@article {pmid42425298,
year = {2026},
author = {Sun, X and Zhang, Y and Lu, R and Dai, T and Qiu, J and Jia, X and Wang, G and Pan, Z and Xu, S and Sima, Y},
title = {Integrated mapping and gene editing identify BmCCP as a regulator of cocoon shape and spinning behaviour in Bombyx mori.},
journal = {Journal of insect physiology},
volume = {173},
number = {},
pages = {105033},
doi = {10.1016/j.jinsphys.2026.105033},
pmid = {42425298},
issn = {1879-1611},
mesh = {Animals ; *Bombyx/genetics/growth & development/metabolism/physiology ; *Insect Proteins/genetics/metabolism ; Gene Editing ; Chromosome Mapping ; *Silk/metabolism ; CRISPR-Cas Systems ; Larva/growth & development/genetics ; },
abstract = {The cocoon shape in Bombyx mori (silkworm) is a construction trait shaped by cocoon-spinning behaviour, but the molecular regulation pathways remain poorly understood. Here, quantitative phenotyping, bulked segregant analysis sequencing (BSA-seq), brain transcriptomics and CRISPR/Cas9 mutagenesis were combined to identify a regulator of cocoon morphogenesis and to assess potential roles in spinning behaviour. Using representative strains L6J5 and J8, which produce short oval and long peanut-shaped cocoons, respectively, showed that cocoon shape, quantified by the cocoon aspect ratio, is a quantitative trait. BSA-seq mapped the trait to a 5.04-Mb candidate interval on chromosome 4 containing 213 annotated genes. Integration of the mapping results with brain transcriptomes from individuals with extreme cocoon phenotypes identified Bombyx mori cocoon shape-correlated protein (BmCCP) as the sole overlapping candidate gene. BmCCP was more highly expressed in the brain of strain J8 than L6J5 at the wandering stage, and the locus contained multiple associated polymorphisms. CRISPR/Cas9-mediated knockout of BmCCP in strain J8 significantly increased the cocoon aspect ratio and cocoon size. In the widely used experimental strain DaZao, which has not been artificially selected for cocoon shape, BmCCP deficiency likewise increased cocoon size and significantly reduced the larval spinning rate, while crosses with J8 further supported the role of BmCCP in regulating the cocoon aspect ratio. Together, these results identify BmCCP as a regulator of cocoon morphogenesis and provide a foundation to investigate the relationship between the cocoon morphology and spinning behaviour of the silkworm.},
}

Animals
*Bombyx/genetics/growth & development/metabolism/physiology
*Insect Proteins/genetics/metabolism
Gene Editing
Chromosome Mapping
*Silk/metabolism
CRISPR-Cas Systems
Larva/growth & development/genetics

@article {pmid42435068,
year = {2026},
author = {Zhang, C and Josyula, NK and Cornejo-Corona, I and Watson, R and Cediel-Becerra, JD and Chevrette, MG and Devarenne, TP and Straight, PD},
title = {Tandem-sgRNA Provides an Effective Reverse Genetic Approach for Suppression of Streptomyces Biosynthetic Gene Clusters and Secondary Metabolism.},
journal = {ACS synthetic biology},
volume = {15},
number = {8},
pages = {3382-3395},
doi = {10.1021/acssynbio.6c00274},
pmid = {42435068},
issn = {2161-5063},
support = {GM141700/GM/NIGMS NIH HHS/United States ; },
mesh = {*Streptomyces/genetics/metabolism ; *Multigene Family/genetics ; *Secondary Metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Reverse Genetics/methods ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; },
abstract = {Bacterial biosynthetic gene clusters (BGCs) encode secondary metabolites with diverse biological activities; however, most BGC products remain uncharacterized. One approach to identifying products and their metabolism is to use reverse genetics to identify metabolite-associated phenotypes. CRISPR interference (CRISPRi) offers a promising approach to disrupt BGC functions in high-GC genomes, typical of Streptomyces species. In many of these organisms, single-guide RNA (sgRNA)-mediated CRISPRi often results in incomplete product suppression, resulting in partial phenotypes that are unsuitable for functional studies. Using Streptomyces sp. Mg1, we found that a tandem-sgRNA configuration for CRISPRi improved the efficiency of target metabolite suppression. We engineered strains to express two sgRNAs to target the same promoter region within a BGC, resulting in greater than 80% metabolite suppression across diverse secondary metabolite classes. We used tandem-sgRNA CRISPRi to identify phenotypes associated with the loss of polyketide linearmycins, the siderophore desferrioxamine, the terpene β-carotene, and an uncharacterized nonribosomal peptide synthetase (NRPS). This approach revealed that β-carotene depletion substantially reduced intrinsic cellular autofluorescence. Targeting the unknown NRPS produced developmental phenotypes and enabled the identification of the biosynthetic genes for the antibiotic lavendomycin, revealing a noncollinear organization of genes in the BGC. We suggest that tandem-sgRNA CRISPRi provides an efficient reverse genetics platform for the functional characterization of Streptomyces BGCs, enabling the correlation of metabolites with the gene function, identification of associated phenotypes, and prioritization of cryptic BGCs for natural product discovery.},
}

*Streptomyces/genetics/metabolism
*Multigene Family/genetics
*Secondary Metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Reverse Genetics/methods
CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics

@article {pmid42435429,
year = {2026},
author = {Zhou, Q and Xu, B and Wang, Y and Yang, X and Wang, L and Zheng, X and Lou, Y and Zheng, M and Xu, F and Xiao, X},
title = {LAMP-Based Two-DNA-Fragment Fusion and Its Application in Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {15},
number = {8},
pages = {3300-3309},
doi = {10.1021/acssynbio.6c00187},
pmid = {42435429},
issn = {2161-5063},
support = {JS2026005//Wenzhou Medical University/ ; 22578338//National Natural Science Foundation of China/ ; LTGY24H200005//Natural Science Foundation of Zhejiang Province/ ; 2024YFC2309905//National Key Research and Development Program of China/ ; NA//Key Discipline of Zhejiang Province in Medical Technology/ ; WKJ-ZJ-26018//Medical Science and Technology Project of Zhejiang Province/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; *DNA, Bacterial/genetics/analysis ; CRISPR-Cas Systems/genetics ; *DNA/genetics ; Molecular Diagnostic Techniques ; },
abstract = {Loop-mediated isothermal amplification (LAMP) continuously generates strand-displaced single-stranded DNA intermediates, providing the possibility of assembling DNA fragments. Here, we developed a novel two-DNA-fragment fusion technique, termed fusion LAMP, which is an isothermal DNA-fusion strategy that enables the fusion of two independent DNA fragments within a single amplification reaction. By combining fusion LAMP with CRISPR/Cas13a, we further established an "AND-gate" nucleic acid detection platform, termed Fusion LAMP-Coupled CRISPR/Cas13a (FLCC), which enables concurrent detection of two targets by reading the fusion product-triggered fluorescence signals. This platform generates signals only when two targets are present simultaneously. To prove this concept, we then employed FLCC to identify the methicillin-resistant Staphylococcus aureus (MRSA). This method achieved a limit of detection of 10 copies/μL of MRSA genomic DNA and showed no cross-reactivity with closely related bacterial strains. Furthermore, we validated its feasibility by detecting 19 clinical isolates, demonstrating a simple and accurate approach for MRSA detection. Collectively, the FLCC platform ensures identifying pathogens accurately and provides a promising diagnostic approach for detecting complex genetic targets.},
}

*Nucleic Acid Amplification Techniques/methods
*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification
*DNA, Bacterial/genetics/analysis
CRISPR-Cas Systems/genetics
*DNA/genetics
Molecular Diagnostic Techniques

@article {pmid42454678,
year = {2026},
author = {Feldmann, D and van Beljouw, SPB and Haagsma, AC and Kalogeropoulos, K and Muralidharan, A and Brouns, SJJ},
title = {Craspase Protease Activation Is Sensitive to Oncogenic Single-Nucleotide RNA Mismatches.},
journal = {ACS chemical biology},
volume = {21},
number = {8},
pages = {1877-1882},
doi = {10.1021/acschembio.6c00241},
pmid = {42454678},
issn = {1554-8937},
support = {101003229//European Commission/ ; OCENW.XS23.1.006//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; SUMMIT.1.004//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; 15602//KWF Kankerbestrijding/ ; 4257-00010B//Danmarks Frie Forskningsfond/ ; NNF16OC0020670//Novo Nordisk Fonden/ ; },
mesh = {Humans ; Enzyme Activation ; *RNA/genetics ; *Base Pair Mismatch ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Peptide Hydrolases/metabolism/genetics ; },
abstract = {The type III-E CRISPR-controlled protease Craspase is distinguished from other type III systems by its single-subunit RNA-guided protein complex and direct coupling of RNA recognition to protease activation without second messenger signaling, making it an attractive development platform for bioengineering and therapeutics. Here, we identify five positions within the CRISPR RNA (crRNA) of Craspase from Candidatus "Scalindua brodae" (Sb-Craspase) that are sensitive to single-nucleotide mismatches. We leverage these positions to design crRNAs that selectively target clinically relevant single-nucleotide variants (SNVs) in oncogenic RNA transcripts. Using this approach, Sb-Craspase is selectively activated by the "undruggable" KRAS G12D SNV, while the wild-type transcript does not induce protease activation. Collectively, our results establish a framework for designing crRNAs to target clinically relevant SNVs, laying the groundwork for Craspase-based diagnostics and therapeutics against otherwise intractable oncogenic mutations.},
}

Humans
Enzyme Activation
*RNA/genetics
*Base Pair Mismatch
Polymorphism, Single Nucleotide
CRISPR-Cas Systems
*Peptide Hydrolases/metabolism/genetics

