@article {pmid41884948,
year = {2026},
author = {Ekstrand, F and Ruhrmann, S and Bacos, K and Bartel, S and Jellema, P and Rots, MG and Ling, C and Prinz, CN},
title = {Nanopore Electroporation: A New Delivery Method Within the Field of Epigenetic Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {28},
pages = {e13858},
doi = {10.1002/smll.202513858},
pmid = {41884948},
issn = {1613-6829},
support = {682206//ERC CoG NanoPokers/ ; //Swedish Research Council (VR)/ ; //Novo Nordisk foundation/ ; //Region Skåne/ ; Dnr 2009-1039//Strategic Research Area Exodiab/ ; //The Crafoord Foundation/ ; //NanoLund/ ; //The Swedish Diabetes Foundation/ ; ITM-17//The Swedish foundation for Strategic Research/ ; IRC15-0067//The Swedish foundation for Strategic Research/ ; },
mesh = {*Electroporation/methods ; *Nanopores ; *Gene Editing/methods ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Animals ; Humans ; Insulin-Secreting Cells/metabolism ; Insulin/genetics/metabolism ; Epigenome Editing ; },
abstract = {Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR-dCas9-based tools offer this potential, yet β-cells are notoriously difficult to transfect, and efficient, non-viral delivery methods are lacking. Here, we developed nanopore-mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β-cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel-associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin-1 gene (Ins1) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult-to-transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR-dCas9-based epigenetic editors in pancreatic β-cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.},
}

*Electroporation/methods
*Nanopores
*Gene Editing/methods
*Epigenesis, Genetic/genetics
CRISPR-Cas Systems/genetics
Animals
Humans
Insulin-Secreting Cells/metabolism
Insulin/genetics/metabolism
Epigenome Editing

@article {pmid41950922,
year = {2026},
author = {Kang, T and Bleris, L},
title = {Cellular-state control using ribozyme-scaffolded miRNA-sensing and CRISPR-mediated actuation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101379},
doi = {10.1016/j.crmeth.2026.101379},
pmid = {41950922},
issn = {2667-2375},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *RNA, Catalytic/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Epithelial-Mesenchymal Transition/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; },
abstract = {Cellular transitions between states are fundamental to development, adaptation, and pathological processes, but monitoring and guiding these transitions using endogenous signals remain challenging. MicroRNAs (miRNAs) represent a powerful modality, as distinct cell states are characterized by unique miRNA expression signatures. Here, we introduce a state-specific miRNA-directed CRISPR system for detecting and responding to epithelial-to-mesenchymal transition (EMT), a critical process in development, wound healing, and cancer metastasis. This system leverages EMT-specific miRNAs to regulate activation of type II polymerase-driven ribozyme-single-guide RNA (sgRNA) constructs, which direct CRISPR-based effectors to modulate gene expression. Using this approach, we demonstrate selective elimination of cells that have undergone mesenchymal transition and dynamic filtering of cell populations. This system provides a versatile platform for precise activation of CRISPR-Cas9 effectors using endogenous, state-specific cues. Integrating miRNA signatures with CRISPR technology to monitor, modulate, and reprogram cell-state transitions paves the way for applications in regenerative medicine, cancer therapy, and beyond.},
}

*MicroRNAs/genetics/metabolism
Humans
*RNA, Catalytic/metabolism/genetics
*CRISPR-Cas Systems/genetics
Epithelial-Mesenchymal Transition/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line, Tumor

@article {pmid42000058,
year = {2026},
author = {Carvalho, C and Hérivaux, A and Mello, T and Bastide, F and Thomas, O and Guillemette, T and Saulnier, P and Souza Dos Santos, AL and Papon, N and Bouchara, JP},
title = {TRXR2, a thioredoxin reductase-encoding gene, contributes to protection against the oxidative stress and virulence in Scedosporium apiospermum.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108498},
doi = {10.1016/j.micpath.2026.108498},
pmid = {42000058},
issn = {1096-1208},
mesh = {*Oxidative Stress ; *Scedosporium/pathogenicity/genetics/enzymology/drug effects ; Virulence/genetics ; Animals ; Hyphae ; *Thioredoxin-Disulfide Reductase/genetics/metabolism ; *Thioredoxin Reductase 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Fungal Proteins/genetics/metabolism ; Spores, Fungal ; Cell Wall/metabolism/ultrastructure ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Antifungal Agents/pharmacology ; Larva/microbiology ; Gene Expression Regulation, Fungal ; Benzene Derivatives ; },
abstract = {Scedosporium apiospermum has received an increased attention over the past decades, especially because of its low susceptibility to current antifungals and its clinical relevance in cystic fibrosis. Nevertheless, little is still known about its pathogenic mechanisms. During the inflammatory reaction, macrophages and neutrophils release antimicrobial compounds, especially reactive oxygen species (ROS). To cope with ROS, pathogens have developed various strategies, including synthesis of some immunoprotective secondary metabolites, and enzymatic mechanisms relying on antioxidant enzymes. A recent transcriptomic study showed that the TRXR2 gene, encoding one of its two thioredoxin reductases (TrxRs), is overexpressed upon exposure to oxidative stress. To investigate the role of TrxR2, the encoding gene was disrupted using the CRISPR-Cas9 technology. An ultrastructural study revealed that TRXR2 gene knock-out induced the loss of the outer cell wall layer of hyphae, thereby increasing their surface hydrophobicity. The effects on the tolerance of the fungus to chemically-induced oxidative stresses were also investigated, revealing a higher susceptibility to cumene hydroperoxide. Additionally, an overexpression of BoyT gene (encoding the other TrxR) was seen as a compensatory mechanism, but bioinformatic analysis also suggested distinct roles for the two TrxRs in relation with distinct subcellular localizations. Finally, conidia exhibited a reduced capacity to adhere to epithelial cells and a diminished virulence in the Galleria mellonella model. In conclusion, these results suggest that TrxR2 plays a contributory role in the pathogenicity of S. apiospermum, but is not a major determinant of its virulence, which justifies future studies on the other components of the thioredoxin system.},
}

*Oxidative Stress
*Scedosporium/pathogenicity/genetics/enzymology/drug effects
Virulence/genetics
Animals
Hyphae
*Thioredoxin-Disulfide Reductase/genetics/metabolism
*Thioredoxin Reductase 2/genetics/metabolism
Reactive Oxygen Species/metabolism
Fungal Proteins/genetics/metabolism
Spores, Fungal
Cell Wall/metabolism/ultrastructure
CRISPR-Cas Systems
Gene Knockout Techniques
Antifungal Agents/pharmacology
Larva/microbiology
Gene Expression Regulation, Fungal
Benzene Derivatives

@article {pmid42019502,
year = {2026},
author = {Vereecke, N and Behrmann, M and Khare, A and Dekker, JP},
title = {Improved Cas9-targeted nanopore sequencing facilitates ultra-deep analysis of genomic variation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101410},
doi = {10.1016/j.crmeth.2026.101410},
pmid = {42019502},
issn = {2667-2375},
mesh = {*High-Throughput Nucleotide Sequencing/methods ; *Nanopore Sequencing/methods ; Staphylococcus aureus/genetics ; *CRISPR-Cas Systems/genetics ; Bacteroides fragilis/genetics ; *Nanopores ; *Genomics/methods ; *CRISPR-Associated Protein 9/metabolism ; Genome, Bacterial ; *Genetic Variation ; },
abstract = {We present nanopore adapter-enriched Cas9-targeted sequencing (nAECATS), a method permitting inexpensive, ultra-deep, selective long-read sequencing of targeted regions in native, unamplified DNA. This method modifies previous Cas9-targeted sequencing approaches through the inclusion of a bead-based capture step that exploits the poly(T)8 stretch within the R10.4.1 ligation adapter for additional purification. Testing on a 10 kb Bacteroides fragilis genomic region achieved 90% on-target yield with 51,000× coverage from a single Flongle flow cell (353-fold increase versus whole-genome sequencing). Applied to a variable-length (>20 kb) Staphylococcus aureus genomic target containing dynamic gene amplifications conferring antibiotic resistance, nAECATS achieved 46,000× coverage and 74% on-target yield, revealing 2-4 tandem amplifications at single-cell resolution. While efficiency decreased with longer fragments (up to 41 kb), substantial enrichment improvements were demonstrated. We anticipate that nAECATS ultra-deep sequencing will find broad application for a wide range of biological questions in pro- and eukaryotic (epi)genomics and microbiology.},
}

*High-Throughput Nucleotide Sequencing/methods
*Nanopore Sequencing/methods
Staphylococcus aureus/genetics
*CRISPR-Cas Systems/genetics
Bacteroides fragilis/genetics
*Nanopores
*Genomics/methods
*CRISPR-Associated Protein 9/metabolism
Genome, Bacterial
*Genetic Variation

@article {pmid42053710,
year = {2026},
author = {Panchuk, IO and Grigorieva, OV and Kurshakova, EV and Nagieva, SE and Shchagina, OA and Levchenko, OA and Pozhitnova, VO and Voronina, ES and Tabakov, VY and Smirnikhina, SA and Lavrov, AV},
title = {Generation of an Induced Pluripotent Stem Cell Line from a Duchenne Muscular Dystrophy Patient Carrying the DMD p.Ser429Ter (c.1286C>G) Nonsense Mutation.},
journal = {Bulletin of experimental biology and medicine},
volume = {180},
number = {4},
pages = {548-554},
pmid = {42053710},
issn = {1573-8221},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Muscular Dystrophy, Duchenne/genetics/pathology/metabolism ; Kruppel-Like Factor 4 ; *Codon, Nonsense/genetics ; Cell Differentiation ; *Dystrophin/genetics/metabolism ; Cell Line ; Male ; Fibroblasts/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; CRISPR-Cas Systems ; Cellular Reprogramming ; Sendai virus/genetics ; },
abstract = {An induced pluripotent stem cell (iPSC) line was successfully generated from dermal fibroblasts of a patient with Duchenne muscular dystrophy (DMD) harboring the pathogenic nonsense variant c.1286C>G (p.Ser429Ter) in the DMD gene using non-integrating Sendai virus reprogramming. The iPSC clone exhibited typical pluripotent stem cell morphology, expressed key pluripotency markers (OCT4, SSEA4, NANOG, and TRA-1-60), and retained trilineage differentiation potential. The cell line had a normal karyotype, and elimination of reprogramming vectors (OCT3/4, SOX2, KLF4, and c-MYC) was confirmed. This isogenic cell model provides a valuable platform for investigating DMD pathogenesis associated with this specific mutation and for developing targeted therapeutic approaches, including CRISPR/Cas9-mediated gene correction.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Muscular Dystrophy, Duchenne/genetics/pathology/metabolism
Kruppel-Like Factor 4
*Codon, Nonsense/genetics
Cell Differentiation
*Dystrophin/genetics/metabolism
Cell Line
Male
Fibroblasts/metabolism/cytology
Octamer Transcription Factor-3/genetics/metabolism
CRISPR-Cas Systems
Cellular Reprogramming
Sendai virus/genetics

@article {pmid42080223,
year = {2026},
author = {Zhao, S and Huang, S and Li, N and Wang, N and Yu, Z and Liang, X and Yang, Z and Guo, H},
title = {Piperazine-Derived Diamine Lipid Nanoparticles Targeting to the Liver for Delivering Clustered Regularly Interspaced Short Palindromic Repeat Editing of PCSK9 to Durably Maintain Plasmatic Low-Density Lipoprotein Cholesterol in Low Levels.},
journal = {ACS applied bio materials},
volume = {9},
number = {10},
pages = {4465-4476},
doi = {10.1021/acsabm.6c00032},
pmid = {42080223},
issn = {2576-6422},
mesh = {*Proprotein Convertase 9/genetics/metabolism ; *Nanoparticles/chemistry ; Humans ; Animals ; Mice ; *Gene Editing ; *Liver/metabolism/drug effects ; *Cholesterol, LDL/blood ; *Piperazine/chemistry ; *Lipids/chemistry ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; Materials Testing ; Mice, Inbred C57BL ; Hep G2 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cardiovascular diseases remain a leading cause of morbidity and mortality worldwide, and durable suppression of low-density lipoprotein cholesterol (LDL-C) via genome editing represents a promising therapeutic strategy. Here, we report the rational design of a piperazine-derived bis-tertiary amine ionizable lipid (M10) and its optimized lipid nanoparticle formulation (M10-F4) for efficient and liver-targeted CRISPR/Cas9 delivery. Compared with benchmark lipids such as SM-102, M10 enables a reduced molar ratio of ionizable lipid while maintaining high nucleic acid encapsulation efficiency (>80%) and forming stable, spherical nanoparticles. The piperazine-based multi-cationic core confers an optimized apparent pKa of 6.56, facilitating endosomal escape through enhanced protonation under acidic conditions. Confocal microscopy in HepG2 and Huh-7 cells reveals efficient cellular uptake and enhanced cytosolic release of RNA cargo with minimal lysosomal entrapment. In vivo, M10-F4 exhibits strong liver tropism following systemic administration. A single intravenous dose mediates robust PCSK9 gene editing in C57BL/6 mice, resulting in sustained reductions of circulating PCSK9 and LDL-C levels under both normal and high-fat diet conditions for up to 48 days, accompanied by decreased hepatic PCSK9 expression. Importantly, acute safety evaluation in BALB/c mice showed no obvious signs of short-term systemic toxicity, including stable body weight, minimal induction of inflammatory cytokines (IL-6, TNF-α, and CXCL-10), no significant elevation of liver enzymes, and normal gross organ morphology. Collectively, this work establishes M10-F4 as a molecularly engineered, liver-targeted LNP platform in which ionizable lipid architecture and formulation composition enable effective in vivo genome editing with favorable tolerability, highlighting the importance of rational materials design at the materials-bio interface for cardiometabolic gene-editing applications.},
}

*Proprotein Convertase 9/genetics/metabolism
*Nanoparticles/chemistry
Humans
Animals
Mice
*Gene Editing
*Liver/metabolism/drug effects
*Cholesterol, LDL/blood
*Piperazine/chemistry
*Lipids/chemistry
*Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Particle Size
Materials Testing
Mice, Inbred C57BL
Hep G2 Cells
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42119627,
year = {2026},
author = {Guo, K and Yu, S and Yang, N and Wang, X and Liu, Q and Fu, Y and Liu, J},
title = {Serial passage is associated with virulence attenuation in Neospora caninum and transcriptomic remodeling of invasion- and chronic-stage-associated pathways.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108549},
doi = {10.1016/j.micpath.2026.108549},
pmid = {42119627},
issn = {1096-1208},
mesh = {*Neospora/pathogenicity/genetics/growth & development ; Virulence/genetics ; Animals ; Gene Expression Profiling ; *Transcriptome ; Serial Passage ; Cattle ; Protozoan Proteins/genetics ; Host-Parasite Interactions/genetics ; Coccidiosis/parasitology/veterinary ; CRISPR-Cas Systems ; },
abstract = {N. caninum is an obligate intracellular apicomplexan parasite and a major cause of abortion in cattle worldwide. However, the molecular basis underlying virulence attenuation during long-term in vitro passage remains poorly understood. Here, we performed comparative transcriptomic profiling between the virulent Nc1-09 strain and its attenuated derivative Nc1 to investigate transcriptional changes associated with virulence reduction. A total of 487 differentially expressed genes (DEGs) were identified. Functional enrichment analyses revealed prominent changes in pathways related to protein modification, host-parasite interaction, motility, and metabolism. Notably, multiple members of the surface antigen glycoprotein (SRS) family displayed marked transcriptional shifts, consistent with altered host cell interaction and immune-related functions. CRISPR/Cas9-mediated disruption of three representative DEGs (NCLIV_016320, NCLIV_024950, and NCLIV_022830) did not reproduce the attenuated phenotype, suggesting that disruption of these individual genes is insufficient to account for the full attenuated phenotype under the conditions tested. Gene set enrichment analysis further demonstrated systematic downregulation of protein modification-associated pathways in the attenuated strain. Overall, the data indicate that virulence attenuation in Neospora caninum （N. caninum） is associated with coordinated transcriptional remodeling across multiple functional pathways rather than discrete gene loss.},
}

*Neospora/pathogenicity/genetics/growth & development
Virulence/genetics
Animals
Gene Expression Profiling
*Transcriptome
Serial Passage
Cattle
Protozoan Proteins/genetics
Host-Parasite Interactions/genetics
Coccidiosis/parasitology/veterinary
CRISPR-Cas Systems

@article {pmid42144912,
year = {2026},
author = {Guo, X and Tian, T and Zhou, X},
title = {Split crRNAs Enhance Cas12a Diagnostic Performance.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70383},
doi = {10.1002/cbic.70383},
pmid = {42144912},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {CRISPR RNA (crRNA) is the key guide molecule in the CRISPR-Cas12a system, directing the Cas protein to recognize target sequences. It consists of a repeat-derived stem loop that binds Cas12a and helps stabilize the ribonucleoprotein complex, and a spacer region that base-pairs with the target and determines recognition specificity. Recently, multiple studies have shown that crRNAs can be split and reassembled in vitro in diverse ways. These split-and-reconfigured strategies have enabled detection schemes that are more flexible than full-length crRNAs, cover a broader range of targets, and achieve higher signal-to-background ratios. Here, we focus on split crRNA strategies for CRISPR-Cas12a and systematically summarize existing split crRNA-based detection platforms. We outline their design principles, reaction mechanisms, and performance features, and we synthesize how these approaches improve key metrics-including target scope, sensitivity, specificity, and controllability. Finally, we discuss the major advantages and current limitations of split crRNA strategies and highlight directions for further design optimization and translational applications. Schematic overview of split crRNA strategies for enhancing CRISPR-Dx performance. These advances are mainly reflected in four aspects: broadening the target range, thereby enabling Cas12a to be applied to the detection of short RNAs, structured RNAs, and certain non-nucleic acid targets; improving sensitivity by enhancing detection signals through reassembly-dependent activation, cascade amplification, or auxiliary activation strategies; increasing specificity by strengthening the discrimination of single-nucleotide differences through stepwise recognition and conditional assembly; and enhancing controllability by achieving on-demand activation of Cas12a activity via light, enzymes, small molecules, or proximity effects.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid42145154,
year = {2026},
author = {Dong, R and Zhang, Y and Yuan, G and Yue, X and Ding, Y and Zeng, X and Xiao, H},
title = {A Novel Strategy to Produce CAR-γδ T Cells via Site-Directed Gene Integration by a Combination of CRISPR/Cas9 and AAV.},
journal = {Cancer medicine},
volume = {15},
number = {5},
pages = {e71918},
doi = {10.1002/cam4.71918},
pmid = {42145154},
issn = {2045-7634},
support = {82170141//National Natural Science Foundation of China/ ; 82470209//National Natural Science Foundation of China/ ; },
mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; Genetic Vectors/genetics ; Cell Line, Tumor ; },
abstract = {Chimeric antigen receptor (CAR)-αβ T cells are commonly employed in tumor therapy but hindered by some limitations. γδ T cells are promising substrates for CAR therapy for their major histocompatibility complex (MHC)-unrestricted recognition manner and innate immune function. Here, we established a novel method for generating CAR-γδ T cells. We utilized the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) method to interrupt the TCR delta chain constant region (TRDC) sequence, followed by the site-directed insertion of the CAR sequence into the TRDC locus via homologous complementation mediated by adeno-associated virus (AAV) gene delivery. We optimized electroporation parameters for Cas9/ribonucleoproteins (RNP) delivery and infection conditions for CAR-gene carrying AAV in γδ T cells. These optimizations facilitated efficient TCR knockout and site-directed CAR insertion, ultimately yielding functional CAR-γδ T cells. In vitro experiments demonstrated that these newly prepared CAR-γδ T cells could stimulate cytokine production, kill tumor cells as well as exhibit robust proliferative potential and memory-like phenotype. These state-of-the-art CAR-γδ T cells could reduce tumor burden and extend the survival period of tumor-bearing mice.},
}

*Dependovirus/genetics
Animals
*CRISPR-Cas Systems
Humans
Mice
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism
*T-Lymphocytes/immunology/metabolism
Genetic Vectors/genetics
Cell Line, Tumor

@article {pmid42145193,
year = {2026},
author = {Yin, XY and Wang, SZ},
title = {High-throughput identification methods of genomic functional variation in post-GWAS era and their application in agricultural animals.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {5},
pages = {451-470},
doi = {10.16288/j.yczz.25-319},
pmid = {42145193},
issn = {0253-9772},
mesh = {Animals ; *Genome-Wide Association Study/methods ; *Genetic Variation ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-wide association study (GWAS) has identified a large number of genetic variations that are significantly associated with human diseases and animal and plant economic traits. However, the majority of these variants are located in non-coding regions of the genome, which makes it challenging to accurately pinpoint functional variants of biological significance from a vast number of candidate loci. In the post-GWAS era, high-throughput analytical approaches, such as high-throughput reporter gene analysis, CRISPR/Cas9-based gene editing technologies, and epigenetic analyses, have become powerful tools for systematically uncovering functional variants in the genome. These methods not only enable efficient identification of functional variants but also help elucidate the mechanisms through which they regulate gene expression, thereby clarifying the molecular basis underlying trait formation or disease pathogenesis. In this review, we systematically summarize current high-throughput strategies for identifying functional genomic variants, highlight their applications and recent advances in major agricultural animal species, and outline future research directions, with the aim of providing a reference for subsequent studies in related fields.},
}

Animals
*Genome-Wide Association Study/methods
*Genetic Variation
Gene Editing
*Genomics/methods
CRISPR-Cas Systems

@article {pmid42148306,
year = {2026},
author = {Jezierski, A and Huang, J and Desbiens, L and Benabdallah, B and McComb, S and Beausejour, C},
title = {Induced pluripotent stem cells as platforms for engineering NK cell immunotherapies.},
journal = {Frontiers in cell and developmental biology},
volume = {14},
number = {},
pages = {1810206},
pmid = {42148306},
issn = {2296-634X},
abstract = {Human induced pluripotent stem cells (iPSCs) are transforming adoptive cell therapy by combining unlimited self-renewal, broad differentiation potential, and high amenability to genome engineering. These attributes make iPSCs a versatile source for the development of standardized immune effector cells at industrial scale, enabling a shift from patient- or donor-restricted cell products toward true off-the-shelf immunotherapies that can be improved through iterative genome engineering. iPSC-derived natural killer (iNK) cells are the most clinically advanced and exemplify the platform's advantages over conventional autologous or donor-sourced approaches. Unlike autologous therapies, which require labor-intensive and expensive personalized clinical-grade manufacturing, and are constrained by variable quality and genetic intractability of donor products, iPSC technology supports the creation of renewable, clonally defined master cell banks as uniform starting material for NK-cell therapy products. Advances in CRISPR/Cas-based editing now permit multiplex introduction of functional traits, enhanced cytokine signaling, antibody-dependent cytotoxicity, checkpoint resistance, optimized trafficking, safety switches, and increasing signal complexity, directly at the pluripotent or progenitor stages; ultimately allowing for fully-programmable iNK cells with customizable potency and persistence. Early clinical studies of iNK products validate the feasibility, safety, and therapeutic potential of this approach, but also underscore the need for continued refinement of differentiation protocols, manufacturing pipelines, and regulatory standards to ensure efficacy, genomic stability, phenotypic maturity, and long-term safety. This review outlines current breakthroughs and future directions of iNK cell therapies, emphasizing how programmable iPSC chassis platforms are enabling modular and off-the-shelf targeted immunotherapies.},
}

@article {pmid42148710,
year = {2026},
author = {Du, T and He, J and Wang, Y and Xie, X and Chen, J and Xu, Z and Zhou, C and Sun, W},
title = {An Evolutionarily Conserved Function of Grainyhead in Orchestrating Insect Wing Development.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {10},
pages = {e71927},
doi = {10.1096/fj.202600640R},
pmid = {42148710},
issn = {1530-6860},
support = {CSTB2025NSCQ-GPX0734//CSTC | Natural Science Foundation of Chongqing Municipality/ ; 32070499//MOST | National Natural Science Foundation of China (NSFC)/ ; 2025CDJ-IAISYB-022//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; },
mesh = {Animals ; *Wings, Animal/growth & development/metabolism ; *Insect Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics/growth & development/metabolism ; *Bombyx/genetics/growth & development/metabolism ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; },
abstract = {The development of insect appendages is governed by deeply conserved genetic programs, even as developmental strategies diverge widely across taxa. In this study, we identify the conserved transcription factor Grainyhead (Grh) as a crucial regulator of insect wing development. In the silkworm Bombyx mori, CRISPR/Cas9-mediated knockout of BmGrh did not compromise larval viability but resulted in severe wing defects in adults, including crumpled and non-expandable wings. Similarly, tissue-specific knockdown of DmGrh in Drosophila melanogaster wing imaginal discs led to pupal lethality, a sex-biased eclosion rate, and adults exhibiting crumpled wings with disrupted wing hair polarity. Comparative transcriptomics revealed that loss of DmGrh function predominantly downregulates genes associated with cuticle structure and extracellular matrix organization in both species. In Drosophila, chromatin immunoprecipitation further confirmed that DmGrh directly binds to regulatory regions of these downregulated cuticle-related genes. Through functional screening, we identified the cuticle protein gene cpr65Ea as a key downstream effector, whose knockdown recapitulated the wing morphogenesis and eclosion defects observed in DmGrh RNA interference individuals. Moreover, extending our investigation to an agricultural pest, RNAi-mediated silencing of SfGrh in the white-backed planthopper (Sogatella furcifera) impaired wing expansion and completely abolished flight ability. Our findings establish Grh as an evolutionarily conserved regulator of insect wing development and highlight its potential as a novel genetic target for pest management by disrupting flight capacity and dispersal.},
}

