR/nCas9-assisted cytidine base editor system (pHaloBE) through host-specific adaptations, achieving multiplex editing of nine target sites. These tools were applied to engineer cellular morphology in one step, and successively construct polyhydroxyalkanoate (PHA) copolymers P34HB and PHBV biosynthetic pathways. This integrated toolkit resolves long-standing genetic manipulation bottlenecks in H. bluephagenesis and provides a systematic framework for engineering complex phenotypes in other non-model organisms.},
}
@article {pmid42191086,
year = {2026},
author = {Lu, Y and Zhong, S and Chen, L and Li, Z and Zhang, M and Wang, Y and Cao, J},
title = {CRISPR/Cas9-induced chemR23 knock-out improves arterial inflammation in atherosclerotic mice.},
journal = {Experimental cell research},
volume = {460},
number = {2},
pages = {115084},
doi = {10.1016/j.yexcr.2026.115084},
pmid = {42191086},
issn = {1090-2422},
mesh = {Animals ; *Atherosclerosis/pathology/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Knockout ; Mice, Inbred C57BL ; *Inflammation/pathology/genetics/metabolism ; Male ; Macrophages/metabolism/pathology ; Plaque, Atherosclerotic/pathology/genetics ; Disease Models, Animal ; Lipid Metabolism/genetics ; Diet, High-Fat/adverse effects ; *Receptors, Chemokine/genetics ; },
abstract = {BACKGROUND AND PURPOSE: Emerging evidence implicates chemerin, a chemoattractant protein, in the pathogenesis of atherosclerosis (AS). Yet, the role of its receptor, Chemokine-Like Receptor 1 (ChemR23), in AS remains enigmatic. Leveraging CRISPR/Cas9 genome editing, this study delineates the impact of ChemR23 ablation in an AS murine model, aiming to unravel its mechanistic involvement in AS pathophysiology.<br><br>METHODS: Employing CRISPR/Cas9, we orchestrated a targeted knockout of the ChemR23 gene in C57BL/6J wild-type (WT) mice, followed by an eight-month high-fat dietary regimen. High-fat diet-fed WT mice and their standard diet counterparts constituted the experimental and baseline cohorts, respectively. We deployed commercial assay kits to quantify lipid metabolism markers and inflammatory mediators. Histopathological changes in arterial plaques and lipid deposition were appraised using Hematoxylin &amp; Eosin and Oil Red O staining. Western blotting, Immunohistochemistry, and Immunofluorescence staining were harnessed to interrogate plaque inflammatory signaling and autophagy-related protein expression. Macrophage polarization dynamics were dissected via flow cytometry, and foam cell gene expression profiles were ascertained through quantitative real-time PCR.<br><br>RESULTS: ChemR23 knockout conferred ameliorative effects on lipid metabolic aberrations and plaque stability in AS mice, evidenced by diminished lipid accrual in plaques. It attenuated the activation of the NF-&#x3ba;B inflammatory cascade, fostered an M2 macrophage polarization bias, and impeded the macrophage-to-foam cell transition. Notably, ChemR23 ablation suppressed autophagic activities within the plaques.<br><br>CONCLUSION: Targeted ChemR23 gene disruption in mice manifests as a modulator of inflammatory and autophagic pathways, thereby mitigating AS exacerbation. This positions ChemR23 as a promising molecular candidate for strategic AS therapeutics.},
}
@article {pmid42191275,
year = {2026},
author = {Vuong, HL and Thi Thu Le, H},
title = {Advances in protein engineering.},
journal = {International review of cell and molecular biology},
volume = {402},
number = {},
pages = {61-87},
doi = {10.1016/bs.ircmb.2025.11.002},
pmid = {42191275},
issn = {1937-6448},
mesh = {*Protein Engineering/methods ; Humans ; Animals ; Synthetic Biology ; CRISPR-Cas Systems/genetics ; Proteins/genetics/chemistry ; },
abstract = {Protein engineering (PE) has been applied to various medicines, food, and environments. Contributions of proteins have been reported with remarkable results in protein therapeutics, antibody engineering, enzyme synthesis, and more specific functions in industrial processes. Therefore, this chapter highlights the most recent PE advances in a battle against mainly human diseases and biomedical sciences. The application of PE will be reviewed, focusing on developing innovative techniques, including evolution, rational design, semi-rational design, and hybrid approaches to protein design in applications. In addition, we provide key achievements of PE in CRISPR/Cas systems, high-through data, and synthetic biology with updated results. Current challenges of using PE, ethical considerations, and various approaches for protein therapeutics are also discussed. In this chapter, the updated findings provide a comprehensive overview of the transformative potential of PE for researchers in the application areas of human disease, especially in cancer therapeutics.},
}
@article {pmid42193394,
year = {2026},
author = {Siringan, MJ and Chen, X and Huo, J},
title = {RNA-Loaded Nanoparticles for Targeted Lung Delivery.},
journal = {Biomedicines},
volume = {14},
number = {5},
pages = {},
pmid = {42193394},
issn = {2227-9059},
abstract = {The lung represents a promising yet underexploited target for RNA therapeutics due to its large surface area and accessibility via non-invasive inhalation delivery. Despite rapid advances in RNA-based modalities, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and CRISPR-Cas systems, efficient pulmonary delivery remains a major challenge. Multiple biological barriers, such as mucus and surfactant layers, mucociliary clearance, immune surveillance, and limited cellular uptake of negatively charged nucleic acids, significantly restrict therapeutic efficacy. In addition, aerosolization processes may introduce mechanical stress, compromising RNA integrity. Nanoparticle-based delivery systems have emerged as a central strategy to address these limitations. By protecting RNA cargo, enhancing mucus penetration, and promoting cellular internalization, engineered nanoparticles enable more effective pulmonary delivery. In this review, we adopt a barrier-centered perspective to examine the key biological obstacles to lung-targeted RNA delivery and highlight recent advances in nanoparticle-mediated strategies, with a focus on lipid nanoparticles, polymeric systems, and inorganic nanomaterials. We further discuss design principles that govern RNA stability, transport, and intracellular release and critically compare the strengths, limitations, and translational potential of each platform, including considerations of toxicity, biodegradability, and clinical readiness. Finally, we outline emerging clinical applications of RNA-loaded nanoparticles, using lung cancer as a representative disease model, and discuss remaining challenges and future directions. Continued innovation in nanoparticle engineering and delivery strategies is expected to accelerate the clinical translation of RNA therapeutics for pulmonary diseases.},
}
@article {pmid42193866,
year = {2026},
author = {Siddika, A and Rousseau, J and Veillette, F and Bouchard, C and Lu, Y and Tremblay, JP},
title = {Gene Editing Strategies for Duchenne Muscular Dystrophy: From Molecular Mechanisms to Clinical Translation.},
journal = {Cells},
volume = {15},
number = {10},
pages = {},
pmid = {42193866},
issn = {2073-4409},
support = {Defeat Duchenne Foundation (53320215); VCGS (492510)//Defeat Duchenne Foundation ; VCGS (CIHR)/ ; },
mesh = {*Muscular Dystrophy, Duchenne/genetics/therapy ; Humans ; *Gene Editing/methods ; *Translational Research, Biomedical ; Animals ; *Genetic Therapy/methods ; Dystrophin/genetics ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) remains a major challenge in genetic medicine due to the difficulty of achieving durable, body-wide restoration of dystrophin in post-mitotic muscle tissues. Although current therapies-including exon skipping and micro-dystrophin gene replacement-have demonstrated clinical feasibility, their benefits are limited by incomplete efficacy, mutation specificity, and the need for repeated or high-dose interventions. These limitations highlight the need for strategies capable of directly and permanently correcting the underlying genetic defect. Recent advances in genome editing have positioned CRISPR-based technologies as promising candidates for this objective. Rather than functioning as a single approach, gene-editing platforms encompass a spectrum of strategies-including exon deletion, exon reframing, base editing, and prime editing-each with distinct advantages depending on the mutational context. In particular, the emergence of precision editing tools has enabled controlled nucleotide-level modifications, expanding the range of correctable mutations while reducing reliance on double-strand DNA breaks. In this review, we adopt a comparative and translational perspective to evaluate gene-editing strategies for DMD. We examine how different approaches align with specific mutation types, summarize key findings from preclinical studies, and analyze the major barriers to clinical implementation, including delivery efficiency, immune responses, editing durability, and genomic safety. We further discuss emerging innovations in editing technologies and delivery systems that aim to address these limitations. Collectively, this work reframes gene editing as a decision-oriented and application-driven therapeutic framework. Continued integration of advances in genome engineering, delivery platforms, and muscle biology will be essential to translate these technologies into safe, effective, and durable treatments capable of altering the clinical trajectory of DMD.},
}
@article {pmid42193938,
year = {2026},
author = {Silva, FRD and Dias, PRF and Pavan, ICB and Oliveira, AP and Basei, FL and Santos, LED and Sousa, LM and Consonni, SR and Oliveira, AG and Silveira, LR and Kobarg, J},
title = {NEK6 Knockout Causes Defects in Mitochondrial Morphology and Respiration.},
journal = {Cells},
volume = {15},
number = {10},
pages = {},
pmid = {42193938},
issn = {2073-4409},
mesh = {Humans ; *Mitochondria/metabolism/ultrastructure ; *NIMA-Related Kinases/metabolism/genetics/deficiency ; *Gene Knockout Techniques ; Cell Respiration ; Cell Line, Tumor ; CRISPR-Cas Systems ; },
abstract = {The family of Nek kinases has 11 human members that are conserved in their kinase domains but diverse in their regulatory domains. Functionally, they can be associated with diverse aspects of cell cycle regulation, from mitosis and primary cilia function to centrosome disjunction in the G2 phase and checkpoints of the DNA damage response. However, novel functional contexts have emerged in recent years, including regulatory roles of Neks 1, 4, 5, and 10 in mitochondrial metabolic and morphological homeostasis. We recently generated, by CRISPR-Cas9 technology, a DU-145 prostate cancer cell line, with an NEK6 gene knockout. Here, we focus on a detailed characterization of changes in this cell line, in mitochondrial respiration function and morphology. DU-145 NEK6 knockout cells exhibited reduced mitochondrial respiration and a fragmented phenotype in electron microscopy, with reduced mitochondrial cristae numbers. Alterations in mitochondrial architecture and respiration were correlated with increased expression of anaerobic glycolytic proteins (HK2, PFKP, and LDHA) and decreased expression of PDH, an enzyme of aerobic glycolysis. Molecular analysis by Western blot revealed decreased levels of mitochondrial mass and biogenesis protein markers (TOM20, TFAM), without alterations in other markers such as VDAC1/3 or mtDNA copy number in the NEK6 knockout cells. Furthermore, the regulators of mitochondrial fusion/fission are altered in the knockout cells (decrease in the Long-OPA1:Short-OPA1 ratio and DRP1 total level), which is associated with an increase in endoplasmic reticulum-mitochondria contact at ≤20 nm observed in transmission electron microscopy (TEM) image analysis. Using analysis of TEM micrographs, we found an increase in the autophagic structures (autophagosome, amphisome, and autolysosome), with mitochondria as cargo in some structures, which was correlated with a decrease in LC3A/B and an increase in the BECLIN1 total level, and with an increase in acidic vesicles approximation, suggesting that reduction in TOM20 and TFAM without alterations in VDAC1/3 and mtDNA copy number might be related to mitochondrial degradation through autophagy. Together, our data suggest a new role for NEK6 in regulating mitochondrial homeostasis, where its loss alters mitochondrial morphology and respiration, and could be associated with an increase in the degradation of the dysfunctional mitochondria through autophagy.},
}
@article {pmid42196146,
year = {2026},
author = {Kim, S and Kim, GN and Jeong, YJ and Cho, J and Jang, M and Hong, J and Sung, YH},
title = {CRISPR-Cpf1-Mediated Gene-Editing System Based on a Single Bidirectional Promoter.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
pmid = {42196146},
issn = {1422-0067},
support = {2018R1A2B6002192//Ministry of Science and ICT/ ; 20012477//Ministry of Trade, Industry and Energy/ ; RS-2023-00283544//Korea Drug Development Fund/ ; },
mesh = {*Promoter Regions, Genetic ; *Gene Editing/methods ; Dependovirus/genetics ; *CRISPR-Cas Systems ; Animals ; Genetic Vectors/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Endonucleases/genetics ; Acidaminococcus/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Recent advances in gene therapy have highlighted the potential of CRISPR-based gene-editing systems combined with adeno-associated virus (AAV) vectors. However, the limited packaging capacity of AAV remains a significant challenge for the simultaneous expression of Cas effector proteins and guide RNAs within a single vector. To address this limitation, we developed a compact AAV vector that enables the co-expression of Acidaminococcus sp. Cas12a (AsCpf1) and CRISPR RNAs (crRNAs) using a single bidirectional promoter derived from the mouse H1 promoter. Our single bidirectional H1 promoter supported indel formation comparable to that achieved by dual-promoter systems and facilitated scalable genome editing with single-, dual-, and triple-target configurations. Genome editing was successfully accomplished both in vitro and in vivo following AAV delivery. This study shows that our engineered compact AAV vector platform is capable of simultaneously delivering AsCpf1 and multiplexed crRNAs.},
}
@article {pmid42196223,
year = {2026},
author = {Ma, S and Li, Y and Fei, T},
title = {CRISPR Screening in Hepatocellular Carcinoma: From Tumor Progression to Immune Evasion and Therapeutic Resistance.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
pmid = {42196223},
issn = {1422-0067},
support = {2023A1515140084//Guangdong Basic and Applied Basic Research Foundation/ ; 32470673//National Natural Science Foundation of China/ ; B16009//the 111 Project/ ; 2022JH13/10200026//the Construction Project of Liaoning Provincial Key Laboratory, China/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/therapy/immunology/pathology ; *Liver Neoplasms/genetics/immunology/therapy/pathology ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems ; Animals ; Disease Progression ; *Tumor Escape/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Immune Evasion/genetics ; },
abstract = {Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and a leading cause of cancer-related mortality worldwide. Despite advances in targeted therapies and immunotherapies, clinical outcomes remain poor owing to profound molecular heterogeneity, intrinsic therapeutic resistance, and complex immune evasion mechanisms. Although genomic profiling has identified recurrent alterations in HCC, large-scale functional validation of candidate drivers and vulnerabilities remains challenging. CRISPR (clustered regularly interspaced short palindromic repeats)-based screening technologies have transformed this landscape by enabling systematic interrogation of gene function in physiologically relevant contexts. In this review, we summarize recent studies that have applied CRISPR screening approaches in HCC research. These efforts have uncovered multilayered dependency programs that govern ferroptosis resistance, metabolic reprogramming, epigenetic regulation, tumor suppressor networks, immune evasion, and resistance to targeted therapies. We also discuss the major limitations of current studies, including model bias, incomplete representation of HCC heterogeneity, and technical constraints intrinsic to pooled screening. Overall, integration of CRISPR screening with patient-derived models, single-cell readouts, and precision editing technologies is expected to accelerate mechanistic discovery and biomarker-guided therapeutic prioritization for HCC.},
}
@article {pmid42196225,
year = {2026},
author = {Zaman, W and Park, S},
title = {Advances in Functional Genomics and Biotechnology for Enhancing Therapeutic Potential of Medicinal Plants.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
pmid = {42196225},
issn = {1422-0067},
mesh = {*Plants, Medicinal/genetics/metabolism ; *Biotechnology/methods ; *Genomics/methods ; Metabolic Engineering/methods ; Synthetic Biology/methods ; Gene Editing ; CRISPR-Cas Systems ; Humans ; },
abstract = {Medicinal plants have long served as a primary source of bioactive compounds with essential therapeutic applications. Recent advances in functional genomics and plant biotechnology now enable precise manipulation of metabolic pathways to enhance the production of specialized metabolites with medicinal value. However, an integrative understanding of how genomic discovery can be linked with pathway engineering, scalable production systems, and healthcare applications remains insufficiently developed. This knowledge gap limits the effective translation of molecular insights into the sustainable production of medicinally important compounds. The novelty of this review lies in its integrated framework linking functional genomic discovery with pathway engineering, synthetic biology, artificial intelligence-assisted prediction, and scalable production systems for medicinal plant-derived therapeutics. This review aims to provide a comprehensive overview of cutting-edge approaches in medicinal plant research, emphasizing high-throughput RNA sequencing, CRISPR/Cas9 gene editing, synthetic biology, and metabolic engineering for optimizing the production of key bioactive compounds, including artemisinin, cannabinoids, ginsenosides, and taxol. It further examines how these tools collectively support metabolite discovery, pathway elucidation, yield improvement, and biotechnological production in major medicinal plant systems. We explore the application of genomic and biotechnological approaches in plants such as Artemisia annua, Cannabis sativa, Panax ginseng, and Taxus baccata to enhance metabolite yields and promote sustainable production. The review highlights case studies that demonstrate how genetic modification, metabolic engineering, and synthetic pathway design have been successfully employed to increase the synthesis of key medicinal compounds. Moreover, we discuss the integration of artificial intelligence and machine learning to predict gene-metabolite relationships, support personalized phytochemical therapies, and facilitate sustainable, large-scale production. Finally, the review addresses the implications of these innovations for the pharmaceutical industry, healthcare, and agriculture, while also highlighting sustainable and scalable directions for future medicinal plant biotechnology.},
}
@article {pmid42196288,
year = {2026},
author = {De, N and Bhadra, J and Momin, MSA and Mitra, K and Bhunia, D and Sannigrahi, A},
title = {Therapeutic Innovations for Monkeypox Inhibition.},
journal = {International journal of molecular sciences},
volume = {27},
number = {10},
pages = {},
pmid = {42196288},
issn = {1422-0067},
support = {EEQ/2022/000548//Science and Engineering Research Board/ ; },
mesh = {Humans ; *Antiviral Agents/therapeutic use/pharmacology ; Peptide Nucleic Acids/therapeutic use ; Animals ; CRISPR-Cas Systems ; Biocompatible Materials/therapeutic use ; },
abstract = {This review investigates biomaterial-based strategies for improved treatment of MPXV. We focus on emerging synthetic biomedical approaches to combating the virus. These include peptide nucleic acids, CRISPR-based systems, and small-molecule therapeutics. These methods work by targeting and blocking viral proteins and enzymes. Such synthetic platforms may help reduce viral transmission and minimize side effects. They also offer potential solutions to challenges such as viral resistance in humans. In addition, biomaterials contribute to the development of more stable and effective vaccines. Combining these biomaterials with mRNA technology provides a promising framework for future vaccine development. Overall, this review underscores biomaterial-driven antiviral systems as a major frontier in translational medicine with profound implications for global health and pandemic awareness.},
}
@article {pmid42198588,
year = {2026},
author = {Camacho-Aguilar, P and Delgado-Nungaray, JA and Reynaga-Delgado, E and Gonzalez-Reynoso, O and Rodriguez-Anaya, LZ and Muñoz Miranda, LA and Rincón Enríquez, G and Higuera-Ciapara, I and Figueroa-Yáñez, LJ},
title = {New Insights into CRISPR-like Arrays in Helicobacter pylori: An Exploratory Analysis from Genomic Data.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {5},
pages = {},
pmid = {42198588},
issn = {2076-0817},
support = {1309039//SECIHTI/ ; 1267568//SECIHTI/ ; },
mesh = {*Helicobacter pylori/genetics ; *Genome, Bacterial ; Genomic Islands ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics ; Genomics/methods ; Antigens, Bacterial/genetics ; Computational Biology/methods ; CRISPR-Cas Systems ; Humans ; Helicobacter Infections/microbiology ; },
abstract = {Helicobacter pylori (H. pylori) is a highly adaptable gastric pathogen with marked genomic plasticity. Whilst functional CRISPR-Cas systems provide adaptive immunity in many bacteria, they have not been identified in H. pylori, unlike CRISPR-like sequences. In this study, eight H. pylori genomes were analysed using the bioinformatics tools CRISPRCasFinder, CRISPRCasTyper, and CRISPRloci. A total of 25 CRISPR-like arrays were identified, showing high conservation (88%) both between and within strains, suggesting that these arrays are not random remnants but rather organised structures possibly involved in cellular processes. Notably, a structural association was observed between the CRISPR-like sequences and the cag pathogenicity island (CagA-PAI). Conversely, CagA-PAI instability in specific strains was observed in the presence of the TnpA and TnpB transposons. Furthermore, in strain 29CaP, CRISPR-like assemblies were located in genomic proximity to the prophage Helico 1961P, leading to the hypothesis of a compensatory or regulatory effect in the absence of CagA-PAI. Taken together, these findings indicate that CRISPR-like arrays in H. pylori characterise a genomic architecture within regions of high plasticity. This study provides a solid exploratory foundation for future functional research on the adaptive and pathogenic evolution of H. pylori.},
}
@article {pmid42198599,
year = {2026},
author = {Abdulrahman, B and Rahimi Aqdam, S and Mosca, M and Ahmed-Hassan, H and Razcon-Echeagaray, M and Popa, L and Gilch, S and Babelhadj, B and Vaccari, G and Schätzl, HM},
title = {A Neuronal Cell Line Model for Studying Camel Prions.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {5},
pages = {},
pmid = {42198599},
issn = {2076-0817},
support = {R01 NS121016/NS/NINDS NIH HHS/United States ; 201900008//Alberta Innovates/ ; 201600009//Alberta Innovates/ ; },
mesh = {Animals ; *Camelus ; *Neurons/metabolism/pathology ; Cell Line ; *Prion Diseases/metabolism/pathology/veterinary ; *Prions/metabolism/genetics ; Mice ; Brain/pathology/metabolism ; Gene Knockout Techniques ; },
abstract = {Prion diseases are fatal neurodegenerative disorders that affect humans and animals, caused by the conformational conversion of the normal cellular prion protein (PrP[C]) into its misfolded, infectious isoform PrP[Sc]. Recently, camel prion disease (CPrD) was identified in dromedary camels (Camelus dromedarius) in Algeria. Due to the potential implications for animal and human health, as well as the possible socio-economic impact in Mediterranean regions where camels play a pivotal role as a source of food, in-depth characterization of camel prions is important to increase our understanding of camel prion disease. We developed a neuronal cell line model for studying the molecular features of camel prion infection. We genetically edited mouse neuronal CAD5 cells to generate CAD5 PrP knockout (KO) cells. We then used lentiviral transduction to generate CAD5 cells expressing camel PrP (CAD5-camel-PrP). Following infection of these cells with a CPrD-positive camel brain homogenate, we observed PrP[Sc] signals at various passages, as indicated by immunoblotting analysis. RT-QuIC (Real-Time Quaking-Induced Conversion) assays further supported these findings, demonstrating transient prion conversion activity in the CPrD-infected CAD5-camel-PrP cells. Taken together, our data describe the first neuronal cell line permissive to camel prion infection, a novel in vitro tool for mechanistic studies of camel prion disease.},
}
@article {pmid42199442,
year = {2026},
author = {Xia, L and Tang, C and Tong, D and He, Q},
title = {Research advances in the application of microfluidic chip technology for rapid detection of antibiotic-resistant bacteria.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1819856},
pmid = {42199442},
issn = {2235-2988},
mesh = {*Bacteria/drug effects/isolation &amp; purification/genetics ; Humans ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Microfluidics/methods/instrumentation ; Microbial Sensitivity Tests/methods ; *Microfluidic Analytical Techniques/methods ; Rapid Diagnostic Tests ; *Lab-On-A-Chip Devices ; *Bacterial Infections/diagnosis/microbiology ; Point-of-Care Systems ; },
abstract = {The escalating global burden of antimicrobial resistance (AMR) necessitates diagnostic strategies that can overcome the limitations of conventional culture-based methods, which often require several days to generate clinically actionable results. Such delays are associated with increased mortality, inappropriate antibiotic use, and continued transmission of resistant pathogens. In this context, microfluidic chip technology has emerged as a promising platform for rapid, miniaturized, and increasingly automated point-of-care diagnostics. Recent advances have enabled integrated lab-on-a-chip systems that combine bacterial isolation, phenotypic antimicrobial susceptibility testing, and genotypic resistance detection within closed and self-contained architectures, thereby reducing contamination risk and operator dependence. In addition, these platforms are increasingly capable of operating at single-cell resolution, allowing the detection of heteroresistance and resistant subpopulations that may be overlooked by conventional bulk assays. A major advantage of microfluidic systems is their ability to bridge phenotypic and genotypic diagnostics by enabling real-time monitoring of bacterial growth, metabolic activity, and morphological responses to antibiotics while simultaneously incorporating on-chip nucleic acid amplification for resistance gene detection. This integrated approach improves the interpretation of discrepancies between genetic determinants and functional resistance. Studies to date have demonstrated high sensitivity and specificity in complex clinical matrices, including blood, urine, and sputum, with turnaround times reduced from days to less than one hour in some applications. Furthermore, the integration of CRISPR-Cas systems, nanomaterial-enhanced biosensing, and machine learning has further improved analytical performance and data interpretation. Nevertheless, important translational challenges remain, including scalable manufacturing, regulatory standardization, and integration into routine clinical workflows. Future microfluidic platforms are expected to support multiplexed, intelligent antimicrobial susceptibility testing capable of simultaneous pathogen identification, resistance profiling, and therapeutic guidance, thereby advancing precision diagnostics for AMR management.},
}
@article {pmid42199538,
year = {2026},
author = {Wizrah, MSI},
title = {CRISPR-Cas systems as next-generation antimicrobials: a systemic review of mechanisms, delivery strategies, and translational challenges.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1747931},
pmid = {42199538},
issn = {1664-302X},
abstract = {INTRODUCTION: The rapid global increase in multidrug-resistant (MDR) bacteria has compromised the effectiveness of conventional antibiotics, stressing the urgent need for alternative antimicrobial strategies. CRISPR-Cas systems, originally evolved as bacterial adaptive immune mechanisms, provide programmable and highly specific tools for targeting antimicrobial resistance (AMR) determinants.<br><br>OBJECTIVE: This systematic review aims to evaluate the antibacterial mechanisms, delivery strategies, preclinical evidence, safety considerations, and translational potential of CRISPR-Cas systems for combating MDR bacterial infections.<br><br>METHODS: A systematic literature search was conducted in PubMed, Scopus, Cochrane Library, and Web of Science up to January 2026 in accordance with PRISMA 2020 guidelines. Eligible studies included original in vitro and in vivo experimental or preclinical investigations assessing CRISPR-Cas systems (Cas9, Cas12, Cas13, or related effectors) for antibacterial activity or antibiotic resensitization. Data were extracted on CRISPR effector type, bacterial target, delivery platform, and therapeutic outcome. Due to methodological heterogeneity, results were synthesized narratively.<br><br>RESULTS: Most studies reported effective killing or resensitization of MDR bacteria through chromosomal double-strand break induction, resistance plasmid curing, integron disruption, or RNA-targeted cleavage. Cas9 was the most frequently employed effector, followed by Cas12 and Cas13. Delivery strategies included bacteriophages, conjugative plasmids, and nanoparticle-based systems, with phage-mediated delivery demonstrating the most consistent efficacy in complex environments and animal models. Notably, a CRISPR-enhanced engineered bacteriophage cocktail (LBP-EC01) has advanced to clinical evaluation.<br><br>DISCUSSION: Overall, the evidence supports CRISPR-Cas antimicrobials as a promising precision-based approach for addressing AMR. However, major barriers remain, including limited host range, instability in physiological environments, emergence of escape mutations, and insufficient data on off-target effects and long-term safety. Addressing these challenges through optimized delivery platforms, multiplex targeting strategies, and standardized safety and regulatory frameworks will be essential for clinical translation.<br><br>https://www.crd.york.ac.uk/PROSPERO/view/CRD420261319789, identifier CRD4201319789.},
}
@article {pmid42201448,
year = {2026},
author = {Abuhassan, Q and Al-Ameer, HJ and Gajjar, TB and Hanumanthayya, M and Shukla, SK and Panigrahi, R and Bainsal, N and Khaydarova, D},
title = {CRISPR/Cas‑driven biosensing: molecular mechanisms and advances in diagnostics.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42201448},
issn = {1573-4978},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; DNA/genetics ; Electrochemical Techniques/methods ; },
abstract = {The advent of CRISPR-based technologies for DNA diagnostics represents a new epoch in providing technologies for DNA sequence-specific detection of any DNA target. By using the programmable, precise targeting capabilities associated with Cas effector proteins (e.g., Cas9 and Cas12), scientists have been able to repurpose these proteins with their inherent properties: high programmability, single base specificity, and collateral cleavage activity to develop novel, highly capable biosensors for DNA analysis. Unlike previous reviews, this work provides a systematic and mechanism-based classification of CRISPR/Cas biosensors, highlighting recent advances beyond conventional descriptive summaries. This review provides a comprehensive overview of the rapid evolution and application of CRISPR/Cas-based biosensors as a novel strategy for detecting a wide range of human health biomarkers, from nucleic acids to proteins and small molecules. First, we describe the principle of CRISPR/Cas system. Then, we critically analyze and compare the integration of CRISPR/Cas systems with distinct signal transduction strategies, with a dedicated focus on optical (photoelectrochemical, electrochemiluminescence, and fluorescence) and electrochemical readout platforms. Key technological breakthroughs, including ultra-sensitive detection in the attomolar-femtomolar range, advanced amplification strategies (e.g., RCA and EXPAR), and multiplex detection capabilities, are highlighted. Finally, we emphasize the clinical relevance, scalability challenges, and translational potential of these platforms, providing insights into their application in early disease diagnosis, real-time monitoring, and point-of-care testing. Overall, this review offers a critical perspective on current limitations and future directions, positioning CRISPR-based biosensors as promising tools for next-generation precision diagnostics and improved global health outcomes.},
}
@article {pmid42201536,
year = {2026},
author = {Chen, X and Zhan, B and Shi, R and Chen, J and Lin, Z and Li, Z},
title = {Identification of Closantel as a small-molecule inhibitor of the compact CRISPR-Cas RNA editor Cas13bt3.},
journal = {Molecular diversity},
volume = {},
number = {},
pages = {},
pmid = {42201536},
issn = {1573-501X},
support = {32471255//National Natural Science Foundation of China/ ; 2024J02006//Natural Science Foundation of Fujian Province/ ; },
abstract = {The type VI CRISPR-Cas systems are widely employed for programmable RNA editing, and the ultra-compact Cas13bt3 ribonuclease offers particular advantages for cellular delivery due to its minimal molecular size. However, its therapeutic potential is hindered by nonspecific collateral RNA cleavage activity and the lack of small-molecule inhibitors to enable spatiotemporal regulation of its function. Here, we performed a high-throughput screen for Cas13bt3 inhibitors using a fluorescence resonance energy transfer (FRET)-based RNA cleavage assay. From a library of 17,760 compounds, we identified Closantel as a specific Cas13bt3 inhibitor, with an IC50 of 7.48 µM. Biochemical assays confirmed that Closantel abrogates both on-target and collateral RNA cleavage by Cas13bt3, while exerting negligible inhibitory activity against Cas13a, a closely related Cas13 ortholog. Combined molecular docking and electrophoretic mobility shift assay (EMSA) analyses further revealed that Closantel binds to the cavity of Cas13bt3 that accommodates the direct repeat region of crRNA, thereby competitively interfering with crRNA-Cas13bt3 binding. Finally, to minimize nonspecific RNA cleavage of Cas13bt3, we engineered a K748A mutant that retains robust on-target RNA cleavage activity with reduced collateral activity in vitro. Our findings provide a selective small-molecule chemical probe for Cas13bt3 and an optimized variant with improved targeting precision, collectively advancing the utility of Cas13bt3 for precise RNA editing applications.},
}
@article {pmid42201952,
year = {2026},
author = {Shen, Y and Yeung, AT and Wang, B and Yeh, CT and Ditchfield, P and Korn, E and Han, C},
title = {Tuning mitotic recombination with patterned DNA nicks for precision mosaic analysis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2531265123},
pmid = {42201952},
issn = {1091-6490},
support = {R24 OD031953/OD/NIH HHS/United States ; R24OD031953//HHS | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *Mitosis/genetics ; *Mosaicism ; CRISPR-Cas Systems ; *Recombination, Genetic ; Drosophila/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Deoxyribonuclease I/genetics/metabolism ; Drosophila melanogaster/genetics ; Crossing Over, Genetic ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR/Cas9-based mosaic analysis is a powerful tool for in vivo genetics but is limited by cytotoxicity and mutagenesis associated with DNA double-strand breaks. Here, we establish Cas9-derived nickases as safer and more reliable alternatives for inducing mitotic recombination in Drosophila. We demonstrate that single-strand nicks are sufficient to generate mosaic clones and systematically dissect the parameters governing this process. We find that clone frequency can be controlled by the gRNA nicking pattern, with two distant nicks on the same DNA strand synergistically enhancing recombination by over ninefold compared to a single nick. Based on these findings, we propose a mechanistic model for nick-induced crossover and provide a versatile toolkit for generating tissue-specific nickases. This work establishes nickase-based mosaic analysis by gRNA-induced crossing-over as a superior method for high-fidelity clonal analysis, enabling more precise investigation of gene function in development and disease.},
}
@article {pmid42201953,
year = {2026},
author = {Hong, Y and Yu, Z and Zhu, W and Sun, J and Zhu, Z and Wang, Z and Cao, M and Lang, Z and Lyu, YX and Liu, P and Zhu, JK},
title = {Multiplex gene editing enables the multibiofortification of essential vitamins and other health-promoting phytonutrients in tomato.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2603937123},
pmid = {42201953},
issn = {1091-6490},
mesh = {*Solanum lycopersicum/genetics/metabolism/chemistry ; *Gene Editing/methods ; Animals ; *Biofortification/methods ; Humans ; Plants, Genetically Modified/genetics ; *Vitamins/metabolism ; Mice ; *Phytochemicals/metabolism ; CRISPR-Cas Systems ; },
abstract = {Dietary deficiencies in essential micronutrients and other phytonutrients represent a global health and economic burden, contributing to "hidden hunger" and chronic diseases. While genome editing has been employed to improve individual nutritional traits in crops, multibiofortification through simultaneous modification of multiple distinct metabolic pathways is more challenging. Here, we designed a multiplex CRISPR-Cas strategy to edit five key genes in tomato: Sl7-DR2, SlGAD3, SlSGR1, SlGGP1, and SlGGP2. This approach successfully generated quintuple mutant (5m) tomato lines simultaneously biofortified with seven health-promoting compounds: vitamin D3 (from 0 to 0.70 μg/g dry weight), vitamin C (up to 2.53-fold), provitamin A/β-carotene (up to 3.86-fold), α-carotene (up to 2.47-fold), lutein (up to 3.26-fold), lycopene (up to 7.07-fold), and γ-aminobutyric acid (GABA, up to 5.26-fold). Notably, these multibiofortified tomatoes exhibited no significant trade-offs in plant growth or fruit quality. Extracts from 5m tomatoes showed enhanced suppression of colorectal cancer cell proliferation in vitro. This antiproliferative effect was validated in vivo, where dietary supplementation with 5m tomato powder significantly inhibited tumor growth in a mouse xenograft model. Our work demonstrates an effective strategy for developing a next generation of "functional foods" through multibiofortification, creating a single, nutrient-dense crop that combats both micronutrient malnutrition and chronic diseases.},
}
@article {pmid42202045,
year = {2026},
author = {Zhang, J and Chen, L and Zhu, X and Cai, Y and Wei, S and Zhou, X and Shi, Y and Liu, C and Huang, C and Bi, S and Wu, F and Zhou, X and Hong, J and Wang, Y},
title = {Coordinated regulation using small-molecule drugs enables controlled therapeutic genome editing and enhanced genomic precision in situ.},
journal = {Science translational medicine},
volume = {18},
number = {851},
pages = {eadx7857},
doi = {10.1126/scitranslmed.adx7857},
pmid = {42202045},
issn = {1946-6242},
mesh = {Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Mice ; *Genomics ; Dependovirus/genetics ; },
abstract = {Achieving precise temporal control over genome editing is essential for safety but remains a challenge, especially when using small-molecule drugs as external regulators over systems like clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated systems). Consequently, controlled therapeutic in situ editing that maintains both precision and efficacy has yet to be demonstrated. Here, we report the PRINCE system, in which nuclease proteins and guide RNAs are both inducible, to deliver programmable nucleases under control more effectively. PRINCE demonstrated temporal precision in human cell cultures over a 2-year period, even after stable genomic integration. The design principles of PRINCE were broadly applicable from CRISPR-Cas9 to a prime editor and also compact programmable nucleases, and the latter platform was named "Little Prince." Upon administration of drug inducers, Little Prince, delivered in a single adeno-associated virus vector in situ to humanized mouse models, ameliorated pathological phenotypes of hypercholesterolemia (average reductions of 45 and 47% in serum total cholesterol and low-density lipoprotein cholesterol, respectively) and neovascular age-related macular degeneration, with significantly reduced lesion size and leakage (P < 0.0001). Last, we demonstrated a consistent and marked reduction in off-target activity across the PRINCE and Little Prince systems in comparison with constitutive editors, with fewer off-target sites and substantially lower editing frequencies, irrespective of nuclease type, delivery method, or genomic target. These results position PRINCE and Little Prince as controlled genome editing platforms with potential for in vivo, particularly in situ, therapeutic applications.},
}
@article {pmid42202049,
year = {2026},
author = {Galtier, M and Krawczyk, A and Fuche, FJ and Charpenay, LH and Stzepourginski, I and Pignotti, S and Arraou, M and Terrasse, R and Brödel, AK and Poquet, C and Prevot, G and Spadoni, D and Buhot, B and Muench, K and Havránek, J and Cárdenas Ramírez, P and Rouquette, M and Decrulle, A and Kerbarh, O and Lieberman, E and Bramorski, C and Grienenberger, A and Hessel, EM and Salzano, G and Garry, DJ and Leveau, A and Duportet, X and Bikard, D and Fernandez-Rodriguez, J},
title = {Treatment of Shiga toxin-producing E. coli infection by CRISPR-Cas-targeted cleavage of the Shiga toxin gene in animal models.},
journal = {Science translational medicine},
volume = {18},
number = {851},
pages = {eadw8114},
doi = {10.1126/scitranslmed.adw8114},
pmid = {42202049},
issn = {1946-6242},
mesh = {Animals ; *Escherichia coli Infections/microbiology/therapy ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; *Shiga-Toxigenic Escherichia coli/genetics/pathogenicity ; *Shiga Toxin/genetics ; Mice ; Humans ; Female ; },
abstract = {Escherichia coli is not only a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extraintestinal infections. Shiga toxin-producing E. coli (STEC) poses a notable public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome, a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections because of their potential to induce prophages carrying Shiga toxin (stx) genes, triggering toxin production. Here, we developed a CRISPR-based antimicrobial strategy using a Cas12 nuclease to selectively eliminate O157 STEC clinical isolates, cleaving more than 99% of stx variants, and prevent toxin release. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a nonreplicative DNA payload to E. coli O157, preventing its dissemination. Our therapeutic candidate, EB003, reduced bacterial burden in a murine STEC colonization model. Moreover, EB003 mitigated clinical symptoms, abrogated Stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses in an infant rabbit disease model. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.},
}
@article {pmid42202254,
year = {2026},
author = {Liu, Z and Zhang, R and Chang, C and Xu, D and Mao, D and Zhu, X and Xu, H},
title = {Detection of Ultralow-Frequency ctDNA Mutations Using a Dual Hairpin-Competition CRISPR/Cas14a System.},
journal = {Analytical chemistry},
volume = {98},
number = {22},
pages = {16682-16693},
pmid = {42202254},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/genetics/blood ; *Mutation ; Polymerase Chain Reaction ; },
abstract = {Circulating tumor DNA (ctDNA) mutation profiling is essential for guiding targeted therapy and monitoring cancer recurrence, yet its clinical adoption is constrained by overwhelming wild-type DNA background and the limited sensitivity of existing platforms. Here, we introduce a dual hairpin-competition CRISPR/Cas14a (DHCC) system that integrates two sequential layers of hairpin competition: selective enrichment of mutant DNA during asymmetric PCR, followed by suppression of nonspecific sgRNA binding during Cas14a detection. This design dramatically enhances mutant-wild-type discrimination, elevating the discrimination factor from 2.48 to 145─a 58-fold improvement. While previous Cas14a methods achieve detection limits of 0.5-0.1% variant allele frequency (VAF), DHCC delivers a 250-fold sensitivity gain, routinely detecting four clinically relevant mutations (EGFR T790M, L858R, G719A, and NRAS Q61K) at VAFs as low as 0.002%. In multiplexed format, sensitivities of 0.005-0.01% VAF are maintained. Clinical validation using 22 plasma ctDNA samples demonstrated 100% concordance with droplet digital PCR for EGFR L858R detection. Compared to ddPCR and next-generation sequencing, DHCC substantially reduces turnaround time and cost while operating on standard qPCR instruments, eliminating the need for specialized infrastructure. By combining ultrahigh sensitivity, PAM independence, multiplexing preamplification capability, and practical affordability, DHCC provides an accessible platform for ctDNA-based liquid biopsy in clinical settings.},
}
@article {pmid42202397,
year = {2026},
author = {Han, S and Lin, X and Lei, Y and Liu, Z and Li, J and Ou, X},
title = {Universal primer-based RPA combined with parallel CRISPR/Cas12a decoding for rapid multi-species meat authentication.},
journal = {Food chemistry},
volume = {520},
number = {},
pages = {148905},
doi = {10.1016/j.foodchem.2026.148905},
pmid = {42202397},
issn = {1873-7072},
mesh = {*Meat/analysis/classification ; Animals ; *Food Contamination/analysis ; DNA Primers/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Swine ; },
abstract = {Rapid meat authentication is critical for food safety. Here, we report a two-step assay employing universal primer-based RPA amplification followed by species-specific CRISPR/Cas12a detection for the identification of 11 common meat species. A single universal primer pair enables broad amplification, while specific crRNAs allow parallel single-target CRISPR reactions. The assay takes approximately 40 min at constant temperature, costing ∼$4 per test with dual fluorescence and lateral flow strips. High specificity without cross-reactivity was observed, and detection limits ranged from 10[0] to 10[4] copies/μL. In binary meat mixtures, the fluorescence assay achieved adulteration detection limits of 0.05-0.5% (w/w), while the lateral flow format showed 0.05-5% (w/w) depending on the species. The method was verified using commercially processed products. This laboratory-validated strategy simplifies primer design and provides a promising platform for the qualitative screening of multiple meat targets, while further validation is required to assess its field robustness.},
}
@article {pmid42203510,
year = {2026},
author = {Pirzada, MUR and Powell-Rodgers, G and Richee, J and Norppa, AJ and Jungers, CF and Colijn, S and Frilander, MJ and Stratman, AN and Djuranovic, S},
title = {Loss of U11/U12 spliceosome gene ZCRB1 leads to aberrant ciliogenesis and WNT signaling.},
journal = {Life science alliance},
volume = {9},
number = {8},
pages = {},
pmid = {42203510},
issn = {2575-1077},
support = {R01 GM136823/GM/NIGMS NIH HHS/United States ; R01 GM112824/GM/NIGMS NIH HHS/United States ; R35 GM137976/GM/NIGMS NIH HHS/United States ; K99 HL171944/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Wnt Signaling Pathway/genetics ; *Spliceosomes/genetics/metabolism ; *Cilia/metabolism/genetics ; Zebrafish/genetics ; RNA Splicing/genetics ; *RNA-Binding Proteins/genetics/metabolism ; *RNA, Small Nuclear/genetics/metabolism ; Introns/genetics ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {The U12-dependent, or minor, spliceosome processes only 0.5% of human introns, and yet, it is known to profoundly influence gene expression and cellular signaling. The ZCRB1 protein is a core component of the U12 mono-snRNP, but its functional significance to minor splicing, gene regulation, and biological signaling cascades remains poorly understood. Using CRISPR-Cas9 and siRNA-targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant abnormal splicing events and altered expression of minor intron-containing genes. RNA-sequencing and targeted analyses of minor intron-containing genes indicate direct mis-splicing and expression of genes involved in ciliogenesis, with a coinciding up-regulation of WNT signaling pathway components. CRISPR-Cas12a knockdown of zcrb1 in zebrafish embryos leads to developmental patterning and body axis abnormalities, disrupted ciliogenesis, and up-regulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome, via ZCRB1, in cellular and developmental processes across species, shedding light on the molecular crosstalk that integrates splicing regulation, ciliogenesis, and WNT signaling.},
}
@article {pmid42204342,
year = {2026},
author = {Walker, MWG and Richard, E and Wiegand, T and Wang, J and Yang, Z and Casas-Ciniglio, AA and Hoffmann, FT and Shahnawaz, H and Gaudet, RG and Arpaia, N and Fernández, IS and Sternberg, SH},
title = {Temperate phages enhance bacterial host fitness via RNA-guided flagellar remodelling.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42204342},
issn = {2058-5276},
support = {CAREER 2239685//National Science Foundation (NSF)/ ; DGE-2036197//National Science Foundation (NSF)/ ; SF349247//Simons Foundation/ ; U24GM129539//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2AI177904//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01CA259634//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Bacterial flagella drive motility and play crucial roles in host-pathogen interactions, as flagellin is recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors called TldR that regulate flagellin expression, but the importance of this regulation to host fitness was unclear. Here we use a human clinical Enterobacter isolate encoding a Flagellin Remodeling prophage (FRφ) to show that FRφ exploits TldR and its flagellin isoform to alter the flagellar composition and phenotypic properties of its host. This transformation enhances bacterial motility and mammalian immune evasion, and cryo-EM structures reveal distinct flagellin architectures underlying physiological changes. FRφ also improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodelling in a host-associated environment. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.},
}
@article {pmid42204874,
year = {2026},
author = {Patra, S and Shand, H and Ghorai, S},
title = {Beyond conventional therapies: immunotherapeutic strategies targeting HPV-associated cervical cancer.},
journal = {Immunotherapy},
volume = {18},
number = {5-6},
pages = {503-523},
pmid = {42204874},
issn = {1750-7448},
mesh = {Humans ; Female ; *Uterine Cervical Neoplasms/therapy/immunology/virology ; *Immunotherapy/methods ; *Papillomavirus Infections/immunology/therapy ; *Human Papillomavirus Viruses/immunology ; Papillomavirus Vaccines/immunology/therapeutic use ; Animals ; },
abstract = {Cervical cancer continues to represent a significant global health challenge, primarily due to persistent infection with high-risk human papillomavirus (HPV) types. While prophylactic HPV vaccines have substantially reduced infection rates, their inability to address established infections or HPV-driven malignancies highlights a critical therapeutic gap. Conventional treatment modalities, such as chemotherapy, radiotherapy, and surgery remain the cornerstone of cervical cancer management; however, these approaches are often associated with nonspecific toxicity, diminished quality of life, treatment resistance, and elevated recurrence rates. Notably, conventional therapies are not specifically designed to target persistent HPV infection, and viral clearance may not be consistently achieved. This narrative review synthesizes studies retrieved from PubMed, Scopus, Web of Science, and Google Scholar published up to March 2026. It critically evaluates the limitations of current therapeutic strategies and emphasizes emerging non-conventional immunotherapeutic approaches designed to address HPV persistence and tumor immune evasion. Particular attention is given to nanocarrier-based therapeutic platforms, siRNA-mediated E6/E7 silencing and CRISPR-based disruption of the HPV genome, are presented as promising methods for precise molecular intervention. Additionally, advances in therapeutic vaccines, immune checkpoint inhibition, and γδ T-cell-based immunotherapy are also explored as potential strategies to restore HPV-specific immune surveillance and achieve sustained clinical responses.},
}
@article {pmid42205057,
year = {2026},
author = {Liu, Y and Yue, X and Li, B and Yang, H and Wang, Y and Ge, S and Liu, S},
title = {Target-to-signal conversion and spatial enrichment cascade boost CRISPR/Cas12a biosensing for trace-level pathogen detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {23},
pages = {4851-4857},
doi = {10.1039/d6ay00441e},
pmid = {42205057},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Salmonella typhimurium/isolation &amp; purification/genetics ; Limit of Detection ; DNA, Catalytic/chemistry/metabolism ; },
abstract = {S. typhimurium is a widely distributed and highly pathogenic Gram-negative bacterium capable of causing outbreaks of foodborne illness through the ingestion of contaminated food. Consequently, the development of a rapid and accurate detection strategy for S. typhimurium is of paramount importance. This study constructed a CRISPR/Cas12a-activated DNA nanowire biosensor based on DNAzyme-catalysed amplification for the sensitive detection of Salmonella typhimurium. Specific cleavage by DNAzymes generates single-stranded DNA to activate the HCR, enabling the serial HCR signal enrichment of three DNA hairpin structures, whose branched ends bear PAM sequences. Subsequent introduction of CRISPR/Cas12a cleaves the DNA NW, restoring quenched fluorescence signals to achieve ultra-sensitive detection of Salmonella typhimurium. Under optimal conditions, this biosensor exhibits a linear detection range of 10[1]-10[5] cfu mL[-1], with an estimated limit of detection (LOD) of 6.26 cfu mL[-1]. The method offers advantages of straightforward operation, rapid response, and high sensitivity. Furthermore, by modifying the recognition module and primer sequences, this biosensor holds promise for highly sensitive detection of multiple foodborne pathogens.},
}
@article {pmid42206360,
year = {2026},
author = {Ramirez-Chamorro, L and Bonhomme, F and Wolff, ALI and Stouf, M and Lecointe, F and Hollenstein, M and Krupovic, M and De Paepe, M and Bhoobalan-Chitty, Y},
title = {A pair of DNA glucosyltransferases elevate counter-defense in bacteriophage T4.},
journal = {Nucleic acids research},
volume = {54},
number = {10},
pages = {},
pmid = {42206360},
issn = {1362-4962},
support = {ANR-20-CE12-0008-02//ANR/ ; NNF21OC0067491//Novo Nordisk Fonden/ ; },
mesh = {*Bacteriophage T4/enzymology/genetics ; *Glucosyltransferases/metabolism/genetics ; DNA, Viral/metabolism ; Escherichia coli/virology/genetics ; *Viral Proteins/metabolism/genetics ; 5-Methylcytosine/analogs &amp; derivatives/metabolism ; Glycosylation ; },
abstract = {Bacteriophages encode diverse nucleotide-modification pathways to evade host restriction-modification (RM) and CRISPR-Cas systems. On the other hand, modifications can also serve as a target for host defense systems, illustrating the complexity of the defense and counter defense landscape. Bacteriophage T4 encodes two glucosyltransferases (GTs), α-GT and β-GT, that post-replicatively add a glucose moiety to the hydroxymethylated deoxycytosines (5-hmC) on phage DNA in the α- and β-conformation, respectively. Among all fully sequenced phages, only six closely related phages encode both α-GT and β-GT. Here, through biochemical and genetic analysis, we show that β-GT has higher catalytic activity, whereas α-GT is more strongly expressed. During T4 infection, these factors determine the contributions of both GTs, with 66% of all 5-hmC α-glucosylated and 33% β-glucosylated. Encoding a single GT is sufficient to overcome the Escherichia coli RM systems, unless the glucosylation levels decrease below 80%, which constitute a complete protection threshold. However, when encountering a host encoding DNA glycosylase Brig1, in addition to type I and type IV RM systems, a second GT is necessary to enable Brig1 escapers to resist RM systems. These results demonstrate that encoding multiple GTs serves as a counter-defense mechanism when simultaneously confronted with several antiphage defense systems.},
}
@article {pmid42206605,
year = {2026},
author = {Waller, MA and D'Araujo, TY and Denes, CE and Neely, GG},
title = {Dissecting conserved molecular mechanisms of biological toxin activity through CRISPR screening.},
journal = {Biochemical Society transactions},
volume = {54},
number = {6},
pages = {601-620},
pmid = {42206605},
issn = {1470-8752},
support = {GNT2020532//National Health and Medical Research Council/ ; DP220103530//Australian Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Toxins, Biological/genetics/metabolism ; Humans ; Animals ; Intracellular Space/metabolism ; },
abstract = {Toxins, substances that are produced by living organisms with the potential to cause harm, demonstrate great diversity in their structure, function, and origin. Though some toxins have been repurposed for use as novel therapeutics, research tools, or for application in agriculture, the mechanism of action for many toxins remains uncharacterised. Pooled CRISPR screens offer a high-throughput and unbiased method for rapid annotation of the host cell genome and identification of factors mediating or modifying intoxication. In this review, we provide a brief overview of CRISPR screening before detailing how screens have been used to characterise toxins from various biological kingdoms. We highlight certain cell entry factors and intracellular processes as conserved targets of various toxins. Finally, we highlight limitations in the methods of CRISPR screens used thus far and make recommendations as to how screen design can be modified to more completely characterise toxin activity and elucidate systemic effects of intoxication.},
}
@article {pmid42206706,
year = {2026},
author = {Hu, Y and Zhu, H and Diao, Y and Zhou, L and Bai, C},
title = {[Development and preliminary clinical evaluation of a CRISPR-AaCas12b-based nucleic acid detection method for Mycobacterium tuberculosis].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {42},
number = {5},
pages = {428-434},
pmid = {42206706},
issn = {1007-8738},
mesh = {*Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *Bacterial Proteins/genetics/metabolism ; *Alicyclobacillus/genetics ; Humans ; *Tuberculosis/diagnosis/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Objective To express and purify Alicyclobacillus acidiphilus Cas12b (AaCas12b) protein, and establish a rapid clustered regularly interspaced short palindromic repeats (CRISPR)-based assay for Mycobacterium tuberculosis (Mtb) detection, thereby providing a novel tool for clinical diagnosis of tuberculosis (TB). Methods Following construction and transformation of the recombinant expression vector into E. coli BL21 (DE3), soluble expression of the recombinant AaCas12b protein was induced with low-concentration isopropyl β-D-thiogalactopyranoside (IPTG) at low temperature, followed by purification using nickel-affinity chromatography. Subsequently, a specific recombinase polymerase amplification (RPA) assay targeting the IS6110 insertion sequence of Mtb was developed, and the corresponding single guide RNA (sgRNA) was prepared via in vitro transcription. Then a novel CRISPR-AaCas12b detection assay for Mtb was established, and its diagnostic performance was systematically evaluated against conventional clinical methods including acid-fast staining, T-SPOT.TB assay, Mycobacterium culture, and GeneXpert assay. Results A prokaryotic expression and purification system for recombinant AaCas12b was successfully established, yielding AaCas12b protein with activity at a concentration of 13.42 mg/mL and a recovery rate of 61.45%. A specific RPA-CRISPR-AaCas12b detection system was established targeting the IS6110 sequence, with a limit of detection (LOD) of 1.5 CFU/mL for Mtb. In clinical sample validation, the system achieved a sensitivity of 95.83% (95% CI: 79.97%~99.27%), a specificity of 92.31% (95% CI: 75.88%~97.88%), and an accuracy of 94.00% (95% CI: 83.78%~97.93%). Conclusion The established AaCas12b protein preparation system is efficient and stable, enabling the production of low-cost, high-activity tool enzymes. The RPA-CRISPR-AaCas12b TB diagnostic assay targeting the IS6110 sequence exhibits excellent specificity and sensitivity, providing a technical reference and experimental basis for the further development of rapid TB diagnostic platforms.},
}
@article {pmid42207849,
year = {2026},
author = {Wu, Y and Shen, F and Hu, Q and Yu, Z and Ye, Z and Ding, X},
title = {Recent advances in integrated CRISPR/Cas biosensing for aquatic food safety: overcoming the matrix interference and the difficulties in point-of-care testing.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-22},
doi = {10.1080/10408398.2026.2678519},
pmid = {42207849},
issn = {1549-7852},
abstract = {Rapid, sensitive, and on-site detection of pathogenic bacteria in aquatic-product and aquaculture-associated samples is critical for food safety. Although CRISPR/Cas has emerged as a powerful biosensing tool, its practical application is hindered by complex aquatic matrices and the difficulty of integrating pretreatment, amplification, and signal readout into point-of-care testing (POCT) workflows. This review examines integrated CRISPR/Cas biosensing strategies for pathogenic bacterial detection in aquatic matrices. It outlines the enzymatic features of core effectors, reviews pretreatment methods for complex samples, and summarizes how CRISPR/Cas is combined with isothermal amplification to improve analytical sensitivity. It also compares biosensing platforms from the perspective of matrix interference and field applicability. The analysis indicates that isothermal amplification is essential for detecting low-abundance targets in complex aquatic matrices. Among current strategies, ratiometric fluorescence, magnetic separation-assisted sensing, and signal-on electrochemical reporting show particular promise because they improve calibration, reduce matrix background, and limit nonspecific signal loss, respectively. Future progress depends on standardized sample processing, quantitative multiplexing, and automated "sample-in, result-out" systems for real-world deployment.},
}
@article {pmid42208154,
year = {2026},
author = {Crudele, M and Barnaba, NF and Di Cosmo, D and Mascia, T and Lum, KY and Cowled, MS and Larsen, TO and Faretra, F and Coleman, JJ and De Miccolis Angelini, RM},
title = {Characterization of the chloromonilicin biosynthetic gene cluster in the brown rot fungus Monilinia fructicola by a CRISPR/Cas9 transformation system.},
journal = {Microbiological research},
volume = {310},
number = {},
pages = {128558},
doi = {10.1016/j.micres.2026.128558},
pmid = {42208154},
issn = {1618-0623},
mesh = {*Multigene Family ; Polyketide Synthases/genetics/metabolism ; *Ascomycota/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; Plant Diseases/microbiology ; Gene Expression Profiling ; Fruit/microbiology ; Transformation, Genetic ; Biosynthetic Pathways/genetics ; },
abstract = {Monilinia fructicola is a major pathogen responsible for brown rot, causing substantial yield losses, particularly in stone fruit. Genomic analysis identified a type I polyketide synthase gene cluster, approximately 55 kb in length and consisting of twenty-four genes, putatively involved in chloromonilicin biosynthesis. These include a polyketide synthase (PKS), flavin-dependent halogenase, N-acetyltransferases, methyltransferases, a lactamase-like protein, scytalone dehydratase, and genes related to oxidoreduction, transcription factors, and transporters. To elucidate the product of the cluster, targeted gene disruption was achieved using a CRISPR/Cas9 ribonucleoprotein complex and polyethylene glycol-mediated transformation in the reference strain Mfrc123. Mutants with disruption in the ChmN core gene (encoding the PKS) were analysed by sequencing, digital droplet PCR to assess the insertion copy number of the hygromycin resistance cassette, and gene expression profiling. Phenotypic analysis indicated no significant differences between mutants and the WT strain in conidia production, germination rate, colony growth, or virulence on inoculated fruits. Metabolomic profiling using UHPLC-MS/MS demonstrated that the mutants did not produce chloromonilicin or other cluster-associated metabolites, such as chloromonilinic acids C and D, and 4-chloropinselin, indicating that the gene cluster is essential for their biosynthesis. In the WT strain, production of these metabolites increased following co-culture with Penicillium expansum compared to monoculture and remained low after fruit inoculation, suggesting a role in competition rather than pathogenesis. Gene expression analysis further showed upregulation of ChmN in response to various plant-associated microbes, but not to other Monilinia species.},
}
@article {pmid42209465,
year = {2026},
author = {Ghiotto, G and Zampieri, G and Orellana, E and Chatzis, A and Kougias, PG and Camargo, A and Roux, S and Campanaro, S and Kyrpides, NC and Treu, L},
title = {Single nucleotide variants drive evolutionary phage-host arms race in anaerobic carbon dioxide-converting microbiome.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-73084-2},
pmid = {42209465},
issn = {2041-1723},
abstract = {Microbial bioconversions are shaped by environmental perturbations and the adaptation of resident microbiomes. Prokaryotes coexist with bacteriophages, yet their coevolutionary trajectories remain underexplored. Here, we investigate the effects of a cultivation vessel leak on an anaerobic consortium performing carbon dioxide reduction. Using time-series shotgun metagenomic sequencing, we reconstruct microbial and viral genomes to track community shifts. We further apply single-nucleotide variant profiling and CRISPR array analysis to monitor viral microdiversity and host defense mechanisms. After bioaugmentation restores bioconversion efficiency, the consortium undergoes pronounced restructuring, with new dominant taxa emerging from the rare biosphere. We identify patterns consistent with phage predation selectively removing certain species, while others exhibit resilience to infection. This shift aligns with a widespread viral outbreak and a transient increased frequency of single nucleotide variants in bacterial CRISPR-Cas defense genes. Expansion of CRISPR spacers further supports that CRISPR-mediated processes influence microbial resilience. Concurrently, phages infecting resilient hosts exhibited adaptive evolution, marked by high genetic heterogeneity. Selective pressure varies across their genomes, targeting infectivity genes and protospacer-adjacent motifs. These findings highlight a dynamic evolutionary arms race driven by the selection of beneficial genetic variants, providing a mechanistic framework for multi-omics investigations, and informing biotechnological applications, including phage-based microbiome manipulation.},
}
@article {pmid42211656,
year = {2026},
author = {Nuevo, JJM and Fortaleza, JAG and Cabuhat, KSP and Ong, CJN and Jalova, AC and Mortel, FA and Bacalzo, GD},
title = {CRISPR-driven strategies to disrupt methicillin-resistant Staphylococcus aureus biofilms: a review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1784529},
pmid = {42211656},
issn = {2235-2988},
mesh = {*Biofilms/drug effects/growth &amp; development ; *Methicillin-Resistant Staphylococcus aureus/genetics/drug effects/physiology ; *CRISPR-Cas Systems ; Humans ; Quorum Sensing ; Anti-Bacterial Agents/pharmacology ; Staphylococcal Infections/microbiology/therapy ; Virulence Factors/genetics ; },
abstract = {Methicillin-resistant Staphylococcus aureus exhibits heightened tolerance to antimicrobial therapy through restricted drug penetration, extracellular polymeric substance-mediated protection, metabolic heterogeneity, and persister cell formation, limiting the effectiveness of current treatment strategies. CRISPR-Cas systems have emerged as programmable antimicrobial tools capable of targeting resistance genes, virulence determinants, and regulatory pathways; however, existing approaches remain largely gene-centric and insufficiently integrated with the biological complexity of biofilm-associated infections. This review aims to provide a comprehensive and integrative analysis of CRISPR-based strategies for targeting MRSA biofilms by linking molecular CRISPR mechanisms with key biofilm processes and evaluating their translational potential. CRISPR-Cas systems have emerged as programmable antimicrobial platforms with the ability to selectively target resistance genes, virulence factors, and regulatory networks. In MRSA biofilms, these systems are increasingly being explored for their potential to disrupt biofilm-associated determinants and weaken the molecular basis of persistence. Recent advances involving Cas9, Cas12a, and Cas13 highlight the potential of CRISPR-based targeting to interfere with resistance mechanisms, quorum sensing pathways, and structural components relevant to biofilm stability. Emerging in vivo studies, particularly those using engineered bacteriophages and localized delivery systems, provide early evidence that CRISPR-based strategies can reduce bacterial burden and impair biofilm integrity under physiologically relevant conditions. Nevertheless, significant barriers remain, including limited penetration into mature biofilms, delivery inefficiency, off-target activity, immunogenicity, resistance evolution, and regulatory uncertainty. Ultimately, CRISPR-based interventions represent a promising but still developing approach for the control of MRSA biofilm-associated infections, requiring further refinement in delivery design, target selection, and translational validation.},
}
@article {pmid42211740,
year = {2026},
author = {Matsugi, E and Kishi, K and Kishi, A and Nagase, K and Nigorikawa, K and Nomura, W},
title = {SauCas9-based cell cycle-dependent genome editing via AAV delivery.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201751},
pmid = {42211740},
issn = {3117-387X},
abstract = {The CRISPR-Cas system is a widely used genome editing technology with diverse applications. Although homology-directed repair (HDR) offers precise gene editing, its efficiency is typically lower than that of non-homologous end joining (NHEJ). Building on our previous cell cycle-dependent genome editing system for Streptococcus pyogenes Cas9 (SpyCas9), we adopted this approach for Staphylococcus aureus Cas9 (SauCas9) to enable efficient adeno-associated virus (AAV) delivery. To enhance HDR efficiency and editing accuracy in the context of AAV delivery, we developed a cell cycle-dependent genome editing system. We screened 10 anti-CRISPR (Acr) candidates and identified AcrIIA5, A13, A14, A15, and C1 as potent inhibitors of SauCas9. The fusion of these Acrs with the Cdt1(30-120) fragment restricted SauCas9 activity to the S/G2 phases, where HDR is predominant. Although AcrIIA11 and AcrIIA16 alone showed weak inhibition, their Cdt1 fusions (AcrIIA11+Cdt1 and AcrIIA16+Cdt1) showed a 2-fold increase in HDR efficiency within the AAV delivery system. This AAV-based, cell cycle-dependent SauCas9 system, which leverages optimized Acr-Cdt1 fusions, holds promise for improving the efficiency and accuracy of in vivo genome editing. Its small size is ideal for AAV packaging and may offer fewer off-target effects.},
}
@article {pmid42212714,
year = {2026},
author = {Zhang, F and Yan, D and Hou, J and Yang, D and Xiong, Y and Wen, M and Zhu, X},
title = {Writing Big in Plant Genomes: Advances, Challenges and Strategies for Targeted Large-Fragment DNA Insertion.},
journal = {Plant, cell &amp; environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70634},
pmid = {42212714},
issn = {1365-3040},
support = {2023YFA0913500//National Key Research and Development Program of China/ ; 25HHWCSS0007//Haihe Laboratory of Sustainable Chemical Transformations/ ; 32230012//National Natural Science Foundation of China/ ; 32470363//National Natural Science Foundation of China/ ; 32201738//National Natural Science Foundation of China/ ; },
abstract = {Precise genome editing has transformed plant biology and crop improvement by enabling targeted modification of endogenous loci. Beyond gene knockout and base editing, the site-specific insertion of exogenous DNA, particularly large DNA fragments, has become a central goal for engineering complex traits, reconstructing metabolic pathways and constructing plant artificial chromosomes. A diverse toolkit is now available for targeted DNA integration, including nuclease-dependent strategies, serine and tyrosine recombinases, transposon-derived systems, and CRISPR/Cas-coupled insertion platforms. Here, we review the mechanistic principles and recent advances of these four major tools, highlighting their capacities, insertion precision and compatibility with plant systems. We compare their strengths and limitations in terms of insertion-size capacity, integration efficiency, target site flexibility and technical complexity. Emerging innovations such as AI-guided nuclease and recombinase design, fusion of Cas with recombinases or viral replication proteins and RNA-guided transposition offer promising solutions to overcome these constraints. Together, these advances are rapidly expanding the landscape of targeted DNA insertion in plants and will reinforce future applications in molecular breeding, metabolic pathway engineering and the construction of synthetic genomic architectures.},
}
@article {pmid42212930,
year = {2026},
author = {Zhang, Y and Hao, L and Li, Q and Zhou, Z and Liu, D and Qiu, H and Yang, W and Zhang, B},
title = {Ultrasensitive Wash-Free Homogeneous CRISPR Assay Using Spatial Proximity Chemiluminescence Reporter.},
journal = {ACS nano},
volume = {20},
number = {22},
pages = {16001-16015},
doi = {10.1021/acsnano.5c22763},
pmid = {42212930},
issn = {1936-086X},
mesh = {*Luminescent Measurements/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reactive Oxygen Species/metabolism ; Limit of Detection ; Luminescence ; },
abstract = {CRISPR-based diagnostics are promising platforms for point-of-care (POC) testing, but are often hindered by limited sensitivity and complex workflows. Here, we present a spatial proximity chemiluminescence (SPC) reporter that converts CRISPR-Cas12a trans-cleavage activity into a highly sensitive, excitation-free signal. Mechanistically, the intact SPC reporter ensures efficient intramolecular reactive oxygen species (ROS) transfer by spatially confining a catalytic donor and a luminescent acceptor. Upon target-activated Cas12a cleavage, this spatial proximity is disrupted, drastically attenuating ROS transfer and reducing oxidized luminescent acceptors for chemiluminescence. The SPC-CRISPR exhibits an attomolar-level limit of detection with an optimal nearly 50,000-fold sensitivity enhancement, and features an 8-log dynamic range suitable for target quantification. This platform exhibits robust resistance to matrix interference, ensuring high accuracy while requiring only minimal sample preprocessing. We demonstrate SPC-CRISPR is compatible with recombinase polymerase amplification to develop a single-tube reaction with a sensitivity of 1 copy/μL. Extensive clinical validation demonstrated 99.2% overall accuracy for HPV16 identification in 126 cervical swabs, alongside 86.4% accuracy for miR-19a profiling in 22 bladder cancer plasma samples. Furthermore, this wash-free homogeneous workflow is embedded in a portable and sealed microfluidic-based device for sample-to-result diagnostics, showing 100% concordance with qPCR. SPC-CRISPR integrates enhanced sensitivity and simplified operation, holding great potential for POC molecular diagnostics.},
}
@article {pmid42213189,
year = {2026},
author = {Arjunan, NK and Thiruvengadam, V},
title = {Harnessing CRISPR-Cas technology for insect pest control: current advances and future perspectives.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42213189},
issn = {1573-4978},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Insect Control/methods ; *Pest Control, Biological/methods ; Gene Drive Technology ; *Insecta/genetics ; *Pest Control/methods ; },
abstract = {Insect pests pose a significant threat to agriculture, public health, and global biodiversity, prompting the development of innovative control strategies. CRISPR-Cas technology, a revolutionary genome-editing tool, has emerged as a promising approach to tackle these challenges. This review comprehensively explores the current advances in using CRISPR-Cas systems for insect pest control, highlighting key methodologies, successes, and potential hurdles. The fundamental principles of CRISPR-Cas technology, emphasizing its precision and versatility in gene editing, have been outlined. We then delve into various pest-control applications, such as gene drive systems designed to spread genetic modifications through pest populations, thereby reducing their reproductive capacity or increasing their susceptibility to environmental factors. Successful applications of CRISPR-based strategies in insect species such as Aedes aegypti, where gene editing has been used to suppress vector populations and reduce disease transmission potential, and Spodoptera frugiperda, where targeted gene disruption has improved pest management studies, are also highlighted. Furthermore, the ecological and ethical implications of deploying CRISPR-Cas based interventions are discussed, including concerns about unintended consequences, resistance development, and impacts on non-target species. Regulatory frameworks and public acceptance are also examined as critical components for the successful implementation of these technologies. The review concludes with a forward-looking perspective on the future of CRISPR-Cas in pest control, considering advancements in delivery methods, precision editing, and integration with other pest management strategies. We propose potential research directions and highlight the need for interdisciplinary collaboration to optimize the use of CRISPR-Cas technology for sustainable, effective insect pest management. This article serves as a comprehensive resource for researchers, policymakers, and stakeholders, providing a detailed overview of the current state of CRISPR-Cas technology in insect pest control and its future potential to transform pest management practices.},
}
@article {pmid42214852,
year = {2026},
author = {Xiao, S and Song, J and Chen, H and Yin, W and Yan, X and Hu, K and Shi, J and Yang, M},
title = {Digital droplet microfluidics integrating DNA walkers and CRISPR-Cas13a for simultaneous surface protein and miRNA profiling in single exosomes.},
journal = {Biosensors &amp; bioelectronics},
volume = {310},
number = {},
pages = {118854},
doi = {10.1016/j.bios.2026.118854},
pmid = {42214852},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *MicroRNAs/genetics/isolation &amp; purification/analysis ; *Erb-b2 Receptor Tyrosine Kinases/genetics/isolation &amp; purification ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; *Epithelial Cell Adhesion Molecule/genetics/isolation &amp; purification/analysis ; DNA/chemistry ; Equipment Design ; },
abstract = {Tumor-derived exosomes carry multi-scale molecular signatures (e.g., surface proteins and nucleic acids) that reflect tumor heterogeneity, yet simultaneously profiling these biomarkers in single intact vesicles remains technically challenging. Herein, we developed a digital droplet microfluidic platform that integrates a DNA walker and a CRISPR/Cas13a system for the simultaneous detection of surface proteins (EpCAM, HER2) and miRNA (miR-21) at the single exosome level. This platform employed engineered liposome nanoprobes (eLipo-NPs) with EpCAM aptamers and hairpin probes (HPs) functionalized on their outer membranes, and encapsulated a CRISPR/Cas13a system within their lumen. Upon co-encapsulation with single exosomes into droplets, EpCAM-mediated membrane fusion redistributed HPs across the hybrid membrane and delivered CRISPR/Cas13a into the exosomes. The membrane-anchored DNA walker then bound HER2 and drove cyclic DNAzyme cleavage of HPs to restore red fluorescence. At the same time, crRNA-guided Cas13a recognized miR-21 and triggered trans-cleavage of reporters to generate green fluorescence. Digital counting of dual-positive droplets enabled quantitative single-exosome analysis with a limit of detection (LOD) of 10 particles/μL and a detection time of 60 min. Clinical validation using plasma-derived exosomes from 24 breast cancer patients and 14 healthy donors demonstrated distinct distributions among HER2-positive, HER2-negative, and healthy control groups, with the percentage of dual-positive droplets significantly correlated with clinical HER2 status, highlighting the platform's potential for liquid biopsy and precision oncology.},
}
@article {pmid42215224,
year = {2026},
author = {Yang, Z and Xiao, S and Chen, X and Li, Y and Bai, W and Liu, Q},
title = {Recent advances in CRISPR-based detection of foodborne pathogens: Mechanistic foundations, technological advances, and biosensing integration.},
journal = {Food microbiology},
volume = {139},
number = {},
pages = {105144},
doi = {10.1016/j.fm.2026.105144},
pmid = {42215224},
issn = {1095-9998},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; *Foodborne Diseases/microbiology ; Food Safety ; *Bacteria/genetics/isolation &amp; purification/classification ; Food Contamination/analysis ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {CRISPR-based biosensing has emerged as a rapid, sensitive, and field-deployable platform for foodborne pathogen detection, thereby effectively addressing the intrinsic limitations of conventional detection methodologies. By integrating CRISPR/Cas effectors with nucleic acid amplification (NAA) reactions and multiple signal transduction modes, these biosensing platforms exhibit considerable potential for the specific detection of key foodborne pathogens in complex food matrices. In this review, the molecular mechanisms underlying CRISPR/Cas-mediated pathogen detection, pivotal technological milestones, and the integration of CRISPR-based systems with diversified read-out strategies and advanced biosensing formats are systematically summarized. Furthermore, the persisting bottlenecks impeding the widespread application of these systems-including food matrix-induced inhibition, the lack of standardized detection devices, and limited multiplex throughput-are highlighted. Finally, future directions are outlined, where artificial intelligence (AI)-driven sequence mining and rational multiplex design are expected to accelerate the development of next-generation food safety surveillance systems, with concrete implementations in signal processing and decision-support workflows.},
}
@article {pmid42216279,
year = {2026},
author = {Alhabsi, A and Ayala, FM and Pan, J and Wang, YL and Mourad, AMI and Dracatos, P and Wulff, BBH and Alagoz, Y},
title = {Potential unlocked: an atlas of cloned wheat genes for genome engineering and breeding.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.71305},
pmid = {42216279},
issn = {1469-8137},
support = {//King Abdullah University of Science and Technology (KAUST)/ ; //King Abdullah University of Science and Technology (KAUST) Center of Excellence in Sustainable Food Security seed funding/ ; 202208320272//China Scholarship Council/ ; },
abstract = {Bread wheat (Triticum aestivum) production is increasingly threatened by biotic and abiotic stresses. Developing varieties with improved stress tolerance and desirable end-use qualities is crucial for meeting growing global demand. Genome-editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR/Cas)-derived systems, represent powerful tools to accelerate trait discovery and crop improvement. Here, we present a comprehensive atlas of cloned wheat genes and discuss examples and strategies for using it to identify candidate targets for CRISPR/Cas-mediated improvement. Finally, we recommend ways to integrate gene editing into breeding timelines and accelerate the incorporation of desirable alleles.},
}
@article {pmid42216525,
year = {2026},
author = {Zhang, C and Feng, H and Li, L and Lin, J and Xu, L and Zhu, J and Liu, Y},
title = {Ligand-Induced Trigger RNA Cleavage Enables Programmable Gene Expression Regulation via Strand Displacement in Prokaryotic and Eukaryotic Cells.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {31},
number = {5},
pages = {51369},
doi = {10.31083/FBL51369},
pmid = {42216525},
issn = {2768-6698},
support = {2024B1515040028//Guangdong Basic and Applied Basic Research Foundation/ ; 22477144//National Natural Science Foundation of China/ ; 22222706//National Natural Science Foundation of China/ ; 22377034//National Natural Science Foundation of China/ ; 2020YFA0211200//National Key R&amp;D Program of China/ ; 2022YFC2804101//National Key R&amp;D Program of China/ ; //GBRCE for Functional Molecular Engineering/ ; PA250207//Open Fund of Hubei Key Laboratory of Pollutant Analysis and Resource Technology/ ; },
mesh = {Humans ; RNA, Catalytic/genetics/metabolism ; Ligands ; HEK293 Cells ; *Eukaryotic Cells/metabolism ; *Gene Expression Regulation ; Escherichia coli/genetics/metabolism ; CRISPR-Cas Systems ; *RNA Cleavage ; Aptamers, Nucleotide/genetics ; *Prokaryotic Cells/metabolism ; },
abstract = {BACKGROUND: Synthetic RNA circuits offer powerful tools for reprogramming cellular behavior, but constructing ligand-responsive RNA switches that function reliably inside living cells remains challenging. Existing cis-acting designs often lack modularity and programmability due to tight coupling between sensing and output domains.<br><br>METHODS: We developed a generalizable strategy termed Ligand-Induced Trigger RNA Cleavage (LITC). This approach integrates an aptamer-embedded hammerhead ribozyme (aptazyme) as a trans-acting trigger. The aptazyme sequence is inserted into the spacer region of an RNA trigger strand, separating its toehold and displacement domains. Ligand-induced aptazyme self-cleavage inactivates the trigger, thereby controlling downstream toehold-mediated strand displacement reactions. We validated this system in both prokaryotic (E. coli) and eukaryotic (HEK-293T) cells using translation-controlling toehold switches and gRNA switches within the CRISPR/Cas9 system.<br><br>RESULTS: The LITC strategy successfully enabled programmable, dose-dependent regulation of gene expression. A spacer inserted between toehold and displacement domains did not impair trigger function. Embedding self-cleaving ribozymes (HHR, sTRSV) constitutively silenced trigger activity. Using a theophylline-responsive aptazyme, we achieved ligand-controlled regulation of a toehold switch, with different communication modules (CMs) yielding varied regulatory performance and theophylline concentrations up to 4 mM providing graded control. Furthermore, this approach was extended to control CRISPR interference (CRISPRi) in E. coli and CRISPR activation (CRISPRa) of the endogenous ASCL1 gene in HEK-293T cells, demonstrating cross-system portability.<br><br>CONCLUSIONS: The LITC platform provides a general, modular, and transferable strategy for small-molecule control of toehold-mediated strand displacement reactions. It enables precise conditional regulation of RNA-based devices, including translation switches and CRISPR-Cas9 systems, across both prokaryotic and eukaryotic cells, thereby offering a versatile framework for constructing intelligent genetic circuits.},
}
@article {pmid42217400,
year = {2026},
author = {Wakaba, P and Muramatsu, A and Imagawa, T and Furuse, Y and Saito, N and Uno, N},
title = {CRISPR-Cas12a biosensing via transcription of crRNA from PCR or LAMP products for pathogen detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {310},
number = {},
pages = {118860},
doi = {10.1016/j.bios.2026.118860},
pmid = {42217400},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Mycobacterium tuberculosis/isolation &amp; purification/genetics/pathogenicity ; *Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; Humans ; *DNA, Bacterial/genetics/isolation &amp; purification/analysis ; *CRISPR-Associated Proteins/genetics ; Molecular Diagnostic Techniques/methods ; Transcription, Genetic ; Bacterial Proteins/genetics ; *Endodeoxyribonucleases/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 12a (Cas12a) is an RNA-guided nuclease that has been adapted for nucleic acid-based molecular diagnostics. However, the widespread adoption of Cas12-based molecular diagnostics has been limited by dependency of a protospacer adjacent motif (PAM) sequence within the target double-stranded DNA (dsDNA) required to activate Cas12a. To eliminate PAM dependency, we explored a noncanonical activation mode by designing tailed primers for PCR to generate crRNA from amplified target dsDNA products via transcription. The transcribed crRNA, along with a dsDNA activator, then activates Cas12a. We validated this method and named it PCR followed by Transcription And CRISPR-Cas12a (PCR-TRAC). We then developed loop-mediated isothermal amplification (LAMP)-TRAC using tailed primers. Both methods detected Mycobacterium tuberculosis genomic DNA extracted from clinical samples. LAMP-TRAC was more sensitive and faster than PCR-TRAC, detecting as little as four copies/μL of M. tuberculosis genomic DNA within 1 h. We envision that our CRISPR-Cas12-based diagnostic approach could be expanded to become a universal platform for identifying various other nucleic acid targets.},
}
@article {pmid42217635,
year = {2026},
author = {Zhen, Z and Shuhua, L and Baihe, M and Xin, C and Meiliang, G and Fanxin, L and Lianrui, L},
title = {Advances in rapid microbiological testing for animal diseases: A review.},
journal = {Journal of microbiological methods},
volume = {246},
number = {},
pages = {107564},
doi = {10.1016/j.mimet.2026.107564},
pmid = {42217635},
issn = {1872-8359},
mesh = {Animals ; Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Rapid Diagnostic Tests ; *Animal Diseases/diagnosis/microbiology ; Point-of-Care Systems ; Humans ; CRISPR-Cas Systems ; Livestock/microbiology ; Sensitivity and Specificity ; Point-of-Care Testing ; },
abstract = {Animal pathogenic microorganisms destabilize livestock economies and jeopardize human health through zoonotic transmission and foodborne illness. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site or point-of-care (POC) monitoring required to prevent disease outbreaks and manage animal health effectively. By integrating latest research advances, this study reviews advances in rapid detection technologies for animal pathogens, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification (LAMP); recombinase polymerase amplification (RPA)). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification from clinical livestock samples and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption from days to less than one hour while challenges regarding sample preparation from complex clinical matrices such as whole blood, serum, tissue homogenates, and fecal samples remain. The future of animal health surveillance lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors for farm-level early warning. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for POC veterinary diagnostics, while qPCR/dPCR (dPCR)remain indispensable for quantitative regulatory applications such as disease certification and vaccine efficacy monitoring. We propose a structured technology selection framework to guide researchers and veterinary practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements for different livestock species and production systems.},
}
@article {pmid42217941,
year = {2026},
author = {Liu, D and Ma, G and Bai, L and Guo, K and Wang, T and Jiang, Z and Qian, F and Wang, Y and Pang, Y and Zou, W and Wang, R},
title = {STAR-CRISPR: a one-pot ultraspecific CRISPR strategy for rapid, visualized SNV detection and genotyping in point-of-care diagnostics.},
journal = {Talanta},
volume = {309},
number = {},
pages = {130036},
doi = {10.1016/j.talanta.2026.130036},
pmid = {42217941},
issn = {1873-3573},
mesh = {Humans ; *Point-of-Care Systems ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; *Pancreatic Neoplasms/genetics/diagnosis ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; *Genotyping Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Genotype ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Single nucleotide variation (SNV), as a key biomarker for disease diagnosis and personalized treatment, faces challenges in rapid and accurate detection. This study developed a single-tube accelerated recognition of SNVs strategy named STAR-CRISPR, which could accomplish SNV detection within only 20 min. This method integrated isothermal amplification and CRISPR/Cas12b cleavage system in one pot, and results could be directly identified by the naked eye. This method could accurately distinguish single-base differences, and could detect as low as 1% mutations against high background interference. We verified the proposed method by testing 70 clinical samples of idiopathic chronic pancreatitis, pancreatic cancer and acute myeloid leukemia. Results showed 100% consistency with next-generation sequencing results, demonstrating good accuracy and reliability of the proposed method. To further facilitate point-of-care diagnosis, we developed integrated miniature microfluidic chips, which greatly simplified sample identification and enabled logical interpretation of results. The combined STAR-CRISPR and microfluidic platform not only identifies SNVs but also supports simultaneous visual genotyping of wild-type, homozygous, and heterozygous mutations. Consequently, the proposed strategy is accurate, rapid, and versatile, holding significant potential for next-generation molecular diagnostics.},
}
@article {pmid42217942,
year = {2026},
author = {Chen, B and Yang, H and Zhao, J and Wang, Y and Li, H and Wang, C and Guo, L and Xu, J},
title = {Template-independent poly-adenine elongation enables multivalent CRISPR/Cas12a activation for amplified lateral flow biosensing.},
journal = {Talanta},
volume = {309},
number = {},
pages = {130054},
doi = {10.1016/j.talanta.2026.130054},
pmid = {42217942},
issn = {1873-3573},
mesh = {Humans ; *Bacterial Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *CRISPR-Cas Systems ; *DNA Nucleotidylexotransferase/metabolism/analysis ; *Endodeoxyribonucleases/metabolism ; Limit of Detection ; *Poly A/metabolism/chemistry ; },
abstract = {Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that plays a critical role in immune system development and serves as an important biomarker for acute lymphoblastic leukemia. However, current methods for TdT activity analysis often rely on sophisticated instrumentation and lack simple and portable detection formats. Herein, we report a TdT-enabled multivalent CRISPR/Cas12a lateral flow assay for sensitive and instrument-free detection of TdT activity. In this strategy, TdT-catalyzed poly-adenine (poly-A) extension converts enzymatic activity into adenine-rich DNA scaffolds, which recruit multiple crRNA molecules to trigger multivalent activation of Cas12a. This design effectively bridges TdT activity with CRISPR/Cas12a signal amplification. The activated Cas12a subsequently induces trans-cleavage of a reporter probe, and the cleavage event is translated into a visual signal on a lateral flow strip. The proposed assay enables sensitive detection of TdT with a limit of detection of 0.016 U/mL and a visual detection limit of 0.05 U/mL. In addition, the assay exhibits high specificity toward TdT over other polymerases and demonstrates satisfactory performance in human serum samples with recoveries ranging from 98.8% to 103.7%. This work expands the applicability of CRISPR/Cas12a systems to enzyme activity sensing and provides a simple and practical platform for point-of-care detection of TdT.},
}
@article {pmid42218072,
year = {2026},
author = {Teng, Z and Prieto-Vivas, JE and Verstrepen, KJ and Wang, Q and Dai, Z},
title = {Beyond natural evolution: multi-scale in vivo mutagenesis toolkits for synthetic evolution.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.05.008},
pmid = {42218072},
issn = {1879-3096},
abstract = {Our current industrial, agricultural, and medical practices exploit the extraordinary biodiversity generated through billions of years of natural evolution. Despite their high fitness in native habitats, biomolecules and organisms are often not optimally suited for industrial and medical use. Synthetic evolution leverages technologies such as DNA synthesis, CRISPR-Cas engineering, and synthetic biology to enable continuous in vivo mutagenesis of biomolecules or organisms to improve specific desirable characteristics. This review presents the latest mutagenesis toolkits classified by mutational scale: genome-wide, medium-scale, and site-specific, each tailored to different application scenarios. We discuss the mechanisms and capabilities underlying each scale, analyze current limitations, and highlight the untapped potential of next-generation gene-editing technologies, high-throughput screening, and artificial intelligence in advancing synthetic evolution.},
}
@article {pmid42219100,
year = {2026},
author = {Wang, H and Zhang, W and Liu, D and Mao, X and Yang, Y and Zhang, Y and Shangguan, P and Wang, Y and Wang, Z and Liu, Y and Zhang, Q},
title = {enCas7-11S3: A compact Cas7-11 variant with enhanced RNA cleavage and minimal collateral activity.},
journal = {International journal of biological macromolecules},
volume = {369},
number = {},
pages = {152796},
doi = {10.1016/j.ijbiomac.2026.152796},
pmid = {42219100},
issn = {1879-0003},
mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *RNA Cleavage ; *RNA/metabolism/genetics ; },
abstract = {In the CRISPR-Cas system, the RNA-targeting single-protein effectors include the Class 2 Type VI Cas13 and Class 1 Type III-E Cas7-11. Cas7-11 from Desulfonema ishimotonii is a large single-protein effector containing four Cas7 domains and one Cas11 domain, and its substantial size hinders delivery via a single adeno-associated virus (AAV) vector. To address this limitation, a compact Cas7-11 variant (Cas7-11S) was engineered by deleting the insertion (INS) domain. However, this truncation reduced target RNA cleavage activity compared to full-length Cas7-11. Here, we engineered Cas7-11S to improve its RNA cleavage efficiency while further reducing its size. We designed a dual-fluorescence reporter system in mammalian cells to evaluate the RNA cleavage efficiency of Cas7-11S. We found that adding a nucleocytoplasmic shuttling signal to the C-terminus of Cas7-11S greatly improved its RNA cleavage efficiency. We then systematically screened Cas7-11S mutants in HEK293T cells and identified variants with stronger RNA cleavage activity, as well as truncated variants with more compact structures. Finally, by combining these beneficial modifications, we generated an enhanced Cas7-11S variant called enCas7-11S3 that is both more compact and exhibits higher RNA cleavage activity. Importantly, enCas7-11S3 retains almost no collateral activity, broadening the potential application of Cas7-11 in RNA editing.},
}
@article {pmid42219400,
year = {2026},
author = {Awan, MJA and Akram, A and Naqvi, RZ and Akhtar, M and Siddique, S and Buzdar, MI and Amin, I and Mansoor, S},
title = {Transgene-free plant genome editing via viral delivery of miniature CRISPR-Cas12f.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42219400},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Nicotiana/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; *CRISPR-Associated Proteins/genetics ; Oxidoreductases/genetics ; Genetic Vectors ; Transgenes ; Plant Viruses ; },
abstract = {Efficient delivery of CRISPR reagents into plant cells remains a major bottleneck, particularly for transgene-free genome editing. RNA viral vectors such as Tabacco Rattle Virus (TRV) provide an attractive platform for transient expression but are limited by their small cargo capacity. Recently discovered miniature nucleases like Cas12f, due to their compact size and high activity, offer a promising alternative for viral delivery systems. In this study, a plant codon-optimized Acidibacillus sulfuoxidans Cas12f (AsCas12f), fused with nuclear localization signals, was cloned into the TRV genome to develop a viral vector-based delivery platform. Two sgRNAs targeting the Nicotiana benthamiana PHYTOENE DESATURASE (NbPDS) gene were delivered through Agrobacterium-mediated infiltration. To enhance mobility and transcript abundance, mobile RNA elements such as modified Flowering Locus T (mFT), truncated FLOWERING LOCUS T (tFT), and transfer RNA of methionine (tRNA[Met]), and the Pea early browning virus (PeBV) promoter were incorporated into Cas12f and sgRNA constructs. TRV-mediated delivery of CRISPR-Cas12f induced targeted mutagenesis in N. benthamiana systemic leaves, confirmed by persistent photobleaching and Sanger sequencing. Both tFT- and tRNA-tagged Cas12f constructs improved editing efficiency, with the tRNA fusion showing enhanced activity. Expression of Cas12f and sgRNA under the PeBV promoter further enhanced mutation frequency, with pTRV2-PeBV::Cas12f-tRNA and pTRV2-PeBV::mFT-sgRNA constructs achieving the highest efficiency. This study establishes a compact TRV-based CRISPR-Cas12f platform enabling efficient, transgene-free genome editing in plants. The system bypasses tissue culture-dependent transformation and provides a biosafety-compliant strategy for scalable, non-transgenic crop improvement.},
}
@article {pmid42219636,
year = {2026},
author = {Zhu, Y and Ma, S and Du, J and Yang, T and Chen, Y and He, X and Yu, X and Zhou, Q and Wu, J},
title = {Photothermal PCR within 6 min based on pullulan-coated nanoplasmas for ultrafast nucleic acid analysis.},
journal = {Analytica chimica acta},
volume = {1412},
number = {},
pages = {345667},
doi = {10.1016/j.aca.2026.345667},
pmid = {42219636},
issn = {1873-4324},
mesh = {*Glucans/chemistry ; *Polymerase Chain Reaction/methods ; Gold/chemistry ; Temperature ; *Metal Nanoparticles/chemistry ; *DNA, Bacterial/analysis/genetics ; },
abstract = {BACKGROUND: Photothermal PCR based on specific nanomaterials has attracted attention due to their efficient photothermal conversion. However, these nanomaterials absorb polymerases, thereby inhibiting the PCR. Owing to their small size and highly active surface atoms, they are prone to aggregation and sedimentation, resulting in uneven heating within the reaction system. In addition, the fluorescent groups used for detection are susceptible to photobleaching under excitation light. Therefore, there is a need for stable photothermal nanomaterials and compatible photothermal PCR detection strategies.<br><br>RESULTS: This paper presents a coated-type nanoplasmas-based photothermal PCR method and platform for ultra-fast nucleic acid analysis. Pullulan-coated AuNRs were introduced, and pullulan-AuNRs nanoplasmas were prepared to prevent the aggregation of AuNRs and the adsorption of the polymerase. A photoelectric platform for ultrafast photothermal PCR was built. The photothermal effect of AuNRs was excited by an infrared laser, causing the PCR solution to rapidly heat up. By regulating the on-off cycle of the excitation light source and the fan through temperature feedback, the photothermal conversion of AuNRs was alternately excited, achieving ultra-fast photothermal PCR within 6&#x202f;min. The CRISPR/Cas detection technology was coupled with the photothermal PCR to detect the amplified products and output fluorescence signals within 4&#x202f;min, enabling ultra-fast detection of Salmonella DNA as low as 38 copies/reaction.<br><br>SIGNIFICANCE: The pullulan-coated photothermal nanomaterials were introduced, which solved the problems of instability and incompatibility with the PCR system. The one-tube photothermal PCR-CRISPR assay maintains airtight conditions while avoiding interference between the excitation light source and the detection light source. This method achieves ultrafast detection within 10&#x202f;min in a simple, rapid, accurate, and contamination-free manner.},
}
@article {pmid42221583,
year = {2026},
author = {David Hanna, LB and Steinig, E and Bond, K and Lim, CK and Ramachandran, PS},
title = {Enrichment techniques for clinical metagenomics.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1723747},
pmid = {42221583},
issn = {2235-2988},
mesh = {*Metagenomics/methods ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; Sensitivity and Specificity ; Polymerase Chain Reaction/methods ; CRISPR-Cas Systems ; },
abstract = {Metagenomic next-generation sequencing (mNGS) offers a powerful, hypothesis-free approach for pathogen detection in clinical samples, allowing the identification of both known and novel microorganisms. However, the predominance of host nucleic acid in most samples poses a significant challenge, often overshadowing low-abundance pathogen sequences and increasing the cost of mNGS due to the high sequencing depth required. Enrichment techniques which selectively amplify pathogen-specific sequences can help to overcome this challenge, improving the sensitivity, specificity, and overall efficiency of mNGS - albeit while compromising the hypothesis-free nature and breadth of shotgun mNGS. As such, they can augment the use of mNGS in clinical scenarios where a more targeted approach is needed. This review provides a comprehensive analysis of the main enrichment techniques currently employed in the field, including PCR-based enrichment, CRISPR-Cas9 enrichment, molecular inversion probes (MIP), nanopore adaptive sequencing (AS), and hybridisation capture-based methods. We evaluate each method on a range of metrics including methodology, cost, sensitivity, specificity, and ease of integration into clinical workflows, as well as describing their application to date for purposes including pathogen detection, antimicrobial resistance profiling, and whole-genome sequencing across diverse clinical sample types. Current limitations and future directions for refinement and implementation of these techniques are also discussed. By summarising the current landscape and latest advancements in mNGS enrichment strategies, this review aims to guide the optimisation of mNGS workflows in clinical diagnostics and highlight key areas for future research.},
}
@article {pmid42223080,
year = {2026},
author = {An, SY and Kim, I and Hong, SH and Kim, EH and Suh, JY},
title = {AcrIIA8 is a putative phage structural protein of the HTJ2 family that does not inhibit Streptococcus pyogenes Cas9.},
journal = {Protein science : a publication of the Protein Society},
volume = {35},
number = {7},
pages = {e70651},
pmid = {42223080},
issn = {1469-896X},
support = {RS-2025-23525174//National Research Foundation of Korea/ ; RS-2024-00440614//National Research Foundation of Korea/ ; BDB-2025-04-04230007//Korea Institute of Marine Science &amp; Technology Promotion/ ; },
mesh = {*Streptococcus pyogenes/enzymology/genetics/virology ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry/metabolism ; *Viral Structural Proteins/chemistry/metabolism/genetics ; *Bacteriophages/chemistry ; },
abstract = {Anti-CRISPR (Acr) proteins are phage-encoded anti-defense factors that suppress CRISPR-Cas immunity in bacteria. AcrIIA8 was previously identified as an inhibitor of Streptococcus pyogenes Cas9 (SpyCas9) through functional assays of metagenomic libraries. Here, we report that AcrIIA8 does not inhibit SpyCas9 in biochemical assays under a range of buffer conditions and temperatures. The solution structure and dynamics of AcrIIA8 reveal a six-stranded β-barrel fold with flexible β1-β2 and β2-β3 loops, characteristic of phage virion-assembly proteins. In addition, genomic context analysis places AcrIIA8 and its homologs within conserved prophage morphogenetic regions at the position expected for type II head-tail joining (HTJ2) proteins. We further detected no interaction between AcrIIA8 and SpyCas9 in NMR titration experiments, suggesting that they do not specifically associate. Taken together, these findings argue against assigning AcrIIA8 as a SpyCas9 inhibitor and instead support its annotation as a putative phage structural protein of the HTJ2 family.},
}
@article {pmid42223605,
year = {2026},
author = {Ojaroodi, AF and Ehtiati, S and Hadinia, F and Sani, MZ and Ghadimi-Moghadam, A and Movahedpour, A},
title = {Biosensors as transformative tools for multiplex detection of respiratory viral pathogens.},
journal = {Archives of microbiology},
volume = {208},
number = {8},
pages = {},
pmid = {42223605},
issn = {1432-072X},
mesh = {*Biosensing Techniques/methods/instrumentation ; Humans ; *Respiratory Tract Infections/diagnosis/virology ; *Viruses/isolation &amp; purification/genetics/classification ; Point-of-Care Systems ; *Virus Diseases/diagnosis/virology ; Rapid Diagnostic Tests ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Respiratory viral infections remain a major global health challenge, highlighting the need for rapid, sensitive, and accessible diagnostic approaches. Conventional diagnostic techniques, including reverse transcription-polymerase chain reaction (RT-PCR), viral culture, and enzyme-linked immunosorbent assays (ELISA), provide high analytical performance but are often limited by infrastructure requirements, cost, and turnaround time, restricting their use in decentralized or resource-limited settings. This review critically examines recent advances in biosensor technologies for respiratory virus detection, with a particular focus on electrochemical biosensors, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based biosensors detection systems, and emerging point-of-care (POC) diagnostic platforms. The manuscript discusses biosensor design strategies, detection mechanisms, analytical performance, and clinical applicability across major respiratory viral pathogens. In addition, current challenges related to clinical validation, scalability, standardization, and cost-effectiveness are analyzed. Overall, current evidence indicates that biosensor-based diagnostic platforms have significant potential to complement existing laboratory-based methods, particularly in decentralized and rapid testing settings. However, further large-scale clinical validation, regulatory standardization, and integration into healthcare workflows are required before widespread clinical implementation can be achieved.},
}
@article {pmid42225261,
year = {2026},
author = {Kim, H and Seo, Y and Kho, H and Singh, SS and Lee, J and Lee, H and Hwang, JW and Riew, TR and Koh, S and Choi, JY and Roh, HW and Son, SJ and Kim, GT and Cho, SK and Jin, HS and Jeong, SY and Lee, KI and Lee, JY and Kim, BG},
title = {A novel mouse model of cerebral microbleeds by targeted Col4a1 editing in adult brain microvessels.},
journal = {Brain : a journal of neurology},
volume = {},
number = {},
pages = {},
doi = {10.1093/brain/awag048},
pmid = {42225261},
issn = {1460-2156},
support = {RS-2019-NR040055//National Research Foundation of Korea (NRF) research programs/ ; RS-2021-NR056919//National Research Foundation of Korea (NRF) research programs/ ; RS-2023-00244748//National Research Foundation of Korea (NRF) research programs/ ; RS-2023-00245169//National Research Foundation of Korea (NRF) research programs/ ; NRF2021M3H1A104892211//Korea Initiative for fostering University of Research and Innovation (KIURI) Program of the National Research Foundation (NRF) funded by the Korean government/ ; M2023C046000102//Ajou University Medical Center/ ; 202500300001//Ajou University Medical Center/ ; 2024-ER0505-01//National Institute of Health (NIH)/ ; },
abstract = {Cerebral small vessel disease is a leading cause of cognitive decline and stroke in the elderly, with cerebral microbleeds (CMBs) serving as a key imaging biomarker. Despite their clinical significance, the pathophysiological mechanisms underlying cerebral small vessel disease remain poorly understood owing to a lack of appropriate animal models. We performed targeted deletion of Col4a1 in brain microvessels of adult mice using brain endothelium-specific adeno-associated virus (AAV)-BR1 vectors with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). Eight-week-old Cas9 transgenic mice received retro-orbital injections of AAV-BR1 containing single guide RNA (sgRNA) targeting Col4a1 or control Rosa26 sequences. Animals underwent longitudinal behavioural testing, including novel object recognition, Y-maze and rotarod tests, over 6 months. Brain pathology was assessed using T2*-weighted MRI, histological analysis and electron microscopy. For human studies, we analysed MRI and genomic data from 836 participants from the BICWALZS biobank, examining associations between genetic variants and CMB burden using linear regression and χ2 analyses. T2*-weighted MRI revealed numerous CMBs with distributions remarkably similar to human CMBs, appearing within 3 months post-injection. CMB burden increased progressively over 6 months in a dose-dependent manner. Behaviourally, mice exhibited progressive cognitive decline and motor incoordination. Histological examinations revealed haemosiderin deposits corresponding to MRI-detected CMBs, without macroscopic intracerebral haemorrhage or white matter changes. Ultrastructural analysis demonstrated significant basement membrane thinning in Col4a1-depleted microvessels. CMB accumulation was associated with widespread astrocytic reactivity extending beyond microbleed sites, whereas microglial activation remained localized. In human subjects, we identified significant associations between four genetic variants of TIMP2, an endogenous inhibitor of the matrix-degrading enzyme MMP2 and CMB burden, with odds ratios of 1.50-1.96 for increased microbleed susceptibility. This work provides the first animal model demonstrating that selective disruption of collagen IV in adult brain microvessels is sufficient to generate CMBs with high penetrance and dose-dependent tunability. Our findings establish that basement membrane integrity is critical for preventing microbleed formation and suggest that dysregulated collagen IV homeostasis underlies sporadic human CMB development.},
}
@article {pmid42226661,
year = {2026},
author = {Guo, A and Bell, AG and Myhrvold, C},
title = {Towards deployable CRISPR-based nucleic acid detection.},
journal = {Progress in biomedical engineering (Bristol, England)},
volume = {8},
number = {2},
pages = {},
pmid = {42226661},
issn = {2516-1091},
support = {R01 AI182281/AI/NIAID NIH HHS/United States ; T32 GM148739/GM/NIGMS NIH HHS/United States ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Nucleic Acid Amplification Techniques/methods ; SARS-CoV-2/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Molecular Diagnostic Techniques/methods ; Point-of-Care Systems ; *COVID-19 Nucleic Acid Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Rapid Diagnostic Tests ; },
abstract = {Deployable diagnostics are necessary for the control and treatment of infectious diseases, with significant unmet needs revealed during the COVID-19 pandemic. Nucleic acid diagnostics remain among the most sensitive and specific forms of detection, yet their reliance on laboratory equipment and trained personnel limits their deployment in resource limited settings. CRISPR-based diagnostics are uniquely positioned to enable rapid, affordable, and highly accurate nucleic acid testing at both the point-of-care and the point-of-need. In this review, we discuss advances toward deployable CRISPR-based diagnostics. We begin by examining innovations in sample processing methods, emphasizing strategies that reduce equipment requirements and enhance compatibility across diverse sample types and pathogens. We then explore developments in one-pot isothermal and amplification-free approaches, comparing the benefits and tradeoffs associated with each, as well as multiplexing strategies for simultaneous detection of multiple pathogens. Finally, we consider additional factors that impact assay deployability, including reagent lyophilization to minimize cold chain dependence and readout technologies that enable detection in resource-limited settings. We conclude by outlining remaining challenges and opportunities for future progress.},
}
@article {pmid42226679,
year = {2026},
author = {Han, L and Wu, N and Li, Q and Li, Y and Zhang, Y and Chen, X and Zhou, H and Chen, F and Sun, T},
title = {Xylt2 Knockout Eliminates O-Xylosylation and Enhances Bone Morphogenetic Protein 2 Production in Chinese Hamster Ovary Cells.},
journal = {Biotechnology journal},
volume = {21},
number = {6},
pages = {e70256},
doi = {10.1002/biot.70256},
pmid = {42226679},
issn = {1860-7314},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Pentosyltransferases/genetics/metabolism ; UDP Xylose-Protein Xylosyltransferase ; *Bone Morphogenetic Protein 2/genetics/metabolism/biosynthesis ; Gene Knockout Techniques ; Humans ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Cricetinae ; Protein Processing, Post-Translational ; Heparan Sulfate Proteoglycans/metabolism ; },
abstract = {O-xylosylation of glycine-serine (GS) linkers in multispecific antibodies or fusion proteins introduces product heterogeneity, posing critical challenges for biomanufacturing quality control and elevating potential immunogenicity risks. This post-translational modification is primarily catalyzed by xylosyltransferase 2 (Xylt2) in chinese hamster ovary (CHO) cells. To address this, we generated Xylt2-deficient CHO cells via both clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and zinc finger nuclease (ZFN)-mediated gene knockout, which resulted in the complete elimination of O-xylosylation as verified by mass spectrometry in a GS linker-containing bispecific antibody. Furthermore, because heparan sulfate proteoglycans (HSPGs), key cell-surface receptors for ligand binding and internalization, rely on O-xylosylation for their proper biosynthesis and function, the Xylt2 knockout also enhanced the titer of human bone morphogenetic protein 2 (hBMP2), which undergoes HSPG-dependent cellular uptake, with up to a 2.5-fold increase from 9.62 to 34.09 µg/mL in engineered cells compared to wild-type CHO controls. Collectively, our results demonstrate that Xylt2-deficient cell lines provide a genetic approach to produce recombinant proteins without O-xylosylation, as well as for enhancing the titer of hBMP2.},
}
@article {pmid42227122,
year = {2026},
author = {Litvinova, IS and Zubkova, AE and Yudkin, DV},
title = {[Genetically Modified Pigs as Organ Donors: Challenges and Prospects].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {2},
pages = {216-234},
doi = {10.7868/S3034555326020024},
pmid = {42227122},
issn = {0026-8984},
mesh = {Animals ; *Animals, Genetically Modified/genetics ; *Transplantation, Heterologous/methods ; Humans ; Swine/genetics ; *Organ Transplantation ; *Tissue Donors/supply &amp; distribution ; Gene Editing ; },
abstract = {Organ transplantation is a treatment method for various organ failures and other severe pathologies, applied in critical cases to save a patient's life. However, there is a severe shortage of donor organs world-wide, resulting in hundreds of thousands of patients being unable to receive the organs they need in time. One possible solution to this problem is xenotransplantation-transplanting organs from animals to humans. Experiments in xenotransplantation began in the mid-1960s, with primates considered as the first potential donors. However, for a number of reasons, they proved unsuitable as a source of organs, while pigs turned out to be the best donors. The development of modern genetic engineering and genome editing methods has led to a new perspective on these animals as a source of human organs. Various genetic modifications have significantly reduced the immune response of the recipient to the graft and improved survival. To date, several successful transplants of organs and tissues from wild-type and genetically modified pigs to humans have been carried out worldwide, and a few companies are developing specialized lines of animals for xenotransplantation. In this review, we provide a detailed overview of the history of xenotransplantation worldwide, as well as all the genetic modifications introduced into the genome of pigs the organs of which have been used successfully for human transplantation, the role of these genetic modifications, and the mechanisms by which they are introduced.},
}
@article {pmid42227365,
year = {2026},
author = {Nooreen, Z and Verma, N and Narwariya, SS and Bhise, MR and Wal, A and Verma, R and Khan, A and Teli, S},
title = {Harnessing CRISPR-Cas Technology for Precision Antimicrobial Targeting.},
journal = {Current topics in medicinal chemistry},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115680266395401251105000157},
pmid = {42227365},
issn = {1873-4294},
abstract = {INTRODUCTION: Antibiotics have made major contributions to medicine, but misuse has resulted in antimicrobial resistance (AMR), which is caused by organisms such as ESKAPE and poses a danger to world health, which can potentially claim many more lives by 2050. Traditional antibiotics do not eliminate these resistant microbes, necessitating the development of new treatments. CRISPR-Cas systems, which target specific bacterial genes, present a viable strategy for combating AMR. CRISPR-Cas technologies provide a viable option for precision antimicrobial targeting by selectively inactivating resistance genes and virulence factors.<br><br>METHOD: The relevant data were obtained by reading several sources, including review papers from various publications from 2015 to 2024, to ensure that the study is inclusive, current, and relevant to new developments and trends in deep learning applications for the CRISPR-Cas system that included keywords like antimicrobial resistance, CRISPR-Cas system, and ESKAPE. Additionally, information was gathered from online sources.<br><br>RESULT: This system has demonstrated encouraging promise in targeting antimicrobial-resistant (AMR) bacteria and viruses, especially when combined with other delivery systems as conjugative plasmids, bacteriophages, and nanoparticles. Its capacity to specifically break foreign DNA has been shown in studies to either kill bacteria or suppress the production of harmful genes. CRISPRCas technologies have been effectively used to fight AMR pathogens. Furthermore, it has been demonstrated that CRISPR efficiently targets viral genomes, such as those of SARS-CoV-2 and Hepatitis B, offering great therapeutic promise in the treatment of viral-borne diseases.<br><br>DISCUSSION: CRISPR-Cas technology inhibits certain resistance and virulence genes, providing a precise, tailored approach to address antibiotic resistance. Compared to conventional antibiotics, it efficiently eradicates infections while preserving good bacteria. Its ability to combat a variety of drug-resistant bacteria and viruses has increased because of recent developments in delivery systems, including conjugative plasmids, bacteriophages, and nanoparticles. This precise method might lessen dependency on traditional medicines and transform infection control.<br><br>CONCLUSION: It is a novel yet precise tool for genome editing, that provides an innovative method to combat antimicrobial resistance by efficiently aiding in antibacterial targeting. It selectively eliminates dangerous pathogens while maintaining beneficial microbiota by enabling precise gene editing. By effectively treating resistant infections and promoting individualized care, this precision holds the potential to transform antimicrobial treatments.},
}
@article {pmid42229170,
year = {2026},
author = {Koo, C and Lee, D and Lee, B and Kim, S and Lee, J and Kwon, J},
title = {Base editing reveals context-dependent regulation of adhesion, anoikis, and motility by BAP1 in renal cell models.},
journal = {Biochemical and biophysical research communications},
volume = {827},
number = {},
pages = {154092},
doi = {10.1016/j.bbrc.2026.154092},
pmid = {42229170},
issn = {1090-2104},
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *Anoikis/genetics ; Humans ; *Cell Movement/genetics ; Cell Adhesion/genetics ; *Kidney Neoplasms/genetics/pathology ; *Carcinoma, Renal Cell/genetics/pathology ; *Gene Editing ; CRISPR-Cas Systems ; *Kidney/cytology/pathology/metabolism ; Cell Line, Tumor ; Cell Line ; Mutation ; },
abstract = {BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 function, reinstated anchorage dependence, and re-sensitized cells to anoikis. Here, we investigated whether disruption of Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Using adenine base editing, we introduced an inactivating Glu31Gly mutation into HK-2 cells, generating two independent isogenic BAP1-mutant clones, and established a BAP1-knockout clone by CRISPR/Cas9 as an additional control. Glu31Gly mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair, comparable to knockout cells. Despite the clear functional inactivation of BAP1, the Glu31Gly and knockout HK-2 cells neither acquired anchorage-independent growth nor anoikis resistance; instead, detached cells displayed increased apoptosis. In KMRC-20 cells, restoration of BAP1 enhanced both migration and invasion, whereas BAP1 inactivation or loss in HK-2 cells increased invasion but reduced migration, indicating distinct context-dependent roles for BAP1 in normal versus malignant renal cells. These findings demonstrate that BAP1 inactivation alone is insufficient to confer anchorage-independent survival in normal kidney epithelial cells and suggest that additional oncogenic alterations are required during kidney tumorigenesis. Our study further highlights the utility of precise base editing for dissecting the functional consequences of clinically relevant cancer mutations.},
}
@article {pmid42229577,
year = {2026},
author = {Saberian, M and Roosta, A and Afrisham, R},
title = {CRISPR-dCas9 epigenetic reprogramming in cancer: platforms, immuno-modulation and delivery challenges.},
journal = {Gene},
volume = {1005},
number = {},
pages = {150246},
doi = {10.1016/j.gene.2026.150246},
pmid = {42229577},
issn = {1879-0038},
mesh = {Humans ; *Neoplasms/genetics/therapy/immunology ; *CRISPR-Cas Systems/genetics ; Epigenome Editing ; *Epigenesis, Genetic ; Immunotherapy/methods ; Animals ; *Gene Editing/methods ; Cellular Reprogramming/genetics ; },
abstract = {CRISPR-dCas9 (catalytically dead Cas9) has revolutionized targeted epigenetic editing, offering locus-specific modulation of gene expression without altering DNA sequence. Beyond conventional approaches, novel strategies are rapidly emerging. These include combinatorial epigenetic reprogramming (co-recruiting multiple chromatin modifiers to a single locus), precision enhancer targeting (modulating oncogenic cis-regulatory elements), epigenetic modulation of immune pathways (reprogramming tumor or immune cells to boost anti-tumor immunity), and next-generation delivery systems for dCas9-based tools. This review synthesizes peer-reviewed literature (2015-2025) to highlight promising, yet still preclinical, advances in combinatorial reprogramming, enhancer targeting, immune-modulatory epigenetic approaches and delivery strategies, and to identify gaps that must be addressed prior to clinical translation. We highlight multi-effectors platforms (e.g. SunTag-like arrays, SSSavi modular docking, CRISPRoff memory writers) that amplify and diversify chromatin modifications. Precision enhancer editing systems (e.g. enCRISPRa/enCRISPRi) enable direct activation or silencing of distal regulatory elements in cancer cells. Epigenetic immunotherapy approaches use dCas9-activators to upregulate NK/T-cell ligands (MICA/MICB) and antigen-presentation genes (MHC I/II) in tumor cells. Finally, we survey innovations in dCas9 delivery that address in vivo challenges. Our review critically evaluates these advances, identifies gaps (off-target effects, context-dependence), and outlines future directions toward precision epigenetic therapies for diverse cancers.},
}
@article {pmid42229658,
year = {2026},
author = {Khera, HK and Kanan, S and Varghenese, A},
title = {BfCas12a-driven CRISPR-Cas assay for detection of SARS-CoV-2 nucleic acids.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {152850},
doi = {10.1016/j.ijbiomac.2026.152850},
pmid = {42229658},
issn = {1879-0003},
abstract = {The 2019 COVID-19 pandemic highlighted the urgent need for adaptable point-of-care diagnostics capable of swiftly detecting emerging pathogens. While RT-PCR has caught the attention as most used molecular test during pandemic recent advancements in alternate technologies include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated protein) based diagnostic tools tailored for pathogen detection. This study introduces an innovative CRISPR nucleic acid diagnostic, BCS-NABing (BfCas12a Sensor for Nucleic Acid Based-testing), utilizing Cas12a from Butyrivibrio fibrosolvens MD2001. BCS demonstrates similar sensitivity, detecting SARS-CoV-2 in synthetic as well as in patient-derived samples as LbCas12a. The technology employs lateral flow and fluorescence readouts, showcasing BCS-NABing is amenable to both. BCS-NABing significantly expands the nucleic acid diagnostic toolkit, offering a versatile platform adaptable to future pandemics and other infectious and genetic diseases, revolutionizing rapid and precise pathogen identification at the point of care.},
}
@article {pmid42230546,
year = {2026},
author = {Gao, Y and Chen, Y and Hu, Z and Wang, Z and Sun, D and Wu, C and Shuai, Q and Yan, Y},
title = {DOTAP-Engineered Lipid Nanoparticles Enable Fibroblast-Targeted CRISPR/Cas9 Delivery for HSP47 Silencing and Pulmonary Fibrosis Therapy.},
journal = {ACS applied materials &amp; interfaces},
volume = {18},
number = {23},
pages = {32391-32405},
doi = {10.1021/acsami.6c05610},
pmid = {42230546},
issn = {1944-8252},
mesh = {Animals ; *Fibroblasts/metabolism ; Mice ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Fatty Acids, Monounsaturated/chemistry ; *Quaternary Ammonium Compounds/chemistry ; *HSP47 Heat-Shock Proteins/genetics/metabolism/antagonists &amp; inhibitors ; Mice, Inbred C57BL ; *Idiopathic Pulmonary Fibrosis/therapy ; Humans ; Gene Silencing ; Cell Line ; *Pulmonary Fibrosis/therapy ; Liposomes ; },
abstract = {Heat shock protein 47 (HSP47) regulates collagen synthesis and fibrosis, which makes it a promising target for controlling pulmonary fibrosis. However, challenges such as cell-selective and efficient drug delivery continue to impede the verification and translation of HSP47-targeted therapies. The CRISPR/Cas9 system can effectively inhibit the overexpression of HSP47 within fibrotic lesions while significantly reducing off-target effects. This study develops a class of lipid nanoparticles (LNPs) capable of codelivering Cas9 mRNA and sgHSP47 to fibroblasts. By incorporation of an additional cationic effector molecule, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), these LNPs achieve differential delivery across various cell lines and markedly enhance uptake efficiency in fibroblasts. This system mediates effective HSP47 knockdown in L929 cells and the lungs of C57BL/6 mice, leading to reduced collagen deposition and fibroblast activation in bleomycin-induced idiopathic pulmonary fibrosis (IPF) mice. This LNP platform holds substantial potential for gene editing-based IPF therapy and provides valuable insights for the efficient and selective delivery of CRISPR/Cas9 systems.},
}
@article {pmid42230639,
year = {2026},
author = {Yuan, B and Tian, Y and Lin, WB and Bi, C and Zhang, Y and Jin, Y and Maatouk, B and Khashab, NM and Li, M},
title = {Cyanine-modified ssODNs enhance CRISPR-Cas9 HDR in stem cell embryo models via chromatin and chemical modulation.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-73901-8},
pmid = {42230639},
issn = {2041-1723},
support = {BAS/1/1080-01-01//King Abdullah University of Science and Technology (KAUST)/ ; },
abstract = {While CRISPR-Cas9 has transformed biomedical research, precise genome editing via homology-directed repair (HDR) remains limited by low efficiency and cytotoxicity, particularly in sensitive systems. Here, we show that simple 5' cyanine modifications of single-stranded oligodeoxynucleotides (ssODNs) markedly enhance HDR efficiency across human stem cell models, providing a 2-3-fold survival advantage. This approach enables in situ HDR in 3D human embryo models (blastoids), achieving up to 30% HDR-a 2.5-3-fold improvement over unmodified ssODNs-without compromising viability or developmental potential. Edited blastoids recapitulate key implantation features, including epiblast outgrowths surrounded by hypoblasts and trophoblasts. Mechanistically, enhanced HDR is associated with upregulation of HDR-related genes, including linker histone H1.0, which binds 5'Cy5-ssODNs and promotes HDR. Additionally, 5'Cy5 increases stability and nuclear availability of ssODNs. Molecular dynamics and free energy analyses suggest that 5'Cy5 promotes compact, circular-like conformations, validated by atomic force microscopy, and enhances donor-target duplex stability. Together, these findings establish cyanine-modified ssODNs as a simple, biocompatible strategy to improve precise genome editing.},
}
@article {pmid42231015,
year = {2026},
author = {Yamaguchi, K and Tomizawa, E and Kanematsu, N and Sakaguchi, K and Kasai, T and Ebisawa, R and Hasumi, A and Kawakatsu, K and Osakabe, Y and Osakabe, K and Nakatsuka, T},
title = {Engineering plant architecture in the ornamental species Eustoma grandiflorum by knockout of strigolactone biosynthesis.},
journal = {Plant cell reports},
volume = {45},
number = {6},
pages = {},
pmid = {42231015},
issn = {1432-203X},
support = {JP16H06279 (PAGS)//JSPS KAKENHI/ ; },
mesh = {*Lactones/metabolism ; Plants, Genetically Modified ; Gene Knockout Techniques ; Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems/genetics ; Phenotype ; Plant Shoots/genetics/growth &amp; development ; Dioxygenases/genetics/metabolism ; Flowers/genetics ; },
abstract = {The targeted mutation of the strigolactone biosynthetic gene, CCD8, through genome-editing CRISPR-Cas9 induces dwarfism and enhanced branching in Eustoma grandiflorum. In ornamental plants, modifying shoot architecture is a major breeding objective for diverse applications; however, achieving this goal using conventional techniques remains challenging. Strigolactones are plant hormones that inhibit shoot branching. We aimed to generate a strigolactone-deficient mutant of Eustoma grandiflorum using CRISPR-Cas9 genome editing. Four guide RNAs targeting CAROTENOID CLEAVAGE DIOXYGENASE 8 (EgCCD8) were designed, and transgenic E. grandiflorum plants were produced. Three independent lines carried deletions or insertions in all EgCCD8 alleles, indicating high editing efficiency. Two lines exhibited increased branching and pronounced dwarfism. In one of these lines, plant height in T1 null segregants was reduced to 47% of wild-type (WT) levels. Branch number increased 5.0-fold compared with the WT, and flower bud formation increased 1.7-fold alongside enhanced branching. These findings demonstrate that the targeted mutation of CCD8 through genome editing induces dwarfism and enhanced branching without compromising other ornamental traits.},
}
@article {pmid42231680,
year = {2026},
author = {Wang, L and Zhao, XY and Tang, H and Chen, TT and Chu, X},
title = {An Exosome RNA In Situ Detection Platform Based on a Regulated CRISPR/Cas12a Activity System and Its Application in Tumor Progression Monitoring and Therapeutic Efficacy Evaluation.},
journal = {Analytical chemistry},
volume = {98},
number = {23},
pages = {17385-17394},
doi = {10.1021/acs.analchem.6c02295},
pmid = {42231680},
issn = {1520-6882},
mesh = {*Exosomes/metabolism/genetics ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; Disease Progression ; Liposomes/chemistry ; Cell Line, Tumor ; *RNA/analysis ; Aptamers, Nucleotide/chemistry ; Biomarkers, Tumor ; },
abstract = {Tumor-derived exosomes, serving as promising biomarkers, hold substantial potential for reflecting the progression of diseases and assessing the efficacy of antitumor therapies. In this study, we developed a liposome-based platform capable of both precise recognition of tumor-derived exosomes and highly sensitive in situ analysis of the internal RNA. Leveraging surface-anchored DNA tags and their hybridization with two allosteric aptamers targeting exosomal marker CD63 and tumor marker PD-L1, the platform can specifically capture tumor-derived exosomes and facilitate the membrane fusion. Upon mixing of the vesicular contents, an internal functional duplex converter undergoes strand displacement with the target mRNA, releasing an uncaged strand. This strand eliminates the inhibition of the Cas12a/crRNA assembly imposed by an elongation-caged activator, thereby restoring the transcleavage activity of Cas12a. The activated Cas12a cleaves reporter probes to generate a fluorescent signal, enabling highly sensitive in situ detection of tumor-derived exosomal RNA. The constructed platform enables dynamic monitoring of disease progression in tumor-bearing mice by quantifying the relative levels of tumor-derived exosomes in serum and further distinguishes therapeutic outcomes among different drug treatments. This study highlights the significant potential of the proposed platform in tumor diagnosis and the evaluation of antitumor therapeutic efficacy.},
}
@article {pmid42234680,
year = {2026},
author = {Hemberg, M and Hansen, AL and Storgaard, J and Blay-Cadanet, J and Pedersen, A and Thielke, AL and Holm, CK},
title = {MAVS is important for antiviral defense against influenza A virus in a human respiratory epithelium model.},
journal = {PloS one},
volume = {21},
number = {6},
pages = {e0350839},
pmid = {42234680},
issn = {1932-6203},
mesh = {Humans ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; *Influenza A virus/physiology/immunology ; *Respiratory Mucosa/virology/immunology/metabolism ; *Influenza, Human/immunology/virology ; Virus Replication ; Interferon Type I ; CRISPR-Cas Systems ; Interferons/metabolism ; Cells, Cultured ; Animals ; },
abstract = {The respiratory epithelium is an important immunological barrier and the first line of defense against influenza A virus (IAV). In mice and in various cellular systems, induction of type I interferons (IFNα/β) during IAV infections is known to depend on cytosolic RNA sensors retinoic acid-induced gene I (RIG-I) and melanoma differentiation-association gene 5 (MDA5) and their common adaptor protein mitochondrial antiviral-signaling adaptor protein (MAVS). Until now, it has not been possible to directly assess the importance of MAVS for induction of IFNs and for resistance to IAV infection in primary human respiratory epithelium. Here, we used CRISPR-Cas9 to establish MAVS-deficient cultures of primary human respiratory epithelium using the air-liquid interphase culture system. Using this setup, we show that MAVS is indeed required for the induction of type I and type III IFNs and subsequently for the induction of IFN-stimulated genes in response to IAV infection in this respiratory epithelium model. Finally, we demonstrate that MAVS is important for restricting viral replication in this model. In conclusion, this study demonstrates that MAVS plays a non-redundant protective role during IAV infection in primary human respiratory epithelium.},
}
@article {pmid42235367,
year = {2026},
author = {Simonneau, B and Mienanzambi, S and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {Generation of two iPSC lines each carrying a stop codon mutation, c.366T &gt; A (p.Y122X) and c.1657C &gt; T (p.R553X), in the CFTR gene from the parental line PCIi033-A using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104024},
doi = {10.1016/j.scr.2026.104024},
pmid = {42235367},
issn = {1876-7753},
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; *Codon, Terminator/genetics ; Cell Differentiation ; Mutation ; Base Sequence ; },
abstract = {Cystic fibrosis is a recessive genetic disease due to mutations in the CFTR gene. Approximately 80% of patients carry the CFTR-F508del mutation and may benefit from the triple therapy Kaftrio®. However, patients with other rare mutations that prevent the production of the CFTR protein, such as nonsense mutations, have no available treatments. With CRISPR/Cas tools, we generate two iPSC lines bearing stop-codon mutations (c.366T > A and c.1657C > T) in the commercialized iPSC PCIi033-A. Both cell lines retained the characteristics of iPSCs. Differentiation of those iPSCs into lung epithelia could be a promising strategy for studying CFTR defects and developing readthrough strategies.},
}
@article {pmid42236579,
year = {2026},
author = {Thevis, M and Thomas, A and Naumann, N},
title = {[Gene doping: current test methods and analytical challenges].},
journal = {Bundesgesundheitsblatt, Gesundheitsforschung, Gesundheitsschutz},
volume = {},
number = {},
pages = {},
pmid = {42236579},
issn = {1437-1588},