@article {pmid42477478,
year = {2026},
author = {Tokgün, O and İnci, K and Gültekin, A and Çelikkaya, B and İrep, N and Akça, H and Tokgün, PE},
title = {Targeting RAB27A-mediated small extracellular vesicle secretion via CRISPR-Cas9 negatively affects proliferation and metastasis in both in vitro and in vivo SCLC models.},
journal = {Cancer gene therapy},
volume = {33},
number = {8},
pages = {1000-1013},
pmid = {42477478},
issn = {1476-5500},
mesh = {Humans ; Animals ; Cell Proliferation ; *rab27 GTP-Binding Proteins/genetics/metabolism ; Mice ; *Lung Neoplasms/pathology/genetics/metabolism ; *Small Cell Lung Carcinoma/genetics/pathology/metabolism/therapy ; *CRISPR-Cas Systems ; *Extracellular Vesicles/metabolism/genetics ; Cell Line, Tumor ; Female ; Neoplasm Metastasis ; Xenograft Model Antitumor Assays ; },
abstract = {Small cell lung cancer (SCLC) comprises 15% of lung cancers with a capacity for early and distant metastatic development, high proliferative capacity, and poor survival rates. Ionizing radiation and chemotherapy are effective against early-stage SCLC. This sensitivity wanes over time, however, making treatment difficult. Different types of neoplasms have demonstrated the pivotal role of small extracellular vesicles (sEVs) in disease progression. However, the role of sEVs development in SCLC remains unclear. In this study, the impact of sEVs secretion in SCLC cells was investigated using the CRISPR-Cas9 system to target the RAB27A. The effects of sEVs release inhibition on tumour growth and metastasis were evaluated using micro-PET-CT analysis. A reduction in cellular proliferation as a consequence of sEVs release, along with diminished expression of proteins and RNA (CD9, CD63, and Tsg101) implicated in sEVs secretion in silenced SCLC cells (p < 0.001, p < 0.0001) was detected. The suppression of sEVs release exhibited significant adverse effects on tumor development and metastatic dissemination in the in vivo tumor model. The present study suggests that the targeting of RAB27A could be a viable cancer therapy for SCLC. Targeting the exosomal pathway has the potential to enhance treatment efficacy, and SCLC may depend on sEVs secretion.},
}

Humans
Animals
Cell Proliferation
*rab27 GTP-Binding Proteins/genetics/metabolism
Mice
*Lung Neoplasms/pathology/genetics/metabolism
*Small Cell Lung Carcinoma/genetics/pathology/metabolism/therapy
*CRISPR-Cas Systems
*Extracellular Vesicles/metabolism/genetics
Cell Line, Tumor
Female
Neoplasm Metastasis
Xenograft Model Antitumor Assays

@article {pmid42567105,
year = {2026},
author = {Hassannia, M and Amirifar, P},
title = {CRISPR-Cas9 gene editing approaches in colorectal cancer: Current progress and future prospects.},
journal = {Cancer treatment and research communications},
volume = {48},
number = {},
pages = {101341},
doi = {10.1016/j.ctarc.2026.101341},
pmid = {42567105},
issn = {2468-2942},
mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods ; Animals ; },
abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by genetic heterogeneity and the accumulation of mutations in key oncogenes and tumor suppressor genes. CRISPR-Cas9 technology has greatly advanced genetic research by enabling precise genome editing. This review focuses on the innovative applications of CRISPR-Cas9 in CRC research, particularly its role in identifying novel therapeutic targets, elucidating mechanisms of drug resistance, and uncovering metabolic and stem cell pathway alterations in tumorigenesis. We highlight the diverse CRISPR systems, including Cas9, Cas12, Cas13, and advanced variants such as CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), base editing, and prime editing, which have expanded gene knockout studies and enhanced our understanding of CRC. Despite these breakthroughs, challenges such as off-target effects and delivery limitations remain. Ongoing efforts to refine CRISPR technology aim to enhance its precision and clinical applicability, ultimately paving the way for more effective and personalized treatment strategies for CRC. In this review, we explore these advances and focus on the latest developments in CRISPR-based approaches for CRC treatment.},
}

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

@article {pmid42567230,
year = {2026},
author = {Dewar, CE and King, EFB and Rojas, F},
title = {Optimising electroporation protocols for Trypanosoma brucei using the Amaxa 4D-nucleofector system.},
journal = {Molecular and biochemical parasitology},
volume = {267},
number = {},
pages = {111767},
doi = {10.1016/j.molbiopara.2026.111767},
pmid = {42567230},
issn = {1872-9428},
mesh = {*Trypanosoma brucei brucei/genetics ; *Transfection/methods ; *Electroporation/methods/instrumentation ; Cell Survival ; Animals ; CRISPR-Cas Systems ; },
abstract = {Stable transfection of Trypanosoma brucei remains a cornerstone for functional genetic studies in this model parasite. Although the Amaxa Nucleofector II system dramatically improved transfection efficiency in both monomorphic and pleomorphic bloodstream forms, the more recent 4D Nucleofector platform offers enhanced programmability and buffer flexibility that have yet to be systematically evaluated for T. brucei. Here, we benchmark a range of 4D Nucleofector programs to determine optimal parameters for transfection efficiency, cell viability, and reproducibility in bloodstream forms. Using a CRISPR/Cas9 expressing cell line, we compare stable transfection efficiencies across programs. We further demonstrate the advantages of the 16-well Nucleocuvette™ Strip format, enabling simultaneous processing of multiple experimental conditions in 20 µL reactions, reducing DNA, cell, and reagent requirements while increasing experimental throughput. Our results provide a standardized framework for future genetic manipulation of T. brucei using the 4D-Nucleofector X Unit, facilitating robust and reproducible transfection across life-cycle stages and strains.},
}

*Trypanosoma brucei brucei/genetics
*Transfection/methods
*Electroporation/methods/instrumentation
Cell Survival
Animals
CRISPR-Cas Systems

@article {pmid42609829,
year = {2026},
author = {Al-Azzani, H and Aliouat, H and Cheng, H and Dagah, OMA and Al-Subari, MH and Zhou, W and Wu, S},
title = {Antibacterial Immunotherapy: Mechanistic Insights, Emerging Therapeutic Strategies, and Clinical Translation.},
journal = {Infection and drug resistance},
volume = {19},
number = {},
pages = {619916},
pmid = {42609829},
issn = {1178-6973},
abstract = {Antimicrobial resistance (AMR) continues to compromise the effectiveness of conventional antibacterial therapy, driving the development of therapeutic strategies that extend beyond direct antibiotic-mediated bacterial killing. Multidrug-resistant (MDR) pathogens evade treatment through diverse mechanisms, including enzymatic drug inactivation, target modification, efflux pump overexpression, biofilm formation, and persisters development. AMR results in chronic and recurrent infections, prolonged hospitalization, increased healthcare costs, and elevated morbidity and mortality, underscoring the need for innovative therapeutic approaches that target both the pathogen and the host. To bridge the dynamic interplay between bacterial pathogens and the host immune system with emerging therapeutic innovations, this narrative review first examines the biological mechanisms underlying bacterial resistance. It then explores therapeutic strategies beyond conventional antibiotics, providing an overview of current approaches and their limitations, including drug repurposing, bacteriophage therapy, and CRISPR-Cas technology. The review subsequently focuses on antibacterial immunotherapy, discussing a broad range of emerging approaches, including probiotics, monoclonal antibodies, cell-based therapies, host-directed therapies, aptamers, nanotechnology-based platforms, cytokine-based therapies, and antimicrobial peptides. An integrated overview of preclinical evidence, clinical studies, and FDA-approved therapies is presented to assess the translational potential of immunotherapy strategies in combating AMR. Scientific, regulatory, manufacturing, and implementation challenges that influence their successful translation into clinical practice are discussed throughout. By integrating the biological basis of host-pathogen interactions with emerging antibacterial therapeutics and their translational development, this review provides a comprehensive framework for evaluating innovative strategies against antimicrobial resistance. In contrast to modality-focused reviews, it offers a unified perspective that highlights the complementary roles of pathogen-targeted and host-directed interventions and identifies future opportunities to improve the prevention and management of multidrug-resistant bacterial infections.},
}

@article {pmid42379321,
year = {2026},
author = {Wei, Y and Zhu, G},
title = {Targeted gene editing of heterodisulfide reductase mediated electron bifurcation optimises heterodisulfide reductase-ferredoxin-adenosine triphosphate axis for enhanced methanogenesis in anaerobic granular sludge.},
journal = {Bioresource technology},
volume = {459},
number = {},
pages = {135281},
doi = {10.1016/j.biortech.2026.135281},
pmid = {42379321},
issn = {1873-2976},
mesh = {*Methane/biosynthesis/metabolism ; *Sewage/microbiology ; *Adenosine Triphosphate/metabolism ; *Oxidoreductases/genetics/metabolism ; Anaerobiosis ; *Ferredoxins/metabolism ; *Gene Editing/methods ; *Electrons ; CRISPR-Cas Systems/genetics ; },
abstract = {The thermodynamic bottleneck of syntrophic propionate oxidation constrains the efficiency and stability of anaerobic digestion (AD), which depends on flavin-based electron bifurcation (FBEB) mediated by heterodisulfide reductase (Hdr). Five CRISPR-Cas9 engineered Methanobacterium formicicum strains targeting Hdr, nickel homeostasis, and flavin metabolism were evaluated in 10 % and 20 % granular sludge systems to rewire electron flux toward the energy-conserving ferredoxin (Fd) reduction pathway. Within each experimental tier, results are reported against matched controls. In the enzyme-supplementation tier, the heterodisulfide reductase ABC subunit-F420-reducing hydrogenase A subunit (HdrABC-MvhA) supplement produced the highest cumulative methane yield and elevated intracellular adenosine triphosphate (ATP) to 28.31 ± 1.60 nmol/L versus 16.27 ± 0.90 nmol/L in the matched wild-type control (1.74 ± 0.10-fold; P < 0.01, n = 3). In the genome-editing tier, the Δhpt-nikR strain achieved the highest cumulative methane yield under high sludge loading. Because N[5]-methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) and A1A0-ATP synthase activities were not directly measured, and the Hdr activity increment falls within the variance of crude-extract assays, these co-occurring changes are interpreted as correlative support for a putative "Hdr-Fd-ATP" working model rather than direct demonstration of a defined energy-conservation pathway. The engineered strains enriched hydrogenotrophic methanogens (Methanobacterium, 1.08-1.13-fold) and increased the predicted genomic abundance of electron-bifurcation, CO2-reduction, and methyl-transfer pathway genes, as inferred from 16S rRNA-based functional prediction. This study provides correlative evidence that CRISPR-based metabolic engineering of methanogens can modulate the Hdr-Fd-ATP axis within heterogeneous granular sludge communities, establishing a mechanistic framework for in situ bioaugmentation strategies targeting intracellular energy-conservation bottlenecks.},
}

*Methane/biosynthesis/metabolism
*Sewage/microbiology
*Adenosine Triphosphate/metabolism
*Oxidoreductases/genetics/metabolism
Anaerobiosis
*Ferredoxins/metabolism
*Gene Editing/methods
*Electrons
CRISPR-Cas Systems/genetics

