@article {pmid40399693,
year = {2026},
author = {Ishiguro, S and Ishida, K and Sakata, RC and Ichiraku, M and Takimoto, R and Yogo, R and Kijima, Y and Mori, H and Tanaka, M and King, S and Tarumoto, S and Tsujimura, T and Bashth, O and Masuyama, N and Adel, A and Toyoshima, H and Seki, M and Oh, JH and Archambault, AS and Nishida, K and Kondo, A and Kuhara, S and Aburatani, H and Klein Geltink, RI and Yamamoto, T and Shakiba, N and Takashima, Y and Yachie, N},
title = {A multi-kingdom genetic barcoding system for precise clone isolation.},
journal = {Nature biotechnology},
volume = {44},
number = {4},
pages = {616-629},
pmid = {40399693},
issn = {1546-1696},
mesh = {Humans ; Animals ; Mice ; HEK293 Cells ; *DNA Barcoding, Taxonomic/methods ; Clone Cells ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Single-Cell Analysis ; Genes, Reporter ; },
abstract = {Cell-tagging strategies with DNA barcodes have enabled the analysis of clone size dynamics and clone-restricted transcriptomic landscapes in heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here we present a multi-kingdom genetic barcoding system, CloneSelect, which enables a target cell clone to be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first stably tagged with DNA barcodes and propagated so that their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored during the experiment using CRISPR base editing. CloneSelect is scalable and compatible with single-cell RNA sequencing. We demonstrate the versatility of CloneSelect in human embryonic kidney 293T cells, mouse embryonic stem cells, human pluripotent stem cells, yeast cells and bacterial cells.},
}

Humans
Animals
Mice
HEK293 Cells
*DNA Barcoding, Taxonomic/methods
Clone Cells
Gene Editing/methods
CRISPR-Cas Systems/genetics
Single-Cell Analysis
Genes, Reporter

@article {pmid40624252,
year = {2026},
author = {Wu, Y and Xiao, YL and Tang, W},
title = {High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase.},
journal = {Nature biotechnology},
volume = {44},
number = {4},
pages = {587-601},
pmid = {40624252},
issn = {1546-1696},
support = {RSG-22-043-01-ET//American Cancer Society (American Cancer Society, Inc.)/ ; SSP-2021-113//Kinship Foundation/ ; R21GM141670//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2022-74685//David and Lucile Packard Foundation (David & Lucile Packard Foundation)/ ; R21 GM141670/GM/NIGMS NIH HHS/United States ; },
mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; Escherichia coli/genetics/enzymology ; Humans ; *Adenosine Deaminase/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; },
abstract = {Base editors (BEs), covalent fusions of a cytosine or adenine deaminase with a nuclease-impaired CRISPR protein, mediate site-specific conversion of C:G to T:A (CBEs) or A:T to G:C (ABEs) in the genome. Existing BEs modify all cytosines or adenines within the editing window, which limits their precision. Here we engineer nucleotide and context specificity of the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to pinpoint cytosine editing. Strategically sampling multiple nucleic-acid-recognition hotspots through directed evolution, we develop 16 TadA-derived NCN-specific deaminases that cover every possible -1 and +1 context for a target cytosine, providing on-demand deaminase choices for editor customization. We apply these variants to (1) correct disease-associated T:A-to-C:G transitions documented by ClinVar, achieving greater accuracy than conventional CBEs in 81.5% of cases, and (2) model two cancer-driver mutations-KRAS[G12D] (ACC) and TP53[R248Q] (CCG)-in vitro. Our approach offers a general strategy to access precise base editors for potential clinical applications.},
}

*Cytosine/metabolism
*Gene Editing/methods
Escherichia coli/genetics/enzymology
Humans
*Adenosine Deaminase/genetics/metabolism
CRISPR-Cas Systems/genetics
Mutation

@article {pmid40883228,
year = {2026},
author = {Nazarov, A and Partosh, T and Krsticevic, F and Rallis, D and Arien, Y and Ostrovsky, G and Kramer, RM and Halon, E and Handler, AM and Baxter, SW and Gazit, Y and Mathiopoulos, KD and Pines, G and Papathanos, PA},
title = {CRISPR/Cas9-mediated mutagenesis of the white-eye gene in the tephritid pest Bactrocera zonata.},
journal = {Insect science},
volume = {33},
number = {2},
pages = {476-490},
doi = {10.1111/1744-7917.70150},
pmid = {40883228},
issn = {1744-7917},
support = {//US-Israel Binational Agricultural Research and Development Fund - BARD/ ; //Hebrew University of Jerusalem and Zelman Cowen Academic Initiatives - ZCAI/ ; //HORIZON-CL6-2021-FARM2FORK-01-04/ ; },
mesh = {Animals ; *Tephritidae/genetics ; *CRISPR-Cas Systems ; Male ; Gene Editing ; Mutagenesis ; *Insect Proteins/genetics/metabolism ; },
abstract = {Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops. The Sterile Insect Technique (SIT), a species-specific and environmentally friendly pest control method, significantly benefits from the availability of Genetic Sexing Strains (GSSs) that enable efficient mass production of males for sterile release. However, no GSS currently exists for B. zonata limiting SIT applications targeting this important invasive pest. Here, we report two key advancements toward GSS development in this species. First, we present a high-quality, chromosome-level genome assembly from male B. zonata, identifying two scaffolds derived from the Y chromosome, which represent potential targets for future male-specific genetic engineering. Second, we demonstrate the feasibility of CRISPR/Cas9 genome editing in B. zonata by generating stable, homozygous white-eye mutants through targeted disruption of the conserved white-eye gene. This visible, recessive phenotype serves as a proof-of-concept for developing selectable markers in this species. Together, these results provide foundational genomic and genetic tools to support the development of GSSs in B. zonata, advancing the potential for sustainable, genetics-based pest control strategies.},
}

Animals
*Tephritidae/genetics
*CRISPR-Cas Systems
Male
Gene Editing
Mutagenesis
*Insect Proteins/genetics/metabolism

@article {pmid40883657,
year = {2026},
author = {Okada, A and Okamoto, M and Nguyen, TNM and Fung, E and Nguyen, H and Crisp, P and Choo, A and Baxter, SW},
title = {The shibire[ts4] mutation causes temperature sensitive paralytic and lethal phenotypes in the Queensland fruit fly, Bactrocera tryoni.},
journal = {Insect science},
volume = {33},
number = {2},
pages = {533-546},
doi = {10.1111/1744-7917.70148},
pmid = {40883657},
issn = {1744-7917},
support = {D44003//International Atomic Energy Agency Coordinated Research Project/ ; HG14033//International Atomic Energy Agency Coordinated Research Project/ ; //This project was part of the National SITplus program and supported by Horticulture Innovation using the research and development levy funds from the vegetable, apple and pear, citrus, strawberry, table grape, cherry and summerfruit industries, with co-investment from South Australian Research and Development Institute (SARDI) and Primary Industries and Regions South Australia (PIRSA), and contributions from the Australian Government./ ; },
mesh = {Animals ; *Tephritidae/genetics/growth & development/physiology ; Female ; Male ; Phenotype ; Larva/genetics/growth & development ; *Insect Proteins/genetics/metabolism ; Mutation ; Temperature ; Pupa/growth & development/genetics ; CRISPR-Cas Systems ; },
abstract = {Bactrocera tryoni, the Queensland fruit fly, is among the most damaging insect pests to the Australian horticultural industry as larvae infest ripening fruits or vegetables prior to harvest. Genetic biocontrol using Sterile Insect Technique (SIT) programs have been used to successfully suppress populations, via mass release of factory-reared sterile males that mate with wild females. Bi-sex flies are currently used for releases, although the efficiency of these control programs could be improved through using genetic sexing strains that eliminate females early during development, as they are not required for SIT. Here we used CRISPR/Cas9 mutagenesis to modify two nucleotides in the B. tryoni gene shibire, which created a proline to serine amino acid substitution and produced a temperature sensitive phenotype. Shibire is an essential GTPase required in endocytosis and synaptic vesicle recycling, and classical mutagenic screens in the vinegar fly Drosophila melanogaster previously identified temperature sensitive alleles including shi[ts4] that results in adult paralysis. In B. tryoni, the shi[ts4] mutant strain exhibited similar adult paralytic phenotypes when exposed to high temperatures, as well as temperature dependent lethality at egg, larval and pupal stages when subjected to heat treatment above standard rearing temperatures. These temperature sensitive phenotypes could be adapted to develop a SIT genetic sexing strain for conditional elimination of females prior to sterile releases, to improve efficiency and reduce costs.},
}

Animals
*Tephritidae/genetics/growth & development/physiology
Female
Male
Phenotype
Larva/genetics/growth & development
*Insect Proteins/genetics/metabolism
Mutation
Temperature
Pupa/growth & development/genetics
CRISPR-Cas Systems

@article {pmid41235931,
year = {2026},
author = {Ioannidou, C and Gregoriou, ME and Schetelig, MF and Drosopoulou, E and Mathiopoulos, KD and Bourtzis, K},
title = {CRISPR/Cas9-based white pupae mutant lines in Bactrocera spp. for sterile insect technique applications.},
journal = {Insect science},
volume = {33},
number = {2},
pages = {463-475},
doi = {10.1111/1744-7917.70190},
pmid = {41235931},
issn = {1744-7917},
support = {//Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture/ ; 101059523//European Union´s Horizon Europe Research and Innovation Program REACT/ ; //Two postgraduate programs of the Department of Biochemistry and Biotechnology of the University of Thessaly ("Biotechnology-Quality Assessment in Nutrition and the Environment" and "Applications of Molecular Biology-Genetics-Diagnostic Biomarkers")/ ; //"Synthetic Biology: from omics technologies to genomic engineering (OMIC-ENGINE)" (MIS 5002636) which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure", funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund)/ ; },
mesh = {Animals ; *Tephritidae/genetics/growth & development ; *CRISPR-Cas Systems ; Male ; *Pest Control, Biological/methods ; Pupa/genetics/growth & development ; Female ; Gene Editing ; },
abstract = {The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae. The sterile insect technique (SIT) is a biological control method used to suppress populations of the invasive Bactrocera fruit flies by releasing sterilized male insects that compete with wild males for mates, reducing reproduction and eventually pest numbers. The effectiveness of the SIT against insect pests can be enhanced through male-only releases, achieved via genetic sexing strains (GSS) that enable early-stage sex separation. To overcome limitations faced when developing a new GSS through the classical genetic approach, a novel "neoclassical approach" has been proposed, focusing on the identification of genetic markers, the induction of desired phenotypes through genome editing, and the linkage of selectable markers to male sex. In this study, we evaluated the white pupae gene as a selectable marker for GSS development in 3 Bactrocera species. The white pupae orthologous genes have been identified, and, through CRISPR/Cas9 mutagenesis, the 3rd exon of the white pupae gene was knocked out resulting in white pupae lines in Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae species. These results demonstrate the applicability of CRISPR/Cas9-mediated disruption of the conserved white pupae gene as a selectable marker in multiple Bactrocera species, supporting the development of genetic sexing systems for SIT-based pest management.},
}

Animals
*Tephritidae/genetics/growth & development
*CRISPR-Cas Systems
Male
*Pest Control, Biological/methods
Pupa/genetics/growth & development
Female
Gene Editing

@article {pmid41292010,
year = {2026},
author = {Chen, C and Kassim, Y and Xu, X and He, X and Lin, H and Dang, Y and Shi, Y and Wang, H and Wang, S and Zhang, K},
title = {Gene editing and multi-omics approaches to study early embryogenesis in cattle.},
journal = {Reproduction, fertility, and development},
volume = {38},
number = {1},
pages = {},
doi = {10.1071/RD25167},
pmid = {41292010},
issn = {1448-5990},
mesh = {Animals ; Cattle/embryology ; *Gene Editing/veterinary ; *Embryonic Development/genetics ; Proteomics ; *Metabolomics/methods ; Genomics/methods ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Single-Cell Analysis ; Multiomics ; },
abstract = {Advancements in multi-omics profiling and targeted molecular functional tools have led to significant progress in our understanding of early embryonic development in mammals. This is very relevant in the beef and dairy industries for exploring the etiology of early embryo loss. This review highlights how state-of-the-art single-cell and integrative low-input omics technologies, including single-cell RNA-seq, ATAC-seq, metabolomics, and proteomics, have uncovered complex developmental dynamics during the first week of bovine embryogenesis. This review also provides a concurrent overview of the application of functional tools, including CRISPR-Cas9, RNA editing, base editing, and Trim-Away, in the analysis of critical genes/proteins during the various stages of early embryo development in cattle. The integration of high-dimensional molecular profiling with targeted manipulations enables researchers to analyze key developmental events with unparalleled resolution. It is evident that these approaches provide a more comprehensive mechanistic depiction of early embryogenesis, thereby informing translational efforts in improving bovine fertility.},
}

Animals
Cattle/embryology
*Gene Editing/veterinary
*Embryonic Development/genetics
Proteomics
*Metabolomics/methods
Genomics/methods
CRISPR-Cas Systems
Gene Expression Regulation, Developmental
Single-Cell Analysis
Multiomics

@article {pmid41808208,
year = {2026},
author = {Zhang, Y and Wang, M and Bi, C and Li, M},
title = {Targeted native long-read sequencing of DNA methylation alterations following CRISPR-Cas9-induced double-strand breaks in human cells.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {41808208},
issn = {1756-0500},
support = {BAS/1/1080-01-01//King Abdullah University of Science and Technology/ ; },
mesh = {Humans ; *DNA Methylation/genetics ; *CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; 5-Methylcytosine/analogs & derivatives/metabolism ; Epigenesis, Genetic ; Sequence Analysis, DNA/methods ; Nanopore Sequencing ; },
abstract = {OBJECTIVES: CRISPR-Cas9 nucleases are widely used to introduce targeted DNA double-strand breaks (DSBs) for genome engineering, but the long-term impact of these lesions on local epigenetic information remains poorly characterized. In a companion research article, we used Cas9-assisted targeted nanopore sequencing (CTS) to reveal that CRISPR-Cas9-induced DSBs can disrupt local epigenetic maintenance across multiple genomic contexts and cell systems. Here, we present a structured description of the raw and minimally processed datasets underlying the study. These datasets provide base-resolution measurements of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at the differentially methylated regions (DMRs) of several imprinted loci, two heterochromatic regions, a cancer-associated promoter epimutation region, and the SNRPN DMR at early/late passages of a clonal line. They enable re-analysis and methodological benchmarking of DSB-associated epigenetic instability.

DATA DESCRIPTION: We provide aligned BAM files and per-CpG methylation calls for multiple genomic contexts under both CRISPR-targeted and non-targeting control conditions. Specifically, the collection includes: (i) imprinted loci in human embryonic stem cells (hESCs), including small nuclear ribonucleoprotein polypeptide N (SNRPN), paternally expressed 10 (PEG10), and KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), (ii) heterochromatic regions in hESCs, including urothelial cancer associated 1 (UCA1), and cysteine rich C-terminal 1 (CRCT1)), (iii) the epimutation locus of MutL homolog 1 (MLH1) in RKO cells, and (iv) the DMR of SNRPN locus in early- and late-passage derivatives of a single hESC clone. For each collection, there is a dataset that includes both the raw aligned Nanopore sequencing reads (BAM) deposited in the NCBI Sequence Read Archive (SRA) and the corresponding processed per-CpG 5mC/5hmC matrices deposited in Zenodo. All higher-level analyses in the research article-such as DMR calling, haplotype-resolved analyses, and structural variant (SV) characterization-are fully reproducible using these deposited data. Additional processed analyses are comprehensively documented in the companion article and are therefore not duplicated here. Together, these datasets offer a rich resource for benchmarking long-read methylation analysis workflows and further investigation of DSB-associated epigenetic instability across diverse genomic contexts.},
}

Humans
*DNA Methylation/genetics
*CRISPR-Cas Systems/genetics
*DNA Breaks, Double-Stranded
5-Methylcytosine/analogs & derivatives/metabolism
Epigenesis, Genetic
Sequence Analysis, DNA/methods
Nanopore Sequencing

@article {pmid41834297,
year = {2026},
author = {Zhu, L and Xiong, W and Yang, S and Qi, Q and Liu, X and Zhou, X and Tian, T},
title = {Dynamic Control of RNA Structure and Function through Bioorthogonal Staudinger Chemistry.},
journal = {ACS chemical biology},
volume = {21},
number = {4},
pages = {835-843},
doi = {10.1021/acschembio.6c00067},
pmid = {41834297},
issn = {1554-8937},
mesh = {*RNA/chemistry/metabolism ; Azides/chemistry ; Humans ; CRISPR-Cas Systems ; Nucleic Acid Conformation ; *Imidazoles/chemistry ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/chemistry ; },
abstract = {Here, we report a reversible chemical strategy for regulating RNA function through a Staudinger reaction-mediated postsynthetic modification. We designed a bifunctional azide reagent, 1,3-diazidopropan-2-yl 1H-imidazol-1-carboxylate (DAPIC), which specifically modifies the 2'-hydroxyl of RNA, thereby disrupting RNA structure and function. Treatment with 2-diphenylphosphinoethylamine (DPPEA) reactivates the modified RNA through an efficient Staudinger reduction. This approach enables reversible modulation of RNA folding, hybridization, and protein-binding interactions, and can be applied to guide RNAs in the CRISPR-Cas9 system. DAPIC modification completely abrogates Cas9-mediated DNA cleavage, which is restored in a DPPEA concentration-dependent manner both in vitro and in living cells. Compared with monoazide derivatives, DAPIC exhibits enhanced reactivity and reduced reagent requirements. This Staudinger-based RNA regulation platform establishes a robust and generalizable chemical tool for conditional gene editing and studies of RNA function in complex biological environments.},
}

*RNA/chemistry/metabolism
Azides/chemistry
Humans
CRISPR-Cas Systems
Nucleic Acid Conformation
*Imidazoles/chemistry
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/chemistry

@article {pmid41875383,
year = {2026},
author = {Wootan, CM and Lutterman, J and Springer, N and Xu, X and Zhang, F},
title = {DNA repair under heat: DNA polymerase λ modulates heat stress-induced mutagenesis in plants.},
journal = {The Plant cell},
volume = {38},
number = {4},
pages = {},
doi = {10.1093/plcell/koag090},
pmid = {41875383},
issn = {1532-298X},
support = {//University of Minnesota Multifunctional Agriculture Initiative/ ; IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; //University of California, Davis/ ; CA-D-PLB-2850-H//California Agricultural Experiment Station/National Institute of Food and Agriculture/ ; },
mesh = {*DNA Repair/genetics ; *Heat-Shock Response/genetics ; *Mutagenesis/genetics ; *Arabidopsis/genetics/physiology ; *DNA Polymerase beta/metabolism/genetics ; Hot Temperature ; Gene Expression Regulation, Plant ; *Arabidopsis Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Mutation rates often rise under environmental stress, a process known as stress-induced mutagenesis. Among abiotic factors, heat stress is a potent driver that elevates mutation rates and enhances genetic variation, yet the underlying mechanisms in eukaryotes remain unclear. Here, we investigated how heat stress influences DNA repair and mutagenesis both locally and globally in Arabidopsis thaliana using CRISPR-Cas9-induced DNA breaks, whole genome sequencing, and single-cell transcriptomic analysis. Heat stress greatly enhanced CRISPR editing efficiency across different chromatin contexts, reaching up to a 29.9-fold increase in heterochromatic regions. Moreover, heat stress consistently shifted repair outcomes toward one base pair insertions, independent of chromatin state. We identified a heat-inducible, error-prone DNA polymerase, DNA polymerase λ (DNA Polλ), as the key mediator of these repair profile changes. Genome-wide analyses of somatic mutations further revealed that the heat-induced increase in mutagenesis also depends on DNA Polλ. Single-cell transcriptomic profiling showed that DNA Polλ expression is tightly regulated and enriched in the central zone of the shoot apical meristem. Such tissue-specific patterns suggest that DNA Polλ serves dual functions: maintaining genome integrity in essential stem cell populations while also enabling stress-induced mutagenesis that can be transmitted to progeny.},
}

*DNA Repair/genetics
*Heat-Shock Response/genetics
*Mutagenesis/genetics
*Arabidopsis/genetics/physiology
*DNA Polymerase beta/metabolism/genetics
Hot Temperature
Gene Expression Regulation, Plant
*Arabidopsis Proteins/metabolism/genetics
CRISPR-Cas Systems/genetics

@article {pmid41911342,
year = {2026},
author = {Chi, H and McMahon, SA and Graham, S and White, MF},
title = {The CRISPR ring nuclease Csx15 oligomerises on cyclic nucleotide binding to regulate antiviral defence.},
journal = {The Biochemical journal},
volume = {483},
number = {5},
pages = {699-712},
doi = {10.1042/BCJ20260019},
pmid = {41911342},
issn = {1470-8728},
support = {101018608//EC | European Research Council (ERC)/ ; },
mesh = {Protein Multimerization ; *Nucleotides, Cyclic/metabolism/chemistry ; Protein Binding ; *Adenine Nucleotides/metabolism/chemistry ; Binding Sites ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Oligoribonucleotides ; },
abstract = {Prokaryotic type III CRISPR systems signal infection by generating cyclic oligoadenylate (cOA) second messengers, which activate defence proteins allosterically, providing immunity. cOA molecules are typically degraded by extrinsic, stand-alone ring nuclease (RN) enzymes with phosphodiesterase activity or by the intrinsic RN activity of the effectors themselves. Viruses and plasmids also encode RNs, which can function as anti-CRISPRs. Eight different families of extrinsic RNs are currently known. Here, we report the structural and biochemical analysis of one of these families: Csx15. We show that Csx15 is a dimeric protein of the CRISPR-associated Rossmann fold (CARF) superfamily with the ability to bind cyclic tetra-adenylate (cA4) molecules in a shared binding site formed by the head-to-tail stacking of dimers in a filament conformation. Some family members are non-enzymatic, relying on the sequestration (sponging) of cA4 to regulate the host immune response, while others act as canonical RNs, slowly degrading cA4.},
}

Protein Multimerization
*Nucleotides, Cyclic/metabolism/chemistry
Protein Binding
*Adenine Nucleotides/metabolism/chemistry
Binding Sites
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
CRISPR-Cas Systems
Oligoribonucleotides

@article {pmid41928679,
year = {2026},
author = {Li, D and Feng, J and Wang, X and Zhao, E and Liang, Y and Li, K and Li, Y},
title = {An Anaerobic Fluorescent Reporter System and CRISPR-Cas12a Enable High-Throughput Metabolic Engineering of Clostridium butyricum.},
journal = {ACS synthetic biology},
volume = {15},
number = {4},
pages = {1669-1680},
doi = {10.1021/acssynbio.6c00158},
pmid = {41928679},
issn = {2161-5063},
mesh = {*Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium butyricum/genetics/metabolism ; Anaerobiosis ; Gene Editing/methods ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Genes, Reporter ; High-Throughput Screening Assays/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Clostridium butyricum is an important probiotic and industrial organism with significant potential for anaerobic bioproduction. However, the lack of efficient genetic tools, particularly for high-throughput screening under strict anaerobic conditions, has hindered its metabolic engineering. To address this, we first established a highly efficient conjugation method, significantly improving exogenous DNA transformation efficiency. Leveraging time-resolved transcriptomic data, we then mapped the dynamic activity of native promoters and developed a robust anaerobic fluorescent protein reporter system. This system overcomes the oxygen-dependent limitation of traditional reporters like GFP, enabling high-throughput and quantitative screening of promoter strength in live anaerobes. Furthermore, we constructed a CRISPR-Cas12a-based genome editing platform for scarless gene manipulation and a two-plasmid curing strategy to generate markerless and genetically stable engineered strains. The power of this integrated toolkit was demonstrated by engineering the acetaldehyde metabolic pathway. Overexpression of aldehyde dehydrogenase (ALDH) resulted in a 79.29% increase in enzyme activity, indicating an enhanced catalytic capacity for acetaldehyde oxidation. This proof-of-concept module, together with the anaerobic fluorescent reporter and CRISPR-Cas12a platform, supports a streamlined workflow for genetic part characterization and metabolic engineering in C. butyricum under strict anaerobic conditions.},
}

*Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
*Clostridium butyricum/genetics/metabolism
Anaerobiosis
Gene Editing/methods
Plasmids/genetics/metabolism
Promoter Regions, Genetic/genetics
Green Fluorescent Proteins/genetics/metabolism
Genes, Reporter
High-Throughput Screening Assays/methods
Bacterial Proteins/genetics/metabolism

@article {pmid41968243,
year = {2026},
author = {Valero, C and Tindale, J and Bromley, MJ and van Rhijn, N},
title = {Marker-Free CRISPR/Cas9 RNP Transformation for Gene Disruption in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {23-30},
pmid = {41968243},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; Genetic Engineering/methods ; },
abstract = {CRISPR-based genetic engineering has resulted in the possibility of far more efficient and challenging genetic engineering in filamentous fungal pathogens. In this respect, selection marker-free CRISPR-Cas9 transformation has been possible in fungi such as Aspergillus fumigatus, one of the most prevalent fungal pathogens of humans. Here, we describe a protocol to perform this technique, which can be widely adapted to use multiple strains and other species.},
}