abstract = {Gene doping refers to the use of gene therapeutic substances or technologies for the purpose of illicit sport performance enhancement. A wide variety of doping analytical detection methods are currently under development, ranging from PCR (Polymerase Chain Reaction) to high-resolution mass spectrometry to clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (CRISPR/Cas)-based detection methods. Due to the diverse approaches in gene therapy clinical research, it can be assumed that a wide variety of methods will also be needed to detect cases of gene doping. The increasing availability of unapproved gene doping-related products on the open consumer market underscores the urgent need to establish new detection methods for routine doping control analysis.},
}
@article {pmid42236690,
year = {2026},
author = {Price, JDW and Vizeacoumar, FS and Abuhussein, O and Maranda, V and Zhang, Y and Adachi, H and Nguyen, K and Kyrylenko, L and Rangel-Pozzo, A and Dong, H and Gong, L and Materi, A and Walke, P and Ganapathysamy, A and Denomy, C and Freywald, T and Dahiya, R and Elhasasna, H and Saxena, A and Vizeacoumar, JP and Patel, H and Rajamanickam, K and de Oliveira, DM and Lazell-Wright, M and Morales, AM and Aggarwal, A and Xu, JL and Alli, N and Munhoz, EP and Gao, P and Salsman, J and Dahiya, DK and Kola, NS and Gonzalez-Lopez, C and Thibault, P and Levin, M and Dellaire, G and Jette, N and Groot, G and Köbel, M and Lee, CH and Hopkins, L and Krishnan, A and Ahmed, S and Eskiw, C and Barakat, KH and Thakur, A and Wu, Y and DePinho, RA and Mai, S and Yu, YT and Wong, JMY and Freywald, A and Vizeacoumar, FJ},
title = {Epigenetic control of telomeric RNA maintains heterochromatin in telomerase-driven cancers.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {42236690},
issn = {2059-3635},
support = {PJT-156401//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; FBD-187665//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; PJT-156017//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; PJT-156401//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; 2021-OG-837261//Cancer Research Society (Soci&#xe9;t&#xe9; de Recherche sur le Cancer)/ ; 1160056//Cancer Research Society (Soci&#xe9;t&#xe9; de Recherche sur le Cancer)/ ; 2018-OG-23052//Cancer Research Society (Soci&#xe9;t&#xe9; de Recherche sur le Cancer)/ ; 2021-OG-876486//Cancer Research Society (Soci&#xe9;t&#xe9; de Recherche sur le Cancer)/ ; CFI-33364//Canada Foundation for Innovation (Fondation canadienne pour l'innovation)/ ; },
mesh = {Humans ; *Telomerase/genetics ; *Epigenesis, Genetic/genetics ; *Neoplasms/genetics/pathology ; *Heterochromatin/genetics ; *Telomere/genetics ; *Methyltransferases/genetics ; *RNA/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; },
abstract = {Tumor heterogeneity presents a major clinical challenge. Reactivation of telomerase (hTERT) is a near-universal hallmark of cancer, yet direct inhibition of hTERT has shown limited therapeutic benefit. To uncover tractable telomerase-related vulnerabilities, we applied a synthetic dosage lethality (SDL) strategy to identify genes required only when hTERT is overexpressed. We performed genome-wide CRISPR/Cas9 and shRNA screens across multiple isogenic cell line pairs differing in hTERT expression. From these datasets, 100 high-confidence candidates were prioritized and validated using an arrayed in vitro CRISPR screen and a pooled in vivo CRISPR screen across diverse cancer models, non-malignant cells, and patient-derived organoids. Through this pipeline, we identified FTSJ3, an RNA 2'-O-methyltransferase, as a top SDL target of hTERT. Depletion of FTSJ3 selectively impaired the viability of hTERT-positive cancer cells while sparing normal cells. Mechanistically, FTSJ3 installs 2'-O-methylation on the telomeric RNA TERRA, a modification essential for TERRA stability and function. Loss of FTSJ3 destabilizes TERRA, disrupts recruitment of the histone methyltransferase SUV39H1, and diminishes H3K9 trimethylation and HP1 assembly at sub-telomeric regions. This breakdown of repressive telomeric chromatin leads to genome instability and apoptosis specifically in hTERT-positive cells. These findings highlight SDL as a powerful strategy for uncovering targetable vulnerabilities and establish FTSJ3 as a central regulator of heterochromatin stability in telomerase-active cancers. Targeting FTSJ3 enzymatic activity offers a promising therapeutic entry point, acting upstream of TERRA to eliminate telomerase-driven malignancies selectively.},
}
@article {pmid42237969,
year = {2026},
author = {Alharbi, AG},
title = {Translational Barriers to AAV and CRISPR Gene Therapy in Diabetes.},
journal = {Saudi medical journal},
volume = {47},
number = {4},
pages = {626-652},
pmid = {42237969},
issn = {1658-3175},
mesh = {Humans ; *Genetic Therapy/methods ; *Diabetes Mellitus/therapy/genetics ; *Dependovirus/genetics ; *Translational Research, Biomedical ; Animals ; *CRISPR-Cas Systems ; Gene Transfer Techniques ; Clinical Trials as Topic ; Genetic Vectors ; },
abstract = {Gene therapy targets diabetes pathophysiology rather than symptoms, yet clinical translation is slower than preclinical success. This review synthesizes 143 registered trials (ClinicalTrials.gov, 2010-2025) identifying systematic implementation barriers. Despite >15 years of development, 83% of trials remain in Phase I-II. Only zimislecel achieved Phase III outcomes (83% insulin independence at 12 months, n=12, requiring immunosuppression). VM202 for diabetic neuropathy failed Phase III despite positive extension results. Three systematic barriers emerged: (1) human transduction efficiency is 6-8.7-fold lower than preclinical models; (2) pre-existing immunity excludes 58-78% of candidates; (3) manufacturing capacity serves <2.5% of target population (40-400 years to treat at maximum capacity). These constraints explain why ex vivo cell therapies advanced to efficacy trials while in vivo gene delivery remains in Phase I despite longer development timelines. Research priorities should emphasize non-viral delivery systems offering scalable manufacturing and universal hypoimmune donor cells to address access barriers.},
}
@article {pmid42238783,
year = {2026},
author = {Carson, RM and Needham, PM and Mendoza, PJF and Nugen, SR},
title = {Scarless one-tube genome assembly via computationally optimized uracil-DNA glycosylase reactions.},
journal = {RSC chemical biology},
volume = {},
number = {},
pages = {},
pmid = {42238783},
issn = {2633-0679},
abstract = {Synthetic biology enables the creation of systems such as bacteriophage (phage)-based biosensors, leveraging the innate specificity and efficiency of phages to rapidly identify pathogens. However, the current genome assembly and editing methods, including Gibson Assembly, Golden Gate Assembly, and CRISPR-Cas systems, have limitations that can hinder speed and flexibility, especially when complex modifications are needed. This study introduces a novel means for generating engineered bacteriophages through a one-pot, modular in vitro genome assembly platform utilizing uracil-DNA glycosylase, which allows genome modification without requiring extended overlaps, the removal of restriction enzyme sites, a Cas system, or homologous recombination. The design also minimizes the risk of secondary structure formation (e.g., hairpins), allowing for a more efficient assembly of fragments. To demonstrate functional genome engineering, we incorporated a NanoLuc luciferase reporter gene into the T7 genome, producing a recombinant phage capable of detecting E. coli, a strategy consistent with our previous work on waterborne pathogen detection. This platform enables rapid and flexible synthetic genome construction with high functional assembly efficiency, with broad applications in phage engineering, biosensing, and synthetic biology.},
}
@article {pmid42239233,
year = {2026},
author = {Finocchio, G and Oberli, S and Lampe, G and Schmitz, M and Sternberg, SH and Jinek, M},
title = {Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42239233},
issn = {2692-8205},
abstract = {CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex[1,2]. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools[3-6]. The type I-F Pseudoalteromonas CAST (PseCAST) displays the highest activity in mammalian cells to date[7] and has been the subject of extensive directed evolution[8], but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation[9]. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the PseCAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa PseCAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.},
}
@article {pmid42239534,
year = {2026},
author = {Hu, Y and Zhao, D and Diao, Y and Bai, C and Zhou, K and Huang, F and Li, R and Hao, X and Liu, H and Liu, J and Zhou, L},
title = {Simultaneous molecular detection of Mycobacterium tuberculosis and multidrug resistance using CRISPR-AaCas12b-based nucleic acid assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1844184},
pmid = {42239534},
issn = {2235-2988},
mesh = {*Mycobacterium tuberculosis/genetics/isolation &amp; purification/drug effects ; *Tuberculosis, Multidrug-Resistant/diagnosis/microbiology ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; Rapid Diagnostic Tests ; Rifampin/pharmacology ; Antitubercular Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; DNA-Directed RNA Polymerases/genetics ; Mutation ; Microbial Sensitivity Tests ; Isoniazid/pharmacology ; Catalase ; },
abstract = {OBJECTIVES: To address the unmet need for rapid, accurate diagnosis of Mycobacterium tuberculosis (MTB) and multidrug-resistant tuberculosis (MDR-TB), we developed and validated a clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas)-based diagnostic assay.<br><br>METHODS: A multiplex-recombinase polymerase amplification (RPA) coupled CRISPR-Alicyclobacillus acidiphilus Cas12b (AaCas12b) assay was established for simultaneous detection of MTB by targeting the specific insertion sequence IS6110 and the two most common drug resistance mutations, rpoB 1349C>T for rifampicin resistance and katG 944G>C for isoniazid resistance. The assay supported dual-readout signal detection using both a fluorescent platform and lateral flow chromatography (LFC). Its diagnostic performance was evaluated in 48 clinical samples using WHO-recommended GeneXpert MTB/RIF, phenotypic drug susceptibility testing (pDST), and sequencing as reference standards.<br><br>RESULTS: The multiplex-RPA CRISPR-AaCas12b assay showed a limit of detection (LoD) of 1.5 CFU/mL for MTB detection, with a sensitivity of 97.1% and a specificity of 100% using culture as the reference standard, and a total turnaround time of 30&#xa0;min (20&#xa0;min for RPA and 10&#xa0;min for CRISPR cleavage). For MDR-TB-related mutations, the assay achieved a sensitivity of 94.1% and a specificity of 100% for rpoB 1349C>T, and 94.7% sensitivity and 93.1% specificity for katG 944G>C, using sequencing as the reference standard. Notably, the LFC-integrated assay maintained comparable diagnostic accuracy with a total turnaround time of 35&#xa0;min (20&#xa0;min for RPA, 5&#xa0;min for CRISPR cleavage, and 10&#xa0;min for lateral flow strip reading).<br><br>CONCLUSION: The established multiplex-RPA CRISPR-AaCas12b assay enables simple, accurate, and sensitive detection of MTB and common mutations associated with MDR-TB. With a rapid, simplified workflow and low resource requirements, this approach holds considerable potential for point-of-care testing in resource-limited settings, thus facilitating improved surveillance and control of TB and drug-resistant TB.},
}
@article {pmid42240620,
year = {2026},
author = {Zhou, J and Liu, W and Nie, X and Wang, X and Hu, Y and Wan, C and Liao, Y and Pan, S},
title = {Development of a multifunctional nucleic acid response platform utilizing the Cas12a and Cas13a integrated targeting system.},
journal = {Nucleic acids research},
volume = {54},
number = {10},
pages = {},
pmid = {42240620},
issn = {1362-4962},
support = {82103613//National Natural Science Foundation of China/ ; 82103613//National Natural Science Foundation of China/ ; 2023B22//Tongji Hospital Scientific Research Cultivation Program/ ; 32500005//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA/metabolism/genetics ; RNA/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; },
abstract = {Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system has ushered in a transformative era in biodetection, leveraging its simplicity and efficiency to enable Cas protein-based signaling systems for applications in early tumor screening, viral detection, and molecular logic circuits. However, the constrained compatibility of CRISPR/Cas-based signaling systems with diverse input types limits their versatility, primarily due to the restricted activation mechanisms of the Cas protein. Herein, we developed the Cas12a and Cas13a Integrated Targeting (CACIT) system, which harnesses DNA/RNA strand displacement reactions to integrate the enzymatic capabilities of Cas12a and Cas13a. This system supports simultaneous DNA and RNA inputs, offering exceptional programmability and cost-effectiveness. By employing strand displacement reactions, the CACIT system achieves synchronized activation of Cas12a and Cas13a. We have demonstrated that the CACIT system excels in single-nucleotide-variant (SNV) detection, viral RNA detection, machine learning-driven nucleic acid concentration response modeling, logic operations, and intracellular imaging. As a streamlined and versatile signaling platform, the CACIT system expands the scope of CRISPR/Cas activation strategies. With its inherent simplicity and compatibility, this system facilitates integration with diverse nanodevices. Further, this system provides a highly programmable, multifunctional computational module for molecular networks, heralding new possibilities for artificial signaling systems.},
}
@article {pmid42242804,
year = {2026},
author = {Qian, J and Lu, J and Chen, X and Shen, H and Lu, F},
title = {Programmable Fc-encoded DNA tile-cube capture enables a thrombin-activated ratiometric ECL/SERS biosensor via a PAM-engineered toehold switch and CRISPR/Cas12a cleavage.},
journal = {Analytica chimica acta},
volume = {1415},
number = {},
pages = {345709},
doi = {10.1016/j.aca.2026.345709},
pmid = {42242804},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Thrombin/analysis ; *CRISPR-Cas Systems/genetics ; Spectrum Analysis, Raman ; Electrochemical Techniques/methods ; *DNA/chemistry ; Humans ; Luminescent Measurements ; Electrodes ; *CRISPR-Associated Proteins/metabolism/chemistry ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; Dendrimers ; },
abstract = {BACKGROUND: Accurate thrombin detection is important for coagulation-related assessment, but reliable quantification at ultralow levels remains challenging because matrix interference, electrode-to-electrode variation, and single-channel signal drift can compromise analytical accuracy. Herein, we developed a thrombin-responsive ratiometric electrochemiluminescence/surface-enhanced Raman scattering (ECL/SERS) biosensor integrating Fc-encoded DNA tiles, a DNA-cube capture scaffold, and a PAM-engineered toehold-switch-regulated CRISPR/Cas12a module on a Ti3C2/CsPbBr3@PDA@Au-modified electrode.<br><br>RESULTS: An intentionally cleavable linker probe (LP) serves as the bridge for retaining Fc-rich DNA tiles near the electrode. Without thrombin, intact LP enables tile capture, causing ECL quenching and strong Fc SERS output. With thrombin, split-aptamer proximity assembly activates the toehold switch and Cas12a/crRNA, leading to LP cleavage, Fc-tile depletion, ECL recovery, and SERS attenuation. The anti-correlated signals were integrated as Q&#x202f;=&#x202f;IECL/ISERS. The biosensor showed a detection range from 1&#x202f;&#xd7;&#x202f;10[-7] to 1&#x202f;&#xd7;&#x202f;10[-1]&#x202f;nM and a detection limit of approximately 0.064&#x202f;fM. Synthetic cleaved LP standards confirmed that LP cleavage can be directly converted into ratiometric ECL/SERS switching. Serum spike-recovery tests gave recoveries of 96.8%-104.0%.<br><br>SIGNIFICANCE: This work establishes a programmable capture-release strategy that converts thrombin recognition into CRISPR/Cas12a-mediated LP cleavage and deterministic interfacial reconfiguration, providing a sensitive, internally referenced, and extensible platform for protein biosensing.},
}
@article {pmid42242806,
year = {2026},
author = {Geng, Y and Zhao, F and Yang, J and Liu, K and Wu, L},
title = {An amplification-free CRISPR/Cas12a-nanopipette electrochemical sensor for in situ discrimination of Salmo salar and Oncorhynchus mykiss.},
journal = {Analytica chimica acta},
volume = {1415},
number = {},
pages = {345713},
doi = {10.1016/j.aca.2026.345713},
pmid = {42242806},
issn = {1873-4324},
mesh = {Animals ; *Salmo salar/genetics ; *Electrochemical Techniques/instrumentation/methods ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation/methods ; *Oncorhynchus mykiss/genetics ; *DNA/analysis/genetics ; Limit of Detection ; },
abstract = {BACKGROUND: Salmo salar (S. salar) and Oncorhynchus mykiss (O. mykiss) exhibit highly similar morphological characteristics, which frequently leads to market adulteration. This phenotypic resemblance poses significant challenges for accurate on-site species identification using conventional analytical methods.<br><br>RESULTS: In this study, an amplification-free electrochemical biosensor was constructed by integrating the CRISPR/Cas12a recognition system with a single nanopipette, enabling precise identification of S. salar DNA without the need for complex pretreatment. This strategy is based on target DNA-induced activation of the trans-cleavage activity of Cas12a, which cleaves the DNA reporter molecules immobilized on the inner wall of the nanopipette, leading to alterations in surface charge and enabling electrochemical readout via ion current rectification (ICR). Under optimized conditions, the sensor exhibited a detection limit as low as 0.11 pM, effectively distinguished O. mykiss DNA, and demonstrated favorable reproducibility and stability.<br><br>SIGNIFICANCE: This work provides a low-waste, on-site analytical tool for the authentication of aquatic species, enabling direct in situ detection within salmon tissue sections without the need for nucleic acid amplification or complex sample pretreatment, thereby effectively filling a technical gap in rapid and accurate species identification.},
}
@article {pmid42242974,
year = {2026},
author = {Liao, S and He, Y and Liu, G and Li, Y and Tang, X and Zheng, X and Qi, Y and Zhang, T and Zhang, Y},
title = {Boosting genome editing of non-coding sequences in plants with glycosylase-mediated multi-nucleotide deletion editors.},
journal = {Science bulletin},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.scib.2026.05.046},
pmid = {42242974},
issn = {2095-9281},
abstract = {Compared with protein-coding sequences, non-coding sequences-such as promoters, untranslated regions (UTRs), microRNAs, and other regulatory ncRNAs-constitute the vast majority of the plant genome. They serve as critical regulators of gene expression and therefore represent promising targets for crop improvement. However, their study and manipulation remain challenging due to the lack of efficient tools for generating large genomic deletions. Here, we report that CRISPR-Cas9-based glycosylase base editors (gBEs) function primarily as highly efficient multi-nucleotide deletion editors (gMDEs) in plants, a role distinct from their predominant base-editing activity in mammals. This functional shift is likely driven by a preferential AP lyase repair pathway for glycosylase-generated abasic sites (apurinic/apyrimidinic or AP sites) in plant cells. Unlike its parental system, CRISPR-Cas9, gMDEs efficiently generate 6-20 bp deletions across protospacers in both rice and soybean. We demonstrate their versatility by generating a continuum of plant height variation through promoter editing of OsD18 and boosting grain size by disrupting regulatory elements in both the 5' and 3' UTRs of OsGLW7, which function through distinct regulatory mechanisms. This work establishes gMDEs as a versatile and precise genome editing platform for inducing multi-nucleotide deletions in plants, making them efficient tools for genetic perturbation, especially of non-coding sequences.},
}
@article {pmid42243670,
year = {2026},
author = {Klepper, AM and Akhgari, A and Rischer, H},
title = {dSaCas9 enables enhanced transcriptional activation in Nicotiana benthamiana compared to its dSpCas9 ortholog.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-09174-6},
pmid = {42243670},
issn = {1471-2229},
abstract = {The SaCas9 from Staphylococcus aureus has been shown to be more effective than the commonly used SpCas9 from Streptococcus pyogenes for the generation of mutations in plants and has boosted CRISPR/Cas systems. CRISPRa/Cas systems on the other hand have been focused on the use of SpCas9. Building on foundational work, we developed a modular CRISPRa/Cas ortholog system based on a single plasmid infiltration for the use and comparison of both SaCas9 and SpCas9 for enhanced gene expression. Utilizing the multi-kingdom Golden Gate cloning platform, we integrated various elements to create a widely adaptable system.For the first time in the current study, we demonstrate that the dSaCas9 effector induces stronger activation of reporter gene expression by targeted promoters than dSpCas9 in plants. As both effectors are sharing a common target site for the tested promoter, they could be directly compared with each other showing that the dSaCas9-effector exhibited a stronger effect in the expression of the reporter genes. This allows fine-tuning the expression of genes by using different effectors. In addition, we could show on the example of the pNOS promoter and a truncated version of the pNOS promoter, that a lower basic expression can lead to an increased relative induction.},
}
@article {pmid42244457,
year = {2026},
author = {Adams, AB and Tector, M and Burlak, C and Estrada, J and Reyes, L and Copsel, S and Muniz, C and Novara Gennuso, V and Martucci, M and Iwakoshi, N and Dryden, M and Faber, D and Ray, B and Haver, H and Hariharan, J and Vianna, R and Tector, AJ},
title = {Swine Leukocyte Antigen DR Deletion is a Viable Option for Donor Pigs Used in Renal Xenotransplantation.},
journal = {Annals of surgery},
volume = {},
number = {},
pages = {},
doi = {10.1097/SLA.0000000000007095},
pmid = {42244457},
issn = {1528-1140},
support = {UO1 AI126322//National Institute of Allergy and Infectious Disease/ ; },
abstract = {OBJECTIVE: Swine Leukocyte Antigen-DR knockout (SLA-DR KO) pigs were created and evaluated for safety/infectious profile and the ability to function in a preclinical model of xenotransplantation.<br><br>BACKGROUND: SLA-DR is the dominant class II MHC antigen in pigs. It is unclear whether it is feasible/safe to delete SLA-DR in donor pigs to be used in kidney xenotransplantation.<br><br>METHODS: SLA DR KO pigs were created on the &#x3b1;-gal Sda (GGTA1/B4GALNT2) deficient genetic background using CRISPR/Cas and somatic cell nuclear transfer. Pigs were evaluated for 55 potential zoonotic pathogens using digital droplet PCR assays. Four GGTA1/B4GALNT2/SLA-DR KO pig kidneys were transplanted into immunosuppressed rhesus monkeys. Renal function was monitored to evaluate whether these prototype kidneys could provide life supporting renal function in a preclinical model.<br><br>RESULTS: SLA-DR KO pigs were produced and are healthy more than 16 months later, devoid of 55 pathogens with zoonotic infectious potential. Recipients survived 7, 126, >365, and >365 days. Serum creatinine was maintained in long term survivors (Cr 0.8&#xa0;mg/dL in both). Early graft losses (7 and 126&#xa0;d) occurred because of donor specific pre-transplant SLA antibodies detected using a SLA bead crossmatch assay.<br><br>CONCLUSIONS: SLA-DR KO pigs are viable and safe to consider as potential donors in clinical trials. The SLA DR KO pig kidneys provided good long-term graft function in a preclinical model if the donor was not sensitized to other SLA antigens present in the donor pig. The SLA-DR KO prototype is promising for evaluation in pig-to-human clinical xenograft trials.},
}
@article {pmid42244531,
year = {2026},
author = {Kunwar, S and Hallmark, T and Manna, S and Keiser, D and Naegle, B and Thomas, A and Beisel, CL and Jackson, RN},
title = {Target RNA-triggered CRISPR-Cas12a2 Preferentially Cleaves Collateral DNA over RNA.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.01.05.631166},
pmid = {42244531},
issn = {2692-8205},
abstract = {CRISPR-Cas systems often rely on collateral cleavage of nucleic-acid substrates to combat recognized mobile genetic elements. Of the CRISPR-associated (Cas) RNA-guided effector nucleases, Cas12a2 stands out as the only known example exhibiting rapid collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA, after activating upon binding cognate RNA. However, little is known about the underlying mechanisms of collateral cleavage. Here, we show, using enzyme kinetics and inhibition assays, that Cas12a2 preferentially cleaves collateral DNA over RNA substrates, even when RNA substrates are more abundant. Additionally, using enzyme mutants, enzyme kinetics, and plasmid cleavage assays, we determine that the dsDNA cleavage mechanism relies on the 'aromatic clamp' residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can readily cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while an RNA-targeting Cas13a cannot. This work provides foundational kinetic and biochemical insights into the collateral cleavage mechanism and substrate preferences of Cas12a2, with immediate implications for understanding Cas12a2-based immunity and developing Cas12a2-based technologies.},
}
@article {pmid42244906,
year = {2026},
author = {Zhang, Z and Huang, Z and Wang, Y and Li, Z and Wen, Z and Gu, Y},
title = {Transposon-based genome editing of industrial microorganisms: advances, challenges, and prospects.},
journal = {Synthetic and systems biotechnology},
volume = {14},
number = {},
pages = {388-398},
pmid = {42244906},
issn = {2405-805X},
abstract = {As mobile genetic elements, transposons play a crucial role in the adaptive evolution and genome engineering of industrial microorganisms. Their applications range from high-throughput functional genomics, enabling systematic genotype-phenotype mapping via transposon sequencing, to the construction of random integration libraries for chassis development and directed evolution. Despite the emergence of precise editing tools such as CRISPR-Cas, transposon technology remains indispensable in non-model industrial strains owing to its operational simplicity and high efficiency. Recent discoveries of novel transposon systems, along with their functional enhancement, have further expanded their utility. Looking ahead, integrating transposon technology with strategies such as AI-assisted design and CRISPR-Cas-based systems will greatly advance our ability to decipher and engineer industrial microbial cell factories. This review summarizes the principles, challenges, and opportunities of transposon-associated technologies in industrial microorganisms, offering new insights into their roles in industrial biotechnology.},
}
@article {pmid42245112,
year = {2026},
author = {Kumar, A and Muthuramalingam, P and Verma, L and Kumar, R and Kumar, N and Misra, J and Ravi, K and Shin, H and Ramesh, M},
title = {Modern genomic and omics-based technologies for millet breeding and genetic improvement.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1782766},
pmid = {42245112},
issn = {1664-462X},
abstract = {Millets are a diverse group of small seeded grasses that have long served as vital staple foods and forage crops across a wide range of agro-ecological regions. Known for their exceptional adaptability to marginal and resource poor environments, millets have historically supported farming communities in arid and semi-arid regions. Despite these advantages, they remain underutilized in modern agriculture due to limited genomic resources, harsh growing conditions, and insufficient technological support for their improvement. However, growing concerns over climate change, malnutrition and the need for sustainable agriculture have renewed global scientific interest in millet research and breeding. Recent breakthroughs in molecular biology such as marker-assisted selection (MAS), Genome-wide association studies (GWAS), genomic selection (GS), genetic engineering, omics technologies, speed breeding, and machine learning (ML) have significantly transformed the landscape of millet improvement. Advances in MAS, high-throughput genotyping, transcriptomics, proteomics, metabolomics, and phenomics have enabled more profound insights into the genetic architecture of key agronomic traits. These tools have facilitated the identification of genes, regulatory networks, and metabolic pathways governing drought tolerance, nutrient use efficiency, disease resistance and other essential stress responses. The integration of next-generation sequencing and comparative genomics has further expanded millet research through the development of reference genomes, pangenomes, and comprehensive germplasm characterizations. Pangenomic approaches, in particular, have uncovered structural variations and novel alleles that contribute to phenotypic diversity, offering valuable targets for breeding climate-resilient cultivars. High-resolution phenomic platforms have enhanced the precision of trait evaluation, enabling rapid screening of large populations under diverse environmental conditions. Additionally, genome editing technologies, especially CRISPR/Cas systems and multiplex CRISPR/Cas, have opened new avenues for precise genetic improvement by enabling targeted gene modification to enhance stress resilience and yield traits. Therefore, these integrated omics-driven and molecular breeding strategies are reshaping the millet improvement. With modern biotechnological innovations, researchers are now better equipped to develop high-yielding, nutrient-rich and climate-resilient millet cultivars. These advancements position millets as strategic crops that can strengthen global food and nutritional security while promoting sustainable agricultural systems in the face of mounting environmental challenges.},
}
@article {pmid42247138,
year = {2026},
author = {Pei, T and Yang, W and Lei, Y and Qu, Z and Gao, Y and Zhang, M and Xu, T and Wen, Q and Liu, Q},
title = {CRISPR/Cas‑based epigenome editing for osteogenic lineage commitment.},
journal = {Cell and tissue research},
volume = {404},
number = {3},
pages = {},
pmid = {42247138},
issn = {1432-0878},
mesh = {*Epigenome Editing ; Humans ; *Osteogenesis/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Cell Lineage/genetics ; Mesenchymal Stem Cells/cytology ; },
abstract = {Bone regeneration remains constrained by incomplete osteogenic commitment of mesenchymal stem cells (MSCs), underscoring the need for precise lineage control. CRISPR/Cas-based epigenome editing provides programmable access to chromatin regulators without altering the DNA sequence, and catalytically inactive Cas9 (dCas9) fused to transcriptional activators, repressors, or chromatin modifiers enables locus-specific modulation of key osteogenic networks, including RUNX2, OSX, and BMP2, while suppressing inhibitory loci such as PPARG, SOST, and DKK1. Multiplex strategies further allow the concurrent activation of osteogenic genes and repression of adipogenic or Wnt antagonists, reshaping lineage allocation in vitro and in vivo. Delivery innovations-from AAV vectors and lipid nanoparticles to biomaterial scaffolds and extracellular vesicles-support local and systemic applications with increasing precision, while whole-genome chromatin profiling and high-fidelity Cas variants reduce off-target risk, and CRISPRoff/on platforms provide reversible and heritable control of transcriptional states. Proof-of-concept studies in small animals demonstrate bone repair in preclinical models, with emerging large-animal data highlighting translational potential. Remaining challenges include payload size, immunogenicity, durability of epigenetic states, GMP-grade manufacturing, and regulatory classification. Looking ahead, advances such as AI-guided gRNA libraries, mechano-responsive scaffolds, and long-term tracking of epigenetic memory may yield durable "smart" osteo-epigenetic therapies. Collectively, CRISPR/dCas9-based epigenome editing is progressing from mechanistic exploration toward clinically viable strategies for skeletal regeneration.},
}
@article {pmid42247307,
year = {2026},
author = {Liao, ZE and He, Z},
title = {Somatic cell reprogramming into stem cells: approaches, mechanisms, and therapeutic applications.},
journal = {Asian journal of andrology},
volume = {},
number = {},
pages = {},
doi = {10.4103/aja20268},
pmid = {42247307},
issn = {1745-7262},
abstract = {Somatic cell reprogramming technology can reverse fate determinations of the differentiated cells by regulating their epigenetic and gene expression programs. This reprogramming enables the acquisition of pluripotency to differentiate into mature and functional cells, which provides sufficient cells for regenerative and reproductive medicine. Significant progress has recently been made by peers and us in reprogramming somatic cells, e.g., Sertoli cells, fibroblasts, and peripheral blood mononuclear cells into functional stem cells. In this review, we systematically summarize the development and optimization of multiple core methods for cell reprogramming, including somatic cell nuclear transfer (SCNT), transcription factor-induced reprogramming, chemical reprogramming, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas)-based reprogramming. We also address the epigenetic remodeling mechanisms that are involved in somatic cell reprogramming, including the dynamic processes of chromatin accessibility regulation, DNA demethylation, histone modifications, and the regulation of non-coding RNAs (ncRNAs). Moreover, we discuss current challenges and perspectives in this field. Significantly, stem cells derived from somatic cells have great applications in regenerative medicine for treating various kinds of diseases, such as infertility, diabetes, neurodegenerative diseases, and chimeric antigen receptor T-cell (CAR-T) immunotherapy with attention to cell maturity, heterogeneity, and safety. Our in-depth understanding of approaches, mechanisms, and applications of somatic cell reprogramming into stem cells is essential for cell therapy and tissue engineering.},
}
@article {pmid42247893,
year = {2026},
author = {Li, Z and Huang, J and Li, Y and Cao, F and Gao, X and Lin, Y and Li, Y},
title = {Type VI secretion system: Central regulator of antimicrobial resistance dynamics via indirect mechanisms.},
journal = {Microbiological research},
volume = {311},
number = {},
pages = {128574},
doi = {10.1016/j.micres.2026.128574},
pmid = {42247893},
issn = {1618-0623},
abstract = {Multidrug resistance (MDR) in bacteria poses a significant global threat to public health. Elucidating the core molecular regulatory mechanisms underlying MDR is crucial for developing novel intervention strategies. In Gram-negative bacteria, the phage-derived Type VI Secretion System (T6SS) functions as a versatile "molecular weapon". Beyond its classical role in interbacterial antagonism, T6SS acts as a key indirect regulatory hub for modulating bacterial antimicrobial resistance (AMR) in a strain-specific and environment-dependent manner. Although T6SS does not directly participate in the expression of antibiotic resistance genes (ARGs) or the catalytic activity of AMR-related enzymes, it profoundly influences the development and dissemination of AMR across strains and species through multiple indirect mechanisms. This review systematically analyzes four core T6SS-mediated mechanisms: (1) secretion of AMR-associated effectors and biofilm modulation to establish resistant phenotypes; (2) formation of synergistic regulatory networks with biofilm development, oxidative stress response, efflux pumps, and other secretion systems, which specifically enhances bacterial antibiotic tolerance (distinct from antibiotic resistance phenotypes); (3) acceleration of horizontal gene transfer (HGT) of ARGs through natural transformation, plasmid conjugation, and outer membrane vesicle (OMV)-mediated transport; (4) targeted interbacterial killing enabling antimicrobial-resistant strains to overcome colonization resistance, gain ecological advantages, and exacerbate clinical infections. Building on this framework, novel anti-AMR strategies targeting T6SS are outlined, including direct disruption of T6SS assembly and function, interference with upstream regulators (e.g., quorum sensing), optimization of CRISPR-Cas gene editing, and engineered T6SS-targeted delivery platforms. By dissecting the T6SS-driven AMR network and its clinical translational potential, this review provides a foundation for designing next-generation therapies to reverse AMR and block ARG transmission and also discusses existing bottlenecks limiting the clinical translation of T6SS-targeted therapies, while identifying critical future research directions such as deciphering species-specific mechanisms and enhancing targeted delivery efficiency.},
}
@article {pmid42247991,
year = {2026},
author = {Chen, H and Jiang, Z},
title = {CrisprFusion: A feature fusion model with multi-type input features for sgRNA activity prediction.},
journal = {Computational biology and chemistry},
volume = {124},
number = {Pt 1},
pages = {109137},
doi = {10.1016/j.compbiolchem.2026.109137},
pmid = {42247991},
issn = {1476-928X},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Deep Learning ; *Gene Editing ; Humans ; },
abstract = {The CRISPR/Cas9 system enables precise and efficient genome editing, but its efficacy heavily relies on sgRNA activity. Although deep learning has been widely applied to sgRNA activity prediction, existing methods often integrate multiple biological features without a well-designed fusion strategy. To tackle this issue, we present CrisprFusion, a deep learning framework that explicitly encodes four biological features through a four-branch input structure. The core of our model is a novel Multi-Grain Cross Attention Fusion Module, which performs fusion at two levels: branch-level gating for adaptive reweighting of different modalities, and token-level alignment for capturing position-specific interactions along the 23-nt sgRNA sequence. We evaluate CrisprFusion on seven high-throughput datasets with six representative baselines. Our method achieves consistent and superior average performance across all datasets and remains competitive in cross-cell-line validation on four functional screens. Ablation experiments verify the effectiveness of the proposed fusion module, and attention visualization reveals the importance of individual biological features. Overall, CrisprFusion offers an effective and interpretable approach for multimodal biological feature integration in sgRNA activity prediction.},
}
@article {pmid42248117,
year = {2026},
author = {der Auweraer, SV and Roth, MB and Vlahos, K and Howden, SE and Lockhart, PJ and Payne, JM and Brems, H and Bozaoglu, K},
title = {Generation and characterization of four iPSC and isogenic gene-corrected lines from Legius syndrome patients.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104026},
doi = {10.1016/j.scr.2026.104026},
pmid = {42248117},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Cell Differentiation ; Leukocytes, Mononuclear/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Legius syndrome is an autosomal dominant disorder caused by variants in SPRED1. In this study, we generated four induced pluripotent stem cell (iPSC) lines derived from patients with Legius syndrome by reprogramming peripheral blood mononuclear cells. Using CRISPR/Cas9 or prime editing, the pathogenic variants were corrected to generate isogenic control lines. All patient and isogenic control lines exhibited a normal morphology and karyotype, expressed pluripotency markers, and possessed trilineage differentiation potential. This is the first established human iPSC model developed for Legius syndrome and is a valuable resource for investigating the molecular mechanisms underlying this condition.},
}
@article {pmid42249743,
year = {2026},
author = {Tian, Y and Cao, Y and Pan, Z and Xu, L and Fan, Z and Mo, Y and Zhu, X and Zhang, X and Li, H and Zeng, S and Ren, F},
title = {CRISPR-Cas13a/Cas12a Assisted Dual Portable and Visualized HDV and HBV Detection.},
journal = {Journal of medical virology},
volume = {98},
number = {6},
pages = {e70976},
pmid = {42249743},
issn = {1096-9071},
support = {KZ202010025035//Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan/ ; CX24PY23//Chinese Institutes for Medical Research, Beijing/ ; DFL20221503//Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration/ ; L234046//Beijing Natural Science Foundation-Changping Innovation Joint Fund/ ; 02-13//High-Level Public Health Technical Talents Project of Beijing/ ; PYZ24152//Scientific Research Cultivation Fund of Capital Medical University/ ; BJYAYY-YN2024-16//Scientific Research Project of Beijing Youan Hospital, Capital Medical University/ ; BJYAYY-YN2025-05//Scientific Research Project of Beijing Youan Hospital, Capital Medical University/ ; 2025046//Wang Baoen Liver Fibrosis Research Fund of China Foundation for Hepatitis Prevention and Control/ ; SF2026-2G-1154//Capital Health Development Scientific Research Special Project/ ; 2025KF10006//Project of State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/ ; 2026BMALA049//Beijing Medical Association Special Research Project for Medical Innovation and Development/ ; },
mesh = {Humans ; *Coinfection/diagnosis/virology ; *CRISPR-Cas Systems/genetics ; DNA, Viral/genetics ; *Hepatitis B/diagnosis/virology ; *Hepatitis B virus/isolation &amp; purification/genetics ; *Hepatitis D/diagnosis/virology ; *Hepatitis Delta Virus/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; RNA, Viral/genetics ; Sensitivity and Specificity ; },
abstract = {Co-infection with both hepatitis B virus (HBV) and hepatitis D virus (HDV) can aggravate the severity of the end-stage liver disease and accelerate its progression. However, no combined diagnostic method for HDV and HBV nucleic acids exists. In this study, we have developed a highly sensitive and specific dual detection method for HDV RNA and HBV DNA using the CRISPR-Cas system. We established a dual detection method combining CRISPR-Cas12a with recombinase polymerase amplification (RAA) for HBV, and CRISPR-Cas13a with RT-RAA for HDV. Validation was performed using specimens from 70 co-infected patients. RAA primers and crRNAs were designed and optimized to establish a dual fluorescence detection method (DF) and lateral flow strip-based dual detection (DL) within the same CRISPR-Cas13a/Cas12a system for HDV RNA and HBV DNA. The system demonstrated 100% specificity, and both DF and DL methods exhibited a sensitivity of 10 copies/μL for synthetic positive plasmids and samples. Peak fluorescence detection was achieved with T7 RNA polymerase, while the best detection efficiency was at ssRNA: ssDNA ratio of 1:1.5. In the validation of plasma samples from 70 co-infected clinical patients, the positive concordance rates for RT-RAA-CRISPR-Cas13a/Cas12a DF and DL were 85.7% (60/70) and 82.9% (58/70), respectively. We developed a CRISPR-Cas13a/Cas12a-based dual assay for sensitive, specific, and accurate detection of HDV RNA and HBV DNA, offering an effective tool for the early detection, treatment, and monitoring of HDV and HBV infections.},
}
@article {pmid42251734,
year = {2026},
author = {Sengodan, K and Xian, C and Shirk, P and Howell, J and Moola, AK and Palli, SR},
title = {CRISPR/Cas9-Mediated Knockout of White Gene Produces Eye Color Mutants in the Southern Green Stinkbug, Nezara viridula.},
journal = {Archives of insect biochemistry and physiology},
volume = {122},
number = {2},
pages = {e70175},
doi = {10.1002/arch.70175},
pmid = {42251734},
issn = {1520-6327},
support = {IIP-1821936//Center for Arthropod Management Technologies/ ; 2353057000//US Department of Agriculture/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Hemiptera/genetics ; *Eye Color/genetics ; Gene Knockout Techniques ; Mutation ; *Insect Proteins/genetics ; *Eye Proteins/genetics ; },
abstract = {CRISPR/Cas9 gene editing is a transformative tool for genetic studies in non-model organisms like the southern green stinkbug, Nezara viridula. However, current protocols depend on embryonic microinjection of CRISPR/Cas9, which remains technically difficult. An alternative method of delivering Cas9 ribonucleoprotein directly into female ovaries has been tested in only a few insect species, such as mosquitoes and whiteflies. Here, we developed a simple technique for gene editing by injecting Cas9 ribonucleoprotein into adult Nezara viridula females using ReMOT control or BAPC delivery methods previously described. We observed gene editing of the eye color marker white using the ReMOT method and HhKV ligand. These results demonstrate proof of concept for creating germline mutations in N. viridula. The protocol presented in this study could help advance genetic research in hemipteran pest species.},
}
@article {pmid42251763,
year = {2026},
author = {Byiringiro, I and Contiliani, DF and Davies, C and Gurel, F and Creste, S and Qi, Y},
title = {Improving iSpyMacCas9 multiplex genome editing in rice by CRISPR-combo-mediated BBM1 activation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {5},
pages = {e70980},
pmid = {42251763},
issn = {1365-313X},
support = {IOS-2132693//National Science Foundation/ ; IOS-2224203//National Science Foundation/ ; 21010111//Foundation for Food and Agriculture Research/ ; 2020/07045-3//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2021/13478-2//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2022/11738-0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Transcriptional Activation/genetics ; Plants, Genetically Modified ; Genome, Plant/genetics ; Gene Expression Regulation, Plant ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The recently developed CRISPR-Combo technology enables simultaneous targeted mutagenesis and transcriptional activation in plants. However, its reliance on SpCas9 limits its use at AT-rich genomic loci, such as promoter regions commonly targeted for transcription activation. To overcome this limitation, we explored the usage of Cas12b and iSpyMacCas9 in the CRISPR-Combo architecture for simultaneous genome editing and gene activation. We tested these expanded CRISPR-Combo systems for hormone-free regeneration of rice plants by transcriptional activation of a morphogenic gene, OsBBM1, while knocking out the genes of interest. The Cas12b-Combo system induced mild OsBBM1 upregulation (~3-fold), which did not affect the genome editing efficiency. By contrast, iSpyMacCas9-Combo achieved approximately 12-fold OsBBM1 transcriptional activation, supporting hormone-free regeneration at a high rate (42%). As a result, iSpyMacCas9-Combo conferred higher genome editing efficiency, including improved multiplex editing, than the standard iSpyMacCas9 system, either with or without hormones during rice regeneration. Hence, our data prove iSpyMacCas9-Combo to be a more efficient system for genome editing in rice, especially at low-efficiency target sites, when coupled with OsBBM1 transcriptional activation. These findings establish iSpyMacCas9-Combo as a useful addition to the CRISPR-Combo toolkit, expanding its genomic targeting scope and enabling more efficient genome editing by activation of an appropriate endogenous gene such as OsBBM1 in rice.},
}
@article {pmid42251880,
year = {2026},
author = {Han, Y and Wu, H and Gao, S and Wang, Y and Ma, Q},
title = {A one-pot RPA-T7/crDNA-CRISPR/Cas13a assay for portable and ultrasensitive detection of Helicobacter pylori and clarithromycin resistance mutations.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {152949},
doi = {10.1016/j.ijbiomac.2026.152949},
pmid = {42251880},
issn = {1879-0003},
abstract = {The high global prevalence of Helicobacter pylori (H. pylori) infection and rising clarithromycin resistance demand rapid, sensitive, point-of-care diagnostic tools. Although CRISPR/Cas systems have revolutionized nucleic acid detection, challenges such as unstable pre-synthesized crRNA and multi-step procedures limit their practical application. Here, we report a one-pot assay, termed RT-CRISPR (RPA-T7/crDNA-mediated CRISPR), that integrates recombinase polymerase amplification (RPA) with T7 transcription and CRISPR/Cas13a detection in a single closed tube. A key innovation is the use of a T7 promoter primers-crDNA hybrid chain (T7/crDNA) as a template for in situ synthesis of crRNA by T7 RNA polymerase, eliminating exogenous labile crRNA. The assay detects the H. pylori 16S rRNA gene at 1 copy/μL and the A2143G clarithromycin resistance mutation at 5 copies/μL, with no cross-reactivity against seven other gastrointestinal pathogens. It reliably detects the A2143G mutation at a ratio as low as 0.5% in mixed DNA (total 1× 10[4] copies/μL), highlighting its ability to identify low-abundance resistance mutations in heterogeneous samples. The entire assay, from DNA extraction to result, completes within 40 min using a portable fluorescence detector. Clinical validation with 103 samples showed 100% concordance with qPCR. The reagents can be lyophilized into stable pellets, maintaining performance after two months at room temperature. RT-CRISPR offers a simple, rapid, and robust platform for molecular diagnosis of H. pylori and drug resistance, holding promise for point-of-care testing and large-scale screening.},
}
@article {pmid42252785,
year = {2026},
author = {Jiang, J and Yan, Y},
title = {Compositional Optimization of CRISPR/Cas9 Lipid Nanoparticles for Efficient Knockdown of Target Genes.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {11},
pages = {e70413},
doi = {10.1002/cbic.70413},
pmid = {42252785},
issn = {1439-7633},
support = {Grant 22275167//National Natural Science Foundation of China/ ; 22405240//National Natural Science Foundation of China/ ; Grant LZ25B040002//Zhejiang Provincial Natural Science Foundation of China/ ; LTGY24B040001//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; Animals ; *Gene Knockdown Techniques ; Mice ; Gene Editing ; Liposomes ; },
abstract = {Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.},
}
@article {pmid42257904,
year = {2026},
author = {Ngolong Ngea, GL and Doganiero, S and Jimdjo Kouasseu, C and Palmieri, D and Castoria, R and Ianiri, G},
title = {Recent Advances in the Comprehension of Molecular and Genetic Mechanisms Underlying Yeast Biocontrol Efficacy Against Fungal Pathogens in Agriculture.},
journal = {Phytopathology},
volume = {},
number = {},
pages = {},
doi = {10.1094/PHYTO-03-26-0078-RVW},
pmid = {42257904},
issn = {0031-949X},
abstract = {Recent advances in biotechnologies have enabled scientists to uncover biological processes across multiple research fields. Still, the molecular and genetic mechanisms underlying the biological control efficacy of Yeast Biocontrol Agents (YBCAs) against fungal plant pathogens remain incompletely elucidated. This review focuses on recent insights into the regulatory basis and molecular interplay underlying successful disease control of YBCAs. It provides a detailed description of core antagonistic molecular mechanisms-nutrient and iron competition, mycoparasitism via cell wall degradation, antifungal compound production, oxidative stress resistance, biofilm formation and colonization, and induction of the host defense response-and integrates genomic, transcriptomic, proteomic, and metabolomic evidence to elucidate each mechanism. Further, how engineering-based approaches that leverage omics data and functional genetics can overcome obstacles to effectively transferring YBCA effectiveness from laboratory conditions to the field was also discussed. Finally, greater use of CRISPR-Cas technology is recommended to understand better how master transcription factors coordinate multiple mechanisms simultaneously; these factors are crucial for the synergistic antifungal effect, which is critical for developing highly effective YBCAs. Ultimately, the mechanism-based perspective provides a unified conceptual framework for understanding YBCA efficacy and guides the rational design of next-generation biocontrol agents for sustainable agriculture.},
}
@article {pmid42258486,
year = {2026},
author = {Pilarski, J and Stadler, T and Seidel, S},
title = {Assessing the inference of single-cell phylogenies and population dynamics from CRISPR lineage recordings.},
journal = {PLoS computational biology},
volume = {22},
number = {6},
pages = {e1014370},
pmid = {42258486},
issn = {1553-7358},
mesh = {*Phylogeny ; *Cell Lineage/genetics ; Bayes Theorem ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computer Simulation ; *Single-Cell Analysis/methods ; Computational Biology ; Cell Division/genetics ; Population Dynamics ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Multicellular organisms develop from a single cell by repeated rounds of cell division, differentiation, and death, which can be represented as a single-cell phylogenetic tree. Genetic lineage tracing allows us to investigate this development by tracking the ancestry of individual cells as populations grow and change over time. However, accurate reconstruction of the cell phylogeny and quantification of the corresponding phylodynamic parameters - cell division, differentiation, and death rates - from this tracking data remains challenging and needs to be systematically evaluated. We perform simulations and assess, using the Bayesian framework, the joint inference of time-scaled cell phylogenies and phylodynamic parameters from CRISPR lineage recordings with random or sequential edits. Principally, we characterize the inference improvements as the recorder capacity increases. We observe more accurate phylogenetic reconstruction from sequential compared to random recordings, but no substantial improvement in phylodynamic inference when using the additional information contained in the order of edits. Overall, we find that CRISPR lineage recordings carry a strong signal on the rates of cell division when appropriate models are used. However, we detect biases in the inferred rates of cell division and death under phylodynamic model misspecification, i.e., when fitting classic memoryless birth-death processes to synchronous cell divisions. Moreover, for scenarios when cells differentiate into distinct types, we demonstrate that Bayesian phylodynamic analysis of sparse end-point measurements can resolve these cell differentiation trajectories by lineage and time. Under prototypical dynamics, we recover cell type-specific division and death rates, and cell type transition rates in over 80% of simulations. Overall, this simulation study explores how much information on cellular development can be extracted from state-of-the-art genetic lineage tracing data using phylogenetic and phylodynamic methodology.},
}
@article {pmid42258532,
year = {2026},
author = {Kammerdiener, EK and Hren, AP and Harrison, R and Charles, S and Klingeman, D and Wiser, TD and Eckert, CA and Alexander, WG},
title = {Empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread functionality and rules for gRNA design.},
journal = {Nucleic acids research},
volume = {54},
number = {11},
pages = {},
pmid = {42258532},
issn = {1362-4962},
support = {//Center for Bioenergy Innovation/ ; ERKP886//U.S. Department of Energy/ ; //Laboratory Directed Research and Development Program/ ; //Oak Ridge National Laboratory/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Genome, Bacterial ; *Escherichia coli K12/genetics ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; },
abstract = {The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional. These nonfunctional gRNAs exhibit strong spacer self-interaction, which can either be excluded using a simple design rule or "repaired" during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million empirically evaluated E. coli gRNAs as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.},
}
@article {pmid42258544,
year = {2026},
author = {Bai, C and Zhu, H and Bayona, LM and Du, C and van Wezel, GP},
title = {Inducible CRISPRi enables efficient and high-fidelity genome editing in Streptomyces.},
journal = {Nucleic acids research},
volume = {54},
number = {11},
pages = {},
pmid = {42258544},
issn = {1362-4962},
support = {101055020//China Scholarship Council/ ; /ERC_/European Research Council/International ; },
mesh = {*Streptomyces coelicolor/genetics ; *CRISPR-Cas Systems ; *Genome, Bacterial ; Multigene Family ; Genes, Essential ; Homologous Recombination ; *Streptomyces/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; },
abstract = {Streptomycetes are prolific producers of bioactive natural products, but many of the biosynthetic gene clusters (BGCs) are silent in the laboratory. Genetic manipulation is important to unlock their full potential. CRISPR-Cas-based genome editing has greatly advanced genetic engineering in Streptomyces. However, several challenges remain, including Cas nuclease toxicity, unintended genomic rearrangements, and elimination of the delivery plasmid. Here, we present a novel genome editing strategy that harnesses cumate-inducible CRISPR interference (CRISPRi) to transiently knockdown essential genes such as divIVA or dnaA as counterselectable marker. This enforces loss of the vector backbone, promotes homologous recombination, and yields markerless mutants by loss of the antibiotic resistance cassette during the final recombination step. We demonstrate the versatility of the ICE system (Inducible CRISPRi targeting an Essential gene) by (i) deleting four BGCs in Streptomyces coelicolor M145, (ii) inserting both a promoter and a large BGC, and (iii) introducing precise single-nucleotide substitutions. Furthermore, deletion of the prodigiosin BGC elicited expression of a poorly expressed BGC for prolinolexin lipopeptides in Streptomyces roseifaciens DSM 106196T. Considering that different essential genes may be targeted, we anticipate that inducible CRISPRi-based counterselection may be adaptable to genome editing strategies in a broad range of microbial systems.},
}
@article {pmid42258545,
year = {2026},
author = {Hashemloo, MA and Killelea, T and Mamić, T and Ireland, TH and Lou-Hing, A and Kemm, F and Dimude, JU and Žagar, M and Ivančić-Baće, I and Rudolph, CJ and Bolt, EL},
title = {Visualizing the interplay of Cas1-Cas2 with DNA replication-repair that creates CRISPR-Cas immunity.},
journal = {Nucleic acids research},
volume = {54},
number = {11},
pages = {},
pmid = {42258545},
issn = {1362-4962},
support = {BB/T006625-1//BBSRC/ ; BB/T007168/1//BBSRC/ ; A18658//Nottingham Impact Accelerator Programme/ ; IP-2022-10-7882//The Croatian Science Foundation/ ; DOK-NPOO-2023-10-9630//The Croatian Science Foundation/ ; //University of Nottingham Gold/ ; },
mesh = {*DNA Replication ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *DNA Repair ; *Escherichia coli Proteins/metabolism/genetics ; Escherichia coli/genetics/immunology ; *Bacterial Proteins/metabolism/genetics ; Endonucleases ; },
abstract = {Prokaryotic CRISPR-Cas systems rely on the Cas1-Cas2 protein complex to capture new DNA from mobile genetic elements (MGEs), to form immunological memory that defends against the MGEs. However, the mechanisms by which Cas1-Cas2 locates suitable DNA substrates inside cells remain unclear, limiting our understanding of how CRISPR-Cas immunity arises de novo. We directly visualized functional, DNA-bound Cas1-Cas2 complexes in bacteria, revealing the processes that license Cas1-Cas2 to capture DNA. Visible DNA-bound Cas1-Cas2 complexes formed only when replisomes are actively advancing, accumulating at post-replicative DNA gaps behind replication forks-structures arising during normal genome duplication, which are normally repaired by homologous recombination. Replication stress, which increases replicative DNA gap frequency, enhanced visible Cas1-Cas2 DNA binding. DNA capture by Cas1-Cas2 was strongly stimulated in cells lacking the RecFOR complex, which normally directs DNA gaps to repair. The RecBCD recombination initiator complex was essential for DNA capture by Cas1-Cas2 in these cells. The findings support a model in which naïve CRISPR-Cas adaptation is licensed by abundant replication-dependent DNA repair intermediates, prior to their repair by recombination. This identifies the mechanism co-ordinating Cas1-Cas2 with essential DNA replication and repair processes that all cells need, including when they are hijacked to replicate parasitic MGEs.},
}
@article {pmid42258724,
year = {2026},
author = {Taranenko, D and Kotovskaya, O and Kuznedelov, K and Yanovskaya, D and Demkina, A and Fardeeva, S and Mamontov, V and Vierra, K and Burman, N and Li, D and Wang, M and Wiedenheft, B and Severinov, K and Semenova, E and Isaev, A},
title = {A census of anti-CRISPR proteins reveals AcrIE9 and AcrIE13 as inhibitors of the Escherichia coli K12 type IE CRISPR-Cas system.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {24},
pages = {e2529903123},
pmid = {42258724},
issn = {1091-6490},
support = {24-74-10089//Russian Academy of Sciences (RAS)/ ; GM104071//HHS | NIH (NIH)/ ; GM104071//HHS | NIH (NIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli K12/genetics/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species' core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales; this module often co-occurs with an AcrIE13 protein with an unusual HTH-like architecture.},
}
@article {pmid42259419,
year = {2026},
author = {Dimopoulos, D and Dafou, D and Sklaviadis, T and Xanthopoulos, K},
title = {Current genetic approaches for the treatment of prion diseases.},
journal = {Neuroscience},
volume = {610},
number = {},
pages = {136-146},
doi = {10.1016/j.neuroscience.2026.06.004},
pmid = {42259419},
issn = {1873-7544},
abstract = {Prion diseases are fatal neurodegenerative disorders caused by the misfolding of the host-encoded prion protein (PrP) into a pathogenic conformer (PrP[Sc]). Despite decades of investigation, no therapy has proven effective, largely due to rapid disease progression and the absence of druggable intermediates. Recent molecular advances, however, have established PrP itself as a viable therapeutic substrate. Experimental ablation or suppression of Prnp in mice -the gene encoding PrP- confers complete resistance to prion infection in animal models, providing a strong genetic rationale for PrP- lowering interventions. This review focuses on current genetic approaches aiming at reducing PrP expression. Antisense oligonucleotides (ASOs) and RNA-interference (RNAi) vectors have demonstrated potent, durable suppression of Prnp transcripts and extended survival in prion diseases murine models, while genome- and epigenome-editing platforms, including CRISPR-Cas and dCas9-based repressors, now permit permanent or reversible transcriptional control of Prnp with increasing precision. While these technologies are conceptually transformative, translational application faces major challenges, including early diagnosis, brain-wide delivery, biomarker validation and ethical implementation of presymptomatic therapy in Prnp mutation carriers. Integration of validated cerebrospinal biomarkers such as PrP and neurofilament light chain, adaptive trial designs and international registries will be essential for clinical development. Together, these advances position genetic approaches focusing on PrP-lowering as a promising paradigm for preventive treatment of prion diseases and as a model for rational gene-targeted therapies in other rapidly progressive neurodegenerative disorders.},
}
@article {pmid42259648,
year = {2026},
author = {Yin, X and Xiong, Q and Shu, T and Yan, S and Zhao, J and Wang, Z and Deng, G and Liu, Y and Zhu, L and Zhu, C},
title = {Digital Hydrogel Fluorescent-Enhancing Microspheres via CRISPR/Cas12a for Amplification-Free Relative Quantification of Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c01288},
pmid = {42259648},
issn = {1520-6882},
abstract = {CRISPR/Cas systems hold great promise for molecular diagnostics, but their amplification-free applications are hampered by weak signals and poor quantification. Here, we developed CrisprDEM, a CRISPR/Cas12a-based digital hydrogel fluorescent-enhancing microsphere system that integrates hydrogel microsphere confinement, microfluidic digital imaging, and machine learning for ultrasensitive and quantitative nucleic acid detection without amplification. Hydrogel microspheres (HMs) efficiently captured and spatially concentrated CRISPR/Cas12a reaction reporters, achieving a 1000-fold signal amplification compared with homogeneous methods. The design of the microfluidic chip arranged the microspheres into a single-layer array, making each microsphere an independent digital reporting unit. The Intelligent Bead Analysis Software enabled automatic analysis and relative quantification via a positive bead ratio (PBR). As a proof-of-concept, we selected the respiratory adenovirus as the detection target. We optimized the CRISPR/Cas12a-microsphere enrichment reaction system and characterized the morphologies and chemical properties of the microspheres before and after enrichment. The results demonstrated that CrisprDEM technology exhibited a detection sensitivity of 10 aM for respiratory adenovirus, exhibiting no cross-reactivity with other respiratory viruses, indicating a high specificity. In the validation of 20 clinical samples, the detection results were consistent with the gold-standard real-time quantitative polymerase chain reaction (qPCR), and the PBR value showed a good linear relationship with the cycle threshold (Ct) value, enabling a relative quantification. This system expands the toolbox for amplification-free CRISPR diagnostics and holds the potential for point-of-care and early infection detection.},
}
@article {pmid42259773,
year = {2026},
author = {Luo, G and Ma, F and Yang, Y and Yang, C and Li, X and Xie, J and Xiong, K and Chen, P and Ma, K and Zhao, Z and Tang, BZ and Duo, Y},
title = {Engineering an AIEgen-based platform integrating CRISPR/Cas9 to remodel the tumor microenvironment and reinforce photo-immunotherapy against glioblastom.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {42259773},
issn = {2059-3635},
mesh = {Humans ; *Tumor Microenvironment/genetics/drug effects/immunology ; Animals ; *Glioblastoma/genetics/therapy/pathology/immunology ; *Immunotherapy ; *CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Brain Neoplasms/genetics/therapy/pathology/immunology ; *Photochemotherapy ; Nanoparticles/chemistry ; Blood-Brain Barrier ; *5'-Nucleotidase/genetics ; Neutrophils ; },
abstract = {Glioblastoma remains one of the most lethal brain tumors. Although immunotherapy and other therapeutic modalities has achieved significant therapeutic success in several malignancies, its efficacy in glioblastoma remains limited primarily due to the complex tumor microenvironment (TME) and physiological barriers such as the blood-brain barrier (BBB). In this context, nanomedicine and gene editing have emerged as promising strategies due to their unique ability to cross the BBB and protect therapeutic agents through intrinsic physicochemical properties. To overcome the physiological barriers for better therapeutic outcomes. Here, a novel aggregation-induced emission luminogen (AIEgen), NDA-DPE, was synthesized, exhibiting NIR-I to NIR-II fluorescence and dual photothermal (PTT) and photodynamic (PDT) properties through restricted intramolecular motion. Bone-derived neutrophil-based biomimetic nanoparticles (bNe@AIE/Cas9-CD73) were then prepared by integrating NDA-DPE with CRISPR/Cas9-mediated CD73 gene silencing. The neutrophil encapsulation enabled efficient BBB penetration and targeted accumulation in glioblastoma tissue. CRISPR/Cas9-CD73 downregulated CD73 expression, disrupted the ATP-adenosine axis, and reshped the immunosuppressive TME into an immuno-supportive one, increasing the therapeutic sensitivity of tumor cells. Under NIR-II excitation, bNe@AIE/Cas9-CD73 achieved fluorescence-guided PTT and PDT, inducing immunogenic cell death (ICD), stimulating immune-cell recruitment, and activating systemic antitumor immunity. bNe@AIE/Cas9-CD73 demonstrated a potent gene-photothermal-photodynamic-immune synergistic effect, significantly inhibiting glioblastoma growth and establishing a promising nanoplatform for effective and targeted glioblastoma treatment.},
}
@article {pmid42260913,
year = {2026},
author = {Shahid, M and Ilyas, T and Shafi, Z},
title = {Rhizobacterial Exopolysaccharides in Soil-Plant Systems: Molecular Mechanisms, Engineering Approaches, and Translational Challenges.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {23},
pages = {17694-17713},
doi = {10.1021/acs.jafc.6c02760},
pmid = {42260913},
issn = {1520-5118},
mesh = {*Polysaccharides, Bacterial/metabolism/genetics ; Soil Microbiology ; *Plants/microbiology/metabolism ; Rhizosphere ; *Bacteria/metabolism/genetics/isolation &amp; purification/classification ; Soil/chemistry ; },
abstract = {Plant productivity has become increasingly affected by various abiotic and biotic factors such as drought, salinity, metal toxicity, heat/cold stresses, and pathogen pressure that disrupt soil-plant interactions. Plant growth-promoting rhizobacteria (PGPR)-secreted exopolysaccharides (EPSs) play a significant role in maintaining rhizosphere stability through promoting soil aggregation, increasing the soil water retention capacity, and proper ion management. In addition, EPSs provide extracellular binding sites for toxic metals and facilitate the formation of stress-tolerant biofilms. Production of EPSs is under the strict control of sophisticated regulatory systems, linking environmental conditions and adaptive mechanisms at the genetic level. Novel advancements in omics and genome editing techniques could be used in the development of improved EPS-secreting strains with enhanced stress-resistance capabilities. Potential applications include PGPR formulations for seed coating, bioinoculants, and soil treatments; however, strain heterogeneity and environmental variability represent important challenges.},
}
@article {pmid42261185,
year = {2026},
author = {Jahangiri-Sisakht, A and Safari, L and Alipanahi, R},
title = {CRISPR-MBTF: a multi-branch transformer fusion framework for CRISPR-Cas9 off-target prediction.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {3},
pages = {},
pmid = {42261185},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Deep Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a revolutionary genome editing technology derived from a bacterial adaptive immune system that uses a single guide RNA (sgRNA) to direct the Cas9 enzyme to specific DNA sequences for precise genetic modifications. Its ease of use and efficiency has accelerated advancements in genetic research and therapeutic development. However, unintended cleavage at off-target sites remains a significant concern, limiting the safety and broader applicability of CRISPR-based editing. Accurate computational prediction of off-target locations is therefore essential to mitigate potential risks and improve experimental design. In this study, we introduce CRISPR multi-branch transformer fusion (CRISPR-MBTF), a novel deep learning-based framework employing a multi-branch Transformer architecture combined with an attention-based fusion mechanism to model the intricate biological context influencing CRISPR activity. By capturing subtle sequence patterns and contextual dependencies, our model achieves enhanced predictive performance compared to existing approaches. Additionally, interpretability analyses uncover biologically meaningful patterns and highlight influential sequence regions, offering valuable insights into the determinants of CRISPR specificity. This work presents a robust and interpretable tool to support the design of safer and more effective genome editing strategies.},
}
@article {pmid42261595,
year = {2026},
author = {Saeed, M and Arham, M and Zafar, I and Jamal, A and Hussian, M and Usman, M and Bahwerth, FS and Noman, M and Hossain, MB},
title = {Harnessing Deep Learning Models for Guide RNA Optimization and Off-Target Prediction in CRISPR Systems.},
journal = {Biotechnology journal},
volume = {21},
number = {6},
pages = {e70255},
doi = {10.1002/biot.70255},
pmid = {42261595},
issn = {1860-7314},
mesh = {*Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Recurrent Neural Networks ; Humans ; Predictive Learning Models ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Convolutional Neural Networks ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome and transcriptome editing technologies have emerged as powerful tools for therapeutic, agricultural, and industrial applications. However, their broader clinical and translational use remains limited by variable guide RNA (gRNA) or single-guide RNA (sgRNA) efficiency and unintended off-target activity, which may lead to genotoxic effects and major safety concerns. To address these challenges, recent research has increasingly shifted from heuristic scoring approaches and traditional machine learning (ML) methods toward deep learning (DL) models capable of learning complex sequence-function relationships from large-scale experimental datasets generated by assays such as GUIDE-seq (Genome-wide Unbiased Identification of Double-stranded Breaks Enabled by Sequencing), CIRCLE-seq (Circularization for In Vitro Reporting of Cleavage Effects by Sequencing), and CHANGE-seq (Cumulative and Homology-independent Analysis of Nuclease Genome-wide Effects by Sequencing). This review critically examines recent advances in DL approaches for gRNA optimization and off-target prediction in CRISPR systems. We discuss the development of convolutional neural networks (CNNs), recurrent neural networks (RNNs), transformer-based architectures, and foundation models designed to improve prediction accuracy, specificity, and generalizability across diverse biological contexts.},
}
@article {pmid42262436,
year = {2026},
author = {Alvi, AA and Hussain, M and Noureen, S and Malik, ZA and Zahoor, S and Jamil, A and Mohsin, MA and Azeem, A and Javaid, H and Hassan, Z},
title = {CRISPR-based gene editing for antimicrobial resistance control in human medicine.},
journal = {Archives of microbiology},
volume = {208},
number = {9},
pages = {},
pmid = {42262436},
issn = {1432-072X},
mesh = {Humans ; *Anti-Bacterial Agents/pharmacology ; *Bacteria/genetics/drug effects ; Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; *Gene Editing/methods ; Clinical Trials as Topic ; },
abstract = {Antimicrobial resistance (AMR) has already become one of the most urgent threats to the public health of this century. In 2019 alone, it directly causes about 1.27 million deaths and it was estimated that 1.91 million people will die yearly by 2050 should present trends persist. The traditional antibiotic development pipelines have been shown to be structurally insufficient to meet the rate at which bacterial populations have developed, diversified and spread resistance determinants, typically by horizontal gene transfer. In this context, CRISPR-Cas gene editing has become a focused antimicrobial approach that can selectively target resistance genes, virulence factors, and mobile genetic elements without the broad-spectrum collateral damage associated with conventional antibiotics. The review assesses CRISPR-Cas systems, namely Cas9, Cas12a, Cas3, and Cas13 in the context of two complementary mechanistic strategies namely selective killing of pathogens and antibiotic resensitization by the targeted disruption of gene resistance. We compare the impact of key delivery systems, such as bacteriophage vectors, lipid nanoparticles, and conjugative plasmids, evaluating them based on their therapeutic activity, host selectivity, and possible translation. The present state of clinical translations is discussed, including the two most advanced clinical-stage candidates SNIPR001 (Phase I/II, NCT05277350) and LBP-EC01 (Phase 2/3, NCT05488444). We also address the open issues that include off-target editing, host immune reactions, bacterial counter-resistance, regulatory ambiguity, and scalability of manufacturing. Lastly, we provide priority research directions, such as the combination antimicrobial strategies, AI-assisted CRISPR design, and next-generation delivery engineering, none of which will be resolved before routine clinical application of CRISPR-based antimicrobials is achieved.},
}
@article {pmid42263664,
year = {2026},
author = {Ladisa, F and Morelli, E and Soncini, D and Garibotto, M and Munshi, NC and Fulciniti, M and Cea, M},
title = {CRISPR application in hematological disorders: from bench to bedside.},
journal = {Blood advances},
volume = {},
number = {},
pages = {},
doi = {10.1182/bloodadvances.2025017417},
pmid = {42263664},
issn = {2473-9537},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas genome editing has advanced from an experimental tool to a clinically validated therapeutic platform in hematology. Landmark successes in inherited blood diseases including sickle cell disease, β-thalassemia, and severe combined immunodeficiency, have demonstrated that precise and durable genetic modifications can be safely and effectively implemented in human hematopoietic cells, positioning hematology at the forefront of translational genome editing. Beyond monogenic disease, CRISPR-based approaches are transforming both the biological understanding and treatment of hematologic malignancies by enabling systematic interrogation of cancer dependencies, functional mapping of genetic vulnerabilities, and mechanism-driven target validation, including in vivo and immune-relevant contexts. In parallel, therapeutic applications are emerging through the development of engineered cellular therapies, including edited autologous and allogeneic immune effector cells designed to enhance antitumor efficacy, persistence, and immune evasion. This review synthesizes recent CRISPR-based advances across benign and malignant hematologic diseases. We compare major editing modalities, including nuclease-mediated disruption, base editing, prime editing, and CRISPR-based transcriptional modulation, and highlight key preclinical studies alongside emerging clinical trial data. We also discuss translational challenges that currently limit broader clinical adoption, including delivery and manufacturing scalability, off-target and genotoxicity risks, tumor and immune heterogeneity, and the long-term durability and fitness of edited cell populations. Finally, we outline priorities for the next phase of the field, emphasizing how continued innovation in CRISPR technologies may enable increasingly precise, durable, and mechanism-informed therapeutic strategies in hematology.},
}
@article {pmid42263803,
year = {2026},
author = {Rambabu, I and Baskar, G and Suliman, M and Saeed, M and Radhakrishnan, M and Balu, R and Palaniyandi, T},
title = {Engineered CRISPR-Cas systems for transcriptional regulation and precision molecular diagnostics: advances, challenges, and emerging microfluidic integration.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {591},
number = {},
pages = {121145},
doi = {10.1016/j.cca.2026.121145},
pmid = {42263803},
issn = {1873-3492},
abstract = {CRISPR-Cas technology has evolved rapidly from a bacterial adaptive immune system to transformative use in molecular diagnostic and genomic engineering. Beyond traditional genome-editing capabilities, newly engineered versions of CRISPR/Cas can be used for programmable transcriptional regulation, epigenetic modification, molecular imaging, and ultrasensitive nucleic acid detection. Specifically, catalytic-inactive Cas proteins like dCas9 and dCas12 retain their ability to bind specific sequences on DNA but do not cleave it. Therefore, these proteins can be reversibly regulated by either CRISPRi or CRISPRa to alter gene expression. Thus, they represent powerful tools for both functional genomic studies and synthetic biological applications. Advances in CRISPR engineering have recently greatly increased the diagnostic potential of Cas12 and Cas13 enzymes. For example, collateral cleavage activity allowed the creation of CRISPR-based diagnostic platforms (SHERLOCK, DETECTR and FELUDA), which can detect target DNA/RNA sequences at high sensitivity and specificity. Moreover, they were demonstrated to work in detecting several infectious pathogens (SARS-CoV-2, Zika virus, and M. tuberculosis) and thus have significant value in point-of-care testing, especially when there is limited availability of resources. CRISPR systems are also being combined with increasing frequency with epigenetic regulators, fluorescence microscopy methods, biosensors, and lab-on-a-chip platforms that incorporate microfluidics to provide improved molecular analysis and automated diagnosis. The purpose of this review is to describe how engineered CRISPR-Cas systems have been developed from primarily genome editing tools into multi-functional platforms for transcriptional regulation, epigenetic engineering, diagnostics, imaging, and emerging microfluidic integrations. Additionally, this review will address some of the current challenges that exist with using CRISPR-based technologies, including off-target effects, delivery efficiency, diagnostic standardization, scaling up production, and translating these technologies clinically.},
}
@article {pmid42263833,
year = {2026},
author = {Li, F and Zhu, H and Yao, R and Jiang, X and Li, G},
title = {3D DNA walker-based biosensors: From programmable trajectory to detection of foodborne pathogens.},
journal = {Biotechnology advances},
volume = {91},
number = {},
pages = {108947},
doi = {10.1016/j.biotechadv.2026.108947},
pmid = {42263833},
issn = {1873-1899},
abstract = {3D DNA walker, characterized by its programmable molecular recognition capability, efficient signal amplification, and precise track controllability, has emerged as a novel intelligent sensing element for constructing highly sensitive biosensors to detect foodborne pathogens. This review systematically summarizes recent advances in the application of 3D DNA walkers for foodborne pathogen detection, with a specific focus on construction strategies, driving mechanisms, and the design and application of corresponding biosensors. First, the two core construction strategies of 3D DNA walkers are thoroughly analyzed from the perspective of track carrier materials, covering functionalized carriers based on nanomaterials and structurally precise tracks constructed on nucleic acid scaffolds. Subsequently, five major driving mechanisms are investigated in depth, including nuclease activation, DNAzyme-driven cleavage, strand displacement-mediated dynamic regulation, CRISPR/Cas-mediated cleavage, and environmental factor responsiveness, thereby comprehensively elucidating the motion control principles underlying the 3D DNA walker operation. Furthermore, the development of optical, electrochemical, and multi-mode biosensors based on 3D DNA walkers and their applications in detecting foodborne pathogens are systematically summarized. Finally, current limitations of the technology are discussed along with a forward-looking perspective on its future development. This review aims to provide theoretical insights for promoting the innovative development of 3D DNA walker technology and facilitate its large-scale application and technological breakthroughs in food safety monitoring and on-site rapid diagnostics.},
}
@article {pmid42265375,
year = {2026},
author = {Bargoti, T and Nain, V and Singh, D},
title = {Prime editing: evolution of CRISPR-Cas system for a robust next-generation genome editing in plants.},
journal = {Planta},
volume = {264},
number = {1},
pages = {},
pmid = {42265375},
issn = {1432-2048},
support = {CRG/2020/003753//SERB-CRG/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Plants/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing is a propulsive and versatile genome engineering technology that enables precise installation of all possible 12 base-to-base conversions, targeted insertions, deletions, and combinatorial modifications without inducing double-strand break (DSB) or requiring exogenous donor DNA template. Since its inception, prime editing has been rapidly adopted across plant systems, offering a powerful platform for functional genomics, trait improvement, and precision molecular breeding. This review comprehensively traces the evolution of prime editors (PEs) from first-generation PE1 to advanced variants such as PE7 and TwinPE. We detail the key technological milestones, including innovations in protein engineering, prime editing guide RNA (pegRNA) architectural improvement, and strategic modulation of host DNA repair mechanisms aimed at enhancing editing efficiency, precision, and versatility. Further, we provide an in-depth overview of plant-adapted prime editing systems, focusing on codon optimization, promoter refinement, pegRNA scaffold engineering, and the integration of plant-compatible Cas9 and reverse transcriptase variants. Special emphasis is given to the application of prime editing in diverse crop species. By consolidating recent advances and highlighting emerging trends, this review presents a forward-looking perspective on the deployment of prime editors (PEs) as transformative tool for precision genome engineering and sustainable crop improvement.},
}
@article {pmid42266926,
year = {2026},
author = {Ji, T and Fang, X and Gao, Y and Yu, K and He, J and Wang, L and Zhu, W and Huang, G and Gao, X},
title = {Visual detection platform based on RPA-CRISPR/Cas12a for Klebsiella pneumoniae and Carbapenem-resistant Klebsiella pneumoniae in clinical and food safety settings.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1817859},
pmid = {42266926},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/genetics/isolation &amp; purification/drug effects ; Humans ; *Food Safety ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Klebsiella Infections/microbiology/diagnosis ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; beta-Lactamases/genetics ; DNA-Directed RNA Polymerases/genetics ; Carbapenems/pharmacology ; Bacterial Proteins/genetics ; DNA Primers/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {INTRODUCTION: The rise of Klebsiella pneumoniae (KP) and carbapenem-resistant KP (CRKP) poses grave threats to public health and food safety, creating an urgent demand for rapid point-of-care testing (POCT). Traditional detection methods are limited by laboratory barriers, making them unsuitable for POCT implementation.<br><br>METHODS: Herein, a one-tube assay integrating recombinant polymerase amplification (RPA) with CRISPR/Cas12a technology was developed for the rapid, sensitive and specific detection of KP and blaOXA-48-carrying CRKP. Specific primers targeting the KP-specific rpoB gene and carbapenem-resistance gene blaOXA-48 were designed, and optimal primer pairs were screened via agarose gel electrophoresis. CrRNA sequences were designed according to RPA amplicons, and the components of the CRISPR/Cas12a reaction were optimized. A two-step reaction system was initially evaluated, followed by the establishment of an integrated one-tube RPA-CRISPR/Cas12a assay. A total of 66 clinical specimens and artificially contaminated food samples were used for method validation, with microbial culture and qPCR as reference methods.<br><br>RESULTS: The two-step assay was capable of detecting bacterial suspensions at a concentration of 100 CFU/mL. The one-tube system could be completed within 1 hour at 37 &#xb0;C. This assay avoided aerosol contamination and allowed visual result readout under blue light. In the validation test, the detection results of the one-tube assay were consistent with those obtained by microbial culture and qPCR.<br><br>DISCUSSION: This study constructed a dual-target RPA-CRISPR/Cas12a platform for the visual detection of KP and blaOXA-48-positive CRKP under blue light. This assay reduces reliance on sophisticated equipment and professional personnel. It can serve as a promising POCT tool for clinical diagnosis and food safety surveillance, and provides evidence for the timely formulation of rational antimicrobial treatment strategies.},
}
@article {pmid42267662,
year = {2026},
author = {},
title = {Corrigendum to: "Subtypes of Type I-E CRISPR-Cas Systems Distribution in Human Escherichia coli Isolates from China".},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {25731599261457400},
doi = {10.1177/25731599261457400},
pmid = {42267662},
issn = {2573-1602},
}
@article {pmid42267671,
year = {2026},
author = {Brown, RA and Dangel, AW and Saini, A and Collins, PL and Colonna, M and Oltz, EM},
title = {CRISPRi screening identifies SON and MAP4K1 as regulators of type III cytokine expression in innate lymphoid cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {6},
pages = {},
doi = {10.1093/jimmun/vkag110},
pmid = {42267671},
issn = {1550-6606},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Immunity, Innate ; Interleukin-22 ; *Lymphocytes/immunology/metabolism ; Interleukins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Interleukin-17/genetics/metabolism ; Gene Expression Regulation ; *Cytokines/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive "type III" responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1β. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12Rβ1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. Knockdown of nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12Rβ1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFN-γ, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.},
}
@article {pmid42268478,
year = {2026},
author = {Khajouei, F and Ghaemi, A and Abnous, K and Taghdisi, SM and Ramezani, M and Alibolandi, M},
title = {CRISPR-Based Gene Therapy for Brain Disease.},
journal = {Molecular neurobiology},
volume = {63},
number = {1},
pages = {},
pmid = {42268478},
issn = {1559-1182},
mesh = {Humans ; *Genetic Therapy/methods ; Animals ; *Brain Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Neurological disorders are complex and often very challenging for patients. Many of these conditions result from mutations in genes that are essential for normal function. Most existing treatments only alleviate symptoms, highlighting the urgent need for more effective therapeutic strategies. In the current drug development landscape, gene therapy offers hope as a promising approach. Specifically, CRISPR-Cas9 technology enables precise gene editing across diverse cell types and organisms. An increasing number of research groups are investigating innovative therapies and the molecular mechanisms behind neurological diseases. This review highlights the use of CRISPR-based gene therapies for various brain diseases, including multiple sclerosis, Alzheimer's, Parkinson's disease, epilepsy, stroke, and brain tumors. It consistently recognizes significant challenges in clinical applications, including overcoming the blood-brain barrier (BBB), managing off-target effects, ensuring efficient delivery, and addressing immunogenicity and ethical concerns.},
}
@article {pmid42269591,
year = {2026},
author = {Christensen, OP and Markham, A and Kang, H and Gabriel, E and Pers, TH},
title = {Causal effect estimation from trans-regulatory single-cell CRISPR screens.},
journal = {Cell genomics},
volume = {6},
number = {6},
pages = {101251},
pmid = {42269591},
issn = {2666-979X},
mesh = {*Single-Cell Analysis/methods ; Humans ; Single-Cell Gene Expression Analysis ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; Transcriptome/genetics ; },
abstract = {Recent advances in single-cell transcriptomics and CRISPR-based genome editing have enabled large-scale perturbation experiments with genome-wide expression readouts. Single-cell CRISPR screens offer the opportunity to move beyond correlation and estimate causal effects of genetic perturbations on gene expression at scale. These approaches promise to substantially deepen insights into cellular functions and disease mechanisms. However, interpreting statistical associations as causal effects requires additional assumptions beyond those needed for standard statistical analyses. In this minireview, we introduce key concepts and principles for causal effect estimation in trans-regulatory single-cell CRISPR studies. We describe a set of assumptions under which estimates from existing statistical methods admit a causal interpretation and provide a concise overview of these approaches. Finally, through an illustrative example, we demonstrate how violations of these assumptions can bias estimated effects.},
}
@article {pmid42269836,
year = {2026},
author = {Hamann, V and Hook, S and Sujariyakul, P and Ramalingam, R and Sgodda, M and Klefenz, I and Stalke, A and Yuan, Q and Steinbrück, L and Rovai, A and Ruhe, M and Chung, BM and Steinemann, D and Sharma, AD and Cantz, T and Lin, PJC and Wedemeyer, H and Ott, M and Krooss, SA},
title = {In vivo base editing alleviates hepatic iron accumulation and fibrosis in models of HFE-related hereditary hemochromatosis.},
journal = {Journal of hepatology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jhep.2026.05.022},
pmid = {42269836},
issn = {1600-0641},
abstract = {BACKGROUND &amp; AIMS: HFE-related hereditary hemochromatosis is caused by loss-of-function mutations in the HFE gene, leading to excessive intestinal iron absorption and hepatic deposition. The C282Y variant in homozygosity accounts for 80-90% of diagnosed cases. If untreated, iron accumulation can cause liver fibrosis, cirrhosis, and hepatocellular carcinoma.<br><br>METHODS: We employed lipid nanoparticles (LNPs) to deliver base editor mRNA and sgRNA for in vivo correction of the HFE C282Y mutation in a murine model under iron challenge. Additionally, patient-derived induced pluripotent stem cells (iPSCs) and hepatocyte-like cells were edited using the same approach.<br><br>RESULTS: Base editing achieved a conversion rate of 73.6 &#xb1; 4.9% in cultured murine hepatocytes and up to 67% in vivo. No off-target effects were detected at genomic sites with one or two mismatches, as confirmed by next-generation sequencing. Treated mice showed significantly reduced hepatic iron overload despite continued high dietary iron intake. Transcriptomic analysis revealed decreased signatures associated with fibrosis and cancer. For preclinical evaluation, iPSCs from C282Y homozygous patients were differentiated into hepatocyte-like cells. LNP-mediated base editing achieved up to 63.8 &#xb1; 0.8% correction in these cells, again without detectable off-target activity.<br><br>CONCLUSIONS: These results provide proof of concept that base editing of the C282Y variant is both safe and efficient in vivo and in human-derived cells, effectively reducing hepatic iron accumulation and preventing fibrotic remodeling.<br><br>IMPACT AND IMPLICATIONS: HFE C282Y-related hemochromatosis lacks causal therapies and carries a risk of iron-driven liver disease, supporting the need for precise in vivo gene correction. Here, LNP-mediated ABE delivery enabled efficient editing in hepatocytes and iPSC-derived models, reducing hepatic iron and normalizing biomarkers without detectable safety concerns. These findings highlight translational potential for non-viral gene correction, though further validation in larger and long-term studies is required.},
}
@article {pmid42271576,
year = {2026},
author = {Manikandan, DC and Sathiyabama, M},
title = {Agro-nanotechnology: A comprehensive overview of its role in groundnut production.},
journal = {Journal of the science of food and agriculture},
volume = {},
number = {},
pages = {},
doi = {10.1002/jsfa.70783},
pmid = {42271576},
issn = {1097-0010},
abstract = {Groundnut (Arachis hypogaea L.) is an economically important oilseed crop cultivated worldwide for its nutritional and industrial value. However, its productivity and quality are frequently constrained by several challenges, including abiotic and biotic stresses, post-harvest losses, and aflatoxin contamination. In recent years, agro-nanotechnology has emerged as a promising approach to address these limitations by improving nutrient delivery, enhancing plant defense responses, and supporting advanced agricultural strategies. Nanoparticles have been reported to improve nodulation and rhizosphere interactions by influencing plant-microbiome dynamics, thereby contributing to enhanced crop growth and stress tolerance. However, direct evidence remains limited, and several observations are derived from related crop systems. Recent advances have highlighted the integration of nanotechnology with CRISPR-Cas genome editing systems, enabling targeted and DNA-free delivery of gene-editing components for crop improvement. It has been proposed that such approaches could contribute to improved oleic acid content, reduced allergenicity, and enhanced disease resistance in groundnut. Nano-remediation strategies have also shown potential in mitigating pesticide residues and heavy metal contamination, thereby reducing the risk of aflatoxin accumulation. Key developments in this field include nano-formulations for precise nutrient management, modulation of plant-microbiome interactions, and nanoparticle-mediated delivery systems for genome editing technologies. Nevertheless, several challenges remain, including regulatory uncertainties, potential environmental risks, nanoparticle toxicity, and the lack of standardized field-scale evaluations. Addressing these limitations through interdisciplinary research, robust risk-assessment frameworks, and crop-specific regulatory policies will be essential for the responsible implementation of agro-nanotechnology. Overall, this review highlights the emerging role of agro-nanotechnology in addressing key constraints in groundnut production, while emphasizing the need for further groundnut-specific validation and field scale applicability. © 2026 Society of Chemical Industry.},
}
@article {pmid42272755,
year = {2026},
author = {Shan, C and Liu, C and Jin, C and Tian, D},
title = {Multilayer regulation of CRISPR systems: integrating anti-CRISPR proteins, CRISPRi/a, and quorum sensing networks.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1844116},
pmid = {42272755},
issn = {2235-2988},
mesh = {*Quorum Sensing ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacteriophages/genetics ; Gene Editing/methods ; },
abstract = {The CRISPR-Cas system has evolved into a highly efficient platform for genome editing and programmable gene regulation, demonstrating broad application potential in microbiology, biotechnology, and medicine. However, traditional CRISPR tools typically rely on constitutively active nuclease activity; this constant activation state is prone to off-target effects and cytotoxicity, and lacks precise spatiotemporal regulation in complex biological environments. Therefore, developing strategies to achieve fine-tuned and context-dependent regulation of CRISPR activity has become a critical issue in this field that urgently needs to be addressed. Recent studies have demonstrated that various endogenous and exogenous regulatory modules can modulate the activity of the CRISPR-Cas system at different biological levels. Among these, anti-CRISPR proteins (Acr), which are natural inhibitory factors derived from bacteriophages, can suppress the nuclease activity of Cas proteins at the protein level by directly interfering with their function; The CRISPR interference/activation (CRISPRi/a) system, on the other hand, relies on catalytically inactivated Cas proteins to achieve sequence-specific regulation of target gene transcription; furthermore, quorum sensing (QS) networks dynamically regulate the expression of relevant genes by sensing cell density and environmental signals, thereby influencing the functional state of the CRISPR system at the population level. Based on the aforementioned regulatory mechanisms, this paper provides a comprehensive, literature-based overview of the molecular basis and recent advances in the applications of Acr proteins, the CRISPRi/a system, and QS networks in CRISPR-Cas regulation. Building on this, we propose a hierarchical regulatory framework: QS networks serve as upstream environmental sensing modules that drive CRISPRi/a-mediated programmable transcriptional regulation, while Acr proteins act as downstream rapid-response elements that finely tune CRISPR activity. This multi-tiered regulatory system holds promise for the dynamic optimization and precise control of CRISPR systems, offering new design concepts for constructing adaptive, programmable genetic regulatory networks, and demonstrating significant application potential in fields such as microbial engineering, anti-infective strategies, and precision gene regulation. The regulatory mechanism of Acr proteins on CRISPR activity has been experimentally validated in several studies. Nevertheless, the integration of Acr proteins with other regulatory modules such as CRISPRi/a systems or QS networks remains in the exploratory stage and requires further empirical research to confirm their functionality in complex biological systems.},
}
@article {pmid42273222,
year = {2026},
author = {Hanke, A and Schütz, L and Walz, M and Wunsch, N and Kreis, L and Hohenwarter, L and Baßler, A and Wassenegger, M and Krczal, G and Koch, A and Uslu, VV},
title = {Exogenous 24nt siRNAs induce AGO4A-dependent silencing via promoter DNA methylation and H3K9me2 deposition.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1826532},
pmid = {42273222},
issn = {1664-462X},
abstract = {Transcriptional Gene Silencing (TGS) is an essential process in plants for development, gene regulation, defense against viruses, and genome integrity. TGS is predominantly established by RNA-directed DNA methylation (RdDM), a self-reinforcing mechanism in which sRNAs guide transcriptional suppressors to target genomic loci by sequence complementarity, and methylated DNA in turn facilitates sRNA genesis. Recently, exogenous application of promoter targeting long dsRNAs was associated with promoter methylation without any detectable gene silencing. A plethora of sRNAs of different sizes and types form as cleavage products of precursor dsRNAs such as pre-miRNAs, inverted-repeats, viral replication intermediates, or exogenous dsRNAs. Due to the complexity of sRNA products, the features of the sRNAs, which trigger de novo RdDM, remain enigmatic. Here, we demonstrated that in planta delivery of chemically synthesized 24-nucleotide(nt) small interfering RNAs (siRNAs), targeting the 35S promoter of GFP-expressing Nicotiana benthamiana (Nb) 16c line, was sufficient to induce RdDM, H3K9me2 deposition, and also TGS. Using CRISPR/Cas-mediated gene editing, we showed that exogenous 24nt siRNA-triggered TGS is dependent on ARGONAUTE 4A (NbAGO4A) but not on NbAGO4B. Exogenously administered 24nt siRNAs could provide the means to investigate such initiation events, while allowing functional dissection of siRNA classes and their modifications in planta.},
}
@article {pmid42273272,
year = {2026},
author = {Ye, L and Wang, X and Cui, J and Chen, X and Tao, P and Liu, Y and Zhang, Y and Xue, F and Dai, J and Tang, F},
title = {Structural Remodeling of Phage φPNJ-6 Hoc Promotes Adhesion to the Intestinal Epithelium.},
journal = {Transboundary and emerging diseases},
volume = {2026},
number = {},
pages = {2424208},
pmid = {42273272},
issn = {1865-1682},
mesh = {Animals ; *Intestinal Mucosa/virology/microbiology ; Escherichia coli/virology ; Mucin-2/metabolism ; *Capsid Proteins/genetics/chemistry/metabolism ; Mice ; *Virus Attachment ; },
abstract = {Although the mechanisms underlying bacteriophage-host interactions have been extensively elucidated, the "nonlytic" interactions between bacteriophage and the host intestinal microenvironment remain an emerging field. Our previous work demonstrated that the Escherichia coli T4-like bacteriophage φPNJ-6 adheres to the mucin MUC2 via the capsid protein Hoc. However, whether a rigid lock-and-key model fully explains this interaction remains unclear. In this study, we employed CRISPR-Cas12a-mediated precise deletion to remove the key Hoc residues involved in MUC2 binding (aa29-33) from the φPNJ-6 genome, generating the mutant strain Hoc[Δ29-33]-PNJ-6. Unexpectedly, both in vitro and in vivo experiments revealed that the engineered bacteriophage Hoc[Δ29-33]-PNJ-6 exhibited significantly enhanced adhesion. Structural analysis of Hoc showed that the deletion altered its conformation and increased the number of L-fucose-binding sites. These changes confer stronger fucose affinity to Hoc[Δ29-33], thereby promoting intestinal colonization by φPNJ-6. Overall, these results indicate that the Hoc-MUC2 interaction does not conform to a simple lock-and-key model and that structural remodeling of Hoc significantly affects the adhesion capacity of φPNJ-6. Our findings provide a strategy for designing long-term mucosa-resident therapeutic bacteriophages and expand the theoretical framework of bacteriophage-host microenvironment interactions.},
}
@article {pmid42275032,
year = {2026},
author = {Yuan, S and Tan, D and Zhu, D and Balcazar, JL and Wang, H and Friman, VP and Sun, M and Hu, F},
title = {Global transmission and distribution of phage-encoded cholera toxin genes constrained by toxin-repression genes and anti-phage defense systems.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrag139},
pmid = {42275032},
issn = {1751-7370},
abstract = {Cholera is a severe diarrheal disease caused by toxigenic Vibrio cholerae, whose virulence depends on lysogenic infection by CTXφ bacteriophages encoding the cholera toxin genes (ctxA and ctxB) and associated accessory genes (ace and zot). However, the global distribution and transmission dynamics of phage-encoded cholera toxin genes across environments remain poorly understood. To address this, we performed a large-scale bioinformatic analysis of publicly available whole genomes. We show that both phages and bacteria carrying toxin genes are globally distributed across human-associated, freshwater, fish, and mammalian habitats, with Vibrio and Aeromonas being the dominant bacterial taxa and Inoviridae is the most prevalent phage family. Phage-mediated horizontal gene transfer (HGT) of toxin genes occurred in both Vibrio and non-Vibrio species, with the highest transfer between Inoviridae and V. cholerae occuring predominantly among bacteria from the same habitat. Temporal analysis revealed an increase in candidate HGT events after 2000, peaking at 377845 events during 2010-2019. HGT events negatively correlated with the presence of CRISPR-Cas system and toxin-repression genes (hns, hapR, and tsrA) in host bacteria. Experimental validation indicated that H-NS and HapR inhibit phage infection by repressing phage release. Together, our results suggest that CRISPR-Cas phage defense system and toxin-repression mechanisms could constrain the spread of toxin-carrying phages, with potential implications for the occurrence and severity of cholera outbreaks worldwide.},
}
@article {pmid42275882,
year = {2026},
author = {Yang, Y and Wu, Y and Xu, X and Ihsan, A and Han, L and Wang, X},
title = {CRISPR screen identifies MAP2K3 as a key target for oleuropein to alleviate deoxynivalenol-induced hepatic injury.},
journal = {Phytomedicine : international journal of phytotherapy and phytopharmacology},
volume = {158},
number = {},
pages = {158388},
doi = {10.1016/j.phymed.2026.158388},
pmid = {42275882},
issn = {1618-095X},
mesh = {Animals ; Iridoid Glucosides/pharmacology ; *Trichothecenes/toxicity ; Mice ; *Chemical and Drug Induced Liver Injury/drug therapy ; Apoptosis/drug effects ; *Iridoids/pharmacology ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {INTRODUCTION: Deoxynivalenol (DON), a widely prevalent mycotoxin in temperate climates, causes significant hepatic injury upon the consumption of contaminated cereals. However, the key targets and mechanisms underlying DON-induced inflammatory hepatic injury remain unclear, thus hindering the development of targeted therapeutics.<br><br>OBJECTIVES: This study aimed to identify the key host targets mediating DON-induced inflammatory hepatic injury and its underlying molecular mechanism, as well as to discover potential therapeutic agents for alleviating this damage.<br><br>METHODS: We established a J774A.1-CRISPR-Cas9 whole-genome knockout library to screen for host genes essential for DON-induced apoptosis. Structure-based virtual screening was employed to identify MAP2K3 inhibitors, and the mechanism of oleuropein (Ole) action was explored using both in vivo and in vitro models.<br><br>RESULTS: CRISPR screening revealed that MAP2K3 is crucial for DON-induced apoptosis. Mechanistically, MAP2K3 mediates inflammatory hepatic injury by activating the p38/p53/caspase-8/caspase-9/caspase-3 pathway. Virtual screening identified Ole as a direct MAP2K3 inhibitor, which binds to key amino acid residues (Lys149, Ser194, Tyr230). Ole effectively inhibited DON-induced hepatic damage in vitro. In the DON-induced murine hepatitis model, Ole demonstrated robust therapeutic effects against DON-induced hepatitis, and its efficacy was superior to that of N-acetylcysteine (NAC).<br><br>CONCLUSION: This study demonstrates that MAP2K3 is a key target mediating DON-induced inflammatory hepatic injury and confirms oleuropein (Ole) as a potential therapeutic agent. Together, these findings advance our understanding of the underlying mechanisms and propose a new treatment strategy.},
}
@article {pmid42276271,
year = {2026},
author = {Hiya-Kawaguchi, U and Kashiwakura, Y and Baatartsogt, N and Lodoi, K and Togashi, T and Sato, T and Tsuchida, K and Batjargal, K and Hayakawa, M and Sato, R and Sato, Y and Ohmori, T},
title = {Non-viral delivery of a base editor enables personalized correction of hemophilia B nonsense variants in a mouse model.},
journal = {Journal of thrombosis and haemostasis : JTH},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jtha.2026.06.003},
pmid = {42276271},
issn = {1538-7836},
abstract = {BACKGROUND: Hemophilia B is caused by loss-of-function variants in the F9 gene encoding factor IX (FIX). While adeno-associated virus (AAV) vector-based gene therapy can restore FIX expression for over a decade, it does not restore the pathogenic variants and faces limitations related to immunogenicity and re-dosing.<br><br>AIM: To develop a variant-specific, non-viral base editing strategy using lipid nanoparticles (LNPs) for the precise personalized correction of pathogenic nonsense variations in F9.<br><br>METHODS: Editing efficiency was evaluated in HEK293 cells harboring each F9 variant treatable by A&#x2022;T to G&#x2022;C base editing. Functional and molecular restoration in vivo was assessed in hemophilia B model mice created with AAV8 vectors expressing F9 variants. LNPs harboring adenine base editor (Cas9 nickase conjugated with ABE8e) mRNA and the corresponding guide RNA were intravenously injected into the mice.<br><br>RESULTS: We created HEK293 stably expressing six correctable nonsense variants: c.1067G>A, c.1068G>A, c.1222C>T, c.1292G>A, c.1358G>A, and c.1359G>A. Transfection of the cells with a base editor consisting of SpRY, an engineered SpCas9 with broader proximal protospacer adjacent motif compatibility, and ABE8e, together with the guide RNA sequence, successfully induced A>G conversion at all target sites. The administration of LNPs harboring the base editor mRNA and guide RNA to the hemophilia B model restored the pathological variants (62.8% &#xb1; 14.4% for c.1068G>A, 35.9% &#xb1; 3.8% for c.1222C>T, and 70.6% &#xb1; 2.7% for c.1292G>A) and increased plasma FIX activity.<br><br>CONCLUSION: The variant-specific, non-viral base editing platform represents a truly curative intervention for severe hemophilia B caused by single-nucleotide variants.},
}
@article {pmid42276705,
year = {2026},
author = {Radoua, A and Alrustom, B and Wang, J and Bordessoules, M and Chluba, J and Plenchette, S and Micheau, O},
title = {Efficient generation of isogenic FADD[-/-], RIPK1[-/-] and Caspase-8[-/-] cells using a the ptARgenOM non-viral CRISPR-Cas9 system.},
journal = {Methods in cell biology},
volume = {208},
number = {},
pages = {115-147},
doi = {10.1016/bs.mcb.2026.02.005},
pmid = {42276705},
issn = {0091-679X},
mesh = {*Caspase 8/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Fas-Associated Death Domain Protein/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/genetics/deficiency ; *Gene Knockout Techniques/methods ; Mice ; Humans ; Green Fluorescent Proteins/genetics ; Apoptosis/genetics ; Genetic Vectors ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; },
abstract = {The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effective method based on ptARgenOM, an all-in-one mammalian expression vector designed for efficient delivery of the CRISPR-Cas9 system. This vector co-expresses the guide RNA (gRNA) and Cas9 endonuclease, which is fused to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein (EGFP) and the puromycin N-acetyltransferase. This design enables transient, expression-dependent antibiotic selection and fluorescence-based enrichment of successfully transfected cells, facilitating the rapid generation of isogenic KO populations or clones. The method is particularly well-suited, though not limited, to functional studies involving intracellular components of the cell death machinery, including both the extrinsic and intrinsic apoptotic signaling pathways. We illustrate the utility of this system by targeting and deleting FADD, Caspase-8, and RIPK1. This approach can be easily adapted to any intracellular target protein, offering a robust platform for gene function analysis in mammalian cells.},
}
@article {pmid42276707,
year = {2026},
author = {Tonietto, M and Jäger, V and Maitz, K and Flasch, B and Reinisch, A and Kargl, J and Dengler, MA and Jost, PJ},
title = {Murine models of lung cancer as a platform to investigate cell death.},
journal = {Methods in cell biology},
volume = {208},
number = {},
pages = {165-202},
doi = {10.1016/bs.mcb.2026.02.009},
pmid = {42276707},
issn = {0091-679X},
mesh = {Animals ; *Lung Neoplasms/pathology/genetics/immunology ; Mice ; Disease Models, Animal ; *Cell Death/genetics ; Tumor Microenvironment/genetics ; Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems/genetics ; Humans ; Apoptosis/genetics ; Gene Editing ; Proto-Oncogene Proteins p21(ras)/genetics ; },
abstract = {Alterations in programmed cell death pathways play a critical role in cancer development and maintenance. Yet the detailed mechanisms contributing to tumor initiation, progression, and therapeutic response in lung cancer remain incompletely understood. Also, models to study how changes in the cell death machinery impact tumor-immune interactions are limited. To address this, we describe two complementary murine models of lung adenocarcinoma that enable functional interrogation of cell death pathways in vivo. The first model is a clinically relevant, genetically engineered mouse model (GEMM) driven by Kras[G12D] activation and Tp53 loss, in which somatic CRISPR-Cas9-mediated gene editing permits tumor cell-specific knockout of candidate genes, facilitates in-depth studies of programmed cell death and immune signaling within 19 weeks. The second approach illustrates how a syngeneic orthotopic transplantation model can be used to study target genes and pathways that influence the tumor microenvironment and immunogenic cell death in a two-week timeframe. Together, these methods provide reproducible and versatile tools to investigate how modulation of cell death pathways impacts lung cancer development and progression and affects the tumor immune microenvironment, thus providing important information to guide the development of novel therapeutic strategies in lung cancer.},
}
@article {pmid42277013,
year = {2026},
author = {Wang, Q and Gong, Y and Wang, L and Lv, N and Du, X and Zhang, J and Xin, Y and Nikoloski, Z and Li-Beisson, Y and Sun, L and Ma, B and Wang, X and Xu, J},
title = {High-throughput Raman-activated cell sorting of microalgal genome-wide edited library revealed a regulatory pathway for carotenoid synthesis.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74304-5},
pmid = {42277013},
issn = {2041-1723},
support = {32370097//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Functional genomics have been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated cell sorting (RACS). The CRISPR/Cas-generated library consisted of 3567 microalgal mutants derived from 2397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unraveled mutations in the violaxanthin de-epoxidase (noVDE) or in the proteasome assembly chaperone 4 (noPAC4) genes. Knocking out all five known noVDEs revealed that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidence suggested two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this forward-genetics approach can greatly accelerate the discovery of genes and pathways.},
}
@article {pmid42277643,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M},
title = {Evolutionary interplay: virulence, endolysin-like hydrolases, and defense correlations in the Erwinia amylovora pangenome.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-026-05295-y},
pmid = {42277643},
issn = {1471-2180},
abstract = {Erwinia amylovora, the causative agent of fire blight, poses a significant threat to global pome fruit production. This study presents a comprehensive genomic analysis of 317 E. amylovora strains and 227 Erwinia phages to elucidate virulence evolution, phage-host dynamics, and the genomic signatures of the co-evolutionary arms race. Our analysis suggests that a substantial portion of E. amylovora's virulence factors (VFs) share evolutionary origins with diverse plant, human, and animal pathogens, underscoring widespread horizontal gene transfer. We identified bacterial phage hydrolases‑like proteins that share phylogenetic and domain-level similarities with phage endolysins. These observations are consistent with the possibility that some bacterial hydrolases originated from phage-derived ancestors, although functional repurposing remains to be experimentally validated. Crucially, our analysis identifies systematic, non-random associations between bacterial defense systems (e.g., RM, CRISPR-Cas, TA) and mobile anti-defense genes. Statistical correlations show strong patterns of co-occurrence and mutual exclusivity, which are consistent with an ongoing phage-bacteria arms race. These patterns provide a genomic basis for generating hypotheses about co-evolutionary dynamics. These findings may advance our understanding of E. amylovora pathogenicity and phage interactions, offering foundational insights for developing targeted phage-based biocontrol strategies against this devastating plant pathogen. Experimental validation of the predicted virulence factors and defense correlations is warranted to confirm their biological roles.},
}
@article {pmid42277912,
year = {2026},
author = {Chang, L and Xu, W and Wang, X and Xue, Y and Zhang, Y and Zhu, X and Zhang, Y and Liang, T and Liu, W},
title = {CRISPR diagnostics: from trans-nuclease activity to cancer diagnosis.},
journal = {Cell &amp; bioscience},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13578-026-01603-1},
pmid = {42277912},
issn = {2045-3701},
support = {2019YFC1316000//National Key Research and Development Program of China/ ; 81830089//National Natural Science Foundation of China/ ; 82188102//National Natural Science Foundation of China/ ; LQ23H200004//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {The field of nucleic acid-based testing experienced a decade-long stagnation since the development of quantitative polymerase chain reaction (qPCR) in 1992 and isothermal amplification methods in the early 2000s. However, in 2016, the discovery of trans-nuclease activity in CRISPR-Cas systems revolutionized the molecular diagnostics for nucleic acids. A typical CRISPR diagnostic workflow comprises three phases: (1) target recognition through CRISPR RNA (crRNA)-guided hybridization; (2) signal transduction via trans-cleavage of engineered reporters (e.g., fluorophore-quencher oligonucleotides), and (3) signal readout using fluorescence, electrochemical, or colorimetric platforms. Emerging shortly prior to the COVID-19 pandemic, CRISPR diagnostics quickly gained prominence as a field-deployable alternative to qPCR due to its rapidity (< 1 h), minimal equipment requirements, and field adaptability. This technological paradigm underwent rigorous validation and refinement alongside the rapid evolution of SARS-CoV-2 detection, which facilitated its adaptation for cancer diagnosis. Recent advancements in sensitivity (attomolar-level detection) and specificity (single-nucleotide discrimination) have enabled transformative applications in cancer diagnostics, including: (1) identification of nucleic acid biomarkers, such as high-frequency somatic mutations, circulating nucleic acids and miRNAs; and (2) detection of non-nucleic acid biomarkers, including epigenetic aberrations, proteins, small molecules and metabolite biomarkers. This review chronicles the decadal evolution of CRISPR diagnostics, with particular emphasis on recent advancements of its application in cancer diagnosis. We critically evaluate persistent technical limitations, including PAM sequence restriction, suboptimal sensitivity and specificity, quantitative constraints, and unmet point-of-care testing (POCT) in complex biological matrices. Additionally, we discuss prospective solutions to address these challenges.},
}
@article {pmid42278292,
year = {2026},
author = {Tuerxun, K and Ding, Z and Luo, X and Zhou, S},
title = {Fermentation Process Optimization for High 2-Phenylethanol Aroma Whisky.},
journal = {International journal of molecular sciences},
volume = {27},
number = {11},
pages = {},
pmid = {42278292},
issn = {1422-0067},
support = {51878291//National Natural Science Foundation of China/ ; },
mesh = {*Fermentation ; *Saccharomyces cerevisiae/genetics/metabolism ; *Phenylethyl Alcohol/metabolism/analysis ; *Wine/analysis ; CRISPR-Cas Systems ; *Odorants/analysis ; Transaminases/genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {2-Phenylethanol (2-PE) is a key aromatic alcohol contributing to the rose-like odor in brewed wines, primarily synthesized by yeast metabolism with a typical yield of less than 100 mg/L. To enhance the 2-PE content in brewed wines, this study used CRISPR-Cas9 gene editing technology to delete the ARO8 gene (encoding aromatic transaminase I) in Saccharomyces cerevisiae SY. The single-factor experiments were performed to optimize the fermentation process, and the 2-PE content in the brewed wine was measured by high-performance liquid chromatography. The results demonstrated that the 2-PE content in whisky fermented by the SY-A8 was 0.73 g/L, increasing 23.73% compared to SY. The fermentation conditions of SY-A8 were optimized through single-factor experiments and the Box-Behnken design. The optimal conditions were a sugar concentration of 46.30 g/L, a fermentation time of 6 days, and an L-phenylalanine concentration of 1.43 g/L. The high 2-phenylethanol aroma whisky was brewed with a higher 2-phenylethanol content of 3.68 g/L in a 1 L fermenter at the optimal conditions. In conclusion, the modification of Saccharomyces cerevisiae by CRISPR-Cas9 gene editing combined with fermentation process optimization provides an effective technical strategy for improving the 2-PE content in whisky, thereby providing a research perspective for the flavor enhancement of whisky and other brewed wines.},
}
@article {pmid42278622,
year = {2026},
author = {Zhao, YY and Evans, CE},
title = {Contemporary Endothelial Genome Editing Technologies: Towards Precision Genetic Medicine for Vascular Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {11},
pages = {},
pmid = {42278622},
issn = {1422-0067},
support = {1R01HL133951-21/NH/NIH HHS/United States ; 2R01HL164014-22/NH/NIH HHS/United States ; 3R01HL162299-22/NH/NIH HHS/United States ; 4R01HL172447-23/NH/NIH HHS/United States ; 24TPA1285575//American Heart Association/ ; 23SCEFIA1155876//American Heart Association/ ; 5P20GM103499-23/NH/NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Vascular Diseases/genetics/therapy ; *Endothelial Cells/metabolism ; Animals ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Endothelium, Vascular/metabolism ; },
abstract = {Endothelial dysfunction is a key characteristic of many diseases, including atherosclerosis, hypertension, heart failure, stroke, cancer, acute respiratory distress syndrome (ARDS), peripheral vascular disease, coronavirus 2019 (COVID-19), and pulmonary arterial hypertension (PAH). To improve understanding of the roles of endothelial cells (ECs) in health and disease, EC-specific genome editing technologies have been developed in recent years. Therapeutic strategies that aim to restore a healthy endothelial monolayer include the inhibition of endothelial genes that cause EC injury and dysfunction and the induction or activation of endothelial genes that drive EC repair and regeneration. In this review, we describe established recombinase-mediated genetic modification technologies and emerging EC-specific genome editing technologies including viral and non-viral delivery of the CRISPR/Cas9 genome editing system, and we summarize the strengths and limitations of each technology. We then discuss possible avenues for future research, including the development of organ-specific EC genome editing technologies. In short, EC-specific genome editing technologies can be used to modulate gene expression selectively in ECs and even within a specific vascular bed and/or distinctive EC subtype, and, in doing so, greatly improve the understanding of vascular biology and help develop precision genetic medicine targeting the disease-causing vascular bed(s) to effectively treat diseases caused by vascular endothelial dysfunction.},
}
@article {pmid42279769,
year = {2026},
author = {Pan, X and Ding, X},
title = {Advances in Foodborne Pathogen Detection: From Conventional Confirmation to Integrated and Intelligent Platforms.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {11},
pages = {},
pmid = {42279769},
issn = {2304-8158},
support = {2023YFD2402800//China Youth Development Foundation/ ; BE2023725//Jiangsu Province Science and Technology Department/ ; 82373629//National Natural Science Foundation of China/ ; },
abstract = {Foodborne pathogens pose a major challenge for public health, food safety regulation, and industrial quality control. Effective surveillance, outbreak tracing, and early warning for foodborne microbial contamination require rapid, reliable detection methods. Conventional culture-based methods are still essential for regulatory confirmation since they recover viable isolates and support downstream verification. However, their long turnaround time, labor-intensive procedures, and limited throughput restrict their use in rapid screening and on-site testing. In recent years, immunological assays, nucleic acid amplification and recognition methods, biosensors, microfluidic systems, CRISPR-Cas platforms, mass spectrometry, sequencing technologies, and artificial intelligence-assisted analysis have expanded the detection toolbox. These methods improve speed, sensitivity, portability, and multiplexing capacity, but their performance still depends on food-matrix properties, sample pretreatment, and application conditions. This review compares representative methods in terms of analytical principle, sample pretreatment, sensitivity, specificity, assay time, viable-cell discrimination, field applicability, and standardization potential. In our opinion, culture-based methods are central for confirmation, while emerging technologies are better suited for rapid screening, integrated analysis, and point-of-need testing. Nevertheless, matrix interference, limited validation in naturally contaminated samples, insufficient viable/dead-cell discrimination, and weak cross-platform consistency remain key barriers to routine use.},
}
@article {pmid42282866,
year = {2026},
author = {Xie, S and Liu, S and Schwarz, S and Sun, H and Xu, Q and Chai, J and Lin, L and Du, S and Hou, J and Song, Y and Brenciani, A and Zhu, Y and Zhang, W},
title = {Toxin gene profiles, antibiotic resistance, and genetic diversity of Clostridium perfringens from food-producing animals: a whole-genome sequencing study with implications for food safety.},
journal = {Current research in food science},
volume = {12},
number = {},
pages = {101460},
pmid = {42282866},
issn = {2665-9271},
abstract = {Clostridium perfringens is a significant zoonotic foodborne pathogen. To systematically assess the potential risks associated with food-producing animals as a reservoir of C. perfringens in the early stages of the food production chain, we conducted whole-genome sequencing (WGS) and phenotypic analysis of 91 clinical C. perfringens isolates collected from pigs, chickens, cows, ducks, and geese across different regions of China. The results revealed that the isolates harbored a rich repertoire of toxin genes, with 71.43% (65/91) carrying greater than or equal to 10 toxin genes. Besides the classic type A, type C, which causes animal enterotoxemia, was most prevalent in pigs (45.76%). Notably, the and necrotic B-like (NetB) toxin, typically associated with avian necrotic enteritis, was also detected in isolates from cows and geese, suggesting potential cross-host transmission of toxin types. Antimicrobial susceptibility testing revealed a severe resistance situation, particularly among porcine isolates, which showed the highest resistance rates to clindamycin, penicillin, and tetracycline, with widespread multidrug resistance (MDR). Genomic analysis further identified 14 types of antimicrobial resistance (AMR) genes. The tetracycline resistance gene tetA(P) had an extremely high carriage rate of 94.51%, and AMR genes were most enriched in porcine isolates. Multilocus sequence typing (MLST) identified 59 sequence types (STs), 42 of which were newly discovered, demonstrating high genetic diversity. Major clonal complexes (CCs) showed certain host and geographic clustering. Furthermore, while the restriction-modification (RM) system was present in all isolates, the distribution of other defense systems like CRISPR-Cas was strain-specific. This study revealed that C. perfringens from Chinese food-producing animals is characterized by high virulence, extensive antimicrobial resistance, and high genetic diversity. It highlighted that pigs may serve as a crucial reservoir and evolutionary hub for virulent MDR isolates, posing a continuous threat to food safety and public health, and underscored the necessity for enhanced monitoring at the farm level.},
}
@article {pmid42283176,
year = {2026},
author = {Hussain, MS and Babu, MA and Roopashree, R and Lal, M and Anand, T and Rekha, A and Goyal, K and Ali, H and Singh, TG and Singh, SK and Dua, K and Gupta, G},
title = {Regulatory Networks of ncRNAs and NF-κB in Glioblastoma: Implications for Therapeutics.},
journal = {Current neuropharmacology},
volume = {},
number = {},
pages = {},
doi = {10.2174/011570159X382254251121114451},
pmid = {42283176},
issn = {1875-6190},
abstract = {Glioblastoma (GBM) is the most malignant form of primary brain tumor, exhibiting rapid growth, increased blood vessel growth, therapy resistance, and severe immune suppression. Constant activation of the nuclear factor κB- (NF-κB) signaling axis underlies many of these cancer traits. Concurrently, non-coding RNAs (ncRNAs), notably microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), have emerged as critical modulators of GBM pathways. This review explains how specific ncRNAs use NF-κB signaling to regulate glioma cell survival, invasion, and therapeutic responses. We synthesized current evidence for miR-21 and miR-181 family members in promoting NF-κB-driven gene expression patterns, described lncRNAs, such as MALAT1 and HOTAIR, which support NF-κB complexes, and highlighted circRNAs, including circKPNB1 and circEZH2, that act as competing RNAs to modulate NF-κB activity. We evaluated preclinical strategies targeting ncRNA-NF-κB interactions, including antisense oligonucleotides, small interfering RNAs, locked nucleic acids, CRISPR-Cas approaches, and smallmolecule- inhibitors, with an emphasis on delivery systems, target specificity, and tumor diversity. Finally, we propose a comprehensive model of ncRNA-NF-κB crosstalk in GBM pathobiology and outline practical approaches to exploit these networks for personalized treatment.},
}
@article {pmid42283788,
year = {2026},
author = {Heydarov, RN and Romanova, KA and Ushtanit, AI and Mikhailovich, VM},
title = {The genomic landscape of Klebsiella pneumoniae in Russia.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {7},
pages = {},
pmid = {42283788},
issn = {1573-0972},
support = {25-24-00721//Russian Science Foundation/ ; 25-24-00721//Russian Science Foundation/ ; 25-24-00721//Russian Science Foundation/ ; 25-24-00721//Russian Science Foundation/ ; },
mesh = {*Klebsiella pneumoniae/genetics/isolation &amp; purification/drug effects/pathogenicity/classification ; Plasmids/genetics ; *Genome, Bacterial/genetics ; beta-Lactamases/genetics ; Russia/epidemiology ; *Klebsiella Infections/microbiology/epidemiology ; Bacterial Proteins/genetics ; Virulence Factors/genetics ; Humans ; Whole Genome Sequencing ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Genomics ; Virulence/genetics ; CRISPR-Cas Systems ; },
abstract = {Klebsiella pneumoniae is a critical priority pathogen known for its extensive antimicrobial resistance (AMR) and capacity to cause severe infections. The traditional dichotomy between multidrug-resistant (MDR) and hypervirulent strains is rapidly eroding, driving the emergence of highly lethal convergent phenotypes. While global surveillance heavily emphasizes KPC-producing lineages, the specific genomic architecture driving this convergence in the Russian Federation remains insufficiently resolved. In this study, we analyzed population structure represented in a curated dataset 264 K. pneumoniae genomes, comprising 18 newly sequenced clinical isolates and 246 public assemblies from 2015 to 2024, using whole-genome sequencing, pangenome reconstruction, resistome, virulence factors and plasmidome profiling, and CRISPR-Cas typing. Our analysis revealed that high-risk sequence types ST395 (56.1%) and ST147 (8.7%) dominate the regional landscape. Carbapenemase genes were detected in 76.5% of isolates, primarily driven by blaOXA-48 on IncL/M plasmids within ST395 and blaNDM variants in ST147. Crucially, 46.2% of isolates harbored convergent plasmids, predominantly large, mosaic IncFIB/IncHI1B cointegrates, that simultaneously encode hypervirulence determinants like the aerobactin synthesis locus alongside resistance genes including blaNDM-1 and blaCTX-M-15. Additionally, we identified a heavy enrichment of plasmid-borne Type IV-A3 CRISPR-Cas systems in the dominant ST395 clone. The regional dominance of ST395 and ST147, coupled with the extensive horizontal integration of both resistance and virulence, represents a formidable public health threat. These findings underscore the critical need for localized genomic surveillance to effectively monitor evolving convergent pathogens and guide tailored antimicrobial stewardship.},
}
@article {pmid42283883,
year = {2026},
author = {Taheri, S and Azarpira, N and Mahmoodi, S and Gila, F and Dara, M},
title = {CRISPR/Cas9-mediated knockout of TopBP1 shifts the Bax/Bcl-2 balance toward apoptosis in MCF7 breast cancer cells.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42283883},
issn = {1573-4978},
mesh = {Humans ; *bcl-2-Associated X Protein/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics/metabolism ; Female ; Apoptosis/genetics ; *Breast Neoplasms/genetics/metabolism ; *Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; MCF-7 Cells ; *Nuclear Proteins/genetics/metabolism ; Gene Knockout Techniques/methods ; *Carrier Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Exons ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Breast cancer (BC) is the most prevalent cancer among women and the second leading cause of cancer-related deaths globally, after lung cancer. Despite advances in treatment, BC remains a major contributor to cancer mortality worldwide, underscoring the need for innovative therapeutic approaches. The TopBP1 (DNA Topoisomerase II Binding Protein 1) gene, involved in DNA damage response and cell cycle regulation, has been associated with cancer progression and resistance to chemotherapy. This study investigates the potential of using CRISPR/Cas9 technology to knockout the TopBP1 gene as a novel strategy in breast cancer research.<br><br>METHODS AND RESULTS: A pair of guide RNAs (gRNAs) was specifically designed to target the TopBP1 gene, inducing the deletion of exon 4. These gRNAs were transfected into the MCF7 breast cancer cell line, and the efficacy of genomic editing was validated using PCR and Sanger sequencing. Subsequent analyses employing real-time PCR and Western blotting were conducted to investigate the downstream effects of this genetic modification on gene expression. The CRISPR/Cas9 system successfully knocked out exon 4 of the TopBP1 gene in MCF7 breast cancer cells, as validated by PCR and Sanger sequencing. Real-time PCR analysis revealed a significant increase in Bax expression and a decrease in Bcl-2 expression in the knockout cells compared to controls. These changes indicate enhanced apoptotic activity following TopBP1 knockout, suggesting that MCF7 cells may become more sensitive to apoptosis. Overall, the findings support the hypothesis that targeting TopBP1 could play a critical role in promoting cell death in breast cancer, potentially offering a new therapeutic strategy.<br><br>CONCLUSIONS: This study successfully employed the CRISPR/Cas9 system to knockout exon 4 of the TopBP1 gene in MCF7 breast cancer cells, resulting in reduced TopBP1 expression. The subsequent increase in the pro-apoptotic Bax gene and decrease in the anti-apoptotic Bcl-2 gene suggest that targeting TopBP1 could enhance apoptosis in breast cancer cells, offering a promising alternative to conventional treatments. Further research is necessary to fully explore the therapeutic potential of this approach.},
}
@article {pmid42285217,
year = {2026},
author = {Leon Agudelo, JA and Martínez Vallejo, H},
title = {Therapeutic applications of gene editing using CRISPR-Cas9 in the posterior segment: review of the literature.},
journal = {Archivos de la Sociedad Espanola de Oftalmologia},
volume = {},
number = {},
pages = {502582},
doi = {10.1016/j.oftale.2026.502582},
pmid = {42285217},
issn = {2173-5794},
abstract = {CRISPR-Cas gene editing has become increasingly relevant in the treatment of several ophthalmic diseases. Its ability to make precise modifications at the DNA or RNA level has enabled targeted approaches for specific mutations involved in conditions such as Leber congenital amaurosis type 10 (LCA10), certain forms of retinitis pigmentosa, and age-related macular degeneration. This article provides an organized overview of the biological basis of CRISPR-Cas technology and highlights key advances from preclinical studies and early clinical trials. Technical limitations and ongoing safety challenges are also discussed. Programs such as EDIT-101, EDIT-103, and HG202 stand out as important milestones in the evolution of ocular gene editing.},
}
@article {pmid42286276,
year = {2026},
author = {Wójcicki, M and Cieślik, M and Górski, A and Jończyk-Matysiak, E},
title = {Giving Antibiotics a Second Chance: Evolutionary Trade-Offs and Phage-Driven Restoration of Antibiotic Susceptibility.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42286276},
issn = {1179-190X},
abstract = {Antimicrobial resistance poses a critical and escalating threat to global public health, driven by the widespread and often unjustified use of antibiotics and the rapid dissemination of resistance determinants. With the antibiotic discovery pipeline largely depleted, alternative and complementary strategies are urgently needed to preserve the effectiveness of existing antimicrobials. Bacteriophages-viruses that specifically infect bacteria-have re-emerged as promising tools not only for direct bacterial eradication but also for reshaping bacterial evolutionary trajectories. This review examines the concept of phage-driven restoration of antibiotic susceptibility, focusing on evolutionary trade-offs that arise when bacteria adapt to phage pressure. Resistance to bacteriophages frequently involves modifications of surface structures, capsules, or efflux systems, changes that often incur fitness costs manifested as reduced virulence, impaired biofilm formation, or increased antibiotic sensitivity. Experimental studies and clinical case reports demonstrate that phage-antibiotic synergy can suppress bacterial growth more effectively than monotherapy, limit resistance emergence, and resensitize multidrug-resistant pathogens to previously ineffective antibiotics. Particular attention is given to mechanisms involving efflux pump targeting, capsule loss, biofilm disruption, and temperate phage-antibiotic interactions. In addition, emerging strategies that combine bacteriophages with CRISPR-Cas systems enable precise targeting and removal of resistance genes, offering a highly selective means to restore antibiotic efficacy and curb horizontal gene transfer. Together, these findings highlight bacteriophages as powerful evolutionary and therapeutic tools capable of giving antibiotics a "second chance". Integrating phage-based approaches into antibiotic stewardship frameworks may represent a sustainable path forward in combating multidrug-resistant bacterial infections.},
}
@article {pmid42287453,
year = {2026},
author = {Wong, MTJ and Zulkifli, ND and Ravichandran, T and Vasodavan, K and Al-Shaibah, O and Himel, GMS and Achuthan, A and Anthonysamy, MA and Theva Das, K},
title = {The evolution of AI-integrated genome editing and its challenges.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {37},
number = {1},
pages = {},
pmid = {42287453},
issn = {1432-1777},
mesh = {*Gene Editing/methods ; *Artificial Intelligence ; Humans ; Animals ; Machine Learning ; CRISPR-Cas Systems/genetics ; Genomics/methods ; },
