@article {pmid41317251,
year = {2026},
author = {Kheirandish, A and Sorourian, S and Behbahani, AB and Ahmadi, MKB and Dehbidi, GR and Rahimi, E and Safari, F},
title = {Optimizing Recombinant Protein Production in CHO Cells by Silencing the Caspase 8 Associated Protein 2 Gene via the CRISPR-Cas9 System.},
journal = {Molecular biotechnology},
volume = {68},
number = {6},
pages = {2837-2846},
pmid = {41317251},
issn = {1559-0305},
mesh = {Animals ; CHO Cells ; Cricetulus ; *CRISPR-Cas Systems ; *Recombinant Proteins/genetics/biosynthesis/metabolism ; Cell Survival/drug effects ; Gene Silencing ; Cricetinae ; Apoptosis ; Butyric Acid/pharmacology ; *Apoptosis Regulatory Proteins/genetics ; },
abstract = {Chinese hamster ovary (CHO) cells play a crucial role in biopharmaceutical production due to their ability to produce complex proteins. Enhancing the productivity of CHO cells is essential for meeting the growing demand for biologics. Caspase 8-Associated Protein 2 (CASP8AP2), a key regulator of apoptosis and cell survival, has been identified as a potential target to increase CHO cell productivity. To this end, CRISPR-mediated homology-independent targeted integration (HITI) was used to silence CASP8AP2. Results of the cell viability assay revealed that CASP8AP2-deficient clones (C2, C3, and C4) were more resistant to sodium butyrate (NaBu) compared to native cells, with IC50 values of 11.83, 12.77, 10.25, and 8.55 mM, respectively. Protein production assays showed a significant increase in JRed and luciferase expression in silenced clones (C2 and C4) compared to wild-type cells, with up to 1.2- and 1.9-fold increases for JRed, and 1.4- and 1.7-fold increases for luciferase, respectively. These findings could be attributed to the clones experiencing cell cycle arrest specifically during the S phase. While these results demonstrate proof-of-principle using reporter proteins, future validation with therapeutic biologics such as implementing monoclonal antibodies in bioreactor settings could confirm scalability for industrial bioprocessing. Transcriptomic analyses would further elucidate downstream effects on apoptosis and metabolism pathways. The results suggest that targeting CASP8AP2 could be a promising strategy for improving bioprocess efficiency and yield in CHO cell-based production systems.},
}

Animals
CHO Cells
Cricetulus
*CRISPR-Cas Systems
*Recombinant Proteins/genetics/biosynthesis/metabolism
Cell Survival/drug effects
Gene Silencing
Cricetinae
Apoptosis
Butyric Acid/pharmacology
*Apoptosis Regulatory Proteins/genetics

@article {pmid41495582,
year = {2026},
author = {Hossain, H and Sayeed, SSB and Islam, S and Ahmad, T and Brishty, KA and Chowdhury, MSR and Alam, MS and Mahmud, MS and Rahman, MM},
title = {Prime Editing, CRISPR-Cas9, and NanoCas Genome Editing for Cancer Treatment.},
journal = {Molecular biotechnology},
volume = {68},
number = {6},
pages = {2630-2658},
pmid = {41495582},
issn = {1559-0305},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; Animals ; *Genetic Therapy/methods ; },
abstract = {Over the past two decades, genome editing has advanced dramatically from Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) to more refined systems such as CRISPR-Cas9, prime editing, and nanoCas technologies. These innovations have opened new frontiers in cancer treatment. This review aims to critically examine and compare recent advances in these genome editing platforms, with a focus on their molecular mechanisms, delivery challenges, oncological applications, and clinical prospects. We systematically explore how CRISPR-Cas9 enables gene knockouts, high-throughput functional genomic screens, and immune editing, while acknowledging its limitations due to off-target effects and genotoxicity. In contrast, base and prime editors offer precise, double-strand breaks (DSBs) free alternatives, suitable for correcting oncogenic mutations such as TP53, KRAS, and EGFR. Prime editing, although versatile, faces delivery and efficiency challenges. The emergence of nanoCas systems, derived from compact Cas orthologs, provides promising delivery advantages for in vivo applications. We also examine how tumor microenvironment, cell-type specificity, and immune barriers impact editing efficacy and safety. Strategies such as high-fidelity variants, optimized guide RNAs, and stimuli-responsive nanoparticles are discussed to enhance precision and minimize risk. Conclusively, integrating these genome editing tools into oncology requires addressing translational barriers while harnessing their precision and therapeutic potential for next-generation cancer treatments.},
}

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

@article {pmid41840308,
year = {2026},
author = {Madhi, M and Gholizadeh, P},
title = {CRISPR-Cas9: Genome Engineering and Future Vaccine Applications.},
journal = {Molecular biotechnology},
volume = {68},
number = {7},
pages = {3068-3090},
pmid = {41840308},
issn = {1559-0305},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Animals ; *Vaccines/genetics/immunology ; *Genetic Engineering/methods ; Vaccinology/methods ; Vaccine Development ; },
abstract = {The CRISPR-Cas9 system, a transformative genome engineering tool derived from prokaryotic adaptive immunity, is reshaping the landscape of biological research and therapeutic development. This review provides a critical synthesis of its rapidly evolving, yet underexplored, application in rational vaccine design. We analyze how CRISPR-Cas9 and its derivative platforms (including base editing, prime editing, and CRISPRi/a) are being repurposed from therapeutic gene editing to become indispensable assets in vaccinology. This transition is powered by the convergence of CRISPR-mediated precision with synthetic biology, enabling the rapid engineering of novel vaccine vectors and attenuated strains, the precise optimization of antigen sequences for enhanced breadth and potency, and the direct modulation of host immune responses. Notwithstanding this potential, significant technical and translational hurdles persist, including off-target editing risks, delivery inefficiencies in vivo, and unresolved regulatory pathways for genetically modified vaccines. We detail these mechanisms and evaluate the current preclinical and clinical landscape, while addressing persistent challenges in safety, delivery, and scalability. By delineating these advances and obstacles, this review outlines a forward-looking framework for leveraging CRISPR technology to create programmable, precision vaccines against emerging and re-emerging pathogens, moving the field beyond empirical methods toward a new paradigm of rational immunization.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
Animals
*Vaccines/genetics/immunology
*Genetic Engineering/methods
Vaccinology/methods
Vaccine Development

@article {pmid41854240,
year = {2026},
author = {Zhang, Y and Luo, X and Li, H and Jin, S and Zhang, X and Wang, M},
title = {Single-Cell CRISPR: An Efficient Strategy for Decoding Plant Cis-Regulatory Complexity.},
journal = {Plant biotechnology journal},
volume = {24},
number = {7},
pages = {4454-4467},
doi = {10.1111/pbi.70647},
pmid = {41854240},
issn = {1467-7652},
support = {2023A01//Xinjiang Uyghur Autonomous Region/ ; //Tianchi Talent Introduction Plan of Xinjiang Uyghur Autonomous Region/ ; 2021YFF1000900//National Key Research and Development Program of China/ ; },
mesh = {*Single-Cell Analysis/methods ; *Plants/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Plant/genetics ; Single-Cell Gene Expression Analysis ; Gene Regulatory Networks/genetics ; *Regulatory Sequences, Nucleic Acid/genetics ; },
abstract = {The generation of complex traits involves the coordinated interplay of multiple gene networks. Elucidating the function of transcriptional cis-regulatory elements (CREs) in regulating gene expression is crucial for understanding complex regulatory pathways and improving our ability to modify macro-phenotypes. While traditional bulk sequencing approaches rely on tissue or cell population aggregates, single-cell transcriptomics provides a more precise perspective by capturing cell-type-specific information. The integration of single-cell technology with genome-wide genetic screening, particularly through the single-cell CRISPR (scCRISPR) system, enables the identification of critical regulatory elements and provides novel insights into gene-expression control mechanisms. Here, we summarise recent advances in diverse strategies for functional genome analysis using the scCRISPR system, with an emphasis on its potential to revolutionise single-cell genetic screening of CREs. We also explore the challenges and opportunities for applying these approaches in plant research.},
}

*Single-Cell Analysis/methods
*Plants/genetics
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation, Plant/genetics
Single-Cell Gene Expression Analysis
Gene Regulatory Networks/genetics
*Regulatory Sequences, Nucleic Acid/genetics

@article {pmid41909927,
year = {2026},
author = {Liu, H and Farmer, R and Payyavula, R and Kudithipudi, C and Timko, MP},
title = {Functional Characterisation of HAIRPLUS (NtHAP) Genes Involved in Trichome Development and Specialised Metabolism of Tobacco.},
journal = {Plant biotechnology journal},
volume = {24},
number = {7},
pages = {4640-4655},
doi = {10.1111/pbi.70655},
pmid = {41909927},
issn = {1467-7652},
support = {GI15099//Altria Client Services, LLC/ ; },
mesh = {*Trichomes/metabolism/genetics/growth & development ; *Nicotiana/genetics/metabolism/growth & development ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *Genes, Plant ; RNA Interference ; Nicotine/metabolism ; Diterpenes/metabolism ; CRISPR-Cas Systems ; Phylogeny ; },
abstract = {Plant glandular trichomes are specialised epidermal structures capable of synthesising, storing and secreting numerous varieties of secondary metabolites in different classes and are central to plant defence and the biosynthesis of high-value metabolites. In this study, we characterised the HAIRPLUS (HAP) gene family and uncovered its role as a conserved regulator of trichome development and metabolism in tobacco. Four homologues, NtHAP1a, NtHAP1b, NtHAP2a and NtHAP2b, were identified and functional studies using RNAi and CRISPR-Cas9 revealed that NtHAPs act as negative regulators of glandular trichome development. Suppression of NtHAPs resulted in increased trichome density and enlarged glandular heads, as well as enhanced accumulation of diterpenoids (e.g., neophytadiene) and increased nicotine levels. Additionally, NtHAP1 appeared to have a stronger effect on trichome density. This study establishes the NtHAP genes as key negative regulators of glandular trichome development in tobacco, expanding their functional scope from trichome morphogenesis to metabolic regulation and highlighting their evolutionary conservation across Solanaceae. These findings pave the way for both fundamental research into trichome biology and practical applications in metabolic engineering and crop improvement, such as pest resistance.},
}

*Trichomes/metabolism/genetics/growth & development
*Nicotiana/genetics/metabolism/growth & development
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified
*Genes, Plant
RNA Interference
Nicotine/metabolism
Diterpenes/metabolism
CRISPR-Cas Systems
Phylogeny

@article {pmid41919983,
year = {2026},
author = {Kaur, N and Raffan, S and Clark, SJ and Musa, S and Scherf, K and Elmore, JS and Curtis, TY and Honan, E and Halford, NG},
title = {Field Trials and Baking Studies of Ultra-Low Asparagine, Genome Edited (CRISPR/Cas9) and Mutant (TILLING) Wheat.},
journal = {Plant biotechnology journal},
volume = {24},
number = {7},
pages = {4704-4715},
doi = {10.1111/pbi.70661},
pmid = {41919983},
issn = {1467-7652},
support = {BB/T017007/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/W007134/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Triticum/genetics/metabolism ; *Asparagine/metabolism/analysis ; Acrylamide/analysis/metabolism ; *CRISPR-Cas Systems/genetics ; Bread/analysis ; Plants, Genetically Modified ; Mutation ; Aspartate-Ammonia Ligase/genetics/metabolism ; Gene Knockout Techniques ; Mutagenesis ; Cooking ; Genome, Plant/genetics ; },
abstract = {Field trials were conducted of wheat (Triticum aestivum) cv. Cadenza in which asparagine synthetase gene, TaASN2, had been knocked out, either on its own or together with a partial knockout of the related gene, TaASN1, using CRISPR/Cas9. Chemical mutagenesis (TILLING) TaASN2 nulls in the Claire background were also included. The main aim was to assess the free asparagine content of the grain and the conversion of free asparagine to acrylamide, a toxic contaminant, in bread, toast and biscuits. Over 2 years of trials combined, the TaASN2 and TaASN1/2 CRISPR knockouts resulted in a reduction of free asparagine in the grain of 59% and 93%, respectively, compared with Cadenza. The reduction in the TaASN2 total knockout TILLING line compared with Claire was 50%. Yield was not affected in the edited lines but was reduced in the TILLING lines. Acrylamide in bread made from a TaASN1/2 CRISPR line was below detection levels, while in a TaASN2 CRISPR line it was 14% of the Cadenza control. Even after 4 min of toasting, acrylamide levels remained at 8% and 23%, respectively, of the control. The concentration in bread made from the TILLING TaASN2 knockout was 21% that for the Claire control, rising to 46% after 4 min of toasting. Acrylamide in biscuits made from a TaASN1/2 CRISPR line was reduced by 93% compared with the control. The relationship between acrylamide and colour was altered in the edited and mutant lines compared with the controls, with less acrylamide forming for the same degree of colour.},
}

*Triticum/genetics/metabolism
*Asparagine/metabolism/analysis
Acrylamide/analysis/metabolism
*CRISPR-Cas Systems/genetics
Bread/analysis
Plants, Genetically Modified
Mutation
Aspartate-Ammonia Ligase/genetics/metabolism
Gene Knockout Techniques
Mutagenesis
Cooking
Genome, Plant/genetics

@article {pmid42002657,
year = {2026},
author = {Sichani, AS and Hassani, M and Gila, F and Shafieipour, N and Dabbaghipour, R and Heidari, Z and Sisakht, M and Hassani, M and Fallahi, J},
title = {A Comprehensive Review on CRISPR-Based Screening and Its Applications.},
journal = {Molecular biotechnology},
volume = {68},
number = {7},
pages = {3051-3067},
pmid = {42002657},
issn = {1559-0305},
support = {43016329//Shahid Beheshti University of Medical Sciences/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-based tools have quickly moved from specialist techniques to routine instruments in biology and medicine, and they are now central to large-scale loss-of-function and perturbation screens. In this review, we focus on how pooled CRISPR screens are used to interrogate gene function in living cells, most often through cell fitness or simple selectable markers, and contrast this with arrayed formats that trade throughput for richer molecular readouts, such as transcriptome-wide changes. We bring together current strategies for library design, delivery, and selection and show how different Cas nucleases, including Cas9, Cas12, and Cas13, broaden the range of genome and transcriptome perturbations that can be assayed. We then discuss recent applications in drug response, viral infection, and cancer biology and consider how improvements in high-content technologies, data analysis, and emerging diagnostic uses are likely to shape the next generation of CRISPR-based screening studies.},
}

Humans
*CRISPR-Cas Systems
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing/methods
*Gene Editing/methods

@article {pmid42205057,
year = {2026},
author = {Liu, Y and Yue, X and Li, B and Yang, H and Wang, Y and Ge, S and Liu, S},
title = {Target-to-signal conversion and spatial enrichment cascade boost CRISPR/Cas12a biosensing for trace-level pathogen detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {23},
pages = {4851-4857},
doi = {10.1039/d6ay00441e},
pmid = {42205057},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Salmonella typhimurium/isolation & purification/genetics ; Limit of Detection ; DNA, Catalytic/chemistry/metabolism ; },
abstract = {S. typhimurium is a widely distributed and highly pathogenic Gram-negative bacterium capable of causing outbreaks of foodborne illness through the ingestion of contaminated food. Consequently, the development of a rapid and accurate detection strategy for S. typhimurium is of paramount importance. This study constructed a CRISPR/Cas12a-activated DNA nanowire biosensor based on DNAzyme-catalysed amplification for the sensitive detection of Salmonella typhimurium. Specific cleavage by DNAzymes generates single-stranded DNA to activate the HCR, enabling the serial HCR signal enrichment of three DNA hairpin structures, whose branched ends bear PAM sequences. Subsequent introduction of CRISPR/Cas12a cleaves the DNA NW, restoring quenched fluorescence signals to achieve ultra-sensitive detection of Salmonella typhimurium. Under optimal conditions, this biosensor exhibits a linear detection range of 10[1]-10[5] cfu mL[-1], with an estimated limit of detection (LOD) of 6.26 cfu mL[-1]. The method offers advantages of straightforward operation, rapid response, and high sensitivity. Furthermore, by modifying the recognition module and primer sequences, this biosensor holds promise for highly sensitive detection of multiple foodborne pathogens.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Salmonella typhimurium/isolation & purification/genetics
Limit of Detection
DNA, Catalytic/chemistry/metabolism

@article {pmid42247991,
year = {2026},
author = {Chen, H and Jiang, Z},
title = {CrisprFusion: A feature fusion model with multi-type input features for sgRNA activity prediction.},
journal = {Computational biology and chemistry},
volume = {124},
number = {Pt 1},
pages = {109137},
doi = {10.1016/j.compbiolchem.2026.109137},
pmid = {42247991},
issn = {1476-928X},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Deep Learning ; *Gene Editing ; Humans ; },
abstract = {The CRISPR/Cas9 system enables precise and efficient genome editing, but its efficacy heavily relies on sgRNA activity. Although deep learning has been widely applied to sgRNA activity prediction, existing methods often integrate multiple biological features without a well-designed fusion strategy. To tackle this issue, we present CrisprFusion, a deep learning framework that explicitly encodes four biological features through a four-branch input structure. The core of our model is a novel Multi-Grain Cross Attention Fusion Module, which performs fusion at two levels: branch-level gating for adaptive reweighting of different modalities, and token-level alignment for capturing position-specific interactions along the 23-nt sgRNA sequence. We evaluate CrisprFusion on seven high-throughput datasets with six representative baselines. Our method achieves consistent and superior average performance across all datasets and remains competitive in cross-cell-line validation on four functional screens. Ablation experiments verify the effectiveness of the proposed fusion module, and attention visualization reveals the importance of individual biological features. Overall, CrisprFusion offers an effective and interpretable approach for multimodal biological feature integration in sgRNA activity prediction.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Deep Learning
*Gene Editing
Humans

@article {pmid42288047,
year = {2026},
author = {Maire, A and Laurenceau, R and Rolhion, N and Bikard, D},
title = {In situ genetic modification of gut bacteria.},
journal = {Current opinion in microbiology},
volume = {92},
number = {},
pages = {102766},
doi = {10.1016/j.mib.2026.102766},
pmid = {42288047},
issn = {1879-0364},
abstract = {The crucial role of the gut microbiome in human health has driven a need to understand bacterial function within their complex native ecosystem. However, traditional functional genomic methods require isolating, cultivating, and modifying bacteria in vitro before their reintroduction in vivo. This process often necessitates the use of axenic animals or antibiotic treatments, creating artificial conditions that disrupt key microbial interactions and can obscure relevant phenotypes. This review highlights emerging tools for precise, in situ genetic manipulation of bacteria directly within the gut. We cover diverse technologies, including DNA delivery systems (e.g. engineered temperate phages, phagemids, and conjugative plasmids), and genetic perturbation strategies (e.g. CRISPR-Cas tools and transposons). These methods offer the opportunity to engineer unculturable microbes in their natural habitat and conduct genetic screens to investigate the role of specific genes and pathways. Finally, we explore the potential therapeutic applications of in situ microbiome editing.},
}

@article {pmid42288957,
year = {2026},
author = {Pal, P and Sarkar, S and Rajak, J and Maitra, S and Almohaimeed, HM and Sami Aggad, W and Almars, AI and Mitra, AK and Uti, DE and Dhara, B},
title = {Engineering Lactobacillus for therapeutic delivery and biosensing: lessons from niche adaptation.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {1-16},
doi = {10.1080/17460913.2026.2686558},
pmid = {42288957},
issn = {1746-0921},
abstract = {Lactobacillus species are renowned for their probiotic properties and niche adaptability, driven by unique genomic traits, stress-response mechanisms, and biofilm formation. This versatility makes them exceptional candidates for advanced biotechnological applications. Their biocompatibility and immunomodulatory effects allow them to serve as live biotherapeutic products. Through genetic engineering and encapsulation, Lactobacillus can be programmed to deliver recombinant proteins and vaccines, cytokines and anti-inflammatory molecules, targeted enzymes, and peptides. Beyond therapy, these bacteria can be engineered into biosensors to detect pathogens, toxins, and clinical biomarkers. By integrating CRISPR-Cas systems and reporter genes into whole‑cell or cell‑free platforms, they offer robust solutions for food safety, environmental monitoring, and diagnostics. While challenges in stability and regulation persist, advancements in synthetic biology are transforming Lactobacillusfrom a simple probiotic into a precise, multifunctional tool for improving global health and environmental oversight.},
}

@article {pmid41766608,
year = {2026},
author = {Yin, J and Wen, H and Zeng, J and Sun, Y and Chen, X and Jin, L and Song, Y and Xia, S},
title = {CRISPR-based genome editing in human embryos: a review of efficiency, safety, and ethical implications.},
journal = {Biology of reproduction},
volume = {114},
number = {6},
pages = {1775-1790},
doi = {10.1093/biolre/ioag056},
pmid = {41766608},
issn = {1529-7268},
mesh = {Humans ; *Gene Editing/ethics/methods ; Animals ; *CRISPR-Cas Systems ; *Embryo, Mammalian ; },
abstract = {Programmable gene editing tools, particularly CRISPR/Cas9 and its advanced derivatives (base and prime editors), have revolutionized biomedical research and offer unprecedented potential for studying human embryogenesis and correcting monogenic diseases. This review systematically examines the evolution and challenges of these technologies in human embryos and mammalian models. We trace key methodological advancements, from initial studies hampered by low efficiency and mosaicism to refined strategies like RNP delivery and base editing that improved precision. A critical shift occurred with the discovery that CRISPR/Cas9 can cause severe on-target damage, such as large deletions and chromosomal loss, redirecting the field's focus toward safety. We present a comparative analysis of editing efficiencies across species (human, mouse, primate, pig, cow, and rabbit) and tools (Cas9, BEs, and PEs), consistently demonstrating the superiority of RNP for precise editing. Fundamental barriers to clinical translation are discussed, including the trade-off between efficiency and mosaicism, persistent off-target effects, and profound ethical concerns. The review concludes that while somatic gene therapy advances rapidly, heritable genome editing remains premature due to unresolved risks. Future progress depends on developing safer editors, understanding on-target consequences, and adhering to rigorous ethical standards.},
}

Humans
*Gene Editing/ethics/methods
Animals
*CRISPR-Cas Systems
*Embryo, Mammalian

@article {pmid42036673,
year = {2026},
author = {Jiang, CQ and Song, Z and Yan, ZC and Liu, Y and Zhang, JJ and Yue, B and Qiu, M and Hu, YJ},
title = {MXene-based CRISPR/Cas9 nanoplatform targeting FABP5 for ROS amplification and synergistic photothermal/photodynamic therapy of cervical cancer.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42036673},
issn = {1477-3155},
support = {62405299//the Young Scientists Fund of the National Natural Science Foundation of China/ ; ZR2024QE315//Youth Foundation of Shandong Natural Science Foundation/ ; 62375249//National Natural Science Foundation of China/ ; ZR2022JQ22//Natural Science Foundation of Shandong Province/ ; tsqn201909054//Taishan Scholar Project/ ; 202341006//Fundamental Research Funds for the Central Universities/ ; TJYXZDXK-3-029C//Tianjin Key Medicine Discipline (Specialty) Construction Project/ ; },
mesh = {Female ; *Uterine Cervical Neoplasms/therapy/metabolism/pathology ; Humans ; *Photochemotherapy/methods ; *Reactive Oxygen Species/metabolism ; Animals ; *CRISPR-Cas Systems/genetics ; *Fatty Acid-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Mice ; Gene Editing ; Apoptosis/drug effects ; Oxidative Stress/drug effects ; Photothermal Therapy/methods ; Nitrites ; Transition Elements ; },
abstract = {Cervical cancer remains a leading cause of cancer-related mortality among women worldwide, underscoring the need for more effective therapeutic strategies. Photodynamic therapy (PDT) has gained attention in tumor treatment owing to its high selectivity and minimal invasiveness. However, PDT is often compromised by the intrinsic antioxidant defense systems of cervical cancer cells. Herein, we developed a gene editing photonic nanoplatform, MXene@PEI-FABP5 (MPF), which integrates the photothermal/photodynamic properties of MXene with CRISPR/Cas9-mediated FABP5 gene editing to achieve synergistically enhanced antitumor effects. Fatty acid binding protein 5 (FABP5), highly expressed in cervical cancer, plays a pivotal role in regulating lipid peroxidation and oxidative stress tolerance. By delivering the CRISPR/Cas9 system using MXene into tumor cells, FABP5 expression was effectively silenced, thereby disrupting cellular antioxidant defenses at the genetic level. Meanwhile, under 808 nm laser irradiation, MXene generated robust hyperthermia and reactive oxygen species (ROS), jointly amplifying oxidative stress and inducing cell death predominantly through apoptosis. Both in vitro and in vivo results demonstrated that MPF achieved an impressive tumor inhibition rate of ~96% while maintaining excellent biosafety. This work presents a "gene editing and photothermal/photodynamic" hybrid therapeutic paradigm, offering a promising avenue to overcome the limitations of conventional PDT and improve cervical cancer treatment outcomes.},
}

Female
*Uterine Cervical Neoplasms/therapy/metabolism/pathology
Humans
*Photochemotherapy/methods
*Reactive Oxygen Species/metabolism
Animals
*CRISPR-Cas Systems/genetics
*Fatty Acid-Binding Proteins/genetics/metabolism
Cell Line, Tumor
Mice
Gene Editing
Apoptosis/drug effects
Oxidative Stress/drug effects
Photothermal Therapy/methods
Nitrites
Transition Elements