*Aspergillus fumigatus/genetics
*CRISPR-Cas Systems/genetics
*Transformation, Genetic
Gene Editing/methods
*Ribonucleoproteins/genetics
Genetic Engineering/methods

@article {pmid41968244,
year = {2026},
author = {Guo, Y and Scharf, DH},
title = {CRISPR-Cas9 and Microhomology-Mediated End-Joining for Biosynthetic Pathway Reconstruction in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {31-45},
pmid = {41968244},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Biosynthetic Pathways/genetics ; Plasmids/genetics ; *DNA End-Joining Repair ; Genetic Vectors/genetics ; Gene Editing/methods ; },
abstract = {We have recently engineered Aspergillus fumigatus using an AMA1-based episomal expression CRISPR vector and a microhomology-mediated end-joining repair system. We adopted A. fumigatus as a heterologous expression host to investigate the biosynthetic pathways of epipolythiodioxopiperazine-type compounds. In the present chapter, we describe the assembly of the CRISPR-Cas9 vector and microhomology template plasmid, their transformation into A. fumigatus, and metabolite detection.},
}

*Aspergillus fumigatus/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Biosynthetic Pathways/genetics
Plasmids/genetics
*DNA End-Joining Repair
Genetic Vectors/genetics
Gene Editing/methods

@article {pmid41968255,
year = {2026},
author = {Campanella, JEM and Arentshorst, M and Ram, AFJ and van den Hondel, CAMJJ and Malavazi, I},
title = {Molecular Biosensing of the Aspergillus fumigatus Cell Wall Integrity Pathway Using the (p)agsA::luc (Luciferase) Reporter System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {199-223},
pmid = {41968255},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/metabolism ; *Cell Wall/metabolism/genetics ; *Biosensing Techniques/methods ; *Genes, Reporter ; *Luciferases/genetics/metabolism ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Promoter Regions, Genetic ; Luminescent Measurements ; Gene Expression Regulation, Fungal ; },
abstract = {In this protocol, we describe a robust luciferase-based biosensor assay to monitor the activity of the cell wall integrity (CWI) pathway in Aspergillus fumigatus in real time. The method relies on the stable integration of a markerless, synthetic reporter cassette (p)agsA::luc at the aft4 Safe Haven (Sh) genomic locus using CRISPR-Cas9. This cassette comprises a modified A. niger agsA promoter containing three tandem RlmA-binding sites, which drives the expression of the luciferase gene. Upon exposure to cell wall stress, the endogenous transcription factor RlmA activates the reporter, generating a luminescent signal proportional to promoter activity. The protocol includes the construction of aft4 locus-specific CRISPR-Cas9 plasmids, A. fumigatus transformation and candidate selection, as well as the setup of the luminescence bioassay in white 96-well microplates. This system enables highly sensitive, nondestructive, and time-resolved quantification of CWI pathway activation during early fungal growth or biofilm under various genetic or chemical perturbations. Moreover, it supports comparative studies across wild-type and mutant strains, offering a powerful platform for dissecting stress response signaling and identifying antifungal compounds that target the CWI pathway.},
}

*Aspergillus fumigatus/genetics/metabolism
*Cell Wall/metabolism/genetics
*Biosensing Techniques/methods
*Genes, Reporter
*Luciferases/genetics/metabolism
CRISPR-Cas Systems
Fungal Proteins/genetics/metabolism
Promoter Regions, Genetic
Luminescent Measurements
Gene Expression Regulation, Fungal

@article {pmid41968752,
year = {2026},
author = {Zhang, X and Li, X and Li, X and Peng, C and Wang, W},
title = {A Pangenome Framework Reveals Conserved Pathogenicity-Associated Biosynthetic Gene Clusters in Fusarium equiseti, a Root Rot Pathogen of Panax notoginseng.},
journal = {Molecular plant pathology},
volume = {27},
number = {4},
pages = {e70260},
pmid = {41968752},
issn = {1364-3703},
support = {2025J0492//Yunnan Provincial Department of Education Science Research Fund Project/ ; 22377105//National Natural Science Foundation of China/ ; 202401AV070003//National Nature Science Foundation of Yunnan Province/ ; 202505AF350066//Yunnan provincial science and technology talent/ ; //Xingdian Talent Support Program-Yunling Scholat (W.-G. Wang)/ ; },
mesh = {*Fusarium/genetics/pathogenicity/metabolism ; *Panax notoginseng/microbiology ; *Multigene Family/genetics ; *Plant Roots/microbiology ; *Plant Diseases/microbiology ; Genome, Fungal/genetics ; Virulence/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Panax notoginseng (Chinese ginseng, sanqi) is an economically and medicinally important herb whose cultivation is increasingly threatened by destructive soilborne root rot. Although Fusarium species represent the dominant pathogens in this disease complex, the virulence determinants underlying Fusarium-P. notoginseng interactions remain largely unknown, particularly the contribution of secondary metabolite (SM) biosynthetic gene clusters (BGCs) in medicinal plants. Here, we present a pangenome-informed and experimentally validated framework linking Fusarium SM to disease development. By constructing a genus-scale BGC landscape from 54 Fusarium genomes, integrating it with infection-stage transcriptomics of Fusarium equiseti P26, and establishing an efficient CRISPR/Cas9-based genome-editing system, we functionally prioritized and interrogated conserved, infection-induced BGCs from dozens of predicted clusters. This integrative pipeline identified two broadly conserved and infection-essential BGCs that are indispensable for disease development, including an evolutionarily conserved ICS-NRPS hybrid cluster and a ferrichrome-type siderophore NRPS cluster widely distributed across Fusarium species. Notably, the pathogenic contribution of the ICS-NRPS pathway was independently corroborated in Fusarium oxysporum, supporting a conserved role for this BGC class across the genus. Together, our findings provide the first pangenome-guided gene-to-phenotype dissection of SM-associated virulence in P. notoginseng root rot, establishing conserved SM biosynthetic backbones as causal pathogenicity determinants and tractable targets for disrupting Fusarium disease development across BGC-rich fungal genomes.},
}

*Fusarium/genetics/pathogenicity/metabolism
*Panax notoginseng/microbiology
*Multigene Family/genetics
*Plant Roots/microbiology
*Plant Diseases/microbiology
Genome, Fungal/genetics
Virulence/genetics
Biosynthetic Pathways/genetics
CRISPR-Cas Systems/genetics

@article {pmid41972732,
year = {2026},
author = {Rutkowska, A and Strózik, T and Wasiak, T and Ciunowicz, D and Kapelan, N and Szczepaniak, N and Sosnowski, J and Goślińska, W and Bartkowiak, J and Budny-Lewandowska, A and Antończyk, P and Markiewicz, M and Gustaw, P and Filiks, K and Jaskólska, M and Stoczyńska-Fidelus, E},
title = {CRISPR-Cas9 Therapeutics in Early Clinical Development: Delivery and Molecular Diagnostics.},
journal = {Cells},
volume = {15},
number = {7},
pages = {},
pmid = {41972732},
issn = {2073-4409},
support = {2024/ABM/05/KPO/KPOD.07.07-IW.07-0242/24-00//LEK-AM Pharmaceutical Company Ltd/ ; 503/0-154-01/503-01-001//Medical University of Lodz/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; *Pathology, Molecular/methods ; *Genetic Therapy/methods ; *Molecular Diagnostic Techniques ; *Gene Transfer Techniques ; },
abstract = {CRISPR-Cas9 has progressed from an experimental tool to a therapeutic modality, marked by the first regulatory approvals of an ex vivo-edited autologous CD34+ hematopoietic stem cell product that induces fetal hemoglobin (CASGEVY/exa-cel). In this narrative review, we synthesize modality-specific molecular diagnostic strategies used across early CRISPR clinical translation. In parallel, early clinical experience has begun to demonstrate the feasibility of in vivo editing, including subretinal delivery for CEP290-associated inherited retinal degeneration (EDIT-101 programme) and hepatocyte-targeted lipid nanoparticles (LNPs) for liver-derived targets such as transthyretin and plasma prekallikrein (KLKB1). As translation expands across hematologic, metabolic, ocular and oncology indications, development is increasingly constrained by the predictability and safety of editing outcomes, delivery-determined biodistribution and exposure time, and immune recognition of bacterial Cas9 orthologs and delivery components. We summarize diagnostic readouts for confirming patient genotype, quantifying on-target editing and expression changes, assessing off-target and structural outcomes using orthogonal assays, and monitoring clonal dynamics and immune responses during long-term follow-up. We also discuss how these readouts interface with CMC controls and regulatory expectations for advanced therapy medicinal products (ATMPs), highlighting the need for fit-for-purpose, standardized testing frameworks in early trials.},
}

Humans
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Animals
*Pathology, Molecular/methods
*Genetic Therapy/methods
*Molecular Diagnostic Techniques
*Gene Transfer Techniques

@article {pmid41972830,
year = {2026},
author = {Chokwassanasakulkit, T and Ranasinghe, V and Woods, E and Nguyen, LQ and McMillan, NAJ},
title = {What If Trojan Horse Nanoparticles Could Change the Game for HPV Gene-Targeted Therapies?.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70916},
pmid = {41972830},
issn = {1096-9071},
support = {2027649//National Health and Medical Research Council (NHMRC)/ ; 2027569//National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Nanoparticles/administration & dosage ; *Papillomavirus Infections/therapy/virology ; *Genetic Therapy/methods ; Uterine Cervical Neoplasms/therapy/virology ; Female ; RNA, Small Interfering/administration & dosage/genetics ; *Papillomaviridae/genetics ; Animals ; CRISPR-Cas Systems ; },
abstract = {Human papillomavirus (HPV) is a common sexually transmitted infection linked to various cancers, particularly cervical cancer, primarily driven by high-risk strains like HPV16 and HPV18. While vaccines are effective in preventing new infections, they do not address existing cases, highlighting the need for innovative therapies. Gene-targeted approaches, such as CRISPR/Cas and siRNA, show promise in inhibiting HPV oncogenes. Recent advancements in Trojan horse nanoparticles (NPs) offer a strategy for delivering these therapies directly to HPV-infected cells. These NPs improve stability and targeted delivery, enhancing the biodistribution of CRISPR/Cas systems and siRNAs while protecting them from degradation. However, challenges like immune responses and regulatory hurdles persist. Therefore, this review emphasizes the potential of Trojan horse NPs in treating HPV-related cancers, identifies critical areas for future research, and provides updates on gene-targeted therapy encapsulated NPs in preclinical and clinical trials.},
}

Humans
*Nanoparticles/administration & dosage
*Papillomavirus Infections/therapy/virology
*Genetic Therapy/methods
Uterine Cervical Neoplasms/therapy/virology
Female
RNA, Small Interfering/administration & dosage/genetics
*Papillomaviridae/genetics
Animals
CRISPR-Cas Systems

@article {pmid41973392,
year = {2026},
author = {Shojaei Baghini, S and Esfahani, K and Rad, N and Arezoumandi, M and Taghipour, E and Salmanian, AH},
title = {Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {41973392},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Araceae/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Plants, Genetically Modified/genetics/growth & development ; RNA, Transfer/genetics ; },
abstract = {Lemna minor (commonly known as duckweed) is a fast-growing aquatic plant recognized as a promising green bioreactor for recombinant protein production. Its rapid proliferation, high protein yield, environmental adaptability, and edibility make it highly attractive for biotechnological applications. It is essential to develop and expand genetic tools tailored to this species to maximize these advantages and further unlock its biotechnological potential. A key strategy for achieving this goal is the implementation of advanced genome editing technologies, such as the CRISPR/Cas9 system. Although multiplex CRISPR/Cas9 gene editing has previously been successfully applied in Lemna aequinoctialis, the capability of the endogenous plant tRNA processing system for multiplex editing in L. minor using the polycistronic tRNA-sgRNA (PTG)/Cas9 system has not yet been explored. In this study, a PTG construct was engineered to include four sgRNAs designed to simultaneously target two plant-specific glycosyltransferase genes: α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT). As anticipated, the PTG-Cas9 system successfully induced frameshift mutations, characterized by insertions and deletions (indels), in regenerated L. minor plants derived from transformed calli. Validation via PCR and RT-PCR analysis, followed by sequencing of the target loci, confirmed the presence of indels at the target sites. Furthermore, western blot analyses utilizing antibodies specific to XylT and FucT in two homozygous lines (lines 44 and 217) revealed truncated XylT proteins in both lines. Moreover, an in-frame FucT protein was detected in line 217, whereas FucT expression was absent in line 44. This study marked the first successful demonstration of PTG-Cas9 system for multiplex genome editing in L. minor, paving the way for advanced genetic engineering in this species.},
}

*CRISPR-Cas Systems/genetics
*Araceae/genetics
*Gene Editing/methods
*Gene Knockout Techniques/methods
*Plants, Genetically Modified/genetics/growth & development
RNA, Transfer/genetics

@article {pmid41973928,
year = {2026},
author = {Lian, G and Khabazeh, A and Sheen, V},
title = {A modified CRISPR/Cas9 approach in silencing the triplication in Down syndrome: A treatment path XISTs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {16},
pages = {e2517953123},
doi = {10.1073/pnas.2517953123},
pmid = {41973928},
issn = {1091-6490},
support = {1R01HD109794-01//Foundation for the NIH (FNIH)/ ; 5R21NS115593-02//Foundation for the NIH (FNIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Long Noncoding/genetics ; *Down Syndrome/genetics/therapy ; Gene Silencing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {Down syndrome (DS) is one of the most common developmental human genetic disorders and is due to triplication of chromosome 21 (HSA21). Although previous studies using epigenetic suppression of HSA21 by the long noncoding RNA XIST showed a potential for DS treatment, integration efficiency of XIST by conventional zinc finger nucleases is too low to allow for practical implementation. Here, we report a modified CRISPR/Cas9 approach, which enhances the efficiency of XIST gene integration. First, a codon-optimized λ-phage exonuclease (exo) was fused with Cas9 to create 5'- and 3'-end overhangs at cutting sites of donor DNA and acceptor chromosome DNA. Second, four sgRNAs, two of which selectively targeted each the acceptor or donor DNA, were assembled tandemly into one Cas9 plasmid (PX459) to increase the Cas9-cutting efficiency and promote donor DNA integration. Third, sgRNAs were designed by searching for unique single nucleotide polymorphism nucleotides distinct between the three HSA21 copies, as a protospacer adjacent motif site to specifically target one HSA21 copy. Fourth, donor DNA plasmid containing XIST was modified to disable replication and inhibit transcription function and allow for inducible expression. Our modified CRISPR method significantly enhanced the integration efficiency (20 to 40%) of long XIST gene (14 kb) into an extra chromosome 21 (HSA21), as was identified with PCR, cell cloning, immunostaining, and FISH. RNA sequencing results showed that imbalance of gene transcription across extra HSA21 can be partially corrected by XIST gene integration. The modified CRISPR method with XIST paves a road for therapeutic treatment for DS.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA, Long Noncoding/genetics
*Down Syndrome/genetics/therapy
Gene Silencing
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
Genetic Therapy/methods

@article {pmid41974871,
year = {2026},
author = {Vitiello, A and Boccellino, M and Zovi, A and Bassetti, M},
title = {Antimicrobial resistance and gene therapy: emerging molecular strategies for a global health threat.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {41974871},
issn = {1476-5462},
abstract = {Antimicrobial resistance (AMR) is one of the most serious and pressing health challenges facing modern medicine. Despite advances in antimicrobial stewardship, diagnostics and infection prevention, the rapid emergence and spread of resistant pathogens continues to limit treatment options and increase morbidity, mortality and healthcare costs. The discovery of new innovative antimicrobial therapies remains of paramount importance. In this context, gene therapy is gaining attention as a complementary strategy that can directly target the molecular genetic determinants of antimicrobial resistance. Recent advances in RNA-based technologies and CRISPR-Cas systems have enabled increasingly precise manipulation of microbial genomes, opening up the possibility of restoring antimicrobial susceptibility, reducing virulence and limiting the spread of resistance genes.},
}

@article {pmid41977119,
year = {2026},
author = {Abubakar, S and Abdulsalam, L and Fatty, L and Kanwal, R and Naeem, M and Ahmad, I},
title = {Emerging CRISPR Approaches for Countering Immune Evasion: Insight from Recent Studies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977119},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology/genetics ; Gene Editing/methods ; Immunotherapy/methods ; *Tumor Escape/genetics ; Animals ; *Immune Evasion/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {Cancer immunotherapy has recently become an essential approach for treating cancer, showing considerable promise as a substitute for surgery, radiation therapy, and conventional chemotherapy. It primarily aims to boost the host's natural defense system to combat cancer malignancies by utilizing components of immune checkpoint blockades (ICBs), mainly programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), along with elements of adoptive cellular therapies (ACTs) like Chimeric Antigen Receptor (CAR) therapy, T Cell Receptor (TCR) therapy and Tumor-Infiltrating Lymphocyte (TIL) therapy. However, cancer cells tend to undermine the effectiveness of cancer immunotherapeutic strategies by employing one or more immune evasion mechanisms. This review briefly highlights how key mechanisms of cancer immune evasion confer resistance to immunotherapy and how the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR)/Cas9 systems, as gene-editing tools, are poised to enhance cancer immunotherapy for treating challenging cancers. We emphasize that (CRISPR/Cas9) systems can be used to explore and positively alter the genes of the immune system, boosting the effectiveness of cancer immunotherapy by editing immune checkpoints, TILs, and CAR-T cells, and disrupting genes, facilitating tumors' evasion of the immune system. Furthermore, we highlight the growing interest in emerging base editor technology to engineer natural killer (NK) cells to overcome NK-cell-based immunotherapy challenges, particularly human leukocyte antigens (HLA)-mediated limitations, and to engineer CAR-T cells for improved immunotherapy outcomes.},
}

Humans
*CRISPR-Cas Systems
*Neoplasms/therapy/immunology/genetics
Gene Editing/methods
Immunotherapy/methods
*Tumor Escape/genetics
Animals
*Immune Evasion/genetics
Immunotherapy, Adoptive/methods

@article {pmid41977403,
year = {2026},
author = {Ma, S and Li, Y and Fei, T},
title = {Functional CRISPR Screens Define Genetic Drivers for Cancer Transformation and Progression from Non-Cancerous Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977403},
issn = {1422-0067},
support = {2023A1515140084//Guangdong Basic and Applied Basic Research Foundation/ ; 32470673//National Natural Science Foundation of China/ ; B16009//the 111 Project/ ; 2022JH13/10200026//the Construction Project of Liaoning Provincial Key Laboratory, China/ ; },
mesh = {Humans ; Animals ; Mice ; *Cell Transformation, Neoplastic/genetics ; *CRISPR-Cas Systems ; *Liver Neoplasms/genetics/pathology ; Female ; *Carcinoma, Hepatocellular/genetics/pathology ; Disease Progression ; Gene Expression Regulation, Neoplastic ; *Breast Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution.},
}

Humans
Animals
Mice
*Cell Transformation, Neoplastic/genetics
*CRISPR-Cas Systems
*Liver Neoplasms/genetics/pathology
Female
*Carcinoma, Hepatocellular/genetics/pathology
Disease Progression
Gene Expression Regulation, Neoplastic
*Breast Neoplasms/genetics/pathology
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41490267,
year = {2026},
author = {Carturan, A and Angelos, MG and Guruprasad, P and Patel, RP and Pajarillo, R and Lee, A and Espie, D and Zhang, Y and Chiang, YH and Xie, W and Rodriguez, JL and Harris, J and Devi, P and Afolayan-Oloye, OI and Xu, J and Sussman, JH and Elghawy, O and Yang, A and Barsouk, A and Cho, JH and Shaw, CE and Singh, E and Ugwuanyi, O and Paruzzo, L and Stella, F and Liu, S and Nason, S and Imparato, A and Rotolo, A and Lemoine, J and Barrett, DM and Posey, A and Rook, AH and Pillai, V and Bagg, A and Pileri, SA and Liu, D and Tan, K and Schuster, SJ and Teachey, DT and Porazzi, P and Ruella, M},
title = {Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T-cell therapies.},
journal = {Blood},
volume = {147},
number = {16},
pages = {1842-1856},
doi = {10.1182/blood.2025031409},
pmid = {41490267},
issn = {1528-0020},
support = {R01 FD008168/FD/FDA HHS/United States ; R37 CA262362/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *CD2 Antigens/immunology/genetics/metabolism ; *Receptors, Chimeric Antigen/immunology/genetics ; Animals ; Mice ; *T-Lymphocytes/immunology ; *Hematologic Neoplasms/therapy/immunology ; CD58 Antigens/immunology ; CRISPR-Cas Systems ; },
abstract = {Patients with T-cell lymphomas and leukemias have overall poor outcomes because of the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. The development of chimeric antigen receptor (CAR) T cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective antitumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, with anti-CD2 CAR T cells engineered to lack CD2 expression (CART2) would improve manufacturability and preclinical efficacy. Optimized CD2-knockout CART2 cells, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, costimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel programmed cell death protein 1 (PD-1):CD2 switch receptor that rescues intracellular CD2 signaling, particularly when programmed death-ligand 1 is engaged, thereby improving in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T-cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T cells and provides an effective strategy to overcome CD2-signaling deficiencies.},
}

Humans
*Immunotherapy, Adoptive/methods
*CD2 Antigens/immunology/genetics/metabolism
*Receptors, Chimeric Antigen/immunology/genetics
Animals
Mice
*T-Lymphocytes/immunology
*Hematologic Neoplasms/therapy/immunology
CD58 Antigens/immunology
CRISPR-Cas Systems

@article {pmid41512493,
year = {2026},
author = {Ahmad, R and Gastoldi, G and Wong, ST and Baeza Garcia, A and Caljon, G},
title = {The EamA metabolite transporter does not affect antileishmanial drug susceptibility.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {30},
number = {},
pages = {100632},
pmid = {41512493},
issn = {2211-3207},
mesh = {*Leishmania infantum/drug effects/genetics/metabolism ; *Antiprotozoal Agents/pharmacology ; *Protozoan Proteins/genetics/metabolism ; Drug Resistance/genetics ; Animals ; CRISPR-Cas Systems ; *Membrane Transport Proteins/genetics/metabolism ; Macrophages/parasitology ; Gene Editing ; Mice ; Humans ; Parasitic Sensitivity Tests ; Leishmaniasis, Visceral/parasitology/drug therapy ; },
abstract = {Leishmaniasis is a major neglected tropical disease, exists in 98 countries and constitutes a global public health threat. As chemotherapy is confronted with drug resistance and treatment failure, understanding the underlying mechanisms and continued drug discovery efforts are needed in the fight against leishmaniasis. A previous cosmid-based overexpression study suggested a role for EamA (LINF_020008400), annotated as a putative drug-metabolite transporter, in resistance to novel antileishmanial oxaboroles. To assess fitness cost and drug susceptibility, gene deficient Leishmania infantum lines were generated using CRISPR-Cas9 gene editing and overexpression from the ssu locus was achieved using the pLEXSY system. While in vitro parasite growth and survival were unchanged compared to control lines, the intracellular burden of the null mutant was lower. In vitro exposure to current antileishmanial drugs and several novel leads revealed an unchanged drug sensitivity profile in extracellular and intramacrophage assays. Similarly, the overexpression lines showed a significantly lower infection rate, but their drug susceptibility profiles showed no significant differences from the control. Collectively, these data suggest that -under the tested conditions- LINF_020008400 is not essential for parasite fitness, host cell infectivity and survival following exposure to antiparasitic drugs.},
}

*Leishmania infantum/drug effects/genetics/metabolism
*Antiprotozoal Agents/pharmacology
*Protozoan Proteins/genetics/metabolism
Drug Resistance/genetics
Animals
CRISPR-Cas Systems
*Membrane Transport Proteins/genetics/metabolism
Macrophages/parasitology
Gene Editing
Mice
Humans
Parasitic Sensitivity Tests
Leishmaniasis, Visceral/parasitology/drug therapy

@article {pmid41741641,
year = {2026},
author = {Yang, L and Renauer, PA and Tang, K and Saskin, J and Zhou, L and Zou, C and Lee, SH and Fox, M and Johnson-Noya, S and Weiss, B and Deng, S and Fang, P and Chen, B and Sferruzza, G and Fooladi, S and Zhao, K and Park, D and Zhang, F and Tu, J and Chen, J and Moliterno, J and Gunel, M and Peng, L and Chen, S},
title = {OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumours.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {740-751},
pmid = {41741641},
issn = {1476-4687},
mesh = {Humans ; Animals ; *Killer Cells, Natural/immunology/metabolism/cytology/transplantation ; Mice ; Female ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Neoplasms/therapy/immunology/pathology ; Tumor Microenvironment/immunology ; *Immunotherapy, Adoptive/methods ; Male ; Cell Line, Tumor ; *Receptors, G-Protein-Coupled/genetics/metabolism/immunology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; CRISPR-Cas Systems/genetics ; },
abstract = {Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumours but remain limited because of poor tumour infiltration, persistence and resistance in the tumour microenvironment[1-4]. Here, to identify gain-of-function targets that enhance CAR-NK cell efficacy, we performed an unbiased in vivo CRISPR activation screen followed by a barcoded targeted in vivo open reading frame screen in primary human CAR-NK cells. We identified and comprehensively validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NK cells with OR7A10 cDNA (a CRISPR-independent method with a simple manufacturing strategy) enhanced their proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness and tumour microenvironment resistance. Moreover, exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors was reduced. OR7A10 gain-of-function CAR-NK cells displayed strong in vivo efficacy across multiple solid tumour models. For example, 100% complete response with long-term tumour control and survival benefit in an orthotopic breast cancer mouse model were achieved. These findings establish OR7A10-engineered CAR-NK cells as a highly potent and scalable off-the-shelf therapeutic for solid tumours.},
}