@article {pmid42463075,
year = {2026},
author = {Cheng, Y and Niu, S and Zhang, Z and Qian, H and Huang, Z and Jing, H and Liao, L and Man, C and Gao, H and Chen, Q and Du, L and Chen, S and Wang, F},
title = {TBC1D14 positively regulates autophagy induced by Brucella melitensis vaccine strain BA0711 in Sheep Leydig Cells.},
journal = {Microbial pathogenesis},
volume = {219},
number = {},
pages = {108664},
doi = {10.1016/j.micpath.2026.108664},
pmid = {42463075},
issn = {1096-1208},
mesh = {Animals ; *Autophagy/genetics ; *Brucella melitensis/immunology ; Male ; *GTPase-Activating Proteins/genetics/metabolism ; Sheep ; *Leydig Cells/microbiology/metabolism/immunology ; Gene Knockout Techniques ; *Brucellosis/veterinary/immunology ; CRISPR-Cas Systems ; Microscopy, Electron, Transmission ; },
abstract = {Brucella is an intracellular Gram-negative bacterium that primarily infects the host reproductive and immune systems, inducing autophagy and facilitating pathogen replication. TBC (Tre2-Bub2-Cdc16) domain-containing proteins are important in membrane trafficking, cell polarity, and signal transduction as regulators of Rab small GTPases. Previously, we demonstrated that B. melitensis M5-90 modulates the expression of miR-146b-5p, which targets TBC1D14, in RAW264.7 cells. In this study, CRISPR-Cas9 was used to generate TBC1D14-knockout (KO) Sheep Leydig cells (SLCs), and B. melitensis BA0711 treatment experiment was conducted at a multiplicity of infection (MOI) of 100. After confirming that SLCs retain autophagic activity, Western blot, autophagy flux assays, transmission electron microscopy (TEM), and RT-qPCR were performed to identify the function of TBC1D14. We found that autolysosome fluorescence was significantly enhanced in NC-SLCs compared with TBC1D14-KO-SLCs following BA0711 treatment at 8 and 12 hpi. Notably, autophagy flux in NC-SLCs remained consistently higher than that in KO-SLCs from 8 hpi onwards. Consistently, Western blot revealed a decreased LC3-II/LC3-I ratio, and TEM confirmed a reduced number of autolysosomes in KO-SLCs at 12 hpi. These results indicate that TBC1D14 positively regulates autophagy in SLCs by BA0711 stimulation. Although transcriptomic and proteomic analyses indicated activation of autophagy- and phagocytosis-related pathways during BA0711 exposure, this response was significantly attenuated in TBC1D14-KO cells. Furthermore, TBC1D14 knockout led to downregulation of phagosome-related proteins and altered chemotaxis pathways, whereas re-expression of TBC1D14 in KO cells upregulated RAB28. In conclusion, this study demonstrates that TBC1D14 positively regulates autophagy and provides insights into the response of SLCs treated by B. melitensis BA0711. Furthermore, it suggests that RAB28 may serve as a downstream effector of TBC1D14.},
}

Animals
*Autophagy/genetics
*Brucella melitensis/immunology
Male
*GTPase-Activating Proteins/genetics/metabolism
Sheep
*Leydig Cells/microbiology/metabolism/immunology
Gene Knockout Techniques
*Brucellosis/veterinary/immunology
CRISPR-Cas Systems
Microscopy, Electron, Transmission

@article {pmid42492860,
year = {2026},
author = {Lee, JH and Lee, ES and Xiang, XR and Kyung, SM and Seo, J and Lee, W and Park, HE and Shin, MK and Yoo, HS},
title = {Roles of mdh and MAP1981c in Mycobacterium avium subsp. paratuberculosis intracellular survival within bovine monocyte-derived macrophages via CRISPR interference.},
journal = {Microbial pathogenesis},
volume = {219},
number = {},
pages = {108724},
doi = {10.1016/j.micpath.2026.108724},
pmid = {42492860},
issn = {1096-1208},
mesh = {Animals ; *Mycobacterium avium subsp. paratuberculosis/genetics/pathogenicity/growth & development ; Cattle ; *Macrophages/microbiology/immunology ; Paratuberculosis/microbiology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Profiling ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Microbial Viability ; Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and is capable of persisting within macrophages despite the activation of host immune defenses. Although this intracellular persistence is a key determinant of MAP pathogenicity, the bacterial factors and host responses that regulate this process remain poorly understood. In this study, we established the first CRISPR interference (CRISPRi) platform applied to bovine monocyte-derived macrophages (MDM) to evaluate the functions of MAP genes involved in intracellular survival and to perform an integrative analysis of host transcriptomic responses. MAP mutants were targeted to two genes (mdh and MAP1981c). The optimal concentration of anhydrotetracycline (ATc) was determined to be 2 μg/ml by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to 72 h. The gene expression profiles and intracellular MAP levels were investigated using RNA-seq and colony-forming units, respectively. The survival rates of the MAP mutants significantly decreased with the time course of infection in MAP-mdhKD and MAP1981cKD (KD, knockdown). RNA-seq-based gene expression profiling suggested that target gene silencing in MAP mutants led to altered expression of host genes involved in lipid metabolism, T-cell activation reduction, and antimicrobial response in bovine MDM, contributing to reduced intracellular survival of MAP. Our study demonstrates that the downregulation of mdh and MAP1981c in MAP significantly alters the host transcriptomic landscape in bovine MDM, revealing their critical roles in subverting host immune defenses for intracellular persistence.},
}

Animals
*Mycobacterium avium subsp. paratuberculosis/genetics/pathogenicity/growth & development
Cattle
*Macrophages/microbiology/immunology
Paratuberculosis/microbiology
*Bacterial Proteins/genetics/metabolism
Gene Expression Profiling
Host-Pathogen Interactions
CRISPR-Cas Systems
Microbial Viability
Gene Expression Regulation, Bacterial
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42576388,
year = {2026},
author = {Zhang, Y and Yang, Y and Liu, Z and Li, Y and Xue, Y and Zhang, Z and Chen, G and Liu, J and He, M and Lu, T and Zhang, Y and Zhao, D and Yang, K and Miao, L and Gao, F and Guo, Y},
title = {The DreAM-plus integrative RNA switch enhances transient AAV expression and reduces side effects of gene editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2026.08.006},
pmid = {42576388},
issn = {1525-0024},
abstract = {Uncontrolled long-term adeno-associated virus (AAV) expression prohibits therapeutic strategies that require more precise and dynamic regulation. For example, long-lasting expression of gene editors by AAV could augment off-target effects and immunogenicity. Drug-inducible RNA switches are desirable tools to achieve transient AAV expression. However, current RNA switches only target a single mechanism such as transcription or RNA splicing, exhibiting limited capacity in transgene regulation. Here, we report DreAM-plus, a multilayer RNA switch that integrates an aptamer-based poly(A) regulator (pA), a drug-elicitable alternative splicing module (DreAM), and an engineered P2A element with conditional upstream open reading frames (uORFs). The pA-DreAM concatenation enhanced gene inducibility by up to 5-fold more than pA or DreAM alone, with 1.4- to 6.3-fold further improvement by uORFs. DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, and IsDra2 TnpB) with a temporal resolution of less than 24 h, which significantly mitigated off-target effects by 1.4- to 2.8-fold. With lipid-nanoparticle-delivered pre-existing immunity in mice, DreAM-plus attenuated AAV-delivered Cas-specific CD8[+] T cell immune toxicity in the liver and heart. Therefore, the inducible RNA switches could be synergistically integrated to build sophisticated genetic cassettes for enhanced safety of AAV-mediated gene editing.},
}

@article {pmid42603302,
year = {2026},
author = {He, R and Zhang, C and Zhang, J and Zhang, K and Yin, W and Qiao, B and Zhang, L and Zhang, S and Qiao, J and Liu, Y},
title = {A One-Pot Reverse Transcriptase-Mediated, Pre-amplification-Free CRISPR/Cas12a Assay for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01202},
pmid = {42603302},
issn = {2379-3694},
support = {2025AFB825//Natural Science Foundation of Hubei Province/ ; WHPU-EIRF-2606//Wuhan Polytechnic University/ ; 2023DJC136//Department of Science and Technology of Hubei Province/ ; },
abstract = {CRISPR/Cas12a holds great promise for biosensing and diagnostics, but conventional methods suffer from low catalytic efficiency, high background, and reliance on pre-amplification. Direct detection of structured RNAs also remains challenging. Herein, we report the development of a reverse transcriptase and LNA probe (LNA-p)-mediated CRISPR/Cas12a positive feedback system (RTLC) for highly efficient, one-pot detection of both DNA and RNA. Without pre-amplification or thermal cycling, the assay achieves a 0.5 aM detection sensitivity within 27 min, exhibits single-base resolution, and allows direct detection of RNAs up to 985 nt in length. Together, RTLC is successfully validated in practical samples by detecting lncRNA HULC and miR-21, offering a robust, versatile tool for high-performance nucleic acid diagnostics.},
}

@article {pmid42604377,
year = {2026},
author = {Su, L and Mara, P and Edgcomb, V and Teske, A},
title = {From CRISPR-Cas to Argonautes: Defense systems of bacteria and archaea in the hydrothermal deep subsurface.},
journal = {ISME communications},
volume = {6},
number = {1},
pages = {ycag200},
pmid = {42604377},
issn = {2730-6151},
abstract = {Microbial defense systems are central to virus-host interactions and thus to microbial survival but remain poorly studied in microbial communities of extreme environments. Here we examine the repertoire and distribution of microbial defense genes in hydrothermally influenced deep subsurface sediments and rocks of the Guaymas Basin (Gulf of California). Restriction-modification and abortive infection systems were broadly distributed across the examined sediment depths, and clustered, regularly interspaced short palindromic repeats-Cas systems were primarily detected within temperate surficial sediments. Prokaryotic Argonaute genes were found mostly in archaeal MAGs at elevated temperatures up to 81.8°C. Overall defense gene repertoire declines downcore, as temperature increases and phylogenetic host range narrows, with phylogeny as the decisive control factor. We suggest that these defense systems, together with DNA repair mechanisms, protein maintenance activities, and RNA modification pathways, constitute a survival toolkit for the hydrothermally influenced subsurface, where energy limitation and temperature extremes select for resilient microbial communities.},
}

@article {pmid42605670,
year = {2026},
author = {Le, S and Thach, T},
title = {Structure- and deep learning-guided engineering of a size-minimized CRISPR/Cas.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70707},
pmid = {42605670},
issn = {1742-4658},
abstract = {Multidomain proteins play central roles in cellular regulation, yet their intrinsic flexibility and structural instability often hinder optimization for biotechnological applications. Here, we present an integrated structure-guided and deep learning-assisted engineering strategy that combines structure modeling with Protein Message Passing Neural Network (ProteinMPNN)-based sequence design to generate an ultracompact CRISPR activator (uCRISPRa) derived from the miniature CRISPR/Cas12f. Structural and computational analyses identified flexible, nonessential regions within both Cas12f and its single-guide RNA (sgRNA), enabling rational truncation and sequence redesign while preserving DNA-targeting capability. When delivered as mRNA encapsulated in lipid nanoparticles, uCRISPRa achieved selective activation of olfr544 among more than a thousand homologous olfactory receptor genes in skeletal muscle cells, leading to enhanced mitochondrial biogenesis. These findings demonstrate that the integration of structure-based protein engineering with deep learning sequence optimization provides a powerful framework for developing compact and efficient CRISPR effectors, offering broad potential for precise gene regulation and functional studies of complex macromolecular systems.},
}