@article {pmid42045967,
year = {2026},
author = {Zhang, Y and Wang, Y and Ma, D and Xiao, G and Feng, L and Cai, J and Xu, Y and Wang, Y and Liu, X and Tian, J and Zuo, Z and Lan, J and Shen, B and Ding, S},
title = {Machine learning-advanced hydrogel-based transcription-coupled positive-feedback CRISPR/Cas13a analysis for novel microRNA signatures in differential diagnosis of non-small cell lung cancer.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42045967},
issn = {1477-3155},
support = {82502846//National Natural Science Foundation of China/ ; 2025ZNSFSC1562//Natural Science Foundation of Sichuan Province/ ; 25QNMP119//Health Commission of Sichuan Province Medical Science and Technology Program/ ; 25QNMP041//Health Commission of Sichuan Province Medical Science and Technology Program/ ; 2023CQBSHTB3028//Special Funding for Postdoctoral Research Projects in Chongqing/ ; },
mesh = {*Carcinoma, Non-Small-Cell Lung/diagnosis/genetics/blood ; Humans ; *Machine Learning ; *Lung Neoplasms/diagnosis/genetics ; *MicroRNAs/genetics/blood ; *Hydrogels/chemistry ; *CRISPR-Cas Systems/genetics ; Diagnosis, Differential ; Biomarkers, Tumor/blood/genetics ; Biosensing Techniques/methods ; },
abstract = {MicroRNAs (miRNAs) hold significant potential as biomarkers for the precise diagnosis of non-small cell lung cancer (NSCLC). However, miRNAs remain underused due to their low abundance, high heterogeneity, and complex matrix interference in liquid biopsies, as well as the requirement for specialized techniques. Herein, we devised a hydrogel-based transcription-coupled positive-feedback CRISPR/Cas13a (TCPFC) enhanced electrochemiluminescent (ECL) analyzer for advanced analysis of plasma miRNA signatures via machine learning (ML). Initially, three NSCLC-associated miRNA signatures (miR-203b, miR-450b, and miR-642a) were identified from plasma miRNA datasets and validated using RT-qPCR. An Au@ACZ-SA-PEG hydrogel emitter was engineered to deliver a robust ECL output on a glassy carbon electrode. Additionally, the TCPFC strategy utilized transcription-coupled positive-feedback CRISPR/Cas13a to achieve cascade signal amplification. Concurrently, collateral cleavage eliminated dopamine quenchers, thereby restoring the ECL signal ("OFF-ON") for readout and achieving attomolar-level detection. The integration of ML algorithms with the hydrogel-based TCPFC-ECL platform yielded differential diagnosis accuracies of 100.00% (train, n = 110) and 92.73% (test, n = 110), effectively distinguishing healthy controls (HC) from patients with stages I/II and III/IV NSCLC. Consequently, this biosensing platform demonstrates considerable promise as a practical tool for the precise diagnosis of NSCLC.},
}

*Carcinoma, Non-Small-Cell Lung/diagnosis/genetics/blood
Humans
*Machine Learning
*Lung Neoplasms/diagnosis/genetics
*MicroRNAs/genetics/blood
*Hydrogels/chemistry
*CRISPR-Cas Systems/genetics
Diagnosis, Differential
Biomarkers, Tumor/blood/genetics
Biosensing Techniques/methods

@article {pmid42167468,
year = {2026},
author = {Yang, M and Gao, J and Han, Y and Ji, G and Liu, Z},
title = {In vivo evidence for the role of CRFB5 in zebrafish resistance to grass carp reovirus (GCRV) infection.},
journal = {Developmental and comparative immunology},
volume = {180},
number = {},
pages = {105631},
doi = {10.1016/j.dci.2026.105631},
pmid = {42167468},
issn = {1879-0089},
mesh = {Animals ; *Zebrafish/immunology/virology ; *Reoviridae Infections/immunology ; *Reoviridae/immunology/physiology ; Immunity, Innate/genetics ; *Fish Diseases/immunology/virology ; *Zebrafish Proteins/genetics/metabolism ; *Receptors, Interferon/genetics/metabolism ; *Carps/immunology/virology ; *Fish Proteins/genetics/metabolism ; Disease Resistance/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Liver/immunology/metabolism ; Interferons/metabolism ; Animals, Genetically Modified ; },
abstract = {The interferon (IFN) system is central to vertebrate antiviral innate immunity, whose activation requires the binding of IFNs to specific cell membrane receptors. Teleost IFN receptors CRFB5 is a key component of this receptor complex. However, there is no solid in vivo genetic evidence to verify its physiological function in antiviral defense. Using a zebrafish model, we found that crfb5 expression was rapidly up-regulated in the liver at 4 h post-infection with grass carp reovirus (GCRV). We then constructed crfb5 homozygous knockout zebrafish (crfb5[-/-]) using CRISPR/Cas9 technology. Challenge with GCRV showed that crfb5[-/-] zebrafish had a significantly lower survival rate than wild-type (WT) zebrafish. Compared with WT, GCRV-infected crfb5[-/-] zebrafish showed suppressed mxa expression but elevated levels of ifnφ3 and il6. Collectively, our in vivo data demonstrate that CRFB5 plays a key role in zebrafish anti-GCRV immune response and the maintenance of inflammatory homeostasis by regulating the transcriptional balance of key immune factors.},
}

Animals
*Zebrafish/immunology/virology
*Reoviridae Infections/immunology
*Reoviridae/immunology/physiology
Immunity, Innate/genetics
*Fish Diseases/immunology/virology
*Zebrafish Proteins/genetics/metabolism
*Receptors, Interferon/genetics/metabolism
*Carps/immunology/virology
*Fish Proteins/genetics/metabolism
Disease Resistance/genetics
CRISPR-Cas Systems
Gene Knockout Techniques
Liver/immunology/metabolism
Interferons/metabolism
Animals, Genetically Modified

@article {pmid42171238,
year = {2026},
author = {Chu, Z and Yong, H and Li, Z and Wang, X and Li, B and Yan, C and Li, Q and Tao, Y and Zhou, D and Geng, S},
title = {Effective gene editing of melanoma by delivery of Cas9 mRNA with highly branched poly(β-amino ester).},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {23},
pages = {7142-7149},
doi = {10.1039/d5tb02556g},
pmid = {42171238},
issn = {2050-7518},
mesh = {Humans ; Animals ; *RNA, Messenger/genetics/chemistry/metabolism ; Mice ; *Gene Editing/methods ; *Melanoma/genetics/therapy/pathology ; *Polymers/chemistry ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; Cell Line, Tumor ; },
abstract = {Melanoma, the most aggressive form of skin cancer, still lacks clinically reliable treatments. CRISPR/Cas9 gene editing offers a promising approach for melanoma therapy; however, the efficient and safe delivery of CRISPR/Cas9 components to melanoma cells remains a formidable challenge. Here, we report the development of highly branched poly(β-amino ester) (HPAE) for the efficient delivery of Cas9 mRNA to melanoma cells and for transdermal application in vivo. HPAE is synthesized via a one-pot "A2 + B4 + C2" type Michael addition strategy and is capable of efficiently condensing mRNA to form nanosized polyplexes with a moderate positive charge. In human embryonic kidney cells (HEK293T), mouse melanoma cells (B16F10), and human melanoma cells (A2058 and A375), up to 76.3%, 74.6%, 16.5%, and 57.1% mRNA transfection efficiency was achieved without obvious cytotoxicity. Moreover, repeated dosing further enhanced mRNA transfection efficiency, resulting in sustained gene expression. Importantly, HPAE enables the effective co-delivery of Cas9 mRNA and sgRNA to melanoma cells, facilitating up to 25.5% gene knockout. HPAE also demonstrates strong transdermal capability, resulting in high in vivo mRNA expression following direct topical administration. This study demonstrates the feasibility of treating melanoma through the transdermal application of the CRISPR/Cas9 system, enabled by the efficient mRNA delivery provided by HPAE.},
}

Humans
Animals
*RNA, Messenger/genetics/chemistry/metabolism
Mice
*Gene Editing/methods
*Melanoma/genetics/therapy/pathology
*Polymers/chemistry
*CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems/genetics
HEK293 Cells
Cell Line, Tumor

@article {pmid42279769,
year = {2026},
author = {Pan, X and Ding, X},
title = {Advances in Foodborne Pathogen Detection: From Conventional Confirmation to Integrated and Intelligent Platforms.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {11},
pages = {},
pmid = {42279769},
issn = {2304-8158},
support = {2023YFD2402800//China Youth Development Foundation/ ; BE2023725//Jiangsu Province Science and Technology Department/ ; 82373629//National Natural Science Foundation of China/ ; },
abstract = {Foodborne pathogens pose a major challenge for public health, food safety regulation, and industrial quality control. Effective surveillance, outbreak tracing, and early warning for foodborne microbial contamination require rapid, reliable detection methods. Conventional culture-based methods are still essential for regulatory confirmation since they recover viable isolates and support downstream verification. However, their long turnaround time, labor-intensive procedures, and limited throughput restrict their use in rapid screening and on-site testing. In recent years, immunological assays, nucleic acid amplification and recognition methods, biosensors, microfluidic systems, CRISPR-Cas platforms, mass spectrometry, sequencing technologies, and artificial intelligence-assisted analysis have expanded the detection toolbox. These methods improve speed, sensitivity, portability, and multiplexing capacity, but their performance still depends on food-matrix properties, sample pretreatment, and application conditions. This review compares representative methods in terms of analytical principle, sample pretreatment, sensitivity, specificity, assay time, viable-cell discrimination, field applicability, and standardization potential. In our opinion, culture-based methods are central for confirmation, while emerging technologies are better suited for rapid screening, integrated analysis, and point-of-need testing. Nevertheless, matrix interference, limited validation in naturally contaminated samples, insufficient viable/dead-cell discrimination, and weak cross-platform consistency remain key barriers to routine use.},
}

@article {pmid42282866,
year = {2026},
author = {Xie, S and Liu, S and Schwarz, S and Sun, H and Xu, Q and Chai, J and Lin, L and Du, S and Hou, J and Song, Y and Brenciani, A and Zhu, Y and Zhang, W},
title = {Toxin gene profiles, antibiotic resistance, and genetic diversity of Clostridium perfringens from food-producing animals: a whole-genome sequencing study with implications for food safety.},
journal = {Current research in food science},
volume = {12},
number = {},
pages = {101460},
pmid = {42282866},
issn = {2665-9271},
abstract = {Clostridium perfringens is a significant zoonotic foodborne pathogen. To systematically assess the potential risks associated with food-producing animals as a reservoir of C. perfringens in the early stages of the food production chain, we conducted whole-genome sequencing (WGS) and phenotypic analysis of 91 clinical C. perfringens isolates collected from pigs, chickens, cows, ducks, and geese across different regions of China. The results revealed that the isolates harbored a rich repertoire of toxin genes, with 71.43% (65/91) carrying greater than or equal to 10 toxin genes. Besides the classic type A, type C, which causes animal enterotoxemia, was most prevalent in pigs (45.76%). Notably, the and necrotic B-like (NetB) toxin, typically associated with avian necrotic enteritis, was also detected in isolates from cows and geese, suggesting potential cross-host transmission of toxin types. Antimicrobial susceptibility testing revealed a severe resistance situation, particularly among porcine isolates, which showed the highest resistance rates to clindamycin, penicillin, and tetracycline, with widespread multidrug resistance (MDR). Genomic analysis further identified 14 types of antimicrobial resistance (AMR) genes. The tetracycline resistance gene tetA(P) had an extremely high carriage rate of 94.51%, and AMR genes were most enriched in porcine isolates. Multilocus sequence typing (MLST) identified 59 sequence types (STs), 42 of which were newly discovered, demonstrating high genetic diversity. Major clonal complexes (CCs) showed certain host and geographic clustering. Furthermore, while the restriction-modification (RM) system was present in all isolates, the distribution of other defense systems like CRISPR-Cas was strain-specific. This study revealed that C. perfringens from Chinese food-producing animals is characterized by high virulence, extensive antimicrobial resistance, and high genetic diversity. It highlighted that pigs may serve as a crucial reservoir and evolutionary hub for virulent MDR isolates, posing a continuous threat to food safety and public health, and underscored the necessity for enhanced monitoring at the farm level.},
}

@article {pmid42283176,
year = {2026},
author = {Hussain, MS and Babu, MA and Roopashree, R and Lal, M and Anand, T and Rekha, A and Goyal, K and Ali, H and Singh, TG and Singh, SK and Dua, K and Gupta, G},
title = {Regulatory Networks of ncRNAs and NF-κB in Glioblastoma: Implications for Therapeutics.},
journal = {Current neuropharmacology},
volume = {},
number = {},
pages = {},
doi = {10.2174/011570159X382254251121114451},
pmid = {42283176},
issn = {1875-6190},
abstract = {Glioblastoma (GBM) is the most malignant form of primary brain tumor, exhibiting rapid growth, increased blood vessel growth, therapy resistance, and severe immune suppression. Constant activation of the nuclear factor κB- (NF-κB) signaling axis underlies many of these cancer traits. Concurrently, non-coding RNAs (ncRNAs), notably microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), have emerged as critical modulators of GBM pathways. This review explains how specific ncRNAs use NF-κB signaling to regulate glioma cell survival, invasion, and therapeutic responses. We synthesized current evidence for miR-21 and miR-181 family members in promoting NF-κB-driven gene expression patterns, described lncRNAs, such as MALAT1 and HOTAIR, which support NF-κB complexes, and highlighted circRNAs, including circKPNB1 and circEZH2, that act as competing RNAs to modulate NF-κB activity. We evaluated preclinical strategies targeting ncRNA-NF-κB interactions, including antisense oligonucleotides, small interfering RNAs, locked nucleic acids, CRISPR-Cas approaches, and smallmolecule- inhibitors, with an emphasis on delivery systems, target specificity, and tumor diversity. Finally, we propose a comprehensive model of ncRNA-NF-κB crosstalk in GBM pathobiology and outline practical approaches to exploit these networks for personalized treatment.},
}

@article {pmid42283788,
year = {2026},
author = {Heydarov, RN and Romanova, KA and Ushtanit, AI and Mikhailovich, VM},
title = {The genomic landscape of Klebsiella pneumoniae in Russia.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {7},
pages = {},
pmid = {42283788},
issn = {1573-0972},
support = {25-24-00721//Russian Science Foundation/ ; 25-24-00721//Russian Science Foundation/ ; 25-24-00721//Russian Science Foundation/ ; 25-24-00721//Russian Science Foundation/ ; },
mesh = {*Klebsiella pneumoniae/genetics/isolation & purification/drug effects/pathogenicity/classification ; Plasmids/genetics ; *Genome, Bacterial/genetics ; beta-Lactamases/genetics ; Russia/epidemiology ; *Klebsiella Infections/microbiology/epidemiology ; Bacterial Proteins/genetics ; Virulence Factors/genetics ; Humans ; Whole Genome Sequencing ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Genomics ; Virulence/genetics ; CRISPR-Cas Systems ; },
abstract = {Klebsiella pneumoniae is a critical priority pathogen known for its extensive antimicrobial resistance (AMR) and capacity to cause severe infections. The traditional dichotomy between multidrug-resistant (MDR) and hypervirulent strains is rapidly eroding, driving the emergence of highly lethal convergent phenotypes. While global surveillance heavily emphasizes KPC-producing lineages, the specific genomic architecture driving this convergence in the Russian Federation remains insufficiently resolved. In this study, we analyzed population structure represented in a curated dataset 264 K. pneumoniae genomes, comprising 18 newly sequenced clinical isolates and 246 public assemblies from 2015 to 2024, using whole-genome sequencing, pangenome reconstruction, resistome, virulence factors and plasmidome profiling, and CRISPR-Cas typing. Our analysis revealed that high-risk sequence types ST395 (56.1%) and ST147 (8.7%) dominate the regional landscape. Carbapenemase genes were detected in 76.5% of isolates, primarily driven by blaOXA-48 on IncL/M plasmids within ST395 and blaNDM variants in ST147. Crucially, 46.2% of isolates harbored convergent plasmids, predominantly large, mosaic IncFIB/IncHI1B cointegrates, that simultaneously encode hypervirulence determinants like the aerobactin synthesis locus alongside resistance genes including blaNDM-1 and blaCTX-M-15. Additionally, we identified a heavy enrichment of plasmid-borne Type IV-A3 CRISPR-Cas systems in the dominant ST395 clone. The regional dominance of ST395 and ST147, coupled with the extensive horizontal integration of both resistance and virulence, represents a formidable public health threat. These findings underscore the critical need for localized genomic surveillance to effectively monitor evolving convergent pathogens and guide tailored antimicrobial stewardship.},
}

*Klebsiella pneumoniae/genetics/isolation & purification/drug effects/pathogenicity/classification
Plasmids/genetics
*Genome, Bacterial/genetics
beta-Lactamases/genetics
Russia/epidemiology
*Klebsiella Infections/microbiology/epidemiology
Bacterial Proteins/genetics
Virulence Factors/genetics
Humans
Whole Genome Sequencing
Drug Resistance, Multiple, Bacterial/genetics
Anti-Bacterial Agents/pharmacology
Genomics
Virulence/genetics
CRISPR-Cas Systems

@article {pmid42283883,
year = {2026},
author = {Taheri, S and Azarpira, N and Mahmoodi, S and Gila, F and Dara, M},
title = {CRISPR/Cas9-mediated knockout of TopBP1 shifts the Bax/Bcl-2 balance toward apoptosis in MCF7 breast cancer cells.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42283883},
issn = {1573-4978},
mesh = {Humans ; *bcl-2-Associated X Protein/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics/metabolism ; Female ; Apoptosis/genetics ; *Breast Neoplasms/genetics/metabolism ; *Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; MCF-7 Cells ; *Nuclear Proteins/genetics/metabolism ; Gene Knockout Techniques/methods ; *Carrier Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Exons ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Breast cancer (BC) is the most prevalent cancer among women and the second leading cause of cancer-related deaths globally, after lung cancer. Despite advances in treatment, BC remains a major contributor to cancer mortality worldwide, underscoring the need for innovative therapeutic approaches. The TopBP1 (DNA Topoisomerase II Binding Protein 1) gene, involved in DNA damage response and cell cycle regulation, has been associated with cancer progression and resistance to chemotherapy. This study investigates the potential of using CRISPR/Cas9 technology to knockout the TopBP1 gene as a novel strategy in breast cancer research.

METHODS AND RESULTS: A pair of guide RNAs (gRNAs) was specifically designed to target the TopBP1 gene, inducing the deletion of exon 4. These gRNAs were transfected into the MCF7 breast cancer cell line, and the efficacy of genomic editing was validated using PCR and Sanger sequencing. Subsequent analyses employing real-time PCR and Western blotting were conducted to investigate the downstream effects of this genetic modification on gene expression. The CRISPR/Cas9 system successfully knocked out exon 4 of the TopBP1 gene in MCF7 breast cancer cells, as validated by PCR and Sanger sequencing. Real-time PCR analysis revealed a significant increase in Bax expression and a decrease in Bcl-2 expression in the knockout cells compared to controls. These changes indicate enhanced apoptotic activity following TopBP1 knockout, suggesting that MCF7 cells may become more sensitive to apoptosis. Overall, the findings support the hypothesis that targeting TopBP1 could play a critical role in promoting cell death in breast cancer, potentially offering a new therapeutic strategy.

CONCLUSIONS: This study successfully employed the CRISPR/Cas9 system to knockout exon 4 of the TopBP1 gene in MCF7 breast cancer cells, resulting in reduced TopBP1 expression. The subsequent increase in the pro-apoptotic Bax gene and decrease in the anti-apoptotic Bcl-2 gene suggest that targeting TopBP1 could enhance apoptosis in breast cancer cells, offering a promising alternative to conventional treatments. Further research is necessary to fully explore the therapeutic potential of this approach.},
}

Humans
*bcl-2-Associated X Protein/genetics/metabolism
CRISPR-Cas Systems/genetics
*DNA-Binding Proteins/genetics/metabolism
Female
Apoptosis/genetics
*Breast Neoplasms/genetics/metabolism
*Proto-Oncogene Proteins c-bcl-2/genetics/metabolism
MCF-7 Cells
*Nuclear Proteins/genetics/metabolism
Gene Knockout Techniques/methods
*Carrier Proteins/genetics/metabolism
Gene Expression Regulation, Neoplastic
Exons
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42285217,
year = {2026},
author = {Leon Agudelo, JA and Martínez Vallejo, H},
title = {Therapeutic applications of gene editing using CRISPR-Cas9 in the posterior segment: review of the literature.},
journal = {Archivos de la Sociedad Espanola de Oftalmologia},
volume = {},
number = {},
pages = {502582},
doi = {10.1016/j.oftale.2026.502582},
pmid = {42285217},
issn = {2173-5794},
abstract = {CRISPR-Cas gene editing has become increasingly relevant in the treatment of several ophthalmic diseases. Its ability to make precise modifications at the DNA or RNA level has enabled targeted approaches for specific mutations involved in conditions such as Leber congenital amaurosis type 10 (LCA10), certain forms of retinitis pigmentosa, and age-related macular degeneration. This article provides an organized overview of the biological basis of CRISPR-Cas technology and highlights key advances from preclinical studies and early clinical trials. Technical limitations and ongoing safety challenges are also discussed. Programs such as EDIT-101, EDIT-103, and HG202 stand out as important milestones in the evolution of ocular gene editing.},
}

@article {pmid42286276,
year = {2026},
author = {Wójcicki, M and Cieślik, M and Górski, A and Jończyk-Matysiak, E},
title = {Giving Antibiotics a Second Chance: Evolutionary Trade-Offs and Phage-Driven Restoration of Antibiotic Susceptibility.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42286276},
issn = {1179-190X},
abstract = {Antimicrobial resistance poses a critical and escalating threat to global public health, driven by the widespread and often unjustified use of antibiotics and the rapid dissemination of resistance determinants. With the antibiotic discovery pipeline largely depleted, alternative and complementary strategies are urgently needed to preserve the effectiveness of existing antimicrobials. Bacteriophages-viruses that specifically infect bacteria-have re-emerged as promising tools not only for direct bacterial eradication but also for reshaping bacterial evolutionary trajectories. This review examines the concept of phage-driven restoration of antibiotic susceptibility, focusing on evolutionary trade-offs that arise when bacteria adapt to phage pressure. Resistance to bacteriophages frequently involves modifications of surface structures, capsules, or efflux systems, changes that often incur fitness costs manifested as reduced virulence, impaired biofilm formation, or increased antibiotic sensitivity. Experimental studies and clinical case reports demonstrate that phage-antibiotic synergy can suppress bacterial growth more effectively than monotherapy, limit resistance emergence, and resensitize multidrug-resistant pathogens to previously ineffective antibiotics. Particular attention is given to mechanisms involving efflux pump targeting, capsule loss, biofilm disruption, and temperate phage-antibiotic interactions. In addition, emerging strategies that combine bacteriophages with CRISPR-Cas systems enable precise targeting and removal of resistance genes, offering a highly selective means to restore antibiotic efficacy and curb horizontal gene transfer. Together, these findings highlight bacteriophages as powerful evolutionary and therapeutic tools capable of giving antibiotics a "second chance". Integrating phage-based approaches into antibiotic stewardship frameworks may represent a sustainable path forward in combating multidrug-resistant bacterial infections.},
}

@article {pmid42287453,
year = {2026},
author = {Wong, MTJ and Zulkifli, ND and Ravichandran, T and Vasodavan, K and Al-Shaibah, O and Himel, GMS and Achuthan, A and Anthonysamy, MA and Theva Das, K},
title = {The evolution of AI-integrated genome editing and its challenges.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {37},
number = {1},
pages = {},
pmid = {42287453},
issn = {1432-1777},
mesh = {*Gene Editing/methods ; *Artificial Intelligence ; Humans ; Animals ; Machine Learning ; CRISPR-Cas Systems/genetics ; Genomics/methods ; },
abstract = {Artificial Intelligence (AI) is poised to revolutionize the field of genome editing by enhancing precision, efficiency, and accessibility. AI-driven approaches are already improving the design of CRISPR-based systems by enabling more accurate identification of target sequences and predicting off-target effects. Machine learning (ML) algorithms can analyze vast genomic data, as well as identify patterns and mutations that might be overlooked by traditional methods. Taking together, utilizing AI/ML tools allow for the enhancement of every step in genome editing. Recent advances also demonstrated that AI-powered tools can facilitate the simulation and modeling of genetic modifications, predicting their effects on cellular behavior and phenotypes. This allows for a more rapid prediction of the genome editing effects, without the need for wet lab. Additionally, AI can accelerate drug discovery and therapeutic development by streamlining the identification of genetic targets and optimizing gene therapies. The integration of AI with genome editing promises to democratize access to cutting-edge technologies, enabling researchers to design and plan for complex genetic modifications with minimal technical expertise. Drawing from various examples, this paper dives into the advancements and applications of AI in genome editing, its limitations, as well as future directions and opportunities in this field.},
}

*Gene Editing/methods
*Artificial Intelligence
Humans
Animals
Machine Learning
CRISPR-Cas Systems/genetics
Genomics/methods

@article {pmid40796977,
year = {2026},
author = {Naert, T and Yamamoto, T and Han, S and Röck, R and Horn, M and Bethge, P and Vladimirov, N and Voigt, FF and Figueiro-Silva, J and Bachmann-Gagescu, R and Vleminckx, K and Helmchen, F and Lienkamp, SS},
title = {Precise, predictable genome integrations by deep-learning-assisted design of microhomology-based templates.},
journal = {Nature biotechnology},
volume = {44},
number = {6},
pages = {1023-1036},
pmid = {40796977},
issn = {1546-1696},
support = {804474//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 310030_189102//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 1294725N//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; 3G0A6922//Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)/ ; },
mesh = {Humans ; Animals ; *Deep Learning ; Mice ; HEK293 Cells ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Xenopus ; Genome/genetics ; },
abstract = {Precise CRISPR-based DNA integration and editing remain challenging, largely because of insufficient control of the repair process. We find that repair at the genome-cargo interface is predictable by deep learning models and adheres to sequence-context-specific rules. On the basis of in silico predictions, we devised a strategy of base-pair tandem repeat repair arms matching microhomologies at double-strand breaks. These repeat homology arms promote frame-retentive cassette integration and reduce deletions both at the target site and within the transgene. We demonstrate precise integrations at 32 loci in HEK293T cells. Germline-transmissible transgene integration and endogenous protein tagging in Xenopus and adult mouse brains demonstrated precise integration during early embryonic cleavage and in nondividing, differentiated cells. Optimized repair arms also facilitated small edits for scarless single-nucleotide or double-nucleotide changes using oligonucleotide templates in vitro and in vivo. We provide the design tool Pythia to facilitate precise genomic integration and editing for experimental and therapeutic purposes for a wide range of target cell types and applications.},
}