abstract = {Artificial Intelligence (AI) is poised to revolutionize the field of genome editing by enhancing precision, efficiency, and accessibility. AI-driven approaches are already improving the design of CRISPR-based systems by enabling more accurate identification of target sequences and predicting off-target effects. Machine learning (ML) algorithms can analyze vast genomic data, as well as identify patterns and mutations that might be overlooked by traditional methods. Taking together, utilizing AI/ML tools allow for the enhancement of every step in genome editing. Recent advances also demonstrated that AI-powered tools can facilitate the simulation and modeling of genetic modifications, predicting their effects on cellular behavior and phenotypes. This allows for a more rapid prediction of the genome editing effects, without the need for wet lab. Additionally, AI can accelerate drug discovery and therapeutic development by streamlining the identification of genetic targets and optimizing gene therapies. The integration of AI with genome editing promises to democratize access to cutting-edge technologies, enabling researchers to design and plan for complex genetic modifications with minimal technical expertise. Drawing from various examples, this paper dives into the advancements and applications of AI in genome editing, its limitations, as well as future directions and opportunities in this field.},
}
@article {pmid42288047,
year = {2026},
author = {Maire, A and Laurenceau, R and Rolhion, N and Bikard, D},
title = {In situ genetic modification of gut bacteria.},
journal = {Current opinion in microbiology},
volume = {92},
number = {},
pages = {102766},
doi = {10.1016/j.mib.2026.102766},
pmid = {42288047},
issn = {1879-0364},
abstract = {The crucial role of the gut microbiome in human health has driven a need to understand bacterial function within their complex native ecosystem. However, traditional functional genomic methods require isolating, cultivating, and modifying bacteria in vitro before their reintroduction in vivo. This process often necessitates the use of axenic animals or antibiotic treatments, creating artificial conditions that disrupt key microbial interactions and can obscure relevant phenotypes. This review highlights emerging tools for precise, in situ genetic manipulation of bacteria directly within the gut. We cover diverse technologies, including DNA delivery systems (e.g. engineered temperate phages, phagemids, and conjugative plasmids), and genetic perturbation strategies (e.g. CRISPR-Cas tools and transposons). These methods offer the opportunity to engineer unculturable microbes in their natural habitat and conduct genetic screens to investigate the role of specific genes and pathways. Finally, we explore the potential therapeutic applications of in situ microbiome editing.},
}
@article {pmid42288957,
year = {2026},
author = {Pal, P and Sarkar, S and Rajak, J and Maitra, S and Almohaimeed, HM and Sami Aggad, W and Almars, AI and Mitra, AK and Uti, DE and Dhara, B},
title = {Engineering Lactobacillus for therapeutic delivery and biosensing: lessons from niche adaptation.},
journal = {Future microbiology},
volume = {21},
number = {8},
pages = {795-810},
doi = {10.1080/17460913.2026.2686558},
pmid = {42288957},
issn = {1746-0921},
abstract = {Lactobacillus species are renowned for their probiotic properties and niche adaptability, driven by unique genomic traits, stress-response mechanisms, and biofilm formation. This versatility makes them exceptional candidates for advanced biotechnological applications. Their biocompatibility and immunomodulatory effects allow them to serve as live biotherapeutic products. Through genetic engineering and encapsulation, Lactobacillus can be programmed to deliver recombinant proteins and vaccines, cytokines and anti-inflammatory molecules, targeted enzymes, and peptides. Beyond therapy, these bacteria can be engineered into biosensors to detect pathogens, toxins, and clinical biomarkers. By integrating CRISPR-Cas systems and reporter genes into whole‑cell or cell‑free platforms, they offer robust solutions for food safety, environmental monitoring, and diagnostics. While challenges in stability and regulation persist, advancements in synthetic biology are transforming Lactobacillusfrom a simple probiotic into a precise, multifunctional tool for improving global health and environmental oversight.},
}
@article {pmid42289911,
year = {2026},
author = {Hara, H and Uosaki, H and Nakahara, F and Inoue, M and Hanazono, Y},
title = {Stepwise Evaluation of Plasmid- and Adeno-Associated Virus-Based Knock-In Using A Triple-Reporter Platform.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {31},
number = {4},
pages = {e70129},
doi = {10.1111/gtc.70129},
pmid = {42289911},
issn = {1365-2443},
support = {JP18am0301002//Japan Agency for Medical Research and Development/ ; JP22ae0201007//Japan Agency for Medical Research and Development/ ; JP22bm0804018//Japan Agency for Medical Research and Development/ ; JP25bm1323001//Japan Agency for Medical Research and Development/ ; //Sumitomo Pharma Co. Ltd/ ; },
mesh = {Animals ; *Dependovirus/genetics ; *Gene Knock-In Techniques/methods ; *Plasmids/genetics ; Mice ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Genes, Reporter ; Mouse Embryonic Stem Cells/metabolism ; Electroporation ; },
abstract = {Homology-directed repair (HDR)-mediated knock-in efficiency is a composite of double-strand break (DSB) induction and repair pathway selection during targeted insertion. Thus, optimization of HDR-mediated knock-in presents significant challenges. To address this, we employed a mouse embryonic stem cell-based triple-reporter platform to examine how donor design and experimental parameters are associated with DSB induction and the balance between HDR-mediated knock-in and end-joining-mediated targeted insertion (EJ-TI). Our analysis reveals that donor design directly impacts DSB induction: co-electroporation of donor plasmids with Cas9/gRNA reduced cleavage efficiency, and Homology-Independent Targeted Integration (HITI) sequences further reduced this efficiency, thereby biasing overall knock-in efficiency. When normalized to DSB levels, HITI increased total targeted insertion yield (knock-in + EJ-TI) with shorter arms and favored HDR-mediated knock-in over EJ-TI with longer arms. Conversely, under adeno-associated virus (AAV) donor conditions, HITI showed donor-format dependency. It increased knock-in and decreased EJ-TI only with a self-complementary AAV donor, but had little effect with a single-stranded AAV donor. These results highlight the importance of separately evaluating DSB induction, total targeted insertion yield, and the balance between HDR-mediated knock-in and EJ-TI using a triple-reporter platform when optimizing knock-in strategies.},
}
@article {pmid42290770,
year = {2026},
author = {Chen, F and Hu, X and Xiao, Q and Tan, L and Liu, W},
title = {A mini review on advances in diagnostic techniques for Schistosoma japonicum detection and its epidemiological features among humans and wild rodents in China.},
journal = {Frontiers in veterinary science},
volume = {13},
number = {},
pages = {1857648},
pmid = {42290770},
issn = {2297-1769},
abstract = {Schistosomiasis is a zoonotic parasitic disease caused by Schistosoma japonicum infection, representing a significant public health concern for both animals and humans in China. A thorough understanding of the epidemiological features and diagnostic techniques associated with schistosomiasis is crucial for conducting prevention, control, and eradication strategies. This review provides a comprehensive update on the current diagnostic technologies and the prevalence of S. japonicum in human and wild rodent populations in China, focusing on literature published from 2015 to the present. In general, diagnostic methods include morphological identification, serological assays, and molecular techniques. While traditional methods like Kato-Katz remain widely used, emerging tools such as PCR-based assays, isothermal amplification (LAMP, RPA, RAA), and CRISPR/Cas systems offer enhanced sensitivity and suitability of field deployment. Serological tests (IHA, ELISA) are valuable for large-scale screening but face specificity challenges. A descriptive aggregation of 37 studies involving 46,910,186 human serum samples revealed an overall seroprevalence rate of 1.54% (95% CI: 1.53-1.54), with significant variation across 10 provinces (0.08% in Fujian to 4.95% in Yunnan). Higher seroprevalence was observed in males, local residents, and individuals engaged in farming or fishing. Concurrently, a narrative synthesis of 24 studies across seven provinces showed a substantially higher prevalence of 8.97% (95% CI: 8.50-9.44) in 14,381 wild rodents, with Rattus norvegicus showing the highest infection rate (37.44%). In conclusion, despite significant control progress, S. japonicum remains endemic in specific regions, with wild rodents serving as critical reservoir hosts. Integrating sensitive molecular diagnostics into surveillance programs and targeting rodent reservoirs are essential for achieving the national goal of schistosomiasis elimination by 2030.},
}
@article {pmid42291797,
year = {2026},
author = {Dunne, VL and Wright, TC and Toner, A and Wilson, ML and Savage, KI and O'Sullivan, JM and Prise, KM},
title = {Impact of ataxia-telangiectasia mutated (ATM) loss on radiobiological and immune response to radium-223 in prostate cancer in vitro models.},
journal = {Clinical and translational radiation oncology},
volume = {59},
number = {},
pages = {101206},
pmid = {42291797},
issn = {2405-6308},
abstract = {INTRODUCTION: Personalised medicine approaches are redefining the therapeutic landscape for men with metastatic castration-resistant prostate cancer (mCRPC). While poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated clinical benefit in patients with BRCA1/2 mutant tumours, the therapeutic efficacy of these inhibitors in Ataxia-telangiectasia mutated (ATM)-mutated prostate cancer patients remains modest, highlighting the need for alternative treatment strategies. This study aimed to elucidate the impact of ATM loss on radiobiological and immune responses to different radiation modalities in prostate cancer models.<br><br>METHODS: Isogenic CRISPR Cas-mediated ATM-deficient and wild-type (WT) cells were treated with X-rays or Radium-223 dichloride ([223]Ra). Cellular radiosensitivity was measured using clonogenic assays and 53BP1 immunofluorescence assessed DNA damage. Cell cycle distribution and apoptosis were analysed by flow cytometry. Innate immune activation was assessed using cGAS immunofluorescence and quantitative PCR of CCL5, CXCL10 and IFIT2.<br><br>RESULTS: [223]Ra significantly increased radiosensitivity in comparison to X-rays which was amplified by ATM loss. [223]Ra exposure in ATM deficient cells induced significantly greater levels of DNA damage as measured by persistent 53BP1 foci at 24h, distinct G2 accumulation and elevated levels of apoptosis in PC-3 and DU145 ATM-deficient cells in comparison to either X-rays or WT counterparts (p < 0.05). Furthermore, [223]Ra triggered cGAS positive micronuclei and upregulation of STING driven inflammatory genes, particularly in ATM-deficient cells (p < 0.05).<br><br>CONCLUSIONS: These findings demonstrate that ATM-deficiency enhances radiobiological response and amplifies immune activation to [223]Ra, supporting further pre-clinical evaluation of ATM loss as a determinant of response and therapeutic target to optimise [223]Ra based strategies for mCRPC.},
}
@article {pmid42292392,
year = {2026},
author = {Wong, K and Calnan, C and Benson, MJ},
title = {CRISPR-screen informed engineered T cell therapies.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1839064},
pmid = {42292392},
issn = {1664-3224},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; Tumor Microenvironment/immunology ; *Neoplasms/therapy/immunology/genetics ; *T-Lymphocytes/immunology/transplantation/metabolism ; Animals ; *CRISPR-Cas Systems ; *Lymphocytes, Tumor-Infiltrating/immunology/transplantation/metabolism ; Antigens, Neoplasm/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Adoptive T cell therapies can deliver curative responses for refractory patients with B cell malignancies, yet clinical activity in solid tumors remains inconsistent. Tumor-intrinsic barriers dominating this inconsistency include the immunosuppressive solid tumor microenvironment (TME) imposing chronic inhibitory cues to T cells and the scarcity of patient-shared and uniformly expressed tumor-restricted antigens for T cells to target. CRISPR-based forward genetics screens enable mapping of the functional genome regulating T cell anti-tumor activity. Here, we review recent insights from pooled CRISPR knockout screens in T cells to define convergent targets and pathways regulating T cell anti-tumor function and align the pharmacology of engineered T cells with sequential barriers they encounter within the TME. We additionally propose a framework for CRISPR screen-enabled target prioritization and present an example of how these principles can be applied to the functional enhancement of T cells through TIL (Tumor Infiltrating Lymphocyte) therapy, which utilizes a patient's personalized immune response against solid tumor antigens.},
}
@article {pmid42293525,
year = {2026},
author = {Nagaraja, PK and Mitra, SD and Murugesan, D and Muninarayanaswamy, PKA and Geddam, S and Venugopal, N and Tewari, R and Ramamurthy, AS and Skariah, S and Nayakvadi, S and Shome, BR and Shome, R},
title = {Genomic profiling of ESBL/AmpC-producing Escherichia coli from backyard poultry: resistome, virulome, plasmidome, and CRISPR-Cas insights.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1836952},
pmid = {42293525},
issn = {1664-302X},
abstract = {INTRODUCTION: Antimicrobial resistance (AMR) is a major One Health concern driven by many factors including unregulated animal farming. This study aimed to perform a comprehensive phenotypic and genotypic characterization of Escherichia coli isolated from backyard poultry reared in households in rural setup in southern India.<br><br>METHODS: A total of 48 cloacal samples were collected from poultry birds across 12 epidemiological units using a 30-cluster sampling strategy. E. coli isolation was performed using standard microbiological methods and confirmed by species-specific multiplex PCR. Of the 48 samples, 45 yielded confirmed E. coli isolates, among which 18 ESBL- and/or AmpC-positive isolates were selected for whole genome sequencing (WGS). Genomic analyses included detection of antimicrobial resistance genes, virulence-associated genes, mobile genetic elements, plasmid replicons, biofilm-associated genes, multilocus sequence typing, serotyping, CH typing, Clermont phylogrouping, CRISPR-Cas profiling, and SNP-based phylogenetic analysis.<br><br>RESULTS: A total of 48 cloacal samples yielded 45 confirmed isolates of which 18 detected with ESBL genes including bla SHV (28.8%), bla TEM (26.6%), and blaCTX-M (20%) along with plasmid-mediated AmpC genes (11.11%). Resistome profiling revealed diverse ARGs such as bla CTX-M-15, bla DHA-1, qnrS/B, tet(A), sul1/2/3, dfrA variants, and mphA/B, in addition to intrinsic efflux systems (acr, emr, mdt, mar). Virulome analysis showed conserved genes associated with adhesion (fim, csg, ecp), iron acquisition (ent, fep, fes, ybt), and stress response (gad, hlyE, ompT, iss). CRISPR-Cas analysis revealed a high prevalence of Type I-E arrays (83.3%) coexisting with multiple plasmids. MLST and CH typing revealed high genetic diversity across 12 sequence types, including ST10, ST48, ST3107, and ST226. SNP-based phylogeny placed isolates mainly within commensal phylogroups A and B1, with relatedness to global strains. All sequenced isolates were multidrug-resistant, with significant &#x3b2;-lactam resistance enrichment (Z&#x202f;=&#x202f;3.46, p&#x202f;<&#x202f;0.001). ARG distribution differed significantly among phylogroups (p&#x202f;=&#x202f;0.036), and Simpson's diversity index (D&#x202f;=&#x202f;0.94) indicated marked clonal heterogeneity. Strong plasmid-ARG associations (Cram&#xe9;r's V&#x202f;>&#x202f;0.5) suggested plasmid-mediated resistome structuring.<br><br>DISCUSSION: Backyard poultry-derived E. coli showed multidrug resistance, genetic diversity, and virulence-associated traits, highlighting backyard poultry as a potential reservoir for AMR dissemination at the human-animal-environment interface.},
}
@article {pmid42293685,
year = {2026},
author = {Shen, Y and Lu, SM and Yang, L and Li, Y and Zhang, Y and Liang, LG},
title = {Engineered bacteria in disease diagnosis and therapy: A synthetic biology perspective.},
journal = {Synthetic and systems biotechnology},
volume = {14},
number = {},
pages = {459-470},
pmid = {42293685},
issn = {2405-805X},
abstract = {Synthetic biology is an interdisciplinary field that integrates knowledge and techniques from modern biology and many other disciplines to design and construct novel biological systems or to modify existing life forms. Its core technologies include gene editing (e.g., CRISPR/Cas9), DNA assembly, in vivo directed evolution, and integration with artificial intelligence. The development of these technologies has greatly advanced the application of synthetic biology in medicine. In disease diagnosis, engineered bacteria have shown considerable promise. They can be designed to sense disease-specific signals and produce detectable reporter outputs, thereby establishing new paradigms for early diagnosis and real-time disease monitoring. For example, bacteria engineered via synthetic biology have been developed as "living sensors" to detect disease biomarkers. In therapeutic applications, synthetic biology offers a fresh perspective on using microorganisms to treat diseases. Researchers can design and construct microorganisms with tailored functions for targeted drug delivery, immunotherapy, and microbiome modulation. These applications not only improve the precision and efficacy of treatments but also offer innovative solutions to overcome the limitations of conventional therapeutic approaches. However, despite their considerable potential, the clinical translation of engineered bacteria still faces numerous challenges, such as ensuring stable in vivo colonization, controlling immunogenicity, standardizing large-scale production, and establishing robust regulatory and ethical frameworks. This review summarizes engineering strategies aimed at enhancing the safety and efficacy of bacterial therapies, with the goal of optimizing bacterial functions and expanding their potential in diagnostics and precision medicine.},
}
@article {pmid42295254,
year = {2026},
author = {Griffiths-Sanhueza, C and Trujillo, MG and Moraga, F and Urrutia, &#xcd;M and Ramirez-Araya, S and Bastías, R and Olivares-Pacheco, J and García, K and Plaza, N},
title = {Draft genome sequence of Psychrobacter sp. strain TPE 89C isolated from Antarctic seawater.},
journal = {Microbiology resource announcements},
volume = {},
number = {},
pages = {e0024126},
doi = {10.1128/mra.00241-26},
pmid = {42295254},
issn = {2576-098X},
abstract = {We report the draft genome sequence of Psychrobacter sp. strain TPE 89C, isolated from Antarctic seawater (25 m depth). The 3.41-Mbp assembly (42.4% GC, 484× coverage) contains a putative Type I-F CRISPR-Cas system with 86 spacers. This resource supports future research on microbial defense and adaptation in extreme environments.},
}
@article {pmid42295496,
year = {2026},
author = {Mohmad, A and V, MH and Malla, BA and Malla, WA and Rather, MM},
title = {Theileria annulata diagnostics: past, present and future.},
journal = {Tropical animal health and production},
volume = {58},
number = {5},
pages = {},
pmid = {42295496},
issn = {1573-7438},
mesh = {Animals ; *Theileriasis/diagnosis/parasitology ; *Theileria annulata/isolation &amp; purification/genetics ; Cattle ; Sensitivity and Specificity ; },
abstract = {Bovine tropical theileriosis, caused by Theileria annulata, poses a significant threat to livestock health and productivity, particularly in endemic regions. Early and accurate diagnosis is crucial for effective disease management and control. Molecular techniques, such as conventional PCR (cPCR), reverse line blot (RLB) and real-time PCR (qPCR), offer high sensitivity and specificity but are limited by cost, technical expertise, and the requirement for specialized laboratory infrastructure. Multiplex PCR (mPCR), a variant of standard cPCR, offers the advantage of simultaneously detecting two or more pathogens by specific amplification of the target loci in a single reaction. The assay uses a locus specific primer combination, but primer optimization is essential to minimize inter-primer competition and non-specific amplifications, restrict assay for large scale epidemiological studies. Sherlock assay is highly sensitive and specific, but having risk of cross- contamination, necessitating stringent controls. Serological assays, including ELISA and lateral flow-based immunoassays, provide cost-effective and rapid alternatives; however, variability in sensitivity, cross-reactivity, and reliance on antigen quality remain challenges. Future advancements should prioritize on developing highly specific, field-deployable and cost-efficient diagnostic platforms, such as synthetic peptides based various T. annulata immunodominant antigens and CRISPR- Cas based nucleic acid detection systems. Serologically, developing lateral flow assay (LFA) including in-silico predicted immunodominant peptides may yield next-generation pen-side test to detect T. annulata infection in field settings. Exploring these developments is essential for improving the early detection and control of tropical bovine theileriosis. Hence, the current overview provides a comprehensive analysis of current molecular and serological diagnostic approaches and next generation diagnostics for bovine tropical theileriosis.},
}
@article {pmid42295939,
year = {2026},
author = {Zhuang, T and Long, Y and Xu, Y and Li, Q and Liu, Z and Zhong, X and Wu, D and Ma, Y and Su, W and Li, X and Ke, Z and Guo, C},
title = {A CRISPR/Cas13a-Based One-Step System for Rapid Detection of Emerging Viruses: Deployment during the Chikungunya Outbreak in Guangdong Province, China.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00452},
pmid = {42295939},
issn = {2379-3694},
abstract = {Early and accurate diagnosis of Chikungunya virus (CHIKV) infection is critical for controlling its outbreaks. CRISPR/Cas-based detection offers promise for pathogen identification, yet one-step CRISPR/Cas systems are limited by suboptimal sensitivity, field-deployability, and adaptability to complex clinical samples, hindering their use in rapid outbreak response. Here, we developed a CRISPR/Cas13a-based One-Step System for Rapid Detection of Emerging Viruses (CRISPR-CORE) and applied it during the CHIKV outbreak in Guangdong Province, China. Multidimensional optimizations enabled the CRISPR-CORE system to achieve a limit of detection of 5 copies/μL within 40 min. An extraction-free RNA release protocol for CHIKV in blood samples and a premixed reagent approach were implemented. Furthermore, a portable fluorescence detector was used to enhance user-friendliness in point-of-care (POC) settings. The clinical CHIKV genomic information was identified through hybrid capture sequencing, informing the design of highly specific CRISPR RNA (crRNA). Clinical validation across three regions yielded 92.6% sensitivity and 100% specificity, underscoring the applicability and reliability of the CRISPR-CORE system. Our system demonstrates its suitability for CHIKV outbreak detection. It facilitates rapid and POC testing for emerging viruses in resource-limited settings. Furthermore, it provides a universal strategy for the prevention and control of infectious diseases.},
}
@article {pmid42296013,
year = {2026},
author = {Al-Kubaisy, SH and Hussein, RA and Al-Ouqaili, MTS},
title = {Representative Targeted Molecular And Genomic Characterization Of Virulence Genes In Staphylococcus aureus From Diabetic Foot Infections.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {231},
pages = {},
doi = {10.3791/71373},
pmid = {42296013},
issn = {1940-087X},
mesh = {*Diabetic Foot/microbiology ; Humans ; *Staphylococcal Infections/microbiology ; *Staphylococcus aureus/genetics/pathogenicity/drug effects/isolation &amp; purification ; Cross-Sectional Studies ; Microbial Sensitivity Tests ; Methicillin-Resistant Staphylococcus aureus/genetics/pathogenicity ; Virulence/genetics ; *Virulence Factors/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Whole Genome Sequencing/methods ; },
abstract = {Diabetic foot infections (DFIs) represent a major public health concern, and methicillin-resistant Staphylococcus aureus (MRSA) is among the most clinically significant pathogens. This study investigated the prevalence of virulence genes (cna and hlg), antimicrobial resistance profiles, and representative genomic features of multidrug-resistant S. aureus isolates recovered from patients with DFIs. A cross-sectional observational study was conducted on 125 patients with diabetic foot infections between January and December 2024. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method and cefoxitin screening according to Clinical and Laboratory Standards Institute (CLSI) guidelines, with S. aureus ATCC 25923 used as the quality-control strain. Vancomycin susceptibility was confirmed by minimum inhibitory concentration (MIC) testing. Polymerase chain reaction (PCR) was used to detect the virulence genes (cna and hlg) and blaOXA-group I genes. Whole-genome sequencing (WGS) was performed on two representative isolates, including one multidrug-resistant (MDR) isolate and one extensively drug-resistant (XDR) isolate, using a de novo sequencing approach to generate draft genome assemblies. Among 125 clinical specimens, bacterial growth was observed in 90 samples (72%), of which 45 isolates (50%) were identified as S. aureus. Among these isolates, 35/45 (77.78%) were classified as MRSA, and 36/45 (80%) were multidrug resistant. The hlg gene was detected in all isolates, whereas the cna gene was identified in 13/45 (28.89%) isolates. No blaOXA-group I genes were detected. Genomic analysis identified multiple resistance-associated genes, including blaZ, tet(38), norA, and vanT, together with CRISPR-Cas elements and plasmid-associated resistance determinants. These findings highlight the high prevalence of multidrug-resistant S. aureus in DFIs and support the importance of continued genomic surveillance of clinically relevant resistant strains.},
}
@article {pmid42296184,
year = {2026},
author = {Zhang, L and Zhang, Y and Chi, Y and Ren, Y and Zhao, Q and Zhao, D},
title = {Lentiviral SgRNA Delivery for CRISPR/Cas9 Editing in Cre-Dependent Cas9 Knock-in Primary Mouse Hepatocytes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {231},
pages = {},
doi = {10.3791/70727},
pmid = {42296184},
issn = {1940-087X},
mesh = {Animals ; *Hepatocytes/cytology/metabolism/physiology ; *Lentivirus/genetics ; Mice ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/administration &amp; dosage ; *Integrases/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice, Transgenic ; *Gene Editing/methods ; },
abstract = {Over the past few decades, CRISPR/Cas9-mediated genome editing has transformed functional studies in cell line models by making genetic manipulation highly efficient and feasible. However, extending this technology to primary hepatocytes remains a major challenge. While cultured primary hepatocytes are indispensable for disease modeling and drug development as they retain key metabolic functions absent in cell lines, their limited in vitro lifespan and negligible proliferative capacity pose fundamental barriers to efficient genome editing. Here, we describe an effective two-step perfusion protocol that enables the isolation of primary hepatocytes with high cell viability (>90%) and high yield (approximately 1 × 10[7] hepatocytes per adult mouse). Following isolation, lentiviral sgRNA transduction is typically performed within 3-4 h, and genome editing outcomes are assessed 5-7 days post-infection. Using hepatocytes from transgenic LSL-Cas9-EGFP mice, in which the Cas9 cassette was activated by a lentiviral vector co-expressing Cre recombinase and sgRNA, achieving up to 80% allele-level gene knockout efficiency in monolayer cultures. In addition, we achieved approximately 12% gene KO efficiency in three-dimensional hepatocyte organoids (HEOs), which more closely recapitulate the architecture and functional characteristics of native liver tissue. In this protocol, a successful experiment is defined by three criteria: sufficient hepatocyte yield (>5 × 10[6] cells per mouse) with high viability (>85%), efficient single-guide RNA (sgRNA) delivery into hepatocytes, and validated target gene disruption at the genomic level. This protocol demonstrates the feasibility of efficient in vitro genome manipulation in both monolayer-cultured hepatocytes and HEOs, providing a robust platform for genetic modeling and functional studies of liver diseases.},
}
@article {pmid42296196,
year = {2026},
author = {Cheng, L and Lu, X and Sun, X and Wen, Z and Zhang, X and Miao, L and Wang, P},
title = {CRISPR/Cas9-mediated Endogenous Fluorescent Tagging of Germline-specific Genes in Caenorhabditis elegans.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {231},
pages = {},
doi = {10.3791/70879},
pmid = {42296196},
issn = {1940-087X},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; *Germ Cells/metabolism/physiology ; *Gene Knock-In Techniques/methods ; },
abstract = {Protein localization in the Caenorhabditis elegans (C. elegans) germline is central to interpreting gene function during gametogenesis, yet conventional transgene approaches often yield variable expression and can be silenced in germ cells. Here, a practical CRISPR/Cas9 workflow inserting a fluorescent tag into an endogenous locus is described, enabling the generation of stable knock-in alleles that report protein distribution under native regulation. The protocol covers key stages of the procedure: selecting a tagging strategy appropriate for the target protein, delivering CRISPR reagents by gonadal microinjection into young adult hermaphrodites, and recovering injected animals for screening. Knock-in candidates are identified through PCR-based genotyping across two generations to isolate homozygous worms and verify the edited allele. Finally, confocal microscopy is used to verify germline fluorescence and assess subcellular localization in vivo. The workflow is designed to be reproducible and broadly applicable to germline-enriched genes, providing a straightforward route to establish homozygous tagged strains for developmental and cell-biological analyses.},
}
@article {pmid42297998,
year = {2026},
author = {Mishra, MK and Pamu, S and Guptha, PM and Vanangamudi, M and Mittapalli, SK and Dash, PP and Thakor, V and Yanadaiah, P and Patyar, S},
title = {Integrating HTS and CRISPR/Cas for next-generation nucleic and non-nucleic acid diagnostics.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {42297998},
issn = {1617-4623},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; Gene Editing/methods ; *High-Throughput Screening Assays/methods ; },
abstract = {The synergy between HTS and CRISPR/Cas is changing how genes, biomarkers, and diseases are studied. Very useful due to the ability to scan entire molecular libraries in a single assay and its extreme rapidity. CRISPR/Cas systems, however, are crucial for achieving control and specificity, properties essential for precise genetic editing and targeted detection. HTS could be combined with CRISPR in two ways: HTS would expand the search space, and CRISPR would narrow it. This perspective highlights recent advances in which both platforms have been used together - for example, to find genetic variants and molecular markers in cancer, infectious diseases, and even biosensors to track the environment and metabolism. We discuss technical advances as well as practical issues that make the use of CRISPR/Cas more challenging in the clinical environment, including off-target activity, reproducibility, and the increasing complexity and dimensionality of data.},
}
@article {pmid42298089,
year = {2026},
author = {Alshorman, J and Mehran, MJ and Miyanda Tembo, K and Mostafavi, N and Bolideei, M and Wang, Y},
title = {Applications of genome editing technologies in the treatment of human diseases.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42298089},
issn = {1476-5462},
abstract = {Genome editing has progressed from a laboratory capability for targeted DNA manipulation to a clinically relevant strategy for correcting, silencing, or regulating genes implicated in human disease. In this Review, we synthesize the mechanisms, capabilities, and constraints of the principal programmable platforms-zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas systems-and highlight how base editors, prime editors, and epigenetic editors expand the range of achievable outcomes beyond double-strand break-dependent repair to precise nucleotide substitutions, small insertions/deletions, and transcriptional modulation. We compare genome-editing cargo formats, including plasmid DNA, viral-vector DNA, mRNA, guide RNA, and ribonucleoprotein complexes, together with the delivery modalities used to transport them, including AAV, adenoviral and herpesviral vectors, lipid nanoparticles (LNPs), electroporation, and virus-like particles. We then consolidate key biomedical applications enabled by these technologies, spanning endogenous gene tagging, high-throughput functional variant screening, molecular recording, and the generation of genetically faithful disease models. Across oncology, respiratory, hematologic, cardiovascular, metabolic, neurodegenerative, viral, ocular, and immune disorders, genome editing is advancing both ex vivo and in vivo interventions, including engineered cellular immunotherapies, hematopoietic stem and progenitor cell editing for hemoglobinopathies, and emerging liver-directed programs for lipid and coagulation targets. Finally, we discuss priorities for broad clinical implementation: improving editing fidelity and PAM flexibility, increasing performance in non-dividing cells, enabling tissue-selective delivery to difficult organs (for example, lung and central nervous system), and addressing manufacturing scalability, long-term monitoring, and equitable global access.},
}
@article {pmid42298143,
year = {2026},
author = {Uddin, MM and Khan, SMZA},
title = {A modality-aware CRISPR actionability framework for functional prioritization of genome-wide significant type 2 diabetes loci.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {42298143},
issn = {1617-4623},
mesh = {*Diabetes Mellitus, Type 2/genetics ; *Genome-Wide Association Study/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Transcription Factor 7-Like 2 Protein/genetics ; *Gene Editing/methods ; Kcnj11 Channel ; Potassium Channels, Inwardly Rectifying/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Islets of Langerhans/metabolism ; Genetic Loci ; },
abstract = {Genome-wide association studies (GWAS) have identified numerous loci associated with Type 2 Diabetes (T2D), yet translating statistical signals into experimentally testable hypotheses remains a central challenge in post-GWAS biology. The predominance of non-coding regulatory variants complicates target gene assignment and raises uncertainty regarding optimal clustered regularly interspaced short palindromic repeats (CRISPR) perturbation strategy. Here, we present a structured CRISPR Actionability Framework that integrates genomic context, pancreatic islet enhancer overlap, tissue-specific expression validation, and locus clarity into a quantitative CRISPR Actionability Score (CAS). We applied this framework to ten genome-wide significant T2D loci and assigned modality-aware CRISPR strategies (knockout versus CRISPR interference). CAS values ranged from 4 to 10, enabling tiered prioritization into high, moderate, and lower experimental priority classes. High-priority loci included SLC30A8, TCF7L2, and KCNJ11, which demonstrated strong regulatory or coding evidence combined with islet expression support. By explicitly linking genomic architecture to perturbation modality, this framework provides a transparent and reproducible bridge between statistical genetics and functional genome editing. This approach establishes a scalable template for rational CRISPR target selection in complex disease research.},
}
@article {pmid42298891,
year = {2026},
author = {Ding, Z and Wei, Y and Han, Y and Gu, P},
title = {Inducible CRISPR/Cas systems in precision oncology: Current applications and future perspectives.},
journal = {Clinical and translational medicine},
volume = {16},
number = {6},
pages = {e70720},
pmid = {42298891},
issn = {2001-1326},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods/trends ; *Neoplasms/therapy/genetics ; *Gene Editing/methods/trends ; Animals ; *Medical Oncology/methods/trends ; },
abstract = {BACKGROUND: Inducible CRISPR/Cas systems enable spatiotemporal control of genome editing in response to chemical, optical, biological, or physical stimuli. By restricting genome-editing activity to defined conditions, these systems may reduce off-target exposure and immune burden while improving tumor-selective control, making them attractive tools for precision oncology.<br><br>MAIN BODY: This review summarizes the molecular mechanisms, design principles, and current applications of inducible CRISPR/Cas systems in cancer research and therapy. These platforms are classified into chemically inducible, optogenetic, tumor microenvironment-responsive, physically triggered, and logic-gated systems. Regulatory strategies are discussed at multiple levels, including transcriptional control, post-translational regulation, guide RNA engineering, and stimulus-responsive delivery. Key applications include functional genomic screening, cancer modeling, therapeutic gene editing, immunotherapy enhancement, and combinatorial treatment strategies. We also examine current delivery approaches, including viral vectors, lipid nanoparticles, stimulus-responsive nanocarriers, and biomimetic platforms.<br><br>CONCLUSION: Inducible CRISPR/Cas systems represent a promising platform for next-generation precision cancer therapy. However, substantial optimization and rigorous preclinical validation remain necessary to address challenges related to leaky expression, induction efficiency, tissue penetration, immunogenicity, and long-term safety before clinical translation can be realized.<br><br>KEY POINTS: Inducible CRISPR/Cas systems enable conditional genome editing in precision oncology. Chemical, optical, TME-responsive, physical, and logic-gated systems offer distinct control features. Delivery, leakiness, immunogenicity, and safety remain key translational barriers. Ex vivo immune-cell engineering and locoregional delivery may offer nearer-term clinical routes.},
}
@article {pmid42299600,
year = {2026},
author = {Liao, J and Teng, L and Hui, C and Liu, X and Huang, J and Li, Y and Qin, H and Song, Y},
title = {Development of a one-pot RPA-CRISPR/Cas12a assay for rapid multiplex detection of respiratory pathogens.},
journal = {Medicine},
volume = {105},
number = {24},
pages = {e49303},
pmid = {42299600},
issn = {1536-5964},
support = {2024-MS-292//Liaoning Provincial Natural Science Foundation/ ; },
mesh = {Humans ; *Respiratory Tract Infections/diagnosis/microbiology/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Influenza B virus/isolation &amp; purification/genetics ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; Influenza A virus/isolation &amp; purification/genetics ; Mycoplasma pneumoniae/isolation &amp; purification ; Reproducibility of Results ; Recombinases ; Klebsiella pneumoniae/isolation &amp; purification ; },
abstract = {Respiratory tract infections impose a substantial clinical and public health burden, and timely etiological identification remains challenging. Rapid panel testing is therefore needed for common respiratory pathogens. In this study, we developed a closed-tube, one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay using a multichamber 8-strip tube format to enable parallel fluorescence readout while reducing the risk of aerosol contamination. The assay targets 9 pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae. Analytical performance, including limit of detection, specificity, and repeatability, was evaluated using viral recombinant plasmids and bacterial genomic DNA. Clinical evaluation was performed using available respiratory specimens and bacterial isolates, with reverse-transcription quantitative polymerase chain reaction for viral targets and conventional bacterial identification for bacterial targets as reference methods. The assay provided results within approximately 50 to 60 minutes. The limits of detection for viral targets were 1 copy/μL for influenza A virus and adenovirus and 2 copies/μL for influenza B virus and respiratory syncytial virus. No cross-reactivity was observed under the tested conditions. Repeatability was satisfactory, with coefficients of variation below 15% across targets. In a clinical evaluation of 34 specimens or isolates, the one-pot assay showed 100% overall agreement with the reference methods. These findings indicate that the closed-tube, one-pot RPA-CRISPR/Cas12a platform enables rapid and parallel detection of common respiratory pathogens and may support point-of-care testing and deployment in resource-limited settings.},
}
@article {pmid42299924,
year = {2026},
author = {Chen, H and Li, Q and Mao, X},
title = {A novel CRISPR/Cas14a-synergized wood-based platform for the ultrasensitive detection of aflatoxin B1.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {22},
pages = {4584-4588},
doi = {10.1039/d6ay00717a},
pmid = {42299924},
issn = {1759-9679},
mesh = {*Aflatoxin B1/analysis ; *CRISPR-Cas Systems/genetics ; *Wood/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; },
abstract = {A CRISPR/Cas14a-synergized wood-based platform (CWP) is designed here to detect aflatoxin B1 (AFB1) at an LOD of 0.67 fmol L[-1] with high sensitivity and no preamplification. Once the assay is performed, AFB1 is optically identified through an aptamer competition process, which triggers the trans-cleavage activity of Cas14a, resulting in significant color changes on the wood surface upon introducing chromogenic substrates. This method can be conveniently extended to the detection of disease-related protein biomarkers with low detection thresholds.},
}
@article {pmid42301653,
year = {2026},
author = {Almeida, EA and Arora, M and Mehndiratta, M and Agarwal, R},
title = {CRISPR/Cas in gynecologic cancers: A review of experimental and therapeutic applications.},
journal = {Indian journal of cancer},
volume = {63},
number = {1},
pages = {1-8},
doi = {10.4103/ijc.ijc_14_25},
pmid = {42301653},
issn = {1998-4774},
mesh = {Humans ; Female ; *Genital Neoplasms, Female/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {Gynecological malignancies-including cervical, ovarian, and endometrial cancers-remain a major global health challenge, contributing significantly to cancer-related morbidity and mortality among women. Despite advances in conventional treatments such as surgery, chemotherapy, radiotherapy, and immunotherapy, issues such as drug resistance, tumor recurrence, and limited efficacy in advanced-stage disease necessitate novel therapeutic strategies. The emergence of CRISPR/Cas-based genome editing has revolutionized cancer research by enabling precise, efficient, and programmable modifications of specific genomic loci. In gynecologic oncology, CRISPR/Cas systems have been employed to dissect oncogenic mechanisms, identify therapeutic targets, and develop innovative treatment modalities. In cervical cancer, CRISPR-mediated targeting of HPV E6 and E7 oncogenes has shown potential in restoring tumor suppressor pathways and enhancing chemosensitivity. In ovarian cancer, gene editing has been used to modulate chemoresistance, tumor angiogenesis, and metastasis through the knockout of key regulators such as DNMT1, EGFL6, and BRCA1/2. Similarly, in endometrial cancer, CRISPR tools have elucidated mechanisms of hormonal resistance and facilitated the development of in vivo models via somatic gene editing. This review highlights recent advances in the application of CRISPR/Cas technology to gynecologic malignancies, discussing its potential as both a therapeutic and research platform while acknowledging current limitations and translational hurdles.},
}
@article {pmid42301915,
year = {2026},
author = {Kunwar, S and Hallmark, T and Manna, S and Keiser, D and Naegle, B and Thomas, A and Beisel, CL and Jackson, RN},
title = {Target RNA-triggered CRISPR-Cas12a2 preferentially cleaves collateral DNA over RNA.},
journal = {Nucleic acids research},
volume = {54},
number = {11},
pages = {},
pmid = {42301915},
issn = {1362-4962},
support = {/NH/NIH HHS/United States ; R35GM138080/GM/NIGMS NIH HHS/United States ; 101158249//European Research Council Proof-of-Concept award/ ; //Helmholtz Center for Infection Research (HZI) Singh-Chhatwal-Postdoctoral Fellowship/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *DNA Cleavage ; *CRISPR-Cas Systems ; *RNA/metabolism/chemistry ; *DNA/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; DNA, Single-Stranded/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry ; Kinetics ; },
abstract = {CRISPR-Cas systems often rely on collateral cleavage of nucleic-acid substrates to combat recognized mobile genetic elements. Of the CRISPR-associated (Cas) RNA-guided effector nucleases, Cas12a2 stands out as the only known example exhibiting rapid collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA, after activating upon binding cognate RNA. However, little is known about the underlying mechanisms of collateral cleavage. Here, we show, using enzyme kinetics and inhibition assays, that Cas12a2 preferentially cleaves collateral DNA over RNA substrates, even when RNA substrates are more abundant. Additionally, using enzyme mutants, enzyme kinetics, and plasmid cleavage assays, we determine that the dsDNA cleavage mechanism relies on the 'aromatic clamp' residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can readily cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while an RNA-targeting Cas13a cannot. This work provides foundational kinetic and biochemical insights into the collateral cleavage mechanism and substrate preferences of Cas12a2, with immediate implications for understanding Cas12a2-based immunity and developing Cas12a2-based technologies.},
}
@article {pmid42303719,
year = {2026},
author = {Zhang, R and Bai, PJ and Jiang, W and Liu, X and Zhang, S and Wang, Z and Shao, L},
title = {Mouse model of X-linked Alport syndrome with K229X mutation in the COL4A5 gene.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-56834-6},
pmid = {42303719},
issn = {2045-2322},
abstract = {X-linked Alport syndrome (XLAS) is a hereditary glomerular basement membrane (GBM) disease caused by COL4A5 mutations, leading to end-stage renal disease. With unclear pathogenesis and limited treatments, reliable animal models are urgently needed. In this study, the mutation K229X in COL4A5 detected in XLAS patients was introduced into mice model by CRISPR/Cas. The clinical manifestations and pathological changes in the K229X mice were characterized through urinary and serum tests, histopathology, immunofluorescence, and transmission electron microscopy. In K229X male mice, we observed significant hematuria and proteinuria, along with azotemia, and noted a marked decrease in the expression of COL4A5 at both the mRNA and protein levels within the kidneys. Pathological examination revealed glomerulosclerosis, increased mononuclear cells in the renal interstitium, interstitial fibrosis, and absence of α5 collagen IV, with histological abnormalities in the glomeruli, renal tubules, and interstitium progressing with age. Electron microscopy found irregular thickening of the GBM, accompanied by irregular layering. The phenotypic and pathological features of this mouse model are consistent with those observed in XLAS patients and other previously established mouse models. This K229X mouse model is of significant importance for exploring the pathogenic mechanisms of XLAS and researching potential therapeutic approaches.},
}
@article {pmid42305150,
year = {2026},
author = {Gherbawy, YA and Al-Harthi, H and El-Dawy, E and Gaber, M and Pet, I and Wang, L and Hu, P and Hussein, M},
title = {The molecular revolution in fungal diagnostics: bridging gaps across clinical, agricultural, and environmental mycology.},
journal = {Mycology},
volume = {17},
number = {2},
pages = {350-377},
pmid = {42305150},
issn = {2150-1203},
abstract = {Fungi play essential roles in human health, agriculture, and ecosystems, yet their diversity has long been underestimated due to reliance on morphology and culture. Molecular innovations-DNA barcoding, multilocus and whole-genome sequencing, NGS, and rapid nucleic acid diagnostics (qPCR, LAMP, CRISPR/Cas)-have revolutionized fungal taxonomy and detection. The ITS region now serves as a universal barcode, often complemented by other loci or genomic data. High-throughput and portable tools enable near real-time identification, supported by AI-driven bioinformatics for species recognition and resistance prediction. Despite progress, challenges remain in database accuracy, primer design, and standardization. Integrating molecular taxonomy, AI, and global collaboration promises scalable, reliable frameworks for fungal surveillance and control across clinical, agricultural, and environmental domains.},
}
@article {pmid42305815,
year = {2026},
author = {Goberna, MF and Lezcano, EG and Castro Alegría, A and Nara Pereira, E and Quintana, SA and Arrúa, AA and Fernández Ríos, D},
title = {Genome editing between wonder and rejection.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1832039},
pmid = {42305815},
issn = {2296-4185},
}
@article {pmid42307050,
year = {2026},
author = {Cao, L and Zhao, L and Zhou, Y and Ye, Q and Tan, WS},
title = {Attenuation of Triple Selection Markers to Enhance mAb Expression in Tyrosine-Based Selection Systems via an Inducible CRISPRi Platform.},
journal = {Biotechnology journal},
volume = {21},
number = {6},
pages = {e70265},
doi = {10.1002/biot.70265},
pmid = {42307050},
issn = {1860-7314},
mesh = {Animals ; *Antibodies, Monoclonal/genetics/metabolism/biosynthesis ; CHO Cells ; Cricetulus ; *Tyrosine/metabolism ; *CRISPR-Cas Systems/genetics ; Doxycycline/pharmacology ; Promoter Regions, Genetic ; },
abstract = {Chinese hamster ovary (CHO) cells are the predominant host for monoclonal antibody (mAb) production, where the choice of selection system is critical. While metabolic selection systems are favored over antibiotic-based ones to avoid purification and safety issues, traditional single-marker systems poorly regulate the light-to-heavy chain (LC/HC) ratio, which is key for yield. Although novel multi-marker systems can address this, they often lack strategies to enhance expression. To overcome these limitations, our study employed a triple-marker system based on the tyrosine biosynthetic pathway to regulate the LC/HC ratio and eliminate the need for alkaline concentrated tyrosine feeding. Furthermore, we introduced an inducible CRISPR interference (iCRISPRi) platform, which integrates SV40 promoter-targeting CRISPRi with a Tet-on system, to enhance its efficiency. This strategy upregulated three fluorescent reporter genes, resulting in a selected mini-pool with 84.84% higher cell-specific productivity and 87.50% higher final titer in tyrosine-free perfusion culture. Mechanistically, doxycycline (Dox)-induced dCas9 expression inhibited the transcription of SV40-driven markers, enriching cells with high marker integration under tyrosine deprivation and consequently increasing HC and LC gene copy numbers to boost mAb yields. This iCRISPRi strategy serves as a potential enhancer for SV40 promoter-driven systems, offering novel insights for optimizing metabolic selection.},
}
@article {pmid42307052,
year = {2026},
author = {Li, X and Wu, S and Tian, P},
title = {Base Editors for Engineering Industrial Microorganisms: Types, Applications, and Future Perspectives.},
journal = {Biotechnology journal},
volume = {21},
number = {6},
pages = {e70266},
doi = {10.1002/biot.70266},
pmid = {42307052},
issn = {1860-7314},
support = {22278022//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Industrial Microbiology/methods ; Bacteria/genetics ; *Metabolic Engineering ; },
abstract = {Base editing encompasses technologies that enable the direct conversion of one nucleotide base into another, typically by employing deaminases fused to programmable DNA- or RNA-binding scaffolds such as CRISPR-dCas9/nCas9. Deaminase-free editing strategies are also emerging as promising alternatives. Unlike conventional CRISPR-Cas9 systems that rely on DNA double-strand breaks (DSBs) and subsequent homology-directed repair or non-homologous end joining (NHEJ), base editors operate independently of these pathways, thus enabling continuous in vivo genome evolution and phenotypic diversification. While previous reviews have summarized base editing in plants and animals, the present review focuses specifically on base editors tailored for industrial microorganisms. We provide a comprehensive overview of deaminase-dependent and glycosylase-dependent base editors, emphasizing their applications in sculpting microbial genomes, redirecting metabolic flux, and enhancing stress tolerance. In addition, we summarize recent advances in in situ bacterial base editing, an emerging frontier for industrial strain improvement. Finally, we dissect the current limitations of these tools and propose actionable strategies to enhance editing performance and broaden their applicability. By integrating these perspectives, this review aims to guide future development and deployment of base editing technologies in both model and non-model industrial microorganisms, ultimately advancing their roles in biomanufacturing, biomonitoring, and beyond.},
}
@article {pmid42307111,
year = {2026},
author = {Hill, E and Raban, R and Akbari, OS},
title = {Beyond pesticides: next-generation genetic biocontrol technologies for sustainable population suppression of agricultural insect pests.},
journal = {Fly},
volume = {20},
number = {1},
pages = {2682509},
doi = {10.1080/19336934.2026.2682509},
pmid = {42307111},
issn = {1933-6942},
mesh = {Animals ; *Pest Control, Biological/methods ; *Insecta/genetics ; CRISPR-Cas Systems ; Gene Drive Technology ; Agriculture ; Male ; },
abstract = {Chemical insecticides have long been used to control agricultural pests, but their widespread application has driven resistance and caused significant ecological and health impacts. CRISPR/Cas9-based genetic biocontrol technologies, including the precision-guided sterile insect technique (pgSIT) and homing gene drives (HGDs), offer targeted alternatives for suppressing pest populations with reduced environmental cost. pgSIT produces sterile males without radiation and achieves high mating competitiveness without multigenerational persistence. In contrast, HGDs bias inheritance to enable sustained population suppression through disruption of essential fertility or viability genes, albeit with greater ecological and regulatory considerations. Experimental applications in multiple agricultural pest species demonstrate robust suppression efficacy. Emerging innovations, including temperature-inducible pgSIT systems, may further streamline mass-rearing and deployment. Together, these approaches have the potential to reduce crop losses and reliance on chemical insecticides while lowering long-term management costs. Their successful integration into agricultural systems will depend on rigorous risk assessment, regulatory oversight, and stakeholder engagement.},
}
@article {pmid42307461,
year = {2026},
author = {Solouki, K and Sohail, M},
title = {Nanomaterials for Subcellular Organelle Targeting: Unlocking New Avenues for Enhanced Therapeutic Effectiveness.},
journal = {Journal of drug targeting},
volume = {},
number = {},
pages = {1-51},
doi = {10.1080/1061186X.2026.2691784},
pmid = {42307461},
issn = {1029-2330},
abstract = {Subcellular organelle targeting is changing the way nanomedicine is designed, moving the field beyond simple cellular entry toward more precise intracellular localization, controlled cargo release, and functional activity within disease-relevant compartments. This review critically discusses nanomaterial-based strategies for targeting the nucleus, mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, and cytoskeleton-associated trafficking pathways. Its main novelty is the use of a cross-organelle, mechanism-based framework that links nanocarrier physicochemical properties with intracellular transport biology, rather than examining each organelle or delivery platform separately. Lipid nanoparticles, polymeric carriers, dendrimers, inorganic nanomaterials, biomimetic systems, and engineered extracellular vesicles are compared according to their targeting mechanisms, cargo compatibility, therapeutic potential, and translational limitations. Particular attention is given to nuclear import mediated by NLS-, CPP/TAT-, and aptamer-based strategies; mitochondrial delivery shaped by membrane potential, membrane fusion, and redox-responsive release; lysosomal targeting for pH- and enzyme-activated therapies; and ER/Golgi-directed delivery through retrograde trafficking, retention motifs, and modulation of stress-related pathways. The review also brings together several emerging directions, including stimuli-responsive release, biomimetic surface engineering, extracellular vesicle scalability, CRISPR/Cas delivery, base and prime editing, and targeted protein degradation, all of which may support more programmable forms of intracellular therapy. Importantly, it separates true organelle localization from transient trafficking or nonspecific perinuclear accumulation, emphasizing the need for stronger and more reliable validation methods. Key barriers remain, including inefficient endosomal escape, off-target intracellular accumulation, organelle-specific toxicity, long-term safety concerns, reproducibility, scalable manufacturing, and regulatory classification. Overall, this review frames organelle-directed nanomedicine as a rational design strategy for improving therapeutic precision, while also stressing that clinical translation will depend on clear evidence of durable, safe, and measurable therapeutic benefit at the organelle level.},
}
@article {pmid42308031,
year = {2026},
author = {Doenier, J and Tolleter, D and Frail, S and Finazzi, G and Burlacot, A and Yeh, E},
title = {A genome-wide CRISPR screen reveals how diatoms thrive in dynamic light.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {25},
pages = {e2532811123},
doi = {10.1073/pnas.2532811123},
pmid = {42308031},
issn = {1091-6490},
support = {na//Burroughs Wellcome Fund (BWF)/ ; na//Carnegie Institution for Science (CIS)/ ; DE-SC0019417//DOE | Advanced Research Projects Agency - Energy (ARPA-E)/ ; na//Chan Zuckerberg Initiative (CZI)/ ; 833184//EC | European Research Council (ERC)/ ; },
mesh = {*Diatoms/genetics/physiology/radiation effects ; *Light ; Photosynthesis/genetics ; *CRISPR-Cas Systems ; Genome ; },
abstract = {Diatoms are a highly diverse algal group with outsized impact on global primary production and marine carbon sequestration. They are red lineage phototrophs of complex endosymbiotic origin and therefore evolutionarily divergent from plants and other green lineage phototrophs that typically serve as photosynthesis models. To accelerate the discovery of unique diatom biology, we developed a genome-wide CRISPR/Cas9 screen in the marine diatom, Phaeodactylum tricornutum. Dynamic light conditions are common in nutrient-rich, well-mixed marine environments in which diatoms thrive. The P. tricornutum mutant library was grown in different light regimes, including both high light and fluctuating light. We identified a broad set of genes required for survival specifically in dynamic light, including effectors of cyclic electron flow (CEF) and enzymes catalyzing posttranslational modifications of Calvin cycle enzymes. Among genes of unknown function identified, we demonstrated that the red lineage-exclusive gene STROBE1 is a CEF potentiator required for CEF-dependent generation of a trans-thylakoid proton gradient. STROBE1 and other genes identified in this screen reveal unexpected mechanisms underlying the adaptation of diatoms to dynamic light environments. This genome-wide genetic screen in P. tricornutum will accelerate the unbiased discovery of novel gene functions in these ecologically important organisms.},
}
@article {pmid42309797,
year = {2026},
author = {Liu, T and Jiang, HJ and Wang, XL and Yu, YY and Wang, F and Lin, JJ and Yang, HQ},
title = {A novel strategy to enhance precise targeting of the RNA base editor mxABE.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {6},
pages = {628-637},
doi = {10.16288/j.yczz.25-303},
pmid = {42309797},
issn = {0253-9772},
mesh = {Animals ; *RNA Editing ; Humans ; Mice ; *Muscular Dystrophy, Duchenne/therapy/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {Single nucleotide variations (SNVs) represent the most common form of pathogenic mutations in humans, while base editing technology offers an ideal solution for treating such pathogenic variants. RNA editing has become a hotspot in current gene therapy due to its reversible action, which avoids long-term risks by not permanently altering the genome, and the compact size of the editors. Among these, the mini-dCas13X.1-mediated RNA adenine base editing (mxABE) system demonstrates highly efficient RNA base editing; however, it still suffers from non-target nucleotide editing caused by the bystander editing effect, which constitutes a major off-target risk. In this study, by deleting the nucleotide opposite the non-target adenosine in the sgRNA sequence, we effectively reduced the bystander editing effect of the mxABE system. In vitro results showed that this strategy successfully controlled the bystander editing rate below 5% while maintaining highly efficient on-target editing of approximately 70%. In a murine model of DMD (Duchenne muscular dystrophy), a single administration of AAV (adeno-associated virus)-delivered mxABE system demonstrated significant therapeutic efficacy in the tibialis anterior muscle, with successful elimination of off-target adenosine bystander editing. This study provides a novel precision-targeting strategy for treating monogenic genetic diseases using the mxABE RNA editing system.},
}
@article {pmid42309824,
year = {2026},
author = {Jiang, T and Guo, J and Jiao, Y and Xu, M and He, J and Liu, X and Qin, G and Liu, X and Chen, Y and Cong, P and He, Z},
title = {Cellular and molecular characterization of the testicular development of GDF9-edited pigs.},
journal = {Reproduction, fertility, and development},
volume = {38},
number = {9},
pages = {},
doi = {10.1071/RD26060},
pmid = {42309824},
issn = {1448-5990},
mesh = {Animals ; Male ; *Growth Differentiation Factor 9/genetics/metabolism ; *Testis/metabolism/growth &amp; development ; Swine ; Leydig Cells/metabolism ; Sertoli Cells/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; SOX9 Transcription Factor/metabolism ; },
abstract = {CONTEXT: Growth differentiation factor (GDF) 9 is a member of the transforming growth factor (TGF)-β superfamily, which plays an important role in mammalian ovarian follicular development and female fertility. However, its regulatory role in the male reproductive system remains largely unknown.<br><br>AIMS: This study aimed to investigate the regulatory mechanism of GDF9 in porcine testicular development.<br><br>METHODS: We established a GDF9-edited pig model through CRISPR-Cas9 technology and somatic cell nuclear transplantation. The testicular development of one edited piglet at birth and one at Day 14 were characterized via Western blotting, haematoxylin-eosin staining, and immunohistochemistry at cellular and molecular levels.<br><br>KEY RESULTS: Based on the preliminary analysis of one edited piglet at birth and one at Day 14, we did not observe changes to the gross morphological and anatomical structures of porcine testes at early developmental stages. However, the developmental process or cellular functions of Sertoli cells, Leydig cells and spermatogonia may be affected, as reflected by the altered expression patterns of SOX9, AR, DHRS9, DDX4 and ANXA2 in the testes. Noticeably, the altered expression of StAR may indicate an impact of GDF9 editing on the steroid hormone synthesis functions of porcine Leydig cells.<br><br>CONCLUSIONS: These findings indicate that GDF9 may affect porcine testicular development at the early postnatal stage.<br><br>IMPLICATIONS: These preliminary results provide a valuable foundation for future comprehensive studies toelucidate the regulatory mechanism of GDF9 in porcine testicular development.},
}
@article {pmid42312293,
year = {2026},
author = {Muhammed, A and Tripathi, J and Verma, H},
title = {Comparative genomic and network analysis of Methanocaldococcus genus reveals genetic diversity and adaptive metabolic traits.},
journal = {3 Biotech},
volume = {16},
number = {7},
pages = {276},
pmid = {42312293},
issn = {2190-572X},
abstract = {UNLABELLED: A detailed comparative genomic analysis was conducted on eight Methanocaldococcus strains, focusing on their phylogeny, metabolic pathways, adaptive traits and biotechnological potential. Functional attributes consists of conserved pathway of methane metabolism alongside variable gene clusters associated with the sulfur and nitrogen cycling. Phylogenomic analyses using Average Nucleotide Identity (ANI), Genome to Genome Distance Calculator (GGDC) and EasyCGTree revealed high genetic variability across the genus, with M. infernus ME and M. indicus SL43 being the most divergent members. CRISPR-Cas and phage analyses showed that the strains consist of a substantial number of CRISPR arrays with diverse spacer sequences. Protein-protein interaction networks were constructed on the methane, suphur and nitrogen metabolic pathway proteins, which highlighted MtrA and Mer proteins as hub-bottleneck proteins of the network. This in-silico study of the genus Methanocaldococcus provides insights that inform future experimental investigations into archaeal metabolism, evolutionary adaptation and extremophile biology.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-026-04865-1.},
}
@article {pmid42315622,
year = {2026},
author = {Peng, C and Zhang, C and Jiang, T},
title = {Validation of a Qualitative Detection Method for Influenza A Virus RNA Based on the RT-RPA-CRISPR/Cas13a System.},
journal = {Current microbiology},
volume = {83},
number = {8},
pages = {},
pmid = {42315622},
issn = {1432-0991},
support = {No. SYWD2025119//Suzhou Municipal Science and Technology Bureau/ ; },
mesh = {*Influenza A virus/genetics/isolation &amp; purification ; *RNA, Viral/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Influenza, Human/diagnosis/virology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Seasonal influenza A virus (IAV) frequently causes outbreaks, creating an urgent need for rapid, cost-effective detection. Existing methods have limitations: antigen tests lack sensitivity, RT-qPCR (the gold standard) requires bulky thermal cyclers, isothermal techniques like LAMP suffer from complex primer design and non-specific amplification, and many CRISPR-based diagnostics integrate nanotechnology or microfluidics, increasing cost and complexity without systematic clinical validation. To address these issues, we adapted RT-RPA combined with CRISPR-Cas13a into a simplified qualitative IAV detection platform. The workflow uses isothermal RT-RPA for amplification, Cas13a for specific recognition and trans-cleavage, and a lateral flow strip for visual readout. A rapid nucleic acid release reagent simplifies sample pretreatment. This approach retains the high sensitivity and specificity of nucleic acid testing while eliminating complex equipment. Preliminary performance evaluation showed that the platform produced a clear signal at 10[1] copies/mL of viral genome and exhibited no cross-reactivity with other respiratory viruses. Using RT-qPCR as the gold standard, testing of 78 clinical samples achieved 100% concordance. In summary, this study provides a practical simplification of an established RT-RPA-CRISPR/Cas13a workflow for IAV detection, supported by preliminary clinical validation, and demonstrates its suitability for rapid testing in low-resource settings.},
}
@article {pmid42318126,
year = {2026},
author = {Liu, Y and Chen, C and Zhu, H and Yang, J},
title = {From QTL mapping to genome editing: advances and integrated strategies for improving aluminum tolerance in crops.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1862097},
pmid = {42318126},
issn = {1664-462X},
abstract = {Aluminum (Al) toxicity in acidic soils remains one of the most serious constraints on global crop production, limiting productivity across nearly one-third of the world's potentially arable land. As pressure grows to cultivate marginal lands under climate change and food security challenges, improving Al tolerance has become an urgent priority in crop science and sustainable agriculture. This review provides a timely synthesis of recent advances in the genetic and molecular dissection of Al tolerance, highlighting the progression from classical biparental QTL mapping to genome-wide association studies and, more recently, CRISPR/Cas-based precision editing. Major breakthroughs, including the identification of key ALMT and MATE transporters, the expansion of STOP1-centered regulatory networks, and the discovery of the first Al receptor, have greatly deepened our understanding of plant adaptation to acid soils. We further examine how high-throughput phenotyping, marker-assisted selection, genomic selection, and gene pyramiding are accelerating the translation of genetic discoveries into breeding practice. Importantly, emerging genome-editing strategies now enable targeted and potentially transgene-free improvement of endogenous tolerance genes. By integrating molecular breeding with agronomic approaches such as liming and nutrient management, this review outlines a forward-looking framework for developing resilient crop varieties and achieving more sustainable productivity on acidic soils worldwide.},
}
@article {pmid42318429,
year = {2026},
author = {Zhou, X and Ma, T and Ding, X and Luo, Z and Yin, C and Lu, Z},
title = {The evolving landscape of gene editing therapies for human genetic diseases: a twenty-year bibliometric analysis.},
journal = {Frontiers in medicine},
volume = {13},
number = {},
pages = {1872028},
pmid = {42318429},
issn = {2296-858X},
abstract = {BACKGROUND/OBJECTIVES: Despite the transformative potential of gene editing technologies, a systematic mapping of their translational trajectory from bench to bedside remains scarce. This bibliometric analysis aims to chart the bench-to-bedside evolution, identify leading disease targets and therapeutic strategies, and uncover emerging clinical trends and challenges.<br><br>METHODS: We analyzed 1,571 peer-reviewed articles and reviews explicitly addressing gene editing therapies for human genetic diseases, published between 2005 and 2025 and retrieved from Web of Science Core Collection and Scopus. Science mapping was conducted using CiteSpace and VOSviewer to visualize collaboration networks, thematic clusters, and research fronts.<br><br>RESULTS: Publication output followed a three-phase exponential growth: engineered nucleases (2005-2012), CRISPR revolution (2013-2018), and precision translation (2019-2025). The United States and China dominated productivity; Harvard and the University of California were key institutional hubs. Hematologic disorders-particularly sickle cell disease and &#x3b2;-thalassemia-constituted the primary disease focus. Emerging frontiers include base and prime editing, epigenetic modulation, multiplex editing, and AI-assisted design. Collaboration networks remain predominantly national, with limited global integration.<br><br>CONCLUSIONS: This study delineates the rapid evolution of gene editing therapies, highlighting robust clinical translation for hematological conditions while exposing critical gaps in non-hematopoietic tissues, delivery efficiency, long-term safety, and equitable access. These findings provide a strategic roadmap to broaden the therapeutic reach of gene editing across diverse disease domains.},
}
@article {pmid42319549,
year = {2026},
author = {Garchery, C and Benejam, J and Grau, A and Gricourt, J and Pelpoir, E and Causse, M},
title = {Mammalian growth factors enhance regeneration in transgenic tomato lines.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {42319549},
issn = {1573-9368},
support = {ANR-24-PESV-0001//ANR/ ; },
mesh = {*Solanum lycopersicum/genetics/growth &amp; development/drug effects ; *Plants, Genetically Modified/genetics/growth &amp; development/drug effects ; *Regeneration/genetics ; Animals ; *Intercellular Signaling Peptides and Proteins/pharmacology/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Genome editing technologies are now available for many crop species, greatly enhancing our ability to investigate gene function and transforming the field of plant transgenesis. However, the capacity to regenerate whole plants from cell culture remains a major limiting factor in many crops. Even in species with regeneration potential, certain genotypes remain recalcitrant. The physiological state of plant cells plays a central role in growth and development and is closely associated with kinase-mediated signaling networks. Notably, several defense-related genes activated during cellular repair processes following transgenesis share significant homology with mammalian defense genes. In this study, we evaluated whether supplementation with three mammalian growth factors could enhance regeneration efficiency in tomato. We selected two cytokines and a pro-inflamatory factor showing homology with plant kinase genes. We compared the percentage of transgenic plants generated through CRISPR-Cas9-mediated mutagenesis of four genes involved in sugar and organic acid metabolism across six tomato lines exhibiting varying regeneration capacities. Over three years of transformation experiments, we demonstrated that the addition of mammalian growth factors during transgenesis significantly improved regeneration frequency, particularly in recalcitrant tomato genotypes. Furthermore, growth factor supplementation not only enhanced transformation efficiency in difficult-to-transform lines but also increased the production of stable secondary lines.},
}
@article {pmid42319574,
year = {2026},
author = {Yi Lin Lee, MC and Camargo, JA and Caetano Vilas Boas, G and Silva, KS and Silva, LP and Mioshi, CM and Pimenta, R and Silva, IA and Ramos Moreira Leite, K and Nahas, WC and Dos Reis, ST},
title = {CRISPR/Cas9-mediated miR-21 editing in high-grade urothelial carcinoma cells and its biological effects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42319574},
issn = {1573-4978},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Cell Movement/genetics ; *Urinary Bladder Neoplasms/genetics/pathology ; Gene Expression Regulation, Neoplastic/genetics ; *Gene Editing/methods ; PTEN Phosphohydrolase/genetics ; Cell Proliferation/genetics ; RNA-Binding Proteins/genetics ; Apoptosis Regulatory Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: Urothelial carcinoma, the predominant form of bladder cancer, represents a global public health challenge due to its high rates of recurrence and progression. At the molecular level, microRNA-21 (miR-21) has been characterized as an "oncomir" because of its ability to negatively regulate tumor suppressor genes, thereby promoting tumor survival and progression. In this context, the CRISPR/Cas9 system has emerged as a precise genome-editing tool.<br><br>OBJECTIVE: To investigate the biological effects of miR-21 modulation using CRISPR/Cas9-mediated genome editing in the T24 high-grade invasive urothelial carcinoma cell line.<br><br>METHODS: The CRISPR/Cas9 system was delivered as a ribonucleoprotein (RNP) complex. Editing efficiency was assessed using quantitative reverse transcription PCR (RT-qPCR). Functional effects were evaluated through gene expression assays, cell migration assays, as well as Matrigel invasion assays. The presence of the Cas9 protein was confirmed by immunofluorescence.<br><br>RESULTS: CRISPR/Cas9 treatment targeting miR-21 showed a trend toward reduced miR-21 expression (p&#x2009;=&#x2009;0.0563), although this did not reach statistical significance. A statistically significant increase in MASPIN (p&#x2009;<&#x2009;0.0001) and PDCD4 (p&#x2009;=&#x2009;0.0239), as well as a trend toward increased PTEN expression (p&#x2009;=&#x2009;0.055), was observed following treatment. Functionally, a significant reduction in the migratory capacity of edited cells was observed after 48 h (p&#x2009;=&#x2009;0.0334). The presence of Cas9 was successfully confirmed in transfected cells.<br><br>CONCLUSION: These findings suggest that CRISPR/Cas9-mediated modulation of miR-21 may influence tumor suppressor pathways and reduce the migratory potential of urothelial carcinoma cells.},
}
@article {pmid42320754,
year = {2026},
author = {Li, P and Lv, B and Zhang, L and Xu, X and Zhang, Z and Li, W and Zhang, T and Miao, X and Pan, X and Luo, Y and Mao, W and Lu, H and Song, J},
title = {Endonuclease-Assisted Selective Exponential Amplification (ESEA) for Ultra-Sensitive Enrichment and Detection of Low-abundance Mutant Alleles in Lung Cancer.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmoldx.2026.05.008},
pmid = {42320754},
issn = {1943-7811},
abstract = {Lung cancer is one of the most prevalent and lethal malignancies worldwide. Despite recent advancements in precision medicine, early detection and therapeutic monitoring of lung cancer remain challenging. Here, we present an Endonuclease-Assisted Selective Exponential Amplification (ESEA) platform that selectively depletes wild-type alleles through programmable endonuclease digestion (CRISPR-Cas or restriction enzymes) while simultaneously amplifying mutant alleles, enabling robust and cost-effective detection at mutant allele frequencies (MAF) as low as 0.00625%. To address the PAM-site limitations inherent in CRISPR-based enrichment strategies, we introduce a primer-directed restriction site programming approach that expands the theoretical coverage to over 94% of mutations listed in COSMIC. The ESEA system offers high sensitivity and cost-effectiveness compared with conventional methods, and enables multiplexed detection of key lung cancer hotspot mutations, including EGFR L858R, EGFR exon 19 deletions, EGFR T790M, BRAF V600E, as well as hotspots in KRAS and PIK3CA. In a small-sample-size test, the ESEA system achieved 100% sensitivity and specificity in pleural effusion samples (n=5) and a 100% ctDNA detection rate in patients with extracranial lesions and disease progression (n=6). These results highlight its potential as a cost-effective, highly sensitive, and robust platform for dynamic, real-time companion diagnostics, as well as non-invasive monitoring of treatment response and tumor evolution.},
}
@article {pmid42321594,
year = {2026},
author = {Muniz, MI and de Oliveira Martins, E and Carzaniga, T and Marni, S and Buscaglia, M and Weber, G},
title = {Mesoscopic Calculation of Single Mismatches in RNA/DNA Hybrids: Strong Hydrogen Bonds of dTrG Affect CRISPR Off-Target Binding.},
journal = {The journal of physical chemistry. B},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jpcb.6c01938},
pmid = {42321594},
issn = {1520-5207},
abstract = {In nature, complementary DNA-RNA hybrids occur in several biological contexts, mainly related to transcription, replication, and gene regulation. Despite their importance, mismatches in DNA-RNA hybrids are not as well characterized as in the case of DNA-DNA or RNA-RNA pairing. Of particular concern are strong mismatches between DNA and RNA strands that may favor off-target binding in CRISPR-Cas gene editing. Here, we investigate the effect of single internal mismatches on the thermal stability of RNA/DNA sequences using a mesoscopic approach based on published melting temperatures, as well as new measurements, to estimate hydrogen bonds and stacking interaction potentials. Our results show that only dTrG base pairs have substantial hydrogen bonding, even larger than the Watson-Crick dTrA pair, while all others are negligible. However, we show that in many cases stacking provides some stability to the otherwise weaker mismatches. Our results provide an explanation for the stability of dTrG as well as why it has CRISPR editing efficiencies similar to those of perfect matches.},
}
@article {pmid42322299,
year = {2026},
author = {Wolabu, TW and Mahmood, K and Birhan, T and Daruvuri, SM and Jerez, IT and Wu, Y and Xu, X and Tadege, M and Wen, J and Udvardi, M},
title = {Mutating alfalfa NAP1 and NAP2 transcription factors by multiplex CRISPR/Cas9 genome editing leads to delayed senescence with improved forage biomass, nutritional quality, and salinity tolerance.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {6},
pages = {e70996},
doi = {10.1111/tpj.70996},
pmid = {42322299},
issn = {1365-313X},
mesh = {*Medicago sativa/genetics/physiology/metabolism ; CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Biomass ; Gene Editing/methods ; *Transcription Factors/genetics/metabolism ; Plants, Genetically Modified ; *Salt Tolerance/genetics ; Plant Senescence/genetics ; Gene Expression Regulation, Plant ; Nutritive Value ; Mutation ; },
abstract = {Alfalfa (Medicago sativa L.) is one of the most valuable forage crops due to its high biomass yield potential and nutritional values. However, forage biomass and quality are affected by different environmental and genetic factors, including developmental and stress-induced senescence, management, and harvesting stage. The CRISPR/Cas9 technology targeting stress-responsive transcription factors (TFs) offers great potential to enhance plant resilience against abiotic stresses and to develop high-quality and high-yielding forage crops. Here, we employed a multiplex CRISPR/Cas9-mediated gene-editing approach to simultaneously target two alfalfa homologous NAC TFs family genes (MsNAP1 and MsNAP2) in one construct to enhance vegetative growth and attain improved biomass by delaying leaf senescence. Two guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Seventy-five putative loss-of-function alfalfa plants targeting both MsNAP1 and MsNAP2 were generated. Phenotypic and genotypic analyses revealed that Msnap1 and Msnap2 double mutants with delayed leaf senescence produced greater forage biomass, with increases of up to 83% in fresh weight and 42% in dry weight compared to the empty vector control. Furthermore, acid detergent fiber and neutral detergent fiber content were reduced with a concomitant increase in crude protein content, total digestible nutrients, and relative feed value in both leaves and stems, indicating significant improvement in forage quality. Our findings suggest that CRISPR/Cas9-edited Msnap1 and msnap2 double mutant lines could be used as valuable genetic resources to generate elite transgene-free alfalfa cultivars with delayed leaf senescence and improved forage biomass and quality.},
}
@article {pmid42322398,
year = {2026},
author = {Wang, X and Yao, Q and Luo, Y and Wang, Z and Feng, L and Hu, S and Yin, Z and Lei, J and Xie, W and Sun, Q},
title = {Establishment of rapid identification methods of menstrual blood based on SHERLOCK technology.},
journal = {International journal of legal medicine},
volume = {},
number = {},
pages = {},
pmid = {42322398},
issn = {1437-1596},
support = {2024JB040//Central level public welfare research institutes basic research business fund project/ ; 2022YFC3341002//Key Technologies Research and Development Program/ ; },
abstract = {The accurate determination of the tissue origin of body fluid-like forensic samples at crime scenes holds significant value for crime scene reconstruction and case trials. mRNA is the most commonly used molecular marker for body fluid identification. In this study, short crRNA fragments targeting the menstrual blood-specific genes MMP10 and STC1 were designed and screened. A SHERLOCK detection method based on CRISPR/Cas technology was established. Two signal detection methods, SHERLOCK-Fluorescence and SHERLOCK-LFA, were evaluated using 86 samples from five body fluid types (semen, peripheral blood, menstrual blood, saliva, and vaginal secretions). The SHERLOCK-Fluorescence method showed a sensitivity of 10[-][3] ng and could detect trace menstrual blood in mixed stains, aged, and mock degraded (UV-irradiated) samples. Compared to traditional RNA detection methods, it offered higher sensitivity, specificity, and simplicity. The SHERLOCK-LFA method, with a sensitivity of 10[-][1] ng, was also effective for detecting menstrual blood and is suitable for rapid, on-site forensic detection. Both methods provide new approaches for the rapid identification of menstrual blood in forensic science.},
}
@article {pmid42322576,
year = {2026},
author = {Almakrami, M and Ahmed, Z and Alali, GM and Alqurashi, A},
title = {CRISPR systems in cancer therapeutics and diagnostics.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {72},
number = {3},
pages = {8-18},
doi = {10.14715/cmb/2025.72.3.2},
pmid = {42322576},
issn = {1165-158X},
mesh = {Humans ; *Neoplasms/diagnosis/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Biomarkers, Tumor/genetics ; Animals ; Genetic Therapy/methods ; Immunotherapy ; },
abstract = {Cancer is the leading cause of morbidity and mortality globally, underscoring the need for precise diagnostic approaches and effective therapeutic strategies. The invention of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has transformed cancer research by enabling accurate and efficient genome editing. This review provides a comprehensive overview of CRISPR-based applications in cancer therapeutics and diagnostics, beginning with an introduction to cancer biology and the CRISPR/Cas9 mechanism of action. The therapeutic potential of CRISPR is discussed with a focus on targeting oncogenes, restoring tumor suppressor gene function, and advancing cancer immunotherapy through approaches such as CAR-T cell engineering. The use of CRISPR-based screening platforms to identify genes associated with drug resistance is also examined, along with current challenges including off-target effects, delivery limitations, and ethical considerations, as well as future directions for clinical translation. In addition, the diagnostic capabilities of CRISPR technologies are highlighted, including the detection of circulating tumor DNA, cancer-associated extracellular vesicles, protein biomarkers, and microRNAs. Recent advances in CRISPR/Cas-based strategies aimed at improving detection sensitivity and specificity are summarized. Overall, this review highlights the expanding role of CRISPR systems in cancer diagnosis and therapy and emphasizes their potential to contribute to the development of more personalized and effective cancer management strategies.},
}
@article {pmid42323028,
year = {2026},
author = {Ates, A and Onal, M and Senyigit, M and Gundogdu, G and Aytan, F and Baspinar, Y},
title = {Lipoplex as a useful tool for resensitization of methicillin-resistant Staphylococcus aureus to erythromycin via CRISPR-Cas technology.},
journal = {International journal of pharmaceutics},
volume = {},
number = {},
pages = {127105},
doi = {10.1016/j.ijpharm.2026.127105},
pmid = {42323028},
issn = {1873-3476},
abstract = {The global escalation of antimicrobial resistance (AMR) necessitates innovative therapeutic interventions. Methicillin-resistant Staphylococcus aureus (MRSA) remains a primary clinical threat due to its extensive resistance profile. In this study, CRISPR-Cas9 technology was employed to target the ermA gene, which is responsible for erythromycin resistance in clinical MRSA isolates. A cationic liposome (Lip) composed of cholesterol, DOTAP, and DOPE was developed to deliver the pCasSA plasmid. Particle characterization revealed stable liposomes (Lips) (<200 nm, PDI < 0.3, ZP>+30 mV). pCasSA was loaded into the Lip, to form a lipoplex (Lpx), a complex of Lip and pCasSA, via electrostatic interactions. Cytotoxicity studies using mouse fibroblast L929 cells revealed that cell viabilities remained at approximately 93% and 83% following treatment with the highest concentration of Lip and Lpx formulations, respectively. Successful gene editing was confirmed via Sanger sequencing and RT-qPCR, showing a 75% reduction in ermA expression when Lpx wascombined with sonoporation. Phenotypic assays demonstrated a significant restoration of susceptibility. The minimum inhibitory concentration (MIC) of erythromycin decreased 8-fold (from 8 mg/L to 1 mg/L), and inhibition zones increased 2.5-fold (from 12 mm to 30 mm). These findings suggest that Lpx-mediated CRISPR-Cas9 delivery is a highly efficient strategy for antibiotic resensitization, potentially restoring the clinical utility of existing antimicrobials.},
}
@article {pmid42323568,
year = {2026},
author = {Alves, CPP and Pinto, OHB and Pappas, GJ and Mota, SS and Rahlff, J and Krüger, RH},
title = {Taxonomic and functional diversity of the microbiome associated with the freshwater sponge Metania sp. (Haplosclerida: Metaniidae) from the Brazilian Cerrado, a metagenomic approach.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {42323568},
issn = {1471-2180},
mesh = {Animals ; Brazil ; *Porifera/microbiology ; *Microbiota/genetics ; *Metagenomics/methods ; *Bacteria/classification/genetics/isolation &amp; purification ; Fresh Water/microbiology ; *Archaea/classification/genetics/isolation &amp; purification ; Symbiosis ; Phylogeny ; Metagenome ; RNA, Ribosomal, 16S/genetics ; Biodiversity ; Sequence Analysis, DNA ; },
abstract = {BACKGROUND: Sponges, the oldest metazoans on the planet, have an evolutionary history shaped by symbiotic associations with microorganisms. Although well studied in marine sponges, these associations are poorly understood in freshwater species. This study explored the taxonomic diversity and functional potential of the microbiome of the freshwater sponge Metania sp. and its distinction from the surrounding water, using a metagenomic approach. The samples were collected in the Brazilian Cerrado.<br><br>RESULTS: Taxonomic assignment identified 17 phyla, including bacterial and archaeal, with 19 sequence variants successfully assigned to the species level. Bacteria comprised 16 phyla, with a predominance of Pseudomonadota, Actinomycetota, and Bacteroidota in both microbiomes. The sponge microbiome is distinct from the water microbiome (PERMANOVA; F&#x2009;=&#x2009;21.6, p&#x2009;=&#x2009;0.04), sharing only 27% of the identified taxa. Functional prediction resulted in 7,201 KEGG Orthologs (KOs), assigned to 117 significantly enriched metabolic pathways. Although 95 pathways are shared, differential abundance analysis identified 1,024 KOs more abundant in the sponge microbiome and 1,275 in the water. The presence of bacterial defense systems such as CRISPR-Cas in the sponge microbiome suggests a crucial role in protecting against phages while maintaining symbiosis. In contrast, the water microbiota is enriched with pathways linked to environmental adaptation, such as secondary metabolite biosynthesis and pollutant degradation. Although the water microbiome harbored 1.3 times more biosynthetic gene clusters (BGCs), the sponge microbiome also demonstrated biotechnological potential for producing secondary metabolites, especially antimicrobial.<br><br>CONCLUSIONS: These findings demonstrate that the freshwater sponge Metania sp. hosts a complex and functionally specialized microbial community that plays fundamental roles in adaptation, nutrition, and defense, highlighting the critical importance of symbiotic associations for the host.},
}
@article {pmid41298934,
year = {2025},
author = {Lee, SY and Birkholz, N and Lee, JH and Fineran, PC and Park, HH},
title = {Regulation of anti-CRISPR operons by structurally distinct families of Aca proteins.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1698},
pmid = {41298934},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; PE25150//Korea Polar Research Institute (KOPRI)/ ; },
mesh = {*Operon ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; *Viral Proteins/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Binding ; Bacteriophages/genetics ; Crystallography, X-Ray ; Models, Molecular ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages and mobile genetic elements, driving an evolutionary arms race in which phages deploy anti-CRISPR (Acr) proteins. Acr proteins are often co-encoded in operons with anti-CRISPR-associated (Aca) proteins, which coordinate the regulation of acr gene expression. Here, we reveal the molecular basis of DNA binding that mediates transcriptional repression by two distinct Aca family members: Aca7 and Aca11. Crystal structures of Aca7 and Aca11 highlight conserved helix-turn-helix (HTH) motifs within α-helix bundles, providing a universal DNA-binding platform. Aca7 forms a symmetrical dimer to recognize a 19-bp inverted repeat (IR) within the acrIF11-aca7 operon. Strikingly, Aca11 binds 22-bp IRs in two distinct promoters, suggesting that Aca proteins can control multiple target operons. Mutagenesis and electrophoretic mobility shift assays (EMSAs) confirm that dimerization and sequence-specific IR recognition are essential for DNA binding. Despite mechanistic similarities, these and other Aca proteins exhibit notable differences. Structural comparisons across Aca families reveal that while monomer structures are generally similar with conserved HTH motifs, the structures of their dimeric functional units vary significantly. These structural differences might be essential for Aca proteins to bind to various promoters and regulate the expression of different Acr proteins.},
}
@article {pmid41298993,
year = {2025},
author = {Heu, CC and Benowitz, KM and Matzkin, LM and Allan, CW and LeRoy, DM and Li, X and Tabashnik, BE and Carrière, Y and Fabrick, JA},
title = {Editing the kinesin-12 gene affects responses to Bt toxin Cry1Ac in Helicoverpa zea.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {45378},
pmid = {41298993},
issn = {2045-2322},
support = {2020-33522-32268//USDA, National Institute of Food and Agriculture, Biotechnology Risk Assessment Research Grants Program/ ; 2020-67013-31924//USDA, National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; 2020-22620-023-000D//USDA, Agricultural Research Service/ ; },
mesh = {Animals ; Bacillus thuringiensis Toxins ; *Endotoxins/pharmacology ; *Gene Editing ; *Hemolysin Proteins/pharmacology ; *Kinesins/genetics ; *Moths/genetics/drug effects ; *Bacterial Proteins/pharmacology ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Bacillus thuringiensis/genetics ; *Insect Proteins/genetics ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage key insect pests. However, the evolution of resistance to Bt proteins in at least 11 pest species has reduced the effectiveness of Bt crops. Resistance to crystalline (Cry) Bt proteins including Cry1Ac produced by Bt cotton is a major problem in Helicoverpa zea (also known as bollworm and corn earworm), one of the most economically damaging pests in the United States. A previous genome-wide association study identified a nonsense point mutation in a kinesin-12 gene that was associated with resistance to Cry1Ac in a lab-selected strain of H. zea. Here, we used CRISPR/Cas9 gene editing to knock out the kinesin-12 gene in a Cry1Ac-susceptible laboratory strain, which caused a 4.0-fold increase in resistance to Cry1Ac. Conversely, gene editing that repaired the natural kinesin-12 nonsense mutation in a lab-selected resistant strain increased susceptibility to Cry1Ac by 3.8-fold. These complementary results provide compelling evidence that kinesin-12 plays a role in the mode of action of Cry1Ac against H. zea.},
}
@article {pmid41299842,
year = {2025},
author = {Shi, C and Yu, Z and Tan, H and Li, W and Wang, Y and Wang, Y and Zhang, Q and Man, Y},
title = {One-Pot CRISPR-Based Isothermal Amplification for Nucleic Acid Detection: A Comparative Review of Different Strategies.},
journal = {ACS sensors},
volume = {10},
number = {12},
pages = {9108-9134},
doi = {10.1021/acssensors.5c00806},
pmid = {41299842},
issn = {2379-3694},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Nucleic Acids/analysis/genetics ; Humans ; Molecular Diagnostic Techniques ; },
abstract = {Nucleic acid detection plays an important role in pathogen monitoring and disease diagnosis. CRISPR one-pot assays combined with isothermal amplification are emerging as promising point-of-care technologies that simplify workflows while increasing sensitivity and specificity. However, the incompatibility inherent in the one-pot reaction of isothermal amplification and CRISPR detection limits their practical application. This review comprehensively analyzes diverse advanced one-pot CRISPR-based isothermal amplification strategies developed to overcome this fundamental challenge. These strategies primarily encompass physical separation strategies (utilizing lid-bottom, internal ledge, nested tube, and membrane approaches), phase separation strategies (employing glycerol, sucrose, and gel matrices), reaction system optimization strategies (fine-tuning reaction parameters and incorporating specialized additives), non-PAM and suboptimal PAM strategies, improved Cas enzyme strategies (enhanced Cas12 and Cas13 variants), light-controlled approaches (PC-oligonucleotides, NPOM-dt modification, and acylation modification), and microfluidic chip integration strategies (centrifugal microfluidic chips, droplet microfluidic chips, and microarray chips). These methodological approaches have achieved important advances in simplifying operational processes, enhancing sensitivity, shortening detection cycles, and minimizing cross-contamination risks. The review further synthesizes critical insights regarding current opportunities, technical challenges, and future directions for one-pot CRISPR-based isothermal amplification technologies in nucleic acid detection, providing valuable guidance for researchers and practitioners in this evolving field.},
}
@article {pmid41300710,
year = {2025},
author = {Iksat, N and Madirov, A and Zhanassova, K and Masalimov, Z},
title = {Artificial Intelligence-Assisted CRISPR/Cas Systems for Targeting Plant Viruses.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
pmid = {41300710},
issn = {2073-4425},
support = {grant No. BR21882269//the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Artificial Intelligence ; *Plant Viruses/genetics ; *Gene Editing/methods ; *Plant Diseases/virology/genetics ; Crops, Agricultural/genetics/virology ; },
abstract = {Plant viral infections continue to pose a significant and ongoing threat to global food security, especially in the context of climatic instability and intensive agricultural practices. The CRISPR/Cas system has emerged as a powerful tool for developing virus-resistant crops by enabling precise modifications to viral genomes or plant susceptibility factors. Nonetheless, the efficacy and dependability of CRISPR-based antiviral approaches are limited by challenges in guide RNA design, off-target effects, insufficiently annotated datasets, and the intricate biological dynamics of plant-virus interactions. This paper summarizes the latest advancements in the incorporation of artificial intelligence (AI) methodologies, including machine learning and deep learning algorithms, into the CRISPR design and optimization framework. It examines how convolutional and recurrent neural networks, transformer architectures, and generative models like AlphaFold2, RoseTTAFold, and ESMFold can be used to predict protein structures, score sgRNAs, and model host-virus interactions. AI-enhanced methods have been proven to improve target specificity, Cas protein performance, and in silico validation. This paper aims to establish a foundation for next-generation genome editing strategies against plant viruses and promote the adoption of AI-powered CRISPR technologies in sustainable agriculture.},
}
@article {pmid41300715,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Daurov, D and Daurova, A and Shamekova, M and Zhambakin, K},
title = {The Enhancement of Fungal Disease Resistance in Major Staple Crops Using CRISPR-Cas Technology.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
pmid = {41300715},
issn = {2073-4425},
support = {BR21882269//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Crops, Agricultural/genetics/microbiology ; Gene Editing/methods ; Fungi/pathogenicity/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Fungal pathogens represent a major constraint to global agricultural productivity, causing a wide range of plant diseases that severely affect staple crops such as cereals, legumes, and vegetables. These infections result in substantial yield losses, deterioration of grain and produce quality, and significant economic impacts across the entire agri-food sector. Among phytopathogens, fungi are considered the most destructive, causing a wide range of diseases such as powdery mildew, rusts, fusarium head blight, smut, leaf spot, rots, late blight, and other fungal pathogens. Traditional plant protection methods do not always provide long-term effectiveness and environmental safety, which requires the introduction of innovative approaches to creating sustainable varieties. CRISPR-Cas technology opens up new opportunities for targeted genome editing, allowing the modification or silencing of susceptibility genes and thus increasing plant resistance to fungal infections. This review presents current achievements and prospects for the application of CRISPR-Cas technology to increase the resistance of major agricultural crops to fungal diseases. The implementation of these approaches contributes to the creation of highly productive and resistant varieties, which is crucial for ensuring food security in the context of climate change.},
}
@article {pmid41300819,
year = {2025},
author = {Kansal, R},
title = {Curing Sickle Cell Disease by Allogeneic Hematopoietic Stem Cell (HSC) Transplantation Toward In Vivo HSC Gene Therapy.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
pmid = {41300819},
issn = {2073-4425},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *Hematopoietic Stem Cell Transplantation/methods ; *Genetic Therapy/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Transplantation, Homologous ; Animals ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Sickle cell disease comprises a group of prevalent inherited disorders defined by an underlying sickle cell allele that forms sickle hemoglobin. The incidence of this disease is rising, with more than 500,000 children born with it globally. The disease carries significant morbidity and mortality. Its only curative treatment was an allogeneic hematopoietic stem cell (HSC) transplant (HSCT) until late 2023, when two one-time gene therapies were approved for treating patients aged 12 years or older with severe sickle cell disease. This work aims to inform readers about these two gene therapies: one lentiviral-based and the other nonviral. The latter is based on the Nobel Prize-winning discovery of clustered, regularly interspaced, short, palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 proteins and single-guide RNA (sgRNA)-based genome editing. Both approved gene therapies require an autologous HSCT with ex vivo genetically edited autologous hematopoietic stem and progenitor cells. Therefore, access to these gene therapies is limited to specialized centers with expertise in HSCTs. This review is meant for students, researchers, and clinical practitioners. It explains the basis for both approved gene therapies, their mechanisms of action, differences, risks, and other lentiviral-based and CRISPR-Cas9-based ex vivo gene therapies for sickle cell disease in clinical development. Additionally, it discusses the current state of preclinical studies for in vivo HSC gene therapy for sickle cell disease, which utilize advanced genome editing technologies developed after CRISPR-Cas9-sgRNA-based genome editing. In vivo HSC gene therapy, after it is clinically developed, would eliminate the need for an HSCT in receiving gene therapy and vastly increase access for numerous patients worldwide, even in low-income countries with the most significant disease burden.},
}
@article {pmid41301473,
year = {2025},
author = {Gibril, BAA and Chai, X and Xu, J},
title = {From Correlation to Causation: Defining Gene and RNA Function in Poultry Muscle Biology Using In Vivo Genetic Tools.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
pmid = {41301473},
issn = {2218-273X},
support = {20242BCE50051//Science and Technology Research Project of the Education Department of Jiangxi Province/ ; },
mesh = {Animals ; *Muscle, Skeletal/metabolism ; Muscle Development/genetics ; *Poultry/genetics ; Muscular Diseases/genetics ; Transcriptome ; CRISPR-Cas Systems ; *RNA/genetics/metabolism ; },
abstract = {A central challenge in functional genomics is understanding the difference between correlative transcriptomic observations and definitive causal understanding of gene function in vivo. Poultry skeletal muscle, a system of significant agricultural and biological importance, demonstrates this challenge. While transcriptomic studies have cataloged extensive RNA expression dynamics during muscle development and in growth-related myopathies like wooden breast, establishing causative roles for these molecules is lacking. This review synthesizes how advanced genetic tools are now enabling a shift from correlation to causation in avian muscle biology. We detail how viral vectors (e.g., adenovirus, lentivirus, and RCAS) and CRISPR/Cas9 systems have provided direct in vivo validation of the functional roles of specific mRNAs, miRNAs, lncRNAs, and circRNAs in regulating myogenesis, hypertrophy, and atrophy. We contrast this success in fundamental biology with the study of myopathies, which remains largely descriptive. Here, a wealth of transcriptomic data has identified dysregulated pathways, including ECM remodeling, metabolism, and inflammation, but functional validation for most candidates is absent. We argue that the critical next step is to apply this established functional genomics toolkit to disease models. By defining causal mechanisms, this research will not only address a major agricultural issue but also provide a model for using genetic tools to dissect complex traits in a post-genomic era.},
}
@article {pmid41301547,
year = {2025},
author = {Mohammed, A and Ibrahim, NA and Basher, NS},
title = {Protein Engineering and Drug Discovery: Importance, Methodologies, Challenges, and Prospects.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
pmid = {41301547},
issn = {2218-273X},
support = {IMSIU-DDRSP2501//Imam Mohammad ibn Saud Islamic University/ ; },
mesh = {*Drug Discovery/methods ; *Protein Engineering/methods ; Humans ; Animals ; Recombinant Proteins/therapeutic use/chemistry/genetics ; },
abstract = {Protein engineering is a rapidly evolving field that plays a critical role in transforming drug discovery and development. This innovative field harnesses the unique structural and functional properties of engineered proteins, such as monoclonal antibodies, nanobodies, therapeutic enzymes, and cytokines, to address complex diseases more effectively than traditional small-molecule drugs. These biologics not only enhance therapeutic specificity but also minimize adverse effects, marking a significant advancement in patient care. However, the journey of protein engineering is not without challenges. Issues related to protein folding, stability, and potential immunogenicity pose significant complications. Additionally, navigating the complex regulatory landscape can delay the transition from laboratory to clinical application. Addressing these hurdles requires the integration of cutting-edge technologies, including phage and yeast display technology, CRISPR, and advanced computational modeling, which enhance the predictability and efficiency of protein design. In this review, we explore the multifaceted impact of protein engineering on modern medicine, highlighting its potential to transform treatment paradigms, methodologies, challenges, and the successful development and approval of recombinant protein-based therapies. By navigating the complexities and leveraging technological advancements, the field is poised to unlock new therapeutic possibilities, ultimately improving patient outcomes and transforming healthcare.},
}
@article {pmid41301629,
year = {2025},
author = {Wang, Y and Li, L and Liang, Y and Xu, K and Ye, Y and He, M},
title = {Phage Therapy for Acinetobacter baumannii Infections: A Review on Advances in Classification, Applications, and Translational Roadblocks.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {11},
pages = {},
pmid = {41301629},
issn = {2079-6382},
support = {82302568//National Natural Science Foundation of China/ ; 2308085QH283//Anhui Provincial National Science Foundation/ ; 2022xkjT012//Basic and Clinical Collaboration Enhancement Program Foundation of Anhui Medical University/ ; },
abstract = {The global spread of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe public health threat, driving growing interest in phage-based precision antibacterial strategies. This systematic review synthesizes recent advances in the field of A. baumannii phage. Modern taxonomy, based on whole-genome phylogeny, has reclassified the majority of A. baumannii phages into the class Caudoviricetes, revealing distinct evolutionary clades that correlate with host tropism and biological properties, superseding the traditional morphological families (Myoviridae, Siphoviridae, Podoviridae). To overcome limitations of natural phage therapy, such as narrow host range, cocktail therapies (ex vivo resistance mutation rates < 5%) and phage-antibiotic synergism (enabling antibiotic efficacy at 1/4 minimum inhibitory concentration) have significantly enhanced antibacterial efficacy. Preclinical models demonstrate that phage therapy efficiently clears pathogens in pneumonia models and promotes the healing of burn wounds and diabetic ulcers via immunomodulatory mechanisms. Technical optimizations include nebulized inhalation delivery achieving 42% alveolar deposition, and thermosensitive hydrogels enabling sustained release over 72 h. Genetic engineering approaches, such as host range expansion through tail fiber recombination and CRISPR/Cas-mediated elimination of lysogeny, show promise. However, the genetic stability of engineered phages requires further validation. Current challenges remain, including limited host spectrum, the absence of clinical translation standards, and lagging regulatory frameworks. Future efforts must integrate metagenomic mining and synthetic biology strategies to establish a precision medicine framework encompassing resistance monitoring and personalized phage formulation, offering innovative solutions against CRAB infections.},
}
@article {pmid41301838,
year = {2025},
author = {Dziedzic, A and Kubina, R and Skonieczna, M and Madej, M and Fiegler-Rudol, J and Abid, M and Nadhim, D and Tanasiewicz, M},
title = {CRISPR Genome Editing in Personalized Therapy for Oral and Maxillofacial Diseases: A Scoping Review.},
journal = {Biomedicines},
volume = {13},
number = {11},
pages = {},
pmid = {41301838},
issn = {2227-9059},
abstract = {Background: CRISPR/Cas genome editing is emerging as a powerful tool in oral and maxillofacial medicine, with potential applications in personalized therapies for conditions that currently lack durable treatments. Objectives: This scoping review aimed to map existing evidence on CRISPR-based applications in oral and maxillofacial fields, rather than to assess treatment effectiveness. Methods: A systematic search of PubMed, Scopus, Web of Science, and ClinicalTrials.gov (2012-2024) identified studies and registered trials involving CRISPR with oral health relevance. Eligible articles included peer-reviewed experimental reports and clinical trials. Results: From 1437 records, 121 studies met inclusion criteria: 106 preclinical reports and 15 clinical or translational studies. Investigated domains included oral cancer therapy, hereditary craniofacial syndromes, regenerative strategies, infectious disease models, and pathogen detection. Early clinical efforts focus mainly on CRISPR-edited T-cell immunotherapies in oncology. Major barriers include off-target effects, delivery challenges, regulatory complexity, and ethical concerns. Conclusions: CRISPR-based bioengineering shows strong promise for precision care in oral and maxillofacial medicine. However, current evidence remains largely preclinical and heterogeneous. No clinical recommendations can yet be made, and translation will depend on rigorous late-phase trials, ethical oversight, and health-economic evaluation.},
}
@article {pmid41302913,
year = {2025},
author = {Ayaz, S and Kong, WW and Wang, J and Liu, SH and Xu, JP},
title = {Host Immunity Mechanisms Against Bacterial and Viral Infections in Bombyx mori.},
journal = {Insects},
volume = {16},
number = {11},
pages = {},
pmid = {41302913},
issn = {2075-4450},
abstract = {The domesticated silkworm, Bombyx mori, is a highly valued biodiversity and economic asset, acclaimed for its silk production, besides making important contributions to various scientific disciplines. However, the sericulture industry faces ongoing threats from bacterial and viral infections, which severely impact silkworm health and silk yield. This review provides a comprehensive overview of the innate immune response of B. mori against bacterial and viral pathogens, emphasizing the fundamental molecular and cellular defense mechanisms. We explore the humoral and cellular immune response using antimicrobial peptides (AMPs), pattern recognition receptors (PRRs) like peptidoglycan recognition protein (PGRP), and glucan recognition protein (GRP), which activate canonical signaling pathways. The review further highlights the molecular mechanisms underlying the silkworm's defense against viruses, incorporating RNA interference (RNAi), apoptosis, and distinct signaling pathways such as Toll and Imd, JAK/STAT, and STING. We also discussed the viral suppression strategies and modulation of host metabolism during infection. Furthermore, the review explores the recent use of CRISPR-Cas gene editing to enhance disease resistance, presenting a promising avenue for mitigating pathogen-induced losses in sericulture. By elucidating these mechanisms, the work provides a synthesis that is critical in terms of developing particular interventions and developing more resistant silkworm strains to ensure that the industry of sericulture becomes viable and productive.},
}
@article {pmid41303344,
year = {2025},
author = {Zhang, X and Che, J and Li, Z and Bao, B and Fan, C},
title = {The Mutation of piezo1 Weakens the Intermuscular Bones in Zebrafish and Crucian Carp.},
journal = {International journal of molecular sciences},
volume = {26},
number = {22},
pages = {},
pmid = {41303344},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 32170514//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Carps/genetics ; *Ion Channels/genetics/metabolism ; *Mutation ; *Zebrafish Proteins/genetics/metabolism ; *Bone and Bones/metabolism ; CRISPR-Cas Systems ; *Fish Proteins/genetics/metabolism ; },
abstract = {Intermuscular bones (IBs), unique skeletal features found only in teleost fishes, pose significant challenges to food processing and consumption. While recent studies have identified several key genetic regulators of IB development, the role of mechanosensory mechanisms remains largely unexplored. This study investigated the role of Piezo1, a critical mechanosensitive ion channel, in IB formation using zebrafish and crucian carp models. Our findings demonstrated that piezo1 was expressed in the myoseptum of zebrafish, and CRISPR/Cas9-mediated knockout of this gene resulted in shorter and smaller IBs. Similar knockout experiments in crucian carp confirmed the conserved role of Piezo1 across cyprinid species. These results established Piezo1 as a key regulator of IB development, providing new insights into the molecular mechanisms underlying this process and suggesting potential strategies for breeding IB-free fish strains through modulation of mechanosensory pathways.},
}
@article {pmid41304743,
year = {2025},
author = {Zhang, H and Li, Y and Li, J and Li, X and Li, T},
title = {Base and Prime Editing for Inherited Retinal Diseases: Delivery Platforms, Safety, Efficacy, and Translational Perspectives.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
pmid = {41304743},
issn = {1999-4923},
support = {U22A20311//he National Natural Science Foundation of China/ ; 82571246//the National Natural Science Foundation of China/ ; 82388101//the National Natural Science Foundation of China/ ; 23J41900200//Science and Technology Commission of Shanghai Municipality/ ; SHWSRS(2025)_071//Shanghai "Rising Stars of Medical Talents" Youth Development Program/ ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous spectrum of disorders that lead to progressive and irreversible vision loss. Gene therapy is the most promising emerging treatment for IRDs. While gene augmentation strategies have demonstrated clinical benefit and results within the first approved ocular gene therapy, their application is restricted by adeno-associated virus (AAV) packaging capacity and limited efficacy for dominant mutations. Recent breakthroughs in precision genome editing, particularly base editing (BE) and prime editing (PE), have provided alternatives capable of directly correcting pathogenic variants. BE enables targeted single-nucleotide conversions, whereas PE further allows for precise insertions and deletions, both circumventing the double-strand DNA cleavage or repair processes typically induced by conventional CRISPR-Cas editing systems, thereby offering advantages in post-mitotic retinal cells. Preclinical investigations across murine and non-human primate models have demonstrated the feasibility, molecular accuracy, and preliminary safety profiles of these platforms in targeting IRD-associated mutations. However, critical challenges remain before clinical application can be realized, including limited editing efficiency in photoreceptors, interspecies variability in therapeutic response, potential risks of off-target effects, and barriers in large-scale vector manufacturing. Moreover, the delivery of genome editors to the outer retina remains suboptimal, prompting intensive efforts in capsid engineering and the development of non-viral delivery systems. This review synthesizes the current progress in BE and PE optimization, highlights innovations in delivery platforms that encompass viral and emerging non-viral systems and summarizes the major barriers to clinical translation. We further discuss AI-driven strategies for the rational design of BE/PE systems, thereby outlining their future potential and perspectives in the treatment of IRDs.},
}
@article {pmid41304764,
year = {2025},
author = {Alidriss, OM and AlSudais, H and Alhumaidan, OS and Altwaijry, HD and Bakhsh, A and Almuhanna, Y and Alkudmani, ZS and Alqarni, IA and Alenazi, D and Aljasham, AT and Jamous, YF},
title = {Targeted Drug Delivery Strategies in Overcoming Antimicrobial Resistance: Advances and Future Directions.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
pmid = {41304764},
issn = {1999-4923},
abstract = {Antimicrobial resistance (AMR) is a present, pressing global public health crisis associated with rising morbidity and mortality rates due to previously curable infectious disease. Targeted drug delivery is an important approach to address AMR due to its ability to improve the therapeutic performance of antibiotics without leading to any adverse effects or organ toxicities. In this review we explore molecular mechanisms of AMR and drawbacks of conventional antibiotic therapies and discuss unique drug delivery approaches to compensate these. Nanoparticulate carrier systems, stimuli-responsive systems, antibody-drug conjugates, and CRISPR-Cas systems are some of the carrier method designs that are promising for tackling hard to treat infections related to pathogenic strains and biofilms due to their features. Many of these are among the most significant advances in the field. However, there are many challenges to be overcome, with biological limitations, scaling and regulatory challenges, etc., before they can be employed in commercial applications. Materials are being developed, and an approach standardized and applicable to future work is in development to improve the efficiency of targeted delivery systems. Controlled drug delivery, which could be the answer to an increasing AMR problem, will not only help in alerting awareness among individuals but will also help in prolonging the activity of antibiotics by providing synergistic interdisciplinary solutions. This review emphasizes the complementary role of targeted drug delivery in transitioning from laboratory investigations to clinical therapy. It addresses underrepresented aspects, including new materials, scalability, regulatory considerations, and ethical implications, while offering a roadmap for translating innovations into next-generation antimicrobials.},
}
@article {pmid41304797,
year = {2025},
author = {Sharma, A and Sharma, V and Sharma, S and Sharma, S and Sharma, M and Sivanesan, I},
title = {Advanced Nanosystems and Emerging Therapies: Innovations in Tuberculosis Treatment and Drug Resistance.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
pmid = {41304797},
issn = {1999-4923},
abstract = {Tuberculosis (TB) remains a significant worldwide health challenge due to the limitations of conventional treatments and the rising incidence of drug-resistant Mycobacterium tuberculosis strains. This review consolidates the advancements in nanotechnology-based therapeutics, inhalable formulations, CRISPR-Cas tools, host-directed therapies (HDTs), and nanoparticle-based vaccine development aimed at enhancing TB management. Novel nanocarriers such as liposomes, solid-lipid nanoparticles (SLNs), dendrimers, and polymeric nanoparticles (NPs) offer enhanced bioavailability of drugs, sustained release, as well as targeted delivery to infected macrophages, thereby reducing systemic toxicity and dosing frequency. Inhalable nanomedicines provide localized delivery to the pulmonary site, enhancing the concentration of the drug at the primary site of infection. CRISPR-Cas technology is emerging as a transformative approach to disabling drug-resistant genes and enhancing diagnostic precision. HDTs, including agents like vitamin D and metformin, show potential in modulating host immune responses and enhancing pathogen clearance. Nanoparticle-based vaccines, including mRNA and antigen-conjugated platforms, aim to overcome the limitations of the BCG vaccine by enhancing antigen presentation and eliciting stronger, longer-lasting immunity. Collectively, these modalities mark a shift toward more personalized, effective, and less toxic TB therapies. However, challenges such as regulatory approval, safety, scalability, and accessibility remain. This review highlights the integrated potential of nanomedicine, gene editing, and immunomodulation to transform TB care and combat drug resistance, paving the way for more robust and durable treatment strategies.},
}
@article {pmid41304933,
year = {2025},
author = {Yue, Y and Xu, Z and Soteyome, T and Premarathna, M and Yin, X and Liu, J},
title = {Phage Encapsulation and Delivery Technology: A Strategy for Treating Drug-Resistant Pathogenic Microorganisms.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {11},
pages = {},
pmid = {41304933},
issn = {1424-8247},
abstract = {Antimicrobial resistance (AMR) is one of the most critical challenges to global public health in the 21st century, posing a significant threat to healthcare systems and human health due to treatment failure and high mortality. The World Health Organization (WHO) estimates that, without effective interventions, AMR-associated infections could cause 10 million deaths annually and economic losses of up to 100 trillion US dollars by 2050. The rapid spread of drug-resistant strains, especially in hospital and community settings, has significantly reduced the efficacy of traditional antibiotics. With the continuous advancements in relevant research, bacteriophage (Phage) therapy is constantly innovating in the antimicrobial field. The application of frontier technologies, such as phage cocktails and engineered phages, has significantly enhanced the broad spectrum and high efficiency of phage therapy, which is gradually becoming a new generation of tools to replace antibiotics and effectively combat pathogenic bacteria. However, phage therapy is facing several challenges, including phage inactivation by gastric acid, enzymes, ultraviolet light, and mechanical stress, as well as the potential risk of bacterial phage resistance. Advanced encapsulation technologies such as electrospun fibers, liposomes, chitosan nanoparticles, and electrospray provide solutions to these problems by protecting phage activity and enabling controlled release and targeted delivery. This review addresses phage therapeutic studies of Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes, summarizes the recent advances in phage research, and details the current development and applications of encapsulated phage technologies across various delivery modes.},
}
@article {pmid41305485,
year = {2025},
author = {Iftehimul, M and Hasan, NA and Bass, D and Bashar, A and Haque, MM and Santi, M},
title = {Combating White Spot Syndrome Virus (WSSV) in Global Shrimp Farming: Unraveling Its Biology, Pathology, and Control Strategies.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
pmid = {41305485},
issn = {1999-4915},
support = {2022/21/Other//Ocean Country Partnership Programme (OCPP), Blue Planet Fund/ ; },
mesh = {*White spot syndrome virus 1/pathogenicity/immunology/physiology/genetics ; Animals ; *Aquaculture/methods ; *Penaeidae/virology/immunology ; Immunity, Innate ; Viral Vaccines/immunology ; },
abstract = {White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and waterborne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures such as augmented biosecurity, selective breeding, and immunostimulants have, despite extensive research, achieved only limited success. New biotechnological tools such as RNA interference, CRISPR-Cas gene editing, and nanotechnology offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control.},
}
@article {pmid41305525,
year = {2025},
author = {Verma, N and O'Mahony, A and Mohammad, R and Keiser, D and Mosman, CW and Holden, D and Starr, K and Bauer, J and Bauer, B and Suntisukwattana, R and Atthaapa, W and Tantituvanont, A and Nilubol, D and Gladue, DP},
title = {The First CRISPR-Based Therapeutic (SL_1.52) for African Swine Fever Is Effective in Swine.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
pmid = {41305525},
issn = {1999-4915},
mesh = {Animals ; *African Swine Fever/therapy/virology ; Swine ; *African Swine Fever Virus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Viral ; },
abstract = {African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90-100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection.},
}
@article {pmid41305866,
year = {2026},
author = {Naderi, S and Williamson, J and Sun, H and Joshi, S and Spera, RJ and Zaib, S and Sharma, S and Sun, C and Brodovskiy, A and Zawar, I and Kapur, J},
title = {Hydroxycarboxylic Acid Receptor 2 Mediates β-hydroxybutyrate's Antiseizure Effect in Mice.},
journal = {Annals of neurology},
volume = {99},
number = {3},
pages = {809-824},
pmid = {41305866},
issn = {1531-8249},
support = {K23 AG084893/AG/NIA NIH HHS/United States ; R01 NS120945/NS/NINDS NIH HHS/United States ; R37 NS119012/NS/NINDS NIH HHS/United States ; R37N119012//United States National Institute of Health (NINDS)/ ; R01NS120945//United States National Institute of Health (NINDS)/ ; },
mesh = {Animals ; Mice ; *3-Hydroxybutyric Acid/pharmacology/therapeutic use ; *Seizures/drug therapy/metabolism ; *Receptors, G-Protein-Coupled/metabolism/genetics ; Mice, Knockout ; Male ; Hippocampus/drug effects/metabolism ; Mice, Inbred C57BL ; *Anticonvulsants/pharmacology/therapeutic use ; Diet, Ketogenic ; },
abstract = {OBJECTIVE: The ketogenic diet, a high-fat, low-carbohydrate regimen, is often used to treat drug-resistant seizures and is being studied for Alzheimer's disease and other neuropsychiatric disorders. However, its mechanism of action remains unclear. β-hydroxybutyrate, a primary circulating ketone body produced by the ketogenic diet, may mediate its effects on seizures by binding to a recently identified Gi-coupled receptor: hydrocarboxylic acid receptor 2 (HCAR2).<br><br>METHODS: RNAscope in situ hybridization assay and real-time quantitative polymerase chain reaction were used to assess HCAR2 expression in the mouse brain. We generated HCAR2[-]/[-] using the CRISPR-Cas technique on an S129 mouse background. Whole-cell current-clamp was performed to measure the passive and active membrane properties of hippocampal dentate granule cells. The voltage-clamp was performed to record synaptic currents. Two complementary in vivo mouse models-continuous hippocampal stimulation to induce status epilepticus (SE) and kindling-were used to induce seizures.<br><br>RESULTS: HCAR2 was localized in dentate granule cells and microglia. In mice with HCAR2, &#x3b2;-hydroxybutyrate reduced neuronal excitability by hyperpolarizing the resting membrane potential, raising the action potential threshold, and reducing the firing frequency of dentate granule cells. &#x3b2;-hydroxybutyrate suppressed excitatory synaptic transmission. These effects were nullified in HCAR2[-]/[-] mice. HCAR2[-]/[-] mice showed no cognitive impairment. Moreover, &#x3b2;-hydroxybutyrate did not affect seizures in HCAR2[-]/[-] mice. However, it diminished both the duration and severity of seizures in HCAR2[+]/[+] mice.<br><br>INTERPRETATION: These findings demonstrate that HCAR2 mediates &#x3b2;-hydroxybutyrate's antiseizure effects by regulating neuronal excitability and synaptic transmission. These studies propose a new mechanism for the antiseizure action of the ketogenic diet. ANN NEUROL 2026;99:809-824.},
}
@article {pmid41306592,
year = {2025},
author = {Shi, L and Chen, H and Zhang, Z and Wang, Y and Ren, W and Huang, J},
title = {Evolving HPV diagnostics: current practice and future frontiers.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1681779},
pmid = {41306592},
issn = {2235-2988},
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; Female ; *Molecular Diagnostic Techniques/methods/trends ; *Papillomaviridae/genetics/isolation &amp; purification ; Uterine Cervical Neoplasms/virology/diagnosis ; Early Detection of Cancer/methods ; High-Throughput Nucleotide Sequencing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Polymerase Chain Reaction/methods ; Mass Screening/methods ; },
abstract = {Human papillomavirus (HPV) infection serves as a primary causative agent of cervical cancer, highlighting the importance of early screening and detection in mitigating the incidence and mortality rates of HPV-related diseases. Over the past decades, HPV detection technologies have evolved considerably, transitioning from traditional methods to more advanced, patient-centered approaches. This review provides a comprehensive overview of both established and emerging HPV detection strategies, with a particular focus on their clinical applicability, technical advantages, and limitations. Conventional methods such as hybrid capture and PCR-based assays remain the backbone of clinical screening, offering robust sensitivity and specificity. However, their reliance on invasive sampling and centralized laboratory infrastructure limits accessibility and patient compliance, particularly in low-resource settings. To address these limitations, emerging technologies-including CRISPR/Cas systems, droplet digital PCR (ddPCR), next-generation sequencing (NGS), isothermal amplification techniques (IAT) and artificial intelligence (AI) combined with hpv screening offer enhanced accuracy, rapid turnaround, and the potential for point-of-care deployment. In parallel, innovations in sampling such as self-collected vaginal swabs and liquid biopsy using urine, blood, or extracellular vesicles are improving test acceptability and broadening screening coverage. By summarizing current progress and highlighting ongoing challenges, this review aims to guide the development of more precise, non-invasive, and scalable HPV detection strategies to reduce the global burden of HPV-related disease, support global prevention efforts, and guide public health policies.},
}
@article {pmid41307501,
year = {2025},
author = {Sahu, S and Boukherroub, R and Ritzenthaler, C and Szunerits, S},
title = {Emerging technologies for in-field plant virus detection: innovations and future directions.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
pmid = {41307501},
issn = {1465-2099},
mesh = {*Plant Viruses/isolation &amp; purification/genetics ; *Plant Diseases/virology ; Biosensing Techniques/methods ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Nanotechnology/methods ; },
abstract = {Plant virus infections pose a substantial threat to crop quality and productivity, contributing to considerable economic losses in global agriculture annually. Traditionally, laboratories have widely adopted serological techniques, such as ELISA, and molecular methods, including quantitative PCR, for virus diagnostics. More recently, sophisticated next-generation sequencing approaches have been introduced to improve the efficiency and reliability of virus detection and identification. However, the development of sensitive, rapid and low-cost methods for the on-site detection, quantification and identification of plant viruses remains an ongoing challenge and is still in its early days. Point-of-care technologies have not fully realized their potential in agriculture due to numerous challenges, such as the elevated cost of development, lack of standardized validation and insufficient field testing. Therefore, future success depends on addressing these technical, economic and regulatory hurdles, as well as considering the specific user needs within the agricultural context. In this mini-review, recent advancements in biosensing for on-site plant virus monitoring, involving nanotechnology-based sensors, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, electrochemical and modern field-effect transistor-based sensors offering high sensitivity, speed and portability, are discussed. These technologies, when integrated with smartphone applications and/or machine learning modules, could enable real-time, field-deployable diagnostics for early disease management and sustainable agriculture. The aim is to raise awareness among plant virologists about this panel of emerging diagnostic concepts that could help improve current methods, ultimately facilitating the management of plant viral diseases.},
}
@article {pmid41307588,
year = {2025},
author = {Kumar, V and Verma, P},
title = {Advances in microbial biotechnology for sustainable wastewater reclamation: recent trends and future prospects.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {478},
pmid = {41307588},
issn = {1573-0972},
mesh = {*Wastewater/microbiology/chemistry ; *Biotechnology/methods/trends ; *Water Purification/methods ; Biodegradation, Environmental ; Bacteria/metabolism/genetics ; Microalgae/metabolism ; Bioelectric Energy Sources ; Waste Disposal, Fluid/methods ; Microbial Consortia ; },
abstract = {The growing demand for freshwater, coupled with the increasing volume of industrial and municipal wastewater, has intensified the need for sustainable and eco-friendly reclamation strategies. Recent advancements in microbial biotechnology have emerged as promising tools for developing cost-effective, efficient, and environmentally sustainable wastewater treatment (WWT) strategies for reuse and safe disposal. This mini-review explores current innovations, such as microbial consortia, bioaugmentation, and the microalgae-bacteria nexus, which have shown promising results in nutrient removal, enhanced degradation of complex pollutants (including emerging contaminants), and biomass valorization. Moreover, bioelectrochemical systems, such as microbial fuel cells (MFC) and microbial electrolysis cells (MEC), have revolutionized WWT by facilitating pollutant degradation while simultaneously generating bioelectricity or biohydrogen. This article also critically examines the role of CRISPR-based tools and 'omics' approaches, which have enabled the development of novel microbial strains and degradative pathways, enhancing wastewater reclamation in challenging environments. Furthermore, advancements through the integration of multi-omics and artificial intelligence, digital twins, and Internet of Things (IoT) for microbial optimization and real-time process control are discussed. The review highlights the role of microbial systems in resource recovery, supporting a circular economy by transforming wastewater into valuable bioresources. Additionally, this review addresses the major challenges and proposes future research directions for effective wastewater treatment. The novelty of this manuscript is that no single review explores the cutting-edge microbial biotechnologies for wastewater reclamation, uniquely integrating CRISPR-Cas genome editing, multi-omics analyses, and artificial intelligence-driven optimization to advance pollutant degradation and real-time process control in one place. This study concludes that by implementing multi-omics and artificial intelligence (AI)-driven optimization process for wastewater treatment can be effective towards wastewater treatment while simultaneously minimizing the environmental pollution.},
}
@article {pmid41308487,
year = {2026},
author = {Rodríguez-Estévez, D and Gil-Durán, C and Silva, R and Palma, D and Vaca, I and Chávez, R},
title = {CRISPR/Cas9-mediated development of Penicillium roqueforti strains deficient in roquefortine C and mycophenolic acid enables toxin-free blue cheese production.},
journal = {International journal of food microbiology},
volume = {446},
number = {},
pages = {111535},
doi = {10.1016/j.ijfoodmicro.2025.111535},
pmid = {41308487},
issn = {1879-3460},
mesh = {*Penicillium/genetics/metabolism ; *Cheese/microbiology/analysis ; *Mycophenolic Acid/metabolism ; *CRISPR-Cas Systems ; *Mycotoxins/biosynthesis ; Food Microbiology ; *Indoles/metabolism ; Heterocyclic Compounds, 4 or More Rings ; Piperazines ; },
abstract = {Penicillium roqueforti, a key fungus in the manufacture of blue-veined cheeses, can produce mycotoxins such as roquefortine C and mycophenolic acid. The production of these metabolites is highly strain- and condition-dependent. In industrial manufacture, hypotoxigenic P. roqueforti strains are typically used as controlled adjunct starters under standardized conditions, resulting in minimal mycotoxin accumulation, whereas naturally matured or artisan cheeses display more variable strain composition and ripening environments, which can elevate risk. In this context, the development of strains incapable of mycotoxin biosynthesis represents an important step toward safer cheese products. Here, we report the generation of P. roqueforti strains lacking the ability to synthesize roquefortine C and mycophenolic acid using CRISPR/Cas9. Single and double mutants deficient in one or both mycotoxins were obtained. Laboratory-scale cheeses produced under artisan-like conditions with these engineered strains contained no detectable levels of the target mycotoxins, in contrast to cheeses made with the wild-type strain. All mutants retained the ability to colonize cheese but displayed altered fungal biomass production compared to the native strain. These differences were consistent in curd and laboratory media and were not associated with changes in lipolytic or proteolytic activities. Further analyses revealed that while the absence of mycophenolic acid did not affect NaCl sensitivity, the lack of roquefortine C increased sensitivity to salt. Collectively, these results demonstrate the feasibility of producing mycotoxin-free blue cheeses using strains deficient in roquefortine C and mycophenolic acid biosynthesis, thereby laying the foundation for developing mycotoxin-free cheeses with engineered atoxigenic P. roqueforti strains.},
}
@article {pmid41308567,
year = {2025},
author = {Downton, P and Bates, N and Woods, S and Adamson, A and Sergouniotis, PI},
title = {Genome editing of a low-penetrance albinism-associated variant in TYR in patient-derived pluripotent stem cells.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103855},
doi = {10.1016/j.scr.2025.103855},
pmid = {41308567},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monophenol Monooxygenase/genetics/metabolism ; *Gene Editing/methods ; *Albinism/genetics/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; Male ; },
abstract = {TYR encodes tyrosinase, the enzyme catalysing the initial steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). TYR c.1205G>A (p.Arg402Gln) is a common genetic variant associated with several pigmentation traits. Notably, when this variant is encountered in specific haplotypic backgrounds in the homozygous state, it predisposes to albinism. We generated an induced pluripotent stem cell (iPSC) line from an affected individual carrying such a homozygous genotype (UMANi255-A), and then used CRISPR-Cas9 to correct the TYR c.1205G>A variant (UMANi255-A-1). The resulting iPSC lines demonstrate capacity for multi-lineage differentiation, providing a useful in vitro model for studying pigmentation biology.},