@article {pmid42608492,
year = {2026},
author = {Majewska, Z and Jursza, G and Dolzblasz, A},
title = {Dexamethasone-inducible LhGR/pOp system: simple and flexible spatiotemporal control of gene expression.},
journal = {Planta},
volume = {264},
number = {4},
pages = {},
pmid = {42608492},
issn = {1432-2048},
support = {BPIDUB.4610.174.2022//Centre of Excellence and Applied Sport Science Research, Queensland Academy of Sport/ ; 501/73/10110/MPK 2599150000/2026//funding provided by the Department of Plant Developmental Biology/ ; },
mesh = {*Dexamethasone/pharmacology ; *Arabidopsis/genetics/drug effects ; *Gene Expression Regulation, Plant/drug effects ; Plants, Genetically Modified/genetics ; *Arabidopsis Proteins/genetics/metabolism ; },
abstract = {Our review illustrates how the dexamethasone-inducible LhGR/pOp system has been used across numerous tissues/organs and plant species, and summarizes the resources and inducer application procedures established to date. The establishment of groundbreaking molecular biology tools has enabled the rapid advancement of research based on the model plant Arabidopsis, which currently strongly benefits non-model but economically important species. Spatiotemporal control of transgene expression via the chemically inducible system GR-LhG4/pOp has proven to be a particularly powerful, universal and non-invasive experimental approach. This review characterizes the mechanism of action of GR-LhG4/pOp and synthesizes information on available GR-LhG4 transgenic lines and inductor (dexamethasone) application procedures across various plant tissues. Moreover, feasible experimental approaches are depicted that range from classical ones aiming at selected genes overexpression or silencing to highly innovative ones, like those integrating the GR-LhG4/pOp system with CRISPR-Cas for targeted ablation of specific cell types. Information about the existing GR-LhG4/pOp lines from non-Arabidopsis plants is also provided, in hope that this sophisticated but universal research tool will become more exploited in crop research.},
}

*Dexamethasone/pharmacology
*Arabidopsis/genetics/drug effects
*Gene Expression Regulation, Plant/drug effects
Plants, Genetically Modified/genetics
*Arabidopsis Proteins/genetics/metabolism

@article {pmid42609038,
year = {2026},
author = {Candelotti, AM and Garzillo, G and Bartolini, S and Di Censo, C and Peruzzi, G and Stabile, H and Fionda, C and Sciumè, G and Pietropaolo, G},
title = {Rapid and Highly Efficient CRISPR-Cas9 RNP Genome Editing in Primary ILC2s.},
journal = {European journal of immunology},
volume = {56},
number = {8},
pages = {e70254},
pmid = {42609038},
issn = {1521-4141},
support = {IG-28719//the Italian Association for Cancer Research (AIRC)-Investigator Grant "Mirella Rizzo"/ ; PNRR-MAD-2022-12375947//the Italian Association for Cancer Research (AIRC)-Investigator Grant "Mirella Rizzo"/ ; //Next Generation EU-PNRR M6C2 - Investimento 2.1 Valorizzazione e potenziamento della ricerca biomedica del SSN/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; Mice ; Humans ; },
abstract = {CRISPR-mediated gene editing enables efficient genetic manipulation of ILC2s through ex vivo or in vivo activation and Cas9 RNP delivery. This platform provides a robust approach to dissect gene function in ILC2s, with minimal manipulation.},
}

Animals
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics
Mice
Humans

@article {pmid40945831,
year = {2025},
author = {Duan, X and Zhou, Z and Zeng, X and Chen, J and Mao, A},
title = {Single-step purification of functional Cas9 protein via the ubiquitin expression system.},
journal = {International journal of biological macromolecules},
volume = {328},
number = {Pt 1},
pages = {147590},
doi = {10.1016/j.ijbiomac.2025.147590},
pmid = {40945831},
issn = {1879-0003},
mesh = {*Ubiquitin/genetics/metabolism/chemistry ; Escherichia coli/genetics/metabolism ; *CRISPR-Associated Protein 9/isolation & purification/genetics/metabolism/chemistry ; Streptococcus pyogenes/enzymology/genetics ; CRISPR-Cas Systems ; Chromatography, Affinity ; Recombinant Fusion Proteins/isolation & purification/genetics ; *Gene Expression ; },
abstract = {The CRISPR/Cas9 system serves as a powerful platform for precise genome editing, with CRISPR/Cas9 ribonucleoprotein (RNP) complexes exhibiting superior editing efficiency compared to alternative delivery modalities. However, current methods for Cas9 production typically involve multiple purification steps. Here, we developed a streamlined single-step purification strategy for preparation of functional Streptococcus pyogenes Cas9 (SpCas9) with dispensable tag removal after inducible expression in Escherichia coli. Notably, N-terminal Ubiquitin (Ub) fusion preserved both accurate nuclear localization and remarkable protein stability, enabling robust production of over 8 mg/L of >95 % pure Ub-Cas9 via single metal affinity chromatography. Comprehensive endonuclease activity assays confirmed that the purified Ub-Cas9 maintained robust DNA cleavage capacity both in vitro and in vivo. Our work provides a convenient and efficient platform for production of highly purified Ub-Cas9 protein for widespread applications in gene function study.},
}

*Ubiquitin/genetics/metabolism/chemistry
Escherichia coli/genetics/metabolism
*CRISPR-Associated Protein 9/isolation & purification/genetics/metabolism/chemistry
Streptococcus pyogenes/enzymology/genetics
CRISPR-Cas Systems
Chromatography, Affinity
Recombinant Fusion Proteins/isolation & purification/genetics
*Gene Expression

@article {pmid42323083,
year = {2026},
author = {Byrnes, C and Clarke, BA and Zhu, H and Lee, YT and Majumder, S and Kono, M and Proia, RL},
title = {1-Deoxysphingolipids require very-long-chain ceramide synthesis to induce ER stress and cytotoxicity.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {8},
pages = {113281},
pmid = {42323083},
issn = {1083-351X},
mesh = {Humans ; *Ceramides/biosynthesis/chemistry ; *Endoplasmic Reticulum Stress/drug effects ; *Sphingolipids/metabolism/chemistry ; Cell Line, Tumor ; Cell Survival/drug effects ; CRISPR-Cas Systems ; },
abstract = {Sphingolipids play key roles in cellular systems both as membrane components and as signaling molecules. Their biosynthesis, which occurs in the endoplasmic reticulum (ER), begins with the condensation of an amino acid, typically serine, and a fatty acyl-CoA. Under certain pathological conditions, alanine can be substituted for serine in the condensation reaction, producing 1-deoxysphingolipids, which lack the 1-hydroxyl group on the sphingoid base. Unlike typical sphingolipids, 1-deoxysphingolipids are unable to accept a head group modification, which alters their metabolic processing and prevents their canonical degradation. The accumulation of these "headless" 1-deoxysphingolipids causes neurotoxicity in various neurological and metabolic disorders. Here, we conducted a genome-wide CRISPR-Cas9 screen to identify pathways leading to 1-deoxysphinganine-induced toxicity in SH-SY5Y cells, a model used to study neurotoxic responses. Our top genetic hits highlighted the pathway involved in synthesizing ceramides with very-long-chain fatty acids (C22-C26). Using CRISPR-Cas9-modified SH-SY5Y cells with loss-of-function (LOF) mutations in the TECR or CERS2 genes-both critical for producing very-long-chain ceramides-we validated that this pathway was essential for 1-deoxysphinganine-mediated toxicity. Furthermore, we demonstrated that the ceramide synthesis pathway is required for 1-deoxysphinganine to trigger ER stress, as evidenced by significantly increased expression of the unfolded protein response in WT, but not TECR or CERS2 LOF mutant, SH-SY5Y cells exposed to 1-deoxysphinganine. Collectively, the data support a model in which ceramide synthase-dependent conversion of 1-deoxysphinganine to very-long-chain 1-deoxyceramide species is required for full ER-stress induction and cytotoxicity. The findings highlight potential therapeutic targets for neuropathological diseases caused by 1-deoxysphingolipid accumulation.},
}

Humans
*Ceramides/biosynthesis/chemistry
*Endoplasmic Reticulum Stress/drug effects
*Sphingolipids/metabolism/chemistry
Cell Line, Tumor
Cell Survival/drug effects
CRISPR-Cas Systems

@article {pmid42459132,
year = {2026},
author = {Wen, Z and Wei, C and Shen, M and Yu, S},
title = {Signal logic gate mediated by a controllable CRISPR/Cas12a system for simultaneous detection of DNA mutation and methylation.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {32},
pages = {6823-6830},
doi = {10.1039/d6ay01060a},
pmid = {42459132},
issn = {1759-9679},
mesh = {*DNA Methylation/genetics ; Humans ; Septins/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Promoter Regions, Genetic ; Colorectal Neoplasms/genetics/diagnosis ; DNA Mutational Analysis/methods ; },
abstract = {Early detection of both genetic mutations and epigenetic modifications is critical for cancer diagnosis, but current methods often require separate assays or suffer from bisulfite-induced DNA damage. Here, we present a controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction. The strategy converts target information into two distinct ssDNA activators (T1 for methylation and T2 for mutation) via orthogonal enzymatic cascades (GlaI- and FEN1-mediated cleavage followed by strand displacement amplification). By limiting crRNA concentrations, the CRISPR/Cas12a trans-cleavage activity produces three well-resolved fluorescence kinetic states: low (mutation only), medium (methylation only), and high (both targets), respectively, which could be distinguished using three predefined threshold values. Validation with clinical samples from colorectal cancer patients and healthy controls showed complete concordance with Sanger sequencing and qPCR. And the proposed method successfully detected Septin9 methylation in peripheral blood. This isothermal, single-tube, bisulfite-free strategy offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.},
}

*DNA Methylation/genetics
Humans
Septins/genetics
*CRISPR-Cas Systems/genetics
*Mutation
Proto-Oncogene Proteins p21(ras)/genetics
Promoter Regions, Genetic
Colorectal Neoplasms/genetics/diagnosis
DNA Mutational Analysis/methods