Humans
Animals
*Deep Learning
Mice
HEK293 Cells
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Xenopus
Genome/genetics

@article {pmid41950758,
year = {2026},
author = {Han, L and Wang, SG and Teng, M and Zheng, LP and Ge, SY and Mao, Q and Han, F and Yang, L and Chai, SJ and Luo, Q and Yao, Y and Yu, ZH and Luo, J},
title = {Research note: A novel double-gene deleted vaccine against hypervirulent MDV variant generated by CRISPR/Cas9-based gene editing.},
journal = {Poultry science},
volume = {105},
number = {7},
pages = {106872},
pmid = {41950758},
issn = {1525-3171},
mesh = {Animals ; *Chickens ; CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Marek Disease/prevention & control/virology/immunology ; *Marek Disease Vaccines/immunology/genetics ; *Poultry Diseases/prevention & control/virology/immunology ; *Herpesvirus 2, Gallid/immunology/genetics ; Gene Deletion ; Virulence ; },
abstract = {Marek's disease (MD), an important avian immunosuppressive and neoplastic diseases, has resulted in huge economic losses for the poultry industry worldwide. Over the past 50 years, the long-term prevalence of this disease and the growing immune pressure of MD vaccination have triggered persistently increased virulence. The emerging hypervirulent variant of MDV (HV-MDV) overcomes the protection conferred by commercial vaccines, highlighting the urgent need to develop novel, highly efficient MD vaccines. To address this challenge, using CRISPR/Cas9-based gene editing, we generated a novel candidate vaccine, SQ01ΔmeqΔLAT, which features double deletions of both oncogene meq and LAT-clustered miRNAs. Comprehensive experiments confirmed the gene deletions, genetic stability and retention of the replication kinetics of the mutant, without revealing any adverse impact on the expression of essential viral genes. Further animal experiments showed no histopathological lesions or neoplastic changes in SQ01ΔmeqΔLAT-challenged birds, demonstrating favourable safety to chicken hosts. More importantly, the SQ01ΔmeqΔLAT vaccine achieved a high protection index (PI) of 88.9% against HV-MDV strain HNSQ01; this was an improvement compared to CVI988, which provided a PI value of only 70.6%. Our data provide an important basis for the development of highly efficient MD vaccines and shed new light on the design of herpesvirus vaccines, simultaneously targeting both viral protein-coding genes and non-coding RNAs.},
}

Animals
*Chickens
CRISPR-Cas Systems
*Gene Editing/veterinary
*Marek Disease/prevention & control/virology/immunology
*Marek Disease Vaccines/immunology/genetics
*Poultry Diseases/prevention & control/virology/immunology
*Herpesvirus 2, Gallid/immunology/genetics
Gene Deletion
Virulence

@article {pmid42035477,
year = {2026},
author = {Noronha, S and Liu, Y and Geneti, G and Li, H and Wu, X and Sun, D and Gujar, V and Furusawa, T and Lobanov, A and Cam, M and Pal, LR and Nair, NU and Day, CP and Ruppin, E and Ghosh, C and Hu, J and Ramamoorthy, B and Kumar, S and Andresson, T and Chan, K and O'Neill, M and Chari, R and Pommier, Y and Rivero, JD and Weyemi, U and Kebebew, E and Boufraqech, M},
title = {CRISPR-Based Gene Dependency Screens Reveal Mechanism of BRAF Inhibitor Resistance in Anaplastic Thyroid Cancer.},
journal = {Molecular carcinogenesis},
volume = {65},
number = {7},
pages = {874-887},
pmid = {42035477},
issn = {1098-2744},
mesh = {Humans ; *Proto-Oncogene Proteins B-raf/antagonists & inhibitors/genetics ; *Thyroid Carcinoma, Anaplastic/genetics/drug therapy/pathology ; *Drug Resistance, Neoplasm/genetics ; *Thyroid Neoplasms/genetics/drug therapy/pathology ; Cell Line, Tumor ; *Protein Kinase Inhibitors/pharmacology ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; Imidazoles/pharmacology ; Oximes/pharmacology ; Unfolded Protein Response/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; },
abstract = {Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors. Using gene essentiality score across cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.},
}

Humans
*Proto-Oncogene Proteins B-raf/antagonists & inhibitors/genetics
*Thyroid Carcinoma, Anaplastic/genetics/drug therapy/pathology
*Drug Resistance, Neoplasm/genetics
*Thyroid Neoplasms/genetics/drug therapy/pathology
Cell Line, Tumor
*Protein Kinase Inhibitors/pharmacology
Transcriptional Coactivator with PDZ-Binding Motif Proteins
Imidazoles/pharmacology
Oximes/pharmacology
Unfolded Protein Response/drug effects
Gene Expression Regulation, Neoplastic/drug effects
CRISPR-Cas Systems

@article {pmid42093418,
year = {2026},
author = {Cho, HM and Ryu, YC and Cho, MK and Hwang, BH},
title = {Functional peptide-assisted delivery of CRISPR/Cas9: recent progress on gene editing.},
journal = {Biomaterials science},
volume = {14},
number = {12},
pages = {3132-3156},
doi = {10.1039/d6bm00231e},
pmid = {42093418},
issn = {2047-4849},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Peptides/chemistry/metabolism ; Animals ; *Gene Transfer Techniques ; *Cell-Penetrating Peptides/chemistry ; },
abstract = {The CRISPR/Cas9 system has transformed the field of genome engineering by enabling precise and programmable modification of genomic sequences. Despite its remarkable potential, efficient and safe delivery of CRISPR/Cas9 components remains a significant bottleneck, particularly in translational and therapeutic applications. In the past decade (2015-2025), peptide-based delivery systems have gained increasing attention as versatile non-viral alternatives due to their modular design, inherent biocompatibility and potential for tissue-specific targeting. This review provides a critical synthesis of recent advances in functional peptide-based delivery strategies for CRISPR/Cas9. We focus on key categories of peptides-cell-penetrating peptides, cell-targeting peptides, endosomal escape peptides, and nuclear localization signals-and their multifunctional or hybrid combinations. By identifying key design principles and failure modes, this review discusses how these platforms can be optimized to support the development of next-generation CRISPR therapeutics, ultimately moving closer to safe and effective genome editing in future clinical applications.},
}

*CRISPR-Cas Systems/genetics
Humans
*Peptides/chemistry/metabolism
Animals
*Gene Transfer Techniques
*Cell-Penetrating Peptides/chemistry

@article {pmid42186844,
year = {2026},
author = {Zhao, Y and Sun, H and Wan, Y and Zhang, Z and Xiao, L and Han, T and Hao, Z and Li, N and Pang, C and Zhang, W and Wan, J},
title = {Research on a highly sensitive aptamer sensor for Vibrio alginolyticus based on CRISPR-Cas13a and T7 transcription cascade amplification.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {22},
pages = {4701-4711},
doi = {10.1039/d6ay00035e},
pmid = {42186844},
issn = {1759-9679},
mesh = {*Vibrio alginolyticus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {Vibrio alginolyticus is a prevalent aquatic pathogen that poses significant risks to public health. Thus, the development of rapid and highly sensitive detection methods is imperative. In this study, we developed a novel aptasensor leveraging the CRISPR/Cas13a system. By integrating a triple-amplification strategy comprising "aptamer competition recognition-T7 transcription amplification-Cas13a trans-cleavage", this platform enables efficient and specific detection of V. alginolyticus. The biosensor employs a V. alginolyticus-specific aptamer (Apt) as the recognition element. In the presence of the target bacterium, Apt binds to a surface membrane protein, resulting in the release of a blocking strand (Block). This triggers a conformational change in a hairpin probe (HP), thereby exposing the T7 promoter sequence. Subsequently, T7 RNA polymerase initiates an isothermal transcription reaction, producing abundant RNA products. These RNAs activate the trans-cleavage activity of Cas13a, which cleaves a fluorescent reporter probe to generate a quantifiable signal. This method eliminates the need for nucleic acid extraction and sophisticated instrumentation. It achieves a detection limit as low as 2 CFU mL[-1], and demonstrates high specificity by effectively distinguishing closely related species (e.g., Vibrio parahaemolyticus). When applied to simulated seawater and seafood samples, the recovery rates ranged from 94.61% to 106.56%, indicating robust anti-interference capacity and reproducibility. This work establishes a highly sensitive and specific biosensing technology for the on-site rapid detection of aquatic pathogens, offering promising applications in environmental monitoring and food safety.},
}

*Vibrio alginolyticus/isolation & purification/genetics
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry/genetics
*CRISPR-Cas Systems/genetics
Transcription, Genetic
*Nucleic Acid Amplification Techniques/methods
Limit of Detection

@article {pmid42231680,
year = {2026},
author = {Wang, L and Zhao, XY and Tang, H and Chen, TT and Chu, X},
title = {An Exosome RNA In Situ Detection Platform Based on a Regulated CRISPR/Cas12a Activity System and Its Application in Tumor Progression Monitoring and Therapeutic Efficacy Evaluation.},
journal = {Analytical chemistry},
volume = {98},
number = {23},
pages = {17385-17394},
doi = {10.1021/acs.analchem.6c02295},
pmid = {42231680},
issn = {1520-6882},
mesh = {*Exosomes/metabolism/genetics ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; Disease Progression ; Liposomes/chemistry ; Cell Line, Tumor ; *RNA/analysis ; Aptamers, Nucleotide/chemistry ; Biomarkers, Tumor ; },
abstract = {Tumor-derived exosomes, serving as promising biomarkers, hold substantial potential for reflecting the progression of diseases and assessing the efficacy of antitumor therapies. In this study, we developed a liposome-based platform capable of both precise recognition of tumor-derived exosomes and highly sensitive in situ analysis of the internal RNA. Leveraging surface-anchored DNA tags and their hybridization with two allosteric aptamers targeting exosomal marker CD63 and tumor marker PD-L1, the platform can specifically capture tumor-derived exosomes and facilitate the membrane fusion. Upon mixing of the vesicular contents, an internal functional duplex converter undergoes strand displacement with the target mRNA, releasing an uncaged strand. This strand eliminates the inhibition of the Cas12a/crRNA assembly imposed by an elongation-caged activator, thereby restoring the transcleavage activity of Cas12a. The activated Cas12a cleaves reporter probes to generate a fluorescent signal, enabling highly sensitive in situ detection of tumor-derived exosomal RNA. The constructed platform enables dynamic monitoring of disease progression in tumor-bearing mice by quantifying the relative levels of tumor-derived exosomes in serum and further distinguishes therapeutic outcomes among different drug treatments. This study highlights the significant potential of the proposed platform in tumor diagnosis and the evaluation of antitumor therapeutic efficacy.},
}

*Exosomes/metabolism/genetics
Animals
Humans
Mice
*CRISPR-Cas Systems/genetics
Disease Progression
Liposomes/chemistry
Cell Line, Tumor
*RNA/analysis
Aptamers, Nucleotide/chemistry
Biomarkers, Tumor

@article {pmid42276707,
year = {2026},
author = {Tonietto, M and Jäger, V and Maitz, K and Flasch, B and Reinisch, A and Kargl, J and Dengler, MA and Jost, PJ},
title = {Murine models of lung cancer as a platform to investigate cell death.},
journal = {Methods in cell biology},
volume = {208},
number = {},
pages = {165-202},
doi = {10.1016/bs.mcb.2026.02.009},
pmid = {42276707},
issn = {0091-679X},
mesh = {Animals ; *Lung Neoplasms/pathology/genetics/immunology ; Mice ; Disease Models, Animal ; *Cell Death/genetics ; Tumor Microenvironment/genetics ; Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems/genetics ; Humans ; Apoptosis/genetics ; Gene Editing ; Proto-Oncogene Proteins p21(ras)/genetics ; },
abstract = {Alterations in programmed cell death pathways play a critical role in cancer development and maintenance. Yet the detailed mechanisms contributing to tumor initiation, progression, and therapeutic response in lung cancer remain incompletely understood. Also, models to study how changes in the cell death machinery impact tumor-immune interactions are limited. To address this, we describe two complementary murine models of lung adenocarcinoma that enable functional interrogation of cell death pathways in vivo. The first model is a clinically relevant, genetically engineered mouse model (GEMM) driven by Kras[G12D] activation and Tp53 loss, in which somatic CRISPR-Cas9-mediated gene editing permits tumor cell-specific knockout of candidate genes, facilitates in-depth studies of programmed cell death and immune signaling within 19 weeks. The second approach illustrates how a syngeneic orthotopic transplantation model can be used to study target genes and pathways that influence the tumor microenvironment and immunogenic cell death in a two-week timeframe. Together, these methods provide reproducible and versatile tools to investigate how modulation of cell death pathways impacts lung cancer development and progression and affects the tumor immune microenvironment, thus providing important information to guide the development of novel therapeutic strategies in lung cancer.},
}

Animals
*Lung Neoplasms/pathology/genetics/immunology
Mice
Disease Models, Animal
*Cell Death/genetics
Tumor Microenvironment/genetics
Tumor Suppressor Protein p53/genetics
CRISPR-Cas Systems/genetics
Humans
Apoptosis/genetics
Gene Editing
Proto-Oncogene Proteins p21(ras)/genetics

@article {pmid42277013,
year = {2026},
author = {Wang, Q and Gong, Y and Wang, L and Lv, N and Du, X and Zhang, J and Xin, Y and Nikoloski, Z and Li-Beisson, Y and Sun, L and Ma, B and Wang, X and Xu, J},
title = {High-throughput Raman-activated cell sorting of microalgal genome-wide edited library revealed a regulatory pathway for carotenoid synthesis.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74304-5},
pmid = {42277013},
issn = {2041-1723},
support = {32370097//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Functional genomics have been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated cell sorting (RACS). The CRISPR/Cas-generated library consisted of 3567 microalgal mutants derived from 2397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unraveled mutations in the violaxanthin de-epoxidase (noVDE) or in the proteasome assembly chaperone 4 (noPAC4) genes. Knocking out all five known noVDEs revealed that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidence suggested two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this forward-genetics approach can greatly accelerate the discovery of genes and pathways.},
}

@article {pmid42277643,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M},
title = {Evolutionary interplay: virulence, endolysin-like hydrolases, and defense correlations in the Erwinia amylovora pangenome.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-026-05295-y},
pmid = {42277643},
issn = {1471-2180},
abstract = {Erwinia amylovora, the causative agent of fire blight, poses a significant threat to global pome fruit production. This study presents a comprehensive genomic analysis of 317 E. amylovora strains and 227 Erwinia phages to elucidate virulence evolution, phage-host dynamics, and the genomic signatures of the co-evolutionary arms race. Our analysis suggests that a substantial portion of E. amylovora's virulence factors (VFs) share evolutionary origins with diverse plant, human, and animal pathogens, underscoring widespread horizontal gene transfer. We identified bacterial phage hydrolases‑like proteins that share phylogenetic and domain-level similarities with phage endolysins. These observations are consistent with the possibility that some bacterial hydrolases originated from phage-derived ancestors, although functional repurposing remains to be experimentally validated. Crucially, our analysis identifies systematic, non-random associations between bacterial defense systems (e.g., RM, CRISPR-Cas, TA) and mobile anti-defense genes. Statistical correlations show strong patterns of co-occurrence and mutual exclusivity, which are consistent with an ongoing phage-bacteria arms race. These patterns provide a genomic basis for generating hypotheses about co-evolutionary dynamics. These findings may advance our understanding of E. amylovora pathogenicity and phage interactions, offering foundational insights for developing targeted phage-based biocontrol strategies against this devastating plant pathogen. Experimental validation of the predicted virulence factors and defense correlations is warranted to confirm their biological roles.},
}

@article {pmid42277912,
year = {2026},
author = {Chang, L and Xu, W and Wang, X and Xue, Y and Zhang, Y and Zhu, X and Zhang, Y and Liang, T and Liu, W},
title = {CRISPR diagnostics: from trans-nuclease activity to cancer diagnosis.},
journal = {Cell & bioscience},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13578-026-01603-1},
pmid = {42277912},
issn = {2045-3701},
support = {2019YFC1316000//National Key Research and Development Program of China/ ; 81830089//National Natural Science Foundation of China/ ; 82188102//National Natural Science Foundation of China/ ; LQ23H200004//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {The field of nucleic acid-based testing experienced a decade-long stagnation since the development of quantitative polymerase chain reaction (qPCR) in 1992 and isothermal amplification methods in the early 2000s. However, in 2016, the discovery of trans-nuclease activity in CRISPR-Cas systems revolutionized the molecular diagnostics for nucleic acids. A typical CRISPR diagnostic workflow comprises three phases: (1) target recognition through CRISPR RNA (crRNA)-guided hybridization; (2) signal transduction via trans-cleavage of engineered reporters (e.g., fluorophore-quencher oligonucleotides), and (3) signal readout using fluorescence, electrochemical, or colorimetric platforms. Emerging shortly prior to the COVID-19 pandemic, CRISPR diagnostics quickly gained prominence as a field-deployable alternative to qPCR due to its rapidity (< 1 h), minimal equipment requirements, and field adaptability. This technological paradigm underwent rigorous validation and refinement alongside the rapid evolution of SARS-CoV-2 detection, which facilitated its adaptation for cancer diagnosis. Recent advancements in sensitivity (attomolar-level detection) and specificity (single-nucleotide discrimination) have enabled transformative applications in cancer diagnostics, including: (1) identification of nucleic acid biomarkers, such as high-frequency somatic mutations, circulating nucleic acids and miRNAs; and (2) detection of non-nucleic acid biomarkers, including epigenetic aberrations, proteins, small molecules and metabolite biomarkers. This review chronicles the decadal evolution of CRISPR diagnostics, with particular emphasis on recent advancements of its application in cancer diagnosis. We critically evaluate persistent technical limitations, including PAM sequence restriction, suboptimal sensitivity and specificity, quantitative constraints, and unmet point-of-care testing (POCT) in complex biological matrices. Additionally, we discuss prospective solutions to address these challenges.},
}

@article {pmid42278292,
year = {2026},
author = {Tuerxun, K and Ding, Z and Luo, X and Zhou, S},
title = {Fermentation Process Optimization for High 2-Phenylethanol Aroma Whisky.},
journal = {International journal of molecular sciences},
volume = {27},
number = {11},
pages = {},
pmid = {42278292},
issn = {1422-0067},
support = {51878291//National Natural Science Foundation of China/ ; },
mesh = {*Fermentation ; *Saccharomyces cerevisiae/genetics/metabolism ; *Phenylethyl Alcohol/metabolism/analysis ; *Wine/analysis ; CRISPR-Cas Systems ; *Odorants/analysis ; Transaminases/genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {2-Phenylethanol (2-PE) is a key aromatic alcohol contributing to the rose-like odor in brewed wines, primarily synthesized by yeast metabolism with a typical yield of less than 100 mg/L. To enhance the 2-PE content in brewed wines, this study used CRISPR-Cas9 gene editing technology to delete the ARO8 gene (encoding aromatic transaminase I) in Saccharomyces cerevisiae SY. The single-factor experiments were performed to optimize the fermentation process, and the 2-PE content in the brewed wine was measured by high-performance liquid chromatography. The results demonstrated that the 2-PE content in whisky fermented by the SY-A8 was 0.73 g/L, increasing 23.73% compared to SY. The fermentation conditions of SY-A8 were optimized through single-factor experiments and the Box-Behnken design. The optimal conditions were a sugar concentration of 46.30 g/L, a fermentation time of 6 days, and an L-phenylalanine concentration of 1.43 g/L. The high 2-phenylethanol aroma whisky was brewed with a higher 2-phenylethanol content of 3.68 g/L in a 1 L fermenter at the optimal conditions. In conclusion, the modification of Saccharomyces cerevisiae by CRISPR-Cas9 gene editing combined with fermentation process optimization provides an effective technical strategy for improving the 2-PE content in whisky, thereby providing a research perspective for the flavor enhancement of whisky and other brewed wines.},
}

*Fermentation
*Saccharomyces cerevisiae/genetics/metabolism
*Phenylethyl Alcohol/metabolism/analysis
*Wine/analysis
CRISPR-Cas Systems
*Odorants/analysis
Transaminases/genetics/metabolism
Saccharomyces cerevisiae Proteins/genetics/metabolism

@article {pmid42278622,
year = {2026},
author = {Zhao, YY and Evans, CE},
title = {Contemporary Endothelial Genome Editing Technologies: Towards Precision Genetic Medicine for Vascular Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {11},
pages = {},
pmid = {42278622},
issn = {1422-0067},
support = {1R01HL133951-21/NH/NIH HHS/United States ; 2R01HL164014-22/NH/NIH HHS/United States ; 3R01HL162299-22/NH/NIH HHS/United States ; 4R01HL172447-23/NH/NIH HHS/United States ; 24TPA1285575//American Heart Association/ ; 23SCEFIA1155876//American Heart Association/ ; 5P20GM103499-23/NH/NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Vascular Diseases/genetics/therapy ; *Endothelial Cells/metabolism ; Animals ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Endothelium, Vascular/metabolism ; },
abstract = {Endothelial dysfunction is a key characteristic of many diseases, including atherosclerosis, hypertension, heart failure, stroke, cancer, acute respiratory distress syndrome (ARDS), peripheral vascular disease, coronavirus 2019 (COVID-19), and pulmonary arterial hypertension (PAH). To improve understanding of the roles of endothelial cells (ECs) in health and disease, EC-specific genome editing technologies have been developed in recent years. Therapeutic strategies that aim to restore a healthy endothelial monolayer include the inhibition of endothelial genes that cause EC injury and dysfunction and the induction or activation of endothelial genes that drive EC repair and regeneration. In this review, we describe established recombinase-mediated genetic modification technologies and emerging EC-specific genome editing technologies including viral and non-viral delivery of the CRISPR/Cas9 genome editing system, and we summarize the strengths and limitations of each technology. We then discuss possible avenues for future research, including the development of organ-specific EC genome editing technologies. In short, EC-specific genome editing technologies can be used to modulate gene expression selectively in ECs and even within a specific vascular bed and/or distinctive EC subtype, and, in doing so, greatly improve the understanding of vascular biology and help develop precision genetic medicine targeting the disease-causing vascular bed(s) to effectively treat diseases caused by vascular endothelial dysfunction.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems
*Vascular Diseases/genetics/therapy
*Endothelial Cells/metabolism
Animals
*Precision Medicine/methods
*Genetic Therapy/methods
Endothelium, Vascular/metabolism

@article {pmid40098415,
year = {2026},
author = {Guo, Y and Ge, T and Wang, Q and Liu, TX and Li, Z},
title = {An RPA-CRISPR/Cas12a based platform for rapid, sensitive, and visual detection of Apis mellifera filamentous virus.},
journal = {Insect science},
volume = {33},
number = {3},
pages = {746-758},
doi = {10.1111/1744-7917.70024},
pmid = {40098415},
issn = {1744-7917},
support = {2022JQ-154//Natural Science Basic Research Program of Shaanxi Province/ ; 2452021101//Ministry of Education of the People's Republic of China Chinese Universities Scientific Fund/ ; NWAFU//Ministry of Education of the People's Republic of China Chinese Universities Scientific Fund/ ; },
mesh = {Bees/virology ; Animals ; *CRISPR-Cas Systems ; *DNA Viruses/isolation & purification/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Recombinases ; *Insect Viruses/isolation & purification/genetics ; },
abstract = {Apis mellifera filamentous virus (AmFV) is an emerging DNA virus significantly affecting honey bee health. AmFV infections weaken bee resistance to other pathogens, and can cause tissue lysis and death. Early, accurate detection of AmFV is crucial for timely intervention and preventing large-scale outbreaks. Current AmFV detection relies largely on polymerase chain reaction (PCR)-based methods. To enable rapid field detection of AmFV, we developed a rapid and ultrasensitive detection platform using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 12a (Cas12a) technology. A CRISPR RNA (crRNA1) specifically targeting the AmFV Bro gene was designed, ensuring no cross-reactivity with other insect DNA viruses or uninfected honey bees. After optimization of the reaction time, the platform generated results within 35 min: 20 min for the RPA reaction and 15 min for CRISPR-mediated cleavage. Two visualization approaches, fluorescence-based and lateral flow dipstick, were used to display the detection results. The detection sensitivity of both approaches was as few as 10 copies of the AmFV genome. Validation with field-collected honey bee samples demonstrated consistency with conventional PCR, revealing widespread latent AmFV infections in the field. Taken together, we successfully developed an RPA-CRISPR/Cas12 platform for rapid, specific, and sensitive detection of AmFV in Apis mellifera and Apis cerana. This platform holds promise as a simple, accurate, and cost-effective tool for point-of-care AmFV diagnosis in the field.},
}

Bees/virology
Animals
*CRISPR-Cas Systems
*DNA Viruses/isolation & purification/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Recombinases
*Insect Viruses/isolation & purification/genetics