Animals
*Wings, Animal/growth & development/metabolism
*Insect Proteins/genetics/metabolism
*Drosophila melanogaster/genetics/growth & development/metabolism
*Bombyx/genetics/growth & development/metabolism
Gene Expression Regulation, Developmental
*Transcription Factors/genetics/metabolism
Female
CRISPR-Cas Systems
Male

@article {pmid42149441,
year = {2026},
author = {Klijnhout, JA and Senders, EAW and van den Bogaard, EH and Smits, JPH},
title = {Genetic Engineering of Human Keratinocytes Using CRISPR/Cas9 Ribonucleoprotein Complexes or Modified Cas9-Encoding mRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3031},
number = {},
pages = {87-101},
pmid = {42149441},
issn = {1940-6029},
mesh = {Humans ; *Keratinocytes/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Messenger/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Cells, Cultured ; },
abstract = {CRISPR/Cas9 is a straightforward genome-editing technique that is implemented across disciplines and research areas. However, in keratinocytes, CRISPR/Cas9 can be particularly difficult due to variable genome-editing efficiency, reduced cell viability, and difficulties during (sub)cloning of gene-edited keratinocyte populations. Here, we provide a step-by-step detailed protocol for the genetic manipulation of human (primary) keratinocytes, including widely accepted procedures for the analysis of CRISPR/Cas9 efficiency, (sub)cloning procedures to select heterozygous or homozygous keratinocytes, and off-target genome-editing analysis.},
}

Humans
*Keratinocytes/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Messenger/genetics
*Ribonucleoproteins/genetics/metabolism
*Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Cells, Cultured

@article {pmid42150000,
year = {2026},
author = {Hu, Q and Lin, J and Cui, H and Zhao, X and Wu, M and Wei, S and Deng, M and Wang, B and Liu, T and Gao, X and Huang, Q and Liang, Y and Liu, S and Yang, H and Lin, HB and Li, G},
title = {Cas13-Mediated RNA Base Editing for the Treatment of Hereditary Hypertrophic Cardiomyopathy.},
journal = {Circulation},
volume = {153},
number = {20},
pages = {1598-1602},
doi = {10.1161/CIRCULATIONAHA.125.076905},
pmid = {42150000},
issn = {1524-4539},
}

@article {pmid40335752,
year = {2026},
author = {Kannan, S and Altae-Tran, H and Zhu, S and Xu, P and Strebinger, D and Oshiro, R and Faure, G and Moeller, L and Pham, J and Mears, KS and Ni, HM and Macrae, RK and Zhang, F},
title = {Evolution-guided protein design of IscB for persistent epigenome editing in vivo.},
journal = {Nature biotechnology},
volume = {44},
number = {5},
pages = {759-770},
pmid = {40335752},
issn = {1546-1696},
mesh = {*Gene Editing/methods ; Humans ; *Protein Engineering/methods ; *Epigenome/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Human ; CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.},
}

*Gene Editing/methods
Humans
*Protein Engineering/methods
*Epigenome/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Human
CRISPR-Cas Systems
Epigenome Editing

@article {pmid40555761,
year = {2026},
author = {Niu, X and Tang, W and Liu, Y and Mo, B and Yu, Y and Liu, Y and Wei, W},
title = {Prime editor-based high-throughput screening reveals functional synonymous mutations in human cells.},
journal = {Nature biotechnology},
volume = {44},
number = {5},
pages = {832-844},
pmid = {40555761},
issn = {1546-1696},
support = {NSFC 31930016//National Natural Science Foundation of China (National Science Foundation of China)/ ; tsqn202312362//Taishan Scholar Foundation of Shandong Province/ ; },
mesh = {Humans ; *High-Throughput Screening Assays/methods ; *Silent Mutation/genetics ; Machine Learning ; Mutation/genetics ; RNA Splicing/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Synonymous mutations are generally considered neutral, while their roles in the human genome remain largely unexplored. Here we use the PEmax system to create a library of 297,900 engineered prime-editing guide RNAs and perform extensive screening to identify synonymous mutations affecting cell fitness. Unlike recent findings in yeast, group-level analyses show that synonymous mutations diverge from nonsynonymous mutations in fitness effects yet exhibit similar phenotypic distributions relative to negative controls. Following rigorous quality control, only a small subset demonstrated measurable effects. For these functional mutations, we develop a specialized machine learning tool and uncover their impact on various biological processes such as messenger RNA splicing and transcription, supported by multifaceted experimental evidence. We find that synonymous mutations can alter RNA folding and affect translation, as demonstrated by PLK1_S2. By integrating screening data with our model, we predict clinically deleterious synonymous mutations. This research deepens our understanding of synonymous mutations, providing insights for clinical disease studies.},
}

Humans
*High-Throughput Screening Assays/methods
*Silent Mutation/genetics
Machine Learning
Mutation/genetics
RNA Splicing/genetics
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41692169,
year = {2026},
author = {Hu, Y and Wang, Y and Wang, S and Jiao, Y and Tao, R and Yao, S},
title = {Reporter-based screening identifies small-molecule CBL0137 as an enhancer of CRISPR cytosine base editor and prime editor via p53 activation and NF-κB inhibition.},
journal = {New biotechnology},
volume = {93},
number = {},
pages = {54-74},
doi = {10.1016/j.nbt.2026.02.002},
pmid = {41692169},
issn = {1876-4347},
mesh = {Humans ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/metabolism ; *NF-kappa B/metabolism/antagonists & inhibitors ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; *Genes, Reporter ; HEK293 Cells ; },
abstract = {Recently developed CRISPR base editors (BEs) and prime editors (PEs) enable precise genome editing without inducing double-strand breaks, making them highly promising tools for therapeutic applications. However, their efficiency remains a major barrier to clinical translation, particularly at difficult-to-target sites. To address this limitation, we used a high-throughput GFP reporter system responsive to cytosine base editor (CBE) activity to screen small molecules involved in DNA damage response, cell cycle, and apoptosis pathways. This screen identified CBL0137 as a candidate that significantly enhanced editing efficiency at both the reporter and endogenous target sites, with up to an 80 % improvement. Mechanistic studies revealed that CBL0137 acts through activation of the p53 pathway and inhibition of NF-κB. Interestingly, the enhancement was largely specific to CBEs, while in PEs, CBL0137 selectively improved multi-site mutations and fragment insertions without affecting single-point edits or deletions. Collectively, these results identify CBL0137 as a selective enhancer of specific genome editing activities, providing a strategy to increase editing efficiency and advancing the translational potential of next-generation genome editing tools for therapeutic applications.},
}

Humans
*Gene Editing/methods
*Tumor Suppressor Protein p53/metabolism
*NF-kappa B/metabolism/antagonists & inhibitors
*CRISPR-Cas Systems/genetics
*Cytosine/metabolism
*Genes, Reporter
HEK293 Cells

@article {pmid41881989,
year = {2026},
author = {Ren, X and Zheng, L and Liu, Y and Maliskova, L and Tam, TW and Sun, Y and Liu, H and Cui, X and Lee, J and Takagi, MA and Li, B and Ren, B and Wang, W and Shen, Y},
title = {CRISPR tiling deletion screens reveal functional enhancers and allelic compensation effects (ACE) on SIN3A transcription.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41881989},
issn = {2041-1723},
support = {UM1HG009402//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30DK063720//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; S101S10OD021822-01//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; 1S10OD028511-01//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Humans ; *Enhancer Elements, Genetic/genetics ; *Repressor Proteins/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Transcription, Genetic ; *Sin3 Histone Deacetylase and Corepressor Complex/genetics/metabolism ; Cell Differentiation/genetics ; Promoter Regions, Genetic ; Alleles ; *Dosage Compensation, Genetic ; Neurons/metabolism ; Gene Expression Regulation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identify a total of 39 functional enhancers for four dosage-sensitive genes, APP, FMR1, MECP2, and SIN3A, using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. More importantly, we discover that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription are stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A, and potentially other dosage-sensitive genes.},
}

Humans
*Enhancer Elements, Genetic/genetics
*Repressor Proteins/genetics/metabolism
Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Transcription, Genetic
*Sin3 Histone Deacetylase and Corepressor Complex/genetics/metabolism
Cell Differentiation/genetics
Promoter Regions, Genetic
Alleles
*Dosage Compensation, Genetic
Neurons/metabolism
Gene Expression Regulation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41882347,
year = {2026},
author = {Akdoğan, E and Lundgren, SM and Kamber, RA and Bassik, MC and Collins, SR},
title = {Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41882347},
issn = {2399-3642},
support = {P30CA093373//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; DP2HD094656//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01 GM148769/GM/NIGMS NIH HHS/United States ; S10OD018223//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; DP2 HD094656/HD/NICHD NIH HHS/United States ; R01GM148769//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CA259218//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Receptors, Formyl Peptide/metabolism/genetics ; Endocytosis ; *CRISPR-Cas Systems ; Signal Transduction ; ADP-Ribosylation Factor 6 ; HEK293 Cells ; *Cell Membrane/metabolism ; },
abstract = {Chemoattractants generate strong chemotactic and cytotoxic responses in immune cells by activating cognate receptors. Cell surface receptor levels control sensitivity, which is critical for achieving effective responses without excessive inflammation. The surface levels of the attractant receptor FPR1 are controlled through a balance of delivery and removal, which responds to receptor activation and other stimuli. While this regulation is critical for appropriate responses, the underlying mechanisms remain unclear, including the roles of classic endocytosis regulators. We address these questions using both focused and genome-scale approaches. We find that the receptor kinase GRK6 acts in parallel with GRK2 and GRK3 to trigger internalization, and that internalization uses a β-arrestin-independent pathway, as well as pathways involving β-arrestin1 and 2. Moreover, we use an integrated analysis of two parallel CRISPR/Cas9 screens to classify regulators of FPR1 biogenesis, surface expression, recycling, and endocytosis. We identify the formin mDia1 and the small GTPase ARF6 as specific regulators of FPR1 internalization, which we confirm using chemical inhibitors in primary human neutrophils. Finally, we find that ARF6 contributes to the β-arrestin-independent pathway. Together, our results provide a systems overview of the control of FPR1 surface levels and offer insights into alternative endocytosis mechanisms used by chemoattractant receptors.},
}

Humans
*Receptors, Formyl Peptide/metabolism/genetics
Endocytosis
*CRISPR-Cas Systems
Signal Transduction
ADP-Ribosylation Factor 6
HEK293 Cells
*Cell Membrane/metabolism

@article {pmid41882878,
year = {2026},
author = {Yang, Y and Han, Z and Li, G and Li, Z and Shao, C and Li, W and Wang, J and Luan, J and Cui, Y and Han, J},
title = {Biallelic inactivation of EXT1 in patient-derived iPSCs confirms the "Two-hit" hypothesis in hereditary multiple osteochondromas.},
journal = {Bioscience trends},
volume = {20},
number = {2},
pages = {245-253},
doi = {10.5582/bst.2026.01046},
pmid = {41882878},
issn = {1881-7823},
mesh = {Humans ; *N-Acetylglucosaminyltransferases/genetics/metabolism ; Exostosin 1 ; *Exostoses, Multiple Hereditary/genetics/pathology ; *Induced Pluripotent Stem Cells/metabolism ; Cell Differentiation/genetics ; Mutation ; CRISPR-Cas Systems ; Chondrocytes/metabolism/cytology ; Chondrogenesis/genetics ; Alleles ; Organoids ; },
abstract = {Hereditary Multiple Osteochondromas (HMO) is a rare autosomal dominant skeletal disorder caused by heterozygous loss-of-function mutations in EXT1 or EXT2, which encode glycosyltransferases essential for heparan sulfate (HS) biosynthesis. Whether haploinsufficiency alone suffices or biallelic inactivation is required for osteochondroma formation remains a central unresolved question. In this study, we employed CRISPR/Cas9 combined with PiggyBac transposon technology to introduce a second pathogenic mutation (c.1883+1G>T) into patient-derived induced pluripotent stem cells (iPSCs) carrying a heterozygous EXT1 c.1126C>T mutation. This approach enabled the generation of isogenic iPSC lines: wild-type (WT), single-mutant (SM), and double-mutant (DM). These iPSCs were differentiated through induced mesenchymal stem cells (iMSCs) into chondrocytes. Biallelic EXT1 mutation in DM cells led to significant upregulation of SOX9, COL2A1, and ACAN, elevated glycosaminoglycan (GAG) levels, and markedly reduced HS, whereas SM cells remained indistinguishable from WT. Three-dimensional (3D) chondrogenic organoid cultures revealed that DM organoids were enlarged and structurally disorganized, partially recapitulating key histopathological features of osteochondromas. Transcriptomic analysis identified the Wnt signaling pathway as the most significantly enriched pathway among differentially expressed genes following EXT1 loss. Collectively, these findings provide direct human cellular evidence that complete EXT1 inactivation-not haploinsufficiency-drives aberrant chondrogenesis, likely through impaired sequestration of morphogen ligands, thereby supporting the Two-hit pathogenic model.},
}

Humans
*N-Acetylglucosaminyltransferases/genetics/metabolism
Exostosin 1
*Exostoses, Multiple Hereditary/genetics/pathology
*Induced Pluripotent Stem Cells/metabolism
Cell Differentiation/genetics
Mutation
CRISPR-Cas Systems
Chondrocytes/metabolism/cytology
Chondrogenesis/genetics
Alleles
Organoids

@article {pmid42001795,
year = {2026},
author = {Radszuweit, P and Fitzel, R and Bruestl, S and Hentrich, T and Korkmaz, F and Mankel, B and González-Menéndez, I and Rudat, S and Marschalek, R and Erkner, E and Keppeler, H and Schairer, R and Luib, L and Mezger, M and Quintanilla-Martinez, L and Schulze-Hentrich, J and Lengerke, C and Schneidawind, D and Schneidawind, C},
title = {Characterizing the impact of MLL fusion variants and fusion partners on leukemia plasticity using a human CRISPR-engineered MLL-rearranged leukemia model.},
journal = {Neoplasia (New York, N.Y.)},
volume = {77},
number = {},
pages = {101308},
pmid = {42001795},
issn = {1476-5586},
mesh = {Humans ; *Myeloid-Lymphoid Leukemia Protein/genetics ; *Oncogene Proteins, Fusion/genetics ; Animals ; Mice ; *Histone-Lysine N-Methyltransferase/genetics ; *CRISPR-Cas Systems ; *Gene Rearrangement ; *Leukemia/genetics/pathology/metabolism ; Disease Models, Animal ; Gene Expression Profiling ; Cell Line, Tumor ; },
abstract = {Acute leukemias involving KMT2A (MLL) rearrangements are aggressive hematologic malignancies associated with a poor prognosis, especially in infants. The majority of MLL breakpoints are located within the breakpoint cluster region spanning exons 8-14, with AFF1 (AF4) and MLLT3 (AF9) being the most frequent fusion partners. To study the contribution of different fusion partners and breakpoint locations to leukemogenesis, we created a human CRISPR/Cas9-based model. We introduced MLL::AF4 or MLL::AF9 fusions with MLL breakpoints in intron 9 or 11, respectively, into human cord blood-derived CD34[+] cells from the same donor. Compared to healthy control cells, all MLL-rearranged cells showed increased proliferation and stemness, as well as an altered immunophenotype characterized by the upregulation of leukemic markers. Transcriptomic profiling revealed breakpoint- and partner-specific gene expression patterns that influence the characteristics of the disease. Notably, even after prolonged in vitro culture MLL(intron 9)::AF9 cells displayed robust colony formation in semisolid media and engrafted robustly in NOD scid gamma mice. The cells still exhibited high lineage plasticity, switching from a myeloid to a B-lymphoid identity in vivo. In conclusion, this model enables the mechanistic dissection of MLL fusion variants in vitro and in vivo, providing a foundation for developing targeted therapies for MLL-rearranged leukemias.},
}

Humans
*Myeloid-Lymphoid Leukemia Protein/genetics
*Oncogene Proteins, Fusion/genetics
Animals
Mice
*Histone-Lysine N-Methyltransferase/genetics
*CRISPR-Cas Systems
*Gene Rearrangement
*Leukemia/genetics/pathology/metabolism
Disease Models, Animal
Gene Expression Profiling
Cell Line, Tumor

@article {pmid42102651,
year = {2026},
author = {Walus, M and Kida, E and Golabek, AA},
title = {Lysosomal storage, mitochondrial pathology, and autophagy in knockout of tripeptidyl peptidase 1 in human neuroblastoma cells in vitro.},
journal = {Molecular genetics and metabolism},
volume = {148},
number = {2},
pages = {110130},
doi = {10.1016/j.ymgme.2026.110130},
pmid = {42102651},
issn = {1096-7206},
mesh = {Humans ; Tripeptidyl-Peptidase 1 ; *Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics/deficiency/metabolism ; *Autophagy/genetics ; *Mitochondria/pathology/metabolism/genetics ; *Lysosomes/metabolism/pathology/genetics ; *Neuronal Ceroid-Lipofuscinoses/genetics/pathology/metabolism ; *Aminopeptidases/genetics/metabolism/deficiency ; *Serine Proteases/genetics/metabolism/deficiency ; Cell Line, Tumor ; Gene Knockout Techniques ; *Neuroblastoma/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {Deficiency of tripeptidyl-peptidase 1 (TPP1; EC 3.4.14.9), a lysosomal enzyme encoded by the CLN2 gene, is associated with the lysosomal storage disorder - classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease). The classic form of CLN2 disease leads to the accumulation of autofluorescent lysosomal storage and a massive loss of neurons with gliosis in the brain. The major component of the storage is subunit c of mitochondrial ATP synthase, a highly hydrophobic, 75-aminoacid polypeptide. We developed an in vitro model of CLN2 disease by knocking out CLN2 in the human neuroblastoma cells SH-SY5Y by using CRISPR/cas9 technology. We focused on defining the pattern of deposition of subunit c, factors contributing to subunit c accumulation, and subcellular morphometry to identify differences between the model cells knockout (KO) and controls. Implementation of acetone for cell fixation allowed us to: i. identify higher levels of subunit c in the mitochondria of KO cells than controls; ii. characterize in detail subunit c inclusions, also present in controls; iii. identify other mitochondrial proteins colocalizing with subunit c in inclusions; and iv. detect mitochondrial pathology in degenerating cells often accompanied by deposition of subunit c. Differentiation of cells with retinoic acid and brain-derived neurotrophic factor led to a substantial increase in the levels of subunit c and to significant differences in the levels of autophagy-related proteins between KO and control cells. Inhibition of induced autophagy by bafilomycin A1 (Baf.A1) decreased subunit c levels in controls but not in KO cells, whereas the levels of subunit c were unaffected by Baf.A1 treatment upon basal autophagy. Finally, subcellular morphometry showed differences in the number and size of vesicular structures immunostained for autophagy-related proteins between KO cells and controls upon both induced and basal autophagy, further supporting the association of TPP1 deficiency with autophagy.},
}

Humans
Tripeptidyl-Peptidase 1
*Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics/deficiency/metabolism
*Autophagy/genetics
*Mitochondria/pathology/metabolism/genetics
*Lysosomes/metabolism/pathology/genetics
*Neuronal Ceroid-Lipofuscinoses/genetics/pathology/metabolism
*Aminopeptidases/genetics/metabolism/deficiency
*Serine Proteases/genetics/metabolism/deficiency
Cell Line, Tumor
Gene Knockout Techniques
*Neuroblastoma/genetics/pathology
CRISPR-Cas Systems

@article {pmid42138076,
year = {2026},
author = {Hale, AT and Kundishora, AJ and Kalailingam, P and Barak, T and Duy, PQ and Ramundo, CM and Fan, B and Li, Q and Brastianos, PK and Shankar, GM and Alper, SL and Kleinstiver, BP and Musolino, PL and Kahle, KT},
title = {Towards precision medicine for brain arteriovenous malformations.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
pmid = {42138076},
issn = {1558-8238},
mesh = {Humans ; *Precision Medicine ; *Intracranial Arteriovenous Malformations/genetics/therapy/metabolism/pathology ; Animals ; Mutation ; Proto-Oncogene Proteins B-raf/genetics/metabolism ; },
abstract = {Recent advances in cerebrovascular genomics, single-cell biology, pharmacology, and gene editing technology are transforming our understanding of brain arteriovenous malformations (bAVMs) - a leading cause of pediatric hemorrhagic stroke. Once considered static anatomical defects, bAVMs are now recognized as dynamic, genetically driven lesions resulting from somatic mutations in KRAS, BRAF, and pathways involved in arteriovenous specification, angiogenesis, and vascular remodeling. By integrating human genetics, animal models, and endovascular innovations, researchers have uncovered convergent mechanisms that link endothelial Ras/MAPK hyperactivation to abnormal vessel growth and higher rupture risk. These insights provide a foundation for precision medicine approaches that combine molecular diagnostics - such as liquid or endoluminal biopsies - with mutation-specific pharmacotherapies and emerging CRISPR-based gene editing strategies. We suggest that genotype-guided interventions, tailored by spatial and developmental cerebrovascular context, could ultimately reclassify bAVMs from surgically incurable malformations to treatable molecular conditions.},
}

Humans
*Precision Medicine
*Intracranial Arteriovenous Malformations/genetics/therapy/metabolism/pathology
Animals
Mutation
Proto-Oncogene Proteins B-raf/genetics/metabolism

@article {pmid42143446,
year = {2026},
author = {Martin, L and Rostami, S and Schuster, I and Qin, PZ and Rajan, R},
title = {Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.},
journal = {Biochemical and biophysical research communications},
volume = {823},
number = {},
pages = {153760},
doi = {10.1016/j.bbrc.2026.153760},
pmid = {42143446},
issn = {1090-2104},
abstract = {CRISPR-Cas systems comprise a CRISPR RNA (crRNA)-guided CRISPR-associated (Cas) nuclease for providing immune protection. The complementary base pairing between crRNA and the invader genome leads to the formation of an "R-loop", which triggers the nuclease activity of the Cas protein, effectively neutralizing the invasion. This molecular mechanism has been repurposed for genome applications using Cas9 and Cas12a. Cas12a has several favorable features for applications including its smaller size, crRNA processing ability, and creation of staggered double-stranded DNA (dsDNA) cleavage. Gene editing with these Cas proteins, however, has some setbacks due to off-target and non-specific DNA cleavages. To increase the specificity in DNA cleavage, we introduced proline/alanine substitutions at different positions along a conserved arginine/lysine-rich "bridge helix" (BH) of Cas12a that plays an integral role in mediating conformational changes needed for DNA cleavage. Cleavage kinetics analyses reveal that enhanced helical integrity of the BH of Francisella novicida Cas12a provided by alanine substitutions increases DNA cleavage efficiency, while reducing the ability of the variants to discriminate DNA mismatches. Proline substitutions demonstrate an opposite effect by reducing the efficiency of cleaving on-target DNA, but almost completely abolishing linearization of a target with a mismatch in the middle of the R-loop. These results parallel those reported for Cas9 and show that balancing the helicity of BH through rational amino acid substitutions can finetune Cas12a's off-target profiles. This may provide a strategy for enhancing specificity of Cas12a in genome manipulation.},
}