@article {pmid42600477,
year = {2026},
author = {Yuan, J and Shen, M and Ding, L and Yang, X and Wu, Y and Yu, S},
title = {Orthogonal Cas13a/Cas12a cascade for one-pot amplification-free detection of miRNA-21.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130431},
doi = {10.1016/j.talanta.2026.130431},
pmid = {42600477},
issn = {1873-3573},
abstract = {MicroRNAs (miRNAs) are promising biomarkers for clinical diagnosis and disease monitoring. However, current CRISPR/Cas-based miRNA sensors generally require reverse transcription or nucleic acid amplification to improve sensitivity, which complicates the workflow and increases the risk of contamination, nonspecific amplification, and false-positive results. Herein, we developed a one pot amplification-free Cas13a/Cas12a cascade platform based on a designed dual-functional molecular bridge probe, Conv HP-3, which served as both a substrate for Cas13a-mediated trans-cleavage and an activator for Cas12a-mediated trans-cleavage, thereby linking target recognition to cascade signal amplification for miRNA-21 detection. Following the introduction of miRNA-21, Cas13a was specifically activated through target-crRNA recognition and cleaved the Conv HP-3 probe to release a Cas12a-activating DNA fragment. This fragment subsequently triggered Cas12a-mediated cleavage of the ssDNA reporter, generating a markedly enhanced fluorescence signal. This one-pot Cas13a/Cas12a cascade fluorescence biosensor enabled quantitative detection of miRNA-21 over a concentration range of 1-1000 pmol/L within 60 min, with a low detection limit of 0.66 pM. Notably, the assay achieved average recoveries ranging from 95.97% to 108.59%, with a variation between 0.6% and 1.78%, demonstrating its good accuracy and precision. This biosensing platform shows great promise for the rapid and sensitive detection of miRNAs in clinical applications.},
}

@article {pmid42600889,
year = {2026},
author = {Shiroshita, K and Stolz, A and Malouf, C and Tran, VL and Li, J and Weiss, MJ and Naldini, L},
title = {Unlocking the Curative Potential of Gene Transfer and Editing for Hematopoietic Disorders.},
journal = {Experimental hematology},
volume = {},
number = {},
pages = {105493},
doi = {10.1016/j.exphem.2026.105493},
pmid = {42600889},
issn = {1873-2399},
abstract = {Hematopoietic stem and progenitor cells (HSPCs) gene therapy may transform the therapeutic landscape for inherited hematological disorders and already offers potentially curative options for some diseases, including inborn errors of immunity and β-hemoglobinopathies. Its continued success relies on further refinement of gene transfer technologies, gene editing tools such as CRISPR-Cas, and optimized ex vivo HSPC manipulation protocols that ensure robust, long-term engraftment and clonal diversity with reduced-toxicity, non-genotoxic conditioning strategies. Here, we review recent developments and refinements in gene transfer and editing technologies for HSPCs, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar. Future directions must prioritize integrating technological innovation with the development of equitable and simplified models to reduce costs and ensure that these life-saving cellular therapies reach patients worldwide. Teaser abstract Hematopoietic stem and progenitor cells (HSPCs) gene therapy is advancing rapidly, with lentiviral gene transfer, genome editing, and emerging in vivo delivery approaches expanding the therapeutic landscape for inherited hematologic disorders. Recent clinical successes have demonstrated the potential for durable correction, while ongoing refinements continue to improve safety, efficacy, and feasibility. Key challenges remain in genotoxicity, conditioning toxicity, manufacturing scalability, and equitable global access.},
}

@article {pmid42601370,
year = {2026},
author = {Iqbal, G and Pinto, N and Pawaskar, D and Sonwane, AA and Rasal, KD and Singh, LS and Chikkathimmashetty, NN and Goswami, M},
title = {Design and transfection of CRISPR/Cas9 constructs for the myostatin gene in Labeo rohita muscle cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42601370},
issn = {2045-2322},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Transfection/methods ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Muscle Cells/metabolism ; *Cyprinidae/genetics ; },
abstract = {Myostatin (mstn) is a negative regulator of skeletal muscle growth and is considered as an important target for enhancing aquaculture production. The present study aimed to design and validate single-guide RNAs (sgRNAs) and CRISPR/Cas9 constructs for exon 1 of the mstnb gene in Labeo rohita, and to evaluate their transfection efficiency in the L. rohita dorsal muscle (LRDM) cell line at the 10th, 20th, and 30th passages. sgRNAs were designed and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector using BbsI restriction digestion and ligation. Successful insertion and correct orientation of the sgRNAs were confirmed through Sanger sequencing. LRDM cells were revived and maintained in L-15 medium supplemented with 10% fetal bovine serum. Transfection was performed at the 10th, 20th, and 30th passages. Distinct GFP-positive cells were observed at all passages for both sgRNA constructs, indicating the ability of the developed cell line to successfully express the constructs across different passages. The study successfully established CRISPR/Cas9 plasmid constructs for the mstnb gene in L. rohita and demonstrated their transfection in LRDM cell line across multiple passages. These findings provide a basis for future studies on genome editing approaches using CRISPR/Cas9 constructs in fish muscle cell lines and highlight the potential application of CRISPR/Cas9 technology for genetic engineering applications in fish muscle cells.},
}

Animals
*Myostatin/genetics
*CRISPR-Cas Systems/genetics
*Transfection/methods
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
*Muscle Cells/metabolism
*Cyprinidae/genetics

@article {pmid42602771,
year = {2026},
author = {Hina, A and Abbasi, A and Chaudhry, A and Sanaullah, T and Arshad, M and Sarwar, HM and Sarfaraz, S and Karikari, B and Kavhiza, NJ},
title = {A review of flavonoids at the crossroads of plant defense: integrating biotic and abiotic stress tolerance through AI- and CRISPR/Cas-guided metabolic reprogramming.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1865665},
pmid = {42602771},
issn = {1664-462X},
abstract = {Flavonoids are multifunctional phenylpropanoid-derived metabolites that occupy a central position in plant adaptation to environmental stress. Beyond their established roles in antioxidant protection, they contribute to defense against pathogens and herbivores, signaling processes, and physiological acclimation to adverse environmental conditions. Although flavonoid responses to individual biotic or abiotic stresses have been extensively investigated, considerably less attention has been given to how flavonoid-associated regulatory networks function when multiple stresses occur simultaneously. This gap is particularly important because crops in agricultural systems are routinely exposed to overlapping biotic and abiotic challenges that generate distinct physiological, transcriptional, and metabolic responses. This review synthesizes current knowledge of flavonoid biosynthesis, structure-activity relationships, and the regulatory mechanisms governing flavonoid accumulation under diverse stress conditions. Particular emphasis is placed on the reorganization of flavonoid-associated networks under combined stress, including signaling crosstalk, pathway competition, metabolic trade-offs, and flux allocation that collectively shape adaptive responses. This review further evaluates how artificial intelligence can support identification of regulatory targets and pathway bottlenecks, how integration with CRISPR/Cas technologies may facilitate more precise manipulation of flavonoid biosynthesis, and how iterative Design-Build-Test-Learn (DBTL) frameworks could improve predictive flavonoid engineering through continuous integration of computational prediction and experimental validation. By integrating advances in stress biology, computational prediction, genome engineering, and iterative DBTL frameworks, this review outlines a roadmap for predictive reprogramming of flavonoid networks under combined stress and the development of crops with improved resilience to increasingly complex environmental conditions.},
}

@article {pmid42286694,
year = {2026},
author = {Xu, J and Xu, J and Cao, C and Shu, Y and Shen, L and He, X and Huangfu, Q and Sun, C and Wang, W and Wei, J and Cai, M and Wang, B and Li, A and Liu, Y and Wen, J},
title = {IDMME and IDMDE: inducible CRISPR-dCasRx platforms for spatiotemporal RNA m[5]C editing.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42286694},
issn = {1474-760X},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; 82371630//National Natural Science Foundation of China/ ; },
mesh = {*5-Methylcytosine/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA Methylation ; *RNA Editing ; Animals ; Epigenesis, Genetic ; *Gene Editing/methods ; Epigenome Editing ; Carcinoma, Renal Cell/genetics ; Cell Line, Tumor ; },
abstract = {RNA 5-methylcytosine (m[5]C) is a dynamic epigenetic mark implicated in tumorigenesis, however, existing editors lack spatiotemporal regulation and reversibility. Here, we develop abscisic acid (ABA)-inducible CRISPR-dCasRx systems for programmable m[5]C methylation (IDMME) and demethylation (IDMDE). These editors enable site-specific, low off-target m[5]C modification through ligand-dependent assembly of split effector domains. We further integrate photocaged ABA to achieve light-controlled activation. Application in renal carcinoma models shows that targeted m[5]C editing modulates transcript function and suppresses tumor growth in vitro and in vivo. This platform provides a versatile, spatiotemporally controllable approach for dissecting RNA epigenetic mechanisms and advancing RNA-based therapeutic strategies.},
}

*5-Methylcytosine/metabolism
Humans
*CRISPR-Cas Systems
RNA Methylation
*RNA Editing
Animals
Epigenesis, Genetic
*Gene Editing/methods
Epigenome Editing
Carcinoma, Renal Cell/genetics
Cell Line, Tumor