@article {pmid41238927,
year = {2025},
author = {Gundogdu, M and Islek, Z},
title = {Genetic Manipulation Tools in Leishmania: From CRISPR/Cas9 to Vaccine Strategies for Disease Control.},
journal = {Acta parasitologica},
volume = {70},
number = {6},
pages = {217},
pmid = {41238927},
issn = {1896-1851},
abstract = {PURPOSE: Gene editing technologies have emerged as a crucial approach totackling parasitic infections. Recent research underscores the potential of protozoan parasites, including Leishmania, to utilize gene editing strategies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins)(CRISPR/Cas9) to edit parasite genomes, modify virulence factors, and alter host immune responses. This review aims to explore the potential of CRISPR/Cas9 in the study of Leishmania and Leishmaniasis, focusing on its applications in understanding mechanisms and developing novel editing strategies. METHODS: The immunological response of the mammalian host and the nature of the Leishmania parasites play essential roles in the formation and progression of parasitic diseases. Among alternative treatment strategies, CRISPR/Cas9 has attracted attention as a promising tool for introducing protective or therapeutic mutations in Leishmaniasis. This review will provide recent insights into the role of gene editing, especially CRISPR/Cas9, in host-pathogen interactions, intercellular communication, immunomodulation, and pathogenesis of Leishmania. RESULTS: Current findings reveal that CRISPR/Cas9 enables efficient modification of Leishmania genomes, providing valuable insights into parasite biology, host-pathogen interactions, intercellular communication, immunomodulation, and disease pathogenesis. Additionally, immunotherapeutic models are being investigated to explore the potential applications of CRISPR/Cas9 in theranostics. CONCLUSION: CRISPR/Cas9 has attracted attention as a promising tool for introducing protective or therapeutic mutations in Leishmaniasis. Integrating CRISPR/Cas9 with Leishmania-related research opens new avenues for disease control and understanding, while biologically inspired immunomodulatory strategies highlight its emerging role in next-generation parasite-targeted immunotherapy.},
}

@article {pmid41269653,
year = {2026},
author = {Islam, MM and Han, K and Woo, K and Shin, WS and Oh, MH},
title = {Comprehensive in silico analysis of Acinetobacter isolates from South Korea reveals genomic diversity, antimicrobial resistance, virulence factors, and evolutionary dynamics.},
journal = {Genes & genomics},
volume = {48},
number = {1},
pages = {147-162},
pmid = {41269653},
issn = {2092-9293},
support = {2022R1F1A1071415 and NRF-RS-2023-00275307//Ministry of Education/ ; },
abstract = {BACKGROUND: Multidrug-resistant Acinetobacter strains pose a significant threat to healthcare systems and have become a growing concern. A state-of-the-art understanding of this formidable pathogen—including its antibacterial resistance profile, virulence factors, genomic diversity, and evolutionary dynamics—is crucial for implementing effective responses during a pandemic. OBJECTIVE: To explore the genomic diversity, antimicrobial resistance (AMR) profile, virulence factors, and evolutionary trends of multidrug-resistant Acinetobacter isolates collected in South Korea from 2010 to 2022, we conducted a comprehensive in silico analysis of 74 complete genome sequences. METHODS: 74 Acinetobacter complete genomes were annotated using Prokka (v1.14.6), and a pangenome was constructed using Roary (v3.13). The genomes were systematically analyzed for antibiotic resistance genes (ARGs), virulence factor genes (VFGs), mobile genetic elements (MGEs), CRISPR/cas, and prophage using various in silico tools. Multi-locus sequence typing (MLST) was conducted using the Pasteur scheme via the MLST 2.0 web server. To assess genetic diversity, eBURST and whole-genome SNP-based phylogenetic analyses were employed. We performed a targeted analysis of carbapenem-resistant Acinetobacter strains, investigating the relationship between prophages and CRISPR/Cas systems. RESULT: Pan-genome analysis showed an open genome structure (α = 0.4921) in Acinetobacter, indicating ongoing genetic evolution. A total of 77 unique resistance genes, linked to six resistance mechanisms and 21 drug classes, were identified, including blaOXA-23, efflux pumps (AdeIJK, AdeFGH), and carbapenemases. Additionally, 68 virulence factors associated with adherence, biofilm formation, iron uptake, immune evasion, and serum resistance etc. were prevalent. Mobile genetic elements, such as Tn6207, Tn6209, Tn2006, Tn2008, and ISAba1/blaOXA-23 combinations, were identified, suggesting mechanisms for the spread of resistance genes. Seventeen types of prophage were identified, and a low prevalence of CRISPR sequence implies susceptibility to phage predation. The ST2 genotype was dominant, and SNP-based phylogeny showed significant genomic diversity. CONCLUSION: This study provides a comprehensive understanding of the resistance, virulence, and mobile genetic element profile of Acinetobacter isolates from South Korea, laying the groundwork for future antimicrobial resistance research and intervention strategies, especially the bacteriophage treatment.},
}

@article {pmid41606801,
year = {2026},
author = {Mashreghi, M and Rezazade Bazaz, M and Jaafari, MR},
title = {CRISPR-Cas9 lipid nanoparticles for targeting cyclin-dependent kinases in the tumor microenvironment.},
journal = {Biotechnology progress},
volume = {42},
number = {3},
pages = {e70105},
doi = {10.1002/btpr.70105},
pmid = {41606801},
issn = {1520-6033},
support = {996134//National Institute for Medical Research Development/ ; },
mesh = {*Nanoparticles/chemistry ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Tumor Microenvironment/drug effects/genetics ; *Lipids/chemistry ; *Cyclin-Dependent Kinase 4/genetics/antagonists & inhibitors/metabolism ; Cell Line, Tumor ; *Cyclin-Dependent Kinases/metabolism/genetics/antagonists & inhibitors ; Mice ; Apoptosis/drug effects ; Gene Therapy Agents ; Liposomes ; },
abstract = {Due to the significant roles of cyclin-dependent kinases 4 and 6 (CDK4/6) in cancer progression, this study aimed to introduce clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) plasmid DNA (pDNA) encapsulated in lipid nanoparticles (LNPs) as a novel CDK4/6 inhibitor using a gene knock-out strategy for treating cancer. pDNA-LNP was prepared and characterized using a microfluidic system. The results indicated the hydrodynamic diameter of the pDNA-LNP was 90.0 ± 0.1 nm with the PDI of 0.1 and a negative zeta-potential. The cytotoxicity results demonstrated statistically significant differences at doses of 0.250, 500, and 1 μg of pDNA with the capabilities of pDNA-LNP in the induction of apoptosis, as depicted by the Annexin-V-FITC-PI method. Real-time quantitative PCR (qPCR) also indicated a significant reduction in the expression levels of both CDK4 and 6 in the cells that were treated with pDNA-LNP. The in vivo anti-tumor activities of pDNA-LNP have demonstrated that the formulation has the potential to decrease tumor size and improve survival parameters, including median survival time (MST), which was increased from 31 days for the PBS group to 51 days for the pDNA-LNP group at 0.5 μg. On the other hand, the dose of 1 μg had shown signs of toxicity, indicating the need to optimize dosing in future studies. In summary, these findings indicate that CRISPR-Cas9 encapsulated in the LNP can suppress tumor growth and offer a promising strategy for future cancer treatment approaches.},
}

*Nanoparticles/chemistry
Animals
Humans
*CRISPR-Cas Systems/genetics
*Tumor Microenvironment/drug effects/genetics
*Lipids/chemistry
*Cyclin-Dependent Kinase 4/genetics/antagonists & inhibitors/metabolism
Cell Line, Tumor
*Cyclin-Dependent Kinases/metabolism/genetics/antagonists & inhibitors
Mice
Apoptosis/drug effects
Gene Therapy Agents
Liposomes

@article {pmid41762232,
year = {2026},
author = {Deepika, and Sharma, S and Kumar, P and Rathore, V and Chauhan, A and Kumar, A and Dogra, RK and Handa, A},
title = {Insights into physiological, biochemical and molecular mechanisms of abiotic stress tolerance in Persian walnut (Juglans regia L.).},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {41762232},
issn = {1615-6102},
abstract = {Walnut (Juglans regia) is an economically and nutritionally valuable tree species that often encounters diverse abiotic stresses such as drought, salinity, cold, heat and heavy metal toxicity. These stresses adversely affect its growth, productivity and survival by altering physiological functions, disturbing cellular homeostasis and triggering oxidative damage. In response, J. regia deploy a multifaceted adaptive system comprising morphological changes, biochemical adjustments and intricate molecular signaling pathways. The review aims to analyze current knowledge on the physiological, morphological changes observed under stress with biochemical defense mechanism. These include the crucial antioxidant defense system (increase in Superoxide Dismutase (SOD), Peroxidase (POD) and Catalase (CAT) activity), the accumulation of protective soluble solutes and amino acids and the biosynthesis of secondary metabolites through the Methylerythritol Phosphate (MEP) pathway involved in mitigating oxidative stress caused by Reactive Oxygen Species (ROS). Crucially, we synthesize the understanding of molecular regulation that underpins stress adaptation. This encompasses stress-responsive gene expression including Jr (Juglans regia) VHAG1 (V-ATPase H+ transporting ATPase subunit G1), JrDREB (Dehydration-Responsive Element Binding protein), JrRD29 (Responsive to Dehydration 29), transcriptional regulation by myeloblastosis (MYB), Dehydration-responsive element-binding proteins (DREB) and WRKY-TF families and their interaction with hormonal (abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene) which play a pivotal role in signal transduction and stress tolerance. These mechanisms employed by walnut under abiotic stresses, highlighting key genetic and hormonal pathways that can be targeted for the development of stress-resilient cultivars and ensuring sustainable production under changing climatic conditions. Recent advancements in genomics, transcriptomics and proteomics got attention that provides new insights into the regulatory networks and candidate genes conferring stress resilience. Furthermore, it explores biotechnological approaches for improving stress tolerance, highlighting the prospects of latest high-throughput techniques, including molecular breeding, genetic engineering, Next-Generation Sequencing (NGS), microRNA (miRNA)-based regulation and Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas) gene editing. This integrated review connects multi-level stress response mechanisms, highlighting potential molecular markers and biotechnological interventions for accelerating walnut improvement and ensuring sustainable production under changing climatic conditions.},
}

@article {pmid41794282,
year = {2026},
author = {Xue, Z and Lan, J and Zhao, Y and Yu, P and Liu, L and Lu, B and Yang, F},
title = {A novel rat model harboring two BDNF gene mutations exhibiting autism-like behaviors and cognitive impairments.},
journal = {Neuropharmacology},
volume = {291},
number = {},
pages = {110911},
doi = {10.1016/j.neuropharm.2026.110911},
pmid = {41794282},
issn = {1873-7064},
mesh = {Animals ; *Brain-Derived Neurotrophic Factor/genetics/metabolism ; *Disease Models, Animal ; Male ; Rats ; Social Behavior ; Receptor, trkB/metabolism/genetics ; *Autism Spectrum Disorder/genetics ; Hippocampus/metabolism ; Mutation ; Cognition Disorders/genetics ; *Autistic Disorder/genetics ; Rats, Sprague-Dawley ; Flavones/pharmacology ; Prefrontal Cortex/metabolism ; CRISPR-Cas Systems ; },
abstract = {Autism spectrum disorder (ASD) is a type of neurodevelopmental disorder that occurs most frequently in early childhood, affecting approximately 1% of the global population. Currently, the elusive nature of the pathological mechanisms underlying ASD precludes the existence of a definitive, effective treatment approach. In this study, we have successfully generated a novel ASD rat model utilizing CRISPR/Cas9 technology, offering a promising platform for further investigation and potential therapeutic interventions. The model is characterized by two crucial point mutations occurring at key enzyme cleavage sites of brain-derived neurotrophic factor (BDNF), thereby causing disruptions in enzyme cleavage processes. The phenotypes of this rat model faithfully recapitulate the salient deficits frequently encountered in ASD patients, exhibiting impairments in social behavior, cognition, and anxiety, along with neuronal abnormalities with key brain regions, notably the hippocampus (HPC) and medial prefrontal cortex (mPFC). Through preliminary RNA-seq analysis, we found changes in gene expression patterns related to synapses and neuronal excitability in these areas, providing new insights into the pathogenesis of ASD. Furthermore, our utilization of 7,8-dihydroxyflavone (7,8-DHF), a robust enhancer for the upregulation of both BDNF and TrkB mRNA and simultaneously activates the BDNF-TrkB signaling pathway, appears to strengthen the BDNF-TrkB signaling cascade. This intervention modifies firing patterns of neuronal spikes and synaptic transmission, which may contribute to the amelioration of ASD-like social interaction behavior exhibited in BDNF[met/leu] rats. Our research not only deepens our understanding of the pathogenesis of ASD, but also present encouraging avenue for early intervention strategies and treatments.},
}

Animals
*Brain-Derived Neurotrophic Factor/genetics/metabolism
*Disease Models, Animal
Male
Rats
Social Behavior
Receptor, trkB/metabolism/genetics
*Autism Spectrum Disorder/genetics
Hippocampus/metabolism
Mutation
Cognition Disorders/genetics
*Autistic Disorder/genetics
Rats, Sprague-Dawley
Flavones/pharmacology
Prefrontal Cortex/metabolism
CRISPR-Cas Systems

@article {pmid41814945,
year = {2026},
author = {Lee, S and Kim, S and Chong, J and Kim, MG and Lim, K and Lee, NE and Koo, J and Kang, J and Lee, H},
title = {A Cryoprotectant-Compatible Nanoporous Platform for Stable and Scalable Delivery of Biopharmaceuticals.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {33},
pages = {e10532},
pmid = {41814945},
issn = {1521-4095},
support = {RS-2022-NR072331//National Research Foundation of Korea/ ; RS-2024-00403376//National Research Foundation of Korea/ ; RS-2023-KH135060//Korea Health Industry Development Institute, Ministry of Health & Welfare, Republic of Korea/ ; RS-2024-00438476//Korea Health Industry Development Institute, Ministry of Health & Welfare, Republic of Korea/ ; //KIST Institutional Program/ ; },
mesh = {*Cryoprotective Agents/chemistry ; Animals ; Humans ; Freeze Drying ; *Nanopores ; Gene Editing ; Ribonucleoproteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 ribonucleoproteins (RNPs) represent a promising class of biopharmaceuticals for treating genetic and complex diseases. However, their clinical translation is limited by instability during storage and delivery. Lyophilization offers a potential solution, though conventional approaches often compromise structural integrity and bioactivity under non-cryogenic conditions. Here, we have developed a nanostructured delivery platform, designated Nano Banker & Blowball (NB[2]), which features a blowball-like architecture and tunable nanoscale pores. These pores are designed to protect RNPs and enable controlled release. The freeze-dried formulation (FNB[2]) integrates optimized cryoprotectants and a surface-engineered nanoparticle design, preserving morphology and function without excessive excipients. FNB[2] exhibits rapid rehydration and retains ∼70% of gene editing activity post-lyophilization, enabling robust functional gene editing in vitro and in vivo. It also maintains long-term stability and supports efficient cellular uptake, enabling administration via multiple routes. FNB[2] represents a scalable and robust platform for genetic therapeutics, vaccines, and biologics, particularly well-suited for resource-limited and emergency medical applications.},
}

*Cryoprotective Agents/chemistry
Animals
Humans
Freeze Drying
*Nanopores
Gene Editing
Ribonucleoproteins/chemistry/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41837831,
year = {2026},
author = {Awinashe, M and Viswaja, K and Pathath, AW and Subair, FP and Varshney, A and Mishra, P and Mulla, M and Mulla, M},
title = {CRISPR-cas9-Mediated Gene Editing to Reverse Oncogenic Mutations in Oral Squamous Cell Carcinoma.},
journal = {Annals of African medicine},
volume = {25},
number = {4},
pages = {805-809},
doi = {10.4103/aam.aam_864_25},
pmid = {41837831},
issn = {0975-5764},
mesh = {Humans ; *Mouth Neoplasms/genetics/pathology/therapy ; *Carcinoma, Squamous Cell/genetics/therapy/pathology ; *Mutation ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing/methods ; Cell Proliferation/genetics ; Oncogenes ; },
abstract = {INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a genetically driven malignancy characterized by a high burden of oncogenic mutations that contribute to aggressive tumor behavior, therapeutic resistance, and poor survival outcomes. Conventional treatment modalities largely target downstream molecular pathways without correcting the underlying genetic aberrations, underscoring the need for precision-based therapeutic strategies. This study aimed to assess the feasibility and functional impact of CRISPR-Cas9-mediated gene editing in reversing oncogenic mutations associated with OSCC using an in vitro experimental model.

MATERIALS AND METHODS: A controlled in vitro experimental study was conducted using the established human OSCC cell lines harboring mutations in key oncogenic genes. Cells were divided into control, mock-transfected, and CRISPR-Cas9-edited groups. Target-specific single-guide RNAs were designed to correct oncogenic mutations using advanced CRISPR-based editing platforms. Gene-editing efficiency was validated by molecular assays, while functional outcomes were assessed using cell proliferation analysis. Statistical evaluation was done using the one-way ANOVA with significance set at P < 0.05.

RESULTS: CRISPR-Cas9-edited OSCC cells demonstrated a significant reduction in cell proliferation compared to control and mock-transfected groups (P < 0.001). No significant variation was found between control and mock-transfected cells, confirming minimal procedural influence.

CONCLUSION: CRISPR-Cas9-mediated correction of oncogenic mutations effectively suppresses malignant cellular proliferation in OSCC, highlighting its promising role as a precision therapeutic strategy in oral cancer management.},
}

Humans
*Mouth Neoplasms/genetics/pathology/therapy
*Carcinoma, Squamous Cell/genetics/therapy/pathology
*Mutation
*CRISPR-Cas Systems
Cell Line, Tumor
*Gene Editing/methods
Cell Proliferation/genetics
Oncogenes

@article {pmid41865101,
year = {2026},
author = {Prakash, S and Mishra, C and Sinha, M and Kumari, H and Kumar, A},
title = {The evolution of next-generation lateral flow assays for bacterial and fungal diagnostics.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41865101},
issn = {1436-5073},
abstract = {Infectious diseases driven by increasingly resistant bacterial and fungal pathogens demand diagnostics that are faster and more accessible than conventional culture-based methods. This review traces the evolution of lateral flow assays (LFAs) from simple qualitative strips to sophisticated, molecularly enhanced diagnostic platforms. This review synthesizes literature mostly published between 2010 and 2025, identified through PubMed, Scopus, and Web of Science using search terms including 'lateral flow assay,' 'point-of-care diagnostics,' 'CRISPR diagnostics,' 'nanozyme biosensor,' 'antimicrobial resistance,' 'Candida auris,' and 'invasive aspergillosis. It highlights how advances in materials science (including quantum dots and nanozymes), isothermal amplification (RPA, LAMP), and CRISPR/Cas-based recognition have pushed LFAs toward laboratory-comparable sensitivity while preserving their simplicity. The clinical impact of these next-generation LFAs is illustrated using high-threat pathogens such as MRSA, Candida auris, and invasive Aspergillus, where rapid, point-of-care identification improves outcomes and supports antimicrobial stewardship. The review also examines the digital transformation of LFAs through smartphone-based readouts and artificial intelligence, which enable quantitative analysis and real-time epidemiological surveillance, even in remote settings. Despite ongoing challenges, including the hook effect, cross-reactivity, and regulatory fragmentation, the emerging technologies described here suggest that LFAs can help decouple high-quality infectious disease diagnostics from centralized laboratories, supporting a more equitable, global access to precision microbiological testing.},
}

@article {pmid41870761,
year = {2026},
author = {Dhaouadi, S and Titouche, Y and Dhaouadi, F and Akkou, M and Elandoulsi, RB},
title = {Mammaliicoccus sciuri as a sentinel for antimicrobial resistance and virulence: genomic epidemiology, transmission dynamics, and control in the one health era.},
journal = {Veterinary research communications},
volume = {50},
number = {3},
pages = {},
pmid = {41870761},
issn = {1573-7446},
abstract = {Mammaliicoccus sciuri (M. sciuri), a Gram-positive bacterium belonging to the group of coagulase-negative staphylococci (CoNS), has been taxonomically reclassified from the genus Staphylococcus to the newly established genus Mammaliicoccus. Its adaptability across diverse niches supports its relevance to One Health. Widely distributed in livestock and diverse environments, M. sciuri has been increasingly reported as an opportunistic pathogen and genetic reservoir for antimicrobial resistance (AMR) and virulence determinants of relevance to human and veterinary medicine. This review provides a synthesis of existing literature regarding the epidemiology, ecological niches, clinical relevance of the M. sciuri species, as well as the genomic and genetic features that underpin its contribution to the dissemination of resistance and virulence factors. Furthermore, it examined the global spread of methicillin-resistant M. sciuri (MR-M. sciuri) clones, characterized through phylogenetic analyses, sequence types (STs), and their complex transmission dynamics. The molecular basis of its resistance mechanisms is explored, with attention directed toward the genetic environments surrounding the mecA and mecC genes within the SCCmec cassettes, as well as the involvement of mobile genetic elements and heavy metal resistance systems. The genetic repertoire of M. sciuri, including the presence of CRISPR–Cas systems associated with adaptive immunity and genome plasticity, was also examined. Moreover, the review delves into the duality of M. sciuri as it can produce antimicrobials such as bacteriocins, which are active against bacteria and fungi. Finally, this review outlines control strategies, including genomic surveillance and stricter antimicrobial regulations, to address MDR M. sciuri strains in the human-animal-environment interface.},
}

@article {pmid41913088,
year = {2026},
author = {Zhang, S and Sun, W and Xiao, T and Wang, Y and Wu, X and Chen, H and Chen, M and Zhang, J},
title = {TopCas: Topology-Gated Cas12a via DNA-RNA Chimeric Circular crRNA for Amplification-Free Nucleic Acid Detection and Conditional Gene Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {33},
pages = {e75046},
doi = {10.1002/advs.75046},
pmid = {41913088},
issn = {2198-3844},
support = {2025ZD0551200//National Science and Technology Major Project on the Prevention and Treatment of Cancers, Cardiovascular and Cerebrovascular Diseases, Respiratory and Metabolic Diseases/ ; 2022YFC2603800//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; RNA/genetics ; *DNA/genetics ; *Bacterial Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {Controlled activation of CRISPR-Cas12a is critical for achieving conditional gene editing and molecular diagnostics. As an indispensable component for forming an active complex, CRISPR RNA (crRNA) represents a key route to regulate Cas12a activity. Here, we establish TopCas (Topology-gated Cas12a via DNA-RNA Chimeric Circular crRNA) as a platform for preamplification-free nucleic acid detection and conditional gene editing. Within TopCas, the circular crRNA sterically constrains Cas12a's nuclease activity until target-activated complexes trans-cleave the DNA segment of the chimeric crRNA, converting the circular guide into its linear form and initiating an autocatalytic cascade that culminates in fluorophore release and signal amplification. By the same mechanism, the system conditionally activates Cas12a's gene-editing function (cis-cleavage) exclusively in the presence of specific nucleic acid targets (e.g., viral DNA or RNA). We demonstrate that TopCas affords high specificity and sensitivity in nucleic acid detection, supports accurate detection in clinical viral nucleic acid samples, and shows potential for in vivo real-time molecular imaging, while also demonstrating the feasibility of conditional gene editing. This innovative chimeric circular crRNA-Cas12a system not only provides a new tool for precise disease diagnostics but also offers a promising strategy for personalized therapeutic intervention.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
Humans
RNA/genetics
*DNA/genetics
*Bacterial Proteins/genetics
Endodeoxyribonucleases

@article {pmid41968146,
year = {2026},
author = {Nguyen, LT and Rakestraw, NR and Pizzano, BLM and Iyyappan, R and Young, CB and Huang, Y and Beerensson, KT and Fang, A and Antal, SG and Anamisis, KV and Peggs, CMD and Yan, J and Jing, Y and Lewis, JG and Burdine, RD and Adamson, B and Jiang, Z and Toettcher, JE and Myhrvold, C and Jain, PK},
title = {Efficient genome editing with chimeric oligonucleotide-directed editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41968146},
issn = {2041-1723},
support = {R35 GM147788/GM/NIGMS NIH HHS/United States ; U01 DK127429/DK/NIDDK NIH HHS/United States ; U01DK127429//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35GM147788//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01HD113698//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R61 AI181016/AI/NIAID NIH HHS/United States ; T32GM148739//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01HD102533//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; T32GM136583//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32 GM148739/GM/NIGMS NIH HHS/United States ; R61AI181016//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; },
mesh = {*Gene Editing/methods ; Humans ; Animals ; HEK293 Cells ; *Oligonucleotides/genetics ; CRISPR-Cas Systems/genetics ; Mice ; Cattle ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report several nCas9-DNA polymerase fusion proteins and their engineered versions to create a simple and efficient two-component chimeric oligonucleotide-directed editing (CODE) system. CODE contains a derivative of Bst DNA polymerase engineered for increased thermostability and processivity as well as a chimeric pegRNA (cpegRNA) for programmable search and replace genome editing. Additionally, CODEMax(exo+) features a 5' to 3' exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate that CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci in HEK293T cells with plasmid- and RNP-based delivery. We also show that CODEMax can successfully modify mouse and bovine embryos with up to 9.3% precise editing. Further optimization of CODEMax systems may enhance editing outcomes in embryos and other challenging contexts. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.},
}

*Gene Editing/methods
Humans
Animals
HEK293 Cells
*Oligonucleotides/genetics
CRISPR-Cas Systems/genetics
Mice
Cattle
CRISPR-Associated Protein 9/genetics/metabolism
DNA-Directed DNA Polymerase/genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid41996722,
year = {2026},
author = {Han, K and Qin, Z and Hu, S and Wu, H and Lei, W},
title = {CRISPR/Cas9-mediated generation of a REPS2 knockout human embryonic stem cell line.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103986},
doi = {10.1016/j.scr.2026.103986},
pmid = {41996722},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), an Xchromosome-linked gene, is broadly expressed in brain, adrenal gland, and other tissues. REPS2 encodes a protein that forms part of a complex involved in the regulation of growth factor receptor endocytosis, and has been associated with a variety of diseases. We generated a REPS2 knockout human embryonic stem cell line using CRISPR/Cas9 genome editing system, which remained typical stem cell morphology, a normal karyotype, and pluripotency, and demonstrated the capacity to differentiate into all three germ layers. The REPS2 knockout hESC line provides a valuable tool for modeling REPS2-associated pathological process.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Cell Line
*Gene Knockout Techniques
Cell Differentiation