Humans
Animals
*Killer Cells, Natural/immunology/metabolism/cytology/transplantation
Mice
Female
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Neoplasms/therapy/immunology/pathology
Tumor Microenvironment/immunology
*Immunotherapy, Adoptive/methods
Male
Cell Line, Tumor
*Receptors, G-Protein-Coupled/genetics/metabolism/immunology
Xenograft Model Antitumor Assays
Cell Proliferation
CRISPR-Cas Systems/genetics

@article {pmid41791393,
year = {2026},
author = {Rapp, J and Verhülsdonk, A and Garcke, A and Stadelmann, A and Farke, N and Troßmann, F and Kronenberger, T and Alvarado, A and Petras, D and Link, H},
title = {The metabolome of an E. coli CRISPRi library identifies benefits of minimal metabolite levels and targets for engineering.},
journal = {Cell systems},
volume = {17},
number = {4},
pages = {101518},
doi = {10.1016/j.cels.2025.101518},
pmid = {41791393},
issn = {2405-4720},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolome/genetics ; *Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Metabolic Networks and Pathways/genetics ; Gene Library ; Metabolomics/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Tandem Mass Spectrometry ; },
abstract = {Metabolite concentration changes can have broad consequences on the function and robustness of metabolic networks. Here, we measured the metabolome response of 1,515 CRISPR interference (CRISPRi) E. coli strains targeting all genes in the iML1515 metabolic model. Metabolites that are hardly measurable in wild-type E. coli accumulated in specific CRISPRi strains, indicating that they are normally maintained at low levels. We confirmed metabolite accumulation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated putative reference spectra for 102 metabolites for which no MS[2] data had previously been available. We show that minimal metabolite levels are beneficial because they (1) enable substrate level regulation of enzyme activity, (2) prevent competitive inhibition, and (3) suppress side reactions. However, minimal metabolite pools also limit flux through engineered pathways. For example, low levels of farnesyl diphosphate (frdp) constrained a synthetic carotenoid pathway, and we show that the knockdown of octaprenyl diphosphate synthase (IspB) increased frdp levels and carotenoid production. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Escherichia coli/metabolism/genetics
*Metabolome/genetics
*Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
Metabolic Networks and Pathways/genetics
Gene Library
Metabolomics/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Tandem Mass Spectrometry

@article {pmid41825300,
year = {2026},
author = {Low, YC and McKnight, CL and Elliott, DA and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent embryonic stem cell TAFAZZIN hESC model (WAe009-A-3H) of Barth syndrome.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103948},
doi = {10.1016/j.scr.2026.103948},
pmid = {41825300},
issn = {1876-7753},
mesh = {Humans ; *Barth Syndrome/pathology/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism ; Female ; *Transcription Factors/genetics/metabolism ; Cell Line ; Acyltransferases/genetics ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Cell Differentiation ; },
abstract = {Barth syndrome is among the most common mitochondrial diseases presenting with cardiomyopathy. We have generated a human embryonic stem cell (hESC) model of Barth syndrome (TAFAZZIN[Δ3] C15) in a female background (H9 hESC) using CRISPR/Cas9 gene editing, with compound heterozygous variants in TAFAZZIN that result in exon 3 skipping in all stable transcripts. This cell line displayed characteristics consistent with pluripotent stem cells, including typical colony morphology, expression of pluripotency markers, trilineage potential, and a normal karyotype. This TAFAZZIN[Δ3] C15 line could be used for investigation of disease mechanisms in mitochondrial cardiomyopathy and preclinical therapeutic screening.},
}

Humans
*Barth Syndrome/pathology/genetics/metabolism
*Human Embryonic Stem Cells/metabolism
Female
*Transcription Factors/genetics/metabolism
Cell Line
Acyltransferases/genetics
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism
Cell Differentiation

@article {pmid41826696,
year = {2026},
author = {Aird, EJ and Rabl, J and Knuesel, T and Groen, K and Awwad, SW and Korablev, B and Scherpe, L and Al-Herz, W and Hupfer, R and Recher, M and Jackson, SP and Hale, BG and Corn, JE},
title = {An SP110-SP100 axis is a critical regulator of promyelocytic leukaemia body dynamics and mitotic fidelity.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {684-695},
pmid = {41826696},
issn = {1476-4679},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 310030_188858//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 320030_232029//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; ALTF 144-2021//European Molecular Biology Organization (EMBO)/ ; Women's Postdoctoral Career Development Award in Science//Weizmann Institute of Science/ ; Outstanding Postdoctoral Women Fellowship//Council for Higher Education of Israel | Israeli Centers for Research Excellence (I-CORE)/ ; DRCPGM\100005//Cancer Research UK (CRUK)/ ; SEBINT-2024/100003//Cancer Research UK (CRUK)/ ; },
mesh = {Humans ; *Mitosis ; *Autoantigens/metabolism/genetics ; *Promyelocytic Leukemia Protein/metabolism/genetics ; *Antigens, Nuclear/metabolism/genetics ; *Leukemia, Promyelocytic, Acute/genetics/metabolism/pathology ; CRISPR-Cas Systems ; DNA Damage ; },
abstract = {Stimulation of the innate immune system by foreign RNA elicits a potent interferon response and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Here, using genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we discover that promyelocytic leukaemia (PML) nuclear body-localized speckled protein 110 (SP110) is a potent inhibitor of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a toxic activity of SP100, a major constituent of PML bodies. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, leading to micronucleus formation, DNA damage and genotoxic cell death. A combination of cryo-electron microscopy, AlphaFold modelling and cellular biochemistry reveals that SP110 dissolves toxic SP100 oligomers via necessary and sufficient direct interactions between their caspase activation and recruitment domains. These data reveal the critical roles of SP100 and SP110 in governing the disassembly of PML bodies during mitosis, as well as the repercussions if this process is misregulated.},
}

Humans
*Mitosis
*Autoantigens/metabolism/genetics
*Promyelocytic Leukemia Protein/metabolism/genetics
*Antigens, Nuclear/metabolism/genetics
*Leukemia, Promyelocytic, Acute/genetics/metabolism/pathology
CRISPR-Cas Systems
DNA Damage

@article {pmid41826699,
year = {2026},
author = {Skafar, V and de Souza, I and Ghosh, B and Ferreira Dos Santos, A and Porto Freitas, F and Chen, Z and Sun, S and Donate Castillo, M and Nepachalovich, P and Seufert, L and Bothe, S and Tschuck, J and Mathur, A and Nunes-Alves, A and Buhr, J and Aponte-Santamaría, C and Schmitz, W and Mack, M and Eilers, M and Bargou, R and Chaufan, M and Kaur, M and Palma, M and Ubellacker, JM and Elling, U and Augustin, HG and Hadian, K and Meierjohann, S and Proneth, B and Conrad, M and Fedorova, M and Alborzinia, H and Friedmann Angeli, JP},
title = {Riboflavin metabolism shapes FSP1-driven ferroptosis resistance.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {696-706},
pmid = {41826699},
issn = {1476-4679},
mesh = {*Ferroptosis/drug effects ; Humans ; *Riboflavin/metabolism/pharmacology ; Antioxidants/metabolism ; Lipid Peroxidation/drug effects ; Cell Line, Tumor ; CRISPR-Cas Systems ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; },
abstract = {Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell's antioxidant capacity.},
}

*Ferroptosis/drug effects
Humans
*Riboflavin/metabolism/pharmacology
Antioxidants/metabolism
Lipid Peroxidation/drug effects
Cell Line, Tumor
CRISPR-Cas Systems
Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics

@article {pmid41831412,
year = {2026},
author = {Liu, J and Luo, S and Chen, S and Chen, G and Zhu, Y and Lou, Y and Fan, R and Zhang, Y and Pan, J and Zhu, C and Xu, L and Li, L},
title = {Dual-function CRISPR/Cas12a assisted strand displacement reaction with RuHex-loaded DNA condensates for ultrasensitive electrochemical detection of hepatocellular carcinoma mRNA.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118610},
doi = {10.1016/j.bios.2026.118610},
pmid = {41831412},
issn = {1873-4235},
mesh = {Humans ; *Liver Neoplasms/genetics/diagnosis ; *Carcinoma, Hepatocellular/genetics/diagnosis ; *Biosensing Techniques/methods ; *RNA, Messenger/genetics/isolation & purification ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Ruthenium/chemistry ; DNA/chemistry ; CRISPR-Associated Proteins/chemistry ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins ; },
abstract = {Hepatocellular carcinoma (HCC) typically develops in a clinically silent manner, and the suboptimal sensitivity and specificity of currently available diagnostic biomarkers remain significant obstacles to its accurate and early detection. To improve molecular diagnostic performance, we developed a dual-function CRISPR/Cas12a assisted strand displacement reaction (dCas12a-SDR) with hexaammine ruthenium(III) chloride (RuHex)-loaded DNA condensate for ultrasensitive and highly specific detection of HCC-associated mRNAs. Upon target recognition, the previously sequestered Cas12a activation site within the electrode-immobilized capture probe is exposed, thereby inducing hybridization between the accessible single-stranded domains retained in RuHex-loaded DNA condensates (RuDC) and the displacement strand (Ds), which ultimately leads to the release of electroactive Ds-RuDC condensates and the effective activation of Cas12a. Activated Cas12a then removes the activation site via cis-cleavage, releasing the target to enter subsequent reaction cycles; concurrently, the activated system initiates trans-cleavage of adjacent capture probes and Ds-RuDC assemblies on the electrode that harbor trans-cleavage motifs, thereby promoting the release of RuDC from the electrode interface. This cascade ultimately leads to a pronounced reduction in the electrochemical signal. Owing to this target-triggered dual cis- and trans-cleavage mechanism mediated by Cas12a, the biosensor achieves highly efficient signal amplification. The platform affords a limit of detection as low as 39.2 aM for PD-L1 mRNA and exhibits excellent specificity, stability, and reproducibility. Moreover, by jointly detecting a panel of HCC-associated mRNAs (PD-L1, GPC3, EpCAM, and FGA), the platform successfully discriminated healthy individuals from patients with early-stage hepatocellular carcinoma in clinical serum samples. Collectively, this platform provides a powerful tool for molecular diagnosis of hepatocellular carcinoma.},
}

Humans
*Liver Neoplasms/genetics/diagnosis
*Carcinoma, Hepatocellular/genetics/diagnosis
*Biosensing Techniques/methods
*RNA, Messenger/genetics/isolation & purification
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
Ruthenium/chemistry
DNA/chemistry
CRISPR-Associated Proteins/chemistry
Endodeoxyribonucleases/chemistry
Bacterial Proteins

@article {pmid41855944,
year = {2026},
author = {Miao, Y and Wang, C and Peng, Y and Sun, X and Zheng, Z and Zhang, Q and Cheng, W and Li, J},
title = {PAM-assembled CRISPR-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for detecting prostate cancer exosomes.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118635},
doi = {10.1016/j.bios.2026.118635},
pmid = {41855944},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; Male ; *Prostatic Neoplasms/diagnosis/blood/genetics ; *Exosomes/chemistry/genetics/pathology ; Colorimetry/methods ; CRISPR-Cas Systems/genetics ; *Glutamate Carboxypeptidase II/isolation & purification ; Aptamers, Nucleotide/chemistry ; Antigens, Surface ; Limit of Detection ; Prostate-Specific Antigen ; Tetraspanin 30/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {Prostate specific membrane antigen (PSMA)-positive exosomes hold significant potential for the diagnosis and risk assessment of prostate cancer. However, accurate detection is severely hindered by their low abundance in blood and interference from similarly sized particles. In this study, we have developed a protospacer adjacent motif (PAM)-assembled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for the highly sensitive detection of PSMA-positive exosomes. In this work, two split strands respectively containing CD63 and PSMA aptamers are utilized to bind CD63 and PSMA on the exosome surface, forming a template that induces the opening of a hairpin DNA (HP DNA). A PAM site forms via hairpin-to-double-stranded structure transition. CRISPR-Cas12a recognizes PAM, activates to cleave FAM-labeled probes for fluorescence, while cerium dioxide nanozyme (CeO2 NZ) (with phosphatase-mimicking activity) hydrolyzes cleavage products. Hydroxyl radicals from hydrolysis oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to induce colorimetry. CRISPR-Cas12a-nanozyme dual recognition significantly improves prostate cancer exosome detection selectivity and sensitivity. Under optimized conditions, the limits of detection for the fluorescence and colorimetric modes reach 49 particles/μL and 63 particles/μL, respectively. By mutually validating dual detection modes, this biosensing technology accurately distinguishes prostate cancer patients from healthy individuals, holding great promise for early diagnosis.},
}

Humans
*Biosensing Techniques/methods
Male
*Prostatic Neoplasms/diagnosis/blood/genetics
*Exosomes/chemistry/genetics/pathology
Colorimetry/methods
CRISPR-Cas Systems/genetics
*Glutamate Carboxypeptidase II/isolation & purification
Aptamers, Nucleotide/chemistry
Antigens, Surface
Limit of Detection
Prostate-Specific Antigen
Tetraspanin 30/chemistry
Fluorescent Dyes/chemistry

@article {pmid41863957,
year = {2026},
author = {Yang, Y and Huang, Z and Peng, X and Cui, F and Hu, X and Ren, H},
title = {Single-microsphere biosensors based on UiO-66 confinement-enhanced fluorescence emission for sensitive detection of antibiotics and pathogenic bacteria.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118632},
doi = {10.1016/j.bios.2026.118632},
pmid = {41863957},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Staphylococcus aureus/isolation & purification/pathogenicity ; *Anti-Bacterial Agents/analysis/isolation & purification ; Limit of Detection ; *Chloramphenicol/analysis/isolation & purification ; Fluorescent Dyes/chemistry ; Microspheres ; Food Contamination/analysis ; Food Microbiology ; CRISPR-Cas Systems ; Fluorescence ; Humans ; Polystyrenes/chemistry ; },
abstract = {To meet the urgent need for sensitive detection of foodborne pathogens and antibiotic residues-key to preventing outbreaks and curbing antimicrobial resistance-we developed a stable fluorescent probe, H4TCPE@UiO-66 (H@U). The probe retains strong fluorescence under harsh conditions (18.70% intensity change at pH 3, 11.36% at 80 °C) and exhibits a 6.90-fold signal amplification over H4TCPE. In addition, H@U is able to be a sensitive probe integrated with millimetre-scale polystyrene microspheres (mPS) and realize rapid detection of chloramphenicol within 20 min with a detection limit of 33.52 pg/mL, representing a 26.61-fold improvement in sensitivity over the enzyme-linked immunosorbent assay. Building on this, we harnessed the trans-cleavage activity of CRISPR/Cas12a to develop a CRISPR/Cas12a-enhanced H@U mPS (H@U-C mPS) biosensor for the quantitative detection of Staphylococcus aureus, achieving a linear range of 10-10[5] CFU/mL (two orders of magnitude lower than qPCR) and a detection limit of 6.97 CFU/mL. Furthermore, the practical applicability of H@U-C mPS biosensors was successfully demonstrated through validation using 30 real-world samples. Together, these platforms offer a robust, rapid, and highly sensitive strategy for monitoring chemical hazards and microbial in food safety and clinical diagnostics.},
}

*Biosensing Techniques/methods
*Staphylococcus aureus/isolation & purification/pathogenicity
*Anti-Bacterial Agents/analysis/isolation & purification
Limit of Detection
*Chloramphenicol/analysis/isolation & purification
Fluorescent Dyes/chemistry
Microspheres
Food Contamination/analysis
Food Microbiology
CRISPR-Cas Systems
Fluorescence
Humans
Polystyrenes/chemistry

@article {pmid41873591,
year = {2026},
author = {Jürgens, K and Menzel, L and Klinke, N and Schäper, L and Ratnavadivel, S and Walter, S and Milting, H and Meyer, H and Paululat, A},
title = {An ARVC-5 Drosophila knock-in model reveals new functions of Tmem43 in lipid homeostasis.},
journal = {Biology open},
volume = {15},
number = {4},
pages = {},
doi = {10.1242/bio.062326},
pmid = {41873591},
issn = {2046-6390},
support = {PA517/16-1//DFG/ ; MI1146/6-1//DFG/ ; MI1146/6-1::PA517/16-1//Deutsche Forschungsgemeinschaft/ ; //Osnabrück University/ ; },
mesh = {Animals ; *Lipid Metabolism/genetics ; *Homeostasis ; Disease Models, Animal ; Gene Knock-In Techniques ; Mitochondria/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Energy Metabolism ; Lipidomics ; Mutation ; Proteomics ; CRISPR-Cas Systems ; },
abstract = {Arrhythmogenic right ventricular cardiomyopathy type 5 is caused by the missense mutation S358L in the gene TMEM43 in humans. To date, the molecular mechanisms underlying the disease remain poorly understood. We established a CRISPR/Cas9 knock-in Drosophila model carrying the orthologous Tmem43p.S333L mutation to investigate these mechanisms in vivo. The resulting flies were viable but displayed reduced lifespan, smaller body size, lipid droplet accumulation, and mitochondrial defects. Proteomic and lipidomic profiling revealed a dosage-dependent misregulation of the energy metabolism, concomitant with reduced fatty acid synthesis and ß-oxidation rates, altered peroxisomal pathways, and changes in membrane phospholipid composition. Notably, phosphatidylethanolamine (PE) and phosphatidylinositol (PI) levels were elevated, while triacylglycerols were reduced. Ultrastructural analyses confirmed mitochondrial degradation in the muscle tissue of corresponding mutants. These findings establish Tmem43p.S333L knock-in flies as a robust in vivo model of ARVC-5 and support a role for TMEM43 in linking lipid homeostasis to mitochondrial energy metabolism and integrity. Mutation-derived impairments in these processes result in cardiomyopathy.},
}

Animals
*Lipid Metabolism/genetics
*Homeostasis
Disease Models, Animal
Gene Knock-In Techniques
Mitochondria/metabolism
*Drosophila Proteins/genetics/metabolism
*Drosophila/genetics/metabolism
*Membrane Proteins/genetics/metabolism
Energy Metabolism
Lipidomics
Mutation
Proteomics
CRISPR-Cas Systems

@article {pmid41905104,
year = {2026},
author = {Koutala, E and Cantarini, C and Raymond, K and Lebrin, F},
title = {Generation of ALK1 p.Gly48Glu mutant LUMCi029-A-3 for modeling Hereditary hemorrhagic telangiectasia type 2.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103974},
doi = {10.1016/j.scr.2026.103974},
pmid = {41905104},
issn = {1876-7753},
mesh = {Humans ; *Telangiectasia, Hereditary Hemorrhagic/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Activin Receptors, Type II/genetics/metabolism ; *Mutation/genetics ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; },
abstract = {Hereditary hemorrhagic telangiectasia type 2 (HHT2) is an autosomal dominant vascular disorder caused by pathogenic variants in ACVRL1, which encodes activin receptor-like kinase 1 (ALK1). Here, we report the generation and characterization of an isogenic human induced pluripotent stem cell (hiPSC) line carrying a heterozygous ACVRL1 c.143G > A (p.Gly48Glu) mutation. The mutation was introduced using CRISPR/Cas9-mediated genome editing and confirmed by PCR and Sanger sequencing. The edited line retained normal karyotype, pluripotency, and trilineage differentiation capacity. This hiPSC line represents a relevant in vitro model for HHT2 disease modelling and drug testing.},
}

Humans
*Telangiectasia, Hereditary Hemorrhagic/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*Activin Receptors, Type II/genetics/metabolism
*Mutation/genetics
CRISPR-Cas Systems
Cell Line
Gene Editing

@article {pmid41916193,
year = {2026},
author = {Luo, Y and Vallone, VF and Blanc, E and Miller, DC and He, J and Stachelscheid, H and Beule, D and Scheffel, J and Siebenhaar, F},
title = {CRISPR/Cas9 engineered and whole-genome characterized KIT D816V-mutant human iPSC lines.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103975},
doi = {10.1016/j.scr.2026.103975},
pmid = {41916193},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Proto-Oncogene Proteins c-kit/genetics/metabolism ; *Mutation/genetics ; Cell Line ; Gene Editing ; Whole Genome Sequencing ; Genome, Human ; },
abstract = {We report on the generation of human induced pluripotent stem cell (iPSC) lines, BIHi005-A-86 and BIHi005-A-87, carrying the KIT D816V mutation associated with Indolent Systemic Mastocytosis (ISM). To overcome the confounding genetic backgrounds of existing leukemic models, we introduced this gain-of-function mutation into the healthy BIHi005-A line using CRISPR/Cas9 editing. The resulting clones were validated via whole-genome sequencing (WGS) to confirm specific on-target editing and lack of predicted or disease-relevant off-target effects, while maintaining genomic stability. Together with the parental line, this resource provides an isogenic controlled platform for investigating KIT D816V-driven pathogenesis.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Proto-Oncogene Proteins c-kit/genetics/metabolism
*Mutation/genetics
Cell Line
Gene Editing
Whole Genome Sequencing
Genome, Human

@article {pmid41935495,
year = {2026},
author = {Zhang, J and Zhang, W and Yan, J and Zhang, Y and Zheng, J and Liu, Y and Gu, N and Han, K},
title = {A proximity induced strand displacement amplification and CRISPR/Cas12a-based SERS assay for ultrasensitive detection of dengue virus.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118651},
doi = {10.1016/j.bios.2026.118651},
pmid = {41935495},
issn = {1873-4235},
mesh = {*Dengue Virus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; *Dengue/virology/diagnosis ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue virus (DENV) is a mosquito-borne single-stranded RNA virus that causes dengue fever and can progress to severe disease, posing a significant public-health threat. Rapid and highly sensitive detection is crucial for the early diagnosis of DENV, which, however, suffers from difficult discrimination from similar symptoms of other virus infection, such as Chikungunya or Zika virus, as well as insufficient specificity and time-consuming procedures. Here, we present a surface-enhanced Raman scattering (SERS) biosensor that integrates strand displacement amplification (SDA) with CRISPR/Cas12a for ultrasensitive detection of DENV. In the absence of DENV, SDA and Cas12a stay inactive and the SERS probes retain an aggregation state due to the crosslinking of single-stranded Linker, exhibiting strong SERS signals. In the presence of target sequences, SDA generates abundant single-stranded Trigger-strands, activating Cas12a through crRNA recognition, cleaving the Linker required to enable the crosslinking and leaving the SERS nanoprobes in a monodispersed state, demonstrating weak SERS signals. This method shows exceptional sensitivity for Dengue virus type 1 (DENV-1), with a limit of detection as low as 4.68 fM. It also provides excellent specificity by accurately distinguishing DENV-1 from other pathogens, while maintaining reliable performance. The modular design of this strategy endows the system capability to be adapted for different targets, making it not only a promising tool for the highly sensitive diagnosis of DENV-1, but also a versatile platform for the precise recognition of other pathogens.},
}

*Dengue Virus/isolation & purification/genetics
*Biosensing Techniques/methods
*Spectrum Analysis, Raman/methods
*Dengue/virology/diagnosis
*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41935496,
year = {2026},
author = {Wu, Y and Lv, B and Zhou, R and Zhang, H and Li, D},
title = {Highly sensitive closed-tube detection of alkaline phosphatase based on phosphate-mediated CRISPR/Cas12a regulation.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118659},
doi = {10.1016/j.bios.2026.118659},
pmid = {41935496},
issn = {1873-4235},
mesh = {*Alkaline Phosphatase/blood/isolation & purification ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Phosphates/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; },
abstract = {Recently, we reported that a 5'-terminal phosphate at the junction of split activators inhibits the trans-cleavage activity of Cas12a. In this work, we discovered that phosphate-mediated Cas12a inhibition has two key features: strong position dependence and notable dual-phosphate enhancement. AlphaFold 3 (AF3) structural predictions indicate that the inhibition is mediated by interactions between the phosphate and specific local residues on Cas12a. On the basis of these findings, we developed a highly sensitive alkaline phosphatase (ALP) biosensor by harnessing the principle that ALP controls Cas12a activation through dephosphorylating activators. Without the need for upstream signal amplification, the biosensor achieves a high sensitivity of 6.07 × 10[-6] U/L for ALP across a broad dynamic range of 7.5 × 10[-6] to 2.5 × 10[-2] U/L. Furthermore, the assay is readily extendable to the evaluation of ALP inhibitors. Notably, a closed-tube assay system with lyospheres (CASL) was developed. Our biosensor equipped with the CASL enables closed-tube ALP detection in human serum, effectively preventing environmental contamination and interference. Owing to its simple closed-tube workflow and excellent performance, this biosensor holds great potential for point-of-care clinical diagnostics. Beyond its application, the phosphate-mediated CRISPR/Cas12a regulation strategy elucidated herein offers deeper mechanistic insight and a versatile blueprint for future biosensor design.},
}