@article {pmid42361799,
year = {2026},
author = {He, W and Huang, JW and Wang, Y and Hayward, SB and Leuzzi, G and Fu, R and Wang, S and Vaitsiankova, A and Chen, Y and Bedford, MT and Guerois, R and Ciccia, A and Xu, H},
title = {Deciphering protein mutation-phenotype linkages from CRISPR-based tiling mutagenesis screens.},
journal = {Cell systems},
volume = {17},
number = {8},
pages = {101651},
pmid = {42361799},
issn = {2405-4720},
support = {R01 CA197774/CA/NCI NIH HHS/United States ; R35 GM137927/GM/NIGMS NIH HHS/United States ; R35 GM153387/GM/NIGMS NIH HHS/United States ; R35 GM156417/GM/NIGMS NIH HHS/United States ; },
mesh = {*Mutagenesis/genetics ; Phenotype ; Mutation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-based high-throughput mutagenesis screens enable systematic mapping of mutations to phenotypes, yet deciphering mutation-phenotype links remains challenging. Here, we present ProTiler-Mut, a versatile computational framework that leverages tiling mutagenesis screens, which introduce variants across entire protein sequences, to analyze mutation effects at the levels of residues, substructures, and protein-protein interactions (PPIs). Applying ProTiler-Mut to multi-condition base-editing (BE) screens targeting DNA damage response proteins and T cell regulators, we define a separation-of-function (SoF) category beyond the conventional loss-of-function (LoF) and gain-of-function (GoF) classes, where SoF mutations show the strongest enrichment for ClinVar-annotated pathogenic variants. ProTiler-Mut also identifies candidate substructures that enable functional inference of unscreened pathogenic mutations and prioritizes candidate phenotype-associated PPIs potentially disrupted by functional variants. Using ProTiler-Mut, in cells with elevated programmed cell death 1 (PD-1) expression, we identify pathogenic GoF mutations that constitute a substructure that may disrupt mitogen-activated protein kinase (MAPK)1-RSK1 interactions and lead to MAPK activation. Finally, we show that ProTiler-Mut is applicable across different mutagenesis screening platforms. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Mutagenesis/genetics
Phenotype
Mutation/genetics
Humans
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42372725,
year = {2026},
author = {Hu, Y and Li, Q and Li, Y and Zeng, Y and Zheng, L and Shen, J and Gao, X and Zhao, GP and Zhao, W and Dai, L},
title = {Targeted genomic editing of human gut Bacteroides species based on CRISPR-associated transposases.},
journal = {Cell systems},
volume = {17},
number = {8},
pages = {101650},
doi = {10.1016/j.cels.2026.101650},
pmid = {42372725},
issn = {2405-4720},
mesh = {Humans ; *Bacteroides/genetics ; *Gene Editing/methods ; *Transposases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Gut Bacteroides are abundant and critical to human health, yet most are genetically cumbersome, non-model microbes. A widely applicable editing tool for Bacteroides is essential for gut microbiome manipulation. Here, we develop STIB (ShCAST-based transient insertion system for Bacteroides), an efficient genome-editing tool derived from CRISPR-associated transposases that enables rapid and site-specific insertions independent of homologous recombination. By fusing a nicking homing endonuclease to the transposase and an ATPase to Cas12k, we systematically optimize STIB to minimize plasmid cointegration and achieve >97% on-target insertion. STIB exhibits broad applicability across different genomic loci in diverse Bacteroides species, including non-model species. Finally, we apply STIB to achieve species- and site-specific editing of distinct Bacteroides species within a complex synthetic gut microbiota. Overall, STIB expands the toolbox for the functional investigation and engineering of the human microbiome. A record of this paper's transparent peer review process is included in the supplemental information.},
}

Humans
*Bacteroides/genetics
*Gene Editing/methods
*Transposases/genetics/metabolism
CRISPR-Cas Systems/genetics
*Gastrointestinal Microbiome/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42595755,
year = {2026},
author = {Saxena, S and Kabra, M and Abdeen, AA and Tabima, DM and Sinha, D and Rawding, PA and Zhu, M and Xie, R and Kulkarni, T and Hanstad, GM and Fernandez Zepeda, MA and Gamm, DM and Pattnaik, BR and Gong, S and Saha, K},
title = {Genome-wide CRISPR screening identifies cellular factors controlling nonviral genome editing efficiency.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42595755},
issn = {2041-1723},
support = {R35 GM119644/GM/NIGMS NIH HHS/United States ; U19 NS132296/NS/NINDS NIH HHS/United States ; U19NS132296//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R35GM119644//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Connexin 26 ; Connexins/genetics/metabolism ; Potassium Channels, Inwardly Rectifying/genetics/metabolism ; Receptors, GABA-A/genetics/metabolism ; Mutation ; HEK293 Cells ; Genome, Human ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
Connexin 26
Connexins/genetics/metabolism
Potassium Channels, Inwardly Rectifying/genetics/metabolism
Receptors, GABA-A/genetics/metabolism
Mutation
HEK293 Cells
Genome, Human
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42597245,
year = {2026},
author = {Ye, R and Liu, M and Zhang, X and Liang, Q and Cao, Y and Zhu, C and Yu, H and Zhang, S and Wang, Y},
title = {Rapid and ultrasensitive detection of Candida tropicalis using multiple cross-displacement amplification combined with CRISPR/Cas12a.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1796886},
pmid = {42597245},
issn = {2235-2988},
mesh = {*Candida tropicalis/genetics/isolation & purification ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Candidiasis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; DNA, Fungal/genetics ; Multiplex Polymerase Chain Reaction ; Rapid Diagnostic Tests ; Endodeoxyribonucleases/genetics ; CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; DNA, Ribosomal Spacer/genetics ; Bacterial Proteins ; },
abstract = {BACKGROUND: Candida tropicalis (C. tropicalis), classified as a World Health Organization "critical priority" pathogen, causes life-threatening invasive infections with high mortality due to diagnostic delays. Therefore, the development of rapid fungal detection platforms is an urgent scientific and clinical priority.

METHODS: We designed multiple cross-displacement amplification (MCDA) primers to target the internal transcribed spacer 2 (ITS II) gene of C. tropicalis for specific amplification. Subsequently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-crRNA complex bound to the amplified products, activating its trans-cleavage activity to generate a detectable fluorescent signal. Finally, clinical samples were used to validate the detection results, which were further compared with those obtained using fungal culture and multiplex polymerase chain reaction (Multiplex PCR).

RESULTS: Under optimized conditions, the C. tropicalis-MCDA-CRISPR/Cas12a assay was completed within approximately 53 min, with a limit of detection of 30 fg of genomic DNA per reaction. The assay showed no cross-reactivity with non-C. tropicalis pathogens. Clinical validation using 128 specimens demonstrated a sensitivity of 100.0% (95% CI: 93.4-100.0%) and a specificity of 97.3% (95% CI: 90.6-99.7%) against a composite reference standard, with near-perfect agreement (κ= 0.968).

DISCUSSION: The C. tropicalis-MCDA-CRISPR/Cas12a assay is an efficient, accurate, and practical diagnostic tool suitable for use in resource-limited laboratories.},
}

*Candida tropicalis/genetics/isolation & purification
Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Candidiasis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
DNA Primers/genetics
DNA, Fungal/genetics
Multiplex Polymerase Chain Reaction
Rapid Diagnostic Tests
Endodeoxyribonucleases/genetics
CRISPR-Associated Proteins/genetics
*Molecular Diagnostic Techniques/methods
DNA, Ribosomal Spacer/genetics
Bacterial Proteins

@article {pmid42597591,
year = {2026},
author = {Hermain, S and K S, H and Nimbagal, RN},
title = {Beyond chemotherapy: The rise of nucleic acid nanoformulations in personalized lung cancer therapy.},
journal = {Cancer pathogenesis and therapy},
volume = {4},
number = {5},
pages = {349-361},
pmid = {42597591},
issn = {2949-7132},
abstract = {Lung cancer remains the leading cause of cancer-related mortality worldwide, driven by complex crosstalk among genetic, molecular, and environmental factors. Conventional treatments, including immunotherapies and targeted inhibitors, face three main challenges: tumor heterogeneity, drug resistance, and systemic toxicity. Nucleic acid therapeutics (NATs) encompass a diverse array of DNA- and RNA-based tools, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), antisense oligonucleotides (ASOs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems. These tools are central to developing precision oncology approaches that operate through direct gene regulation, mutation correction, and immune system reprogramming. The clinical application of NATs currently faces three main obstacles, which include their vulnerability to enzymatic degradation, their limited ability to penetrate tissues, and their tendency to cause off-target effects. The field has progressed through the implementation of nanoformulation techniques, which utilize lipid-based polymeric and metallic carriers together with exosomes and DNA origami, and hybrid nanostructures as new platforms to enhance the stability of drugs and their cellular absorption and targeted delivery to tumors. The scientists developed functionalized nanocarriers by combining targeting ligands with materials that could respond to specific environmental changes, which allowed them to manage drug distribution and release patterns throughout the tumor microenvironment. This review focuses on establishing a direct connection between nucleic acid design and nanotechnology through an analysis of mechanistic details and progress in preclinical and clinical research, and the difficulties encountered during the progress to practical applications. The research demonstrates how artificial intelligence and bioinspired nanocarriers and multi-omics data integration create new opportunities for developing personalized adaptive nanogenetic treatment methods, which will treat lung cancer. The current advancements indicate that we are approaching a transformative era in which nanomedicine and nucleic acid therapeutics will enable safe genetic alterations of cancer through targeted therapeutic applications.},
}

@article {pmid42598860,
year = {2026},
author = {Chilamkurthy, R and Sudhakar, S and Pater, AA and Bosmeny, MS and Stabile, F and Katolik, A and Harikrishna, S and El Azzouzi, F and Turk, R and Damha, MJ and Leslie, S and Korolev, S and Pradeepkumar, PI and Gagnon, KT},
title = {CRISPR RNA architecture steers Cas9 catalysis and fidelity via a guide repeat clasp motif.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42598860},
issn = {1362-4962},
support = {R01 GM135646/GM/NIGMS NIH HHS/United States ; R01 GM154323/GM/NIGMS NIH HHS/United States ; 1R01GM135646/NH/NIH HHS/United States ; R01GM154323/NH/NIH HHS/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Biocatalysis ; Base Sequence ; },
abstract = {A widely adopted modification of CRISPR-Cas9 is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). Here we find that these guide architectures induce differential catalysis, gene editing, and specificity. Spacer sequence and RNA structural features could not predict guide architecture editing preference across 255 endogenous targets. We used cryo-EM and molecular dynamics to identify a new Cas9 structural motif, the guide repeat clasp (GRC), that checks guide RNA repeat structure and coordinates with R-loop sensing checkpoint mechanisms to help license cleavage. Limited mutagenesis of GRC residues significantly altered Cas9 editing and specificity, supporting a key role in catalysis. To further understand the role of the GRC and guide RNA repeat dynamics, we created guide repeat-truncated sgRNAs, or grtRNAs, which conferred some dgRNA properties onto sgRNA, including generally lower off-target editing for targets with PAM-proximal mismatches. dgRNAs and grtRNAs could be combined with a new high-fidelity Cas9 variant called ZiFY, rationally designed to reduce editing of targets with PAM-distal mismatches, to generate broader mismatched target discrimination. These results uncover a previously unknown mechanism that steers Cas9 catalysis and demonstrate the potential to improve Cas9 fidelity by modulating guide repeat interactions.},
}

*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism
*CRISPR-Cas Systems
Biocatalysis
Base Sequence