@article {pmid42000542,
year = {2026},
author = {Kerkhof, LMC and Pepers, BA and van der Graaf, LM and Santiago-Aranda, A and Voesenek, BJB and Reits, EAJ and Buijsen, RAM and van Roon-Mom, WMC},
title = {Generation of an isogenic human induced pluripotent stem cell line for spinocerebellar ataxia type 1.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103987},
doi = {10.1016/j.scr.2026.103987},
pmid = {42000542},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Spinocerebellar Ataxias/pathology/metabolism/genetics ; Cell Line ; Ataxin-1/genetics/metabolism ; Cell Differentiation ; CRISPR-Cas Systems/genetics ; },
abstract = {Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in exon 8 of the ATXN1 gene. In this study, an isogenic human induced pluripotent stem (hiPS) cell SCA1 line was generated using CRISPR/Cas9 genome editing. Characterization revealed an expanded repeat containing 54 CAG repeats in one allele and an unmodified second allele. The isogenic hiPS cell line showed a typical hiPS cell morphology, expressed pluripotency markers and was able to differentiate into all three germ layers.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Spinocerebellar Ataxias/pathology/metabolism/genetics
Cell Line
Ataxin-1/genetics/metabolism
Cell Differentiation
CRISPR-Cas Systems/genetics

@article {pmid42020735,
year = {2026},
author = {White, MT and Wang, K and Zhang, H and Eckhard, U and Hullahalli, K and Chen, J and Wu, S and Geis, AL and Zhang, J and Queen, J and Gomis-Ruth, FX and Waldor, MK and Dong, M and Sears, CL},
title = {A pro-carcinogenic bacterial toxin binds claudin-4 to cleave E-cadherin.},
journal = {Nature},
volume = {654},
number = {8118},
pages = {504-512},
pmid = {42020735},
issn = {1476-4687},
mesh = {*Cadherins/metabolism/chemistry ; *Claudin-4/metabolism/genetics ; Humans ; *Bacterial Toxins/metabolism ; Protein Binding ; Animals ; Bacteroides fragilis/enzymology/pathogenicity/metabolism ; *Proteolysis/drug effects ; *Metalloendopeptidases/metabolism ; CRISPR-Cas Systems/genetics ; Antigens, CD/metabolism ; Colorectal Neoplasms/microbiology/metabolism ; Epithelial Cells/metabolism ; },
abstract = {The human colon is colonized by trillions of bacteria that play substantial roles in human health and disease[1]. Epidemiological and experimental studies suggest that certain colonic bacteria can stimulate the development and progression of colorectal cancer[2]. One such bacterium, enterotoxigenic Bacteroides fragilis, drives colon tumour formation through the action of a single toxin, the B. fragilis toxin (BFT)[3,4]. BFT is a metalloprotease that binds to a colonic epithelial cell receptor and causes cleavage of the E-cadherin ectodomain, leading to epithelial barrier disruption, inflammation and increased cellular proliferation[4-6]. However, the identity of the BFT receptor is unknown and the molecular mechanism of BFT-initiated E-cadherin cleavage is not well understood. Here we identify claudin-4 as a BFT receptor through a genome-wide CRISPR screen and demonstrate that claudin-4 binding promotes BFT-mediated cleavage of cell surface E-cadherin. Our work both sheds light on BFT's mechanism of action and opens avenues for the development of anti-BFT therapies, which may prove useful for colorectal cancer prevention and treatment of acute enterotoxigenic B. fragilis infection.},
}

*Cadherins/metabolism/chemistry
*Claudin-4/metabolism/genetics
Humans
*Bacterial Toxins/metabolism
Protein Binding
Animals
Bacteroides fragilis/enzymology/pathogenicity/metabolism
*Proteolysis/drug effects
*Metalloendopeptidases/metabolism
CRISPR-Cas Systems/genetics
Antigens, CD/metabolism
Colorectal Neoplasms/microbiology/metabolism
Epithelial Cells/metabolism

@article {pmid42033935,
year = {2026},
author = {Yao, F and Ziqing, Z and Jingxi, H and Yuxin, X and Tiancheng, Z and Qi, X and Guangjin, P},
title = {Generation of an IL2 knock-in human induced pluripotent stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103996},
doi = {10.1016/j.scr.2026.103996},
pmid = {42033935},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Interleukin-2/genetics/metabolism ; *Gene Knock-In Techniques ; Cell Differentiation ; Cell Line ; Animals ; },
abstract = {Interleukin-2 (IL2) is a critical cytokine that drives T-cell proliferation, activates NK cells, and holds significant therapeutic value. Its established clinical importance in cancer immunotherapy lies in sustaining the expansion, persistence, and effector function of adoptive cell therapies. Here, we established a stable IL2 knock-in human induced pluripotent stem cell (iPSC) line via CRISPR/Cas9 technology. This engineered line constitutively secretes IL2, maintains a normal karyotype, and retains typical pluripotent characteristics, including gene expression and in vivo differentiation potential. It thus provides a reliable model for studying IL2 signaling, immune crosstalk, and therapeutic screening.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Interleukin-2/genetics/metabolism
*Gene Knock-In Techniques
Cell Differentiation
Cell Line
Animals

@article {pmid42045938,
year = {2026},
author = {Dinçer, C and Fussing, B and Garnett, MJ and Coelho, MA},
title = {BEstimate: a computational tool for the design and interpretation of CRISPR base editing experiments.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42045938},
issn = {1474-760X},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Software ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Computational Biology/methods ; },
abstract = {CRISPR base editors enable scalable targeted DNA mutagenesis and are a powerful tool for analysing the function of variants of uncertain significance and disease modelling. Existing guide RNA (gRNA) design tools lack comprehensive functional annotation of target sequences. Here we developed BEstimate, a flexible computational pipeline that systematically specifies base editor gRNA target sites, generates on-target activity and off-target predictions, and provides functional, structural and clinical annotations of installed variants. BEstimate supports custom gRNA design against variant alleles and reversion of disease variants. BEstimate is a freely available, versatile tool for designing gRNA libraries and analysing base editor screens.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Software
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
*Computational Biology/methods

@article {pmid42070526,
year = {2026},
author = {Jerred, C and Ramachandran, H and Hildebrandt, B and Zink, A and Ventura, N and Rossi, A and Prigione, A},
title = {Generation of an iPSC line IUFi004-A-13 with homozygous NDUFS1 mutation for the study of Leigh syndrome.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104002},
doi = {10.1016/j.scr.2026.104002},
pmid = {42070526},
issn = {1876-7753},
mesh = {Humans ; *Leigh Disease/genetics/pathology/metabolism ; *NADH Dehydrogenase/genetics/metabolism ; Homozygote ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Mutation ; *Electron Transport Complex I/genetics ; Cell Differentiation ; CRISPR-Cas Systems ; Mutation, Missense ; },
abstract = {NDUFS1 is a critical component of mitochondrial respiratory chain Complex I (CI). Pathogenic variants of NDUFS1 can cause Leigh syndrome (LS), a severe pediatric mitochondrial disorder. To model NDUFS1-linked LS, we generated an iPSC line with homozygous missense mutations in exon 8 using CRISPR/Cas9. The cell line demonstrated typical morphology, expression of iPSC markers, ability to differentiate into all three germ layers, and genomic integrity. This model will enable the study of LS caused by CI in an isogenic context.},
}

Humans
*Leigh Disease/genetics/pathology/metabolism
*NADH Dehydrogenase/genetics/metabolism
Homozygote
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Line
Mutation
*Electron Transport Complex I/genetics
Cell Differentiation
CRISPR-Cas Systems
Mutation, Missense

@article {pmid42103732,
year = {2026},
author = {Barazas, M and van Schendel, R and Tijsterman, M},
title = {A mutational scar-based genome-wide map of DNA double-strand break repair.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103732},
issn = {2041-1723},
support = {2021-2/13905//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 2024-3/16583//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; DNA Polymerase theta ; *DNA Repair/genetics ; *Mutation ; CRISPR-Cas Systems ; DNA End-Joining Repair/genetics ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; Werner Syndrome Helicase/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Genome alterations arise from inaccurate DNA repair and accumulate as distinct mutational signatures. Here, we systematically interrogate the contribution of every protein-coding gene to double-strand break (DSB) repair by generating high-resolution outcome profiles following gene knockouts. Using a CRISPR/Cas9-based, massively parallel bulk screening approach, we establish a comprehensive catalogue of MUtational Scars of Induced DNA Cleavage (MUSIC) that maps the full landscape of DSB repair factors. Our analysis identifies and validates gene clusters - including nearly all known components and several previously unrecognised factors - associated with non-homologous end-joining, the 53BP1 pathway, homology-directed repair, and polymerase theta (POLQ)-mediated end-joining. By focusing on pathway-specific repair outcomes, we uncover an unexpected role for the WRN helicase in suppressing inverted templated insertions, a poorly understood POLQ-associated mutational signature. Finally, dissection of MUSIC features reveals unanticipated functional distinctions among genes within the same DSB pathway, providing mechanistic insight and enabling further investigation into chromosomal break repair.},
}

*DNA Breaks, Double-Stranded
Humans
DNA Polymerase theta
*DNA Repair/genetics
*Mutation
CRISPR-Cas Systems
DNA End-Joining Repair/genetics
Tumor Suppressor p53-Binding Protein 1/metabolism/genetics
Werner Syndrome Helicase/genetics/metabolism
DNA-Directed DNA Polymerase/genetics/metabolism
Gene Knockout Techniques

@article {pmid42107826,
year = {2026},
author = {Xu, T and Guo, Z and Li, Y and Lyu, H},
title = {Determining optimal sgRNA coverage and screening duration for pooled CRISPR screens: A quantitative framework.},
journal = {Methods (San Diego, Calif.)},
volume = {253},
number = {},
pages = {49-60},
doi = {10.1016/j.ymeth.2026.04.016},
pmid = {42107826},
issn = {1095-9130},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Reproducibility of Results ; Gene Library ; },
abstract = {CRISPR-based loss-of-function screening has emerged as a powerful tool for systematically characterizing gene functions. However, standardized quantification metrics for sgRNA coverage-a critical parameter determining genome-wide screening reliability and resource efficiency, remain undefined. In this study, we first conducted systematic sgRNA coverage tests in HeLa cells to determine the optimal coverage for CRISPR[iBAR] knockout libraries. Furthermore, we incorporated multiple timepoints to monitor sgRNA-mediated gene knockout dynamics. Here, balancing data quality, time efficiency, and cost, we identify 15 days and 800 × coverage as the optimal screening parameters for standard iBAR‑based CRISPR screens. Data from varying coverage levels can also serve as essential references for screening under different experimental conditions. Longitudinal analysis revealed that extending the screening period beyond 15 days maintains stable sgRNA distribution patterns within the cell population. This study establishes key parameter benchmarks to ensure CRISPR knockout screening efficacy and reproducibility, providing a solid foundation for downstream drug screening and target identification.},
}

Humans
*CRISPR-Cas Systems/genetics
HeLa Cells
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Reproducibility of Results
Gene Library

@article {pmid42129543,
year = {2026},
author = {Bilanovic, J and Bortolatto, J and Duan, S and Bjørnsdottir, V and Teetz, AK and Thoms, A and Fischbach, J and Schmidt, F and Nyberg, WA and Hartweger, H and Escolano, A and Thomas, PG and Victora, GD and Bilate, AM and Jacobsen, JT},
title = {One-step generation of T-cell receptor knock-in mice in the TCRβ locus.},
journal = {The EMBO journal},
volume = {45},
number = {12},
pages = {4321-4336},
pmid = {42129543},
issn = {1460-2075},
support = {101042650//EC | European Research Council (ERC)/ ; },
mesh = {Animals ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; Mice, Transgenic ; Dependovirus/genetics ; T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Transgenic mouse models expressing predefined T-cell receptors (TCRs) have been instrumental in advancing our understanding of T-cell biology. However, these traditional models rely on random genomic insertion of large constructs, require labor-intensive embryo manipulation, and frequently result in aberrant TCR expression and phenotypes. These limitations render traditional models insufficient to meet the mounting demands for rapid and precise model systems to evaluate TCR specificities. In this study, we developed a streamlined method that uses adeno-associated virus (AAV) and CRISPR/Cas9-mediated genome editing to precisely integrate pre-rearranged TCRα/β sequences into the mouse TCRβ (Trb) locus, enabling the rapid generation of TCR knock-in mice with physiological TCR expression and functional T-cell differentiation upon antigenic challenge. This approach bypasses the need for screening multiple founders for faithful TCR expression, enhancing the versatility and utility of monoclonal TCR mice in basic immunology and preclinical research, such as in the fields of cancer immunotherapy and vaccine development.},
}

Animals
*Receptors, Antigen, T-Cell, alpha-beta/genetics/metabolism
*Gene Knock-In Techniques/methods
Mice
Mice, Transgenic
Dependovirus/genetics
T-Lymphocytes/immunology
CRISPR-Cas Systems
Gene Editing/methods

@article {pmid42185540,
year = {2026},
author = {Oberlin, S and Tay, NQ and Xue, A and Mosadeghi, R and Pimentel, H and McManus, MT},
title = {Multiplexed perturbation enables scalable pooled screens.},
journal = {Nature methods},
volume = {23},
number = {6},
pages = {1163-1173},
pmid = {42185540},
issn = {1548-7105},
support = {5U01CA272546-03//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 188001//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; T32HG002536//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Intercellular Adhesion Molecule-1/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; HEK293 Cells ; },
abstract = {CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
Intercellular Adhesion Molecule-1/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockdown Techniques
HEK293 Cells

@article {pmid42208154,
year = {2026},
author = {Crudele, M and Barnaba, NF and Di Cosmo, D and Mascia, T and Lum, KY and Cowled, MS and Larsen, TO and Faretra, F and Coleman, JJ and De Miccolis Angelini, RM},
title = {Characterization of the chloromonilicin biosynthetic gene cluster in the brown rot fungus Monilinia fructicola by a CRISPR/Cas9 transformation system.},
journal = {Microbiological research},
volume = {310},
number = {},
pages = {128558},
doi = {10.1016/j.micres.2026.128558},
pmid = {42208154},
issn = {1618-0623},
mesh = {*Multigene Family ; Polyketide Synthases/genetics/metabolism ; *Ascomycota/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; Plant Diseases/microbiology ; Gene Expression Profiling ; Fruit/microbiology ; Transformation, Genetic ; Biosynthetic Pathways/genetics ; },
abstract = {Monilinia fructicola is a major pathogen responsible for brown rot, causing substantial yield losses, particularly in stone fruit. Genomic analysis identified a type I polyketide synthase gene cluster, approximately 55 kb in length and consisting of twenty-four genes, putatively involved in chloromonilicin biosynthesis. These include a polyketide synthase (PKS), flavin-dependent halogenase, N-acetyltransferases, methyltransferases, a lactamase-like protein, scytalone dehydratase, and genes related to oxidoreduction, transcription factors, and transporters. To elucidate the product of the cluster, targeted gene disruption was achieved using a CRISPR/Cas9 ribonucleoprotein complex and polyethylene glycol-mediated transformation in the reference strain Mfrc123. Mutants with disruption in the ChmN core gene (encoding the PKS) were analysed by sequencing, digital droplet PCR to assess the insertion copy number of the hygromycin resistance cassette, and gene expression profiling. Phenotypic analysis indicated no significant differences between mutants and the WT strain in conidia production, germination rate, colony growth, or virulence on inoculated fruits. Metabolomic profiling using UHPLC-MS/MS demonstrated that the mutants did not produce chloromonilicin or other cluster-associated metabolites, such as chloromonilinic acids C and D, and 4-chloropinselin, indicating that the gene cluster is essential for their biosynthesis. In the WT strain, production of these metabolites increased following co-culture with Penicillium expansum compared to monoculture and remained low after fruit inoculation, suggesting a role in competition rather than pathogenesis. Gene expression analysis further showed upregulation of ChmN in response to various plant-associated microbes, but not to other Monilinia species.},
}

*Multigene Family
Polyketide Synthases/genetics/metabolism
*Ascomycota/genetics/metabolism/pathogenicity
*CRISPR-Cas Systems
Plant Diseases/microbiology
Gene Expression Profiling
Fruit/microbiology
Transformation, Genetic
Biosynthetic Pathways/genetics

@article {pmid42229170,
year = {2026},
author = {Koo, C and Lee, D and Lee, B and Kim, S and Lee, J and Kwon, J},
title = {Base editing reveals context-dependent regulation of adhesion, anoikis, and motility by BAP1 in renal cell models.},
journal = {Biochemical and biophysical research communications},
volume = {827},
number = {},
pages = {154092},
doi = {10.1016/j.bbrc.2026.154092},
pmid = {42229170},
issn = {1090-2104},
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *Anoikis/genetics ; Humans ; *Cell Movement/genetics ; Cell Adhesion/genetics ; *Kidney Neoplasms/genetics/pathology ; *Carcinoma, Renal Cell/genetics/pathology ; *Gene Editing ; CRISPR-Cas Systems ; *Kidney/cytology/pathology/metabolism ; Cell Line, Tumor ; Cell Line ; Mutation ; },
abstract = {BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 function, reinstated anchorage dependence, and re-sensitized cells to anoikis. Here, we investigated whether disruption of Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Using adenine base editing, we introduced an inactivating Glu31Gly mutation into HK-2 cells, generating two independent isogenic BAP1-mutant clones, and established a BAP1-knockout clone by CRISPR/Cas9 as an additional control. Glu31Gly mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair, comparable to knockout cells. Despite the clear functional inactivation of BAP1, the Glu31Gly and knockout HK-2 cells neither acquired anchorage-independent growth nor anoikis resistance; instead, detached cells displayed increased apoptosis. In KMRC-20 cells, restoration of BAP1 enhanced both migration and invasion, whereas BAP1 inactivation or loss in HK-2 cells increased invasion but reduced migration, indicating distinct context-dependent roles for BAP1 in normal versus malignant renal cells. These findings demonstrate that BAP1 inactivation alone is insufficient to confer anchorage-independent survival in normal kidney epithelial cells and suggest that additional oncogenic alterations are required during kidney tumorigenesis. Our study further highlights the utility of precise base editing for dissecting the functional consequences of clinically relevant cancer mutations.},
}

*Ubiquitin Thiolesterase/genetics/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
*Anoikis/genetics
Humans
*Cell Movement/genetics
Cell Adhesion/genetics
*Kidney Neoplasms/genetics/pathology
*Carcinoma, Renal Cell/genetics/pathology
*Gene Editing
CRISPR-Cas Systems
*Kidney/cytology/pathology/metabolism
Cell Line, Tumor
Cell Line
Mutation

@article {pmid42248117,
year = {2026},
author = {der Auweraer, SV and Roth, MB and Vlahos, K and Howden, SE and Lockhart, PJ and Payne, JM and Brems, H and Bozaoglu, K},
title = {Generation and characterization of four iPSC and isogenic gene-corrected lines from Legius syndrome patients.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104026},
doi = {10.1016/j.scr.2026.104026},
pmid = {42248117},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Cell Differentiation ; Leukocytes, Mononuclear/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Legius syndrome is an autosomal dominant disorder caused by variants in SPRED1. In this study, we generated four induced pluripotent stem cell (iPSC) lines derived from patients with Legius syndrome by reprogramming peripheral blood mononuclear cells. Using CRISPR/Cas9 or prime editing, the pathogenic variants were corrected to generate isogenic control lines. All patient and isogenic control lines exhibited a normal morphology and karyotype, expressed pluripotency markers, and possessed trilineage differentiation potential. This is the first established human iPSC model developed for Legius syndrome and is a valuable resource for investigating the molecular mechanisms underlying this condition.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Line
Cell Differentiation
Leukocytes, Mononuclear/metabolism/cytology
CRISPR-Cas Systems

@article {pmid42251763,
year = {2026},
author = {Byiringiro, I and Contiliani, DF and Davies, C and Gurel, F and Creste, S and Qi, Y},
title = {Improving iSpyMacCas9 multiplex genome editing in rice by CRISPR-combo-mediated BBM1 activation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {5},
pages = {e70980},
pmid = {42251763},
issn = {1365-313X},
support = {IOS-2132693//National Science Foundation/ ; IOS-2224203//National Science Foundation/ ; 21010111//Foundation for Food and Agriculture Research/ ; 2020/07045-3//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2021/13478-2//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2022/11738-0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Transcriptional Activation/genetics ; Plants, Genetically Modified ; Genome, Plant/genetics ; Gene Expression Regulation, Plant ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The recently developed CRISPR-Combo technology enables simultaneous targeted mutagenesis and transcriptional activation in plants. However, its reliance on SpCas9 limits its use at AT-rich genomic loci, such as promoter regions commonly targeted for transcription activation. To overcome this limitation, we explored the usage of Cas12b and iSpyMacCas9 in the CRISPR-Combo architecture for simultaneous genome editing and gene activation. We tested these expanded CRISPR-Combo systems for hormone-free regeneration of rice plants by transcriptional activation of a morphogenic gene, OsBBM1, while knocking out the genes of interest. The Cas12b-Combo system induced mild OsBBM1 upregulation (~3-fold), which did not affect the genome editing efficiency. By contrast, iSpyMacCas9-Combo achieved approximately 12-fold OsBBM1 transcriptional activation, supporting hormone-free regeneration at a high rate (42%). As a result, iSpyMacCas9-Combo conferred higher genome editing efficiency, including improved multiplex editing, than the standard iSpyMacCas9 system, either with or without hormones during rice regeneration. Hence, our data prove iSpyMacCas9-Combo to be a more efficient system for genome editing in rice, especially at low-efficiency target sites, when coupled with OsBBM1 transcriptional activation. These findings establish iSpyMacCas9-Combo as a useful addition to the CRISPR-Combo toolkit, expanding its genomic targeting scope and enabling more efficient genome editing by activation of an appropriate endogenous gene such as OsBBM1 in rice.},
}

*Oryza/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plant Proteins/genetics/metabolism
Transcriptional Activation/genetics
Plants, Genetically Modified
Genome, Plant/genetics
Gene Expression Regulation, Plant
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42252785,
year = {2026},
author = {Jiang, J and Yan, Y},
title = {Compositional Optimization of CRISPR/Cas9 Lipid Nanoparticles for Efficient Knockdown of Target Genes.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {11},
pages = {e70413},
doi = {10.1002/cbic.70413},
pmid = {42252785},
issn = {1439-7633},
support = {Grant 22275167//National Natural Science Foundation of China/ ; 22405240//National Natural Science Foundation of China/ ; Grant LZ25B040002//Zhejiang Provincial Natural Science Foundation of China/ ; LTGY24B040001//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; Animals ; *Gene Knockdown Techniques ; Mice ; Gene Editing ; Liposomes ; },
abstract = {Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.},
}

Humans
*Nanoparticles/chemistry
*Lipids/chemistry
*CRISPR-Cas Systems/genetics
HeLa Cells
Animals
*Gene Knockdown Techniques
Mice
Gene Editing
Liposomes

@article {pmid42258486,
year = {2026},
author = {Pilarski, J and Stadler, T and Seidel, S},
title = {Assessing the inference of single-cell phylogenies and population dynamics from CRISPR lineage recordings.},
journal = {PLoS computational biology},
volume = {22},
number = {6},
pages = {e1014370},
doi = {10.1371/journal.pcbi.1014370},
pmid = {42258486},
issn = {1553-7358},
mesh = {*Phylogeny ; *Cell Lineage/genetics ; Bayes Theorem ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computer Simulation ; *Single-Cell Analysis/methods ; Computational Biology ; Cell Division/genetics ; Population Dynamics ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Multicellular organisms develop from a single cell by repeated rounds of cell division, differentiation, and death, which can be represented as a single-cell phylogenetic tree. Genetic lineage tracing allows us to investigate this development by tracking the ancestry of individual cells as populations grow and change over time. However, accurate reconstruction of the cell phylogeny and quantification of the corresponding phylodynamic parameters - cell division, differentiation, and death rates - from this tracking data remains challenging and needs to be systematically evaluated. We perform simulations and assess, using the Bayesian framework, the joint inference of time-scaled cell phylogenies and phylodynamic parameters from CRISPR lineage recordings with random or sequential edits. Principally, we characterize the inference improvements as the recorder capacity increases. We observe more accurate phylogenetic reconstruction from sequential compared to random recordings, but no substantial improvement in phylodynamic inference when using the additional information contained in the order of edits. Overall, we find that CRISPR lineage recordings carry a strong signal on the rates of cell division when appropriate models are used. However, we detect biases in the inferred rates of cell division and death under phylodynamic model misspecification, i.e., when fitting classic memoryless birth-death processes to synchronous cell divisions. Moreover, for scenarios when cells differentiate into distinct types, we demonstrate that Bayesian phylodynamic analysis of sparse end-point measurements can resolve these cell differentiation trajectories by lineage and time. Under prototypical dynamics, we recover cell type-specific division and death rates, and cell type transition rates in over 80% of simulations. Overall, this simulation study explores how much information on cellular development can be extracted from state-of-the-art genetic lineage tracing data using phylogenetic and phylodynamic methodology.},
}

*Phylogeny
*Cell Lineage/genetics
Bayes Theorem
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Computer Simulation
*Single-Cell Analysis/methods
Computational Biology
Cell Division/genetics
Population Dynamics
Cell Differentiation/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42258532,
year = {2026},
author = {Kammerdiener, EK and Hren, AP and Harrison, R and Charles, S and Klingeman, D and Wiser, TD and Eckert, CA and Alexander, WG},
title = {Empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread functionality and rules for gRNA design.},
journal = {Nucleic acids research},
volume = {54},
number = {11},
pages = {},
pmid = {42258532},
issn = {1362-4962},
support = {//Center for Bioenergy Innovation/ ; ERKP886//U.S. Department of Energy/ ; //Laboratory Directed Research and Development Program/ ; //Oak Ridge National Laboratory/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Genome, Bacterial ; *Escherichia coli K12/genetics ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; },
abstract = {The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional. These nonfunctional gRNAs exhibit strong spacer self-interaction, which can either be excluded using a simple design rule or "repaired" during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million empirically evaluated E. coli gRNAs as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
Genome, Bacterial
*Escherichia coli K12/genetics
*Escherichia coli/genetics
*CRISPR-Cas Systems