*Alkaline Phosphatase/blood/isolation & purification
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Phosphates/chemistry/metabolism
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Bacterial Proteins/chemistry/genetics/metabolism
*Endodeoxyribonucleases/chemistry/genetics/metabolism

@article {pmid41946842,
year = {2026},
author = {Akana, RV and Yoe, J and Laveroni, O and Sun, C and Kim, YM and Jerby, L},
title = {High-content CRISPR activation screens identify synthetically lethal RNA-based mechanisms to sensitize cancer cells to targeted T cell cytotoxicity.},
journal = {Nature genetics},
volume = {58},
number = {4},
pages = {841-853},
pmid = {41946842},
issn = {1546-1718},
support = {1019508.01//Burroughs Wellcome Fund (BWF)/ ; OPP1113682//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; Receptors, Antigen, T-Cell/immunology/genetics ; *T-Lymphocytes/immunology ; *RNA/genetics ; Animals ; Mice ; *Melanoma/genetics/immunology ; *Neoplasms/genetics/immunology ; *Cytotoxicity, Immunologic/genetics ; },
abstract = {T cells recognize their target cells through the T cell receptor (TCR). Combining gain-of-function, single-cell and optical high-content screens, we identified RNA-based mechanisms that selectively sensitize target cells to TCR-specific T cell cytotoxicity. First, CRISPR activation screens in melanoma cells identify functionally diverse regulators of TCR-specific cytotoxicity, including SAFB, KHDRBS1, MYC, CD44, WNT3A, WNT1 and others. Expressing sensitizing hits in cancer and virally infected cells restores TCR-specific cytotoxicity. Next, we developed in situ Perturb-seq for optical pooled genetic screens with in situ detection of perturbations and spatial transcriptomic readouts. Perturb-seq and in vivo-in situ Perturb-seq show that the hits converge on shared cell-autonomous and intercellular mechanisms, map gene-environment interactions and reveal that Wnt ligands activate T cells. Introducing a scalable approach to decode gene function at the cell and tissue level, the study uncovered context-specific gene functions to restore targeted T cell-based elimination of dysfunctional cells via synthetically lethal, RNA-based interventions.},
}

Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line, Tumor
Receptors, Antigen, T-Cell/immunology/genetics
*T-Lymphocytes/immunology
*RNA/genetics
Animals
Mice
*Melanoma/genetics/immunology
*Neoplasms/genetics/immunology
*Cytotoxicity, Immunologic/genetics

@article {pmid41950725,
year = {2026},
author = {Clémençon, M and Brogard, J and Rozen, M and Hourton, C and García, RM and Goureau, O and Bigou, S and Reichman, S},
title = {Generation of human P347L RHO-associated retinitis pigmentosa iPSC lines by a mutation insertion in the RHODOPSIN gene carrying the RHO c.1040C > T variant using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103968},
doi = {10.1016/j.scr.2026.103968},
pmid = {41950725},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Rhodopsin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutation ; Cell Line ; Cell Differentiation ; },
abstract = {The P347L RHODOPSIN-related retinal dystrophy leads an autosomal dominant Retinitis Pimentosa. Here we describe the generation of two isogenic human induced pluripotent stem cell (hiPSC) lines carrying the mutation c.1040C > T, p.Pro347Leu in the RHODOPSIN gene using CRISPR/Cas9 engineering from a control hiPSC clone. The two generated hiPSC lines can be differentiated in all the three germ layers, showed pluripotency makers expression and presented a normal karyotype. These hiPSCs will provide a new cell tool to better understand physiopathological mechanisms of retinitis pigmentosa and for the development of innovative treatment.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Retinitis Pigmentosa/genetics/pathology/metabolism
*Rhodopsin/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Mutation
Cell Line
Cell Differentiation

@article {pmid41961863,
year = {2026},
author = {Emond, A and Laflamme, C and Therrien, M and Liao, M and Maios, C and Labarre, A and Drapeau, P and Parker, JA},
title = {Characterization of a C9orf72 Knockout Danio rerio model for ALS and cross-species validation of potential therapeutics screened in Caenorhabditis elegans.},
journal = {PloS one},
volume = {21},
number = {4},
pages = {e0346613},
pmid = {41961863},
issn = {1932-6203},
mesh = {Animals ; *Zebrafish/genetics ; *Caenorhabditis elegans/genetics ; *C9orf72 Protein/genetics ; *Amyotrophic Lateral Sclerosis/genetics/drug therapy/pathology ; Disease Models, Animal ; Gene Knockout Techniques ; Humans ; CRISPR-Cas Systems ; Motor Neurons/pathology/metabolism ; Larva ; Animals, Genetically Modified ; Phenotype ; },
abstract = {Intronic hexanucleotide repeat expansions in the C9orf72 gene represent the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This expansion decreases C9orf72 expression in affected patients, indicating that loss of C9orf72 function (LOF) acts as a pathogenic mechanism. Several models using Danio rerio (zebrafish) for C9orf72 depletion have been developed to explore disease mechanisms and the consequences of C9orf72 LOF. However, inconsistencies exist in reported phenotypes, and many have yet to be validated in stable germline ablation models. To address this, we created a zebrafish C9orf72 knockout model using CRISPR/Cas9. The C9orf72 LOF model demonstrates, in a generally dose-dependent manner, increased larval mortality, persistent growth reduction, and motor deficits. Additionally, homozygous C9orf72 LOF larvae exhibited mild overbranching of spinal motoneurons. To identify potential therapeutic compounds, we performed a screen on an established Caenorhabditis elegans (C. elegans) C9orf72 homologue (alfa-1) LOF model, identifying 12 compounds that enhanced motility, reduced neurodegeneration, and alleviated paralysis phenotypes. Motivated by the shared motor phenotype, 2 of those compounds were tested in our zebrafish C9orf72 LOF model. Pizotifen malate was found to significantly improve motor deficits in C9orf72 LOF zebrafish larvae. We introduce a novel zebrafish C9orf72 knockout model that exhibits phenotypic differences from depletion models, providing a valuable tool for in vivo C9orf72 research and ALS therapeutic validation. Furthermore, we identify pizotifen malate as a promising compound for further preclinical evaluation.},
}

Animals
*Zebrafish/genetics
*Caenorhabditis elegans/genetics
*C9orf72 Protein/genetics
*Amyotrophic Lateral Sclerosis/genetics/drug therapy/pathology
Disease Models, Animal
Gene Knockout Techniques
Humans
CRISPR-Cas Systems
Motor Neurons/pathology/metabolism
Larva
Animals, Genetically Modified
Phenotype

@article {pmid41964260,
year = {2026},
author = {Muthusamy, B and Nizan, S and Bar-Ziv, A and Perl-Treves, R},
title = {Functional Validation of the Melon Fom-1 Gene, Controlling Resistance to Fusarium oxysporum Races 0 and 2, by CRISPR/Cas9 Mutagenesis.},
journal = {Molecular plant pathology},
volume = {27},
number = {4},
pages = {e70258},
pmid = {41964260},
issn = {1364-3703},
support = {1137/16//Israel Science Foundation/ ; 873-0074-11//Chief Scientist grant for Biotechnology, Ministry of Agriculture, Israel/ ; },
mesh = {*Fusarium/pathogenicity/physiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; *Plant Proteins/genetics/metabolism ; *Genes, Plant ; *Cucumis melo/genetics/microbiology ; },
abstract = {Functional validation of NLR genes is critical for confirming their specific roles and developing durable disease-resistant crops. The Fom-1 gene of Cucumis melo, controlling resistance to races 0 and 2 of F. oxysporum f. sp. melonis (FOM), had been identified by map-based cloning as MELO3C022146, which encodes a TIR-NBS-LRR (TNL) protein. It resides in a head-to-head orientation adjacent to another TNL gene, Prv, controlling resistance to papaya ring spot virus (PRSV). In this study we validated the function of Fom-1 in mediating FOM resistance by applying CRISPR/Cas9 editing to the resistant cultivar, Védrantais. Two gRNAs were designed to target exons 1 and 2, respectively, and mutations were introduced at both target sites, resulting in truncated open reading frames in both alleles. Inoculation assays of T1 plants with FOM races 2 and 0 revealed a breakdown of resistance, manifested by leaf necrosis and wilting, and susceptibility was stably inherited in the T2 generation. This proved that MELO3C022146, the candidate gene for Fom-1, is responsible for resistance in melon cultivar Védrantais. Future studies will address the molecular functions of this gene as well as possible interactions between Fom-1 and its neighbour R-gene, Prv.},
}

*Fusarium/pathogenicity/physiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
*CRISPR-Cas Systems/genetics
Mutagenesis/genetics
*Plant Proteins/genetics/metabolism
*Genes, Plant
*Cucumis melo/genetics/microbiology

@article {pmid41965285,
year = {2026},
author = {Yang, X and Cai, J and Wang, J and Meng, Y and Shi, Y and Cai, H},
title = {CRISPR-Cas9 knockout screens to identify drug resistance genes in acute myeloid leukemia.},
journal = {Methods in cell biology},
volume = {205},
number = {},
pages = {199-216},
doi = {10.1016/bs.mcb.2026.01.006},
pmid = {41965285},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; *Leukemia, Myeloid, Acute/genetics/drug therapy/pathology ; Humans ; *Drug Resistance, Neoplasm/genetics ; *Gene Knockout Techniques/methods ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; },
abstract = {Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.},
}

*CRISPR-Cas Systems/genetics
*Leukemia, Myeloid, Acute/genetics/drug therapy/pathology
Humans
*Drug Resistance, Neoplasm/genetics
*Gene Knockout Techniques/methods
Cell Line, Tumor
Antineoplastic Agents/pharmacology

@article {pmid41965303,
year = {2026},
author = {Zhai, X and Yang, B and Deng, F and Gulati, S and Mckinnirey, F and Wu, X and Li, Y and Goldys, EM},
title = {CbAgo-enriched Cas12a biosensor for cancer mutations screening.},
journal = {Analytica chimica acta},
volume = {1403},
number = {},
pages = {345420},
doi = {10.1016/j.aca.2026.345420},
pmid = {41965303},
issn = {1873-4324},
mesh = {Humans ; *Biosensing Techniques/methods ; Mutation ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/genetics/metabolism ; *Pancreatic Neoplasms/genetics/diagnosis ; Circulating Tumor DNA/genetics/blood ; Proto-Oncogene Proteins p21(ras)/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA Mutational Analysis/methods ; Bacterial Proteins ; },
abstract = {BACKGROUND: Accurate detection of low-frequency DNA mutations in body fluids is essential for cancer monitoring and treatment evaluation. However, the high abundance of wild-type DNA often masks rare mutant signals, making sensitive detection particularly challenging.

RESULTS: We developed a screening strategy termed the CbAgo-enriched Cas12a mutation screening system (CECMS). By integrating the single-nucleotide resolution of CbAgo with the trans-cleavage activity of CRISPR-Cas12a, this system selectively eliminates wild-type DNA while enriching targeted mutant alleles. CECMS achieves up to 100-fold higher sensitivity at 37 °C compared with conventional Cas12a biosensors, enabling reliable detection of variant allele frequencies (VAFs) as low as 0.01%. In undiluted serum spiked samples for circulating tumor DNA (ctDNA) detection, the method successfully detected pancreatic cancer-associated KRAS G12D mutations at a VAF of 0.1%.

SIGNIFICANCE: By leveraging CbAgo-mediated enrichment, the capability of exposing rare SNV for downstream detection is markedly improved. With its high efficiency and ease of use, CECMS holds strong potential as a convenient tool for clinical cancer diagnostics and monitoring.},
}

Humans
*Biosensing Techniques/methods
Mutation
CRISPR-Cas Systems
*Endodeoxyribonucleases/genetics/metabolism
*Pancreatic Neoplasms/genetics/diagnosis
Circulating Tumor DNA/genetics/blood
Proto-Oncogene Proteins p21(ras)/genetics
*CRISPR-Associated Proteins/genetics/metabolism
DNA Mutational Analysis/methods
Bacterial Proteins

@article {pmid41965876,
year = {2026},
author = {Seibert, M and Kurrle, N and Kaleab, S and Wempe, F and von Metzler, I and Serve, H and Schnütgen, F},
title = {Endogenous protein tagging coupled with a CRISPR screening approach identifies UBE3C as a potential MYC oncogene regulator.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41965876},
issn = {2045-2322},
mesh = {Humans ; *Proto-Oncogene Proteins c-myc/genetics/metabolism ; *CRISPR-Cas Systems ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Cell Line, Tumor ; *Multiple Myeloma/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The transcription factor MYC is a key regulator of cellular proliferation and metabolism and is frequently dysregulated in malignancies such as multiple myeloma (MM). Despite its clinical relevance, direct therapeutic targeting of MYC remains limited, emphasizing the need to identify upstream regulators that control endogenous MYC expression. To systematically uncover such regulators, we developed a genome-wide CRISPR-Cas9 loss-of-function screening approach, employing a custom-engineered MM reporter cell line (RPMI8226-F11), in which oncogenic MYC protein was endogenously tagged with EGFP (referred to as GFP). This fluorescent readout enabled a direct, quantitative assessment of endogenous MYC expression levels. A pooled genome-wide sgRNA library was introduced, and cells were sorted based on GFP fluorescent intensity to reflect varying MYC levels. Next-generation sequencing of sgRNA distributions across sorted populations enabled the identification of candidate MYC regulators. Validation of screen hits, including the established MYC activator IRF4 and repressor FBXW7, confirmed the reliability of our system. To further dissect regulatory networks, we performed an overrepresentation analysis of target genes, which revealed the enrichment of Mediator complex subunits among MYC activators and ubiquitin-proteasome pathway components among MYC repressors. Functional validation of prioritized hits-MED30 (Mediator complex) and UBE3C (E3 ubiquitin ligase)-demonstrated a strong impact on endogenous MYC levels. Notably, the knockout of UBE3C markedly increased MYC expression, whereas its paralogs, UBE3A and UBE3B, showed no measurable effect, suggesting a specific regulatory role for UBE3C in MM cells. Together, our study provides a comprehensive CRISPR screen-based resource for the discovery of MYC regulators and highlights UBE3C as a potential therapeutic node for modulating MYC expression in MM.},
}

Humans
*Proto-Oncogene Proteins c-myc/genetics/metabolism
*CRISPR-Cas Systems
*Ubiquitin-Protein Ligases/genetics/metabolism
Cell Line, Tumor
*Multiple Myeloma/genetics/metabolism/pathology
Gene Expression Regulation, Neoplastic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41966744,
year = {2026},
author = {Tang, Q and Song, XH and Guo, AN and Chen, Y and Shao, JW},
title = {CRISPR-mediated regulation of apoptosis in cancer: Molecular targets, mechanisms, and translational challenges.},
journal = {Biochemical and biophysical research communications},
volume = {817},
number = {},
pages = {153719},
doi = {10.1016/j.bbrc.2026.153719},
pmid = {41966744},
issn = {1090-2104},
abstract = {The CRISPR/Cas system, owing to its high gene-editing efficiency and relatively low off-target effects, has emerged as a pivotal technological platform in cancer research. By precisely modulating oncogenic signaling pathways and apoptosis-related molecules, CRISPR provides a critical tool for elucidating and reprogramming the regulatory mechanisms of apoptosis in tumor cells. However, despite substantial progress in preclinical studies, achieving efficient and selective induction of apoptosis in tumor cells remains a major translational challenge. Increasing evidence indicates that CRISPR-based strategies can achieve more effective antitumor outcomes by reprogramming tumor cell sensitivity to apoptosis, rather than relying solely on single-gene editing. This review systematically summarizes the molecular targets and underlying mechanisms of CRISPR-mediated regulation of tumor cell apoptosis, with particular emphasis on key apoptotic signaling pathways and representative research advances. It highlights the pivotal role of targeting survival-associated genes in suppressing tumor progression and promoting apoptotic cell death. Furthermore, this review discusses the potential synergistic mechanisms of CRISPR in combination with chemotherapy or immunotherapy, as well as the value of CRISPR-based functional screening in identifying apoptosis-regulatory targets and drug resistance-associated mechanisms. Finally, we analyze the key challenges facing the clinical translation of CRISPR-mediated apoptosis regulation and propose future research directions and conceptual frameworks to optimize CRISPR-based anticancer strategies and facilitate their clinical application.},
}

@article {pmid41968241,
year = {2026},
author = {Martin-Vicente, A and Fortwendel, JR},
title = {Cas9 Ribonucleoproteins (RNPs) for Gene Deletion.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {3-12},
pmid = {41968241},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Deletion ; *Aspergillus fumigatus/genetics ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Homologous Recombination ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Targeting/methods ; },
abstract = {Genetic manipulations in pathogenic microorganisms, like Aspergillus fumigatus, allow us to study the function of genes in pathogenicity or other important traits. Classic genetic engineering tools in filamentous fungi rely on DNA fragment sub-cloning or fusion PCR to build a gene deletion cassette containing extended flanking regions of homology to promote efficient recombination. However, the introduction of CRISPR technology into fungal genetics has greatly simplified and accelerated the genome editing process. Here, we describe a simple and universal, one-step CRISPR-Cas9-mediated genetic tool employing repair templates containing microhomology regions. This gene targeting system displays high homologous recombination efficiency rates and can be easily implemented across different genetic backgrounds.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Gene Deletion
*Aspergillus fumigatus/genetics
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
Homologous Recombination
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Targeting/methods

@article {pmid41968242,
year = {2026},
author = {Handelman, M and Werner, H and Osherov, N},
title = {Generation of Seamless Point Mutations with Cas9 RNP and pTel-hygR Plasmid in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {13-21},
pmid = {41968242},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/drug effects ; *CRISPR-Cas Systems ; Drug Resistance, Fungal/genetics ; *Plasmids/genetics ; *Point Mutation ; Antifungal Agents/pharmacology ; Fungal Proteins/genetics ; Gene Editing/methods ; Cytochrome P-450 Enzyme System/genetics ; Triazoles/pharmacology ; },
abstract = {The environmental mold Aspergillus fumigatus is a common human fungal pathogen that causes a wide range of diseases. The antifungal triazoles that inhibit the Cyp51 enzyme involved in ergosterol biosynthesis are used to treat A. fumigatus infections. However, triazole resistance is an increasing concern due to mutations in the genes cyp51A, hmg1, and others, and efflux pumps overexpression. The process of verifying mutations is time-consuming, even with CRISPR-Cas9 methods, as it still requires constructing repair templates with selectable markers. This study presents a faster and more efficient method to introduce mutations conferring triazole resistance in A. fumigatus by using in vitro assembled CRISPR-Cas9, along with a recyclable selectable marker. With this approach, we successfully introduced triazole resistance-conferring mutations in A. fumigatus genes (cyp51A, cyp51B, and hmg1), both individually and in combination. The technique has the potential to introduce mutations for resistance to other antifungals, toxic metals, and environmental stressors, thus enhancing the ability to generate dominant mutations in A. fumigatus.},
}

*Aspergillus fumigatus/genetics/drug effects
*CRISPR-Cas Systems
Drug Resistance, Fungal/genetics
*Plasmids/genetics
*Point Mutation
Antifungal Agents/pharmacology
Fungal Proteins/genetics
Gene Editing/methods
Cytochrome P-450 Enzyme System/genetics
Triazoles/pharmacology

@article {pmid40814875,
year = {2026},
author = {Rana, AJ and Hussain, MS and Hanbashi, A and Kamli, F and Khan, G and Qadri, M and Al-Qahtani, SA and Khan, MG and Burle, SS and Jakhmola, V and Gupta, G},
title = {CRISPR-Edited Cell Lines: A New Era in Functional Oncology Research.},
journal = {Current pharmaceutical design},
volume = {32},
number = {13},
pages = {1027-1034},
pmid = {40814875},
issn = {1873-4286},
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/pathology/drug therapy ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {The use of CRISPR-Cas9 to engineer cancer cell lines has made it possible to precisely examine how cancer cells react to different drugs and therapies. Some of the key improvements are in the use of Mediator Complex Subunit 12 (MED12)-knockout cells to study cell resistance to BRAF inhibitors, CRISPR models of epithelial-mesenchymal transition for breast cancer, and pharmacogenomic analysis in various cancer cell lines. CRISPR is used in immunotherapy to help Chimeric Antigen Receptor T (CAR-T) cells function better by disrupting the immune checkpoints like Programmed Cell Death Protein 1 (PD-1) and Cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) and to adapt T cells to react with various antigens. As a result of these innovations, it is now possible to track how cancers like non-small cell lung cancer (NSCLC) and ovarian cancer evolve, change their epigenetic features, and find strategies to reverse their resistance. Moving forward, integrating AI analytics, single-cell multi-omics, patient-derived organoids, and CRISPR mechanisms will help improve precision oncology and speed up effective treatment planning.},
}

Humans
*Gene Editing
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy/pathology/drug therapy
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid41101730,
year = {2026},
author = {Zhang, H and Tonini, MR and Martires, LCM and Jenkins, HN and Pratt, CB and Gordon, EN and Zhao, S and Choi, AH and Meier, SR and Khendu, T and Liu, S and Shen, B and Stowe, H and Pashiardis, K and Pan, X and Bandi, M and Zhang, M and Yu, Y and Min, C and Huang, A and Andersen, JN and Nicholson, HE and Teng, T},
title = {Tumor suppressor collateral damage screens reveal mRNA homeostasis protein HBS1L as a novel vulnerability in ch9p21 driven FOCAD deleted cancer.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1873},
number = {1},
pages = {120070},
doi = {10.1016/j.bbamcr.2025.120070},
pmid = {41101730},
issn = {1879-2596},
mesh = {*GTP-Binding Proteins/genetics/metabolism ; *RNA, Messenger/metabolism ; Chromosome Deletion ; Chromosomes, Human, Pair 9 ; Genes, Tumor Suppressor ; Synthetic Lethal Mutations ; *Endonucleases/genetics/metabolism ; *Nuclear Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Humans ; *Tumor Suppressor Proteins/genetics/metabolism ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Animals ; Mice ; Female ; },
abstract = {Chromosomal deletion of tumor suppressor genes often occurs in an imprecise manner, leading to co-deletion of neighboring genes. This collateral damage can create novel dependencies specific to the co-deleted context. One notable example is the dependency on PRMT5 activity in tumors with MTAP deletion, which co-occurs with CDKN2A/B loss, leading to the development of MTA-cooperative PRMT5 inhibitors. To identify additional collateral damage context/target pairs for chromosome 9p and other common loci of chromosomal deletions, we conducted a combinatorial CRISPR screen knocking out frequently co-deleted genes in combination with a focused target library. We identified the gene encoding the ribosome rescue factor PELO as synthetic lethal with the SKI complex interacting protein FOCAD, which is frequently co-deleted alongside MTAP and CDKN2A/B on chromosome 9p. A genome-wide screen in FOCAD isogenic cells further identified the ribosome rescue GTPase and PELO binding partner HBS1L as the top synthetic lethal target for FOCAD loss. Analysis of publicly available data and genetic manipulation of HBS1L using orthogonal modalities validated this interaction. HBS1L dependency in FOCAD-deleted cells was rescued by FOCAD re-expression, and FOCAD intact cells could be rendered HBS1L-dependent by FOCAD knockout, demonstrating the context specificity of this interaction. Mechanistically, HBS1L loss led to translational arrest and activated the unfolded protein response in FOCAD-deleted cells. In vivo, HBS1L deletion eliminated growth of FOCAD-deleted tumors. Here we propose a model where the FOCAD/SKI complex and HBS1L/PELO work together to resolve aberrant mRNA-induced ribosomal stalling, making the HBS1L/PELO complex an intriguing novel target for treating FOCAD-deleted tumors.},
}

*GTP-Binding Proteins/genetics/metabolism
*RNA, Messenger/metabolism
Chromosome Deletion
Chromosomes, Human, Pair 9
Genes, Tumor Suppressor
Synthetic Lethal Mutations
*Endonucleases/genetics/metabolism
*Nuclear Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Knockout Techniques
Humans
*Tumor Suppressor Proteins/genetics/metabolism
*Neoplasms/genetics/pathology
Cell Line, Tumor
Xenograft Model Antitumor Assays
Animals
Mice
Female