@article {pmid42599326,
year = {2026},
author = {Musini, A and Yata, VK and Bukke, SPN and Akhila, K and Shivani, T and Narapureddy, BR and Thalluri, C and Mohamed, AOA and Adam, MDM},
title = {Phage and CRISPR based precision antimicrobials: a dual strategy against multidrug-resistant bacteria.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42599326},
issn = {1573-4978},
support = {RGP2/639/46//Deanship of Research and Graduate Studies at King Khalid University (Large Research Project)/ ; },
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacteria/genetics/drug effects/virology ; Humans ; *Phage Therapy/methods ; Anti-Bacterial Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Anti-Infective Agents/pharmacology ; },
abstract = {The rapid global emergence of multidrug-resistant (MDR) bacterial pathogens has significantly reduced the effectiveness of conventional antibiotics, creating an urgent need for alternative antimicrobial strategies. Among emerging precision therapeutics bacteriophage therapy and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems have shown to have strong potential through highly specific bacterial targeting mechanisms. Bacteriophages have the ability to replicate themselves and penetrate biofilms, and the ability of CRISPR-Cas systems to edit the genes responsible for antimicrobial resistance, virulence factors, and the mobile genetic elements that underlie bacterial resistance. The recent advancement enabled the integration of these technologies through CRISPR-armed bacteriophages, which utilize bacteriophages as delivery mechanisms for CRISPR and address the large populations of MDR bacteria. Compared to administering CRISPR and bacteriophage independently, the current data suggest that the use of these two methods synergistically will lead to greater efficacy of delivery, specific targeting of resistance determinants, decreased risk of resistance development, and minimal impact on the body's beneficial microorganisms. While the potential combination of these approaches holds great promise to help combat the issue of MDR bacteria, there are still numerous barriers to overcome in order to implement these methods which include narrow phage host range, bacterial escape mechanisms, off-target CRISPR activity, anti-CRISPR proteins, host immune responses, and unresolved manufacturing and regulatory limitations. This review critically examines bacteriophage-based antimicrobials, CRISPR-Cas therapeutic systems, and their emerging integration as CRISPR-armed phages, highlighting their comparative advantages, current limitations, and future potential as promising targeted antimicrobial approach platforms requiring further clinical validation.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems/genetics
*Drug Resistance, Multiple, Bacterial/genetics
*Bacteria/genetics/drug effects/virology
Humans
*Phage Therapy/methods
Anti-Bacterial Agents/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
*Anti-Infective Agents/pharmacology

@article {pmid42599328,
year = {2026},
author = {Mengistu, DA and Mekasha, YT and Molla, FW},
title = {Non-Drug therapeutic strategies to combat antimicrobial resistance in livestock: a review.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42599328},
issn = {1573-4978},
mesh = {Animals ; *Livestock/microbiology/genetics ; Humans ; Probiotics/therapeutic use ; Phage Therapy ; CRISPR-Cas Systems ; *Drug Resistance, Microbial ; Anti-Infective Agents ; Fecal Microbiota Transplantation ; Anti-Bacterial Agents ; Genetic Engineering/methods ; },
abstract = {BACKGROUND: Antimicrobial resistance (AMR) represents an escalating global public health crisis, projected to cause 10 million deaths annually by 2050. Pathogens such as bacteria, viruses, fungi, and parasites evade treatments in animals and humans due to overuse and misuse of antimicrobials, particularly antibiotics, in medical and veterinary practices. This drives a worldwide surge in resistant infections, disproportionately burdening livestock health and productivity.

OBJECTIVES: This review examines the global rise of AMR and its impacts on livestock and public health, underscoring the need for non-drug therapeutic alternatives.

CONCLUSION: Promising alternatives include genetic engineering and CRISPR-Cas systems, phage therapy, probiotics, antimicrobial peptides, fecal microbiota transplantation, phytotherapy and essential oils, immunomodulators, nanotechnology, biofilm disruptors, acidifiers, vaccination strategies, and precision livestock farming. These approaches target resistance mechanisms without relying on conventional drugs. Despite challenges like knowledge gaps, regulatory barriers, financial limitations, and policy gaps, stakeholders must prioritize accelerated research, regulatory reforms, investments, and international collaboration. Rapid integration of these technologies into human and veterinary medicine is vital to mitigate AMR's health, economic, and social impacts.},
}

Animals
*Livestock/microbiology/genetics
Humans
Probiotics/therapeutic use
Phage Therapy
CRISPR-Cas Systems
*Drug Resistance, Microbial
Anti-Infective Agents
Fecal Microbiota Transplantation
Anti-Bacterial Agents
Genetic Engineering/methods

@article {pmid42599397,
year = {2026},
author = {Sun, J and Ni, Y and Guo, M and Yu, T},
title = {A Multiplex CRISPR-Cas12a-Based Hydrogel Microarray Platform for the Simultaneous Detection of Common Adenovirus Types in Clinical Samples.},
journal = {ACS infectious diseases},
volume = {12},
number = {8},
pages = {2815-2824},
doi = {10.1021/acsinfecdis.6c00407},
pmid = {42599397},
issn = {2373-8227},
mesh = {Humans ; *CRISPR-Cas Systems ; Hydrogels/chemistry ; Sensitivity and Specificity ; *Adenoviridae/genetics/isolation & purification/classification ; *Microarray Analysis/methods ; CRISPR-Associated Proteins/genetics ; *Adenoviridae Infections/diagnosis/virology ; Rapid Diagnostic Tests ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Adenovirus infections are a leading cause of respiratory and gastrointestinal diseases, representing a significant global health challenge. Rapid and accurate detection of adenovirus types is essential for timely diagnosis and effective management. Traditional diagnostic methods, such as PCR, are often time-consuming and require complex laboratory infrastructure, limiting their application in resource-limited settings. In this study, we present a CRISPR/Cas12a-based assay integrated with a hydrogel microarray for the simultaneous detection of six common adenovirus types (1, 2, 3, 4, 7, and 14). After amplification of adenoviral DNA using recombinase polymerase amplification (RPA), the amplified DNA enters the hydrogel, where it activates the Cas12a-crRNA complex trapped within the gel. This activation leads to the cleavage of an ssDNA reporter, generating a fluorescent signal. The use of a hydrogel microarray enables efficient and multiplexed detection of adenovirus types in a single assay. The method demonstrated high sensitivity, with detection limits ranging from 10 to 50 copies/μL across the six adenovirus types. It also showed excellent specificity, with no cross-reactivity observed with other respiratory viruses. Clinical validation with 30 human adenovirus samples revealed 100% specificity and high concordance with qPCR results. This CRISPR/Cas12a-based hydrogel microarray platform offers a rapid, cost-effective, and highly specific diagnostic tool with significant potential for clinical and public health applications.},
}

Humans
*CRISPR-Cas Systems
Hydrogels/chemistry
Sensitivity and Specificity
*Adenoviridae/genetics/isolation & purification/classification
*Microarray Analysis/methods
CRISPR-Associated Proteins/genetics
*Adenoviridae Infections/diagnosis/virology
Rapid Diagnostic Tests
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42599816,
year = {2026},
author = {Sun, T and Yuan, A and Xie, W and Jiang, G and Peng, H},
title = {CRISPR RNA Engineering Enables Single Nucleotide Polymorphism Discrimination in Nucleic Acid Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e00004},
doi = {10.1002/anie.202600004},
pmid = {42599816},
issn = {1521-3773},
support = {22276199//National Natural Science Foundation of China/ ; 2023YFA0915102//National Key Research and Development Program of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 22306195//Youth Fund from National Natural Science Foundation of China/ ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic systems have emerged as powerful platforms for sensitive nucleic acid detection, yet their clinical reliability is frequently constrained by off-target activation and insufficient discrimination of closely related sequences. This review synthesizes current advances aimed at enhancing the specificity of CRISPR diagnostics, with particular emphasis on the pivotal role of CRISPR RNA (crRNA) engineering. We detail how structural determinants of crRNA, including spacer length optimization, intentional mismatch design, secondary-structure modulation, chemical modification, strand-displacement gating, and synergistic design frameworks, govern CRISPR-mediated target recognition and define the energetic and kinetic thresholds for accurate cleavage. Key engineering strategies encompassing computational prediction and modeling, high-throughput screening, and hybrid guide architectures are systematically examined for their capacity to elevate single-nucleotide discrimination, stabilize reaction performance, and enable robust multiplexed detection for pathogen profiling and mutation identification. Despite rapid progress, outstanding challenges persist, including interference from complex clinical matrices, lack of unified evaluation standards, and scalability barriers that hinder clinical translation. Addressing these limitations through integrated crRNA design, system-level optimization, and standardized benchmarking will be essential for realizing the promise of CRISPR diagnostics. Ultimately, these advances are poised to support ultrasensitive, highly specific, and portable point-of-care testing, thereby accelerating precision medicine and strengthening infectious disease surveillance and management.},
}

@article {pmid42600441,
year = {2026},
author = {Zhang, X and Wang, Z and Guo, Z and Wang, H and Chen, Y and Dong, Z and Zeng, L and Yu, D and Hong, M and Wang, J and Tian, G and Yang, W},
title = {A Novel CRISPR/Cas13a sensor for highly sensitive detection of DENV based on AuPt/g-C3N4@GO and Ag@NU-1000.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119122},
doi = {10.1016/j.bios.2026.119122},
pmid = {42600441},
issn = {1873-4235},
abstract = {Dengue virus, an arthropod-borne pathogen, poses a significant global public health concern, necessitating the development of sensitive and rapid diagnostic assays. This paper introduces a novel biosensing platform designed for the ultrasensitive detection of dengue viral RNA. The platform integrates recombinase polymerase amplification, CRISPR/Cas13a-based recognition, and electrochemiluminescence resonance energy transfer (ECL-RET). The sensor employs a highly efficient luminescent emitter, which is a ternary composite of gold-platinum nanoparticles supported on graphitic carbon nitride and graphene oxide. A suitable energy acceptor, consisting of silver-decorated metal-organic frameworks, effectively quenches the emitter's signal via resonance energy transfer. Upon the introduction of target nucleic acids, RPA rapidly generates numerous amplicons. These amplicons are specifically recognized by the CRISPR/Cas13a system, which subsequently activates the collateral cleavage activity of Cas13a. This enzymatic activity cleaves a DNA linker that tethers the acceptor to the electrode surface, leading to the release of the acceptor and a subsequent restoration of the electrochemiluminescence intensity. The recovered signal exhibits a linear proportionality to the target concentration across a broad dynamic range, spanning from 0.1 fg/mL to 100 ng/mL. The detection limits for the four dengue serotypes range from 1.09 to 2.5 fg/mL. The assay demonstrates excellent specificity, showing no cross-reactivity with Zika, Japanese encephalitis, or West Nile viruses, and also exhibits good reproducibility. By combining isothermal amplification, specific CRISPR targeting, and a sensitive luminescence readout, this work presents a straightforward and robust method for the early diagnosis of dengue and for outbreak surveillance. Furthermore, this platform can be readily adapted for the detection of other pathogens of interest.},
}