@article {pmid42259773,
year = {2026},
author = {Luo, G and Ma, F and Yang, Y and Yang, C and Li, X and Xie, J and Xiong, K and Chen, P and Ma, K and Zhao, Z and Tang, BZ and Duo, Y},
title = {Engineering an AIEgen-based platform integrating CRISPR/Cas9 to remodel the tumor microenvironment and reinforce photo-immunotherapy against glioblastom.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {42259773},
issn = {2059-3635},
mesh = {Humans ; *Tumor Microenvironment/genetics/drug effects/immunology ; Animals ; *Glioblastoma/genetics/therapy/pathology/immunology ; *Immunotherapy ; *CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Brain Neoplasms/genetics/therapy/pathology/immunology ; *Photochemotherapy ; Nanoparticles/chemistry ; Blood-Brain Barrier ; *5'-Nucleotidase/genetics ; Neutrophils ; },
abstract = {Glioblastoma remains one of the most lethal brain tumors. Although immunotherapy and other therapeutic modalities has achieved significant therapeutic success in several malignancies, its efficacy in glioblastoma remains limited primarily due to the complex tumor microenvironment (TME) and physiological barriers such as the blood-brain barrier (BBB). In this context, nanomedicine and gene editing have emerged as promising strategies due to their unique ability to cross the BBB and protect therapeutic agents through intrinsic physicochemical properties. To overcome the physiological barriers for better therapeutic outcomes. Here, a novel aggregation-induced emission luminogen (AIEgen), NDA-DPE, was synthesized, exhibiting NIR-I to NIR-II fluorescence and dual photothermal (PTT) and photodynamic (PDT) properties through restricted intramolecular motion. Bone-derived neutrophil-based biomimetic nanoparticles (bNe@AIE/Cas9-CD73) were then prepared by integrating NDA-DPE with CRISPR/Cas9-mediated CD73 gene silencing. The neutrophil encapsulation enabled efficient BBB penetration and targeted accumulation in glioblastoma tissue. CRISPR/Cas9-CD73 downregulated CD73 expression, disrupted the ATP-adenosine axis, and reshped the immunosuppressive TME into an immuno-supportive one, increasing the therapeutic sensitivity of tumor cells. Under NIR-II excitation, bNe@AIE/Cas9-CD73 achieved fluorescence-guided PTT and PDT, inducing immunogenic cell death (ICD), stimulating immune-cell recruitment, and activating systemic antitumor immunity. bNe@AIE/Cas9-CD73 demonstrated a potent gene-photothermal-photodynamic-immune synergistic effect, significantly inhibiting glioblastoma growth and establishing a promising nanoplatform for effective and targeted glioblastoma treatment.},
}

Humans
*Tumor Microenvironment/genetics/drug effects/immunology
Animals
*Glioblastoma/genetics/therapy/pathology/immunology
*Immunotherapy
*CRISPR-Cas Systems/genetics
Mice
Cell Line, Tumor
*Brain Neoplasms/genetics/therapy/pathology/immunology
*Photochemotherapy
Nanoparticles/chemistry
Blood-Brain Barrier
*5'-Nucleotidase/genetics
Neutrophils

@article {pmid42261185,
year = {2026},
author = {Jahangiri-Sisakht, A and Safari, L and Alipanahi, R},
title = {CRISPR-MBTF: a multi-branch transformer fusion framework for CRISPR-Cas9 off-target prediction.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {3},
pages = {},
pmid = {42261185},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Deep Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a revolutionary genome editing technology derived from a bacterial adaptive immune system that uses a single guide RNA (sgRNA) to direct the Cas9 enzyme to specific DNA sequences for precise genetic modifications. Its ease of use and efficiency has accelerated advancements in genetic research and therapeutic development. However, unintended cleavage at off-target sites remains a significant concern, limiting the safety and broader applicability of CRISPR-based editing. Accurate computational prediction of off-target locations is therefore essential to mitigate potential risks and improve experimental design. In this study, we introduce CRISPR multi-branch transformer fusion (CRISPR-MBTF), a novel deep learning-based framework employing a multi-branch Transformer architecture combined with an attention-based fusion mechanism to model the intricate biological context influencing CRISPR activity. By capturing subtle sequence patterns and contextual dependencies, our model achieves enhanced predictive performance compared to existing approaches. Additionally, interpretability analyses uncover biologically meaningful patterns and highlight influential sequence regions, offering valuable insights into the determinants of CRISPR specificity. This work presents a robust and interpretable tool to support the design of safer and more effective genome editing strategies.},
}

*CRISPR-Cas Systems
Computational Biology/methods
Deep Learning
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42261595,
year = {2026},
author = {Saeed, M and Arham, M and Zafar, I and Jamal, A and Hussian, M and Usman, M and Bahwerth, FS and Noman, M and Hossain, MB},
title = {Harnessing Deep Learning Models for Guide RNA Optimization and Off-Target Prediction in CRISPR Systems.},
journal = {Biotechnology journal},
volume = {21},
number = {6},
pages = {e70255},
doi = {10.1002/biot.70255},
pmid = {42261595},
issn = {1860-7314},
mesh = {*Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Recurrent Neural Networks ; Humans ; Predictive Learning Models ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Convolutional Neural Networks ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome and transcriptome editing technologies have emerged as powerful tools for therapeutic, agricultural, and industrial applications. However, their broader clinical and translational use remains limited by variable guide RNA (gRNA) or single-guide RNA (sgRNA) efficiency and unintended off-target activity, which may lead to genotoxic effects and major safety concerns. To address these challenges, recent research has increasingly shifted from heuristic scoring approaches and traditional machine learning (ML) methods toward deep learning (DL) models capable of learning complex sequence-function relationships from large-scale experimental datasets generated by assays such as GUIDE-seq (Genome-wide Unbiased Identification of Double-stranded Breaks Enabled by Sequencing), CIRCLE-seq (Circularization for In Vitro Reporting of Cleavage Effects by Sequencing), and CHANGE-seq (Cumulative and Homology-independent Analysis of Nuclease Genome-wide Effects by Sequencing). This review critically examines recent advances in DL approaches for gRNA optimization and off-target prediction in CRISPR systems. We discuss the development of convolutional neural networks (CNNs), recurrent neural networks (RNNs), transformer-based architectures, and foundation models designed to improve prediction accuracy, specificity, and generalizability across diverse biological contexts.},
}

*Deep Learning
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Recurrent Neural Networks
Humans
Predictive Learning Models
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Convolutional Neural Networks

@article {pmid42266926,
year = {2026},
author = {Ji, T and Fang, X and Gao, Y and Yu, K and He, J and Wang, L and Zhu, W and Huang, G and Gao, X},
title = {Visual detection platform based on RPA-CRISPR/Cas12a for Klebsiella pneumoniae and Carbapenem-resistant Klebsiella pneumoniae in clinical and food safety settings.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1817859},
pmid = {42266926},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/genetics/isolation & purification/drug effects ; Humans ; *Food Safety ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Klebsiella Infections/microbiology/diagnosis ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; beta-Lactamases/genetics ; DNA-Directed RNA Polymerases/genetics ; Carbapenems/pharmacology ; Bacterial Proteins/genetics ; DNA Primers/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {INTRODUCTION: The rise of Klebsiella pneumoniae (KP) and carbapenem-resistant KP (CRKP) poses grave threats to public health and food safety, creating an urgent demand for rapid point-of-care testing (POCT). Traditional detection methods are limited by laboratory barriers, making them unsuitable for POCT implementation.

METHODS: Herein, a one-tube assay integrating recombinant polymerase amplification (RPA) with CRISPR/Cas12a technology was developed for the rapid, sensitive and specific detection of KP and blaOXA-48-carrying CRKP. Specific primers targeting the KP-specific rpoB gene and carbapenem-resistance gene blaOXA-48 were designed, and optimal primer pairs were screened via agarose gel electrophoresis. CrRNA sequences were designed according to RPA amplicons, and the components of the CRISPR/Cas12a reaction were optimized. A two-step reaction system was initially evaluated, followed by the establishment of an integrated one-tube RPA-CRISPR/Cas12a assay. A total of 66 clinical specimens and artificially contaminated food samples were used for method validation, with microbial culture and qPCR as reference methods.

RESULTS: The two-step assay was capable of detecting bacterial suspensions at a concentration of 100 CFU/mL. The one-tube system could be completed within 1 hour at 37 °C. This assay avoided aerosol contamination and allowed visual result readout under blue light. In the validation test, the detection results of the one-tube assay were consistent with those obtained by microbial culture and qPCR.

DISCUSSION: This study constructed a dual-target RPA-CRISPR/Cas12a platform for the visual detection of KP and blaOXA-48-positive CRKP under blue light. This assay reduces reliance on sophisticated equipment and professional personnel. It can serve as a promising POCT tool for clinical diagnosis and food safety surveillance, and provides evidence for the timely formulation of rational antimicrobial treatment strategies.},
}

*Klebsiella pneumoniae/genetics/isolation & purification/drug effects
Humans
*Food Safety
*Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification
Sensitivity and Specificity
*CRISPR-Cas Systems
*Klebsiella Infections/microbiology/diagnosis
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
beta-Lactamases/genetics
DNA-Directed RNA Polymerases/genetics
Carbapenems/pharmacology
Bacterial Proteins/genetics
DNA Primers/genetics
Anti-Bacterial Agents/pharmacology

@article {pmid42267671,
year = {2026},
author = {Brown, RA and Dangel, AW and Saini, A and Collins, PL and Colonna, M and Oltz, EM},
title = {CRISPRi screening identifies SON and MAP4K1 as regulators of type III cytokine expression in innate lymphoid cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {6},
pages = {},
doi = {10.1093/jimmun/vkag110},
pmid = {42267671},
issn = {1550-6606},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Immunity, Innate ; Interleukin-22 ; *Lymphocytes/immunology/metabolism ; Interleukins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Interleukin-17/genetics/metabolism ; Gene Expression Regulation ; *Cytokines/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive "type III" responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1β. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12Rβ1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. Knockdown of nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12Rβ1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFN-γ, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.},
}

Animals
Mice
*Protein Serine-Threonine Kinases/genetics/metabolism
*Immunity, Innate
Interleukin-22
*Lymphocytes/immunology/metabolism
Interleukins/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
Interleukin-17/genetics/metabolism
Gene Expression Regulation
*Cytokines/metabolism/genetics
CRISPR-Cas Systems

@article {pmid42268478,
year = {2026},
author = {Khajouei, F and Ghaemi, A and Abnous, K and Taghdisi, SM and Ramezani, M and Alibolandi, M},
title = {CRISPR-Based Gene Therapy for Brain Disease.},
journal = {Molecular neurobiology},
volume = {63},
number = {1},
pages = {},
pmid = {42268478},
issn = {1559-1182},
mesh = {Humans ; *Genetic Therapy/methods ; Animals ; *Brain Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Neurological disorders are complex and often very challenging for patients. Many of these conditions result from mutations in genes that are essential for normal function. Most existing treatments only alleviate symptoms, highlighting the urgent need for more effective therapeutic strategies. In the current drug development landscape, gene therapy offers hope as a promising approach. Specifically, CRISPR-Cas9 technology enables precise gene editing across diverse cell types and organisms. An increasing number of research groups are investigating innovative therapies and the molecular mechanisms behind neurological diseases. This review highlights the use of CRISPR-based gene therapies for various brain diseases, including multiple sclerosis, Alzheimer's, Parkinson's disease, epilepsy, stroke, and brain tumors. It consistently recognizes significant challenges in clinical applications, including overcoming the blood-brain barrier (BBB), managing off-target effects, ensuring efficient delivery, and addressing immunogenicity and ethical concerns.},
}

Humans
*Genetic Therapy/methods
Animals
*Brain Diseases/therapy/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid42269591,
year = {2026},
author = {Christensen, OP and Markham, A and Kang, H and Gabriel, E and Pers, TH},
title = {Causal effect estimation from trans-regulatory single-cell CRISPR screens.},
journal = {Cell genomics},
volume = {6},
number = {6},
pages = {101251},
pmid = {42269591},
issn = {2666-979X},
mesh = {*Single-Cell Analysis/methods ; Humans ; Single-Cell Gene Expression Analysis ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; Transcriptome/genetics ; },
abstract = {Recent advances in single-cell transcriptomics and CRISPR-based genome editing have enabled large-scale perturbation experiments with genome-wide expression readouts. Single-cell CRISPR screens offer the opportunity to move beyond correlation and estimate causal effects of genetic perturbations on gene expression at scale. These approaches promise to substantially deepen insights into cellular functions and disease mechanisms. However, interpreting statistical associations as causal effects requires additional assumptions beyond those needed for standard statistical analyses. In this minireview, we introduce key concepts and principles for causal effect estimation in trans-regulatory single-cell CRISPR studies. We describe a set of assumptions under which estimates from existing statistical methods admit a causal interpretation and provide a concise overview of these approaches. Finally, through an illustrative example, we demonstrate how violations of these assumptions can bias estimated effects.},
}

*Single-Cell Analysis/methods
Humans
Single-Cell Gene Expression Analysis
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Animals
Transcriptome/genetics

@article {pmid42275032,
year = {2026},
author = {Yuan, S and Tan, D and Zhu, D and Balcazar, JL and Wang, H and Friman, VP and Sun, M and Hu, F},
title = {Global transmission and distribution of phage-encoded cholera toxin genes constrained by toxin-repression genes and anti-phage defense systems.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrag139},
pmid = {42275032},
issn = {1751-7370},
abstract = {Cholera is a severe diarrheal disease caused by toxigenic Vibrio cholerae, whose virulence depends on lysogenic infection by CTXφ bacteriophages encoding the cholera toxin genes (ctxA and ctxB) and associated accessory genes (ace and zot). However, the global distribution and transmission dynamics of phage-encoded cholera toxin genes across environments remain poorly understood. To address this, we performed a large-scale bioinformatic analysis of publicly available whole genomes. We show that both phages and bacteria carrying toxin genes are globally distributed across human-associated, freshwater, fish, and mammalian habitats, with Vibrio and Aeromonas being the dominant bacterial taxa and Inoviridae is the most prevalent phage family. Phage-mediated horizontal gene transfer (HGT) of toxin genes occurred in both Vibrio and non-Vibrio species, with the highest transfer between Inoviridae and V. cholerae occuring predominantly among bacteria from the same habitat. Temporal analysis revealed an increase in candidate HGT events after 2000, peaking at 377845 events during 2010-2019. HGT events negatively correlated with the presence of CRISPR-Cas system and toxin-repression genes (hns, hapR, and tsrA) in host bacteria. Experimental validation indicated that H-NS and HapR inhibit phage infection by repressing phage release. Together, our results suggest that CRISPR-Cas phage defense system and toxin-repression mechanisms could constrain the spread of toxin-carrying phages, with potential implications for the occurrence and severity of cholera outbreaks worldwide.},
}

@article {pmid42276271,
year = {2026},
author = {Hiya-Kawaguchi, U and Kashiwakura, Y and Baatartsogt, N and Lodoi, K and Togashi, T and Sato, T and Tsuchida, K and Batjargal, K and Hayakawa, M and Sato, R and Sato, Y and Ohmori, T},
title = {Non-viral delivery of a base editor enables personalized correction of hemophilia B nonsense variants in a mouse model.},
journal = {Journal of thrombosis and haemostasis : JTH},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jtha.2026.06.003},
pmid = {42276271},
issn = {1538-7836},
abstract = {BACKGROUND: Hemophilia B is caused by loss-of-function variants in the F9 gene encoding factor IX (FIX). While adeno-associated virus (AAV) vector-based gene therapy can restore FIX expression for over a decade, it does not restore the pathogenic variants and faces limitations related to immunogenicity and re-dosing.

AIM: To develop a variant-specific, non-viral base editing strategy using lipid nanoparticles (LNPs) for the precise personalized correction of pathogenic nonsense variations in F9.

METHODS: Editing efficiency was evaluated in HEK293 cells harboring each F9 variant treatable by A•T to G•C base editing. Functional and molecular restoration in vivo was assessed in hemophilia B model mice created with AAV8 vectors expressing F9 variants. LNPs harboring adenine base editor (Cas9 nickase conjugated with ABE8e) mRNA and the corresponding guide RNA were intravenously injected into the mice.

RESULTS: We created HEK293 stably expressing six correctable nonsense variants: c.1067G>A, c.1068G>A, c.1222C>T, c.1292G>A, c.1358G>A, and c.1359G>A. Transfection of the cells with a base editor consisting of SpRY, an engineered SpCas9 with broader proximal protospacer adjacent motif compatibility, and ABE8e, together with the guide RNA sequence, successfully induced A>G conversion at all target sites. The administration of LNPs harboring the base editor mRNA and guide RNA to the hemophilia B model restored the pathological variants (62.8% ± 14.4% for c.1068G>A, 35.9% ± 3.8% for c.1222C>T, and 70.6% ± 2.7% for c.1292G>A) and increased plasma FIX activity.

CONCLUSION: The variant-specific, non-viral base editing platform represents a truly curative intervention for severe hemophilia B caused by single-nucleotide variants.},
}

@article {pmid42276705,
year = {2026},
author = {Radoua, A and Alrustom, B and Wang, J and Bordessoules, M and Chluba, J and Plenchette, S and Micheau, O},
title = {Efficient generation of isogenic FADD[-/-], RIPK1[-/-] and Caspase-8[-/-] cells using a the ptARgenOM non-viral CRISPR-Cas9 system.},
journal = {Methods in cell biology},
volume = {208},
number = {},
pages = {115-147},
doi = {10.1016/bs.mcb.2026.02.005},
pmid = {42276705},
issn = {0091-679X},
mesh = {*Caspase 8/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Fas-Associated Death Domain Protein/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/genetics/deficiency ; *Gene Knockout Techniques/methods ; Mice ; Humans ; Green Fluorescent Proteins/genetics ; Apoptosis/genetics ; Genetic Vectors ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; },
abstract = {The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effective method based on ptARgenOM, an all-in-one mammalian expression vector designed for efficient delivery of the CRISPR-Cas9 system. This vector co-expresses the guide RNA (gRNA) and Cas9 endonuclease, which is fused to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein (EGFP) and the puromycin N-acetyltransferase. This design enables transient, expression-dependent antibiotic selection and fluorescence-based enrichment of successfully transfected cells, facilitating the rapid generation of isogenic KO populations or clones. The method is particularly well-suited, though not limited, to functional studies involving intracellular components of the cell death machinery, including both the extrinsic and intrinsic apoptotic signaling pathways. We illustrate the utility of this system by targeting and deleting FADD, Caspase-8, and RIPK1. This approach can be easily adapted to any intracellular target protein, offering a robust platform for gene function analysis in mammalian cells.},
}

*Caspase 8/genetics
Animals
*CRISPR-Cas Systems/genetics
*Fas-Associated Death Domain Protein/genetics/deficiency
*Receptor-Interacting Protein Serine-Threonine Kinases/genetics/deficiency
*Gene Knockout Techniques/methods
Mice
Humans
Green Fluorescent Proteins/genetics
Apoptosis/genetics
Genetic Vectors
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Transfection

@article {pmid41955489,
year = {2026},
author = {Jiang, B and Zhang, T and Lu, Y and Zhou, S and Xiao, W and Pan, S and Shi, N and Sheng, Y and Hu, J},
title = {CRISPR/Cas13a: Compensatory Target Activation Mechanism.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {32},
pages = {e24156},
pmid = {41955489},
issn = {2198-3844},
support = {22576127//National Natural Science Foundation of China/ ; 2023YFC2417200//National Key Research and Development Program of China/ ; 25ZR1401116//Sci-Tech Innovation Initiative Science and Technology Commission of Shanghai Municipality/ ; 24ZR1423000//Sci-Tech Innovation Initiative Science and Technology Commission of Shanghai Municipality/ ; 2025-YJRC-03//Putuo Hospital, Shanghai University of Traditional Chinese Medicine Start-up Grant/ ; 2019QN01Y725//Guangdong Provincial Pearl River Talents Program/ ; 202206010108//Science and Technology Program of Guangzhou/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; MicroRNAs/genetics ; Humans ; },
abstract = {CRISPR/Cas13a is a powerful RNA-targeting platform for molecular diagnostics, but conventional single-effector systems typically require contiguous RNA targets longer than ∼20-28 nt, limiting sensitivity and target flexibility. CRISPR/Cas13a-CTAM is presented as a compensatory target activation mechanism that facilitates synergistic Cas13a activation through two independently programmable short RNA effectors. By functionally decoupling allosteric activation and binding stabilization, CRISPR/Cas13a-CTAM supports robust activation by ultra-short RNA targets as short as 13 nt, substantially expanding the detectable target range. Compared with traditional single-effector Cas13a assays, CRISPR/Cas13a-CTAM achieves a detection limit of 1 fM for a 13-nt RNA target, representing an approximately tenfold sensitivity improvement. Notably, a single-nucleotide mismatch within the 13-nt target induces up to a 35-fold reduction in apparent cleavage rate, corresponding to a sevenfold enhancement in mismatch discrimination. The dual-effector architecture further enables simultaneous dual-target detection, demonstrated by dual miRNA profiling related to COVID-19 and combined detection of exosome membrane proteins. Moreover, the weakly activating effector was utilized as an anchoring module to achieve the first functional immobilization of Cas13a on a sensing surface, enabling in situ electrochemical miRNA detection. By overcoming the reliance on long RNA targets, CRISPR/Cas13a-CTAM provides a sensitive, programmable platform for RNA diagnostics and integrated biosensor development.},
}

*CRISPR-Cas Systems/genetics
MicroRNAs/genetics
Humans

@article {pmid42202397,
year = {2026},
author = {Han, S and Lin, X and Lei, Y and Liu, Z and Li, J and Ou, X},
title = {Universal primer-based RPA combined with parallel CRISPR/Cas12a decoding for rapid multi-species meat authentication.},
journal = {Food chemistry},
volume = {520},
number = {},
pages = {148905},
doi = {10.1016/j.foodchem.2026.148905},
pmid = {42202397},
issn = {1873-7072},
mesh = {*Meat/analysis/classification ; Animals ; *Food Contamination/analysis ; DNA Primers/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Swine ; },
abstract = {Rapid meat authentication is critical for food safety. Here, we report a two-step assay employing universal primer-based RPA amplification followed by species-specific CRISPR/Cas12a detection for the identification of 11 common meat species. A single universal primer pair enables broad amplification, while specific crRNAs allow parallel single-target CRISPR reactions. The assay takes approximately 40 min at constant temperature, costing ∼$4 per test with dual fluorescence and lateral flow strips. High specificity without cross-reactivity was observed, and detection limits ranged from 10[0] to 10[4] copies/μL. In binary meat mixtures, the fluorescence assay achieved adulteration detection limits of 0.05-0.5% (w/w), while the lateral flow format showed 0.05-5% (w/w) depending on the species. The method was verified using commercially processed products. This laboratory-validated strategy simplifies primer design and provides a promising platform for the qualitative screening of multiple meat targets, while further validation is required to assess its field robustness.},
}

*Meat/analysis/classification
Animals
*Food Contamination/analysis
DNA Primers/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
Swine

@article {pmid41772232,
year = {2026},
author = {Tinoco, AI and Henderson, CF and Meier, EK and Swinhoe, N and Cleves, PA},
title = {Efficient genome editing using CRISPR-Cas9 in reef-building corals.},
journal = {Nature protocols},
volume = {21},
number = {6},
pages = {2851-2879},
pmid = {41772232},
issn = {1750-2799},
support = {2128073//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Anthozoa/genetics ; *CRISPR-Cas Systems ; Coral Reefs ; RNA, Guide, CRISPR-Cas Systems/genetics ; Microinjections ; },
abstract = {Coral reefs are one of the most biodiverse and productive ecosystems on Earth. However, corals are currently under threat from increasing ocean temperatures driven by climate change. Despite the known importance of these fragile ecosystems, our understanding of the molecular mechanisms driving ecologically important traits has been constrained by a lack of genetic tools for functional characterization. To address this limitation, we have developed straightforward and efficient methods to genetically modify corals and study gene function throughout various life history stages using CRISPR-Cas9-based mutagenesis. In this protocol, we first describe how to spawn and collect gametes from the coral Acropora millepora during seasonal spawning events. Next, we describe a method for microinjection of one-cell coral zygotes with CRISPR-Cas9 reagents. We include considerations about effective single-guide RNA design, methods for identifying successfully injected animals, strategies for rearing mutant larvae and juveniles, and methods for the detection and quantification of genomic modifications. This protocol is currently the only way to perform gene editing in corals and takes ~2-4 weeks to complete and has been successfully applied to study genes controlling heat tolerance in coral larvae and skeleton formation in coral juveniles. These technical advances set the foundation for a new field using reverse genetics to study ecologically important traits in corals, such as the establishment of symbiosis and its breakdown upon heat stress.},
}

Animals
*Anthozoa/genetics
*CRISPR-Cas Systems
Coral Reefs
RNA, Guide, CRISPR-Cas Systems/genetics
Microinjections

@article {pmid41935970,
year = {2026},
author = {Chen, YW and Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC},
title = {Mismatch-Driven CRISPR/Cas12a Biosensing of UV-Induced DNA Lesions for Environmental Solar Exposure Surveillance.},
journal = {Environmental science & technology},
volume = {60},
number = {22},
pages = {15930-15939},
doi = {10.1021/acs.est.5c12461},
pmid = {41935970},
issn = {1520-5851},
mesh = {*Ultraviolet Rays ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *DNA Damage ; Sunlight ; },
abstract = {Monitoring environmentally relevant ultraviolet (UV) radiation is critical for understanding its biological impacts on ecosystems and human health. However, conventional UV dosimeters lack the molecular sensitivity to detect DNA-level damage that initiates such effects. Here, we present a CRISPR/Cas12a-based biosensing platform capable of quantifying solar UV exposure through the detection of UV-induced thymine dimers in DNA activators. This system harnesses mismatch-driven suppression of Cas12a activity, enabling a reduction in the fluorescence signal in response to UV-induced molecular lesions. The impact of thymine arrangement and the dimerization position of the activators on sensitivity were investigated. UV-induced diminution in Cas12a's trans-cleavage efficiency (kcat/Km) was also characterized, revealing a 1.67-fold decrease as the UVB dose increased from 0 to 2 J/cm[2]. Under optimized conditions, the sensor achieved a detection limit of 0.029 J/cm[2] for UVB and demonstrated high sensitivity to UVC. Field validation under natural sunlight showed a strong correlation with reference radiometric measurements, validating the biosensor's accuracy and environmental relevance. The system's sensitivity to low lesion densities, straightforward mechanism, and simple operation highlights its potential for environmental surveillance, human health risk assessment, and ecological monitoring in response to solar UV radiation.},
}

*Ultraviolet Rays
*Biosensing Techniques
*CRISPR-Cas Systems
*DNA Damage
Sunlight

@article {pmid41962729,
year = {2026},
author = {Liu, Y and Chen, S and Zhang, C and Xue, H and Abubakar, MU and Yin, S and Yang, X and Fan, B and Tai, P and Xiong, M and Li, J and He, B},
title = {Asy-RPA/PCR combined with One-crRNA-CRISPR/Cas12a for simultaneous detection of multiple Clarithromycin resistance mutations in Helicobacter pylori.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {74},
number = {},
pages = {102942},
doi = {10.1016/j.nano.2026.102942},
pmid = {41962729},
issn = {1549-9642},
mesh = {*Helicobacter pylori/genetics/drug effects ; *Clarithromycin/pharmacology ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Drug Resistance, Bacterial/genetics ; *Polymerase Chain Reaction/methods ; Humans ; Anti-Bacterial Agents/pharmacology ; Polymorphism, Single Nucleotide ; Rapid Diagnostic Tests ; Helicobacter Infections/microbiology/drug therapy/genetics ; },
abstract = {METHODS: Genetic testing for Clarithromycin resistance-associated single-nucleotide variations (SNVs) in H. pylori could be applied for formulating individual eradication plan. In this study, we integrated asymmetric recombinase polymerase amplification (Asy-RPA) with a single crRNA for CRISPR/Cas12a-designated the ARoRC system-to circumvent protospacer adjacent motif (PAM) dependency.