@article {pmid41159851,
year = {2026},
author = {Coppola, MR and Bellitto, D and Asgari, E and Bazzurro, V and Casucci, G and Piacente, F and Bozzo, M and Ceresa, D and Parisi, C and Winata, C and Candiani, S and Tonetti, M},
title = {Zebrafish as a model for Catel-Manzke syndrome-identification and characterization of the zebrafish TGDS ortholog.},
journal = {The FEBS journal},
volume = {293},
number = {8},
pages = {2248-2269},
doi = {10.1111/febs.70307},
pmid = {41159851},
issn = {1742-4658},
support = {GMR22T1065//Fondazione Telethon/ ; PRIN 2022 - grant 22ETJJES//Ministero dell'Università e della Ricerca/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Gene Expression Regulation, Developmental ; Humans ; },
abstract = {Catel-Manzke syndrome (CMS) is a rare genetic disorder associated with mutations in the TDP-glucose 4,6-dehydratase (TGDS) gene, the function of which in vertebrates remains unclear. This study investigated the zebrafish ortholog tgds to assess its suitability for modeling the disease. During development, the tgds transcript exhibits a conserved biphasic expression pattern with an initial maternal contribution followed by a second wave of expression after gastrulation. Recombinant zebrafish Tgds expressed in Escherichia coli demonstrated UDP-D-glucose 4,6-dehydratase (EC4.2.1.76) activity, similar to TGDS orthologs in lower eukaryotes, where it acts as the first step in the L-rhamnose biosynthetic pathway. This finding suggests the presence of a yet unidentified pathway in vertebrates. Furthermore, CMS-associated mutations in conserved residues significantly impair enzyme activity and stability. CRISPR/Cas9-mediated F0 knockout of tgds resulted in a range of developmental defects in zebrafish. In particular, craniofacial cartilage alterations, associated with a decrease in sulfate glycosaminoglycan content, mirrored some skeletal features observed in humans with CMS. These findings establish the zebrafish as a relevant model to further explore CMS pathogenesis and the in vivo function of tgds.},
}

Animals
*Zebrafish/genetics/embryology/metabolism
Disease Models, Animal
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
Mutation
Gene Expression Regulation, Developmental
Humans

@article {pmid41851456,
year = {2026},
author = {Nyberg, WA and Bernard, PL and Ngo, W and Wang, CH and Ark, J and Rothrock, A and Borgo, GM and Kimmerly, GR and Jung, JH and Allain, V and Hamilton, JR and Baldwin, A and Stickels, R and Wyman, S and Khan, SH and Lang, S and Marsh, D and Almudhfar, N and Novick, C and Mortazavi, Y and Zhang, S and AbdElwakil, MM and Sandoval, LR and Hwang, S and Chu, SN and Jung, H and Liu, C and Sharma, D and McCreary, T and Li, Z and Satpathy, AT and Carnevale, J and Rutishauser, RL and Cromer, MK and Roybal, KT and Dodgson, SE and Doudna, JA and Asokan, A and Eyquem, J},
title = {In vivo site-specific engineering to reprogram T cells.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {712-721},
pmid = {41851456},
issn = {1476-4687},
mesh = {Animals ; Mice ; *T-Lymphocytes/metabolism/cytology/immunology ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; CRISPR-Cas Systems/genetics ; Humans ; Transgenes/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; Female ; *Cellular Reprogramming/genetics ; Genetic Vectors/genetics ; *Cell Engineering/methods ; Immunotherapy, Adoptive/methods ; Male ; },
abstract = {Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise[1]. However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR-Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.},
}

Animals
Mice
*T-Lymphocytes/metabolism/cytology/immunology
Receptors, Chimeric Antigen/genetics/metabolism/immunology
CRISPR-Cas Systems/genetics
Humans
Transgenes/genetics
*Gene Editing/methods
Dependovirus/genetics
Female
*Cellular Reprogramming/genetics
Genetic Vectors/genetics
*Cell Engineering/methods
Immunotherapy, Adoptive/methods
Male

@article {pmid41854269,
year = {2026},
author = {Fernandes, LGV and Putz, EJ and Nally, JE},
title = {The Leptospira immunoglobulin-like protein LigB from Leptospira borgpetersenii serovar Arborea is not required for either acute or chronic infection.},
journal = {Infection and immunity},
volume = {94},
number = {4},
pages = {e0066225},
doi = {10.1128/iai.00662-25},
pmid = {41854269},
issn = {1098-5522},
mesh = {Animals ; *Leptospirosis/microbiology/immunology ; *Leptospira/genetics/pathogenicity/immunology ; *Bacterial Proteins/genetics/metabolism ; Virulence Factors/genetics/metabolism ; Rats ; Virulence ; Persistent Infection/microbiology ; CRISPR-Cas Systems ; },
abstract = {Leptospirosis is a globally important zoonotic disease caused by more than 40 pathogenic Leptospira spp. that are responsible for more than 1 million human cases and almost 60,000 deaths annually. The disease also affects many companion, domestic, and wild animal species. Leptospiral immunoglobulin-like proteins (Lig), particularly LigA and LigB, are well-established surface membrane proteins that have been extensively studied due to their interactions with the host immune system. Silencing expression of both ligA and ligB in L. interrogans serovar Copenhageni results in attenuation of virulence, confirming their role as virulence factors. To examine the role of Lig proteins in other pathogenic species of Leptospira, we applied the CRISPR-Prime Editing (CRISPR-PE) strategy to engineer a one-nucleotide frameshift deletion in ligB, generating a knockout mutant in L. borgpetersenii serogroup Ballum serovar Arborea strain LR131, a pathogenic species that lacks ligA. Despite a complete loss of LigB expression, the mutant strain maintained its ability to cause acute lethal infection in hamsters and the ability to establish renal colonization in rats. These findings demonstrate that ligB alone is dispensable for both acute and chronic infection in the L. borgpetersenii strain LR131 background. This work represents the first targeted disruption of ligB in L. borgpetersenii as facilitated by CRISPR-PE and prompts a reevaluation of LigB as a universal virulence determinant in different genetic backgrounds. These insights are critical for advancing our understanding of leptospiral pathogenesis and guiding the design of broadly protective subunit vaccines.},
}

Animals
*Leptospirosis/microbiology/immunology
*Leptospira/genetics/pathogenicity/immunology
*Bacterial Proteins/genetics/metabolism
Virulence Factors/genetics/metabolism
Rats
Virulence
Persistent Infection/microbiology
CRISPR-Cas Systems

@article {pmid41954981,
year = {2026},
author = {Sentmanat, MF and Wang, ZT and Kouranova, E and Peters, ST and Chan, WC and Lin, J and Miao, Y and White, JM and Wallace, M and Cui, X},
title = {Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954981},
issn = {1362-4962},
support = {//Alvin J. Siteman Cancer Center/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Knock-In Techniques/methods ; *Dependovirus/genetics ; *Gene Editing/methods ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Genome ; },
abstract = {Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles, imprecision in the insert and donor concatenation, genotypes of non-KI alleles, and more importantly, uniquely identifies and localizes random integration of the full or partial donor(s). Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models.},
}

Animals
*CRISPR-Cas Systems
Mice
*Gene Knock-In Techniques/methods
*Dependovirus/genetics
*Gene Editing/methods
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Genome

@article {pmid41954985,
year = {2026},
author = {Zhang, S and Liu, Y and Wu, W and Liu, Z and He, Q and Wang, T and Yang, J and Yin, H and Yuan, Z and Zhang, H},
title = {Allosteric activation mechanism of the type VII CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954985},
issn = {1362-4962},
support = {32571682//National Natural Science Foundation of China/ ; 25JCZDJC00740//Natural Science Foundation of Tianjin Municipal Science and Technology Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; RNA/metabolism/chemistry/genetics ; Catalytic Domain ; Models, Molecular ; Ribonucleases/chemistry/metabolism/genetics ; },
abstract = {Type VII CRISPR-Cas system, evolutionarily associated with type III systems, utilizes a Cascade complex formed by Cas5 and catalytically inactive Cas7 copies for target RNA binding, but instead incorporates a specialized Cas14 ribonuclease for target cleavage. Here, we report a high-quality cryo-EM structure at the target engagement state with a shortened crRNA and elucidate how the recruited Cas14 captures the target RNA and undergoes target-mediated activation. The signature Cas14 is homologous to eukaryotic CPSF73 and prokaryotic RNase J, comprising two conserved subdomains, MβL and β-CASP. Different from canonical type III systems, 5'-end target RNA, rather than 3'-end, is bent into the positively charged binding channel formed by the two subdomains to access the conserved catalytic pocket on Cas14. Two special structural features, α1 helix from Cas7 and α10 helix from Cas14, promote the bent target RNA docking into the catalytic pocket of Cas14 nuclease in concert. A dual-functional loop, displaced by the entering target RNA, induces a closed-to-open transition between the two subdomains for nuclease activation. More importantly, the flipped dual-functional loop also maintains the stabilization of incoming target RNA. Altogether, our work provides a more comprehensive understanding of type VII system mechanism, laying a mechanistic foundation for RNA-targeting tool development.},
}

*CRISPR-Cas Systems/genetics
Allosteric Regulation
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
Cryoelectron Microscopy
RNA/metabolism/chemistry/genetics
Catalytic Domain
Models, Molecular
Ribonucleases/chemistry/metabolism/genetics

@article {pmid41956064,
year = {2026},
author = {Wang, M and Sternberg, SH},
title = {'Insane in the membrane': 2',3'-cGAMP triggers filamentous phage defense.},
journal = {Cell host & microbe},
volume = {34},
number = {4},
pages = {556-558},
doi = {10.1016/j.chom.2026.03.009},
pmid = {41956064},
issn = {1934-6069},
mesh = {*Nucleotides, Cyclic/metabolism ; *Inovirus/physiology ; Signal Transduction ; Humans ; Membrane Proteins/metabolism ; Nucleotidyltransferases/metabolism ; *Cell Membrane/metabolism/virology ; *Bacteria/metabolism/virology ; Animals ; },
abstract = {In this issue, Tak et al. report that bacteria produce 2',3'-cGAMP, a signaling molecule once considered unique to metazoans. This cyclic dinucleotide activates a SAVED-domain effector that polymerizes into membrane-disrupting filaments, inducing abortive infection and preventing phage replication, broadening the evolutionary scope of cGAS-STING-like signaling.},
}

*Nucleotides, Cyclic/metabolism
*Inovirus/physiology
Signal Transduction
Humans
Membrane Proteins/metabolism
Nucleotidyltransferases/metabolism
*Cell Membrane/metabolism/virology
*Bacteria/metabolism/virology
Animals

@article {pmid41956072,
year = {2026},
author = {Decloquement, M and Macauley, MS},
title = {Revealing cancer glycome drivers using CRISPR activation screens.},
journal = {Cell genomics},
volume = {6},
number = {4},
pages = {101215},
pmid = {41956072},
issn = {2666-979X},
mesh = {Humans ; *Neoplasms/genetics/metabolism ; Glycosylation ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glycomics/methods ; },
abstract = {In this study, Wisnovsky and colleagues[1] investigate the genetic determinants underlying cancer-associated glycome remodeling. Using genome-wide CRISPR-based approaches, they identified regulators of cell surface glycosylation. Specifically, genetic networks are discovered that upregulate cancer-related ligands of the sialic acid-binding immunomodulatory Siglecs.},
}

Humans
*Neoplasms/genetics/metabolism
Glycosylation
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Glycomics/methods

@article {pmid41959582,
year = {2026},
author = {Carrasco, E and Gutierrez-Marcos, J},
title = {Novel genome editing approaches to manipulate apical meristem activity for crop yield.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1743528},
pmid = {41959582},
issn = {1664-462X},
abstract = {Meristem function underlies organogenesis and yield potential in crop species, and its regulation depends on the crosstalk of genetic and hormonal networks that balance stem-cell niche maintenance and differentiation. During the shoot apical meristem (SAM) transition, developmental reprogramming shifts the meristem from a vegetative to a reproductive state, referred to as inflorescence meristem (IM). Major regulatory events in this transition include the cytokinin-gibberellin crosstalk, that regulate the expression of the CLAVATA/WUSCHEL (CLV/WUS) negative feedback loop and key transcription factor families like KNOTTED-LIKE HOMEOBOX (KNOX) and SHOOT MERISTEMLESS (STM). Despite the basic principles of apical meristem differentiation are well-described nowadays, major phenotypic bottlenecks were reached in major staple crops during the artificial selection process, known as domestication, leading to a final reduction in total crop yield. This review aims to describe the key processes and genes that play a role in this transition and how they can be artificially targeted to overcome these limitations. Major bioengineering approaches are covered, ranging from classical random mutagenesis with chemicals like ethyl methanesulfonate (EMS) to targeted genome editing using diverse Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR/Cas) systems. Finally, emerging strategies such as epibreeding are considered as promising tools to achieve precise, reversible modulation of meristem activity and to unlock new routes for crop yield enhancement.},
}

@article {pmid41123029,
year = {2026},
author = {Burnase, N and Kumar, B and Kumar, PS and Dharshini, RS and Das, R and Barapatre, A},
title = {Advances in Antiviral Strategies for Oral Herpes Infections in Immunocompromised Patients.},
journal = {Oral diseases},
volume = {32},
number = {2},
pages = {300-321},
doi = {10.1111/odi.70090},
pmid = {41123029},
issn = {1601-0825},
mesh = {Humans ; *Immunocompromised Host ; *Antiviral Agents/therapeutic use ; *Herpesviridae Infections/drug therapy/immunology ; Genetic Therapy ; *Stomatitis, Herpetic/drug therapy/immunology ; Drug Resistance, Viral ; RNA Interference ; CRISPR-Cas Systems ; *Herpes Simplex/drug therapy ; },
abstract = {OBJECTIVE: This review explores conventional antiviral therapies and innovative treatment strategies for managing oral herpes simplex virus (HSV) or human herpesvirus (HHV) infections, particularly in immunocompromised patients. It aims to evaluate clinical outcomes, address challenges such as drug resistance, and assess the sustainability of current and emerging therapeutic approaches.

SUBJECT: A descriptive review of current treatment strategies for oral HSV infections in immunosuppressed individuals is presented. Relevant studies are analyzed for their contributions, highlighting therapeutic advancements, limitations of existing drugs, and novel approaches in development.

RESULTS: Oral herpesvirus infections can lead to considerable morbidity and mortality in immunocompromised patients. Resistance to standard antivirals often develops due to prolonged or inconsistent use and impaired cell-mediated immunity. To overcome these issues, innovative strategies like gene therapy, RNA interference (RNAi), and CRISPR-Cas9 have emerged. These approaches aim to suppress viral gene expression, strengthen innate immunity, and genetically modify host cells to resist viral entry and replication.

CONCLUSION: Emerging antiviral therapies show promise in addressing the limitations of conventional treatments, particularly drug resistance and non-specific targeting. These novel approaches offer more effective, precise, and safer options for treating oral viral infections in immunosuppressed populations at heightened risk for severe outcomes.},
}

Humans
*Immunocompromised Host
*Antiviral Agents/therapeutic use
*Herpesviridae Infections/drug therapy/immunology
Genetic Therapy
*Stomatitis, Herpetic/drug therapy/immunology
Drug Resistance, Viral
RNA Interference
CRISPR-Cas Systems
*Herpes Simplex/drug therapy

@article {pmid41296247,
year = {2026},
author = {Aksoy, MO and Rozynek, J and Stachowiak, M and Szczerbal, I},
title = {The role of the CEBPB gene in porcine adipogenesis: a study using CRISPR/Cas9-edited mesenchymal stem cells.},
journal = {Journal of applied genetics},
volume = {67},
number = {2},
pages = {477-486},
pmid = {41296247},
issn = {2190-3883},
support = {2018/29/B/NZ2/00956//Narodowe Centrum Nauki/ ; },
mesh = {Animals ; *Adipogenesis/genetics ; *Mesenchymal Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; Swine/genetics ; *CCAAT-Enhancer-Binding Protein-beta/genetics ; *Gene Editing ; Cell Differentiation/genetics ; Promoter Regions, Genetic ; Adipocytes/cytology/metabolism ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a powerful tool for gene editing and the regulation of gene expression. It enables the introduction of targeted mutations, thereby facilitating functional studies of specific genes in various cellular processes. In this study, we aimed to generate a deletion in the promoter region of the CEBPB gene, which encodes a transcription factor involved in adipogenesis, and to evaluate the impact of this modification on the adipogenic differentiation potential of porcine mesenchymal stem cells (MSCs). A 575-bp deletion was introduced in the target region, resulting in the generation of both homozygous and heterozygous mutant cells. Adipogenic differentiation was assessed by quantifying transcript levels of adipocyte marker genes (GATA2, CEBPA, PPARG, and FABP4) at days 0, 4, 6, 8, and 10 of the differentiation process. Disruption of CEBPB expression led to the downregulation of these adipogenic markers, indicating impaired adipocyte differentiation. Additionally, to assess the proliferative capacity of the edited cells, the expression levels of proliferation-associated genes (CCND1, MCM2, and PCNA) were measured. A reduction in their transcript levels was observed in both homozygous and heterozygous mutant cells. These findings indicate that both homozygous and heterozygous deletions in the CEBPB promoter completely block adipogenesis and alter MSC proliferation, highlighting the pivotal role of CEBPB not only in adipogenic differentiation but also in the regulation of cell proliferation in porcine mesenchymal stem cells. These results provide new insights into the molecular mechanisms underlying adipose tissue development and have implications for pig breeding strategies aimed at optimizing carcass composition, as well as for biomedical research focused on adipose tissue biology.},
}

Animals
*Adipogenesis/genetics
*Mesenchymal Stem Cells/cytology/metabolism
*CRISPR-Cas Systems/genetics
Swine/genetics
*CCAAT-Enhancer-Binding Protein-beta/genetics
*Gene Editing
Cell Differentiation/genetics
Promoter Regions, Genetic
Adipocytes/cytology/metabolism

@article {pmid41885168,
year = {2026},
author = {Hanzelova, P and Baird, C and Keshinro, B and Kadhom, R and Lalonde, RL and Akimenko, MA},
title = {An essential role for actinotrichia in zebrafish fin patterning and courtship behavior.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {16},
pages = {},
doi = {10.1242/dev.204990},
pmid = {41885168},
issn = {1477-9129},
support = {RGPIN-2024-06801//Natural Sciences and Engineering Research Council of Canada/ ; PJT 166139/CAPMC/CIHR/Canada ; PJT 166139/CAPMC/CIHR/Canada ; //University of Ottawa/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics/physiology ; *Animal Fins/embryology/metabolism ; Male ; *Zebrafish Proteins/genetics/metabolism ; *Body Patterning/genetics ; Female ; *Courtship ; CRISPR-Cas Systems ; *Microfilament Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Cell Movement ; *Sexual Behavior, Animal/physiology ; },
abstract = {A key difference between tetrapod limb buds and teleost fin buds is the presence of rigid actinotrichia fibers that guide the migrating cells contributing to ray formation. Major structural components of actinotrichia are encoded by fish-specific actinodin (And) genes, which were lost in tetrapods. To investigate the consequences of this loss during the fin-to-limb transition, we generated deletions in zebrafish and1 and and2 using CRISPR/Cas9 mutagenesis. Double mutants (and1-/-and2-/-) lack actinotrichia. Embryos and larvae have reduced fin fold size, with disorganized cell migration. In adults, all fin fold-derived skeletal structures are disrupted, including the rays of all fins, as well as the caudal fin endoskeleton. Surprisingly, double mutant males fail to breed, despite being fertile. Video analysis revealed that defects in the fins of males impair their ability to stimulate egg release. Our findings highlight the role of actinotrichia in both fin patterning and zebrafish courtship. We propose that actinodin gene maintenance is under strong selection in fish with similar courtship. We speculate that the loss of actinodin genes and a shift in courtship strategy may have coincided during tetrapod evolution.},
}

Animals
*Zebrafish/embryology/genetics/physiology
*Animal Fins/embryology/metabolism
Male
*Zebrafish Proteins/genetics/metabolism
*Body Patterning/genetics
Female
*Courtship
CRISPR-Cas Systems
*Microfilament Proteins/genetics/metabolism
Gene Expression Regulation, Developmental
Cell Movement
*Sexual Behavior, Animal/physiology

@article {pmid41911023,
year = {2026},
author = {Ye, J and Tang, X and Chen, Y and Guo, R and Zhang, W and Wang, M and Yang, X and Deng, W and Zhang, Y and Feng, X and Hu, N and Zhang, D},
title = {Conformationally Regulated CRISPR/Cas12a Activation Enabled by a Programmable DNA Dumbbell for Electrochemical SNP Genotyping.},
journal = {Analytical chemistry},
volume = {98},
number = {14},
pages = {10814-10823},
doi = {10.1021/acs.analchem.6c00333},
pmid = {41911023},
issn = {1520-6882},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *DNA/chemistry/genetics ; *Genotyping Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; Genotype ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Single-nucleotide polymorphism (SNP) genotyping is crucial for genetic research and precision medicine, yet reliable discrimination of single-base variants in complex genomic backgrounds remains analytically challenging. Although CRISPR/Cas12a-based biosensing offers high sequence specificity, its intrinsic mismatch tolerance often leads to nonspecific activation by wild-type sequences, thereby compromising SNP fidelity. Herein, we report a highly sensitive electrochemical sensing platform based on a programmable DNA dumbbell (Dum) probe that functions as a conformational energy-barrier regulator of CRISPR/Cas12a activation. The closed-loop dumbbell architecture sterically shields the crRNA-activating sequence, establishing a high activation threshold that suppresses nonspecific Cas12a triggering. Only precise SNP hybridization induces a thermodynamically favorable conformational rearrangement, releasing the mediator and transitioning the CRISPR system from an inactive to an active state. To further enhance analytical sensitivity and reliability, nucleic acid-functionalized FeCo nanozymes were incorporated as catalytic signal transducers, enabling a self-validating dual-mode signal electrochemical readout through intrinsic metal redox and H2O2 electrocatalysis. The resulting platform achieved reliably discriminates mutation abundances down to 0.1%. Validation using soybean genomic DNA samples demonstrates the robustness and practical applicability of the proposed strategy. This work establishes a conformational energy-barrier-regulated CRISPR activation paradigm, providing a generalizable analytical framework for high-fidelity SNP genotyping in molecular breeding and clinical diagnostics.},
}

*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*DNA/chemistry/genetics
*Genotyping Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics
*Biosensing Techniques/methods
Genotype
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins

@article {pmid41952451,
year = {2026},
author = {Mengistu, G},
title = {CRISPR-Cas Systems in Human Disease Therapy: Advances, Clinical Applications, Limitations, and Future Directions.},
journal = {The journal of gene medicine},
volume = {28},
number = {4},
pages = {e70091},
doi = {10.1002/jgm.70091},
pmid = {41952451},
issn = {1521-2254},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/trends ; Precision Medicine ; Animals ; },
abstract = {CRISPR-Cas systems have emerged as versatile platforms for targeted genome and transcriptome engineering, enabling precise manipulation of disease-associated genetic pathways. Continued advances in CRISPR technologies including base editing, prime editing, and epigenome modulation have expanded therapeutic possibilities beyond nuclease-mediated DNA cleavage, allowing programmable gene correction and regulation. Early clinical studies demonstrate sustained therapeutic benefit in selected monogenic disorders and highlight the feasibility of both ex vivo and in vivo editing strategies. However, clinical translation remains constrained by challenges such as off-target activity, delivery inefficiency, immune responses to Cas proteins, editing heterogeneity, and uncertainties regarding long-term safety. This review critically synthesizes recent advances in CRISPR-Cas systems for human disease therapy, integrating molecular innovations, delivery strategies, clinical progress, and ethical considerations. By evaluating both technological achievements and unresolved limitations, this article outlines key priorities for advancing CRISPR-based therapeutics toward safe, effective, and equitable precision medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Therapy/methods/trends
Precision Medicine
Animals

@article {pmid41952472,
year = {2026},
author = {Martinkienė, J and Cui, T and Ciotta, G and Kupčinskas, J and Pažėraitis, D},
title = {RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.},
journal = {BioTechniques},
volume = {78},
number = {1-12},
pages = {149-157},
doi = {10.1080/07366205.2026.2649857},
pmid = {41952472},
issn = {1940-9818},
mesh = {*DNA, Single-Stranded/isolation & purification/genetics/chemistry ; Chromatography, High Pressure Liquid/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Chromatography, Reverse-Phase/methods ; },
abstract = {BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8[+] T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.},
}

*DNA, Single-Stranded/isolation & purification/genetics/chemistry
Chromatography, High Pressure Liquid/methods
Humans
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Chromatography, Reverse-Phase/methods