@article {pmid42143634,
year = {2026},
author = {Mahendrarajan, V and Sankaranarayanan, GN and Muthukaliannan, GK and Easwaran, N},
title = {Triphala Modulates the Membrane Vesicle Transcriptome of Enterococcus durans VIT3 to Influence Antibiotic Response and Probiotic Functions.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42143634},
issn = {1559-0305},
support = {SG20210254//VIT University/ ; },
abstract = {Membrane vesicles (MVs) are critical mediators of bacterial communication, physiology, and host interactions. This study explores how triphala, a polyherbal formulation, modulates the RNA cargo of MVs in Enterococcus durans VIT3 isolate under varied stress conditions. The isolated MVs from all treatment conditions (triphala, antibiotics, or sequential combinations) were spherical, intact, with negative zeta potential and sizes ranging from 56.4 to 462.3 nm. Cytotoxicity assays indicated no toxicity toward CaCo2 cells under the tested conditions. Transcriptomic profiling suggested that triphala pretreatment enhanced relative expression of genes linked to oxidative stress defense (ohrB, sodA), envelope integrity (dlt operon, pbp, mprF), and CRISPR-Cas-associated genes (cas1). In contrast, antibiotic first regimens suppressed stress and defense-associated genes, underscoring treatment order effects. Notably, triphala antibiotic sequence condition showed coordinated expression patterns within ATP synthase, arginine deiminase, and CRISPR loci, suggesting operon-level transcriptional organization of metabolic and stress-response pathways. STRING network analysis further supported clustering of genes showing relatively higher expression into functionally related molecules. Collectively, these findings suggest a potential role for triphala in shaping MV RNA cargo, offering a conceptual framework for synbiotic strategies aimed at supporting microbial stress resilience. All transcriptomic observations are based on pooled MV samples, which represent exploratory expression trends rather than statistically inferred differential expression or functional outcomes.},
}

@article {pmid42144412,
year = {2026},
author = {Kasapoglu, MZ and Acar, I and Gumustop, I and Erol, I and Kurt, IC and Ortakci, F},
title = {Pangenomics of Limosilactobacillus fermentum reveals genomic diversity and bacteriocin activity against Staphylococcus aureus and Escherichia coli.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-51219-1},
pmid = {42144412},
issn = {2045-2322},
support = {MGA-2024-45355//Bilimsel Araştırma Projeleri Birimi, İstanbul Teknik Üniversitesi/ ; },
abstract = {Limosilactobacillus fermentum is a versatile lactic acid bacterium with significant probiotic and biotechnological potential, yet the genomic determinants underlying its ecological adaptation and therapeutic applications remain underexplored. This study performed a comparative genomic analysis of 52 L. fermentum strains, with a focus on the novel strain ATT-06 isolated from traditional Turkish shalgam. We uncovered substantial genetic diversity, evidenced by an open pan-genome (18,647 genes) and a small core genome (718 genes). CRISPR-Cas systems were prevalent (46 strains), with Type-IE and Type-IIA being most common and mutually exclusive from Type-IC. Strain ATT-06, which uniquely produced gamma-aminobutyric acid at 17.74 µg/mL, harbored a Type-IIA CRISPR system and a single prophage. In silico molecular docking and dynamics simulations revealed that the bacteriocin Lactococcin, encoded by strain ATA-LTC-Lf170503, exhibited strong binding affinities (ΔG: -8.1 to -13.0 kcal/mol) against Rho proteins of Staphylococcus aureus and Escherichia coli, outperforming Acidocin A. These findings highlight the genomic plasticity and adaptive mechanisms of L. fermentum, and position strain ATT-06 as a promising probiotic candidate with potential neuroactive and antimicrobial applications.},
}

@article {pmid41104514,
year = {2026},
author = {Hu, Y and Zeng, Z and Ming, X and Yin, S and Guan, Y and Gao, L and Li, D},
title = {Construction of pathogenic Sec16a mutation mouse model using CRISPR/Cas9.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {3},
pages = {537-545},
doi = {10.1002/ame2.70082},
pmid = {41104514},
issn = {2576-2095},
support = {HZKY20220076//The Chunhui Program of the ministry of Education/ ; 21JC1402200//the Shanghai Municipal Commission of Science and Technology/ ; 22YF1437700//the Shanghai Municipal Commission of Science and Technology/ ; 23HC1400700//the Shanghai Municipal Commission of Science and Technology/ ; 32200732//National Natural Science Foundation of China/ ; 2022YFC3400200//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Disease Models, Animal ; *Mutation ; Mice ; *Vesicular Transport Proteins/genetics ; Humans ; Mice, Inbred C57BL ; Male ; },
abstract = {BACKGROUND: SEC16A is a pivotal protein that facilitates the transport of proteins from the endoplasmic reticulum to the Golgi apparatus. Utilizing the protein structure function database, a potentially pathogenic mutation site (NM_014866.1: c.4606C>G(p.L1536V)) was pinpointed within the conserved central core region of the human SEC16A protein, a component integral to the COPII complex assembly.

METHODS: Leveraging information on human gene mutations and aligning human and mouse protein amino acid sequences, the Sec16aL1551V/L1551V mouse model was successfully developed using CRISPR/Cas9 technology.

RESULTS: Two behavioral experiments, namely novel object recognition and cued fear conditioning, revealed that Sec16aL1551V/L1551V mice demonstrated a phenotype of neurological impairment, evidenced by diminished abilities in learning and memory. Furthermore, while undergoing tail suspension, the Sec16aL1551V/L1551V mice displayed a distinctive limb clasping behavior, a characteristic typically associated with mouse models of chronic neurodegenerative diseases.

CONCLUSION: The Sec16aL1551V/L1551V mouse model developed in this study providing a powerful tool for better understanding of the pathogenic mechanisms of Sec16a gene mutations in brain dysfunction diseases.},
}

Animals
*CRISPR-Cas Systems/genetics
*Disease Models, Animal
*Mutation
Mice
*Vesicular Transport Proteins/genetics
Humans
Mice, Inbred C57BL
Male

@article {pmid41700779,
year = {2026},
author = {Alberio, V and Savy, V and Felipe, MY and Fernandez-Martín, R and Salamone, DF},
title = {Simultaneous CRISPR-on activation of TFAP2C and SMARCA4 promotes development of trophoblast-fate cells in bovine embryos†.},
journal = {Biology of reproduction},
volume = {114},
number = {5},
pages = {1598-1609},
doi = {10.1093/biolre/ioag041},
pmid = {41700779},
issn = {1529-7268},
support = {PICT 2018-04632//ANPCyT from the Ministry of Science and Technology, Argentina/ ; },
mesh = {Animals ; *Trophoblasts/physiology/metabolism/cytology ; Cattle/embryology ; *Transcription Factor AP-2/genetics/metabolism ; Gene Expression Regulation, Developmental ; Female ; *Embryonic Development/genetics ; *Transcription Factors/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; Cell Differentiation/genetics ; *Nuclear Proteins/genetics/metabolism ; Embryo, Mammalian ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Pregnancy ; },
abstract = {Assisted reproductive techniques are widely used to produce domestic animal embryos for commercial or research purposes. In cattle, abnormal trophoblast cell differentiation during embryo development causes pregnancy and placentation failures. The CRISPR-on system has been successfully used in bovine embryos to individually activate early trophoblast lineage genes TFAP2C and SMARCA4. This study evaluates the effect of CRISPR-on activation of early and advanced trophoblast-related genes in bovine zygotes to promote trophectoderm differentiation. In one experiment, RNA was microinjected to simultaneously activate TFAP2C and SMARCA4; in another, circular DNA was microinjected to induce CDX2 or GATA3 expression separately. Controls included groups without small guide RNAs (sgRNAs, SHAM) and non-injected embryos (IVF). RNA microinjection transiently and simultaneously increased TFAP2C, SMARCA4, and downstream gene expression, producing a prolonged effect beyond individual gene activation. This led to a significant increase in trophectoderm cells at the blastocyst stage. Individual activation of CDX2 and GATA3 was effective, also inducing upstream genes without altering trophectoderm cell percentages. Neither RNA nor DNA microinjection affected blastocyst production compared to IVF. This work demonstrates the successful increase of trophoblast cells in bovine embryos using CRISPR-on, offering a useful strategy for IVF or SCNT embryos. The CRISPR-dCas9VP160 system may also aid understanding of trophoblast lineage signaling during development.},
}

Animals
*Trophoblasts/physiology/metabolism/cytology
Cattle/embryology
*Transcription Factor AP-2/genetics/metabolism
Gene Expression Regulation, Developmental
Female
*Embryonic Development/genetics
*Transcription Factors/genetics/metabolism
*DNA Helicases/genetics/metabolism
Cell Differentiation/genetics
*Nuclear Proteins/genetics/metabolism
Embryo, Mammalian
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Pregnancy

@article {pmid41712528,
year = {2026},
author = {Magdy, M and Tinker-Kulberg, R and Josephs, EA},
title = {Polyvalent Guide RNAs Enhance the CRISPR-Mediated Suppression of a Human Coronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1738-1750},
doi = {10.1021/acssynbio.5c00574},
pmid = {41712528},
issn = {2161-5063},
support = {R35GM133483/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Coronavirus 229E, Human/genetics ; Gene Editing/methods ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {While CRISPR enzymes have become important tools for targeted gene editing in mammalian cells, they can also be used to specifically target and deplete viral nucleic acids to treat infections; this can be accomplished by delivering an RNA-targeting CRISPR effector like Cas13 along with a guide RNA (gRNA) that recognizes sequences from the genomes of single-stranded RNA (ssRNA) viruses. Previously, we hypothesized that by designing individual gRNAs able to target multiple, similar-but-not-identical viral sequences simultaneously ("polyvalent" guide RNAs or pgRNAs), gRNA's polyvalency would overcome any deficits caused by mispairing between the gRNA and the viral targets and, hence, still increase Cas13's antiviral potency and prevent mutagenic escape. We subsequently demonstrated this was the case using a model of viral infection in plants; however, it was not determined whether this strategy would also work against a human virus. Here, pgRNAs were designed to target multiple RNA sequences within human coronavirus 229E (hCoV-229E) and delivered along with Cas13 into a human lung epithelial cell line infected by hCoV-229E. CRISPR antiviral treatments using pgRNAs exhibited significant viral suppression in a CRISPR-dependent manner─more so than their single-target gRNA counterparts, even when multiple single-target gRNAs were used simultaneously. This improvement was also observed even as Cas13 with those same pgRNAs exhibited less "collateral" or nonspecific RNase activity relative to their single-target counterparts, which could imply that they may have greater specificity and safety profiles as therapeutic agents. Our findings demonstrate a computational and experimental pipeline by which pgRNAs, created using an unconventional gRNA design strategy, can be generated and validated to target human viruses using CRISPR antiviral biotechnologies more effectively.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Coronavirus 229E, Human/genetics
Gene Editing/methods
RNA, Viral/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid41874563,
year = {2026},
author = {Fang, J and Simon, JM and Wang, T and Gao, Y and Bi, X and Hu, L and Liao, C and Zhang, C and Adachi, Y and Zhou, J and Liu, H and Liang, Q and Nathan, JA and Mani, R and Brugarolas, J and Zhang, Q},
title = {Genome-wide CRISPR screen identifies a cytokine-enhancer circuit driving HIF-2α activation in renal cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI201639},
pmid = {41874563},
issn = {1558-8238},
support = {P50 CA196516/CA/NCI NIH HHS/United States ; },
mesh = {*Basic Helix-Loop-Helix Proteins/genetics/metabolism/biosynthesis ; Humans ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; STAT3 Transcription Factor/metabolism/genetics ; Mice ; Animals ; Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Janus Kinase 1/metabolism/genetics ; *CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Cytokines/metabolism/genetics ; Signal Transduction ; *Neoplasm Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; },
abstract = {Resistance to HIF-2α inhibitors such as belzutifan underscores the need to better understand how HIF-2α is transcriptionally regulated in clear cell renal cell carcinoma (ccRCC). Here, we uncover a cytokine-driven enhancer mechanism that sustains HIF-2α expression through the JAK1/STAT3 signaling pathway. Using a genome-wide CRISPR screen in von Hippel-Lindau-deficient (VHL-deficient) ccRCC cells, we identified SOCS3 as a key negative regulator of HIF-2α. Mechanistically, loss of SOCS3 activates JAK1/STAT3 signaling, leading to the recruitment of STAT3 to distal enhancers upstream of endothelial PAS domain-containing protein (EPAS1) that physically loop to its promoter to drive HIF-2α transcription. This cytokine-enhancer circuit was recapitulated in samples from patients with ccRCC and functionally validated using CRISPR interference (CRISPRi), which disrupted enhancer-promoter looping and reduced tumor growth in HIF-2α-dependent models. SOCS3 overexpression or pharmacologic inhibition of JAK1/STAT3 markedly suppressed HIF-2α expression and tumor progression both in vitro and in vivo. Unlike prior studies focusing on VHL/HIF occupancy-driven enhancer activation, this work defines a trans-acting cytokine-JAK1/STAT3 pathway that transcriptionally controls EPAS1. Together, these findings reveal a targetable enhancer mechanism that sustains HIF-2α expression and suggest that combined inhibition of JAK1/STAT3 and HIF-2α may overcome therapeutic resistance in kidney cancer.},
}

*Basic Helix-Loop-Helix Proteins/genetics/metabolism/biosynthesis
Humans
*Kidney Neoplasms/genetics/metabolism/pathology
*Carcinoma, Renal Cell/genetics/metabolism/pathology
STAT3 Transcription Factor/metabolism/genetics
Mice
Animals
Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
Janus Kinase 1/metabolism/genetics
*CRISPR-Cas Systems
*Enhancer Elements, Genetic
*Cytokines/metabolism/genetics
Signal Transduction
*Neoplasm Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Von Hippel-Lindau Tumor Suppressor Protein/genetics

@article {pmid41915438,
year = {2026},
author = {Ma, Z and Jia, W and Zhou, X and Liu, J and Li, Q and Chang, R and Shiqi, G and Yuan, N and Chen, Z and Lan, P},
title = {In vivo CRISPR screens identify CBX4 as an epigenetic regulator for cancer immunotherapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI200564},
pmid = {41915438},
issn = {1558-8238},
mesh = {Animals ; Mice ; Humans ; *Epigenesis, Genetic/immunology ; *Immunotherapy ; *Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Polycomb-Group Proteins/genetics/immunology ; *Ligases/genetics/immunology ; Cell Line, Tumor ; *Neoplasm Proteins/genetics/immunology ; *Neoplasms/genetics/immunology/therapy ; *Liver Neoplasms/immunology/genetics/therapy ; },
abstract = {Epigenetic dysregulation is associated with immune evasion and immune checkpoint blockade (ICB) resistance. Here, using in vivo CRISPR/Cas9 screens targeting epigenetics-related factors in mouse tumor models treated with ICB, we identified chromobox 4 (CBX4) as a key negative regulator of the immune tumor microenvironment (TME). Single-cell RNA-seq and spatial transcriptomics analyses of patients receiving neoadjuvant anti-programmed cell death protein 1 (anti-PD-1) therapy revealed high CBX4 expression in both tumor cells and immunosuppressive tumor-associated macrophage subpopulations, with preferential accumulation in nonresponders. Deficiency of CBX4 in macrophages or tumor cells induced robust antitumor immunity and increased infiltration and the cytotoxic activity of CD8+ T cells and NK cells, thereby heightening the sensitivity of ICB treatment. Mechanistically, CBX4 targeted H3K9me3- and H3K27me3-marked endogenous retroelements such as RLTR4-Mm-int. Loss of CBX4 derepressed retrotransposons, activating cytosolic RNA-sensing pathways and triggering the type I IFN response, ultimately leading to a robustly inflamed TME. Moreover, we uncovered a negative correlation between CBX4 expression, immune responses, and retrotransposon levels, and were able to determine the prognosis of patients with hepatocellular carcinoma (HCC) undergoing ICB therapy. Our study establishes CBX4 as an epigenetic immune checkpoint through the epigenetic silencing of retrotransposons, remodeling the immune TME and thus providing a promising therapeutic target to enhance tumor immunogenicity and overcome immunotherapy resistance.},
}

Animals
Mice
Humans
*Epigenesis, Genetic/immunology
*Immunotherapy
*Tumor Microenvironment/immunology/genetics
*CRISPR-Cas Systems
*Polycomb-Group Proteins/genetics/immunology
*Ligases/genetics/immunology
Cell Line, Tumor
*Neoplasm Proteins/genetics/immunology
*Neoplasms/genetics/immunology/therapy
*Liver Neoplasms/immunology/genetics/therapy

@article {pmid41926741,
year = {2026},
author = {Driscoll, CS and Kim, J and Knott, JG},
title = {CRISPR-mediated editing of cis-regulatory elements in early mouse embryos: a tool for studying pluripotency gene regulation.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {5},
pages = {},
doi = {10.1093/reprod/xaag042},
pmid = {41926741},
issn = {1741-7899},
support = {HD095371//National Institutes of Child Health and Development/ ; //Michigan State University AgBioResearch/ ; HD087166//T32 doctoral fellowship from the NICHD/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; Female ; *Transcription Factor AP-2/genetics/metabolism ; *Blastocyst/metabolism ; *Embryonic Development/genetics ; Octamer Transcription Factor-3/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Pluripotent Stem Cells/metabolism ; },
abstract = {In brief Cis-regulatory elements and transcription factor binding motifs play crucial roles in regulating the spatial and temporal patterns of gene expression during development. This study tested the utility of CRISPR/Cas9 as a tool to interrogate the function of transcription factor AP2 gamma motifs in pluripotency gene expression during mouse preimplantation embryo development. Abstract Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) is a highly efficient tool that enables the generation of gene knockouts, knock-ins, and single base substitutions in a variety of organisms. Recently, we used CRISPR to examine the activity of cis-regulatory elements (CREs) in mouse preimplantation embryos. However, there is limited information on the feasibility of using CRISPR in preimplantation embryos to interrogate the function of select transcription factor (TF) binding motifs located within critical CREs in pluripotency genes. In the current study we employed CRISPR to disrupt TF AP2 gamma (TFAP2C) binding motifs located within key CREs involved in the regulation of Pou5f1 and Sox2 expression in early embryos. Microinjection of ribonucleoprotein complexes containing Cas9 and single guide RNAs (sgRNAs) targeting TFAP2C motifs located within a distal enhancer and proximal promoter substantially impaired Pou5f1 and Sox2 expression, respectively. Quantification of the editing efficiencies at each targeted CRE revealed that the targeting sgRNA sequences and the number of sgRNAs injected influenced the overall editing rates. Lastly, we investigated whether TFAP2C-induced activation of Sox2 expression in 2-cell embryos required TFAP2C motifs located within the Sox2 proximal promoter. CRISPR-mediated editing of these motifs diminished the activation of Sox2 expression. In summary, these findings indicate that CRISPR/Cas9 is a feasible approach for editing TF motifs in preimplantation embryos and provide evidence that TFAP2C directly contributes to Pou5f1 and Sox2 expression in preimplantation embryos.},
}

Animals
Mice
*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Expression Regulation, Developmental
Female
*Transcription Factor AP-2/genetics/metabolism
*Blastocyst/metabolism
*Embryonic Development/genetics
Octamer Transcription Factor-3/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*Pluripotent Stem Cells/metabolism

@article {pmid41957993,
year = {2026},
author = {Duhalde, MA and Martino, RA and Smania, A and Alvarez, HM and Hernández, MA},
title = {CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous Rhodococcus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1926-1942},
doi = {10.1021/acssynbio.6c00019},
pmid = {41957993},
issn = {2161-5063},
mesh = {*Rhodococcus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cytidine/genetics/metabolism ; Plasmids/genetics ; Genome, Bacterial/genetics ; DNA End-Joining Repair/genetics ; },
abstract = {Oleaginous Rhodococcus strains can degrade diverse compounds and synthesize and accumulate large amounts of intracellular lipids, making them attractive platforms for biotechnological applications. However, efficient genome editing in Rhodococcus remains challenging, and new molecular tools are needed to advance the understanding of its metabolism, stress responses, and cellular physiology. Here, we describe the nonhomologous end joining (NHEJ) system in rhodococci and implement an efficient genome-editing system based on a CRISPR/Cas9 nuclease approach that utilizes this repair mechanism, eliminating the need for donor DNA templates. In addition, we report the first implementation of a cytidine base-editing (CBE) system, enabling precise single-nucleotide substitutions (C•G → T•A) in oleaginous Rhodococcus strains. Both strategies rely on a dual-plasmid CRISPR platform, resulting in two plasmid sets: pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA. These systems enabled high rates of INDEL formation and C•G → T•A base conversions, with efficiencies of 70-80% and 75-85% at native genomic targets, respectively. Finally, for the CBE system, we implemented a Csy4-mediated sgRNA-processing module to support multiplex genome editing, enabling the simultaneous modification of multiple loci. Together, these tools outperform recombination-based approaches and will facilitate the study of complex metabolic pathways and the development of genetic strategies for biotechnological applications in Rhodococcus, while also being transferable to other actinobacteria.},
}

*Rhodococcus/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Cytidine/genetics/metabolism
Plasmids/genetics
Genome, Bacterial/genetics
DNA End-Joining Repair/genetics

@article {pmid41974167,
year = {2026},
author = {Peng, Z and Liu, Z and Wei, S and Zhu, Y and Jia, X and Xiao, W and Dong, Z and Hu, H and Lu, C and Pan, M and Chen, P},
title = {Genome-Wide CRISPR/Cas9 Screening Reveals BmM-ALP Orchestrates the Antioxidant Response and Metabolic Adaptations for Heat Resistance in Bombyx mori.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1968-1979},
doi = {10.1021/acssynbio.6c00053},
pmid = {41974167},
issn = {2161-5063},
mesh = {Animals ; *Bombyx/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Reactive Oxygen Species/metabolism ; *Antioxidants/metabolism ; *Thermotolerance/genetics ; Adaptation, Physiological/genetics ; *Insect Proteins/genetics/metabolism ; Heat-Shock Response/genetics ; Animals, Genetically Modified ; },
abstract = {Climate change-induced heat stress represents an increasing threat to biodiversity, engendering the need to understand evolutionary adaptations to thermal extremes. Using Bombyx mori as an economically important insect and research model, we employed genome-wide CRISPR/Cas9 screening integrated with high-throughput sequencing to systematically identify adaptations for heat resistance. The analysis identified the Bombyx mori membrane-bound alkaline phosphatase-like gene (BmM-ALP) as a vital thermoregulator. BmM-ALP triggered a pleiotropic protective cascade by significantly decreasing reactive oxygen species (ROS), suppressing apoptosis, and reprogramming mitochondrial metabolism via the phosphorylation of Vitamin B1 (VB1), suggesting a potential role in sustaining oxidative phosphorylation and enhancing energy metabolism under stress. Transgenic validation experiments confirmed the consistency of this strategy, and the BmM-ALP-OE strain displayed significantly enhanced thermal tolerance and prolonged survival under high-temperature stress. Beyond establishing BmM-ALP as a key gene of heat resistance in silkworms, this study uncovered a regulatory axis linking BmM-ALP to Vitamin B1 metabolism, illustrating a novel connection between energy homeostasis and thermal adaptation. The findings provide new insights that can be applied to breeding resistant strains in agriculture and biodiversity conservation in the context of global warming.},
}

Animals
*Bombyx/genetics/metabolism/physiology
*CRISPR-Cas Systems/genetics
Reactive Oxygen Species/metabolism
*Antioxidants/metabolism
*Thermotolerance/genetics
Adaptation, Physiological/genetics
*Insect Proteins/genetics/metabolism
Heat-Shock Response/genetics
Animals, Genetically Modified

@article {pmid41979903,
year = {2026},
author = {Stuckless, EE and Gai, LS and Slattery, SS and Dempsey, KH and Browne, TS and Gloor, GB and Edgell, DR},
title = {PHYCUT: Scalable Multiplex CRISPR/Cas9 Editing for Genome Engineering in the Diatom Phaeodactylum tricornutum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1850-1865},
doi = {10.1021/acssynbio.5c00843},
pmid = {41979903},
issn = {2161-5063},
mesh = {*Diatoms/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Glycosylation ; Plasmids/genetics ; Fucose/metabolism ; },
abstract = {Diatoms are globally significant microalgae that contribute ∼20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom Phaeodactylum tricornutum has emerged as a promising synthetic biology platform for the bioproduction of recombinant proteins, supported by a human-like N-linked glycosylation pathway. However, its α(1,3)-linked core fucose is potentially immunogenic in humans and thus limits its biopharmaceutical applications. One hurdle to efficient genome engineering in P. tricornutum is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (Phaeodactylum tricornutum Csy4-Cas9 multiplex tool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multiguide RNA arrays. To highlight PHYCUT applications, we demonstrate multiplex editing of all three FucT genes responsible for α(1,3) fucosylation in P. tricornutum, yielding strains with reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the P. tricornutum glycosylation pathway to support next-generation algal biotechnology.},
}

*Diatoms/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Glycosylation
Plasmids/genetics
Fucose/metabolism