RESULTS: The ARoRC platform detected all targeted mutations with 100% agreement compared to Sanger sequencing. Assay sensitivity was determined as follows: A2143G (10[-2] ng/μL), A2142C (2.58 × 10[-3] ng/μL), A2142G (2.49 × 10[-3] ng/μL), and A2142G + A2143G (2.39 × 10[-3] ng/μL), enabling Asy-PCR-CRISPR/Cas12a detection suitable for fecal samples. The assay achieved visual results within 1 h using lateral flow strips, with no cross-reactivity to WT or non-target sequences.

DISCUSSION: We developed a rapid, ultrasensitive, and portable assay for detecting Clarithromycin resistance-associated mutations in H. pylori. The robustness of the platform in complex matrices such as feces, along with its dual readout capability (fluorescence and lateral flow), supports its potential for point-of-care (POC) application.},
}

*Helicobacter pylori/genetics/drug effects
*Clarithromycin/pharmacology
*CRISPR-Cas Systems/genetics
*Mutation
*Drug Resistance, Bacterial/genetics
*Polymerase Chain Reaction/methods
Humans
Anti-Bacterial Agents/pharmacology
Polymorphism, Single Nucleotide
Rapid Diagnostic Tests
Helicobacter Infections/microbiology/drug therapy/genetics

@article {pmid41974708,
year = {2026},
author = {Lai, S and Keller, MP and Zhang, J and Fang, Z and Xie, Y and Weng, C and Zhang, S and Zhang, S and Gao, P and Ke, L and Wang, Y and Mitok, KA and Clark, L and Schueler, KL and Liu, H and Hatipoglu, B and Hatzoglou, M and Chen, Y and Shalev, A and Jin, F and Attie, AD and Li, Y},
title = {Proinsulin regulators identified with CRISPR screen and in vivo mouse QTL mapping.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41974708},
issn = {2041-1723},
support = {R01 DK131437/DK/NIDDK NIH HHS/United States ; R01HG012384//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01 HG012384/HG/NHGRI NIH HHS/United States ; R01DK131437//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; P30 AG092586/AG/NIA NIH HHS/United States ; R01CA267872//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01HG009658//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01DK113185//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; R01 DK113185/DK/NIDDK NIH HHS/United States ; UG3NS132061//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *Quantitative Trait Loci/genetics ; *Proinsulin/metabolism/blood/genetics ; Mice ; Golgi Apparatus/metabolism ; Humans ; Protein Disulfide-Isomerases/genetics/metabolism ; Insulin-Secreting Cells/metabolism ; Insulin/metabolism ; Chromosome Mapping ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exocytosis ; Cell Line ; Secretory Vesicles/metabolism ; },
abstract = {Altered proinsulin levels in β-cells and bloodstream are hallmarks of diabetes and other diseases, but our knowledge about the proinsulin regulators remains limited. Here we perform a genome-wide CRISPR screen to identify 84 proinsulin regulators that alter intracellular proinsulin/insulin ratio in a mouse β-cell line. The proinsulin regulators are distinct from the insulin regulators from a previous orthogonal CRISPR screen. Functional annotation of the proinsulin regulators highlights Golgi as the primary organelle for proinsulin storage and regulation. Trafficking towards the Golgi increases the intra-cellular proinsulin/insulin ratio, while trafficking away from the Golgi, including exocytosis and Golgi-to-ER retrograde transport, decreases the intracellular proinsulin levels. We also map mouse quantitative trait loci (QTLs) associated with plasma proinsulin levels and use the CRISPR screen results to pinpoint the causal genes within the QTL loci. Interestingly, protein disulfide isomerase Pdia6 is the strongest hit from both CRISPR screen and the in vivo QTL mapping. Knocking down Pdia6 significantly reduce proinsulin accumulation in Golgi and secretory granules. Intriguingly, Pdia6-depletion in both human and mouse β-cells does not affect the folding status of proinsulin but causes significantly impaired proinsulin production through a UPR-independent mechanism. Taken together, our genetic profiles provide mechanistic insights into the regulation of proinsulin/insulin homeostasis.},
}

Animals
*Quantitative Trait Loci/genetics
*Proinsulin/metabolism/blood/genetics
Mice
Golgi Apparatus/metabolism
Humans
Protein Disulfide-Isomerases/genetics/metabolism
Insulin-Secreting Cells/metabolism
Insulin/metabolism
Chromosome Mapping
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Exocytosis
Cell Line
Secretory Vesicles/metabolism

@article {pmid42010284,
year = {2026},
author = {Zhao, J and Zhang, J and Gao, M and Miao, Z and Zhang, Y and Guo, Y and Fan, Z and Tian, J and Yang, L and Jiang, N and Ma, J and Jiao, J and Pan, J and Ma, X},
title = {Photoactivatable CRISPR/Cas13d via upconversion nanoparticles for deep tissue RNA engineering and orthopedic therapy.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42010284},
issn = {2041-1723},
support = {82572860//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82102639//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302347//National Natural Science Foundation of China (National Science Foundation of China)/ ; 25JCYBJC01510, 22JCQNJC00850//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 25YDTPJC00330//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Animals ; Mice ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; *Tissue Engineering/methods ; Polyethyleneimine/chemistry ; *RNA/genetics ; Blue Light ; Bone and Bones ; Osteocytes/metabolism ; },
abstract = {Spatiotemporal control of RNA therapeutics remains a fundamental challenge limiting clinical translation. Here, we develop a photoactivatable CRISPR/Cas13d (paCas13d) system that enables non-invasive, light-controlled RNA manipulation in deep tissues. Through structure-guided engineering, we identify optimal split sites within RfxCas13d and create light-switchable fragments using CRY2PHR/CIBN optogenetic dimerization. To overcome the limited tissue penetration of blue light, we engineer polyethylenimine-functionalized upconversion nanoparticles (UCNPs-PEI) that serve dual roles as gene carriers and photon transducers, converting tissue-penetrating near-infrared (NIR) to blue light. The UCNPs-PEI@paCas13d system achieves precise spatiotemporal control of RNA targeting within bone tissue in vivo. In a murine steroid-associated osteonecrosis model, NIR-activated paCas13d achieves robust TET3 knockdown, disrupting the TET3-5hmC-PTEN axis that drives glucocorticoid-induced osteocyte apoptosis. This targeted intervention prevents bone deterioration, with treated mice showing preserved trabecular architecture, enhanced bone volume, and favorable shifts in bone turnover markers, while maintaining systemic glucocorticoid efficacy. Our platform combines the programmability of CRISPR/Cas13d with non-invasive optical control, offering a versatile approach for treating diseases requiring localized RNA modulation while minimizing systemic effects.},
}

Animals
Mice
*Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
Humans
*Tissue Engineering/methods
Polyethyleneimine/chemistry
*RNA/genetics
Blue Light
Bone and Bones
Osteocytes/metabolism

@article {pmid42080370,
year = {2026},
author = {Wang, Z and Li, J and Yue, Z and He, B and Zhang, W and Fang, Z and Xia, Q and Liu, Y and Li, Y},
title = {A Modular and Programmable Cas13d Platform for RNA Single Nucleotide Variant Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {32},
pages = {e23680},
pmid = {42080370},
issn = {2198-3844},
support = {32471528//National Natural Science Foundation of China/ ; 82404929//National Natural Science Foundation of China/ ; zz-RCPY-23-25//State Key Laboratory of Systems Medicine for Cancer/ ; zz-94-25-22//State Key Laboratory of Systems Medicine for Cancer/ ; SB2023-13//State Key Laboratory of Systems Medicine for Cancer/ ; zz-GZR-25-09//State Key Laboratory of Systems Medicine for Cancer/ ; 23QC1400600 to Y.L//Shanghai Rising-Star Program/ ; DFYC-LYJ2022 to Y.L//Shanghai Oriental Talent Youth Program/ ; 10000015Z155080000004//National Clinical Key Specialty Construction Project/ ; YG2023QNA09//Shanghai Jiao Tong University Star Program Medical, Industrial Cross Research Fund/ ; QiankeherencaiXKBF[2025]025)//Guizhou Provincial Science and Technology Projects/ ; XK202401//Jiading district medical key discipline construction project/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/genetics ; },
abstract = {CRISPR-based nucleic acid diagnostics have shown broad potential, yet reliable single-nucleotide variant (SNV) discrimination remains limited by flanking sequence requirements that constrain targetability, and an inherent specificity-sensitivity trade-off where mismatch designs used to suppress wild type recognition often penalize enzymatic activity. Here we develop a scenario-guided Cas13d framework that supports pre-defined operating modes tailored to distinct analytical goals. Leveraging the minimal protospacer flanking site constraints of Cas13d, we first map mismatch-sensitive windows to derive rule-based crRNA designs that improve allelic discrimination. We then restore assay performance through structure-guided engineering of a miniaturized Cas13d scaffold by internally inserting auxiliary RNA binding domains (RBDs). Systematic benchmarking across representative oncology hotspots delineates two practical regimes comprising an ultra-sensitive, amplification-free mode in which a dual-RBD variant paired with optimized mismatched crRNAs achieves ∼0.6% variant allele fraction (VAF) detection, and a robust amplified mode incorporating optional loop-mediated isothermal amplification coupling that favors simpler architectures to balance performance and background across broader low-VAF ranges. In an evaluation of 45 clinical tumor RNA specimens spanning pancreatic, cholangiocarcinoma, and colorectal cancers, the assay correctly classified mutation status with full concordance for KRAS G12D, IDH1 R132C and BRAF V600E, with a subset of positive cases corroborated by orthogonal RT-ddPCR. A prospective IDH1 R132C clinical-matrix spike-in further supported sub-1% detection without pre-amplification. Collectively, this work establishes a configurable Cas13d toolkit and a rule-guided strategy for deploying CRISPR-based RNA SNV diagnostics with application-specific performance objectives.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Cas Systems/genetics
*RNA/genetics

@article {pmid42172545,
year = {2026},
author = {Jiang, Y and Wen, H and Xu, J and Peng, W and Zhou, J and Mao, H and Gu, Z and He, Y},
title = {Dual-Gene CRISPR Editing via Peptide Dendrimers Regulates Redox Balance for Diabetic Wound Repair.},
journal = {Biomacromolecules},
volume = {27},
number = {6},
pages = {3647-3661},
doi = {10.1021/acs.biomac.6c00110},
pmid = {42172545},
issn = {1526-4602},
mesh = {Animals ; *Wound Healing/genetics/drug effects ; Mice ; *Dendrimers/chemistry/pharmacology ; Oxidation-Reduction ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Oxidative Stress ; Kelch-Like ECH-Associated Protein 1/genetics/antagonists & inhibitors ; NF-E2-Related Factor 2/genetics/metabolism ; *Peptides/chemistry ; *Diabetes Mellitus, Experimental/genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Nanoparticles/chemistry ; Genetic Therapy/methods ; Hypoxia-Inducible Factor-Proline Dioxygenases/genetics ; },
abstract = {The management of chronic diabetic wounds, plagued by persistent oxidative stress, remains a major clinical challenge. We devised a CRISPR/Cas9-based gene therapy to fundamentally reprogram this pathological microenvironment. A single system was engineered for the simultaneous knockdown of Keap1 and PHD2, key negative regulators of the Nrf2 and HIF-1α pathways, respectively. This payload was delivered by multifunctional peptide-modified lysine dendrimers (MsRNPs), which self-assembled into stable, positively charged nanoparticles that effectively complexed with DNA. The MsRNPs showed excellent biocompatibility and mediated efficient cellular uptake and gene editing in vitro, leading to reduced ROS levels. Consequently, a single topical application of the polyplexes in a diabetic mouse model robustly accelerated wound closure, enhanced collagen deposition, and promoted angiogenesis, driven by the synergistic activation of Nrf2 and HIF-1α. This study establishes a novel combinatorial gene-editing strategy and a versatile nanoplatform for treating oxidative stress-related pathologies.},
}

Animals
*Wound Healing/genetics/drug effects
Mice
*Dendrimers/chemistry/pharmacology
Oxidation-Reduction
*Gene Editing/methods
*CRISPR-Cas Systems
Oxidative Stress
Kelch-Like ECH-Associated Protein 1/genetics/antagonists & inhibitors
NF-E2-Related Factor 2/genetics/metabolism
*Peptides/chemistry
*Diabetes Mellitus, Experimental/genetics
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism
Nanoparticles/chemistry
Genetic Therapy/methods
Hypoxia-Inducible Factor-Proline Dioxygenases/genetics

@article {pmid42190792,
year = {2026},
author = {Zhang, YH and Yuan, Y and Chen, BT and Yang, N and Chen, TJ and Lin, YT and Na, XM and Wang, SH and Xiong, YN and Zhu, MX and Chen, LZ and Mokwatlo, SC and Ouyang, P and Ling, C},
title = {Engineering complex phenotypes in Halomonas bluephagenesis TD01 via large-fragment manipulation and multiplex base editing.},
journal = {Metabolic engineering},
volume = {96},
number = {},
pages = {405-419},
doi = {10.1016/j.ymben.2026.05.008},
pmid = {42190792},
issn = {1096-7184},
mesh = {*Halomonas/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Polyhydroxyalkanoates/biosynthesis/genetics ; },
abstract = {Halomonas bluephagenesis is a representative platform strain of next generation industrial biotechnology (NGIB), enabling contamination-resistant open fermentation due to inherent tolerance to high salinity and alkalinity. However, progress in strain development has been constrained by limited genome engineering tools, particularly for large-fragment manipulation and multiplex base editing. Herein, we developed a counterselection marker-based single-plasmid system (pHaloFM) that leverages native homologous recombination to enable sequential insertion of fragments up to 8 kb and deletion of regions up to 50 kb. Additionally, we re-engineered a CRISPR/nCas9-assisted cytidine base editor system (pHaloBE) through host-specific adaptations, achieving multiplex editing of nine target sites. These tools were applied to engineer cellular morphology in one step, and successively construct polyhydroxyalkanoate (PHA) copolymers P34HB and PHBV biosynthetic pathways. This integrated toolkit resolves long-standing genetic manipulation bottlenecks in H. bluephagenesis and provides a systematic framework for engineering complex phenotypes in other non-model organisms.},
}

*Halomonas/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Metabolic Engineering/methods
Polyhydroxyalkanoates/biosynthesis/genetics

@article {pmid42202254,
year = {2026},
author = {Liu, Z and Zhang, R and Chang, C and Xu, D and Mao, D and Zhu, X and Xu, H},
title = {Detection of Ultralow-Frequency ctDNA Mutations Using a Dual Hairpin-Competition CRISPR/Cas14a System.},
journal = {Analytical chemistry},
volume = {98},
number = {22},
pages = {16682-16693},
doi = {10.1021/acs.analchem.6c02495},
pmid = {42202254},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/genetics/blood ; *Mutation ; Polymerase Chain Reaction ; },
abstract = {Circulating tumor DNA (ctDNA) mutation profiling is essential for guiding targeted therapy and monitoring cancer recurrence, yet its clinical adoption is constrained by overwhelming wild-type DNA background and the limited sensitivity of existing platforms. Here, we introduce a dual hairpin-competition CRISPR/Cas14a (DHCC) system that integrates two sequential layers of hairpin competition: selective enrichment of mutant DNA during asymmetric PCR, followed by suppression of nonspecific sgRNA binding during Cas14a detection. This design dramatically enhances mutant-wild-type discrimination, elevating the discrimination factor from 2.48 to 145─a 58-fold improvement. While previous Cas14a methods achieve detection limits of 0.5-0.1% variant allele frequency (VAF), DHCC delivers a 250-fold sensitivity gain, routinely detecting four clinically relevant mutations (EGFR T790M, L858R, G719A, and NRAS Q61K) at VAFs as low as 0.002%. In multiplexed format, sensitivities of 0.005-0.01% VAF are maintained. Clinical validation using 22 plasma ctDNA samples demonstrated 100% concordance with droplet digital PCR for EGFR L858R detection. Compared to ddPCR and next-generation sequencing, DHCC substantially reduces turnaround time and cost while operating on standard qPCR instruments, eliminating the need for specialized infrastructure. By combining ultrahigh sensitivity, PAM independence, multiplexing preamplification capability, and practical affordability, DHCC provides an accessible platform for ctDNA-based liquid biopsy in clinical settings.},
}

Humans
*CRISPR-Cas Systems/genetics
*Circulating Tumor DNA/genetics/blood
*Mutation
Polymerase Chain Reaction

@article {pmid42212930,
year = {2026},
author = {Zhang, Y and Hao, L and Li, Q and Zhou, Z and Liu, D and Qiu, H and Yang, W and Zhang, B},
title = {Ultrasensitive Wash-Free Homogeneous CRISPR Assay Using Spatial Proximity Chemiluminescence Reporter.},
journal = {ACS nano},
volume = {20},
number = {22},
pages = {16001-16015},
doi = {10.1021/acsnano.5c22763},
pmid = {42212930},
issn = {1936-086X},
mesh = {*Luminescent Measurements/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reactive Oxygen Species/metabolism ; Limit of Detection ; Luminescence ; },
abstract = {CRISPR-based diagnostics are promising platforms for point-of-care (POC) testing, but are often hindered by limited sensitivity and complex workflows. Here, we present a spatial proximity chemiluminescence (SPC) reporter that converts CRISPR-Cas12a trans-cleavage activity into a highly sensitive, excitation-free signal. Mechanistically, the intact SPC reporter ensures efficient intramolecular reactive oxygen species (ROS) transfer by spatially confining a catalytic donor and a luminescent acceptor. Upon target-activated Cas12a cleavage, this spatial proximity is disrupted, drastically attenuating ROS transfer and reducing oxidized luminescent acceptors for chemiluminescence. The SPC-CRISPR exhibits an attomolar-level limit of detection with an optimal nearly 50,000-fold sensitivity enhancement, and features an 8-log dynamic range suitable for target quantification. This platform exhibits robust resistance to matrix interference, ensuring high accuracy while requiring only minimal sample preprocessing. We demonstrate SPC-CRISPR is compatible with recombinase polymerase amplification to develop a single-tube reaction with a sensitivity of 1 copy/μL. Extensive clinical validation demonstrated 99.2% overall accuracy for HPV16 identification in 126 cervical swabs, alongside 86.4% accuracy for miR-19a profiling in 22 bladder cancer plasma samples. Furthermore, this wash-free homogeneous workflow is embedded in a portable and sealed microfluidic-based device for sample-to-result diagnostics, showing 100% concordance with qPCR. SPC-CRISPR integrates enhanced sensitivity and simplified operation, holding great potential for POC molecular diagnostics.},
}

*Luminescent Measurements/methods
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Reactive Oxygen Species/metabolism
Limit of Detection
Luminescence

@article {pmid42214852,
year = {2026},
author = {Xiao, S and Song, J and Chen, H and Yin, W and Yan, X and Hu, K and Shi, J and Yang, M},
title = {Digital droplet microfluidics integrating DNA walkers and CRISPR-Cas13a for simultaneous surface protein and miRNA profiling in single exosomes.},
journal = {Biosensors & bioelectronics},
volume = {310},
number = {},
pages = {118854},
doi = {10.1016/j.bios.2026.118854},
pmid = {42214852},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *MicroRNAs/genetics/isolation & purification/analysis ; *Erb-b2 Receptor Tyrosine Kinases/genetics/isolation & purification ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; *Epithelial Cell Adhesion Molecule/genetics/isolation & purification/analysis ; DNA/chemistry ; Equipment Design ; },
abstract = {Tumor-derived exosomes carry multi-scale molecular signatures (e.g., surface proteins and nucleic acids) that reflect tumor heterogeneity, yet simultaneously profiling these biomarkers in single intact vesicles remains technically challenging. Herein, we developed a digital droplet microfluidic platform that integrates a DNA walker and a CRISPR/Cas13a system for the simultaneous detection of surface proteins (EpCAM, HER2) and miRNA (miR-21) at the single exosome level. This platform employed engineered liposome nanoprobes (eLipo-NPs) with EpCAM aptamers and hairpin probes (HPs) functionalized on their outer membranes, and encapsulated a CRISPR/Cas13a system within their lumen. Upon co-encapsulation with single exosomes into droplets, EpCAM-mediated membrane fusion redistributed HPs across the hybrid membrane and delivered CRISPR/Cas13a into the exosomes. The membrane-anchored DNA walker then bound HER2 and drove cyclic DNAzyme cleavage of HPs to restore red fluorescence. At the same time, crRNA-guided Cas13a recognized miR-21 and triggered trans-cleavage of reporters to generate green fluorescence. Digital counting of dual-positive droplets enabled quantitative single-exosome analysis with a limit of detection (LOD) of 10 particles/μL and a detection time of 60 min. Clinical validation using plasma-derived exosomes from 24 breast cancer patients and 14 healthy donors demonstrated distinct distributions among HER2-positive, HER2-negative, and healthy control groups, with the percentage of dual-positive droplets significantly correlated with clinical HER2 status, highlighting the platform's potential for liquid biopsy and precision oncology.},
}

Humans
*Exosomes/chemistry/genetics
*MicroRNAs/genetics/isolation & purification/analysis
*Erb-b2 Receptor Tyrosine Kinases/genetics/isolation & purification
CRISPR-Cas Systems/genetics
*Biosensing Techniques/instrumentation
*Epithelial Cell Adhesion Molecule/genetics/isolation & purification/analysis
DNA/chemistry
Equipment Design

@article {pmid42217942,
year = {2026},
author = {Chen, B and Yang, H and Zhao, J and Wang, Y and Li, H and Wang, C and Guo, L and Xu, J},
title = {Template-independent poly-adenine elongation enables multivalent CRISPR/Cas12a activation for amplified lateral flow biosensing.},
journal = {Talanta},
volume = {309},
number = {},
pages = {130054},
doi = {10.1016/j.talanta.2026.130054},
pmid = {42217942},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *DNA Nucleotidylexotransferase/metabolism/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Associated Proteins/metabolism ; *Poly A/metabolism/chemistry ; Limit of Detection ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that plays a critical role in immune system development and serves as an important biomarker for acute lymphoblastic leukemia. However, current methods for TdT activity analysis often rely on sophisticated instrumentation and lack simple and portable detection formats. Herein, we report a TdT-enabled multivalent CRISPR/Cas12a lateral flow assay for sensitive and instrument-free detection of TdT activity. In this strategy, TdT-catalyzed poly-adenine (poly-A) extension converts enzymatic activity into adenine-rich DNA scaffolds, which recruit multiple crRNA molecules to trigger multivalent activation of Cas12a. This design effectively bridges TdT activity with CRISPR/Cas12a signal amplification. The activated Cas12a subsequently induces trans-cleavage of a reporter probe, and the cleavage event is translated into a visual signal on a lateral flow strip. The proposed assay enables sensitive detection of TdT with a limit of detection of 0.016 U/mL and a visual detection limit of 0.05 U/mL. In addition, the assay exhibits high specificity toward TdT over other polymerases and demonstrates satisfactory performance in human serum samples with recoveries ranging from 98.8% to 103.7%. This work expands the applicability of CRISPR/Cas12a systems to enzyme activity sensing and provides a simple and practical platform for point-of-care detection of TdT.},
}

*CRISPR-Cas Systems
*DNA Nucleotidylexotransferase/metabolism/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Associated Proteins/metabolism
*Poly A/metabolism/chemistry
Limit of Detection
Rapid Diagnostic Tests
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism/genetics

@article {pmid42229577,
year = {2026},
author = {Saberian, M and Roosta, A and Afrisham, R},
title = {CRISPR-dCas9 epigenetic reprogramming in cancer: platforms, immuno-modulation and delivery challenges.},
journal = {Gene},
volume = {1005},
number = {},
pages = {150246},
doi = {10.1016/j.gene.2026.150246},
pmid = {42229577},
issn = {1879-0038},
mesh = {Humans ; *Neoplasms/genetics/therapy/immunology ; *CRISPR-Cas Systems/genetics ; Epigenome Editing ; *Epigenesis, Genetic ; Immunotherapy/methods ; Animals ; *Gene Editing/methods ; Cellular Reprogramming/genetics ; },
abstract = {CRISPR-dCas9 (catalytically dead Cas9) has revolutionized targeted epigenetic editing, offering locus-specific modulation of gene expression without altering DNA sequence. Beyond conventional approaches, novel strategies are rapidly emerging. These include combinatorial epigenetic reprogramming (co-recruiting multiple chromatin modifiers to a single locus), precision enhancer targeting (modulating oncogenic cis-regulatory elements), epigenetic modulation of immune pathways (reprogramming tumor or immune cells to boost anti-tumor immunity), and next-generation delivery systems for dCas9-based tools. This review synthesizes peer-reviewed literature (2015-2025) to highlight promising, yet still preclinical, advances in combinatorial reprogramming, enhancer targeting, immune-modulatory epigenetic approaches and delivery strategies, and to identify gaps that must be addressed prior to clinical translation. We highlight multi-effectors platforms (e.g. SunTag-like arrays, SSSavi modular docking, CRISPRoff memory writers) that amplify and diversify chromatin modifications. Precision enhancer editing systems (e.g. enCRISPRa/enCRISPRi) enable direct activation or silencing of distal regulatory elements in cancer cells. Epigenetic immunotherapy approaches use dCas9-activators to upregulate NK/T-cell ligands (MICA/MICB) and antigen-presentation genes (MHC I/II) in tumor cells. Finally, we survey innovations in dCas9 delivery that address in vivo challenges. Our review critically evaluates these advances, identifies gaps (off-target effects, context-dependence), and outlines future directions toward precision epigenetic therapies for diverse cancers.},
}

Humans
*Neoplasms/genetics/therapy/immunology
*CRISPR-Cas Systems/genetics
Epigenome Editing
*Epigenesis, Genetic
Immunotherapy/methods
Animals
*Gene Editing/methods
Cellular Reprogramming/genetics

@article {pmid42242804,
year = {2026},
author = {Qian, J and Lu, J and Chen, X and Shen, H and Lu, F},
title = {Programmable Fc-encoded DNA tile-cube capture enables a thrombin-activated ratiometric ECL/SERS biosensor via a PAM-engineered toehold switch and CRISPR/Cas12a cleavage.},
journal = {Analytica chimica acta},
volume = {1415},
number = {},
pages = {345709},
doi = {10.1016/j.aca.2026.345709},
pmid = {42242804},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Thrombin/analysis ; *CRISPR-Cas Systems/genetics ; Spectrum Analysis, Raman ; Electrochemical Techniques/methods ; *DNA/chemistry ; Humans ; Luminescent Measurements ; Electrodes ; *CRISPR-Associated Proteins/metabolism/chemistry ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; Dendrimers ; },
abstract = {BACKGROUND: Accurate thrombin detection is important for coagulation-related assessment, but reliable quantification at ultralow levels remains challenging because matrix interference, electrode-to-electrode variation, and single-channel signal drift can compromise analytical accuracy. Herein, we developed a thrombin-responsive ratiometric electrochemiluminescence/surface-enhanced Raman scattering (ECL/SERS) biosensor integrating Fc-encoded DNA tiles, a DNA-cube capture scaffold, and a PAM-engineered toehold-switch-regulated CRISPR/Cas12a module on a Ti3C2/CsPbBr3@PDA@Au-modified electrode.