@article {pmid41954204,
year = {2026},
author = {Zheng, S and Wang, C and Huang, Y and Meng, X and Zhao, D and Guan, E and Ma, F and Chen, A and Zheng, L},
title = {Dual-Mode CRISPR/Cas13a Assay for the Detection of Human Metapneumovirus in Clinical Respiratory Samples.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70921},
doi = {10.1002/jmv.70921},
pmid = {41954204},
issn = {1096-9071},
support = {2025YFC2311704//National Key R&D Program of China/ ; },
mesh = {Humans ; *Metapneumovirus/isolation & purification/genetics ; Sensitivity and Specificity ; *Paramyxoviridae Infections/diagnosis/virology ; *Respiratory Tract Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; Reproducibility of Results ; *Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction ; RNA, Viral/genetics ; },
abstract = {Human metapneumovirus (HMPV) is a significant pathogen, causing widespread acute respiratory infections (ARIs). Rapid and accurate detection is crucial for timely diagnosis and outbreak control. To address the limitations of real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based HMPV detection, we developed a novel method integrating recombinase-aided amplification (RAA) with CRISPR-Cas13a technology. Based on the alignment of 335 HMPV whole-genome sequences, a conserved region of the nucleocapsid (N) gene was selected for RAA primer and CRISPR RNA (crRNA) design. The concentrations of Cas protein, crRNA, and probe were optimized for specificity, sensitivity, and repeatability. A dual-mode platform was established, combining a fluorescence assay for quantitative detection and a lateral flow assay (LFA) for visual, on-site readout. Both methods were validated using 106 clinical samples and compared with RT-qPCR. Both methods demonstrated high specificity (no cross-reactivity with other respiratory viruses), a detection limit of 1 copy/μL, and excellent repeatability. Clinical validation showed complete concordance (κ = 1, p < 0.001) between the fluorescence assay and RT-qPCR, and high agreement for LFA (κ = 0.924, p < 0.001), with a sensitivity of 94.23%, specificity of 98.15%, positive predictive value (PPV) of 98%, and negative predictive value (NPV) of 94.6%. This study presents an RAA-CRISPR/Cas13a dual-mode platform for HMPV detection, providing a robust molecular diagnostic framework that paves the way for the future development of rapid testing assays.},
}

Humans
*Metapneumovirus/isolation & purification/genetics
Sensitivity and Specificity
*Paramyxoviridae Infections/diagnosis/virology
*Respiratory Tract Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
Reproducibility of Results
*Nucleic Acid Amplification Techniques/methods
Real-Time Polymerase Chain Reaction
RNA, Viral/genetics

@article {pmid41954977,
year = {2026},
author = {Duan, X and Zhang, H and Lv, J and Fan, X and Liu, X and Wang, Q and Zhang, F and Zhang, Q and Liu, L and Liang, L and Liu, R},
title = {PE-STAR: prime editing with SOS-triggered and RecJ-augmented repair enables high-efficiency editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954977},
issn = {1362-4962},
support = {2023YFC3402300//National Key R&D Program of China/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project/ ; 22278058//National Natural Science Foundation of China/ ; 22578048//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics/metabolism ; *SOS Response, Genetics/genetics ; *DNA Repair ; CRISPR-Cas Systems ; Exodeoxyribonucleases/genetics/metabolism ; Plasmids/genetics ; },
abstract = {Prime editing enables precise genome modifications without DNA double-strand breaks, yet bacterial applications are limited by low efficiency and small edit sizes. Here, we develop PE-STAR, Prime Editing with SOS-Triggered and RecJ-Augmented Repair, to enhance prime editing in Escherichia coli. Removing three inhibitory 3'→5' exonucleases (SbcB, ExoX, and XseA) improved edited-strand retention, and extending post-transformation outgrowth increased editing efficiency. RecJ overexpression strengthened 5'-directed processing during flap resolution and gap expansion, biasing repair toward incorporation of the reverse-transcribed edited strand. To enrich edited cells, we integrated an SOS-responsive counter-selection circuit that links PE3-associated dual nicking to LexA-dependent gRNA expression targeting a plasmid encoding the toxin CcdB, thereby eliminating unedited cells. PE-STAR achieved up to 80%-90% editing efficiency for short-fragment modifications, representing up to 16-fold improvement across loci. The platform supported insertions, deletions, and replacements of up to 46 bp with high efficiency. Furthermore, installing an attB site by prime editing, followed by Bxb1 integrase recombination, enabled chromosomal integration of 3.2 and 8.0 kb cassettes with 100% recombination efficiency among screened colonies, including GFP reporter and riboflavin biosynthetic pathway. PE-STAR expands both the efficiency and functional scope of bacterial prime editing for programmable genome engineering.},
}

*Gene Editing/methods
*Escherichia coli/genetics
*Escherichia coli Proteins/genetics/metabolism
*SOS Response, Genetics/genetics
*DNA Repair
CRISPR-Cas Systems
Exodeoxyribonucleases/genetics/metabolism
Plasmids/genetics

@article {pmid41168408,
year = {2026},
author = {Ke, Z and Zhang, Q and Chen, X and Jin, R and Han, G and Cao, Q and Zhu, K and Wei, S and Chen, J and Wang, Q and Zhang, M and Huang, W and Li, K and Xiong, K and Lu, K and Chen, Y and Cao, C},
title = {CRISPR/Cas9 library screening uncovered CCT2 as a critical driver of acquired resistance to EGFR-targeted therapy by stabilizing TMX1 in non-small cell lung cancer.},
journal = {Cell death and differentiation},
volume = {33},
number = {4},
pages = {860-876},
pmid = {41168408},
issn = {1476-5403},
mesh = {*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/metabolism/pathology ; Humans ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; *Drug Resistance, Neoplasm/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; ErbB Receptors/metabolism/antagonists & inhibitors ; Animals ; Mice ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; Mice, Nude ; Protein Stability ; *Thioredoxins/metabolism ; *Membrane Proteins/metabolism ; },
abstract = {In the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), the emergence of acquired resistance remains a significant challenge. Elucidating the underlying mechanisms of resistance is crucial for developing novel strategies to overcome or delay therapeutic escape. To this end, this study aimed to identify key drivers of EGFR-TKIs resistance and explore actionable targets for intervention. We investigated resistance mechanisms by integrating CRISPR/Cas9-based genome-wide screening with tandem mass tag (TMT) proteomic analysis, and virtually screened bioactive small molecule libraries to identify compounds capable of restoring EGFR-TKIs sensitivity. The multi-omics approach revealed that CCT2 is a critical mediator of resistance to third-generation EGFR-TKIs in lung cancer, with higher expression of CCT2 observed in resistant cells compared to sensitive cells. Mechanistically, CCT2 recruits tripartite motif-containing protein 28 (TRIM28) to catalyze SUMO2 modification of thioredoxin-related transmembrane protein 1 (TMX1), inhibiting its ubiquitination and enhancing protein stability. This post-translational modification (PTM) promotes TMX1-dependent reactive oxygen species (ROS) clearance, thereby conferring resistance. Importantly, pharmacological inhibition with the compound HY-10127, identified through virtual screening, effectively restored EGFR-TKIs sensitivity in resistant cell lines and delayed the development of resistance in xenograft models. The findings establish the CCT2/TRIM28/TMX1/ROS axis as a novel resistance mechanism in EGFR-mutated lung cancer, and targeting this pathway with HY-10127 represents a promising strategy to overcome resistance to third-generation EGFR-TKIs, providing preclinical rationale for clinical translation. These discoveries advance our understanding of molecular resistance mechanisms and offer potential therapeutic targets for improving lung cancer prognosis.},
}

*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/metabolism/pathology
Humans
*Lung Neoplasms/drug therapy/genetics/pathology/metabolism
*Drug Resistance, Neoplasm/drug effects/genetics
*CRISPR-Cas Systems/genetics
ErbB Receptors/metabolism/antagonists & inhibitors
Animals
Mice
Protein Kinase Inhibitors/pharmacology/therapeutic use
Cell Line, Tumor
Mice, Nude
Protein Stability
*Thioredoxins/metabolism
*Membrane Proteins/metabolism

@article {pmid41546459,
year = {2026},
author = {Niu, C and Fang, S and Zeng, B and Liu, D and Huang, K and Zhang, G and Qian, N and Li, D and Ye, C},
title = {Development of a PCR-Cas12a-LFD visual detection system for highly sensitive and specific detection of Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp. in tobacco.},
journal = {Pest management science},
volume = {82},
number = {5},
pages = {4169-4182},
doi = {10.1002/ps.70528},
pmid = {41546459},
issn = {1526-4998},
support = {//Youth Support Program of Zhengzhou Tobacco Research Institute, China National Tobacco Corporation (CNTC) (No. 702024CR0070)/ ; },
mesh = {*Nicotiana/microbiology ; *Plant Diseases/microbiology ; *Pseudomonas/isolation & purification/genetics ; *Alternaria/isolation & purification/genetics ; *Phytophthora/isolation & purification/genetics ; *Ralstonia/isolation & purification/genetics ; *Polymerase Chain Reaction/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; },
abstract = {Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp. are the major pathogens responsible for tobacco diseases and severe threats to the sustainable development of the tobacco industry. Traditional detection methods, such as plate isolation and polymerase chain reaction (PCR), have improved in sensitivity and timeliness but remain limited by operational complexity, heavy reliance on high expertise requirements for result interpretation. This study focuses on the trans-cleavage activity of the CRISPR-Cas12a system and develops a series of novel detection methods for Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp., respectively. By screening specific genomic targets, designing highly sensitive primers and CRISPR RNA (crRNA), and integrating PCR with lateral flow dipstick (LFD), the PCR-Cas12a-LFD detection system was established with a sensitivity of 100 pg μL[-1] for Ralstonia sp., 10 pg μL[-1] for Phytophthora sp., 5000 pg μL[-1] for Alternaria sp., and 0.1 pg μL[-1] for Pseudomonas sp., respectively, and excellent specificity without cross-reactivity. Furthermore, the combination of this method with LFD enables visual interpretation of results. This study provides an efficient and visualized diagnostic tool for the early prevention and control of tobacco diseases during field cultivation while offering a theoretical reference for the molecular detection of other plant pathogens. © 2026 Society of Chemical Industry.},
}

*Nicotiana/microbiology
*Plant Diseases/microbiology
*Pseudomonas/isolation & purification/genetics
*Alternaria/isolation & purification/genetics
*Phytophthora/isolation & purification/genetics
*Ralstonia/isolation & purification/genetics
*Polymerase Chain Reaction/methods
*CRISPR-Cas Systems
Sensitivity and Specificity

@article {pmid41778754,
year = {2026},
author = {Hoyle, C and Pino, RD and Lai, SM and Green, JP and Adamson, A and Coutts, G and Lawrence, CB and Travis, M and Brough, D and Lopez-Castejon, G},
title = {Inflammatory responses following CRISPR modification of the nuclear localisation sequence in endogenous interleukin-1 alpha.},
journal = {Disease models & mechanisms},
volume = {19},
number = {4},
pages = {},
doi = {10.1242/dmm.052705},
pmid = {41778754},
issn = {1754-8411},
support = {Fellowship//Dowager Countess Eleanor Peel Trust/ ; BB/Y004876/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; SRB1013//Dowager Countess Eleanor Peel Trust/ ; MR/T016515/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *Interleukin-1alpha/metabolism/genetics/chemistry ; *Nuclear Localization Signals/genetics/metabolism ; *Inflammation/genetics/pathology ; Lipopolysaccharides/pharmacology ; *Cell Nucleus/metabolism/drug effects ; Macrophages/metabolism/drug effects ; Mice ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Peritonitis/genetics/pathology ; },
abstract = {Interleukin (IL)-1α is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1β, pro-IL-1α is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1α is proposed to regulate its secretion. To date, studies on the nuclear role of pro-IL-1α have used overexpression systems. Here, we generated a mouse strain in which the endogenous Il1a gene was edited using CRISPR to disrupt the NLS, yielding a mutated NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1α RNA expression levels in response to lipopolysaccharide (LPS) but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1α in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1α release were detected, potentially due to increased levels of pro-IL-1α. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1α nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1α function.},
}

Animals
*Interleukin-1alpha/metabolism/genetics/chemistry
*Nuclear Localization Signals/genetics/metabolism
*Inflammation/genetics/pathology
Lipopolysaccharides/pharmacology
*Cell Nucleus/metabolism/drug effects
Macrophages/metabolism/drug effects
Mice
Mice, Inbred C57BL
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Peritonitis/genetics/pathology

@article {pmid41926379,
year = {2026},
author = {Boy, RL and Zampieri, RA and Aoki, JI and Coelho, AC and Floeter-Winter, LM and Laranjeira-Silva, MF},
title = {Glycosomal Aquaglyceroporin 1 dual role in iron homeostasis and antimony susceptibility in Leishmania amazonensis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {4},
pages = {e0014141},
doi = {10.1371/journal.pntd.0014141},
pmid = {41926379},
issn = {1935-2735},
mesh = {Animals ; *Antimony/pharmacology ; Mice ; Homeostasis ; *Iron/metabolism ; Macrophages/parasitology ; *Leishmania mexicana/drug effects/metabolism/genetics ; *Antiprotozoal Agents/pharmacology ; *Protozoan Proteins/genetics/metabolism ; Female ; *Aquaporin 1/genetics/metabolism ; Leishmaniasis/parasitology ; Mice, Inbred BALB C ; *Leishmania/drug effects/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major, localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9-mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (SbIII), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in SbIII IC50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance.},
}

Animals
*Antimony/pharmacology
Mice
Homeostasis
*Iron/metabolism
Macrophages/parasitology
*Leishmania mexicana/drug effects/metabolism/genetics
*Antiprotozoal Agents/pharmacology
*Protozoan Proteins/genetics/metabolism
Female
*Aquaporin 1/genetics/metabolism
Leishmaniasis/parasitology
Mice, Inbred BALB C
*Leishmania/drug effects/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41946722,
year = {2026},
author = {Malone, HA and Myers, JA and Gruss, EG and Morgan, MA and Friske, JD and McCarty, TC and Navarro, JJ and Robinson, S and Halliburton, RL and Kietlinska, SJ and De Luna Vitorino, FN and Hansen, BS and Pruett-Miller, SM and Garcia, BA and Roussel, MF and Partridge, JF and Roberts, CWM},
title = {PHIP suppresses NuRD to enable the growth of SWI/SNF-mutant cancers.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41946722},
issn = {2041-1723},
support = {R01-CA-273455//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-172152//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-113794//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; F31-CA-278355//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P01-CA-96832//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01-HD-106051//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P01-CA-196539//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Neoplasms/genetics/metabolism/pathology ; *Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism/genetics ; Cell Line, Tumor ; *Transcription Factors/genetics/metabolism ; Mutation ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; Chromatin Assembly and Disassembly/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Chromatin/metabolism ; Promoter Regions, Genetic ; Transcriptional Activation ; Ubiquitination ; },
abstract = {SWI/SNF chromatin remodeling complexes are perturbed in 20% of all cancers and in several developmental disorders, yet the mechanisms by which these mutations dysregulate transcription and drive disease are poorly understood. To both elucidate these mechanisms and identify vulnerabilities caused by these mutations, we leverage genome-wide CRISPR-Cas9 screening in hundreds of cancer cell lines and identify the chromatin reader protein PHIP as a specific dependency in cancers with broadly disrupted SWI/SNF function. Mechanistically, we reveal that PHIP cooperates with SWI/SNF to facilitate transcriptional activation by ubiquitinating and suppressing subunits of the repressive Nucleosome Remodeling and Deacetylase (NuRD) complex. We demonstrate that loss of SWI/SNF results in NuRD complexes accumulating at promoters where they would otherwise cause widespread transcriptional silencing if not antagonized by PHIP. Collectively, we identify PHIP as a regulator of the interplay between distinct chromatin regulators that function in development and disease and as a targetable vulnerability in cancers with broad SWI/SNF inactivation.},
}

Humans
*Neoplasms/genetics/metabolism/pathology
*Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism/genetics
Cell Line, Tumor
*Transcription Factors/genetics/metabolism
Mutation
*Chromosomal Proteins, Non-Histone/genetics/metabolism
Chromatin Assembly and Disassembly/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Chromatin/metabolism
Promoter Regions, Genetic
Transcriptional Activation
Ubiquitination

@article {pmid41946927,
year = {2026},
author = {Fang, GQ and Deng, Y and Lyu, XY and Yin, CQ and Song, J and Zhang, Y and Zhong, J and Shen, EZ and Song, CQ},
title = {Boosting prime editing with engineered non-canonical pegRNAs.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41946927},
issn = {2157-846X},
support = {32471552//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNA (pegRNA), with the reverse transcription template and primer binding site (RTT-PBS) attached to the 3' ends of CRISPR-Cas guide RNAs. Although PE ribonucleoprotein (RNP) delivery holds great therapeutic potential, its weak genomic editing capability limits therapeutic applications. Here we present structure-guided engineering of the PE complex using non-canonical pegRNAs (npegRNAs), with the RTT-PBS integrated within the single guide RNA loops, to improve PE efficiency. This approach demonstrates enhanced precise editing rates across various genomic sites and cell types, and improves therapeutic gene correction in a tyrosinaemia mouse model. Cas9-associated npegRNAs are more resistant to exonuclease degradation, probably enhancing the PE complex's targeting efficiency in living cells. Using PE RNP delivery, npegRNAs achieve increased average editing yields of 26.8-fold over canonical pegRNAs and 5.9-fold over engineered pegRNAs (epegRNAs). Furthermore, npegRNA-mediated RNPs increased the efficiency of installing disease-relevant mutations up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, our findings demonstrate a robust PE strategy and highlight the potential of npegRNAs for therapeutic PE applications.},
}

@article {pmid41949766,
year = {2026},
author = {Mostafa, K and Scarano, A and Abdulla, MF and Hacıkamiloğlu, S and Kurt, O and Santino, A and Kavas, M},
title = {A rapid Agrobacterium rhizogenes-mediated transient expression for assessing sgRNA efficiency in CRISPR-Act3.0 in tomato.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {41949766},
issn = {1432-203X},
support = {121O463//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Agrobacterium/genetics ; Plants, Genetically Modified ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Fatty Acids/metabolism ; Transformation, Genetic ; Promoter Regions, Genetic ; },
abstract = {CRISPR-Act3.0 is a robust tool for modulating fatty acid profiles in plants. We demonstrate that Agrobacterium rhizogenes-mediated transformation provides a rapid, cost-effective, and equipment-independent platform for validating sgRNA efficiency and metabolic outcomes within a short time. The CRISPR-Act3.0 system offers a powerful strategy for activating endogenous gene expression in plants. However, the labor-intensive and time-consuming nature of stable transformation often hinders the rapid validation of multiple sgRNAs. In this study, we optimized a rapid Agrobacterium rhizogenes-mediated transient expression system in tomato to evaluate sgRNA efficiency within the CRISPR-Act3.0 framework. As a proof-of-concept, we targeted four genes involved in fatty acid biosynthesis: SlFATA, SlFATB-01, SlFATB-02, and SlFATB-03. To ensure precise control, we utilized the root-specific pSMB promoter to drive the CRISPRa components. Our results demonstrate that this system can successfully induce significant transcriptional activation and alter fatty acid compositions specifically increasing palmitic acid levels by up to 45%-within approximately 30 days. This approach bypasses the requirement for whole-plant stable transformation during the initial screening phase and prevents potential pleiotropic effects by restricting activation to root tissues. Overall, this study provides a highly efficient diagnostic pipeline for functional genomics and metabolic engineering in tomato, offering a significant advantage for rapid trait evaluation before committing to stable transgenic line production.},
}

*Solanum lycopersicum/genetics/metabolism
*Agrobacterium/genetics
Plants, Genetically Modified
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Fatty Acids/metabolism
Transformation, Genetic
Promoter Regions, Genetic

@article {pmid41951520,
year = {2026},
author = {He, Y and Ma, Y and Wu, Y and Tang, X and Liu, S and Yin, D and Zheng, X and Qi, Y and Zhang, Y and Zhang, T},
title = {Harnessing diverse tRNAs and AI-guided mining for compact and efficient plant multiplex genome editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.02.016},
pmid = {41951520},
issn = {1879-3096},
abstract = {The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.},
}

@article {pmid41951915,
year = {2026},
author = {Zhu, Y and Moerner, WE and Qi, LS},
title = {CRISPR-Cas-based live cell imaging of genome dynamics.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {41951915},
issn = {1471-0064},
abstract = {The 3D architecture and dynamics of the genome are crucial for regulation of genome stability, transcription and cellular function. CRISPR-based live imaging technologies have enabled real-time visualization of specific genomic loci and transcripts in living cells. These tools harness customized guide RNAs and nuclease-deactivated Cas effectors to achieve precise genomic targeting, and recent methodological advances provide the 3D spatiotemporal resolution required to decipher real-time chromatin communication. These methods are elucidating the biophysical properties of chromatin, linking dynamic enhancer-promoter interactions directly to transcription, and revealing the role of 3D genome dynamics in basic cellular processes and disease. Here, we summarize the development of CRISPR-based live-cell imaging techniques, highlight the complementary 3D microscopy and analysis methods compatible with these methods, and offer perspectives on their applications to uncover fundamental principles that govern genome dynamics and function.},
}

@article {pmid41952056,
year = {2026},
author = {Salum, YM and Chen, J and Dang, J and Qiao, Q and Chen, T and Lin, T and Wei, H and He, W},
title = {Leveraging RNAi and CRISPR/Cas9-based strategies for target gene characterization and control of western flower thrips (Frankliniella occidentalis): Advances and perspectives.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70782},
pmid = {41952056},
issn = {1526-4998},
support = {//Major Project of Science and Technology of Fujian Province (2024NZ029029)/ ; //Fujian Provincial Science and Technology Project (2023L3021 and 2025N0081)/ ; },
abstract = {Frankliniella occidentalis, the western flower thrips, is among the most destructive agricultural pests worldwide and a major vector of orthotospoviruses. Escalating insecticide resistance has intensified efforts to develop molecularly informed control strategies. RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing have emerged as principal tools for functional genomics in this species, enabling targeted analysis of genes involved in development, metabolism, and insecticide resistance. This review synthesizes recent advances in RNAi and CRISPR applications in F. occidentalis, with particular emphasis on delivery strategies, editing efficiencies, and resulting phenotypes. RNAi studies demonstrate growing technical feasibility through injection, feeding-based approaches, and plant-mediated systems, and emerging CRISPR/Cas investigations confirm the possibility of targeted gene knockouts affecting resistance traits and visible genetic markers. Despite these advances, genetic manipulation of F. occidentalis remains technically challenging, primarily due to biological and technical constraints, including inefficient delivery of ribonucleoprotein to the germline, the extremely small and fragile nature of eggs, and narrow developmental windows for manipulation. We discuss these barriers and highlight methodological innovations required to improve RNAi stability, embryo manipulation, and genome editing efficiency. Finally, we propose how these molecular and genetic tools may be incorporated into existing integrated pest management, emphasizing their potential as complementary, species-specific strategies rather than standalone control solutions toward sustainable F. occidentalis management. © 2026 Society of Chemical Industry.},
}

@article {pmid41952275,
year = {2026},
author = {Das, U and Prasad, SS and Sahoo, T and Paramanik, S and Halder, A and S, P},
title = {Unveiling the potential of banana (Musa spp.) improvement through genetic manipulation: current trends and future implications.},
journal = {Plant signaling & behavior},
volume = {21},
number = {1},
pages = {2656013},
doi = {10.1080/15592324.2026.2656013},
pmid = {41952275},
issn = {1559-2324},
mesh = {*Musa/genetics/microbiology ; Gene Editing ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Disease Resistance/genetics ; Plant Breeding ; Plant Diseases/microbiology/genetics ; },
abstract = {Banana (Musa spp.) is a globally important fruit crop and a staple food for millions of people. However, its narrow genetic diversity and clonal propagation make it highly vulnerable to pests, diseases, and abiotic stresses. Genetic improvement is limited by sterility, and triploid plants are reproduced clonally in most cultivated varieties, preventing traditional breeding based on genetic advancements. Transgenesis and gene editing are among the genetic engineering techniques used to increase yield, improve quality, and enhance resilience. Advances in Agrobacterium-mediated transformation and CRISPR/Cas tools have enabled the development of bananas with enhanced resistance to Fusarium wilt tropical race 4 (TR4), Black Sigatoka, and bacterial wilt, along with increased provitamin A content, longer shelf life, and reduced postharvest losses. DNA-free genome editing provides a promising approach to overcoming certain regulatory barriers and enhancing public acceptance. While challenges such as genotype-specific transformation efficiency, regulatory hurdles, and public perception persist, genetic manipulation holds the potential to both preserve and improve global banana production. This review synthesizes recent progress, key targets, and future prospects for the genetic improvement of banana.},
}

*Musa/genetics/microbiology
Gene Editing
Plants, Genetically Modified/genetics
Genetic Engineering/methods
Disease Resistance/genetics
Plant Breeding
Plant Diseases/microbiology/genetics