}
@article {pmid41309229,
year = {2025},
author = {Song, BS and Baek, YH and Kim, EH and Kwon, HI and Kim, AH and Lee, SH and Son, YB and Kim, SH and Song, MS and Choi, YK and Park, SJ},
title = {Development of an RT-LAMP-CRISPR/Cas12a assay for rapid and specific detection of Bandavirus dabieense.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {11},
pages = {e2506013},
doi = {10.71150/jm.2506013},
pmid = {41309229},
issn = {1976-3794},
support = {2022R1C1C1004704//National Research Foundation of Korea/ ; RS-2023-00301974//Ministry of Education/ ; //Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry/ ; RS-2022-IP322088//Ministry of Agriculture, Food and Rural Affairs/ ; },
mesh = {*Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; RNA, Viral/genetics ; *RNA Viruses/genetics/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.},
}
@article {pmid41309382,
year = {2025},
author = {Dao, TO and Park, HE and Lee, JH and Kim, KM and Trinh, MP and Kang, HL and Yoo, HS and Shin, MK},
title = {Advances and Challenges in Mycobacterial Genetic Engineering: Techniques for Knockout, Knockdown and Overexpression.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2507051},
pmid = {41309382},
issn = {1738-8872},
mesh = {*Genetic Engineering/methods ; *Gene Knockout Techniques/methods ; *Mycobacterium/genetics ; CRISPR-Cas Systems ; *Gene Knockdown Techniques/methods ; DNA Transposable Elements ; Homologous Recombination ; Gene Transfer Techniques ; },
abstract = {Genetic engineering of mycobacteria is challenging due to their hydrophobic cell wall structure and slow growth rates. Despite these obstacles, significant progress has been made to develop genetic engineering tools to study gene function and pathogenesis in these organisms. This review comprehensively explores the current methodologies employed in the genetic modification of mycobacteria, focusing on gene knockout, knockdown, and overexpression systems. Techniques covered include homologous recombination, recombineering, transposon mutagenesis, CRISPR-Cas systems, conditional expression strategies, and phage-mediated gene delivery. The mechanism, advantages, and limitations of those methods are critically analyzed, with particular emphasis on the adaptability of these tools to various mycobacterial species. By providing a detailed comparative analysis of available genetic tools, this review is a practical guide for researchers aiming to develop targeted and efficient genetic modifications in Mycobacterium species, accelerating discoveries in pathogenesis, drug resistance, and vaccine development.},
}
@article {pmid41309578,
year = {2025},
author = {Arana, S and Du, PP and Vaughan-Jackson, A and Enright, N and Spees, K and Valbuena, R and Garcia, CA and Nguyen, T and Venida, A and Seczynska, M and Bintu, L and Lehner, PJ and Prolo, LM and Bassik, MC},
title = {Reduced Cas9 transgene silencing by incorporation of intron sequences.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10656},
pmid = {41309578},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; R01 HG011866/HG/NHGRI NIH HHS/United States ; U54 CA261719/CA/NCI NIH HHS/United States ; R01HG011866//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Introns/genetics ; *Transgenes/genetics ; *Gene Silencing ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Chromatin/metabolism/genetics ; Animals ; Cell Line ; },
abstract = {Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.},
}
@article {pmid41309688,
year = {2025},
author = {Zhang, F and Peng, Y and Fan, D and Song, G and Gao, X and Tian, Y},
title = {Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse embryos.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10693},
pmid = {41309688},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Embryo, Mammalian/metabolism ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {IscB, as the putative ancestor of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein known to function in eukaryotic cells but requires a complex TAM (NWRRNA). Here, we characterize a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtain enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we find that enDelIscB-T5E displays robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we establish efficient miniature base editors (ICBE and IABE). Finally, we efficiently generate mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E into mouse embryos. Collectively, our work presents a set of enDelIscB-based miniature genome-editing tools with great potential for diverse applications in vivo.},
}
@article {pmid41309901,
year = {2025},
author = {Moroi, K and Yamamoto, T and Kurita, T},
title = {Double-strand break-free and transgene-free genome editing in the microalga Nannochloropsis oceanica using removable vectors containing the CRISPR base editing system.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42431},
pmid = {41309901},
issn = {2045-2322},
mesh = {*Gene Editing/methods ; *Microalgae/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plasmids/genetics ; DNA Breaks, Double-Stranded ; Transgenes ; *Stramenopiles/genetics ; },
abstract = {The accumulation of lipids by algae makes them attractive for carbon-neutral fuel production; however, the industrial-scale production of algal lipids has yet to be achieved. Currently, researchers are trying to improve the lipid productivity of algal strains using genome editing for molecular breeding with CRISPR-Cas9, which allows the efficient alteration of genomic information. However, CRISPR-based gene modification via double-strand breaks sometimes induces unintended large deletions that are toxic to host cells. Here, we applied the cytidine base editor combined with an episomal vector backbone containing a centromere and autonomous replication sequence to the microalga Nannochloropsis oceanica. The cytosine base editor introduces cytidine-to-thymidine base substitutions using deaminase without double-strand breaks, and an episomal vector enables plasmid removal after base substitution. We succeeded in inducing cytidine-to-thymidine substitution at the six target sites of five endogenous genes. The base substitution activity ranged from 29.2% to 47.6% on cytidine bases at the 16th to 19th positions from the protospacer adjacent motifs. The removal of base editor plasmids was also detected, which is essential for constructing transgene-free strains. Our results provide insights into the applicability of further technologies in the genetic modification of microalgae.},
}
@article {pmid41310261,
year = {2025},
author = {Spaans, GW and van der Berg, JP and Bouwman, LMS and Kleter, GA},
title = {Advancements in genomic crop techniques and considerations for regulation and food safety.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {49},
pmid = {41310261},
issn = {1573-9368},
mesh = {*Crops, Agricultural/genetics/growth &amp; development ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Food Safety ; Gene Editing ; Plant Breeding ; *Genomics/methods ; Food, Genetically Modified ; Mutagenesis ; Humans ; Genome, Plant ; },
abstract = {Advancements in genomic crop techniques have led to the development of new genetic technologies, such as base- and prime editing, but improvements have been made to existing conventional techniques as well. Fields in which these advancements occur include targeted mutagenesis, conventional random mutagenesis, and developments with null segregants, e.g., crops from which transgenic elements have been crossed out. In this review, we describe the developments in these three fields and provide considerations concerning regulatory and safety aspects. Because of differences in legislation of modern biotechnology between countries or regions, regulatory challenges are to be expected given the ongoing developments in genomic crop techniques. Moreover, the nature of the mutations induced with these newly developed techniques is not different from those induced with conventional techniques, making the modified crop plants indistinguishable from non-modified counterparts of the same crop species. Thus, enforcement of regulations cannot solely rely on technical analytical methods. Also, potential off-target or unintended effects in the primary mutants remain underexplored. Yet, these do not raise safety concerns owing to the experience with the crop breeding practice of iterative cycles for desirable traits selection, as well as the segregation and discard of unwanted phenotypes. Given that regulation will always change after innovation and developments within the sector advance rapidly, we advocate that both authorities and the breeding sector pro-actively implement a food safety culture. Such a safety culture will help developers of genomic technologies in crops to identify potential food safety issues at an early stage of development of future products.},
}
@article {pmid41310509,
year = {2025},
author = {Ekrami, A and Taheri, B and Daneshfar, S and Moradi, M and Ghorbani, A and Akhash, N and Jafarzadeh, Z and Farshadzadeh, Z and Saki, M},
title = {Occurrence of CRISPR-Cas genes and lack of association with antibiotic resistance in Shigella isolates collected from patients with diarrhea in Ahvaz, southwest Iran.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1666},
pmid = {41310509},
issn = {1471-2334},
abstract = {BACKGROUND: So far, few studies have examined the association between CRISPR-Cas and antibiotic resistance in Shigella isolates. Hence, this study sought to address this issue in Shigella species isolated from stool samples of patients with diarrhea in Ahvaz, southwest Iran. METHODS: In this cross-sectional study, stool samples were collected from 103 children (3–14 years) with diarrhea admitted to Abuzar Hospital affiliated to the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. All samples were cultured on the xylose lysine desoxycholate agar and hektoen enteric agar and incubated at 37 °C for 24 h. Primary identification of the Shigella species was performed by biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance rates were evaluated by the Kirby-Bauer disc diffusion. The prevalence of 12 CRISPR-Cas genes was investigated by PCR. RESULTS: Overall, 83 Shigella isolates were identified by standard biochemical tests. Finally, 72 Shigella isolates including 45 S. flexneri (46.8%) and 27 (53.2%) S. sonnei were confirmed by PCR. The most effective antibiotics were trimethoprim/sulfamethoxazole (n = 48, 66.7%), imipenem (n = 46, 63.9%), and ceftazidime (n = 43, 59.7%), respectively. All isolates harbored at least one of the CRISPR-Cas genes. Occurrence of CRISPR-Cas genes was as follows: CRa (100.0%), CRb (100.0%), CRc (100.0%), CSe2 (100.0%), CRf (95.8%), CSe1_Cas3 (87.5%), Cas2_Cas (84.7%), Cas (81.9%), CRd (80.6%), Cas7 (76.4%), Cas6e_Cas5 (72.2%), and CRe (48.6%). There was no significant association between the occurrence of CRISPR-Cas elements and resistance to any antibiotic in Shigella isolates (P-value ≥ 0.9999), except for Cas gene with ceftriaxone and cefepime. CONCLUSION: This study revealed high resistance rates of various antibiotics in Shigella isolates. However, there was no significant association between the existences of CRISPR-Cas genes with antibiotic resistance. Further investigation with higher sample size is needed to confirm this observation.},
}
@article {pmid41313207,
year = {2026},
author = {Wang, Y and Qin, Z and Wang, Q and Yang, Y and Gu, C and Yu, F and Wu, Y and Zhang, Lx},
title = {An RPA-CRISPR/Cas12a-based rapid and sensitive nucleic acid method for detection of Toxoplasma gondii in tissue and blood samples.},
journal = {Microbiology spectrum},
volume = {14},
number = {1},
pages = {e0155025},
pmid = {41313207},
issn = {2165-0497},
support = {231111111500//Key Research and Development Special Project of Henan Province of China/ ; 2022YFD1800200, 2023YFD1801200//National Basic Research Program of China/ ; },
mesh = {*Toxoplasma/genetics/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Protozoan/genetics ; *Toxoplasmosis/diagnosis/parasitology/blood ; Limit of Detection ; *Toxoplasmosis, Animal/diagnosis/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Toxoplasma gondii is a zoonotic pathogen that can infect humans and a wide range of warm-blooded animals, posing a significant threat to human health and the livestock industry. The development of a time-saving, highly sensitive, and specific method for the detection of T. gondii in tissue and blood samples is crucial to the monitoring, prevention, and control of toxoplasmosis. In this study, we evaluated the efficiency of a previously described method, termed REPORT, that integrates recombinase polymerase amplification with CRISPR/Cas12a for the detection of T. gondii nucleic acids. We evaluated the limit of detection (LOD) and specificity of the extended REPORT method using prepared target DNA in addition to tissue and blood samples. Furthermore, we validated the accuracy of T. gondii detection in clinical samples using the REPORT-based method in comparison with nested PCR based on the B1 gene. Sensitivity tests showed that the LOD of the REPORT-based fluorescence method and the lateral flow strip method were 3.7 copies /μL for target DNA, 3.1 tachyzoites/g for tissue samples, and five tachyzoites/mL for blood samples. Specificity tests suggested that the REPORT method had good specificity and did not cross-react with several common parasites. The method performed well for clinical DNA samples, demonstrating its ability for use in on-site detection.IMPORTANCEToxoplasma gondii can infect over 200 species of warm-blooded animals, including humans, posing not only a significant threat to public health systems but also causing substantial economic losses to the global livestock industry. Current diagnostic methods are slow, equipment-dependent, and impractical for field use. This study addresses these limitations by developing REPORT, a rapid, ultrasensitive nucleic acid test combining recombinase polymerase amplification and CRISPR/Cas12a. The REPORT detects T. gondii in tissue and blood samples within 1 h at low cost, requiring only a portable heater. Its visual results (fluorescence or test strips) enable on-site use without specialized training, achieving 100% accuracy versus nested PCR. With a sensitivity of 3.1 parasites per gram of tissue and five parasites per milliliter of blood, this method revolutionizes toxoplasmosis screening in resource-limited clinics, farms, and food safety inspections, empowering timely interventions to curb transmission and improve public health outcomes.},
}
@article {pmid41313840,
year = {2026},
author = {Mohammad, SI and Kareem, AK and Vasudevan, A and Rekha, MM and Jabir, MS and Nayak, P and AlKhafaje, Z and Arora, V and Kadhum, W and Chennakesavulu, K},
title = {Genome editing of immune checkpoints: CRISPR-mediated PD-1 inhibition in cancer.},
journal = {Seminars in oncology},
volume = {53},
number = {1},
pages = {152438},
doi = {10.1016/j.seminoncol.2025.152438},
pmid = {41313840},
issn = {1532-8708},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/immunology ; *Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; *Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; B7-H1 Antigen/antagonists &amp; inhibitors/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is a primary mechanism by which tumors evade immune surveillance, limiting the efficacy of cytotoxic T lymphocytes (CTLs) and tumor-infiltrating lymphocytes (TILs). Although immune checkpoint blockade therapies have revolutionized cancer treatment, their efficacy is restricted by acquired resistance, T-cell exhaustion, and tumor heterogeneity. The advent of CRISPR-Cas9 genome editing provides a precise and versatile approach to disrupt PD-1 or PD-L1, directly enhancing anti-tumor immune responses. Preclinical studies demonstrate that ex vivo PD-1 knockout in primary human T cells or TILs enhances proliferation, cytokine production, and cytotoxicity, resulting in improved tumor clearance in xenograft and humanized mouse models. In chimeric antigen receptor (CAR) T cell therapy, CRISPR-mediated disruption of PD-1 improves effector function, persistence, and resistance to exhaustion, with universal and allogeneic CAR-T platforms benefiting from multiplex genome editing. Direct PD-L1 knockout in tumor cells, often facilitated via nanoparticle- or biomaterial-assisted delivery, reshapes the immunosuppressive tumor microenvironment, promotes T cell infiltration, and enhances the efficacy of adoptive cellular therapy. Combination approaches integrating PD-1 editing with viral antigen targeting, long noncoding RNA (lncRNA) modulation, or conventional checkpoint blockade demonstrate synergistic anti-tumor effects. Clinically, early-phase trials in non-small cell lung cancer, mesothelin-positive solid tumors, and hematological malignancies establish the feasibility, safety, and preliminary efficacy of PD-1-deficient T cells. Despite these promising outcomes, challenges such as off-target effects, delivery efficiency, immunogenicity, long-term persistence, and regulatory considerations remain. This review aims to comprehensively evaluate preclinical and clinical studies investigating CRISPR-mediated PD-1/PD-L1 inhibition across various cancers, summarize mechanistic insights, and highlight translational opportunities and challenges for clinical implementation.},
}
@article {pmid41314751,
year = {2025},
author = {Fatima, M and Tariq, I and Tariq, A and Talib, S and Fatima, M and Shehzadi, M and Aqib, AI},
title = {Pharmacogenomics and CRISPR-based therapies.},
journal = {Progress in brain research},
volume = {297},
number = {},
pages = {319-343},
doi = {10.1016/bs.pbr.2025.08.009},
pmid = {41314751},
issn = {1875-7855},
mesh = {Humans ; *Pharmacogenetics/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {Pharmacogenomics and CRISPR-based treatments are two areas of precision medicine that are advancing together. Pharmacogenomics involves studying how differences in someone's genes can change the effect of medications on them. Pharmacogenomics helps reduce adverse reactions to drugs and improve healing by choosing and measuring drugs according to a patient's genetic information. Additionally, CRISPR-Cas systems now serve as leading genome editing tools that allow precise alterations at given points of the genome. CRISPR technology's use in pharmacogenomics creates new opportunities for modifying gene expression, fixing harmful mutations, and creating innovative treatment approaches. A more proactive approach to illness treatment is supported by this synergy, in which genetic factors serve as both direct targets for intervention and a basis for medication selection. This chapter examines the theoretical and practical frameworks that link CRISPR-based treatments with pharmacogenomics, emphasizing recent uses in pharmacoresistance, cancer, and monogenic diseases. To guarantee safe and fair deployment, it also covers the ethical, legal, and technical issues that need to be resolved. When combined, these technologies hold the potential to revolutionize medicine by facilitating individualized and curative drugs.},
}
@article {pmid41314912,
year = {2026},
author = {Chen, L and Ouyang, W and Hu, Y and Peng, L and Chen, P and Guo, W and Yang, H and Xu, J and Pan, M and Xu, D and Wang, X and Zhang, C and Chen, S and Hao, Q and Yuan, S and Huang, Y and Shan, Z and Yang, Z and Xia, R and Hewezi, T and Chen, H and Tran, LP and Zhou, X and Cao, D},
title = {Creating artificial miR2118a/b to boost yield and broad-spectrum resistance in soybean via CRISPR/Cas9-targeted mutation.},
journal = {Trends in biotechnology},
volume = {44},
number = {4},
pages = {1149-1166},
doi = {10.1016/j.tibtech.2025.10.022},
pmid = {41314912},
issn = {1879-3096},
mesh = {*Glycine max/genetics/growth &amp; development/microbiology ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Disease Resistance/genetics ; Mutation ; Plant Diseases/genetics/microbiology ; Pseudomonas syringae ; Gene Expression Regulation, Plant ; },
abstract = {While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops.},
}
@article {pmid41315077,
year = {2025},
author = {Loedige, KW and White, AL and McMurrough, TA and Stead, BE and Edgell, DR},
title = {A buffer-tuning strategy to profile domain-specific activity of chimeric I-TevI/CRISPR gene editors in vitro.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42742},
pmid = {41315077},
issn = {2045-2322},
support = {RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; ALLRP 571374 - 21//Mitacs/ ; ALLRP 571374 - 21//Mitacs/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Buffers ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Domains ; DNA Cleavage ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; DNA/metabolism ; },
abstract = {Protein-DNA interactions can be manipulated in vitro by changing buffer conditions. Here, we develop a methodology to map the cleavage preferences of chimeric gene editors that are fusions of the I-TevI nuclease domain to CRIPSR nucleases by manipulating in vitro salt concentrations. We found that DNA cleavage by the I-TevI (Tev) nuclease domain at CNNNG sites was de-coupled from the gRNA-targeted site in low salt buffers. For TevCas12a, this non-targeted cleavage activity was enriched at Tev CNNNG cleavage motifs optimally positioned within a 30-bp window upstream of a Cas12a TTTV PAM site. Non-targeted cleavage did not require Cas12a nuclease activity or specific Cas12a gRNA targeting. Similar non-targeted products were observed in low salt buffer conditions for TevSaCas9, Tev-meganuclease and Tev-zinc finger editors. Cas12a and SaCas9 activity at gRNA-directed sites and sites with multiple mismatches were also sensitive to buffer salt concentration. Oxford Nanopore sequencing revealed a remarkably similar Tev CNNNG cleavage preference at different salt concentrations and in different fusion contexts, emphasizing the robustness and specificity of Tev activity. More generally, our work highlights the sensitivity of gene editors to in vitro reaction conditions and how these conditions can be leveraged to functionally dissect the activity of individual domains of chimeric gene editors.},
}
@article {pmid41315225,
year = {2025},
author = {Wen, HP and Yu, C and Bi, S and Jiang, LH and Wang, ZG and Yao, Z and Pang, DW and Liu, SL},
title = {Programmable targeted RNA degradation via dCas13d-directed chaperone-mediated autophagy (dCasCMA).},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10738},
pmid = {41315225},
issn = {2041-1723},
support = {22293032//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22374138//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21977054//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24JCZDJC01240//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 23JCYBJC01880//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Humans ; Animals ; *Chaperone-Mediated Autophagy/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA Stability/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; HEK293 Cells ; Autophagy ; },
abstract = {RNA-targeted degradation technologies offer significant promise for treating diseases by selectively disrupting gene expression. However, a robust method to specifically, efficiently, and programmability degrade targeted RNAs in mammalian cells is still in demand. Here, we present a versatile platform, dCas13d-directed chaperone-mediated autophagy (dCasCMA), which integrates the precise targeting capabilities of dCas13/CRISPR with the degradation efficiency of chaperone-mediated autophagy (CMA) to achieve efficient degradation of specific RNAs. By combining dCas13d with a CMA-targeting motif and customizable guide RNA (gRNA), the platform allows for accurate targeting of both exogenous and endogenous RNAs in cells. Moreover, the incorporation of multiplexed gRNA expression arrays enables the simultaneous degradation of multiple RNA targets during viral pathogenesis in live cells and in vivo. Our findings emphasize the platform's modular design, which enables flexible combinations of dCCTM components with user-defined gRNA sequences. This versatility positions it as a promising tool for developing innovative therapies for various diseases.},
}
@article {pmid41315365,
year = {2025},
author = {Wang, Y and Liao, Y and Sun, Y and Mitra, B and Guo, R and Piedras, BI and White, S and Tang, HY and Asara, JM and Tempera, I and Lieberman, PM and Gewurz, BE},
title = {The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10731},
pmid = {41315365},
issn = {2041-1723},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; PF-24-1250090-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; P01CA269043//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P01 CA120964/CA/NCI NIH HHS/United States ; P01 CA269043/CA/NCI NIH HHS/United States ; R01AI164709//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; PF-23-1144614-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1194768-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1308318-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; R00 DE031016/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Lysosomes/metabolism ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Intracellular Membranes/metabolism ; Signal Transduction ; Phosphatidylinositol 3-Kinases/metabolism ; Cell Proliferation ; },
abstract = {Most Epstein-Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we perform a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, are among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. The CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associate with one another and with lysosome outer membranes to inhibit mTORC1. Rather than perturbing mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block the interaction between mTORC1 and Rheb and also with mTORC1 substrates S6 and 4E-BP1. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.},
}
@article {pmid41316351,
year = {2025},
author = {Qin, Z and Wang, Y and Sun, M and Wang, Q and Duan, J and Gu, C and Zhang, X and Yu, F and Wu, Y and Xu, H and Li, J and Zhang, L},
title = {Development of a field-deployable RPA-CRISPR/Cas12a assay for the detection of Cyclospora cayetanensis in human feces.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {4},
pmid = {41316351},
issn = {1756-3305},
support = {232102110088//Henan Province Scientific and Technological Project/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Project of Henan Province/ ; },
mesh = {*Cyclospora/isolation &amp; purification/genetics ; Humans ; *Feces/parasitology ; *Cyclosporiasis/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Cyclospora is an emerging intestinal pathogenic protozoan transmitted through foodborne and waterborne routes. At least 19 countries in the world have recorded outbreaks of cyclosporiasis, mainly associated with the consumption of contaminated fresh agricultural products. The lack of a sensitive immediate test is one of the major obstacles to the rapid diagnosis of cyclosporiasis. The target interference mechanisms of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been adapted into versatile and efficient genome manipulation and disease-curing technologies, while also being promising for point-of-care testing (POCT) applications. It can serve as an excellent rapid and specific detection tool.<br><br>METHODS: The recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system were combined to develop a detection method for C. cayetanensis (termed RECCT-Cay) via visual observation of fluorescent readings under blue light and field diagnosis using lateral flow strip (LFS) biosensors.<br><br>RESULTS: The detection limit of the established RECCT-Cay was 7 copies/&#x3bc;L. Under simulated clinical conditions, the detection limit was 30 oocysts per gram of stool. At the same time, the established detection platform can distinguish C. cayetanensis from the closely related Eimeria spp. The results of our constructed assay were compared with nested PCR, and the detection results of 30 clinical stool samples were consistent, with three samples positive for C. cayetanensis. Based on the RECCT-Cay detection principle, a portable suitcase-sized device has been designed, which can conduct rapid on-site detection of clinical samples.<br><br>CONCLUSIONS: The RECCT-Cay platform features rapid speed, high sensitivity, and the capability for field detection, making it a promising tool for use in remote areas.},
}
@article {pmid41316685,
year = {2026},
author = {Megarani, DV and Yang, L and Siler, HJ and Quijano Cardé, EM and Martyniuk, CJ and Hick, PM and Becker, JA and Soto, E and Surachetpong, W and Yanong, RPE and Subramaniam, K},
title = {One-Pot RT-LAMP CRISPR/Cas12b Platform for Rapid Detection of Tilapia Lake Virus.},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70087},
doi = {10.1111/jfd.70087},
pmid = {41316685},
issn = {1365-2761},
support = {2023-67015-39481//National Institute of Food and Agriculture/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; *RNA Virus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/veterinary/methods ; Aquaculture ; *RNA Viruses/isolation &amp; purification ; *Tilapia ; },
abstract = {Tilapia Lake Virus (TiLV) is a significant threat to global tilapia aquaculture, highlighting the need for rapid and accurate diagnostic methods to manage outbreaks and minimise economic losses. This study presents the development and partial validation of a one-pot assay integrating RT-LAMP with the CRISPR/Cas12b system for sensitive and specific TiLV detection. This assay amplifies viral RNA using RT-LAMP, while CRISPR/Cas12b enables a real-time detectable signal. Targeting a conserved region in TiLV segment four, the assay achieves results within 75 min at 62°C, with easy visualisation using a portable fluorescence viewer. It demonstrated high sensitivity, with a 95% limit of detection of 79.6 copies (95% CI: 48-132 copies), and high specificity, with no cross-reaction to other fish RNA or DNA viruses. Based on a validation panel of 261 samples from 9 source populations, the assay exhibited 92% diagnostic sensitivity (95% CI: 87%-96%) and 100% diagnostic specificity (95% CI: 97%-100%). When assessed as a non-lethal sample, gills provided a reliable and less invasive alternative despite lower viral loads compared to internal organs. Therefore, this partially validated one-pot assay is potentially practical for enhancing TiLV detection and disease management in aquaculture systems, especially in field settings and resource-limited laboratories.},
}
@article {pmid41317251,
year = {2026},
author = {Kheirandish, A and Sorourian, S and Behbahani, AB and Ahmadi, MKB and Dehbidi, GR and Rahimi, E and Safari, F},
title = {Optimizing Recombinant Protein Production in CHO Cells by Silencing the Caspase 8 Associated Protein 2 Gene via the CRISPR-Cas9 System.},
journal = {Molecular biotechnology},
volume = {68},
number = {6},
pages = {2837-2846},
pmid = {41317251},
issn = {1559-0305},
mesh = {Animals ; CHO Cells ; Cricetulus ; *CRISPR-Cas Systems ; *Recombinant Proteins/genetics/biosynthesis/metabolism ; Cell Survival/drug effects ; Gene Silencing ; Cricetinae ; Apoptosis ; Butyric Acid/pharmacology ; *Apoptosis Regulatory Proteins/genetics ; },
abstract = {Chinese hamster ovary (CHO) cells play a crucial role in biopharmaceutical production due to their ability to produce complex proteins. Enhancing the productivity of CHO cells is essential for meeting the growing demand for biologics. Caspase 8-Associated Protein 2 (CASP8AP2), a key regulator of apoptosis and cell survival, has been identified as a potential target to increase CHO cell productivity. To this end, CRISPR-mediated homology-independent targeted integration (HITI) was used to silence CASP8AP2. Results of the cell viability assay revealed that CASP8AP2-deficient clones (C2, C3, and C4) were more resistant to sodium butyrate (NaBu) compared to native cells, with IC50 values of 11.83, 12.77, 10.25, and 8.55 mM, respectively. Protein production assays showed a significant increase in JRed and luciferase expression in silenced clones (C2 and C4) compared to wild-type cells, with up to 1.2- and 1.9-fold increases for JRed, and 1.4- and 1.7-fold increases for luciferase, respectively. These findings could be attributed to the clones experiencing cell cycle arrest specifically during the S phase. While these results demonstrate proof-of-principle using reporter proteins, future validation with therapeutic biologics such as implementing monoclonal antibodies in bioreactor settings could confirm scalability for industrial bioprocessing. Transcriptomic analyses would further elucidate downstream effects on apoptosis and metabolism pathways. The results suggest that targeting CASP8AP2 could be a promising strategy for improving bioprocess efficiency and yield in CHO cell-based production systems.},
}
@article {pmid41317788,
year = {2026},
author = {Brogan, DJ and Lin, CP and Benetta, ED and Wang, T and Chen, F and Li, H and Lin, C and Komives, EA and Akbari, OS},
title = {Synthetic Type III-E CRISPR-Cas Effectors for Programmable RNA-targeting.},
journal = {Journal of molecular biology},
volume = {438},
number = {2},
pages = {169566},
doi = {10.1016/j.jmb.2025.169566},
pmid = {41317788},
issn = {1089-8638},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Domains ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.},
}
@article {pmid41317988,
year = {2026},
author = {Arya, SK and Goodman, CL and Palli, SR},
title = {The expanding toolkit of insect cell culture: a new era in biotechnology.},
journal = {Current opinion in insect science},
volume = {74},
number = {},
pages = {101465},
doi = {10.1016/j.cois.2025.101465},
pmid = {41317988},
issn = {2214-5753},
support = {R01 GM070559/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Insecta/genetics/cytology ; *Biotechnology/methods ; *Cell Culture Techniques/methods ; Cell Line ; Baculoviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {Insect cell culture has become an essential platform in modern biotechnology, valued for its safety, scalability, and ability to perform complex post-translational modifications. This review highlights the latest and most important advances in the field. We focus on efforts at developing and engineering new insect cell lines, innovations in expression systems, especially the baculovirus expression vector system and the transformative impact of CRISPR/Cas9-based genome editing. Additionally, we explore breakthroughs that improve the efficiency of recombinant protein production and discuss key challenges such as viral contamination and expression instability. Collectively, these developments mark an important step forward in insect cell biotechnology and are expected to enhance the efficiency and scalability of producing vaccines and biopharmaceuticals. Together, these innovations illustrate a transition from cataloging cell line development to understanding the mechanisms and engineering principles driving these advances. This review not only summarizes recent progress but also provides perspective on how foundational lepidopteran models have guided innovations now extending into dipteran, hemipteran, and hymenopteran systems, shaping the future of insect biotechnology.},
}
@article {pmid41318543,
year = {2025},
author = {Pernaci, C and Johnson, A and Gillette, S and Warden, AS and McCormick, C and Weiser-Novak, S and Ramirez, G and Broersma, EH and Mishra, P and Sivakumar, A and Cherqui, S and Coufal, NG},
title = {Microgliopathy as a primary mediator of neuronal death in models of Friedreich's Ataxia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {81},
pmid = {41318543},
issn = {2041-1723},
support = {R01NS135162//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; TRAN1-13983//California Institute for Regenerative Medicine (CIRM)/ ; EDUC2-08388//California Institute for Regenerative Medicine (CIRM)/ ; R01 AG086443/AG/NIA NIH HHS/United States ; S10 OD025060/OD/NIH HHS/United States ; R01 NS135162/NS/NINDS NIH HHS/United States ; },
mesh = {*Friedreich Ataxia/pathology/genetics/metabolism ; Animals ; *Microglia/pathology/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Frataxin ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Cell Death ; Mitochondria/metabolism/pathology ; *Neurons/pathology/metabolism ; Purkinje Cells/pathology/metabolism ; Trinucleotide Repeat Expansion ; Iron/metabolism ; Male ; Female ; White Matter/pathology ; CRISPR-Cas Systems ; },
abstract = {Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by a GAA repeat expansion in the frataxin (FXN) gene, leading to a severe reduction of the mitochondrial FXN protein, crucial for iron metabolism. While microglial inflammation is observed in FRDA, it remains unclear whether immune dysfunction is a primary disease mediator or a secondary reactionary phenotype. Utilizing patient-derived induced pluripotent stem cells (iPSCs), we report an intrinsic microglial phenotype of stark mitochondrial defects, iron overload, lipid peroxidation, and lysosomal abnormalities. These factors drive a pro-inflammatory state that contributes to neuronal death in co-culture systems. In a murine xenograft model, transplanted human FRDA microglia accumulate in white matter and the Purkinje cell layer, resulting in Purkinje neuron loss in otherwise healthy brains. Notably, CRISPR/Cas9-mediated correction of the GAA repeat reverses microglial defects and mitigates neurodegeneration. Here, we suggest that microglial dysfunction serve as a disease driver and a promising therapeutic target in FRDA.},
}
@article {pmid41319833,
year = {2026},
author = {Hu, H and Ke, X and Xiao, H and Shang, X and Yin, Q and Li, J and Li, X and Hu, Z and Qian, P and Wang, M},
title = {Genomic assembly, rescue, and characterization of a functional pseudorabies virus.},
journal = {Virologica Sinica},
volume = {41},
number = {1},
pages = {97-106},
pmid = {41319833},
issn = {1995-820X},
mesh = {Animals ; *Herpesvirus 1, Suid/genetics ; *Genome, Viral ; Chlorocebus aethiops ; Vero Cells ; Swine ; Mice ; Pseudorabies/virology ; Gene Editing ; Mice, Inbred BALB C ; CRISPR-Cas Systems ; Swine Diseases/virology ; },
abstract = {With recent advances in synthetic biology methods, the genomes of several large DNA viruses have been de novo synthesized and assembled, leading to the functional rescue of the respective viruses. Pseudorabies virus (PRV), a large DNA virus belonging to the family Herpesviridae, causes severe diseases in swine, resulting in significant economic losses to the global pig farming industry. Genome editing is crucial for attenuating virulence and developing safer vaccines for PRV. However, its complex repetitive sequences and extremely high GC-rich genome pose significant challenges for genetic manipulation. In this study, we developed a PRV genome assembly platform using yeast-based transformation-associated recombination (TAR) technology. The genome of a prevalent genotype II variant strain, PRV-GX-2011 (GenBank number PV405324.1), was divided into nine A-level fragments and cloned into vectors via TAR. Subsequently, three B-level fragments were generated by recombining three A-level fragments each. In vitro CRISPR/Cas9-mediated editing was introduced to insert an egfp gene into the non-coding intergenic region between UL23 and UL22 genes. Infectious viruses were rescued by co-transfection of linearized B-level fragments in Vero cells, and an isolated virus, PRV-GX-Syn1, was purified via plaque assay. While PRV-GX-Syn1 exhibited reduced viral titer and smaller plaque size compared to the parental strain, its morphological characteristics remained indistinguishable from the parental virus. In BALB/c mice, PRV-GX-Syn1 caused lethal infection, producing lung pathology comparable to the parental strain. This TAR-based platform offers faster and more flexible genomic modification of PRV, facilitating both basic research and PRV-based vaccine vectors.},
}
@article {pmid41319963,
year = {2026},
author = {Kong, H and Wang, S and Zhuo, C and Zhong, Q and Xu, Y and Lao, YH and Lv, S and Xie, X and Yuan, Q and Li, K and Tao, Y and Li, M},
title = {Nanovesicles integrating PD-1-mediated targeting and CRISPR/Cas9-based CD47 editing for dual immune checkpoint blockade.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114480},
doi = {10.1016/j.jconrel.2025.114480},
pmid = {41319963},
issn = {1873-4995},
mesh = {*CD47 Antigen/genetics ; CRISPR-Cas Systems ; Animals ; Gene Editing ; *Immune Checkpoint Inhibitors/administration &amp; dosage ; *Programmed Cell Death 1 Receptor/genetics/immunology ; Humans ; B7-H1 Antigen/immunology ; Mice ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; Mice, Inbred C57BL ; Female ; Immunotherapy/methods ; },
abstract = {Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment, yet many tumors evade immune surveillance through multiple suppressive mechanisms. In particular, the adaptive immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and the innate "don't eat me" signal CD47/signal-regulatory protein alpha (SIRPα) represent two distinct pathways that cancers exploit to avoid T-cell attack and macrophage phagocytosis, respectively. Herein, we present BITE (Biomimetic Immune Targeting and Editing), a genetically engineered biomimetic nanoplatform designed to concurrently blockade both pathways by combining PD-1-mediated tumor targeting with CRISPR/Cas9 gene editing of CD47. BITE nanovesicles display PD-1 on their surface, enabling selective binding to PD-L1-expressing tumor cells and local disruption of PD-1/PD-L1 signaling. Simultaneously, they deliver a CRISPR/Cas9 payload that knocks out the CD47 gene in tumor cells, abolishing the anti-phagocytic signal and thus activating innate immune clearance. We demonstrate that BITE efficiently homes to PD-L1-positive tumors in vitro and in vivo, achieves significant CD47 gene disruption in tumor cells, and triggers robust phagocytosis by macrophages. In a mouse tumor model, dual checkpoint blockade by BITE reshapes the tumor microenvironment, yielding increased infiltration of CD4[+] T cells, CD8[+] T cells, and M1 macrophages; treatment with BITE induces pronounced tumor regression and extended survival, outperforming single-target controls. Our results establish a proof-of-concept for this dual-function nanovesicle approach, highlighting its potential to engage both adaptive and innate immunity synergistically. The BITE platform offers a versatile and targeted strategy to overcome immune resistance in cancer, representing a promising therapeutic avenue in biomedical engineering and nanomedicine.},
}
@article {pmid41321825,
year = {2025},
author = {Senthilraja, G and Sandhya, M and Priyadharshini, E and Anand, T and Kavitha, M and Tharmalingam, N},
title = {Targeting effector proteins of plant pathogens as a strategy for durable plant disease resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1681047},
pmid = {41321825},
issn = {1664-302X},
}
@article {pmid41323291,
year = {2025},
author = {Li, Y and Hall-Ponselè, AM},
title = {Plant Cell Strain Improvement Through Engineering Biology for Industrial Plant Cell Culture.},
journal = {Engineering biology},
volume = {9},
number = {1},
pages = {e70002},
pmid = {41323291},
issn = {2398-6182},
abstract = {Plant cell culture (PCC) presents a promising and sustainable alternative to traditional agricultural methods for producing specialty bioactive compounds. However, its widespread industrial application has been hindered by challenges such as low yields, cell line instability and inconsistent product quality. engineering biology (EB) offers a powerful toolkit to overcome these limitations by systematically improving plant cell lines. This review focuses on the application of EB principles to enhance PCC for the production of high-value bioactives from an industry-oriented perspective. We explore three core pillars of the EB toolkit: (1) Multiomics and in silico design, which leverage comprehensive data integration and predictive modelling for rational target identification; (2) gene manipulation and pathway bioengineering, encompassing precise genome editing (e.g., CRISPR/Cas), synthetic gene circuits and directed evolution for targeted metabolic reprogramming and (3) biosensors for high-throughput screening and real-time monitoring, enabling rapid testing and optimisation of engineered cell lines. The synergistic integration of these tools within the iterative design-build-test-learn (DBTL) cycle is highlighted as a key strategy for accelerating strain improvement. Ultimately, the convergence of these EB approaches is transforming PCC into a robust platform for producing pharmaceuticals, functional foods and green chemicals, contributing to a biobased economy with a minimal ecological footprint.},
}
@article {pmid41324601,
year = {2025},
author = {Bhattacharya, S and Goyal, K and Satpati, P},
title = {Thermodynamics of PAM Recognition by Cas9 of Streptococcus pyogenes.},
journal = {Journal of chemical information and modeling},
volume = {65},
number = {24},
pages = {13328-13337},
doi = {10.1021/acs.jcim.5c01934},
pmid = {41324601},
issn = {1549-960X},
mesh = {*Streptococcus pyogenes/enzymology ; *Thermodynamics ; *CRISPR-Associated Protein 9/metabolism/chemistry ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; Gene Editing ; Base Sequence ; },
abstract = {The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of SpCas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. SpCas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. SpCas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of SpCas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of SpCas9 to differentiate between noncanonical and canonical PAMs (ΔΔG) is directly related to the number of direct interactions between SpCas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔG. The calculated ΔΔG adequately explains the observed differences in DNA cleavage activity of SpCas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in SpCas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.},
}
@article {pmid41324747,
year = {2025},
author = {Priyanka, SS and Iqbal, G and Nidarshan, NC and Kumari, K and Vanjre, S and Rasal, K and Sonwane, A and Brahmane, M and Goswami, M},
title = {Avenues of genome editing for color trait improvement in ornamental fishes: current status and future perspectives.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {51},
pmid = {41324747},
issn = {1573-9368},
mesh = {Animals ; *Gene Editing/methods ; *Fishes/genetics/growth &amp; development ; *Pigmentation/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics/growth &amp; development ; },
abstract = {Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.},
}
@article {pmid41325351,
year = {2025},
author = {Jansen van Vuuren, J and Matthews, MC and Robène, I and Rozsasi, S and Campa, M and Burger, J and Viljoen, A and Mostert, D},
title = {Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68841},
pmid = {41325351},
issn = {1940-087X},
mesh = {*Fusarium/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Recombinases/genetics/chemistry/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; DNA, Fungal/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.},
}
@article {pmid41325824,
year = {2026},
author = {Uddin, N and Ullah, MW and Zhu, D and Li, X and Yang, S and Xie, X},
title = {Engineering lignin pathway, plant cell wall modification, and genome editing for advanced renewable bioenergy and material applications.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108772},
doi = {10.1016/j.biotechadv.2025.108772},
pmid = {41325824},
issn = {1873-1899},
mesh = {*Lignin/biosynthesis/genetics/metabolism ; *Cell Wall/metabolism/genetics ; *Gene Editing ; *Plants/genetics/metabolism ; *Metabolic Engineering ; *Plant Cells/metabolism ; *Biofuels ; CRISPR-Cas Systems ; },
abstract = {Lignin biosynthesis and plant cell wall engineering are central to plant structural integrity and biomass utility. Recent advances in molecular and synthetic biology have opened opportunities to tailor lignin contents, composition, and polymer structure for renewable bioenergy and sustainable biomaterial applications. This review provides an integrative perspective on biosynthesis, regulation, and engineering of lignin. It summarizes the current progress in understanding the genetic, transcriptional, epigenetic, and metabolic networks that control lignin formation, with a focus on emerging tools such as CRISPR/Cas genome editing, synthetic promoters, and metabolic rewiring. Beyond cataloguing current knowledge, it critically analyzes the trade-offs involved in lignin modification for biomaterials, addressing unresolved challenges such as monolignol transport, metabolic flux control, and species-specific regulatory divergence. Engineered lignin and modified plant cell walls hold significant potential for biorefineries, advanced polymers, pharmaceuticals, and carbon sequestration, yet their translation from the laboratory to the field remains limited. Engineered lignin offers real-world applications across diverse industries, including bioenergy, bioplastics, carbon fiber composites, pharmaceuticals, and sustainable construction materials, thereby reinforcing its pivotal role in advancing a circular bioeconomy. The review further proposes future research directions that integrate multi-omics, single-cell technologies, machine learning, and field-based validation to enable precision lignin engineering. Strategic advances in this field will support next-generation bioenergy systems, advanced biomaterials, and the transition to a circular bioeconomy.},
}
@article {pmid41325825,
year = {2026},
author = {Zhang, Y and Deng, S},
title = {Geminivirus vectors: From gene silencing to synthetic biology.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108771},
doi = {10.1016/j.biotechadv.2025.108771},
pmid = {41325825},
issn = {1873-1899},
mesh = {*Geminiviridae/genetics ; *Synthetic Biology/methods ; *Genetic Vectors/genetics ; *Gene Silencing ; Gene Editing ; Genetic Engineering ; },
abstract = {Geminiviruses, the largest plant DNA virus family, cause devastating diseases in crops worldwide. These viruses possess distinctive features, such as the stem-loop structure and replication protein (Rep), which enable the creation of functional geminiviral replicons (GVRs) in plants. Over three decades, geminiviruses have been developed into vectors for virus-induced gene silencing (VIGS), high-level protein expression, and genome editing. This review introduces the genomic structure, Rep protein domains and functions, as well as the historical applications of geminiviruses, then highlights their prominent roles in VIGS and synthetic biology. As VIGS vectors, bipartite geminiviruses utilize AV1 gene replacement, while monopartite species rely on satellite DNAs to insert target sequences, enabling gene silencing in diverse plants. In synthetic biology, GVRs facilitate high-level protein expression through autonomous replication and enhance CRISPR/Cas genome editing efficiency in crops. Additionally, gene regulatory elements, including tissue-specific promoters and gene expression enhancement sequences from geminiviral genomes or satellite DNA expand their utility in genetic engineering. Finally, this review provides an outlook on the future development of geminivirus vectors. GVRs can work as plasmid-like DNAs for supporting diverse and creative designs in plant synthetic biology. The stem-loop structure and Rep are not unique to geminiviruses, a fact that suggests potential cross-kingdom applications of GVRs beyond plants. Vast viral resources enable further acceleration of GVR applications through resource mining and optimization. Moreover, attenuated or engineered geminiviral strains hold promise as "plant vaccines" via cross-protection. Collectively, geminivirus vectors bridge fundamental viral research with practical innovations in crop improvement, biomanufacturing, and synthetic biology.},
}
@article {pmid41326076,
year = {2026},
author = {Niu, RC and Zeng, QH and Wang, WJ and Hu, J and Liu, TX and Zhang, SZ},
title = {Multi-omics analyses identify the modulators COX6A1 and NAL as regulators of silk cocoon formation in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {216},
number = {Pt 1},
pages = {106764},
doi = {10.1016/j.pestbp.2025.106764},
pmid = {41326076},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics/metabolism/growth &amp; development ; *Silk/biosynthesis/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Larva/metabolism/genetics/growth &amp; development ; Proteomics ; CRISPR-Cas Systems ; *Electron Transport Complex IV/genetics/metabolism ; Transcriptome ; Multiomics ; },
abstract = {The diamondback moth (DBM) is a major global pest of cruciferous crops. Silk production, essential for DBM larval locomotion and pupal attachment, is governed by fibroin heavy chain (FibH), fibroin light chain (FibL), and fibrohexamerin (P25). However, the regulatory mechanisms and downstream key genes involved in silk production in DBM remain poorly understood. To address this, we integrated transcriptomic and proteomic data from CRISPR/Cas9 generated PxFibH, PxFibL, and PxP25 mutants to investigate the impact of silk gene deletions in the silk gland and identify modulators of silk formation. In the transcriptomic analysis, we identified 1994, 913, and 1266 differentially expressed genes (DEGs) in the three mutant strains, respectively. GO and KEGG enrichment analysis revealed significant involvement in pathways such as oxidation-reduction process, transmembrane transport, enzyme activity, and extracellular matrix (ECM) receptor interaction. At the proteomic level, 604, 210, and 266 differentially expressed proteins (DEPs) were identified in the three mutants, respectively. GO and KEGG enrichment analysis of these DEPs consistently highlighted energy metabolism, hydrolase activity, and catalytic activity pathways. Integrated multi-omics analyses identified three conserved regulator genes: cytochrome c oxidase subunit 6A1 (COX6A1), N-acetylneuraminate lyase (NAL), and protein phosphatase 1 regulatory subunit 14B (PPP1R14B). CRISPR/Cas9 knockout of PxCOX6A1 resulted in incomplete cocoon formation, along with increased larval mortality, prolonged development, and reduced oviposition. PxNAL knockout was lethal, while heterozygotes exhibited decreased cocoon formation, pupal weight, and fecundity. This study reveals FibH/FibL/P25-dependent metabolic networks regulating silk production and identifies COX6A1 and NAL as novel targets for environmentally sustainable pest control strategies.},
}
@article {pmid41328347,
year = {2026},
author = {Li, Q and Bao, Q and Zhao, S and Wu, F and Li, Y and Wang, K and Li, W and Gao, H},
title = {Advancements in CRISPR-based therapies for ocular pathologies: from disease mechanisms to intervention strategies.},
journal = {Theranostics},
volume = {16},
number = {1},
pages = {156-192},
pmid = {41328347},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Eye Diseases/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Eye diseases caused by genetic mutations affect over 2.2 billion people worldwide. The development of CRISPR technology has opened exciting possibilities for how we diagnose and treat these conditions. However, designing effective CRISPR systems, managing potential risks, and considering the ethical questions around gene therapy in clinical practice are major challenges. To move forward successfully, it's important to evaluate how practical CRISPR-based treatments are for eye diseases from a clinical perspective, while also understanding how CRISPR systems work. In this review, we start by covering the basic principles behind CRISPR technology and explore its different types. Next, we look at various ways CRISPR is being used in eye research and treatments, from early studies to new clinical approaches. Lastly, we address the regulatory environment and ethical issues involved, discussing existing rules, safety concerns, and guidelines for genetic modifications in medical settings. Our goal is to share new insights into innovative treatments for eye diseases and to support the safe use of CRISPR in clinical eye care. This review aims to be a helpful resource for researchers, doctors, and regulators working on CRISPR-based therapies.},
}
@article {pmid41328409,
year = {2026},
author = {Zhang, H and Song, Y and Liu, W and Zheng, X and An, X and Li, C and Chen, W and Wang, H and Zhang, Y},
title = {Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages.},
journal = {Synthetic and systems biotechnology},
volume = {12},
number = {},
pages = {59-70},
pmid = {41328409},
issn = {2405-805X},
abstract = {Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed defect-complementation homologous recombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two lox sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb gp0.4-0.7 and 1.02-kb gp4.3-4.7), achieved gene insertion (3.08-kb lacZ), and introduced a single-base substitution (TGA to TAA) in the stop codon of gp9 within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 10[5] PFU mL[-1] (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.},
}
@article {pmid41328592,
year = {2025},
author = {Geng, Y and Jiang, C and Zhang, H and Yang, H and Peng, Y and Chen, Y and Hu, C and Liu, H and Li, S and Chen, H and Xie, S and Guo, A},
title = {Genome-scale CRISPR screen identifies host factors associated with bovine parainfluenza virus 3 infection.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2589554},
pmid = {41328592},
issn = {2150-5608},
mesh = {Animals ; Cattle ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; CRISPR-Cas Systems ; Virus Replication ; *Host-Pathogen Interactions/genetics ; Cell Line ; *Respirovirus Infections/virology/veterinary/genetics ; Wnt-5a Protein/genetics/metabolism ; *Cattle Diseases/virology ; Virus Internalization ; Gene Knockout Techniques ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV-3) is a major pathogen associated with the bovine respiratory disease complex. However, the limited understanding of host factors crucial for BPIV-3 replication has hindered the development of effective preventive and therapeutic strategies. To tackle this critical issue, we constructed a bovine genome-wide CRISPR/Cas9 knockout library in Madin-Darby bovine kidney cells, which was then used to systematically identify and characterize the host genes essential for BPIV-3a replication. Subsequently, 10 genes were validated using both RT-qPCR and viral titration assays. Furthermore, through gene knockout or knockdown and rescue experiments, we identified three key genes required for BPIV-3a replication: Wnt family member 5A (WNT5A), solute carrier family 16 member 13 (SLC16A13), and selenoprotein N (SELENON). However, their effects on viral adhesion and internalization varied. WNT5A was involved in both processes, SLC16A13 participated solely in internalization, while SELENON had no significant impact on either. Beyond BPIV-3a, these three genes were also found to be essential for the infection of BPIV-3c and Bovine enterovirus. In conclusion, this study offers novel insights into the molecular mechanisms governing the replication and pathogenesis of BPIV-3a, BPIV-3c, and bovine enterovirus within host cells, thereby providing a foundation for identifying potential targets in the development of novel antiviral strategies.},
}
@article {pmid41328622,
year = {2026},
author = {Ma, C and Zhou, Y and Jiang, N and Ren, X and Xu, C and Su, N and Fan, Y and Liu, W},
title = {Development of an RPA-CRISPR-Cas12a Fluorescence Assay for Rapid and Sensitive Detection of Tilapia Parvovirus (TiPV).},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70095},
doi = {10.1111/jfd.70095},
pmid = {41328622},
issn = {1365-2761},
support = {32202994//the National Natural Science Foundation of China/ ; 2023TD46//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2024XT0602//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Tilapia ; *Parvovirus/isolation &amp; purification/genetics ; },
abstract = {Tilapia parvovirus (TiPV) is an emerging pathogen associated with high mortality rates in farmed tilapia, highlighting the urgent need for rapid and accurate diagnostic tools. In this study, we established an RPA-CRISPR/Cas12a detection system targeting the TiPV NS1 gene. The assay conditions were systematically optimised, including 15-min RPA amplification at 39°C, with reagent concentrations of 200 nM Cas12a, 250 nM crRNA and 200 nM ssDNA reporter. Specificity tests showed no cross-reactivity with other tilapia pathogens (TiLV, S. agalactiae) and other aquatic pathogens (LMBRaV, YcCV, GCRV II, WSSV, CyHV-2, SVCV). Sensitivity evaluation revealed a limit of detection (LoD) of 1.97 × 10[1]copies/μL, which was 100-fold more sensitive than PCR (1.97 × 10[3]copies/μL). Clinical validation with 20 tilapia samples demonstrated a 50% positive detection rate for RPA-CRISPR/Cas12a, 15% higher than PCR (35%). This integrated method combines the advantages of RPA and CRISPR-based signal transduction, offering a field-applicable solution for TiPV monitoring in resource-limited aquaculture environments.},
}
@article {pmid41328758,
year = {2026},
author = {Plewnia, A and Hoenig, BD and Lötters, S and Heine, C and Erens, J and Böning, P and Bending, GD and Krehenwinkel, H and Williams, MA},
title = {The Emergence of a CRISPR-Cas Revolution in Ecology: Applications, Challenges, and an Ecologist's Overview of the Toolbox.},
journal = {Molecular ecology resources},
volume = {26},
number = {1},
pages = {e70086},
pmid = {41328758},
issn = {1755-0998},
support = {//University of Warwick/ ; NE/S010270/1//Natural Environment Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Ecology/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated nucleases) systems allow researchers to detect, capture, and even alter parts of an organism's genome. However, while the use of CRISPR-Cas has revolutionised many fields in the life sciences, its full potential remains underutilised in ecology and biodiversity research. Here we outline the emerging applications of CRISPR-Cas in ecological contexts, focusing on three main areas: nucleic acid detection, CRISPR-enhanced sequencing, and genome editing. CRISPR-based nucleic acid detection of environmental DNA samples is already reshaping species monitoring, providing highly sensitive and non-invasive tools for both scientists and the public alike, with reduced costs and minimal experience required. Further, CRISPR-enhanced sequencing, including Cas-mediated target enrichment, enables efficient recovery of ecologically relevant loci and supports diverse applications such as amplification-free metagenomics. Finally, while genome editing on wild species remains largely theoretical in ecology, these tools are already being used in controlled settings to study adaptation and resilience in the face of ongoing global stressors. Together, the applications of CRISPR-Cas are paving the way for more affordable, accessible, and impactful applications for species conservation, and promise to improve our ability to tackle the ongoing global biodiversity crisis.},
}
@article {pmid41329281,
year = {2025},
author = {Kolesov, DE and Orlova, NA and Vorobiev, II},
title = {Generation of Long-Lived CHO Cells Suitable for Production of Afucosylated Antibodies and Fc-Fusion Proteins.},
journal = {Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections},
volume = {525},
number = {1},
pages = {359-367},
pmid = {41329281},
issn = {1608-3105},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Immunoglobulin Fc Fragments/genetics/metabolism ; *Fucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; *Recombinant Fusion Proteins/genetics/biosynthesis ; *Antibodies/metabolism ; Gene Knockout Techniques ; },
abstract = {Using genome editing, we created a homozygous α-(1,6)-fucosyltransferase (FUT8[-]/[-]) knockout in apoptosis-resistant CHO 4BGD cells, yielding the new 4BGD-F cell line. Combining CRISPR/Cas9 with paired gRNAs and non-specific puromycin selection yielded a cell population with an exceptionally high FUT8 knockout frequency, obviating the need for metabolic enrichment with lentil lectin (Lens culinaris agglutinin, LCA). Despite impaired clonogenicity of the knockout cells, we successfully isolated multiple clonal cell lines harboring extensive biallelic FUT8 deletions. Isolated clones with biallelic deletions retained key parental line characteristics: viability >90% in 17-day fed-batch cultures at high densities (>15 × 10[6] cells/mL), and rapid selectability using both dihydrofolate reductase and glutamine synthetase systems. Mass spectrometric analysis of the test protein GLP1-Fc secreted by 4BGD-F cells confirmed the absence of N-glycan fucosylation. The CHO 4BGD-F cell line provides a valuable platform for producing afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.},
}
@article {pmid41329461,
year = {2026},
author = {Nguyen, AH and Quang, MT},
title = {CRISPR/Cas9 Genome Editing in Oncology: Mechanisms, Therapeutic Platforms and Translational Challenges.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2201-2229},
pmid = {41329461},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; Translational Research, Biomedical ; },
abstract = {The CRISPR/Cas9 genome editing technology has had a significant impact on cancer research and therapeutic development, providing unprecedented precision in manipulating cancer-associated genes. Although this review focuses on Cas9, we situate it within the broader CRISPR landscape that includes DNA-targeting effectors (Cas9/Cas12), RNA-targeting systems such as Cas13, and type III systems with dual DNA and RNA activity, modalities that expand both experimental and therapeutic possibilities. This comprehensive review examines the current applications of CRISPR/Cas9 in oncology, including its mechanisms and the challenges associated with its clinical translation. Knockout, interference, and activation CRISPR screening platforms have transformed functional genomics by systematically interrogating gene function, identifying therapeutic vulnerabilities, and clarifying resistance mechanisms across diverse cancer phenotypes. This technology has also reshaped cancer modeling, enabling precise recapitulation of disease-relevant mutations from engineered cell lines to patient-derived xenografts that capture tumor heterogeneity and microenvironmental interactions. Notably, the integration of CRISPR/Cas9 with CAR-T therapy has enabled multiplex editing to eliminate alloreactivity, overcome checkpoint-mediated exhaustion, and engineer universal CAR-T cells. Emerging in vivo strategies that directly generate or reprogram CAR-T cells in patients via targeted viral and nonviral delivery underscore accelerating translational momentum. However, significant challenges, including off-target mutagenesis, delivery barriers, p53-mediated selective pressure favoring potentially oncogenic populations, and Cas9 immunogenicity, continue to hinder clinical translation. These limitations necessitate high-fidelity nucleases, optimized guide designs, and improved delivery systems. The future of CRISPR/Cas9 in cancer therapy will depend on technological innovation, comprehensive safety frameworks, and rigorous clinical evaluation as next-generation editing modalities advance toward transformative precision oncology.},
}
@article {pmid41329618,
year = {2025},
author = {Udemezue, VC and Shaikh, KM and Vorontsova, M and Valgepea, K},
title = {Optimization of Plasmid Curing from Genetically Engineered Clostridium autoethanogenum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4967-4972},
pmid = {41329618},
issn = {2161-5063},
mesh = {*Plasmids/genetics ; *Clostridium/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; Gene Editing/methods ; *Genetic Engineering/methods ; Electroporation ; },
abstract = {Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO2 as their sole carbon source with H2. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14-100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.},
}
@article {pmid41330004,
year = {2025},
author = {Coşar, B and Kılıç, P and İşeri, &#xd6;D},
title = {The intersection of CAR-T immunotherapy with emerging technologies.},
journal = {Cytokine &amp; growth factor reviews},
volume = {86},
number = {},
pages = {238-259},
doi = {10.1016/j.cytogfr.2025.11.001},
pmid = {41330004},
issn = {1879-0305},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology/genetics ; *Neoplasms/therapy/immunology ; Animals ; Cytokines/immunology ; *T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing ; Tumor Microenvironment/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy is a transformative modality in cancer immunotherapy that employs genetically engineered T-cells to eliminate malignant cells selectively. Its efficacy and limitations are governed by cytokine- and growth factor-mediated signaling networks that shape T-cell activation, proliferation, differentiation, and persistence. This review traces the molecular evolution of CAR-T architecture across generations, highlighting how synthetic modulation of cytokine and co-stimulatory pathways enhances potency while reducing exhaustion and toxicity. We discuss strategies that incorporate cytokine engineering, metabolic reprogramming, and logic-gated activation to counteract the immunosuppressive tumor microenvironment. Recent technological advances-such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based cytokine pathway editing, induced pluripotent stem cell (iPSC)-derived "off-the-shelf" CAR-T platforms, and extracellular vesicle (EV)-mediated cytokine delivery-are reshaping adoptive immunotherapy. Framing CAR-T development through the lens of cytokine and growth factor biology, we outline how integrating these pathways enables safer, more durable, and scalable next-generation therapies for hematologic and solid tumors.},
}
@article {pmid41330077,
year = {2026},
author = {Fathpour, H and Fouladi, M and Jafarpour, F and Moradi-Hajidavaloo, R and Izadi, T and Shiralian-Esfahani, H and Kues, W and Nasr-Esfahani, MH and Hajian, M and Eghbalsaied, S},
title = {Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.},
journal = {Research in veterinary science},
volume = {198},
number = {},
pages = {105992},
doi = {10.1016/j.rvsc.2025.105992},
pmid = {41330077},
issn = {1532-2661},
mesh = {Animals ; *Myostatin/genetics/metabolism ; *Goats/genetics ; *Fibroblasts/metabolism ; *CRISPR-Cas Systems ; Gene Editing/veterinary ; },
abstract = {Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.},
}
@article {pmid41330274,
year = {2025},
author = {Macklin, BL and Runyon, WV and Feliciano, CM and Dierks, PH and Kelly, KR and Watry, HL and Judge, LM and Conklin, BR},
title = {Generation of WTD, a control human iPSC line for genetic research.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103872},
doi = {10.1016/j.scr.2025.103872},
pmid = {41330274},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Mutation ; },
abstract = {The establishment of well characterized control iPSC lines is essential for robust, reproducible research across laboratories. We used CRISPR/Cas9 to derive an isogeneic control line from a patient-derived iPSC line carrying a mutation in the NEFL gene (E396K). After correction of the E396K mutation, UCSFi003-A (WTD) exhibits multi-lineage differentiation potential, a normal karyotype, no large genomic abnormalities, and has consents for public distribution of cells and genomic data.},
}
@article {pmid41330302,
year = {2026},
author = {Xu, W and Lin, Y and Huang, Z and Li, Y and Lu, Y and Liu, M and Cui, S and Zhang, T and Shi, N and Sheng, Y and Hu, J},
title = {Split proximity circuit initiated CRISPR-Cas12a system profiling exosomal surface proteins for early cancer detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118280},
doi = {10.1016/j.bios.2025.118280},
pmid = {41330302},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/diagnosis/blood/genetics ; *Early Detection of Cancer/methods ; Female ; Epithelial Cell Adhesion Molecule/genetics/isolation &amp; purification ; *Biomarkers, Tumor/genetics/blood ; Limit of Detection ; Mucin-1/genetics ; Aptamers, Nucleotide/chemistry ; },
abstract = {Early diagnosis of breast cancer is critical for improving prognosis, but traditional methods have limitations. Herein, we propose an SPC-CRISPR system for the sensitive and specific detection of multiple breast cancer exosomal proteins without prior exosome isolation. This system couples CRISPR system with an enzyme-free amplification method to achieve dual-signal amplification. SPC-CRISPR is based on a split proximity circuit (SPC) that triggers catalytic hairpin assembly (CHA), converting protein signals on the surface of exosomes into nucleic acid signals, and the CRISPR-Cas12a system enabling further signal amplification and output. The system targets phosphatidylserine (PS), MUC1, and EpCAM on exosomes: Tim4-modified magnetic beads capture PS-expressing exosomes, and dual-aptamers recognize MUC1 and EpCAM, enabling SPC assembly and subsequent amplification. In buffer and cell-derived exosomes, the SPC-CRISPR system showed a detection limit of 10 particles/μL (R[2] = 0.990). Clinical tests utilizing merely 1 μL of serum samples successfully distinguished breast cancer patients from healthy donors (AUC = 0.9778, accuracy = 91.23 %), detected stage 0 breast cancer patients against healthy controls (accuracy = 92.59 %), and differentiated metastatic from non-metastatic cases (p < 0.001). The combination of high sensitivity, minimal sample requirements, and an exosome isolation-free workflow positions the SPC-CRISPR system as a promising tool for the clinical early detection and classification of breast cancer, with broader applicability to other cancers by swapping the corresponding aptamers.},
}
@article {pmid41330380,
year = {2026},
author = {Feng, C and Peets, EM and Zhou, Y and Crepaldi, L and Usluer, S and Dunham, A and Braunger, JM and Su, J and Strauss, ME and Muraro, D and Xian Cheam, KA and Bonder, MJ and Nogales, EG and Cooper, S and Bassett, A and Leonard, S and Gu, Y and Fussing, B and Burke, D and Parts, L and Stegle, O and Velten, B},
title = {A genome-scale single-cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines.},
journal = {Cell genomics},
volume = {6},
number = {2},
pages = {101076},
pmid = {41330380},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation/genetics ; *Genome, Human/genetics ; Quantitative Trait Loci/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Population-scale resources of genetic, molecular, and cellular information form the basis for understanding human genomes, charting the heritable basis of disease and tracing the effects of mutations. Pooled perturbation assays, probing the effect of many perturbations coupled with single-cell RNA sequencing (scRNA-seq) readout, are especially potent references for interpreting disease-linked mutations or gene-expression changes. However, the utility of existing maps has been limited by the comprehensiveness of perturbations conducted and the relevance of their cell-line context. Here, we present a genome-scale CRISPR interference perturbation map with scRNA-seq readout across many genetic backgrounds in human pluripotent cells. We map trans expression changes induced by knockdowns and characterize their variation across donors, with expression quantitative trait loci linked to higher genetic modulation of perturbation effects. This study pioneers population-scale CRISPR perturbations with high-dimensional readouts, which will fuel the future of effective modulation of cellular disease phenotypes.},
}
@article {pmid41330665,
year = {2026},
author = {Bao, Y and Ding, W and Zhang, L and Wang, W and Liu, J and Qu, Y and Zhu, L and Zhang, K and Zhong, G and Han, R and Shen, Q and Wang, B and Gu, X and Cao, Y and Sun, W},
title = {A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344830},
doi = {10.1016/j.aca.2025.344830},
pmid = {41330665},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Genotype ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification ; *Recombinases/metabolism ; Papillomavirus Infections/virology/diagnosis ; DNA, Viral/genetics ; },
abstract = {Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a-an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity-for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 min under isothermal conditions (37 °C). Clinical validation demonstrated 100 % concordance with qPCR and identified three additional low viral-load positives (Ct > 35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This 'broad-spectrum capture and precision typing' paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity.},
}
@article {pmid41330674,
year = {2026},
author = {Hou, L and Ruan, F and Zhao, K and Li, B},
title = {Boosting split-crRNA CRISPR/Cas12a activity by 3'-end extension of DNA activator for direct microRNA sensing.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344841},
doi = {10.1016/j.aca.2025.344841},
pmid = {41330674},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *DNA/chemistry/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The unique trans-cleavage activity of CRISPR/Cas12a has been extensively utilized in the domain of biosensing. Nevertheless, the detection of miRNAs using the traditional CRISPR/Cas12a system requires nucleic acid amplification or reverse transcription to convert miRNA into DNA, which increases reaction time and the risk of contamination.<br><br>RESULTS: This study presents a split-crRNA CRISPR/Cas12a-based biosensing for direct detection of miRNA. The target miRNA-375 was utilized as the spacer region of the crRNA, facilitating its binding to the truncated scaffold RNA, thereby resulting in the formation of a complete crRNA. More importantly, we discovered that the cleavage activity of split-crRNA CRISPR/Cas12a was significantly enhanced by extending sequences at the 3'-end of the DNA activator. Compared with the conventional split-crRNA CRISPR/Cas12a system, the split-crRNA CRISPR/Cas12a with 24-nucleotide random sequence extension at the 3'-end of the DNA activator exhibited a 6.4-fold increase in activity. The enhancement mechanism of 3'-end extension of DNA activator was discussed. This proposed split-crRNA CRISPR/Cas12a system was applied to detect miRNA-375 with a linear range of 5 pM-1&#xa0;nM, and the detection limit was estimated to be 0.6 pM (3&#x3c3;). Furthermore, this system was used to detect miRNA-375 in 10&#xa0;% diluted human serum, achieving satisfactory recovery rate (98&#xa0;%-106&#xa0;%).<br><br>SIGNIFICANCE: This finding indicates that it is feasible to enhance the activity of the split-crRNA CRISPR/Cas12a by extending the 3'-end of the DNA activator, thereby achieving highly sensitive direct detection of miRNA. It is a simple yet effective strategy for enhancing the sensitivity of direct miRNA detection.},
}
@article {pmid41330849,
year = {2026},
author = {Boob, AG and Zhang, C and Pan, Y and Zaidi, A and Whitaker, RJ and Zhao, H},
title = {Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus.},
journal = {Trends in biotechnology},
volume = {44},
number = {4},
pages = {1167-1186},
doi = {10.1016/j.tibtech.2025.11.003},
pmid = {41330849},
issn = {1879-3096},
mesh = {*Sulfolobus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; *Chromosomes, Archaeal/genetics ; Synthetic Biology ; },
abstract = {Sulfolobus islandicus, an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.},
}
@article {pmid41330932,
year = {2025},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Sung, YW and Chung, WS and Kim, JY},
title = {The evolving landscape of precise DNA insertion in plants.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10428},
pmid = {41330932},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant ; Plants, Genetically Modified/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; *Mutagenesis, Insertional/methods ; Gene Targeting/methods ; },
abstract = {Precise DNA insertion into plant genomes is central to advancing crop improvement and synthetic biology. CRISPR-Cas systems have enabled programmable DNA integration using tools such as gene targeting (GT), prime editing (PE), and recombinase- or transposase-based platforms. These tools are transitioned from theoretical concepts to practical applications, supporting applications like in-locus protein tagging, regulatory element engineering, and multi-gene stacking. Key challenges persist, such as inefficient large-fragment insertion, delivery barriers, and regulatory hurdles. This review traces the evolution from random to CRISPR-Cas-based systems, analyzes current limitations, and discusses emerging solutions paving the way for predictable DNA insertion in modern plant biotechnology.},
}
@article {pmid41331675,
year = {2025},
author = {Wang, M and Zhang, Y and Bi, C and Li, M},
title = {CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {411},
pmid = {41331675},
issn = {1474-760X},
support = {BAS/1/1080-01-01//KAUST Office of Sponsored Research/ ; 5932//KAUST Center of Excellence for Smart Health/ ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; MutL Protein Homolog 1/genetics ; Human Embryonic Stem Cells/metabolism ; Genomic Imprinting ; DNA Repair ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing enables precise genetic modifications by introducing targeted DNA double-strand breaks (DSBs). While Cas9-induced DSBs are known to cause unintended on-target mutations, their impact on the epigenetic landscape remains unexplored.<br><br>RESULTS: Here, we investigate how Cas9-induced DSBs affect DNA methylation patterns in human embryonic stem cells (hESCs). We induce DSBs at differentially methylated regions of imprinted genomic loci and perform high-coverage, long-read native DNA sequencing to simultaneously obtain genetic variant and base-resolution methylation data in a haplotype-resolved manner. Our findings reveal that DSBs cause significant changes in DNA methylation at target sites through mechanisms including homologous recombination, large structural variations, or defective methylation maintenance during DNA repair. Notably, these epigenetic changes can occur either together with or independently of genetic alterations. Beyond imprinted loci, Cas9-induced DSBs significantly disrupt DNA methylation patterns of the MLH1 epimutation alleles in colorectal cancer cells, and hypermethylated heterochromatin loci in hESCs. Clonal analysis indicates that the aberrant methylation changes are stable during in vitro passaging. Intriguingly, significant changes in DNA methylation levels are also detected around endogenous deletions in unedited genomic regions, suggesting that methylation alterations are not unique to Cas9 nuclease activity but represent a general outcome of DSB repair in human cells.<br><br>CONCLUSIONS: This study underscores the importance of assessing and mitigating unintended epigenetic consequences in genome editing applications, as such changes can profoundly affect gene regulation and cellular function.},
}
@article {pmid41331925,
year = {2025},
author = {Matsumoto, D and Kubota, K and Sato, Y and Kato-Inui, T and Nigorikawa, K and Miyaoka, Y and Nomura, W},
title = {Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin.},
journal = {Journal of biomedical science},
volume = {32},
number = {1},
pages = {102},
pmid = {41331925},
issn = {1423-0127},
support = {JP23K13844//KAKENHI/ ; JP24K09445//KAKENHI/ ; JP20H03442//KAKENHI/ ; JP20K21253//KAKENHI/ ; JP22H02201//KAKENHI/ ; JP23K23468//KAKENHI/ ; },
mesh = {Humans ; *Diphtheria Toxin/genetics ; *CRISPR-Associated Protein 9/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Mutation ; },
abstract = {BACKGROUND: In gene therapy via genome editing, it is essential to precisely repair disease-associated gene sequences without introducing random mutations. However, achieving highly accurate genome editing remains challenging owing to the low efficiency of homology-directed repair (HDR)-mediated gene repair, which relies on template DNA. Therefore, if Cas9 mutants capable of enhancing HDR can be identified, they could enable more precise gene therapies.<br><br>METHOD: In this research project, we developed a screening system that uses the acquisition of diphtheria toxin resistance as an indicator of HDR efficiency in human cells and EGFP disruption as an indicator of off-target effect.<br><br>RESULTS: By screening a library of SpCas9 variants with random mutations introduced into its nuclease domain, we identified a novel SpCas9 mutant with higher HDR efficiency than wild-type Cas9.<br><br>CONCLUSION: We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.},
}
@article {pmid41332134,
year = {2026},
author = {Inuzuka, T and Mouzannar, K and Zhang, M and Umarova, R and Park, SB and Uchida, T and Ma, CD and Liang, TJ},
title = {A CRISPR-based genome-wide loss-of-function screen defines a role of host metabolism in regulating hepatitis B virus infection.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1616-1632},
pmid = {41332134},
issn = {1525-0024},
support = {Z01 DK054500/ImNIH/Intramural NIH HHS/United States ; Z01 DK054504/ImNIH/Intramural NIH HHS/United States ; Z99 DK999999/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Hepatitis B virus/genetics/physiology ; *Hepatitis B/metabolism/virology/genetics ; *CRISPR-Cas Systems ; Hep G2 Cells ; Virus Replication/genetics ; Hepatocytes/metabolism/virology ; *Host-Pathogen Interactions/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Hepatitis B virus (HBV) co-opts and interacts with an extensive array of host factors for productive infection. Herein, we develop an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host dependency factors. HepG2[NTCP/Cas9] cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR, and NTCP. Among the novel candidate genes, knockout of 12 genes significantly decreased HBV replication markers. Validation using siRNA knockdown in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV proviral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication. Our study demonstrates the value of the HBV reporter system in identifying previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development.},
}
@article {pmid41332531,
year = {2025},
author = {Zhang, P and Xue, B and Xie, Y and Li, K and Yang, H and Sun, P and Zhang, L},
title = {OSM-11 modulates salinity-stress tolerance in Caenorhabditis elegans.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41332531},
issn = {2692-8205},
support = {P40 OD010440/OD/NIH HHS/United States ; },
abstract = {Most terrestrial animals exhibit narrow salinity tolerance compared to their marine counterparts. Previous studies identified osm-11 (which encodes a Notch co-ligand) mutations as a driver of hyper-saline tolerance in Caenorhabditis elegans, but mechanistic insights remained unclear. This study employs RNA sequencing and CRISPR/Cas-9 genome editing to demonstrate that osm-11 mutations enhance salinity stress resistance through upregulation of fatty acid metabolism (acdh-12, acs-17) and cytochrome P450 pathways (ugt-15), while suppressing calcium signaling. Furthermore, we demonstrated that acdh-12 mutation impairs salinity-stress tolerance by activating ferroptosis and mitophagy, accompanied by down-regulated oxidative phosphorylation and up-regulated autophagic pathways. Morphological observations show that mitochondrial fragmentation contributes to wild-type nematode mortality under high salinity, while enlarged lipid droplets in wild-types correlate with reduced β-oxidation gene expression (dhs-28, daf-22), whose knockout disrupts tolerance in mutants. These findings unravel the multi-pathway regulatory network of osm-11-mediated salinity tolerance, providing mechanistic insights for developing protective strategies against environmental salinity stressors impacting animal survival.},
}
@article {pmid41336948,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, J and Jeong, H and Park, J and Kang, T and Jeong, KH},
title = {On-chip Nanoplasmonic RT-RPA and CRISPR/Cas12a Assay for Point-of-care Molecular Diagnostics.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-4},
doi = {10.1109/EMBC58623.2025.11253682},
pmid = {41336948},
issn = {2694-0604},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; *Point-of-Care Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques ; *Pathology, Molecular/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate nucleic acid detection at point-of-care (POC) is essential for advancing effective disease diagnosis and management. Here, we report a handheld nanoplasmonic all-in-one setup for on-chip recombinase polymerase amplification (RPA) and real-time fluorescence detection by CRISPR/Cas12a reaction. The all-in-one setup consists of AuNIs-based nanoplasmonic cavity (AuNIs-NC), a disposable plastic-on-polymer (PoP) cartridge, and fluorescence microlens array (FMLA) camera. The AuNIs-NC allows uniform and efficient photothermal heating under white LED illumination due to strong broadband light absorption and internal reflection by randomly distributed AuNIs and thin Al film. This setup allows the RPA and CRISPR/Cas 12a reactions in a single chamber of PoP cartridge, with fluorescence signals monitored by a FMLA camera. The experimental result demonstrates rapid SARS-CoV-2 E gene plasmid DNA detection within 20 min, achieving a detection sensitivity of 10 copies/ul. Testing with 16 clinical samples shows a linear trend with RT-qPCR, indicating the platform's reliable sensitivity and specificity. This compact platform offers affordable and reliable molecular diagnosis, facilitating rapid and scalable POC testing for a range of infectious diseases.Clinical Relevance- This on-chip real-time RT-RPA and CRISPR/Cas12a assay provides rapid and precise molecular diagnostics at POC using fully integrated plasmonic system.},
}
@article {pmid41337296,
year = {2025},
author = {Cimolato, C and Letrari, S and Chiacchiera, AF and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Phage-Mediated CRISPRi System to Inhibit Antibiotic Resistances in Bacteria.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-7},
doi = {10.1109/EMBC58623.2025.11253443},
pmid = {41337296},
issn = {2694-0604},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteria/genetics/drug effects/virology ; *Drug Resistance, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Humans ; },
abstract = {Antimicrobial resistance (AMR) poses a critical threat to global health, rendering traditional antibiotics increasingly ineffective and amplifying the urgency for innovative solutions. Among promising alternatives, synthetic biology emerges as a powerful tool to combat AMR. This work proposes an innovative strategy based on engineering bacteriophages to deliver CRISPR interference (CRISPRi) systems into antibiotic-resistant pathogens to precisely silence target resistance genes. A comprehensive mathematical model is developed and simulated to capture the dynamics of phage-mediated CRISPRi delivery. By explicitly incorporating mutations that affect CRISPRi functionality, the study evaluates system performance and its potential for long-term therapeutic efficacy. This model serves as a critical framework for optimizing future CRISPRi-based interventions and advancing synthetic biology-driven approaches to tackle AMR.Clinical relevance- This paper provides a quantitative modeling framework to evaluate key parameters affecting engineered phage therapy efficiency, supporting rational design and phage posology optimization.},
}
@article {pmid41338106,
year = {2026},
author = {Paenkaew, S and Euppayo, T and Tungtrakanpoung, R and Teapunvong, W and Nganvongpanit, K and Buddhachat, K},
title = {Rapid and specific detection of Babesia vogeli using RPA/CRISPR-Cas12a: A feasible field-friendly diagnostic for canine babesiosis.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110660},
doi = {10.1016/j.vetpar.2025.110660},
pmid = {41338106},
issn = {1873-2550},
mesh = {Animals ; Dogs ; *Babesiosis/diagnosis/parasitology ; *Dog Diseases/diagnosis/parasitology ; *Babesia/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Recombinases/metabolism ; },
abstract = {Babesia vogeli is a protozoan parasite causing canine babesiosis, a tick-borne disease prevalent in tropical and subtropical regions. Its microscopic identification is challenging due to morphological similarity with other Babesia spp., and serological assays often yield inaccurate results. To address this issue, we developed a rapid, equipment-minimal diagnostic method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a (RPA/CRISPR-cas12a) for B. vogeli-specific detection. The RPA assay enables DNA amplification for both B. vogeli and Hepatozoon canis, while CRISPR/Cas12a using gRNA_Bab ensures specificity for B. vogeli, even in co-infections and other pathogens. This approach detects as few as 10[5] copies within two hours for both readout platforms such as fluorescence and lateral flow dipstick (LFD). Forty canine blood samples were detected by RPA/CRISPR-cas12a to examine its performance. Results showed high concordance with qPCR-high resolution melting (HRM) (Cohen's kappa: 0.93 for fluorescence, 0.81 for LFD), outperforming conventional PCR. The clinical sensitivity and specificity of RPA/CRISPR-cas12a were 100 % and 96.8 %, respectively and the concordance with qPCR-HRM was 97.5 %. RPA/CRISPR-cas12a for Babesia spp. detection provided a simple, rapid, and accurate method, demonstrating promise for point-of-care diagnosis of canine babesiosis in resource-limited settings. This method showed high potential as a practical diagnostic tool in veterinary clinics, with accelerated surveillance to control outbreaks of Babesia-associated canine babesiosis.},
}
@article {pmid41338219,
year = {2026},
author = {Yang, Z and Zhang, L and Jiang, X and Yang, X and Ma, K and Yoo, D and Lu, Y and Zhang, S and Chen, J and Nie, Y and Bian, X and Han, J and Fu, L and Zhang, J and Ventura, M and Zhang, G and Sun, Q and Eichler, EE and Mao, Y},
title = {Incomplete lineage sorting of segmental duplications defines the human chromosome 2 fusion site early during African great ape speciation.},
journal = {Cell genomics},
volume = {6},
number = {1},
pages = {101079},
pmid = {41338219},
issn = {2666-979X},
support = {R01 HG002385/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Hominidae/genetics ; *Segmental Duplications, Genomic/genetics ; *Chromosomes, Human, Pair 2/genetics ; *Genetic Speciation ; Chromosome Inversion/genetics ; Evolution, Molecular ; CRISPR-Cas Systems ; },
abstract = {All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Here, we show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the turnover of subterminal repetitive DNA. We characterized the fusion site at the single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. One of these SDs shares homology to a hypomethylated SD spacer sequence present in the subterminal heterochromatin of Pan but is completely absent subtelomerically in both humans and orangutans. CRISPR-Cas9-mediated depletion of the fusion site in human neural progenitor cells alters the expression of genes, indicating a potential regulatory consequence to this human-specific karyotypic change. Overall, this study offers insights into how complex regions subject to ILS may contribute to speciation.},
}
@article {pmid41338874,
year = {2026},
author = {Villegas, NK and Tran, MH and Keller, A and Plesa, C},
title = {BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {9-20},
doi = {10.1177/25731599251401526},
pmid = {41338874},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; *Genes, Synthetic/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Modern gene synthesis platforms enable investigations of protein function and genome biology at an unprecedented scale. Yet, the proportion of error-free constructs in diverse gene libraries decreases with length due to the propagation of oligo synthesis errors. To rescue these error-free constructs, we developed Barcode-Assisted Retrieval CRISPR-Activated Targeting (BAR-CAT), an in vitro method that uses multiplexed dCas9-single-guide RNA (sgRNA) complexes to extract barcodes corresponding to error-free constructs. After a 15-min incubation and wash regimen, three low-bundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to a 384-gene DropSynth gene library, BAR-CAT enriched 12 targets up to 122-fold and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.},
}
@article {pmid41339636,
year = {2025},
author = {Agnarelli, A and Buckley-Benbow, L and Ozgencil, M and Lad, M and Ampah, KK and Kalinka, A and Belan, O and Maslen, S and Skehel, MJ and Walter, D and Day, M and Bellelli, R},
title = {The genetic and biochemical basis of human leading strand synthesis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {412},
pmid = {41339636},
issn = {2041-1723},
mesh = {Humans ; *DNA Replication/genetics ; Genomic Instability ; *DNA Polymerase II/metabolism/genetics/chemistry ; Proliferating Cell Nuclear Antigen/metabolism ; DNA/biosynthesis/genetics ; Iron/metabolism ; Iron-Sulfur Proteins/metabolism ; CRISPR-Cas Systems ; },
abstract = {The maintenance of genome stability requires efficient leading strand synthesis by DNA Polymerase Epsilon (Polε). By performing CRISPR genetic screens in cells lacking the POLE4 subunit of Polε we define a genetic map of the factors required to support Polε function in the absence of its accessory subunits. A set of genes involved in iron metabolism emerge as required to sustain Iron Sulphur Cluster (ISC)-dependent Polε activity. We then dissect a synthetic lethal interaction between POLE3-POLE4 and the CHTF18-RFC2/5 complex. By combining cell biology, structural modelling and biochemistry, we define the existence of two tiers of regulation of Polε processivity: leading strand-specific loading of PCNA by CHTF18-RFC2/5 and "gripping" of newly synthesised dsDNA by POLE3-POLE4. The combined loss of these functions is incompatible with leading strand synthesis and viability. In summary, we describe the biochemical basis of human leading strand synthesis and the consequence of its dysfunction in genome stability.},
}
@article {pmid41339642,
year = {2025},
author = {Yang, WJ and Liu, BY and Xue, L},
title = {Knockout of protein arginine methyltransferase 1 inhibited cell growth and promoted cell migration in human bronchial epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {43069},
pmid = {41339642},
issn = {2045-2322},
support = {2018BFC360//Fund for Key Laboratory Construction of Hubei Province/ ; 31101047//National Natural Science Foundation of China/ ; CZQ22013//"the Fundamental Research Funds for the Central Universities", South-Central MinZu University/ ; PTZ24018//i Medical Biology International Science and Technology Cooperation Base/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *Cell Proliferation/genetics ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Epithelial Cells/metabolism/cytology ; *Bronchi/cytology/metabolism ; Cell Line ; Apoptosis/genetics ; Gene Knockout Techniques ; *Repressor Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Previous studies have demonstrated that PRMT1 was involved in the progression of multiple lung diseases. However, its specific function within the bronchial epithelium was still limited and needed further exploration. In the present study, human bronchial epithelial cell line 16HBE was chosen to elucidate the biological role of PRMT1 in lung epithelium. Cell proliferation, cell-cycle distribution, cell apoptosis, and cell motility capacity were systematically evaluated following CRISPR/Cas9-mediated knockout of PRMT1. We showed that knockout of PRMT1 in 16HBE inhibited cell proliferation, redistributed cell cycle, promoted cell apoptosis, and accelerated cell migration via a series of regulated cyclins, cyclin-dependent kinase regulators, and EMT markers. Taken together, these findings identify PRMT1 as a potential modulator of epithelial cell proliferation, survival, and motility in the human bronchial epithelium, offering new insights into its possible role in epithelial remodeling during pulmonary disorders.},
}
@article {pmid41340056,
year = {2025},
author = {Braun, S and Knackfuß, K and Ziesmann, T and Mlinzk, L and Goerg, A and Frankenheim, J and Walter, A and Schneider-Brachert, W and Distler, U and Fritsch, J},
title = {Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {520},
pmid = {41340056},
issn = {1478-811X},
mesh = {Humans ; *Caspase 8/metabolism ; *ADAM Proteins/metabolism/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; *Proteolysis ; *Membrane Proteins/metabolism/genetics/deficiency ; *Necroptosis ; *Receptors, Tumor Necrosis Factor, Type I/metabolism ; Enzyme Activation ; Jurkat Cells ; U937 Cells ; Signal Transduction ; },
abstract = {BACKGROUND: Cell death and survival processes must be tightly regulated to ensure proper tissue homeostasis and prevent excessive inflammation and tissue damage. Death receptors, including TNF-R1, can induce either immunogenic (necroptosis) or non-immunogenic (apoptosis) cell death and relay proliferative / cell survival signaling by activating NFκB and MAPK cascades. In a recent report, we identified the metalloproteinase ADAM15 as a possible TNF-responding enzyme, leading to the hypothesis that it regulates either cell survival or death cascades.<br><br>METHODS: CRISPR/Cas-9 was used to knock out the adam15 gene. Loss of gene expression was validated by Western blot and flow cytometry in U937 and Jurkat cells. NF&#x3ba;B, MAPK signaling, and cell death cascades were monitored by Western blot, flow cytometry, and enzyme assays. A bottom-up proteome analysis was performed to elucidate cellular processes affected by ADAM15 loss. The subcellular localization of ADAM15 was monitored by microscopy and immuno-magnetic fractionation.<br><br>RESULTS: We identified ADAM15 as a regulator of necroptosis, leaving apoptosis and cell survival signaling unaffected. Loss of ADAM15 resulted in abrogated necroptosis, as evidenced by the application of death ligands TNF, TRAIL, FasL, and TL1a, as well as the BH3 mimetic Obatoclax. We observed enhanced basal Caspase-8 activity, which was not cytotoxic, and partial RIPK1 proteolysis. The loss of ADAM15 was verified in a proteome screen, which revealed alterations in various molecular pathways, including autophagy, organelle trafficking, and sorting. We observed ADAM15 in intracellular compartments, which in part have a lysosomal protein signature. We observed enhanced surface expression of TNF-R1, proposing it as a possible ADAM15 substrate.<br><br>CONCLUSIONS: ADAM15 is a previously unknown regulator of necroptosis, likely due to its role in modulating intracellular organelle sorting processes. Its proteolytic activity and possible scaffolding capacity for recruiting adaptor molecules make it a veritable drug target. The activation or deactivation of ADAM15 may be exploited to modulate various disease conditions.},
}
@article {pmid41341502,
year = {2025},
author = {Gao, Y and Chen, J},
title = {Fast but accurate: a systematic review and meta-analysis on diagnostic performance of MRSA detection in clinical samples by using CRISPR-based rapid molecular methods.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1703247},
pmid = {41341502},
issn = {1664-302X},
abstract = {BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to its multidrug resistance and association with severe infections. Conventional culture methods are time-consuming, usually requiring 48-72 h to obtain results, while conventional molecular methods such as PCR or qPCR, though faster, still require trained personnel and specialized instruments, which may delay timely clinical treatment and infection control. CRISPR-based methods have emerged as promising alternative tools for MRSA detection, but their real-world performance still requires comprehensive assessment. This meta-analysis aimed to systematically evaluate the diagnostic accuracy and timeliness of CRISPR/Cas systems for MRSA detection in clinical samples.<br><br>METHODS: A systematic search of PubMed, Embase, Web of Science, and Cochrane Library was conducted using search terms related to MRSA, CRISPR/Cas, diagnostic accuracy, and rapid detection. Studies reporting sensitivity and specificity with extractable 2&#x202f;&#xd7;&#x202f;2 contingency tables were included. Quality was assessed via QUADAS-2. Meta-disc 1.4.0 and Stata 16.0 were used for statistical analysis, including pooled sensitivity, specificity, likelihood ratios, diagnostic odds ratios (DOR) and summary receiver operating characteristic (SROC). Median detection time and subgroup analyses were also conducted.<br><br>RESULTS: Twelve studies were included. The results showed that the CRISPR-based methods showed a pooled sensitivity of 99% (95% CI: 97-100%) and specificity of 100% (95% CI: 99-100%), with a PLR of 32.68 (95% CI: 15.45-69.15), NLR of 0.03 (95% CI: 0.02-0.07), and DOR of 664.25 (95% CI: 234.59-1880.84). The median detection time across included studies was 60&#x202f;min (IQR: 41.25-98.75&#x202f;min).<br><br>CONCLUSION: CRISPR-based molecular assays demonstrated exceptional accuracy and rapid detection capability for MRSA in clinical settings, significantly outperforming conventional methods. However, potential publication bias and methodological limitations warrant cautious interpretation of these results.<br><br>PROSPERO ID: CRD420251115439.},
}
@article {pmid41341583,
year = {2025},
author = {Jiang, Z and Jia, B and Hu, N and Zhang, M and Xiao, H and Chen, G and Yu, J and Li, X and Shen, B and Feng, J and Wang, J},
title = {In Vivo engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1679421},
pmid = {41341583},
issn = {1664-3224},
mesh = {Animals ; Mice, Transgenic ; Humans ; Mice ; *Enterotoxins/immunology ; *Antibodies, Neutralizing/immunology/genetics/biosynthesis ; Female ; CRISPR-Cas Systems ; Genetic Engineering ; Antibodies, Monoclonal/immunology/genetics ; Glycosylation ; },
abstract = {Transgenic animal bioreactors provide a complementary strategy to traditional mammalian cell culture systems for the production of therapeutic human monoclonal antibodies (mAbs). Here we present a CRISPR/Cas9-mediated breakthrough in creating two novel genetically engineered (GE) mouse models with species-specific chromosomal integration of human anti-staphylococcal enterotoxin B (SEB) mAb genes at either the ROSA26 or Hipp11 (H11) safe-harbor loci - evolutionarily conserved genomic safe harbors (GSH). These genetically optimized animals demonstrated broad tissue capability for glycosylation-competent human antibodies, achieving exceptional secretion levels reaching 208 mg/L in serum, 43 mg/L in mammary secretions, 24 mg/L in saliva on average. The transgenic lines maintained this antibody production stability for >140 weeks without compromising animal viability, while preserving germline transmission fidelity through six successive generations. Furthermore, the highly glycosylated human antibodies derived from these genetic engineered mice exhibited high binding affinity to SEB (KD=0.108 nM for ROSA26; 0.154 nM for H11), providing comprehensive protection against SEB intoxication in vivo. This study opens avenues for utilizing transgenic animal bioreactors for large-scale production of fully human antibodies or disease-resistant livestock in the foreseeable future.},
}
@article {pmid41342880,
year = {2026},
author = {Kama, Y and Hirano, KI and Masuhara, K and Endo, Y and Suzuki, Y and Fujimoto, M and Matsuda, T and Yahata, T and Kato, M and Hozumi, K and Tanaka, T and Hosokawa, H},
title = {Notch interaction with RUNX factors regulates initiation of the T-lineage program.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {2},
pages = {},
pmid = {41342880},
issn = {1540-9538},
support = {JP19H03692//Japan Society for the Promotion of Science/ ; JP24K22062//Japan Society for the Promotion of Science/ ; JP24K02485//Japan Society for the Promotion of Science/ ; JP23K15307//Japan Society for the Promotion of Science/ ; //Tokai University/ ; //Chugai Foundation for Innovative Drug Discovery Science/ ; //Uehara Memorial Foundation/ ; //Naito Foundation/ ; //Takeda Science Foundation/ ; //SENSHIN Medical Research Foundation/ ; //Princess Takamatsu Cancer Research Fund/ ; //Foundation for Promotion of Cancer Research/ ; //Chemo-Sero-Therapeutic Research Institute/ ; //Vehicle Racing Commemorative Foundation/ ; //Kobayashi Foundation/ ; //Ichiro Kanehara Foundation for the Promotion of Medical Science and Medical Care/ ; //TERUMO Life Science Foundation/ ; //Mitsubishi Foundation/ ; 35-y10//Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics/ ; //Tokai University School of Medicine/ ; //Kyushu University/ ; },
mesh = {*T-Lymphocytes/metabolism/physiology ; *Cell Lineage/genetics ; Gene Knock-In Techniques ; Mice, Transgenic ; *CCCTC-Binding Factor ; *Core Binding Factor Alpha 2 Subunit/metabolism ; CRISPR-Cas Systems ; *Receptor, Notch1/metabolism ; Lymphoid Progenitor Cells/metabolism/physiology ; Mediator Complex/metabolism ; E1A-Associated p300 Protein/metabolism ; Transcriptional Activation/physiology ; Protein Binding ; Protein Domains ; Male ; Female ; Animals ; Mice ; Trans-Activators ; },
abstract = {Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)- and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1-binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex reorganization and genomic redeployment in initiating T-lineage program.},
}
@article {pmid41344099,
year = {2025},
author = {Ryu, YC and Bao, G and Hwang, BH},
title = {Peptide-assisted lipofection enables efficient non-viral delivery of large CRISPR/Cas9 constructs for genome editing applications.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {193},
number = {},
pages = {118838},
doi = {10.1016/j.biopha.2025.118838},
pmid = {41344099},
issn = {1950-6007},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; *Transfection/methods ; Plasmids/genetics ; *Gene Transfer Techniques ; *Peptides/chemistry ; Mice ; *Lipids/chemistry ; Genetic Therapy/methods ; Cell Survival ; Genetic Vectors ; },
abstract = {Efficient and safe delivery of large genetic constructs such as CRISPR/Cas9 plasmids remains a critical bottleneck in gene therapy. In this work, the peptide-assisted lipofection (PAL) system was developed as a breakthrough non-viral vector for gene delivery, specifically designed for large plasmids including CRISPR/Cas9 constructs. This system achieved exceptional transfection efficiency up to 98.7 % in HEK293T cells, surpassing conventional delivery methods such as electroporation and standard lipofections. PAL successfully delivered large plasmids up to 29 kb while maintaining high cell viability and achieved 44.1 % indel formation efficiency in gene editing experiments. Fluorescence microscopy verified PAL's efficient endosomal escape and nuclear targeting abilities. The superior performance of the PAL is attributed to its cellular uptake and endosomal escape enhanced by transfection-assisting peptide. In vivo studies in mouse models showed sustained gene expression in liver tissue, demonstrating superior performance compared to naked plasmid delivery. These results establish PAL as a versatile and promising platform for gene therapy and genome editing, offering a safer alternative to viral vectors for large genetic payload delivery.},
}
@article {pmid41344241,
year = {2026},
author = {Peng, Y and Xu, J and Chen, B and Zeng, D and Yu, X and Chen, W},
title = {Ultrasensitive detection of lead ion in tea samples using a versatile and robust multi-DNAzyme DNA machine mediated CRISPR/Cas12a signal amplification system.},
journal = {Food chemistry},
volume = {499},
number = {},
pages = {147352},
doi = {10.1016/j.foodchem.2025.147352},
pmid = {41344241},
issn = {1873-7072},
mesh = {*DNA, Catalytic/genetics/chemistry ; *Tea/chemistry ; *Lead/analysis ; *Food Contamination/analysis ; CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Camellia sinensis/chemistry/genetics ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; Ions/analysis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Lead ion (Pb[2+]) contamination in tea poses serious threats to food safety. We present a novel detection platform integrating a multifunctional DNAzyme machine with the CRISPR/Cas12a system. Upon target Pb[2+] binding, the multi-DNAzyme activates the DNA machine, initiating multiple isothermal cycles that generate a poly-A sequence. This sequence activates Cas12a, triggering trans-cleavage of the hairpin fluorescent probes and significant signal amplification. The self-sustained amplification of DNA machine eliminates the need for any auxiliary probes, greatly simplifying the assay design. This platform achieves excellent sensing perfromances with detection limit of 67.53 pM, wide linear range (0.01-100 nM), satisfied specificity and high recovery rates above 96 % in real tea samples. This integration of DNAzyme recognition with CRISPR/Cas12a signal enhancement provides a rapid, and cost-effective method for Pb[2+] detection. This strategy offers practical potential for food safety monitoring and environmental assessment, and future research may expand its applicability to other heavy metal contaminants.},
}
@article {pmid41344285,
year = {2026},
author = {Dasanayaka, BP and Pathirana, SL and Jayawardana, A and Handunnetti, SM and Fernando, N and Galhena, BP and Weerasena, SJ and Nitsche, A and Iddamaldeniya, SS and Dietzsch, AK},
title = {A roadmap in detecting frequently reported bovine babesiosis: From blood smear to CRISPR.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110662},
doi = {10.1016/j.vetpar.2025.110662},
pmid = {41344285},
issn = {1873-2550},
mesh = {Animals ; Cattle ; *Babesiosis/diagnosis/parasitology/blood ; *Cattle Diseases/diagnosis/parasitology/blood ; *CRISPR-Cas Systems ; *Babesia/isolation &amp; purification/genetics ; Babesia bovis/isolation &amp; purification/genetics ; Real-Time Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity ; },
abstract = {Current diagnosis of Babesia bovis and B. bigemina relies on direct microscopy, nucleic acid detection, and serology. Light-microscopic analysis of Giemsa-stained smears still serves as the primary diagnostic modality at the point of care. However, carrier cattle, particularly those harbouring B. bovis, often carry parasites at levels far below the detection threshold, and such levels are sensitive only to DNA-based detection approaches. Early probe-hybridization techniques have been largely replaced by conventional Polymerase Chain Reaction (PCR), nested formats, and real-time quantitative PCR (qPCR)-which enable species-specific discrimination within closed-tube systems, thereby minimizing contamination risk. Species‑level identification is essential for clinical management, surveillance, and experimental studies. Duplex TaqMan qPCRs simultaneously distinguish B. bovis from B. bigemina, surpassing the analytical sensitivity offered by nested PCR (nPCR) methodologies. Reverse-line-blot (RLB) hybridisation broadens the diagnostic scope by concurrently detecting co-infections of B. bovis and B. bigemina and mixed haemoparasitic species in one workflow. However, the analytical sensitivity of RLB remains inferior to that of qPCR in detecting low-density and carrier-state infections of B. bigemina. RLB remains useful for retrospective genotyping when amplification is impractical or fails. Field-ready isothermal approaches have expanded the scope of molecular diagnostics beyond laboratory settings, facilitating field-level application and rapid on-site detection. Coupling Loop-mediated isothermal amplification (LAMP) with a lateral-flow dipstick (LFD) (LAMP-LFD) enables pen-side direct visual detection. Antibody tests are essential tools for herd-level surveillance. A recent chimeric Enzyme-Linked Immunosorbent Assay (ELISA) that combines three immunodominant B. bovis antigens broaden strain coverage and boosts diagnostic reliability. Recombinase polymerase amplification coupled to CRISPR-Cas12a cleavage has achieved single-target detection of B. bigemina from tick salivary-gland DNA, paving the way for innovative pen-side platforms, once cost and technical hurdles are overcome. Importantly, vector-based detection using appropriate tissues bearing an optimum level of ticks is species-dependent. B. bigemina sporozoites concentrate in nymph/adult salivary glands, whereas B. bovis sporozoites are produced mainly in larval salivary glands, so monitoring programs should stratify sampling accordingly.},
}
@article {pmid41344296,
year = {2025},
author = {Landi, E and Zondag, R and Dehnen, JA and Albert, S and Dickman, MM and LaPointe, VLS and van Bokhoven, H},
title = {Biallelic excision of the CTG18.1 expansion in two Fuchs endothelial corneal dystrophy-derived iPSC lines and one control (SCTCi046-A-1, SCTCi047-A-1 and SCTCi041-A-1) using an episomal vector-based CRISPR/Cas9 approach.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103881},
doi = {10.1016/j.scr.2025.103881},
pmid = {41344296},
issn = {1876-7753},
mesh = {Humans ; *Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Cell Line ; Transcription Factor 4/genetics ; Alleles ; Plasmids/genetics ; },
abstract = {An expanded CTG repeat in intron 2 of the transcription factor 4 (TCF4) gene is the main cause of Fuchs endothelial corneal dystrophy (FECD), a complex corneal disease. The prevailing paradigm is that the expanded repeat exerts toxic effects, resulting in corneal endothelium degeneration. Here we explored the use of CRISPR/Cas9-mediated, non-homologous end-joining (NHEJ) for disease-modeling purposes, by performing a biallelic excision of the CTG18.1 expansion in two FECD- and one control-derived induced pluripotent stem cell lines (iPSCs). The three Δ/Δ CTG18.1 lines generated by this study provide a platform to investigate the CTG18.1 contribution to FECD pathogenesis.},
}
@article {pmid41344324,
year = {2026},
author = {Djamshidi, M and Hill, A and Heshmatzad, K and Langley, J and Krowicki, H and Ali, M and Yang, Y and Tanida, R and Abdul-Careem, MF and Billon, P and Riabowol, K},
title = {FAME-CRISPR improves CRISPR-Cas9 genome editing via HDAC inhibition and engineered virus-like particle delivery.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101248},
pmid = {41344324},
issn = {2667-2375},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Histone Deacetylase Inhibitors/pharmacology ; *Virion/genetics ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {CRISPR-mediated gene editing using engineered virus-like particles (eVLPs) can achieve high efficiency, but performance varies with reduced effectiveness often seen in primary cells or when generating polyclonal models at scale. We developed a faster, accurate and 4-fold more efficient CRISPR-Cas9 (FAME-CRISPR) method using pan-histone deacetylase inhibitors with eVLP transduction compared to previous reports using other histone deacetylase inhibitors. Combined optimization of pan-HDACi treatment with eVLP enhanced double-strand break (DSB)-mediated CRISPR and base editing gave significantly edited populations within 2- to 3-cell mean population doublings, reducing the need for post-editing selection in immortalized cancer cells and in primary diploid fibroblasts that have limited replicative lifespans.},
}
@article {pmid41344457,
year = {2026},
author = {Liu, L and Huang, X and Wan, S and Gao, Z and Liu, H},
title = {Ectodysplasin A regulates the development of scale and intermuscular bone in teleosts.},
journal = {International journal of biological macromolecules},
volume = {337},
number = {Pt 2},
pages = {149465},
doi = {10.1016/j.ijbiomac.2025.149465},
pmid = {41344457},
issn = {1879-0003},
mesh = {Animals ; Zebrafish/genetics/growth &amp; development ; *Ectodysplasins/genetics/metabolism ; *Fishes/genetics/growth &amp; development ; *Bone and Bones/metabolism ; Phylogeny ; *Animal Scales/growth &amp; development/metabolism ; Signal Transduction ; Gene Expression Regulation, Developmental ; *Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Most bony fish are extensively covered by scales, which play crucial roles in locomotion, balance, and sensory perception. However, the molecular mechanisms underlying fish scales development remain poorly understood. In this study, comparative genomic analyses were performed between scaled and scaleless fish species, leading to the identification of key genes (eda, wnt3a, gsk3bb, etc.) involved in scale formation. Phylogenetic and sequence analyses of the eda gene revealed that the Eda protein in Monopterus albus lacks a transmembrane domain, disrupting Eda/Edar binding and potentially driving scale degeneration. Using CRISPR/Cas9 technology, we generated zebrafish eda[-/-] mutants, which exhibited a complete absence of scales, fin rays, pharyngeal teeth, and gill rakers. Notably, the intermuscular bones in these mutants showed significantly reduced length, and simplified morphology, indicating impaired growth. Furthermore, quantitative PCR (qPCR) analysis demonstrated that eda deficiency disrupts the Eda/Edar/NF-κB signaling pathway. Our findings provide significant insights into the molecular regulatory mechanisms underlying the development of skin appendages (e.g., scales) and intermuscular bones.},
}
@article {pmid41344486,
year = {2026},
author = {Huang, Y and Li, L and Do, CW and Luo, Q and Zheng, Z and Xiong, W},
title = {Lipid nanoparticle-mediated CRISPR/Cas9 delivery enables efficient trabecular meshwork gene editing in mice.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {389},
number = {},
pages = {114499},
doi = {10.1016/j.jconrel.2025.114499},
pmid = {41344486},
issn = {1873-4995},
mesh = {Animals ; *Trabecular Meshwork/metabolism ; *Gene Editing/methods ; *Nanoparticles/administration &amp; dosage/chemistry ; *CRISPR-Cas Systems ; *Lipids/chemistry/administration &amp; dosage ; Mice, Inbred C57BL ; Mice ; RNA, Messenger/administration &amp; dosage/genetics ; *Gene Transfer Techniques ; Humans ; Calcium-Binding Proteins/genetics ; Green Fluorescent Proteins/genetics ; Extracellular Matrix Proteins/genetics ; Male ; CRISPR-Associated Protein 9/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) enable efficient mRNA delivery, yet their potential for ocular gene editing remains largely unexplored. Here, we systematically evaluated three LNP formulations containing distinct ionizable lipids, DLin-MC3-DMA, ALC0315, and SM102, for gene delivery to ocular tissues. Among them, SM102-based LNP encapsulating GFP mRNA (SM102-GFP) exhibited the highest transfection efficiency across three cultured ocular cells in vitro. Following intravitreal injection in mice, SM102-GFP achieved selective and robust expression in the trabecular meshwork (TM) without detectable retinal transfection. GFP expression in TM peaked at one week post-injection, declined by three weeks, and could be effectively re-induced by a second dosing of the same vector. Compared with adeno-associated viral (AAV) and adenoviral (Ad) vectors, SM102-GFP showed superior TM specificity and reduced retinal inflammation. Co-delivery of SpCas9 mRNA and sgRNA via SM102-based LNPs enabled efficient CRISPR-mediated knockout of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp knockout induced sustained intraocular pressure elevation and anterior chamber deepening with open angles, recapitulating features of primary open-angle glaucoma. Chronic ocular hypertension further led to Müller gliosis and ganglion cell complex thinning, indicative of progressive retinal stress. These findings establish SM102-based LNPs as a safe and efficient platform for TM-targeted gene editing and glaucoma modeling.},
}
@article {pmid41344769,
year = {2026},
author = {Liu, X and Zheng, Y and Chen, Z and Wang, S and Liao, H and Jia, J and Wang, G and Wang, J and Yuan, C and Guo, X and Yin, Y and Hu, Q},
title = {Rapid and visual detection of Listeria monocytogenes by combining one-pot LAMP-CRISPR/Cas12b with lateral flow assay.},
journal = {Food microbiology},
volume = {135},
number = {},
pages = {104977},
doi = {10.1016/j.fm.2025.104977},
pmid = {41344769},
issn = {1095-9998},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Food Contamination/analysis ; Food Microbiology/methods ; Swine ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.},
}
@article {pmid41345100,
year = {2025},
author = {Bai, M and Zhang, J and Lin, W and Zhou, Y and Jiang, M and Wu, H and Peng, C and Lin, J and He, F and Kuang, H and Guan, Y},
title = {A flanking-nicks prime editor (FLICK-PE) system to boost prime editing in dicots.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {337},
pmid = {41345100},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *Glycine max/genetics/drug effects ; *Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Glyphosate ; Glycine/analogs &amp; derivatives/pharmacology ; CRISPR-Cas Systems/genetics ; Herbicide Resistance/genetics ; 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications to mammalian cells and monocot staple crops, but remains relatively challenging in dicot plants. Here, we develop a Flanking-Nicks Prime Editor (FLICK-PE) system that boosts editing efficiency in soybean and tobacco. We show that optimization for PE by adding a nicking sgRNA could dramatically enhance intended-editing efficiency in soybean. Inspired by this observation, we design a FLICK-PE strategy to confer a pair of nicks flanking the target site. In soybean, FLICK-PE achieves on average a 15.7-fold increase in intended-editing efficiency compared to PE2, and a 2.2-fold increase compared to PE3. Using FLICK-PE, we efficiently engineer glyphosate resistance in soybean by introducing TAP-IVS mutations in EPSPS1a, achieving three amino-acid substitutions and an intended editing efficiency of 21.1%. This approach yields stable edited soybean varieties with vigorous glyphosate tolerance and minimal growth penalties in a field trial. FLICK-PE also demonstrates efficacy in tobacco, underscoring its broad applicability and versatility for rapid, precision breeding in agriculturally vital crops.},
}
@article {pmid41345278,
year = {2026},
author = {de la Rosa, C and Kendirli, A and Baygün, S and Bauernschmitt, F and Thomann, AS and Kisioglu, I and Beckmann, D and Carpentier Solorio, Y and Pfaffenstaller, V and Tai, YH and Mehraein, N and Sanchez, P and Spieth, L and Gerdes, LA and Beltran, E and Dornmair, K and Simons, M and Peters, A and Schmidt-Supprian, M and Kerschensteiner, M},
title = {In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation.},
journal = {Nature neuroscience},
volume = {29},
number = {2},
pages = {493-509},
pmid = {41345278},
issn = {1546-1726},
support = {259373024//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 408885537//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 239283807//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Macrophages/metabolism ; Mice ; Cytokines/metabolism/genetics ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Multiple Sclerosis/immunology/genetics ; Humans ; *Neuroinflammatory Diseases/genetics ; CRISPR-Cas Systems ; *Encephalomyelitis, Autoimmune, Experimental/immunology ; },
abstract = {Here we established an in vivo CRISPR screening pipeline using genetically editable progenitor cells to dissect macrophage regulation in mouse models of multiple sclerosis (MS). Screening over 100 cytokine receptors and signaling molecules identified interferon-γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor and transforming growth factor-β as essential regulators of macrophage polarization in vivo. Single-cell transcriptomics confirmed that transferred progenitor cells generate all blood-derived CNS myeloid cell populations, enabling Perturb-seq analysis of cytokine actions in neuroinflammation. Combined with biosensor expression, our approach allows monitoring cytokine effects on myeloid cell migration, debris phagocytosis and oxidative activity in vivo. Comparative transcriptomic analyses revealed conserved neuroinflammatory cytokine signatures across myeloid populations, CNS compartments and species, elucidating cytokine cues shaping myeloid function in the cerebrospinal fluid and parenchyma of individuals with MS. This versatile pipeline thus provides a scalable framework for high-resolution analysis of macrophage states and uncovers the cytokine signals that underlie their regulation in MS and MS models.},
}
@article {pmid41345283,
year = {2026},
author = {Saydam, S and Dinçer, P},
title = {Precision rewriting of muscle genetics: therapeutic horizons of base and prime editing in skeletal muscle disorders.},
journal = {Gene therapy},
volume = {33},
number = {3},
pages = {249-259},
pmid = {41345283},
issn = {1476-5462},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Muscular Diseases/genetics/therapy ; CRISPR-Cas Systems ; *Muscle, Skeletal/metabolism ; Animals ; Mutation ; },
abstract = {Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.},
}
@article {pmid41346237,
year = {2025},
author = {Pandit, B and Hanson, E and Dagci, H and Yang, Q and Yigit, MV and Royzen, M},
title = {Effects of N[6]-Methyladenosine (m[6]A) and 5-Methylcytosine (m[5]C) Modifications in the Guide Region of CRISPR RNA on Cas12a Nuclease Activity.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2551-2556},
pmid = {41346237},
issn = {1520-4812},
support = {R21 HG012257/HG/NHGRI NIH HHS/United States ; R35 GM156250/GM/NIGMS NIH HHS/United States ; },
mesh = {*5-Methylcytosine/chemistry/metabolism ; *Adenosine/analogs &amp; derivatives/chemistry/metabolism ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N[6]-methyladenosine (m[6]A) and 5-methylcytosine (m[5]C), introduced into the different positions of the guide region of a crRNA. Melting temperature (Tm) analysis showed that m[6]A had a minimal impact on RNA-DNA duplex stability. In contrast, the incorporation of m[5]C residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a's nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater Tm increase with m[5]C, m[6]A incorporation led to a faster fluorescence recovery rate than that with m[5]C.},
}
@article {pmid41346247,
year = {2026},
author = {Singh, V and Mishra, M and Singla-Pareek, SL and Roy, JK and Pareek, A},
title = {Lysine Matters: Genetic and Biotechnological Innovations to Combat Protein Malnutrition.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {3},
pages = {1509-1529},
doi = {10.1111/pce.70316},
pmid = {41346247},
issn = {1365-3040},
support = {//This study was supported by Department of Biotechnology, Ministry of Science and Technology, India./ ; },
mesh = {*Lysine/metabolism/deficiency ; Crops, Agricultural/genetics/metabolism ; *Biotechnology/methods ; Gene Editing ; Metabolic Engineering ; Plants, Genetically Modified ; Plant Breeding ; *Protein Deficiency/prevention &amp; control ; CRISPR-Cas Systems ; Biofortification ; },
abstract = {Lysine deficiency in staple crops like maize, rice, and wheat remains a major cause for global protein malnutrition, underscoring the urgent need for effective biofortification strategies. This review critically examines recent advances in enhancing lysine content, spanning conventional breeding and metabolic engineering to cutting-edge precision genome editing. While conventional breeding, exemplified by Quality Protein Maize, has improved lysine levels, it is often constrained by yield and quality trade-offs. Metabolic engineering strategies, including overexpression of lysine biosynthetic genes, suppression of catabolic genes, and modification of storage proteins, have achieved substantial lysine enrichment but face regulatory and consumer acceptance challenges due to their transgenic nature. The advent of CRISPR/Cas technology now enables precise, transgene-free editing of key enzymes such as DHDPS, AK, and LKR/SDH offering a powerful alternative, though concerns regarding off-target effects and pleiotropy remain. While integrating multi-omics with AI-driven predictive modelling can optimise metabolic flux for higher lysine yield, coupling next-generation genome editing with speed breeding offers a transformative route to develop high-lysine, high-yielding crops for sustainable nutritional security.},
}
@article {pmid41346702,
year = {2026},
author = {Patra, C and Hussein, Z and Ace, VD and Misnik, EV and Rybalko, DS and Salimova, AA and Ereshko, DS and Dubovichenko, MV and Nour, MAY and Drozd, VS and Kolpashchikov, DM},
title = {The efficacy of oligonucleotide-based gene therapeutics in gene silencing.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {599-616},
pmid = {41346702},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Silencing ; *Oligonucleotides, Antisense/therapeutic use/genetics ; RNA, Small Interfering/genetics/therapeutic use ; Animals ; *Oligonucleotides/genetics/therapeutic use ; },
abstract = {Oligonucleotide-based gene therapeutics (OGTs) have emerged as a promising strategy for treating a variety of diseases, offering a tool for gene modulation at the mRNA level. Despite significant progress in OGTs development, their efficacy in both experimental and clinical settings has often fallen short of expectations. Current estimates suggest that less than 1% of transfected OGTs are released into the cytosol, significantly limiting the interaction with target RNA. Moreover, data suggests that only about 2% of the tested siRNAs achieve the expected 70% target gene knockdown in vitro. Clinically approved OGTs appear to be effective only against genetic disorders that lack effective alternative treatment, and even in these cases their therapeutic contribution remains marginal. Notably, the majority of approved OGTs, as well as those currently in clinical trials, are antisense oligonucleotides (ASOs) despite cell culture data showing that small interfering RNAs (siRNAs) exhibit greater potency. The delayed commercialization of siRNAs, despite high research interest, may be attributed to passenger stand-dependent off target effect and the immaturity of their design and modification strategies. This review critically evaluates the factors influencing therapeutic efficacy of OGTs and highlights the persistent gap between theoretical promise and clinical reality.},
}
@article {pmid41347244,
year = {2025},
author = {Daraghmeh, DN and AbuIriban, RW and Nawawreh, N and Abuamro, AM and Alassar, MM and Daraghma, SN and Alhajahmed, NM and Thandar, Y},
title = {Advancements in alternative approaches to address antimicrobial resistance in bacterial pneumonia: a comprehensive review.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1704931},
pmid = {41347244},
issn = {1664-302X},
abstract = {PURPOSE: This review explores both current and emerging alternative treatment approaches to combat AMR specifically in the context of bacterial pneumonia, highlighting therapies that extend beyond conventional antibiotics.<br><br>METHODS: PubMed, Embase, and Google Scholar were searched for full-text, English-language articles, with emphasis on publications from 2020 to 2025. Earlier seminal studies were also included when necessary to provide historical, mechanistic, or conceptual context. The review focuses was on alternative strategies that have shown effectiveness in preclinical or clinical settings to combat AMR in relation to bacterial pneumonia.<br><br>RESULTS: Emerging strategies to tackle AMR in bacterial pneumonia involve several innovative approaches including stem cells, bacteriophage therapy, metal based nanoparticles (e.g., silver, copper, and gold). The adjunctive use of probiotics and herbal medicine has demonstrated potential in enhancing clinical outcomes and modulating host immunity. Moreover, gene editing technologies like CRISPR-CAS and various vaccination programs are being investigated for their roles in prevention and resistance management. While these methods show promise, many are still in the early stages of development and encounter challenges related to standardization, safety, and regulatory approval.<br><br>CONCLUSION: Alternative therapies present exciting possibilities for addressing AMR in bacterial pneumonia. However, to effectively translate these innovations into clinical practice, we need thorough research, international collaboration, and supportive policy frameworks. By combining these strategies with antimicrobial stewardship initiatives, we can help maintain antibiotic effectiveness and enhance patient outcomes.},
}
@article {pmid41348151,
year = {2025},
author = {Cheng, Y and Gao, W and Shi, S and Han, F and Dong, H},
title = {Identification of the orange pigment in Nonomuraea gerenzanensis and development of a pigment-free mutant.},
journal = {AMB Express},
volume = {16},
number = {1},
pages = {4},
pmid = {41348151},
issn = {2191-0855},
support = {2024TSGC0896//Shandong Province Science and Technology-based Small and Medium-sized Enterprises Innovation Capacity Enhancement Project/ ; },
abstract = {The secondary metabolite A40926, a precursor to the glycopeptide antibiotic dalbavancin, is synthesized by the rare actinomycete Nonomuraea gerenzanensis (N. gerenzanensis) within the pharmaceutical industry. The biosynthesis of A40926 is accompanied by the production of an orange pigment, which poses significant challenges and incurs high costs in the purification process of A40926. To identify this orange pigment, a comprehensive analysis was conducted, including the examination of the biosynthetic gene cluster, potential biosynthetic pathways, purification processes, and structural identification. Additionally, the ispF gene, which encodes the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and is implicated in the biosynthesis of orange pigment, was deleted using the CRISPR/Cas9 system. To enhance A40926 production in the ΔIspF mutant, the overexpression of the cyclic AMP receptor protein (Crp) was implemented to assess its regulatory impact on A40926 biosynthesis. Consequently, the orange pigment produced by N. gerenzanensis was identified as lycopene, synthesized via the methylerythritol phosphate (MEP) pathway. Although the ΔIspF mutant was unable to biosynthesize the orange pigment, its production of A40926 was adversely affected and was lower than that of the original strain. Consequently, the overexpression of the global regulator Crp significantly enhanced A40926 production, achieving a yield of 841.1 mg/L. The investigation of pigment-free mutants presented in this study offers valuable insights for effectively reducing production costs within the microbial pharmaceutical industry.},
}
@article {pmid41348871,
year = {2025},
author = {Puppala, AK and Nielsen, AC and Regan, M and Mancinelli, GE and De Pooter, RF and Arnovitz, S and Harding, C and McGregor, M and Balanis, NG and Clarke, R and Merrill, BJ},
title = {Programmable multistep CRISPR gene activation via control of RNA polymerase III termination.},
journal = {Science advances},
volume = {11},
number = {49},
pages = {eadt1532},
pmid = {41348871},
issn = {2375-2548},
support = {R01 GM139894/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA Polymerase III/metabolism/genetics ; *CRISPR-Cas Systems ; *Transcriptional Activation ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; },
abstract = {Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have only modest capabilities for stepwise gene regulation. Here, we develop a sequential genetic system that transcriptionally activates endogenous genes in a preprogrammed, stepwise manner. This system uses the removal of an RNA polymerase III termination sequence to trigger both the transcriptional activation and DNA endonuclease activities of a Cas9-VPR protein, driving progression through a cascade of gene activation events. The system's functionality in human cells, including iPSCs, enables the development of a path for cellular programming by controlling the sequential order of gene activation to influence cellular states.},
}
@article {pmid41349356,
year = {2025},
author = {Shahid, N and Hammond, JR},
title = {Characterization of genetically modified human embryonic kidney 293 cells lacking equilibrative nucleoside transporter subtype 2, or both subtypes 1 and 2, and the impact of their loss on sensitivity to chemotherapeutic purine/pyrimidine analogs.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {53},
number = {12},
pages = {100203},
pmid = {41349356},
issn = {1521-009X},
mesh = {Humans ; HEK293 Cells ; *Equilibrative Nucleoside Transporter 1/genetics/metabolism ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; CRISPR-Cas Systems ; *Purines/pharmacology/metabolism ; *Pyrimidines/pharmacology/metabolism ; *Antineoplastic Agents/pharmacology ; Biological Transport ; Gene Knockout Techniques ; },
abstract = {Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs. SIGNIFICANCE STATEMENT: A panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.},
}
@article {pmid41349512,
year = {2025},
author = {Kosaka, Y and Lopez, B and Kishimoto, N and Jacob, S and Montenont, E and Huallanca, R and Coughenour, G and Di Paola, J and Ross, J and Lee, K and Rondina, MT and Bray, PF and Rowley, JW},
title = {Functional classification of platelet gene variants using CRISPR HDR in CD34[+] cell-derived megakaryocytes.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2888-2901},
pmid = {41349512},
issn = {1537-6605},
support = {R01 HL166805/HL/NHLBI NIH HHS/United States ; R01 HL142804/HL/NHLBI NIH HHS/United States ; R01 HL139825/HL/NHLBI NIH HHS/United States ; I01 CX001696/CX/CSRD VA/United States ; K24 HL155856/HL/NHLBI NIH HHS/United States ; R01 HL144957/HL/NHLBI NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; *Megakaryocytes/metabolism ; *Blood Platelets/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD34/metabolism/genetics ; Integrin beta3/genetics ; Gene Editing/methods ; Integrin alpha2/genetics ; *Genetic Variation ; Hematopoietic Stem Cells/metabolism ; Thrombasthenia/genetics ; },