@article {pmid42014015,
year = {2026},
author = {Li, R and Xie, L and Hu, J and Liu, B and Zhang, H and Qian, H},
title = {Genome-wide CRISPR knockout screening identifies novel disease-associated genes in retinal pigment epithelium cells.},
journal = {Experimental eye research},
volume = {268},
number = {},
pages = {111032},
doi = {10.1016/j.exer.2026.111032},
pmid = {42014015},
issn = {1096-0007},
mesh = {*Retinal Pigment Epithelium/metabolism/pathology ; Humans ; Gene Knockout Techniques ; *Retinal Degeneration/genetics/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome-Wide Association Study ; Cell Survival ; *CRISPR-Cas Systems ; *Gene Expression Regulation/physiology ; Cells, Cultured ; *Eye Proteins/genetics ; },
abstract = {Dysfunction and degeneration of retinal pigment epithelium (RPE) cells are common pathological features observed in various retinal degenerative diseases. It has been proposed to treat these diseases by either protecting RPE cells or replacing them with new RPE cells derived from stem cells. However, the development of effective therapeutic strategies is still limited due to the insufficient understanding of the pathogenic factors involved in retinal degeneration and their impact on the function and survival of RPE cells. In this study, we employed genome-scale CRISPR knockout (KO) screening in human RPE cells to identify genes critical for RPE cell survival. Over 300 genes were identified, including well-established housekeeping genes as well as several candidate genes previously linked to retinal degeneration, many of which still lack comprehensive investigation. Among these, we further validated PRPF38B, which was both enriched in our screening and highlighted in a prior family-based linkage study, as essential for RPE cell survival, thus confirming the effectiveness of our approach. As a component of the spliceosome, we found that PRPF38B is crucial for functions specific to RPE cells, offering new insights into its role in retinal degeneration. Our study presents a novel approach for investigating risk genes associated with retinal diseases and may inspire future research on RPE cells and vision disorders.},
}

*Retinal Pigment Epithelium/metabolism/pathology
Humans
Gene Knockout Techniques
*Retinal Degeneration/genetics/metabolism/pathology
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome-Wide Association Study
Cell Survival
*CRISPR-Cas Systems
*Gene Expression Regulation/physiology
Cells, Cultured
*Eye Proteins/genetics

@article {pmid42044361,
year = {2026},
author = {Huang, J and Zhang, C and Li, J and Ren, H and Yang, K and Zhang, Y},
title = {Comparative Evaluation of Engineered Bacteria and Yeast for Oral Delivery of CRISPR/Cas9 Systems in Colon Cancer Therapy.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1915-1925},
doi = {10.1021/acssynbio.6c00006},
pmid = {42044361},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Colonic Neoplasms/therapy/genetics ; Mice ; *Escherichia coli/genetics/metabolism ; Humans ; Administration, Oral ; Gene Editing/methods ; Cell Line, Tumor ; Female ; Genetic Therapy/methods ; Mice, Inbred BALB C ; },
abstract = {Colorectal cancer (CRC) poses a serious threat to human health. CRISPR/Cas9 technology offers new therapeutic strategies for the management of this disease, but its oral application is severely hindered by the limitations of suitable delivery systems. Herein, we develop and compare two separate orally delivered, genetically and chemically modified CRISPR/Cas9 delivery platforms based on E. coli BL21 and P. pastoris X33, which upon colonization in the intestine, secreted extracellular vesicles carrying the Cas9 protein and ART1-targeting sgRNA for tumor-specific gene disruption. Arginine ADP-ribosyltransferase 1 (ART1) plays a crucial role in the biological regulation of colon cancer, which was for the first time to the best of our knowledge, employed in vivo as a target gene in this study. Furthermore, we employed polydopamine (PDA) coating and gastrointestinal synthetic epithelial lining systems to facilitate microbial viability and intestinal retention, establishing on site cell factories for sustained CRISPR secretion. In subcutaneous tumor-bearing murine models, both delivery systems demonstrated comparable antitumor efficacy with significant tumor suppression. Taken together, the genetically modified microbial platform using bacterial and yeast strategies shows great potential and broad therapeutic versatility, offering a promising CRISPR-based solution for CRC treatment.},
}

*CRISPR-Cas Systems/genetics
Animals
*Colonic Neoplasms/therapy/genetics
Mice
*Escherichia coli/genetics/metabolism
Humans
Administration, Oral
Gene Editing/methods
Cell Line, Tumor
Female
Genetic Therapy/methods
Mice, Inbred BALB C

@article {pmid42052944,
year = {2026},
author = {Chen, Z and Hong, W and Wei, X and Li, Y and Feng, T and Ke, X and Li, X and Wang, Y and Hang, H and Tian, X and Chu, J},
title = {A Versatile tRNA-gRNA Array-Based CRISPR/Cas9 Platform Enabling Multiplex Genome Editing and Large-Fragment Engineering in Acremonium chrysogenum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1955-1967},
doi = {10.1021/acssynbio.6c00052},
pmid = {42052944},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Acremonium/genetics/metabolism ; *RNA, Transfer/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Cephalosporins/biosynthesis ; Metabolic Engineering/methods ; Gene Knockout Techniques ; },
abstract = {Cephalosporin C (CPC)-derived antibiotics have played a vital role in improving human health and extending life expectancy. Acremonium chrysogenum remains the only microorganism capable of industrial-scale CPC production to date. However, the lack of efficient multiplex genome-editing tools has limited studies on its gene function, high-yield mechanisms as well as metabolic engineering. To overcome this limitation, a rapid and efficient CRISPR/Cas9-based multiplex genome-editing system was developed, driven by endogenous tRNA promoters, enabling one-step multilocus knockout, large-fragment DNA deletion, and gene overexpression in A. chrysogenum. Given that many strains lack visible phenotypes associated with specific genes, we introduced a visually distinguishable red phenotype by expressing the heterologous protein mCherry under a strong promoter. In the wild-type strain, 20 endogenous tRNA promoters were evaluated and compared to the heterologous Aspergillus nidulans PgpdA and Aspergillus fumigatus U6 promoters. The endogenous tRNA[Val] promoter showed the highest knockout efficiency (95.5%). The tRNA-gRNA array-based CRISPR/Cas9 system enabled double- and triple-site knockouts without donor DNA in industrial strain, with efficiencies of 50.0-83.3% and 14.3%, respectively. This is the first demonstration of simultaneous triple-site knockout in A. chrysogenum, especially in industrial strain. Using this system, we successfully deleted a 50.7-kb DNA fragment containing the sorbicillinoids biosynthetic gene cluster with nearly 100% efficiency and achieved overexpression of the key gene pcbAB involved in CPC biosynthesis in high-yield strain, increasing CPC titer from 5.59 g/L to 6.92 g/L with an improvement of 23.8%. Overall, this tRNA-gRNA array-based CRISPR/Cas9 multiplex gene-editing system provides an efficient and versatile platform for functional genomics and industrial strain engineering in A. chrysogenum.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Acremonium/genetics/metabolism
*RNA, Transfer/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics
Cephalosporins/biosynthesis
Metabolic Engineering/methods
Gene Knockout Techniques

@article {pmid42068556,
year = {2026},
author = {Ban, H and Rondthaler, SN and Lebovich, M and Lora, MA and Ugbesia, B and Andrews, LB},
title = {Cross-Strain Transferability of CRISPRi Systems and Design Rules from Laboratory to Clinical Escherichia coli Strains.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1993-2010},
doi = {10.1021/acssynbio.6c00075},
pmid = {42068556},
issn = {2161-5063},
support = {NSF DMR-1904901//NSF National Science Foundation/ ; NSF EES-1824090//NSF UMass ADVANCE program National Science Foundation/ ; NSF CBET-1943695//NSF National Science Foundaion/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to nonmodel strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different Escherichia coli strains, including a laboratory K-12 strain (MG1655) and three nonmodel strains that are clinical isolates (probiotic Nissle 1917, uropathogenic CFT073, and uropathogenic UMN026). We evaluated the repression in each strain using sets of guide RNAs (gRNAs) targeting along the gene sequence and assayed cytotoxicity of expressing each dCas protein. Growth toxicity from expression of the different dCas proteins notably differed and showed high variation between some host strains. We also observed variable repression among the strains and notably poorer repression in multiple clinical strains. Therefore, we developed a dual gRNA CRISPRi system for enhanced gene silencing among the strains, which achieved up to 824-fold repression in CFT073. The results demonstrate that strain-specific design considerations can arise when a CRISPRi genetic system is transferred to a closely related bacterial strain. These findings provide insight into the relationships between criteria used for CRISPRi genetic design and in vivo activity across nonmodel E. coli strains, providing guidelines for diverse applications of these tools.},
}

*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid42084552,
year = {2026},
author = {Huang, X and Liu, M and Chen, Y and Fang, L and Cao, Y},
title = {Genome-Scale CRISPRi Screening Identifies Gene Targets for Enhanced Octanoic Acid Tolerance and Production in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1771-1778},
doi = {10.1021/acssynbio.6c00105},
pmid = {42084552},
issn = {2161-5063},
mesh = {*Escherichia coli/genetics/metabolism/drug effects ; *Caprylates/metabolism/pharmacology ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; NADP/metabolism ; Genome, Bacterial ; },
abstract = {Medium-chain fatty acids (MCFAs) are valuable precursors for biofuels and other commodity chemicals; however, the microbial biosynthesis of these compounds is severely constrained by cytotoxic effects. Here, we employed a genome-scale CRISPR interference (CRISPRi) library to systematically identify gene targets whose repression enhanced the tolerance to octanoic acid (C8) in Escherichia coli. Among the identified targets, repression of ygaM, gluQ, gatY, and talA enabled a 1.1- to 1.7-fold increase in C8 production relative to the parental strain MS-1. Mechanistic analyses revealed that the enhanced tolerance was associated with improved membrane properties, reduced reactive oxygen species (ROS) levels, and a shorter cell morphology. Further metabolic engineering to optimize NADPH availability increased C8 production to 1083 mg/L, representing a 2.3-fold increase over that of the MS-1 strain. This study provides new insights into engineering robust E. coli strains for MCFAs production and highlights the utility of genome-scale CRISPRi screening for identifying genetic determinants of microbial stress tolerance.},
}

*Escherichia coli/genetics/metabolism/drug effects
*Caprylates/metabolism/pharmacology
Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
Escherichia coli Proteins/genetics/metabolism
Reactive Oxygen Species/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
NADP/metabolism
Genome, Bacterial

@article {pmid42136289,
year = {2026},
author = {Sorourian, S and Behbahani, AB},
title = {CRISPR-Cas Systems and CHO in Biopharmaceuticals: Unlocking New Possibilities in Gene Editing.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010408149251016112221},
pmid = {42136289},
issn = {1873-4316},
abstract = {The CRISPR-Cas system has significantly advanced genome editing, offering superior efficiency, precision, and ease of use compared to traditional technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). CHO cells are a widely used mammalian cell line for large-scale therapeutic protein manufacturing due to their ability to produce human-like glycosylation patterns and grow in serum-free media. Recent CRISPR-based CHO cell engineering enables precise genetic modifications, improving productivity, stability, scalability, and reducing costs. This article highlights the transformative role of CRISPR technologies in addressing genetic disorders and expanding the frontiers of multiple scientific fields. It offers a comprehensive analysis of several CRISPR-Cas systems, including Cas9, Cas12, Cas13, and Cas14, emphasizing their unique structural features and functional capabilities. While Cas9 has dominated many genomeediting applications, the use of Cas13 in Chinese Hamster Ovary (CHO) cells has opened up promising RNA-targeting strategies. Moreover, the compact Cas14 system presents notable potential for applications requiring ultra-precise genome manipulation. With their critical role in therapeutic protein production, CHO cells have greatly benefited from CRISPR-enabled engineering, leading to measurable improvements in productivity, stability, and cost-efficiency. Key advancements in CRISPR delivery platforms, including both viral and nonviral vectors, are discussed alongside ongoing challenges such as off-target effects and regulatory considerations. Emerging trends such as base editing, prime editing, and the integration of artificial intelligence for system optimization are also explored. Altogether, the discussion underscores the pivotal contribution of CRISPR technologies to CHO cell engineering and their broader impact on the future of biopharmaceutical manufacturing.},
}

@article {pmid42137038,
year = {2026},
author = {Koshi, N and Kobayashi, M and Ezura, H and Miura, K},
title = {Enhancement of parthenocarpy and fruit set through genome editing in tomato variety for processing use.},
journal = {Plant biotechnology (Tokyo, Japan)},
volume = {43},
number = {1},
pages = {127-131},
pmid = {42137038},
issn = {1342-4580},
abstract = {Tomatoes are extremely important plants that are cultivated worldwide, with various varieties grown in different regions. The traits required can vary depending on the region and intended use. Parthenocarpy, a trait that confers numerous advantages, reduces the labor required for pollination and minimizes the incidence of poor fruit set owing to temperature fluctuations. Mutations in SlIAA9 induce parthenocarpy in tomatoes, and the introduction of this trait into processed varieties via genome editing suggests its potential to markedly shorten the breeding timeline. Genome editing has gained considerable attention as a breeding technique because it enables precise mutations in specific genes. However, only a few recent studies have reported examples of genome editing in Japanese tomato varieties for processing. In this study, we employed a genome-editing technique targeting SlIAA9 to induce parthenocarpy in the Japanese tomato variety Natsunokoma for processing purposes, thereby reducing the labor required for pollination. The null-segregant Sliaa9 mutant exhibited enhanced parthenocarpy and fruit set. These results suggest that improvements in fruit-bearing and parthenocarpic traits enhance the quality of tomato varieties that are mainly used for processing.},
}

@article {pmid42137600,
year = {2026},
author = {Schönberg, PY and Muñoz-Ovalle, Á and Saleh, HA and Crespo, E and Kuhnert, R and Michen, S and Loureiro, L and Temme, A and Feldmann, A and Buchholz, F},
title = {Epigenetic editing balances TCR suppression and persistence in CAR T cells.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201712},
pmid = {42137600},
issn = {3117-387X},
abstract = {Allogeneic chimeric antigen receptor (CAR) T cell therapies offer a scalable, off-the-shelf option for cancer treatment, but their clinical use is limited by the risk of graft-versus-host disease (GvHD), mediated by the endogenous T cell receptor (TCR). Conventional strategies to eliminate TCR expression rely on genome editing tools such as CRISPR-Cas9 or base editing, which introduce permanent DNA changes and pose safety concerns. Here, we present an epigenetic editing approach that enables efficient, specific, and reversible silencing of the CD3ε gene, a critical component of the TCR complex, without altering the genome. We systematically optimized the epigenetic editor and guide RNA in a cell line and achieved robust TCR silencing in primary T and CAR T cells while preserving CAR expression, activation, and effector function. Transcriptome analysis confirmed minimal off-target effects. In vivo observations suggest the epigenetically silenced T cells to prevent GvHD while persisting longer than TCR-knockout cells, supporting the notion that transient TCR suppression may help balance safety and long-term efficacy. Our findings establish epigenetic editing as a non-genotoxic alternative to genome editing, offering a flexible and safer route to generate next-generation allogeneic CAR T cells.},
}

@article {pmid42137606,
year = {2026},
author = {Campbell, JM and Korpela, DM and Han, H and Zhao, S and Webster, DA and Nguyen, YAH and Koes, N and Aune, RO and Dagan, H and Milliken, R and Watts, JK and Murthy, N and Carlson, DF},
title = {Swine reporter model for preclinical evaluation and characterization of gene delivery vectors.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201729},
pmid = {42137606},
issn = {3117-387X},
abstract = {Delivery of gene therapy vectors efficiently targeted to any somatic cell remains a key barrier for the development of genetic medicines. While rodent models provide insights into vector biodistribution and cellular tropism, their anatomical and physiological differences from humans limit their translational potential and studies in large animal models are often required. In this study, we developed a swine reporter model (SRM-1) to evaluate both viral and non-viral vector delivery in a large animal system. The SRM-1 model harbors a tdTomato reporter at the ROSA26 locus that can be activated by Cre recombinase or CRISPR-Cas reagents and allows for tracking of gene delivery vectors in vivo. To evaluate this model, we administered adeno-associated virus serotype 9 (AAV9) and lipid nanoparticles (LNPs) carrying messenger RNA (mRNA) systemically and found successful in vivo reporter activation across a variety of tissues. Intracerebroventricular (i.c.v.) administration of LNP-mRNA was also performed and demonstrated localized activation in cortical brain cells. In addition to systemic biodistribution studies, this model has utility for testing clinically relevant local administration methods, surgical and non-surgical, of delivery vectors. Our findings support the SRM-1 model as a valuable tool for advancing gene therapies from preclinical testing to clinical application.},
}

@article {pmid42137980,
year = {2026},
author = {Xiong, W and Zhou, E and Qi, Q and Liu, X and Zhang, K and Huang, S and Li, M and Wang, C and Zhou, X and Tian, T},
title = {Harnessing a single molecule for dual bioorthogonal regulation of RNA function and m6A methylation.},
journal = {Nucleic acids research},
volume = {54},
number = {9},
pages = {},
doi = {10.1093/nar/gkag438},
pmid = {42137980},
issn = {1362-4962},
support = {22377094//National Natural Science Foundation of China/ ; 22177089//National Natural Science Foundation of China/ ; 22037004//National Natural Science Foundation of China/ ; 91853119//National Natural Science Foundation of China/ ; 91753201//National Natural Science Foundation of China/ ; 22177088//National Natural Science Foundation of China/ ; 22377095//National Natural Science Foundation of China/ ; 22407104//National Natural Science Foundation of China/ ; 22407106//National Natural Science Foundation of China/ ; 22407107//National Natural Science Foundation of China/ ; 22377094//National Natural Science Foundation of China/ ; 2022YFA1502902//National Key Research and Development Program of China/ ; },
mesh = {Methylation ; Click Chemistry/methods ; *Adenosine/analogs & derivatives/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Humans ; *RNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Azides/chemistry ; RNA Processing, Post-Transcriptional ; },
abstract = {This study presents a multifunctional RNA regulation strategy that enables RNA molecules to undergo both bioorthogonal ligation and cleavage reactions within the same system. Using guide RNA (gRNA) as an example, we demonstrate on-demand inactivation via click chemistry and subsequent reactivation by light exposure. Applied to CRISPR-mediated, site-specific RNA methylation, this technology enables continuous, multistep programmable control, overcoming the one-way limitation of traditional methods. Results show that gRNA can stably tolerate both azide and photodegradable groups, facilitating efficient targeting of M3M14-dCas9 and dCas13b-M3M14 methylation systems to introduce m6A at specific RNA sites. Small molecules can turn off methylation through in situ click chemistry, while 365-nm light exposure rapidly restores gRNA function, allowing precise control over RNA methylation. This strategy highlights the efficiency and flexibility of progressive bioorthogonal RNA modulation and paves the way for multisite, dynamic regulation in complex biological systems.},
}

Methylation
Click Chemistry/methods
*Adenosine/analogs & derivatives/metabolism/chemistry
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
Humans
*RNA/metabolism/chemistry/genetics
CRISPR-Cas Systems
Azides/chemistry
RNA Processing, Post-Transcriptional

@article {pmid42138081,
year = {2026},
author = {Chang, LC and Eyler, CE and Lee, CL},
title = {Chromosomal instability induced by CRISPR/Cas9: implications for pancreatic cancer therapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI206028},
pmid = {42138081},
issn = {1558-8238},
mesh = {Humans ; *Pancreatic Neoplasms/genetics/therapy/pathology/metabolism ; *Chromosomal Instability ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; },
abstract = {Clinical management of pancreatic cancer (PC) remains severely limited, primarily due to the complex tumor microenvironment. Emerging DNA damage-targeted strategies have demonstrated considerable therapeutic potential in PC. In this issue of the JCI, Teh et al. employed cancer-specific multitarget sgRNAs to induce DNA double-strand breaks (DSBs), resulting in lethal effects in PC cells. Integrative bioinformatic and cytogenetic analyses revealed that CRISPR/Cas9-mediated DSBs provoked persistent chromosomal instability, ultimately leading to chromosome catastrophe and cell death. Compared with equivalent radiation-induced DSBs, these sgRNAs exhibited superior cytotoxicity and were able to eliminate cells resistant to a specific sgRNA via subsequent targeting at distinct genomic sites, highlighting a promising and innovative precision therapeutic approach for clinical treatment of PC.},
}

Humans
*Pancreatic Neoplasms/genetics/therapy/pathology/metabolism
*Chromosomal Instability
*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded

@article {pmid42138085,
year = {2026},
author = {Teh, SSK and Kotwal, A and Bennett, A and Halper-Stromberg, E and Morsberger, L and Zamani, S and Shi, Y and Skaist, A and Zhu, Q and Bowland, K and Liang, H and Hruban, RH and Hung, CF and Anders, RA and Roberts, NJ and Scharpf, RB and Goldstein, M and Zou, YS and Eshleman, JR},
title = {Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI190121},
pmid = {42138085},
issn = {1558-8238},
mesh = {*Pancreatic Neoplasms/genetics/pathology/therapy/metabolism ; *CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded ; Cell Line, Tumor ; Animals ; Mice ; Gene Editing ; },
abstract = {While radiation is an effective oncologic therapy, killing cancer by inducing DNA double-strand breaks (DSBs), it lacks specificity for neoplastic cells. We have previously adapted the CRISPR/Cas9 gene-editing technology as a cancer-specific treatment modality targeting somatic mutations in pancreatic cancer (PC). However, its tumoricidal potential remains unclear, especially in comparison with therapeutic doses of radiation. Here, we demonstrate that CRISPR/Cas9-induced DSBs are more cytotoxic in PCs than a comparable number of radiation-induced DSBs. We observed more than 90% tumor growth inhibition by targeting 9 sites with cancer-specific sgRNAs. Through both bioinformatics and cytogenetics analyses, we found that CRISPR/Cas9-induced DSBs triggered ongoing chromosomal rearrangements, with 87% of structural variants not directly produced from the initial CRISPR/Cas9-induced DSBs, and chromosomal instability peaking before cell death. By comparing the cytotoxicity of CRISPR/Cas9- and radiation-induced DSBs, we demonstrated that the number of DSBs required to achieve equitoxic effects was approximately 3 times higher for radiation than CRISPR/Cas9. Finally, we showed that PC cells that had survived CRISPR/Cas9 targeting retained susceptibility to subsequent CRISPR/Cas9-induced DSBs at different genomic sites with more than 87% growth inhibition. Together, our data support the therapeutic potential of CRISPR/Cas9 as an anticancer strategy.},
}

*Pancreatic Neoplasms/genetics/pathology/therapy/metabolism
*CRISPR-Cas Systems
Humans
*DNA Breaks, Double-Stranded
Cell Line, Tumor
Animals
Mice
Gene Editing

@article {pmid42138264,
year = {2026},
author = {Gao, M and Jiang, T and Si, W and Rong, Y and Hu, Y and He, X and Liu, BF and Chen, P and Yang, Y and Deng, Y and Wang, FB},
title = {Harnessing CRISPR-Cas12 and Microfluidics Chips for Multiplex Respiratory Pathogens Diagnosis.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03226},
pmid = {42138264},
issn = {2379-3694},
abstract = {Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.},
}

@article {pmid42138716,
year = {2026},
author = {Lefrançois, G and Lavallée, E and Rowell, MC and Bourdeau, V and Mohebali, F and Bertomeu, T and Duman, AM and Nikolova, M and Tyers, M and Gravel, SP and Schmitzer, AR and Ferbeyre, G},
title = {The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
doi = {10.7554/eLife.102680},
pmid = {42138716},
issn = {2050-084X},
support = {TFRI Project #1123//Terry Fox Research Institute/ ; Operating Grant 2016//Cancer Research Society/ ; 935858//Cancer Research Society/ ; 840633//Cancer Research Society/ ; RGPIN-2021-03128//Natural Sciences and Engineering Research Council of Canada/ ; Ganotec/Marc-André Pigeon Fund//Cancer Research Society/ ; 1054571//Cancer Research Society/ ; },
mesh = {*Metformin/pharmacology/metabolism ; Humans ; Cell Line, Tumor ; *Mitochondrial Proton-Translocating ATPases/metabolism/genetics ; *Cell Proliferation/drug effects ; Oxidative Phosphorylation/drug effects ; *Antineoplastic Agents/pharmacology ; Mitochondria/drug effects/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Hypoglycemic Agents/pharmacology ; },
abstract = {Here, we identify the subunit e of F1F0-ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F1F0-ATP synthase dimers, which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro, and disabling its expression by CRISPR-Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide-treated cells, including mitochondrial morphology alterations, reduction of the NAD[+]/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers, and a compensatory increase in glycolysis. Notably, metformin disrupts F1F0-ATP synthase oligomerization, leading to the accumulation of vestigial assembly intermediates in pancreatic and osteosarcoma cancer cells, a phenotype also observed upon ATP5I inactivation in pancreatic cancer cells. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and antiproliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F1F0-ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.},
}

*Metformin/pharmacology/metabolism
Humans
Cell Line, Tumor
*Mitochondrial Proton-Translocating ATPases/metabolism/genetics
*Cell Proliferation/drug effects
Oxidative Phosphorylation/drug effects
*Antineoplastic Agents/pharmacology
Mitochondria/drug effects/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems
*Hypoglycemic Agents/pharmacology