@article {pmid41796459,
year = {2026},
author = {Matsuoka, T and Oda, K and Iwashita, K and Watanabe, J},
title = {Identification of DeuA, an Aspergillus oryzae-derived deuterolysin-like metalloprotease, as the predominant thermostable protease in soy sauce.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {6},
pages = {445-449},
doi = {10.1016/j.jbiosc.2026.02.010},
pmid = {41796459},
issn = {1347-4421},
mesh = {*Aspergillus oryzae/enzymology/genetics ; *Soy Foods/microbiology/analysis ; *Metalloproteases/genetics/metabolism/chemistry ; Fermentation ; Enzyme Stability ; *Fungal Proteins/genetics/metabolism/chemistry ; *Peptide Hydrolases/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {The thermostable proteases present in soy sauce can degrade proteins in processed foods, such as boiled eggs and fish cakes, leading to undesirable textural changes in the product. In this study, we identified DeuA, a deuterolysin-like metalloprotease from Aspergillus oryzae, as the major contributor to thermostable protease activity during soy sauce fermentation. Using CRISPR/Cas9-based genome co-editing, we generated deuA-knockout mutants and mutants of the related gene deuB, which encodes a deuterolysin-like metalloprotease, and evaluated their enzymatic activity levels under solid-state culture conditions mimicking soy sauce koji fermentation. The ΔpyrGΔdeuA strain exhibited a marked reduction in thermostable protease activity, with residual activity barely detectable in both the koji extracts and the final soy sauce, whereas knockout of deuB had no significant effect. These results indicate that DeuA is the predominant contributor to thermostable protease activity in soy sauce. The knockout of deuA did not affect other key brewing parameters such as the nitrogen or sugar contents, indicating the potential of this gene as a target for strain improvement. Our findings establish DeuA as an essential thermostable protease in soy sauce and provide a foundation for the development of brewing strains with improved industrial applicability that will not affect the textural stability of processed foods.},
}

*Aspergillus oryzae/enzymology/genetics
*Soy Foods/microbiology/analysis
*Metalloproteases/genetics/metabolism/chemistry
Fermentation
Enzyme Stability
*Fungal Proteins/genetics/metabolism/chemistry
*Peptide Hydrolases/metabolism/genetics
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid41812798,
year = {2026},
author = {Huang, G and Zhuge, B and Du, X and Wang, M and Zong, H},
title = {TSA-ultrasound synergy enhances CRISPR-Cas9 gene editing efficiency in diploid yeast.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107449},
doi = {10.1016/j.mimet.2026.107449},
pmid = {41812798},
issn = {1872-8359},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Diploidy ; *Hydroxamic Acids/pharmacology ; Gene Knockout Techniques ; *Candida/genetics/drug effects ; Sonication/methods ; Chromatin ; },
abstract = {The CRISPR-Cas9 system is invaluable for microbial engineering. However, its efficiency remains limited in numerous microorganisms, especially in polyploid yeasts where the compact chromatin structures pose significant physical barriers, which are major constraints in microbial engineering. To address this limitation, we developed and validated a standardized synergistic protocol using the diploid industrial yeast Candida glycerinogenes as a model. This protocol combines TSA-induced chromatin decondensation with brief, low-intensity ultrasonication to enhance Cas9 accessibility. Key parameters were systematically optimized, and 200 nM Trichostatin A (TSA) (10 h) followed by 200 W sonication (3 min) were established as the optimal condition set. Validation results showed that the protocol more than doubled single-gene knockout efficiencies (GPD1, TRP1) compared with conventional methods. Importantly, it enabled complex edits that were previously unattainable in C. glycerinogenes, including the precise deletion of a 7.8-kb fragment and the editing of an 11.4-kb region for functional genomics. In summary, this study establishes a simple and effective workflow that overcomes chromatin-based barriers in the polyploid industrial yeast C. glycerinogenes, providing a practical tool for genetic engineering and functional genomics in this and potentially other recalcitrant yeasts.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Diploidy
*Hydroxamic Acids/pharmacology
Gene Knockout Techniques
*Candida/genetics/drug effects
Sonication/methods
Chromatin

@article {pmid41887392,
year = {2026},
author = {Wang, H and Shen, T and Yang, S and Zhou, X and Cao, P and Yu, H and He, K and Fu, M and Yu, H and Liu, X and Zhou, T and Wang, J and Huang, M and Qian, X and Wang, X and Wang, Q and Liu, L and Fan, Z and Zhang, Y and Lin, F},
title = {Genome-wide CRISPR/Cas9 screen identified MCL1 as a senolytic target for clearing palbociclib-induced senescent and PD-L1-positive cells in colorectal cancer.},
journal = {Cancer letters},
volume = {647},
number = {},
pages = {218446},
doi = {10.1016/j.canlet.2026.218446},
pmid = {41887392},
issn = {1872-7980},
mesh = {Humans ; *Colorectal Neoplasms/drug therapy/genetics/pathology/metabolism ; *Pyridines/pharmacology ; *Piperazines/pharmacology ; Animals ; *Cellular Senescence/drug effects/genetics ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/antagonists & inhibitors/metabolism ; CRISPR-Cas Systems ; Mice ; Xenograft Model Antitumor Assays ; *B7-H1 Antigen/metabolism/genetics ; Apoptosis/drug effects ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Mice, Nude ; Gene Expression Regulation, Neoplastic/drug effects ; Female ; },
abstract = {Colorectal cancer (CRC) is the most prevalent digestive system malignancy worldwide. The development of targeted therapeutics specifically effective for CRC is currently in dire need. Preclinical studies showed that CDK4/6 inhibitor palbociclib suppressed the growth of CRC, but whether this effect is durable is unclear. In this study, we aimed to evaluate the roles of palbociclib-induced senescence and find a new strategy to maximize its effectiveness in CRC treatment. Animal and cellular experiments revealed that palbociclib-induced senescence and the senescence-associated secretory phenotype (SASP) caused drug resistance, anti-apoptosis, PD-L1 upregulation and inhibition of CD8[+] T cells' function. Using CRISPR/Cas9 screening, we identified MCL1 as a senolytic target to eliminate palbociclib-induced senescent CRC cells in the presence of palbociclib. Mechanically, palbociclib-induced senescent cells upregulated ZHX2 and its transcriptional target MCL1, rendered their resistance to apoptosis and T cell-mediated cytotoxicity, whereases combining palbociclib with MCL1 inhibitor markedly induced apoptosis in senescent cells by activating both extrinsic and intrinsic apoptotic pathways. Lastly, we proposed a seno-therapy consisting of a palbociclib pre-treatment plus a combination treatment of palbociclib and MCL1 inhibitor and found it effectively inhibited tumor growth and improved the survival of CRC xenografted mice. Besides its senolytic effect, seno-therapy also reduced PD-L1-positive cells and enhancing the cytotoxic functions of CD8[+] T cells. In conclusion, co-targeting CDK4/6 and MCL1 efficiently eliminates palbociclib-induced senescent CRC cells and offers a promising CDK4/6 inhibitor-based strategy for CRC treatment, ensuring prolonged tumor suppression and reducing the risk of progression or recurrence.},
}

Humans
*Colorectal Neoplasms/drug therapy/genetics/pathology/metabolism
*Pyridines/pharmacology
*Piperazines/pharmacology
Animals
*Cellular Senescence/drug effects/genetics
*Myeloid Cell Leukemia Sequence 1 Protein/genetics/antagonists & inhibitors/metabolism
CRISPR-Cas Systems
Mice
Xenograft Model Antitumor Assays
*B7-H1 Antigen/metabolism/genetics
Apoptosis/drug effects
Cell Line, Tumor
Drug Resistance, Neoplasm
Mice, Nude
Gene Expression Regulation, Neoplastic/drug effects
Female

@article {pmid41887586,
year = {2026},
author = {Menestreau, M and Frostegård, Å and Kjos, M},
title = {Strain-specific challenges in applying CRISPR/Cas9-based genome editing in the novel genus Stutzerimonas.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107478},
doi = {10.1016/j.mimet.2026.107478},
pmid = {41887586},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Genome, Bacterial ; Denitrification/genetics ; },
abstract = {Recently reclassified from the genus Pseudomonas, Stutzerimonas comprises metabolically versatile bacteria widely distributed across diverse environments and with a capacity to perform complete denitrification. Here, we evaluated the applicability of CRISPR/Cas9-based genome editing in Stutzerimonas species. Using a two-plasmid pCasPA/pACRISPR system, we achieved efficient deletion of the denitrification-associated narG and dnrE genes in Stutzerimonas decontaminans 19SMN4. On the other hand, Cas9-associated toxicity significantly limited transformation in Stutzerimonas perfectomarina ZoBell. These results highlight both the potential and the limitations of CRISPR/Cas9 editing in Stutzerimonas, emphasizing that genome editing efficiency and tolerance may vary even among closely related strains.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Plasmids/genetics
*Genome, Bacterial
Denitrification/genetics

@article {pmid41905455,
year = {2026},
author = {Meng, Y and Zhang, Z and Jia, K and Wang, W and Wang, H and Hu, Z and Liu, J},
title = {Membrane protein-focused CRISPR screen identifies ATP2A2 as a druggable transcriptional co-regulator of CCND1 (cyclin D1) in lung adenocarcinoma.},
journal = {Life sciences},
volume = {394},
number = {},
pages = {124358},
doi = {10.1016/j.lfs.2026.124358},
pmid = {41905455},
issn = {1879-0631},
mesh = {Humans ; *Lung Neoplasms/genetics/pathology/metabolism/drug therapy ; *Adenocarcinoma of Lung/genetics/metabolism/pathology/drug therapy ; Animals ; *Cyclin D1/genetics/metabolism ; Mice ; Cell Proliferation ; A549 Cells ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Apoptosis ; Cell Line, Tumor ; Mice, Nude ; Carcinoma, Non-Small-Cell Lung/genetics/pathology/metabolism ; Cell Movement ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {INTRODUCTION: Lung adenocarcinoma (LUAD) is one of the most commonly seen non-small cell lung cancer (NSCLC). Recent progress has highlighted cyclin-dependent kinase (CDK) inhibitors for treating diverse cancers including NSCLC. However, acquired resistance of CDK inhibitors has prompted the exploration of druggable regulatory or compensatory pathways in cell cycle as alternative or combination therapies of CDK inhibitors.

AIM: This study aims to discover the upstream regulatory proteins in LUAD using a membrane protein-focused CRISPR screen in A549 cells and dissect their functions.

MATERIALS AND METHODS: The top hit ATP2A2, an endoplasmic reticulum (ER)-localized protein, was analyzed for its function in LUAD using cell proliferation assay, migration and invasion assays and apoptosis assay. The mechanistic studies of ATP2A2 were conducted using RT-qPCR, Western blotting, RNA-Seq, cell cycle assay, protein immunoprecipitation, mass spectrometry, immunofluorescence microscopy and others.

KEY FINDINGS: The correlation of ATP2A2 expression with NSCLCs was confirmed with public database and collected clinical samples. ATP2A2 promoted LUAD development by involving in cell cycle regulation. ATP2A2 interacted with another ER protein HACD3 and co-regulated the transcription of CCND1 gene (cyclin D1), which forms complex with CDK4/6. ATP2A2 and HACD3 promoted CCND1 expression by activating NF-κB signaling and the nuclear translocation of p65 protein, which is a known transcription factor of CCND1. Curcumin, an ATP2A2-targeting natural product, could inhibit LUAD both in vitro and in xenografted mouse model.

SIGNIFICANCE: This study revealed ATP2A2-HACD3-NF-κB as a regulatory axis of cyclin D1 expression and shed light on developing ATP2A2-targeted medications for LUAD treatment.},
}

Humans
*Lung Neoplasms/genetics/pathology/metabolism/drug therapy
*Adenocarcinoma of Lung/genetics/metabolism/pathology/drug therapy
Animals
*Cyclin D1/genetics/metabolism
Mice
Cell Proliferation
A549 Cells
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Apoptosis
Cell Line, Tumor
Mice, Nude
Carcinoma, Non-Small-Cell Lung/genetics/pathology/metabolism
Cell Movement
Clustered Regularly Interspaced Short Palindromic Repeats
Female

@article {pmid41905499,
year = {2026},
author = {Fu, T and Wang, F and Ren, J and Chen, S and Peng, N and Zhang, L and Pu, K and Li, Q},
title = {An extraction-free HLPCR-Cas12a assay for ultra-sensitive and rapid detection of Acinetobacter baumannii and Klebsiella pneumoniae following craniotomy.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107480},
doi = {10.1016/j.mimet.2026.107480},
pmid = {41905499},
issn = {1872-8359},
mesh = {*Acinetobacter baumannii/isolation & purification/genetics ; Humans ; *Klebsiella pneumoniae/isolation & purification/genetics ; *Craniotomy/adverse effects ; Sensitivity and Specificity ; *Acinetobacter Infections/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; *Klebsiella Infections/diagnosis/microbiology ; CRISPR-Cas Systems ; Postoperative Complications/microbiology/diagnosis ; },
abstract = {Postoperative intracranial infection represents a significant complication in neurosurgical procedures, with Acinetobacter baumannii (Ab) and Klebsiella pneumoniae (Kp) frequently identified as common drug-resistant pathogens. In response to this challenge, we have developed an efficient, extraction-free detection method that facilitates the rapid and highly sensitive identification of Ab and Kp. This method employs a single-tube format that integrates heat lysis with PCR, allowing for the concurrent bacterial lysis and target gene amplification. Detection of the amplified products is achieved through the Cas12a/crRNA complex, which, upon activation, exhibits trans-cleavage activity that cleaves reporters to produce a signal detectable by microplate readers or lateral flow test strips (LFTS). The Heat Lysis-PCR-CRISPR/Cas12a (HLPCR-CRISPR/Cas12a) detection platform unifies lysis and amplification in one step, streamlining the process. It exhibits high specificity (no cross-reactivity) and achieves detection limits of 10[2] CFU/μL (fluorescence) and 10[3] CFU/μL (LFTS). When validated on clinical CSF samples, it delivered results in 90 min with perfect concordance with quantitative PCR (qPCR). Combining speed, high sensitivity, specificity, and operational simplicity, this platform provides a powerful diagnostic tool for early detection of Ab and Kp in postoperative intracranial infections.},
}

*Acinetobacter baumannii/isolation & purification/genetics
Humans
*Klebsiella pneumoniae/isolation & purification/genetics
*Craniotomy/adverse effects
Sensitivity and Specificity
*Acinetobacter Infections/diagnosis/microbiology
*Polymerase Chain Reaction/methods
*Klebsiella Infections/diagnosis/microbiology
CRISPR-Cas Systems
Postoperative Complications/microbiology/diagnosis

@article {pmid41921842,
year = {2026},
author = {Liao, YJ and Li, YG and Li, YL},
title = {CRISPR/Cas9-mediated lgp2 knockout and cross-species rescue reveal the immunoregulatory role of LGP2 in zebrafish.},
journal = {Fish & shellfish immunology},
volume = {173},
number = {},
pages = {111308},
doi = {10.1016/j.fsi.2026.111308},
pmid = {41921842},
issn = {1095-9947},
mesh = {Animals ; *Zebrafish/genetics/immunology ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/immunology ; Gene Knockout Techniques/veterinary ; *Immunity, Innate/genetics ; *Fish Diseases/immunology ; Gene Expression Profiling/veterinary ; *Gene Expression Regulation/immunology ; Species Specificity ; },
abstract = {LGP2 is a key regulator within the RIG-I-like receptor (RLR) pathway with dual roles in antiviral immunity, but its function under basal conditions and its evolutionary variation among fish species require further investigation. In this study, we constructed a zebrafish lgp2 knockout model using CRISPR/Cas9. Knockout lgp2 led to upregulation of key RLR signaling components (mda5, mavs), type I interferons (ifnphi1, ifnphi3, ifnphi4), and the interferon-stimulated gene mxa. Transcriptomic profiling of 3 dpf embryos identified 3186 differentially expressed genes (DEGs), including significantly elevated expression of five claudin-family genes. Protein-protein interaction (PPI) analysis of DEGs indicated that irf7 and claudin b exhibit an interaction at the protein level. Gene ontology analysis showed that LGP2 was enriched in terms such as helicase activity and ATPase activity, and was also significantly enriched in extracellular structure terms. Comparative sequence analysis revealed conserved DEXDc, HELICc and CTD domains in LGP2 from zebrafish, grass carp and barbel chub, with notable species-specific sequence variations. Functional rescue experiments demonstrated that all three LGP2 orthologs could restore lgp2 expression and modulate downstream interferon responses in lgp2[-/-] embryos; however, induction of the transcription factor irf7 was specifically mediated only by zebrafish-derived LGP2. These results provide insights into the functional diversity of LGP2 in teleost immunity and inform strategies for selective breeding aimed at disease resistance.},
}

Animals
*Zebrafish/genetics/immunology
CRISPR-Cas Systems
*Zebrafish Proteins/genetics/immunology
Gene Knockout Techniques/veterinary
*Immunity, Innate/genetics
*Fish Diseases/immunology
Gene Expression Profiling/veterinary
*Gene Expression Regulation/immunology
Species Specificity

@article {pmid41939725,
year = {2026},
author = {Xu, H and Liu, R and Zhou, H and Kong, B and Shen, K and Zhao, T and Du, X and Zhang, H and Song, H and Guo, D and Gu, X and Wang, Q and Lee, CW and Yin, G and Zhang, Y and Chen, W},
title = {Engineered small extracellular vesicles as bioactive materials: Integrating engineering strategies for cargo loading and targeted delivery systems.},
journal = {Bioactive materials},
volume = {59},
number = {},
pages = {96-134},
pmid = {41939725},
issn = {2452-199X},
abstract = {Small extracellular vesicles (sEVs) are increasingly regarded as a unique class of bioactive materials whose intrinsic membrane composition and nanoscale architecture provide a versatile platform for therapeutic engineering. Rather than passive carriers, sEVs can be actively programmed through diverse strategies to achieve efficient loading, precise targeting, and functional integration with synthetic systems. Endogenous modulation of donor cells-via genetic editing, priming with bioactive glass, cytokine stimulation, or hypoxic cues-enables selective packaging of nucleic acids, proteins, and metabolites into secreted vesicles. Exogenous techniques, including electroporation, sonication, and extrusion, allow controlled incorporation of therapeutic drugs or genome-editing complexes such as CRISPR/Cas. In parallel, surface modifications based on Lamp2b-fusion scaffolds, aptamers, antibodies, and click chemistry confer tissue tropism and extend circulation time. Integration with nanomaterials, scaffolds, and microfluidic platforms further enhances stability, scalability, and reproducibility, positioning sEVs at the intersection of biology and materials science. This review highlights recent advances in engineering sEVs as programmable bioactive materials and discusses their potential to transform regenerative medicine, oncology, and precision therapeutics.},
}

@article {pmid41940952,
year = {2026},
author = {Wang, N and Tang, J and Hou, X and Quan, C and Mai, J and He, C and Chen, R and Tao, B and Gu, Y and Jia, D and Fu, T and Zou, J and Zhao, L and Wen, J and Shen, J},
title = {BnaCIPK9 homoeologs mediate the dosage-dependent regulation of seed oil in allotetraploid Brassica napus L.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {4},
pages = {},
pmid = {41940952},
issn = {1432-2242},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022YFD1200804//National Key Research and Development Program of China/ ; 2021HSZD004//Hubei Hongshan Laboratory research funding/ ; 2662023PY004//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Brassica napus/genetics/metabolism ; *Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Plant Oils/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Tetraploidy ; *Gene Dosage ; Haplotypes ; Arabidopsis/genetics ; Genes, Plant ; },
abstract = {CRISPR/Cas9 mutagenesis and overexpression lines analyses revealed CIPK9 homoeologs function in oil regulation of allotetraploid Brassica napus, cooperative multicopy interactions, and a superior haplotype on chromosome A10. Rapeseed (Brassica napus, B. napus), a globally significant allopolyploid oilseed crop, fulfills substantial annual vegetable oil demand. Evolutionary adaptation in this species is underpinned by gene duplication and homoeolog retention, enhancing plasticity under dynamic environmental stresses. This study focuses on BnaCIPK9, a regulator of seed oil content. Phylogenomic and structural analyses demonstrate that BnaCIPK9 homoeologs underwent duplication followed by consistent evolutionary retention within the Brassica lineage, exhibiting remarkable sequence and structural conservation. Expression profiling revealed tissue-partitioned functional specialization among homoeologs, with BnaA10.CIPK9 and BnaC05.CIPK9 showing seed-preferential expression. CRISPR/Cas9 knockout in B. napus and heterologous overexpression in Arabidopsis demonstrate these homoeologs act as dosage-dependent regulators of oil accumulation, dependent on their distinct expression patterns. They further exhibit expression-driven functional diversification in abiotic stress responses during seedling development. Population genomics reveal differential evolutionary trajectories among duplicates, with intensified selection on chromosome A10 driving adaptive divergence. Crucially, haplotype-trait association identifies hap.qCIPK9.A10.0 as a major haplotype linked to elevated oil content. This work elucidates how homoeolog subfunctionalization fine-tunes critical agronomic traits, oil biosynthesis, and stress resilience, in polyploid crops, establishing haplotype-assisted breeding as imperative for developing crop cultivars. Favorable haplotypes, exemplified by hap.qCIPK9.A10.0, offer precise targets for high-oil crop breeding improvement.},
}

*Brassica napus/genetics/metabolism
*Seeds/genetics/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
*Plant Oils/metabolism
Phylogeny
CRISPR-Cas Systems
Tetraploidy
*Gene Dosage
Haplotypes
Arabidopsis/genetics
Genes, Plant

@article {pmid41942919,
year = {2026},
author = {Behera, L and Samal, KC and C, P and Agrawal, PK and Achary, VMM and Dash, M and Mishra, A and Rani, M and Masika, FB and Goud, GSD and Kesawat, MS and Samantaray, S},
title = {An improved Agrobacterium-mediated transformation method for genome editing using CRISPR/Cas9 in elite indica rice (Oryza sativa L.).},
journal = {BMC genomics},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12864-026-12800-0},
pmid = {41942919},
issn = {1471-2164},
}

@article {pmid41943368,
year = {2026},
author = {Sangeetha, VJ and Pawase, PA and Vasanthkumar, SS and Patrimath, SS and Bashir, O},
title = {Precision-engineered starch: Integrating metabolic engineering and cell-free synthetic biology for sustainable bioplastics and functional foods.},
journal = {Carbohydrate polymers},
volume = {381},
number = {},
pages = {125146},
doi = {10.1016/j.carbpol.2026.125146},
pmid = {41943368},
issn = {1879-1344},
mesh = {*Metabolic Engineering/methods ; *Starch/chemistry/biosynthesis/genetics/metabolism ; *Synthetic Biology/methods ; *Functional Food ; Amylose/chemistry/metabolism ; Gene Editing ; },
abstract = {This review consolidates recent advances that reposition starch development from downstream modification towards deliberate biosynthetic engineering. It critically examines the starch biosynthetic pathway as a foundation for rational design, highlighting the coordinated roles of granule-bound starch synthase, soluble starch synthases, starch branching enzymes, and debranching enzymes in defining granule structure. Clear structure-function-nutrition relationships are delineated, demonstrating how amylose-amylopectin ratio, chain-length distribution, and phosphate esterification govern physicochemical behaviour, digestibility, glycemic response, and resistant starch formation. Insights derived from naturally occurring starch mutants are integrated to elucidate genotype-phenotype-function linkages. It further evaluates the recent progress in planta metabolic engineering, with particular emphasis on CRISPR-Cas-based genome editing as a precise strategy for generating food-relevant starch phenotypes without foreign DNA insertion. Representative applications include high-amylose, low-glycemic staple crops and starches with engineered branching patterns to enhance processing performance. In parallel, emerging cell-free synthetic biology platforms are presented as complementary systems for producing structurally defined glucans with high purity and reproducibility. Collectively, these developments establish engineered starch as a next-generation bio-based material aligned with nutritional quality, technological functionality and sustainability objectives.},
}

*Metabolic Engineering/methods
*Starch/chemistry/biosynthesis/genetics/metabolism
*Synthetic Biology/methods
*Functional Food
Amylose/chemistry/metabolism
Gene Editing

@article {pmid41943836,
year = {2026},
author = {Xu, J and Cheng, L and Ma, S and Gan, C and Chai, J and Zheng, X and Hu, L and Ling, M and Zhang, M and Zhao, B and Cheng, H},
title = {In vivo CRISPR/Cas9 Screening Reveals that UBE2L3 Modulates Autophagic Flux through TSC2 Ubiquitination and Potentiates PD-1 Blockade in Triple-Negative Breast Cancer.},
journal = {International journal of biological sciences},
volume = {22},
number = {6},
pages = {2950-2969},
pmid = {41943836},
issn = {1449-2288},
mesh = {*Triple Negative Breast Neoplasms/metabolism/genetics ; *Ubiquitin-Conjugating Enzymes/metabolism/genetics ; Humans ; *Autophagy/genetics/physiology ; *Tuberous Sclerosis Complex 2 Protein/metabolism/genetics ; Ubiquitination ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Female ; Animals ; Mice ; *Programmed Cell Death 1 Receptor/metabolism/antagonists & inhibitors ; Mice, Nude ; },
abstract = {Triple-negative breast cancer (TNBC), a distinct breast cancer subtype, poses significant challenges to conventional therapeutic approaches, and effective targeted therapies are limited. CRISPR/Cas9 library screening has demonstrated unprecedented efficiency and revolutionary potential in the identification of therapeutic targets. In this study, we performed In vivo CRISPR/Cas9 library screening and identified the E2 ubiquitin-conjugating enzyme UBE2L3 as a critical regulatory factor in the progression of TNBC. Loss of UBE2L3 restricted tumor cell growth by modulating autophagy in TNBC cells. Mechanistically, UBE2L3 downregulation led to increased tuberous sclerosis complex 2 (TSC2) expression, suppressing mTOR activity and altering autophagic processes in tumor cells. This regulation was mediated through the interaction between UBE2L3 and the E3 ubiquitin ligase SMURF2, which together control TSC2 protein ubiquitination and degradation. Autophagy and the tumor microenvironment are closely associated, and we observed that UBE2L3 knockdown in TNBC tumors significantly increased CD8+ T lymphocyte infiltration and enhanced tumor sensitivity to anti-PD-1 therapy. Collectively, our findings provide a theoretical foundation for considering UBE2L3 as a potential therapeutic target in TNBC.},
}