abstract = {The interpretation of genetic variants in inherited diseases, such as inherited platelet disorders (IPDs), remains a major clinical challenge, as most are classified as variants of uncertain significance (VUSs). A key barrier to functional evaluation is the lack of accessible, lineage-appropriate assays that reliably reflect native gene regulation and cell-specific biology. To address this gap, we developed CRIMSON HD (CRISPR-edited megakaryocytes [MKs] for surveying platelet variant functions through homology-directed repair [HDR]), a CRISPR-Cas9 HDR-based genome-editing platform applicable to CD34[+] cell-derived blood lineages and optimized for evaluating platelet-associated variants. Using this system, we modeled known and candidate disease-associated variants in integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3), which encode the platelet αIIb/β3 integrin and are causative in Glanzmann thrombasthenia (GT). We introduced precise variants into primary human MKs derived from CD34[+] hematopoietic stem and progenitor cells, achieving >90% editing efficiency. Edited MKs faithfully recapitulated both expression and functional phenotypes of known type I, II, and III GT variants. CRIMSON HD enabled functional evaluation and reclassification of several GT VUSs, including αIIb Gly201Ala, a population variant now shown to cause near-complete loss of αIIb/β3 expression; αIIb Ala777Asp, which results in intermediate αIIb/β3 expression and impaired agonist-induced integrin binding; and β3 Arg119Gln, previously linked to the loss of anti-HPA1a antibody binding in fetal and neonatal alloimmune thrombocytopenia (FNAIT), now shown to impair integrin surface expression. These findings demonstrate the importance of lineage-specific, physiologically relevant assays for the functional classification of platelet-related variants, providing mechanistic information and clinically meaningful insights for individuals with IPDs.},
}
@article {pmid41349515,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Capture first, then deliver!.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {12},
pages = {2008-2009},
doi = {10.1016/j.str.2025.11.001},
pmid = {41349515},
issn = {1878-4186},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; DNA/metabolism/chemistry ; *Integrases/chemistry/metabolism ; Protein Binding ; },
abstract = {In this issue of Structure, Henriques et al.[1] present structural snapshots that capture distinct conformational states of the type I-F Cas1-Cas2/3 integrase complex, illustrating that foreign DNA binding triggers a large-scale domain rearrangement that enables prespacer delivery to the CRISPR array.},
}
@article {pmid41350682,
year = {2025},
author = {Lyu, G and Li, P and Lang, W},
title = {A review of recent studies on CRISPR/Cas9-mediated genome editing in a variety of muscle-related genetic disorders.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1381},
pmid = {41350682},
issn = {1479-5876},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Muscular Diseases/genetics/therapy ; *Genetic Diseases, Inborn/genetics ; Genetic Therapy ; Muscular Dystrophies/genetics ; },
abstract = {The human body is capable of mutating a single gene to produce a wide range of debilitating disorders. Genomic editing for disease prevention via phenotypic reversal was a significant challenge prior to the development of clustered regulatory interspaced short tandem repeats (CRISPR) and CRISPR-associated protein (Cas) systems. Gene therapy-editing a patient's DNA to correct a particular mutation-and treating human diseases that have not responded to conventional medicine are two areas where CRISPR/Cas9 technology shows the most promise as a therapeutic tool. This powerful instrument has shown great promise in muscle-related illnesses, offering new insights into muscle biology and developing more effective treatment techniques. Discoveries about the hereditary causes of the majority of inherited myopathies and muscular dystrophies (MDs) have emerged over the last two decades. Additionally, skeletal muscles weaken and degenerate over time due to a group of hereditary disorders known as MDs. The field of skeletal muscle diseases and associated genetic alterations is seeing remarkable progress in developing therapeutic vectors to fix these mutations. Myopathies, MDs, and neuromuscular disorders are just a few examples of the many genetic abnormalities related to muscles that have sparked renewed interest in the potential of genome editing as a therapeutic tool due to its efficiency, adaptability, and relative ease of use in targeted genome editing. Consequently, CRISPR/Cas9 has garnered much interest and is used more often in therapeutic techniques due to its potential capacity to cure various human ailments. To pave the way for more effective and personalized therapies, this review article provides a thorough overview of the revolutionary role of CRISPR/Cas9 in improving our understanding and treatment of genetic disorders related to muscles by combining present knowledge with future perspectives.},
}
@article {pmid41351274,
year = {2025},
author = {Hou, H and Li, Y and Su, N and Ding, Y and Shang, C and Li, X and Xiong, Z and Sun, Y and Zhan, W and Wang, Y and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {Slmsh1-induced heritable enhancement of traits for tomato breeding improvement.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {5},
pages = {e70607},
doi = {10.1111/tpj.70607},
pmid = {41351274},
issn = {1365-313X},
support = {CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; CARS-23-B08//China Agriculture Research System/ ; SWU-KF25027//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/growth &amp; development ; *Plant Breeding/methods ; *Plant Proteins/genetics/metabolism ; Fruit/genetics/growth &amp; development ; Droughts ; Quantitative Trait, Heritable ; Phenotype ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Vegetable grafting is a horticultural technique employed to develop specialized plant varieties by effectively enhancing resistance to both biotic and abiotic stresses, as well as improving fruit quality and yield. However, these advantageous traits are generally non-heritable. The MSH1 gene induced heritable enhancement-through-grafting (HEG) effect on growth vigor, demonstrating promising application potential. In this study, we employed the msh1 mutant tomato as a rootstock to induce heritable superior traits and combined this approach with hybridization techniques to enhance tomato cultivars. Three Slmsh1 mutants were generated using CRISPR/Cas9 which exhibited a dwarf phenotype with whitened spots. By grafting several distinct inbred lines onto Slmsh1, we observed significant HEG, drought stress tolerance, and fruit quality. Under drought conditions, Slmsh1-grafted tomato seedlings exhibited increased biomass and enhanced drought tolerance through the regulation of antioxidant enzyme activities. Differential expression and methylation analyses of the graft progeny revealed that these heritable enhanced traits (HETs) are likely attributable to epigenetic modifications in the expression of ROS-scavenging- and hormone-related genes. Furthermore, to explore practical applications, we crossed inbred lines with HETs and evaluated the growth, yield, and fruit quality of the resulting hybrid combinations. The results indicated that these hybrid combinations improved fruit yield and quality, enhancing the total soluble solids, soluble sugar, and soluble protein content. These findings suggest that Slmsh1-grafted progenies enhanced plant biomass and drought resistance, while their hybrid combinations positively influenced root growth, yield, and fruit quality, providing new insights into the synergistic integration of genome editing and conventional breeding.},
}
@article {pmid41352420,
year = {2026},
author = {Tang, M and Liang, R and Wu, Z and Chen, C and He, B and Zhou, N and Wang, S and Xiao, X and Li, G and Jiang, Y and Gong, G and Zhou, Y},
title = {Deciphering OCT4A-dose-dependent transcriptional profiles associated with tumorigenic potential in somatic cancer cells.},
journal = {SLAS technology},
volume = {36},
number = {},
pages = {100381},
doi = {10.1016/j.slast.2025.100381},
pmid = {41352420},
issn = {2472-6311},
mesh = {*Octamer Transcription Factor-3/genetics/metabolism ; Humans ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; *Carcinogenesis/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; *Transcriptome ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Prognosis ; },
abstract = {AIMS: The transcription factor OCT4A, a well-established master pluripotency factor, exerts regulatory effects on cell fate determination that are closely associated with its protein levels. This study aims to uncover the downstream gene profile features relevant to tumorigenic potential mediated by OCT4A under varying protein abundance in somatic cancer cells (SCCs).<br><br>MATERIALS AND METHODS: CRISPR-Cas9-mediated knockout and doxycycline-inducible OCT4A expression systems were established in cervical (HeLa) and hepatocellular (HepG2, Huh7) cancer cells. Single-cell sequencing, spatial transcriptomic and survival analysis data were used to elucidate the expression pattern of OCT4 in somatic cancer tissues and its prognostic relevance. The plate colony formation assay was performed to assess the tumorigenic capacity of SCCs, and Bulk RNA sequencing coupled with weighted gene co-expression network analysis (WGCNA) identified dose-relevant downstream pathways. Functional enrichment, survival modeling, and RT-qPCR validation were used to construct OCT4A-dose-dependent transcriptional regulatory networks.<br><br>KEY FINDINGS: OCT4 transcript, is heterogeneously present and confined to a small subset of tumor cells within somatic cancer tissues, with a significantly higher proportion of OCT4-positive cells in tumor tissues compared to paired paraneoplastic tissues and is significantly correlated with poor prognosis in SCCs. Endogenous low-level OCT4A positively regulates tumorigenic capacity predominantly through targeting non-coding genes, whereas high-level OCT4A suppresses tumorigenic capacity primarily via protein-coding genes in SCCs. A prognostic model based on high-level OCT4A-regulated protein-coding genes was associated with favorable clinical outcomes, aligning with in vitro phenotypic results.<br><br>SIGNIFICANCE: Our findings further confirm in SCCs that the functional pleiotropy of OCT4A is closely linked to its protein abundance, and further systematically elucidate the molecular signatures of OCT4A-regulated downstream gene networks associated with tumorigenic phenotypes at differential protein levels, providing novel insights for its translational exploitation in both oncological intervention and regenerative medicine strategies.},
}
@article {pmid41352695,
year = {2026},
author = {Pradhan, K and Anoop, S},
title = {CRISPR 2.0: Expanding the genome engineering Toolbox for epigenetics, RNA editing, and molecular diagnostics.},
journal = {Gene},
volume = {979},
number = {},
pages = {149938},
doi = {10.1016/j.gene.2025.149938},
pmid = {41352695},
issn = {1879-0038},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *RNA Editing/genetics ; *Pathology, Molecular/methods ; Epigenomics/methods ; Animals ; Genetic Engineering/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Non-canonical CRISPR systems adaptation has led to genome editing through nucleases, and the development of transcriptional and epigenetic regulation, transcriptome editing, and molecular diagnostics has resulted in a diversified set of tools-CRISPR 2.0. In this review, the author summarizes the mechanisms and recent engineering advances of (i) dCas9-based epigenetic effectors, (ii) RNA-targeting Cas13 systems and engineered RNA editors, (iii) DNA base editors and prime editors, and (iv) CRISPR-powered diagnostic platforms and their translational readiness. There is a critical comparison of the various approaches (e.g., RNAi/ASO versus Cas13-based methods; base editing versus prime editing) along with practical translational considerations such as delivery technologies, safety (off-target/edit windows, mosaicism), and regulatory pathways which are evaluated. Three concise case studies refer to map laboratory evidence to clinical or near-clinical outcomes and the ethical and governance discussion is widened to include global access, intellectual property and equity in deployment. Finally, the authors classify technologies according to their level of readiness - diagnostics and some ex-vivo therapeutic approaches are already in or very close to clinical use, chosen in-vivo editing methods are undergoing early trials, and AI-assisted nuclease design is still mostly theoretical but is getting better fast. This comprehensive viewpoint is intended to help researchers and physicians understand which CRISPR tools are most likely to be translated soon and where more validation is required.},
}
@article {pmid41352906,
year = {2026},
author = {Fakhr, ZA and Xie, W and Zeng, S and Cai, S},
title = {Site accessibility-driven CRISPR/Cas13a activation for amplification-free RNA biosensing.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344858},
doi = {10.1016/j.aca.2025.344858},
pmid = {41352906},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *RNA/analysis ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Kinetics ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis-Menten kinetic parameters.<br><br>RESULTS: Three ciRS-7-specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (&#x394;S&#xa0;=&#xa0;-862, -813, and -615&#xa0;cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA-target pair. Michaelis-Menten analysis showed kcat values of 1.39, 1.31, and 1.16 s[-1] for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity.<br><br>SIGNIFICANCE: Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.},
}
@article {pmid41352908,
year = {2026},
author = {Li, L and Tang, Z and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344860},
doi = {10.1016/j.aca.2025.344860},
pmid = {41352908},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *RNA, Viral/analysis/genetics ; Hepatitis B virus/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; HIV/genetics ; },
abstract = {Recently, CRISPR/Cas system has been proposed as a novel tool with simplicity and high accuracy. The CRISPR RNA (crRNA) can be divided into spacer crRNA and handle crRNA without losing its original function. In this work, we have investigated CRISPR Cas12a with split crRNA to detect HBV DNA and HIV RNA in a single tube. In the first step, Cas12a can recognize HBV DNA and initiate its trans-cleavage on FAM-BHQ1 reporter, after 1 h incubation, the fluorescence intensity was correlated with the concentration of HBV DNA. In the second step, the TAMRA BHQ2 ds DNA reporter was introduced in the same tube to bind with remained Cas12a proteins, HIV RNA and handle crRNA. The trans-cleavage from the first step would not interfere with HIV RNA and dsDNA reporter. With the incubation for another hour, HIV RNA can be quantified by the cis-cleavage of TAMRA BHQ2 reporter. we can successively identify the two nucleic acids with the limit of detection of 0.70 pM for HBV DNA, and 0.47 nM for HIV RNA, respectively. This special designed split crRNA can simplify detecting procedure and only need Cas12a protein in a single tube. Next, we expand this strategy in semi-quantifying two kinds of DNA in one tube. Overall, this study overcomes the limitation of conventional CRISPR-based methods and provides a new, inexpensive, and low-threshold approach based on Cas12a with split crRNA.},
}
@article {pmid41352919,
year = {2026},
author = {Guan, X and Wang, S and Wang, P and Zhang, J and Sun, S},
title = {Enhanced chemiluminescence aptasensing with triple cascade amplification for sensitive detection of tumor-derived exosomes.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344873},
doi = {10.1016/j.aca.2025.344873},
pmid = {41352919},
issn = {1873-4324},
mesh = {*Exosomes/chemistry/metabolism ; Humans ; *Aptamers, Nucleotide/chemistry/metabolism ; *Luminescent Measurements/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Mucin-1 ; Alkaline Phosphatase/chemistry/metabolism ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Biomarkers, Tumor ; Tetraspanin 30 ; },
abstract = {BACKGROUND: Tumor-associated exosomes hold significant clinical promise as liquid biopsy biomarkers. However, the accurate detection of these rare exosome subpopulations in clinical samples demands analytical platforms with exceptionally high sensitivity and specificity. While conventional nucleic acid amplification-based methods provide considerable detection sensitivity, they are often hampered by time-consuming procedures, operational complexity, and susceptibility to contamination. Therefore, it is imperative to develop practical exosome measurement platforms that combine high sensitivity, robustness, and rapid analysis capabilities to provide reliable evidence-based support for precision oncology.<br><br>RESULTS: In this work, a triple cascade-amplified aptasensor (TCAA) via functionalized gold nanoparticle (fAuNP), CRISPR/Cas12a, and alkaline phosphatase (ALP) was developed for enhanced chemiluminescence (CL) assay of tumor-derived exosomes without nucleic acid amplification. The target exosomes were initially recognized by CD63 and MUC1 aptamers. fAuNP-conjugated Trigger sequences then activated CRISPR/Cas12a to cleave single-stranded DNA and release ALP. Consequently, the ALP catalyzed substrate to produce CL signals correlating with the concentration of the analyte. By simultaneously integrating the signal amplification capabilities of multiple techniques, this TCAA achieved a limit of detection of 44 particles/&#x3bc;L for MUC1-positive exosomes within 60&#xa0;min with excellent robustness. Compared with the single- and dual-amplification methods, the sensitivity was increased by 40-fold and 6-fold, respectively. Clinical trials showed that the area under the curve of this approach was 0.96, which was higher than that of the commercialized chemiluminescence immunoassay and effectively distinguished breast cancer-derived specimens.<br><br>SIGNIFICANCE: These findings indicate that the TCAA strategy provides a highly sensitive, rapid, and robust tool for the detection of low-abundance tumor exosome subpopulations without nucleic acid amplification. It effectively addresses the limitations of conventional methods and demonstrates high clinical utility. This work offers a reliable and practical platform for non-invasive liquid biopsy, holding great potential for trace-level detection of diverse biomarkers.},
}
@article {pmid41353207,
year = {2025},
author = {Sun, H and Teng, Q and Liu, W and Guo, R and Li, M and Xiong, W and Huang, Q and Yu, Q and Luo, N and Li, Y and Song, J and Gong, S and Shi, X and Yi, C and Liu, K},
title = {CRISPR-free RNA base editing mediated PTC-readthrough restores hearing in mice with Otof nonsense mutation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {413},
pmid = {41353207},
issn = {2041-1723},
support = {no. 81770997//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 82460223//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Codon, Nonsense/genetics ; Mice ; *Deafness/genetics/therapy ; *Genetic Therapy/methods ; Disease Models, Animal ; *Membrane Proteins/genetics/metabolism ; *RNA Editing/genetics ; *Hearing/genetics ; Humans ; Cochlea/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Male ; Female ; Mice, Inbred C57BL ; },
abstract = {The gene therapy achieved by AAV-mediated otoferlin-overexpression is an effective therapeutic strategy for congenital deafness. However, achieving its physiological and endogenous patterns of expression remains challenging. Here, we generate the homologous mutation Otof c.1315 C > T (p.R439*), equivalent to OTOF c.1273 C > T (p.R425*) found in humans with profound deafness, to create a nonsense mutation-induced deaf mouse model. We then deliver the 'RESTART v3' system, which is a CRISPR-free RNA base editor for nonsense mutation suppression, into the cochlea of the mice. We achieve physiological otoferlin expression, and the edited premature termination codon is reverse-mutated to the original amino acid. We observe significant hearing restoration and enhancement of the behavioral auditory startle reflex. Thus, our study presents a successful RNA editing strategy to significantly restore hereditary deafness in mice carrying the specific Otof nonsense mutation, which holds great promise for future clinical translation.},
}
@article {pmid41353342,
year = {2025},
author = {He, L and Yao, Y and You, Y and Wei, X and Ma, Y and Yuan, W and Lang, Z and Zhu, JK},
title = {Versatile molecular tools enabling customizable DNA methylation editing in Arabidopsis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {251},
pmid = {41353342},
issn = {2041-1723},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100458//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics ; *DNA Methylation/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {Tools to edit DNA methylation in a targeted manner are vital for establishing causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we develop a range of highly effective tools for editing DNA methylation in Arabidopsis, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show a diversity of performance features in terms of specificity and efficiency, offering either the capacity to edit DNA methylation in a target-specific manner or the ability to edit DNA methylation genome-wide due to potent off-target effect. Importantly, DNA methylation edited by these tools is inherited in the absence of transgene. These versatile tools pave the way for diverse applications of DNA methylation editing in not only research but also epigenetic breeding of crops.},
}
@article {pmid41353404,
year = {2025},
author = {Burgold, T and Karakoc, E and Gonçalves, E and Barrio-Hernandez, I and Dwane, L and Silva, R and Souster, E and Sharma, M and Beck, A and Koh, GCC and Zalmas, LP and Garnett, MJ and Bassett, AR},
title = {A next-generation dual guide CRISPR system for genetic interaction library screening.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {561},
pmid = {41353404},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 220540/Z/20/A//Wellcome Trust (Wellcome)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; Cell Line, Tumor ; Streptococcus pyogenes/genetics ; Colorectal Neoplasms/genetics ; CRISPR-Associated Protein 9/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Transfer/genetics ; },
abstract = {Pairwise perturbation of gene function using the CRISPR/Cas9 system has potential in screening for genetic interactions and synthetic lethal gene pairs to identify combination therapies for cancer. However, existing dual guide expression systems are cumbersome to clone, often result in a large proportion of undesired guide pairs and have an imbalance of guide expression from the two positions. Here, we demonstrate a next-generation system for dual guide delivery based around a tRNA spacer that allows a single-step cloning strategy, as little as 2% of undesired guide pairs, and highly balanced expression of the two guides. This system allows efficient library-scale screening for hundreds of thousands of genetic interactions using the well-understood Streptococcus pyogenes Cas9 (SpCas9) system. We use this to screen a 100,136 guide pair library in colorectal cancer cells and successfully identify synthetic lethal genetic interactions between paralogs or other known interacting genes, establishing our method for performing efficient large-scale genetic interaction screens. This system is versatile and could be used with most guide RNA vector systems, and for other uses of paired guide delivery, such as improving single gene knockout efficiency or improving guide detection in single cell or optical CRISPR screens.},
}
@article {pmid41353974,
year = {2026},
author = {Madny, MA and Yadav, KS},
title = {Biomimetic oral drug delivery: Translating nature's design into therapeutic innovation.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {259},
number = {},
pages = {115348},
doi = {10.1016/j.colsurfb.2025.115348},
pmid = {41353974},
issn = {1873-4367},
mesh = {Humans ; Administration, Oral ; *Drug Delivery Systems/methods ; *Biomimetic Materials/chemistry ; *Biomimetics/methods ; Animals ; Nanoparticles/chemistry ; },
abstract = {Oral drug delivery, the most patient friendly administration route offers convenience and compliance but faces formidable biological barriers. Enzymatic degradation, mucosal entrapment, efflux transport and extensive first-pass metabolism drastically reduce the effectiveness of sensitive therapeutics including peptides, proteins, nucleic acids and vaccines. Conventional formulations often fail to overcome these challenges highlighting the need for innovative approaches. Biomimetic drug delivery has emerged as a transformative strategy. By emulating structures and functions from cells, membranes, exosomes, viruses and gut microbiota these systems achieve immune evasion, mucus penetration, site-specific targeting and stimulus-responsive release. Such approaches improve formulation stability and in vivo absorption but also promise precise and patient centric therapies. This review provides a comprehensive overview of biomimetic oral systems highlighting their mechanisms, design principles and translational potential. Recent advances include cell membrane-coated nanoparticles for tumor targeting and immune modulation, exosome-inspired carriers for protein and RNA transport, virus-like particles (VLPs) for oral vaccines, and mucoadhesive or mucus-penetrating polymers modeled on pathogen strategies. Complementary pH, enzyme and redox-responsive platforms exploit gastrointestinal (GI) microenvironments to ensure controlled release. Emerging tools such as bioinspired computational modeling, 3D/4D printing, organoid-on-chip models and CRISPR/Cas-based platforms accelerate optimization and clinical translation. Although most technologies remain in preclinical development, early findings demonstrate superior pharmacokinetics, therapeutic efficacy, and safety over conventional systems. This article critically examines biomimetic oral drug delivery addressing advances and underlying mechanisms including regulatory considerations and future directions. They stand poised to form the foundation of next-generation precision therapeutics.},
}
@article {pmid41354630,
year = {2026},
author = {Wang, W and Chen, K and Wang, Z},
title = {Genome-Wide CRISPR Screen Reveals PIK3CA Inhibition Enhances Lipid Nanoparticle-Mediated siRNA Delivery.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {11},
pages = {e17617},
pmid = {41354630},
issn = {2198-3844},
support = {//International Society for Advancement of Cytometry/ ; //Chan Zuckerberg Biohub/ ; //Chan Zuckerberg Initiative/ ; },
mesh = {Humans ; *RNA, Small Interfering/genetics/administration &amp; dosage ; *Nanoparticles/chemistry ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists &amp; inhibitors/metabolism ; Animals ; Mice ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipids/chemistry ; CRISPR-Cas Systems/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are useful carriers for therapeutic siRNA delivery, yet their clinical efficacy remains constrained by insufficient cellular uptake. Here, using a genome-wide CRISPR knockout screen, multiple genetic modulators of LNP uptake is uncovered, with PIK3CA emerging as a top druggable target. Pharmacologic inhibition of PIK3CA with BAY1082439 - a clinically evaluated small molecule - significantly enhances LNP uptake, siRNA delivery, and gene silencing across diverse epithelial cancer cell lines in vitro. Co-administration of BAY1082439 with siRNA-loaded LNPs also better suppressed tumor growth and reduced liver inflammation in vivo, respectively. These findings establish PIK3CA inhibition as a broadly applicable strategy to boost LNP-mediated RNA interference and highlight the promise of combining functional genomics with nanomaterials to advance RNA-based therapeutics.},
}
@article {pmid41354953,
year = {2025},
author = {Nguyen, VT and Van, BTT and Uyen, TN and Tong, NX and Pham, TL and Vy, NHT and Thuy, DT and Thuy, NP and Kobayashi, M},
title = {Functional divergence of zebrafish keap1 paralogs revealed by CRISPR/Cas9-mediated gene editing: a specialized role for keap1b in inflammation.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {53},
pmid = {41354953},
issn = {1573-9368},
support = {108.06-2020.19//National Foundation for Science and Technology Development/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Oxidative Stress/genetics ; *Inflammation/genetics ; Kelch-Like ECH-Associated Protein 1/genetics ; NF-E2-Related Factor 2/genetics ; Signal Transduction/genetics ; Gene Editing ; Carrier Proteins ; },
abstract = {The Keap1/Nrf2 signaling pathway is a master regulator of cellular defense against oxidative and electrophilic stress. In teleosts like zebrafish (Danio rerio), whole-genome duplication resulted in two keap1 paralogs, keap1a and keap1b, whose functional specificities remain incompletely understood. This study investigates the divergent roles of these paralogs by comparing the responses of established keap1a and novel keap1b knockout larvae to distinct chemical stressors. By comparing the responses of keap1b[dl40], keap1a[dl07], and nfe2l2a[dl703] (Nrf2a) larvae to these stressors, we uncovered a striking functional dichotomy. While loss of either paralog conferred resistance to H2O2-induced oxidative stress, keap1b[dl40] larvae, unlike their keap1a[dl07] counterparts, exhibited extreme sensitivity to the lethal effects of CuSO4 exposure, with survival rates plummeting to ~ 25%. This heightened sensitivity to copper sulfate was associated with a blunted transcriptional response of inflammatory markers tnf-a and c3a, suggesting that Keap1b is critical for modulating the Nrf2a-mediated response to inflammatory stress in orchestrating a viable inflammatory response. This work clarifies the non-redundant, vital function of Keap1b in the response to heavy metal-induced stress and provides a valuable genetic resource (keap1b[dl40] null allele) for future studies.},
}
@article {pmid41354981,
year = {2026},
author = {Das, T and Barman, T and Prasad, A},
title = {Precision editing to improve fruit traits: CRISPR/Cas into the picture.},
journal = {Protoplasma},
volume = {263},
number = {3},
pages = {735-746},
pmid = {41354981},
issn = {1615-6102},
mesh = {*Fruit/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Crops, Agricultural/genetics ; Plant Breeding ; },
abstract = {Crop growth, quality, and yield can be adversely affected by various biotic and abiotic stresses. Crop characteristics can be improved with conventional breeding and other variation-based breeding strategies. However, these strategies are time as well as resource consuming and to overcome this, novel approaches are necessary. CRISPR/Cas technique allows to improve desired traits more efficiently and accurately by targeting specific genes. Genome editing has become more versatile with CRISPR/Cas systems and is a valuable tool to protect food security by developing commercial crops optimized for yield and nutritional quality. Researchers are able to target and edit stress response pathway genes to develop crops with increased tolerance to stress. A lack of regeneration protocols and sufficient genome sequencing data has restricted fruit editing to only a few fruits (tomatoes, citrus, apple, kiwi, banana, grapes, strawberries, watermelon, etc.). This review is focused on CRISPR/Cas applications on the nutritional aspects of fruit engineering along with the challenges and opportunities. Another aspect which will be covered is the use of CRISPR/Cas technology to improve fruit resilience to biotic and abiotic stress, but not at the cost of yield. We discuss the pros and cons of using this technology, such as unintended effects on fruit traits or public concerns about GMOs. We conclude that the application of CRISPR/Cas9 technology has the potential to be of great benefit to the agricultural industry not only to improve nutritional aspects but also to help reduce crop losses.},
}
@article {pmid41355773,
year = {2026},
author = {Tian, S and Yao, L and Gong, F and Li, Y and Zhao, Y and Yang, Y},
title = {Rapid and sensitive detection of circulating tumor DNA via a CRISPR/Cas12a-based catalytic hairpin assembly.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {1},
pages = {115-123},
doi = {10.1039/d5ay01624j},
pmid = {41355773},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/blood/genetics ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Biomarkers, Tumor/blood/genetics ; Biosensing Techniques/methods ; Neoplasms/blood/diagnosis/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Cancer is one of the major diseases that endanger the human health. Circulating tumor DNA (ctDNA) is an ideal biomarker for the real-time monitoring of cancer. In the present work, a rapid and sensitive assay coupled with CRISPR/Cas12a and CHA (Cas12a-CHA) was constructed for the detection of ctDNA. We designed and prepared a trigger, which was the substrate of Cas12a. On the addition of ctDNA, crRNA-guided ctDNA activated the trans-endonuclease activity of Cas12a. After being activated, Cas12a exhibited a high trans-cleavage activity on the trigger, which resulted in a decrease in fluorescence. Owing to this design, the Cas12a-CHA assay enabled the sensitive detection of ctDNA with a linear range of 10 fM to 50 pM. Furthermore, a limit-of-detection of 5.8 fM was achieved within 40 min. Besides, the proposed assay had an excellent base mismatch recognition ability and worked well in human serum samples. Conclusively, this detection platform holds significant potential for application in early cancer diagnosis.},
}
@article {pmid41356190,
year = {2026},
author = {Zhong, XY and Yang, YX and Xiong, YF and Ye, GC and Gong, X and Zhong, ML and He, HD and Wang, SG and Xia, QD},
title = {Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1877-1904},
pmid = {41356190},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; Humans ; Animals ; *Molecular Imaging/methods ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Bioimaging technologies visually resolve spatiotemporal dynamics of biomolecules, cells, and tissues, enabling essential insights into gene regulation, disease mechanisms, and drug metabolism. CRISPR/Cas-based fluorescent probes transform CRISPR from "genetic scissors" into "molecular microscopes," providing an indispensable tool for in situ decoding of molecular events in living systems. Their high nucleic acid specificity establishes CRISPR/Cas as a pivotal technology for dynamically monitoring genomic and transcriptomic events at live-cell and in vivo levels. This work systematically outlines design strategies and functional mechanisms of mainstream CRISPR/Cas fluorescent probes for bioimaging, encompassing five categories: fluorescent proteins, synthetic dyes, smart gated probes, nanomaterials, and multimodal integrated probes. Recent advances and persistent challenges in achieving high-sensitivity targeted imaging, effective signal amplification, and precise delivery control are comprehensively examined, including analysis of their advantages, limitations, and adaptability in complex biological environments. Building on breakthroughs in in vivo delivery systems, diverse carriers demonstrate significant potential for enhancing CRISPR/Cas transport efficiency, improving tissue penetration, and enabling spatiotemporal controlled release. Continued innovation drives CRISPR/Cas imaging platforms toward higher sensitivity, enhanced biocompatibility, and multifunctional integration, thereby fostering the convergence and broad application of gene editing and molecular diagnostics.},
}
@article {pmid41356196,
year = {2026},
author = {Zhang, Y and Deng, Q and Xu, Y and Wu, W and Wu, T and Huang, J and Hu, Y and Lin, W and Xu, X and Wu, J},
title = {ROS-responsive cellular vesicles with ferroptosis-targeting siACMSD delivery for acute kidney injury therapy.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1941-1958},
pmid = {41356196},
issn = {1838-7640},
mesh = {*Ferroptosis/drug effects ; Animals ; *Reactive Oxygen Species/metabolism ; *Acute Kidney Injury/therapy/metabolism/drug therapy/pathology ; Mice ; Humans ; Cisplatin/pharmacology ; *Carboxy-Lyases/genetics/metabolism ; Disease Models, Animal ; Cell Line ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; },
abstract = {Background: Acute kidney injury (AKI) is a severe and prevalent nephrotic syndrome which lack of definitive therapies. Alpha-amino-β-carboxymuconic acid-ε-semialdehyde decarboxylase (ACMSD) is a metabolic enzyme mainly expressed in the kidney which exacerbated AKI injury by promoting TCA cycle and inhibiting nicotinamide adenine dinucleotide (NAD[+]) production, whereas lack of effective intervention strategies for ACMSD-targeted therapy. Methods: Herein, we knocked out ACMSD in vitro through CRISPR-Cas9 method, and developed a reactive oxygen species (ROS)-responsive neutrophil-derived cellular vesicles (CVs) drugs (RNAi@ROS-CVs), which efficiently mediated ACMSD knockdown in vivo, exploring the mechanism of ACMSD-induced ferroptosis process in AKI. Results: ACMSD knockout effectively alleviated cisplatin (CP)-induced mitochondrial damage, suppressed TCA cycle progression, promoted NAD[+] synthesis, and inhibited ferroptosis in HK2 cells. In mice AKI model, RNAi@ROS-CVs effectively targeted the injured kidneys, downregulated ACMSD expression in renal tubular epithelial cells, reduced ROS production and lipid peroxidation, and alleviated CP or ischemia/reperfusion (I/R)-induced ferroptosis. Conclusion: These findings highlight the therapeutic potential of ACMSD-targeted knockout in AKI intervention and introduce a versatile and efficient controlled-release drug delivery platform for AKI-targeted therapy, with potential applicability to other acute renal diseases.},
}
@article {pmid41356473,
year = {2025},
author = {Wei, C and Chen, Z},
title = {Comprehensive analysis of phage genomes from diverse environments reveals their diversity, potential applications, and interactions with hosts and other phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1686402},
pmid = {41356473},
issn = {1664-302X},
abstract = {Phages are ubiquitous and diverse, playing a key role in maintaining microbial ecosystem balance. However, their diversity, potential applications, and their interactions with hosts and other phages remain largely unexplored. To address this, we collected 59,652,008 putative viral genomes from our laboratory, 45 public viral datasets, and an integrated public viral genome database (IGN), covering seven habitats. We obtained 741,692 phage genomes with completeness ≥50% (PGD50), and most (93.83%, 695,938/741,692) of these phage genomes were classified into the Caudoviricetes class. We found that 158,522 species-level viral clusters that contained 28.96% (214,814/741,692) phage genomes without any known phage genomes in the IGN, indicating substantial novelty. Global phylogenetic trees for five iterations based on complete phage genomes significantly expanded the known diversity of the virosphere. Genome analysis revealed phage potential divergence with habitat types and highlighted the utilization of alternative genetic codes. Furthermore, 3D structural similarity searches demonstrated significant potential for annotating previously uncharacterized viral proteins. Analysis of CRISPR spacer inferred potential hosts of phages and competitive networks among phages, highlighting virulent phages as promising candidates for phage therapy against pathogenic bacteria. Intriguingly, diverse CRISPR-Cas systems were detected within phage genomes themselves, suggesting their enormous potential as novel gene editing tools. Collectively, this study provides a comprehensive phage genome resource, foundational for future research into phage-host and phage-phage interactions, phage therapy development, and the mining of next-generation genetic tools.},
}
@article {pmid41356798,
year = {2026},
author = {Birappa, G and Perumalsamy, H and Hong, SH and Gowda, DAA and Chandrasekaran, AP and Karapurkar, JK and Rajkumar, S and Balusamy, SR and Jayachandran, A and Baek, KH and Lee, J and Matam, V and Kim, WJ and Kim, KS and Ramakrishna, S and Suresh, B},
title = {Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {986-1005},
pmid = {41356798},
issn = {1838-7640},
mesh = {Humans ; Animals ; *B7-H1 Antigen/metabolism/genetics ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Single-Cell Analysis/methods ; Ubiquitination ; *Carcinogenesis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Sequence Analysis, RNA ; },
abstract = {Background: The expression levels of the programmed death-ligand 1 (PD-L1) protein serves as a prognostic indicator for patients with colorectal cancer (CRC). Advancement of CRC is facilitated by deubiquitinating enzymes (DUBs), which regulate oncoprotein levels via the ubiquitin-proteasomal pathway. The post-translational regulatory mechanisms governing PD-L1 protein abundance on CRC, in relation to different tumor grades and their clinical relevance, remains unknown. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data to identify DUB genes associated with PD-L1 expression in CRC. We used a loss-of-function-based CRISPR/Cas9 library to identify putative DUB genes that regulate the PD-L1 protein level. Immunoprecipitation was used to confirm the interaction between the USP32 and PD-L1 along with its ubiquitination status. A series of in vitro and in vivo carcinogenesis-related experiments were conducted to determine the clinical relevance between USP32 and PD-L1 expression in CRC progression. Results: In this study, we analyzed scRNA-seq data from extensive cohorts of human and mice at the single-cell level to identify DUB genes associated with PD-L1 expression in CRC. Our analysis identified multiple putative DUBs, including USP32 and USP12, as prognostic markers associated with PD-L1 expression, which was found to be elevated in T cells, macrophages, and classical monocytes cell types in patients with CRC. A secondary screening using CRISPR/Cas9-mediated loss-of-function analysis for DUBs found that USP32 modulates PD-L1 protein levels in CRC. Furthermore, we demonstrated that USP32 interacts with, stabilizes, and extends the half-life of PD-L1 by preventing its K-48-linked polyubiquitination as an underlying mechanism that contributes for tumorigenesis. Conclusion: A combination of scRNA-seq analysis and wet-lab experimental validation confirmed that USP32 mediates PD-L1 protein stabilization in colon cancer, identifying it as a potential therapeutic target for CRC. CRISPR/Cas9-mediated targeted knockout of the USP32 gene reduced PD-L1 protein levels and significantly mitigated colorectal cell proliferation and tumorigenesis, both in vitro and in vivo, in a xenograft mouse model, underscoring a novel and alternative approach to the treatment of CRC.},
}
@article {pmid41358836,
year = {2025},
author = {Li, Q and Xu, J and Jiang, J and Gong, L and Mao, X and Wang, F and Yao, P},
title = {Nucleic acid detection method for Chlamydia psittaci based on RPA-CRISPR/Cas12a.},
journal = {Letters in applied microbiology},
volume = {78},
number = {12},
pages = {},
doi = {10.1093/lambio/ovaf138},
pmid = {41358836},
issn = {1472-765X},
support = {BE2023694//Key Research and Development Project of Jiangsu Province/ ; H2023060//Jiangsu Provincial Commission of Health Project/ ; K2024003//Key Project of the Jiangsu Provincial Commission of Health/ ; Ym2023015//Jiangsu Province Preventive Medicine Research Project/ ; Ym2023073//Jiangsu Province Preventive Medicine Research Project/ ; x202339//Jiangsu Provincial Blood Parasite and Endemic Disease Prevention Research Project of China/ ; CE20225041//Changzhou science and technology Foundation/ ; CJ20253132//Changzhou science and technology Foundation/ ; CJ20253133//Changzhou science and technology Foundation/ ; CPHM202401//Nanjing Medical University/ ; CPHM202303//Nanjing Medical University/ ; },
mesh = {*Chlamydophila psittaci/genetics/isolation &amp; purification ; Humans ; *Psittacosis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {In recent years, misdiagnosis or delayed diagnosis of Chlamydia psittaci (C. psittaci) infections has led to frequent outbreaks of severe public health events, such as severe pneumonia and respiratory distress, drawing increasing attention. Rapid and simple detection methods are vital for early intervention to reduce severity and mortality. In this study, we designed highly specific RPA primers and crRNA (CRISPR RNA) based on the highly conserved CPSIT_0429 gene in the C. psittaci genome, and preliminarily established a nucleic acid detection method for C. psittaci using the RPA-CRISPR/Cas12a system. In the two-step assay, the combination of the CPSIT_0429-F1/R1 primer pair and CPSIT_0429-crRNA2 achieved a detection limit of 2 × 10° copies/μL. Incorporating 20% glycerol enabled a one-tube assay with a limit of 2 × 102 copies/μL. Furthermore, the method showed no cross-reactivity with common respiratory pathogens such as influenza virus, SARS-CoV-2, and Streptococcus pneumoniae, demonstrating excellent specificity. Both the two-step and one-tube methods were compared with qPCR-verified C. psittaci positive samples. The results indicated that both assays showed high consistency with qPCR results. The RPA-CRISPR/Cas12a detection method is rapid, accurate, highly sensitive, and specific, providing a reliable platform for early diagnosis and clinical management of C. psittaci infections.},
}
@article {pmid41359128,
year = {2025},
author = {Li, Z and Cheng, Y and Li, C and Wu, Q and Xin, Y},
title = {Harnessing microalgae for bioproducts: innovations in synthetic biology.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {500},
pmid = {41359128},
issn = {1573-0972},
support = {32560020 and 31600059//National Natural Science Foundation of China/ ; RZ2300002678//Start-Up Funds of Hainan University/ ; DC2300001799//Open Project of State Key Laboratory of Marine Resource Utilization in South China Sea/ ; 2018YFA0902500//National Key Research and Development Program of China/ ; },
mesh = {*Microalgae/metabolism/genetics ; *Synthetic Biology/methods ; Biofuels ; *Metabolic Engineering/methods ; Gene Editing ; Lipids/biosynthesis ; CRISPR-Cas Systems ; Metabolic Networks and Pathways ; Photobioreactors ; },
abstract = {Microalgae are increasingly recognized as versatile platforms for sustainable production of biofuels and high-value bioproducts such as lipids, carotenoids and polyunsaturated fatty acids. Rapid progress in synthetic biology is transforming microalgal engineering by enabling precise rewiring of metabolic pathways and overcoming long-standing technical bottlenecks, particularly those related to transformation efficiency, genetic stability and strain scalability. Recent innovations (including CRISPR/Cas genome editing, modular cloning systems, synthetic promoter libraries and dynamic, environment-responsive regulatory circuits) have greatly expanded the genetic toolset available for both model and recalcitrant species. These advances support targeted control of lipid and pigment biosynthesis, improved flux distribution and more robust performance under industrially relevant conditions. When integrated with progress in photobioreactor design, automated cultivation, and process intensification, synthetic biology unlocks new potential for scalable, economically viable microalgal biomanufacturing. This review summarizes these developments, highlights remaining challenges in strain robustness and bioprocess translation, and outlines future pathways toward high-performance microalgal biofactories that can contribute meaningfully to a low-carbon, bio-based economy.},
}
@article {pmid41359384,
year = {2025},
author = {Ichinose, M and Ohta, M and Shimajiri, Y and Akaiwa, Y and Nakamura, I and Shimamoto, M and Makinoda, R and Ozaki, S and Tamai, T and Maekawa, N and Tonomoto, M and Nakamura, T and Yagi, Y and Gutmann, B},
title = {RECODE: a programmable guide-free C-to-U RNA editing tool.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41359384},
issn = {1362-4962},
support = {//EditForce, Inc/ ; },
mesh = {*RNA Editing ; Humans ; Animals ; Mice ; *Cytidine Deaminase/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Uridine/genetics/metabolism ; HEK293 Cells ; *Cytidine/metabolism/genetics ; RNA-Binding Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Programmable RNA cytidine deaminase tools have been developed to convert cytidine-to-uridine (C-to-U) using CRISPR systems with guide RNAs. These tools, however, have limitations such as low editing efficiency, limited targetable sequence flexibility, and off-target RNA editing. Here, we present a novel guide-free C-to-U editing tool, named RECODE (RNA Editor for C-to-U with an Optimized DYW Enzyme), based on the RNA-binding pentatricopeptide repeat proteins, naturally fused to a C-terminal DYW cytidine deaminase domain. The RECODE specificity domain was engineered to enable retargeting, while its length and sequence were optimized to reduce off-target effects. Further optimization of the C-terminal catalytic region increased both the editing activity and the translation of the edited RNA. We showed that RECODE efficiently edits a wide range of targets in human cells, without affecting adjacent cytidines. It achieved over 50% editing efficiency for most sites, except those with an upstream guanine. Furthermore, we showed that RECODE is functional in mice, with high editing efficiency observed in specific tissues such as skeletal muscles using an AAV delivery system, suggesting its therapeutic potential for various diseases.},
}
@article {pmid41359835,
year = {2025},
author = {Roura-Martinez, D and Popa, N and Jaouen, F and Rombaut, C and Lepolard, C and Bachar, D and Borges, A and Cazorla, M and Villet, M and Moreno, S and Marie, H and Gascon, E},
title = {Combination of Cas9 and adeno-associated vectors enables efficient in vivo knockdown of precise miRNAs in the rodent and primate brain.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2513076122},
pmid = {41359835},
issn = {1091-6490},
support = {ANR-22-CE17-0034//Association Nationale de la Recherche et de la Technologie (ANRT)/ ; 6239//Fondation France Alzheimer/ ; 2022//Fondation Recherche Alzheimer/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Brain/metabolism ; Mice ; Genetic Vectors/genetics ; *Gene Knockdown Techniques/methods ; *Dependovirus/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Neural Stem Cells/metabolism ; Neurons/metabolism ; Primates ; Receptors, AMPA/metabolism ; Olfactory Bulb/metabolism ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {microRNAs (miRNAs) are key regulators of multiple biological functions. Although intensively studied, inactivating miRNAs in vivo is particularly challenging, especially in the brain. Here, we designed cell-specific tools aiming at downregulating defined miRNA species in vivo and investigating their function in discrete neuronal networks. Focusing on miR-124, a miRNA highly expressed in the mammalian brain and transcribed from three independent chromosomal loci, we designed and validated different guide RNAs. In vivo, our CRISPR-Cas9 designs strongly downregulate miR-124 levels without affecting the expression of other miRNAs. As a result, levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity. We provide evidence that specific deletion of miR-124 in neural stem cells of the subventricular zone altered migration of newly generated neurons into the olfactory bulb. We also showed that our vectors modified the Ca[2+] permeability of AMPA receptors, a robust functional output downstream of miR-124. We also extended our approach to other miRNAs, mammalian species, and Cas9 proteins, confirming the versatility of CRISPR-Cas9. These tool properties support their potential for elucidating miRNA functions in complex experimental in vivo settings such as brain networks.},
}
@article {pmid41360396,
year = {2025},
author = {Jiang, Q and Ramachandran, A and Avaro, AS and Huyke, DA and Santiago, JG},
title = {Reaction Kinetics of CRISPR trans-Cleavage Controlled Using Isotachophoresis.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {27646-27653},
doi = {10.1021/acs.analchem.5c04301},
pmid = {41360396},
issn = {1520-6882},
mesh = {*Isotachophoresis/methods ; Kinetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-based diagnostics are powerful tools for nucleic acid detection due to their high specificity and programmability. However, assay sensitivity is often limited by the slow kinetics of the trans-cleavage reaction, which typically proceeds at a rate of ∼0.1 to 1 turnover per second. Here, we present a reaction-transport model and experimental study that analyze and accelerate this limiting step using electric-field-driven isotachophoresis (ITP). Building on the work of Ramachandran and Santiago, we develop a model that captures the coupling among ITP focusing, mixing, and preconcentration with CRISPR enzymatic reaction kinetics. Our analysis identifies two key regimes in ITP-coupled CRISPR reactions and derives analytical approximations for the limiting behaviors in each. Compared to a standard, well-mixed assay, we predict a 10- to 100-fold reduction in reaction duration using ITP. We validate the model with experiments across a range of target concentrations. Our work offers a quantitative framework for understanding and optimizing CRISPR trans-cleavage dynamics and provides guidance to design assays that use electric-field-mediated transport.},
}
@article {pmid41361167,
year = {2025},
author = {Fast, L and Omar, M and Kanis, P and Schaffer, T and Chowdhury, D and Rakava, E and Pääbo, S and Riesenberg, S},
title = {Search-and-remove genome editing allows selection of cells by DNA sequence.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10985},
pmid = {41361167},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Neanderthals/genetics ; Mutation, Missense ; CRISPR-Associated Protein 9/metabolism ; Base Sequence ; Cell Line, Tumor ; },
abstract = {The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.},
}
@article {pmid41361700,
year = {2025},
author = {Ain, QU and McCarthy, A and Nadeem, A and Javed, M and Niakan, K and Nashta, AF},
title = {CRISPR/Cas9-mediated generation of GATA3 knockout in Bovine Fibroblast and MDBK cell lines to assess sgRNAs targeting efficiency.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {269},
pmid = {41361700},
issn = {1438-7948},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; *GATA3 Transcription Factor/genetics/metabolism ; Fibroblasts/metabolism/cytology ; Cell Line ; *Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {GATA3 is expressed in the outer cells of the morula stage during embryonic development and is considered a key driver of the regulation of early lineage development in bovines. This research presents an optimised somatic cell validation resource, successfully generating GATA3 knockout (KO) Bovine Fetal Fibroblasts and MDBK cells using CRISPR/Cas9-mediated genome editing for their future implications in vivo studies designed to definitively understand the role of GATA3 in cell lineage specification and bovine embryo development. This involved designing single-guide RNAs (sgRNAs) targeting different regions of the GATA3 gene, cloning them into the px459 plasmid, delivering the CRISPR clone into bovine fibroblast cells and the MDBK cell line, screening for successful targeting and knockouts, and MiSeq analysis to verify successful disruption of the GATA3 gene. A total of eleven guides were designed targeting the functional domains in Exons 4 and 5 and the transcription initiation site in Exon 2. Designed guides were first optimized and screened using an in vitro cleavage assay. The guides with the best cutting efficiencies were then tested in vivo by targeting bovine fetal fibroblast (BFFs) and MDBK cell line followed by MiSeq analysis to verify the successful knockouts. A total of two effective guides were identified targeting the zinc-finger (ZnF) functional domains of the GATA3 gene (sgRNA#5 and sgRNA#8 in Exon 4 and Exon 5, respectively) and one in Exon 2 (sgRNA#1) targeting the transcription initiation site of the GATA3 gene. MiSeq data from targeted bovine cells showed indel frequency of 47.40%, 55.5%, and 42.4% in bovine fetal fibroblasts, 11.03%, 28.9% and 7.3% for MDBK cells for top three sgRNAs. Overall, MiSeq data for 3 selected sgRNAs showed successful disruption of the GATA3 gene, inserting a base pair 2-3 bp upstream of the PAM site, ultimately resulting in a premature stop codon TAA in the downstream region. This study established and validated highly efficient sgRNAs targeting the GATA3 gene, forming a molecular basis for forthcoming functional investigations in bovine embryos to explore gene function and protein-level effects.},
}
@article {pmid41361988,
year = {2026},
author = {Min, YH and Lee, DG and Lee, HY and Yoo, JH and Lee, KH and Shin, YB and Byun, JY},
title = {CRISPR/Cas12a with Antisense Oligonucleotide-Regulated Translational Amplification for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {11},
number = {1},
pages = {394-404},
doi = {10.1021/acssensors.5c03081},
pmid = {41361988},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Oligonucleotides, Antisense/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *DNA, Viral/analysis/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics ; CRISPR-Associated Proteins ; },
abstract = {Highly sensitive nucleic acid testing-assisted early disease detection is crucial for effective disease prevention and management, particularly when targeting low-abundance genetic materials in molecular diagnostics. This study describes CRATE (CRISPR/Cas controlled antisense oligonucleotide (ASO)-mediated translational signal enhancement), a novel ultrasensitive approach for nucleic acid detection by integrating Cas12a trans-cleavage, ASO-controlled gene expression, and cell-free signal protein amplification. This assay leverages the target-induced trans-cleavage of ASO-controlled gene expression for the amplification of signal proteins, with luminescent signal allowing for attomolar-level target DNA detection, as well as antigenic protein application enabling visual detection by lateral flow assay. The CRATE assay improves sensitivity using ASO-modified locked nucleic acid, achieving a 10-aM-level DNA detection. The proof of concept demonstrates 0.1 copies/μL detection of HPV genomic DNA from HPV-positive cancer cells as well as colorimetric lateral flow tests with ∼10 copies/μL sensitivity. The CRATE assay can detect the HBV target in plasma from HBV-positive patients with 100% sensitivity and specificity. With high specificity and accuracy, the CRATE assay retains the potential for detecting any nucleic acid of interest. By integration of precise CRISPR-based cleavage, ASO regulation, and efficient protein signal amplification, this approach provides a promising solution for highly selective and sensitive nucleic acid detection and potential applications in clinical diagnostics and point-of-care testing.},
}
@article {pmid41361999,
year = {2025},
author = {Cheng, Y and Zhang, X and Zhao, P and Zhu, D and Meng, YH and Fu, X and Wang, X},
title = {Enhanced Mannan Production of Saccharomyces cerevisiae by CRISPR/Cas9 and Mannoproteins Characteristics on Wine Astringency Modulation.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32195-32208},
doi = {10.1021/acs.jafc.5c10790},
pmid = {41361999},
issn = {1520-5118},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; *Wine/analysis/microbiology ; *Mannans/metabolism/chemistry ; *Membrane Glycoproteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems ; Taste ; Humans ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Mannose/metabolism/analysis ; },
abstract = {Mannoproteins are critical in modulating wine astringency, yet the specific impacts of their monosaccharide ratio and side-chain structure remain insufficiently explored. This study employed CRISPR/Cas9 to engineer yeast strains producing mannoproteins with either a high mannose-to-glucose ratio (high-yield-mannan strain BSFA12) or a nonbranched N-glycan structure (BY4741-ΔMNN2). The resulting mannoproteins (MPBSFA, MPBY2) were compared against controls (MPBY extracted from Saccharomyces cerevisiae BY4741, a commercial product MP60) using physicochemical analyses (fluorescence quenching, dynamic light scattering, and isothermal titration calorimetry) and sensory evaluation. Both engineered variants demonstrated superior astringency reduction in model and red wines. Our results establish that an increased mannose-to-glucose ratio and reduced N-glycosylation significantly enhance the astringency-mitigating effect, providing a foundation for the industrial-scale production and application of mannoprotein additives.},
}
@article {pmid41362221,
year = {2026},
author = {Yao, X and Wang, L and Su, L and Rao, W and Luo, Z and Li, Y},
title = {Localized CRISPR/Cas13a powered DNA walker for sensitive and high-throughput detection of norovirus.},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {2},
pages = {572-579},
doi = {10.1039/d5tb02131f},
pmid = {41362221},
issn = {2050-7518},
mesh = {*Norovirus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; High-Throughput Screening Assays ; Limit of Detection ; RNA, Viral/analysis ; },
abstract = {The development of sensitive and high-throughput methods for detecting foodborne viruses is crucial for disease prevention and public health protection. In this study, we present a novel localized Cas13a-based DNA walker (LCas13a-DNA walker) for the ultrasensitive, stable, and rapid detection of norovirus (NoV). When the DNA walker was confined in AuNPs, the spatial confinement effect improved the local concentration of reaction substrates, accelerated the reaction speed, and enhanced the sensitivity of the DNA walker. Besides, an original design of uracil-rich hairpin (UH)-modified AuNPs as the walking track significantly improves the stability of the detection system. Meanwhile, employing CRISPR/Cas13a as the driving force streamlines viral RNA recognition and substantially reduces the reaction time down to 30 minutes by eliminating the reverse transcription step. Additionally, a biomimetic array, formed by photonic crystals (PCs), enabled high-throughput signal acquisition with a microplate reader, and concurrently amplified the fluorescence signal. The proposed assay realized ultra-sensitivity of NoV with a detection limit as low as 4.1 pM and a wide linear range from 10 pM to 5 nM. Due to the advantages of high sensitivity, high-throughput, stability, and rapid analysis, this proposed method provides a potential strategy for point-of-care detection of pathogenic viruses in food safety monitoring and disease diagnosis.},
}
@article {pmid41362674,
year = {2026},
author = {Toofan, P and Singh, M and Brooks, A and McLuckie, K},
title = {Non-clinical safety considerations on genome editing using the CRISPR/Cas system.},
journal = {Genes &amp; diseases},
volume = {13},
number = {2},
pages = {101785},
pmid = {41362674},
issn = {2352-3042},
abstract = {Recent advances in gene editing using the CRISPR/Cas system have revolutionized genome editing, opening new horizons for human cellular and gene therapy products. Genome editing technologies are rapidly being adopted in clinical trials. However, critical non-clinical safety considerations are required to address challenges in translating research to the clinic. Here, we review current ex vivo and in vivo genome editing approaches using the CRISPR/Cas system and discuss the practical use of these methods in pre-clinical studies and in the clinic. We also discuss known limitations of genome editing in humans and the mitigation of risk factors associated with it from a non-clinical safety perspective. This review aims to aid researchers in acquiring a perspective that is essential for the safe translation of genome editing to the clinic.},
}
@article {pmid41363172,
year = {2025},
author = {Wen, TT and Xu, L and Jin, R and Liu, Z and Liu, MQ and Dong, CH and Sun, L and Wang, HY and Sun, WJ and Cui, FJ},
title = {Functional Characterization of a β-1,3-Glucanosyltransferase CmGel4 in Cordyceps militaris Using a Precise CRISPR-Cas9 Genome-Editing System.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32078-32093},
doi = {10.1021/acs.jafc.5c08653},
pmid = {41363172},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Cordyceps/genetics/enzymology/chemistry ; Gene Editing ; *Fungal Proteins/genetics/metabolism/chemistry ; beta-Glucans/metabolism ; Amino Acid Sequence ; Glucan Endo-1,3-beta-D-Glucosidase ; },
abstract = {Cordyceps militaris polysaccharides, especially β-glucans, have presented significant antitumor, hypoglycemic, and immunomodulatory activities. However, the enzymes involved in the branching formation of C. militaris β-glucans remain to be elucidated. In the present study, a 1.69-kb β-1,3-glucanosyltransferase CmGel4 gene putatively involved in β-glucan branching was cloned from C. militaris mycelia and bioinformatically analyzed. The encoded 54.12 kDa CmGel4p consisted of 515 amino acid residues and contained a typical GH72[+] structural characteristic of a signal peptide (1-19aa), a GH72 conserved domain (20-334aa), a GPI-anchor site (485aa), and a CBM43/X8 domain (382-458aa). Using the established CRISPR-Cas9 genome-editing system, the full length of 1.69-kb CmGel4 was precisely inserted at a genomic safe-harbor site CmSh1, and the GH72 conserved domain of CmGel4 was successfully deleted in C. militaris genome for the first time. By comparing the mycelial growth and fermentation performance of WT, control, and CmGel4-overexpressed/knockout mutants, β-1,3-glucanosyltransferase gene CmGel4 was shown to play key roles in cell growth and branching of exo-polysaccharides of C. militaris, accompanied by the transcriptional changes of genes such as CmGel4, CmUgp, and CmPgm. These findings provided the proof of β-1,3-glucanosyltransferases vital for formatting cell walls and maintaining cellular integrity, and a fine regulation strategy for precisely remodeling the β-1,3-glucan with high-branched structures in edible fungi.},
}
@article {pmid41364162,
year = {2025},
author = {Murtaza, M and Gupta, P and Choudhary, P and Manzoor, M and Sharma, S and Jaglan, S},
title = {Strategies to decipher silent biosynthetic gene clusters in actinomycetes.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {53},
pmid = {41364162},
issn = {1432-072X},
mesh = {*Actinobacteria/genetics/metabolism ; *Multigene Family/genetics ; *Biosynthetic Pathways/genetics ; Anti-Bacterial Agents/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Actinobacteria have a huge, mainly untapped potential for the production of secondary metabolites. These metabolites are an important source of bioactive compounds. However, a majority of biosynthetic gene clusters (BGCs) are either under-expressed or fully silent under standard laboratory conditions, limiting their potential. The present review article aims to explore the biosynthetic gene clusters (BGCs) of actinobacteria using strategies that aid in unlocking these silent BGCs. The strategies discussed are PCR-Targeted Gene Replacement (PCR-TR); Cre-LoxP recombination system; Transcription factor decoys, Ribosome engineering, and CRISPR/Cas technologies. Besides, elicitors also helped with the identification of these cryptic or silent BGCs and advanced our ability to explore these natural products. Combining experimental and computational platforms provides an opportunity to unlock the hidden chemical diversity in nature, thereby accelerating the identification of new bioactive substances. The new antibiotics explored by all the strategies could help in the fight against antimicrobial resistance (AMR).},
}
@article {pmid41365122,
year = {2026},
author = {Kim, WN and Kim, HU},
title = {Precise DGAT1 base editing and in-frame deletion reveal motif-specific regulation of seed oil biosynthesis in Arabidopsis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110861},
doi = {10.1016/j.plaphy.2025.110861},
pmid = {41365122},
issn = {1873-2690},
mesh = {*Arabidopsis/genetics/metabolism ; *Diacylglycerol O-Acyltransferase/genetics/metabolism ; *Seeds/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Editing ; *Plant Oils/metabolism ; Triglycerides/biosynthesis ; *Sequence Deletion ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triacylglycerol (TAG) biosynthesis and is a key determinant of seed oil content and composition. To dissect the functional contribution of the conserved DGAT1 domains, we employed adenine and cytosine base editors and CRISPR/Cas9-mediated in-frame deletion to generate targeted alleles in Arabidopsis thaliana. A total of 25 single guide RNAs were designed to introduce precise nucleotide substitutions across functional domains, and the edited lines were screened using seed fluorescence and Sanger sequencing. Five base-edited (BE) DGAT1 mutants affecting acyl-CoA/CoA allosteric binding site (S124F, S123R/S124L), thiolase acyl-enzyme intermediate signature motif (L229P), diacylglycerol (DAG)-binding motif (W416C/R419Q, V418I), and an in-frame deletion in the intrinsically disordered N-terminal region (Δ49-76H) were characterized. Amino acid substitutions in the conserved domains led to distinct shifts in seed fatty acid profiles. Loss-of-function-like mutants (S123R/S124L, L229P, W416C/R419Q) reduced 18:1 and 20:1 levels and increased 18:3 levels, whereas putative gain-of-function mutants (S124F, V418I) enhanced 20:1 incorporation and elevated total oil content. Overexpression of DGAT1[S124F] and DGAT1[V418I] in the dgat1 mutant background further increased seed oil accumulation beyond that achieved with wild-type DGAT1. Structural modeling of DGAT1 proteins revealed the location of substituted amino acids and their interactions with surrounding residues, as well as the absence of putative N-terminal regulatory segment. These results demonstrate that precise base editing can modulate DGAT1 activity and TAG composition by targeting functional motifs, providing insights into the structure-function relationships of this key enzyme and offering strategies for metabolic engineering of seed oils.},
}
@article {pmid41365143,
year = {2025},
author = {Queffeulou, M and Fakhfakh, R and Fani, F and Dos Santos, A and Reis Ferreira, G and Bigot, S and Godin, C and Leprohon, P and Papadopoulou, B and Ouellette, M},
title = {CRISPR-Cas13b mediated gene knockdowns in Leishmania infantum.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {29},
number = {},
pages = {100629},
pmid = {41365143},
issn = {2211-3207},
mesh = {*Leishmania infantum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; Antiprotozoal Agents/pharmacology ; Phosphorylcholine/analogs &amp; derivatives/pharmacology ; Protozoan Proteins/genetics ; Antimony/pharmacology ; Luciferases, Firefly/genetics ; RNA, Messenger/genetics ; },
abstract = {Chemotherapy is an effective means to control infections caused by the protozoan parasite Leishmania. However, available treatments are limited, expensive, and associated with considerable toxicity. Genomic strategies have contributed to a better understanding of Leishmania's response to drugs and in the characterization of drug targets. Nonetheless, there is no knockdown system operational for Leishmania. In this study, we show that the CRISPR-Cas13 system can be an effective strategy to knockdown expression levels of both exogenous and endogenous transcripts. We succeeded in effectively knocking down the expression of the firefly luciferase gene integrated in the genome of L. infantum. This Cas13-mediated decrease in mRNA was paralleled with a significant reduction in both the luciferase protein level and its activity. Furthermore, we tested the effectiveness of the Cas13 system to target the endogenous miltefosine transporter (MT) and the aquaglyceroporin 1 (AQP1) genes. Knockdown was effective and parasites with less MT or AQP1 mRNA levels exhibited reduced susceptibility to miltefosine or antimonials, respectively. While further optimization is warranted, this knockdown system has the potential to facilitate numerous studies related to various aspects of Leishmania biology.},
}
@article {pmid41365534,
year = {2025},
author = {Skeate, JG and Lee, CJ and Stewart, C and Fischbach, MJ and Kar, B and Tsai, AK and Kenderian, SS and Stromnes, IM and Largaespada, DA and Moriarity, BS and Rogers, LM},
title = {Functional genomics for improving adoptive T-cell transfer therapies.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {12},
pages = {},
pmid = {41365534},
issn = {2051-1426},
support = {R37 CA276005/CA/NCI NIH HHS/United States ; T32 CA009138/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Genomics/methods ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/transplantation ; Gene Editing/methods ; Animals ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems ; },
abstract = {Adoptive cell therapy (ACT) has shown remarkable success in the treatment of some malignancies, particularly leukemia. However, there are multiple factors that limit the durability of ACT in solid tumors, including dose-limiting toxicities, the immunosuppressive tumor microenvironment, and T-cell exhaustion. As the manufacture and preparation of adoptive T-cell therapies allows time and adequate conditions for ex vivo T-cell engineering, forward genetic screens can identify novel genetic targets that could improve their effectiveness. CRISPR is a commonly used functional genomics tool that has been successfully used to both enhance our understanding of mechanisms of resistance and to discover potential genetic edits to improve ACT. A complementary approach, Sleeping Beauty transposon mutagenesis provides additional opportunities to identify novel genetic edits without being constrained by the annotated human genome. Here, we summarize forward genetic screens and their tools to uncover strategies to enhance ACT. Complementary approaches can be combined and improved on to identify translatable genetic editing strategies through studies that accurately recapitulate disease-specific challenges.},
}
@article {pmid41365538,
year = {2025},
author = {Jung, SC and Oh, H and Eom, W and Jin, YS and Park, SH and Park, K and Koh, HG},
title = {Scarless Genetic Engineering of Saccharomyces cerevisiae for Enhanced Guanosine Monophosphate Production as a Natural Flavor Enhancer.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2508034},
pmid = {41365538},
issn = {1738-8872},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Fermentation ; *Guanosine Monophosphate/biosynthesis/metabolism ; *Flavoring Agents/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Inosine Monophosphate/metabolism ; Pentose Phosphate Pathway/genetics ; },
abstract = {Saccharomyces cerevisiae and Cyberlindnera jadinii are widely utilized in the natural food seasoning industry as sources of flavor enhancing nucleotides such as inosine monophosphate (IMP) and guanosine monophosphate (GMP), which contribute to umami taste and support sodium reduction in food. However, wild type yeast strains produce GMP at levels that are inadequate for industrial scale applications, necessitating metabolic engineering strategies to increase production efficiency. This study employed a CRISPR-Cas9-based scarless genome engineering approach to enhance GMP biosynthesis in S. cerevisiae via promoter replacement. The key genes IMD3 and GUA1, responsible for converting IMP to GMP, were overexpressed to redirect purine flux toward GMP production. To address precursor limitations, ZWF1 and RKI1, involved in the pentose phosphate pathway, were also overexpressed. In parallel, the expression of STB5 and RAP1 was increased to enhance NADPH regeneration and relieve transcriptional bottlenecks. As a result, the final engineered strain SCJ-7 demonstrated a 1.77-fold increase in GMP titer and a 1.40-fold increase in GMP content during flask fermentation compared to the wild-type. In fed-batch fermentation, GMP titer was further improved by 27.6%. These findings demonstrate that combining metabolic flux enhancement with transcriptional regulation provides an effective and scalable strategy for boosting GMP production in S. cerevisiae, offering strong potential for industrial application in the food industry.},
}
@article {pmid41365890,
year = {2025},
author = {Li, W and Liu, S and Fang, X and Zou, J and Jiang, Q and Min, X and Zhu, X and Cao, Y and Gao, X and Han, W and Azhar, M and Xing, X and Li, F and Zhang, Y and Liu, H and Cheng, L and Wang, C and Bao, J},
title = {Efficient high-precision transgene knock-in by Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 (RED-CRISPR).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {538},
pmid = {41365890},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Mice ; Humans ; Recombinational DNA Repair/genetics ; *Gene Editing/methods ; *Recombinases/metabolism/genetics ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications.},
}
@article {pmid41366133,
year = {2025},
author = {Bian, W and Mcquarrie, DWJ and Haussmann, IU and Arnold, R and Soller, M},
title = {Genetic evaluation of CRISPR-Cas9 off-target effects from deleterious mutations on Drosophila male single X chromosome.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {270},
pmid = {41366133},
issn = {1438-7948},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *X Chromosome/genetics ; *Mutation ; Gene Editing/methods ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease protein 9 (Cas9) is a powerful tool used for genome engineering, but concerns remain about off-target effects. Here we evaluate potential deleterious effects of CRISPR-Cas9 by combining sequence analysis and the genetics of the male X chromosome in a Drosophila model. Since males have only one X chromosome deleterious mutations on the X chromosome will manifest in reducing viability or result in visible phenotypes and thus provide sensitive readouts of off-target activity. Our data do not support large scale off-target effects in Drosophila. To optimize sgRNA selection, we incorporated off-target evaluation into the PlatinumCRISPr sgRNA selection tool for a broad range of organisms.},
}
@article {pmid41366211,
year = {2025},
author = {Nuccio, SP and Cadoni, E and Nikoloudaki, R and Galli, S and Ler, AJ and Sanchez-Cabanillas, C and Maher, TE and Fan, E and Guneri, D and Flint, G and Zhu, M and Liu, LS and Fullenkamp, CR and Waller, Z and Magnani, L and Schneekloth, JS and Di Antonio, M},
title = {Chemically modified CRISPR-Cas9 enables targeting of individual G-quadruplex and i-motif structures, revealing ligand-dependent transcriptional perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {385},
pmid = {41366211},
issn = {2041-1723},
support = {Lister Prize 2022//Lister Institute of Preventive Medicine/ ; BB/R011605/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Humans ; Ligands ; *Transcription, Genetic ; Promoter Regions, Genetic ; DNA/chemistry/genetics/metabolism ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Nucleotide Motifs ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Editing/methods ; },
abstract = {The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, limiting their scope. Herein, we describe an Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrates that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA reveals that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4 site. We further demonstrate that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides a platform for investigating G4- and iM-biology with high precision, unveiling the therapeutic relevance of individual DNA structures with selectivity.},
}
@article {pmid41366257,
year = {2025},
author = {Launspach, M and Macos, J and Afzal, S and Hohmann, J and Appis, ML and Pilgram, M and Beez, S and Ohlendorf, E and van der Ven, CFT and Lachiheb, C and Töws, K and Andersch, L and Jens, M and Zirngibl, F and Kath, J and Stecklum, M and Rodriguez-Fos, E and Anders, K and Wagner, DL and Henssen, AG and Kühn, R and Eggert, A and Künkele, A},
title = {Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10987},
pmid = {41366257},
issn = {2041-1723},
mesh = {*Tumor Microenvironment/genetics/immunology ; Humans ; Animals ; Gene Knock-In Techniques ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Genetic Therapy/methods ; Chemokine CXCL10/genetics ; *Cytokines/genetics ; Cell Line, Tumor ; *Neuroblastoma/therapy/immunology/genetics ; Receptors, Chimeric Antigen/genetics ; T-Lymphocytes/immunology ; Chemokine CXCL11/genetics ; *Neoplasms/therapy/immunology/genetics ; Interferon-gamma/genetics ; Precision Medicine ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34[+] HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.},
}
@article {pmid41367203,
year = {2025},
author = {Patel, J and Patel, D and Raval, A},
title = {Artificial Intelligence for Predictive Modeling in CRISPR/Cas9 Gene Editing: a Survey of Methods and Design Strategies.},
journal = {The journal of gene medicine},
volume = {27},
number = {12},
pages = {e70061},
doi = {10.1002/jgm.70061},
pmid = {41367203},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; Algorithms ; },
abstract = {Ongoing developments in genome editing most notably the continued evolution of CRISPR-Cas systems and their orthogonal or modified counterparts have substantively altered both experimental and applied practices in biomedicine, agriculture, and therapeutic design. More recently, the systematic incorporation of artificial intelligence and machine learning methodologies has augmented the specificity, throughput, and explanatory capacity of genome-editing workflows, thereby refining the prediction of on-target efficiencies, the appraisal of off-target liabilities, and the tailoring of molecular therapeutic configurations. The present contribution offers an integrative survey of these computational developments, emphasizing (i) predictive algorithms, (ii) machine-learning and deep-learning frameworks, (iii) data-centric procedural strategies, and (iv) dedicated applications in oncology, neurology, rare-disease research, and precision-medicine contexts. Throughout, we evaluate architectural choices, sequence-encoding representations, and lingering dataset-related biases, while additionally addressing current constraints concerning model interpretability, ethical viability, and the procedural prerequisites for clinical translation. Moreover, we advance a structured taxonomy that organizes AI-mediated genome-editing approaches according to methodological lineage and functional scope, and we delineate extant research lacunae. By combining these elements, we supply a prospective assessment of the means by which artificial intelligence might be further leveraged to support secure, efficacious, and equitably accessible genome engineering outcomes.},
}
@article {pmid41367214,
year = {2025},
author = {Selokar, NL and Singh, P and Jose, B and Gautam, D and Patel, K and Verma, R and De, S and Singh, MK and Singh, D},
title = {A Myostatin (MSTN[-/-]) Knockout Buffalo Produced by CRISPR-Cas9 Mediated Genome Editing and Somatic Cell Nuclear Transfer.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {436-442},
doi = {10.1177/25731599251401528},
pmid = {41367214},
issn = {2573-1602},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Buffaloes/genetics ; *Nuclear Transfer Techniques ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Female ; Animals, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.},
}
@article {pmid41367295,
year = {2026},
author = {Wyer, CAS and Amaro, IA and Pitcher, S and Ponlawat, A and Harrington, LC and Wolfner, MF and Hollis, B and Cator, LJ},
title = {Pickpocket315 affects male mating behavior in the yellow fever mosquito Aedes aegypti.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {2},
pages = {},
pmid = {41367295},
issn = {2160-1836},
support = {NE/S007415/1//Natural Environment Research Council/ ; R01-AI095491/NH/NIH HHS/United States ; //L.C.H and M.F.W and International Atomic Energy Association/ ; //Reproductive Biology of Male Aedes Mosquitoes for SIT Applications/ ; R01 AI095491/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Aedes/genetics/physiology ; Male ; *Sexual Behavior, Animal ; Female ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; RNA Interference ; Mosquito Vectors/genetics ; Copulation ; Yellow Fever/transmission ; },
abstract = {The molecular basis of mating behavior in the important disease vector mosquito, Aedes aegypti, remains poorly characterized. We investigated the functional role of a pickpocket gene, ppk315, in male mating behavior using both RNAi-mediated knockdown and CRISPR/Cas9 approaches. Behavioral assays revealed that RNAi-treated males (dsPPK315) made fewer mating attempts, were less responsive to female acoustic cues, and were less likely to achieve copulation, though their latency to initiate contact when attempts were made was comparable to controls. Males with a CRISPR/Cas9-induced disruption to ppk315 exhibited reduced success in inseminating multiple females, consistent with previous reports from RNAi knockdown males, ruling out off-target effects as the source of behavioral changes. In contrast to the results of behavioral assays with RNAi, ppk315 mutant males (ppk315-/-) attempted copulation as frequently as wild-type males (ppk315+/+) but were slower to contact females. Despite these impairments in one-on-one interactions, both dsPPK315 and ppk315-/- males displayed normal mating success under competitive swarm-like conditions, potentially due to the socially facilitated activation of mating behavior. Collectively, our findings support a role for ppk315 in the initiation of mating behaviors via sensory detection, with context-dependent consequences for reproductive success.},
}
@article {pmid41369349,
year = {2025},
author = {Cheng, X and Wang, D and Zhang, X and Li, L and Liu, Y and Cao, G and Zhang, Y},
title = {Regulation of the Homeostasis of Early Embryo Development in Dairy Cows by Targeted Editing of the PRLR Gene-Mediated Activation of the Anti-Heat Stress Pathway.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369349},
issn = {2073-4409},
support = {No. 2023ZD04050//Guifang Cao/ ; },
mesh = {Animals ; Cattle ; *Gene Editing/methods ; *Heat-Shock Response/genetics ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Receptors, Prolactin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Female ; Reactive Oxygen Species/metabolism ; Fibroblasts/metabolism ; Nuclear Transfer Techniques ; Oocytes/metabolism ; },
abstract = {The intensification of global climate warming exacerbates the issue of heat stress in dairy cows, making the SLICK mutation in the prolactin receptor (PRLR) gene a critical target for enhancing heat tolerance in these animals. This study aims to investigate the effects of CRISPR/Cas9-mediated editing of the PRLR gene on the biological characteristics of bovine fibroblasts and early embryonic development following somatic cell nuclear transfer (SCNT). Using the CRISPR/Cas9 system, we targeted and edited a 20 bp-150 bp region within exon nine of the PRLR gene. After conducting off-target predictions and activity screenings, we identified optimal guide RNA (sgRNA) sequences and established stable transgenic cell lines. Transcriptome sequencing was performed on edited cells to identify key genes and validate their expression profiles. Edited cells were utilized as donor cells for SCNT, during which we assessed oocyte levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial function to analyze embryonic developmental performance. We constructed a cellular stress resistance network aimed at mitigating damage transmission while maintaining embryonic developmental homeostasis. This research provides technical support and theoretical reference for genetic editing breeding programs aimed at improving heat tolerance in dairy cattle.},
}
@article {pmid41369371,
year = {2025},
author = {Gardner-Kay, A and Le, L and Filla, M and Kibiryeva, N and O'Brien, JE and Bittel, DC},
title = {CRISPR Disruption of scaRNA1 Reduces Pseudouridylation in Spliceosomal RNA U2 at U89 and Perturbs the Transcriptome in HEK293T Cells.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369371},
issn = {2073-4409},
support = {Intramural Faculty grant//Kansas City University/ ; },
mesh = {Humans ; HEK293 Cells ; *Spliceosomes/metabolism/genetics ; *Transcriptome/genetics ; *Pseudouridine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Small Nuclear/metabolism/genetics ; RNA Splicing/genetics ; },
abstract = {Small Cajal body-associated RNAs (scaRNAs) are essential for biochemical modification of spliceosomal RNAs and spliceosome function. Changes in scaRNA expression level have been associated with developmental issues, including cancer and congenital heart defects (CHDs), although the mechanism remains unclear. Small Cajal body-associated RNA 1 (scaRNA1) guides pseudouridylation at uridine 89 (Ψ89) of the spliceosomal RNA U2, a highly conserved modification that may be critical for spliceosome function. To investigate the role of scaRNA1 in splicing regulation, CRISPR-Cas9 genome editing was used to introduce targeted deletions in the scaRNA1 locus in HEK293T cells. Edited clones were identified by T7 endonuclease I assay and confirmed by Sanger sequencing. Pseudouridylation at Ψ89 was quantified using CMC-based reverse transcription followed by quantitative PCR, and global mRNA splicing alterations were assessed by RNA sequencing. Clones harboring scaRNA1 disruptions exhibited a significant reduction in Ψ89 pseudouridylation, consistent with impaired scaRNA1 function. Transcriptome analysis (of mRNA from two clones) revealed >300 protein coding genes with significant changes in transcript isoform level, including >100 genes related to RNA-binding activity. These results indicate that scaRNA1 disruption alters spliceosomal function and leads to substantial changes in mRNA splicing. The dysregulated splicing of RNA-binding proteins may impair RNA processing and gene expression programs required for normal development, providing new insight into how noncoding RNA dysfunction may contribute to developmental pathogenesis.},
}
@article {pmid41369550,
year = {2026},
author = {Smith, DJ},
title = {Complementary human gene interaction maps from radiation hybrids and CRISPRi.},
journal = {Physiological genomics},
volume = {58},
number = {1},
pages = {42-57},
doi = {10.1152/physiolgenomics.00075.2025},
pmid = {41369550},
issn = {1531-2267},
support = {C25CR8562//University of California Cancer Research Coordinating Committee/ ; //Norton Simon Research Foundation/ ; },
mesh = {Humans ; Genome-Wide Association Study ; *Gene Regulatory Networks/genetics ; *CRISPR-Cas Systems/genetics ; *Radiation Hybrid Mapping/methods ; *Protein Interaction Maps/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Alleles ; },
abstract = {The only comprehensive human genetic interaction map was constructed using increased gene copy numbers in radiation hybrid (RH) cells. Recently, a second map restricted to essential genes was created using CRISPR interference (CRISPRi)-induced loss-of-function alleles. Here, the two maps are compared to understand their similarities and differences. Both maps showed significant overlap with protein-protein interaction databases and identified a shared set of interacting genes, although the specific gene pairs differed between approaches. Notably, the RH map exhibited strong overlap with genome-wide association study (GWAS) networks, whereas the CRISPRi map did not. These findings demonstrate how gain- and loss-of-function alleles reveal distinct yet complementary genetic interaction landscapes.NEW &amp; NOTEWORTHY This study compared two mammalian genetic interaction networks for cell growth: the radiation hybrid (RH) network used extra gene copies and the CRISPRi network used partial gene suppression. Both networks overlapped with protein-protein interaction data and identified common interacting genes, yet specific gene pair interactions differed dramatically. Only the RH network predicted genome-wide association study (GWAS) networks. As the first comparison of large-scale mammalian genetic interaction networks, this work reveals how gain- and loss-of-function variants capture diverse biological perspectives.},
}
@article {pmid41370122,
year = {2026},
author = {Pfisterer, L and Boyle, C and Cole, A and Mitchell, I and Flanagan, M and Gromley, Z and Gromley, A},
title = {Disruption of the centriolin/Cep110 gene (CNTRL) with CRISPR/Cas9 leads to cell cycle arrest and cell death of rhabdomyosarcoma cells in vitro.},
journal = {Molecular biology of the cell},
volume = {37},
number = {2},
pages = {br4},
pmid = {41370122},
issn = {1939-4586},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Rhabdomyosarcoma/genetics/metabolism/pathology ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; *Cell Cycle Proteins/genetics/metabolism ; Gene Editing/methods ; Centrosome/metabolism ; Cell Death/genetics ; *Microtubule-Associated Proteins/genetics/metabolism ; Cilia/metabolism ; Cell Survival/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; },
abstract = {Rhabdomyosarcoma is the most common pediatric soft tissue cancer, thought to arise from primitive mesenchymal cells that differentiate into skeletal muscle. Previous studies suggest that primary cilia may play a role in the development of rhabdomyosarcoma. Primary cilia are cellular structures that arise from the centrosome and serve important functions in sensory signaling, cell migration, and developmental processes. However, most rhabdomyosarcoma cell lines do not have primary cilia. Because primary cilia are derived from centrosomes, the development of rhabdomyosarcoma may, in fact, be due to the function of centrosome proteins rather than the primary cilia itself. Therefore, this study sought to determine if the centrosomal protein centriolin/Cep110, which is localized to both centrosomes and primary cilia, plays a role in rhabdomyosarcoma biology. The gene editing tool CRISPR/Cas9 was used to disrupt the centriolin/Cep110 gene in the rhabdomyosarcoma cell line CCL-136, and the effects on cell viability and cell cycle progression were assayed. Our results show that loss of centriolin/Cep110 leads to cell cycle arrest and apoptotic cell death in rhabdomyosarcoma cells. These findings suggest that centriolin/Cep110 plays a key role in rhabdomyosarcoma cell proliferation and viability and that this centrosome protein may represent a potential target for future rhabdomyosarcoma therapies.},
}
@article {pmid41370200,
year = {2025},
author = {Shi, X and Hu, C and Jia, L and Lei, Z and Guo, B and Zhou, J and Wang, F},
title = {An SpC editor targeting pre-mRNA splicing for precise CRISPR control and enhanced antitumor efficacy.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41370200},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2024JC-YBQN-0168//Natural Science Basic Research Program of Shaanxi/ ; 2023A1515110886//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA Precursors/genetics/metabolism ; *RNA Splicing/drug effects ; Animals ; Mice ; Cell Line, Tumor ; Spliceosomes/genetics/metabolism ; Macrolides/pharmacology ; Apoptosis/genetics ; Neoplasms/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epoxy Compounds ; },
abstract = {The CRISPR/Cas9 system is a powerful genome editing tool that has the potential to be applied to a variety of biomedical applications. Despite the considerable potential of this gene editing technology, there are numerous safety concerns including the possibility of unpredictable off-target effects. The splicing process, which involves the removal of introns from pre-mRNA and the alignment of exons to produce mature transcripts, is a critical step in gene expression in most eukaryotes. In this study, we present a spliceosome-responsive CRISPR/Cas9 (SpC) editor that utilizes the splicing inhibitor pladienolide B (PB) to regulate pre-mRNA splicing and control the expression of the anti-CRISPR protein AcrIIA4, thereby modulating the activity of the Cas9 nuclease. This approach allows for precise regulation of the gene editing process, thereby effectively mitigating off-target effects. The reliability and robustness of the SpC editor were demonstrated through in vitro and in vivo bioluminescence imaging. Furthermore, a dual-target sgRNA was designed to target the diphtheria toxin A gene, resulting in apoptosis induction and growth inhibition of tumor cells across various types of cancer cells. Our results indicate that this SpC editor has the capacity to precisely regulate tumor cell growth, thus providing new insights and significant implications for cancer gene therapy.},
}
@article {pmid41370232,
year = {2026},
author = {Alok, A and Raman, V and D'Agostino, L and Kshetry, AO and Rai, KM and Wang, C and Gunapati, S and Stupar, RM and Patil, GB and Zhang, F},
title = {Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
pmid = {41370232},
issn = {1532-2548},
support = {IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; #2021-67013-34565//USDA NIFA/ ; //Texas Governor's University Research/ ; },
mesh = {*Glycine max/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Growth Regulators/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Soybean (Glycine max) transformation remains challenging and has not kept pace with rapid advances in genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 (WUS2) and the gene encoding isopentenyltransferase (IPT) achieved higher transformation efficiencies (14.6% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produced heritable transgenic events within 9 to 11 weeks and successfully delivered CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2/IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation frequency, reduces tissue culture requirements, and offers a promising genome-editing platform for soybean improvement.},
}
@article {pmid41370233,
year = {2026},
author = {Nomura, Y and Nomura, J and Tamada, K and Eguchi, N and Torigata, K and Tokumoto, S and Nemoto, A and Shirafuji, T and Yamamoto, K and Hishimoto, A and Nagase, H and Nishikawa, T and Takumi, T},
title = {Isogenic modeling of 1q21.1 reciprocal CNVs in human ES cells reveals divergent neurodevelopmental trajectories.},
journal = {Human molecular genetics},
volume = {35},
number = {2},
pages = {},
doi = {10.1093/hmg/ddaf184},
pmid = {41370233},
issn = {1460-2083},
support = {21&#xa0;K07820//KAKENHI/ ; 24&#xa0;K10078//KAKENHI/ ; 22&#xa0;K15750//KAKENHI/ ; 16H06316//KAKENHI/ ; 16H06463//KAKENHI/ ; 23KK0132//KAKENHI/ ; 24H00620//KAKENHI/ ; 24H01241//KAKENHI/ ; 23H04233//KAKENHI/ ; 24&#xa0;K22036//KAKENHI/ ; //Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science, and Technology/ ; JP21wm0425011//Japan Agency for Medical Research and Development/ ; JPMJPF2018//Japan Science and Technology Agency/ ; JPMJMS2299//Japan Science and Technology Agency/ ; JPMJMS229B//Japan Science and Technology Agency/ ; //Takeda Science Foundation/ ; //Smoking Research Foundation/ ; //Taiju Life Social Welfare Foundation/ ; },
mesh = {Humans ; *DNA Copy Number Variations/genetics ; *Chromosomes, Human, Pair 1/genetics ; Neurogenesis/genetics ; *Neurodevelopmental Disorders/genetics/pathology ; *Human Embryonic Stem Cells/metabolism ; Chromosome Deletion ; Neural Stem Cells/metabolism ; Cell Differentiation/genetics ; Neurons/metabolism ; Chromosome Duplication ; CRISPR-Cas Systems ; Intellectual Disability/genetics ; Cell Line ; Schizophrenia/genetics ; },
abstract = {Copy number variations (CNVs) in the distal 1q21.1 region, both deletion (1q del) and duplication (1q dup) are associated with various neurodevelopmental and neuropsychiatric disorders such as autism spectrum disorder, intellectual disability, epilepsy, and schizophrenia. Besides common phenotypes, 1q del and 1q dup manifest opposite clinical phenotypes, e.g. microcephaly in 1q del and macrocephaly in 1q dup. However, molecular and cellular mechanisms underlying these phenotypes are still elusive. Here, to identify molecular mechanisms associated with neurodevelopmental phenotypes from the viewpoint of neurogenesis and neurodevelopment, we generate isogenic human ES cell (hESC) lines with reciprocal 1q21.1 CNVs using CRISPR/Cas9 system and differentiate them into 2-dimensional (2-D) neurons and neural progenitor cell (NPC) spheroids. Our study recapitulates reciprocal brain size in the NPC spheroids and shows dosage-dependent differentiation changes i.e. more GABAergic components in 1q del and more proliferative state in 1q dup. These results demonstrate that 1q21.1 CNVs dramatically affect cell fate in the early neurodevelopmental periods. This is the first isogenic cell model of human 1q21.1 CNVs, and our findings provide new insights into the underlying mechanisms of neurodevelopmental disorders.},
}
@article {pmid41370671,
year = {2025},
author = {Gao, Z and Lin, K and Gong, Y and Zhao, Y and Zhang, S},
title = {Core-Shell Tripeptide-Lipid/PEI Nanocarriers Enable Efficient Plasmid-Based CRISPR/Cas9 Editing of VEGFR2.},
journal = {Langmuir : the ACS journal of surfaces and colloids},
volume = {41},
number = {50},
pages = {33716-33730},
doi = {10.1021/acs.langmuir.5c02069},
pmid = {41370671},
issn = {1520-5827},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Polyethyleneimine/chemistry ; *Plasmids/genetics/chemistry ; *Vascular Endothelial Growth Factor Receptor-2/genetics ; Animals ; *Lipids/chemistry ; *Nanoparticles/chemistry ; MCF-7 Cells ; Mice ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a versatile genome editing technology that holds tremendous promise for the treatment of various diseases. Although several delivery technologies such as electroporation, viral vectors, and lipid nanoparticles have already shown promise in preclinical and clinical applications for hematological and neuromuscular genetic disorders, in vivo application is still restricted by the inefficient delivery of CRISPR/Cas9 components. Herein, by employing the tripeptide lipid N,N-ditetradecyloxyamidoethyl trimeric ornithine amide (CDO) and polyethylenimine (PEI), we constructed novel ternary systems (pDNA/PEI/CL) for the delivery of pDNA encoding Cas9 and single-guide RNAs (sgRNAs) targeting the VEGFR2 gene. The pDNA/PEI/CL delivery systems were fabricated by condensing pDNA with PEI, followed by coating with cationic liposomes composed of CDO. This system demonstrated high transfection efficiency, successfully delivering CRISPR/Cas9 to A549 and MCF-7 cells with efficiencies of up to 91.0% (n = 3, P < 0.001), while also exhibiting lower cytotoxicity. Notably, the sgRNA1/P1/C1 complex achieved higher genome editing efficiencies than sgRNA3/P1/C1, with 38.6% vs 31.0% in A549 cells (n = 3, P < 0.01) and 26.45% vs 20.18% in MCF-7 cells (n = 3, P < 0.01). Western blot analysis showed that VEGFR2 expression decreased by 48.1% in A549 and 44.3% in MCF-7 cells, while PI3K levels were reduced by 39.6% and 42.8%, respectively. This suppression of the PI3K/Akt signaling pathway led to cell cycle arrest, thereby inhibiting tumor cell proliferation and migration while promoting apoptosis. Furthermore, animal experiments validated the antitumor efficacy, highlighting the translational potential of this platform in cancer therapy. Collectively, these findings highlight the potential of the ternary complex system as a robust and biocompatible CRISPR/Cas9 delivery strategy, offering a promising avenue for gene therapy in cancer and other genetic diseases.},
}
@article {pmid41370881,
year = {2026},
author = {Li, SR and Li, Y and Yang, KB and Wang, SW and Sun, ML and Liu, Z and Zhang, XP and Zhong, Y and Yao, J},
title = {CRISPR/Cas12a coupled with MIRA: A specific and rapid assay for human DNA in challenging forensic matrices.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103393},
doi = {10.1016/j.fsigen.2025.103393},
pmid = {41370881},
issn = {1878-0326},
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Cytochromes b/genetics ; Mice ; Swine ; Chickens ; *DNA Fingerprinting/methods ; DNA ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human DNA detection is crucial in forensic medicine, particularly for trace, degraded, or mixed samples, which demand high sensitivity, specificity, and rapid processing. Traditional methods, such as immunological assays and PCR-based techniques, often suffer from operational complexity, limited sensitivity, or high equipment dependency. To address these challenges, we developed a novel detection system combining multienzyme isothermal rapid amplification (MIRA) with CRISPR-Cas12a for the rapid, specific, and portable human DNA identification. By targeting the human mitochondrial cytochrome b (CYTB) gene and sex-determining Region Y(SRY) gene, we designed MIRA primers and CRISPR-Cas12a crRNA to enable dual recognition and signal amplification. The method involves isothermal amplification at 37°C followed by CRISPR-Cas12a-mediated cleavage, producing detectable fluorescence or lateral flow chromatographic signals. Our system achieves ultra-sensitive detection and high specificity, distinguishing human DNA from non-human sources (e.g., pig, chicken, mouse), and also enables accurate gender identification, further enhancing its utility in forensic and genetic studies. Compared to traditional qPCR, this approach demonstrates superior sensitivity, faster turnaround (≤ 45 min), and minimal equipment requirements, making it ideal for forensic applications. Moreover, the blood, mixed, and degraded samples were used to confirm its robustness, with results interpretable via blue-light fluorescence or colloidal gold test strips. In summary, the MIRA-CRISPR/Cas12a system overcomes the limitations of conventional techniques, offering a rapid, cost-effective, and reliable solution for forensic human DNA identification, with potential extensions to wildlife conservation and food safety testing.},
}
@article {pmid41371153,
year = {2026},
author = {Longhi Cervantes, DS and Leal, GM and Fortirer, JDS and de Oliveira, LF and Navarro, BV and Buckeridge, MS},
title = {microRNAs and stress adaptation in grasses: A systematic review.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110783},
doi = {10.1016/j.plaphy.2025.110783},
pmid = {41371153},
issn = {1873-2690},
mesh = {*MicroRNAs/genetics/metabolism ; *Poaceae/genetics/physiology/metabolism ; *Stress, Physiological/genetics ; *Adaptation, Physiological/genetics ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics/metabolism ; },
abstract = {MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression after transcription, playing crucial roles in plant development and stress adaptation. In grasses, this regulation is vital under isolated biotic and abiotic stress conditions and combined stress scenarios, although many regulatory modules remain unexplored. This systematic review examined 60 studies out of 1823 publications indexed in Scopus, focusing on grass miRNAs with validated targets through Degradome-Seq and/or RACE approaches. Results indicate that miRNA-target modules were validated more often under abiotic stress than biotic or combined stress conditions. The most frequently studied miRNA families include miR156, miR159, miR164, miR169, and miR396, which are commonly linked to various types of stress, whether isolated or combined. Most research has concentrated on major crops such as rice and maize, with limited studies on other agriculturally important grasses. This review highlights advances in miRNA-phytohormone interactions, systemic signaling, and target validation strategies. It also underscores the potential of biotechnological tools such as RNAi, artificial miRNAs, target mimicry, and CRISPR/Cas for engineering more resilient grasses. Integrating multi-omics approaches and an increasing focus on combined stress responses suggest promising strategies for sustainable agriculture, food security, and bioenergy production amidst climate challenges. Together, these advances strengthen the potential of microRNA-based regulation as a key tool for enhancing crop resilience and adaptation.},
}
@article {pmid41371329,
year = {2026},
author = {Chen, B and Gao, J and Sun, H and Zhao, Y and Liu, Y and Qiu, X and Li, Y},
title = {Integrating CRISPR with SERS: Toward intelligent point-of-care diagnostics of the future.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {581},
number = {},
pages = {120782},
doi = {10.1016/j.cca.2025.120782},
pmid = {41371329},
issn = {1873-3492},
mesh = {*Spectrum Analysis, Raman/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Point-of-Care Systems ; Biosensing Techniques ; },
abstract = {In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) system has emerged as a transformative genome-editing platform. Beyond its editing applications, the CRISPR/Cas system has attracted growing interest in molecular diagnostics particularly for nucleic acid detection due to its exceptional sensitivity and target specificity. Meanwhile, surface-enhanced Raman spectroscopy (SERS), which relies on plasmonic nanoparticles or nanostructures, has become a powerful biosensing technology known for its high sensitivity and distinct spectral fingerprinting capability. The integration of CRISPR/Cas-mediated molecular recognition with the ultrasensitive detection of SERS offers a rapid, low-volume, and direct strategy for identifying diverse nucleic acid targets. This synergistic combination has inspired the development of innovative biosensing platforms designed for ultrasensitive and precise molecular diagnostics. In this review, we first outline the fundamental principles of CRISPR/Cas and SERS, then summarize their hybrid applications in nucleic acid detection. Finally, we discuss the current progress, challenges, and future perspectives of CRISPR/Cas-integrated SERS biosensing.},
}
@article {pmid41372121,
year = {2025},
author = {Cheng, KW and Bhave, M and Markhard, AL and Peng, D and Bhatt, KD and Travisano, KA and Medicielo, JV and Anaya, A and Lembirik, S and Njoya, L and Anantpadma, M and Kuhn, JH and Puschnik, AS and Kistler, AL},
title = {Replicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11028},
pmid = {41372121},
issn = {2041-1723},
support = {HHSN272201800013C/OD/NIH HHS/United States ; },
mesh = {*Virus Replication/genetics ; Humans ; *Replicon/genetics ; *Dengue Virus/genetics/physiology ; Chikungunya virus/genetics/physiology ; *CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Ebolavirus/genetics/physiology ; Cell Line ; Animals ; *Host-Pathogen Interactions/genetics ; Membrane Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Hexosyltransferases/genetics/metabolism ; },
abstract = {We describe a viral replicon-based CRISPR knockout (KO) screening approach to specifically identify host factors essential for viral replication which are often missed in live virus screens. We benchmark the replicon screening using a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line and successfully identify host genes known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components), along with additional genes that have not been reported in prior CRISPR KO screens with DENV-2. We extend this replicon screening approach to chikungunya virus (CHIKV), a positive-sense RNA virus, and Ebola virus (EBOV), a negative-sense RNA virus, and identify distinct sets of genes required for replication of each virus. Our findings indicate that viral replicon-based CRISPR screens are a useful approach to identify host factors essential for replication of diverse viruses and to elucidate potential novel targets for host-directed medical countermeasures.},
}
@article {pmid41372159,
year = {2025},
author = {Pan, R and Ren, J and Chen, X and Flores, LF and Gonzalez, RVL and Adonnino, AA and Lofts, B and Waldo, J and Halmai, J and Devinsky, O and Fink, K and Liu, XS},
title = {Editing DNA methylation in vivo.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {527},
pmid = {41372159},
issn = {2041-1723},
support = {R01 NS126185/NS/NINDS NIH HHS/United States ; R01 MH134519/MH/NIMH NIH HHS/United States ; R01NS126185//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01MH134519//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; F30 HD115371/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *DNA Methylation/genetics ; *Gene Editing/methods ; Mice ; DNA Methyltransferase 3A ; Mice, Transgenic ; Promoter Regions, Genetic ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Proprotein Convertase 9/genetics/metabolism ; Liver/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; Proto-Oncogene Proteins/genetics/metabolism ; Male ; DNA-Binding Proteins/genetics/metabolism ; Epigenesis, Genetic ; Neurons/metabolism ; },
abstract = {DNA methylation is a crucial epigenetic mechanism that regulates gene expression. Precise editing of DNA methylation has emerged as a promising tool for dissecting its biological function. However, challenges in delivery have limited most applications of DNA methylation editing to in vitro systems. Here, we develop two transgenic mouse lines harboring an inducible dCas9-DNMT3A or dCas9-TET1 editor to enable tissue-specific DNA methylation editing in vivo. We demonstrate that targeted methylation of the Psck9 promoter in the liver of dCas9-DNMT3A mice results in decreased Pcsk9 expression and a subsequent reduction in serum low-density lipoprotein cholesterol level. Targeted demethylation of the Mecp2 promoter in dCas9-TET1 mice reactivates Mecp2 expression from the inactive X chromosome and rescues neuronal nuclear size in Mecp2[+/-] mice. Genome-wide sequencing analyses reveal minimal transcriptional off-targets, demonstrating the specificity of the system. These results demonstrate the feasibility and versatility of methylation editing, to functionally interrogate DNA methylation in vivo.},
}
@article {pmid41372199,
year = {2025},
author = {Nan, AX and Chickering, M and Bartolome, CL and Shadija, N and Li, D and Estes, BJG and Stetina, JV and Li, W and Andresen, J and Molugu, K and Amunugama, R and Fang, M and Bai, C and Wang, J and Norouzi, D and Cochrane, JC and Gatlin, JT and Dunyak, MT and Kumar, S and Chavez, L and Seth, A and Halperin, S and Finn, JD and Xie, J},
title = {Ligase-mediated programmable genomic integration (L-PGI).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {563},
pmid = {41372199},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; Genomics/methods ; *Ligases/metabolism/genetics ; HEK293 Cells ; },
abstract = {Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.},
}
@article {pmid41372233,
year = {2025},
author = {Macak, D and Kanis, P and Riesenberg, S},
title = {Repurposing clinically safe drugs for DNA repair pathway choice in CRISPR genome editing and synthetic lethality.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11077},
pmid = {41372233},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *Synthetic Lethal Mutations/drug effects ; *Drug Repositioning/methods ; DNA End-Joining Repair/drug effects ; Induced Pluripotent Stem Cells/drug effects/metabolism ; *DNA Repair/drug effects ; Recombinational DNA Repair/drug effects ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/drug effects ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; },
abstract = {We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ, MMEJ), and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1), affecting several key DNA repair proteins, such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling, gene therapy, chimeric antigen receptor immunotherapy, and cancer treatment.},
}
@article {pmid41372414,
year = {2026},
author = {Habtewold, T and Lwetoijera, DW and Hoermann, A and Mashauri, R and Matwewe, F and Mwanga, R and Kweyamba, P and Maganga, G and Magani, BP and Mtama, R and Mahonje, MA and Tambwe, MM and Tarimo, F and Chennuri, PR and Cai, JA and Del Corsano, G and Capriotti, P and Sasse, P and Moore, J and Hudson, D and Manjurano, A and Tarimo, B and Vlachou, D and Moore, S and Windbichler, N and Christophides, GK},
title = {Gene-drive-capable mosquitoes suppress patient-derived malaria in Tanzania.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {442-448},
pmid = {41372414},
issn = {1476-4687},
support = {//Gates Foundation/ ; },
mesh = {Tanzania/epidemiology ; Animals ; *Plasmodium falciparum/isolation &amp; purification/genetics/drug effects/growth &amp; development ; *Anopheles/genetics/parasitology ; Humans ; *Malaria, Falciparum/prevention &amp; control/parasitology/transmission ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; Female ; Animals, Genetically Modified/genetics ; Male ; Child ; CRISPR-Cas Systems/genetics ; },
abstract = {Gene drive technology presents a transformative approach to combatting malaria by introducing genetic modifications into wild mosquito populations to reduce their vectorial capacity. Although effective modifications have been developed, these efforts have been confined to laboratories in the global north. We previously demonstrated that modifying Anopheles gambiae to express two exogenous antimicrobial peptides inhibits the sporogonic development of laboratory-cultured Plasmodium falciparum, with models predicting substantial contributions to malaria elimination in Africa when integrated with gene drive[1-3]. However, the effectiveness of this modification against genetically diverse, naturally circulating parasite isolates remained unknown. To address this critical gap, we adapted our technology for an African context by establishing infrastructural and research capacity in Tanzania, enabling the engineering of local A. gambiae under containment. Here we report the generation of a transgenic strain equipped with non-autonomous gene drive capabilities that robustly inhibits genetically diverse P. falciparum isolates obtained from naturally infected children. These genetic modifications were efficiently inherited by progeny when supplemented with Cas9 endonuclease provided by another locally engineered strain. Our work brings gene drive technology a critical step closer to application, providing a locally tailored and powerful tool for malaria eradication through the targeted dissemination of beneficial genetic traits in wild mosquito populations.},
}
@article {pmid41373007,
year = {2025},
author = {Song, J and Yang, D and Kong, L and Tsai, LK and Zhang, J and Chen, YE and Tsai, RY and Xu, J},
title = {Development of a high-yield Rabbit line for enhanced animal pharming.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {73},
pmid = {41373007},
issn = {0717-6287},
support = {R41 GM110822/GM/NIGMS NIH HHS/United States ; R41GM110822/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Rabbits/genetics ; *Animals, Genetically Modified/genetics ; *Recombinant Proteins/biosynthesis/genetics ; *Milk/chemistry ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; *Caseins/genetics ; Female ; Promoter Regions, Genetic/genetics ; },
abstract = {Animal pharming involves producing recombinant protein drugs using transgenic animals. The United States Food and Drug Administration (FDA) has approved certain drugs produced in the milk of transgenic Rabbits. Traditionally, these pharming Rabbits have been developed using conventional transgenic technology, which often results in an unpredictable success rate, uncontrollable transgene insertion sites, varying copy numbers, and generally low recombinant protein yields, typically 1-2 g/L or lower. We hypothesized that utilizing the promoter of a native major milk protein gene to drive transgene expression could significantly enhance yield. To test this, we developed a rabbit line that expresses tdTomato under the control of the CSN2 gene promoter, responsible for encoding β-casein, the most abundant protein in Rabbit milk. We successfully generated knock-in founder Rabbits using CRISPR/Cas9-mediated knock-in technology, augmented by the homology-directed repair (HDR)-promoting small molecule RS-1. These founder Rabbits were able to transmit the knock-in allele to their offspring, producing both heterozygous and homozygous tdTomato knock-in Rabbits. Remarkably, the recombinant protein yield reached 15-20 g/L in the milk of homozygous animals. Our work demonstrates a promising strategy to enhance recombinant protein production in Rabbit pharming.},
}
@article {pmid41373550,
year = {2025},
author = {Koller, F},
title = {The Potential of NGTs to Overcome Constraints in Plant Breeding and Their Regulatory Implications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373550},
issn = {1422-0067},
support = {3522840500//Federal Agency for Nature Conservation/ ; },
mesh = {*Plant Breeding/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Plants/genetics ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Conventional plant breeding relies on the occurrence of chromosomal crossover and spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way, as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species while at the same time allowing necessary adaptations. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs), which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break in DNA, recombinant enzymatic mutagens (REMs), such as CRISPR/Cas, additionally interfere with cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs introduce genetic changes and combinations that are unknown in the current breeding pool and that are also unlikely to occur as a result of any previously used breeding methods. The resulting genotypes may need to be considered as 'new to the environment'. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity, and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterization can inform the following steps in risk assessment and guide requests for further data.},
}
@article {pmid41373623,
year = {2025},
author = {Petrova, IO and Smirnikhina, SA},
title = {Ex Vivo Gene and Cell Therapy in Hematopoietic Stem Cells.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373623},
issn = {1422-0067},
support = {not applicable//Ministry of Education and Science of Russia/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; *Cell- and Tissue-Based Therapy/methods ; Animals ; *Hematopoietic Stem Cell Transplantation/methods ; Genetic Vectors/genetics ; Lentivirus/genetics ; },
abstract = {Ex vivo cell and gene therapy is a prospective approach to treatment of genetic diseases. To date, one of the most prevalent examples of genetically engineered cell therapies is hematopoietic stem/progenitor cells (HSPCs). This mini review is focused on HSPC therapy methods that have been approved for medical use. Most gene therapy methods rely on the lentiviral integration of the gene into the target cell genome, as lentiviruses are extremely effective, particularly in transduction of non-dividing cells. In this constantly evolving field, it is important to find the balance between safety concerns and efficiency. Analyzing cases of several diseases, for which ex vivo gene therapy was developed, we strive to understand which factors are crucial to success and what the potential drawbacks are. Although in general, viral gene integration demonstrates a considerable therapeutic effect, it has oncogenic potential. Development of self-inactivating vectors was a breakthrough in regard to safety, but the possibility of oncogenesis remains, and strict analysis of integration sites is required.},
}
@article {pmid41374373,
year = {2025},
author = {Sambo, CN and Skepu, A and Nxumalo, NP and Polori, KL},
title = {Diagnostic Advances and Public Health Challenges for Monkeypox Virus: Clade-Specific Insight and the Urgent Need for Rapid Testing in Africa.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {15},
number = {23},
pages = {},
pmid = {41374373},
issn = {2075-4418},
support = {B1B0741B-9691-4333-B77E-3B8D42A7B5FF//Technology Innovation Agency/ ; },
abstract = {Background: Monkeypox (MPX), caused by the Monkeypox virus (MPOX) of the Orthopoxvirus genus, has re-emerged as a significant global health threat. Once confined to Central and West Africa, the 2022-2025 multi-country outbreaks, predominantly caused by Clade IIb, demonstrated sustained human-to-human transmission and global spread. Objective: This review summarizes current knowledge on MPX virology, epidemiology, clinical presentation, and diagnostic technologies, with a focus on innovations supporting rapid and field-deployable detection in resource-limited settings. Methods: The recent literature (2019-2025), including peer-reviewed studies, WHO and Africa CDC reports, and clinical guidelines, was critically reviewed. Data were synthesized to outline key developments in diagnostic methodologies and surveillance approaches. Results: MPX comprises two genetic clades: Clade I (Congo Basin) and Clade II (West African), which differ in virulence and transmission. Clade IIb is associated with sexual and close-contact transmission during recent outbreaks. Clinical manifestations have shifted from classic disseminated rash to localized anogenital lesions and atypical or subclinical infections. RT-PCR remains the diagnostic gold standard, while emerging assays such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR/Cas-based platforms show promise for rapid point-of-care (POC) testing. Complementary serological tools, including ELISA and lateral flow assays, enhance surveillance and immune profiling. Conclusions: The resurgence of MPX highlights the urgent need for accessible, sensitive, and specific diagnostic platforms to strengthen surveillance and outbreak control, especially in endemic and resource-constrained regions.},
}
@article {pmid41375204,
year = {2025},
author = {Li, W and Shi, Y and Li, D and Wang, Y and Sun, Y and Li, H and Han, Y},
title = {A CRISPR Powered Immobilization-Free, Amplification-Free Carbon-Nanotube Field-Effect Transistor (FET) Sensor for Influenza A Virus (IAV).},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {23},
pages = {},
pmid = {41375204},
issn = {1420-3049},
support = {2023YFC2605101//National Key Research and Development Program of China/ ; },
mesh = {*Influenza A virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Transistors, Electronic ; *Nanotubes, Carbon/chemistry ; Humans ; *RNA, Viral/genetics/analysis ; CRISPR-Cas Systems ; Limit of Detection ; *Influenza, Human/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The epidemic of infectious diseases, such as influenza A, has imposed a severe health burden on the population. Early detection, diagnosis, reporting, isolation, and treatment are crucial for the prevention, control, and management of infectious diseases. Nucleic acid testing represents a vital approach for the rapid diagnosis of pathogenic microorganism types. However, current nucleic acid detection methods face notable bottlenecks: traditional CRISPR fluorescence assays require time-consuming pre-amplification of target nucleic acids, while existing carbon-nanotube field-effect transistor (FET)-based platforms, though amplification-free, often necessitate complex chip surface modification and probe immobilization, and suffer from non-reusable chips, all limiting their utility in point-of-care testing (POCT) and large-scale screening. This study reports a CRISPR-based amplification-free RNA detection platform (CRISPR-FET) for the rapid identification of influenza A virus. The CRISPR-FET platform described herein enables the detection of viral RNA without amplification within 20 min, with a limit of detection as low as 1 copy/μL. Secondly, a reporter RNA conjugated with gold particles is used to achieve signal amplification in FET detection; meanwhile, the method eliminates probe immobilization, thereby omitting this step and simplifying chip modification to reduce complex work-flows and pre-treatment costs. The chip's reusability further enhances cost-effectiveness. Additionally, streptavidin-modified magnetic bead adsorption minimizes background errors from excessive reporter RNA and non-target nucleic acids. Finally, validation with 24 clinical samples confirmed the platform's efficacy. By integrating rapidity, simplicity, and high sensitivity, alongside cost advantages from reusable chips, this CRISPR-FET platform meets the critical need for early influenza A diagnosis and holds promise for advancing POCT and large-scale epidemiological screening.},
}
@article {pmid41375334,
year = {2025},
author = {Lai, CM and Xiao, XS and Liu, LW and Lin, XD and Dou, DL and Cai, HY and Mei, ZF and Yang, F and Cheng, Y and Qin, Y},
title = {Nanotechnology Strategies in Plant Genetic Engineering: Intelligent Delivery and Precision Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {23},
pages = {},
pmid = {41375334},
issn = {2223-7747},
support = {2024NZ029029//Major Science and Technology Project of Fujian Province/ ; },
abstract = {Plant genetic engineering is crucial for enhancing crop yield, quality, and resilience to both abiotic and biotic stresses, thereby promoting sustainable agriculture. Agrobacterium-mediated, biolistic bombardment, electroporation, and poly (ethylene glycol) (PEG)-mediated genetic transformation systems are widely applied in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, gene-delivery methods based on nanotechnology have been developed for plant genetic transformation. This nanostrategy demonstrates remarkable transformation efficiency, excellent biocompatibility, effective protection of exogenous nucleic acids, and the potential for plant regeneration. However, the application of nanomaterial-mediated gene-delivery systems in plants is still in its early stages and faces numerous challenges for widespread adoption. Herein, the conventional genetic transformation techniques utilized in plants are succinctly examined. Subsequently, the advancements in nanomaterial-based gene-delivery systems are reviewed. The applications of CRISPR-Cas-mediated genome editing and its integration with plant nanotechnology are also examined. The innovations, methods, and practical applications of nanomaterial-mediated genetic transformation summarized herein are expected to facilitate the progress of plant genetic engineering in modern agriculture.},
}
@article {pmid41376155,
year = {2026},
author = {Jin, X and Wu, X and Song, J and Luo, M and Ye, Q and Ren, C and Song, L and Li, M and Hu, M and An, Y and Su, J and Fu, J and Xu, Q and Luo, M and Liu, F and Liu, M and Li, Q and Yao, S and Chen, L and Yang, Y},
title = {Comparative evaluation of liver-directed knockin strategies with viral and nonviral vectors in mouse inherited disease models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1775-1793},
pmid = {41376155},
issn = {1525-0024},
mesh = {Animals ; *Genetic Vectors/genetics/administration &amp; dosage ; Mice ; Disease Models, Animal ; Dependovirus/genetics ; *Liver/metabolism ; *Gene Knock-In Techniques/methods ; Genetic Therapy/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Recombinational DNA Repair ; *Hemophilia B/genetics/therapy ; Transgenes ; },
abstract = {CRISPR-Cas9-mediated gene knockin has emerged as a promising strategy for early-onset genetic disease intervention. However, the therapeutic efficacy and editing outcomes of different knockin strategies remain incompletely understood. Here, we systematically evaluated three major liver-directed knockin strategies, namely homology-directed repair (HDR), homology-independent targeted integration (HITI), and homology-mediated end joining (HMEJ), using neonatal mouse models of mucopolysaccharidosis type I and hemophilia B. Although all three approaches effectively rescued disease phenotypes, we observed distinct editing outcomes. Notably, the HMEJ approach, delivered via a combined adeno-associated virus-lipid nanoparticle (AAV-LNP) system, exhibited superior integration efficiency (5.8%-5.9%) and fidelity (97%-98%) compared with HDR and HITI. In contrast, whole-genome sequencing indicated that HITI induced a higher risk of random AAV donor integration than HDR or HMEJ. Furthermore, long-read sequencing analyses revealed that the frequencies of inverted terminal repeat (ITR)-mediated transgene integration differed between the 5' and 3' genomic junctions among the three strategies. Specifically, in HDR- and HMEJ-treated mice, ITR-mediated integration events were 7.7- to 19.7-fold more common at the 3' junctions than at the 5' junctions. These findings highlight the comprehensive advantages of the AAV-LNP-mediated HMEJ approach for liver-directed knockin therapy and suggest its strong potential for clinical translation.},
}
@article {pmid41376159,
year = {2026},
author = {Ha, AS and Kalter, N and Rosenberg, M and Acevedo, LA and Liang, B and Liu, W and Paruthiyil, S and Sinha, M and Vu, A and Nguyen, V and Qi, Z and Krishnappa, N and Shu, J and Yu, J and Catanzaro, J and Bluestone, JA and Tang, Q and Urnov, FD and Marson, A and Hendel, A and Herold, KC and Shy, BR and Esensten, JH},
title = {Gene-corrected regulatory T cell therapy for IL2RA deficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1367-1381},
pmid = {41376159},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/metabolism/transplantation ; *Interleukin-2 Receptor alpha Subunit/genetics/deficiency ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Gene Editing ; Male ; Female ; Interleukin-2 ; Mutation ; },
abstract = {Bi-allelic germline deficiency of IL2RA causes a rare autoimmune disease with impaired regulatory T cell (Treg) function and interleukin-2 (IL-2) signaling. Definitive treatment is currently limited to allogeneic hematopoietic stem cell transplantation, which carries significant morbidity and mortality risks. We previously identified a family with three siblings affected by compound heterozygous mutations in their IL2RA gene, resulting in dysfunctional Tregs. Here, we introduce a novel therapeutic approach involving ex vivo generation of gene-corrected autologous regulatory T cells (gcTregs). One of the two disease-causing mutations in patient-derived Tregs was corrected with CRISPR-Cas9-mediated homology-directed repair, restoring IL2RA expression. The resulting gcTregs demonstrated robust suppressive activity in vitro. Clinical-scale manufacturing from a patient with IL2RA deficiency showed efficient gene correction, restored IL2RA expression, and functional equivalence to healthy donor Tregs. This work establishes a Good Manufacturing Practice-compatible manufacturing process for personalized gcTreg therapies, potentially providing a safer treatment option for patients with IL2RA deficiency as well as a framework for treating other inborn errors of immunity.},
}
@article {pmid41377346,
year = {2025},
author = {Abedin, ZU and Waggan, AI and Khan, E and Suleman, MU and Tabassum, SN},
title = {Letter to the Editor: CRISPR-based gene editing for cardiac protection in Barth syndrome.},
journal = {Annals of medicine and surgery (2012)},
volume = {87},
number = {12},
pages = {9163-9164},
pmid = {41377346},
issn = {2049-0801},
abstract = {Barth syndrome is a rare X-linked mitochondrial disorder caused by mutations in the Tafazzin (TAZ) gene. These mutations make it hard for cardiolipin to remodel and mitochondria to work properly. This condition is characterized by growth retardation, neutropenia, skeletal myopathy, and dilated cardiomyopathy, frequently leading to significant morbidity and mortality, with numerous patients necessitating heart transplants. There are no treatments available at this time to fix the genetic problem. Recent progress in gene editing, especially CRISPR-based methods, holds great promise for fixing TAZ mutations. Research utilizing patient-derived cardiomyocytes has demonstrated that the rectification of TAZ mutations reinstates mitochondrial efficiency and enhances cellular functionality. Animal models, including TAZ-knockout mice, have exhibited substantial enhancements in cardiac function, survival rates, and diminished fibrosis subsequent to gene replacement therapy.},
}
@article {pmid41378919,
year = {2026},
author = {Hirata, S and Ozono, T and Kawai, K and Machida, C and Kobayashi, K and Ikeda, Y and Nishimura, T and Kaya, H},
title = {Development of a simple and locus-restricted DNA methylation editing system using direct fusion of a nickase-type SpCas9 and DNA methylation-related enzymes in Arabidopsis thaliana.},
journal = {Plant &amp; cell physiology},
volume = {67},
number = {5},
pages = {739-751},
doi = {10.1093/pcp/pcaf162},
pmid = {41378919},
issn = {1471-9053},
support = {25NIBB333//NIBB Collaborative Research Program/ ; 24NIBB322//NIBB Collaborative Research Program/ ; 23NIBB307//NIBB Collaborative Research Program/ ; 22NIBB306//NIBB Collaborative Research Program/ ; 21-209//NIBB Collaborative Research Program/ ; 20-328//NIBB Collaborative Research Program/ ; 19-331//NIBB Collaborative Research Program/ ; //Okayama University Institute of Plant Science and Resource (IPSR)/ ; 25K01988//Japan Society for the Promotion of Science/ ; 21K06233//Japan Society for the Promotion of Science/ ; //The United Graduate School of Agricultural Sciences/ ; JPMJSP2162//Japan Science and Technology Agency/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *DNA Methylation/genetics ; Arabidopsis Proteins/genetics/metabolism ; *Gene Editing/methods ; Gene Expression Regulation, Plant ; *Deoxyribonuclease I/metabolism/genetics ; Promoter Regions, Genetic/genetics ; Epigenome Editing ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; Transcription Factors/genetics/metabolism ; Plants, Genetically Modified ; Homeodomain Proteins ; },
abstract = {DNA methylation is an important epigenetic modification that regulates gene expression and supports genome stability. DNA methylation editing technology differs from conventional genome editing technology, which introduces mutations into genes, in that it enables changing gene expression without altering the base sequence. In this study, we attempted simple and locus-restricted DNA methylation editing in Arabidopsis thaliana using fusion proteins directly linking a nickase-type SpCas9 protein with DNA methylation-related enzymes. First, fusion of the human Ten-eleven translocation methyl cytosine dioxygenase 1 (TET1) catalytic domain (TET1cd) to nSpCas9 led to removing 5-methylcytosine in the FLOWERING LOCUS WA (FWA) promoter region of the wild-type plant, resulting in increased expression of the FWA gene and consequently, a late-flowering phenotype. Conversely, fusion of a mutant form of the bacterial DNA methyltransferase MQ1 (MQ1v) to nSpCas9 induced de novo DNA methylation in the fwa101-D mutant, in which the FWA promoter region is hypomethylated, and suppressed FWA gene expression, resulting in an early-flowering phenotype compared with the fwa101-D mutant. Of particular importance, our nSpCas9 system achieves targeted DNA methylation editing within a genomic window of ~10-20 kb. The nSpCas9 system features a compact and simplified vector structure due to the DNA methylation-related enzyme directly fusing to nSpCas9. Furthermore, sgRNA can be easily replaced, making it highly flexible. We propose a new method for targeted epigenome editing technology in plants, paving the way for innovative strategies in both basic research on epigenetics and crop development through epigenome editing.},
}
@article {pmid41380236,
year = {2026},
author = {Yin, Z and Yin, H and Zhou, Y and Liu, H},
title = {CRISPR/Cas13a-mediated photoelectrochemical-colorimetric dual-mode biosensor for RNA N-acetyltransferase 10 detection based on Bi2O2S/Ag2S and laccase-like nanozyme.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118298},
doi = {10.1016/j.bios.2025.118298},
pmid = {41380236},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Laccase/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; *Acetyltransferases/isolation &amp; purification/analysis/chemistry ; Humans ; CRISPR-Cas Systems ; Limit of Detection ; Bismuth/chemistry ; },
abstract = {N-Acetyltransferase 10 (NAT10) is a crucial protein that catalyzes RNA acetylation modification and plays a significant role in biological activities. Accurate detection of NAT10 is of great importance in clinical testing and drug development. To achieve this goal, a novel biosensing platform was developed for NAT10 detection, relying on an RNA acetylation-inhibited CRISPR/Cas13a system, a Bi2O2S/Ag2S type-II heterojunction, and a laccase-mimetic nanocatalyst, using a photoelectric-colorimetric dual-mode strategy. Based on the catalysis effect of laccase-mimetic nanocatalyst, its substrates (hydroquinone and 2,4-dichlorophenol) were oxidized to form p-benzoquinone and 2,4-dichloroquinone, respectively. This oxidation process not only impaired the electron-donating ability of hydroquinone but also induced the coupling of 2,4-dichloroquinone with 4-antipyrine to generate a red product-enabling both photoelectrochemical and colorimetric detection of the NAT10 protein. The biosensor exhibited wide linear ranges of 0.1-1000 μg/L (photoelectrochemical mode) and 1-1000 μg/L (colorimetric mode), and the low detection limit of 0.056 and 0.348 μg/L for the two modes, respectively. Additionally, this biosensor was used to investigate the inhibitory effects of plasticizers and phosphorus-containing flame retardants on NAT10. Furthermore, molecular docking simulations were employed to explore the underlying inhibition mechanism. The developed biosensor not only provided a novel detection technique for NAT10, but also offers an alternative method for NAT10 inhibitor screening and a new tool for evaluating the ecotoxicological effects of organic pollutants.},
}
@article {pmid41380779,
year = {2026},
author = {Hill, AC and Schank, MB and Zhang, Y and Sun, N and Wang, L and Zhao, J and Banik, P and Pyburn, JS and Orfield, H and Lightner, JW and Leshaodo, TO and Wu, XY and Ning, S and El Gazzar, M and Moorman, JP and Guo, H and Yao, ZQ},
title = {Suppression of HBV replication and expression by CRISPR/Cas9 ribonucleoproteins.},
journal = {Antiviral research},
volume = {245},
number = {},
pages = {106326},
pmid = {41380779},
issn = {1872-9096},
support = {R01 AI177624/AI/NIAID NIH HHS/United States ; R21 AI157909/AI/NIAID NIH HHS/United States ; },
mesh = {*Hepatitis B virus/genetics/physiology/drug effects ; *Virus Replication/drug effects ; Humans ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/pharmacology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; DNA, Viral/genetics ; Antiviral Agents/pharmacology ; Hep G2 Cells ; Genetic Therapy/methods ; Hepatitis B/therapy/virology ; Cell Line ; CRISPR-Associated Protein 9 ; },