@article {pmid42139236,
year = {2026},
author = {Quiroz-Huanca, A and Vargas-Reyes, M and López, JD and Flores-Jimenez, K and Saldarriaga-Morán, S and Cifuentes, K and Alcántara, R},
title = {Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0337675},
doi = {10.1371/journal.pone.0337675},
pmid = {42139236},
issn = {1932-6203},
mesh = {*beta-Lactamases/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/drug effects/isolation & purification/enzymology ; *Polymerase Chain Reaction/methods ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The β-lactams are critically important broad-spectrum antibiotics, widely used as first-line treatments; however, their effectiveness is increasingly compromised by β-lactamase enzymes. Among these, OXA-type enzymes have expanded to over 400 variants and are highly prevalent in Enterobacteriaceae. Current phenotypic and molecular detection tests have long turnaround times or require specialized equipment, respectively. In this study, we optimize a rapid molecular assay combining a PCR with modified thermal ramp rate (TRR) along with CRISPR-Cas12a fluorescence detection for blaOXA-1-harboring E. coli isolates. Using a commercial DNA Taq polymerase (TRR: 2.2 °C/s, annealing and extension hold time: 1 s), amplification time was reduced from 80 to 30 min, enabling detection within 50 min (PCR: 30 min; CRISPR: 20 min). With a locally produced enzyme (hold: 10 s), amplification time was 44 min. To demonstrate the practical application of the assay, we evaluated spiked poultry fecal samples achieving an analytical sensitivity of 8 CFU/reaction using commercial DNA Taq polymerase. The accelerated PCR:CRISPR workflow delivers results in less than one hour without compromising technical sensitivity (attomoles range), not requiring high technical expertise, and can be implemented in laboratories with basic molecular biology equipment.},
}

*beta-Lactamases/genetics
*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/drug effects/isolation & purification/enzymology
*Polymerase Chain Reaction/methods
Animals
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42141309,
year = {2026},
author = {Qin, L and Liu, D and Wang, Z and Sun, X and Zhao, X},
title = {Molecular biology of Pleurotus mushrooms: genomic resources, genetic manipulation, and regulation mechanisms.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {6},
pages = {},
pmid = {42141309},
issn = {1572-9699},
support = {32402651//National Natural Science Foundation of China/ ; 2024AFB274//Hubei　Provincial　Natural　Science　Foundation　of　China/ ; Q20231511//Youth Talent Project of Scientific Research Plan of Hubei Provincial Education Department of China/ ; K2023058//Scientific Research Fund of Wuhan Institute of Technology/ ; },
mesh = {*Pleurotus/genetics ; *Genomics/methods ; *Genome, Fungal ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; RNA Interference ; },
abstract = {As one of the most widely cultivated edible mushrooms in the world, Pleurotus mushrooms are popular among people for their delicious taste and rich nutritional value. Because of their great economic value, the research on the molecular biology of Pleurotus spp. has been deepening in recent years. The study first summarized the current situation of genomic resources available for this genus. The whole genome sequencing of 14 species, including Pleurotus tuoliensis and Pleurotus ostreatus, provides reference data for mining functional genes. Although the genomic data for Pleurotus mushrooms are continuously increasing, actual instances of successful genetic transformation remain restricted. Research on the regulatory mechanisms of key genes at different developmental stages and under various environmental stresses is insufficient. Then, the application of gene editing methods (CRISPR/Cas9, RNAi, and gene overexpression) in Pleurotus mushrooms was systematically described. RNAi and gene overexpression technologies have become well-established and are routinely used in most Pleurotus mushrooms. However, the application of CRISPR/Cas9 technology is still limited to P. eryngii and P. ostreatus. This limitation is attributed to the difficulties in establishing genetic transformation systems and the low efficiency of homologous recombination. Furthermore, this review explored the value of multi-omics technologies in elucidating the molecular mechanisms of morphogenesis and stress responses. To address the lack of specific antibodies for non-model organisms, we evaluated the application potential of DAP-seq technology in Pleurotus mushrooms and discussed its limitations, including the risk of false positives arising from the absence of a genuine environment in vivo. The purpose of this review is to evaluate the current molecular biology research on Pleurotus spp., and to provide systematic technical support and insights for functional genomics research and the analysis of molecular mechanisms of complex traits in Pleurotus mushrooms.},
}

*Pleurotus/genetics
*Genomics/methods
*Genome, Fungal
Gene Editing
CRISPR-Cas Systems
Gene Expression Regulation, Fungal
RNA Interference

@article {pmid41397899,
year = {2026},
author = {Chowdhury, A and Garcia, BG and Zahoor, MA and ElMawla, NF and Davidson, AR and Wyatt, HDM and Maxwell, KL and Mahassine, A and Gehring, A and Feld, JJ},
title = {A Rapid Assay for Hepatitis C Virus RNA Detection Using Reverse-Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12b-Based Strategy.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {5},
pages = {840-847},
doi = {10.1093/infdis/jiaf609},
pmid = {41397899},
issn = {1537-6613},
mesh = {Humans ; *Hepacivirus/genetics/isolation & purification ; *Hepatitis C/diagnosis/virology ; *RNA, Viral/genetics/blood ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Genotype ; Limit of Detection ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for 2 tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid, and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations.

METHODS: After RNA extraction from plasma, HCV RNA is reverse-transcribed and amplified using loop-mediated isothermal amplification with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout.

RESULTS: HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a, and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/mL (approximately 100 IU/mL). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500 μL of plasma, genotype 1 RNA ≥100 IU/mL was detected within 40-45 minutes, while genotype 3 had an LLOD of 5000 IU/mL. The clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/hepatitis B virus (HBV) coinfection. The specificity was 100%, with no false-positives in 33 HCV-negative samples, including those with HBV or human immunodeficiency virus/HBV coinfection.

CONCLUSIONS: Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45 minutes and hence could be used for efficient screening and diagnosis of HCV infection globally.},
}

Humans
*Hepacivirus/genetics/isolation & purification
*Hepatitis C/diagnosis/virology
*RNA, Viral/genetics/blood
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Genotype
Limit of Detection

@article {pmid41604419,
year = {2026},
author = {Martínez Rivas, FJ and Smith, MA and Zangishei, Z and Alseekh, S and Usadel, B and Plaxton, WC and Fernie, AR},
title = {Malate matters: disrupting bacterial-type phosphoenolpyruvate carboxylase (BTPC) rewires tomato fruit development.},
journal = {Plant physiology},
volume = {201},
number = {1},
pages = {},
pmid = {41604419},
issn = {1532-2548},
support = {452682775//Deutsche Forschungsgemeinschaft/ ; 390686111//Deutsche Forschungsgemeinschaft/ ; //PlantaSyst/ ; 664621//European Union's Horizon 2020 Research and Innovation Programme/ ; 739582//European Union's Horizon 2020 Research and Innovation Programme/ ; 664620//European Union's Horizon 2020 Research and Innovation Programme/ ; RGPIN-2024-06467//Natural Sciences and Engineering Research Council of Canada/ ; //Queen's Research Chair/ ; },
mesh = {*Solanum lycopersicum/growth & development/genetics/enzymology/metabolism ; *Malates/metabolism ; *Fruit/growth & development/genetics/enzymology/metabolism ; *Phosphoenolpyruvate Carboxylase/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant phosphoenolpyruvate carboxylases (PEPCs) are ubiquitously expressed as cytosolic Class-1 PEPC homotetramers composed of 107 kDa plant-type PEPC (PTPC) subunits that are highly sensitive to allosteric inhibition by malate. Class-2 PEPC heterooctameric complexes that are desensitized to malate inhibition also exist in certain sink tissues due to the interaction of a Class-1 PEPC with unrelated 118 kDa bacterial-type PEPC (BTPC) polypeptides. Class-2 PEPCs dynamically associate with the mitochondrial outer envelope and have been hypothesized to support sustained anaplerotic flux and respiratory CO2 refixation in malate-rich sink tissues, including immature tomato fruit. The current study generated CRISPR-Cas9-edited tomato lines with targeted disruption of the BTPC gene and investigated the impact on fruit development, metabolism, and transcriptional regulation. Immunoblotting and co-immunoprecipitation confirmed the absence of BTPC polypeptides and Class-2 PEPC complexes in the edited lines. Fruits from the edited plants were 25% smaller and 40% lighter and required up to 10 additional days to complete ripening compared to the WT. Metabolomic analysis across ripening stages revealed substantial reductions in malate and citrate, with elevated sugars and amino acids, indicating reprogrammed carbon flux. RNA-seq data showed downregulation of genes for cell wall remodeling, sugar transport, and ethylene-responsive transcription factors. These results provide direct evidence that BTPC is essential for organic acid balance, sugar metabolism, and ripening regulation in tomato. Its absence perturbs metabolic homeostasis and developmental progression, positioning BTPC as a strategic target for enhancing fruit quality traits through genetic engineering.},
}

*Solanum lycopersicum/growth & development/genetics/enzymology/metabolism
*Malates/metabolism
*Fruit/growth & development/genetics/enzymology/metabolism
*Phosphoenolpyruvate Carboxylase/metabolism/genetics
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41734599,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {Effect of p53 gene mutation with loss of function on the expression of genes and proteins involved in cell proliferation.},
journal = {Mutation research},
volume = {832},
number = {},
pages = {111931},
doi = {10.1016/j.mrfmmm.2026.111931},
pmid = {41734599},
issn = {1873-135X},
mesh = {Humans ; *Cell Proliferation/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Loss of Function Mutation ; Gene Expression Regulation, Neoplastic ; *Mutation ; Cell Cycle/genetics ; },
abstract = {The tumor suppressor gene TP53 plays a vital role in preserving genomic integrity by regulating cell cycle progression, DNA repair mechanisms, and apoptosis. This study aims to examine how CRISPR/Cas9-induced loss-of-function mutations in the p53 gene influence cellular processes on cell cycle regulation and tumorigenic signaling in HT1080 human fibrosarcoma cells. Successful TP53 gene disruption was confirmed by Sanger sequencing, and its structural modelling using AlphaFold2 and ChimeraX confirmed alterations in the predicted TP53 protein structure compared to that of wild type. Gene expression analyses, conducted via RT-PCR and qPCR, demonstrated a marked decrease in TP53 mRNA expression within the modified cells. Despite the mutation, the edited cells elevated activity of the senescence marker β-galactosidase (SA-β-gal). They decreased the production of collagen, suggesting that the structural disruption caused by CRISPR/Cas9 leads to the loss of functional p53 activity. Western blotting and immunofluorescence assays further revealed a remarkable downregulation of key cell cycle and tumorigenesis-related proteins, including TP53, phosphorylated TP53 (p-TP53), acetylated TP53 (ac-TP53), MMP-2, cyclin D, cyclin E, AKT, BAX, MDM2, and phosphorylated Rb (p-Rb) in the edited cells relative to the wild-type counterpart. Our results suggest that the TP53 mutation may disrupt essential pathways related to cell proliferation and stress responses. This provides new insights into TP53 functionality and underscores its potential as a therapeutic target in cancer biology.},
}

Humans
*Cell Proliferation/genetics
*Tumor Suppressor Protein p53/genetics/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
*Loss of Function Mutation
Gene Expression Regulation, Neoplastic
*Mutation
Cell Cycle/genetics

@article {pmid41882360,
year = {2026},
author = {Smith, QM and Whittle, S and Aramayo, RJ and Rollins, DE and Jalal, ASB and Egharevba, DI and Morris, KL and Pyne, ALB and Rueda, DS},
title = {Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity.},
journal = {Nature},
volume = {653},
number = {8114},
pages = {627-635},
pmid = {41882360},
issn = {1476-4687},
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure ; *CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA, Circular/chemistry/ultrastructure/genetics/metabolism ; *DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics ; *Gene Editing/methods ; Models, Molecular ; Protein Domains ; RNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing tool[1], but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (-)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA-RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (-)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.},
}

*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure
*CRISPR-Cas Systems/genetics
Cryoelectron Microscopy
DNA, Circular/chemistry/ultrastructure/genetics/metabolism
*DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics
*Gene Editing/methods
Models, Molecular
Protein Domains
RNA/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems

@article {pmid41913646,
year = {2026},
author = {Streiber, M and Liu, N and Simon, L and Adermann, F and Bachmann, V and Gath, L and Hoeppener, S and Schubert, S and Werz, O and Lapinte, V and Morille, M and Bauer, M and Press, AT and Schubert, US and Traeger, A},
title = {Extrahepatic Gene Editing In Vivo Using Organic Solvent-Free Lipid Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {27},
pages = {e11489},
pmid = {41913646},
issn = {1613-6829},
support = {13XP5034A//Bundesministerium für Bildung und Forschung/ ; 03RU2U071H//Bundesministerium für Bildung und Forschung/ ; 2021 FGI 0005//Thüringer Aufbaubank/ ; 2023FGR0077//Thüringer Aufbaubank/ ; //Joachim Herz Stiftung/ ; P2024-02-016//Carl-Zeiss-Stiftung/ ; 57604510//German Academic Exchange Service/ ; 2018FGI0025//European Regional Development Fund/ ; ANR- 20-CE09-0011-01//Agence Nationale de la Recherche/ ; 514006196//Deutsche Forschungsgemeinschaft/ ; 316213987//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Nanoparticles/chemistry/ultrastructure ; Humans ; Animals ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Solvents/chemistry ; CRISPR-Cas Systems/genetics ; Polyethylene Glycols/chemistry ; Transfection ; Liposomes ; },
abstract = {Targeted therapy, which modifies genes and their expression, holds great promise for treating a variety of diseases, including cancer, inborn errors of metabolism, and acute and chronic inflammatory and infectious conditions. However, it also presents challenges related to RNA delivery, immune responses, side effects of delivery vectors, and the need for individualized formulations. To overcome these limitations, the choice of lipids and formulation processes might be re-evaluated, with a focus on eliminating critical components, such as poly(ethylene glycol) (PEG) and ethanol. Thus, a purely water-based formulation for lipid nanoparticles was developed, offering a material-efficient, time-saving process with high reproducibility. Initially, a stealth lipid containing poly(2-methyl-2-oxazoline) (PMeOx) was used, and the formulation was later expanded to include approved lipids. These nanoparticles not only efficiently transfect primary human immune cells but also effectively deliver multiple nucleotides in CRISPR-Cas9 applications. Moreover, an in vivo comparison revealed that the nanoparticles exhibited preferential transfection in extrahepatic tissues. This distinguishes them from conventional cholesterol-rich lipid nanoparticles, which primarily target the liver regardless of the application route.},
}

*Nanoparticles/chemistry/ultrastructure
Humans
Animals
*Lipids/chemistry
*Gene Editing/methods
Mice
Solvents/chemistry
CRISPR-Cas Systems/genetics
Polyethylene Glycols/chemistry
Transfection
Liposomes

@article {pmid41931906,
year = {2026},
author = {Pedersen, A and Blay-Cadanet, J and Storgaard, J and Hernaez, B and Thyrsted, J and Bach-Nielsen, CS and Twayana, K and Jørgensen, SE and Rio-Bergé, C and Poulsen, C and Thielke, AL and Thomsen, EA and Kalucka, J and Olagnier, D and Luo, Y and Reggiori, F and Mogensen, TH and Alcamí, A and Hansen, AL and Holm, CK},
title = {NRF2 controls a diverse network of antiviral effectors with p62 acting as a central restriction factor effective across virus families.},
journal = {Redox biology},
volume = {93},
number = {},
pages = {104135},
pmid = {41931906},
issn = {2213-2317},
mesh = {*NF-E2-Related Factor 2/metabolism/genetics ; Humans ; *SARS-CoV-2/physiology ; Virus Replication/drug effects ; *Sequestosome-1 Protein/metabolism/genetics ; Influenza A virus/physiology ; Herpesvirus 1, Human/physiology ; *COVID-19/virology/genetics/metabolism ; Animals ; Autophagy ; Vaccinia virus/physiology ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; Chlorocebus aethiops ; },
abstract = {The transcription factor erythroid 2 (NFE2)-related factor 2 (NRF2) is a key regulator of cellular homeostasis. Recent discoveries have identified agonists of NRF2 as inducers of broad cellular resistance to viral infection including SARS-CoV-2. Nevertheless, it is still unclear to what extent NRF2 itself is an inducer of anti-viral immunity and its downstream antiviral effectors have not been mapped. Here, we first demonstrate through specific genetic activation and silencing that NRF2 restricts SARS-CoV-2 replication. We then used a focused CRISPR-activation screen to map antiviral NRF2-inducible effector genes that restrict replication of SARS-CoV-2, Influenza A virus (IAV), Herpes Simplex virus 1 (HSV1) and Vaccinia virus (VACV). This approach allowed us to identify a range of antiviral effectors each of which restrict members of one or more virus families. Importantly, we identified the NRF2-inducible selective autophagy receptor p62/SQSTM1 as a broadly effective restriction factor across all the tested viruses. Importantly, p62 inhibited SARS-CoV-2 replication in cells treated with the lysosomal inhibitor bafilomycin A1, as well as in cells deficient in the autophagy protein ATG5. Similarly, p62 inhibited replication of HSV1 and IAV independently of ATG5 and ATG16L1 respectively. Thus, NRF2 restricts viral replication through a hitherto underappreciated network of antiviral restriction factors effective across multiple virus families. Importantly, we identify p62 as a broadly acting antiviral effector that restricts viral replication independently of canonical autophagy.},
}

*NF-E2-Related Factor 2/metabolism/genetics
Humans
*SARS-CoV-2/physiology
Virus Replication/drug effects
*Sequestosome-1 Protein/metabolism/genetics
Influenza A virus/physiology
Herpesvirus 1, Human/physiology
*COVID-19/virology/genetics/metabolism
Animals
Autophagy
Vaccinia virus/physiology
Antiviral Agents/pharmacology
CRISPR-Cas Systems
Chlorocebus aethiops

@article {pmid41942711,
year = {2026},
author = {Wang, W and Li, Y and Dong, S and Liu, Y and Guo, C and Su, Y and Tian, W and Hu, X and Wang, Z},
title = {The exonic SNP rs11676272-C risk allele mediates diet-induced obesity and reduces enhancer activation.},
journal = {EMBO reports},
volume = {27},
number = {9},
pages = {2462-2490},
pmid = {41942711},
issn = {1469-3178},
support = {32470645//MOST | National Natural Science Foundation of China (NSFC)/ ; 32070567//MOST | National Natural Science Foundation of China (NSFC)/ ; 32202840//MOST | National Natural Science Foundation of China (NSFC)/ ; C2023201032//| Natural Science Foundation of Hebei Province ()/ ; 050001-5000019//Hebei University ()/ ; ZJ2025118//Zhejiang Province Human Resources and Social Security Department ()/ ; },
mesh = {Animals ; Humans ; *Obesity/genetics/etiology/metabolism ; HEK293 Cells ; Mice ; *Polymorphism, Single Nucleotide ; *Alleles ; *Adenylyl Cyclases/genetics/metabolism ; *Enhancer Elements, Genetic ; Diet, High-Fat/adverse effects ; Genetic Predisposition to Disease ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Gene Expression Regulation ; Male ; },
abstract = {Genome-wide association studies (GWASs) have identified hundreds of obesity-associated SNPs, but establishing their causality remains challenging. Here, we demonstrate that rs11676272, located in the ADCY3 gene, is a functional causal variant for obesity susceptibility. Bioinformatic analyses and dual-luciferase reporter assays indicate that the rs11676272 region may act as a human-gained enhancer regulating ADCY3 expression. In HEK293T cells, CRISPR-Cas9-mediated single-nucleotide editing of rs11676272 (T > C) reduces ADCY3 expression. Moreover, the rs11676272-T allele is preferentially bound by the transcription factor E2F3 to upregulate ADCY3 expression, whereas the rs11676272-C risk allele loses this binding. In vivo, the rs11676272 T > C variant in human ADCY3 (hADCY3) knock-in mice accelerates weight gain under high-fat diet conditions and shortens primary cilia in the ventromedial hypothalamus (VMH). CRISPRa-mediated activation of the hADCY3 promoter region rescues ciliary length in both the VMH and hypothalamic arcuate nucleus of Mut-hADCY3 mice. Our data reveal a causal role for rs11676272 in obesity, offering insight into potential therapeutic strategies.},
}

Animals
Humans
*Obesity/genetics/etiology/metabolism
HEK293 Cells
Mice
*Polymorphism, Single Nucleotide
*Alleles
*Adenylyl Cyclases/genetics/metabolism
*Enhancer Elements, Genetic
Diet, High-Fat/adverse effects
Genetic Predisposition to Disease
Promoter Regions, Genetic
CRISPR-Cas Systems
Gene Expression Regulation
Male

@article {pmid42020604,
year = {2026},
author = {Calvo Fernández, E and Tomassoni, L and Zhang, X and Wang, J and Obradovic, A and Laise, P and Griffin, AT and Vlahos, L and Minns, HE and Morales, DV and Simmons, C and Gallitto, M and Wei, HJ and Martins, TJ and Becker, PS and Crawford, JR and Tzaridis, T and Wechsler-Reya, RJ and Garvin, J and Gartrell, RD and Szalontay, L and Zacharoulis, S and Wu, CC and Zhang, Z and Califano, A and Pavisic, J},
title = {Systematic design of combination therapy by targeting master regulators of coexisting diffuse midline glioma cell states.},
journal = {Nature genetics},
volume = {58},
number = {5},
pages = {1112-1125},
pmid = {42020604},
issn = {1546-1718},
mesh = {Humans ; *Glioma/genetics/drug therapy/pathology ; Animals ; Mice ; Cell Line, Tumor ; Pyrazoles ; *Brain Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology ; Pyrimidines ; Gene Expression Regulation, Neoplastic/drug effects ; Nitriles ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Intratumor heterogeneity fundamentally challenges cancer treatment, as coexisting, molecularly distinct cell states with non-overlapping drug sensitivities can drive therapeutic resistance. We establish and validate a generalizable, network-based framework to systematically identify combination therapies targeting complementary tumor cell states. Applied to diffuse midline glioma (DMG)-a universally fatal pediatric malignancy-this approach identified master regulator protein dependencies in seven coexisting cell states, confirmed by pooled CRISPR-Cas9 assays. Perturbational transcriptional profiles for 372 clinically relevant drugs prioritized candidates predicted to invert state-specific master regulator activity. State-selective drug sensitivity was validated for eight out of nine (89%) drugs in vivo, including avapritinib, ruxolitinib and larotrectinib. Compared with monotherapy, co-administering drugs targeting complementary states significantly prolonged survival across virtually all combinations, with avapritinib plus ruxolitinib extending median survival nearly threefold versus vehicle and 1.5-fold versus avapritinib alone. These findings establish clinically actionable DMG combinations and a tumor-agnostic and mutation-agnostic framework for rational combination therapy design.},
}

Humans
*Glioma/genetics/drug therapy/pathology
Animals
Mice
Cell Line, Tumor
Pyrazoles
*Brain Neoplasms/genetics/drug therapy/pathology
*Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology
Pyrimidines
Gene Expression Regulation, Neoplastic/drug effects
Nitriles
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid42062574,
year = {2026},
author = {Xu, Y and Stubbendieck, RM and Viswanatha, R and Krč, A and Baik, LS and Suh, WS and Hu, Y and Wang, H and Yin, L and Mameli, E and van der Meij, A and Carlson, JR and Doxey, AC and Stenmark, P and Perrimon, N and Currie, CR and Dong, M},
title = {Streptomyces produce a diphtheria toxin-like exotoxin that targets insects.},
journal = {Nature microbiology},
volume = {11},
number = {5},
pages = {1271-1285},
pmid = {42062574},
issn = {2058-5276},
support = {R01AI170835//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI189789//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01NS080833//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Animals ; *Streptomyces/metabolism/genetics ; Drosophila melanogaster/drug effects ; Virulence Factors/metabolism/genetics ; *Diphtheria Toxin/genetics/metabolism/chemistry ; *Insecta/drug effects/microbiology ; Bacterial Proteins/genetics/metabolism/chemistry ; *Exotoxins/metabolism/genetics/chemistry/toxicity ; Phylogeny ; *Insecticides/metabolism ; Peptide Elongation Factor 2/metabolism ; CRISPR-Cas Systems ; Insect Proteins/metabolism ; },
abstract = {Streptomyces and insects engage in complex interactions shaped by millions of years of evolution. While many beneficial relationships are well recognized, it remains unknown whether Streptomyces produce virulence factors targeting insects specifically. Here, through bioinformatic analysis, we identified diphtheria toxin (DT) homologues, which we named Streptomyces antiquus insecticidal proteins (SAIP), within a monophyletic lineage of Streptomyces that emerged more than 100 million years ago. SAIP is cytotoxic to insect cells and lethal to Drosophila melanogaster, suppressing neuronal activity and immune responses in vivo. Structural and functional studies validated that SAIP is homologous to DT and acts by ADP ribosylation of eukaryotic elongation factor 2. CRISPR-Cas9 screening identified the insect protein Flower as the SAIP receptor across a range of insects. Toxigenic Streptomyces can consume dead insects and produce bioactive secondary metabolites while growing on insect carcasses. These findings establish an insecticidal toxin in Streptomyces and demonstrate that Streptomyces have evolved highly specific virulence factors against insects.},
}

Animals
*Streptomyces/metabolism/genetics
Drosophila melanogaster/drug effects
Virulence Factors/metabolism/genetics
*Diphtheria Toxin/genetics/metabolism/chemistry
*Insecta/drug effects/microbiology
Bacterial Proteins/genetics/metabolism/chemistry
*Exotoxins/metabolism/genetics/chemistry/toxicity
Phylogeny
*Insecticides/metabolism
Peptide Elongation Factor 2/metabolism
CRISPR-Cas Systems
Insect Proteins/metabolism

@article {pmid42065511,
year = {2026},
author = {Chen, Y and Tang, D and Zhan, J and Wang, Y and Song, Z},
title = {Study on the detection of prostate cancer using MIRA-CRISPR/Cas12a technology.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3848-3857},
doi = {10.1039/d6ay00025h},
pmid = {42065511},
issn = {1759-9679},
mesh = {Humans ; Male ; *Prostatic Neoplasms/diagnosis/urine/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Antigens, Neoplasm/genetics/urine ; *Molecular Diagnostic Techniques/methods ; Biomarkers, Tumor/urine/genetics ; Sensitivity and Specificity ; },
abstract = {Background: Prostate cancer (PCa) is the most diagnosed cancer among men globally and a leading cause of cancer-related mortality. However, current conventional prostate diagnostic methods fail to meet the growing demands of clinical practice in terms of speed, simplicity, sensitivity, and specificity. To address these limitations, we established a molecular detection system based on MIRA-CRISPR/Cas12a technology. Using reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) to amplify minute PCA3-specific fragments in samples, we employ CRISPR/Cas12a to detect the fluorescence signal released by these fragments, enabling the detection of trace PCA3 molecules in urine. Methods: PCA3 standard strain cultivation and total RNA extraction; establishment and optimization of the MIRA amplification system using primers designed for the PCA3 molecular marker; design of crRNA targeting optimal sites within the detection sequence, combined with CRISPR/Cas12a technology to establish the detection system; preliminary validation of the technology's sensitivity and specificity. Results: the MIRA-CRISPR/Cas12a technology was successfully established for visual detection of PCA3 molecules in prostate cancer. Primer and crRNA sequences within the reaction system were determined. The detection sensitivity for PCA3 molecules in urine reached 1 × 10[0] copies per µL with excellent specificity. Conclusion: the MIRA-CRISPR/Cas12a technology enables specific detection of PCA3 molecules in urine. This technique features high sensitivity, high specificity, visual results, and simple operation. It does not require specialized laboratory UV imaging equipment; results are visible to the naked eye under LED blue light. Following further optimization, it offers a feasible technical solution for rapid molecular screening of prostate cancer.},
}

Humans
Male
*Prostatic Neoplasms/diagnosis/urine/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Antigens, Neoplasm/genetics/urine
*Molecular Diagnostic Techniques/methods
Biomarkers, Tumor/urine/genetics
Sensitivity and Specificity

@article {pmid42084031,
year = {2026},
author = {Ma, R and Xiao, Y and Yu, W and Huo, C and Meng, S and Zhao, Z and Guo, Z and Ren, X and Zhang, H and Li, B and Wang, Y and Liu, S and Huang, J},
title = {A novel high-sensitivity fluorescence detection technology for zearalenone based on the PER-triggered crRNA conformational change and CHA-coordinated energy supply.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3865-3873},
doi = {10.1039/d6ay00474a},
pmid = {42084031},
issn = {1759-9679},
mesh = {*Zearalenone/analysis ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Nucleic Acid Conformation ; Spectrometry, Fluorescence/methods ; Fluorescence ; },
abstract = {Zearalenone (ZEN), frequently encountered in corn, is a hazardous mycotoxin capable of impairing liver and kidney function, compromising immune responses, and potentially inducing carcinogenesis. Current detection methodologies are hampered by elevated costs, complex workflows, limited sensitivity, and poor specificity. There is a pressing need to develop simple, rapid, and ultrasensitive assays that combine high specificity with operational convenience, thereby facilitating precise biotoxin surveillance and control. This study developed a novel biosensing strategy for ultrasensitive detection of zearalenone (ZEN) by engineering a blocked Primer Exchange Reaction (PER) dumbbell-hairpin structure integrated with a Catalytic Hairpin Assembly (CHA)-based DNA machine. We constructed a highly specific and sensitive fluorescence biosensor for zearalenone (ZEN) by integrating a Primer Exchange Reaction (PER) with the trans-cleavage activity of CRISPR/Cas12a. This strategy significantly simplifies the operational procedure compared to conventional techniques. Furthermore, its modular design establishes a versatile and efficient platform adaptable for the detection of various trace analytes, offering a promising proof-of-concept for mycotoxin screening in agricultural products, although further extensive validation across diverse realistic matrices is warranted.},
}

*Zearalenone/analysis
*Biosensing Techniques/methods
CRISPR-Cas Systems
Limit of Detection
Nucleic Acid Conformation
Spectrometry, Fluorescence/methods
Fluorescence

@article {pmid42132069,
year = {2026},
author = {Liu, Z and Qi, J and Sun, L and Su, J and Li, W and Wu, L and Liang, Y and Wei, C and He, F and Han, Y and Sun, Y and Yan, H and Li, H and Xiao, R},
title = {Oxygen-Vacancy-Engineered WOX Nanowire-Based Surface-Enhanced Raman Scattering Biosensor with Lyophilized CRISPR/Cas13a Platform for CHIKV Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00934},
pmid = {42132069},
issn = {2379-3694},
abstract = {The outbreak of Chikungunya virus (CHIKV) has caused widespread acute morbidity with severe polyarthralgia or chronic arthritis, placing a great challenge to public health and socioeconomic development. Establishing a rapid and highly sensitive detection technology is crucial for achieving precise control and prevention. Here, we established a CRISPR/Cas13a-mediated SERS lateral flow immunoassay platform for rapid and highly sensitive detection of CHIKV. One-dimensional nanowires loading Au nanoparticles were used to prepare SERS tags, presenting excellent SERS-enhanced performances and superior applicability to directional flow on the test strip. For the best performance, the structure of WOX nanowires was regulated by adjusting the addition concentration of ascorbic acid during the synthesis process, resulting in a stronger LSPR effect derived from more Au NPs in situ grown on highly reducing WOX. Furthermore, the lyophilized CRISPR/Cas system greatly simplified the workflow. In the optimal conditions, the limit of detection reached 0.56 and 1.03 copies/μL for the CHIKV plasmid and inactivated viruses by this method, respectively. Furthermore, 34 clinical serum samples were accurately diagnosed by our proposed method, 100% consistent with qPCR. This platform with the advantages of simple operation and rapid response provides a reliable technical tool for the early precise identification and efficient monitoring of CHIKV.},
}

@article {pmid42133116,
year = {2026},
author = {Rajan, A and Raveendran, M and Shanmugam, V and Arul, L and Kumar, KK and Subramanian, A and Mannu, J and Eswaran, K},
title = {Engineering crop determinacy: CRISPR/Cas based advances in self-pruning gene function and application.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42133116},
issn = {1573-4978},
support = {BT/INF/22/SP45584/2022//Department of Biotechnology, Government of India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/growth & development ; Gene Editing/methods ; Flowers/genetics/growth & development ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genetic Engineering/methods ; Plant Proteins/genetics ; },
abstract = {The transition from indeterminate to determinate growth represents a key achievement in crop improvement, as it enhances agricultural productivity by synchronizing flowering, facilitating uniform harvest, and improving overall efficiency. In tomato and other crops, this shift is largely governed by mutations in the SELF-PRUNING (SP) gene, a key member of the CENTRORADIALIS (CEN), TERMINAL FLOWER 1 (TFL1), and SELF-PRUNING (SP) (CETS) gene family that regulates the vegetative to reproductive phase transition and influences overall shoot architecture. With increasing labour constraints, climate variability and rising global food security challenges, the ability to engineer optimized plant architectures has become increasingly important. CRISPR-based genome editing provides a precise and efficient strategy to modify SP/TFL1 homologs, enabling targeted transition from indeterminate to compact, determinate growth forms that exhibit synchronized flowering and enhanced mechanical harvestability. These genome editing approaches have been successfully applied across diverse crop species, including tomato, legumes, cotton, cereals and horticultural crops. This review consolidates current understanding of the molecular mechanisms governing determinacy, with emphasis on the central role of SP/TFL1 genes and their interactions with hormonal pathways such as auxin and cytokinin. By integrating these insights with recent advances in CRISPR-based editing platforms, this review provides a practical framework for researchers and breeders aiming to leverage CRISPR technology for next-generation crop improvement. Such strategies hold significant potential for enhancing productivity, resilience and sustainability within modern agricultural systems.},
}

*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/growth & development
Gene Editing/methods
Flowers/genetics/growth & development
Plants, Genetically Modified/genetics
Plant Breeding/methods
Genetic Engineering/methods
Plant Proteins/genetics

@article {pmid42133523,
year = {2026},
author = {Nammi, B and Madugula, SS and Jayasinghe-Arachchige, VM and Pham, T and Liu, J and Wang, S},
title = {Robust CRISPR-Cas Protein Identification using Max-Margin Regularized Transformer Models.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {PP},
number = {},
pages = {},
doi = {10.1109/TCBBIO.2026.3693528},
pmid = {42133523},
issn = {2998-4165},
abstract = {The discovery of CRISPR-Cas system has significantly advanced genome editing, offering vast applications in medical treatments and life sciences research. Despite their immense potential, the existing CRISPR-Cas systems still face challenges concerning size, delivery efficiency, and cleavage specificity. Addressing these challenges requires a deeper understanding of CRISPR-Cas proteins to advance the design and discovery of novel Cas proteins. Here, we study CRISPR-Cas proteins extensively using deep-learning techniques to build classification models that can differentiate between Cas and non-Cas proteins, as well as identify subfamilies Cas9 and Cas12. We developed two types of deep learning models: 1) a transformer encoder-based classification model, trained from scratch; and 2) a large protein language model fine-tuned on ProtBert, pre-trained on more than 200 million proteins. To boost learning efficiency for the model trained from scratch, we introduced a novel margin-based loss function to maximize inter-class separability and intra-class compactness in protein sequence embedding latent space of a transformer encoder. Our results show that the Fine-Tuned ProtBert-based (FTPB) classification model achieved accuracies of 99.06%, 94.42%, 96.80%, 97.57% for Cas9 vs. non-Cas, Cas12 vs.non-Cas, Cas9 vs. Cas12, and multi-class classification of Cas9 vs. Cas12 vs. non-Cas proteins, respectively. The Latent Space Regularized Max-Margin Transformer (LSRMT) model achieved classification accuracies of 99.81%, 99.81%, 99.06%, and 99.27% for the same tasks, respectively. These results demonstrate the effectiveness of the proposed Max-Margin-based latent space regularization in enhancing model robustness and generalization capabilities. Remarkably, the LSRMT model, even when trained on a significantly smaller dataset, outperformed the fine-tuned state-of-the-art large protein model. The high classification accuracies achieved by the LSRMT model demonstrate its proficiency in identifying discriminative features of CAS proteins, marking a significant step towards advancing our understanding of CAS protein structures in future research endeavors.},
}

@article {pmid42134322,
year = {2026},
author = {Lei, L and Kaufmann, MM and Lao, J and Thoulass, G and Ammann, S and Xiao, H and Rhiel, M and Dettmer-Monaco, V and Grünewald, J and Andrieux, G and Alzubi, J and Miller, BR and Weißert, K and Gräßel, L and Schell, C and Illert, AL and Joung, JK and Boerries, M and Cornu, TI and Ehl, S and Erlacher, M and Aichele, P and Cathomen, T},
title = {Genotoxicity profiling reveals distinct platform-and cell type-specific effects in therapeutic gene editing for genetic hyperinflammation.},
journal = {Cell stem cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.stem.2026.04.014},
pmid = {42134322},
issn = {1875-9777},
abstract = {Base editors enable precise correction of point mutations without requiring DNA double-strand breaks, yet platform- and cell type-specific genotoxicities remain incompletely characterized. Here, we applied cytosine base editing (CBE) to disrupt a cryptic splice-site mutation in the Unc13d locus of Jinx mice, a model of familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Efficient editing (62%-89%) in fibroblasts, T cells, and hematopoietic stem cells (HSCs) restored Unc13d splicing, reconstituted cytotoxic T cell function, and protected mice from virus-triggered hyperinflammation after transplantation of edited HSCs. Comparative genotoxicity profiling revealed distinct platform- and cell type-specific patterns: hyperactive CBE induced broader off-target activity and more structural variants than CRISPR-Cas9. Although off-target sequence edits persisted, the stability of CBE-induced chromosomal translocations differed between cell types. These findings establish base editing as a therapeutic strategy for a genetically predisposed hyperinflammatory syndrome and underscore the importance of context-specific safety profiling to guide the clinical translation of genome editors.},
}

@article {pmid41237872,
year = {2026},
author = {Abdul-Rahman, T and Roy, P and Garg, N and Nazir, A and Mehta, KS and Doshi, NA and Hassnain, S and Reddi, R and Kanagala, SG and Ashinze, P and Lavie, CJ and Gupta, R},
title = {Gene editing for inherited cardiac conditions: A new frontier in cardiology.},
journal = {Trends in cardiovascular medicine},
volume = {36},
number = {4},
pages = {232-244},
doi = {10.1016/j.tcm.2025.11.002},
pmid = {41237872},
issn = {1873-2615},
mesh = {Humans ; *Gene Editing/ethics/methods ; *Genetic Therapy/adverse effects/methods ; Genetic Predisposition to Disease ; *Cardiology/methods/trends ; Animals ; *Heart Diseases/genetics/therapy/diagnosis ; Phenotype ; CRISPR-Cas Systems ; Treatment Outcome ; Epigenesis, Genetic ; Mutation ; Heredity ; },
abstract = {Inherited cardiac conditions (ICCs) such as hypertrophic cardiomyopathy and Marfan syndrome pose significant global health challenges, often rooted in complex genetic mutations. Recent advances in gene editing, particularly the CRISPR/Cas9 system, have opened new avenues for precise, personalized interventions. This review examines the current landscape of gene editing in cardiology, with emphasis on its scientific promise, integration with epigenetics, gene therapy, and artificial intelligence, and its potential to transform clinical outcomes. Key gene editing strategies are analyzed for their efficacy and translational potential. The review also explores ongoing clinical trials and emerging research, offering practical insights for future studies. Ethical implications are critically evaluated, with proposed frameworks to address concerns around safety, equity, and long-term consequences. By synthesizing these developments, this review underscores the urgent need for continued interdisciplinary research in the quest to mitigate inherited cardiac diseases.},
}

Humans
*Gene Editing/ethics/methods
*Genetic Therapy/adverse effects/methods
Genetic Predisposition to Disease
*Cardiology/methods/trends
Animals
*Heart Diseases/genetics/therapy/diagnosis
Phenotype
CRISPR-Cas Systems
Treatment Outcome
Epigenesis, Genetic
Mutation
Heredity

@article {pmid41722572,
year = {2026},
author = {Abadie, FMC and Suiter, CC and Smith, NT and Daza, RM and Rominger, MC and Parrish, P and McDiarmid, TA and Lalanne, JB and Martin, B and Calderon, D and Ellison, A and Berger, AH and Shendure, J and Starita, LM},
title = {A multiplex, prime editing framework for identifying drug resistance variants at scale.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101167},
doi = {10.1016/j.xgen.2026.101167},
pmid = {41722572},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Point Mutation/genetics ; *Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; },
abstract = {CRISPR-based genome editing has revolutionized functional genomics, enabling thousands of perturbations to be concurrently assayed in single experiments. However, for methods such as saturation genome editing (SGE), which aims to generate and assay libraries of point mutations, a challenge is that only one region (e.g., one exon) is studied per experiment. Here, we describe prime-SGE, a prime editing-based framework in which libraries of specific point mutations are installed into genes throughout the genome and then functionally assessed by sequencing of prime editing guide RNAs (pegRNAs) rather than the mutations themselves. We apply prime-SGE in two cell lines to assay thousands of point mutations in eight oncogenes for their ability to confer drug resistance to four tyrosine kinase inhibitors. Our prime-SGE strategy, combined with ongoing improvements in prime editing efficiency, opens the door to efficient positive selection screens of large numbers of point mutations at locations throughout the genome.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Point Mutation/genetics
*Drug Resistance, Neoplasm/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Protein Kinase Inhibitors/pharmacology
Cell Line, Tumor

@article {pmid41872214,
year = {2026},
author = {Khadake, RM and Shinde, K and Rode, AB},
title = {Engineering ligands for theophylline riboswitches expands its regulatory dynamic range in prokaryotic and eukaryotic systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41872214},
issn = {2041-1723},
support = {BT/ PR45158/DRUG/134/119/2022//Department of Biotechnology, Ministry of Science and Technology (DBT)/ ; CRG/ 2022/004570//DST | Science and Engineering Research Board (SERB)/ ; },
mesh = {*Theophylline/metabolism/pharmacology/chemistry ; *Riboswitch/genetics ; Ligands ; Aptamers, Nucleotide/metabolism/genetics/chemistry ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Genetic Engineering/methods ; },
abstract = {The theophylline riboswitch has been a foundational tool in synthetic biology for three decades, yet its regulatory performance remains constrained by the modest affinity of its native ligand. Enhancing the dynamic range of riboswitches is critical for precise gene regulation in biotechnological applications. Here, we show that synthetic 4-quinazolinone derivatives, designed through a structure-based approach, are significantly better than theophylline in both binding and functional activation across multiple biological systems. We demonstrate that these derivatives bind the theophylline aptamer with up to 30-fold higher affinity, thereby expanding regulatory performance. In the bacterial system, these ligands enhance "ON" gene expression by up to 380-fold, compared to 75-fold with theophylline. This superior control extends to diverse organisms; in mycobacteria, the activation ratio reached 20-fold, and in eukaryotes, expression increased 11-fold. Furthermore, in riboswitch-mediated conditional CRISPR-Cas9 applications, these ligands achieve 70% genome editing efficiency at 10-fold lower concentrations than theophylline. These results demonstrate that ligand optimization is a crucial driver for enhancing riboswitch performance for advanced biomedical engineering.},
}

*Theophylline/metabolism/pharmacology/chemistry
*Riboswitch/genetics
Ligands
Aptamers, Nucleotide/metabolism/genetics/chemistry
Gene Editing/methods
CRISPR-Cas Systems/genetics
Gene Expression Regulation
Genetic Engineering/methods

@article {pmid41887225,
year = {2026},
author = {Drepanos, LM and Srikanth, S and Kaplan, EG and Shah, ST and Velasco, BE and Merzouk, S and Doench, JG},
title = {Balancing off-target and on-target considerations for optimized CRISPR-Cas9 knockout library design.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101190},
doi = {10.1016/j.xgen.2026.101190},
pmid = {41887225},
issn = {2666-979X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Library ; Mice ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Human/genetics ; },
abstract = {The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations cause library deprecation over time, further motivating an updated genome-wide design effort. Additionally, while on-target efficacy and off-target avoidance are often optimized in isolation, we lack a robust framework for simultaneously weighing and balancing these competing priorities. Here, we present a selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides, allowing the inclusion of those with maximal on-target activity. We create, validate, and make available to the community the Jacquere library for knockout screens of the human genome, as well as its mouse counterpart, Julianna, to facilitate gene function discovery at scale.},
}

*CRISPR-Cas Systems/genetics
Humans
Animals
*Gene Library
Mice
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
Genome, Human/genetics

@article {pmid41985719,
year = {2026},
author = {Liang, G and Li, G and Liang, T and Zhen, Z and Teng, C and Xiong, J and Guangqiang, J and Zheng, M and Pan, Y and Zhong, S and Wu, C and Li, J and Huang, W and Wei, Z},
title = {Construction of an RAA-CRISPR detection platform for differentiation of Brucella abortus A19-∆VirB12 vaccine strain from wild-type strains.},
journal = {Journal of microbiological methods},
volume = {245},
number = {},
pages = {107512},
doi = {10.1016/j.mimet.2026.107512},
pmid = {41985719},
issn = {1872-8359},
mesh = {*Brucella abortus/genetics/isolation & purification/classification ; Animals ; Cattle ; Recombinases/metabolism/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Brucellosis, Bovine/diagnosis/microbiology ; *Brucella Vaccine/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella abortus is a primary etiological agent of bovine brucellosis, a zoonosis posing significant threats to livestock industries and public health. The recently developed A19-∆VirB12 vaccine strain, which carries a deletion of the VirB12 gene, complicates serological differentiation from wild-type infections. This study aimed to establish a rapid, accurate, and economical detection strategy targeting the VirB12 gene to distinguish the A19-∆VirB12 vaccine strain from wild-type B. abortus. We developed a recombinase-aided amplification (RAA) coupled with CRISPR/Cas12a-based detection method. Following optimization, primer pair C and crRNA1 were selected as optimal components, with 150 nM each of Cas12a protein and crRNA identified as the ideal concentrations in a 50 μL reaction. The assay demonstrated high analytical specificity, showing no cross-reactivity with six non-target bacterial pathogens. Sensitivity analysis established a limit of detection of 10[2] copies per reaction. When evaluated on 52 clinical samples, the RAA-CRISPR assay detected two positive samples, outperforming conventional PCR which detected only one. Crucially, the method yielded no positive signal when challenged with the A19-∆VirB12 gene-deficient vaccine strain, while successfully detecting wild-type strains A19 and S2, confirming its discriminatory capability. The entire workflow, comprising RAA amplification (30 min) and CRISPR-mediated cleavage (20 min), can be completed within one hour, with results visualized via fluorescence or lateral flow strips. This study successfully establishes a rapid, sensitive, and specific diagnostic method for distinguishing the A19-∆VirB12 vaccine strain from wild-type B. abortus, offering a practical tool for field surveillance and eradication programs.},
}

*Brucella abortus/genetics/isolation & purification/classification
Animals
Cattle
Recombinases/metabolism/genetics
Sensitivity and Specificity
*CRISPR-Cas Systems
*Brucellosis, Bovine/diagnosis/microbiology
*Brucella Vaccine/genetics
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42003616,
year = {2026},
author = {Theriault, ME and Wong, AI and DeJesus, MA and Pisu, D and Nae Rin Lee, B and Kirukubar, G and Li, S and Wallach, JB and Schnappinger, D and Lê-Bury, G and Russell, DG and Rock, JM},
title = {Utilization of a CRISPRi-based ex vivo challenge model to reveal temporally dependent gene essentiality in intracellular Mycobacterium tuberculosis.},
journal = {mBio},
volume = {17},
number = {5},
pages = {e0061026},
doi = {10.1128/mbio.00610-26},
pmid = {42003616},
issn = {2150-7511},
support = {R01 AI155319/AI/NIAID NIH HHS/United States ; AI155319//National Institute of Allergy and Infectious Diseases/ ; AI162598//National Institute of Allergy and Infectious Diseases/ ; INV-055894//Bill and Melinda Gates Foundation/ ; //Mueller Health Foundation/ ; //Rita Allen Foundation/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/growth & development ; Animals ; Mice ; Macrophages/microbiology ; *Genes, Essential ; *CRISPR-Cas Systems ; *Tuberculosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Inbred C57BL ; Lung/microbiology ; Disease Models, Animal ; },
abstract = {UNLABELLED: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-embB, fadE29, and mbtI-confirmed their temporal significance in vivo. This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality in vivo.