*Triple Negative Breast Neoplasms/metabolism/genetics
*Ubiquitin-Conjugating Enzymes/metabolism/genetics
Humans
*Autophagy/genetics/physiology
*Tuberous Sclerosis Complex 2 Protein/metabolism/genetics
Ubiquitination
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Female
Animals
Mice
*Programmed Cell Death 1 Receptor/metabolism/antagonists & inhibitors
Mice, Nude

@article {pmid41943869,
year = {2026},
author = {Katayama, R and Hayashi, R and Ueda, M and Sakamoto, T and Nakazawa, M},
title = {Elucidation of Enzymatic Routes Underlying Odd-Chain Fatty Acid Synthesis and Propionate Assimilation in Euglena gracilis.},
journal = {The Journal of eukaryotic microbiology},
volume = {73},
number = {3},
pages = {e70078},
doi = {10.1111/jeu.70078},
pmid = {41943869},
issn = {1550-7408},
support = {JPMJGX23B0//Japan Science and Technology Agency/ ; JP24K01897//Japan Society for the Promotion of Science/ ; //Women Researchers Support Ofice of Osaka Metropolitan University/ ; },
mesh = {*Propionates/metabolism ; *Euglena gracilis/metabolism/enzymology/genetics ; *Fatty Acids/biosynthesis ; Gene Knockout Techniques ; Acyl Coenzyme A/metabolism ; Methylmalonyl-CoA Mutase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Wax esters (WEs) produced by Euglena gracilis under anaerobic conditions contain significant proportions of odd-chain fatty acids and fatty alcohols (30%-40%). Propionyl-CoA, the primer for odd-chain fatty acid synthesis, is generally thought to be generated via the methylmalonyl-CoA pathway; however, the enzymatic basis of this pathway in E. gracilis remains unclear. Here, we generated knockout (KO) mutants of candidate enzymes in the methylmalonyl-CoA pathway-succinyl-CoA synthetase (SCS), methylmalonyl-CoA mutase (MCM), methylmalonyl-CoA epimerase (MCE), and propionyl-CoA carboxylase (PCC)-using the CRISPR/Cas9 system and examined their roles in odd-chain fatty acid synthesis and propionate assimilation. The methylmalonyl-CoA pathway exhibited direction-dependent functions. KO of MCE and PCC did not affect the proportion of odd-chain WEs, indicating that these enzymes are not required for odd-chain WE synthesis and suggesting an alternative route converting methylmalonyl-CoA to propionyl-CoA. In contrast, both enzymes were required for propionate assimilation. Functional differentiation was observed between SCSβ isozymes: SCSβ1 contributed to odd-chain WE synthesis, whereas SCSβ2 primarily functioned in propionate assimilation. Partial impairment of propionate assimilation in SCSα or SCSβ2 KO strains suggests involvement of additional routes. These findings improve our understanding of direction-dependent roles in the methylmalonyl-CoA pathway in E. gracilis and support further studies of odd-chain lipid biosynthesis.},
}

*Propionates/metabolism
*Euglena gracilis/metabolism/enzymology/genetics
*Fatty Acids/biosynthesis
Gene Knockout Techniques
Acyl Coenzyme A/metabolism
Methylmalonyl-CoA Mutase/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41945389,
year = {2026},
author = {Li, M and Gao, G and Jiao, Y},
title = {A Hierarchical Screening Strategy for Genome-Edited Events in Polyploid Species: A Case Study on Hexaploid Common Wheat.},
journal = {Current protocols},
volume = {6},
number = {4},
pages = {e70354},
doi = {10.1002/cpz1.70354},
pmid = {41945389},
issn = {2691-1299},
support = {2024YFF1000301//National Key R&D Program of China/ ; tsqn202312306//Taishan Scholars Program/ ; 2025HWYQ-071//Shandong Provincial Natural Science Fund for Excellent Young Scientists Fund Program (Overseas)/ ; SYS202206//Shandong Provincial Natural Science Foundation/ ; ZR2022ZD22//Shandong Provincial Natural Science Foundation/ ; ZR2021ZD30//Shandong Provincial Natural Science Foundation/ ; },
mesh = {*Triticum/genetics ; *Gene Editing/methods ; *Polyploidy ; *Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {Genome editing via CRISPR/Cas9 has been widely adopted in cereal crops. In diploid species such as rice and barley, the generation of knockout mutants is relatively straightforward for functional characterization of the genes of interest due to their single-copy nature in the genome. In contrast, common wheat (Triticum aestivum L.) is a hexaploid species comprising three subgenomes (AABBDD); consequently, most genes are present as three homoeoalleles that retained substantial function redundancy during evolution. The generation of a complete set of single, double, and triple mutants is therefore essential for elucidating homoeoallele-specific functions and dissecting their contributions to the developmental and agronomic traits. Moreover, ensuring germplasm purity through the elimination of residual T-DNA is critical for maintaining stable mutation, particularly in single- and double-mutant lines. Here, we describe a hierarchical screening strategy for efficient identification of a comprehensive series of CRISPR/Cas9-induced mutants. This approach integrates high-throughput DNA isolation, selection of T-DNA-free mutants, maintenance of a uniform genetic background via backcrossing, systematic screening of all mutant combinations, and molecular confirmation of genome edits. This screening pipeline has proven effective in hexaploid common wheat and is readily adaptable to other polyploid species that are amenable to crossing. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Wheat cultivation and leaf sample preparation Basic Protocol 2: High-throughput DNA isolation Basic Protocol 3: Genotyping using an optimized, cost-efficient T7E1 assay Alternate Protocol 1: Genotyping using the KASP assay Support Protocol 1: Screening of T-DNA-free triple mutants.},
}

*Triticum/genetics
*Gene Editing/methods
*Polyploidy
*Genome, Plant
CRISPR-Cas Systems

@article {pmid41946009,
year = {2026},
author = {Sutaoney, P and Singh, P and Malakar, S and Arsi, L and Ghosh, P},
title = {Microbial lipases: Catalyzing sustainable solutions for industrial innovations.},
journal = {Enzyme and microbial technology},
volume = {198},
number = {},
pages = {110869},
doi = {10.1016/j.enzmictec.2026.110869},
pmid = {41946009},
issn = {1879-0909},
abstract = {Microbial lipases are multifaceted biological catalyst that have surfaced as a key driver in various industries and are both eco-friendly and cost efficient.In large scale applications, lipases produced from bacteria, fungi and yeasts function better than their equivalents generated from plants and animals due to their wide substrate specificity, catalytic efficacy and stability under physicochemical circumstances. Recent developments in microbial lipase research, including sources, screening techniques, assay procedures, production methods, purification tactics, and biochemical characterisation, are critically examined in this review.The structural and mechanistic elements that control lipase function-such as lid domains, interfacial activation, and catalytic triads-are given special attention since they all have an impact on the stability, specificity, and industrial performance of the enzyme.Large-scale screening is done to check for the production of lipase in Bacillus sp., Achromobacter sp., Alcaligenes sp., Arthrobacter sp., Pseudomonas sp., and Penicillium sp. Additionally, the combination of synthetic biology, metagenomics, CRISPR-Cas technologies, enzyme engineering, and AI-assisted modelling is emphasized as a revolutionary strategy for identifying and customizing lipases with desired characteristics, including extreme environment microbes and application-specific variants.The review also highlights the growing industrial uses of microbial lipases in the bio-fuel, food and beverage, detergent, textile, leather, pharmaceutical, and medical industries, highlighting their contribution to the development of economically feasible and ecologically safe bioprocesses. All things considered, microbial lipases are an important biotechnological tool for developing sustainable industrial innovation and green chemistry.},
}

@article {pmid41938355,
year = {2026},
author = {Ma, G and Xu, H and Zhang, S and Li, X and Liu, J and Xie, J and Yan, F and Zhou, H},
title = {Enhancing the efficiency of nuclease-based prime editing in rice with the Tf1 reverse transcriptase.},
journal = {aBIOTECH},
volume = {7},
number = {2},
pages = {100026},
pmid = {41938355},
issn = {2662-1738},
abstract = {The development of efficient and precise genome-editing tools is crucial for advancing functional genomics and improving crops. Our previously established nuclease-mediated prime editing (NM-PE) system, which combines the SpCas9 nuclease with prime editing based on microhomology-mediated end joining, enables the seamless insertion of small DNA fragments into plant genomes to add tags to genes of interest. However, the efficiency of 3 × FLAG sequence insertion via NM-PE requires further improvement. Here, we report a significant optimization of this system by replacing the M-MLV reverse transcriptase (RT) with evolved variants of the retrotransposon RT Tf1 derived from the mammalian PE6 system. Through codon optimization, we generated the evoTf1M4 variant, which substantially enhanced the efficiency of NM-PE. The optimized construct rPE20aV3 achieved up to 18.75% precise insertion of a 66-bp 3 × FLAG sequence at endogenous loci, representing a three-fold improvement over the original NM-PE system. Our results demonstrate that Tf1-aided optimization of NM-PE serves as an efficient platform for seamless insertion of a 3 × FLAG sequence in rice, offering broad potential for advanced genome engineering in plants.},
}

@article {pmid40813912,
year = {2026},
author = {Mathis, N and Marquart, KF and Allam, A and Krauthammer, M and Schwank, G},
title = {Systematic pegRNA design with PRIDICT2.0 and ePRIDICT for efficient prime editing.},
journal = {Nature protocols},
volume = {21},
number = {4},
pages = {1523-1543},
pmid = {40813912},
issn = {1750-2799},
support = {185293, 214936//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 201184//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; Humans ; CRISPR-Cas Systems ; Machine Learning ; Chromatin/genetics ; *Computational Biology/methods ; Neural Networks, Computer ; },
abstract = {Prime editing is a versatile genome editing technology that enables precise genetic modifications without inducing DNA double-strand breaks. Owing to numerous variables in the prime editing guide RNA (pegRNA) design, experimentally identifying the most efficient pegRNA for a specific locus and edit is laborious. Therefore, we have developed computational tools to streamline this process. Here we present a comprehensive protocol detailing how to use PRIDICT2.0 and ePRIDICT, machine-learning models that assess the influence of the pegRNA design and chromatin context on prime editing. PRIDICT2.0 is an ensemble of attention-based bidirectional recurrent neural networks that predicts pegRNA efficiencies for replacements, insertions or deletions in different cellular contexts. Compared with other pegRNA design tools, PRIDICT2.0 accommodates larger edits-up to 40 base pairs-across diverse edit types, also allowing the introduction of silent bystander edits that can enhance editing efficiency. ePRIDICT, a gradient-boosting algorithm, further accounts for the local chromatin environments and assesses how the genomic location of the target site affects prime editing rates. Both tools are available at www.pridict.it for individual predictions or can be installed locally for batch processing of multiple edits and target sites. The protocol provides step-by-step instructions on using PRIDICT2.0 and ePRIDICT, covering sequence input, prediction generation and interpretation. Web-based predictions take under a minute, while local installation and batch processing may take up to several hours, depending on the dataset size. By streamlining pegRNA selection and chromatin context analysis, these tools promote the adoption of prime editing in basic and translational research.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*Software
Humans
CRISPR-Cas Systems
Machine Learning
Chromatin/genetics
*Computational Biology/methods
Neural Networks, Computer

@article {pmid40983723,
year = {2026},
author = {Lilja, A and Finkel, Y and Aharon, E and Nachshon, A and Schwartz, M and Stern-Ginossar, N},
title = {Multidimensional analysis of host-virus interactions using the virus-encoded CRISPR-based direct readout system (VECOS).},
journal = {Nature protocols},
volume = {21},
number = {4},
pages = {1429-1465},
pmid = {40983723},
issn = {1750-2799},
support = {CoG-2019-864012//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Cytomegalovirus/genetics/physiology ; Genome, Viral ; *Host-Pathogen Interactions/genetics ; *Host Microbial Interactions/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 technology has transformed the study of gene function, enabling the systematic investigation of host-virus interactions. However, most CRISPR-based screens in the context of viral infections rely on cell survival as a readout, which limits their sensitivity and biases results toward early infection stages. To address these challenges, we developed the virus-encoded CRISPR-based direct readout system (VECOS), a virus-centric approach in which human cytomegalovirus is engineered to express single-guide RNA (sgRNA) libraries directly from its genome. This system allows sgRNA abundance, embedded in the viral genome, to serve as a direct and quantitative readout of gene-perturbation effects on viral propagation. By tracking sgRNA levels at distinct stages of the viral infection cycle, VECOS enables a detailed, multidimensional analysis of virus-host interactions. Here we present a modular detailed Protocol for (1) constructing and reconstituting complex sgRNA libraries in double-stranded DNA viruses using bacterial artificial chromosomes, (2) performing multipassage screens to investigate perturbation effects on various stages of viral infection and (3) analyzing the multipassage and multistage sgRNA abundance measurements utilizing a comprehensive framework for data analysis. Successful implementation of this full Protocol takes 14-22 weeks and requires proficiency in molecular biology, as well as basic familiarity with Unix-based computing and programming in R for data processing. This Protocol offers researchers a robust tool for uncovering the molecular mechanisms that drive viral propagation and host-virus interactions.},
}

*CRISPR-Cas Systems/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
*Cytomegalovirus/genetics/physiology
Genome, Viral
*Host-Pathogen Interactions/genetics
*Host Microbial Interactions/genetics
HEK293 Cells

@article {pmid41674076,
year = {2026},
author = {Arrigo, A and Rao, V and Ratan, A and Kulkarni, SS},
title = {Patient-informed CRISPR screen identifies FLNB as a congenital heart disease and ciliopathy gene.},
journal = {HGG advances},
volume = {7},
number = {2},
pages = {100580},
pmid = {41674076},
issn = {2666-2477},
mesh = {Humans ; Animals ; *Ciliopathies/genetics ; *Heart Defects, Congenital/genetics/diagnosis ; *Filamins/genetics ; *CRISPR-Cas Systems ; Male ; Female ; *Heterotaxy Syndrome/genetics/diagnosis ; Xenopus ; Exome Sequencing ; Mutation ; },
abstract = {Heterotaxy (HTX) is a congenital disorder characterized by abnormal left-right organ placement, often leading to severe congenital heart disease (CHD). Despite advances in sequencing, many CHD and HTX-associated genes remain functionally unvalidated, hindering effective clinical diagnosis and management. Here, we leveraged a high-throughput CRISPR-Cas9 screening approach in the Xenopus model to rapidly evaluate candidate genes identified from whole-exome sequencing of human CHD patients. Our screen identified Filamin B (FLNB), an actin-binding protein previously linked to skeletal disorders but not to ciliopathies or CHD. We identified 5 probands with CHD and HTX, 3 with recessive and 2 with damaging heterozygous variants in FLNB. Disrupting flnb in Xenopus reproduced key features of the human HTX phenotype, including defects in cardiac development and impaired motile cilia function. Rescue experiments confirmed the functional conservation of human FLNB, directly implicating actin cytoskeletal disruption in ciliogenesis and left-right patterning defects. Our results provide crucial evidence linking human FLNB dysfunction to ciliopathies and CHD and HTX.},
}

Humans
Animals
*Ciliopathies/genetics
*Heart Defects, Congenital/genetics/diagnosis
*Filamins/genetics
*CRISPR-Cas Systems
Male
Female
*Heterotaxy Syndrome/genetics/diagnosis
Xenopus
Exome Sequencing
Mutation

@article {pmid41776280,
year = {2026},
author = {Shirai, Y and Kao, JA and Kumar, T and Matsuda, N and Nakagawa, R and Nureki, O and Extavour, CG and Daimon, T},
title = {HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41776280},
issn = {2399-3642},
support = {20H02999, 20K21311, 22K19179, 24H00511, 24K21869//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21J20658//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Overseas Research Fellow//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Long-Term Fellow//Human Frontier Science Program (HFSP)/ ; JPJ008000//Cabinet Office, Government of Japan/ ; IOS-2220747//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Gene Knock-In Techniques/methods ; *Tribolium/genetics/embryology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; DNA End-Joining Repair ; *Oligodeoxyribonucleotides/genetics ; Recombinational DNA Repair ; },
abstract = {Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.},
}

Animals
*Gene Knock-In Techniques/methods
*Tribolium/genetics/embryology
*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
DNA End-Joining Repair
*Oligodeoxyribonucleotides/genetics
Recombinational DNA Repair

@article {pmid41867107,
year = {2026},
author = {Zhuang, S and Li, J and Fang, Z and Zhou, H and Zhang, R and He, J and Zhu, L and Xu, Y and Xu, D and Gu, D and Wang, J},
title = {Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection.},
journal = {ACS infectious diseases},
volume = {12},
number = {4},
pages = {1423-1433},
doi = {10.1021/acsinfecdis.6c00013},
pmid = {41867107},
issn = {2373-8227},
mesh = {*CRISPR-Cas Systems ; *Chikungunya virus/genetics/isolation & purification ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Chikungunya Fever/diagnosis/virology ; *Arboviruses/genetics/isolation & purification ; RNA, Viral/genetics ; *Phosphorothioate Oligonucleotides/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; },
abstract = {The ongoing arthropod-borne Chikungunya virus (CHIKV) highlights the requirements of rapid and accurate diagnostic methods to enhance the epidemic control. CRISPR diagnostic (CRISPR-Dx) technology holds promise, but the development of a highly efficient one-pot diagnostic system usually requires fine-tuning of the balance between isothermal amplification and Cas cleavage procedures. Here, we describe a simple method (psHOLMES) to create one-pot, two-step CRISPR-Dx systems, using photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity. Cas12a activity is first inactivated via binding of ppPS-DNA during the target sequence amplification procedure, which is then reactivated by ultraviolet (UV)-mediated photolysis of ppPS-DNA after amplification, triggering Cas12a trans-cleavage reactions. psHOLMES demonstrates attomolar sensitivity for CHIKV RNA detection and zero cross-reactivity against other related arboviruses. When applied to clinical samples, psHOLMES achieved 100% (50/50) accuracy and could detect CHIKV within 30 min. As traditional efforts for fine-tuning Cas cis-cleavage activity can be omitted, psHOLMES thus enables rapid development of one-pot CRISPR-Dx systems for clinical applications.},
}

*CRISPR-Cas Systems
*Chikungunya virus/genetics/isolation & purification
Humans
Nucleic Acid Amplification Techniques/methods
*Chikungunya Fever/diagnosis/virology
*Arboviruses/genetics/isolation & purification
RNA, Viral/genetics
*Phosphorothioate Oligonucleotides/chemistry
CRISPR-Associated Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
Bacterial Proteins

@article {pmid41875952,
year = {2026},
author = {Li, C and Zhang, L and Xu, Q},
title = {Extraction-free, rapid LAMP-CRISPR/Cas12a assay for detection of pseudorabies virus.},
journal = {Journal of virological methods},
volume = {343},
number = {},
pages = {115387},
doi = {10.1016/j.jviromet.2026.115387},
pmid = {41875952},
issn = {1879-0984},
mesh = {*Herpesvirus 1, Suid/isolation & purification/genetics ; Animals ; Swine ; *Pseudorabies/diagnosis/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; DNA Primers/genetics ; DNA, Viral/genetics ; },
abstract = {This study developed a LAMP-CRISPR/Cas12a detection system for rapid and visual identification of porcine pseudorabies virus (PRV). Optimal sgRNA and LAMP-specific primers were designed based on the conserved sequences of the viral pathogenic gene gG. The combined detection system demonstrated superior sensitivity compared to PCR-CRISPR/Cas12a and qPCR methods, achieving a detection limit of 1.0 × 10[-4] copies/μL for the target plasmid DNA. Specificity testing confirmed the selective identification of PRV without cross-reactivity to other porcine pathogens. Parallel comparison of 26 serum samples between LAMP-CRISPR/Cas12a and PCR-CRISPR/Cas12a systems showed 100% concordance for positive results, with both detecting 12 positive samples. The method eliminates the need for viral nucleic acid extraction and requires only a constant temperature device and/or basic fluorescence detection equipment. Results are obtainable within one hour and are readable by the naked eye. This simple, sensitive, and equipment-independent approach is ideal for on-site rapid diagnosis of pseudorabies in pigs, offering significant applications in clinical diagnosis, epidemiological surveillance, and field testing.},
}

*Herpesvirus 1, Suid/isolation & purification/genetics
Animals
Swine
*Pseudorabies/diagnosis/virology
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Swine Diseases/diagnosis/virology
*CRISPR-Cas Systems
DNA Primers/genetics
DNA, Viral/genetics

@article {pmid41937163,
year = {2026},
author = {Zhou, HR and Doan, DTH and Hartwig, T and Turck, F},
title = {Cis-regulatory architecture downstream of FLOWERING LOCUS T underlies quantitative control of flowering in Arabidopsis thaliana.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41937163},
issn = {1474-760X},
support = {EXC 2048/1 Project ID: 390686111//Cluster of Excellence on Plant Sciences/ ; },
mesh = {*Arabidopsis/genetics/growth & development ; *Flowers/genetics/growth & development ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Enhancer Elements, Genetic ; Gene Editing ; Chromatin/metabolism ; Regulatory Sequences, Nucleic Acid ; },
abstract = {BACKGROUND: The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with its precise expression controlled by complex transcriptional networks. While upstream regulatory regions are well-studied, the role of downstream cis-regulatory elements in modulating FT expression remains poorly characterized.

RESULTS: Systematic dissection of the FT downstream region in its native chromosomal context using CRISPR/Cas9-mediated genome editing provides genetic evidence that a 2.3-kb sequence, encompassing the Block E enhancer immediately adjacent to the FT coding sequence, is essential for proper FT expression and timely flowering. Fine-scale deletions within Block E reveal that a 63-bp sequence containing one CCAAT-box and one G-box, both closely spaced, forms a core functional module, whereas other conserved motifs contribute modestly in a context-dependent manner. Strikingly, a cryptic CCAAT-box module downstream of Block E that becomes active when repositioned. This coincides with increased transcription factor occupancy and local chromatin accessibility.

CONCLUSIONS: Our work reveals that quantitative FT expression and flowering time are governed by the spatial organization and chromatin context of downstream cis-regulatory elements. The positional sensitivity and modular logic of these elements provide framework for understanding and engineering quantitative gene regulation through targeted cis-regulatory design, a concept broadly applicable across diverse developmental systems.},
}

*Arabidopsis/genetics/growth & development
*Flowers/genetics/growth & development
*Arabidopsis Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
CRISPR-Cas Systems
Enhancer Elements, Genetic
Gene Editing
Chromatin/metabolism
Regulatory Sequences, Nucleic Acid

@article {pmid41938130,
year = {2026},
author = {Yin, X and Fan, Z and Tong, Z},
title = {CRISPR-based electrochemiluminescence biosensors: Principles, optimization strategies, and translational challenges - A review of recent progress.},
journal = {Food chemistry. Molecular sciences},
volume = {12},
number = {},
pages = {100392},
pmid = {41938130},
issn = {2666-5662},
abstract = {The integration of CRISPR/Cas systems with electrochemiluminescence (ECL) has emerged as a promising strategy for constructing high-performance biosensing platforms. CRISPR systems, particularly Cas12a and Cas13a, offer programmable recognition of nucleic acid targets and activatable trans-cleavage activity. ECL provides sensitive signal readout with low background and wide dynamic range. As a narrative review, this article provides a comprehensive overview of recent advances in CRISPR-ECL biosensors, with an emphasis on optimization strategies and practical applications. We first discuss the working principles of Cas12a and Cas13a relevant to biosensing, highlighting their distinct kinetic properties, crRNA design considerations, and reaction condition requirements. We then examine optimization approaches at three interconnected levels: nucleic acid probe design (signal-on, signal-off, and auxiliary probes), sensing interface engineering (probe structures, luminophores, electrode materials, and magnetic nanomaterials), and cascade signal amplification (PCR, CHA, RCA, SDA, EDA, and RPA). Through cross-study comparison, we evaluate the strengths and limitations of different approaches and identify critical knowledge gaps. Their applications in detecting disease biomarkers, pathogen nucleic acids, environmental contaminants, and enzyme activities are summarized. Despite remarkable sensitivity achieved, challenges remain in assay time, reproducibility in complex matrices, and clinical validation. From industrialization and global health perspectives, regulatory approval, manufacturing scalability, cost control, and deployment in low-resource settings are also discussed. Finally, future directions toward simplified workflows, enhanced matrix robustness, standardized validation, multiplexed detection, and point-of-care compatible platforms are proposed. This review provides a structured reference and critical perspective for researchers working on CRISPR-ECL biosensing and related fields.},
}