RESULTS: An intentionally cleavable linker probe (LP) serves as the bridge for retaining Fc-rich DNA tiles near the electrode. Without thrombin, intact LP enables tile capture, causing ECL quenching and strong Fc SERS output. With thrombin, split-aptamer proximity assembly activates the toehold switch and Cas12a/crRNA, leading to LP cleavage, Fc-tile depletion, ECL recovery, and SERS attenuation. The anti-correlated signals were integrated as Q = IECL/ISERS. The biosensor showed a detection range from 1 × 10[-7] to 1 × 10[-1] nM and a detection limit of approximately 0.064 fM. Synthetic cleaved LP standards confirmed that LP cleavage can be directly converted into ratiometric ECL/SERS switching. Serum spike-recovery tests gave recoveries of 96.8%-104.0%.

SIGNIFICANCE: This work establishes a programmable capture-release strategy that converts thrombin recognition into CRISPR/Cas12a-mediated LP cleavage and deterministic interfacial reconfiguration, providing a sensitive, internally referenced, and extensible platform for protein biosensing.},
}

*Biosensing Techniques/methods
*Thrombin/analysis
*CRISPR-Cas Systems/genetics
Spectrum Analysis, Raman
Electrochemical Techniques/methods
*DNA/chemistry
Humans
Luminescent Measurements
Electrodes
*CRISPR-Associated Proteins/metabolism/chemistry
Aptamers, Nucleotide/chemistry
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
Dendrimers

@article {pmid41709520,
year = {2026},
author = {Kulshreshtha, A and Ramasamy, M and Irigoyen, S and Padilla, CS and Tu, CK and Laughlin, K and Borneman, J and Mandadi, KK},
title = {High-efficiency genome-editing, transgene evaluation, and antimicrobial efficacy testing using Citrus medica L. hairy roots.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {4},
pages = {e70745},
pmid = {41709520},
issn = {1365-313X},
support = {2025-70029-44033//National Institute of Food and Agriculture/ ; 2024-51181-43464//National Institute of Food and Agriculture/ ; 2021-70029-36056//National Institute of Food and Agriculture/ ; 2019-70016-29066//National Institute of Food and Agriculture/ ; HATCH TEX09621//National Institute of Food and Agriculture/ ; TEX0-7790//National Institute of Food and Agriculture/ ; //Texas A&M AgriLife Institute for Advancing Health Through Agriculture/ ; //Texas A&M Vegetable and Fruit Improvement Center/ ; 124190-96210//Texas A&M AgriLife Research Insect-vectored Disease Seed Grants/ ; },
mesh = {*Citrus/genetics/microbiology ; *Plant Roots/genetics/microbiology ; Plants, Genetically Modified/genetics ; *Plant Diseases/microbiology ; *Gene Editing/methods ; Transformation, Genetic ; Transgenes/genetics ; Disease Resistance/genetics ; Liberibacter ; CRISPR-Cas Systems ; Anti-Infective Agents/pharmacology ; Rhizobiaceae ; },
abstract = {Huanglongbing (HLB) disease, associated with the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), has a significant impact on citrus production worldwide. Conventional biochemical and genetic evaluation studies to identify potential disease resistance strategies have been mainly hindered due to the inability to culture CLas in a defined medium and the general recalcitrance of Citrus cultivars (grapefruits and oranges) to Agrobacterium-mediated plant transformation. We previously demonstrated the utility of plant hairy roots to co-cultivate CLas. In this study, we developed a hairy root transformation system using citron (Citrus medica L.), which is highly amenable to Rhizobium-mediated hairy root transformation. The explant survival and hairy root transformation efficiencies were up to 100% and 73%, respectively, and transgenic roots can be attained in as little as 30-60 days. We demonstrate the utility of this citron-based hairy root transformation for rapid CRISPR/Cas9-mediated gene editing, transgene evaluation, and antimicrobial efficacy testing. The citron-based hairy root transformation system will significantly help the research community to speed-track the assessment of potential HLB disease resistance strategies.},
}

*Citrus/genetics/microbiology
*Plant Roots/genetics/microbiology
Plants, Genetically Modified/genetics
*Plant Diseases/microbiology
*Gene Editing/methods
Transformation, Genetic
Transgenes/genetics
Disease Resistance/genetics
Liberibacter
CRISPR-Cas Systems
Anti-Infective Agents/pharmacology
Rhizobiaceae

@article {pmid41772360,
year = {2026},
author = {Guo, Y and Yu, Z and Fan, S and Zhu, M and Ci, L and Yang, X and Chen, Y and Li, Q and Wang, N and Wang, J and Ye, S and Wang, J and Sun, R and Shen, R},
title = {A Bioluminescence Reporter Mouse Strain for In Vivo Imaging of IFNγ Cell Localization and Function.},
journal = {Immunology},
volume = {178},
number = {3},
pages = {428-438},
doi = {10.1111/imm.70127},
pmid = {41772360},
issn = {1365-2567},
support = {24141901100//Science and Technology Innovation Plan of Shanghai Science and Technology Commission/ ; 24YF2732000//Science and Technology Innovation Plan of Shanghai Science and Technology Commission/ ; 2025NS04//Shanghai Laboratory Animal Research Center/ ; 2025NS05//Shanghai Laboratory Animal Research Center/ ; },
mesh = {Animals ; *Interferon-gamma/genetics/metabolism/immunology ; Mice, Inbred C57BL ; *Luminescent Measurements/methods ; Mice, Transgenic ; Mice ; *Genes, Reporter ; Luciferases/genetics ; Disease Models, Animal ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Humans ; Promoter Regions, Genetic ; },
abstract = {Interferon gamma (IFNγ) is a pivotal inflammatory mediator and immune regulator, but its in vivo spatiotemporal dynamics and functional roles in inflammation and carcinogenesis remain incompletely understood. Here, we developed a C57BL/6J- Ifng-2A-luciferase knock-in mouse strain using CRISPR/Cas9-mediated homology-directed repair, enabling real-time bioluminescence imaging (BLI) of IFNγ-expressing cells by inserting a luciferase cassette under the endogenous Ifng promoter. The validation confirmed that this model is capable of directly detecting Poly(I:C) -induced transient IFNγ, enhancing intratumoral IFNγ signals upon anti-PD-1/CTLA-4 therapy, and dynamically tracking IFNγ expression during imiquimod-induced psoriasis. This transgenic mouse model provides a powerful tool for non-invasive, longitudinal tracking of IFNγ-expressing cells, offering novel insights into IFNγ-mediated immune regulation in inflammation and cancer. It holds promise for identifying IFNγ-related therapeutic targets and predicting responses to immunotherapies.},
}

Animals
*Interferon-gamma/genetics/metabolism/immunology
Mice, Inbred C57BL
*Luminescent Measurements/methods
Mice, Transgenic
Mice
*Genes, Reporter
Luciferases/genetics
Disease Models, Animal
CRISPR-Cas Systems
Gene Knock-In Techniques
Humans
Promoter Regions, Genetic

@article {pmid41865336,
year = {2026},
author = {An, H and Kim, H and Kim, DY and Yoon, HJ and Lee, JY and Eo, WK and Kim, MY and Kim, KH and Cha, HJ},
title = {Transcriptomic analysis of zonula occludens-1 (ZO-1) knockout in ovarian cancer cell lines.},
journal = {Genes & genomics},
volume = {48},
number = {6},
pages = {911-921},
pmid = {41865336},
issn = {2092-9293},
mesh = {Humans ; Female ; *Zonula Occludens-1 Protein/genetics/metabolism ; *Ovarian Neoplasms/genetics/metabolism/pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; *Transcriptome/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {BACKGROUND: Zonula occludens-1 (ZO-1) is a crucial tight junction protein that regulates intercellular permeability and adhesion, thereby preserving the integrity of epithelial and endothelial barriers. ZO-1 is associated with tumorigenesis and the progression of epithelial-mesenchymal transition (EMT), invasion, and metastasis. In our previous study, knockout (KO) of ZO-1 using clustered regularly interspaced short palindromic repeats (CRISPR) reduced proliferation but increased migration and invasion, suggesting that ZO-1 may have a dual role. Therefore, this study aimed to elucidate the role of ZO-1 in ovarian cancer by analyzing transcriptomic changes associated with ZO-1.

OBJECTIVE: This study aims to elucidate the impact of ZO-1 KO on gene expression in ovarian cancer cells by performing comparative RNA sequencing (RNA-seq) analysis on two distinct ZO-1 KO ovarian cancer cell lines, SKOV3 and SNU119.

METHODS: ZO-1 was knocked out in SKOV3 and SNU119 cells using CRISPR-Cas9 technology. After identifying differentially expressed genes (DEGs) through RNA sequencing, Gene Ontology (GO) and pathway enrichment analyses were performed. The selected targets were subsequently validated using reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis to assess both transcript- and protein-level expression changes.

RESULTS: Transcriptomic analysis revealed over 400 DEGs in each cell line. Of these, 14 genes were consistently upregulated in both cell lines, while 24 genes were consistently downregulated. The common DEGs were visualized using a heatmap, and a subset of these genes was further validated by RT-qPCR and Western blot analyses. TGFB2 expression was consistently altered at both the mRNA and protein levels following ZO-1 KO in both cell lines. Similar expression patterns were observed for THBS1, VCAN, ITGB8, SEMA3A, and GAS6. The concordant changes observed in transcriptomic and protein analyses suggest a consistent association between ZO-1 KO and TGFB2 expression.

CONCLUSION: ZO-1 KO in ovarian cancer cells induces substantial transcriptional reprogramming, particularly affecting genes associated with extracellular matrix organization and signaling pathways. Multiple candidate genes showed consistent alterations at both the mRNA and protein levels, supporting the robustness of the observed transcriptional changes. These findings provide a framework for understanding ZO-1-associated regulatory networks in ovarian cancer.},
}

Humans
Female
*Zonula Occludens-1 Protein/genetics/metabolism
*Ovarian Neoplasms/genetics/metabolism/pathology
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition/genetics
*Transcriptome/genetics
Gene Knockout Techniques
CRISPR-Cas Systems
Gene Expression Profiling

@article {pmid42023429,
year = {2026},
author = {Lin, TM and Chang, HF and Lin, TC and Lin, CH and Sun, YL and Lin, CS},
title = {Gene Therapy and Gene Editing in Type 1 Diabetes: CRISPR-Based β-Cell Replacement and Treg Immune Modulation Approaches.},
journal = {Diabetes, obesity & metabolism},
volume = {28},
number = {7},
pages = {5476-5491},
doi = {10.1111/dom.70800},
pmid = {42023429},
issn = {1463-1326},
support = {//Ministry of Education (MOE), Taiwan/ ; NSTC 114-2321-B-A49-001//National Science and Technology Council (NSTC), Taiwan/ ; },
mesh = {*Diabetes Mellitus, Type 1/therapy/immunology/genetics ; Humans ; *Genetic Therapy/methods ; *Insulin-Secreting Cells/immunology/transplantation ; *Gene Editing/methods ; *T-Lymphocytes, Regulatory/immunology ; Animals ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease marked by the destruction of pancreatic β-cells, resulting in lifelong dependence on exogenous insulin. Despite advances in insulin delivery and glucose monitoring technologies, patients remain at risk for acute and long-term complications, underscoring the need for curative strategies. Gene therapy and gene-editing technologies are emerging as transformative approaches capable of restoring β-cell function, modulating immune responses and potentially achieving durable remission.

METHOD: This review synthesizes basic science foundations and clinical trial evidence, focusing on five key protocols (NCT03162237, NCT05210530, NCT05241444, NCT05565248 and NCT06938334).

RESULTS: Strategies include immune modulation (PD-L1, FOXP3), β-cell replacement (CRISPR-edited progenitors, xenotransplantation) and combination approaches. Early-phase clinical trials have demonstrated feasibility and safety; however, long-term efficacy, durability and scalability remain uncertain. Critical challenges include potential off-target effects in CRISPR editing, risks of insertional mutagenesis, safety concerns in xenotransplantation and achieving a balance between immune tolerance and protective immunity. Future directions emphasize combination therapies, personalized medicine and next-generation editing tools such as base and prime editing.

CONCLUSION: Together, these efforts represent a paradigm shift from symptomatic insulin replacement toward curative interventions, while highlighting the considerable translational hurdles that must be overcome before routine clinical application.},
}

*Diabetes Mellitus, Type 1/therapy/immunology/genetics
Humans
*Genetic Therapy/methods
*Insulin-Secreting Cells/immunology/transplantation
*Gene Editing/methods
*T-Lymphocytes, Regulatory/immunology
Animals
*CRISPR-Cas Systems

@article {pmid42119166,
year = {2026},
author = {Huang, D and Sun, D and Ou, C and Zhang, X and Luo, R and Kou, X and Li, X and Li, Y and Le, H and Li, W and You, Y and Gong, C},
title = {A hierarchical self-adjuvanted nanoCRISPR-based vaccine restores endogenous immune recognition and surveillance to amplify adaptive immune responses.},
journal = {Biomaterials},
volume = {334},
number = {},
pages = {124285},
doi = {10.1016/j.biomaterials.2026.124285},
pmid = {42119166},
issn = {1878-5905},
mesh = {Animals ; *Adaptive Immunity ; *Cancer Vaccines/immunology ; Humans ; *Nanoparticles/chemistry ; B7-H1 Antigen/genetics/immunology ; Nanovaccines ; Cell Line, Tumor ; CRISPR-Cas Systems ; Adjuvants, Immunologic ; Mice ; Female ; Mice, Inbred C57BL ; },
abstract = {Tumor vaccines are considered a promising approach in immunotherapy, designed to boost the immune system's capacity to identify tumor-associated antigens and subsequently trigger immune responses against tumors. However, the inherent genetic instability of tumor cells frequently results in decreased expression or loss of antigen and/or major histocompatibility complex (MHC) expression and upregulation of immune checkpoint molecule PD-L1, thus evading endogenous immune recognition and surveillance. Herein, we developed a hierarchical self-adjuvanted nanoCRISPR-based vaccine (HEDERA) loaded with LSD1/PD-L1 dual-editing CRISPR/Cas9 system, seeking to reinstate the endogenous immune detection and monitoring mechanisms to enhance adaptive immune reactions. Knockdown of LSD1 increases the presence of tumor-specific antigens and major histocompatibility complex class I molecules on the surface of cancer cells, thereby restoring immune recognition. Simultaneously, silencing PD-L1 alleviates the "exhaustion" of T cells and reactivates their cytotoxic activity. Moreover, LSD1 knockdown activates the type I interferon pathway to induce a self-adjuvant effect that enhances innate immune responses and thereby strengthens T cell-mediated adaptive immunity. This dual strategy achieves unprecedented efficacy, with 90% primary tumor inhibition, and demonstrates an 87.3% and 90.6% inhibition rate for post-surgical metastatic and recurrent tumors, respectively. Overall, HEDERA overcomes the single-action constraint of traditional tumor vaccines, and avoids combined medication-related poor patient compliance, delivering a more efficient, convenient integrated tumor immunotherapy solution.},
}

Animals
*Adaptive Immunity
*Cancer Vaccines/immunology
Humans
*Nanoparticles/chemistry
B7-H1 Antigen/genetics/immunology
Nanovaccines
Cell Line, Tumor
CRISPR-Cas Systems
Adjuvants, Immunologic
Mice
Female
Mice, Inbred C57BL

@article {pmid42186741,
year = {2026},
author = {Li, J and Ji, C and Yang, W and Han, Y and Zhao, P and Cai, X and Tian, S and Zhu, W and Zhang, J and Xu, J and Yang, W and Li, F and Liu, P},
title = {Engineered CRISPR/Cas12a2 Nanoprobe Imaging in Living Cells for Precise Tumor Diagnosis.},
journal = {Small methods},
volume = {10},
number = {11},
pages = {e70727},
pmid = {42186741},
issn = {2366-9608},
support = {U25C2029//National Natural Science Foundation of China/ ; 32371468//National Natural Science Foundation of China/ ; 22474077//National Natural Science Foundation of China/ ; 23ZR1461400//Shanghai Municipal Natural Science Foundation/ ; 22ZR1459600//Shanghai Municipal Natural Science Foundation/ ; YG2023ZD07//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; YG2024QNB09//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; 20234Y0201//Foundation of Shanghai Municipal Health Commission/ ; 2022JC002//Foundation of Shanghai Municipal Health Commission/ ; TMSK-2024-203//National Key Scientific Infrastructure for Translational Medicine (Shanghai)/ ; 10000015Z155080000004//2024 National Clinical Key Specialty Construction Project/ ; XK202401//Jiading District Medical Key Discipline Construction Project/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; RNA, Messenger/genetics/metabolism ; Mice ; Cell Line, Tumor ; Glutathione ; CRISPR-Associated Proteins/genetics ; Molecular Imaging/methods ; *Neoplasms/diagnosis/diagnostic imaging ; },
abstract = {Messenger RNA (mRNA) imaging in tumor cells plays a crucial role in monitoring the occurrence and development of tumors. However, achieving highly specific and sensitive mRNA imaging remains a significant challenge due to the complex intracellular environment and high background signal. Here, we engineered a CRISPR/Cas12a2 system with an RNA blocking strand that binds to CRISPR RNA (crRNA). After glutathione (GSH) stimulation, the RNA blocking strand is cleaved, allowing the release of crRNA and restoring the capability of CRISPR/Cas12a2 ribonucleoprotein (RNP). Furthermore, we developed a nanoprobe (termed eRNP-FHR) by converging engineered Cas12a2 RNP (eRNP) with framework-hotspot reporters (FHR). FHR features four vertices that modify the sgc8 aptamer to specifically target the protein tyrosine kinase 7 receptor on the surface of tumor cell membranes, link to the eRNP by hybridizing with crRNA, and incorporate fluorescence quenching groups. The eRNP-FHR precisely targets tumor cells through aptamer-mediated endocytosis, specifically recognizes mRNA upon GSH stimulation, and simultaneously cleaves FHR to release a significant fluorescent signal. Excitingly, eRNP-FHR successfully achieved imaging of baculoviral IAP repeat-containing 5 mRNA in pancreatic tumor cells, accurately distinguishing pancreatic tumor cells from normal cells. In a murine pancreatic tumor model, eRNP-FHR exhibited excellent mRNA imaging, highlighting significant potential for precise tumor diagnosis.},
}

Humans
*CRISPR-Cas Systems/genetics
Animals
RNA, Messenger/genetics/metabolism
Mice
Cell Line, Tumor
Glutathione
CRISPR-Associated Proteins/genetics
Molecular Imaging/methods
*Neoplasms/diagnosis/diagnostic imaging

@article {pmid42189082,
year = {2026},
author = {Wu, Y and Jin, R and Lei, T and Liu, J and Chang, Y and Zhang, Z and Li, J and Liu, M},
title = {Aptamer-Coupled Droplet CRISPR/Cas12a Enables Ultrasensitive sPD-L1 Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {22},
pages = {16296-16305},
doi = {10.1021/acs.analchem.6c00779},
pmid = {42189082},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; *B7-H1 Antigen/blood ; Limit of Detection ; Lung Neoplasms/blood/diagnosis ; },
abstract = {Ultrasensitive detection of soluble programmed death-ligand 1 (sPD-L1) in peripheral blood is essential for early cancer diagnosis and immunotherapy monitoring. Conventional enzyme-linked immunosorbent assays lack the requisite sensitivity, whereas PCR quantifies nucleic-acid surrogates rather than the immunologically active protein. Here we report an aptamer-coupled droplet CRISPR/Cas12a (ADC) platform that integrates a structure-switching aptamer with picolitre droplet microfluidics to achieve femtomolar quantification of sPD-L1 within 70 min. Target binding with aptamer displaces a blocking sequence that activates Cas12a trans-cleavage, generating fluorescent droplets without preamplification. Confinement in picolitre droplets accelerates reaction kinetics through elevated local reagent concentrations and suppresses background fluorescence, collectively enhancing sensitivity. The assay exhibits a 0.5 pM limit of detection for sPD-L1, a dynamic range spanning 3 orders of magnitude, and 100% diagnostic accuracy in blinded plasma from lung cancer patients and healthy donors. The modular ADC architecture is readily adaptable to other protein biomarkers, offering a universal strategy for rapid, ultrasensitive liquid-biopsy analysis.},
}

*Aptamers, Nucleotide/chemistry
Humans
*CRISPR-Cas Systems/genetics
*B7-H1 Antigen/blood
Limit of Detection
Lung Neoplasms/blood/diagnosis

@article {pmid42217941,
year = {2026},
author = {Liu, D and Ma, G and Bai, L and Guo, K and Wang, T and Jiang, Z and Qian, F and Wang, Y and Pang, Y and Zou, W and Wang, R},
title = {STAR-CRISPR: a one-pot ultraspecific CRISPR strategy for rapid, visualized SNV detection and genotyping in point-of-care diagnostics.},
journal = {Talanta},
volume = {309},
number = {},
pages = {130036},
doi = {10.1016/j.talanta.2026.130036},
pmid = {42217941},
issn = {1873-3573},
mesh = {Humans ; *Point-of-Care Systems ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; *Pancreatic Neoplasms/genetics/diagnosis ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; *Genotyping Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Genotype ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Single nucleotide variation (SNV), as a key biomarker for disease diagnosis and personalized treatment, faces challenges in rapid and accurate detection. This study developed a single-tube accelerated recognition of SNVs strategy named STAR-CRISPR, which could accomplish SNV detection within only 20 min. This method integrated isothermal amplification and CRISPR/Cas12b cleavage system in one pot, and results could be directly identified by the naked eye. This method could accurately distinguish single-base differences, and could detect as low as 1% mutations against high background interference. We verified the proposed method by testing 70 clinical samples of idiopathic chronic pancreatitis, pancreatic cancer and acute myeloid leukemia. Results showed 100% consistency with next-generation sequencing results, demonstrating good accuracy and reliability of the proposed method. To further facilitate point-of-care diagnosis, we developed integrated miniature microfluidic chips, which greatly simplified sample identification and enabled logical interpretation of results. The combined STAR-CRISPR and microfluidic platform not only identifies SNVs but also supports simultaneous visual genotyping of wild-type, homozygous, and heterozygous mutations. Consequently, the proposed strategy is accurate, rapid, and versatile, holding significant potential for next-generation molecular diagnostics.},
}

Humans
*Point-of-Care Systems
*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems
*Pancreatic Neoplasms/genetics/diagnosis
*Leukemia, Myeloid, Acute/genetics/diagnosis
*Genotyping Techniques/methods
Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Genotype
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation & purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis A Virus Cellular Receptor 1/isolation & purification
*Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems
*Kidney Transplantation
Limit of Detection
Prognosis
Metal Nanoparticles/chemistry
Silver/chemistry
Colorimetry/methods
Metal-Organic Frameworks/chemistry
SELEX Aptamer Technique
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins