@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.},
}

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

@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 & 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.},
}

Humans
Alleles
*CRISPR-Cas Systems/genetics
*Peptides/metabolism
*Antibodies/metabolism/immunology
*Proteomics/methods
Tandem Mass Spectrometry
Cell Line
*HLA Antigens/genetics/metabolism
Proteome/metabolism

@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.},
}

*DNA Methylation
Humans
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
Limit of Detection
CpG Islands
DNA Restriction Enzymes/metabolism
DNA/chemistry/genetics

@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.},
}

Humans
*Lipids/chemistry
*CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry
HEK293 Cells
*Gene Editing/methods
*Transfection/methods
Cell Line, Tumor
Liposomes

@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).},
}

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

@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.},
}

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

@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.},
}

*Biosensing Techniques
Humans
Biomarkers/analysis
*Neurodegenerative Diseases/diagnosis
*Nanostructures
*Mental Disorders/diagnosis

@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.},
}

Animals
Dogs
Female
Cell Line, Tumor
*CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing
*Liquid Biopsy/veterinary
Mutation

@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.},
}

Humans
*Mitochondria/metabolism/genetics
*DNA, Mitochondrial/metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Neurospora crassa/genetics
CRISPR-Cas Systems

@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.},
}

Humans
*Bacteriophages/physiology/genetics
*Phage Therapy/methods
*Sexually Transmitted Diseases, Bacterial/therapy/microbiology
Biofilms
Anti-Bacterial Agents/therapeutic use

@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.},
}

*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

@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&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 & 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.},
}

Humans
*Hepatitis B virus/genetics/isolation & 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

@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.},
}

*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

@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 & 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 & 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.},
}

*Phanerochaete/genetics/metabolism
*Transformation, Genetic
Genetic Vectors/genetics
*Titanium/chemistry
Green Fluorescent Proteins/genetics
CRISPR-Cas Systems
Hygromycin B/pharmacology/analogs & derivatives
*CRISPR-Associated Protein 9/genetics
Genes, Reporter

@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.},
}

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

@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.},
}

*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

@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.},
}

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

@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.},
}

*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

@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.},
}

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

@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 & purification ; Humans ; *CRISPR-Cas Systems ; *Human papillomavirus 18/genetics/isolation & 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.},
}

*Biotin/chemistry
*Human papillomavirus 16/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Human papillomavirus 18/genetics/isolation & purification
Streptavidin/chemistry
Colorimetry/methods
Rapid Diagnostic Tests
*Papillomavirus Infections/diagnosis/virology
Color

@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 & purification/genetics ; Transmissible gastroenteritis virus/isolation & purification/genetics ; Sensitivity and Specificity ; Deltacoronavirus/isolation & purification/genetics ; *Molecular Diagnostic Techniques/veterinary/methods ; Coronavirus Infections/veterinary/diagnosis/virology ; Rotavirus/isolation & 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.},
}

Animals
Swine
*Swine Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/veterinary/methods
Porcine epidemic diarrhea virus/isolation & purification/genetics
Transmissible gastroenteritis virus/isolation & purification/genetics
Sensitivity and Specificity
Deltacoronavirus/isolation & purification/genetics
*Molecular Diagnostic Techniques/veterinary/methods
Coronavirus Infections/veterinary/diagnosis/virology
Rotavirus/isolation & purification/genetics
CRISPR-Cas Systems
Rapid Diagnostic Tests
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@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 & 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.},
}

*Brucella/genetics/isolation & 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

@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.},
}

*Diatoms/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
Adenine Phosphoribosyltransferase/genetics
Mutation
Genetic Engineering/methods
*Gene Editing/methods

@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 & 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.},
}

*Aspergillus oryzae/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
Promoter Regions, Genetic
Metabolic Engineering/methods
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@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.},
}

*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

@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.},
}

*Deep Learning
*CRISPR-Cas Systems/genetics
Humans
Gene Editing
Prediction Algorithms
Animals

@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 & biotechnology},
volume = {42},
number = {5},
pages = {},
pmid = {42068385},
issn = {1573-0972},
support = {SF2350//Lianyungang Social Development Project Key R&D Programme Funding/ ; KD2024KYJJ151//Nanjing Medical University Kanda College Key Projects/ ; },
mesh = {*Vibrio alginolyticus/genetics/isolation & 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.},
}

*Vibrio alginolyticus/genetics/isolation & 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

@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.

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.},
}

*Simian Immunodeficiency Virus/genetics/physiology
Humans
Animals
*Virus Replication/genetics
*CRISPR-Cas Systems
*Simian Acquired Immunodeficiency Syndrome/transmission/virology
HIV-1/genetics
Pan troglodytes

@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&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.},
}

Animals
*Zebrafish/genetics
Neurodevelopment/genetics
*Zebrafish Proteins/genetics
Gene Knockout Techniques
Disease Models, Animal
*Neurodevelopmental Disorders/genetics
CRISPR-Cas Systems
Apoptosis/genetics

@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.},
}

*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

@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 & 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.},
}

*Mycobacterium tuberculosis/genetics/drug effects/isolation & 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

@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.},
}

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

@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 & 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.},
}

*Bacillus subtilis/genetics/metabolism
*Chitosan/metabolism
Glycoside Hydrolases/genetics/metabolism
*CRISPR-Cas Systems
*Chitin/analogs & derivatives/biosynthesis
Fermentation
Oligosaccharides
Bacterial Proteins/genetics/metabolism

@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 & 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.},
}

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

@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.},
}

*Yarrowia/metabolism/genetics/enzymology
*Fatty Acid Elongases/metabolism/genetics
*Fatty Acids/biosynthesis/metabolism
Substrate Specificity
Workflow
CRISPR-Cas Systems

@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.},
}

*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

@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 & 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.},
}

*Treponema pallidum/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Syphilis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Sensitivity and Specificity
DNA, Bacterial/genetics

@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.},
}

*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

@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 & 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.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
*Plant Breeding/methods
Plants, Genetically Modified
Edible Grain/genetics
Mutation

@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.},
}

*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

@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 & 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.},
}

*Vigna/genetics/virology/growth & 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

@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.},
}

*DNA, Single-Stranded/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Recombinational DNA Repair

@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.},
}

*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

@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 & 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.},
}

*Saccharomyces cerevisiae/genetics/growth & development
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Saccharomyces cerevisiae Proteins/genetics

@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.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Neoplasms/therapy/genetics
*Hematologic Neoplasms/therapy/genetics
Genetic Therapy/methods

@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.},
}

*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

@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.},
}

*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

@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.},
}

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

@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&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.},
}

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

@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.},
}

*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

@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.},
}

Humans
Adult
*CRISPR-Cas Systems
Female
Male
*Gene Editing
Young Adult
Middle Aged
*Intention
Surveys and Questionnaires
*Health Knowledge, Attitudes, Practice

@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 & 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.},
}

*Fusarium/genetics/isolation & purification
*CRISPR-Cas Systems
Zea mays/microbiology
DNA, Fungal/genetics/analysis
Rapid Diagnostic Tests
Smartphone
Microscopy, Fluorescence/methods

@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.},
}

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

@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 & 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.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Mutagenesis
Plants, Genetically Modified
Gene Editing
Phenotype

@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 & 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.},
}

*Aspergillus fumigatus/genetics/isolation & 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

@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.},
}

*Solanum lycopersicum/genetics
*Protoplasts/metabolism
*Chromosome Inversion/genetics
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics

@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 & 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.},
}

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 & derivatives/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Drug Resistance, Neoplasm
RNA, Messenger/metabolism
Cell Survival/drug effects

@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//Fundación Científica de la Asociación Española Contra el Cáncer/ ; 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//Dirección General de Universidades e Investigación de la Consejería de Educación e Investigación 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.},
}

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

@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.},
}

*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Cell Line
Algorithms

@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.},
}

*Aspergillus niger/genetics
*Cytosine/metabolism
*Gene Editing/methods
*Adenine/metabolism
CRISPR-Cas Systems

@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 & 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.},
}

*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

@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//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; 22545/PDC/24//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; 23031/GERM/25 supported by FSRM/10.13039/100007801//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región 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.},
}

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

@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 & purification/genetics ; Humans ; *Influenza A Virus, H1N1 Subtype/isolation & 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.},
}

*Spectrum Analysis, Raman/methods
*SARS-CoV-2/isolation & purification/genetics
Humans
*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Gold/chemistry
Colorimetry/methods
Rapid Diagnostic Tests
Limit of Detection
CRISPR-Cas Systems
COVID-19/diagnosis/virology

@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.},
}

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

@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.},
}

Animals
*CRISPR-Cas Systems
Sheep/genetics/embryology
*Cell Lineage/genetics
DNA Transposable Elements/genetics
*Gene Editing/methods
*DNA Barcoding, Taxonomic/methods
Embryonic Development/genetics

@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 1·3·5 Project for Disciplines of Excellence, West China Hospital of Sichuan University/ ; },
mesh = {*Circovirus/isolation & 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.},
}

*Circovirus/isolation & 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

@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 & 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.},
}

*Crops, Agricultural/genetics
*CRISPR-Cas Systems
Genome, Plant
Cytidine Deaminase/genetics/chemistry/metabolism
*Gene Editing

@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 & 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 & 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.},
}

*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

@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.},
}

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

@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 & 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.},
}

*Osteoblasts/metabolism/cytology
Humans
*Vesicular Transport Proteins/genetics/metabolism/antagonists & 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

@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&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.},
}

*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

@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 & purification ; *Babesia/genetics/isolation & 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.},
}

Animals
Dogs
*Ehrlichia canis/genetics/isolation & purification
*Babesia/genetics/isolation & 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

@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.},
}

*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

@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//Fundação para a Ciência 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.},
}

*Dependovirus/genetics
*Glutamate-Ammonia Ligase/genetics/metabolism
*Genetic Vectors/genetics
Humans
Animals
CRISPR-Cas Systems
HeLa Cells
Cricetulus
CHO Cells
Gene Knockout Techniques

@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 & purification ; Bacterial Proteins/genetics ; beta-Lactamases/genetics/analysis/metabolism ; *Methicillin-Resistant Staphylococcus aureus/isolation & purification ; Catalysis ; *Klebsiella pneumoniae/isolation & 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.},
}

*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems/genetics
Pseudomonas aeruginosa/isolation & purification
Bacterial Proteins/genetics
beta-Lactamases/genetics/analysis/metabolism
*Methicillin-Resistant Staphylococcus aureus/isolation & purification
Catalysis
*Klebsiella pneumoniae/isolation & purification
Lab-On-A-Chip Devices
*DNA/chemistry
Rapid Diagnostic Tests
Limit of Detection

@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.},
}

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

@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.},
}

*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

@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.},
}

*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

@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.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
DNA/genetics
Biosensing Techniques/methods
*Genetic Engineering/methods
*Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@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 & 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.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics
*Electrochemical Techniques/methods
*Glycine/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Povidone/chemistry
Limit of Detection

@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.},
}

*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

@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.},
}

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

@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 & 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.},
}

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

@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 & 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.},
}

Animals
Mice, Inbred C57BL
Mice
*Epitopes, T-Lymphocyte/immunology/genetics
*Nanoparticles/chemistry/administration & dosage
Lipids/chemistry
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/immunology/genetics/metabolism
Gene Editing/methods
Female
Interferon-gamma
Liposomes

@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─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.},
}

*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

@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 & 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.},
}

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

@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 & 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.},
}

Humans
*CRISPR-Cas Systems/genetics
*RNA Editing/genetics
*RNA/genetics/metabolism
*Gene Editing/methods
Animals
*CRISPR-Associated Proteins/genetics/metabolism

@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.},
}

*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

@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.

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.

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.},
}

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

@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 & 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.},
}

Animals
*Carps/genetics/growth & development
*Fibroblast Growth Factor 8/genetics/metabolism
*Osteogenesis/genetics/physiology
Gene Knockout Techniques
CRISPR-Cas Systems
Gene Expression Regulation, Developmental

@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.},
}

*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

@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.},
}

Humans
*Biofortification/methods
*Climate Change
CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/metabolism/chemistry
*Gene Editing/methods
Metabolic Engineering
Micronutrients/deficiency/analysis
Nutritive Value

@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 & development ; *Plant Proteins/genetics/metabolism/chemistry ; Amino Acid Sequence ; Gene Expression Regulation, Plant ; *Endonucleases/genetics/metabolism/chemistry ; Plant Leaves/genetics/physiology/growth & 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.},
}

*Glycine max/genetics/enzymology/physiology/growth & development
*Plant Proteins/genetics/metabolism/chemistry
Amino Acid Sequence
Gene Expression Regulation, Plant
*Endonucleases/genetics/metabolism/chemistry
Plant Leaves/genetics/physiology/growth & development
Gene Knockout Techniques
Plants, Genetically Modified
Flowers/genetics/physiology
Plant Senescence/genetics
Reproduction/genetics
Phylogeny
CRISPR-Cas Systems
Phenotype
Mutation

@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.},
}

*CRISPR-Cas Systems/genetics
*Drug Resistance, Bacterial/genetics
*Bacteria/genetics/drug effects
Anti-Bacterial Agents/pharmacology
*Gene Editing/methods

@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.},
}

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

@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 & 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.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Saccharomycetales/genetics/metabolism
Recombinant Proteins/genetics/biosynthesis
Metabolic Engineering/methods