IMPORTANCE: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. This novel approach enabled us to identify how different bacterial metabolic pathways were of greater importance to the bacterium at different time points post-infection. The results provide insights into how the evolving immune response to infection shapes the metabolic and replicative status of the bacterium. This information has significance in the design of therapeutic strategies toward cure.},
}

*Mycobacterium tuberculosis/genetics/growth & development
Animals
Mice
Macrophages/microbiology
*Genes, Essential
*CRISPR-Cas Systems
*Tuberculosis/microbiology
Clustered Regularly Interspaced Short Palindromic Repeats
Mice, Inbred C57BL
Lung/microbiology
Disease Models, Animal

@article {pmid42031246,
year = {2026},
author = {Han, T and Long, K and Hu, W and Liu, M and Guo, M and Huo, D and Hou, C},
title = {Dual-locked probes inhibit off-target circularization: Enhancing specificity in rolling circle transcription for nucleic acid detection.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152180},
doi = {10.1016/j.ijbiomac.2026.152180},
pmid = {42031246},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/genetics ; *Transcription, Genetic ; *Nucleic Acids/genetics/analysis ; Human papillomavirus 16/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; },
abstract = {Rolling Circle Replication (RCR) holds significant potential for detecting low-abundance nucleic acids. However, its practical application is often hindered by nonspecific ligation, which leads to high background signals and false-positive results. Herein, we designed a novel dual-locked circular template, termed no-bilateral-overhang dual-locked probe (ndRC). This probe employed steric hindrance from its dual hairpins lacking overhangs to create a "dual-locked" mechanism that specifically prevents off-target circularization. By integrating this template with exonuclease purification and a CRISPR/Cas12a module, we constructed the dual-locked RCT-CRISPR/Cas12a (DL-RCT-Cas12a) system, in which RCT generates long RNA transcripts containing crRNA repeats. These transcripts then activate Cas12a-mediated trans-cleavage, thereby amplifying the detection signal. The established assay achieved an ultralow detection limit of 40.31 aM, with a detection range from 100 aM to 1 μM. It demonstrated robust performance in complex matrices for the detection of partial HPV16 L1 gene sequences and exhibited a superior ability to discriminate high-concentration targets compared to qPCR. This work presents a versatile strategy to enhance the specificity of highly sensitive detection, improving performance in low-abundance nucleic acid analysis.},
}

CRISPR-Cas Systems/genetics
*Transcription, Genetic
*Nucleic Acids/genetics/analysis
Human papillomavirus 16/genetics
Humans
Limit of Detection
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics

@article {pmid42054542,
year = {2026},
author = {Gu, T and Xue, J and Zhang, Z and Cao, J and Song, J and Li, G and Ming, L and Zhu, Z and Wang, H},
title = {Mechanisms of Resistance to ALS Inhibitors and Bentazone in Fimbristylis littoralis and Rapid Identification of the ALS Trp-574-Leu Mutation Using LAMP-CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {18},
pages = {14311-14321},
doi = {10.1021/acs.jafc.5c17149},
pmid = {42054542},
issn = {1520-5118},
mesh = {*Acetolactate Synthase/genetics/antagonists & inhibitors/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism/antagonists & inhibitors/chemistry ; Herbicide Resistance ; Mutation ; CRISPR-Cas Systems ; *Enzyme Inhibitors/pharmacology ; },
abstract = {Fimbristylis littoralis Gaudich., a harmful sedge weed in Chinese rice paddy, impairs rice productivity and quality. In this study, we identified a resistant population (FL2) displaying multiple resistance to pyrazosulfuron-ethyl and bentazone, alongside cross-resistance to other acetolactate synthase (ALS)-inhibiting herbicides. The other population (FL6) showed exclusive resistance to bensulfuron-methyl. Sequencing demonstrated that FL2 carried a Trp-to-Leu mutation at codon 574 of ALS, whereas no mutations were detected in the psbA gene of bentazone-resistant FL2 or the ALS gene of bensulfuron-methyl-resistant FL6. Studies on nontarget-site resistance (NTSR) mechanisms indicated that FL2's resistance to pyrazosulfuron-ethyl was associated with neither PBO-inhibited P450s nor NBD-Cl-inhibited GSTs. In contrast, FL6's resistance to bensulfuron-methyl and FL2's resistance to bentazone were linked to P450 activity. A loop-mediated isothermal amplification (LAMP) coupled with CRISPR/FnCas12a assay was established for rapid detection of the Trp-574-Leu mutation, facilitating resistance management. These findings provide insights for managing resistant F. littoralis populations.},
}

*Acetolactate Synthase/genetics/antagonists & inhibitors/metabolism
*Herbicides/pharmacology
*Plant Proteins/genetics/metabolism/antagonists & inhibitors/chemistry
Herbicide Resistance
Mutation
CRISPR-Cas Systems
*Enzyme Inhibitors/pharmacology

@article {pmid42061542,
year = {2026},
author = {Yuan, Y and Ni, B and Tian, X and Cui, J and Zhang, Y and Hu, Y and Zheng, D and Zou, Y and Yu, X and Liu, C and Liu, S and Ren, W and Chang, X and Wang, Y and Ge, S and Wei, R and Chen, Y and Wu, X and Li, J and Wang, Z and Huang, B},
title = {Development of a point-of-care diagnostic method for FMDV SAT2 using RT-RAA-CRISPR technology.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152271},
doi = {10.1016/j.ijbiomac.2026.152271},
pmid = {42061542},
issn = {1879-0003},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation & purification ; Animals ; *Foot-and-Mouth Disease/diagnosis/virology ; *CRISPR-Cas Systems ; Cattle ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {In recent years, the foot-and-mouth disease virus (FMDV) serotype SAT2 has expanded beyond its traditionally recognized endemic areas on the African continent, leading to continuous dissemination in Middle Eastern countries and presenting a risk of further spread to Asia. In this study, the highly conserved sequence of the FMDV SAT2 genome was initially selected as the target, and five pairs of reverse transcription-recombinase-aided amplification (RT-RAA) primers were designed. By comparing fluorescence signal intensities, the CRISPR RNA (crRNA) with the highest sensitivity and specificity was identified and subsequently integrated with the CRISPR/Cas13a gene-editing system to establish a novel nucleic acid detection method. This method possesses a robust capacity for differential diagnosis and shows no cross-reaction with other serotypes of FMDV. Moreover, this method demonstrated high specificity and no cross-reactivity with the nucleic acid sequences of various common pathogens in porcine and bovine populations. The test results are readily interpretable and can be directly visualized using a fluorescence reader or lateral flow test strips (LFSs). In simulated clinical samples, this method achieved a concordance rate of 100% with the detection results of fluorescence quantitative RT-PCR. This study successfully developed a highly sensitive and specific FMDV SAT2 nucleic acid detection method based on RT-RAA-CRISPR/Cas13a technology. This method is straightforward to perform, does not require costly experimental equipment, and is suitable for rapid onsite detection, offering a convenient and efficient diagnostic tool for the early diagnosis, prevention, and control of FMDV SAT2 epidemics.},
}

*Foot-and-Mouth Disease Virus/genetics/isolation & purification
Animals
*Foot-and-Mouth Disease/diagnosis/virology
*CRISPR-Cas Systems
Cattle
Swine
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Systems
Sensitivity and Specificity
Recombinases/metabolism

@article {pmid42061934,
year = {2026},
author = {Gunasekaran, H and Najwa, KV and Nidarshan, NC and Porkodi, M and Singh, LS and Rasal, KD and Brahmane, MP and Goswami, M and Sonwane, AA},
title = {Characterization of Trachinotus blochii mstnb gene and construction of cognate gRNA vector.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152225},
doi = {10.1016/j.ijbiomac.2026.152225},
pmid = {42061934},
issn = {1879-0003},
mesh = {Animals ; *Myostatin/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Fishes/genetics ; Amino Acid Sequence ; Base Sequence ; *Fish Proteins/genetics ; },
abstract = {Skeletal muscle development is negatively regulated by the myostatin b (mstnb) gene in finfishes. CRISPR-Cas9-mediated knockout of mstnb has been used to develop fish strains with increased muscle mass. Trachinotus blochii (Silver Pompano), a high-value, moderately sized (~500 g), and cultivable marine finfish, is a promising candidate for mstnb knockout using CRISPR/Cas9. However, limited studies exist on the characterization of T. blochii mstnb and no CRISPR-based knockout studies have been reported in this species. This study aimed to partially characterize the mstnb gene of T. blochii and develop a CRISPR/Cas9-based guide RNA (gRNA) expression vector for its knockout. The T. blochii mstnb sequence available in NCBI was used as a reference to design exon- and intron-specific primers for PCR amplification and sequencing. The sequence revealed three exons and two introns. A predicted 1131 bp open reading frame encodes a 376 amino acid protein containing conserved domains typical of the TGF-β family which includes an N-terminal signal peptide, a propeptide region, a conserved RARR cleavage motif, and a C-terminal GF domain containing nine conserved cysteine residues. Secondary and 3D structure predictions confirmed the protein's functional integrity. Sequence analysis revealed novel putative polymorphisms, including SNPs and a (CA)n microsatellite. Phylogenetic analysis clustered T. blochii with related Trachinotus and other Carangiformes species. A gRNA targeting exon 1 was designed using CRISPOR and successfully cloned into expression vectors. This novel sequence information can aid population-level studies and genetic marker discovery. The constructed gRNA vectors can facilitate CRISPR/Cas9-mediated mstnb knockout in T. blochii to study gene function and develop a fleshy strain.},
}

Animals
*Myostatin/genetics/chemistry
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
*Genetic Vectors/genetics
*Fishes/genetics
Amino Acid Sequence
Base Sequence
*Fish Proteins/genetics

@article {pmid42067160,
year = {2026},
author = {Dai, S and Niu, L and Lv, Y and Zhang, H and Xu, L and Liao, Y and Hu, X and Xie, X and Yan, J and Yan, Y},
title = {Semi-artificial photobiocatalysis via genetically modified sulfur metabolism to in situ assembly of a solar-biohybrid for antibiotic degradation.},
journal = {Bioresource technology},
volume = {454},
number = {},
pages = {134750},
doi = {10.1016/j.biortech.2026.134750},
pmid = {42067160},
issn = {1873-2976},
mesh = {*Sulfur/metabolism ; *Anti-Bacterial Agents/metabolism ; Sulfides/metabolism ; Biodegradation, Environmental ; Nanoparticles/chemistry ; Gene Editing ; CRISPR-Cas Systems/genetics ; Solar Energy ; Tetracycline/metabolism ; },
abstract = {The solar-driven semi-artificial biohybrid system incorporates semiconductor materials with microbial metabolism, affording an innovative strategy for antibiotic degradation via photocatalysis. In this study, the sulfur metabolic pathway of biological cells was rationally engineered using CRISPR-Cas9 and Cre-loxP site-specific gene editing systems, successfully achieving intracellular accumulation of sulfide up to 552.84 ppm. Based on this capability, In(Ⅲ) was adsorbed in situ onto the cell surface, leading to the self-assembly of photosensitive In2S3 nanoparticles (NPs). The resulting inorganic-biological hybrid system of In2S3-cell exhibited a broad-spectrum light-harvesting capability with an ideal optical bandgap of 1.96 eV. Photoelectrochemical analysis confirmed the charge transfer process and the semiconductor biointerface based regeneration mechanism of redox cofactors in the cytosol. Photogenerated electrons directly form ROS for tetracycline oxidation, and are transferred to cells for enhance the regeneration of intracellular reducing cofactors. This light-driven photocatalytic biohybrid system enabled efficient tetracycline degradation of over 98% within 4 h and demonstrated excellent stability over consecutive cycles. Transcriptomic analysis identified key genes involved in solar energy capture, electron transport, and metabolic regulation, elucidating their functional roles in biomanufacturing processes and photocatalytic degradation. This study presents a bottom-up paradigm for the biotic-abiotic system from electronic and molecular perspectives to develop efficient and sustainable technologies for antibiotic remediation and solar energy conversion.},
}

*Sulfur/metabolism
*Anti-Bacterial Agents/metabolism
Sulfides/metabolism
Biodegradation, Environmental
Nanoparticles/chemistry
Gene Editing
CRISPR-Cas Systems/genetics
Solar Energy
Tetracycline/metabolism

@article {pmid42121843,
year = {2026},
author = {Siddika, A and Husseiny, FE and Rousseau, J and Tremblay, JP},
title = {Successful In Vitro Modification of the Dmd Gene Using Prime Editing.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/cells15090740},
pmid = {42121843},
issn = {2073-4409},
support = {53320215//Defeat Duchenne Foundation/ ; CIHR; Application No. 492510/CAPMC/CIHR/Canada ; },
mesh = {*Gene Editing/methods ; *Dystrophin/genetics ; Animals ; Mice ; *Muscular Dystrophy, Duchenne/genetics ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Base Sequence ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5'-TTCT-3' motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.},
}

*Gene Editing/methods
*Dystrophin/genetics
Animals
Mice
*Muscular Dystrophy, Duchenne/genetics
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Myoblasts/metabolism
Base Sequence
Mutation/genetics
CRISPR-Cas Systems/genetics

@article {pmid42121869,
year = {2026},
author = {Siwak, JF and Connelly, JP and Pruett-Miller, SM},
title = {Essential HDRescue: A Co-Targeting Strategy to Enhance Precision Genome Editing by Co-Editing Essential Genes.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/cells15090768},
pmid = {42121869},
issn = {2073-4409},
support = {N/A//American Lebanese Syrian Associated Charities/ ; NA//St. Jude Graduate School of Biomedical Sciences/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair/genetics ; *Genes, Essential/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Induced Pluripotent Stem Cells/metabolism ; Animals ; },
abstract = {Genome editing is widely used and conceptually simple, yet in practice, it is hindered by laborious workflows and high costs. These challenges stem from the difficulty of identifying and isolating cells that contain the desired user-defined modifications, a problem compounded by the wide variability in editing efficiencies across cell types. While homology-directed repair (HDR) provides a mechanism for precise genome modification following nuclease-induced double-strand breaks (DSBs), it is frequently outcompeted by the dominant mutagenic non-homologous end-joining (NHEJ) pathway in mammalian cells. Therefore, we developed a novel enrichment method, Essential HDRescue, to increase the frequency of HDR events at a target site by co-targeting an essential genomic locus. Using both intrinsic positive and negative selection at a common essential gene, we enabled enrichment of precise editing events at a second, unlinked target site. We demonstrated that co-targeting essential genes in cancer cell lines and iPSCs increased HDR rates without the need for an exogenous reporter or selective drug. Analysis of resulting clones revealed that Essential HDRescue produced up to a 6-fold increase in single-allele edits and an ~4-fold increase in homozygous edits relative to single-targeted controls. By harnessing the intrinsic cellular dependencies that arise from DSB repair at essential loci, Essential HDRescue offers a widely applicable method to improve precise genome editing outcomes in mammalian cells, leaving only a minimal, protein-silent scar at the essential gene.},
}

*Gene Editing/methods
Humans
*Recombinational DNA Repair/genetics
*Genes, Essential/genetics
DNA End-Joining Repair/genetics
CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
Induced Pluripotent Stem Cells/metabolism
Animals

@article {pmid42122867,
year = {2026},
author = {Wen, Y and Li, Y and Bao, S and Cao, G and Li, M and Wang, J and Ding, B and Xie, X and Qiu, L},
title = {The Auxin Response Factor TaARF18-A Negatively Regulates Salt Tolerance in Common Wheat (Triticum aestivum L.).},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/plants15091375},
pmid = {42122867},
issn = {2223-7747},
support = {32301817//National Natural Science Foundation of China/ ; 22JCQNJC01470//Natural Science Foundation of Tianjin/ ; 26CXNE010//Gansu Provincial Science and Technology Program Project/ ; KLIBMC2507//Open Fund from the Tianjin Key Laboratory of Intelligent Breeding of Major Crops/ ; },
abstract = {Soil salinization is one of the major abiotic stresses that influences agricultural production and the environment. Auxin response factors (ARFs) are key components of the auxin signal transduction pathway, while their role in wheat salt stress responses remains unclear. In this study, we identified TaARF18 as a negative regulator of salt tolerance in wheat. The coding sequences of TaARF18-A, TaARF18-B, and TaARF18-D were 2106, 2088, and 2088 bp, respectively. TaARF18 is a hydrophilic protein featuring typical Auxin-resp and B3 DNA-binding domains and exhibits relatively high evolutionary conservation among Poaceae species. The expression of TaARF18 was upregulated under salt stress. TaARF18 predominantly accumulated in the nucleus. Silencing of TaARF18 via the BSMV-VIGS approach enhanced salt tolerance in wheat seedlings. In addition, haplotype analysis based on resequencing data from 355 wheat accessions identified 25, 31, and 16 haplotypes for TaARF18-A, TaARF18-B, and TaARF18-D, respectively. Fourteen wheat accessions carrying different haplotypes were evaluated under salt stress, and HapIII of TaARF18-A exhibited the highest level of salt tolerance, which can act as a strong selection locus in global wheat breeding. Our findings provide insight into the function of ARFs in salt stress responses and offer a potential target for CRISPR/Cas-mediated salt-tolerant wheat breeding programs.},
}

@article {pmid42123509,
year = {2026},
author = {Li, Y and Yao, Y and Xu, Z and Xiong, Y and Zhang, C and Yu, L and Gao, H and Fei, T},
title = {Genome-Wide CRISPR Screening Identifies Genetic Modulators of Amyloid Precursor Protein Processing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27093926},
pmid = {42123509},
issn = {1422-0067},
mesh = {Humans ; *Amyloid beta-Protein Precursor/metabolism/genetics ; *CRISPR-Cas Systems ; *Alzheimer Disease/genetics/metabolism ; Amyloid beta-Peptides/metabolism/genetics ; HEK293 Cells ; },
abstract = {The proteolytic processing of the amyloid precursor protein (APP) is a core pathological event in Alzheimer's disease (AD) pathogenesis, yet the global genetic regulatory networks modulating this process have not been fully characterized. To systematically identify novel regulators of APP cleavage, we performed a genome-wide CRISPR/Cas9 knockout screen utilizing an optimized UAS-GAL4-based cellular reporter, and identified genetic modulators governing amyloidogenic and non-amyloidogenic processing. The screen uncovered distinct functional gene clusters regulating the APP, prominently involving cellular metabolism, protein modification, and vesicular trafficking. Specifically, LDHB, PIAS2, CCDC53, and TRIM61 emerged as novel functional modulators. Biochemical validation confirmed that ablating these genes significantly alters the metabolic balance between sAPPα and amyloid-β (Aβ) production. Finally, integration with human AD transcriptomic datasets demonstrated that these identified modulators undergo significant dysregulation in clinics. Together, these findings establish a reporter-based functional screening framework for APP processing and identify candidate regulatory nodes linked to metabolism, protein modification, and vesicular trafficking. These candidates provide a resource for future mechanistic investigation and validation in more disease-relevant AD models.},
}

Humans
*Amyloid beta-Protein Precursor/metabolism/genetics
*CRISPR-Cas Systems
*Alzheimer Disease/genetics/metabolism
Amyloid beta-Peptides/metabolism/genetics
HEK293 Cells

@article {pmid42123512,
year = {2026},
author = {Yuan, Y and Yuan, J and Deng, D and Wu, J and Zhou, X and Jiang, A and Wang, J and Wang, X and Li, M and Long, K and Zhao, L},
title = {CRISPR/Cas9-Mediated Knockout of CGNL1 Confers Resistance to Aflatoxin B1 in Porcine Intestinal Epithelial Cells via Suppressing ROS Generation.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27093928},
pmid = {42123512},
issn = {1422-0067},
support = {XZ202501ZY0147//Tibet Autonomous Region Science and Technology Agency/ ; sccxtd-2025-08-13//the Program for Pig Industry Technology System Innovation Team of Sichuan Province/ ; 32472884//National Natural Science Foundation of China/ ; 32573165//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Aflatoxin B1/toxicity ; *CRISPR-Cas Systems ; Swine ; *Epithelial Cells/metabolism/drug effects ; *Reactive Oxygen Species/metabolism ; Oxidative Stress/drug effects ; *Intestinal Mucosa/metabolism/drug effects ; Gene Knockout Techniques ; Cell Line ; Cell Survival/drug effects ; Signal Transduction ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent and highly toxic mycotoxin in the food and feed chain and can directly injure the intestinal epithelium. Yet, its upstream determinants linking epithelial stress to cytotoxicity remain insufficiently defined. Here, we used porcine intestinal epithelial IPEC-J2 cells to characterize AFB1-induced cytotoxic and transcriptomic responses and to determine the role of the tight-junction scaffold, Cingulin-like 1 (CGNL1), a candidate gene identified through genome-scale CRISPR knockout library screening. The results showed that AFB1 exposure reduced cell viability in a dose-dependent manner and induced oxidative stress. RNA-seq profiling analysis revealed broad transcriptional remodeling, with activation of inflammatory pathways (including NF-κB and JAK-STAT signaling). Based on our constructed CGNL1-knockout IPEC-J2 cell line (CGNL1-KO IPEC-J2) using CRISPR/Cas9, it was found that CGNL1 deficiency markedly alleviated AFB1-induced cytotoxicity and oxidative stress. Comparative transcriptomics analysis showed that CGNL1 knockout attenuated AFB1-triggered aberrant expression of some CGNL1-dependent AFB1-responsive genes related to immune response under AFB1 challenge. Together, these findings identify CGNL1 as a potential modulator of epithelial susceptibility to AFB1 and support its involvement in the regulation of toxin-induced oxidative response.},
}

Animals
*Aflatoxin B1/toxicity
*CRISPR-Cas Systems
Swine
*Epithelial Cells/metabolism/drug effects
*Reactive Oxygen Species/metabolism
Oxidative Stress/drug effects
*Intestinal Mucosa/metabolism/drug effects
Gene Knockout Techniques
Cell Line
Cell Survival/drug effects
Signal Transduction

@article {pmid42123673,
year = {2026},
author = {Jing, L and Roy, D and Kalischuk, M},
title = {Advances in CRISPR Plant Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27094095},
pmid = {42123673},
issn = {1422-0067},
support = {Alliance #44842//Natural Sciences and Engineering Research Council of Canada/ ; 45402//Solanum International Inc./ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; *Plants/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The ability to precisely edit genetic characteristics with a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immunity complex is a revolutionary advance in science. Originally discovered in bacteria as part of a natural defense mechanism against viruses, CRISPR/Cas provides a precise, efficient, and relatively simple method for editing genes in microbes, plants, animals, and humans. The process relies on the Cas protein, an enzyme that cleaves and unwinds DNA at targeted locations. This process is guided by RNA sequences complementary to the DNA or RNA sequence of interest, allowing for changes to the genome through innate non-homologous end joining (NHEJ) and homology-directed repair (HDR). The potential applications of CRISPR/Cas are immense and, in agriculture, is facilitating crop development with resistance to abiotic, biotic, and agronomic characteristics that improve yield, quality, and food security. Gene editing also facilitates the relatively rapid modification of regulatory and complex pathways that enable studies to advance our understanding of gene function. This review provides an update of the fast-evolving CRISPR/Cas modification of important crops to address emerging global population, as well as environmental and climate challenges.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Crops, Agricultural/genetics
Plants, Genetically Modified/genetics
*Plants/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42124669,
year = {2026},
author = {Sanderson, EM and Peralta, J and Nouwens, S and Oriolt, L and Hayes, VM and Kaiser, BK and Meeske, AJ},
title = {Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.02.23.707548},
pmid = {42124669},
issn = {2692-8205},
abstract = {Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri . Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.},
}

@article {pmid42126429,
year = {2026},
author = {Huang, RS and Phung, SK and Sumstad, D and Weis, AJ and Kile, QM and Bendzick, L and Khaw, MJ and Vue, YY and McKenna, DH and Kennedy, PR and Miller, JS and Felices, M},
title = {Reprogramming endogenous NK circuits by highly efficient nonviral genome editing.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {7},
pages = {},
doi = {10.1084/jem.20260192},
pmid = {42126429},
issn = {1540-9538},
support = {R35 CA283892/NH/NIH HHS/United States ; P01 CA111412/NH/NIH HHS/United States ; P01 CA065493/NH/NIH HHS/United States ; P30 CA 77598/NH/NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; *Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Mice ; Gene Regulatory Networks ; Interleukin-12/genetics/metabolism ; Receptors, Chimeric Antigen/genetics/immunology ; },
abstract = {Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.},
}

*Killer Cells, Natural/immunology/metabolism
*Gene Editing/methods
Humans
Animals
CRISPR-Cas Systems
Gene Knock-In Techniques
Mice
Gene Regulatory Networks
Interleukin-12/genetics/metabolism
Receptors, Chimeric Antigen/genetics/immunology

@article {pmid42127676,
year = {2026},
author = {Cheng, M and Chen, X and Cheng, H and Gao, X and Ao, H and Bao, X and Song, X and Tai, Y and Jin, D and Zhang, L},
title = {An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for detection of toxigenic Clostridioides difficile.},
journal = {Biosensors & bioelectronics},
volume = {308},
number = {},
pages = {118779},
doi = {10.1016/j.bios.2026.118779},
pmid = {42127676},
issn = {1873-4235},
abstract = {Clostridioides difficile (C. difficile) infection (CDI) represents a formidable global healthcare challenge, necessitating the development of rapid, accurate, and cost-effective diagnostic platforms to mitigate nosocomial transmission and improve patient outcomes. Compared with the conventional methods, CRISPR/Cas systems featured by specific target reorganization by a single chain RNA, coupled with electrochemical technology enables highly sensitive detection of various biomarkers. However, their application to CDI has remained unexplored due to the lack of tailored crRNAs. Herein, we present the integration of CRISPR/Cas12a with electrochemical transduction for the direct detection of C. difficile. A novel crRNA was engineered to specifically recognize the toxin B gene (tcdB), activating the trans-cleavage activity of Cas12a upon target binding. This cascade triggers the cleavage of immobilized ssDNA reporters on the electrode surface, generating measurable amperometric signal changes. The developed biosensor demonstrates exceptional performance, achieving a detection limit of pM level for tcdB DNA within 40 min, while exhibiting high specificity against non-target pathogens and robust stability over 7 days. This work establishes a rapid and reliable CRISPR-electrochemical diagnostic platform, offering significant potential for point-of-care CDI management.},
}

@article {pmid42128325,
year = {2026},
author = {Cai, Y and Yang, J and Hou, M and Su, W and Liang, F and Zhu, M and Wu, T},
title = {Multi-omics precision diagnosis of brucellosis: Advances in biomarker discovery and clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {121074},
doi = {10.1016/j.cca.2026.121074},
pmid = {42128325},
issn = {1873-3492},
abstract = {Brucellosis, a neglected zoonosis caused by intracellular Brucella bacteria, remains a formidable global public health challenge, especially in developing regions. The notorious ability of Brucella to evade host immunity and establish chronic focal infections limits the utility of traditional diagnostic methods like bacterial culture and serology for early detection, therapeutic monitoring, and disease staging. This review comprehensively synthesizes the ongoing paradigm shift from pathogen-centric detection toward multi-omics precision diagnosis. We critically evaluate advances in nucleic acid amplification technologies (NAATs), charting the progression from quantitative PCR to absolute quantification via droplet digital PCR (ddPCR) and examining the transformative potential of CRISPR-Cas biosensing for ultrasensitive, instrument-free detection. The discussion also encompasses the renaissance of serology through immunoproteomics, which has identified novel serodominant antigens and multi-epitope fusion proteins to address the persistent specificity problems arising from cross-reacting bacteria. Furthermore, we analyze the emerging landscape of host-response biomarkers, integrating transcriptomic, metabolomic, and single-cell RNA sequencing data to delineate distinct immune signatures of acute and chronic infection. Finally, we consider how artificial intelligence (AI) can integrate these multi-dimensional datasets to build predictive diagnostic models. This consolidated multi-omics framework charts a course for precision medicine in brucellosis, aiming to bridge the gap between biomarker discovery and point-of-care clinical application. SUMMARY: Multi-omics technologies (genomics, proteomics, metabolomics, transcriptomics) are advancing brucellosis diagnosis via sensitive detection and accurate biomarkers, and improving treatment through novel strategies like nano-delivery, vaccines, and AI integration.},
}

@article {pmid42128554,
year = {2026},
author = {Xu, W and Cheng, Y and Sun, K and Wu, Y and Xu, Y and Ye, J and Li, P and Wu, H},
title = {Inception-level signal amplification: Cascaded DNAzyme-Cas9 nickase achieves sub-nanomolar kanamycin tracking.},
journal = {Analytica chimica acta},
volume = {1408},
number = {},
pages = {345562},
doi = {10.1016/j.aca.2026.345562},
pmid = {42128554},
issn = {1873-4324},
mesh = {*Kanamycin/analysis/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *Biosensing Techniques/methods ; Milk/chemistry ; *Nucleic Acid Amplification Techniques ; *Deoxyribonuclease I/metabolism/chemistry ; Limit of Detection ; Animals ; *Anti-Bacterial Agents/analysis ; CRISPR-Cas Systems ; Water Pollutants, Chemical/analysis ; },
abstract = {BACKGROUND: Kanamycin's persistent contamination in agricultural products poses significant human health risks due to its nephrotoxicity and bioaccumulation via the food chain. Existing methods for on-site kanamycin monitoring lack sufficient sensitivity and portability, limiting their utility in field settings. The urgent need for rapid detection technologies remains unmet. This work addresses the critical gap in developing a field-deployable platform for ultrasensitive kanamycin residue screening.

RESULTS: We engineered a fluorescence biosensor integrating DNAzyme-assisted Cas9 nickase-based amplification reaction (Cas9nAR). Kanamycin binding induces aptamer conformational changes, triggering Cas9nAR-driven cascade amplification that continuously generates DNAzymes. These cleave reporter probes to enable quantitative detection. The system achieved a broad linear range (1 nM - 5 μM) with an ultralow detection limit (0.3 nM), surpassing conventional methods. It exhibited high specificity against interfering antibiotics and delivered consistent recoveries (97% to 103%) in spiked water and milk samples. Following pretreatment, analysis can be completed within 120 min, validating its operational simplicity and robustness for complex matrices.

SIGNIFICANCE AND NOVELTY: This work reports the first CRISPR-Cas9 nickase/DNAzyme cascade amplification platform for small-molecule detection, establishing a new paradigm that integrates programmable nucleic acid amplification with catalytic signal turnover. By using Cas9nAR-generated ssDNA as an in situ template for autonomous DNAzyme assembly, the biosensor achieves ultrasensitive, and homogeneous detection of kanamycin-addressing a critical gap in field-deployable antibiotic residue monitoring. The modular design offers a generalizable strategy for translating non-nucleic acid recognition events into amplified fluorescent outputs, with broad implications for point-of-need diagnostics in food safety and environmental analysis.},
}

*Kanamycin/analysis/metabolism
*DNA, Catalytic/metabolism/chemistry
*Biosensing Techniques/methods
Milk/chemistry
*Nucleic Acid Amplification Techniques
*Deoxyribonuclease I/metabolism/chemistry
Limit of Detection
Animals
*Anti-Bacterial Agents/analysis
CRISPR-Cas Systems
Water Pollutants, Chemical/analysis

@article {pmid42128971,
year = {2026},
author = {Xie, R and Zhu, C and Liang, X and Lu, K and Wu, J},
title = {Knockout of bsal/cel.2 results in growth retardation, reduced lipid digestion and altered energy metabolism in medaka larvae (oryzias latipes).},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42128971},
issn = {1438-7948},
support = {2024YFD2401502//the National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Oryzias/genetics/growth & development/metabolism ; *Energy Metabolism/genetics ; *Lipid Metabolism/genetics ; *Fish Proteins/genetics/metabolism ; Larva/growth & development/genetics/metabolism ; *Lipase/genetics/metabolism ; CRISPR-Cas Systems ; Lipolysis/genetics ; },
abstract = {To evaluate the significance of bile salt-activated lipase (BSAL) in lipid digestion and metabolism in fish, this study used CRISPR/Cas9 gene editing to generate medaka (Oryzias latipes) bsal mutant lines. Given that the bsal gene comprises three copies (bsal, cel.2, and bsal-like) in the medaka genome, bsal-like variants may lead to functional loss in lipid hydrolysis owing to amino acid residue alterations in the bile salt binding site. Consequently, two types of medaka mutants, bsal[-/-] and bsal[-/-]/cel.2[-/-], were generated for experimental exploration in this study. Compared to wild-type (WT) medaka, the bsal[-]/[-]/cel.2[-]/[-] group showed significant reductions in body length, expression of growth-related genes (gh and igf), total lipase and protease activities, and body composition (cholesterol, triglyceride, and protein levels). The bsal[-]/[-]/cel.2[-]/[-] group also exhibited upregulated expression of lipid synthesis genes (fas, acc1, scd) and downregulated expression of lipolysis-related genes (cpt1, acox1). Notably, key glycolysis genes (pk, gk) and gluconeogenesis-related genes (pck2) were significantly upregulated in the bsal[-]/[-]/cel.2[-]/[-] group. However, the bsal[-/-] group exhibited no significant differences from the WT group in all assays, except for notable reductions in protease activity and expression levels of the cpt1 and gk genes, as well as a significant increase in pck2 gene expression compared to the WT group. Remarkably, the expression level of the cel.2 gene was significantly elevated in the bsal[-/-] group compared to the WT group. In summary, this study demonstrates the pivotal role of the bsal gene in lipid digestion and metabolism in medaka. Furthermore, the presence of multiple copies of the bsal gene aids in fulfilling the demands of lipid digestion in medaka. This conclusion can provide insights into the research on lipid digestion and metabolism in other fish species.},
}

Animals
*Oryzias/genetics/growth & development/metabolism
*Energy Metabolism/genetics
*Lipid Metabolism/genetics
*Fish Proteins/genetics/metabolism
Larva/growth & development/genetics/metabolism
*Lipase/genetics/metabolism
CRISPR-Cas Systems
Lipolysis/genetics

@article {pmid42128985,
year = {2026},
author = {Hou, M and Li, Y and Wu, X and Long, D and Sun, D and Chen, P and Huang, H},
title = {Recent Advances in the Development of CRISPR-Based Live-Cell Molecular Imaging and Sensing.},
journal = {Molecular imaging and biology},
volume = {},
number = {},
pages = {},
pmid = {42128985},
issn = {1860-2002},
support = {2026JJ90110//Natural Science Foundation of Hunan Province Joint Fund for Universities/ ; S202512034126//Hunan Province Undergraduate Innovation Training Program/ ; },
abstract = {Visualizing genome organization and transcriptional dynamics with spatial and temporal precision in living cells is essential for elucidating gene regulation and chromatin-associated disease mechanisms, yet conventional methods confront a fundamental tension between endogenous-sequence targeting and live-cell compatibility. Operator-repressor systems require prior insertion of repetitive arrays at engineered loci, whereas fluorescence in situ hybridization mandates cell fixation and thereby precludes temporal analysis. CRISPR-Cas technologies, originally developed for genome editing, have been re-engineered into a versatile molecular-imaging toolkit capable of interrogating native sequences in living cells. Here, we systematically review CRISPR-based live-cell imaging and sensing platforms, critically evaluating their design principles, mechanistic foundations, and performance limitations. We examine dCas9-based DNA labeling, dCas12a systems for non-repetitive loci, Cas13- and Csm-mediated RNA imaging, novel fluorescent reporters, engineered ribonucleoproteins, and delivery innovations including reagent-based Oligo-LiveFISH. To organize this diverse literature, we distinguish three operationally distinct modalities-live-cell imaging, intracellular sensing, and diagnostic biosensing-and assess each platform through three unifying design trade-offs: sensitivity versus cellular perturbation, multiplexing capacity versus system complexity, and detection threshold versus biological fidelity. Building on this framework, we evaluate the integration of CRISPR imaging with super-resolution microscopy, artificial-intelligence-driven computational analysis, and multimodal spatial omics. Collectively, this synthesis clarifies current capabilities, delineates unresolved constraints, and charts a coherent path toward clinically relevant applications of CRISPR-based live-cell molecular imaging.},
}

@article {pmid42129030,
year = {2026},
author = {Watanabe, K and Ishikawa, M and Ishibashi, K},
title = {Development of Tobamovirus-Resistant Tomato Plants by CRISPR-Cas9-Mediated Knockout of Susceptibility Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {1-10},
pmid = {42129030},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Tobamovirus/pathogenicity/physiology ; Gene Editing/methods ; *Gene Knockout Techniques/methods ; Host-Pathogen Interactions/genetics ; Plants, Genetically Modified/genetics/virology ; },
abstract = {The advent of genome editing technologies such as CRISPR-Cas9 has revolutionized the development of disease-resistant crops, offering precision and efficiency in targeting specific genetic loci responsible for susceptibility. In this protocol, we harness the CRISPR-Cas9 system to disrupt key susceptibility genes in tomato, aiming to fortify resistance against tobamoviruses, particularly the aggressive tomato brown rugose fruit virus (ToBRFV). By systematically knocking out four TOM1 homologs, genes essential for tobamoviral replication, tomato lines with robust and heritable resistance can be developed while minimizing adverse developmental effects. The approach not only underscores the significance of basic research on host-pathogen interactions in modern crop protection but also lays the groundwork for sustainable, gene-driven resistance strategies in commercial tomato breeding.},
}

*Solanum lycopersicum/genetics/virology
*CRISPR-Cas Systems/genetics
*Disease Resistance/genetics
*Plant Diseases/virology/genetics
*Tobamovirus/pathogenicity/physiology
Gene Editing/methods
*Gene Knockout Techniques/methods
Host-Pathogen Interactions/genetics
Plants, Genetically Modified/genetics/virology

@article {pmid42129032,
year = {2026},
author = {Yoshida, T and Ishibashi, K},
title = {Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana Using a Meristem-Invading Virus Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {21-27},
pmid = {42129032},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Meristem/genetics/virology ; CRISPR-Cas Systems ; *Potyvirus/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Gene editing can be achieved using sequence-specific nucleases. This protocol describes a plant gene editing method that eliminates the need for tissue culture by employing a virus-based delivery system. Tobacco ringspot virus (TRSV) can access meristematic tissues in infected plants, enabling the introduction of site-directed mutations into germline cells. This allows for heritable gene modification in the model plant Nicotiana benthamiana without tissue culture.},
}

*Nicotiana/genetics/virology
*Gene Editing/methods
*Genetic Vectors/genetics
*Meristem/genetics/virology
CRISPR-Cas Systems
*Potyvirus/genetics
Plants, Genetically Modified/genetics

@article {pmid42129103,
year = {2026},
author = {Ash, S and Attianese, GMPG and Kosti, P and Semilietof, A and Stefanidis, E and Triboulet, M and Irving, M},
title = {Generation and Characterization of CAR-T Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2998},
number = {},
pages = {439-504},
pmid = {42129103},
issn = {1940-6029},
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Neoplasms/therapy/immunology ; Lymphocyte Activation ; Lentivirus/genetics ; Retroviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.},
}

Humans
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
Animals
Mice
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/metabolism
Cell Line, Tumor
*Receptors, Antigen, T-Cell/genetics/immunology/metabolism
Neoplasms/therapy/immunology
Lymphocyte Activation
Lentivirus/genetics
Retroviridae/genetics
CRISPR-Cas Systems

@article {pmid42129496,
year = {2026},
author = {Duchêne, C and Craig, RJ and Martinho, C and Luthringer, R and Agullo, F and Hipp, K and Escudeiro, P and Alva, V and Haas, FB and Coelho, SM},
title = {Latent endogenous giant viruses drive active infection and inheritance in a multicellular algal host.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42129496},
issn = {2058-5276},
support = {101109906//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; },
abstract = {Endogenous viral elements inserted in host genomes are often regarded as inert relics of past infections. Whether they can retain infective potential and contribute to active viral cycles has remained largely unresolved. Here we demonstrate that giant viral elements in the multicellular alga Ectocarpus can reactivate and drive productive viral infections. Using long-read sequencing and transcriptomics, we identify full-length, transcriptionally active phaeoviruses integrated within the host genome, and we use classical genetics and CRISPR-Cas to demonstrate that these elements are stably inherited through the germline, while their reactivation is precisely regulated by developmental and environmental cues including temperature. We resolve the genomic integration sites and propose a mechanism for phaeovirus integration and replication. Our work provides direct evidence and uncovers the mechanisms by which giant viral elements can reactivate, replicate and transmit both horizontally and vertically in a multicellular eukaryote, establishing a new model of latency, inheritance and evolutionary impact of giant dsDNA viruses.},
}

@article {pmid35851300,
year = {2022},
author = {Droogers, WJ and Willems, J and MacGillavry, HD and de Jong, APH},
title = {Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.},
journal = {eNeuro},
volume = {9},
number = {4},
pages = {},
pmid = {35851300},
issn = {2373-2822},
mesh = {Animals ; *CRISPR-Cas Systems ; *Neurons/metabolism ; *Gene Editing/methods ; Rats ; *Gene Knock-In Techniques/methods ; *Nerve Tissue Proteins/metabolism/genetics ; Synapses/metabolism ; Cells, Cultured ; },
abstract = {CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.},
}

Animals
*CRISPR-Cas Systems
*Neurons/metabolism
*Gene Editing/methods
Rats
*Gene Knock-In Techniques/methods
*Nerve Tissue Proteins/metabolism/genetics
Synapses/metabolism
Cells, Cultured

@article {pmid41062702,
year = {2026},
author = {Casagrande Raffi, G and Kuiken, HJ and Lieftink, C and Bernards, R and Beijersbergen, RL and Wang, L},
title = {Inducible CRISPR-Cas9 screening platform to interrogate non-proliferative cellular states.},
journal = {Nature protocols},
volume = {21},
number = {5},
pages = {1896-1926},
pmid = {41062702},
issn = {1750-2799},
support = {19-051-ASP//Bergmark Foundation/ ; KWF-12539//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Proliferation ; Flow Cytometry/methods ; Cellular Senescence/genetics ; },
abstract = {CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Proliferation
Flow Cytometry/methods
Cellular Senescence/genetics

@article {pmid41258015,
year = {2026},
author = {Longo, GMC and Sayols, S and Roukos, V},
title = {Multilevel characterization of genome editor nuclease activity with BreakTag.},
journal = {Nature protocols},
volume = {21},
number = {5},
pages = {2043-2082},
pmid = {41258015},
issn = {1750-2799},
support = {393547839-SFB 1361//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 402733153-SPP 2202//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 455784893//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; INST 247/845-1 FUGG//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *High-Throughput Nucleotide Sequencing/methods ; DNA Breaks, Double-Stranded ; Humans ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {BreakTag is a scalable next-generation sequencing-based method for the unbiased characterization of programmable nucleases and guide RNAs at multiple levels. BreakTag allows off-target nomination, nuclease activity assessment and the characterization of scission profile, that, in Cas9-based gene editing, is mechanistically linked with the indel repair outcome. The method relies on digestion of genomic DNA by Cas9 and guide RNAs in ribonucleoprotein format, followed by enrichment of blunt and staggered DNA double-strand breaks generated by CRISPR nucleases at on- and off-target sequences. Next-generation sequencing and data analysis with BreakInspectoR allows high-throughput characterization of Cas nuclease activity, specificity, protospacer adjacent motif frequency and scission profile. Here we first describe a detailed BreakTag protocol for the nomination of CRISPR off-targets and multilevel characterization of engineered Cas variants and second, we describe a step-by-step protocol for data analysis using BreakInspectoR. Third, we provide a web interface for XGScission, a machine learning model amenable to training with scission-aware BreakTag data to predict the relative frequency of blunt and staggered double-strand breaks at new sequences unseen by the model. XGScission allows a preselection of target sequences predicted to be cut in staggered configuration that are preferably repaired as single-nucleotide templated insertions. Furthermore, XGScisson can be used to assess sequence determinants of blunt and staggered cleavage by SpCas9 and engineered nuclease variants. As a companion strategy, we describe HiPlex for the generation of hundreds to thousands of single guide RNAs in pooled format for the production of robust BreakTag datasets. The BreakTag library preparation takes ~6 h, and the entire protocol can be completed in ~3 d, including sequencing, data analysis with BreakInspectoR and XGScission model training.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*High-Throughput Nucleotide Sequencing/methods
DNA Breaks, Double-Stranded
Humans
CRISPR-Associated Protein 9/metabolism

@article {pmid41833894,
year = {2026},
author = {Ma, Y and Liao, Y},
title = {CRISPR-mediated cancer therapies: Approaches to direct tumor targeting.},
journal = {Critical reviews in oncology/hematology},
volume = {222},
number = {},
pages = {105277},
doi = {10.1016/j.critrevonc.2026.105277},
pmid = {41833894},
issn = {1879-0461},
mesh = {Humans ; *Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Tumor Microenvironment/genetics ; Animals ; },
abstract = {CRISPR-Cas9 technologies have opened new possibilities for precision cancer treatment, addressing limitations inherent in conventional therapies such as chemotherapy and radiation. This review examines CRISPR-based strategies for direct tumor targeting, including oncogene inactivation, tumor suppressor gene reactivation, and tumor microenvironment (TME) modification. Key advances include KRAS[G12D] inactivation via base editing, in which engineered deaminases introduce precise single-nucleotide changes without generating double-strand breaks; TP53 correction through homologous recombination, which uses a donor DNA template to repair mutant sequences at the targeted locus; and CDKN2A epigenetic remodeling using CRISPR-dCas9-TET1 demethylation, where catalytically inactive Cas9 guides the TET1 demethylase to hypermethylated promoters to restore gene expression. CRISPR screening has identified synthetic lethal interactions, such as PARP1 dependency in BRCA1[-/-] tumors. TME editing strategies, including modification of cancer-associated fibroblasts, demonstrate enhanced antitumor responses. Delivery challenges are being addressed through viral vectors, including adenovirus, AAV, and lentivirus. Non-viral approaches include lipid nanoparticles, gold nanoparticles, exosomes, and stimuli-responsive systems such as MMP-cleavable and hypoxia-responsive nanoparticles. Clinical trials with CRISPR-engineered T-cells (e.g., CTX130) have demonstrated remission rates in hematologic malignancies. However, significant challenges remain, including cytokine release syndrome, immunotoxicity, tumor heterogeneity, and limited delivery efficiency in solid tumors. Overcoming these barriers requires interdisciplinary innovation, ethical oversight, and technological refinement to support the safe and effective integration of CRISPR-based strategies into precision oncology.},
}

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

@article {pmid42046341,
year = {2026},
author = {Wang, Z and Chen, Y and Wang, Y and Li, W and Zhang, X and Zhang, S},
title = {One-Pot Ligation-Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a-Powered Trimode Lateral Flow Assay for Sensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13365-13376},
doi = {10.1021/acs.analchem.5c07208},
pmid = {42046341},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Gold/chemistry ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Platinum/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of microRNAs (miRNAs) holds significant importance for the early diagnosis of cancer. Since current sensitive nucleic acid detection methods like recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/Cas12a are not suitable for detecting short-stranded miRNAs, we introduced a T4 ligase-based ligation process to the RPA-CRISPR/Cas12a system and developed a novel miRNA detection method termed ligation-RPA-CRISPR/Cas12a (LRCC). This assay utilizes a glycerol-enhanced one-pot reaction strategy combined with a lateral flow assay (LFA) to streamline the operation, minimize aerosol contamination, and improve point-of-care testing performance. Kinetic studies have shown that the catalytic efficiency of the glycerol-enhanced one-pot reaction is 3.11 and 2.09 times higher than that of the direct one-pot and stepwise methods, respectively. By synthesizing "three-in-one" Au-Pt nanostars (Au@Pt NSs) as probes and stabilizing them via "click" chemistry modification, this work enabled a trimode detection approach (colorimetric, photothermal, and surface-enhanced Raman spectroscopy (SERS)) with improved accuracy. In the experiment, tetrahedron DNAs were immobilized on the test line of the strip to enhance the capture efficiency of probes, thereby improving the detection sensitivity. The entire detection process was completed in 70 min with detection limits of 23.6 fM for colorimetric (C-LFA), 2.19 fM for photothermal (P-LFA), and 72.29 aM for SERS (S-LFA). The results demonstrate the strong practical applicability of the LRCC strategy, which plays a crucial role in miRNA-based early disease diagnosis.},
}

*MicroRNAs/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Gold/chemistry
Humans
*Recombinases/metabolism
Limit of Detection
Platinum/chemistry
*Endodeoxyribonucleases/metabolism/genetics
Metal Nanoparticles/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid42065492,
year = {2026},
author = {Han, R and Xiao, N and Wu, Z and Zhao, Y and Wang, X and Tang, X},
title = {Sensitive and Robust One-Pot RPA-CRISPR/Cas12a Assay with Elimination of cis-Cleavage.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13307-13318},
doi = {10.1021/acs.analchem.5c06674},
pmid = {42065492},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Limit of Detection ; DNA, Single-Stranded/genetics/chemistry ; *Replication Protein A/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection technology utilizing CRISPR/Cas12a is characterized by high sensitivity, portability, and efficiency, making it a prominent focus in the field of point-of-care testing (POCT). However, it still has limitations in one-pot detection systems. This study developed a one-pot assay based on CRISPR/Cas12a and RPA (11bp+9ss one-pot assay), which nearly eliminates the cis-cleavage activity of Cas12a while retaining its trans-cleavage activity. Specifically, cis-cleavage was abolished by shortening the complementary length between the crRNA and the target to maintain the double-stranded conformation at the cis-cleavage site, whereas trans-cleavage activity was preserved by using an ssDNA complementary to the remaining region. The trans-cleavage activity was applicable to targets within a 200 bp range and under suboptimal PAM conditions. Moreover, rational design of the ssDNA enables effective discrimination of single-base mutations. The 11bp+9ss one-pot assay achieved a limit of detection (LOD) of 1 × 10[0] copies/μL for various targets, demonstrating robust performance even in suboptimal RPA systems. Furthermore, the assay was successfully applied to the detection of Salmonella and Avian Leukosis Virus subgroup J (ALV-J) samples. Overall, the 11bp+9ss one-pot assay exhibits superior sensitivity and robustness, showing great potential for POCT of bacteria and viruses.},
}

*CRISPR-Cas Systems
Limit of Detection
DNA, Single-Stranded/genetics/chemistry
*Replication Protein A/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins
CRISPR-Associated Proteins