abstract = {HBV infection is a global public health problem. The current treatment using nucleotide analogues (NA) can suppress viral replication but cannot eliminate HBV infection due to the persistence of covalently closed circular DNA (cccDNA), which sustains HBV replication and integration into the host cell genome and is refractory to NA treatment. CRISPR/Cas9 has been used to disrupt integrated HBV DNA and minichromosomal cccDNA for HBV suppression, but its expression and delivery require viral or non-viral vectors, which pose safety concerns for human application. We have previously reported the use of synthetic guide RNA (gRNA)/Cas9 ribonucleoprotein (RNP) as a non-viral formulation for HBV gene editing and viral suppression. To formulate highly effective CRISPR/Cas9 modalities for HBV gene therapy, here we designed additional gRNA/Cas9 RNPs and compared their antiviral efficacy in HBV-transfected as well as -infected cells. We found that two selected gRNA/Cas9 RNPs (gRNA5/Cas9, gRNA9/Cas9, and particularly their combinations) elicited the most potent antiviral efficacy, as evidenced by the significant inhibition of HBV DNA, RNA, and protein productions. DNA sequencing of the treated cells revealed moderate to high rates of insertion and deletion (indel) or knock-out (KO) mutations at the HBV target genes. Gene alignment analysis showed a high level of conservation for both gRNA5 and gRNA9 target sequences across major HBV genotypes, indicating that these CRISPR-based gene editing therapeutics have the potential to target different HBV strains worldwide. Thus, these synthetic gRNA/Cas9 RNPs represent promising novel therapeutics that can be developed and utilized for HBV gene disruption and viral eradication.},
}
@article {pmid41380995,
year = {2026},
author = {Roy, S and Nandy, S and Morita, D and Nandy, RK and Veeraraghavan, B and Walia, K and Das, S and Basu, S},
title = {Genomic analysis of a novel high-risk ST5217/ExoU+/O11 clone of carbapenem-resistant OXA-181- and VIM-2-producing Pseudomonas aeruginosa in India.},
journal = {Journal of global antimicrobial resistance},
volume = {46},
number = {},
pages = {158-161},
doi = {10.1016/j.jgar.2025.12.002},
pmid = {41380995},
issn = {2213-7173},
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/isolation &amp; purification/enzymology/classification ; *beta-Lactamases/genetics/metabolism ; India ; Humans ; *Carbapenems/pharmacology ; Pseudomonas Infections/microbiology ; Phylogeny ; *Bacterial Proteins/genetics ; Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Drug Resistance, Multiple, Bacterial/genetics ; Whole Genome Sequencing ; Genomics ; },
abstract = {OBJECTIVES: Studies of carbapenem-resistant Pseudomonas aeruginosa (CRPA)-harbouring OXA-48-like carbapenemases are rare. The study aimed to report the emergence and characterization of a novel high-risk clone of CRPA-harbouring OXA-48-like from India.<br><br>METHODS: Identification, AST, phenotypic detection of carbapenemases and WGS using Ion-Torrent-S5 platform were carried out. Analyses included ResFinder, VFDB, MLST, PAst, Phastest and CRISPR/Cas. SNP-based phylogenetic analysis with global OXA-48-like-harbouring CRPA genomes was carried out by CSI Phylogeny and iTOL for visualization.<br><br>RESULTS: The clinical MDR strain of CRPA AMRIR00655 belonged to a novel sequence type ST5217 and serotype O11. Phenotypic tests followed by WGS revealed the presence of dual carbapenemases, OXA-181 (serine-carbapenemase) and VIM-2 (zinc-carbapenemase), both located on chromosome. blaOXA-181 resides between chromosomal genes encoding RodZ and PAP2 in P. aeruginosa, confirming chromosomal integration.4,261 bp of blaOXA-181-bearing contig-DNA showed 100% homology to K. pneumoniae plasmid pKP3-A. ISEcp1 was present on upstream and on downstream, &#x25b3;lysR, &#x25b3;ereA and repA genes were detected. blaVIM-2 was located within class 1 integron along with aacC6-II, dfrB5, aac(3)-Id, tniC in surrounding regions and 13,242 bp showing 100% identity to P. aeruginosa chromosome. Presence of other ARGs (blaPAO, blaOXA-488,aph(3'')-Ib, aph(6)-Id, crpP, catB7, fosA, sul2) and efflux-pump genes might explain its MDR phenotype. Virulence factors including T3SS (PscF, PopB, PopD, PcrV) and its effectors (ExoT, ExoU, ExoY) indicated the pathogenic potential of ST5217. Core genome analysis showed that ST5217 was closest with other high-risk clones ST1339 and ST773-harbouring blaOXA-48-like.<br><br>CONCLUSIONS: To the best of our knowledge, this is the first report of blaOXA-181-harbouring novel high-risk clone of CRPA ST5217/ExoU+/O11 in India which emphasises the spread of OXA-181 among bacteria other than Enterobacteriaceae-family and warrant close monitoring.},
}
@article {pmid41381092,
year = {2025},
author = {Lane, KR and Jones, SE and Osborne, TH and Geller-McGrath, D and Nwaobi, BC and Chen, L and Thomas, BC and Hudson-Edwards, KA and Banfield, JF and Santini, JM},
title = {Bioleaching Microbial Community Metabolism and Composition Driven by Copper Sulphide Mineral Type.},
journal = {Environmental microbiology reports},
volume = {17},
number = {6},
pages = {e70261},
pmid = {41381092},
issn = {1758-2229},
support = {NE/L002485/1//Natural Environment Research Council/ ; BB/N012674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Hellenic Coppers Mines Ltd/ ; },
mesh = {*Copper/metabolism/chemistry ; *Bacteria/metabolism/genetics/classification/isolation &amp; purification ; *Archaea/metabolism/genetics/classification/isolation &amp; purification ; *Sulfides/metabolism/chemistry ; *Microbial Consortia ; Metagenomics ; *Minerals/metabolism/chemistry ; *Microbiota ; Plasmids/genetics ; },
abstract = {Copper bioleaching is a green technology for the recovery of copper from chalcopyrite (CuFeS2) and chalcocite (Cu2S) ores. Much remains to be learned about how mineral type and surface chemistry influence microbial community composition. Here, we established a microbial consortium from a copper bioleaching column in Cyprus on chalcopyrite and then sub-cultured it to chalcocite to investigate how the community composition shifts due to changes in mineral structure and the absence of mineral-derived Fe. The solution chemistry was determined and microbial communities characterised by genome-resolved metagenomics after 4 and 8 weeks of cultivation. Acidithiobacillus species and strains, a Rhodospirilales, Leptospirillum ferrodiazotrophum and Thermoplasmatales archaea dominated all enrichments, and trends in abundance patterns were observed with mineralogy and surface-attached versus planktonic conditions. Many bacteria had associated plasmids, some of which encoded metal resistance pathways, sulphur metabolic capacities and CRISPR-Cas loci. CRISPR spacers on an Acidithiobacillus plasmid targeted plasmid-borne conjugal transfer genes found in the same genus, likely belonging to another plasmid, evidence of intra-plasmid competition. We conclude that the structure and composition of metal sulphide minerals select for distinct consortia and associated mobile elements, some of which have the potential to impact microbial activity during sulphide ore dissolution.},
}
@article {pmid41381248,
year = {2026},
author = {Adamopoulos, PG and Athanasopoulou, K and Scorilas, A},
title = {A versatile type VI CRISPR-based approach for targeted m[6]A demethylation in mRNAs.},
journal = {Genome research},
volume = {36},
number = {1},
pages = {169-182},
pmid = {41381248},
issn = {1549-5469},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Adenosine/analogs &amp; derivatives/metabolism ; HeLa Cells ; AlkB Homolog 5, RNA Demethylase/genetics/metabolism ; Demethylation ; *CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {Epitranscriptomics, a rapidly evolving field mainly driven by massive parallel sequencing technologies, explores post-transcriptional RNA modifications. N [6]-methyladenosine (m[6]A) has emerged as the most prominent and dynamically regulated modification in human mRNAs, being implicated in the regulation of diverse biological processes, including spermatogenesis, heat shock response, ultraviolet-induced DNA damage response and maternal mRNA clearance. Despite the recognized significance of m[6]A in mRNA regulation, limited studies have focused on the targeted and efficient manipulation of this modification in mRNAs. Here, we present Dem6A-Vec, an "all-in-one" plasmid vector designed for site-specific m[6]A demethylation in human mRNAs. Dem6A-Vec integrates the expression of a catalytically inactive RfxCas13d fused to the m[6]A demethylase ALKBH5 and a U6-driven customizable guide RNA in a single construct, simplifying experimental workflows and enhancing targeting efficiency. Using nanopore direct RNA sequencing, we identify high-confident m[6]A sites in HeLa cells, which serve as targets for Dem6A-Vec. We validate the targeted demethylation of m[6]A sites in the EEF2 and RRAGA genes using the established SELECT-qPCR method, confirming the impacts on mRNA stability and highlighting the tool's precision and versatility. The presented approach is implemented in multiple mRNA sites with diverse methylation stoichiometries, underscoring its adaptability to various transcriptomic contexts. This study provides a robust and scalable method for investigating the functional roles of m[6]A modifications, offering a transformative platform for advancing epitranscriptomic research and potential therapeutic applications.},
}
@article {pmid41381416,
year = {2025},
author = {Cheng, AS and Li, LX and Zhou, JX and Harris, PC and Calvet, JP and Li, X},
title = {In vivo base editing rescues ADPKD in a humanized mouse model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11212},
pmid = {41381416},
issn = {2041-1723},
support = {DK126662//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; R01 DK126662/DK/NIDDK NIH HHS/United States ; K01 DK107729/DK/NIDDK NIH HHS/United States ; DK058816//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK129241//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK059597//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; R01 DK059597/DK/NIDDK NIH HHS/United States ; PR221810//U.S. Department of Defense (United States Department of Defense)/ ; R01 DK129241/DK/NIDDK NIH HHS/United States ; R01 DK058816/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; Mice ; Dependovirus/genetics ; TRPP Cation Channels/genetics ; Kidney/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; *Genetic Therapy/methods ; Liver/pathology/metabolism ; Male ; Promoter Regions, Genetic ; Female ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is a genetic kidney disease, caused by mutations of the PKD1 and PKD2 genes, characterized by the development of renal cysts and extrarenal complications, such as cardiac hypertrophy. Recently, a revolutionary approach, adeno-associated virus (AAV) delivered CRISPR-Cas9 gene editing, has been developed to treat inherited diseases. However, the use of this technology in kidney diseases in vivo is challenged. In this study, we adapt one of the gene editing systems, adenine base editor (ABE9), to develop a broadly expressed and a kidney-specific promoter mediated base editors, and test the effects of these two systems delivered by AAV9 on preventing disease in humanized Pkd1[RC/RC] mice carrying an arginine (R) to cystine (C) mutation that mimics a mutation in ADPKD patients. We show that one dose of the broadly expressed dual ABE9-AAV9 treatment corrects the pathogenic variant in kidneys, hearts and livers, and result in delaying cyst growth, decrease heart hypertrophy and improve liver function. To confirm the specificity of the base editor system in kidneys, we show that one dose of the kidney specific promoter mediated dual-ABE9-AAV9 treatment corrects the Pkd1 gene mutation in the kidney, and not in the heart, resulting in delaying cyst growth in Pkd1[RC/RC] kidneys, supporting a promising strategy of using base editor to target specific organs. Treatment with ABE9 base editors mediated by either the broadly expressed or kidney specific promoter increased the survival rate of Pkd1[RC/null] mice. These preclinical studies support a potential that single-dose genetic therapies may be through the correction of pathogenic variants to prevent ADPKD development in the clinic.},
}
@article {pmid41381501,
year = {2025},
author = {Padilla, R and Shipman, GA and Horth, C and Gravel, M and Bareke, E and Majewski, J},
title = {H3K36 Methylation as a Guardian of Epigenome Integrity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11371},
pmid = {41381501},
issn = {2041-1723},
support = {P01 CA196539/CA/NCI NIH HHS/United States ; PJT-183939//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; P01-CA196539//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Animals ; *Histones/metabolism/genetics ; Humans ; Mice ; Methylation ; *Epigenome/genetics ; Heterochromatin/metabolism/genetics ; Mesenchymal Stem Cells/metabolism ; Methyltransferases/metabolism/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; *Epigenesis, Genetic ; Repressor Proteins/metabolism/genetics ; Mice, Knockout ; Chromatin/metabolism ; Euchromatin/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {H3K36 methylation is a key epigenetic mark with critical roles in development and disease. Here, we systematically dissect its functions using CRISPR-engineered mouse mesenchymal stem cells lacking combinations of the five H3K36 methyltransferases, culminating in quintuple knockout cells devoid of H3K36me2/3. We show that H3K36me2 influences enhancer activity, supports the expression of their target genes, and safeguards active genes from encroachment of the repressive marks, H3K27me2/3. In addition, we find that the loss of H3K36me triggers redistribution of large heterochromatic H3K9me3 domains into euchromatin, in part mediated by SUV39H1, leading to global epigenomic remodelling, constitutive heterochromatin erosion, and a collapse of 3D genome organization. Parallel analyses in human HNSCC cells overexpressing the H3K36M oncohistone reveal conserved disruptions to the epigenome and chromatin architecture. Together, these results establish H3K36 methylation as a pivotal regulator of chromatin state and genomic structure.},
}
@article {pmid41381532,
year = {2025},
author = {Zaada, DSY and Toren, O and Krsticevic, F and Haber, DA and Gildman, D and Galpaz, N and Häcker, I and Schetelig, MF and Marois, E and Arien, Y and Papathanos, PA},
title = {Mosquito sex separation using complementation of selectable traits and engineered neo-sex chromosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11175},
pmid = {41381532},
issn = {2041-1723},
support = {3-1679//Ministry of Science, Technology and Space/ ; 1833/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; ANR-23-CE35-0003//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Female ; Male ; *Aedes/genetics/physiology ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; Genetic Engineering ; *Sex Determination Processes/genetics ; Animals, Genetically Modified ; Pigmentation/genetics ; Phenotype ; Insect Proteins/genetics ; },
abstract = {Effective sex separation remains a critical challenge for mosquito genetic control. Genetic sexing strains (GSS) address this by linking maleness with selectable traits, enabling efficient female removal. Here, we present a versatile platform for GSS development in the invasive Aedes albopictus mosquito that integrates CRISPR-engineered selectable phenotypes with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow pigmentation gene and restore its function in males using nix-containing transgenes, producing a stable strain with yellow females and dark males. Beyond serving as a vivid marker, yellow confers added advantages: GSS females pupate later than wild females, enhancing protandry-based sorting, and lay desiccation-sensitive eggs, lowering accidental female release risk. The strain is compatible with size-based separation, improving sexing accuracy through the integration of natural and engineered dimorphisms. To our knowledge, this represents the first engineered sex-linked selectable trait in mosquitoes based on endogenous genes, establishing a foundation for scalable GSS development.},
}
@article {pmid41381927,
year = {2025},
author = {Yousuf, F and Solanki, M and Singh, SS and Ch, SR and Neeraja, CN and Sundaram, RM and Mangrauthia, SK},
title = {Tissue culture optimization and genome editing for yield improvement of an Indian rice landrace Chittimuthyalu.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {54},
pmid = {41381927},
issn = {1573-9368},
support = {ICAR-EFC Sub-Scheme 10: Enhancing climate resilience and ensuring food security with genome editing tools//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics/growth &amp; development/drug effects ; *Gene Editing/methods ; *Tissue Culture Techniques/methods ; Plant Breeding ; India ; *Plants, Genetically Modified/genetics/growth &amp; development ; Purines/pharmacology ; CRISPR-Cas Systems ; Phenylurea Compounds ; Thiadiazoles ; },
abstract = {Chittimuthyalu, a rice landrace from Southern India, is known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has a poor plant type (tall and weak stem prone to lodging) and is low yielding. The efforts to improve such valuable rice accessions with existing cross-breeding or random mutagenesis often result in undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance-associated genes OsDEP1, OsTB1, OsCKX2, and OsSWEET14. The OsDEP1genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand-grain weight, and ~ 50% increase in total grain yield per plant. The optimized tissue culture protocol and development of further edits in the remaining genes will pave the way for improving the agronomic traits of Chittimuthyalu. This study also highlights much-needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces, which will help bring these hardy, climate-resilient, and nutrient-rich accessions into mainstream cultivation.},
}
@article {pmid41382257,
year = {2025},
author = {Chen, W and Wu, P and Champer, J},
title = {Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs.},
journal = {BMC biology},
volume = {24},
number = {1},
pages = {12},
pmid = {41382257},
issn = {1741-7007},
support = {32270672//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Drive Technology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles through end-joining repair. Using multiple gRNAs to target adjacent sites within a conserved gene can prevent functional resistance by allowing repeated cleavage events, but poor homology during DNA repair may compromise efficiency.<br><br>RESULTS: We first assessed the efficiency of single gRNA drives with truncated homology arms in Drosophila melanogaster mimicking a multiplexed system in which only one site is cleaved. Integrating results into a detailed gRNA multiplexing model, we found that efficiency loss was greater than expected. To mitigate this, we evaluated two new strategies: (1) extended homology arms to span all target sites (with mutations in the PAMs to prevent self-cleavage) and (2) a population-level gRNA multiplexing system involving two or more drives, each carrying two gRNAs. Extended homology arms did not result in notable improvement in conversion efficiency, and the extended region could be lost during drive conversion. The population-level multiplexing gRNAs strategy was more promising, though the intentionally mutated PAM also could not be consistently inherited. Simulations of homing suppression drives applying population-level multiplexed gRNAs increased the success rate of population elimination and reduced the time required for suppression.<br><br>CONCLUSIONS: Future drive designs requiring a larger number of gRNAs could potentially be improved. The design relying on extended homology arms may not represent an optimal strategy. However, population-level multiplexing gRNAs could serve as a promising alternative, enhancing efficiency while maintaining tolerance to functional resistance.},
}
@article {pmid41384994,
year = {2025},
author = {Zhang, J and Liu, J and Bayani, A},
title = {Phage therapy and the microbiome in hematologic malignancies: opportunities, mechanisms, and early evidence.},
journal = {Journal of cancer research and clinical oncology},
volume = {152},
number = {1},
pages = {8},
pmid = {41384994},
issn = {1432-1335},
mesh = {Humans ; *Hematologic Neoplasms/therapy/microbiology/immunology ; *Phage Therapy/methods ; Animals ; *Bacteriophages ; *Gastrointestinal Microbiome ; *Microbiota ; },
abstract = {Hematologic malignancies remain among the most difficult cancers to treat, challenged by profound heterogeneity, treatment-induced immune dysfunction, and the frequent emergence of drug resistance. Beyond tumor-intrinsic mechanisms, dysbiosis of the gut microbiome is increasingly recognized as a critical determinant of therapeutic outcomes, shaping hematopoiesis, immune responses, and drug metabolism. Bacteriophage (phage) therapy has re-emerged as a precision tool capable of selectively eradicating pathogenic taxa while preserving commensal short-chain fatty acid-producing communities. Preclinical and early human studies demonstrate that phages can recalibrate microbial ecosystems, disrupt antibiotic-tolerant biofilms, and enrich metabolites such as butyrate that support mucosal integrity and immune balance. Mechanistically, phage DNA enriched with CpG motifs engages Toll-like receptor 9, activating dendritic cells and enhancing cytotoxic T lymphocyte responses, suggesting dual benefits in infection control and anti-tumor immunity. Emerging applications extend further, with engineered phages serving as vectors for CRISPR-Cas gene editing, targeted cytokine delivery, and nanocarrier platforms for leukemia therapy. Despite translational promise, major hurdles persist, including immunogenicity, horizontal gene transfer, resistance evolution, and regulatory uncertainty. Addressing these challenges through GMP-compliant manufacturing, metagenomics-guided personalization, and AI-optimized cocktail design could establish phage therapy as a microbiome-informed adjunct to overcome drug resistance in blood cancers. However, direct clinical evidence of phage therapy efficacy in hematologic malignancies remains limited, and current data are largely derived from preclinical and compassionate-use contexts.},
}
@article {pmid41385323,
year = {2025},
author = {Zahm, AM and Cranney, CW and Gormick, AN and Rondem, KE and Schmitz, B and Himes, SR and English, JG},
title = {ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41385323},
issn = {1362-4962},
support = {DP2 GM146247/GM/NIGMS NIH HHS/United States ; //NIH/ ; 1DP2GM146247-01/GM/NIGMS NIH HHS/United States ; },
mesh = {*Nanopore Sequencing/methods ; Humans ; SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus/genetics ; COVID-19/virology ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; *Sequence Analysis, DNA/methods ; Dependovirus/genetics ; *Nucleic Acids/genetics/isolation &amp; purification ; Genome, Viral ; },
abstract = {Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform's performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of severe acute respiratory syndrome coronavirus 2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research.},
}
@article {pmid41385544,
year = {2025},
author = {Fu, YZ and Luo, FF and Yang, L and Zhang, YX and Li, JY and Wang, SY and Zhang, Y and Wang, YY},
title = {SPNS1 is an essential cellular factor for EV-A71 by acting as a transporter of viral pocket factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2510020122},
pmid = {41385544},
issn = {1091-6490},
support = {2023YFC2306100//The National Key R&amp;D Program China/ ; 82372230//The National Natural Science Foundation of China/ ; U23A20168//The National Natural Science Foundation of China/ ; No.2022338//The Youth Innovation Promotion Association CAS/ ; },
mesh = {Humans ; Animals ; *Enterovirus A, Human/physiology/metabolism/genetics ; Virus Replication ; *Enterovirus Infections/virology/metabolism/genetics ; Lysosomes/metabolism ; Mice ; Capsid Proteins/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Endosomes/metabolism ; Receptors, Scavenger ; Lysosomal Membrane Proteins ; },
abstract = {Human enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease. Cellular factors critical for EV-A71 infection remain enigmatic. Here, we performed CRISPR/Cas9 screens and identified sphingolipid transporter 1 (SPNS1) as an essential factor for EV-A71. SPNS1 deficiency inhibits infection of EV-A71 and 9 of 11 examined enteroviruses. Mechanistically, the endo/lysosomal localization of SPNS1 and the acidification of the endo/lysosomes are essential for SPNS1 to support EV-A71 infection. SPNS1 deficiency inhibits EV-A71 genomic RNA replication, but barely affects replication of EV-A71 RNA directly transfected into the cytoplasm. SPNS1 interacts with the EV-A71 capsid protein VP1 and entry receptor SCARB2 in the endo/lysosomes, where it acts as a transporter to release the viral pocket factor into the cytosol, leading to uncoating. Animal experiments show that SPNS1 deficiency results in reduced viral loads, pathological effects, and lethality following EV-A71 infection. Our findings collectively identified SPNS1 as a transporter of the EV-A71 viral pocket factor.},
}
@article {pmid41385899,
year = {2026},
author = {Fathy, K and Bharti, J and Khan Sony, S and Nehra, M and Kaul, R and Rawat, B and Sopory, SK and Agrawal, PK and Prakash, A and Kaul, T},
title = {Triumphing over hidden hunger: Redesigning rice (Oryza sativa L.) for enhanced nutraceutical grain composition utilizing multiplexed genome editing.},
journal = {Journal of plant physiology},
volume = {316},
number = {},
pages = {154667},
doi = {10.1016/j.jplph.2025.154667},
pmid = {41385899},
issn = {1618-1328},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Gene Editing/methods ; *Edible Grain/chemistry/genetics ; Cadmium/metabolism/analysis ; *Dietary Supplements ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Zinc/metabolism ; Iron/metabolism ; },
abstract = {Rice, a staple food crop, is consumed by most of the world's population. Micronutrient malnutrition is a severe health issue, leading to diseases such as cancer, anemia, diabetes, heart disease, and disorders in physical and psychological development. We aimed to create rice with low cadmium in the grain but having high cadmium in shoots, safe biofortified protein, high iron, and zinc using CRISPR/Cas9 and breeding technologies instead of adding drugs. The triple gene Knockout rice lines for two iron sensors and one negative regulator gene for cadmium were created to offer high Fe/Zn and low Cd content for breeders. Multiplexed gene editing mediated biolistic transformation of rice callus, and genotyping was used to check the genetic stability of the edited rice lines. Rice lines were found to have enhanced iron, zinc, and protein content, with concentrations varying based on growth conditions. These lines can be used as phytoremediators for cadmium by storing Cd on plant shoots. The rice-edited plants possessed excellent agro-morphological traits, photosynthetic, and physiological performance. The developed edited indica rice lines have crucial agronomic traits with more nutritional value. Compared to the other lines and the wild wildtype, the genome-edited free Cas9 line 2 showed better traits: 13.48 μg/g (iron), 22.9 μg/g (zinc), and a high protein content, which depends on how bioavailable metals and nutrients are in the soil. The line also had 20.60 g of seeds per 1000 g of plant, a total plant yield of 102.76 g, and 101 days of 50 % flowering. This work offers efficient and precise multiple gene-editing in rice with an effective, sustainable strategy for multi-trait enhancement. The developed lines could be used in breeding programs for sustainable solutions for malnutrition worldwide. The experimental results can provide reference and support for the safe use of edited crops as a diet.},
}
@article {pmid41385994,
year = {2026},
author = {Luo, Y and Wang, X and Yang, F and Zhao, Y and Hu, S and Liu, S and Li, S and Luo, G and Sun, Q},
title = {Construction and validation of a rapid semen identification system based on SHERLOCK technology.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103410},
doi = {10.1016/j.fsigen.2025.103410},
pmid = {41385994},
issn = {1878-0326},
mesh = {Humans ; *Semen/chemistry ; Male ; *CRISPR-Cas Systems ; *RNA, Messenger/genetics ; *Forensic Genetics/methods ; DNA Primers ; Sensitivity and Specificity ; },
abstract = {This study developed a rapid detection system for semen-specific mRNA based on CRISPR/Cas13a system to meet the timeliness requirements of forensic on-site body fluid identification. Specific primers and CRISPR RNA (crRNA) short fragments on semen specific mRNA genes were designed and screened, to establish a SHERLOCK detection method based on technology principles of CRISPR/Cas. Furthermore, nucleic acid rapid release agents for treating samples were screend to construct a new detection method in combination with SHERLOCK, and the specificity and sensitivity of the method were tested. The method can rapidly detect the presence of semen from unknown body fluid samples, and the relative fluorescence unit (RFU) value of the semen sample is significantly higher than those of non-semen samples (P < 0.0001), with a sample detection sensitivity of down to 0.25 μL. The construction of the rapid semen detection method using rapid extraction and SHERLOCK reduces operation time, significantly reduces instrument dependence, and provides an innovative solution for forensic on-site rapid body fluid identification.},
}
@article {pmid41386334,
year = {2026},
author = {Svane, N and Kurosawa, T and Schmid, B and Saaby, L and Kristensen, M and Tabata, H and Kubo, Y and Terasaki, T and Brodin, B and Deguchi, Y},
title = {Elucidating the roles of TM7SF3 and LHFPL6 in the putative H[+]/OC antiporter function in the human brain capillary endothelial cell line, hCMEC/D3.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {217},
number = {},
pages = {107409},
doi = {10.1016/j.ejps.2025.107409},
pmid = {41386334},
issn = {1879-0720},
mesh = {Humans ; *Endothelial Cells/metabolism ; Blood-Brain Barrier/metabolism ; *Brain/metabolism/blood supply ; Cell Line ; *Antiporters/metabolism/genetics ; Biological Transport ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: The putative proton/organic cation (H[+]/OC) antiporter has been shown to mediate transport of CNS drug compounds like oxycodone and pyrilamine across the blood-brain barrier (BBB). This transporter has a broad substrate profile and is able to transport substrates against their concentration gradient, making it an interesting target for brain drug delivery. However, the molecular identity of this transporter remains unknown. Recent studies have indicated that the two proteins TM7SF3 and LHFPL6 might be components of this transporter. The present study aimed to investigate the roles of TM7SF3 and LHFPL6 in the H[+]/OC antiporter function to advance understanding of its molecular identity and potential in CNS drug delivery.<br><br>METHODS: CRISPR-Cas9 gene-editing was used to generate three hCMEC/D3 knockout (KO) cell lines: TM7SF3 KO (TM-KO), LHFPL6 KO (LH-KO), and a double KO of TM7SF3 and LHFPL6 (TMLH-KO). The uptake of pyrilamine analogue (EDMPG) and [[3]H]-pyrilamine was assessed in wild type (WT) and KO lines. Quantitative Realtime Polymerase Chain Reaction (qRT-PCR) confirmed successful gene knockouts. Passive diffusion properties and the expression and functionality of known BBB transporters, including LAT1 (SLC7A5), GLUT1 (SLC2A1), and MCT1 (SLC16A1), were also examined.<br><br>RESULTS: The EDMPG uptake was significantly reduced in TM-, LH-, and TMLH-KO cells, suggesting that TM7SF3 and LHFPL6 contribute to the H[+]/OC antiporter function. However, [[3]H]-pyrilamine uptake remained unchanged across all KOs, indicating a TM7SF3- and LHFPL6-independent transport mechanism. This was further supported by the persistent inhibition of [[3]H]-pyrilamine uptake in the presence of known H[+]/OC antiporter substrates. While passive diffusion and GLUT1- and MCT1-mediated transport were unaffected, LAT1-mediated uptake of [[3]H]L-leucine and gabapentin (Neurontin) was significantly reduced in LH- and TMLH-KO cells, correlating with decreased LAT1 mRNA expression in these cells.<br><br>CONCLUSIONS: This study suggests that the H+/OC antiporter operates via two distinct mechanisms: a high-capacity, TM7SF3- and LHFPL6-independent pathway and a low-capacity, TM7SF3- and LHFPL6-dependent pathway. These findings underscore the complexity of the H[+]/OC antiporter molecular composition and highlight the need for further research to fully elucidate its identity.},
}
@article {pmid41387334,
year = {2025},
author = {Song, N and Tian, G and Li, H and Zhang, L and Wang, Y and Zhao, W and Yao, C and Yang, D},
title = {DNA Nanoflowers Efficiently Encapsulate Photodynamic Agents and CRISPR/Cas9 for Synergistic Pancreatic Cancer Therapy.},
journal = {Nano letters},
volume = {25},
number = {51},
pages = {17693-17701},
doi = {10.1021/acs.nanolett.5c04676},
pmid = {41387334},
issn = {1530-6992},
support = {//National Natural Science Foundation of China/ ; //Tianjin Key Medical Discipline Construction Project/ ; },
mesh = {*Pancreatic Neoplasms/drug therapy/pathology/genetics/therapy/metabolism ; *Photochemotherapy/methods ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; NF-E2-Related Factor 2/genetics/metabolism ; Cell Line, Tumor ; *Photosensitizing Agents/chemistry/pharmacology/administration &amp; dosage/therapeutic use ; Chlorophyllides ; *DNA/chemistry ; Reactive Oxygen Species/metabolism ; Porphyrins/chemistry/pharmacology/administration &amp; dosage ; Hemin/chemistry ; Apoptosis/drug effects ; Gene Editing ; Ribonucleoproteins/genetics ; G-Quadruplexes ; },
abstract = {Photodynamic therapy (PDT) holds significant promise for treating pancreatic cancer by utilizing photosensitizers to generate reactive oxygen species (ROS) that induce tumor cell death. However, the therapeutic efficacy of PDT is hindered by inadequate ROS accumulation. Herein, we develop a DNA nanoflower that enables the controlled codelivery of Cas9 ribonucleoprotein (RNP), hemin, and chlorin e6 for synergistic PDT. The Cas9 RNP selectively knocks out the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), thereby increasing cancer cells' sensitivity to ROS. Simultaneously, the G-quadruplex/hemin complex catalyzes the conversion of endogenous H2O2 into O2, alleviating tumor hypoxia and supplying additional oxygen for PDT. This synergistic approach substantially amplifies ROS accumulation by attenuating ROS elimination and enhancing ROS generation, demonstrating high gene editing efficiency, significant Nrf2 down-regulation, elevated apoptosis, and remarkable antitumor efficacy in pancreatic cancer cells and a mouse model, underscoring the potential for precision medicine.},
}
@article {pmid41387457,
year = {2025},
author = {Cheng, F and Soleimani Samarkhazan, H and Khazaei, Y},
title = {CRISPR-engineered microbiome: living therapeutics revolutionize blood cancer immunotherapy.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {17},
pmid = {41387457},
issn = {2055-5008},
mesh = {Humans ; *Immunotherapy/methods ; Animals ; *Hematologic Neoplasms/therapy/immunology/microbiology ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome/genetics ; *Microbiota ; },
abstract = {Blood cancers such as leukemia, lymphoma, and myeloma remain refractory in many patients due to immune escape, antigen heterogeneity, and therapy‑related toxicities. To address these challenges, we review recent strategies that harness CRISPR‑engineered gut commensals as precision "living therapeutics" to modulate host immunity and directly target malignant clones. We frame this review around three principal themes: (1) mechanistic strategies whereby CRISPR-engineered commensals modulate host immunity and directly antagonize malignant clones; (2) the enabling technologies and delivery/containment platforms, CRISPR variants, phage/LNP delivery, genetic circuits and biocontainment, that make living therapeutics feasible; and (3) translational progress, outstanding technical and safety barriers, and ethical/regulatory challenges that must be addressed for clinical deployment. To illustrate these themes, we discuss three concrete therapeutic modalities: engineered microbial secretion of immunomodulators, targeted delivery of tumor-lytic payloads, and engineered production of anticancer metabolites, and how these are enabled by contemporary CRISPR and synthetic-biology toolkits. Selected preclinical models report substantial antitumor effects, often >60% tumor reduction in rodent studies, and restoration of CAR-T cell function in controlled settings; however, effect sizes vary across models, and human translation remains unproven. We also analyze key technical barriers, strain stability, biocontainment, off‑target effects, and propose solutions, including auxotrophic kill-switches and AI‑guided strain optimization. Finally, we outline future directions, from in situ phage delivery to multi‑omics-driven patient stratification. CRISPR‑microbiome editing represents a paradigm shift in hematologic oncology, offering localized, sustained therapy with reduced systemic toxicity.},
}
@article {pmid41387726,
year = {2025},
author = {Srinivasan, R and Sun, T and Sandles, A and Wu, D and Wang, L and Patel, H and Pabalate, R and Bader, M and Heidersbach, A and Ho, C and Xie, S and Ng, A and Haley, B},
title = {Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {504},
pmid = {41387726},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.},
}
@article {pmid41387738,
year = {2025},
author = {Zhang, X and Zhu, T and Zhang, W and Zhang, Y and Zhang, J and Yang, J and Xia, C and Zhao, H and Yu, Y and Wen, C},
title = {Ethylene promotes branch formation but inhibits tendril development in cucumber.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {745},
pmid = {41387738},
issn = {2041-1723},
support = {JKZX202207//Beijing Academy of Agricultural and Forestry Sciences (BAAFS)/ ; },
mesh = {*Ethylenes/pharmacology/metabolism ; *Cucumis sativus/growth &amp; development/genetics/metabolism/drug effects ; Gene Expression Regulation, Plant/drug effects ; Plant Proteins/genetics/metabolism ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; *Plant Growth Regulators/pharmacology/metabolism ; Mutation ; Promoter Regions, Genetic ; },
abstract = {Ethylene coordinates numerous plant growth processes, particularly in cucurbit crops, yet its role in vegetative growth regulation remains largely unexplored. Here, we report the function of ethylene in controlling branch and tendril development in cucumber. We find that ethylene promotes branches formation but inhibits tendrils development in a dose-dependent manner. CRISPR-Cas9-generated gene-edited Csein2 and Csein3/Cseil1 mutants exhibit few branches and more tendrils. Exogenous ethylene can recover the branch/tendril defective phenotypes of the Csein3 and Cseil1 mutants but not those of the Csein2 mutant or the Csein3/Cseil1 double mutant. Transcriptomic and metabolic analyses reveal that CsCYP707A4 and CsTL are the key downstream targets of ethylene signaling. We show that CsEIN3 can bind to its promoters to activate the expression of CsCYP707A4 but inhibit the expression of CsTL, which leads to the opposite effect on branch and tendril development. The study sets the foundation for designing ideal plant architecture to increase production efficiency.},
}
@article {pmid41387770,
year = {2025},
author = {Ge, L and Li, W and Dou, Y and Ma, Y and Sun, M and Chen, X and Feng, X and Li, Y and Yu, Q},
title = {Callus and endosperm green fluorescence reporter-assisted selection system in maize CRISPR/Cas9 gene editing.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {92},
pmid = {41387770},
issn = {1471-2229},
support = {2022SZX13//Science&amp;Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta/ ; ZR2024MC067//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Zea mays/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Endosperm/genetics/metabolism ; *Green Fluorescent Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Genes, Reporter ; },
abstract = {BACKGROUND: Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has emerged as a promising approach for functional gene analysis and genetic improvement. Since stable transformation remains the primary method for implementing this system, the ultimate goal in crop breeding programs would require the selection of transgene-free plants with the CRISPR/Cas expression cassette removed.<br><br>RESULTS: In this study, we developed an endosperm-specific fluorescence reporter-assisted selection system for CRISPR/Cas9 gene editing (pAZS22-eGFP/CRISPR/Cas9) in maize (Zea mays L.), utilizing enhanced green fluorescent protein (eGFP) expressed specifically in the endosperm to facilitate the easy identification of transgenic and transgene-free plants from the T1 generation on. In addition, the 22&#xa0;kDa alpha zein (z1C1_10) promoter from maize, employed in this system, has been shown to be active in both callus and endosperm, thereby being able to enhance the accuracy of transformant identification during the tissue culture process by reducing false positives compared to the traditional selective media methods. Our studies targeting the ZmSnRK2.1 or Dwarf1 (D1) genes demonstrated a reasonable editing efficiency, with rates ranging from 56.3% for T0 plants targeting ZmSnRK2.1, to 87.5% and 100% for T1 plants targeting D1 and ZmSnRK2.1, respectively. In addition, we successfully identified 1 transgene-free homozygous d1 mutant in the T1 generation and 7 transgene-free homozygous snrk2.1 mutants in the T2 generation.<br><br>CONCLUSIONS: The pAZS22-eGFP/CRISPR/Cas9 system provides an efficient tool for gene editing, transformant selection and transgene status identification in maize breeding.},
}
@article {pmid41388295,
year = {2025},
author = {Ji, T and Zhang, Y and Wang, Y and Yuan, K and Wang, M and Ye, J and Zhang, H and Zhang, N and Zhang, H},
title = {AND logic-gated CRISPR/Cas9 and hybridization chain reaction system for precise ctDNA detection.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {43},
pmid = {41388295},
issn = {1477-3155},
support = {2022YFB3808200//National Key Research and Development Program of China/ ; },
mesh = {*Circulating Tumor DNA/genetics/blood/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques/methods ; Biomarkers, Tumor/genetics/blood ; Nucleic Acid Hybridization ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a critical biomarker for liquid biopsies, enabling the non-invasive acquisition of cancer-related information from blood samples. Precise detection of ctDNA, particularly the identification of single-nucleotide variations (SNVs), is crucial for early cancer diagnosis, therapeutic monitoring, and prognostic evaluation. However, current ctDNA detection methods often encounter challenges such as complex procedures, difficult data analysis, and false-positive signals during pre-amplification. In this study, we introduce a novel detection method based on AND logic-gated integration of interspaced short palindromic repeats and associated proteins (CRISPR/Cas9) system with hybridization chain reaction (HCR) isothermal amplification. This strategy enhances the specific and sensitive detection of ctDNA. The incorporation of the AND logic gate effectively minimizes the off-target effects of Cas9 and enables the differentiation of single-nucleotide mutations, such as KRAS G12D, even in complex serum environments. Our system exhibits high sensitivity and specificity, achieving a limit of detection as low as 1 fM and capable of identifying SNVs mutations with allele fractions as low as 0.1% among wild-type sequences. Furthermore, we validated the specificity of our approach by successfully detecting various mutations, including KRAS G12C, KRAS G12D, EGFR T790M and TP53 R273H, in simulated clinical samples. These findings highlight a reliable method for precise ctDNA detection, offering high specificity, selectivity, and accuracy, thus paving the way for potential cancer diagnostic application.},
}
@article {pmid41389042,
year = {2025},
author = {Nie, YG and Zhang, HS and Su, M and Zha, CJ and Yang, K and Ying, ZM},
title = {Proximity-Inducible CRISPR/Cas12a Activity by Scaffold RNA Assembly for Sensing Applications.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {28088-28097},
doi = {10.1021/acs.analchem.5c06530},
pmid = {41389042},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Breast Neoplasms/diagnosis ; *RNA/chemistry/genetics/metabolism ; Adenosine Triphosphate/analysis ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Female ; *Bacterial Proteins/metabolism ; },
abstract = {The programmability and flexibility of the RNA-directed CRISPR/Cas12a system underpin its utility as a potent tool for diagnostic applications. However, existing engineered crRNA strategies are still limited by a narrow target range, inadequate specificity, and operational complexity. To overcome these challenges, a proximity-assembly and activate (PAA) strategy was developed, employing split dumbbell activators with terminally modified target-binding modules that reassemble on scaffold RNA to reconstruct functional crRNA and activate Cas12a trans-cleavage activity. The design allows universal detection of both nucleic acid and non-nucleic acid targets. Notably, owing to its strict target dependency, the assembled crRNA biosensor significantly reduces background signal and suppresses nonspecific leakage. We demonstrated that the PAA system facilitates rapid and highly specific detection of miRNA-21, ATP, and anti-Dig antibody in complex matrices, enabling single-base discrimination among miRNA variants. Moreover, the platform successfully detected endogenous miRNA-21 in serum and cellular samples from breast cancer patients, clearly distinguishing them from healthy controls. This work presents a modular, plug-and-play, and versatile platform for molecular diagnostics, holding considerable potential for advancing clinical diagnostics and precision medicine.},
}
@article {pmid41389205,
year = {2025},
author = {Zobel, M and Damaggio, G and Mignogna, ML and Besusso, D and Scalzo, D and Cossu, A and Trovesi, C and Crosti, M and Cortina, F and Campus, I and Formenti, G and Mazzara, S and Gregoretti, F and Antonelli, L and Oliva, G and Zuccato, C and Colonna, V and Conforti, P and Cereda, M and Rossi, RL and Maestri, S and Scolz, A and Iennaco, R and Cattaneo, E},
title = {A human CAGinSTEM platform for decoding HTT repeats' somatic instability links CAG interruption to HD pathology in neurons.},
journal = {Cell reports},
volume = {44},
number = {12},
pages = {116685},
pmid = {41389205},
issn = {2211-1247},
mesh = {Humans ; *Huntington Disease/genetics/pathology ; *Huntingtin Protein/genetics/metabolism ; *Neurons/metabolism/pathology ; *Genomic Instability ; *Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion ; CRISPR-Cas Systems/genetics ; },
abstract = {Somatic CAG instability in the mutant Huntingtin (HTT) gene is increasingly recognized as a key hallmark of Huntington's disease (HD). Using our novel human CAGinSTEM platform, we manipulated cis genetic elements influencing instability in human HD neurons, monitoring repeat length. Quality-controlled CRISPR-engineered stem cells with increasing CAG lengths and clinical haplotypes were analyzed using third-generation sequencing. Our findings link interruptions in the CAG repeat, especially the loss or duplication of the penultimate CAA of canonical alleles, to significant instability modulation. Notably, four internal CAA interruptions completely abolish CAG instability, reversing HD phenotypes such as altered striatal fate acquisition and nuclear disorganization. This platform highlights the role of cis modifiers, emphasizing the direct influence of HTT DNA repeat composition on CAG instability and providing a robust framework for modeling HTT repeat instability in vitro.},
}
@article {pmid41389602,
year = {2026},
author = {Kumari, P and Gupta, V and Chhikara, A and Dalal, J},
title = {Revolutionizing forensic DNA analysis: The potential of CRISPR-Cas9 technology in genetic investigations.},
journal = {Journal of forensic and legal medicine},
volume = {117},
number = {},
pages = {103047},
doi = {10.1016/j.jflm.2025.103047},
pmid = {41389602},
issn = {1878-7487},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Fingerprinting/methods ; *Forensic Genetics/methods ; *Gene Editing ; },
abstract = {The newest achievements in the field of molecular biology and gene-editing technologies have transformed the paradigm of forensic DNA analysis. However, there are still great difficulties in interpreting degraded, low-template, mixed genetic samples. The review critically evaluates the transformative potential of Clustered Regularly Interspaced Short Palindromic Repeats and an associated protein 9 (CRISPR-Cas9) as an accurate, effective, and cost-efficient system of genome-editing in the field of forensic science. Based on the evidence of the current literature, the paper critically analyzes the mechanisms of CRISPR-Cas9 activity, its RNA-guided specificity, dual-strand cleavage, and high-fidelity targeting, and compares its functionality with other standard methods like the STR and SNP profiling. The review also discusses more complex CRISPR-based diagnostic systems, such as SHERLOCK, DETECTR, and HOLMES that allow the analysis of DNA rapidly, without amplification, and in a portable format. Among major discoveries, there is the ability of CRISPR to increase the accuracy of DNA profiling, resolve mixture, recapitulate damaged genetic material, and reduce the possibility of contamination. In addition to genetic analysis, it has applications in forensic epigenetics, prediction of phenotypes, microbial forensics and environmental trace analysis. The review also covers the ethical, legal and governance implications of implementing CRISPR-based evidence in the judicial process especially in as far as data privacy; admissibility and equity of access are concerned. In general, CRISPR-Cas9 is a paradigm shift in forensic genomics, the one that has the potential to transform personal identification, reconstruction of the crime scene, and the interpretation of molecular evidence. Future efforts should focus on method validation, standardization, and ethical governance to ensure the responsible and sustainable implementation of this technology in forensic practice.},
}
@article {pmid41389662,
year = {2026},
author = {Ma, J and Zhao, CF and Liu, X},
title = {Advances in targeted therapeutics and smart delivery systems based on precision nano-oncology.},
journal = {International immunopharmacology},
volume = {169},
number = {},
pages = {115946},
doi = {10.1016/j.intimp.2025.115946},
pmid = {41389662},
issn = {1878-1705},
mesh = {Humans ; *Neoplasms/therapy ; *Precision Medicine/methods ; Animals ; *Drug Delivery Systems/methods ; *Nanomedicine/methods ; Immunotherapy/methods ; *Antineoplastic Agents/administration &amp; dosage/therapeutic use ; Nanoparticles ; Drug Carriers ; },
abstract = {The convergence of nanotechnology and precision oncology is revolutionizing cancer treatment by enabling highly specific, minimally invasive, and personalized therapeutic strategies. This review explores recent breakthroughs in nano-therapeutics and their pivotal role in overcoming the limitations of conventional cancer therapies. Emphasis is placed on the design and function of nanocarriers that facilitate targeted drug delivery via both passive (EPR effect) and active ligand-mediated mechanisms. Special attention is given to stimuli-responsive systems that release therapeutic agents in response to pH, enzymes, temperature, or redox environments, enhancing spatiotemporal control. The article further discusses the integration of nanotechnology with emerging modalities including immunotherapy, photothermal and photodynamic therapies, gene editing tools (e.g., CRISPR/Cas systems), and multifunctional theranostic platforms. While these innovations offer transformative potential, the review also addresses persistent challenges such as tumor heterogeneity, immune clearance, off-target effects, large-scale manufacturing, and regulatory complexity. By highlighting both promise and hurdles, this article provides a comprehensive lens into the future of precision cancer nanomedicine.},
}
@article {pmid41390353,
year = {2025},
author = {Howe, LJ and Aulchenko, YS and Davey Smith, G and Davies, NM and Esparza-Gordillo, J and Johnson, T and Liu, JZ and Richardson, TG and Sanseau, P and Scott, RA and Seaton, DD and Sharma, A and Cortes, A},
title = {Evaluating transportability of in vitro cellular models to in vivo human phenotypes using gene perturbation data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {513},
pmid = {41390353},
issn = {2041-1723},
mesh = {Humans ; Phenotype ; Lysosomes/metabolism ; *Models, Biological ; Cholesterol, LDL/blood/metabolism ; CRISPR-Cas Systems ; Cholesterol/metabolism ; },
abstract = {Gene perturbation screens (e.g. CRISPR-Cas9) assess the impact of gene disruption on in-vitro cellular phenotypes (e.g., proliferation, anti-viral response). In-vitro experiments can be useful models for in-vivo (organismal) phenotypes (e.g., immune cell anti-viral response and infectious diseases). However, assessing whether an in-vitro cellular model effectively captures in-vivo biology is challenging. An in-vitro model is 'transportable' to an in-vivo phenotype if perturbations impacting the in-vitro phenotype also impact the in-vivo phenotype with mechanism-consistent directionality and effect sizes. We propose a framework; Gene Perturbation Analysis for Transportability (GPAT), to assess model transportability using gene perturbation effect estimates from perturbation screens (in-vitro) and loss-of-function burden tests (in-vivo). In hypothesis-driven analyses, GPAT provides evidence for model transportability of higher lysosomal cholesterol accumulation in-vitro to lower human plasma LDL-cholesterol (P = 0.0006), consistent with the known role of lysosomes in lipid biosynthesis. In contrast, there was limited evidence for other putative in-vitro models. In hypothesis-free analyses, we find evidence for transportability of cancer cell line proliferation to in-vivo human plasma cellular phenotypes (e.g. erythroleukemia proliferation and plasma lymphocyte percentage). Here we show that perturbation data can be used to evaluate transportability of in-vitro cellular models, informing assay prioritisation and supporting novel hypothesis generation.},
}
@article {pmid41390487,
year = {2025},
author = {Carruthers, DN and Kinnunen, PC and Li, Y and Chen, Y and Gin, JW and Yunus, IS and Galliard, WR and Tan, S and Radivojevic, T and Adams, PD and Singh, AK and Sustarich, J and Petzold, CJ and Mukhopadhyay, A and Garcia Martin, H and Lee, TS},
title = {Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11489},
pmid = {41390487},
issn = {2041-1723},
support = {DE-AC0205CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Pseudomonas putida/metabolism/genetics ; *Machine Learning ; *Metabolic Engineering/methods ; *Hemiterpenes/metabolism ; CRISPR-Cas Systems ; Proteomics ; Metabolic Networks and Pathways/genetics ; Automation ; },
abstract = {Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates that ML-driven automated design-build-test-learn cycles, when combined with rigorous data validation, can rapidly enhance titers without specific biological knowledge, suggesting that it can be applied to any host, product, or pathway.},
}
@article {pmid41390513,
year = {2025},
author = {Vermeulen, M and Craig, AW and Babak, T},
title = {Challenges and opportunities for oncology drug repurposing informed by synthetic lethality.},
journal = {NPJ systems biology and applications},
volume = {11},
number = {1},
pages = {143},
pmid = {41390513},
issn = {2056-7189},
support = {PJT 178214//Canadian Institutes of Health Research Project Grant/ ; },
mesh = {Humans ; *Drug Repositioning/methods ; *Synthetic Lethal Mutations/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *Antineoplastic Agents/pharmacology/therapeutic use ; *Neoplasms/genetics/drug therapy ; Gene Knockout Techniques ; Mutation ; },
abstract = {Although two-thirds of cancers arise from loss-of-function mutations in tumor suppressor genes, there are few approved targeted therapies linked to these alterations. Synthetic lethality offers a promising strategy to treat such cancers by targeting vulnerabilities unique to cancer cells with these mutations. To identify clinically relevant synthetic lethal interactions, we analyzed genome-wide CRISPR/Cas9 knock-out (KO) viability screens from the Cancer Dependency Map and evaluated their clinical relevance in patient tumors through mutual exclusivity, a pattern indicative of synthetic lethality. Indeed, we found significant enrichment of mutual exclusivity for interactions involving cancer driver genes compared to non-driver mutations. To identify therapeutic opportunities, we integrated drug sensitivity data to identify inhibitors that mimic the effects of CRISPR-mediated KO. This approach revealed potential drug repurposing opportunities, including BRD2 inhibitors for bladder cancers with ARID1A mutations and SIN3A-mutated cell lines showing sensitivity to nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. However, we discovered that pharmacological inhibitors often fail to phenocopy KO of matched drug targets, with only a small fraction of drugs inducing similar effects. This discrepancy reveals fundamental differences between pharmacological and genetic perturbations, emphasizing the need for approaches that directly assess the interplay of loss-of-function mutations and drug activity in cancer models.},
}
@article {pmid41390669,
year = {2025},
author = {Teske, M and Wertheimer, T and Butz, S and Zwicky, P and Mallona, I and Nopper, SL and Münz, C and Elling, U and Lancrin, C and Becher, B and Grosso, AR and Baubec, T and Schmolka, N},
title = {Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11412},
pmid = {41390669},
issn = {2041-1723},
support = {186012//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; FAN//Universit&#xe4;t Z&#xfc;rich (University of Zurich)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Mesoderm/cytology/metabolism ; Cell Differentiation/genetics ; Cell Lineage/genetics ; *Hematopoietic Stem Cells/cytology/metabolism ; Gene Expression Regulation, Developmental ; Mouse Embryonic Stem Cells/cytology/metabolism ; *Endothelial Cells/cytology/metabolism ; Hematopoiesis/genetics ; },
abstract = {During development, blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm forming haematopoietic progenitors. This study aims to identify the crucial molecular regulators of haemato-endothelial mesoderm formation and to extend our knowledge of the process in an unbiased way. We employ a murine embryonic stem cell model that recapitulates embryonic blood development, and perform targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators. We identify the transcription factors ETV2, LDB1, SMAD1, SIX4 and ZBTB7b as regulators of haemato-endothelial mesoderm commitment. Embryonic stem cells lacking these regulators give rise to mesodermal subsets with a defined lineage differentiation bias, while transcriptome analysis of these cells uncovers the precise impact of each factor on gene expression in the developing mesoderm. Our study reveals molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification and paves the way for future advances in haematopoietic stem cell applications.},
}
@article {pmid41390734,
year = {2025},
author = {Ren, J and Yao, J and Cao, Q and Li, Y and Li, Y and Zhang, Z and Ge, X and Wang, S and Zhang, Y and Wang, X and Zhang, X},
title = {Protein-nucleic acid language model-assisted design of precise and compact adenine base editor.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11207},
pmid = {41390734},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; HEK293 Cells ; *Adenine/metabolism/chemistry ; Mice ; Genetic Therapy/methods ; Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; *Nucleic Acids/genetics/chemistry ; Proprotein Convertase 9/genetics/metabolism ; Mutation ; },
abstract = {Adenine base editors (ABEs) are powerful tools for gene therapy. However, efficient version of ABEs (e.g. ABE8e) always induce excessive bystander and off-target editing events and are large in size, hindering their potential in clinical disease treatment. Here, we develop a pre-trained Protein-Nucleic Acid Constrained Language Model to design ABE8e with high activity, reduced editing window and decreased size. By further engineering, the smallest ABE8e- PNLM-pcABE- with a 27% size reduction, exhibits high activity, precise 3-nt editing window, and reduced off-target events near background level in HEK293T cells. Compared to ABE8e, PNLM-pcABE has up to 133.5-fold precision improvement in pathogenic mutation correction. By PNLM-pcABE, the albino mouse model carrying desired base mutation is nearly 100% obtained via zygotes microinjection and the expression of PCSK9 substantially decreases in mice receiving in vivo delivery with lipid nanoparticle (LNP), indicating their great potential in gene therapy and disease modeling.},
}
@article {pmid41390996,
year = {2026},
author = {Wang, AJ and Du, C and Liu, H and Wang, HL},
title = {Effect of the sdc4 gene knockdown on muscle development in zebrafish.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {281},
number = {},
pages = {111171},
doi = {10.1016/j.cbpb.2025.111171},
pmid = {41390996},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/growth &amp; development/metabolism ; *Syndecan-4/genetics/metabolism ; *Muscle Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; MicroRNAs/genetics/metabolism ; *Muscle, Skeletal/metabolism/growth &amp; development ; Wnt Signaling Pathway ; CRISPR-Cas Systems ; },
abstract = {The growth and regeneration of skeletal muscle are closely related to syndecan-4 (Sdc4), which is a type I transmembrane heparan sulfate proteoglycan belonging to the syndecan family. However, it remains unclear how the sdc4 gene affects fish muscle development. Therefore, an sdc4 knockdown zebrafish line (sdc4[-/-]) was generated by CRISPR/Cas9 technology in this study, and its phenotypes were analyzed. The results revealed that sdc4[-/-] zebrafish exhibited reduced body length and weight compared to the wild-type (WT) at 90 days post fertilization (dpf). Furthemore, sdc4[-/-] zebrafish also showed a significantly larger number of muscle fibers, and significantly reduced individual muscle fiber cross-sectional area. The mRNA expression levels of genes associated with myogenic regulatory factors (MRFs) and the wnt/β-catenin pathway were all significantly downregulated. Based on dual luciferase reporter assays, sdc4 gene expression was regulated by the transcription factor myocyte enhancer factor 2aa (Mef2aa) and miR-141-3p, which bind to its promoter and 3' untranslated region (UTR), respectively. Additionally, the reduced average swimming speed and distance observed in sdc4[-/-] zebrafish at 90 dpf were concomitant with a significant downregulation of mitochondrial respiratory chain complex-related genes and a reduction in ATP concentration. This research aids understanding of sdc4 function in fish and may provide a new perspective for studying the molecular mechanisms of muscle growth and development.},
}
@article {pmid41391580,
year = {2026},
author = {Zarei, S and Hosseiniara, SM and Zijoud, SSH and Hosseiniara, R},
title = {Electrochemical MicroRNA biosensors for kidney Cancer: From biomarker discovery to point-of-care diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {582},
number = {},
pages = {120786},
doi = {10.1016/j.cca.2025.120786},
pmid = {41391580},
issn = {1873-3492},
mesh = {Humans ; *MicroRNAs/analysis/genetics ; *Biosensing Techniques ; *Biomarkers, Tumor/analysis/genetics ; *Kidney Neoplasms/diagnosis/genetics ; *Electrochemical Techniques ; *Point-of-Care Systems ; },
abstract = {Kidney cancer, particularly clear cell renal cell carcinoma (ccRCC), presents a significant clinical burden due to late-stage detection and limited effectiveness of current diagnostic modalities. Minimally invasive strategies, such as liquid biopsy, have emerged as promising alternatives, with microRNAs (miRNAs) gaining attention as stable, disease-specific biomarkers detectable in biofluids. miRNAs function as oncogenes or tumor suppressors, offering advantages over conventional protein biomarkers in early cancer detection and prognostic assessment. Electrochemical biosensors provide a highly sensitive, rapid, and cost-effective platform for miRNA detection, enabling potential point-of-care applications. Recent advances include the integration of nanomaterials, enzymatic and isothermal amplification methods, and CRISPR-Cas systems to enhance specificity and signal sensitivity. Prototype sensors targeting RCC-relevant miRNAs, multiplexed detection for biomarker panels, and smartphone-compatible platforms demonstrate the feasibility of translating these technologies into clinical practice. Despite challenges in assay standardization, pre-analytical variability, and regulatory pathways, electrochemical miRNA biosensors hold transformative potential for non-invasive RCC diagnostics, treatment monitoring, and precision oncology. Continued innovation and clinical validation may establish these platforms as integral tools for personalized patient management.},
}
@article {pmid41391726,
year = {2026},
author = {Liao, XR and Han, D and Qi, LJ and Huang, QY and Gao, QY and He, XY and Guo, T and Lei, JJ and Cheng, SX},
title = {Aptamer-functionalized nanoparticles for CRISPR-Cas9 delivery to circulating malignant cells for therapeutic efficacy evaluation.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114542},
doi = {10.1016/j.jconrel.2025.114542},
pmid = {41391726},
issn = {1873-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry/administration &amp; dosage ; *Aptamers, Nucleotide/administration &amp; dosage/chemistry ; Cell Line, Tumor ; Hyaluronic Acid/chemistry ; *Neoplastic Cells, Circulating/metabolism ; Gene Editing/methods ; *Neoplasms/therapy/genetics ; Hyaluronan Receptors/metabolism ; Epithelial Cell Adhesion Molecule/genetics ; Proto-Oncogene Proteins c-met/genetics ; *Gene Transfer Techniques ; ErbB Receptors/genetics ; Plasmids ; },
abstract = {Genome editing therapies targeting oncogenic pathways represent a promising alternative to small-molecule inhibitors, enabling durable therapeutic responses without inducing drug resistance. However, their success hinges on overcoming tumor heterogeneity, as malignant cells of cancer patients exhibit significant phenotypic variability. To advance personalized research on genome editing efficacy, tailored delivery systems capable of precisely targeting heterogeneous cancer cell populations are essential. Herein, we developed a facile modification strategy to construct a multiplexed surface-functionalized gene delivery system targeting heterogeneous cancer cells for personalized therapeutic studies. The system integrates the EGFR-targeting TuTu22 aptamer with SYL3C-conjugated hyaluronic acid (SYL3C-HA) for EpCAM and CD44 recognition. This triple-targeting platform enables efficient delivery of genome editing plasmid for c-Met knockout in both cancer cell lines and circulating malignant cells (CMCs) from cancer patients. The c-Met knockout not only reduces tumor malignancy but also reverses immune suppression, evidenced by PD-L1 downregulation and restored immune surveillance. By combining gene delivery with an ex vivo patient-derived evaluation platform, this system provides a robust tool for personalized research on the therapeutic strategies for tumor progression inhibition and immunity restoration.},
}
@article {pmid41392542,
year = {2025},
author = {Qiao, Z and Choi, S and Chen, Z and Rodriguez, RM and Wang, Q and Yang, Z and Theuerkauf, SA and Nabhan, JF and Hensch, TK and Buchholz, CJ and Lu, Q},
title = {Targeted Intracellular Delivery via Precision Programming of ARRDC1-Mediated Microvesicles.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {12},
pages = {e70199},
pmid = {41392542},
issn = {2001-3078},
support = {//Vesigen Therapeutics/ ; P42ES030990/NH/NIH HHS/United States ; R01ES029097/NH/NIH HHS/United States ; R01HL139496/NH/NIH HHS/United States ; },
mesh = {Animals ; Mice ; CD8-Positive T-Lymphocytes/metabolism ; Humans ; *Cell-Derived Microparticles/metabolism ; Neurons/metabolism ; *Drug Delivery Systems/methods ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Efficient and cell-specific delivery remains a major barrier to realising the full therapeutic potential of modalities such as mRNA and CRISPR-based gene editors. Here, we report a versatile delivery platform based on engineered ARRDC1-mediated microvesicles (ARMMs) capable of delivering cargo to defined cell populations. By decorating ARMMs with engineered Nipah virus (NiV)-derived fusion and attachment proteins conjugated to cell-specific ligands, we enable selective binding and membrane fusion-mediated cargo release. ARMMs functionalized with anti-CD8 single-chain variable fragment (scFv) delivered protein, mRNA, or CRISPR-Cas9 base editor selectively to CD8[+] T cells. Similarly, ARMMs displaying a designed ankyrin repeat protein (DARPin) targeting the GluA4 receptor enabled delivery to parvalbumin-positive (PV[+]) neurons. In vivo, administration of targeted ARMMs resulted in functional delivery to CD8[+] splenocytes and PV[+] cortical neurons in mice. These findings establish surface-engineered ARMMs as a programmable and modular system for precision delivery of therapeutic macromolecules, with broad applicability in gene and RNA-based medicine.},
}
@article {pmid41394860,
year = {2025},
author = {Andersch, L and Grunewald, L and Stecklum, M and Klironomos, F and Haase, K and Hollek, V and Lam, T and Jung, BA and Winkler, A and Schwiebert, S and Astrahantseff, K and Launspach, M and Jens, M and Henssen, A and Kloke, L and Blüthgen, N and Eggert, A and Schulte, JH and Anders, K and Künkele, A},
title = {Investigating genetic modifications to enhance L1CAM-CAR T cell migration in solid tumors in a 3D bioprinted neuroblastoma model.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1677361},
pmid = {41394860},
issn = {1664-3224},
mesh = {*Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; *Neuroblastoma/immunology/pathology/therapy ; Bioprinting ; *Immunotherapy, Adoptive/methods ; *Cell Movement/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; Receptors, Chimeric Antigen/immunology/metabolism ; Neural Cell Adhesion Molecule L1/immunology/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Membrane Glycoproteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/deficiency/genetics/metabolism ; Xenograft Model Antitumor Assays ; Single-Cell Gene Expression Analysis ; Humans ; Female ; Animals ; Mice ; Cell Line, Tumor ; Coculture Techniques ; Gene Expression Regulation/immunology ; },
abstract = {INTRODUCTION: Effective CAR T cell infiltration into solid tumors remains a major barrier to therapy success. Despite their clinical potential, few studies have evaluated phenotypes of CAR T cells successfully invading the tumor mass following infusion. Phenotypic information would enrich our understanding of the mechanisms governing CAR T cell migration into solid tumors. Here we implemented an in vitro strategy to identify genes driving L1CAM-CAR T cell migration into a 3D tumor mass.<br><br>METHODS: L1CAM-CAR T cells were separated into 2 groups by their capability to infiltrate (or not) a 3D bioprinted neuroblastoma model. Single-cell and bulk RNA sequencing was performed, and infiltrating CAR T cells were compared to noninfiltrating cells to seek genetic drivers of CAR T cell migration. CRISPR/Cas9 technology was used to generate modified L1CAM-CAR T cells.<br><br>RESULTS: Tumor-infiltrating L1CAM-CAR T cells expressed lower levels of the selectin P ligand (SELPLG) glycoprotein and higher levels of the T cell-specific adaptor protein, SH2D2A. Functional characterization of L1CAM-CAR T cells genetically modified to enforce these characteristics demonstrated that neither trait negatively impacted L1CAM-CAR T cell cytotoxicity, activation and cytokine release upon coculture with neuroblastoma target cells. Transgenic SH2D2A expression did not improve CAR T cell migration in an endothelial transmembrane assay. SELPLG knockout benefited CAR T cell in vitro trans-endothelial migration, but did not enhance anti-tumor efficacy in an immunodeficient mouse model.<br><br>DISCUSSION: Our findings reveal a key limitation of murine xenograft models, which are widely used as the gold standard for preclinical CAR T cell testing. The lack of conservation between the human and murine SELPLG proteins likely accounts for the discrepancy between enhanced in vitro migration of SELPLG-deficient L1CAM-CAR T cells and their lack of improved efficacy in the mouse model. This underscores the need for more predictive human-relevant models to better preclinically evaluate CAR T cell function.},
}
@article {pmid41394966,
year = {2025},
author = {Punde, A and Dey, S and Pandire, R and Bhattacharjee, A and Patra, C},
title = {Expanding the CRISPR/Cas toolkit: applications in proteomics and theranostics.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1713700},
pmid = {41394966},
issn = {2296-4185},
abstract = {Conventional methods available for genome editing have proven non-specific, labour-intensive, and time-consuming. In this context, CRISPR/Cas technology represents a significant breakthrough. It is derived from a sophisticated microbial defence system consisting of clustered regularly interspaced short palindromic repeats, or CRISPR, and the RNA-guided DNA endonuclease Cas. Beyond its original role in genome editing, CRISPR continues to play a major role in the field of proteomics, functional genomics, and molecular therapy. Animal models, including mice, Drosophila, zebrafish, etc., have substantially benefited from CRISPR in uncovering protein function through reverse genetics approaches, including knock-in, knockout, CRISPRi, and indel mutation strategies. On the clinical front, CRISPR gene therapy has also seen successes, including applications in sickle cell disease, hypercholesterolemia, and cancer immunotherapy. However, notable challenges remain, including in vivo packaging and delivery efficiency, toxicity, and genomic off-target effects. Ongoing efforts to overcome these include the development of novel delivery formulations (e.g., nanoparticles, exosomes), artificial intelligence-guided experimental design, and miniaturization of Cas proteins. This review focuses on CRISPR/Cas gene editing mechanisms and explores its state-of-the-art applications in the field of proteomics and theranostics.},
}
@article {pmid41395238,
year = {2025},
author = {Zou, Y and Yao, ZW and Xiao, T and Ma, YR and He, J and Chen, LM and Chen, XQ and Chen, N},
title = {Emerging Trichomonad Infections in Companion Animals: Rapid Visual Detection of Pentatrichomonas hominis and Tritrichomonas foetus Using an RPA-CRISPR/Cas12a Assay.},
journal = {Transboundary and emerging diseases},
volume = {2025},
number = {},
pages = {9995679},
pmid = {41395238},
issn = {1865-1682},
mesh = {Animals ; Cats ; *Protozoan Infections, Animal/diagnosis/parasitology ; *Tritrichomonas foetus/isolation &amp; purification/genetics ; Dogs ; *Trichomonadida/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Cat Diseases/parasitology/diagnosis ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Pets ; *Dog Diseases/diagnosis/parasitology ; },
abstract = {Pentatrichomonas hominis (P. hominis) and Tritrichomonas foetus (T. foetus) are prevalent intestinal protozoa. P. hominis is associated with chronic diarrhea in humans and animals, whereas T. foetus causes gastrointestinal disease in companion animals and reproductive-tract infection in cattle. Rapid and accurate identification of these infections at the point-of-care (POC) is crucial for the diagnosis and effective management of zoonotic diseases. In this study, we developed two novel recombinase polymerase amplification (RPA) assays coupled with CRISPR/Cas12a detection. The dual-species assay, using a lateral-flow format, targeted species-specific regions of the 18S rRNA gene of P. hominis and T. foetus, and under ideal conditions, delivered visual results within 40 min for a single sample at 37°C. P. hominis-specific assay: To differentiate P. hominis in mixed infections with T. foetus, a second assay targeted the highly conserved Spo11-1 gene of P. hominis. Optimal crRNA-412 and RPA primers were selected for maximal Cas12a cleavage efficiency. Analytical sensitivity and specificity were compared with conventional nested polymerase chain reaction (PCR) and Sanger sequencing. The results showed that The dual-species assay detected as few as 50 DNA copies/µL of either parasite with no cross-reactivity to Giardia lamblia, Cystoisospora canis, Cryptosporidium spp., Toxoplasma gondii, Toxocara canis, and Toxascaris leonina. Among 70 fecal samples of companion animal (48 dogs and 22 cats), 14 (29.2%) dogs tested positive for P. hominis, and eight cats (36.4%) tested positive for T. foetus by nested PCR. Due to financial and logistical constraints, we selected a smaller subset for subsequent analysis with the RPA-CRISPR/Cas12a lateral-flow strip (LFS) assay, which showed 100% diagnostic concordance with PCR. The Spo11-1 assay achieved a limit of detection of 20 DNA copies/µL and specifically recognized P. hominis among a panel that included seven non-target protozoa and helminths. Validation on 10 additional canine and feline samples (four positives and six negatives) showed complete agreement with nested-PCR results. In conclusion, this CRISPR-based diagnostic approach significantly enhances the efficiency and accuracy of Trichomonads detection, offering a practical, cost-effective solution particularly suitable for veterinary and potentially human healthcare diagnostics in resource-limited settings.},
}
@article {pmid41395656,
year = {2025},
author = {Lin, Z and Pu, Z and Wu, J and Zeng, J and Dou, Q and Mao, M and Zhang, Y},
title = {A Versatile CRISPR/Cas12a Autocatalytic Cascade System via Structure-Switching V-Type Split Probe for Highly Sensitive DNA Diagnostics.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {28079-28087},
doi = {10.1021/acs.analchem.5c06488},
pmid = {41395656},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry/genetics ; *DNA, Viral/analysis/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection of pathogen nucleic acids is critical for controlling infectious disease outbreaks and providing timely treatment. However, current molecular diagnostic applications, including sensitive CRISPR/Cas-based detection systems, rely on target preamplification, which often requires expensive equipment and strict adherence to sometimes complex workflows. Here, we describe a rapid, simple, and amplification-free CRISPR/Cas-based diagnostic system that employs a structure-switching V-shaped DNA probe with a Cas12a recognition sequence split by an ssDNA loop to establish a positive feedback loop and a signal amplification cascade. This approach exhibited an ultralow background signal, rapid production of an exponential signal, and atto-molar sensitivity. It was incorporated into microfluidic and lateral flow assay applications for multiplex detection of distinct papillomavirus strains and point-of-care detection of monkeypox virus infections, respectively. The approach thus has significant potential for rapid and sensitive detection of specific pathogen-derived DNA targets in both clinical laboratory and point-of-care applications.},
}
@article {pmid41396047,
year = {2026},
author = {Matsuoka, T and Kano, S},
title = {Impact of patent-granting differences between Japan and the United States on patent protection for medical methods: insights from genome editing patents.},
journal = {Expert opinion on therapeutic patents},
volume = {36},
number = {2},
pages = {133-144},
doi = {10.1080/13543776.2025.2605318},
pmid = {41396047},
issn = {1744-7674},
mesh = {*Patents as Topic/legislation &amp; jurisprudence ; Japan ; United States ; Humans ; *Gene Editing/legislation &amp; jurisprudence ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Securing patents in multiple countries has become essential for the development of global medical products. However, differences in national patent systems result in varying patentability standards. Although global claim construction strategies have been applied in practice, these approaches have not yet been systematically organized.<br><br>AREA COVERED: This study examines how the patent scope for patent families of international applications related to genome editing technologies filed in 2013, differs between Japan, where medical method patents are prohibited, and the United States, where such patents are permitted.<br><br>EXPERT OPINION: For CRISPR-Cas system patents, claim structures varied significantly, even among the corresponding family patents. To navigate these differences, the following strategies were proposed for filing patents in countries that prohibit medical method patents such as Japan: Convert medical method claims in the U.S. into composition claims that include product inventions, as this process ensures that such claims allow for the enforcement of rights against the suppliers of infringing products.Clearly define the scope of the claimed use-inventions when specifying the characteristics of the product based on its effects.Explicitly describe cells produced by a specific manufacturing method within the claimed rights.},
}
@article {pmid41396964,
year = {2026},
author = {Jiang, C and Liu, Y and Han, W and Zou, D and Chen, K and Jiang, X and Ma, A and Wei, X},
title = {Regulation of Single and Multiple Genes in Bacillus amyloliquefaciens by an Evolution System In Vivo.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {88-98},
doi = {10.1021/acssynbio.5c00480},
pmid = {41396964},
issn = {2161-5063},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Directed Molecular Evolution/methods ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Cytidine Deaminase/genetics/metabolism ; Mutation ; },
abstract = {With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.},
}
@article {pmid41397585,
year = {2026},
author = {Hsu, CY and Polatova, D and Hamad, RH and Patel, PN and Akram, M and Singh, G and Arora, V and Nayak, PP and Kadhem, M and Hamzah, HF},
title = {Phage therapy in cancer treatment: Mechanisms, emerging innovations, and translational progress.},
journal = {Critical reviews in oncology/hematology},
volume = {218},
number = {},
pages = {105085},
doi = {10.1016/j.critrevonc.2025.105085},
pmid = {41397585},
issn = {1879-0461},
mesh = {Humans ; *Phage Therapy/methods ; *Neoplasms/therapy ; *Bacteriophages ; Animals ; Translational Research, Biomedical ; },
abstract = {Bacteriophage therapy has re-emerged as a rapidly advancing field in oncology, bridging antimicrobial precision with tumor-targeted biotherapy. Beyond infection control, phages are now recognized as programmable biological systems capable of eradicating multidrug-resistant (MDR) pathogens, modulating tumor-associated microbiota, activating immune responses, and delivering therapeutic genes or drugs. Preclinical evidence shows that phages can selectively eliminate Fusobacterium nucleatum in oral squamous cell carcinoma, restore microbial balance in colorectal cancer, and enhance immune infiltration via cytokine or antigen display. Engineered constructs including GM-CSF-expressing and MAGE-A1-displaying phages, λ-phage ASPH vaccines, and PEGylated nanocarriers delivering MEG3 or TRAIL have demonstrated strong anti-tumor efficacy across melanoma, hepatocellular, and colorectal cancer models. Additionally, CRISPR-Cas-armed phages precisely remove resistance genes such as bla-CTX-M and mecA, while AI-driven selection pipelines enable data-guided design of personalized phage cocktails. These advances represent a paradigm shift from empirical antibacterial use toward mechanistically engineered, multifunctional phage platforms that integrate microbiome modulation, immune activation, and nanocarrier-mediated gene delivery. Although challenges such as immune clearance, bacterial resistance, and regulatory complexity remain, the convergence of AI, CRISPR, and synthetic biology is accelerating the evolution of phage therapy into a clinically viable precision-oncology strategy. In this context, bacteriophages emerge not merely as antibacterial agents but as intelligent, patient-specific nanomedicines poised to redefine therapeutic boundaries in cancer treatment.},
}
@article {pmid41397613,
year = {2026},
author = {Yin, C and Chen, B and Zheng, X and Wang, N and Wang, J and Li, R and Li, J and Yao, S and Zhai, Y and Song, X},
title = {Portable visual platform integrates polymerase spiral amplification and CRISPR/Cas12a for foodborne bacteria point-of-care testing.},
journal = {Journal of dairy science},
volume = {109},
number = {2},
pages = {1036-1051},
doi = {10.3168/jds.2025-27493},
pmid = {41397613},
issn = {1525-3198},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics ; *Point-of-Care Testing ; CRISPR-Cas Systems ; Animals ; Nucleic Acid Amplification Techniques ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Staphylococcus aureus, a prominent global foodborne pathogen, frequently triggers epidemics with severe public health impacts. Timely and reliable detection of S. aureus is crucial for mitigating the disease burden in low- and middle-income countries. However, conventional laboratory-based detection methods remain impractical in resource-limited settings, highlighting the urgent need for accessible point-of-care solutions. Here, we present an inner-outer-tube (IOT) assay that synergistically integrates the polymerase spiral amplification (PSR) technology for enhanced sensitivity with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a (Cas12a) system for sequence-specific identification. Additionally, we have created a portable all-in-one mobile detection (PAMD) device that combines all the steps needed for testing in the field, allowing for quick visual detection of S. aureus in just 60 min. The PSR-CRISPR/Cas12a-IOT method implemented with the PAMD device achieves a detection limit of 10 cfu/mL without needing extra preparation or costly equipment. The detection platform developed in this work has advantages of ease of operation, manageable costs, and robust performance, making it highly ideal for low-resource contexts and on-site detection scenarios. Furthermore, the PSR-CRISPR/Cas12a-IOT-PAMD detection platform provides global versatility through the interchangeable use of primer sets, hence broadening its applicability to various infections.},
}
@article {pmid41397899,
year = {2026},
author = {Chowdhury, A and Garcia, BG and Zahoor, MA and ElMawla, NF and Davidson, AR and Wyatt, HDM and Maxwell, KL and Mahassine, A and Gehring, A and Feld, JJ},
title = {A Rapid Assay for Hepatitis C Virus RNA Detection Using Reverse-Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12b-Based Strategy.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {5},
pages = {840-847},
doi = {10.1093/infdis/jiaf609},
pmid = {41397899},
issn = {1537-6613},
mesh = {Humans ; *Hepacivirus/genetics/isolation &amp; purification ; *Hepatitis C/diagnosis/virology ; *RNA, Viral/genetics/blood ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Genotype ; Limit of Detection ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for 2 tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid, and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations.<br><br>METHODS: After RNA extraction from plasma, HCV RNA is reverse-transcribed and amplified using loop-mediated isothermal amplification with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout.<br><br>RESULTS: HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a, and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/mL (approximately 100&#x2005;IU/mL). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500&#x2005;&#x3bc;L of plasma, genotype 1 RNA &#x2265;100&#x2005;IU/mL was detected within 40-45&#x2005;minutes, while genotype 3 had an LLOD of 5000&#x2005;IU/mL. The clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/hepatitis B virus (HBV) coinfection. The specificity was 100%, with no false-positives in 33 HCV-negative samples, including those with HBV or human immunodeficiency virus/HBV coinfection.<br><br>CONCLUSIONS: Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45&#x2005;minutes and hence could be used for efficient screening and diagnosis of HCV infection globally.},
}
@article {pmid41397982,
year = {2025},
author = {Okuwa, T and Himeda, T and Kobayashi, K and Nomura, N and Utani, K and Koike, S and Nakamura, A and Higuchi, M},
title = {Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {534},
pmid = {41397982},
issn = {2041-1723},
support = {25K10386//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21K07045//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24K10234//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25fk0108716//Japan Agency for Medical Research and Development (AMED)/ ; S2023-4//Kanazawa Medical University/ ; K2024-3//Kanazawa Medical University/ ; },
mesh = {Humans ; *Integrins/metabolism/genetics ; *Glycosaminoglycans/metabolism ; HeLa Cells ; Virus Internalization ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism ; Virus Attachment ; Host-Pathogen Interactions ; Gene Knockout Techniques ; },
abstract = {Saffold virus (SAFV), a member of the species Cardiovirus saffoldi within the Picornaviridae family, causes acute respiratory and gastrointestinal illnesses as well as hand, foot, and mouth disease. It is also suspected to be associated with neuronal disorders, such as encephalitis and meningitis, in severe cases. Despite its clinical significance, the virus-host interactions underlying SAFV pathogenicity remain largely unknown. Using a genome-wide CRISPR-Cas9 knockout screen, we identify the following receptors for SAFV infection: sulfated glycosaminoglycans (GAGs) and integrin αVβ8. Single knockouts of SLC35B2, an essential gene for sulfated GAG synthesis, or the integrin genes ITGAV or ITGB8 partially reduce SAFV-3 and SAFV-2 susceptibility in HeLa cells, and a double knockout confers complete resistance. Furthermore, we demonstrate that SAFV-3 virions bind directly to sulfated GAGs and integrin αVβ8. Based on these findings, we propose a model of SAFV infection in which sulfated GAGs and integrin αVβ8 act through dual and cooperative pathways to facilitate viral entry.},
}
@article {pmid41397984,
year = {2025},
author = {Chen, J and Hu, L and Vernuccio, R and Shi, N and Tian, J and Zhang, Y and Tian, S and Cao, X and Ha, Z and Lu, J and Battini, L and Raynal, B and Haouz, A and Xue, J and Cai, Q and Zhao, Y and Lu, Y and Smith, GL and Xie, Y and Lu, H and Guardado-Calvo, P and Zhang, P and Zhang, R},
title = {Development of a replication-defective mpox virus platform for fundamental and therapeutic research.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {798},
pmid = {41397984},
issn = {2041-1723},
mesh = {*Virus Replication/drug effects/genetics ; Humans ; *Antiviral Agents/pharmacology/chemistry ; Animals ; CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial/genetics ; Genome, Viral ; *Orthopoxvirus/genetics/drug effects/physiology ; Virion/genetics/drug effects ; *Defective Viruses/genetics/drug effects ; Virus Assembly/drug effects/genetics ; },
abstract = {The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying the mpox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineer replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5 L)-genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. We apply this system to screen a compound library and identify G243-1720, a potent anti-poxvirus inhibitor with broad activity in vitro and in vivo. G243-1720 blocks the formation of extracellular enveloped virions and cell-cell spread. Resistance mutation selection, crystallographic analysis, analytical ultracentrifugation, and mass photometry reveal that, despite its distinct chemical structure, G243-1720 shares a mode of action with tecovirimat, both functioning by affecting dimerization of protein OPG57 (F13). Our findings underscore the potential of G243-1720 as a promising broad-spectrum anti-poxvirus lead compound and demonstrate the utility of replication-defective MPXV particles as a reliable platform for viral biology studies and antiviral development.},
}
@article {pmid41398311,
year = {2025},
author = {Kang, YJ and Ha, HJ and Jin, HB and Lee, SY and Park, HH},
title = {Structural basis of dimerization and cascade formation by Cas5.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2976},
pmid = {41398311},
issn = {2045-2322},
support = {2025//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea/ ; },
mesh = {*Protein Multimerization ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; Models, Molecular ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Conformation ; },
abstract = {CRISPR-Cas systems are essential for prokaryotic adaptive immune mechanisms; however, the structural details of many subtype-specific components remain unclear. Herein, we report the crystal structure and biophysical characterization of Cas5 from Moraxella bovoculi (MboCas5), a component of the type I-C CRISPR-Cas system. We found that M. bovoculi encodes both type I-C and type III-B systems, and that MboCas5 forms a dimer that is stabilized by key interactions, including a salt bridge between R72 and D167. Structural comparisons with other Cas5 homologs and AlphaFold 3 predictions further validated the unique dimer configuration, suggesting that it is conserved across species. Additionally, structural comparison revealed a highly flexible loop region, which likely undergoes conformational changes upon Cascade assembly and might mediate interactions with Cas8 and crRNA. Overall, the findings provided structural and mechanistic insights into Cas5 function and could potentially contribute to our understanding of the assembly of type I-C Cascade complexes.},
}
@article {pmid41398410,
year = {2025},
author = {Chi, H and Hoikkala, V and McMahon, S and Graham, S and Gloster, T and White, MF},
title = {Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {889},
pmid = {41398410},
issn = {2041-1723},
support = {101018608//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Crystallography, X-Ray ; *CRISPR-Cas Systems ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Bacteriophages/genetics ; Nucleotides, Cyclic/metabolism ; Oligoribonucleotides/metabolism ; },
abstract = {Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.},
}
@article {pmid41399197,
year = {2026},
author = {Singh, K and Sharma, S and Kalia, A and Manchanda, P},
title = {Advancement in Mushroom Transformation: From Conventional Techniques to Modern Genetic Engineering.},
journal = {Journal of basic microbiology},
volume = {66},
number = {1},
pages = {e70132},
doi = {10.1002/jobm.70132},
pmid = {41399197},
issn = {1521-4028},
mesh = {*Genetic Engineering/methods ; *Agaricales/genetics/growth &amp; development ; *Transformation, Genetic ; CRISPR-Cas Systems ; Gene Editing ; Agrobacterium/genetics ; },
abstract = {Mushrooms have long been valued for their nutritional, pharmaceutical, and culinary benefits. Recent studies showcased mushrooms as bio-factories for protein production, and as a source of value-added products by employing genetic manipulation and molecular transformation techniques. Advancements in molecular tools and transformation methods have enhanced the efficiency of genetic improvements in mushrooms by both conventional and modern genetic engineering techniques, paving the way for their use in various industrial applications. Genetic transformation in mushrooms involves transferring genes within and across species to understand gene functions and improve mushroom qualities. The techniques involved in transformation includes Agrobacterium-mediated transformation, hybridization, mutation breeding, particle bombardment, protoplast fusion, and CRISPR/Cas9. This review outlines the life cycle of mushrooms, major difficulties in mushroom transformation, various transformation techniques, their history, efficiency, and success rate. It also highlights the potential of genetic engineering to revolutionize mushroom cultivation and their applications.},
}
@article {pmid41399500,
year = {2025},
author = {Swartjes, T and Bouzetos, E and Adiego-Pérez, B and Pool, VD and Staals, RHJ and van der Oost, J and Wu, WY},
title = {Base editing both DNA strands in distinct editing windows with small CRISPR-associated effector Cas12f1.},
journal = {iScience},
volume = {28},
number = {12},
pages = {114033},
pmid = {41399500},
issn = {2589-0042},
abstract = {CRISPR-associated base editors have been established as genome editing tools that enable base conversions in targeted DNA sequences, without generating double-strand breaks. Here, we describe the development of new base editors based on CRISPR-Cas12f1, a miniature Cas protein of only 422 amino acids. Chimeric constructs have been generated by fusing a catalytically inactive dCas12f1, to either a cytosine deaminase or an adenine deaminase. Using these synthetic fusion proteins, systematic analyses have been performed on base editing of a target sequence on a plasmid in Escherichia coli. Interestingly, apart from the previously described base editing of the displaced non-target DNA strand, we also observed efficient editing of the target DNA strand. This effect was not observed for Un1Cas12f1 BEs. In addition to the small size of AsCas12f1 base editors, its unique editing profile makes it a valuable addition to the CRISPR-Cas toolbox.},
}
@article {pmid41400455,
year = {2026},
author = {Mukherjee, S and Kumar, M},
title = {CRISPR: a precise genome editing strategy for the treatment of hepatocellular carcinoma.},
journal = {Expert review of anticancer therapy},
volume = {26},
number = {5},
pages = {599-614},
doi = {10.1080/14737140.2025.2606090},
pmid = {41400455},
issn = {1744-8328},
mesh = {Humans ; *Carcinoma, Hepatocellular/therapy/genetics/pathology ; *Liver Neoplasms/therapy/genetics/pathology ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; Prognosis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Genetic Therapy/methods ; Receptors, Chimeric Antigen ; },
abstract = {INTRODUCTION: The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing tool provides novel therapeutic alternatives by promoting the gene alteration in adaptive T cells or malignant cells to combat Hepatocellular Carcinoma (HCC). More successful cancer treatments are now possible due to the capacity of precisely locating and modifying particular genetic abnormalities that promote malignancy growth and metastasis.<br><br>AREAS COVERED: In this review, we address ongoing clinical trials, the possible similarities between CRISPR-based cancer treatments and current therapeutic choices, and how CRISPR technology can improve treatment outcomes for HCC while using the latest safety measures. Additionally, this analysis sheds light on the existing obstacles and potential future possibilities of applying CRISPR technology to the management of HCC, with a final objective of enhancing patient results and completely changing the field of HCC therapies.<br><br>EXPERT OPINION: The urgent need for innovative therapies is underscored by the poor prognosis associated with severe hepatocellular carcinoma, despite recent advancements in clinical therapies. Through a special emphasis on invivo cancer cell targeting along with the generation of chimeric antigen receptor (CAR) T cells, including T cell receptor (TCR) T cells, this review analyses the uses of CRISPR methods in the therapy of HCC.},
}
@article {pmid41401738,
year = {2026},
author = {Feng, J and Lin, X and Kang, L and Duan, M and Duan, N and Wang, Z and Wu, S},
title = {Integrating a microfluidic chip@LFA biosensor enabled by Pt3Sn@MGO nanocomposites for RAA/CRISPR-Cas12b mediated food adulteration monitoring.},
journal = {Biosensors &amp; bioelectronics},
volume = {296},
number = {},
pages = {118315},
doi = {10.1016/j.bios.2025.118315},
pmid = {41401738},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; Animals ; *Food Contamination/analysis ; *Nanocomposites/chemistry ; Salmon/genetics ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques ; Food Analysis ; Recombinases/chemistry ; Myoglobin/genetics ; },
abstract = {Traditional methods for food species genetic authentication typically involve time-consuming laboratory procedures and inconsistent operations, easily resulting in gene damage and inaccurate diagnostics. Here, by fully integrating reagent flow and reactions through micromachining technology, a standardized and lab-free operational PMMA-based microfluidic chip@ lateral flow assay (LFA) biosensor was developed for salmon adulteration detection. The primers recognizing the myoglobin nuclear gene of salmon were designed and optimized, enabling efficient recombinase-aided amplification (RAA) of target gene and subsequent activation of the CRISPR-Cas12b system. A multifunctional Pt3Sn@MGO nanocomposite was synthesized with enhanced FRET efficiency and photothermal properties, then employed as a signal probe in LFA test strip, achieving fluorescent, photothermal, and colorimetric quantitative detection of salmon contents in mixed samples, with detection limits of 0.007 %, 0.092 %, and 0.153 %, respectively, salmon contents in commercially products were evaluated to verify the practicality. This work presents an integrated, portable, and automation-enabled platform for standardized genetic authentication of food adulteration, which would be utilized as a universal lab-free method for on-site adulteration monitoring and species gene diagnostics by matching conserved genes and primer designs.},
}
@article {pmid41402279,
year = {2025},
author = {Wang, D and Ritz, C and Luo, Y and Suresh, A and Pierce, A and Veo, B and Brunt, B and Dahl, N and Serkova, N and Venkataraman, S and Danis, E and Kus, K and Mazan, M and Rzymski, T and Vibhakar, R},
title = {Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11152},
pmid = {41402279},
issn = {2041-1723},
support = {P30 CA046934/CA/NCI NIH HHS/United States ; },
mesh = {*Medulloblastoma/genetics/metabolism/pathology/drug therapy ; Humans ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Cyclin-Dependent Kinase 8/metabolism/genetics/antagonists &amp; inhibitors ; *Protein Biosynthesis/genetics ; Animals ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Mice ; *Cerebellar Neoplasms/genetics/metabolism/pathology/drug therapy ; Transcription, Genetic ; TOR Serine-Threonine Kinases/metabolism/antagonists &amp; inhibitors ; RNA Polymerase II/metabolism ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening, we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover, we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program, as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets, reduces ribosome biogenesis, and impairs protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore, combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo, leading to more pronounced tumor growth suppression. Overall, our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB.},
}
@article {pmid41402283,
year = {2025},
author = {Sivanandan, S and Leitmann, B and Lubeck, E and Sultan, MM and Stanitsas, P and Ranu, N and Ewer, A and Mancuso, JE and Phillips, ZF and Kim, A and Bisognano, JW and Cesarek, J and Ruggiu, F and Feldman, D and Koller, D and Sharon, E and Kaykas, A and Salick, MR and Chu, C},
title = {A pooled Cell Painting CRISPR screening platform enables de novo inference of gene function by self-supervised deep learning.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {77},
pmid = {41402283},
issn = {2041-1723},
mesh = {*Deep Learning ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Pooled CRISPR screening enables large-scale interrogation of gene functions but typically measures simple phenotypes such as fitness. High-content methods like Perturb-seq extend dimensionality to transcriptomics but are costly and limited in scope. Optical pooled screening (OPS) combines pooled CRISPR screening with imaging to yield scalable, information-rich readouts, yet existing implementations remain pathway-specific. Here we describe an OPS-compatible Cell Painting platform that enables hypothesis-free reverse genetic screening through multiplexed morphological profiling. We validate this technique using a well-defined morphological gene set, compare classical image analysis to self-supervised learning methods using a mechanism-of-action library, and perform discovery screening with a druggable genome library. By combining rich morphological data with deep learning, gene networks emerge without the need for target-specific biomarkers, leading to unbiased discovery of gene functions.},
}
@article {pmid41402624,
year = {2026},
author = {Sinkunas, T and Tamulaitiene, G},
title = {A DNA mimic jams the Cas9 scissors.},
journal = {The FEBS journal},
volume = {293},
number = {9},
pages = {2555-2559},
doi = {10.1111/febs.70374},
pmid = {41402624},
issn = {1742-4658},
support = {S-MIP-20-39//Lietuvos Mokslo Taryba/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Associated Protein 9 ; *CRISPR-Associated Proteins/metabolism/antagonists &amp; inhibitors/chemistry/genetics ; *Endonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry/genetics/antagonists &amp; inhibitors ; },
abstract = {Anti-CRISPR (Acr) proteins are small protein inhibitors that block the RNA-guided nucleic acid (DNA or RNA) targeting activity of CRISPR-Cas enzymes. Despite their shared function, Acr proteins display minimal sequence or structural similarity and employ diverse mechanisms to block nuclease activity. Lee and Park characterized the previously undescribed AcrIIA13b protein, which inhibits Cas9 protein. Structural, biochemical, and mutational analyses revealed that AcrIIA13b acts as a DNA mimic, thereby disabling the Cas9 complex from binding to the DNA target.},
}
@article {pmid41402634,
year = {2025},
author = {Oliynyk, RT and Mahas, A and Karpinski, E and Church, GM},
title = {Plasmid2MC: efficient cell-free generation of high-purity minicircle DNA for genome editing in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1778},
pmid = {41402634},
issn = {2399-3642},
mesh = {*Gene Editing/methods ; *Plasmids/genetics ; Humans ; HEK293 Cells ; Animals ; *DNA, Circular/genetics ; Mice ; CRISPR-Cas Systems ; Cell-Free System ; Mouse Embryonic Stem Cells/metabolism ; },
abstract = {DNA plasmids are widely used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA), which lacks such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce endotoxin-contaminated low-quality mcDNA. To address this challenge, we developed Plasmid2MC, a novel cell-free method utilizing ΦC31 integrase-mediated recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, followed by digestion of the bacterial backbone and all other DNA contaminants, resulting in highly pure and virtually endotoxin-free mcDNA. We demonstrated the application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The method's ease of preparation, high efficiency, and the high purity of the resulting mcDNA make Plasmid2MC a valuable tool for applications requiring bacterial backbone-free circular DNA.},
}
@article {pmid41402770,
year = {2025},
author = {Jiang, M and Zhang, K and Wang, Z and Gao, M and Su, S and He, J and Xu, H and Bo, Z and Jiang, Z and Zhang, C and Hui, JH and Wei, R},
title = {Nanomaterials in gene therapy and genome editing: challenges and emerging directions.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {56},
pmid = {41402770},
issn = {1477-3155},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Nanostructures/chemistry/therapeutic use ; Animals ; CRISPR-Cas Systems ; Nanoparticles/chemistry ; Lipids/chemistry ; },
abstract = {Nanomaterials are redefining the landscape of gene and genome editing, yet their translation to clinical reality remains constrained by multiple unresolved challenges. While they provide structural and functional advantages for delivering nucleic acids and CRISPR/Cas systems across biological barriers, their behavior within living systems is often unpredictable, leading to issues such as off-target editing, immune activation, and inconsistent biodistribution. The design of nanocarriers, whether lipid-based, polymeric, inorganic, must therefore balance efficiency with safety, integrating physicochemical precision with biological adaptability. Recent advances in ionizable lipid nanoparticles demonstrate how fine-tuning charge, surface chemistry, and degradation kinetics can enhance endosomal escape and target specificity, but reproducibility and large-scale manufacturing continue to limit broader application. Moreover, polymeric and exosome-inspired systems promise modularity and targeted reuse, yet they demand clearer understanding of long-term biocompatibility and regulatory acceptance. The future of nanomaterial-enabled genome engineering depends not only on optimizing delivery vehicles but also on establishing predictive models of nano-bio interactions, harmonizing ethical oversight, and developing standardized evaluation pipelines that link nanoscale design to therapeutic outcomes.},
}
@article {pmid41403193,
year = {2025},
author = {Su, S and Zuo, Y and Ma, B and Zhao, Z and Wang, X and Zhang, X and Ignatus, AD and Piñero, JC and Peng, X and Li, F and Chen, M},
title = {Functional Validation of GmGSTs2 in the Resistance to Abamectin in the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae).},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {52},
pages = {33033-33045},
doi = {10.1021/acs.jafc.5c12427},
pmid = {41403193},
issn = {1520-5118},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/pharmacology ; *Moths/genetics/drug effects/enzymology/growth &amp; development ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance ; CRISPR-Cas Systems ; Larva/drug effects/genetics/growth &amp; development/enzymology ; },
abstract = {Abamectin has been used for decades as an insecticide and acaricide in arthropod pest management. However, there is no direct evidence from CRISPR/Cas9 studies confirming the involvement of GSTs in insect resistance to abamectin. The oriental fruit moth, Grapholita molesta, is a destructive pest of fruit trees worldwide. The role of GSTs in the oriental fruit moth remains unclear. In this study, an abamectin-resistant strain (AB-R) was derived from a susceptible laboratory strain (AB-S) of G. molesta. Synergist bioassays showed that the GST inhibitor diethyl maleate (DEM) significantly increased abamectin toxicity in AB-R. Biochemical assays indicated that glutathione S-transferase (GST) activity in AB-R was 1.63-fold higher than in AB-S. Among 25 GST genes examined, GmGSTs2 showed the largest expression difference between AB-R and AB-S and was expressed across developmental stages and body parts. Recombinant GmGSTs2 significantly reduced the effective quantity of abamectin in vitro. CRISPR/Cas9 knockout of GmGSTs2 in both genetic backgrounds increased susceptibility to abamectin and significantly affected the development and survival of G. molesta. The transgenic Drosophila melanogaster strain expressing GmGSTs2 showed an LC50 of 74.12 mg L[-1] (34.59-126.63) versus 25.48 mg L[-1] (12.28-39.82) in W[1118] controls, indicating a 2.91-fold difference. Together, synergism assays, enzyme activity measurements, in vitro metabolism, CRISPR knockout in both resistant and susceptible backgrounds, and a heterologous in vivo assay identify GmGSTs2 as a key metabolic driver of abamectin resistance in G. molesta, providing a practical target for resistance management.},
}
@article {pmid41403730,
year = {2025},
author = {Khari, M and Jain, N and Kaul, S and Pandey, M and Sharma, N},
title = {siRNA and mRNA-Based Preventive and Therapeutic Strategies for HPV-Induced Cervical Cancer.},
journal = {Advanced pharmaceutical bulletin},
volume = {15},
number = {3},
pages = {552-573},
pmid = {41403730},
issn = {2228-5881},
abstract = {Human papillomavirus (HPV), specifically types 16 and 18, is the main cause of cervical cancer and a significant cause of death among women. Specifically, HPV E6 and E7 oncogenes hinder the normal cell cycle regulation, resulting in uncontrolled cell growth and cervical cancer. The available therapy options include surgery, radiotherapy, and chemotherapy, which show success but also demonstrate notable complications. SiRNA (small interfering RNA) and mRNA (messenger RNA) therapies have emerged as precise and effective tools to silence the HPV E6 and E7 oncogenes and stimulate the immune system to fight against HPV infection, respectively, presenting a targeted therapy approach and overcoming the available therapy challenges. Nanoparticles and Pegylated liposomes are the delivery systems that increase the efficacy and safety of siRNA and mRNA therapies. This review critically appreciates the effective targeting of siRNA and mRNA-based therapies by highlighting their key advantages and limitations. Despite being a target-specific and effective approach, there are certain challenges like scale-up, cost-effectiveness, and developing stable delivery systems, which are required to be discussed. In addition, other precision medicine approaches, such as CRISPR/CAS-9, antisense oligonucleotides, or immunotherapy, have also been included as compared to siRNA/mRNA therapies. Their preclinical, patent, and clinical translations have also been discussed exhaustively.},
}
@article {pmid41404141,
year = {2025},
author = {Shilpha, J and Kang, WH},
title = {Molecular and genomic insights into viral resistance in Capsicum spp.: pathogenesis, defense mechanisms, and breeding innovations.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1716114},
pmid = {41404141},
issn = {1664-462X},
abstract = {Plant viruses represent a major challenge to agricultural systems, threatening global food security amid a rising population. Specifically, pepper cultivation (Capsicum annuum L.) is often hindered by various viral diseases, with more than 60 viruses identified as affecting pepper plants. The most efficient strategy for controlling viral diseases is the development of resistant cultivars of peppers. A comprehensive understanding of complex interactions between plant defense mechanisms and the strategies employed by viruses to evade these defenses, coupled with host factors that facilitate viral replication and movement, is essential for developing resistant cultivars. Natural antiviral defense mechanisms in plants are well characterized and include resistance genes, RNA silencing, autophagy-mediated degradation, translational repression, and resistance to viral movement. Recent advances in next-generation sequencing (NGS), genome-wide association studies (GWAS), high-density genotyping platforms and gene-editing tools such as CRISPR/Cas have accelerated the identification of resistance loci and key host factors involved in viral pathogenesis. This review summarizes current molecular and genomic insights into virus-host interactions in Capsicum spp., highlighting their role in advancing marker-assisted selection (MAS) and genomic-assisted breeding. The integration of molecular markers and genome editing into breeding pipelines offers new opportunities for developing durable, broad-spectrum viral resistance in peppers, ultimately supporting sustainable crop production and agricultural resilience.},
}
@article {pmid41404500,
year = {2025},
author = {Hundal, T and Luo, Y and Qie, Y and Gadd, ME and Brim, AD and Vazquez-Rosario, I and Guo, S and Kharfan-Dabaja, MA and Qin, H},
title = {Novel allogeneic CAR T-cell platform involving microhomology-mediated end joining repair and low off-targeting potential.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102778},
pmid = {41404500},
issn = {2162-2531},
abstract = {Several allogeneic chimeric antigen receptor (CAR) T-cell therapies in clinical trials rely on CRISPR-Cas genome editing, but the enzyme's random repair mechanism increases the risk of undesired off-target effects, challenging safe CAR T-cell generation. To address this, we developed a novel CRISPR RNA (crRNA) targeting the T-cell receptor beta constant (TRBC) gene. Combined with AsCas12a Ultra, this crRNA edits primary human T-cells via a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, significantly lowering off-target risks. During evaluation, we sequestered a unique T-cell subset with disrupted T-cell receptor (TCR), retained CD3 expression, and no in vivo alloreactivity. Termed CD3-retained, allogeneically functioning T-cells (CRAFT-cells), these cells exhibited growth kinetics comparable to unedited T-cells. When engineered with CD19- or BAFF-R-targeted CARs, CRAFT CAR T-cells showed strong antigen-specific cytotoxicity and significant ex vivo expansion compared to conventional CD3-disrupted CAR T-cells. Moreover, CRAFT CAR T-cells effectively served as effector cells for bispecific T-cell engagers (BiTEs), enabling CD3-dependent tumor cell killing. Our CRAFT crRNA platform offers a novel strategy to generate safer allogeneic CAR T-cells. The distinct properties of CRAFT CAR T-cells, combined with BiTE therapy, represent a promising and potentially more durable approach for next-generation allogeneic CAR T-cell therapies in clinical applications.},
}
@article {pmid41404502,
year = {2025},
author = {Gao, H and Gao, S and Kan, G and Valentovich, LN and An, Y},
title = {Research progress of base editing and prime editing tools based on the CRISPR/Cas system.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102771},
pmid = {41404502},
issn = {2162-2531},
abstract = {The base editor (BE) and prime editing guide RNA (pegRNA)-based prime editor (PE) technologies relying on the CRISPR/Cas system are very efficient gene editors that have been developed in recent years. The BEs include cytosine base editors (CBEs) that mediate the conversion of C to T, adenine base editors (ABEs) that mediate the conversion of A to G, glycosylase base editors (GBEs) that mediate the conversion of C to G, and the dual-base editors (DBEs) that mediate the simultaneous conversion of C to T and A to G. The BEs and PEs have been successfully applied for genome editing of various animals, plants, and microorganisms due to their advantages of high efficiency and independence of DNA double-strand breaks or donor DNA. The development process and characteristics of various BEs and PEs and their effectiveness of application are systematically introduced to provide a reference for selecting appropriate genome editing technologies. Moreover, the urgent issues that need to be addressed for more efficient and precise editing are summarized and prospected.},
}
@article {pmid41404798,
year = {2025},
author = {Yang, F and Xu, C and Li, C and Xiang, X and Zhao, Y and Hu, C and Rong, H and He, Y and Li, J and Wang, Y and Tang, C and Liu, X and Li, R and Deng, F and Xiang, T},
title = {Amplification-free cancer diagnosis based on inhibition of Cas12a activity by site-specific 5mC-modified cfDNA.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41404798},
issn = {1362-4962},
support = {82172619//National Natural Science Foundation of China/ ; YXQN202421//Outstanding Young Talents Program of Chongqing/ ; 2023nlts007//Enhancing Scientific Research Capabilities of Chongqing University Cancer Hospital/ ; 2024MSXM136//Chongqing Municipal Science and Health Joint Medical Research/ ; },
mesh = {Humans ; *DNA Methylation ; *CRISPR-Cas Systems/genetics ; *Cell-Free Nucleic Acids/genetics/blood/chemistry ; *Neoplasms/diagnosis/genetics/blood ; *CRISPR-Associated Proteins/metabolism/antagonists &amp; inhibitors/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Bacterial Proteins ; },
abstract = {DNA methylation detection holds significant value for cancer diagnosis and recurrence monitoring. However, current methods are often time-consuming, costly, and necessitate specialized techniques. The CRISPR-Cas system, particularly Cas12a, presents a precise and user-friendly platform for disease diagnosis. We developed the CRISPR-Methylated DNA Detection Test (CRISPR-MeDNA Test), a Cas12a-based method for detecting methylation in plasma cell-free DNA (cfDNA). The results reveal that 5mC-modified DNA significantly suppresses the trans-cleavage activity of Cas12a, depending on the methylation site, number, and interval spacing. Simultaneously, methylation of the non-target strand (NTS) suppresses Cas12a activity more strongly than methylation of the target strand (TS), as the NTS plays a critical role in R-loop formation, which is essential for Cas12a cleavage target DNA. Mechanistically, 5mC-modified DNA was found to trigger conformational rearrangements in the Cas12a complex, as predicted by AlphaFold3 modeling and corroborated by FRET assays. Notably, the combination of Cas12a with multiplexed guide RNAs enables effective discrimination between cfDNA from healthy donors and cancer patients without the need for pre-amplification, based on the inhibitory effects of methylated DNA on the Cas12a trans-cleavage activity. This work provides a Cas12a-based detection for a rapid, cost-effective, low-complexity method for 5mC-modified cfDNA in liquid biopsies.},
}
@article {pmid41405812,
year = {2025},
author = {Wang, L and Ren, S and Behan, AA and Buzdar, JA and Arain, MA and Li, Y},
title = {Promising Future of Engineered Probiotics for Antimicrobial Peptides and Protein Production: Prospects and Industrial Challenges.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41405812},
issn = {1867-1314},
abstract = {Antimicrobial resistance (AMR) is a critical global health challenge, necessitating innovative alternatives to conventional antibiotics to combat pathogenic microorganisms. Probiotics, traditionally valued for immunomodulation and gut health are emerging as programmable biotherapeutics capable of producing antimicrobial peptides (AMPs), such as bacteriocins, defensins. Recent advances in synthetic biology, CRISPR-based editing and multi-omics integration has transformed the engineering of probiotics, enabling pathway-level refinement of AMP yield, activity spectrum and physiological stability. Engineered strains can now express heterologous AMPs, target multidrug-resistant pathogens with higher specificity, and maintained functional robustness within the gastrointestinal milieu. Industrial translation is becoming feasible through metabolic engineering, and systems-guided strain design for improved biosynthesis efficiency and scalable production. Despite this progress, key challenges persist, including biosafety risks, ecological impact on native microbiota and regulatory berries for genetically modified organisms. This review synthesizes current advances in omics-informed metabolic rewiring CRISPR-enabled precision engineering, and translational strategies for industrial development of AMP production. Additionally, this review delineates mechanistic insights, engineering toolkits, manufacturing considerations, and conclude by outlining a forward roadmap that integrates molecular design with regulatory and scalability perspectives defining a coherent framework for next generation probiotics antimicrobials against AMR.},
}
@article {pmid41406514,
year = {2026},
author = {Ghodrat, R and Ramachandran, H and Hildebrandt, B and Binder, S and Rossi, A and Reichert, AS},
title = {CRISPR/Cpf1-mediated editing of PINK1 in induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103887},
doi = {10.1016/j.scr.2025.103887},
pmid = {41406514},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Protein Kinases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; },
abstract = {The PTEN induced kinase 1 (PINK1) gene is crucial for mitophagy and mitochondrial quality control. Mutations in the PINK1 gene are associated with several neurological disorders. To decipher the role of PINK1-mediated mitophagy in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with homozygous out-of-frame deletions by targeting exon 6 of the PINK1 gene. The generated homozygous PINK1 mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers.},
}
@article {pmid41406662,
year = {2026},
author = {Schalper, KT and Yang, R and Guan, X and Zhang, J and Schreiber, D and Moon, J and Liu, C},
title = {Programmable CRISPR-mediated gold nanoparticle adhesion for visual colorimetric detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118319},
pmid = {41406662},
issn = {1873-4235},
support = {U01 CA269147/CA/NCI NIH HHS/United States ; },
mesh = {*Colorimetry/methods ; *Gold/chemistry ; *Biosensing Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis/virology ; *DNA, Viral/isolation &amp; purification/genetics/analysis ; *Papillomaviridae/isolation &amp; purification/genetics ; Female ; Limit of Detection ; DNA, Single-Stranded/chemistry ; },
abstract = {While the aggregation behavior of gold nanoparticles (AuNPs) has been extensively studied in biosensing, catalysis, and nanomedicine, their potential for programmable surface adhesion via tunable surface chemistry remains largely untapped. Here, a programmable CRISPR-mediated hydrophobic adhesion phenomenon using streptavidin-coated AuNPs functionalized with Cy5-ssDNA-biotin probes is introduced. Hydrophobic Cy5 moieties on the AuNP surface induce localized aggregation and strong adhesion to hydrophobic surfaces. This unique behavior was leveraged by coupling CRISPR-Cas12a-mediated ssDNA cleavage with Cy5-labeled ssDNA-coated AuNPs to develop a simple, visual-readout colorimetric assay for nucleic acid detection. When combined with recombinase polymerase amplification, the method achieved ultrasensitive detection of human papillomavirus (HPV) DNA down to 10 aM, without the need for complex instrumentation. The platform's clinical utility was validated by detecting HPV DNA in cervical swab samples, highlighting its promise for low-cost, sensitive, and accessible point-of-care diagnostics in resource-limited settings. Unlike conventional aggregation-dispersion systems, this platform introduces a fundamentally distinct signal transduction mechanism based on surface adhesion, defining a new modality within CRISPR-based colorimetric diagnostics and offering a simple, low-cost solution for point-of-care testing.},
}
@article {pmid41406894,
year = {2026},
author = {Heinemann, JA and Hiscox, TC and Zanatta, CB and Kurenbach, B and Walker, S and McCabe, AW and Hoepers, AM and Agapito-Tenfen, SZ},
title = {Genome editing outside of controlled facilities: A review of plausible futures and risks.},
journal = {Ecotoxicology and environmental safety},
volume = {309},
number = {},
pages = {119565},
doi = {10.1016/j.ecoenv.2025.119565},
pmid = {41406894},
issn = {1090-2414},
mesh = {Animals ; Humans ; *Gene Editing/methods/trends ; Risk Assessment ; },
abstract = {Vectors for delivering proteins and/or nucleic acids into the cells of whole organisms, from single to multicellular, are rapidly advancing. Common cargos are nucleic acids needed to express the components of a genome editing reaction, or ribonucleoproteins (RNP) that can act immediately upon delivery. In only 20 years, improvements in associated formulation technologies have decreased the dependence of genome editing on the need for a laboratory or trained personnel, allowing for genome editing outside of controlled facilities. As this happens, both target and non-target organisms may be exposed to active biological agents, necessitating a new framework for risk assessment. Some scientists deny developments for gene editing in uncontrolled environments, leading to scientifically unjustified dismissals of risk.},
}
@article {pmid41407085,
year = {2026},
author = {Qi, H and Yang, Y and Hou, X and Chen, Y and Gong, S},
title = {Sensitivity-improving CRISPR-Cas strategies for non-nucleic acid targets detection.},
journal = {Methods (San Diego, Calif.)},
volume = {246},
number = {},
pages = {151-173},
doi = {10.1016/j.ymeth.2025.12.004},
pmid = {41407085},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Gene Editing/methods ; Nucleic Acids/genetics ; },
abstract = {CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains a critical bottleneck, limiting the sensitivity of such detection methods. To address this challenge, numerous innovative strategies have been proposed to enhance the sensitivity of CRISPR-Cas systems, enabling high-sensitive non-nucleic acid targets detection. This review systematically summarizes the sensitivity-enhancing methodologies for non-nucleic acid targets detection using CRISPR-Cas technologies. We first delineate the working mechanisms of various CRISPR-Cas systems and the signal transduction pathways specific to non-nucleic acid targets. Subsequently, we detail diverse sensitivity-improving approaches, including nucleic acid amplification-facilitated strategies, multimolecular labeling techniques, dual-enzyme cascade methods, and multiplex amplification methodologies. Additionally, the current challenges and future perspectives in this field are discussed, aiming to inspire researchers to develop more ingenious solutions and facilitate real-world applications of CRISPR-Cas system for non-nucleic acid targets detection.},
}
@article {pmid41407557,
year = {2025},
author = {Tyagi, E and Sachan, A and Bhuyan, R and Kumari, P and Prakash, A},
title = {Next-Gen Biofilm Control: Gene Editing and Computational Approaches.},
journal = {APMIS : acta pathologica, microbiologica, et immunologica Scandinavica},
volume = {133},
number = {12},
pages = {e70122},
doi = {10.1111/apm.70122},
pmid = {41407557},
issn = {1600-0463},
mesh = {*Biofilms/drug effects/growth &amp; development ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Phage Therapy ; Bacteriophages ; },
abstract = {Biofilms are microbial communities enclosed in an extracellular polymeric substance (EPS), significantly contributing to antimicrobial resistance (AMR) in medical, industrial, and environmental settings. Their matrix enhances microbial survival, inhibits antibiotic penetration, and facilitates horizontal gene transfer, worsening the AMR crisis. Conventional antimicrobial treatments often fail against biofilms, necessitating novel therapeutic strategies. Emerging biofilm-targeted interventions, such as nanotechnology-based antimicrobials, bacteriophage therapy, and CRISPR-Cas9 gene editing, offer promising solutions. Nanoparticles improve drug delivery, bacteriophages selectively lyse resistant bacterial populations, and CRISPR-Cas9 disrupts AMR-related genes and biofilm virulence factors. Additionally, AI and ML are advancing biofilm prediction models and antimicrobial optimization, paving the way for precision-targeted interventions. This review explores biofilm biology and next-generation biofilm control strategies, with a focus on AI-driven bioinformatics. Future research should focus on clinical translation, regulatory standardization, and scalable implementation in healthcare and industrial settings to combat biofilm-associated AMR.},
}
@article {pmid41407671,
year = {2025},
author = {Bitew, MA and Paredes-Santos, TC and Maru, P and Krishnamurthy, S and Wang, Y and Sangaré, LO and Duley, S and Yamaryo-Botté, Y and Botté, CY and Saeij, JPJ},
title = {A genome-wide CRISPR screen identifies GRA38 as a key regulator of lipid homeostasis during Toxoplasma gondii adaptation to lipid-rich conditions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11177},
pmid = {41407671},
issn = {2041-1723},
support = {R01 AI173803/AI/NIAID NIH HHS/United States ; R01AI173803//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Toxoplasma/genetics/metabolism/pathogenicity/physiology ; Animals ; *Protozoan Proteins/metabolism/genetics ; Homeostasis ; Mice ; *Lipid Metabolism/genetics ; Virulence/genetics ; Phosphatidate Phosphatase/metabolism/genetics ; Toxoplasmosis/parasitology ; Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; Female ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Genome, Protozoan ; },
abstract = {Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we perform a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identify the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 leads to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlate with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii.},
}
@article {pmid41409480,
year = {2025},
author = {Anwar, M and Vinothkanna, A and Jia, AQ},
title = {Fostering plant protection against certain bacterial diseases through quorum-sensing signal molecules: a critical review.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1602573},
pmid = {41409480},
issn = {1664-462X},
abstract = {Quorum sensing (QS) and clustered regularly interspaced short palindromic repeats (CRISPR) systems are envisaged as revolutionary in abating plant bacterial pathogens. Bacterial cell-cell communication and plant pathogen QSSMs (quorum sensing signaling molecules) are dissected for underlying mechanisms in prominent pathogens, viz., Pseudomonas syringae, Erwinia amylovora, and Xanthomonas campestris. Biofilm formation and virulence mechanisms are critically addressed to repurpose potential QS inhibition strategies. CRISPR technologies are combined with CRISPR engineering to produce enhanced disease-resistant varieties, with potential applications. QS-CRISPR interplay for deciphering the key interactive changes in plant health management is prioritized for deliberate future research outcomes. Sustainable agricultural practices are envisaged for successful lab-to-field authentic field trials and large-scale applicability across the globe. Potential technical limitations, the need for stringent agricultural laws, and future innovations are addressed. Moreover, the cost-effectiveness, enhanced crop production, yield, and productivity hindering the above key plant bacterial pathogens are comprehensively addressed against these plant bacterial pathogens. Furthermore, a future outlook characterized by extensive outreach and global implications is substantiated regardless of regional specificity, climate change, and global warming. A decade of research on advancements in adequate plant protection is revisited to incorporate augmented approaches, including artificial intelligence (AI) and machine learning, in sustainable agriculture. The significance of the present review is based on addressing QSSMs and plant protection strategies encompassing modern molecular biological techniques.},
}
@article {pmid41409641,
year = {2024},
author = {Zhang, Z and Ding, S},
title = {Gene editing and reprogramming of human fibroblast cells (hFBs) to human pluripotent stem cells (hiPSCs).},
journal = {Neuromethods},
volume = {210},
number = {},
pages = {39-59},
pmid = {41409641},
issn = {0893-2336},
support = {R01 NS069726/NS/NINDS NIH HHS/United States ; R01 NS123023/NS/NINDS NIH HHS/United States ; },
abstract = {Predictive disease models play significant roles in advancing our knowledge of the pathology of human disease. In this field, animal models have been extensively employed and have provided crucial insights into the pathophysiological mechanisms of human disease. However, they often fail to fully capture many human phenotypes due to significant species differences in genomic responses. Human induced pluripotent stem cells (hiPSCs) are genetically reprogrammed cells that exhibit qualities remarkably similar to those of embryonic stem cells (ESC) and have emerged as a promising source for cell therapy and fundamental research in pathology. The ability to reprogram human fibroblast cells (hFBs) to iPSCs provides an opportunity to model human diseases. However, even hiPSCs from different persons have different genetic background, thus generation of isogenic unaffected control hiPSCs is necessary to study model human disease. Here, we describe methods to generate isogenic hFBs using CRISPR/Cas9 gene editing method, and subsequently reprogram them into iPSCs using commercially available Sendai virus vectors. Specifically, using the CRISPR/Cas9 system and Sendai virus vector, isogenic iPSC lines can be generated. This protocol provides a structured approach for obtaining multiple isogeneic hiPSC lines, which facilitate the modeling of various human diseases.},
}
@article {pmid41410478,
year = {2026},
author = {Wang, X and Yu, G and Luo, Y and Chen, T and Zhang, X and Ye, L and Yang, C and Chen, Q},
title = {The autophagy-related protein PlAtg26b regulates vegetative growth, reproductive processes, autophagy, and pathogenicity in Peronophythora litchii.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2606498},
pmid = {41410478},
issn = {2150-5608},
mesh = {*Autophagy ; Virulence ; Plant Diseases/microbiology/parasitology ; *Autophagy-Related Proteins/genetics/metabolism ; Litchi/microbiology/parasitology ; *Oomycetes/pathogenicity/growth &amp; development/genetics ; CRISPR-Cas Systems ; Reproduction ; Plant Leaves/microbiology ; Mitochondria/metabolism ; Gene Knockout Techniques ; Hyphae/growth &amp; development ; },
abstract = {Peronophythora litchii is an oomycete pathogen responsible for litchi downy blight, a significant threat to global litchi production. Autophagy, a conserved degradation pathway crucial for the growth, development, and pathogenicity of phytopathogenic organisms, remains an area of active investigation. In this study, we characterized the function of the Atg26 homolog PlAtg26b in P. litchii. Using the CRISPR/Cas9 genome editing system, we generated PlATG26b knockout mutants and determined that PlAtg26b localizes to mitochondria under stress conditions. Although deletion of PlATG26b did not impair selective autophagy, it markedly reduced Atg8-PE synthesis, vegetative hyphal growth, asexual and sexual reproduction, and zoospore release. Furthermore, PlATG26b-deficient mutants exhibited significantly reduced virulence on litchi fruits and leaves. Collectively, our findings demonstrate that PlAtg26b plays a pivotal role in the biological development and pathogenicity of P. litchii.},
}
@article {pmid41410797,
year = {2025},
author = {Appolonia, CN and Centore, JT and Shukla, S and Hluck, J and Conlon, RA and Ramakrishnan, P},
title = {A CRISPR/Cas9 assisted strategy for the conditional expression of human NF-kappaB c-Rel cDNA in mouse T cells: design, prospects, and challenges.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {56},
pmid = {41410797},
issn = {1573-9368},
support = {I01 BX005941/BX/BLRD VA/United States ; R01 AI116730/AI/NIAID NIH HHS/United States ; R01 DK128463/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Transgenic ; *Proto-Oncogene Proteins c-rel/genetics ; Promoter Regions, Genetic ; Mice, Inbred NOD ; Gene Editing ; *T-Lymphocytes/metabolism ; DNA, Complementary/genetics ; *Diabetes Mellitus, Type 1/genetics ; NF-kappa B/genetics ; },
abstract = {Nuclear factor-κB protein c-Rel is a critical regulator of autoimmune diabetes. We found that c-Rel O-GlcNAcylation at serine-350 increases with hyperglycemia, which results in increased transcription of proautoimmune Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and decreased transcription of the T regulatory cell transcription factor forkhead box 3 (FOXP3). To further study the translational relevance of c-Rel S350 O-GlcNAcylation in autoimmune diabetes, we sought to generate transgenic non-obese diabetic (NOD) mice conditionally expressing wildtype or mutant S350A human c-Rel cDNA in T cells downstream of the endogenous mouse REL promoter. We used CRISPR-Cas9 gene editing to insert a unique designer cassette containing floxed mouse c-Rel cDNA-STOP sequence to maintain whole body c-Rel expression, followed by a linker and human c-Rel cDNA-STOP sequence. Using comprehensive PCR analyses and high-throughput sequencing, we confirmed successful insertion of the cassette at the mouse REL locus and the expected deletion of the mouse c-Rel cDNA specifically in T cells following CD4-Cre mating. Additional characterization revealed that the knock-in transgenic mice lacked endogenous mouse c-Rel, further confirming desired interference with its natural start codon. Unexpectedly, these mice lacked mouse and human c-Rel protein expression from inserted cDNAs, which mechanistically correlated with increased CpG methylation of the c-Rel promoter region. Thus, our study presents a unique, universal molecular design and method for the generation of conditional knock-in transgenic mice expressing human genes at the endogenous mouse promoter. It also reveals a potential locus-specific challenge that may arise during the development of such novel transgenic mouse models.},
}
@article {pmid41410807,
year = {2025},
author = {Sarroukh, I and Ibriz, M and Yakkou, L and Lebkiri, N and Fokar, M and Iraqi, D and Gaboun, F and Diria, G and Abdelwahd, R},
title = {The Agrobacterium-mediated genetic transformation: a gateway for efficient CRISPR/Cas9 gene editing in leguminous.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {57},
pmid = {41410807},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Transformation, Genetic/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Agrobacterium tumefaciens/genetics ; },
abstract = {Climate change enhances the damaging consequences of abiotic and biotic stressors, leading to severe soil fertility loss and ecosystem degradation worldwide. Leguminous have contributed significantly to replenishing soil nitrogen via symbiotic nitrogen fixation, contributing approximately 15% of nitrogen input, which is crucial for soil health and enhancing crop production. There is an increasing integration of new biotechnological interventions, such as genome editing, including the CRISPR/Cas9 system, and transgenesis, in addition to classical breeding, to make agriculture more resilient. In this review, we examine several elements that influence the genetic transformation system employing Agrobacterium tumefaciens strains in leguminous to make it an ideal vehicle for CRISPR/Cas9 component delivery. The variables investigated in our study included the incubation period, co-cultivation duration, bacterial density, selectable marker, concentration, and growth regulators used. In addition, the selection and efficiency of the explant choice for transformation should be considered in future studies. However, there have been parallel recommendations for the gradual application of selectable markers such as kanamycin.},
}
@article {pmid41410934,
year = {2025},
author = {Deora, S and Deora, GS and Nigam, S and Harish, },
title = {Hacking heterocysts: advances in the genetic regulation of heterocyst differentiation.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {80},
pmid = {41410934},
issn = {1432-072X},
mesh = {Nitrogen Fixation/genetics ; *Gene Expression Regulation, Bacterial ; *Cyanobacteria/genetics/metabolism/growth &amp; development ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Heterocyst differentiation in certain filamentous cyanobacteria is a multifaceted process essential for nitrogen fixation, orchestrated by a sophisticated regulatory network that encompasses several key stages. These include induction, pattern differentiation, commitment, extracellular layer formation, cell-cell communication, and ultimately, nitrogen fixation and metabolism. Key regulators like NtcA and HetR control heterocyst development, while proteins such as PatS, HetN, and PatA modulate pattern formation. Certain non-coding RNAs, such as NsiR1, Yfr1, and NsiR4, also regulate gene expression and contribute to the shutdown of CO2 fixation in differentiating heterocysts. Meanwhile, the heterocysts' unique envelope protects nitrogenase from oxygen, enabling nitrogen fixation. Genetic engineering approaches, including CRISPR-Cas systems, have been employed to increase heterocyst frequency and enhance the production of compounds such as ethanol, butanol and H2. By manipulating genes responsible for heterocyst differentiation, scientists can optimize nitrogen fixation, develop efficient biofertilizers, and unlock opportunities for a more sustainable future in agriculture and biotechnology. This review addresses the current understanding of the regulatory networks and molecular mechanisms that influence the development and function of heterocysts, providing insights into the biology and potential applications of these specialized cells through gene manipulations.},
}
@article {pmid41411128,
year = {2026},
author = {Mochida, T and Fujimoto, N and Asahina, M and Asano, S and Araki, S and Inukai, N and Hotta, A},
title = {Muscle satellite cell editing by LNP-CRISPR-Cas9 to resist muscle injury.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116695},
doi = {10.1016/j.celrep.2025.116695},
pmid = {41411128},
issn = {2211-1247},
mesh = {Animals ; *Satellite Cells, Skeletal Muscle/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Muscle, Skeletal/injuries/metabolism/pathology ; *Nanoparticles/chemistry ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Mice, Inbred mdx ; Male ; Exons/genetics ; Liposomes ; },
abstract = {Muscle satellite cells are essential for skeletal muscle regeneration and represent an attractive therapeutic target for gene delivery in Duchenne muscular dystrophy (DMD). However, efficient in vivo transduction of these cells has remained challenging. Here, we demonstrate that lipid nanoparticle (LNP)-mediated delivery of Streptococcus pyogenes CRISPR-Cas9 mRNA and guide RNA (LNP-CRISPR) induces exon skipping in Pax7-positive satellite cells more efficiently than adeno-associated virus (AAV) vectors following intramuscular or intravenous administration in a DMD mouse model. Furthermore, unlike AAV-CRISPR, LNP-CRISPR-mediated genome editing showed greater resistance to repeated muscle injuries, indicating successful editing of regenerative satellite cells. These results highlight the potential of LNPs as a non-viral platform for durable genome editing in skeletal muscle and lay the foundation for developing safe and sustainable genome-editing therapies for DMD.},
}
@article {pmid41411488,
year = {2026},
author = {Bär, I and Groten, SA and Barraclough, A and Bürgisser, PE and van Kwawegen, C and Lenting, PJ and van Moort, I and Eikenboom, JCJ and Leebeek, FWG and Voorberg, J and van den Biggelaar, M and Bierings, R},
title = {Allele-selective disruption of pathogenic VWF variants in type 2 von Willebrand disease using CRISPR/Cas9.},
journal = {Blood advances},
volume = {10},
number = {5},
pages = {1429-1443},
pmid = {41411488},
issn = {2473-9537},
mesh = {Humans ; *von Willebrand Factor/genetics ; *Alleles ; *CRISPR-Cas Systems ; *von Willebrand Disease, Type 2/genetics/therapy ; Polymorphism, Single Nucleotide ; *Gene Editing/methods ; Genetic Therapy/methods ; Mutation ; },
abstract = {In contrast to major innovations in treating severe hemophilia, the treatment of severe von Willebrand disease (VWD) remains limited to intravenous infusion of von Willebrand factor (VWF) concentrates. To date, no gene therapy-based approaches for the treatment of VWD have been developed, largely owing to the disease's heterogeneous mutational landscape and the challenge of specifically targeting VWF production in endothelial cells. In this study, we developed a novel gene therapy strategy for patients with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF. We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted allele. Variant mapping mass spectrometry that discriminates between wild-type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs. This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.},
}
@article {pmid41411621,
year = {2026},
author = {Tankka, AT and Zhang, Y and Einstein, JM and Zhou, CJ and Pham, VN and Naritomi, JT and Nguyen, GG and Mendez-Molina, AN and Hu, Z and Mizrahi, O and Perelis, M and Sarsam, J and Tan, FE and Kaufman, DS and Yang, J and Antal, CE and Yeo, GW},
title = {Integrative CRISPR Screening and RNA Analyses Discover an Essential Role for PUF60 Interactions with 3' Splice Sites in Cancer Progression.},
journal = {Cancer research},
volume = {86},
number = {7},
pages = {1586-1604},
doi = {10.1158/0008-5472.CAN-25-0453},
pmid = {41411621},
issn = {1538-7445},
support = {R01 CA268179/CA/NCI NIH HHS/United States ; S10 OD025060/OD/NIH HHS/United States ; U24 HG009889/HG/NHGRI NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; F32 HL143978/HL/NHLBI NIH HHS/United States ; P30 CA023100/CA/NCI NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; R01 CA174869/CA/NCI NIH HHS/United States ; U24 HG011735/HG/NHGRI NIH HHS/United States ; RF1 MH126719/MH/NIMH NIH HHS/United States ; R01 CA262794/CA/NCI NIH HHS/United States ; R01 HG011864/HG/NHGRI NIH HHS/United States ; T32 CA067754/CA/NCI NIH HHS/United States ; P30CA23100//National Cancer Institute (NCI)/ ; S10OD026929//National Institutes of Health (NIH)/ ; 1S10OD025060//National Institutes of Health (NIH)/ ; U54 CA209891/CA/NCI NIH HHS/United States ; T32CA067754//National Institutes of Health (NIH)/ ; //Gruss-Lipper Family Foundation/ ; F32 HL143978/HL/NHLBI NIH HHS/United States ; R01 CA174869/CA/NCI NIH HHS/United States ; R01 CA262794/CA/NCI NIH HHS/United States ; R01 CA268179/CA/NCI NIH HHS/United States ; U24 HG009889/HG/NHGRI NIH HHS/United States ; RF1 MH126719/MH/NIMH NIH HHS/United States ; R01 HG011864/HG/NHGRI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; 2023-332369//Chan Zuckerberg Initiative (CZI)/ ; V2024-008//V Foundation for Cancer Research (VFCR)/ ; IRG-19-230-48-IRG//American Cancer Society (ACS)/ ; P30CA023100//National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Animals ; Mice ; Female ; Cell Proliferation/genetics ; Disease Progression ; *RNA Splicing Factors/genetics/metabolism ; *CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Apoptosis/genetics ; *RNA-Binding Proteins/genetics/metabolism ; RNA Splicing ; Xenograft Model Antitumor Assays ; Repressor Proteins ; },
abstract = {UNLABELLED: RNA-binding proteins (RBP) are important regulators of posttranscriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies, such as triple-negative breast cancer (TNBC). In this study, we employed both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates essential for TNBC cell survival. Integrated enhanced cross-linking and immunoprecipitation and RNA sequencing analysis identified that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when misspliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3' splice sites via a substitution in its RNA-binding domain caused widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. Knockdown of PUF60 or disruption of PUF60-RNA interactions inhibited TNBC cell proliferation and shrunk tumor xenografts in multiple models. Together, these findings reveal the molecular mechanism by which PUF60 supports cancer progression.<br><br>SIGNIFICANCE: Functional screening of RNA-binding proteins is an effective strategy for identifying cancer regulators that revealed PUF60-mediated splicing activity as a driver of oncogenic proliferation and a potential therapeutic target.},
}
@article {pmid41412110,
year = {2025},
author = {Snell, JC and Nelson, BJ and Matreyek, KA},
title = {DIALing in elevated expression setpoints with promoter shortening.},
journal = {Cell systems},
volume = {16},
number = {12},
pages = {101482},
doi = {10.1016/j.cels.2025.101482},
pmid = {41412110},
issn = {2405-4720},
mesh = {*Promoter Regions, Genetic/genetics ; *Gene Expression Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; },
abstract = {DIAL is a novel framework for temporal control of transcript abundances in engineered cells. Targeted excision of DNA spacers in transgenic promoters permits controlled transitions of protein expression between setpoints. DIAL expands the repertoire of bioengineering tools for controlling protein expression, cell fates, and biological systems in general.},
}
@article {pmid41412287,
year = {2026},
author = {Su, Z and Liang, Z and Wu, Q and Xu, S and Li, C and Zheng, H and Wu, C and Ji, W and Niu, Y and Yang, Z},
title = {Metal-organic frameworks for CRISPR/Cas9 gene editing delivery: Innovations in therapeutic and diagnostic applications.},
journal = {Acta biomaterialia},
volume = {210},
number = {},
pages = {516-534},
doi = {10.1016/j.actbio.2025.12.030},
pmid = {41412287},
issn = {1878-7568},
mesh = {*Metal-Organic Frameworks/chemistry/therapeutic use ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Gene Transfer Techniques ; *Genetic Therapy/methods ; },
abstract = {CRISPR/Cas gene editing technology demonstrates significant promise in the treatment of various diseases, and a precise, efficient and safe delivery system is a key to realize gene therapy. Although traditional viral vectors can achieve superior transfection efficiency, viruses suffer from low reproduction efficiency and the risk of random gene integration, further limiting their wide application. Notably, metal-organic frameworks (MOFs), with tunable pore structure, easy surface chemical modification, good biocompatibility and physiological stability, have drawn much attention in the domain of targeted delivery of gene editing systems. Compared to lipid nanoparticles (LNPs) and extracellular vesicles (EVs), MOFs offer superior cargo loading (>80 % for proteins) and protect nucleic acids from degradation, while their stimuli-responsive degradation enables controlled release. This review focus on the cutting-edge advances of intelligent-responsive MOFs in delivering gene editing systems to against diseases, including endogenous responses (e.g., ATP, pH, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Additionally, we further summarize the research of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination. This study will help facilitate the clinical translation of MOFs-based CRISPR/Cas delivery systems in the field of therapy and detection of diseases. STATEMENT OF SIGNIFICANCE: This article reviews the cutting-edge advances of intelligent-responsive MOFs in delivering CRISPR/Cas systems to against diseases, including endogenous responses (e.g., pH, ATP, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Importantly, the potential applications of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination also have been discussed. This study will provide insights for the development of MOFs-based CRISPR/Cas delivery systems in the therapy and detection of clinical diseases.},
}
@article {pmid41412367,
year = {2026},
author = {Jose, J and Hamow, K&#xc1; and Éva, C and Moncsek, B and Kyrpa, T and Gamarra Reinoso, L and Bozsó, Z and Bakonyi, J and Balázs, E and Sági, L},
title = {CRISPR/Cas-mediated polyphenol oxidase gene knockout in potato reveals divergent roles in resistance to bacterial wilt and late blight.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {364},
number = {},
pages = {112944},
doi = {10.1016/j.plantsci.2025.112944},
pmid = {41412367},
issn = {1873-2259},
mesh = {*Solanum tuberosum/genetics/microbiology/enzymology/immunology ; *Catechol Oxidase/genetics/metabolism ; *Ralstonia solanacearum/physiology ; *Plant Diseases/microbiology/immunology/genetics ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Phytophthora infestans/physiology ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; },
abstract = {Polyphenol oxidases (PPOs) play a pivotal role in plant immune responses by catalysing the oxidation of phenolic compounds into cytotoxic quinones and melanin and contributing to the fortification of cell walls. Despite their biological significance, the high expression of PPOs in potatoes is not desirable due to their promotion of tuber browning. This study elucidates the relationship between PPO activity and defense mechanisms against the diverse pathogens Ralstonia solanacearum (Rs) and Phytophthora infestans (Pi) while mitigating enzymatic browning. CRISPR/Cas-mediated editing of the tuber- and root-specific PPO genes in the 'Désirée' and 'Balatoni Rózsa' potato cultivars considerably reduced enzymatic activity and browning. Among four PPO-edited mutant lines, three exhibited increased susceptibility to Rs while responses to Pi remained unchanged, underscoring the importance of PPOs in resistance to Rs. The PPO knockouts resulted in significant shifts in metabolite and hormone profiles characterized by elevated levels of dihydrokaempferol, coniferyl alcohol and taxifolin among other metabolites in the roots of Rs-susceptible mutants. Additionally, reduced PPO activity in these lines correlated with increased concentrations of salicylic acid, jasmonic acid and several antimicrobial compounds and alterations in flavonoid regulation. These findings highlight the complex role of PPOs in plant defense, establishing a positive correlation between PPO activity and resistance to Rs, while offering insights into the trade-offs associated with PPO gene editing in potatoes.},
}
@article {pmid41413028,
year = {2025},
author = {Chen, Q and Jiang, X and Yang, B and Deng, Z and Sun, Y},
title = {Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11435},
pmid = {41413028},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Humans ; INDEL Mutation ; HEK293 Cells ; DNA Breaks, Double-Stranded ; },
abstract = {Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known "inhibitor" Acr protein can unexpectedly acting as an "enhancer" of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.},
}
@article {pmid41413662,
year = {2026},
author = {Green, NFO and Sutton, GJ and Pérez-Burillo, J and Wang, J and Bagot, S and Danon, HG and Walsh, K and Gokool, A and Miles, SA and Yang, G and Herring, CA and Liang, Y and Pfundstein, G and Sytnyk, V and Alinejad-Rokny, H and Lister, R and Rosenbluh, J and Gagnon-Bartsch, JA and Voineagu, I},
title = {CRISPRi screening in cultured human astrocytes uncovers distal enhancers controlling genes dysregulated in Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {703-716},
pmid = {41413662},
issn = {1546-1726},
support = {2020814//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Astrocytes/metabolism ; *Alzheimer Disease/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Cells, Cultured ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Machine Learning ; },
abstract = {Genetic variants associated with complex traits often lie in distal enhancers. While candidate enhancers have been mapped genome wide, their functional state and gene targets in specific cell types remain unclear. Here we present AstroREG, a resource of enhancer-gene interactions in human primary astrocytes, generated by combining CRISPR inhibition (CRISPRi), single-cell RNA-seq and machine learning. By functionally testing nearly 1,000 PsychENCODE enhancers, we identified more than 150 regulatory interactions, revealing enhancers that control key astrocyte functions and genes implicated in Alzheimer's disease. The CRISPRi screen also provided valuable ground-truth data from a primary cell type for training and benchmarking prediction models of enhancer activity. We thus developed EGrf, a random forest (RF) model trained on these data, and applied it genome wide to predict regulatory interactions with high specificity. Together, our data provide a comprehensive functional map of enhancer-mediated regulation in a key glial cell type, shedding light on brain function and disease.},
}
@article {pmid41414648,
year = {2026},
author = {Wang, LR and Zhu, ST and Liao, ZH and Wu, N and Nie, ZK and Ye, C and Shi, TQ},
title = {Establishing a CRISPR/Cas9 Genome Editing System Combined with URA3-Blaster in Botrytis cinerea for Enhanced Abscisic Acid Production.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {2},
pages = {2207-2217},
doi = {10.1021/acs.jafc.5c14153},
pmid = {41414648},
issn = {1520-5118},
mesh = {*Botrytis/genetics/metabolism ; *Abscisic Acid/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Metabolic Engineering ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Abscisic acid (ABA) is a key plant growth regulator widely used in agriculture and ecological restoration. Although metabolic engineering of the fungus Botrytis cinerea can enhance ABA production, it has been hindered by inefficient genetic tools. In this study, we first established a recyclable selection marker system in B. cinerea based on orotidine-5'-phosphate decarboxylase. Subsequently, the CRISPR/Cas9 system was optimized, achieving up to 100% editing efficiency, far surpassing traditional homologous recombination. Based on this platform, multiple metabolic engineering strategies were systematically explored to enhance ABA biosynthesis. Increasing acetyl-CoA supply, inhibiting squalene synthesis, and knocking out key secondary metabolism genes Bcpks12 and Bcphs1 all significantly promoted ABA accumulation. Notably, co-overexpression of Bcacly1 and Bcacly2 combined with 1 g/L citrate increased ABA production to 1.36 g/L, representing a 38.66% improvement. Overall, this study provides an efficient genetic toolkit and a solid foundation for the industrial-scale production of ABA via engineered B. cinerea.},
}
@article {pmid41414667,
year = {2025},
author = {Klein, N and Sanchez-Londono, M and Kara, MM and Gomes-Filho, JV and Novak, S and Kholeif, KH and Pekarek, L and Caliskan, N and Randau, L},
title = {Type I-Fv and engineered type IV-A1 CRISPR-Cas effectors facilitate genome reduction in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414667},
issn = {1362-4962},
support = {//German Research Foundation/ ; 360987069//DFG/ ; 505997786//DFG/ ; //Microcosm Earth Center/ ; INST 93/1021-1 FUGG//Helmholtz Association/ ; //Philipps-Universit&#xe4;t Marburg/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; DNA Repair ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Class 1 CRISPR-Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR-Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR-Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR-Cas-mediated genome editing.},
}
@article {pmid41414668,
year = {2025},
author = {Semsey, S and Søndberg, E and Røen, M and Hallström, B and Petersen, A&#xd8; and Alfastsen, L and Bosch, BR and Wohl, B and Clube, J and van der Helm, E and Groendahl, C and Mougiakos, I},
title = {Characterization and engineering of a type IV-A3 CRISPR-Cas system for genome editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414668},
issn = {1362-4962},
support = {//SNIPR Biome/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Klebsiella pneumoniae/genetics ; Genome, Bacterial ; DNA Helicases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas systems have revolutionized genome engineering technologies, but type IV CRISPR-Cas systems and their genome engineering potential have been critically underexplored. In this study, we identified a type IV-A3 CRISPR-Cas system from a clinical Klebsiella pneumoniae isolate and characterized its plasmid targeting activity and capacity to suppress chromosomal and plasmid gene expression in Escherichia coli. We revealed the pivotal role of Csf3 (Cas5) and the dispensable roles of Csf1 (Cas8-like) and Csf4 (DinG helicase) subunits in IV-A3 CRISPR-Cas complex formation. The system prevents plasmid propagation via interplay between DinG helicase activity and strategic protospacer positioning relative to plasmid replication and maintenance components. We enabled the IV-A3 CRISPR-Cas system to introduce lethal, sequence-specific double-stranded (ds)DNA breaks in the E. coli chromosome by fusing the nuclease domain of the I-TevI nuclease to the Cas8 N-terminus. Further, we developed a series of base editors, with various editing efficiencies and windows, by fusing the PmCDA1 cytidine deaminase to the Cas8, Cas5, and DinG subunits. Finally, conjugative transfer of the Cas5-PmCDA1 base editor into E. coli deactivated the tryptophan repressor gene, boosting IAA production. Our study provides new insights into type IV-A3 CRISPR-Cas systems and highlights their potential in genome engineering applications.},
}
@article {pmid41414673,
year = {2025},
author = {Chan, J and Wu, Z and Liu, M and Wang, T and Liu, H and Cao, R and Li, X and Li, X and Zhan, S and Cheng, J and Xu, Y and He, M and Feng, Y and Xu, Q and Sun, Y and Chen, L and Hu, P},
title = {Systematic enhancer mapping and functional analysis in zebrafish with optimized CRISPR interference.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414673},
issn = {1362-4962},
support = {32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; (32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Enhancer Elements, Genetic ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Noncoding cis-regulatory elements, particularly enhancers, are crucial for controlling gene expression. However, the in vivo use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) to study enhancer function has been limited in zebrafish, which is widely used in early development and human disease research. Here, we optimized the CRISPRi system in zebrafish to achieve efficient suppression of tyr expression by fine-tuning component concentrations. Applying this optimized system, we functionally annotated distal enhancers of globin genes. Using Hi-C and histone modification assays, we systematically mapped 434 enhancer-promoter (EP) interactions across the genome. Among these EP loops, CRISPRi perturbation identified previously unreported enhancers with regulatory strengths surpassing known elements, demonstrated by disrupted phenotypes in fin and blood cell development. Additionally, several unreported EP loops were validated, underscoring the robustness of our integrated approach. This study not only provides an optimized CRISPRi system for zebrafish but also introduces a powerful platform that integrates computational and experimental strategies for advancing cis-regulatory element annotation in vertebrate gene regulation.},
}
@article {pmid41414712,
year = {2026},
author = {Congdon, ST and Bennett, J and Opinya, R and Agosto, AR and Dossias, O and Kokko, C and Levesque, AA and Koob, AO and Silver, AC and Thomas-Charles, CA},
title = {Investigating and correcting a rare pathogenic mutation in GDF11.},
journal = {HGG advances},
volume = {7},
number = {1},
pages = {100559},
pmid = {41414712},
issn = {2666-2477},
mesh = {Humans ; HEK293 Cells ; *Growth Differentiation Factors/genetics/metabolism ; *Gene Editing/methods ; *Bone Morphogenetic Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Golgi Apparatus/metabolism ; Codon, Nonsense ; },
abstract = {Single-nucleotide variants (SNVs) and small insertions or deletions (indels) underlie most rare monogenic disorders, yet therapeutic strategies to precisely correct these mutations remain limited. Prime editing enables the repair of such pathogenic variants without introducing double-stranded breaks. Here, we applied CRISPR prime editing to model and correct a de novo GDF11 nonsense mutation (Tyr336∗) identified in a participant from the Undiagnosed Diseases Network with growth delay and multisystem abnormalities. Using HEK293T cells, we generated heterozygous (HET) GDF11 Tyr336∗ clones, which exhibited reduced GDF11 protein levels due to post-translational degradation likely mediated by endoplasmic reticulum- and Golgi-associated quality control pathways. These cells displayed marked Golgi abnormalities, including an increased number of compact, irregularly shaped Golgi structures, findings consistent with Golgi fragmentation and stress. Transcriptomic profiling of HET cells revealed a broad dysregulation of gene networks, including downregulation of metabolic and Golgi-linked biosynthetic genes, and upregulation of cell-adhesion and extracellular matrix genes. These transcriptional shifts paralleled the participant's developmental, neural, and cardiovascular phenotypes. To correct the mutation, we tested multiple bespoke prime editing strategies and identified PE7, in combination with a prime editing guide RNA designed by Pridict, as the most effective ribonucleoprotein complex for rescue. Editing efficiency was further enhanced by introducing an additional silent protospacer-adjacent motif-disrupting mutation, likely preventing both Cas9 re-binding and mismatch repair. Together, these findings support a haploinsufficiency mechanism for the GDF11 Tyr336∗ allele and establish a generalizable framework for disease modeling and allele-specific correction of pathogenic variants in human cells.},
}
@article {pmid41415442,
year = {2025},
author = {Sabol, AL and Mengiste, AA and Sreekanth, V and Singh, P and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, K and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41415442},
issn = {2692-8205},
support = {R01 DK132900/DK/NIDDK NIH HHS/United States ; R35 GM136354/GM/NIGMS NIH HHS/United States ; R01 GM137606/GM/NIGMS NIH HHS/United States ; P30 ES002109/ES/NIEHS NIH HHS/United States ; R01 GM132825/GM/NIGMS NIH HHS/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; },
abstract = {Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}
@article {pmid41415454,
year = {2025},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41415454},
issn = {2692-8205},
support = {R35 GM133730/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}
@article {pmid41416398,
year = {2026},
author = {Hoang, TS and Faraji, F and Mendez-Molina, AN and Adame-Garcia, SR and Sato, K and Ishikawa, T and Vo, PTT and Ramirez, SI and Anguiano Quiroz, PY and Guo, T and Fan, K and Wu, X and Molinolo, AA and Cohen, EEW and Mali, P and Lippman, SM and Gutkind, JS},
title = {Genome-wide CRISPR Screening Reveals a PKA-Driven Resistance Mechanism to Metformin for Oral Cancer Prevention That Can Be Exploited by Combination with NSAIDs.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {2},
pages = {79-92},
pmid = {41416398},
issn = {1940-6215},
support = {T32 CA121938/CA/NCI NIH HHS/United States ; R01 DE035393/DE/NIDCR NIH HHS/United States ; R25 CA221779/CA/NCI NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; U01 CA290479/CA/NCI NIH HHS/United States ; R01 DE026644/DE/NIDCR NIH HHS/United States ; T32 DC000028/DC/NIDCD NIH HHS/United States ; R01DE026644//National Institute of Dental and Craniofacial Research (NIDR)/ ; U01CA290479//National Cancer Institute (NCI)/ ; SU2C-FARF-FFF//Stand Up To Cancer (SU2C)/ ; 308268//Stand Up To Cancer (SU2C)/ ; T32DT4965//Tobacco-Related Disease Research Program (TRDRP)/ ; T32CA121938//National Cancer Institute (NCI)/ ; T32DC000028//National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; 1061310//American Head and Neck Society (AHNS)/ ; //Takeda Science Foundation (TSF)/ ; //Japan Society for the Promotion of Science (JSPS)/ ; //Rotary Foundation (Rotary)/ ; R25CA221779//National Cancer Institute (NCI)/ ; T34DT8340//Tobacco-Related Disease Research Program (TRDRP)/ ; },
mesh = {*Metformin/pharmacology/therapeutic use ; Humans ; *Anti-Inflammatory Agents, Non-Steroidal/pharmacology/therapeutic use ; *Cyclic AMP-Dependent Protein Kinases/metabolism ; *Mouth Neoplasms/prevention &amp; control/genetics/pathology ; *Drug Resistance, Neoplasm/genetics/drug effects ; CRISPR-Cas Systems ; *Squamous Cell Carcinoma of Head and Neck/genetics/prevention &amp; control/pathology/drug therapy ; Cell Line, Tumor ; Mice ; Drug Synergism ; Animals ; Cell Proliferation/drug effects ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; Signal Transduction/drug effects ; Cyclooxygenase 2 Inhibitors/pharmacology ; },
abstract = {UNLABELLED: Head and neck squamous cell carcinoma (HNSCC) is among the 10 most common cancers worldwide and is associated with high morbidity and poor survival. Diminished HNSCC outcomes are often related to delayed diagnosis and treatment of occult progression of premalignant lesions, underscoring the need for effective and low-risk chemoprevention strategies. In this regard, metformin has shown promising clinical activity for HNSCC prevention. In this study, we performed a genome-wide CRISPR/Cas9 screen of metformin-treated HNSCC cells and identified the activation of PKA signaling as the top resistance pathway. We show that metformin mediates PKA activation in HNSCC cells and that PKA inhibition, when combined with metformin treatment, synergistically inhibits HNSCC growth. We found that metformin-induced PKA activation is mediated by a prostaglandin E2 autocrine loop, which can be blocked using cyclooxygenase-2 (COX2) inhibitors. Importantly, COX2 inhibition using nonsteroidal anti-inflammatory drugs (NSAID) combined with metformin treatment synergistically inhibits HNSCC cell growth and prevents the progression of oral premalignant lesions into invasive HNSCC in a model of tobacco-driven oral carcinogenesis. Together, these findings demonstrate that metformin and NSAID combination therapy may represent a promising therapeutic strategy for HNSCC chemoprevention.<br><br>PREVENTION RELEVANCE: Our findings reveal that using metformin for head and neck cancer chemoprevention leads to compensatory activation of a PKA-driven resistance mechanism that can be blocked by cotreatment with NSAIDs. These findings provide a rationale for combining metformin with NSAIDs as a precision head and neck cancer chemoprevention strategy.},
}
@article {pmid41416522,
year = {2025},
author = {Stella, G and Ye, L and Brady, SF and Marraffini, L},
title = {CARF-HAD phosphatase effectors provide immunity during the type III-A CRISPR-Cas response.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41416522},
issn = {1362-4962},
support = {R01GM149834/GF/NIH HHS/United States ; R35 GM122559/GM/NIGMS NIH HHS/United States ; R35GM122559/GF/NIH HHS/United States ; R01 GM149834/GM/NIGMS NIH HHS/United States ; //HHMI/ ; },
mesh = {*CRISPR-Cas Systems ; *Phosphoric Monoester Hydrolases/metabolism/genetics/chemistry ; Protein Domains ; Adenosine Triphosphate/metabolism ; Escherichia coli/genetics/virology ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here, we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain. We named these proteins Chp for CRISPR-associated HAD phosphatase. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP, and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP, and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.},
}
@article {pmid41416796,
year = {2026},
author = {Athipanyasilp, N and Saowpak, S and Chaimayo, C and Angkasekwinai, N and Pattama, A and Athipanyasilp, A and Patchsung, M and Aphicho, K and Uttamapinant, C and Horthongkham, N},
title = {CRISPR-Cas13a SHERLOCK assay for rapid and sensitive detection of chikungunya virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0229825},
pmid = {41416796},
issn = {2165-0497},
support = {R016034012//Faculty of Medicine Siriraj Hospital, Mahidol University/ ; },
mesh = {*Chikungunya virus/genetics/isolation &amp; purification ; *Chikungunya Fever/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Real-Time Polymerase Chain Reaction/methods ; },
abstract = {Chikungunya virus (CHIKV), a major cause of acute febrile illness and joint pain, remains a significant public health threat in tropical regions. Rapid and accurate detection is essential for timely clinical management and outbreak control, particularly in resource-limited settings where real-time PCR (RT-qPCR) is often impractical. We developed and validated a SHERLOCK assay coupled with recombinase polymerase amplification for CHIKV RNA detection. Analytical performance was assessed by determining the limit of detection (LOD), cross-reactivity, clinical sensitivity and specificity, and predictive values. The assay achieved an LOD of 215 copies/reaction with no cross-reactivity against other alphaviruses or flaviviruses. Clinical testing of 146 plasma samples showed a sensitivity and specificity of 94.52% and 100% with lateral-flow readout and 97.26% and 100% with fluorescence readout, respectively. This study establishes a promising CRISPR-Cas13a-based SHERLOCK platform for CHIKV detection, demonstrating high analytical performance, rapid turnaround time, and potential for future adaptation to resource-limited settings.IMPORTANCEEarly and accurate detection of chikungunya virus (CHIKV) is critical for outbreak control, especially in resource-limited settings, where real-time PCR is not feasible. This study demonstrates that the CRISPR-Cas13a-based SHERLOCK platform, combined with RPA, achieves high diagnostic accuracy and a low detection limit, comparable to RT-qPCR. The assay's rapid turnaround time and simple lateral-flow readout make it a promising tool for point-of-care diagnostics during CHIKV outbreaks, potentially improving disease surveillance and clinical decision-making.},
}
@article {pmid41416985,
year = {2026},
author = {Han, DH and Lee, SY and Kim, Y and Oh, J and Park, J and Park, YM and Kim, SG and Kim, TS and Park, JK},
title = {Ultrasensitive Detection of Multiple Foodborne Pathogens Using CRISPR-Cas12a on a Finger-Actuated Microfluidic Device Integrated with a Modular Pressurizing Pump.},
journal = {Analytical chemistry},
volume = {98},
number = {1},
pages = {531-542},
doi = {10.1021/acs.analchem.5c05295},
pmid = {41416985},
issn = {1520-6882},
mesh = {*Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; *Listeria monocytogenes/isolation &amp; purification/genetics ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Food Microbiology ; *Escherichia coli O157/isolation &amp; purification/genetics ; Salmonella/isolation &amp; purification/genetics ; *Microfluidic Analytical Techniques/instrumentation ; Milk/microbiology ; Foodborne Diseases/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Foodborne pathogens pose a serious threat to global health and the economy, causing gastrointestinal illnesses and potentially leading to fatalities. Here, we present a recombinase polymerase amplification (RPA)-CRISPR-Cas12a-based method for the detection of foodborne pathogens using target-specific CRISPR RNAs (crRNAs) on a reusable, reconfigurable finger-actuated microfluidic device. Unlike previous finger-actuated pushbutton-based microfluidic devices, the device incorporates a modular pressurizing pump (MoPP), a standalone, reconfigurable actuation module that not only enhances reusability and reduces cross-contamination risks but also provides a flexible interface that allows user-defined fluidic routing and multiplexed assay workflows. Using a MoPP-integrated finger-actuated microfluidic device, the RPA-CRISPR-Cas12a-based detection of three foodborne pathogens was validated with an optimized crRNA and RPA primer sequence. Genomic DNA (gDNA) extracted from pathogen-spiked milk samples further demonstrated real-world applicability, achieving a limit of detection (LOD) of 1.62, 1.84, and 1.01 CFU/mL for Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes, respectively. The developed microfluidic RPA-CRISPR-Cas12a-based detection platform is expected to be a reconfigurable, user-friendly, and highly sensitive point-of-care testing system for monitoring foodborne pathogens throughout the food supply chain.},
}
@article {pmid41417296,
year = {2025},
author = {Rehman, T and Sharif, A and Khalid, L and Sajid, I},
title = {Whole genome sequencing and genomic characterization of the extensively drug-resistant Acinetobacter baumannii recovered from clinical samples in Lahore, Pakistan.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {217},
pmid = {41417296},
issn = {1573-4978},
mesh = {*Acinetobacter baumannii/genetics/drug effects/isolation &amp; purification/pathogenicity ; Pakistan ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Whole Genome Sequencing/methods ; *Acinetobacter Infections/microbiology/genetics/drug therapy ; Genome, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Genomics/methods ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Extensively drug-resistant (XDR) Acinetobacter baumannii has turned into a significant nosocomial pathogen often resistant to all available classes of antibiotics. It has been identified as a key public health issue due to its capacity to get resistance determinants, virulence genes, and mobile genetic elements. This study hypothesized that the XDR A. baumannii isolated in Pakistan would harbor some of the key genomic determinants of the resistance and pathogenicity.<br><br>METHODOLOGY: A total of 11 A. baumannii were obtained from the patient samples and were identified using microbiological, biochemical, and genomic analysis. The sensitivity to antibiotics was determined by Kirby-Bauer disc diffusion assay. While the whole genome sequencing (WGS) was performed for the single XDR Acinetobacter isolate TAB-4 and its in-silico genome analysis on resistant genes, virulence factors, plasmids, mobile genetic elements, prophages, CRISPR-Cas and biosynthetic gene clusters was conducted.<br><br>RESULTS: The isolated strains were 10 multidrug resistant (MDR) and one (TAB-4) was XDR Acinetobacter strain. The AST showed resistance to nearly all classes of antibiotics, and with limited susceptibility to tetracyclines and aminoglycosides. The genome of TAB-4 strain comprised of 3.94&#xa0;Mb, 120 contigs, and a GC content of 39.14%. The major resistant determinants found were blaOXA-23, blaOXA-69, blaNDM-1, blaADC-25, aminoglycoside modifying enzymes, and efflux pumps (adeABC, adeFGH, adeIJK). Virulence-associated genes (ompA, bap, csuA/B-E, plc) were identified along with five prophage regions, multiple CRISPR arrays, and a betalactone biosynthetic gene cluster.<br><br>CONCLUSION: This study to the best of our knowledge reports the first detailed WGS-based characterization of an XDR A. baumannii from Lahore, Pakistan. These genomic findings offer significant insights into the resistance and virulence factors underlying this challenging clinical issue. Hence, there is an urgent need to find new or alternative treatment methods against high-risk pathogens like XDR A. baumannii.},
}
@article {pmid41417728,
year = {2026},
author = {Huber, A and Djajawi, TM and Rivera, IS and Vervoort, SJ and Kearney, CJ},
title = {CRISPR screens define unified hallmarks of cancer cell-autonomous immune evasion.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116738},
doi = {10.1016/j.celrep.2025.116738},
pmid = {41417728},
issn = {2211-1247},
mesh = {Humans ; *Neoplasms/immunology/genetics/pathology ; *Tumor Escape/genetics ; *CRISPR-Cas Systems/genetics ; Tumor Microenvironment/immunology ; Immunotherapy ; Animals ; *Immune Evasion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Cancer immunotherapy has transformed cancer treatment, yet only a minority of patients achieve durable benefit. Although early efforts to enhance immunotherapy focused on boosting immune effector function, reversing T cell exhaustion, or altering the tumor microenvironment, it is now clear that cancer cell-autonomous mechanisms play a major role in immune escape. Such programs, driven by the cancer cell genome, transcriptome, and epigenome, include desensitization to cytokine signaling, such as interferon (IFN)γ and tumor necrosis factor (TNF); impaired antigen presentation; upregulation of suppressive ligands such as programmed cell death ligand 1 (PD-L1); and epigenetic silencing of immunogenic pathways. The rise of high-throughput functional genomics, especially in vitro and in vivo CRISPR-based screening, has greatly expanded our ability to map these pathways and define how tumors evade CD8[+] T cell-mediated pressure. A deeper understanding of these cancer cell-autonomous immune-evasion mechanisms will be essential for developing new therapeutic strategies that broaden the impact of immunotherapy across diverse cancers.},
}
@article {pmid41417859,
year = {2025},
author = {Bradford, J and Joy, D and Winsen, M and Meurant, N and Wilkins, M and Wilson, LOW and Bauer, DC and Perrin, D},
title = {Democratising high performance computing for bioinformatics through serverless cloud computing: A case study on CRISPR-Cas9 guide RNA design with Crackling Cloud.},
journal = {PLoS computational biology},
volume = {21},
number = {12},
pages = {e1013819},
pmid = {41417859},
issn = {1553-7358},
mesh = {*Cloud Computing ; *Computational Biology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Software ; },
abstract = {Organisations are challenged when meeting the computational requirements of large-scale bioinformatics analyses using their own resources. Cloud computing has democratised large-scale resources, and to reduce the barriers of working with large-scale compute, leading cloud vendors offer serverless computing, a low-maintenance and low-cost model that provides ample resources for highly scalable software applications. While serverless computing has broad use, its adoption in bioinformatics remains poor. Here, we demonstrate the most extensive use of high-performance serverless computing for bioinformatics by applying the available technologies to CRISPR-Cas9 guide RNA (gRNA) design. Our adaptation of the established gRNA design tool, named Crackling, implements a novel, cloud-native and serverless-based, high-performance computing environment using technologies made available by Amazon Web Services (AWS). The architecture, compatible with technologies from all leading cloud vendors, and the AWS implementation, contributes to an effort of reducing the barrier to large computational capacity in bioinformatics and for CRISPR-Cas9 gRNA design. Crackling Cloud can be deployed to any AWS account, and is freely available on GitHub under the BSD 3-clause license: https://github.com/bmds-lab/Crackling-AWS.},
}
@article {pmid41417901,
year = {2025},
author = {Chammas, P and Xie, SQ and Sepulveda-Rincon, LP and Leeke, BJ and Dore, MH and Dormann, D and Wagner, RT and Chang, N and Jones, PL and McManus, MT and Karimi, MM and Young, G and Percharde, M},
title = {CRISPRa-mediated disentanglement of the Dux-MERVL axis in the 2C-like state, totipotency, and cell death.},
journal = {Science advances},
volume = {11},
number = {51},
pages = {eadu9092},
pmid = {41417901},
issn = {2375-2548},
mesh = {Animals ; Mice ; Cell Death/genetics ; *Homeodomain Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *DNA Transposable Elements/genetics ; Zygote/metabolism ; },
abstract = {Transposable elements (TEs) are powerful cis-regulatory drivers of gene expression, particularly during early development when many TEs become de-repressed. MERVL elements are transiently up-regulated in mouse totipotent two-cell (2C) embryos during major zygotic genome activation (ZGA) and 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos. Moreover, sustained Dux activation causes cell toxicity, which may or may not be linked to MERVL. Using a CRISPR activation system, we unpick the relative role of Dux and MERVL in ZGA, totipotent-like characteristics, and cell toxicity. We find that MERVL activation drives a portion of the Dux-dependent transcriptome, sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves the proapoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a previously unknown player in Dux-driven pathology.},
}
@article {pmid41418537,
year = {2026},
author = {Tan, D and Ye, Y and Miao, D and Zhao, C and Wu, S and Shi, J and Yang, J and Fang, K and Lu, F and Lv, Q and Gong, J and Yang, H and Xiao, W and Xiong, Z and Zhang, X and Ruan, H},
title = {UBL3 governs VEGFR inhibitor resistance by activating NOTCH signaling in renal cell carcinoma.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {85},
number = {},
pages = {101332},
doi = {10.1016/j.drup.2025.101332},
pmid = {41418537},
issn = {1532-2084},
mesh = {Humans ; *Carcinoma, Renal Cell/drug therapy/pathology/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Kidney Neoplasms/drug therapy/pathology/genetics ; Signal Transduction/drug effects ; Animals ; Cell Line, Tumor ; Mice ; Xenograft Model Antitumor Assays ; *Receptors, Vascular Endothelial Growth Factor/antagonists &amp; inhibitors ; *Protein Kinase Inhibitors/pharmacology ; Sunitinib/pharmacology ; Pyridines/pharmacology ; Receptors, Notch/metabolism ; Apoptosis/drug effects ; Mice, Nude ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Anilides ; },
abstract = {BACKGROUND: Targeted therapy is the first-line treatment for patients with metastatic renal cell carcinoma (RCC), with vascular endothelial growth factor receptor inhibitors (VEGFRis) constituting the bulk of regimens used. Although the repertoire of VEGFRis for RCC now spans from sunitinib to cabozantinib, resistance to treatments has emerged as a common and prominent challenge. Thus, identifying novel therapeutic targets has become essential for enhancing the antitumor efficacy of current treatments and inhibiting RCC progression.<br><br>METHOD: To investigate the potential mechanisms underlying VEGFRi resistance in RCC, we performed a genome-wide CRISPR/Cas9 library screen under sunitinib and cabozantinib treatment and identified UBL3 as a key driver of VEGFRi resistance in RCC cells. The critical role of UBL3 in promoting VEGFRi resistance was validated using CCK8 assays, flow cytometry, TUNEL assays, and bioinformatics analyses. To elucidate the molecular mechanisms underlying UBL3, we utilized western blotting, RNA sequencing, chromatin immunoprecipitation, small extracellular vesicles (sEVs) isolation, and Astral-DIA proteomics. The contribution of UBL3 to VEGFRi resistance was further confirmed through comprehensive in vitro and in vivo experiments.<br><br>RESULTS: UBL3 was confirmed to suppress apoptosis and promote VEGFRi resistance through NOTCH signaling activation. Further investigations highlighted the importance of NOTCH signaling in VEGFRi resistance in RCC via the NOTCH-PTEN-AKT and NOTCH-FOS pathways and revealed the mechanisms by which UBL3 activated NOTCH signaling. On the one hand, UBL3 formed complex with NOTCH2 and ADAM17 simultaneously, accelerating ADAM17-mediated cleavage of NOTCH2. On the other hand, UBL3-modified NOTCH2 was sorted into sEVs, which were taken up by recipient cells, activating NOTCH signaling and thereby transmitting VEGFRi resistance. Finally, lipid nanoparticle-mediated delivery of the CRISPR/Cas9 knockout system targeting UBL3 effectively restored the sensitivity of RCC tumors to VEGFRis.<br><br>CONCLUSION: This study emphasized the importance of UBL3 in VEGFRi resistance in RCC and proposed that UBL3 activated NOTCH signaling through two distinct pathways, thereby suppressing cancer apoptosis and promoting resistance to VEGFRis. These findings provided a solid scientific foundation and paved the way for the development of novel therapeutic strategies for patients with advanced RCC.},
}
@article {pmid41418729,
year = {2026},
author = {Wang, Y and Zheng, W and Qiu, B and Chen, Q and Yang, T and Zhou, S and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-3F) with Cas9 expression cassette integrated at the AAVS1 locus via CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103882},
doi = {10.1016/j.scr.2025.103882},
pmid = {41418729},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Homologous Recombination/genetics ; Cell Line ; Gene Editing ; Genetic Loci ; Cell Differentiation ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {Cas9, an RNA-guided nuclease, enables precise genome editing by recognizing sgRNA-complementary sequences and cleaving target DNA. In this study, we used CRISPR/Cas9-mediated homologous recombination to integrate a loxP-flanked STOP cassette-controlled Cas9 expression framework (LSL-Cas9) into the AAVS1 safe-harbor locus of human embryonic stem cells. The resulting cell line, SMUDHe010-A-3F, allows Cre-dependent activation of Cas9 but remains inactive in the absence of Cre recombinase. Karyotype and tri-lineage differentiation confirmed genomic stability and pluripotency. This line provides a valuable platform for organoid gene editing and studies of human development and disease.},
}
@article {pmid41418774,
year = {2026},
author = {Ji, R and Chen, Q and Zhang, Y},
title = {Emerging trends in gene and cell therapy: CRISPR in DNA editing and beyond.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102459},
pmid = {41418774},
issn = {2666-3791},
mesh = {Humans ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods/trends ; *Cell- and Tissue-Based Therapy/methods/trends ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-based gene and cell therapies are rapidly transitioning from experimental platforms to clinical reality, exemplified by the recent approval of CRISPR-derived treatments for β-hemoglobinopathies. This review highlights how advances in genome editing technologies, ranging from CRISPR-Cas nucleases to base and prime editors, are expanding the therapeutic landscape beyond traditional gene knockout approaches. We focus on the clinical translation of these tools, drawing on examples from ongoing and completed human trials to illustrate their potential across diverse disease areas. Furthermore, we discuss critical considerations such as delivery challenges, long-term safety, immune responses, and editing specificity, all of which are critical to the safe and effective integration of CRISPR technologies into modern medicine.},
}
@article {pmid41418786,
year = {2026},
author = {Sherman, A and Benvenisty, N},
title = {Genetic screening of long non-coding RNAs in human embryonic stem cells reveals novel regulators of pluripotency.},
journal = {Stem cell reports},
volume = {21},
number = {1},
pages = {102743},
pmid = {41418786},
issn = {2213-6711},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; *Genetic Testing ; Cell Line ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4, which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN, which acts as a putative negative regulator of VRTN, affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.},
}
@article {pmid41418951,
year = {2026},
author = {Chen, J and Bian, X and Zheng, X and Peng, B and Li, R and Du, H and Zhou, L and Wen, Y},
title = {The synergistic effect of DNA nanostructures and CRISPR/Cas system for cancer diagnosis and treatment.},
journal = {International journal of biological macromolecules},
volume = {339},
number = {Pt 1},
pages = {149741},
doi = {10.1016/j.ijbiomac.2025.149741},
pmid = {41418951},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; Humans ; *Neoplasms/diagnosis/therapy/genetics ; Gene Editing/methods ; *Nanostructures/chemistry/therapeutic use ; *DNA/chemistry ; Genetic Therapy/methods ; Animals ; },
abstract = {Cancer remains one of the most formidable global public health challenges, exerting a profound and detrimental impact on human health. Despite substantial advancements in cancer research, the escalating incidence and mortality rates underscore the persistent and growing burden on global healthcare systems. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, heralded as a revolutionary gene-editing tool, holds immense promise for cancer treatment. However, its efficacy is critically contingent upon developing efficient delivery strategies. DNA nanocarriers, characterized by their programmability, sequence specificity, and design flexibility, emerge as a highly effective vehicle for delivering the CRISPR/Cas system, facilitating the precise transportation of gene-editing tools to the cell nucleus. The integration of DNA nanocarriers with CRISPR/Cas technology provides a new paradigm for precise and controllable gene editing. Through programmable spatial assembly, DNA nanocarriers can protect Cas9 ribonucleoprotein complexs (RNPs), facilitate endosomal escape, and co-localize donor DNA to promote homology-directed repair. These synergistic effects bridge molecular programmability and genetic functionality, paving the way for safer and more efficient genome engineering. This review aims to evaluate the application of DNA nanocarriers in cancer diagnosis comprehensively and to explore their potential utility in cancer therapy when combined with the CRISPR/Cas system, offering novel insights and significant scientific contributions to the field.},
}
@article {pmid41419745,
year = {2025},
author = {Ying, Q and Chen, Y and Shen, L and Xu, Y and Tian, R},
title = {SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {911},
pmid = {41419745},
issn = {2041-1723},
support = {82171416//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*X-Box Binding Protein 1/metabolism/genetics ; *Endoribonucleases/metabolism/genetics ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *RNA Splicing/genetics ; Humans ; Signal Transduction/genetics ; Animals ; *Endoplasmic Reticulum Stress/genetics ; Protein Phosphatase 1/metabolism/genetics/antagonists &amp; inhibitors ; Unfolded Protein Response/genetics ; Mice ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq (SPLicing regulator identification through CRISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 (PPP1R15A) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway.},
}
@article {pmid41419797,
year = {2025},
author = {Das, IS and Shi, Q and Dreischhoff, S and Polle, A},
title = {Divergent functions of three Kunitz trypsin inhibitor (KTI) proteins in herbivore defense in poplar.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {153},
pmid = {41419797},
issn = {1471-2229},
mesh = {*Populus/genetics/metabolism/physiology/parasitology ; *Herbivory ; *Plant Proteins/genetics/metabolism ; Animals ; Oxylipins/metabolism ; Gene Expression Regulation, Plant ; Cyclopentanes ; *Plant Defense Against Herbivory/genetics ; Peptides ; },
abstract = {BACKGROUND: Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels.<br><br>RESULTS: Using in-silico database searches, we identified 28 KTIs in the haploid genome of Populus x canescens; 21 of them were responsive to herbivory. The greatest induction by herbivory was observed for KTI_400, KTI_600 and KTI_0882 (P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri.017G153200 orthologue). Mechanical wounding and methyl-jasmonate treatments resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200. Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE, a key enzyme involved in jasmonate biosynthesis. On the contrary, exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTIs. We generated stable CRISPR-Cas12a-mediated knock-out and p35S-mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400&#x2009;+&#x2009;kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced, and their double knockout lines in increased weight gain of the larvae. In contrast, overexpressing or knockout lines of KTI_53200 had no effect on larval weight gain compared with controls.<br><br>CONCLUSION: KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against H. armigera in trees and presents new options to investigate growth-defense theories.},
}
@article {pmid41420106,
year = {2026},
author = {Ge, J and Hirosue, S and Castillon, L and Patel, SA and Wesolowski, L and Dyas, A and Yong, C and de Haan, S and Drost, J and Stewart, GD and Obenauf, AC and Muñoz-Espín, D and Vanharanta, S},
title = {Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities.},
journal = {EMBO molecular medicine},
volume = {18},
number = {2},
pages = {599-619},
pmid = {41420106},
issn = {1757-4684},
support = {C9685/A25177//Cancer Research UK (CRUK)/ ; C62187/A29760//Cancer Research UK (CRUK)/ ; BRC-1215-20014//NIHR | NIHR Cambridge Biomedical Research Centre (NIHR Cambridge BRC)/ ; 955951//European Commission (EC)/ ; MC_UU_12022/7//UKRI | Medical Research Council (MRC)/ ; RP_033_20170303//Kidney Research UK/ ; 338420//Research Council of Finland (AKA)/ ; },
mesh = {*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; CRISPR-Cas Systems ; *Drug Resistance, Neoplasm ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Epithelial Cells ; },
abstract = {The von Hippel-Lindau tumor suppressor (VHL) is a component of a ubiquitin ligase complex that controls cellular responses to hypoxia. Endogenous VHL is also utilized by proteolysis-targeting chimera (PROTAC) protein degraders, a promising class of anti-cancer agents. VHL is broadly essential for cell proliferation, yet it is a key tumor suppressor in renal cell carcinoma. To understand the functional consequences of VHL loss, and to identify targeted approaches for the elimination of VHL null cells, we have used genome-wide CRISPR-Cas9 screening in human renal epithelial cells. We find that, upon VHL loss, the HIF1A/ARNT complex is the central inhibitor of cellular fitness, suppressing mitochondrial respiration, and that VHL null cells show HIF1A-dependent molecular vulnerabilities that can be targeted pharmacologically. Combined VHL/HIF1A inactivation in breast and esophageal cancer cells can also provide resistance to ARV-771, a VHL-based bromodomain degrader that has anti-cancer activity. HIF1A stabilization can thus provide opportunities for early intervention in neoplastic VHL clones, and the VHL-HIF1A axis may be relevant for the development of resistance to the emerging class of PROTAC-based cancer therapies.},
}
@article {pmid41420208,
year = {2025},
author = {Holmlund, H and Yamauchi, Y and Tekayev, M and Jakobs, S and Robin, A and Fujii, W and Ward, MA},
title = {CRISPR/Cas9-mediated knock-in of the murine Y chromosomal genes Zfy1 and Zfy2.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {88},
pmid = {41420208},
issn = {1471-2164},
support = {G12 MD007601/MD/NIMHD NIH HHS/United States ; 17CON-86294//Hawai'i Community Foundation/ ; R01 HD114645/HD/NICHD NIH HHS/United States ; F31 HD111279/HD/NICHD NIH HHS/United States ; HD114645/GF/NIH HHS/United States ; P20 GM103457/GM/NIGMS NIH HHS/United States ; R01 HD072380/HD/NICHD NIH HHS/United States ; NIH F31HD111279/GF/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Male ; Mice ; *Gene Knock-In Techniques ; Spermatogenesis/genetics ; *Y Chromosome/genetics ; Testis/metabolism ; Gene Editing ; Zinc Fingers/genetics ; },
abstract = {BACKGROUND: The Y-linked mouse zinc finger genes Zfy1 and Zfy2 are critical fertility factors in mice but the mechanisms by which they regulate spermatogenesis remain unclear. We recently produced Zfy1/2 double knock-out mice and observed a complete loss in fertility. However, the biochemical mechanism by which Zfy regulates spermatogenesis is unknown, and ZFY expression has not yet been confirmed at the protein level. As both Zfy homologues share ~ 95% sequence similarity, it is difficult to produce an anti-ZFY antibody specific to either homologue.<br><br>RESULTS: To overcome this technical challenge, we used CRISPR/Cas9 genome editing to develop tagged Zfy1 knock-in (XY[Zfy1-HA]), Zfy2 knock-in (XY[Zfy2-FLAG], XY[Zfy2-3xFLAG], and XY[Zfy2-HA]), and Zfy1/2 double knock-in (XY[Zfy1-HA,Zfy2-MYC]) mice. Successful targeting was confirmed by genotyping and sequencing. The knock-in lines were fertile with normal sperm parameters. Using Western blot on testes, knock-in specific bands were detected matching the predicted ZFY expression patterns. Using immunofluorescence on testis sections from knock-in males, ZFY1 and ZFY2 expression was detected in zygotene spermatocytes, and ZFY2 expression was also detected in spermatids step 7-8 and 9.<br><br>CONCLUSIONS: These novel knock-in mice can be used in future investigations to determine how ZFY controls spermatogenesis.},
}
@article {pmid41420495,
year = {2025},
author = {Pan, G and Wang, L and Zhu, H and Wang, H and Zheng, Z},
title = {Utilization of Miniature CRISPR-AsCas12f1 Nuclease for Efficient Genome Editing in Bacillus subtilis.},
journal = {Biotechnology journal},
volume = {20},
number = {12},
pages = {e70168},
doi = {10.1002/biot.70168},
pmid = {41420495},
issn = {1860-7314},
support = {S2023n06020216//Anhui Province Key Research and Development Plan/ ; 2019YFA0904304//National Key Research and Development Program of China/ ; YZJJ2024QN37//Hefei Institutes of Physical Science, Chinese Academy of Sciences Director's Fund/ ; },
mesh = {*Bacillus subtilis/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial ; },
abstract = {To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.},
}
@article {pmid41421070,
year = {2026},
author = {Salodkar, D and Dongarwar, S and Nair, A and Ashtaputre, P and Reddy, S and Somkuwar, S and Begde, D},
title = {Single-Step CRISPR/Cas13a Assay for detection of small RNAs in Saliva : a proof-of-concept study.},
journal = {Cancer genetics},
volume = {300-301},
number = {},
pages = {67-71},
doi = {10.1016/j.cancergen.2025.12.003},
pmid = {41421070},
issn = {2210-7762},
mesh = {Humans ; *Saliva/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *Biomarkers, Tumor/genetics ; Proof of Concept Study ; *MicroRNAs/genetics/analysis ; Sensitivity and Specificity ; *Mouth Neoplasms/genetics/diagnosis ; Leptotrichia/genetics ; Real-Time Polymerase Chain Reaction ; },
abstract = {OBJECTIVE: We describe a proof-of-concept study of a rapid, single-step CRISPR/Cas13a assay using Leptotrichia wadei (LwCas13a) for the detection of small RNA (miRNA) biomarkers in saliva, and compare its performance to real-time PCR (RT-PCR).<br><br>METHODS: The single-step Cas13a assay was evaluated against RT-PCR for its detection efficiency, sensitivity, specificity, and its ability to function in a complex biological matrix. A proof-of-concept test was conducted on patient saliva samples to detect a known oral cancer biomarker, hsa-miR-21-3p RESULTS: The Cas13a assay successfully detected candidate miRNA at picomolar concentrations in both in vitro and saliva samples, demonstrating sensitivity and specificity comparable to RT-PCR. Notably, the assay provided discernible detection of the cancer biomarker directly in patient saliva without the need for RNA extraction or reverse transcription steps.<br><br>CONCLUSION: The proposed single-step CRISPR/Cas13a assay may be developed into a promising platform for developing quick and affordable point-of-care diagnostics for cancer and other diseases, circumventing the need for expensive and time-consuming sample preparation steps.},
}
@article {pmid41421338,
year = {2026},
author = {Du, R and Flynn, MJ and Mahe, K and Honsa, M and Gu, B and Li, D and McGeary, SE and Gradinaru, V and Jungmann, R and Elowitz, MB},
title = {miRNA modules for precise, tunable control of gene expression.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {194-212.e7},
doi = {10.1016/j.molcel.2025.11.028},
pmid = {41421338},
issn = {1097-4164},
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; Mice ; Humans ; Dependovirus/genetics ; Transgenes ; *Gene Expression Regulation ; Gene Editing/methods ; Neurons/metabolism ; CRISPR-Cas Systems ; Gene Dosage ; HEK293 Cells ; },
abstract = {Accurate control of transgene expression is important for research and therapy but is challenging to achieve in most settings. MicroRNA (miRNA)-based regulatory circuits can be incorporated within transgenes for improved control. However, the design principles, performance limits, and applications of these circuits in research and biotechnology have not been systematically determined. Here, combining modeling and experiments, we introduce miRNA-based circuit modules, termed "dosage invariant miRNA-mediated expression regulators" (DIMMERs), that establish precise, tunable control of transgene expression across diverse cell types to facilitate imaging, editing, and gene therapy. The circuits use multivalent miRNA regulatory interactions to achieve nearly uniform, tunable protein expression over two orders of magnitude variation in gene dosage. They function across diverse cell types and can be multiplexed for the independent regulation of multiple genes. DIMMERs reduce off-target CRISPR base editing, improve single-molecule imaging, and allow live tracking of adeno-associated virus (AAV)-delivered transgene expression in mouse cortical neurons. DIMMERs thus enable accurate regulation for research and biotechnology applications.},
}
@article {pmid41421670,
year = {2026},
author = {Fan, X and Li, B and Xu, X and Long, B and Jia, Z and Wang, R and Gao, J and Chen, Y and Peng, M and Zhou, M},
title = {Deciphering the regulatory role of the pfs gene on biofilm formation in Lactobacillus plantarum R: Insights from transcriptome and metabolome.},
journal = {Bioresource technology},
volume = {443},
number = {},
pages = {133833},
doi = {10.1016/j.biortech.2025.133833},
pmid = {41421670},
issn = {1873-2976},
mesh = {*Lactiplantibacillus plantarum/genetics/physiology/metabolism ; *Biofilms/growth &amp; development ; *Transcriptome/genetics ; *Metabolome/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Quorum Sensing/genetics ; *Genes, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Lactobacillus plantarum is a widely recognized probiotic that forms biofilms to enhance environmental tolerance and probiotic properties, but the mechanisms regulating its biofilm formation remain unclear. This study successfully used CRISPR-Cas9 to delete the pfs gene in the high biofilm-producing strain L. plantarum R, and first investigated its role by integrated transcriptomic and metabolomic analyses. The pfs gene participates in the activated methyl cycle and AI-2 synthesis, which is involved in quorum sensing and biofilm formation. Deletion of pfs increased biofilm biomass by 91% and markedly enhanced matrix accumulation, including exopolysaccharides, extracellular proteins and extracellular DNA (eDNA). Transcriptomic analysis revealed significant perturbation of cysteine and methionine metabolism and altered expression of key genes involved in exopolysaccharide synthesis. Metabolomic profiling identified 223 differentially expressed metabolites, primarily associated with carbon flux and EPS precursor pathways. In summary, pfs deletion enhances biofilm formation via metabolic reprogramming rather than classical AI-2 dependent QS pathways. This study provides new insights into the pfs gene-mediated regulation of biofilm formation in L. plantarum and establishes a foundation for future strategies to manipulate biofilm formation in industrial applications.},
}
@article {pmid41422102,
year = {2025},
author = {Zhong, C and Yu, L and Zhao, T and Shen, X and Li, Z and Zhu, W and Hu, Z and Tian, R and Han, Z and He, D},
title = {A Plug-in system for reprogramming the editing patterns of base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {910},
pmid = {41422102},
issn = {2041-1723},
support = {32171465//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32500460//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M734091//China Postdoctoral Science Foundation/ ; },
mesh = {*Gene Editing/methods ; Zebrafish/genetics/embryology ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase's spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE's versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.},
}
@article {pmid41422144,
year = {2025},
author = {Felício, D and Osório, H and Pereira, C and Brandão, AF and Freixo, JP and Carvalho, I and Sousa, AP and Castro-Caldas, M and Sequeiros, J and Lemos, C and Santos, M},
title = {Missense variant in TTBK2 kinase domain causes loss of function and impaired protein phosphorylation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2501},
pmid = {41422144},
issn = {2045-2322},
support = {UI/BD/154402/2023//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UIDB/00215/2020, UIDP/00215/2020, LA/P/0064/2020 , UID/215/2025//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; DL 57/2016 - Norma Transit&#xf3;ria//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Rede Nacional de Espectrometria de Massa/ ; ZGRACA//Ataxia UK/ ; },
mesh = {Humans ; Phosphorylation ; *Mutation, Missense ; *Protein Serine-Threonine Kinases/genetics/metabolism/chemistry ; Protein Domains ; CRISPR-Cas Systems ; *Loss of Function Mutation ; HEK293 Cells ; },
abstract = {Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause spinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.},
}
@article {pmid41422267,
year = {2025},
author = {Fair, T and Pavlovic, BJ and Swope, D and Castillo, OE and Schaefer, NK and Pollen, AA},
title = {Mapping cis- and trans-regulatory target genes of human-specific deletions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11380},
pmid = {41422267},
issn = {2041-1723},
support = {F31 HG011569-01A1//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31 HG011569/HG/NHGRI NIH HHS/United States ; P51 OD011132/OD/NIH HHS/United States ; DP2MH122400-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; DP2 MH122400/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; Pan troglodytes/genetics ; Animals ; CRISPR-Cas Systems ; *Sequence Deletion/genetics ; Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Genome, Human ; Chromatin/metabolism/genetics ; Cell Proliferation/genetics ; Brain/metabolism ; },
abstract = {Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution. Comparative genetic studies of primates have identified thousands of human-specific deletions (hDels), and the cis-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes, we discovered 20 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the brain. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.},
}
@article {pmid41423037,
year = {2026},
author = {Ma, L and Wu, B and Li, S and Zhang, X and Zhao, X and Zhang, J and Zhang, M and Zhang, M and Ma, L and Guo, C and Zhang, T},
title = {CRISPR/Cas9-mediated LmSerpin5 knockout causes midgut dysplasia and leads to embryonic lethality in Locusta migratoria.},
journal = {Journal of insect physiology},
volume = {168},
number = {},
pages = {104925},
doi = {10.1016/j.jinsphys.2025.104925},
pmid = {41423037},
issn = {1879-1611},
mesh = {Animals ; *Locusta migratoria/genetics/growth &amp; development/embryology/immunology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Nymph/growth &amp; development/genetics ; Immunity, Innate/genetics ; Gene Knockout Techniques ; },
abstract = {Serpins play a crucial role in in various physiological processes of insects. Previous studies have suggested that Serpins regulated processes like egg diapause, melanization, and antimicrobial peptide synthesis in Locusta migratoria, but their overall functional characterization remains insufficient. In this study, the functions of LmSerpin5 in regulating developmental processes and innate immunity were investigated via CRISPR/Cas9-mediated knockout. Homozygous LmSerpin5 mutation caused complete embryonic lethality. By contrast, chimeric mutants showed elevated mortality during embryonic-to-first-instar nymph transition, though chitinous tissue development remained unaffected. Additionally, adult mutants exhibited no external malformations but displayed pathological changes in immune organs, including fat body cells with enlarged lipid droplets and nuclei, and midgut absorptive cells lacking brush borders. Furthermore, pro-nymphal midguts exhibited reduced microvilli density, structural defects, and inflammatory intestinal folds. Molecular analysis confirmed upregulation of Toll pathway downstream genes (LmMyd88, LmPelle and LmTube) in mutant tissues, with midgut-specific activation of LmTube and LmPelle linking structural damage to immune dysregulation. These results demonstrated LmSerpin5 maintains homeostasis through dual mechanisms: ensuring embryonic survival and suppressing excessive Toll activation.},
}
@article {pmid41423170,
year = {2026},
author = {Sheri, V and Verma, PK and Lekkala, S and Janga, MR},
title = {Application of digital PCR and CRISPR/Cas13a-based fluorescent assay for accurate and on-site detection of cotton leafroll dwarf virus.},
journal = {Journal of virological methods},
volume = {341},
number = {},
pages = {115332},
doi = {10.1016/j.jviromet.2025.115332},
pmid = {41423170},
issn = {1879-0984},
mesh = {*Plant Diseases/virology ; *Gossypium/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Plant Leaves/virology ; Viral Load ; *Closteroviridae/isolation &amp; purification/genetics ; RNA, Viral/genetics ; Texas ; *Polymerase Chain Reaction/methods ; },
abstract = {Cotton leafroll dwarf virus (CLRDV) is an emerging viral pathogen posing a significant threat to cotton production in the United States. Early and accurate detection is critical for effective disease surveillance and management. Although traditional reverse transcription PCR (RT-PCR) is commonly employed for CLRDV diagnosis, it suffers from limitations in sensitivity, quantification accuracy, and involves labor-intensive workflows. In this study, we evaluated two advanced molecular diagnostic approaches for detecting CLRDV, digital PCR (dPCR) and CRISPR/Cas13a-based fluorescent assay. Symptomatic cotton leaf samples from Lubbock and Brownfield, Texas, were screened and confirmed positive by RT-PCR. Digital PCR analysis enabled absolute quantification of viral load, revealing significantly higher titers in Brownfield (F2) samples and offered improved sensitivity over RT-PCR, particularly in samples with low viral loads. However, dPCR is resource-intensive and requires specialized instrumentation. To address the need for rapid, field-deployable diagnostics, we developed a CRISPR/Cas13a-based assay targeting the conserved ORF3, ORF2, and ORF3a regions of the CLRDV genome. Adapted from the SHERLOCK platform, this fluorescence-based assay uses collateral cleavage activity of Cas13a to enable highly specific visual detection. While the assay successfully enabled direct detection from crude leaf extracts without RNA purification, the sensitivity analysis was conducted using purified, in vitro transcribed RNA. Fluorescence signals were reliably observed with as few as 50 RNA copies, defining the assay's practical limit of detection. While dPCR is optimal for quantitative laboratory analysis, the CRISPR/Cas13a-based assay offers a rapid, sensitive, and cost-effective tool for field-level detection. Together, these complementary tools enhance CLRDV surveillance and management in cotton.},
}
@article {pmid41423250,
year = {2026},
author = {Sharma, A and Pathangey, L and Chirackal, SS and Shim, KG and Fonseca, R and Swaminathan, S},
title = {Ferritin H Knockout Induces Differential Immunomodulatory Drug Responses in Multiple Myeloma Cell Lines.},
journal = {European journal of haematology},
volume = {116},
number = {4},
pages = {391-401},
doi = {10.1111/ejh.70084},
pmid = {41423250},
issn = {1600-0609},
support = {//Paula and Rodger Riney Foundation/ ; },
mesh = {Humans ; *Multiple Myeloma/genetics/metabolism/drug therapy/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; *Ferritins/genetics ; Reactive Oxygen Species/metabolism ; Iron/metabolism ; CRISPR-Cas Systems ; *Immunomodulating Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Oxidative Stress ; Oxidoreductases ; },
abstract = {BACKGROUND: Immunomodulatory agents (IMiDs) are a cornerstone in the successful management of multiple myeloma (MM). However, acquired IMiD resistance leading to disease relapses remains a major barrier. Hydrogen peroxide generation and oxidative stress are key mediators that determine IMiD's effectiveness in MM. Iron plays a key role in the generation of oxidative stress; therefore, cellular iron levels are tightly governed. FTH1 is the major iron storage protein that tightly regulates cellular iron availability. Hence, the present study is targeted to investigate the role of FTH1 in MM and IMiD resistance.<br><br>METHODS: IMiD-sensitive and IMiD-resistant MM cells were analyzed for expression of iron-metabolism genes. CRISPR-cas9-mediated knockout of FTH1 was performed and the after-effects were assessed through multiple experiments.<br><br>RESULTS: Initial analysis showed a positive correlation between FTH1 expression and IMiD resistance in MM cells. FTH1-KO reduced IMiD sensitivity in the KMS11 cell line but had no effect on the RPMI8226 cell line. RNA-seq data showed downregulation of ER-stress and calcium signaling genes after FTH1-KO. Further, KMS11-FTH1KO cells exhibited lower intracellular ROS, labile-iron, and mitochondrial superoxide levels along with increased CD63, suggesting activation of L-ferritin secretory pathways.<br><br>CONCLUSION: Data reveals a link between FTH1, labile iron, ROS, and IMiD resistance in MM cells.},
}
@article {pmid41423621,
year = {2025},
author = {Goswami, SG and Gupta, P and Arvinden, VR and Bhargava, N and Iyer, AR and Saravanakumar, V and Yadav, P and Jha, SK and Singh, S and Kumar, A and Singh, P and Gunda, P and Jain, S and Mehta, P and Nakamura, Y and Kurita, R and Bajaj, A and Ramalingam, S},
title = {CRISPR editing of HPFH3 genotype induces γ-globin expression and reverses sickle cell disease and β-thalassemia phenotypes.},
journal = {Stem cell research &amp; therapy},
volume = {17},
number = {1},
pages = {46},
pmid = {41423621},
issn = {1757-6512},
mesh = {*beta-Thalassemia/genetics/therapy/pathology/metabolism ; *Anemia, Sickle Cell/genetics/therapy/pathology/metabolism ; Humans ; *gamma-Globins/genetics/metabolism ; Animals ; *Gene Editing/methods ; *Fetal Hemoglobin/genetics/metabolism ; CRISPR-Cas Systems ; Genotype ; Mice ; Phenotype ; Hematopoietic Stem Cells/metabolism ; },
abstract = {BACKGROUND: Hereditary persistence of Fetal Hemoglobin (HPFH) is a benign condition known to mitigate symptoms in individuals with co-inherited β-hemoglobinopathies, such as β-thalassemia (BT) and sickle cell disease (SCD), through the reactivation of fetal hemoglobin (HbF). HPFH typically arises from deletions of varying sizes affecting the β-globin gene cluster or point mutations in the promoters of the γ-globin genes. While the therapeutic benefits of point mutations have been extensively studied, the potential of deletional forms of HPFH remains underexplored in preclinical settings.<br><br>METHOD: In this study, we generated benign deletional HPFH3 genotype in SCD and BT patient-derived HSPCs using CRISPR/Cas9 and showed that therapeutically relevant levels of HbF reactivation result in the alleviation of the pathological phenotypes.<br><br>RESULTS: In edited cells derived from SCD patients, we observed reduced sickling and oxidative stress, while in edited from BT cells, restoration of the &#x3b1;-globin/&#x3b2;-globin ratio improved erythroid lineage maturation and reduced ROS levels. Importantly, HPFH3-edited HSPCs retained their genome integrity and showed no detrimental effect on their regeneration or differentiation into erythroid, myeloid, T, and B cell lineages in immunodeficient NBSGW mice post-xenotransplantation. Additionally, we showed a reduced interaction between the LCR and HBB, suggesting that the HPFH3 deletion specifically promoted LCR interactions with HBG1/2, likely due to the absence of the HBB locus.<br><br>CONCLUSIONS: Collectively, our preclinical findings suggest that the generation of the HPFH3 genotype has the potential to significantly enhance HbF levels, offering a promising universal therapeutic strategy for treating both SCD and &#x3b2;-thalassemia.},
}
@article {pmid41423823,
year = {2025},
author = {Stamilla, A and Recchia, D and Stelitano, G and Maci, L and Marturano, MC and De Rossi, E and Chiarelli, LR and Pasca, MR and Degiacomi, G},
title = {Uncovering Insights Into the Biology of Mycobacterium tuberculosis Using Genetic Tools.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70206},
pmid = {41423823},
issn = {2045-8827},
support = {20205B2HZE_003//University of Pavia; Italian Ministry of University and Research/ ; 2022JTPP53//University of Pavia; Italian Ministry of University and Research/ ; PE00000007//NextGenerationEUMUR PNRR/ ; INF-ACT//NextGenerationEUMUR PNRR/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/pathogenicity ; Humans ; *Genetic Engineering/methods ; Tuberculosis/microbiology ; Drug Discovery ; Genome, Bacterial ; },
abstract = {Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most challenging pathogens due to its complex physiology, diverse clinical manifestations, and growing multidrug resistance. The global rise of drug-resistant Mtb strains has prompted the search for innovative genetic and molecular strategies to accelerate drug discovery and vaccine development. Progress in Mtb research has long been hindered by its slow replication rate and impermeable cell envelope, which limit the efficacy of genetic manipulation. This review outlines methodological advances that have transformed the study of Mtb pathogenesis and drug resistance mechanisms. Traditional homologous recombination-based approaches, including allelic exchange and specialized transduction, laid the groundwork for targeted mutagenesis but were limited by low efficiency. The advent of phage-derived recombineering systems, such as the Che9c RecET, has substantially improved the precision and throughput of genetic modification. Hybrid systems such as ORBIT, which combines oligonucleotide-mediated recombineering with Bxb1 integrase, have further enabled rapid and versatile genome engineering across mycobacterial species. Parallel developments in conditional gene expression systems (e.g., the use of TetR/Pip-based promoters) have facilitated the functional analysis of essential genes and the validation of novel drug targets. The advent of CRISPR-Cas technologies has represented a paradigm shift, by enabling programmable, high-fidelity gene regulation and functional genomics even in slow-growing mycobacteria. Together, these genetic innovations are transforming Mtb research by accelerating drug discovery and vaccine design, and shedding light on host-pathogen interactions.},
}
@article {pmid41424172,
year = {2025},
author = {Ikram, M and Farhan, M and Derakhshani, B and Kumar, S and Khan, N and Gupta, R and Usman, B and Liu, P},
title = {Machine Learning and CRISPR-Based Validation Elucidate OsWOX13 Involvement in Rice Heat Stress Tolerance and Flowering.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70714},
doi = {10.1111/ppl.70714},
pmid = {41424172},
issn = {1399-3054},
support = {YSPTZX202206//Special Project for the Academician Team Innovation Center of Hainan Province/ ; ZDYF2022XDNY185//Key Research Program of Hainan Province/ ; },
mesh = {*Oryza/genetics/physiology ; *Machine Learning ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Heat-Shock Response/genetics ; *Flowers/physiology/genetics ; *Thermotolerance/genetics ; CRISPR-Cas Systems/genetics ; Proteomics ; },
abstract = {Rice (Oryza sativa) is a staple food for billions of people globally, but it faces significant yield losses due to heat stress. However, rice responses to heat stress remain understudied as compared to other stress factors. In this study, we combined meta-transcriptomics, machine learning, functional validation, and proteomic analysis to identify the key genes involved in heat stress tolerance in rice. We identified 409 meta-differentially expressed genes (meta-DEGs) between heat-tolerant and susceptible genotypes, which were associated with detoxification, oxidative stress, protein folding, phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction. We trained five machine learning models, of which Random Forest (RF) and eXtreme Gradient Boosting (XGBoost) outperformed the others. Using SHAP analysis, the top 14 genes for each model were identified, including the OsWOX13 gene, which was detected simultaneously across both models, indicating a positive regulator and a strong candidate for heat stress tolerance. Functional validation of OsWOX13 via CRISPR/Cas9-mediated knockout (KO) confirmed its positive role in heat stress, with a delay in flowering and survival rate of ~20% compared to ~60% for WT under heat stress. Physiological and antioxidant enzymatic activities showed a significant (p ≤ 0.05) reduction in ABA accumulation levels, increased MDA accumulation, and decreased SOD and POD activities in KO lines compared to WT. The proteomic analysis identified upregulated heat shock proteins (HSF8, BIP1, BIP5, and HSP81-1) and downregulated flowering-associated proteins (ROC6 and 4CL4) in mutant lines in response to heat stress. These results indicate that OsWOX13 enhances heat tolerance by regulating ABA signaling and antioxidant defense mechanisms. Taken together, this study highlights the efficiency of machine learning models in the identification of stress responsive genes and provides OsWOX13 as a strong positive candidate for heat stress tolerance and breeding climate-resilient rice varieties.},
}
@article {pmid41424849,
year = {2026},
author = {Liu, M and Fu, X and Zhang, H and Pan, J and Jia, Q and Zhang, C and An, F},
title = {Endothelial KSR2 regulated by genetic variation protects against atherosclerosis through AMPKα1 stabilization.},
journal = {Theranostics},
volume = {16},
number = {5},
pages = {2598-2626},
pmid = {41424849},
issn = {1838-7640},
mesh = {Animals ; *Atherosclerosis/genetics/metabolism/pathology ; Mice ; *AMP-Activated Protein Kinases/metabolism/genetics ; Humans ; Polymorphism, Single Nucleotide/genetics ; Mice, Knockout ; Male ; Endothelial Cells/metabolism ; Diet, High-Fat/adverse effects ; Mice, Inbred C57BL ; Apolipoproteins E/genetics ; Mice, Knockout, ApoE ; Disease Models, Animal ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Apoptosis/genetics ; CRISPR-Cas Systems ; },
abstract = {Rationale: The single nucleotide polymorphism (SNP) rs11830157 within the scaffold protein kinase suppressor of Ras 2 (KSR2) locus is strongly associated with the incidence of coronary artery disease (CAD), yet its functional role remains undefined. This study aimed to investigate the potential impact of rs11830157 polymorphism on atherosclerosis and to elucidate the underlying molecular mechanisms. Methods: Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and CRISPR/Cas9 gene-editing techniques were used to investigate the regulatory role of the SNP rs11830157. To assess the role of KSR2 in atherosclerosis, we utilized global KSR2 knockout mice fed a high-fat diet ad libitum, pair-fed global KSR2 and Apoe (Apolipoprotein E) double knockout mice, and mice with endothelial-specific KSR2 overexpression mediated by AAV9-ICAM2. Results: Genetic analyses identified SNP rs12822146, in linkage disequilibrium with rs11830157 and located within an endothelial enhancer, as a regulator of KSR2 expression via differential binding of the transcriptional repressor XBP1s. KSR2 expression was significantly reduced in endothelial cells within atherosclerotic plaques in both humans and mice. Using multiple KSR2 gene-edited mouse models, we demonstrated that endothelial KSR2 protects against atherosclerosis by suppressing inflammation and apoptosis. Mechanistic studies revealed that KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A[CRBN] E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1. The subsequently activated AMPK signaling pathway maintains glycolytic balance in endothelial cells, ultimately exerting anti-inflammatory and anti-apoptotic effects. Conclusions: Our findings provide the first comprehensive molecular explanation of the rs12822146-KSR2-atherosclerosis axis, with important implications for both primary prevention and secondary treatment of CAD.},
}
@article {pmid41424917,
year = {2025},
author = {Sharma, S and Saroha, NK and Sehrawat, A and Tang, G and Singh, D and Teotia, S},
title = {Emerging tools in plant genome editing.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1588089},
pmid = {41424917},
issn = {2673-3439},
abstract = {Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genetic material. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools unlock previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, piggyBac and TnpB, and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems, which could be harnessed for tissue-specific and environmentally-responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically-guided editing, minimizing reliance on biological nucleases and reducing off-target risks. Their modularity and programmability are enabling gene function studies, synthetic pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology, positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive and intelligent crops, tailored to future environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they are proven to be effective elsewhere and hold a great potential to be effective in creating climate-resilient crops.},
}
@article {pmid41425600,
year = {2025},
author = {Fazeli, A and Ullrich, E and Cathomen, T and Bexte, T},
title = {Engineering with care: safety assessment platforms for CRISPR-modified natural killer cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1711414},
pmid = {41425600},
issn = {1664-3224},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Immunotherapy, Adoptive/methods/adverse effects ; },
abstract = {CRISPR-based gene editing has become a transformative tool to enhance immune cell therapies. In particular, engineering natural killer (NK) cells with CRISPR/Cas systems has gained traction due to their ability to mediate strong anti-tumor responses in an MHC-unrestricted, non-alloreactive manner. Early trials show the feasibility and safety of allogeneic NK cells, paving the way as scalable "off-the-shelf" products. CRISPR/Cas9 edits genomes by inducing DNA double-strand breaks (DSBs), mainly repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). While effective, CRISPR carries risks of off-target (OT) activity that may disrupt essential genes, cause chromosomal rearrangements, or trigger oncogenic changes - posing threats to product integrity and patient safety. These concerns intensify with multiplex editing, where multiple loci are modified to improve function, persistence, and immune evasion. Since unmodified NK cells are typically short-lived, many clinical-stage products are engineered to express IL-15 or related constructs, extending their half-life and amplifying risks associated with unintended changes. This underscores the urgent need for robust safety assessments. In this review, we summarize the current landscape of safety assessment platforms for evaluating gene edited NK cells. We highlight predictive in silico tools, biochemical in vitro assays, and emerging cell-based detection systems to identify and quantify CRISPR-induced OT events. Particular attention is given to their suitability, limitations, and practical use in primary NK cells and multiplex editing strategies. Our aim is to support the design of safe, effective editing workflows for NK cell therapies - ensuring rigor as the field advances rapidly toward clinical application.},
}
@article {pmid41427723,
year = {2026},
author = {Ortiz-Severin, J and Geoffroy, P and Aravena, P and Hodar, C and Palma, DE and González, M and Cambiazo, V},
title = {Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0156025},
pmid = {41427723},
issn = {1098-5336},
support = {1211893//ANID-Fondecyt/ ; 2024T2DID//Doctoral Fellowship/ ; ICN2021_044//Millennium Science Initiative Program/ ; },
mesh = {*Piscirickettsia/genetics ; Fish Diseases/microbiology ; Piscirickettsiaceae Infections/microbiology/veterinary ; Animals ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics ; Gene Silencing ; },
abstract = {UNLABELLED: Piscirickettsia salmonis is the causative agent of salmonid rickettsial septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression of dCas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains when the expression of the fur gRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Fe[2+] and Fe[3+] acquisition systems and iron mobilization in the fur1 knockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system in P. salmonis has been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.<br><br>IMPORTANCE: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.},
}
@article {pmid41427901,
year = {2026},
author = {Matsumoto, K and Yamamoto, W and Fukutomi, Y and Koshikawa, S},
title = {Functions of melanin synthesis genes, yellow and tan, in wing pigmentation revealed by CRISPR/Cas9-mediated mutagenesis in Drosophila guttifera.},
journal = {Insect molecular biology},
volume = {35},
number = {3},
pages = {246-256},
doi = {10.1111/imb.70024},
pmid = {41427901},
issn = {1365-2583},
support = {//Japan Society for the Promotion of Science/ ; JP17K19427//KAKENHI/ ; JP24K21982//KAKENHI/ ; JP25K02019//KAKENHI/ ; },
mesh = {Animals ; *Pigmentation/genetics ; CRISPR-Cas Systems ; *Melanins/biosynthesis/genetics ; Wings, Animal/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/metabolism/physiology ; Mutagenesis ; },
abstract = {Colour pattern formation is a key model for studying evolutionary and developmental mechanisms. In the fruit fly Drosophila guttifera, which exhibits distinctive polka-dot wing pigmentation, we investigated the roles of two putative melanin synthesis genes, yellow and tan, using CRISPR/Cas9-mediated genome editing. We established multiple mutant strains with lesions in either gene and found that both genes were essential for normal pigmentation intensity in wing spots, though the patterns themselves persisted. Double mutants showed further reduction in pigmentation, indicating additive effects but not complete loss of patterning. Ectopic expression of wingless failed to induce normal pigmentation in yellow or tan mutants, demonstrating that both genes act downstream of wingless and are required for its pigmentation-inducing function. Furthermore, mosaic phenotypes in G0 individuals revealed quasi-cell-autonomous functions of tan, suggesting that pigmentation in D. guttifera wings depends on local availability of precursors rather than solely on transport via wing veins. This study establishes D. guttifera as a genetically tractable system for functional analyses and contributes to understanding the molecular basis of insect colour pattern formation.},
}
@article {pmid41428463,
year = {2026},
author = {Ye, T and Xue, M and Chen, H and Yue, S and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Allosteric Aptamer CRISPR/Cas Activation Enables Non-competitive ATP Detection and Meat Freshness Assessment.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1592-1601},
doi = {10.1021/acs.jafc.5c12150},
pmid = {41428463},
issn = {1520-5118},
mesh = {*Adenosine Triphosphate/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; Animals ; *Meat/analysis ; *Meat Products/analysis ; },
abstract = {CRISPR/Cas-based aptasensors (Cas-aptasensors) hold great promise for detecting non-nucleic acid targets, yet their intrinsic competitive recognition mechanism imposes a trade-off between transduction efficiency and background leakage. In this study, we developed a Cas-aptasensor that employs a non-competitive recognition mechanism. In our design, the aptamer-target interaction accelerates the toehold-mediated strand displacement reaction and exposes a second toehold domain. The CRISPR/Cas system is ultimately activated via a cascade strand-displacement reaction, which is hindered in the absence of the target and, thus, delays activation. We demonstrated the applicability of this non-competitive Cas-aptasensor for the detection of ATP, achieving a detection limit as low as 1.0 nM within 45 min. Furthermore, we successfully applied this method to ATP detection in complex matrices and to assess the freshness of diverse meat products across different storage temperatures. Overall, this work advances the design of Cas-aptasensors and expands their potential applications in food safety monitoring.},
}
@article {pmid41428486,
year = {2026},
author = {Ribeiro Gomes, AR and Hamel, N and Mastwal, S and Wright, N and Ide, DC and Richie, CT and Usdin, TB and Wang, KH and Leopold, DA},
title = {Targeted gene transfer into developmentally defined cell populations of the primate brain.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116756},
pmid = {41428486},
issn = {2211-1247},
support = {ZIA MH002898/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; *Gene Transfer Techniques ; *Brain/metabolism/embryology ; Callithrix ; Female ; Rats ; Dependovirus/genetics ; Transgenes/genetics ; Gene Editing/methods ; Genetic Vectors ; Male ; CRISPR-Cas Systems ; },
abstract = {The primate brain possesses unique physiological and developmental features, yet its systematic investigation has been hampered by a paucity of transgenic germline models and tools. Here, we present a minimally invasive method to introduce transgenes widely across the primate cerebral cortex using ultrasound-guided fetal intracerebroventricular viral injections (FIVIs). FIVI enables efficient and long-lasting transgene expression following intrauterine delivery of recombinant adeno-associated viruses (rAAVs). In the marmoset, we demonstrate that adjusting gestational timing, rAAV serotype, and transcriptional regulatory elements enables selective targeting of defined cell populations, including layer-restricted labeling and Cre-dependent intersectional access. Pilot experiments in rats further demonstrate the potential of FIVIs for prenatal CRISPR-based gene editing and labeling of peripheral somatosensory and retinal pathways. By mimicking key desirable features of germline transgenic models, this efficient and targeted method for gene transfer into the fetal primate brain expands the experimental opportunities for basic and translational neuroscience research across the lifespan.},
}
@article {pmid41428487,
year = {2026},
author = {Deng, C and Hu, J and Chen, Q and Zhou, S and Ni, J},
title = {Expanded global groundwater microbial diversity reveals bioprospecting potential.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116760},
doi = {10.1016/j.celrep.2025.116760},
pmid = {41428487},
issn = {2211-1247},
mesh = {*Groundwater/microbiology ; Phylogeny ; *Bacteria/genetics/classification ; *Microbiota/genetics ; *Bioprospecting ; Archaea/genetics/classification ; Genome, Bacterial/genetics ; Genome, Archaeal/genetics ; Biodiversity ; Metagenomics ; Metagenome ; },
abstract = {Although the terrestrial subsurface harbors a substantial fraction of Earth's microbial biomass, the genomic diversity of groundwater microbiomes and their potential for bioprospecting remain poorly characterized. Here, we recovered 44,320 bacterial and archaeal genomes from in-house and publicly available metagenomic datasets, establishing a large-scale groundwater microbiota catalog (GWMC) spanning 167 phyla, including four candidate phyla and over 12,000 previously uncharacterized species. This unprecedented phylogenetic diversity was accompanied by a bimodal genome size distribution (0.3-12.8 Mbp), revealing divergent strategies of genomic allocation. By mining extensive genomic resources, we found that small genomes prioritized molecular defense and redox regulation, whereas large genomes frequently harbored greater biosynthetic potential. Notably, we establish the largest selenoprotein catalog to date and highlight groundwater as an overlooked hotspot of microbial selenium metabolism. Overall, this work advances our understanding of microbial diversity in aquifers and uncovers underexplored genomic resources with potential for biotechnology and biomedicine.},
}
@article {pmid41428728,
year = {2025},
author = {Gervais, NC and Rogers, RKJ and Robin, MR and Shapiro, RS},
title = {HyperdCas12a-based multiplexed genetic regulation in Candida albicans.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428728},
issn = {1362-4962},
support = {RGPIN-2018-4914//Natural Sciences and Engineering Research Council of Canada/ ; //NSERC/ ; //Canada Research Chair/ ; RGPIN-2018-4914//NSERC/ ; },
mesh = {*Candida albicans/genetics/metabolism/drug effects ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Ergosterol/biosynthesis ; Fungal Proteins/genetics/metabolism ; Drug Resistance, Fungal/genetics ; },
abstract = {Complex microbial phenotypes involve the combined activity of diverse gene regulatory networks. However, the majority of reverse genetics approaches in microbial pathogenesis research have focused on single-gene perturbation studies, in part due to the lack of available genetic tools in many pathogens. Developing enhanced versions of CRISPR-Cas platforms holds significant promise for improving the scalability of microbial functional genomics research. Here, we demonstrate highly efficient, inducible, and multiplexed activation and repression in the major human fungal pathogen Candida albicans by translating the hyperdCas12a variant to the fungal kingdom. This represents the first application of a CRISPR-Cas12 system in a human fungal pathogen. We profile the effectiveness of our new CRISPR activation and CRISPR interference tools and achieve tunable levels of target modulation. Further, we demonstrate that perturbing combinations of genes in the drug efflux and ergosterol biosynthesis pathways reveals important redundancies and synergistic properties in drug resistance circuitry. Our hyperdCas12a platform is thus an efficient system for the rapid generation of combinatorial mutants that will enable the mechanistic understanding of genetic interactions involved in diverse phenotypes in C. albicans. The enhanced activity with hyperdCas12a in fungi suggests it could be translated to other microbes as a powerful tool for studying genetic interactions.},
}
@article {pmid41428733,
year = {2025},
author = {Yang, Z and Yu, M and Li, P and Li, Z and Teng, Y and Zhou, Y and Zhao, M and Liu, C and Zhao, Z and Wang, Z and Li, J and Jing, Y and Li, Y and Zhao, H and Song, W and Bian, C and Zhao, H and Chen, J and Xin, B and Lai, J},
title = {Casδ, an evolutionary transitional CRISPR system enables efficient genome editing across animals and plants.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428733},
issn = {1362-4962},
support = {//Agriculture Science and Technology/ ; 2023YFD1202900//National Key Research and Development Program of China/ ; PC2023A01004//Pinduoduo-China Agricultural University/ ; //Agriculture Science and Technology/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Evolution, Molecular ; Oryza/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) adaptive immune systems provide sequence-specific mechanisms for targeting foreign DNA or RNA and have been widely used in genome editing and DNA detection. Type V CRISPR-Cas systems are characterized by a single RNA-guided RuvC domain-containing effector, Cas12. Here, through comprehensive mining of large-scale genomic and metagenomic data from microbial sources, we identified a new Class 2 CRISPR-Cas effector superfamily, designated Casδ, comprising three members with protein sizes ranging from 867 to 936 amino acids. Biochemical analyses revealed that Casδ-1 functions as a single RNA-guided endonuclease with specific recognition of 5'-RYR-3' protospacer-adjacent motifs, where R represents A or G, and Y represents T or C. Casδ-1 exhibits robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity. Casδ-1 mediates efficient genome editing across species, achieving up to 60% indel rates in human cells while generating homozygous knockout lines in two agriculturally important monocot species (Oryza sativa and Zea mays) through stable transformation. Structural and evolutionary analyses reveal Casδ as an evolutionary transitional nuclease bridging Cas12n and canonical type V systems, featuring a C-terminal loop that is essential for activity. Collectively, Casδ is an evolutionarily distinct, compact (<1000 aa), tracrRNA-free CRISPR system enabling versatile cross-kingdom genome editing.},
}
@article {pmid41428734,
year = {2025},
author = {Zhu, C and Xiao, D and Wang, Y and Han, H and Qin, C and Liu, S and Chen, X and Xiao, H and Chen, X and Shi, J and Tang, J and Shen, J and Song, H},
title = {Molecular basis of NFIB-mediated regulation of oncogenic transcription.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428734},
issn = {1362-4962},
support = {Z2023033//Hunan Health Commission Key Clinical Specialty Major Research Project/ ; NFPS-JJ-501348//Chinese National Key Clinical Specialty Program/ ; 82272508//National Natural Science Foundation of China/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; 0007/2022/AKP, 0068/2023/ITP2, 0143/2025/ITP2//Macau Science and Technology Development Fund/ ; MYRG-GRG2024-00283-ICMS-UMDF, SRG2023-00054-ICMS//University of Macau/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; },
mesh = {Humans ; *NFI Transcription Factors/genetics/chemistry/metabolism ; *Gene Expression Regulation, Neoplastic ; DNA/metabolism/chemistry/genetics ; Cell Line, Tumor ; *Transcription, Genetic ; CRISPR-Cas Systems ; Protein Binding ; *Neoplasms/genetics/pathology/metabolism ; Cell Proliferation/genetics ; Oncogenes ; Models, Molecular ; Cell Movement/genetics ; Transcriptional Activation ; },
abstract = {The Nuclear Factor I (NFI) family of transcription factors orchestrates key regulatory programs in development, differentiation, and metabolism, with dysregulation implicated in diverse pathological conditions, including cancer. Among the paralogs, NFIB has emerged as an oncogenic driver in multiple tumor types, yet the mechanisms through which it engages DNA and directs oncogenic transcriptional programs remain undefined. Here, using cancer cells with high NFIB expression, we demonstrate that NFIB promotes malignant phenotypes, as CRISPR-Cas9 knockout impairs proliferation, migration, and invasion. Transcriptomic profiling reveals that NFIB regulates a cancer-enriched gene network that includes FGFR3 and PDGFRB. Biophysical analyses show that NFIB, including its DNA-binding domain, functions as a monomer and binds DNA with strict 1:1 stoichiometry. High-resolution crystal structures of NFIB DNA-binding domain bound to ChIP-seq-derived DNA motifs reveal a monomeric binding mode mediated by conserved base-specific interactions with the TGGCA sequence, providing an atomic view of NFIB-DNA recognition. Mutational disruption of key DNA-contacting residues abolishes DNA binding and transcriptional activation, linking atomic-level recognition to oncogenic transcriptional regulation. Together, these findings elucidate the structural mechanism underlying NFIB function in cancer and establish a framework for therapeutic strategies targeting NFIB-driven malignancies.},
}
@article {pmid41430049,
year = {2025},
author = {Dong, Q and Chen, P and Guo, Z and Wei, H and Zeng, Y and Zhang, J and Men, Y and Liu, W and Sun, Y and Yang, J},
title = {Computational design of allulose-responsive biosensor toolbox for auto-inducible protein expression and CRISPRi mediated dynamic metabolic regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11562},
pmid = {41430049},
issn = {2041-1723},
support = {32271545//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Biosensing Techniques/methods ; *Transcription Factors/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Escherichia coli/metabolism/genetics ; Synthetic Biology/methods ; Glucose/metabolism ; },
abstract = {Biosensors based on transcription factors (TFs) have shown extensive applications in synthetic biology. Due to the complex multi-domain structure of effector-TF-DNA, computational design of TFs remains a challenge. Here, we present the successful structure-guided computational design of the access tunnel, ligand binding, allosteric transition process for an allulose-responsive PsiR. It enables a 20-fold increase in sensitivity, reducing the EC50 of PsiR-allulose biosensors (PABs) from 16 mM to 0.8 mM, and delivers a PAB box possessing the detection range from 10 μM to 100 mM. We further validate its broader applicability in enhancing sensitivity of LacI-IPTG biosensor. Based on the developed PABs, we present the inducer-free allulose-mediated auto-inducible protein expression system, and demonstrate an allulose-triggered CRISPR interference circuit for dynamic metabolic regulation. It facilitates a 68% increase in allulose titer and achieves a high yield of 0.43 g/g glucose. This work provides the versatile TF toolbox for developing allulose-triggered regulation circuits in biotechnology application.},
}
@article {pmid41430241,
year = {2025},
author = {Nieto-Sanchez, A and Martinez-Lage, M and Puig-Serra, P and Carpintero, S and Alonso-Yanez, A and Ojeda-Walczuk, P and Ibañez-Navarro, M and Pita, G and Moya, FJ and Moreno, C and Martin, MC and Alonso, R and Nuñez-Torres, R and Sanchez-Arevalo Lobo, VJ and Alonso-Guirado, L and Malats, N and Gonzalez-Neira, A and Fernandez, L and Roda-Navarro, P and Torres-Ruiz, R and Rodriguez-Perales, S},
title = {Selective genome editing of amplified oncogenes triggers immunogenic cell death and tumor remodeling.},
journal = {Molecular cancer},
volume = {25},
number = {1},
pages = {21},
pmid = {41430241},
issn = {1476-4598},
mesh = {Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Mice ; *Oncogenes/genetics ; Cell Line, Tumor ; *Immunogenic Cell Death/genetics ; *Gene Amplification ; *Neoplasms/genetics/pathology/immunology ; Tumor Microenvironment/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Oncogene amplifications fuel some of the most lethal, therapy‑refractory cancers, yet remain clinically untargeted. We report a single‑guide CRISPR/Cas9 strategy that converts the sheer copy‑number excess of oncogene amplicons into an Achilles' heel. A solitary intronic double‑strand break is innocuous in diploid genomes but collapses oncogene amplification‑positive cells across neuroblastoma, small‑cell lung and colorectal carcinoma models, driving > 90% loss of viability, G2/M blockade and catastrophic DNA‑damage signalling. Amplified‑locus cleavage rewires transcription toward cell death activation, necroptosis and cGAS-STING-mediated immunogenic cell death, enabling dendritic‑cell cross‑priming and T‑cell activation and proliferation. In xenografts, delivery of the intronic sgRNA shrinks tumours by 90%, prolongs survival and remodels the innate tumour microenvironment. Deep sequencing confirms negligible off‑target editing, and combination with doxorubicin achieves supra‑additive killing. These findings establish amplification density, not sequence content, as a tractable, tumour‑exclusive target and unveil a dual‑action platform that is simultaneously cytotoxic and immunostimulatory. Editing of tumor amplifications therefore offers a blueprint for translating copy‑number aberrations into precision genome‑editing therapies for treatment‑resistant cancers.},
}
@article {pmid41430372,
year = {2025},
author = {Kim, GE and Lee, SY and Kang, YJ and Bin Jin, H and Park, HH},
title = {AcrIIA19 binds to the WED domain and inhibits various Cas9 orthologs at multiple stages.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {136},
pmid = {41430372},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; Staphylococcus aureus/genetics/enzymology ; Protein Domains ; Streptococcus pyogenes/genetics/enzymology ; Protein Binding ; *Bacterial Proteins/metabolism/chemistry/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Crystallography, X-Ray ; },
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems, providing valuable tools for regulating genome editing. Here, we present the crystal structure of AcrIIA19, a plasmid-encoded Type II-A CRISPR-Cas system inhibitor that targets Cas9. AcrIIA19 adopts a previously uncharacterized fold and forms a stable homodimer. Biochemical assays revealed that AcrIIA19 binds selectively to the wedge (WED) domain of Cas9, a conserved structural interface critical for single guide RNA-DNA duplex stabilization and catalysis. This interaction disrupts Cas9 activity at multiple stages, independent of the order of complex assembly. Notably, AcrIIA19 exhibits broad-spectrum inhibition across divergent Cas9 orthologs, including Streptococcus pyogenes and Staphylococcus aureus Cas9, by exploiting a conserved WED domain vulnerability. Our findings establish AcrIIA19 as a versatile Cas9 inhibitor and highlight the WED domain as a strategic target for developing species-agnostic CRISPR regulatory tools in biotechnology and therapeutic applications.},
}
@article {pmid41431195,
year = {2026},
author = {Spezzani, E and Capelli, L and Di Lena, D and Chamorro-Garcia, A and Ippodrino, R and Porchetta, A and Bertucci, A},
title = {MARPLE: A Proximity-Triggered CRISPR-Cas13 Platform for Ultrasensitive Antibody Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {13},
pages = {e17799},
pmid = {41431195},
issn = {2198-3844},
support = {//National Recovery and Resilience Plan/ ; MUR 2023-2027//'Departments of Excellence' program of the Italian Ministry for University and Research/ ; 31108//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Antibodies/analysis ; Immunoassay/methods ; },
abstract = {Monitoring clinically relevant antibodies-as biomarkers of disease or therapeutic response-is essential for informed clinical decision-making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point-of-care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR-Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity-triggered Linker Exchange), a modular CRISPR-Cas13-based platform for ultrasensitive antibody detection. MARPLE harnesses antibody-induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13-mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets-including anti-digoxigenin, anti-cholesterol, anti-HA, trastuzumab, and anti-MUC1-highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one-pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR-based sensing beyond nucleic acids to protein biomarker detection.},
}
@article {pmid41431758,
year = {2026},
author = {Hwang, S and Ko, H and Lee, HY and Choi, J},
title = {Nanocarriers for the delivery of the CRISPR/Cas9 system.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {3},
pages = {429-448},
pmid = {41431758},
issn = {1748-6963},
support = {//Korean Government/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; *Nanoparticles/chemistry ; *Gene Transfer Techniques ; *Drug Carriers/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeat/associated protein 9 (CRISPR/Cas9) system has been used for the precise manipulation of target DNA, making efficient genome editing in cells a reality. The CRISPR/Cas9 system has shown great potential in biomedical applications, such as disease treatment, transcription regulation, and genome-wide screening, and is opening a new era in biotechnology. However, the efficient and selective delivery of the CRISPR/Cas9 system remains a critical obstacle. Literature search conducted using Web of Science, Scopus, PubMed and Google Scholar for articles published from 2015 to 2024. In this review, we discuss several delivery methods for the CRISPR/Cas9 system, focusing on techniques using nanocarriers. Specifically, we comprehensively discussed the challenges, future directions, and potential of various delivery methods for the CRISPR/Cas9 system.},
}
@article {pmid41431922,
year = {2026},
author = {Chen, K and Zhu, J and Fan, C and Zhou, A and Li, B and Ge, H and Ning, X},
title = {A Nanoimprinted Photothermal Chip for On-Demand Spatiotemporal Activation of CRISPR/Cas9 Gene Editing.},
journal = {Nano letters},
volume = {26},
number = {1},
pages = {532-542},
doi = {10.1021/acs.nanolett.5c05571},
pmid = {41431922},
issn = {1530-6992},
support = {//National Key Research and Development Program of China/ ; //National Natural Science Foundation of China/ ; //Natural Science Foundation of Jiangsu Province/ ; //Innovation Fund of China Acoustics Valley (Suzhou), the Key Research and Development Program of Science and Technology Innovation (Social Development) in Danyang City/ ; //Clinical Medicine Special Research Fund Project of Nantong University/ ; //the Guiding Science and Technology Plan Project of Social Development in Zhenjiang City/ ; //Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Gold/chemistry ; Sheep ; Infrared Rays ; Humans ; },
abstract = {Precise control of gene editing in target cells is essential for CRISPR/Cas9 applications. Here, we present a nanoimprinted photothermal chip (NPC) engineered for on-demand delivery and activation of CRISPR/Cas9 complexes with high spatial and temporal precision. Fabricated by nanoimprint lithography and subsequent surface modification, NPC features a customized PEGylated plasmonic gold nanopillar array, which provides both optimal cellular adhesion and efficient photothermal conversion. Upon NIR irradiation, NPC generates spatially confined thermal microdomains that transiently permeabilizes cell membranes, thereby facilitating cytosolic delivery of CRISPR/Cas9 complexes and synchronously modulating genome-editing kinetics. In vitro studies demonstrate robust gene knockout in both mouse and sheep cell lines while preserving high cell viability and editing fidelity. Remarkably, NPC-mediated PD-1 gene disruption in cytotoxic T cells markedly enhance their antitumor activity. Overall, this work establishes NPC as a transformative platform for precise and controllable CRISPR/Cas9 gene editing with broad therapeutic potential.},
}
@article {pmid41432281,
year = {2026},
author = {Liu, XL and Liu, L and Cheng, L},
title = {Sequence-independent optical regulation of CRISPR/Cas editing using star-shaped crRNA dendrimers.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {6},
pages = {1877-1881},
doi = {10.1039/d5cc06011g},
pmid = {41432281},
issn = {1364-548X},
mesh = {*Dendrimers/chemistry ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; *RNA/chemistry ; Humans ; Bacterial Proteins/metabolism/genetics ; Endodeoxyribonucleases ; },
abstract = {Precise spatiotemporal control of CRISPR/Cas editing is vital for studying dynamic processes and ensuring therapeutic safety. We present a single-site photolabile crRNA dendrimer platform enabling robust, sequence-independent optical regulation of Cas9 and Cas12a. This simple, universal strategy achieves rapid OFF-to-ON control with minimal leakage, advancing programmable, light-responsive genome editing for biomedical applications.},
}
@article {pmid41432353,
year = {2026},
author = {Tang, X and Ju, D and Hu, H},
title = {A Dual CRISPR-Cas/Cre-loxP Genome Engineering Strategy for Stable Uricase Expression in Food-Grade Probiotics.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {331-341},
doi = {10.1021/acssynbio.5c00774},
pmid = {41432353},
issn = {2161-5063},
mesh = {*Probiotics/metabolism ; *CRISPR-Cas Systems/genetics ; *Lactococcus lactis/genetics/enzymology ; Animals ; *Urate Oxidase/genetics/metabolism ; Gene Editing/methods ; Mice ; Integrases/genetics/metabolism ; Genome, Bacterial/genetics ; Genetic Engineering/methods ; },
abstract = {The development of robust, food-grade microbial chassis with tailored metabolic functions is critical for advancing synthetic biology applications in health and nutrition. Here, we report a dual genome engineering strategy that integrates CRISPR-Cas9-mediated knock-in with Cre/loxP-driven genome reduction to streamline the genome of Lactococcus lactis NZ9000 and enable stable expression of a high-activity uricase variant. The resulting strain, NZ9000::UA[T]-ΔD6, demonstrated enhanced enzymatic performance in vitro, achieving 2.34 U/mL activity and complete degradation of ∼500 μM urate within 20 h. Beyond improved catalytic output, this dual-system approach established a genetically stable and biosafe probiotic chassis with moderate colonization capacity in the murine gut. The integration of CRISPR-Cas and Cre/loxP techniques in this work is intended to enhance the expression of heterologous genes in the chassis strain, while providing a versatile platform for the rational design of food-grade probiotics and offering a general strategy for constructing living biotherapeutic agents with targeted metabolic activities.},
}
@article {pmid41432359,
year = {2026},
author = {Fruitet, E and de Fouchier, A and Heckel, DG and Groot, AT},
title = {Multiple CRISPR/Cas9 modifications of an esterase reveal its role in influencing acetate esters in the pheromone blend of a moth.},
journal = {Insect molecular biology},
volume = {35},
number = {2},
pages = {166-176},
doi = {10.1111/imb.70016},
pmid = {41432359},
issn = {1365-2583},
support = {//Instituut voor Biodiversiteit en Ecosysteem Dynamica, Universiteit van Amsterdam/ ; //Universiteit van Amsterdam/ ; //International Max Plank Research School, Max-Planck-Instituts f&#xfc;r chemische &#xd6;kologie/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {*Moths/genetics/metabolism/enzymology ; Animals ; CRISPR-Cas Systems ; Female ; Male ; *Esterases/genetics/metabolism ; *Acetates/metabolism ; Esters/metabolism ; *Insect Proteins/genetics/metabolism ; *Sex Attractants/metabolism ; Quantitative Trait Loci ; },
abstract = {Sexual signalling by pheromones is essential for mate finding and mate choice in moths and plays an important role in reproductive isolation. Acetates (i.e., acetate esters) produced by females of Heliothis (Chloridea) subflexa Fabricius, 1777 (Lepidoptera: Noctuidae) attract conspecific males but repel Heliothis virescens Fabricius, 1777 (Lepidoptera: Noctuidae) males. A QTL (quantitative trait locus) harbouring carboxylesterases and lipases was previously shown to affect acetates, and CRISPR/Cas9-induced knockouts increased acetate amounts by blocking hydrolysis of the esters as expected. A second, unlinked QTL, containing a cluster of three different carboxylesterases (CXEs), unexpectedly yielded decreased acetate amounts. In one of these genes, esterase CXE24, we found a naturally occurring transposable element insertion in exon 8. A CRISPR/Cas9-induced frameshift at the same position yielded the same results. The paradox was resolved by a CRISPR/Cas9-induced frameshift in exon 2 of CXE24 which increased acetate amounts. The frameshift in exon 2 produced a truncated protein lacking the substrate binding site and the catalytic triad, while the frameshift in exon 8 removed only the third residue of the catalytic triad. In silico modelling showed that the exon-8-truncated protein could not hydrolyse the esters by itself, which likely explains the decreased acetate amounts. To place our findings in an evolutionary context, we explored variation in the esterase cluster in 16 species of Lepidoptera with completely sequenced genomes. Geographic and temporal variation in acetates has been observed in H. subflexa, and variation in the frequency of the transposable element could be a possible explanation.},
}
@article {pmid41432570,
year = {2026},
author = {Hu, J and Li, X and Gao, Y and Guo, Y and Liu, Y and Wang, C and Xu, G and Du, C and Liu, S and Zhao, Z and Wang, Y and Wu, Y and Dong, X and Li, C and Wan, J},
title = {Cas9-Embedding Hyperactive TadA8e Confers Efficient and Highly Specific A-To-G Base Editing in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2576-2591},
pmid = {41432570},
issn = {1467-7652},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; //Ministry of Education of the People's Republic of China/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Adenosine Deaminase/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Adenine base editors (ABEs) produce precise A-to-G conversion in the genomic target sites without causing double-strand breaks. However, the hyperactive adenosine deaminase TadA8e raises safety concerns on genome-wide off-target edits. We engineered 11 chimeric proteins for ABEs (CP-ABEs) by embedding hyperactive TadA8e within Cas9 nickase to minimise the sgRNA-independent off-target effects. Four CP-ABEs exhibited robust on-target activity with minimal sgRNA-independent off-target edits. Then we developed four chimeric high-fidelity ABEs (CH-ABEs) to minimise both sgRNA-dependent and sgRNA-independent off-target effects by employing high-fidelity Cas9 variants. The CH-ABEs achieved reductions of up to 7.0-fold and 79.4-fold in the respective off-target edits, while generating 22.0%-72.4% homozygous and biallelic rice mutants. Whole-genome and whole-transcriptome sequencing (WGS/WTS) confirmed the specificity of CH-ABEs. Incorporating Sniper2L into CH-ABEs further enhanced both specificity and on-target activity. Two PAM-less SpRY variants (SpRY-K2, SpRY-KK) expanded the targeting scope of CP-ABEs and boosted activity by 80.0%. Furthermore, we demonstrated that CP-ABE8e-RY[KK] could discriminate paralogous targets in rice and successfully applied it to create herbicide-resistant rice by precisely installing the OsALS-K591E mutation.},
}
@article {pmid41433155,
year = {2026},
author = {Hemani, D and Grissom, JH and Chi, RJ},
title = {Protocol for marker-free genome editing in Saccharomyces cerevisiae using universal donor templates and multiplexed CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104280},
pmid = {41433155},
issn = {2666-1667},
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal/genetics ; },
abstract = {Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers. For complete details on the use and execution of this protocol, please refer to Grissom et al.[1].},
}
@article {pmid41433963,
year = {2026},
author = {Schneider, O and Zehl, M and Miele, M and Pace, V and Brungs, C and Cheng, JF and Hummelbrunner, S and Dirsch, VM and Zotchev, SB},
title = {Heterologous Expression and CRISPR/Cas9-Assisted Manipulation of the Hybrid Gene Cluster Specifying the Biosynthesis of Meroterpenoids and Phenazines.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {137-148},
pmid = {41433963},
issn = {2161-5063},
mesh = {*Terpenes/chemistry/metabolism ; Phenazines/chemistry/metabolism ; CRISPR-Cas Systems/genetics ; Streptomyces/genetics/metabolism ; Anti-Bacterial Agents/biosynthesis ; },
abstract = {A hybrid gene cluster, mfq, predicted to govern the biosynthesis of both meroterpenoids and phenaziterpenes, was cloned from the genome of Streptomyces sp. S4.7 and introduced into the heterologous host Streptomyces coelicolor M1154. The biosynthesis of the meroterpenoids marfuraquinocins C and D, previously isolated from Streptomyces niveus SCSIO 3406, as well as a new congener, marfuraquinocin E, which exhibited antibacterial activity, was activated upon overexpression of the regulatory protein MfqF. However, production of neither phenaziterpenes nor phenazines was detected. The structure of marfuraquinocin E was elucidated, revealing the attachment of a terpene moiety at C-2, in contrast to C-6 as seen in the known congeners A-D. Using the CRISPR/Cas9 system, several genes in the mfq cluster were inactivated, confirming the role of MfqW as a prenyltransferase specific to the meroterpenoid pathway. Both gene overexpression and further knockouts provided the first insights into the complex regulation of this hybrid gene cluster. To restore the presumably deficient phenazine biosynthetic pathway, a gene encoding a PhzF homologue from another gene cluster in S4.7 was heterologously expressed alongside the mfq cluster, leading to the production of 1,6-phenazine dicarboxylic acid upon MfqF overexpression. This work lays the foundation for elucidating the complete biosynthetic pathway of marfuraquinocins and its potential coregulation with that of phenazines.},
}
@article {pmid41435708,
year = {2026},
author = {Fang, J and Chen, Q and Ran, M and Chen, R and Chen, W and Cui, J and Wang, J and Zhong, K and Shi, L and Lu, C and Jiang, H},
title = {RPA-CRISPR/Cas12a-coupled microfluidic biosensor enabling on-site, sensitive quantification of Vibrio parahaemolyticus.},
journal = {Biosensors &amp; bioelectronics},
volume = {296},
number = {},
pages = {118327},
doi = {10.1016/j.bios.2025.118327},
pmid = {41435708},
issn = {1873-4235},
mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; *Nucleic Acid Amplification Techniques/instrumentation ; Limit of Detection ; *CRISPR-Cas Systems/genetics ; Animals ; Seafood/microbiology ; Lab-On-A-Chip Devices ; *Vibrio Infections/microbiology/diagnosis ; Equipment Design ; },
abstract = {Vibrio parahaemolyticus is a major cause of seafood-associated gastroenteritis and aquatic animal diseases, posing persistent threats to public health and aquaculture. Rapid and accurate on-site quantitative detection is essential for risk assessment and early intervention. Although qPCR and digital PCR provide reliable quantification, their reliance on complex instrumentation limits field deployment. RPA-CRISPR-based isothermal assays offer a low-equipment alternative; however, existing approaches lack robust methodological strategies to achieve standard-curve-based quantitative reliability under field-deployable conditions. Here, we propose a methodological framework that enables in-run calibration and variance control for quantitative isothermal amplification by integrating reaction-volume locking and simultaneous standard-curve generation, implemented here within a closed centrifugal microfluidic system. A multi-unit microfluidic platform preloaded with gradient concentration standard plasmids allows concurrent construction of standard curves and sample analysis in a single run, reducing the impact of environmental and batch-to-batch variability. Signal generation was achieved using a one-pot RPA-CRISPR/Cas12a assay, in which balanced amplification and cleavage kinetics were obtained by screening crRNAs targeting suboptimal PAM sites and optimizing reaction conditions. The platform achieved a detection limit of 6.08 copies/μL and a linear quantitative range of 10[0]-10[4] copies/μL (R[2] > 0.96), with performance comparable to qPCR (AUC = 0.984), and acceptable intra- and inter-assay variability under the tested conditions, with relative standard deviations of 2.63-6.07 %, at a cost of approximately $3.30 per test. Validation using spiked and real seafood samples demonstrated reliable on-site quantification. This work establishes a transferable quantitative methodology for RPA-CRISPR-based isothermal assays, advancing field-deployable pathogen detection in aquaculture and food safety.},
}
@article {pmid41435996,
year = {2026},
author = {Gopalakrishnan, R and Kannan, K and Gunasekaran, R and Ramachandran, P and Ganapathy, D and Pitchiah, S},
title = {A comparative review of vector insertion techniques in Saccharomyces cerevisiae.},
journal = {Journal of microbiological methods},
volume = {241},
number = {},
pages = {107378},
doi = {10.1016/j.mimet.2025.107378},
pmid = {41435996},
issn = {1872-8359},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; *Genetic Engineering/methods ; *Genetic Vectors/genetics ; Genome, Fungal ; Homologous Recombination ; *Mutagenesis, Insertional/methods ; *Saccharomyces cerevisiae/genetics ; Synthetic Biology/methods ; },
abstract = {Saccharomyces cerevisiae, a model organism in genetics and molecular biology has been extensively engineered using various vector insertion techniques. This review compares and contrasts three prominent techniques: In vivo homologous recombination (HR), Cre-lox recombination and CRISPR/Cas9. In vivo HR leverages the organism's innate DNA repair machinery for easy vector integration and targeted genome modifications. Cre-lox recombination offers high specificity and efficiency at loxP sites, making it ideal for targeted gene excision or integration. CRISPR/Cas9 has revolutionized genome engineering with its precision and ability to target multiple loci simultaneously. Each technique has its strengths and limitations, including site dependency, off-target effects, and strain-specific variability. This review provides a comprehensive overview of these vector insertion techniques, highlighting their applications, advantages, and limitations in S. cerevisiae genome engineering and synthetic biology.},
}
@article {pmid41436498,
year = {2025},
author = {Jiang, J and Jiang, Z and Luo, Q and Chen, X and Zhuang, J and Chen, J and Mu, Q and Qiu, J and Li, Y and Chen, S and Zhang, P and Yu, K and Chen, S and Liu, GS and Zhuang, J},
title = {Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {128},
pmid = {41436498},
issn = {2041-4889},
support = {82472143//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82372131//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024A1515012562//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; GNT2029648//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {*Topotecan/pharmacology/therapeutic use ; Humans ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Retinoblastoma/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; Cell Line, Tumor ; Topoisomerase I Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Apoptosis/drug effects ; *Retinal Neoplasms/genetics/drug therapy/pathology ; Female ; },
abstract = {The topoisomerase I inhibitor topotecan is an effective chemotherapeutic agent for retinoblastoma; however, treatment resistance remains a major clinical challenge, and its mechanisms remain elusive. Using genome-wide CRISPR-Cas9 knockout screening, we identified ELF2 as a key gene involved in topotecan resistance. Here, we show that surviving retinoblastoma cells exposed to topotecan showed progressively decreased ELF2 expression, accompanied by reduced apoptosis. In a mouse xenograft model, ELF2 disruption diminished the antitumor efficacy of topotecan, with ELF2-knockout cells exhibiting reduced topotecan-induced apoptosis. RNA sequencing further revealed that the MT-CYB pathway, associated with ATP synthesis, contributes to ELF2-mediated resistance. Importantly, clinical analysis demonstrated a correlation between ELF2 expression and tumor volume in retinoblastoma patients treated with topotecan. Together, these findings interrogate the mechanisms underlying topotecan resistance in retinoblastoma and suggest ELF2 as a potential therapeutic target to overcome drug resistance.},
}
@article {pmid41436729,
year = {2025},
author = {Wu, Y and Cai, Z and Cross, D and Noble, JR and Prest, K and Littleboy, J and Cohen, SB and Edlundh, B and Koh, JMS and Xu, R and Noor, Z and Bastami, M and Valentini, S and Richardson, L and Barthorpe, S and Arymanesh, N and Robinson, PJ and Hains, PG and Garnett, MJ and Zhong, Q and Reddel, RR and MacKenzie, KL},
title = {Large-scale drug sensitivity, gene dependency, and proteogenomic analyses of telomere maintenance mechanisms in cancer cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11337},
pmid = {41436729},
issn = {2041-1723},
support = {IIRS-18-164//National Breast Cancer Foundation (NBCF)/ ; GNT1170739//Department of Health | National Health and Medical Research Council (NHMRC)/ ; RG24-05//Cancer Council NSW (Cancer Council New South Wales)/ ; 2017/TPG001, REG171150//Cancer Institute NSW (Cancer Institute New South Wales)/ ; /WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; CMP-01//NSW Ministry of Health (NSW Health)/ ; GNT1170739, H2020-SC1-DTH-2018-1//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/metabolism ; *Telomere Homeostasis/genetics/drug effects ; Cell Line, Tumor ; *Telomere/metabolism/genetics ; Telomerase/metabolism/genetics ; *Proteogenomics/methods ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Proteomics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Replicative immortality is a hallmark of cancer, driven by the activation of telomere maintenance mechanisms, that is yet to be therapeutically exploited. To expedite discoveries that will enable the development of therapeutics that target telomere maintenance mechanisms, this study provides a resource of telomere biology metrics for a pan-cancer panel of 976 cell lines. We generate proteomic data from data-independent-acquisition mass spectrometry for most of these cell lines and integrate pre-existing multi-omic, drug sensitivity, and molecular dependency data from CRISPR/Cas9 knock-out screens. The data illustrate a broad range and heterogeneity in telomere biology, including states that diverge from the binary model of telomere maintenance activation involving either telomerase or the Alternative Lengthening of Telomeres mechanism. Using the telomere biology metrics and multi-omic data, we derive proteomic and transcriptomic predictors of Alternative Lengthening of Telomeres and telomerase activity levels. Our investigations also reveal molecular vulnerabilities associated with the Alternative Lengthening of Telomeres mechanism and drug sensitivity correlating with telomerase activity levels. These findings illustrate opportunities for leveraging this resource to realize the potential for telomere biology-directed cancer therapeutics and companion diagnostics.},
}
@article {pmid41439322,
year = {2026},
author = {Wang, Z and Yang, F and Zeng, S and Sun, R and Hu, Q and Du, Y},
title = {An integrated valved microfluidic platform for rapid and simultaneous nucleic acid detection.},
journal = {Lab on a chip},
volume = {26},
number = {2},
pages = {507-514},
doi = {10.1039/d5lc01096a},
pmid = {41439322},
issn = {1473-0189},
mesh = {Humans ; *Lab-On-A-Chip Devices ; *Nucleic Acid Amplification Techniques/instrumentation ; Human papillomavirus 16/genetics/isolation &amp; purification ; Human papillomavirus 18/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acids/analysis ; *Microfluidic Analytical Techniques/instrumentation ; CRISPR-Cas Systems ; },
abstract = {Applying CRISPR-based diagnostics to point-of-care pathogen detection remains challenging because of the multi-step and time-consuming sample preparation process. This study presents a low-cost, integrated valved microfluidic device that combines recombinase polymerase amplification (RPA), CRISPR signal amplification, and lateral flow readout for simultaneous nucleic acid detection. The core advantage of the platform lies in its ability to sequentially control the entire multi-step assay through simple valve operation, significantly minimizing user intervention. All key reagents, including the RPA mix, Cas12a/crRNA complex, and proteinase K lysis buffer, are pre-lyophilized, ensuring stability and ready-to-use functionality. The platform demonstrates a sensitivity of 20 copies/reaction for HPV16/18 plasmids and accurately genotypes HPV in lysates of cervical cancer cells within one hour, showing complete concordance with quantitative PCR results. This integrated device, achieving a user-friendly protocol and visual readout, provides a powerful tool for nucleic acid-based point-of-care testing and self-testing in resource-limited settings.},
}
@article {pmid41439415,
year = {2026},
author = {Zhao, R and Wan, P and Huang, H and Li, Q and Zeng, Z and Xiong, W},
title = {Harnessing CRISPR-Cas9 and CRISPRi systems to reverse antibiotic resistance in a clinical multidrug-resistant Escherichia coli isolate.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {1},
pages = {},
doi = {10.1093/jac/dkaf442},
pmid = {41439415},
issn = {1460-2091},
support = {2022YFD1800400//National Key R&amp;D Program/ ; },
mesh = {*Escherichia coli/drug effects/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests ; *Anti-Bacterial Agents/pharmacology ; Escherichia coli Proteins/genetics ; Escherichia coli Infections/microbiology ; Humans ; ATP-Binding Cassette, Sub-Family C Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Deletion ; },
abstract = {OBJECTIVES: To evaluate the CRISPR-Cas9 and CRISPR interference (CRISPRi) systems as an antibiotic re-sensitization strategy for reversing multidrug resistance in a clinical Escherichia coli isolate.<br><br>MATERIALS AND METHODS: The CRISPR-Cas9 system was applied for precise deletion of the acrB gene from clinical E. coli isolate GP53 and homologous recombination (HR) was provided for accurate repairs of double-strand breaks. An arabinose-inducible CRISPRi system was developed and optimized using fluorescent reporter strain GH01. Multiple guide RNAs (gRNAs) targeting acrB were designed, and the most effective gRNA was selected based on its transcriptional suppression of gene acrB. The minimum inhibitory concentrations (MICs) of selected antibiotics in GP53&#x394;acrB, CRISPRi strains, WT and WT combined with efflux pump inhibitor PA&#x3b2;N were evaluated.<br><br>RESULTS: The CRISPR-Cas9 system precisely deleted the acrB gene in clinical E. coli isolate GP53 with 11.46% knockout efficiency. The constructed arabinose-inducible CRISPRi system effectively repressed fluorescent protein expression in strain GH01. Although dCas9 expression increased with L-arabinose concentration, the transcriptional repression efficiency of the target gene under 1&#x2005;mM induction reached a significant inhibitory level. The CRISPRi system targeting gene acrB exhibited 44.9%, 5.4% and 23.5% inhibition rates on the transcriptional levels with 1&#x2005;mM L-arabinose for three distinct gRNAs. Both the knockout and CRISPRi strains successfully restored susceptibility of the multidrug-resistant E. coli GP53 to quinolones and tetracyclines, outperforming the effect of PA&#x3b2;N combination therapy.<br><br>CONCLUSIONS: In this study, CRISPR-based systems effectively reversed multidrug resistance in a clinical E. coli isolate, advancing the applications of CRISPR systems in controlling bacterial multidrug resistance.},
}
@article {pmid41440288,
year = {2025},
author = {Hwang, SB and Song, YJ and Park, PG},
title = {A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440288},
issn = {2079-6374},
support = {GCU-202502820001//Gachon University/ ; },
mesh = {*West Nile virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; *West Nile Fever/diagnosis/virology ; Animals ; Humans ; },
abstract = {The West Nile Virus (WNV), transmitted by Culex mosquitoes as a major vector, has been reported worldwide. Also, West Nile neuroinvasive disease (WNND) caused by WNV lineage 1a and 2 neuroinvasive infections has been constantly reported with high fatality rates. Nevertheless, there are no treatments and vaccinations, so diagnosis in the early stages is important. Recently, a molecular diagnostic technique using DNA endonuclease-targeted CRISPR trans reporter (DETECTR) with the CRISPR-Cas12a system integrated with isothermal nucleic acid amplification has newly emerged. In this study, we designed a 2-Step WNV DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and sensitive WNV diagnosis. It successfully detected down to 1.0 × 10[2] RNA copies for both WNV lineage 1a and 2 with demonstrating similar sensitivity to qRT-PCR without cross-reactivity to other viruses. Additionally, we designed a 1-Step WNV DETECTR, incorporating all processing steps into a single tube, capable of detecting down to 1.0 × 10[3] RNA copies for both lineages. Furthermore, we developed a more streamlined method, the 1-Step with Filter WNV DETECTR, which achieved detection limits comparable to the 2-Step method, while reducing the processing time by 5 min. This study also explored the potential of the Punch-it™ NA-Sample Kit as an efficient alternative lysis method by comparing the detection differences across various lysis methods. Through this method, we achieved rapid and simple amplification and detection processes suitable for field diagnostics with high specificity and sufficient sensitivity. Therefore, DETECTR methods presented themselves as promising alternatives to conventional diagnostic tools, potentially overcoming financial and technical constraints in diverse medical settings.},
}
@article {pmid41440293,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Sotnikov, DV and Biketov, SF and Zherdev, AV and Dzantiev, BB},
title = {Advancing Lateral Flow Detection in CRISPR/Cas12a Systems Through Rational Understanding and Design Strategies of Reporter Interactions.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440293},
issn = {2079-6374},
support = {25-16-00246//Russian Science Foundation/ ; 1.1.13//State assignment of the State Research Center of Applied Microbiology and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Kinetics ; },
abstract = {CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a-LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.},
}
@article {pmid41440302,
year = {2025},
author = {Chen, J and Liu, S and Chen, S and Mai, J and Abudukadi, M and Chen, Y and Lu, J and Li, G and Ge, C},
title = {Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR/Cas12a Assay.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440302},
issn = {2079-6374},
mesh = {*Mycoplasma hominis/isolation &amp; purification/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques ; Humans ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor's response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.},
}
@article {pmid41441637,
year = {2025},
author = {Meng, Y and Chen, J and Liu, L},
title = {Functional Coupling and Evolutionary Relationships Between Toxin-Antitoxin Systems and CRISPR-Cas Systems.},
journal = {Toxins},
volume = {17},
number = {12},
pages = {},
pmid = {41441637},
issn = {2072-6651},
support = {20720250095//Fundamental Research Funds for the Central Universities/ ; 20720240046//Fundamental Research Funds for the Central Universities/ ; 32371346//National Natural Science Foundation of China/ ; 32301007//National Natural Science Foundation of China/ ; 2024J011007//Natural Science Foundation of Fujian Province/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; 2023J05008//Natural Science Foundation of Fujian Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Toxin-Antitoxin Systems/genetics ; *Evolution, Molecular ; *Bacteria/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; *Bacterial Toxins/genetics ; },
abstract = {Bacteria encode a broad range of survival and defence systems, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems, restriction-modification systems, and toxin-antitoxin (TA) systems, which are involved in bacterial regulation and immunity. The traditional view holds that CRISPR-Cas systems and TA systems are two independent defense lines in prokaryotes. However, groundbreaking studies in recent years have revealed multi-level functional coupling between them. This review systematically elaborates on this mechanism, focusing on three types of TA systems that mediate the core correlation of CRISPR-Cas systems: CreTA maintains the evolutionary stability of CRISPR-Cas systems through an addiction mechanism; CreR enables self-regulation of CRISPR-Cas expression; and CrePA provides herd immunity by triggering abortive infection after the CRISPR-Cas system has been destroyed by Anti-CRISPRS protein. Additionally, we discuss the evolutionary homology between the type III toxin AbiF and the type VI CRISPR effector Cas13, offering a new perspective for understanding the origin of CRISPR-Cas systems. These findings not only reveal the functional coupling of prokaryotic defense systems but also provide a powerful theoretical framework and practical solutions for addressing stability challenges in CRISPR technology applications.},
}
@article {pmid41441852,
year = {2026},
author = {Tanriverdi, O},
title = {CRISPR-mRNA synergy: toward adaptive cancer immunotherapy.},
journal = {Expert review of anticancer therapy},
volume = {26},
number = {6},
pages = {671-682},
doi = {10.1080/14737140.2025.2610271},
pmid = {41441852},
issn = {1744-8328},
mesh = {Humans ; *Immunotherapy/methods ; *Cancer Vaccines/administration &amp; dosage/immunology ; Animals ; *Neoplasms/therapy/immunology/genetics ; RNA, Messenger/administration &amp; dosage/immunology ; *Gene Editing/methods ; Antigens, Neoplasm/immunology ; mRNA Vaccines/administration &amp; dosage/immunology ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: CRISPR-based genome editing and mRNA vaccine technologies have recently converged to offer new opportunities for precise and adaptable cancer immunotherapy. Their combined use may improve tumor antigenicity while enabling rapid induction of tailored immune responses.<br><br>AREAS COVERED: This review examines how CRISPR-mediated modulation of oncogenic pathways, immune evasion mechanisms, and antigen presentation can enhance the efficacy of mRNA neoantigen vaccines. A structured literature search using PubMed, Web of Science, and Scopus (2013-2025) was conducted to identify preclinical and clinical studies evaluating CRISPR editing, mRNA cancer vaccines, and integrated combination strategies. Evidence from preclinical models demonstrates that CRISPR-driven tumor sensitization such as checkpoint disruption or antigen restoration amplifies T-cell responses elicited by mRNA vaccination. Early-phase clinical trials in melanoma, non - small-cell lung cancer, and pancreatic cancer indicate that sequential CRISPR editing followed by individualized mRNA vaccination is technically feasible and capable of inducing durable immune activity. Challenges related to delivery systems, safety oversight, and ethical considerations are also evaluated.<br><br>EXPERT OPINION: CRISPR - mRNA integration represents a promising path toward adaptive, evolution-aware oncology. As delivery and regulatory frameworks advance, combined genome editing and programmable RNA immunotherapy is likely to become a key pillar of future personalized cancer treatment.},
}
@article {pmid41441922,
year = {2025},
author = {Govender, P and Ghai, M and Karpoormath, R},
title = {Advances in biosensors for bacterial detection and identification.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {1},
pages = {6},
pmid = {41441922},
issn = {1573-0972},
support = {PMDS2205108850//National Research Foundation/ ; },
mesh = {*Biosensing Techniques/methods ; Humans ; *Bacteria/isolation &amp; purification/genetics/classification ; Artificial Intelligence ; Wearable Electronic Devices ; Bacterial Infections/diagnosis/microbiology ; },
abstract = {Bacterial detection and identification is paramount as it plays a key role in safeguarding human health, food safety and security. Over the past decade, biosensors have emerged as a powerful tool for bacterial detection due to their ability to provide rapid, sensitive, specific and cost-effective monitoring of bacteria. Biosensors rely on the interaction between the target analyte and biological recognition elements, which triggers a measurable signal that can be quantified, thus enabling the detection of bacteria. In recent years, nanoparticles have become a focal point in biosensor research due to their unique physical and chemical properties, enhancing their sensitivity, specificity and functionality. Artificial intelligence, microfluidics and wearable biosensor technologies are shaping the next-generation real-time bacterial monitoring tools. AI-based biosensors interpret complex biological signals and provide automated detection of bacterial pathogens. Similarly, wearable biosensors are emerging as a promising option for non-invasive detection and monitoring of wound infections. Additionally, the integration of CRISPR/Cas systems into biosensing platforms has revolutionized molecular diagnostics by enabling highly specific detection of pathogenic bacteria. In forensic sciences, biosensors are being explored for the identification of body fluids based on their unique bacterial signatures, which can assist in crime scene reconstruction and post-mortem interval estimation. Most studies that have reported on biosensors for detection of bacteria, have targeted a single analyte or bacterial species. Given the growing interest and demand for multiplexed biosensors, future research should focus on developing biosensors capable of detecting multiple bacteria simultaneously, without compromising the accuracy. Biosensors with dual functionality will be instrumental in providing an integrated solution to detect, manage and control bacterial pathogens, thereby mitigating any potential threat to human health.},
}
@article {pmid41442816,
year = {2026},
author = {Zhu, Y},
title = {The potential and innovative applications of CRISPR gene editing technology in enzyme gene development.},
journal = {Enzyme and microbial technology},
volume = {195},
number = {},
pages = {110799},
doi = {10.1016/j.enzmictec.2025.110799},
pmid = {41442816},
issn = {1879-0909},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Enzymes/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR gene editing technology is simple in design and highly efficient, making it the most widely used gene editing tool today. At present, CRISPR gene editing technology has shown a certain application value in enzyme development, but its application potential has not been fully developed. CRISPR gene editing technology can not only be used to knockin enzyme genes and knockout genes that are not conducive to enzyme expression, but can also be applied to single-base editing of enzyme genes, tandem sgRNA for multi-enzyme gene editing, sgRNA library for enzyme screening, endogenous enzyme gene modification, transcriptional activation or inhibition of enzyme gene expression, and fluorescence imaging of enzyme genes. Especially, this review innovatively proposes for the first time that CRISPR gene editing technology can be used for site specific fusion of enzyme genes, cell surface display of endogenous enzymes, and knockin of super long DNA for simultaneous expression of multiple enzymes, providing new ideas for maximizing the value of CRISPR gene editing technology in enzyme development in the future.},
}
@article {pmid41443126,
year = {2026},
author = {Rahimi, A and Rahimmanesh, I and Abedpoor, N and Boshtam, M and Bidram, E and Javanmard, SH and Khanahmad, H and Rafiee, L and Bigham, A and Rafienia, M and Karbasi, S and Shariati, L},
title = {The MCM/Lys-Cys nanodevices for the efficient gene delivery: An approach towardsMCP1gene manipulation using CRISPR technology.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {260},
number = {},
pages = {115377},
doi = {10.1016/j.colsurfb.2025.115377},
pmid = {41443126},
issn = {1873-4367},
mesh = {Humans ; *Silicon Dioxide/chemistry ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Chemokine CCL2/genetics ; Cell Proliferation ; Cell Line, Tumor ; Female ; *Cysteine/chemistry ; Breast Neoplasms/genetics/pathology/therapy ; *Lysine/chemistry ; Cell Movement ; Particle Size ; Surface Properties ; Gene Editing ; Plasmids/genetics ; },
abstract = {Breast cancer continues to be the most common malignancy among women worldwide, requiring novel therapeutic approaches. This research investigates an innovative gene delivery strategy employing mesoporous silica nanoparticles (MCM-41) modified with lysine and cysteine (Lys-Cys) for the effective delivery of CRISPR-Cas9 plasmids aimed at the monocyte chemoattractant protein-1 (MCP-1/CCL2) gene. Bioinformatics analysis of the TCGA-BRCA dataset revealed substantial deregulation of CCL2 in breast cancer, underscoring its involvement in tumor growth and inflammation. The MCM/Lys-Cys nanocarrier demonstrated remarkable biocompatibility and effectively encapsulated a plasmid containing GFP, promoting superior cellular uptake in MDA-MB-231 breast cancer cells compared to conventional techniques. Functional experiments demonstrated that CRISPR/Cas9-mediated suppression of CCL2 markedly decreased cell proliferation, migration, and invasion, highlighting the promise of this targeted gene therapy strategy in breast cancer management. The findings indicate that the MCM/Lys-Cys nanosystem presents a viable non-viral approach for precise gene editing, potentially boosting therapeutic efforts against breast cancer by modulating inflammatory pathways.},
}
@article {pmid41443566,
year = {2026},
author = {Ghosh, P and Wadsworth, BC and Terry, L and Evans, TA},
title = {Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {1-9},
doi = {10.1016/j.ydbio.2025.12.015},
pmid = {41443566},
issn = {1095-564X},
support = {P40 OD018537/OD/NIH HHS/United States ; R15 NS098406/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Axon Guidance/physiology/genetics ; *Tribolium/embryology/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Axons/metabolism ; Signal Transduction ; Netrin Receptors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Biological Evolution ; CRISPR-Cas Systems ; },
abstract = {The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance are evolutionarily conserved within insects.},
}
@article {pmid41444327,
year = {2025},
author = {Oh, D and Seok, C and Park, HW and Park, S and Lee, J and Choi, H and Jawad, A and Ham, J and Jang, H and Lee, SC and Oh, BC and Moon, C and Park, KH and Hyun, SH and Kim, D},
title = {Generation and ophthalmological characterization of oculocutaneous albinism type 1 pig models by selection-free genome editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44564},
pmid = {41444327},
issn = {2045-2322},
support = {RS-2025-00518006//National Research Foundation of Korea/ ; 2020R1A2C2101714//National Research Foundation of Korea/ ; RS-2024-00398561 , RS-2024-00399475//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries/ ; 20023068//Ministry of Agriculture, Food and Rural Affairs/ ; HR22C1363//Korea Health Industry Development Institute/ ; },
mesh = {Animals ; *Albinism, Oculocutaneous/genetics/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Swine ; Monophenol Monooxygenase/genetics ; CRISPR-Cas Systems ; Humans ; Electroretinography ; Phenotype ; Melanins/metabolism ; },
abstract = {Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase (TYR) gene, resulting in melanin deficiency and severe visual impairments. Although mouse models provide insights into OCA1 pathogenesis, they exhibit significant anatomical and physiological differences from humans, particularly in ocular structure and function, thereby limiting their ability to recapitulate human OCA1 phenotypes. Therefore, in this study, we generated a porcine OCA1 model by selection-free genome editing via somatic cell nuclear transfer to characterize ophthalmological features and evaluate their translational relevance to human OCA1. Our approach utilized TYR-targeting CRISPR/Cas9 ribonucleoproteins without the need for single-cell-derived clonal expansion, thus streamlining the generation process. After somatic cell nuclear transfer with TYR knockout donor cells, the embryos demonstrated normal in vitro embryonic development comparable to the control, resulting in four healthy OCA1 piglets that exhibited characteristic OCA1 phenotypes with complete melanin loss in ocular and cutaneous tissues. Comprehensive ophthalmological analyses revealed significant structural abnormalities, including marked reduction in retinal layer thickness and elevated intraocular pressure. Remarkably, electroretinography revealed selective impairment of the rod bipolar pathway with reduced b-wave amplitudes and increased oscillatory potentials, indicating disturbances in synaptic processing. Overall, our study demonstrates the efficiency and reliability of selection-free genome editing for generating porcine OCA1 models. Moreover, the ophthalmological findings provide valuable insights for exploring retinal dysfunction and pigmentation mechanisms and advancing the preclinical evaluation of potential therapeutic interventions for human OCA1.},
}
@article {pmid41444344,
year = {2025},
author = {Li, X and Guo, J and Yang, H and Wu, Y and Xie, Z and Li, D},
title = {A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2033},
pmid = {41444344},
issn = {2045-2322},
support = {GJHZ20220913143207014//Science and Technology Innovation Commission of Shenzhen/ ; 12274197//China National Natural Science Fund/ ; 2022B1515020093//Guangdong Scientific and Technological Project/ ; },
mesh = {Humans ; SARS-CoV-2/genetics/isolation &amp; purification ; *Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; *Respiratory Tract Infections/virology/diagnosis ; Coinfection/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Influenza B virus/genetics/isolation &amp; purification ; Influenza A virus/genetics/isolation &amp; purification ; },
abstract = {In recent years, viral co-infections, particularly with respiratory viruses, have resulted in more complex symptoms, a greater disease burden, and increased challenges in clinical decision-making. These complexities underscore the urgent need for improved diagnostic tools in the managing acute respiratory infections. To address the limitations of conventional qPCR and current POCT methodologies, we developed a passively driven microfluidic chip capable of rapidly screening multiple respiratory viruses. This platform is particularly suited for the point-of-care diagnosis of viral co-infections. Our device integrates nucleic acid amplification and CRISPR-based detection within a single, passively operated system. By utilizing a rapid, 10-minute sample preparation protocol and a 35-minute on-chip assay, this platform enables the multiplex detection of influenza A/B, human parainfluenza virus, and SARS-CoV-2. The total assay time from sample to answer is approximately 45 min, with equipment requirements minimized to a heating block. The assay demonstrated a detection sensitivity of about 10 copies/µL for viral RNA in dilution series experiments. The sensitivity of the assay was 98.44% (95% CI: 91.6%-99.96%), and the specificity was 100% (95% CI: 79.4%-100%). The system combines CRISPR-Cas12a-mediated sensing with reverse transcription recombinase polymerase amplification (RPA) for highly specific nucleic acid detection. The chip design utilizes capillary action and gravity-driven flow for autonomous fluid control, while lyophilized reagent preloading ensures storage stability and minimizes user intervention.},
}
@article {pmid41445188,
year = {2026},
author = {Shmuel-Eidelman, M and Cohen-Fultheim, R and Eisenberg, E and Levanon, EY},
title = {Off-target RNA editing hotspots caused by base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2361-2371},
pmid = {41445188},
issn = {1525-0024},
mesh = {*RNA Editing ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Algorithms ; RNA, Guide, CRISPR-Cas Systems/genetics ; Exons ; },
abstract = {Base editors, composed of engineered deaminases fused with Cas proteins and a guide RNA, enable precise, programmable alteration of single nucleotides within the genome and transcriptome. This innovative technology holds promising therapeutic potential for correcting disease-causing point mutations. However, its clinical translation hinges on both high efficacy and accuracy. Non-specific unintended edits by base editors remain a critical challenge. Efforts to mitigate off-target activity have focused mostly on detecting recurrent RNA deaminations at specific sites. Complementarily, our methodology quantifies the total burden of RNA alterations, which is particularly effective for capturing stochastic off-target edits that evade conventional detection. Here, we applied the RNA editing index algorithm to quantify off-target levels across individual genes and identified 2,844 adenine base editors and 1,253 cytosine base editor hotspot genes susceptible to aberrant editing. Exon-level analysis revealed localized regions within genes that are particularly prone to off-target editing, including regions where edits introduce premature stop codons, a critical risk for therapeutic applications. By uncovering these previously unrecognized off-target landscapes, our study deepens our understanding of base editor specificity and provides a framework for optimizing their precision, accelerating the development of safer next-generation editing tools.},
}
@article {pmid41445368,
year = {2026},
author = {Wei, J and Jiang, C and Chen, Y and Yang, X and Li, Q},
title = {Functional characterization of Hsk1 and Chit1 genes in the virulence of Metarhizium guizhouense Xct1 via CRISPR-Cas9-mediated gene editing.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3625-3639},
doi = {10.1002/ps.70484},
pmid = {41445368},
issn = {1526-4998},
support = {//SCCXTD-2024-04/ ; },
mesh = {*CRISPR-Cas Systems ; Virulence/genetics ; Gene Editing ; Animals ; *Metarhizium/genetics/pathogenicity ; *Fungal Proteins/genetics/metabolism ; Spodoptera/growth &amp; development/microbiology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Larva/growth &amp; development/microbiology ; Pest Control, Biological ; },
abstract = {BACKGROUND: The entomopathogenic fungus Metarhizium guizhouense Xct1 exhibits high virulence against early-instar Spodoptera frugiperda larvae (>90% mortality in preliminary studies), yet the molecular mechanisms, particularly the roles of key genes such as the chitin-degrading enzyme (Chit1) and serine/threonine kinase (Hsk1) are poorly understood. Functional studies using CRISPR-Cas9 are lacking, limiting its biocontrol application.<br><br>RESULTS: Chit1 and Hsk1 genes were amplified from M.&#x2009;guizhouense Xct1. Chit1 showed high homology to M.&#x2009;anisopliae, whereas Hsk1 exhibited greater genetic diversity. Expression analysis revealed peak Chit1 expression on Day (D)4 and peak Hsk1 expression on D2. A CRISPR-Cas9 system was established, and knockout of Chit1 resulted in thickened cell walls [119&#x2009;nm versus 87&#x2009;nm in wild-type (WT)] and reduced virulence [median lethal time (LT50)&#x2009;=&#x2009;7.4&#x2009;days versus 4.8&#x2009;days in WT]. Overexpression of Chit1 improved virulence (LT50&#x2009;=&#x2009;3.3&#x2009;days). Hsk1 knockout was lethal, confirming its essential role, while overexpression did not alter virulence (LT50&#x2009;=&#x2009;4.8&#x2009;days).<br><br>CONCLUSIONS: Chit1 is a critical virulence factor, influencing cell-wall integrity and insecticidal activity, while Hsk1 is essential for fungal viability. This study presents the first CRISPR-Cas9-mediated functional analysis of these genes, revealing that Chit1 overexpression enhances biocontrol efficacy against S.&#x2009;frugiperda. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41447470,
year = {2026},
author = {Liu, T and Ye, B and Zhang, Y and Yan, X},
title = {Finely Tuned CRISPRi Module for Upgrading the Performance of Constitutive Promoters in the Bacillus subtilis Protein Expression System.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1046-1052},
doi = {10.1021/acs.jafc.5c03424},
pmid = {41447470},
issn = {1520-5118},
mesh = {*Bacillus subtilis/genetics/metabolism/growth &amp; development ; *Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; Xylose/metabolism ; Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Fermentation ; },
abstract = {Bacillus subtilis is a critical host for protein production, with many industrial strains relying on strong constitutive promoters. However, this kind of promoter typically imposes a heavy burden on the host from the early stage of fermentation, leading to reduced growth rate and biomass. To overcome the drawbacks of these promoters, we developed a xylose-inducible CRISPRi module to dynamically control the activity of these promoters. The strength of this module was finely tuned via promoter engineering and the xylose concentration. The addition of xylose inhibited the target promoter and favored cell growth at an early stage, while the consumption of xylose recovered the strength of the promoter and facilitated protein expression, resulting in better balance between cell growth and protein production. The yield of a target protein was increased by 38% using this module. Our work provides a simple and effective method to upgrade industrial strains driven by strong constitutive promoters.},
}
@article {pmid41447528,
year = {2026},
author = {Liu, P and Yuan, Q and Yang, X and Wang, Q and Chang, T and Bi, Y and Wu, P and Zhang, T and Yang, J and Guo, S and Xue, C and Zheng, Z and Xin, B and Ma, H and Wang, Y},
title = {A synthetic biology toolkit for the plasmid-dependent and thermophilic methylotroph Bacillus methanolicus.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116788},
doi = {10.1016/j.celrep.2025.116788},
pmid = {41447528},
issn = {2211-1247},
mesh = {*Plasmids/genetics/metabolism ; *Bacillus/genetics/metabolism ; *Synthetic Biology/methods ; CRISPR-Cas Systems/genetics ; Gene Editing ; Methanol/metabolism ; Arginine/metabolism ; Metabolic Engineering ; },
abstract = {Bacillus methanolicus, a unique plasmid-dependent and thermophilic methylotroph, is an ideal chassis for one-carbon (C1) biomanufacturing. Despite its evolutionary uniqueness and industrial promise, the synthetic biology toolkit remains limited in comparison to that of conventional model microorganisms. Here, we present a comprehensive toolkit comprising a high-efficiency electroporation protocol, a CRISPR-Cas9 method enabling robust and multiplex genome editing, diverse neutral loci for gene integration, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, restriction-modification machinery, and the functional significance of chromosomal methylotrophic genes. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR-Cas9 editing, we engineered L-arginine feedback regulation to achieve L-arginine overproduction from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and C1 biomanufacturing.},
}
@article {pmid41447548,
year = {2026},
author = {Hefferon, K and Venkataraman, S and Alok, A and Moiketsi, BN and Malik, S and Masisi, K and Rantong, G and Kwape, T and Gaobotse, G and Makhzoum, A},
title = {CRISPR-Cas9 editing of agricultural crops and medicinal plants: toward a cornucopia of natural products.},
journal = {Critical reviews in biochemistry and molecular biology},
volume = {61},
number = {1-3},
pages = {1-16},
doi = {10.1080/10409238.2025.2577956},
pmid = {41447548},
issn = {1549-7798},
mesh = {*Plants, Medicinal/genetics/metabolism ; *Gene Editing/methods ; *Crops, Agricultural/genetics/metabolism ; *CRISPR-Cas Systems ; *Biological Products/metabolism ; Humans ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Plants have been a part of human health since our very beginnings, and many of our modern pharmaceuticals claim their origins from medicinal plants. The range of specialized metabolites synthesized by plants is highly diverse, and metabolic functions have developed over the millennia to cover roles such as defense, adaptation to environmental stress, and even reproduction. These metabolites subsequently play roles in human health and diseases that are both significant and profound. The importance of plant natural products for the pharmaceutical, cosmetic and nutraceutical industries cannot be overstated. However, the fact that these specialized metabolites may be available only in low quantities from plants that are slow growing, endangered, or from fragile environments due to certain biotic and abiotic stresses makes their commercial use challenging despite the scenario that some stresses can enhance the production of secondary metabolites. Genome editing is a technique or technology that comprises of tools like CRISPR/Cas9, TALEN, ZFN. The following review describes the successful use of CRISPR/Cas9 genome editing in engineering medicinal plants, food crops and commercial crops to modulate metabolic pathways involved in the biosynthesis of valuable compounds to improve natural product identification, development and ultimately, commercial viability.},
}
@article {pmid41448060,
year = {2026},
author = {Long, Y and Sun, S and Mei, H and Zhou, D and Zhou, H and Fang, Z and Li, X and Li, N and Zhuang, T and Guo, C},
title = {RT-LAMP-CRISPR/Cas12b-based hand-pressure-actuated microfluidic chip for rapid and portable detection of severe fever with thrombocytopenia syndrome virus.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129277},
doi = {10.1016/j.talanta.2025.129277},
pmid = {41448060},
issn = {1873-3573},
mesh = {*Phlebovirus/genetics/isolation &amp; purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; Pressure ; RNA, Viral/genetics/blood ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic tick-borne virus causing severe viral hemorrhagic fever, posing a significant public health threat. Rapid and accurate detection of SFTSV in resource-limited settings is critical for early diagnosis and effective control of severe fever with thrombocytopenia syndrome (SFTS). Here, we developed the RT-LAMP-CRISPR/Cas12b-based Hand-Pressure-Actuated Microfluidic Chip for Rapid and Portable Detection of Severe Fever with Thrombocytopenia Syndrome Virus (HARD). The hand-pressure-actuated microfluidic chip integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (CRISPR associated proteins, Cas) 12b in order to achieve rapid, low-cost, and contamination-free point-of-care testing. The HARD system achieves a detection limit of 5 copies per reaction, utilizing direct RNA lysis from blood samples and a hand warmer as a heat source, enabling electricity-free operation. Clinical validation with blood samples from vector-borne infectious diseases demonstrated high concordance with laboratory RT-qPCR, with 88.9 % sensitivity, 100 % specificity, and 95 % accuracy. Thus, the HARD platform offers a rapid, portable, and efficient solution for the early diagnosis of SFTSV in resource-limited settings, with potential for broader application in POCT for infectious diseases.},
}
@article {pmid41449724,
year = {2025},
author = {Plesser, E and Goldenberg, L and Kelly, G and Bdolach, E and Arad, T and Bejerano, E and Masok, O and Carmeli-Weissberg, M and Shaya, F and Sherman, A and Eyal, Y and Carmi, N},
title = {Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {6},
pages = {e70654},
pmid = {41449724},
issn = {1365-313X},
mesh = {*Flavanones/metabolism/biosynthesis ; *Citrus/genetics/metabolism ; *Gene Editing ; Plant Leaves/metabolism/genetics ; Fruit/genetics/metabolism ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Taste/genetics ; Hesperidin/metabolism/analogs &amp; derivatives ; },
abstract = {Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.},
}
@article {pmid41450580,
year = {2025},
author = {Graves, LE and Christina, S and Mullany, KL and Alexander, IE and Falhammar, H},
title = {Exploration of the potential of genomic editing in the treatment of congenital adrenal hyperplasia.},
journal = {Frontiers in endocrinology},
volume = {16},
number = {},
pages = {1719376},
pmid = {41450580},
issn = {1664-2392},
mesh = {*Adrenal Hyperplasia, Congenital/genetics/therapy ; *Gene Editing/methods/trends ; Humans ; Steroid 21-Hydroxylase/genetics ; *Genetic Therapy/methods/trends ; Animals ; CRISPR-Cas Systems ; },
abstract = {Despite life-saving glucocorticoids, therapeutic options for congenital adrenal hyperplasia (CAH) remain sub-optimal. Adrenal crisis continues to be the highest cause of mortality in individuals with CAH and even with recommended treatment regimens complications from the disease and treatments themselves persist. These patients have limited treatment options and advanced therapeutics could be a solution. Development of genetic therapies have exponentially increased in recent years. The advent of CRISPR/Cas technology has brought previously inconceivable treatment options to reality. Genomic editing could repair the defective 21-hydroxylase gene and provide a cure for 21-hydroxylase deficiency, the most common CAH variant, eliminating the current need for constant patient intervention. There are a number of technologies within reach for CAH, however, delivery of the genomic editing reagents to the elusive adrenocortical progenitor cells remains challenging. Here we discuss the complexity of CAH genetics, which has implications for choice of genomic editing strategy, and potential future strategies for the development of a cure of CAH.},
}
@article {pmid41451278,
year = {2025},
author = {Narra, M and Ray, A and Polley, B and Yang, H and Bhowmik, PK},
title = {AI-driven advances in plant biotechnology: sharpening the edge of plant tissue culture and genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718810},
pmid = {41451278},
issn = {1664-462X},
abstract = {The advent of artificial intelligence (AI) holds great promise for revolutionizing the fields of plant tissue culture and genome editing. Plant tissue culture is recognized as a powerful tool for rapid multiplication and crop improvement. However, the complex interactions between genetic and environmental factors generate large volumes of data, posing challenges for traditional statistical analysis methods. To address this, researchers are now employing machine learning (ML)-based and artificial neural networks (ANN) approaches to predict and optimize in vitro culture protocols thereby improving precision, sustainability, and efficiency. Integrating AI technologies such as machine learning (ML), artificial neural networks (ANN), and deep learning (DL) can significantly advance the development of data-driven models for CRISPR/Cas9 genome editing. Today, AI-driven methods are routinely applied to enhance precision in predicting on- and off-target sequence locations and editing outcomes. Additionally, predicting protein structures can provide a directed evolution framework that facilitates the creation of improved gene editing tools. However, the application of AI-based CRISPR modeling in plants is not yet fully explored. In this context, we aim to examine representative ML/DL/ANN models of CRISPR/Cas based editing employed in various organisms. This review significantly compiles a diverse set of studies and provides a clear overview of how AI is transforming the fields of plant tissue culture and genome editing. It emphasizes AI's potential to increase the efficiency and precision of biotechnological practices, making them more accessible and cost-effective. While outlining current findings, the paper sets the stage for future research, encouraging further exploration into the integration of AI with plant biotechnology.},
}
@article {pmid41451985,
year = {2026},
author = {Wang, Z and Hu, T and Liu, W and Zhou, H and Lv, X and Li, H and Li, X and Huang, X and He, L},
title = {Development and clinical validation of an ERA-CRISPR/Cas12a assay for the rapid detection of 14 high-risk HPV types.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0303625},
pmid = {41451985},
issn = {2165-0497},
support = {82100344//National Natural Science Foundation of China/ ; 2024A22//Tongji Hospital/ ; },
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Female ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification/classification ; Uterine Cervical Neoplasms/virology/diagnosis ; Reproducibility of Results ; DNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Persistent infection with high-risk human papillomavirus (HR-HPV) is the leading cause of cervical cancer, highlighting the critical need for early detection to improve prevention. Although real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for HR-HPV detection, its dependence on sophisticated equipment, complex procedures, and trained personnel limits accessibility. Here, we developed a simplified assay for 14 HR-HPV types by integrating direct lysis, enzyme-mediated isothermal rapid amplification (ERA), and CRISPR-Cas12a-mediated cleavage into a streamlined workflow that requires only a basic isothermal heating device. The optimized system achieved a sensitivity of 50 copies per reaction with no cross-reactivity, while a refined lysis buffer containing 20% Chelex-100 minimized inhibition from vaginal swab samples, thereby enhancing detection performance. Validation with 152 clinical samples demonstrated 97.62% sensitivity and 100% specificity, confirming the reliability of the method. This user-friendly and cost-effective assay requires minimal equipment, enabling rapid and field-deployable HR-HPV detection, and offers a practical alternative to conventional laboratory-based approaches, particularly in resource-limited settings.<br><br>IMPORTANCE: High-risk human papillomavirus (HR-HPV) is the principal etiological agent of cervical cancer, and early detection remains central to effective disease prevention. Current PCR-based assays, however, rely on specialized laboratories and trained personnel, limiting their deployment in many settings. Here, we report a streamlined CRISPR-Cas12a assay that integrates direct sample lysis, ERA, and CRISPR-based detection into a single workflow operable with only a simple heating device to determine the presence of 14 HR-HPV types. The assay achieves high analytical sensitivity, strong specificity, and robust clinical performance while maintaining low cost and ease of use. This platform enables rapid HR-HPV detection and scalable screening, particularly in resource-constrained environments, with the potential to facilitate earlier intervention and reduce cervical cancer incidence.},
}
@article {pmid41452514,
year = {2025},
author = {Lal, SK and Khatoon, G and Kumar, A and Kumar, K and Kumar, R and Pan, X and Kumar, S and Bhadana, VP and Pandey, A and Kumar, M and Soren, KR and Panditi, V},
title = {Genome Editing Enhanced Abiotic Stress ToleranceIn Cereal Crops.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {13},
pmid = {41452514},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Edible Grain/genetics/growth &amp; development ; *Stress, Physiological/genetics ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Cereals are crucial sources of food for human and animal populations worldwide. Their grain and fodder primarily serve as sources of energy and nutrition. Cereal production is hampered because of the prevalent abiotic stress worldwide. Abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal toxicity significantly reduce global cereal crop production. Previously, traditional breeding and transgenic technology have been promising and potent approaches used to mitigate unfavourable abiotic stresses, enhancing crop production to some extent. The recent advent of more potent genome-editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), has revolutionized the pace of crop improvement programs. Genome-editing technology using engineered nucleases offers significant opportunities for crop improvement. Genome editing tools include Meganucleases, Zinc Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), and CRISPR/CRISPR-associated protein (Cas). Among all genome-editing tools, CRISPR/Cas9 has been widely used to improve crop cultivars due to its specificity, simplicity, robustness, and flexibility. Recent progress in genome-editing technology have improved various plant traits in cereals. Among these traits, cereal genotypes have shown substantial advances in the last decade, particularly in enhanced tolerance to abiotic stress, enabled by genome-editing tools. This review summarizes the recently developed cereal cultivars for abiotic stress tolerance that employ different genome-editing technologies, including the most recent additions, prime editing and base editing. These improved cereal cultivars perform better and maintain higher yields under adverse abiotic stresses.},
}
@article {pmid41453247,
year = {2026},
author = {Park, SE and Jeong, JH and Kim, YG and Park, HH},
title = {Structural analysis of predicted anti-CRISPR, ACZ01644.},
journal = {Biochemical and biophysical research communications},
volume = {797},
number = {},
pages = {153199},
doi = {10.1016/j.bbrc.2025.153199},
pmid = {41453247},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; Crystallography, X-Ray ; Models, Molecular ; *Viral Proteins/chemistry/metabolism/genetics ; Bacteriophages ; Protein Conformation ; DNA Cleavage ; Amino Acid Sequence ; Protein Multimerization ; },
abstract = {The CRISPR-Cas system provides adaptive immunity in bacteria and archaea against invading genetic elements, while anti-CRISPR (Acr) proteins have evolved in phages to counteract this defense. Here, we report the first structural and biochemical characterization of ACZ01644, a protein previously predicted to be an Acr. The crystal structure of ACZ01644 reveals a unique cone-shaped architecture composed of five α-helices and five β-strands forming a compact core, which represents a fold distinct from any known Acr family. Biochemical analyses demonstrated that ACZ01644 assembles as a trimer in solution, suggesting a potential functional relevance of this oligomeric state. However, in vitro assays revealed that ACZ01644 does not inhibit Cas9-mediated DNA cleavage, indicating that its inhibitory activity, if present, may involve other CRISPR subtypes or yet unidentified cofactors. Our findings reveal an unprecedented structural scaffold among putative Acr proteins and provide a foundation for future studies to elucidate its biological role in CRISPR-Cas regulation.},
}
@article {pmid41453683,
year = {2026},
author = {Akhter, R and Kitab, B and Kayesh, MEH and Shimizu, R and Onuma, H and Yamamoto, N and Ogawa, S and Sugiyama, M and Tanaka, Y and Sato, Y and Kohara, M and Tsukiyama-Kohara, K},
title = {Optimization of lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes for in vivo delivery of a CRISPR/Cas9 system targeting hepatitis B virus.},
journal = {Virus research},
volume = {363},
number = {},
pages = {199682},
pmid = {41453683},
issn = {1872-7492},
mesh = {*Hepatitis B virus/genetics/drug effects/physiology ; *CRISPR-Cas Systems ; Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; Mice ; Humans ; *Ribonucleoproteins/administration &amp; dosage/genetics/chemistry ; Virus Replication/drug effects ; *Lipids/chemistry ; Genetic Therapy/methods ; *Hepatitis B, Chronic/therapy/virology ; RNA, Guide, CRISPR-Cas Systems/genetics/administration &amp; dosage ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {Patients with chronic hepatitis B virus (HBV) infection may benefit from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene therapy. We previously identified a guide RNA (WJ11) that suppressed HBV replication in vitro and in vivo; however, we were unable to achieve delivery at clinically feasible doses in vivo using an adeno-associated virus (AAV) vector. Lipid nanoparticle (LNP)-based WJ11/Cas9 ribonucleoprotein-oligonucleotide complex delivery suppressed HBV replication by 2-3-fold more than did AAV-based delivery. In the present study, we investigated the HBV replication-suppressive effects of LNP/WJ11/Cas9 complexes after intravenous administration to persistently HBV genotype C-infected humanized chimeric mice. CL4H6 (ionizable lipid) LNPs were selected as the first candidate for WJ11/Cas9 delivery based on their reported high encapsulation efficiency; however, no significant anti-HBV effect was noted in serum or hepatic tissue. The ionizable lipid candidate CL4F11_ε-3 improved absolute serum HBV values to a certain degree but had no significant effect on hepatic HBV DNA or covalently closed circular (ccc)DNA levels. CL4F11_ζ-2 LNP/WJ11/Cas9, a new complex prepared through structural optimization of the ionizable lipid and heat treatment of WJ11, showed suppressive effect for serum viral load along with a reduction of hepatic HBV DNA, HBV cccDNA, HBsAg, and HBcrAg levels when compared with controls. Therefore, LNP-based delivery of this CRISPR/Cas9 formula holds promise for the treatment of chronic HBV infection.},
}
@article {pmid41454508,
year = {2025},
author = {Li, J and Wang, X and Wang, X and Qu, H and Gao, L and Zhao, Z and Luo, P and Zheng, Y},
title = {A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila.},
journal = {Polish journal of microbiology},
volume = {74},
number = {4},
pages = {484-493},
pmid = {41454508},
issn = {2544-4646},
mesh = {*Legionella pneumophila/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; Legionnaires' Disease/microbiology/diagnosis ; Water Microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Legionella pneumophila is a common environmental bacterium that can cause severe respiratory disease. In this study, a reliable, rapid, and convenient detection method for L. pneumophila was established using a combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology. First, we designed three pairs of RPA primers and two types of crRNA based on the L. pneumophila-specific mip gene. Subsequently, we optimized the primers and amplification time for the RPA reaction, the crRNA for the CRISPR/Cas12a reaction, as well as the concentration of the fluorescent probe. We successfully constructed an RPA-CRISPR/Cas12a fluorescence detection system and a portable RPA-CRISPR/Cas12a LFB. The detection systems achieved a sensitivity of 5 copies/μl and high specificity. One hundred sixty environmental water samples tested by RPA-CRISPR/Cas12a LFB showed no significant difference compared to the qPCR method, providing a reliable tool for future on-site detection.},
}
@article {pmid41454745,
year = {2026},
author = {Mehnath, S},
title = {Engineering stimuli-responsive nanocarriers for CRISPR/Cas9 genome editing: next-generation cancer therapeutics.},
journal = {The Journal of pharmacy and pharmacology},
volume = {78},
number = {3},
pages = {},
doi = {10.1093/jpp/rgaf127},
pmid = {41454745},
issn = {2042-7158},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; Animals ; *Nanoparticles ; Drug Delivery Systems/methods ; Drug Carriers ; Genetic Therapy/methods ; },
abstract = {OBJECTIVES: To highlight recent developments in CRISPR/Cas9 genome-editing strategies for cancer therapy and to evaluate how nanocarrier-based delivery systems enable controlled, spatiotemporal manipulation of genetic information to overcome off-target effects, cytotoxicity, and limitations in clinical translation.<br><br>KEY FINDINGS: CRISPR/Cas9 has emerged as a simple and programmable tool for correcting cancer-associated mutations and regulating adaptive immune responses; however, challenges such as off-target effects, unintended mutations in healthy cells, and cytotoxicity hinder its clinical application. Nanocarriers address these limitations through refined spatiotemporal delivery of Cas9 nuclease and sgRNA using internal and external stimuli-responsive functional groups. These systems improve cancer-cell specificity by engineering guide RNAs, prevent premature clearance, enhance systemic circulation and intracellular delivery, enable nuclear targeting, and regulate Cas9 activity. Stimuli such as light, heat, ultrasound, magnetic fields, pH, redox conditions, glutathione, and oxygen play key roles in controlled activation and release.<br><br>SUMMARY: This review critically evaluates the structural design of nanocarriers, advanced spatiotemporal regulation strategies, and safety and efficacy concerns in CRISPR/Cas9-based cancer therapeutics. It discusses the role of cell-specific promoters, small-molecule stimulation, and stimuli-responsive delivery systems in improving genome-editing precision and therapeutic outcomes. The review also outlines future opportunities for exploiting CRISPR/Cas9 in advanced biomedical applications to enhance the effectiveness of next-generation cancer therapy.},
}
@article {pmid41454886,
year = {2026},
author = {Yang, H and Gao, X and Jin, ZC and Zhang, R and Ning, B and Yan, X},
title = {Simple and Versatile Toolkit for Genetic Manipulation of Bacillus licheniformis.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {262-270},
doi = {10.1021/acssynbio.5c00699},
pmid = {41454886},
issn = {2161-5063},
mesh = {*Bacillus licheniformis/genetics ; Plasmids/genetics ; *Genetic Engineering/methods ; DNA Transposable Elements/genetics ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacillus licheniformis is a spore-forming bacterium with probiotic, environmental, and industrial applications. Many wild strains with diverse functions have been described in recent years. Nevertheless, the lack of efficient and universal genetic manipulation tools hinders the study and engineering of these strains. Here, a versatile and simple genetic manipulation toolkit is established for B. licheniformis. The cornerstone of this toolkit is a conjugative DNA transfer system. This system could effectively transfer temperature-sensitive plasmid pTSMK into all ten tested B. licheniformis strains, with efficiencies ranging from 10[-5] to 10[-3]. Based on this DNA transfer system, the tools for maker-free knockout and knock-in, CRISPRi, as well as transposon mutagenesis, were built. A transposition frequency of 7.68 × 10[-3] was observed. The toolkit developed in this study fulfills most tasks in the engineering of this species and will promote the basic and applied research of B. licheniformis.},
}
@article {pmid41454921,
year = {2025},
author = {Sanjay, G and Seetharam, RN and Singdevsachan, SK and Sathya, M},
title = {Microbial Systems Enhancing CAR-Based Therapies: A Synthetic Biology Paradigm for Next-Generation Cancer Immunotherapy.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {106},
pmid = {41454921},
issn = {1432-0991},
support = {Hoynoza Technologies Pvt. Ltd//Hoynoza Technologies Pvt. Ltd/ ; },
mesh = {Animals ; Humans ; *Immunotherapy/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Synthetic Biology/methods ; },
abstract = {Chimeric antigen receptor (CAR)-based immunotherapies face significant translational challenges in solid tumor applications, particularly regarding manufacturing scalability, tumor targeting specificity, and antigen heterogeneity. This systematic review evaluates microbial systems as innovative platforms to address these limitations through synthetic biology-driven approaches, with a focus on bridging preclinical advances to clinical implementation. Analysis of 389 peer-reviewed studies (2015-2025) reveals that engineered probiotic strains (e.g., Escherichia coli Nissle 1917) achieve selective tumor colonization while functioning as programmable factories for:1. Synthetic antigen production and single-chain variable fragment (scFv) expression,2. Costimulatory domain delivery enabling antigen-agnostic CAR-T activation,3. Tumor microenvironment modulation via immunostimulatory chemokines. Microbial platforms demonstrate superior manufacturing economics (70-90% cost reduction vs. conventional methods) and enhance CAR-T functionality through epigenetic reprogramming by microbial metabolites (e.g., short-chain fatty acids). CRISPR/Cas-engineered genetic circuits further enable precise spatiotemporal control of therapeutic payloads.Microbial systems represent transformative platforms for scalable, programmable CAR immunotherapy with significant potential for solid tumor targeting. Key barriers to clinical translation include biocontainment challenges, incomplete mechanistic understanding of tumor homing specificity, and safety validation requirements. Strategic integration of synthetic biology with microbial chassis offers a viable pathway toward accessible next-generation cancer therapies.},
}
@article {pmid41455263,
year = {2026},
author = {Liu, F and Cheng, AX and Zhang, C and Li, J and Huang, YF and Zhang, YP and Li, CP and Zhao, H},
title = {Amplification-free, CRISPR-Cas12a-mediated fluorescence biosensor using mesoporous silica nanomaterials for ultrasensitive detection of nucleic acid biomarkers.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129289},
doi = {10.1016/j.talanta.2025.129289},
pmid = {41455263},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; *CRISPR-Cas Systems ; Humans ; *Nanostructures/chemistry ; Porosity ; Limit of Detection ; Fluorescent Dyes/chemistry ; Rhodamines/chemistry ; Fluorescence ; ErbB Receptors/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of nucleic acid biomarkers is crucial in many fields, including biomedical diagnosis, veterinary medicine, and food safety. Thus, developing an accurate and cost-effective detection method for nucleic acid biomarkers is essential. Here, we developed a sensitive CRISPR-Cas12a-based fluorescence biosensor using mesoporous silica nanomaterials (MSNs). A large quantity of rhodamine B (RB) was enriched on the MSNs to synthesize RB@MSN nanocomposites, which served as fluorescent probe materials, and Au NPs acted as fluorescence quenching materials. Combined with the high specific recognition capability of the CRISPR-Cas12a system, we detected three important nucleic acids without requiring amplification: the EGFR exon 19 deletion mutation (EGFR 19Del, found in circulating tumor DNA), African swine fever virus (ASFV), and human papilloma virus (HPV). Under optimal conditions and using quantitative analysis, there were strong linear correlations between the concentrations of the targets and their respective fluorescence intensities. The lowest detection limits were 55 aM for EGFR 19Del, 51 aM for ASFV, and 24 aM for HPV. By enriching and encapsulating MSNs with RB, our method avoided the problems of fluorescence modifications in typical CRISPR-Cas12a systems, such as professional outsourcing requirements and easily quenched fluorescence. Moreover, the results exhibited good repeatability and stability. This method provides a novel approach to nucleic acid fluorescence detection using the CRISPR-Cas12a system.},
}
@article {pmid41455873,
year = {2025},
author = {Bibi, R and George, M and Sarkar, K},
title = {RNA-guided STAT3 modification fine tunes the epigenetic and epitranscriptomic regulation of CD4 + T helper cell differentiation during non-small cell lung cancer (NSCLC).},
journal = {Medical oncology (Northwood, London, England)},
volume = {43},
number = {2},
pages = {102},
pmid = {41455873},
issn = {1559-131X},
support = {11019/07/2018-Sch//Ministry of Tribal Affairs, Govt. of India/ ; EMDR/SG/15/2023-5901//Indian Council of Medical Research/ ; },
mesh = {Humans ; *STAT3 Transcription Factor/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/immunology/pathology ; *Lung Neoplasms/genetics/immunology/pathology ; *Epigenesis, Genetic ; Cell Differentiation/genetics ; Tumor Microenvironment/immunology ; *CD4-Positive T-Lymphocytes/immunology ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Transcriptome ; },
abstract = {The accurate control of immune responses in the tumor microenvironment is crucial for augmenting anti-cancer immunity. This work examined the function of STAT3 in modulating epigenetic and epitranscriptomic pathways during the differentiation of CD4 + T helper cells in non-small cell lung cancer (NSCLC). Employing CRISPR/Cas9 genome editing, STAT3 was specifically eliminated in CD4[+]T cells derived from NSCLC patients. Functional investigations demonstrated that the reduction of STAT3 markedly enhanced the production of T helper 1 (TH1) cytokines, notably IFN-γ, while concurrently diminishing immunosuppressive signaling. Epigenetic analysis revealed significant modifications in DNA and RNA methylation patterns, along with heightened R-loop formation-alterations linked to augmented transcriptional activity of anti-tumor immune genes. Moreover, STAT3-deficient CD4[+]T cells demonstrated an enhanced ability to activate cytotoxic T lymphocytes, facilitating the targeted eradication of tumor cells. All of these effects together made the NSCLC microenvironment's immune system better at fighting cancer. Our results identify STAT3 as a crucial regulator of the genetic and epigenetic frameworks that influence T cell functionality in lung cancer. By combining RNA-guided genome editing with immune functional tests, we show that blocking STAT3 in a specific way could bring back strong anti-tumor immunity. This research underscores the therapeutic potential of STAT3-targeted therapies, presenting an innovative approach to alter T cell destiny and improve immune-mediated tumor eradication in non-small cell lung cancer (NSCLC). These methods could lead to the next generation of immunotherapies that improve clinical outcomes by fine-tuning both epigenetic and epitranscriptomic circuits.},
}
@article {pmid41456387,
year = {2026},
author = {Zhang, J and Liu, WJ and Ma, F and Zhang, CY},
title = {Self-priming amplification-integrated orthogonal CRISPR-Cas system for multiplexed profiling of piRNAs in clinical samples.},
journal = {Biosensors &amp; bioelectronics},
volume = {297},
number = {},
pages = {118334},
doi = {10.1016/j.bios.2025.118334},
pmid = {41456387},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/diagnosis ; Nucleic Acid Amplification Techniques/methods ; Female ; *Colorectal Neoplasms/genetics/diagnosis ; Piwi-Interacting RNA ; },
abstract = {CRISPR/Cas-based biosensors hold great diagnostic potential, but they are often limited in clinical practice by insufficient sensitivity and the lack of multiplexed analysis capability. To address these issues, we develop a self-priming amplification-integrated orthogonal CRISPR-Cas (SPA-OCRISPR) system for multiplexed detection of piRNAs. This assay employs an innovative four-way junction probe that seamlessly integrates target recognition with a primer-free self-priming amplification, significantly simplifying the workflow and enhancing the ligation efficiency. High-fidelity SplitR ligase-mediated transduction guarantees the excellent specificity, efficiently eliminating the need for reverse transcription. The orthogonal trans-cleavage activities of Cas12a and Cas13a are harnessed to generate distinct fluorescent signals with minimal cross-interference for multiplexed analysis. This strategy can achieve attomolar-level sensitivity and good specificity. Moreover, it can successfully quantify breast cancer-associated piRNAs (piR-651 and piR-36026) in clinical tissues, and accurately discriminate cancerous samples from healthy ones. Importantly, this strategy exhibits good generality and it can be extended to detect colorectal cancer-related piRNAs (piR-823 and piR-54265) through simply modifying the recognition sequences of split probes, underscoring its broad potential in multiplexed profiling and clinical cancer diagnostics.},
}
@article {pmid41456781,
year = {2026},
author = {Puri, B and Gaikwad, AB},
title = {Targeting LncRNAs with CRISPR/Cas9 for Kidney Therapeutics: A Review.},
journal = {International journal of biological macromolecules},
volume = {339},
number = {Pt 1},
pages = {149932},
doi = {10.1016/j.ijbiomac.2025.149932},
pmid = {41456781},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Long Noncoding/genetics ; Gene Editing/methods ; Animals ; *Kidney Diseases/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Long noncoding RNAs (lncRNAs) have emerged as key players in the pathogenesis of kidney diseases, including acute kidney injury (AKI), AKI-to-chronic kidney disease (CKD) transition, CKD, diabetic kidney disease (DKD), renal cell carcinoma (RCC), polycystic kidney diseases (PKD), and lupus nephritis (LN). Although the roles of lncRNAs in disease progression have been investigated in preclinical models, their underlying mechanisms remain poorly understood. The therapeutic potential of lncRNA-based therapies remains largely unexplored in clinical settings. Recently, an advancement in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology offers a novel strategy for treating sickle cell anemia and β-thalassemia. Additionally, CRISPR/Cas9 is currently being evaluated in clinical trials for various diseases, including kidney diseases like RCC. However, the application of CRISPR/Cas9 to target lncRNAs is still in the early stages. Preclinical experiments have revealed that CRISPR/Cas9 could effectively target lncRNAs in kidney disorders. However, its clinical translation in AKI and CKD conditions remains unclear, and various biological challenges remain to be addressed. This review aims to investigate advancements in CRISPR/Cas9 that target lncRNAs in the kidney, highlighting the limitations and future directions for advancing CRISPR/Cas9-based lncRNA therapy and translating these findings into clinical applications.},
}
@article {pmid41456843,
year = {2026},
author = {Chang, C and Yang, J and Liu, Z and Chen, J and Wang, B and Li, J and Liu, H},
title = {Layer-by-layer coated chitosan-CRISPR/Cas9 mTOR nanoparticles: A novel approach to inhibit lens epithelial cell proliferation and migration for preventing posterior capsule opacification.},
journal = {Experimental eye research},
volume = {264},
number = {},
pages = {110828},
doi = {10.1016/j.exer.2025.110828},
pmid = {41456843},
issn = {1096-0007},
mesh = {*Capsule Opacification/prevention &amp; control/pathology/metabolism ; *Chitosan/pharmacology/chemistry ; Cell Proliferation ; *TOR Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics ; *Epithelial Cells/pathology/metabolism ; *Nanoparticles/chemistry ; Cell Movement ; *CRISPR-Cas Systems ; Animals ; Humans ; Rabbits ; *Lens, Crystalline/cytology ; Posterior Capsule of the Lens/pathology ; Epithelial-Mesenchymal Transition ; Coated Materials, Biocompatible ; Lenses, Intraocular ; },
abstract = {Posterior capsular opacification (PCO) is the most common complication following cataract surgery and a significant cause of vision impairment. PCO arises from the proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs), driven by an activated mTOR signalling pathway. Previous research has demonstrated that inhibiting mTOR activity effectively reduces LEC proliferation and EMT in rabbit models. However, achieving sustained mTOR inhibition remains a challenge. In this study, we encapsulated the CRISPR/Cas9 system targeting mTOR into chitosan nanoparticles (Chi-gRNA) with an average size of 135 nm. These nanoparticles exhibited resistance to DNase I digestion. To prolong release duration, we incorporated these Chi-gRNA nanoparticles onto the surface of intraocular lenses (IOLs) via layer-by-layer (LbL) assembly. The LbL coatings consisted of alternating layers of positively charged polyethyleneimine (PEI) and negatively charged heparin, interspersed with Chi-gRNA nanoparticles over five consecutive cycles. Spectral analysis confirmed the successful integration and coating of nanoparticles, with characteristic peaks validating the electrostatic assembly of the layers. In vitro assays demonstrated that Chi-gRNA-coated IOLs significantly inhibited the proliferation, migration, and adhesion of human lens epithelial cells (hLECs). These findings highlight the potential of LbL-coated IOLs to deliver CRISPR/Cas9 system-targeting mTOR nanoparticles as a novel and effective strategy to prevent PCO in patients undergoing cataract surgery. This approach offers a promising avenue for the long-term management of this prevalent postoperative complication.},
}
@article {pmid41457318,
year = {2026},
author = {Brandt, D and Dörrich, AK and Persicke, M and Kemmler, A and Leonhard, T and Haak, M and Nölting, S and Ruwe, M and Schmid, N and Thormann, KM and Kalinowski, J},
title = {A pentose, as a cytosine nucleobase modification in Shewanella phage Thanatos genomic DNA, mediates enhanced resistance toward host restriction systems.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0133325},
pmid = {41457318},
issn = {1098-5336},
support = {34.EFRE-0300095/1703FI04//European Regional Development Fund/ ; },
mesh = {*Shewanella/virology ; *Cytosine/metabolism/chemistry ; *Bacteriophages/genetics/metabolism ; *DNA, Viral/genetics/metabolism/chemistry ; Genome, Viral ; },
abstract = {UNLABELLED: Co-evolution of bacterial defense systems and phage counter-defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. In order to evade nuclease-based mechanisms that target DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits the recognition and restriction by endonucleases. We found that Shewanella phage Thanatos DNA is insensitive to multiple restriction enzymes and also to Cas I-Fv and Cas9 cleavage. Furthermore, with nanopore sequencing, the phage DNA showed severely impaired basecalling. In addition to an adenine methylation, the data indicated an additional, much more substantial nucleobase modification. Using liquid chromatography-mass spectrometry (LC-MS), we identified an unknown configuration of a deoxypentose attached to cytosine as an undiscovered modification of phage DNA, which is present in Thanatos genomic DNA and likely mediates resistance to restriction endonucleases, as well as reducing Cas nuclease activity significantly. To elucidate the underlying enzyme functions, we identified structural homologs of Thanatos proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of the phage protein TH1_063 by in vitro. Inactivation of TH1_060 leads to an almost complete inhibition of phage propagation, indicating an important role of the cytosine modification in phage survival and/or proliferation.<br><br>IMPORTANCE: Several phages extensively decorate their DNA building blocks, providing an effective protection against various host and phage-produced restriction systems. These modifications allow the phages to distinguish between their own DNA and that of the host, significantly increasing the establishment of the phage chromosome upon entry into the host and subsequent phage proliferation. Several different modifications have been previously identified and characterized. Here, we describe a hitherto unknown cytosine modification, consisting of a deoxypentose-putatively xylose-that provides protection against various bacterial restriction systems, including DNA-targeting CRISPR/Cas systems. Our findings expand the range of DNA modifications that phages use for protection.},
}
@article {pmid41457547,
year = {2025},
author = {Song, S and Yao, D and Cai, Z and Yue, X and Qiao, C and Xue, C},
title = {[Construction of a CRISPR-Cas6-mediated lycopene synthase assembly regulation method].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4745-4758},
doi = {10.13345/j.cjb.250396},
pmid = {41457547},
issn = {1872-2075},
mesh = {Lycopene ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Intramolecular Lyases/genetics/metabolism ; Plasmids/genetics ; Carotenoids/metabolism ; },
abstract = {A CRISPR-Cas6-mediated lycopene synthase assembly regulation strategy was developed to optimize the metabolic pathway of lycopene biosynthesis in Escherichia coli and enhance production efficiency. Leveraging the orthologous properties of EcCas6e and Csy4 within the Cas6 protein family, along with RNA scaffolding, we constructed a protein-RNA complex for enzyme assembly. Sixteen plasmids (LYC-1 to LYC-16) were designed, and the assembly strategy was systematically optimized by varying the gene arrangement, linker length, and RNA scaffold expression. The performance of RNA scaffold-based enzyme assembly was compared with conventional protein linker-based approaches. Lycopene production was quantified via high-performance liquid chromatography (HPLC) to evaluate system performance. The recombinant strain LYC-3-4, which co-localized CrtB and CrtI via EcCas6e-Csy4 protein-RNA complexes, achieved the highest lycopene yield (4.02 mg/L), 58% higher than the control strain LYC-3-5 (2.55 mg/L) with mismatched RNA hybridization regions, and 41% higher than strain LYC-6 (2.86 mg/L), in which the enzymes were expressed separately. This result indicates that protein-RNA-mediated spatial co-localization significantly enhanced the substrate channeling effect, whereas other assembly configurations either failed to improve or even reduced lycopene production. In summary, we exploited the protein assembly capability of CRISPR-Cas6 proteins in combination with RNA scaffolds to achieve efficient enzyme co-localization within the lycopene biosynthetic pathway. This approach offers a convenient, flexible, and scalable tool for enzyme assembly regulation in metabolic engineering, with potential applications in microbial production of lycopene and other valuable metabolites.},
}
@article {pmid41457550,
year = {2025},
author = {Sun, Y and Li, W and Wang, P and Pei, J and Yang, L and Qiu, L and Liu, H},
title = {[CRISPRi-mediated regulation of nitrate metabolism genes in Chlamydomonas reinhardtii enhances lipid accumulation].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4794-4809},
doi = {10.13345/j.cjb.250363},
pmid = {41457550},
issn = {1872-2075},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Lipid Metabolism/genetics ; *Nitrates/metabolism ; Nitrate Reductase/genetics/metabolism ; Nitrogen/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nitrite Reductases/genetics ; },
abstract = {The global energy crisis and environmental pollution are becoming increasingly serious. The development of sustainable and clean renewable energy has become a key direction of scientific research. Microalgae are ideal raw materials for biodiesel production due to their efficient photosynthetic ability, fast growth rate, and rich lipid content. Chlamydomonas reinhardtii, as a model organism of unicellular eukaryotic green algae, has the advantages of a clear genetic background and convenient operation, which makes it an ideal target for the study of lipid metabolism in microalgae. Nitrogen stress can induce lipid accumulation in microalgae, while its molecular mechanism has not been fully elucidated. In this study, we used a CRISPR interference (CRISPRi) system to regulate key genes of nitrogen metabolism in a targeted manner and thus simulated the nitrogen stress environment to investigate its effect on lipid accumulation in C. reinhardtii, aiming to provide a new technological strategy for the efficient production of microalgal lipids. The CRISPRi system was constructed to inhibit the expression of the nitrate reductase gene (CrNIT1) and the nitrite reductase gene (CrNII1) in C. reinhardtii FACHB-2220. We evaluated the effects of nitrogen metabolism inhibition on lipid accumulation by measuring the cell growth, lipid content, and expression levels of key genes. The algal strain ΔNIT1-4 with inhibited CrNIT1 expression showed the CrNIT1 expression 10.27% that of the wild type (WT, and the strain ΔNII1-4 with inhibited CrNII1 expression showed the CrNII1 expression16.02% that of WT, indicating that the CRISPRi system effectively inhibited the transcription of the target genes. Under the condition of nitrogen abundance, the cell density of ΔNIT1-4 and ΔNII1-4 was only 33.7% and 40.2%, respectively, of that of WT. The total lipid content of ΔNIT1-4 and ΔNII1-4 was 34.41% and 33.45% of the dry weight, respectively, which was significantly higher than that of WT. In this study, we successfully simulated the nitrogen stress effect by suppressing the key genes of nitrogen metabolism through the CRISPRi system and significantly improved the lipid accumulation efficiency of C. reinhardtii. This study elucidates the regulatory relationship between nitrogen metabolism and lipid synthesis, providing a theoretical basis and technical support for the industrial application of microalgae in bioenergy production.},
}
@article {pmid41457595,
year = {2025},
author = {Chang, Y and Fan, S and Hao, T and Dai, J and He, W},
title = {[Characterization of cbm2813 encoding the cytochrome P450 enzyme in the biosynthetic gene cluster of carbomycin].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {11},
pages = {4125-4137},
doi = {10.13345/j.cjb.250364},
pmid = {41457595},
issn = {1872-2075},
mesh = {*Multigene Family/genetics ; *Streptomyces/genetics/metabolism/enzymology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Macrolides/metabolism ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/biosynthesis ; },
abstract = {Carbomycin, a 16-membered macrolide antibiotic produced in Streptomyces thermotolerans, comprises two components, carbomycin A (CA) and carbomycin B (CB). CB is converted into CA through epoxidation of the C12-C13 double bond. The gene cbm2813, located in the biosynthetic gene cluster of carbomycin, encodes a cytochrome P450 enzyme considered to catalyze this epoxidation. In this study, the functional and enzymatic properties of the cytochrome P450 enzyme Cbm2813 in the carbomycin biosynthesis gene cluster were characterized by in vivo gene inactivation and in vitro enzymatic reactions. We employed the CRISPR-Cas9 system to delete cbm2813 and obtained the mutant Δcbm2813. The fermentation products of the mutant contained CB but not CA. Complementation of Δcbm2813 restored CA production. Cbm2813 was successfully expressed in Escherichia coli and then purified. In vitro enzyme assays confirmed that Cbm2813 specifically recognized CB but not structurally similar 16-membered macrolide antibiotics, such as josamycin, midecamycin, and isovalerylspiramycin I. Cbm2813 exhibited the maximal activity at pH 5.5 and 36 ℃, with the catalytic efficiency kcat/Km of 4.39×10[3] L/(mol·s). Molecular docking suggested that the C9 carbonyl group of CB coordinated with the heme iron in the active site of the enzyme, ensuring strict substrate specificity. This study expands the toolbox of characterized P450 enzymes and advances the understanding of carbomycin biosynthesis.},
}
@article {pmid41457810,
year = {2026},
author = {Zhao, J and Long, Y and Zhang, Y and Hou, C and Huo, D},
title = {Nano-Mechanical DNA Devices Coupled With CRISPR-Cas12a for CA15-3 Detection.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {10},
pages = {e11023},
doi = {10.1002/smll.202511023},
pmid = {41457810},
issn = {1613-6829},
support = {81772290//National Natural Science Foundation of China/ ; CYB240064//Graduate Scientific Research and Innovation Foundation of Chongqing, China/ ; 2023-K08//Open Research Fund of State Key Laboratory of Digital Medical Engineering/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry ; Humans ; Biosensing Techniques/methods ; *Nanotechnology/methods ; Electrochemical Techniques ; },
abstract = {Accurate monitoring of cancer markers is crucial for clinical treatment and prognosis. CA15-3 activity levels are strongly associated with clinical progression of breast cancer, but their monitoring often relies on large instruments and professionals, and the process is time-consuming and costly. To address these concerns, we proposed an electrochemical biosensing strategy that integrated nano-mechanical DNA devices coupled with the CRISPR-Cas12a to drive molecularly gated functionalized substrates for the ultrasensitive detection of CA15-3. Specifically, Triple helical molecular switch (THMS) as a signal input switch to ensure target recognition specificity and the diffusion-limited 3D DNA walking machine coupled with CRISPR-Cas12a technology as signal amplification means. Based on the bimolecular dynamics model, the rate constants k1 (1.40 × 10[5] M[-1]sec[-1]) and k2 (2.5 × 10[4] M[-1]sec[-1]) of the GNP-PEG(+)/T 3D orbitals modified with positively charged SH-PEG-NH2 are larger than those of unmodified orbitals, proving that nanointerface diffusion restriction effect can accelerate the toehold-mediated chain displacement reaction (TMDR). With the assistance of Co-N/C modified screen-printed electrode (SPE-Co-N/C) sensing interface, the calculated detection limit of CA15-3 is as low as 7.14 × 10[-6] U mL[-1]. The proposed assay, which demonstrated satisfactory selectivity and reproducibility, and correlated highly with ELISA kit results, offered a promising tool for breast cancer early detection and therapeutic monitoring.},
}
@article {pmid41458320,
year = {2025},
author = {Ghiotto, G and Francescato, L and Biancalani, MA and Treu, L and Campanaro, S},
title = {Hydrogen excess drives metabolic reprogramming and viral dynamics in syngas-converting microbiomes.},
journal = {Environmental science and ecotechnology},
volume = {28},
number = {},
pages = {100637},
pmid = {41458320},
issn = {2666-4984},
abstract = {Microbial communities drive essential bioprocesses, including the conversion of synthesis gas into biomethane, a sustainable energy source that supports circular carbon economies. In anaerobic environments, specialized consortia of bacteria and archaea facilitate syngas methanation through syntrophic interactions, where hydrogenotrophic methanogens play a central role in reducing carbon dioxide and monoxide with hydrogen. However, imbalances in gas ratios, particularly excess hydrogen, can disrupt these interactions and impair overall efficiency. Yet, the molecular mechanisms underlying microbial responses to such imbalances remain poorly understood. Here we show that hydrogen excess triggers profound metabolic and viral remodeling in a thermophilic anaerobic microbiome, leading to reduced methane yields and ecological instability. This reprogramming involves transcriptional downregulation of methanogenesis genes in the dominant archaeon Methanothermobacter thermautotrophicus, coupled with upregulation of CRISPR-Cas and restriction-modification systems that correlate with diminished activity of an associated phage, indicating activated host defenses against viral threats. Concurrently, bacterial species such as those from Tepidanaerobacteraceae enhance carbon fixation via the Wood-Ljungdahl pathway, serving as electron sinks to mitigate redox imbalance. These adaptive responses highlight the microbiome's resilience mechanisms under stress, revealing viruses as both stressors and selective forces in syntrophic systems. Such insights advance our understanding of microbiome dynamics in bioconversion processes and guide the engineering of more stable microbial consortia for optimized syngas-to-methane conversion amid variable feedstocks.},
}
@article {pmid41458398,
year = {2025},
author = {Kang, J and Koo, J and Oh, H and Bae, E},
title = {Structural characterization of anti-CRISPR protein AcrIE9.},
journal = {Structural dynamics (Melville, N.Y.)},
volume = {12},
number = {6},
pages = {064701},
pmid = {41458398},
issn = {2329-7778},
abstract = {The arms race between bacteria and bacteriophages has driven the evolution of both CRISPR-Cas systems and anti-CRISPR (Acr) proteins. AcrIE9, a type I-E Acr protein identified in Pseudomonas aeruginosa, inhibits Cascade-mediated DNA binding by interacting with the Cas7e subunit. However, its structural basis and precise inhibitory mechanism have remained unclear. Here, we report the crystal structure of AcrIE9 at 1.73 Å resolution, along with additional structural and biochemical analyses. AcrIE9 exists as both monomer and dimer in solution, while the crystal structure reveals a homodimeric assembly. Each protomer adopts a unique α/β architecture, and structural similarity searches indicate that AcrIE9 represents a previously uncharacterized protein fold. In vitro binding assays using individually purified type I-E Cas subunits from P. aeruginosa did not detect direct interaction with AcrIE9, including with Cas7e. These findings suggest that AcrIE9 may recognize a composite interface formed only within the intact Cascade complex, consistent with the AlphaFold3 prediction of multivalent interactions with Cas7e subunits. Taken together, this study provides the structural characterization of AcrIE9 and supports an inhibitory mechanism involving a multi-subunit binding surface on Cascade.},
}
@article {pmid41459216,
year = {2025},
author = {Sun, Y and Zhao, Q and Li, W and Kwok, LY and Zhang, H},
title = {Genomic diversity and functional adaptation of Limosilactobacillus reuteri isolated from diverse ecological niches.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1732127},
pmid = {41459216},
issn = {1664-302X},
abstract = {Limosilactobacillus reuteri is a widely utilized probiotic, however, the genomic diversity and evolutionary mechanisms underlying its adaptation to various hosts and environments remain incompletely understood. This study employed comparative genomics to analyze 176 L. reuteri genomes from animal (rodents, mammals, ruminants, and birds), human intestinal, and food sources (dairy products, fermented foods; 89 newly sequenced and 92 retrieved, 5 excluded by ANI < 95%). We assessed genomic features, average nucleotide identity, pan/core genomes, carbohydrate-active enzymes, bacteriocin production, CRISPR-Cas systems, and antibiotic resistance genes. The pan-genome consisted of 16,814 genes, while the core genome contained 553 genes. Core-gene phylogeny revealed seven clades, rodents isolates were positioned closer to the root. The clustering trend of fermented foods isolates in the phylogenetic tree may indicate that these strains have undergone convergent evolution or adaptive evolution in a specific environment. CAZymes varied across sources, and the predicted bacteriocin clusters were enriched in animal-derived, particularly in rodent isolates. CAZy functional composition in L. reuteri is shaped by the ecological niche and host environment, reflecting a pattern of host-driven evolutionary adaptation. CRISPR-Cas systems were present in 23.3% of genomes, predominantly in rodents isolates, indicating strong anti-phage capabilities. The heterogeneity of CRISPR-Cas systems among sources suggests that subpopulations of L. reuteri have been subjected to different evolutionary pressures. The predominance of Type II systems agrees with their widespread occurrence in lactobacilli. The presence of multiple probiotic function-related genes across all separation sources confirms the robust probiotic potential of L. reuteri. Antibiotic resistance genes, including tet, ermB, and vatE, were most prevalent among animal-derived isolates, with the highest numbers occurring in mammals and the lowest in rodents. Therefore, strain-specific safety assessments are necessary prior to clinical or food applications. The findings underscore the significance of host-specific adaptations in shaping the genetic and functional profiles of L. reuteri, offering valuable implications for its application in food-derived, human-derived, animal-derived and therapeutics.},
}
@article {pmid41459814,
year = {2026},
author = {Celle, M and Aniorte, S and Issa, AR and Falabregue, M and Jin, H and Sanchez-Mirasierra, I and Ding, S and Soukup, SF and Seugnet, L and Liao, L and Lesca, G and Walter, L and Mollereau, B},
title = {A dwdr45 knock-out drosophila model to decipher the role of autophagy in BPAN.},
journal = {Human molecular genetics},
volume = {35},
number = {3},
pages = {},
doi = {10.1093/hmg/ddaf198},
pmid = {41459814},
issn = {1460-2083},
support = {//French Ministry of Higher Education and Research/ ; 101067877//Marie Sklodowska-Curie Action fellowship/ ; //China Scholarship Council/ ; },
mesh = {Animals ; *Autophagy/genetics ; Disease Models, Animal ; *Drosophila Proteins/genetics/metabolism ; Gene Knockout Techniques ; Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology/metabolism ; *Carrier Proteins/genetics/metabolism ; Phenotype ; Drosophila/genetics ; Humans ; },
abstract = {Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurological disorder characterized by severe cognitive and motor impairments. BPAN is caused by de novo pathogenic variants in the WDR45 gene on the X chromosome. WDR45 gene encodes the protein WDR45/WIPI4, a known regulator of autophagy. A defective autophagy has been observed in cellular models of BPAN disease and is associated with neurological dysfunctions in wdr45 knockout (KO) mice. However, it remains unclear whether the autophagic defect directly contributes to all WDR45 loss-induced phenotypes or whether other WDR45-dependent cellular functions are involved. To investigate this, we generated a CRISPR/Cas9-mediated KO of CG11975 (dwdr45 KO), the Drosophila homolog of WDR45. Our analysis revealed that dwdr45 KO flies display BPAN-like phenotypes, including impaired locomotor function, seizure-like behavior, autophagy dysregulation and iron dyshomeostasis. Additionally, dwdr45 KO flies exhibit shortened lifespan compared to control flies. These findings demonstrate that dwdr45 KO fly is a relevant in-vivo model for investigating the key cellular and molecular mechanisms underlying BPAN-associated phenotypes. Here we showed that induction of autophagy in dwdr45 KO flies improved both the shortened lifespan and the seizure-like behavior, but did not restore locomotor function. This suggests that defective autophagy contributes to some, but not all, aspects of the phenotypes resulting from loss of dWdr45 function.},
}
@article {pmid41461227,
year = {2026},
author = {Su, S and Zhang, X and Wang, X and Qiu, C and Xu, Z and Piñero, JC and Peng, X and Li, F and Zuo, Y and Chen, M},
title = {CRISPR/Cas9-based evidence that overexpression of Gm-mGST1 mediates abamectin resistance in the oriental fruit moth, Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {187},
number = {},
pages = {104472},
doi = {10.1016/j.ibmb.2025.104472},
pmid = {41461227},
issn = {1879-0240},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/pharmacology ; *Moths/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Chloride Channels/genetics/metabolism ; Larva/genetics/growth &amp; development/drug effects ; },
abstract = {Abamectin-based insecticides are widely used in integrated pest management and are particularly effective against fruit borers such as the oriental fruit moth, Grapholita molesta. However, rapid resistance evolution threatens their long-term efficacy. This study elucidates the role of the glutathione S-transferase gene Gm-mGST1 in abamectin resistance in G. molesta. A laboratory-selected resistant strain (AB-R) exhibited an 85.5-fold increase in resistance compared with a susceptible strain (AB-S). Sequencing of glutamate-gated chloride channel (GmGluCl) gene revealed no target-site mutations, implicating a metabolic resistance mechanism. In AB-R, GST enzymatic activity was significantly elevated. GST synergist diethyl maleate (DEM) increased the toxicity of abamectin more strongly in the abamectin-resistant G. molesta strain than in the susceptible strain, indicating that GSTs contribute to abamectin resistance. Gm-mGST1 showed strong and stage-specific overexpression under abamectin exposure. Functional analysis using CRISPR/Cas9 knockout of Gm-mGST1 in the AB-R strain reduced resistance 16.3-fold, providing the definitive evidence that a GST gene directly mediates abamectin resistance in G. molesta. The catalytic activity of recombinant Gm-mGST1 was verified in vitro using CDNB as the substrate. Additionally, abamectin exhibited a certain degree of inhibitory effect on the activity of Gm-mGST1. HPLC analysis further revealed that the peak area of abamectin significantly decreased in the presence of recombinant Gm-mGST1, while ectopic expression in Drosophila melanogaster increased abamectin tolerance by 1.97-fold. There is a significant positive correlation between the abamectin resistance levels and the expression levels of Gm-mGST1 in field populations of G. molesta. These findings identify Gm-mGST1 as a critical gene involved in abamectin resistance and establish it as a potential molecular marker for monitoring resistance in field populations. More broadly, this study sets a precedent for integrating CRISPR/Cas9 gene editing into insecticide resistance research, bridging the gap between correlative evidence and functional validation, and providing a framework for developing GST-targeted resistance management strategies in orchard pests. This study provides evidence using CRISPR/Cas9 to confirm the contribution of GST to abamectin resistance in insects.},
}
@article {pmid41461407,
year = {2026},
author = {Shahid, M},
title = {Molecular engineering and in-silico biotechnological innovations for microbial degradation of persistent pesticides.},
journal = {Pesticide biochemistry and physiology},
volume = {217},
number = {},
pages = {106833},
doi = {10.1016/j.pestbp.2025.106833},
pmid = {41461407},
issn = {1095-9939},
mesh = {*Pesticides/metabolism ; Biodegradation, Environmental ; *Biotechnology ; Metabolic Engineering ; Soil Microbiology ; Bacteria/metabolism/genetics ; Computer Simulation ; },
abstract = {The persistence of recalcitrant pesticides in agricultural soils poses a serious threat to environmental and public health. Conventional remediation methods often have limited efficiency and, sustainability. Whereas, microbial degradation provides an eco-friendly and attractive alternative. This review highlights advances in molecular and biotechnological tools driving microbial pesticide degradation. It also emphasizes key genes, enzymatic pathways, and resilient microbes driving recalcitrant pesticide degradation. This review discusses the integration of next-generation sequencing, multi-omics platforms, CRISPR-Cas editing, synthetic biology, and AI-driven metabolic engineering in advancing microbial pesticide degradation. It also highlights progress in rhizosphere microbiome research, bioinformatics pipelines, and field-scale validation. The transition from lab to field highlights precision bioremediations' potential for sustainable pesticide management.},
}
@article {pmid41461563,
year = {2026},
author = {Riley, SE and Noskova Fairley, M and Xia, S and Cunningham, R and Cholewa-Waclaw, J and Feng, Y and Hansen, CG},
title = {In vivo screen reveals specific roles of Hippo pathway components in development and regeneration.},
journal = {Life science alliance},
volume = {9},
number = {3},
pages = {},
pmid = {41461563},
issn = {2575-1077},
support = {/WT_/Wellcome Trust/United Kingdom ; //MRC Precision Medicine DTP Studentship/ ; 100104/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; C38363/A26931//Cancer Research UK Early Detection Award/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Regeneration/genetics/physiology ; *Zebrafish Proteins/metabolism/genetics ; Signal Transduction/genetics ; Hippo Signaling Pathway ; *Protein Serine-Threonine Kinases/metabolism/genetics ; YAP-Signaling Proteins ; CRISPR-Cas Systems/genetics ; Macrophages/metabolism ; Larva ; Animal Fins/physiology ; Gene Expression Regulation, Developmental ; Transcription Factors/metabolism/genetics ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; },
abstract = {The Hippo signalling pathway is a major regulator of regeneration and development. However, the comparative importance and functional roles of individual Hippo pathway components in vivo are greatly unknown, particularly within the vertebrate lineage. To gain direct and comparable insights, we took advantage of the zebrafish larva model system. We generated individual and combined CRISPR/Cas9 F0 knockouts of a range of core Hippo pathway genes, including upstream regulators, the co-transcriptional regulators Yap1/Taz, and Yap1/Taz target genes. We analysed and compared the resulting developmental and regenerative phenotypes. Our findings highlight that paralogues of core components have distinct, but in some instances overlapping, functions. Intriguingly, we find that Yap1 and Taz have differential roles during development and regeneration. In addition, we characterise and compare two tail fin regenerative paradigms: after both severe and mild injury. These injury paradigms are drastically different and elicit diverse resolution processes. We confirm critical roles of the immune system in the regenerative process. Macrophage recruitment is reduced during severe tail fin regeneration after Yap1 and Taz loss, appearing earlier in yap1 than wwtr1 Crispants and correlating with defective regenerative function. This defective macrophage involvement might therefore be one of the mediators of the deficient regeneration in these two Crispants. Overall, our analysis emphasises distinct requirements and responses of the Hippo pathway during development and across different regenerative paradigms.},
}
@article {pmid41463573,
year = {2025},
author = {Li, Y and Gong, K and Wang, X and Sun, Z and Ding, F},
title = {Heat Shock Transcription Factors as Central Integrators of Plant Stress Responses: From Thermotolerance to Multi-Stress Resilience.},
journal = {Biology},
volume = {14},
number = {12},
pages = {},
pmid = {41463573},
issn = {2079-7737},
support = {ZR2025MS427//Shandong Provincial Natural Science Foundation/ ; 3247180862//National Natural Science Foundation of China/ ; },
abstract = {Heat shock transcription factors (HSFs) have long been recognized for their essential role in mediating thermotolerance via the activation of heat shock proteins (HSPs). Recent studies, however, have significantly broadened this view, revealing that HSFs function as versatile transcriptional regulators orchestrating plant adaptation to a wide range of abiotic and biotic stresses. This review synthesizes current knowledge of HSF structure, activation, and canonical roles in the heat shock response, while emphasizing emerging insights into their diverse functions beyond heat stress. Evidence from both model and crop species demonstrates that many HSFs confer tolerance to a broad range of stresses, including drought, cold, salinity, oxidative stress, and pathogen attack, through intricate crosstalk with hormonal (e.g., ABA, SA, JA) and redox signaling pathways, as well as MAPK-mediated phosphorylation. We also discuss biotechnological strategies such as CRISPR/Cas-mediated genome editing, stress-inducible promoter engineering, and synthetic transcriptional circuits that offer promising avenues for fine-tuning HSF expression and enhancing multi-stress resilience in crops. A deeper understanding of HSF multifunctionality not only advances our comprehension of plant stress biology but also provides a foundation for engineering resilient crops in the context of global climate change.},
}
@article {pmid41465342,
year = {2025},
author = {Sattarov, R and Kuznetsov, A and Klimko, V and Ignatyeva, E and Ivanov, R and Karabelsky, A and Fizikova, A},
title = {The Template-Jumping Editing Approach in F9-Associated Hemophilia B Gene Therapy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465342},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//"Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {*Hemophilia B/therapy/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Factor IX/genetics ; Humans ; CRISPR-Cas Systems ; Mutation ; Animals ; Genetic Vectors/genetics ; },
abstract = {Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients' immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.},
}
@article {pmid41465565,
year = {2025},
author = {Mikhaylova, E and Khusnutdinov, E and Terekhov, M and Pozdeev, D and Gusev, O},
title = {Pig Genome Editing for Agriculture: Achievements and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465565},
issn = {1422-0067},
support = {075-15-2025-014 (075-15-2024-666)//The Ministry of Science and Higher Education of Russian Federation/ ; },
mesh = {*Gene Editing/methods ; Animals ; Swine/genetics ; *Agriculture/methods ; CRISPR-Cas Systems ; Breeding/methods ; *Genome ; },
abstract = {The remaining problems in pig farming may no longer be solved with traditional methods. The search for genetic variants associated with desired characteristics and involvement of animals with superior genetics in breeding programs is rarely effective for polygenic traits and pleiotropic genes. The lack of diversity in the germplasm also limits the use of breeding, but some beneficial mutations that did not occur naturally can be introduced manually via genome editing methods. Mutations discovered in other species, such as cattle, can be reproduced in pigs. Traits that were previously pursued for centuries might be achieved by genome editing in a few years. Enormous progress has been made in producing pigs resistant to viruses and in increasing meat productivity and quality. But there are still pressing problems such as lameness and damaging behaviors that probably cannot be solved without genome editing techniques. Their wider application is complicated by the requirement for large amounts of biomaterial, surgical manipulations and cell culture, as well as by the shift towards biomedical research. This review concentrates on the main achievements and challenges in pig agricultural genetics that can be addressed by genome editing.},
}
@article {pmid41465590,
year = {2025},
author = {Nguyen, PD and Nakanishi, K and Nguyen, HP and Nguyen, HV and Kitao, M and Yoshimoto, M and Kamei, K},
title = {Characterisation of the Novel Cutibacterium acnes Phage KIT09 and First Report of CRISPR-Cas-Independent Bacteriophage Resistance in Phylotype IA1.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465590},
issn = {1422-0067},
support = {JPJSCCB20230005//Japan Society for the Promotion of Science/ ; },
mesh = {*Bacteriophages/genetics/isolation &amp; purification/physiology ; *Propionibacterium acnes/virology/genetics ; *CRISPR-Cas Systems ; Humans ; Mutation ; Genome, Viral ; *Propionibacteriaceae/virology ; },
abstract = {Despite being a commensal bacterium, Cutibacterium acnes has been widely considered a major opportunistic pathogen due to its capacity for biofilm production and inflammatory induction, causing device-related, post-implant infections, and skin inflammatory diseases. In this study, we isolated and characterised the novel bacteriophage Cutibacterium acnes phage KIT09 as a potential antimicrobial candidate for the treatment of Cutibacterium acnes-related infections such as acne vulgaris and postsurgical infections. Subsequently, phage-resistant bacterial mutants were generated through phage KIT09 exposure and characterised. Wastewater samples were collected for the isolation of C. acnes phages, followed by their characterisation using C. acnes National Institute of Technology and Evaluation (NITE) Biological Resources Center (NBRC) 107605 (phylotype IA1). Resistant mutants were isolated after prolonged exposure of the newly isolated phage to host bacteria and then characterised. A novel C. acnes phage, designated KIT09, was isolated, demonstrating prolonged bacteriolysis lasting up to 96 h at a multiplicity of infection of 10, and exhibiting high thermal and pH stability. Following sustained selective pressure by phage KIT09, three phage-resistant bacterial isolates were obtained, forming smaller colonies than the wild-type strain, but maintaining a high phage adsorption capacity (>90% after 20 min). Whole-genome sequencing revealed 12 nucleotide mutations across five genes, including six non-synonymous substitutions. Three genes encoding a two-component histidine kinase, DNA processing protein A (DprA), and a ThuA-containing domain protein were mutated in all resistant isolates. Characterisation of the novel phage KIT09 demonstrated its robust lytic activity and environmental stability against C. acnes phylotype IA1. Isolated resistant mutants retained high phage adsorption, accompanied by recurrent mutations in genes encoding a two-component histidine kinase, DprA, and a ThuA-domain protein, suggesting the presence of alternative, CRISPR-Cas-independent resistance mechanisms in C. acnes.},
}
@article {pmid41466059,
year = {2025},
author = {Chen, W and He, P and Ding, L and Lou, W and Wang, Y and Shi, W and Fan, Z and Sheng, Y and Luo, J and Tan, Z and Wang, Y and Di, W and Ke, X and Yu, B},
title = {EPI-SauriCas9-based mouse ovarian cancer models recapitulating pten deletion in patients.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {159},
pmid = {41466059},
issn = {2399-3642},
support = {82403651//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Female ; *PTEN Phosphohydrolase/genetics ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; Mice ; Disease Models, Animal ; Humans ; Gene Deletion ; Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; },
abstract = {Ovarian cancer remains a deadly gynecological malignancy, with PTEN loss and TP53 mutations frequently implicated in its progression. However, suitable models for studying ovarian cancers with PTEN and TP53 deletions are rare. Here we develop and validate the mouse ovarian epithelium with Pten and Trp53 deletions (MEPP) model using the EPI-SauriCas9 system. We demonstrate the role of Pten loss in promoting tumorigenicity and metastasis. Single-cell RNA sequencing reveals distinct epithelial subpopulations with varying metastatic potential. MEPP also recapitulates key features of human ovarian cancer, including its immune landscape and therapeutic responses. High-throughput drug screening identifies FK228 and thioguanine as promising therapeutic candidates, both of which show in vivo efficacy and are validated in PTEN-deleted organoids. Together, these results establish MEPP as a platform for studying PTEN-deleted ovarian cancer and provide a strategy for generating clinically relevant tumor models through targeted gene editing.},
}
@article {pmid41467478,
year = {2026},
author = {Niazian, M and de Ronne, M and Beauchamp, CJ and Belzile, F and Torkamaneh, D},
title = {CRISPR-induced knockouts reveal a dual role for the soybean NFR5α gene in symbiotic nitrogen fixation and root hair development.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70143},
pmid = {41467478},
issn = {1940-3372},
support = {6548//Genome Canada/ ; 337003//Fonds de recherche du Qu&#xe9;bec - Nature et technologies (FRQNT)/ ; },
mesh = {*Glycine max/genetics/growth &amp; development/microbiology ; *Nitrogen Fixation/genetics ; *Plant Roots/growth &amp; development/genetics ; *Symbiosis/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Root Nodulation/genetics ; },
abstract = {Nitrogen fixation in soybeans, facilitated by symbiotic interactions with rhizobia, is a cornerstone of sustainable agriculture, reducing reliance on synthetic fertilizers. However, the efficiency of symbiotic nitrogen fixation (SNF) varies due to natural genetic variation in SNF-related genes. Our study underscores the pivotal role of the GmNFR5α gene not only in nodulation but also in root hair development, which is crucial for effective nutrient uptake and plant yield. Through detailed genetic analyses and clustered regularly interspaced short palindromic repeats (CRISPR)-based manipulations, we identified and characterized multiple knockout mutants, notably GmNFR5α-KO and combined GmNFR5α+GmROP6-KO, which exhibited significant reductions in root hair density and nodulation. These phenotypic changes correspond with the downregulation of key root hair development genes such as TTG, RHD1, RHD2, and KJK, establishing a clear link between GmNFR5α function and root hair formation. The potential of leveraging these genetic insights to improve nitrogen fixation in legumes and introduce SNF capabilities into cereal crops could revolutionize crop fertilization strategies, offering a sustainable solution to global agricultural challenges.},
}
@article {pmid41467797,
year = {2026},
author = {Ji, HJ and Jang, A-Y and Han, SH and Kim, M-K and Lamien, CE and Wijewardana, V and Ahn, KB and Kim, K-H and Song, JY and Seo, HS},
title = {Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).},
journal = {Journal of clinical microbiology},
volume = {64},
number = {2},
pages = {e0098425},
pmid = {41467797},
issn = {1098-660X},
support = {22202MFDS171//Ministry of Food and Drug Safety/ ; 2018E240600//Korea Disease Control and Prevention Agency/ ; 523140-26//Korea Atomic Energy Research Institute/ ; RS-2022-00164721//National Research Foundation of Korea/ ; CRP D32039//International Atomic Energy Agency/ ; },
mesh = {*Streptococcus pneumoniae/classification/genetics/isolation &amp; purification ; Humans ; *Serotyping/methods ; *Pneumococcal Infections/microbiology ; Serogroup ; *Nanopore Sequencing/methods ; Republic of Korea ; CRISPR-Cas Systems ; Pneumococcal Vaccines ; },
abstract = {Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.},
}
@article {pmid41468058,
year = {2026},
author = {Vasileva, A and Abramova, M and Selkova, P and Arseniev, A and Musharova, O and Malysheva, P and Demkina, A and Khodorkovskii, M and Severinov, K},
title = {Streptococcus uberis Cas9-A Compact Type II-A Nuclease Recognizing a Unique PAM and Functional in Human Cells.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {21-35},
doi = {10.1177/25731599251404417},
pmid = {41468058},
issn = {2573-1602},
mesh = {Humans ; *Streptococcus/genetics/enzymology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/metabolism/genetics ; Animals ; *Bacterial Proteins/genetics/metabolism ; Cattle ; },
abstract = {Several type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 RNA-guided nucleases are commonly used for genome engineering. Their relatively large size and requirements for specific protospacer adjacent motif (PAM) sequences flanking their targets prompt continuous searches for additional more compact Cas9 enzymes with new PAM specificities. Here, we present SuCas9, a compact nuclease from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. SuCas9 thus expands the available genome editing toolset and may find biotechnological and medicinal applications in the future.},
}
@article {pmid41468306,
year = {2026},
author = {Wang, Y and Li, Y and Li, J and Li, M and Qiu, X},
title = {Silencing of optogenetic and chemogenetic transgenes in human iPSCs involves promoter methylation and methylation-independent mechanisms.},
journal = {Epigenetics},
volume = {21},
number = {1},
pages = {2606983},
pmid = {41468306},
issn = {1559-2308},
mesh = {Humans ; *Promoter Regions, Genetic ; *Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; *DNA Methylation ; Optogenetics ; *Transgenes ; *Gene Silencing ; CRISPR-Cas Systems ; DNA Transposable Elements ; Gene Editing ; Chemogenetics ; },
abstract = {The transplantation of neural progenitor cells derived from induced pluripotent stem cells (iPSCs) has therapeutic potential for the treatment of neurological diseases. However, the functional integration of transplanted iPSC-derived neurons into host neural networks remains controversial. Optogenetic and chemogenetic tools offer the means to assess such integration. However, constructing modifiable iPSC-derived neurons requires efficient gene editing. Here, we used CRISPR/Cas9 (targeting the AAVS1 safe harbor) and PiggyBac transposon systems to insert optogenetic and chemogenetic receptors (ChR2/hM4Di) into human iPSCs. While both systems successfully integrated genes into the genomes of HEK293T cells and iPSCs, receptor expression was detected only in HEK293T cells. Bisulfite sequencing revealed extensive methylation of the TRE3G BI promoter (95.3-98.2%) in iPSCs, in contrast to low methylation (5.9%) in HEK293T cells. For PiggyBac, the methylation of CMV/EF1α promoters in iPSCs exhibited integration site-dependent variability (0-95.2%). Notably, even hypomethylated clones failed to show gene expression, suggesting that additional regulatory mechanisms, such as histone modifications or chromatin remodeling, may contribute to transcriptional silencing. Differentiation into neural stem cells does not reverse methylation nor restore protein expression. Our findings demonstrate that the CRISPR/Cas9 and PiggyBac systems enable the integration of optochemical receptor genes into iPSCs. However, promoter methylation or other epigenetic and non-epigenetic gene-silencing mechanisms could pose barriers to efficient protein expression from the integrated transgene in iPSCs.},
}
@article {pmid41468840,
year = {2026},
author = {Shi, T and Jin, X},
title = {Probing neuropsychiatric disorders through in vivo CRISPR screening.},
journal = {Current opinion in genetics &amp; development},
volume = {96},
number = {},
pages = {102424},
pmid = {41468840},
issn = {1879-0380},
support = {R01 HG012819/HG/NHGRI NIH HHS/United States ; R01 MH137042/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Mental Disorders/genetics/pathology ; Mice ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis/methods ; Genetic Predisposition to Disease ; Schizophrenia/genetics ; Brain/pathology/metabolism ; },
abstract = {Although there are many known risk alleles associated with adult-onset psychiatric disorders such as schizophrenia [1-4], bipolar disorder [5-7], and major depressive disorder [8-10], the mechanistic links between these risk alleles and disease pathology, especially on a circuit-level, remain unclear. In vivo pooled CRISPR screening with single‑cell readout (in vivo Perturb‑seq) has begun to fill this gap by mapping causal genes to defined cell states directly in animal tissues [11-14]. Here, we review recent developments and applications of in vivo Perturb-seq in the mouse brain and highlight the potential of utilizing human cellular systems to extend these approaches. Additionally, we discuss how in vivo Perturb-seq can couple genetic perturbation with physiological or environmental perturbations to better model psychiatric diseases with environmental triggers.},
}
@article {pmid41468873,
year = {2026},
author = {Zhang, J and Zhang, X and Xie, X and Han, B and Zhao, F and Yang, X and Zhang, W and Jiang, Y and Zhang, X},
title = {CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform based on a lightful copper nanocluster for highly sensitive detection of mycotoxin.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129326},
doi = {10.1016/j.talanta.2025.129326},
pmid = {41468873},
issn = {1873-3573},
mesh = {*Copper/chemistry ; *Biosensing Techniques/methods ; *Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Limit of Detection ; Food Contamination/analysis ; Fluorescence ; *Mycotoxins/analysis ; },
abstract = {The problem of mycotoxin contamination in foodstuffs has attracted widespread attention and posed a great threat to human health. Therefore, the sensitive and effective detection of mycotoxins is of great importance for preserving public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform was constructed for highly sensitive and fast detection of aflatoxin B1. The copper nanoclusters were synthesized with marker-free DNA single strands within 5 min, showing the outstanding fluorescence properties. With the existence of aflatoxin B1, the released complementary DNA (cDNA) triggered multiple isothermal amplification reaction. Subsequently, the obtained amplification products triggered the trans-cleavage activity of CRISPR/Cas12a system, which degraded the DNA single strands for synthesis of copper nanoclusters, leading to a decreased fluorescent signal. Benefiting from good fluorescence properties of copper nanoclusters, the established biosensing platform for aflatoxin B1 detection had a high specificity and a limit of detection of 47.51 pg/mL in the linear range of 0.05-10 ng/mL. The proposed platform provided a new insight for the detection of non-nucleic acid targets.},
}
@article {pmid41469506,
year = {2025},
author = {Wang, T and Tian, Y and Yin, R and Li, M and Luo, J and Yang, Y and Zhang, C and Chen, H and Wang, Y and Lu, D},
title = {In vivo genome editing with a novel Cj4Cas9.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {152},
pmid = {41469506},
issn = {2399-3642},
support = {82070258//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Animals ; Mice ; *CRISPR-Cas Systems ; Proprotein Convertase 9/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; },
abstract = {Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5'-NNNGRY-3'). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. Seeking to further expand its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications.},
}
@article {pmid41471176,
year = {2025},
author = {Bhowmik, S and Rivu, S and Bari, ML and Ahmed, S},
title = {Genome Mining of Cronobacter sakazakii in Bangladesh Reveals the Occurrence of High-Risk ST83 and Rare ST789 Lineages.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
pmid = {41471176},
issn = {2076-0817},
support = {BIO-34//University Grant Commission, Bangladesh/ ; },
mesh = {Bangladesh/epidemiology ; Humans ; *Cronobacter sakazakii/genetics/isolation &amp; purification/classification/pathogenicity ; *Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/epidemiology ; Virulence Factors/genetics ; Infant ; Food Microbiology ; Plasmids/genetics ; Infant, Newborn ; Whole Genome Sequencing ; Phylogeny ; Infant Formula/microbiology ; },
abstract = {Cronobacter sakazakii is a foodborne pathogen of major concern due to its link with severe neonatal infections through powdered infant formula (PIF). However, its genomic epidemiology in Bangladesh remains uncharacterized. We report the first whole-genome analysis of three isolates from PIF. Two isolates (S41_PIFM and S44_RUTF) belonged to ST83, a lineage repeatedly associated with neonatal meningitis, septicemia, and persistence in PIF production environments, while the third (S43_TF) represented ST789, a recently described and rare lineage of unknown pathogenic potential. Pan-genome and comparative analyses identified 39 virulence determinants, 19 antimicrobial-resistance genes, and diverse mobile genetic elements. ST83 isolates harbored plasmid replicons IncFII(pCTU2) and pESA2, while the ST789 isolate carried insertion sequence ISKpn34, indicating horizontal gene transfer potential. All strains encoded I-E CRISPR-Cas systems. The detection of globally recognized high-risk ST83 clones alongside the novel ST789 lineage highlights emerging public health risks. This study provides the first genomic insights into C. sakazakii in Bangladesh and underscores the urgent need for genomic surveillance and strengthened food safety monitoring to protect infant health in low- and middle-income countries.},
}
@article {pmid41471222,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {From Methylomes to CRISPR Epigenetic Editing: New Paths in Antibiotic Resistance.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
pmid = {41471222},
issn = {2076-0817},
mesh = {*Gene Editing/methods ; *Epigenesis, Genetic ; *Bacteria/genetics/drug effects ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; DNA Methylation ; *Epigenome ; *Drug Resistance, Microbial/genetics ; Gene Expression Regulation, Bacterial ; Epigenome Editing ; },
abstract = {Antibiotic resistance (AR) has long been interpreted through the lens of genetic mutations and horizontal gene transfer. Yet, mounting evidence suggests that epigenetic regulation, including DNA and RNA methylation, histone-like proteins, and small non-coding RNAs, plays a similarly critical role in bacterial adaptability. These reversible modifications reshape gene expression without altering the DNA sequence, enabling transient resistance, phenotypic heterogeneity, and biofilm persistence under antimicrobial stress. Advances in single-molecule sequencing and methylome mapping have uncovered diverse DNA methyltransferase systems that coordinate virulence, efflux, and stress responses. Such epigenetic circuits allow pathogens to survive antibiotic exposure, then revert to susceptibility once pressure subsides, complicating clinical treatment. Parallel advances in CRISPR-based technologies now enable direct manipulation of these regulatory layers. CRISPR interference (CRISPRi) and catalytically inactive dCas9-fused methyltransferases can silence or reactivate genes in a programmable, non-mutational manner, offering a new route to reverse resistance or sensitize pathogens. Integrating methylomic data with transcriptomic and proteomic profiles further reveals how epigenetic plasticity sustains antimicrobial tolerance across environments. This review traces the continuum from natural bacterial methylomes to engineered CRISPR-mediated epigenetic editing, outlining how this emerging interface could redefine antibiotic stewardship. Understanding and targeting these reversible, heritable mechanisms opens the door to precision antimicrobial strategies that restore the effectiveness of existing drugs while curbing the evolution of resistance.},
}
@article {pmid41472024,
year = {2025},
author = {Yi, M and Hu, Y and Fan, B and Pan, Y and Pan, B and Wang, J and Liu, Z},
title = {Advances in Novel Detection Technologies for Occult Hepatitis B Virus Infection: Building an Ultra-Sensitive Barrier for Transfusion Safety.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472024},
issn = {2076-2607},
abstract = {Occult hepatitis B virus infection (OBI), characterized by extremely low viral loads and the persistent intrahepatic presence of cccDNA, poses a profound challenge to global public health security. With a prevalence ranging from 0.06% to over 15% in different donor populations, OBI maintains a risk of transmission and can progress to hepatocellular carcinoma. Its prevention and control have long been limited by the sensitivity constraints of conventional detection methods, highlighting the urgent need for more sensitive diagnostic innovations. Emerging technologies offer distinct breakthroughs: ddPCR facilitates absolute quantification; CRISPR-Cas systems coupled with isothermal amplification enable rapid, point-of-care testing; third-generation sequencing resolves viral integration and mutations; and nanomaterials enhance the signal detection. This review synthesises advancements in OBI diagnostic technologies and provides a comparative overview of their strengths, limitations, and transfusion safety implications, as well as their potential applications in blood transfusion. Recommendations are also proposed to inform the advancement of OBI risk control in blood transfusion and to guide the development of novel diagnostic technologies, particularly relevant to regions with high HBV endemicity, such as China.},
}
@article {pmid41472049,
year = {2025},
author = {Slukin, PV and Fursov, MV and Volkov, DV and Sizova, AA and Detushev, KV and Dyatlov, IA and Fursova, NK},
title = {Diversity of CRISPR-Cas Systems Identified in Urological Escherichia coli Strains.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472049},
issn = {2076-2607},
support = {075-15-2025-525 of 30.05.2025.//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Type I-E and I-F CRISPR-Cas systems were identified in 237 E. coli strains isolated from patients with urinary tract infections (UTIs) between 2004 and 2019. The strains were classified into nine distinct groups (I-IX) based on the presence or absence of cas genes and repeat regions (RRs). Within the type I-E systems, two sequence variants were identified, distinguished by polymorphisms in the casB, cas3, cas7, cas5, and cas6 genes. The direct repeats (DRs) also differed, with I-E-associated RRs ranging from 26 to 32 bp and I-F-associated RRs consistently being 28 bp. We identified 762 unique spacers (29-35 bp in length) across the strain collection, while the number of spacers per strain varied from 1 to 47, and potential DNA targets were determined for 65 spacers, targeting 38 bacteriophage genomes, 19 plasmids, and 8 cas genes of the I-F type CRISPR-Cas system. Multilocus sequence typing (MLST) revealed 68 sequence types and 24 clonal complexes (CCs), with the most prevalent being ST131, CC10, CC69, CC405, CC14, CC38, CC73, and CC648. Significant correlations were observed between specific phylogroups/CCs, the type of CRISPR-Cas system present, and distinct profiles of virulence and antibiotic resistance genes.},
}
@article {pmid41472204,
year = {2025},
author = {Dong, J and He, X and Bao, S and Wei, Z},
title = {Diagnostic Methods for Bovine Coronavirus: A Review of Recent Advancements and Challenges.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472204},
issn = {1999-4915},
support = {KJZC-2024-15//Gansu Provincial Department of Agriculture and Rural Affairs Science and Technology Support Project/ ; },
mesh = {Animals ; Cattle ; *Coronavirus, Bovine/genetics/isolation &amp; purification ; *Cattle Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Coronavirus Infections/diagnosis/veterinary/virology ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.},
}
@article {pmid41472250,
year = {2025},
author = {Tan, C and Xing, S and Xie, X and Chen, X and Liu, X and Wang, W and Liu, L and Zhou, X and Wu, J and Li, C},
title = {Development and Application of a Rapid Field Detection Technology for DENV-2 Based on the HUDSON Nucleic Acid Extraction-Free/RT-RAA/CRISPR-Cas12a System.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472250},
issn = {1999-4915},
support = {2024YFC2607800//Chunxiao-Li/ ; },
mesh = {*Dengue Virus/genetics/isolation &amp; purification/classification ; *Dengue/diagnosis/virology ; *CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; Animals ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue fever has become a major global public health challenge due to its rapidly in-creasing incidence. Rapid on-site detection of dengue virus (DENV) is critical for early diagnosis, timely patient isolation, and outbreak control. In this study, dengue virus serotype 2 (DENV-2), the predominant strain circulating in tropical and subtropical regions, was selected as the target pathogen. We established a one-tube rapid detection assay that integrates the HUDSON nucleic acid extraction-free protocol, reverse transcription recombinase-aided amplification (RT-RAA), and CRISPR/Cas12a-mediated trans cleavage activity. The method achieved a detection limit of 1 × 10[2] copies/μL for simulated infected samples and exhibited no cross-reactivity with other DENV serotypes (DENV-1, DENV-3, DENV-4) or with other arboviruses, including Zika, Japanese encephalitis, yellow fever, and chikungunya viruses. The assay demonstrated high sensitivity and specificity across various sample types, including mosquitoes, rodents, blood, and cultured cells, with results consistent with quantitative PCR (qPCR). Requiring only basic equipment such as a water bath, the system enables on-site detection of DENV-2 within 1 h. This simple, cost-effective, and reliable assay provides a practical tool for field-based DENV-2 surveillance and supports effective public health responses in resource-limited settings.},
}
@article {pmid41472258,
year = {2025},
author = {Gladue, DP and O'Mahony, A},
title = {CRISPR Treatments for AI-Designed Synthetic Viruses: Rapid Programmable Countermeasures for Emerging and Engineered Viruses.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472258},
issn = {1999-4915},
mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Viral ; *Artificial Intelligence ; *Synthetic Biology/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Viruses/genetics ; Animals ; Genetic Engineering/methods ; },
abstract = {The convergence of artificial intelligence and synthetic biology is innovating and accelerating the design of novel viral genomes, expanding both therapeutic opportunities and dual-use risk. This review articulates a countermeasure strategy for emerging and engineered viruses leveraging the programmable CRISPR modality. Building on mounting in vitro and in vivo evidence that Cas9 degrades DNA viruses (e.g., Orthopoxviruses, HSV-1, ASFV), while Cas13 targets RNA viral genomes (e.g., Influenza A, Dengue, RSV), both leading to reduced viremia, diminished disease burden, and alleviated symptoms. Here, we outline a rapid-response pipeline to position CRISPR-based countermeasures in translational and pandemic-response frameworks, linking real-time sequencing to AI-assisted gRNA selection and multiplexed cassette design to achieve viral targeting efficacy. To minimize resistance and off-target risk, we emphasize multi-gRNA cocktails, continuous genomic surveillance, and adaptive gRNA rotation. We also propose governance mechanisms, such as pre-cleared gRNA repositories, transparent design logs, standardized off-target/safety screening, and alignment with evolving nucleic-acid-synthesis screening frameworks to enable emergency deployment while preserving security. Furthermore, compressing the time from sequence to treatment and complementary to vaccines and small-molecule antivirals, CRISPR represents a technologically agile and strategically essential capability to combat both natural outbreaks and AI-enabled biothreats. Collectively, programmable CRISPR antivirals represent an auditable, rapidly adaptable foundation for next-generation biodefense preparedness.},
}
@article {pmid41472363,
year = {2026},
author = {Ajibaye, O and Dada, IS and Mbye, H and Nwankwo, IC and Oriero, E and Amambua-Ngwa, A},
title = {Innovative approach in malaria research: Harnessing CRISPR-Cas9 for antimalarial drug-resistance studies in Africa.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {1909-1920},
pmid = {41472363},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Humans ; *Antimalarials/therapeutic use/pharmacology ; *Drug Resistance/genetics ; Gene Editing/methods ; Africa ; *Plasmodium falciparum/genetics/drug effects ; *Malaria/drug therapy/parasitology/epidemiology/genetics ; Animals ; Malaria, Falciparum/drug therapy/parasitology ; },
abstract = {Malaria remains a major global health challenge, particularly in sub-Saharan Africa, where Plasmodium falciparum is increasingly resistant to frontline antimalarial therapies. CRISPR-Cas9, a groundbreaking genome editing tool, has become a transformative force in biomedical research, offering valuable insights into the genetic mechanisms underlying drug resistance. This review provides a comprehensive overview of the CRISPR-Cas9 system, its origin, evolution, and application in antimalarial drug-resistance research, with particular emphasis on African studies. We examine the role of CRISPR in elucidating resistance pathways, validating molecular markers, and enhancing diagnostic strategies. Additionally, a systematic scoping review highlights the geographic scope and focus of CRISPR-related malaria research conducted across Africa. Significantly low application and adoption were observed. Despite existing technological and regulatory barriers, CRISPR holds strong potential for accelerating antimalarial drug discovery and advancing precision medicine. Its strategic application in malaria-endemic regions could significantly bolster efforts toward disease control and eventual elimination.},
}
@article {pmid41474015,
year = {2026},
author = {Cheng, X and Dong, J and Jain, P and Qin, S and Miao, Y and Liu, K and Theja, MLV and Butch, CJ and Wang, Y and Lane, LA},
title = {Ultrasound Activated Hybrid-Biomimetic Nanocarriers That Combine Tumor-Confined CRISPR/Cas9 Metabolic Reprogramming and Cuproptosis With Anticancer Macrophage Polarization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {10},
pages = {e10436},
doi = {10.1002/smll.202510436},
pmid = {41474015},
issn = {1613-6829},
support = {113-2221-E-038-004//National Science and Technology Council/ ; 114-2221-E-038-019//National Science and Technology Council/ ; TMU111-AE1-B12//Taipei Medical University/ ; 113FRP-28//Taipei Medical University Shuang Ho Hospital Special Research Plan/ ; 114FRP-26//Taipei Medical University Shuang Ho Hospital Special Research Plan/ ; 82127806//National Natural Science Foundation of China/ ; 2022300326//Fundamental Research Funds for the Central Universities/ ; 0213-14380238//Fundamental Research Funds for the Central Universities/ ; 202205033//Nanjing Life and Health Science and Technology Special Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Macrophages/metabolism/drug effects ; Humans ; Animals ; *Nanoparticles/chemistry ; Metal-Organic Frameworks/chemistry ; Cell Line, Tumor ; *Copper/chemistry ; Mice ; *Biomimetic Materials/chemistry ; *Ultrasonic Waves ; *Neoplasms/pathology ; *Drug Carriers/chemistry ; *Biomimetics/methods ; Metabolic Reprogramming ; },
abstract = {Nanomedicine aims to develop nanocarriers that provide strong cell selectivity and efficient intracellular delivery. Additionally, therapeutic strategies are expanding to include metabolic pathways to trigger apoptosis and reduce tumor growth, especially in cases resistant to conventional chemotherapy. Here, we have created nanocarriers with hybrid-biomimetic coatings that, upon ultrasound activation, release encapsulated copper-based metal-organic frameworks (MOFs) and COP1 gene knockout Cas9 ribonucleoproteins (RNPs). This hybrid-membrane coating, which combines tumor and immune cell membranes with perfluorocarbons, enhances tumor-to-normal cell uptake and allows for controlled release and cytolytic entry of the nanocarrier contents. We observe that the RNPs efficiently knockout the COP1 gene, thereby arresting the cancer cell cycle in the G0/G1 phase and promoting mitochondrial respiration over anaerobic glycolysis. This increased respiration makes cancer cells more susceptible to cuproptosis triggered by the MOFs and decreases tumor lactate levels, preventing lactate-driven M2 polarization of tumor-infiltrating macrophages. Furthermore, the nanocarriers' cellular selectivity leaves macrophages unharmed. These effects enable infiltrating macrophages to retain an anticancer M1 polarization and continue to foster a more active immune response. The combination of tumor-specific genetic metabolic reprogramming and enhanced cuproptosis activity, along with increased immune activity, results in significant tumor growth suppression and improved survival rates.},
}
@article {pmid41474367,
year = {2026},
author = {Rasool, HMH and Gong, X and Jin, Y and Liu, M and Yanyan, W and Qu, M and Anxiang, Z and Lingling, X and Li, Z and Zhou, J and Chen, Q},
title = {Development of Visual and Fluorescence Detection Method of Brucella by RPA-CRISPR/Cas12a Assay.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71397},
doi = {10.1096/fj.202503610R},
pmid = {41474367},
issn = {1530-6860},
support = {2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024BEG-02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2024312885//Comprehensive Control Technology For Brucellosis and Hydatic Disease in Xinjiang/ ; CAAS-ZDRW202410//The Agricultural Science and Technology Innovation Program (ASTIP)/ ; },
mesh = {*Brucella/genetics/isolation &amp; purification ; Animals ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Cattle ; Fluorescence ; Limit of Detection ; Humans ; },
abstract = {Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella, including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL-3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time-consuming nature of assays, making them unsuitable for large-scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost-effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA-CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella. Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA-CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA-CRISPR/Cas12a-F and 10 copies/μL for RPA-CRISPR/Cas12a-LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA-CRISPR/Cas12a assay showed high concordance with classical quantitative real-time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.},
}
@article {pmid41474623,
year = {2026},
author = {Banh, DV and Goldberg, GW and Marraffini, LA},
title = {Phage induction of Staphylococcus aureus pathogenicity islands promotes the CRISPR-Cas adaptive immune response.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116776},
pmid = {41474623},
issn = {2211-1247},
support = {DP1 GM128184/GM/NIGMS NIH HHS/United States ; R01 GM149834/GM/NIGMS NIH HHS/United States ; },
mesh = {*Staphylococcus aureus/virology/genetics/immunology/pathogenicity ; *CRISPR-Cas Systems/genetics/immunology ; *Genomic Islands/genetics ; *Adaptive Immunity ; *Staphylococcus Phages/genetics ; Animals ; *Bacteriophages ; Mice ; },
abstract = {Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements carrying virulence genes that spread upon infection by helper phages that induce their transfer. Staphylococci also carry type II and III CRISPR-Cas systems that mount an adaptive immune response against phages through the acquisition of spacer sequences from viral genomes, directing Cas nucleases to their targets. Whether and how SaPIs and CRISPR interact with each other during helper phage infection is not known. Here we report that, as a result of the packaging of incomplete helper phage genomes into SaPI particles, defective viral DNA delivered into new hosts stimulates spacer acquisition in both CRISPR types. Once immunized, staphylococci target the helper phage and prevent SaPI mobilization. Our work reveals an unexpected synergy between CRISPR-Cas systems and SaPIs that enhances antiphage immunity and could favor the retention of beneficial elements within the population.},
}
@article {pmid41474879,
year = {2026},
author = {Cheng, M and Wang, Y and Lin, W and Ye, J and Wu, M and Xiang, B and Liu, L and Sun, B},
title = {A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {1},
pages = {706-716},
doi = {10.1021/acs.analchem.5c05722},
pmid = {41474879},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Light ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/genetics ; },
abstract = {Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied to Mycoplasma pneumoniae detection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research.},
}
@article {pmid41475278,
year = {2026},
author = {Kolanchi, P and Saminathan, N and Selvaraj, D and Krishnamoorthy, A and Palanivelu, K and Aruchalam, A},
title = {Bioprocess and genetic advances enhancing Beauveria bassiana biocontrol efficacy.},
journal = {Microbial pathogenesis},
volume = {211},
number = {},
pages = {108272},
doi = {10.1016/j.micpath.2025.108272},
pmid = {41475278},
issn = {1096-1208},
mesh = {*Beauveria/genetics/pathogenicity/metabolism/physiology ; *Pest Control, Biological/methods ; Virulence ; Animals ; Insecta/microbiology ; *Biological Control Agents ; Gene Editing ; Secondary Metabolism ; },
abstract = {Beauveria bassiana is a widely exploited entomopathogenic fungus that has emerged as a central component of ecologically sustainable pest management. Recent years have witnessed rapid progress across its biological understanding, technological development, and application potential. This review synthesizes contemporary advances spanning infection biology, secondary metabolite biosynthesis, strain development, bioprocess engineering, formulation science, and genetic improvement. At the molecular level, multi-omics studies have elucidated the coordinated regulation of surface adhesion, cuticular penetration, host immune modulation, dimorphic transitions, and toxin production, revealing gene networks that govern virulence, stress tolerance, and ecological adaptation. These insights have informed improved strategies for strain isolation and high-throughput phenotypic screening, enabling the selection of isolates with enhanced pathogenicity, environmental robustness, and endophytic competence. Parallel advances in solid-state and submerged fermentation, supported by agro-industrial substrates and data-driven optimization, have strengthened large-scale production of infective propagules with consistent quality. Such gains are further reinforced by modern formulation approaches, including oil-based dispersions, encapsulation systems, nanoemulsions, and seed-coating technologies, which collectively improve spore stability, persistence, and delivery under heterogeneous field conditions. More recently, CRISPR/Cas-based genome editing and pathway engineering have opened new avenues for precision enhancement of virulence traits, metabolic output, and abiotic stress resilience. Despite these achievements, the broader adoption of B. bassiana remains constrained by variable field performance, slower speed of action relative to chemical insecticides, strain-dependent efficacy, and regulatory and quality-control challenges. By integrating fundamental biology with technological innovation and practical limitations, this review provides a coherent framework for advancing B. bassiana from laboratory optimization to reliable field implementation, underscoring its promise as a next-generation, environmentally aligned biocontrol agent in modern agriculture.},
}
@article {pmid41475346,
year = {2026},
author = {Behera, AK and Kim, JJ and Kordale, S and Pekovic, F and Damodaran, AP and Kumari, B and Vidak, S and Dickson, E and Xiao, MS and Duncan, G and Andresson, T and Misteli, T and Valkov, E and Gonatopoulos-Pournatzis, T},
title = {RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {41-59.e15},
pmid = {41475346},
issn = {1097-4164},
support = {ZIA BC012019/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Progeria/genetics/metabolism/pathology ; *Lamin Type A/genetics/metabolism ; *Alternative Splicing ; *CRISPR-Cas Systems ; Ribonucleoprotein, U1 Small Nuclear/metabolism/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA Precursors/genetics/metabolism ; Zinc Fingers ; },
abstract = {Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin protein levels in patient-derived cells. We further show that ZNF207's zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (snRNP) components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-seq to uncover key regulators and domains of alternative splicing.},
}
@article {pmid41475348,
year = {2026},
author = {Gao, X and Zhu, K and Zhang, W and Wang, L and Wang, L and Hua, L and Niu, T and Qin, B and Yu, X and Zhu, H and Cui, S},
title = {RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.},
journal = {Molecular cell},
volume = {86},
number = {2},
pages = {317-331.e5},
doi = {10.1016/j.molcel.2025.12.005},
pmid = {41475348},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Cryoelectron Microscopy ; *Pectobacterium/genetics/metabolism/enzymology ; *Bacterial Proteins/genetics/metabolism/chemistry ; *RNA, Bacterial/genetics/metabolism/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Models, Molecular ; Nucleic Acid Conformation ; Protein Conformation ; },
abstract = {RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.},
}
@article {pmid41475352,
year = {2026},
author = {Martinho, C and Hoshino, M and Raphalen, M and Bukhanets, V and Kerur, A and Bogaert, KA and Luthringer, R and Coelho, SM},
title = {Efficient CRISPR-Cas genome editing in brown algae.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101273},
pmid = {41475352},
issn = {2667-2375},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Phaeophyceae/genetics ; Polyethylene Glycols/chemistry ; },
abstract = {Brown algae represent the third most complex lineage to have independently evolved multicellularity, distinct from plants and animals. Yet, functional studies of their development and evolution have been limited by the absence of efficient genome editing tools. Here, we present a robust, high-efficiency, and transgene-free CRISPR-based genome editing platform applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a polyethylene glycol (PEG)-mediated ribonucleoprotein (RNP) delivery system that achieves reproducible editing across multiple loci without cloning or specialized equipment. As proof of concept, we recreated the hallmark imm mutant phenotype by precisely editing the IMMEDIATE UPRIGHT (IMM) locus. APT/2-fluoroadenine (2-FA) selection further enhanced specificity with minimal false positives. The method was easily transferable to other species, including kelps. This platform now enables functional genomics in brown algae, providing powerful tools for investigating development, life cycle regulation, and the independent evolution of complex multicellularity.},
}
@article {pmid41475353,
year = {2026},
author = {Marks, D and Garcia, E and Kumar, S and Tyson, K and Koch, C and Ivanov, AP and Edel, JB and Mirza, HB and Flanagan, W and Dunsby, C and French, PMW and McNeish, IA},
title = {Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101270},
pmid = {41475353},
issn = {2667-2375},
support = {FC001999/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Female ; Humans ; *Ovarian Neoplasms/drug therapy/pathology/metabolism/genetics ; *Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *Poly(ADP-ribose) Polymerases/metabolism ; *Poly (ADP-Ribose) Polymerase-1/metabolism/genetics ; },
abstract = {Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood, as current experimental methodologies lack resolution and throughput. To address this, we develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells. This biosensor enables real-time, single-cell analysis of PARP trapping dynamics. Using fluorescence lifetime imaging microscopy (FLIM), we reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe reduced trapping in PARPi-resistant models in vitro and in vivo. This biosensor provides critical insights into PARPi resistance mechanisms, with implications for developing more effective therapies and advancing personalized treatment for ovarian cancer patients.},
}
@article {pmid41475537,
year = {2026},
author = {Tang, W and Ma, M and Song, W and Kou, M and Wang, X and Yan, H and Li, C and Zhang, A and Gao, T and Gao, R and Zhang, Y and Li, Q},
title = {An efficient CRISPR/Cas9-mediated editing of phytoene desaturase in hexaploid sweetpotato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {364},
number = {},
pages = {112967},
doi = {10.1016/j.plantsci.2025.112967},
pmid = {41475537},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oxidoreductases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Polyploidy ; Chlorophyll/metabolism ; },
abstract = {CRISPR/Cas9-mediated gene editing has emerged as a pivotal tool for functional genomics and crop improvement. For the first time, we applied CRISPR/Cas9-mediated editing to the IbPDS gene in the purple-fleshed cultivar 'XZS-8', achieving mutation efficiencies up to 98.18 %. Loss-of-function mutations in IbPDS induced visually discernible albino phenotypes. Hi-TOM sequencing confirmed deletion mutations within the target locus, with editing efficiencies ranging from 17.77 % to 65.90 % (gRNA1) and 87.87-98.18 % (gRNA2). Knockout lines showed significant reductions in chlorophyll a and b content, confirming functional disruption of IbPDS. Collectively, our results demonstrate efficient CRISPR/Cas9-mediated genome editing for generating mutants in the hexaploid sweetpotato.},
}
@article {pmid41476041,
year = {2025},
author = {Xi, W and Xu, Y and Bao, W and Ding, Z and Xiao, H and Yang, J and Yan, X and Ping, Y},
title = {In vivo chemogenetic RNA editing of macrophages by bioengineered viruses for sepsis treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {927},
pmid = {41476041},
issn = {2041-1723},
support = {82425055//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32261143727//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82504685//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M762890//China Postdoctoral Science Foundation/ ; 2025T180980//China Postdoctoral Science Foundation/ ; GZB20240672//China Postdoctoral Science Foundation/ ; LMS25H300002//Natural Science Foundation of Zhejiang Province (Zhejian