@article {pmid42109006,
year = {2026},
author = {Rajabi Zangi, A and Amiri, A and Eskandari, F and Pazooki, P and Heidari, HR and Hamishehkar, H and Javadzadeh, Y},
title = {RNA therapeutics and their delivery methods: a paradigm for haemophilia management.},
journal = {Journal of drug targeting},
volume = {34},
number = {8},
pages = {1394-1413},
doi = {10.1080/1061186X.2026.2638330},
pmid = {42109006},
issn = {1029-2330},
mesh = {Humans ; *Hemophilia A/therapy/genetics ; *RNA, Small Interfering/administration & dosage ; Animals ; Nanoparticles ; RNA, Messenger/administration & dosage ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Lipids/chemistry ; Drug Delivery Systems ; },
abstract = {Haemophilia management is currently undergoing a paradigm shift from traditional protein replacement to RNA modalities and their delivery. This review provides a critical analysis of RNA-based therapeutics (specifically small interfering RNA (siRNA), mRNA and CRISPR/Cas9) as a versatile paradigm distinct from DNA ones. We synthesise clinical and preclinical data to contrast these modalities: siRNA strategies (e.g. fitusiran) have indicated ∼90% reductions in bleeding rates by rebalancing haemostasis independent of factor deficiency; lipid nanoparticles (LNPs)-mRNA platforms offer tuneable, transient factor production without genomic integration risks; and CRISPR-based editing aims for permanent correction (up to 170% coagulation factor IX, FIX expression in preclinical studies) but necessitates rigorous monitoring for off-target effects. Crucially, this article dissects the non-viral delivery landscape determining the clinical viability of these cargos. We evaluate LNPs as the current clinical gold standard for hepatic delivery, contrasting them against emerging polymeric systems, aptamer conjugates and exosomes designed to overcome rate-limiting barriers such as endosomal entrapment and renal clearance. By juxtaposing the immunogenic limitations of viral vectors, we conclude that next-generation RNA therapeutics, enabled by LNPs and GalNAc-conjugation, offer a strategic pathway to overcome the durability gap observed in haemophilia A and expand treatment access to patients currently excluded by viral seroprevalence.},
}

Humans
*Hemophilia A/therapy/genetics
*RNA, Small Interfering/administration & dosage
Animals
Nanoparticles
RNA, Messenger/administration & dosage
*Gene Transfer Techniques
CRISPR-Cas Systems
Genetic Therapy/methods
Lipids/chemistry
Drug Delivery Systems

@article {pmid42269411,
year = {2026},
author = {Zhang, Y and Ma, H and Li, W and Wang, Z and Song, Z and Qi, Y},
title = {A CRISPR/Cas12a-hyperbranched rolling circle amplification sensor for ultrasensitive visual bacteria detection.},
journal = {Talanta},
volume = {310},
number = {},
pages = {129967},
doi = {10.1016/j.talanta.2026.129967},
pmid = {42269411},
issn = {1873-3573},
mesh = {Milk/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Smartphone ; *Biosensing Techniques/methods ; *Staphylococcus aureus/isolation & purification/genetics ; Copper/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Food Microbiology ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The swift spread of pathogenic bacteria via food, air, and water poses severe risks to human health. Conventional detection methods often suffer from time-consuming operations, bulky instruments, and insufficient sensitivity for on-site screening, highlighting an urgent demand for sensitive and visual platforms. Herein, a detection platform (Cas12a-HRCA) was constructed for sensitive pathogen quantification by integrating hyperbranched rolling circle amplification (HRCA), CRISPR/Cas12a system, copper fluorescence nanoparticles (CuNPs) and smartphone-based signal readout. In this strategy, pathogen target-activated CRISPR/Cas12a precisely regulates HRCA initiation, which exponentially generates AT-TA-rich sequences. These products serve as templates for the self-assembly of the fluorescence CuNPs, achieving integrated rapid signal amplification without requiring sample preprocessing steps. Combined with smartphone-based RGB analysis, the platform enables direct, on-site quantitative readout. Cas12a-HRCA demonstrated exceptional performance for S. aureus, with a detection limit of 1 CFU/mL, high specificity, and 91%-106% reliable recovery in spiked milk samples without pre-enrichment or purification, showcasing great potential for point-of-need food safety monitoring. the detection of various pathogenic bacteria in food, clinical or environmental settings.},
}

Milk/microbiology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Animals
Smartphone
*Biosensing Techniques/methods
*Staphylococcus aureus/isolation & purification/genetics
Copper/chemistry
Metal Nanoparticles/chemistry
Limit of Detection
Food Microbiology
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42296867,
year = {2026},
author = {Xie, Z and Wu, Y and Zhou, P and Zhang, Y and Li, X and Li, Y and Cui, H},
title = {Label-free CRISPR/Cas12a biosensor based on G-triplex/Thioflavin T and GlaI-assisted strand displacement amplification for ultrasensitive DNA methylation detection.},
journal = {Talanta},
volume = {310},
number = {},
pages = {130144},
doi = {10.1016/j.talanta.2026.130144},
pmid = {42296867},
issn = {1873-3573},
mesh = {*DNA Methylation ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Benzothiazoles/chemistry ; DNA/genetics ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {DNA methylation represents a pivotal epigenetic biomarker for cancer, and precise profiling of carcinogenesis-associated methylation is essential for early diagnosis and prognostic evaluation. Herein, we report a novel label-free, sensitive DNA methylation biosensor by integrating GlaI-assisted double cascade strand displacement amplification with G-triplex-facilitated CRISPR/Cas12a (G-DCSDA/Cas12a). Instead of conventional fluorescent-quenched (FQ) probes, this assay employed G-triplex/Thioflavin T as a simple and efficient signal reporter for CRISPR/Cas12a. The methylation-specific endonuclease GlaI selectively digested methylated DNA to release free 3'-OH ends, which initiated the subsequent DCSDA and triggered Cas12a activation. The double-template cascade amplification system delivers significantly improved sensitivity in comparison with the single-template strategy. Upon activation, the trans-cleavage activity of Cas12a rapidly disrupted G-triplex/Thioflavin T complexes, generating a distinct fluorescence response. By combining the high specificity of GlaI, efficient signal amplification of DCSDA, and robust collateral cleavage of Cas12a, the G-DCSDA/Cas12a platform achieved ultrahigh sensitivity and selectivity, enabling detection of methylation levels as low as 0.005% in a background of excessive unmethylated DNA. Furthermore, this strategy was successfully integrated into a lateral flow assay (LFA), enabling visual and point-of-care testing (POCT) of DNA methylation. Importantly, the developed biosensor achieved sensitive detection of genomic DNA methylation in real samples and accurately discriminates cancer cells from normal cells, as well as between colorectal cancer tissue and adjacent normal tissue. These results highlighted the significant potential of the G-DCSDA/Cas12a platform for clinical early cancer diagnosis.},
}

*DNA Methylation
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
*Benzothiazoles/chemistry
DNA/genetics
*Nucleic Acid Amplification Techniques/methods

@article {pmid42330823,
year = {2026},
author = {Jiang, H and Yang, J and Li, A and Mou, D and Deng, Y and Lv, X},
title = {Competitive kinetic mechanisms in one-pot isothermal amplification-CRISPR systems: From model construction to performance evaluation.},
journal = {Talanta},
volume = {310},
number = {},
pages = {130184},
doi = {10.1016/j.talanta.2026.130184},
pmid = {42330823},
issn = {1873-3573},
mesh = {Kinetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Hybridization ; },
abstract = {The urgent demand for integrating real-time molecular diagnosis with isothermal amplification and CRISPR-based detection has underscored the critical need for streamlined, single-tube one-pot methodologies. However, such integration is often hindered by temporal incompatibilities, including premature activation of early amplification products by CRISPR components, which leads to primer degradation and reduced amplification efficiency. To address this challenge, we established a dedicated quantitative competitive kinetic framework for enzyme-free isothermal amplification-CRISPR one-pot systems, using hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) as representative models. Two core inhibitory mechanisms were identified: "pre-activation-degradation inhibition", in which early CRISPR activation degrades amplification intermediates, and "substrate competition inhibition" in which hairpin probes compete with reporter probes for CRISPR trans-cleavage. Corresponding kinetic equations were derived to describe these interactions quantitatively. Systematic experimental validation of key parameters such as DNA activator concentration, reporter probe concentration, hairpin probe concentration, and ribonucleoprotein (RNP) complex concentration, confirmed the reliability and predictive accuracy of the proposed models. These results provide mechanistic insights into the factors governing one-pot HCR/CHA-CRISPR coupling and identify conditions that optimize assay performance. Overall, this study offers a theoretical foundation and experimental guidance for probe design, reaction condition optimization, and sensitivity enhancement in enzyme-free isothermal amplification-CRISPR one-pot platforms, and provides a general reference for the rational integration of isothermal amplification and CRISPR-based detection.},
}

Kinetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acid Hybridization

@article {pmid42469290,
year = {2026},
author = {Relova-Hernández, E and Díaz-Bravo, AB and Cantero-Rodríguez, A and Alvarez-Arzola, R and Hernández-Álvarez, N and Carmenate, T and Rojas, G},
title = {CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42469290},
issn = {2045-2322},
mesh = {*Interleukin-2 Receptor alpha Subunit/genetics/metabolism ; Animals ; Humans ; *Interleukin-2 Receptor beta Subunit/metabolism/genetics ; Mice ; Interleukin-2/metabolism ; *Interleukin Receptor Common gamma Subunit/metabolism/genetics ; Cell Line ; Signal Transduction ; Cell Proliferation ; CRISPR-Cas Systems ; STAT5 Transcription Factor/metabolism ; Gene Knockout Techniques ; },
abstract = {Interleukin-2 (IL-2) has pleiotropic roles within the immune system. Its functional versatility depends upon the interactions with diverse arrays of receptor subunits differentially expressed on immune cell subsets. The balance between stimulation of regulatory T cells (Tregs) and effector lymphocytes determines the immunological outcome. While Tregs display constitutively a trimeric IL-2 receptor composed by alpha (also known as CD25), beta and gamma subunits, resting and memory CD8 + T cells express a dimeric beta/gamma receptor. The effects of IL-2 signaling through the trimeric receptor are routinely studied in vitro using either mouse CTLL-2 or human Kit225 lymphoid cell lines. We obtained equivalent IL-2-responsive cellular models displaying dimeric beta/gamma receptors. The purpose was achieved through genetic ablation of CD25 expression on CTLL-2 and Kit225 cells using CRISPR/Cas9 edition. The usefulness of the stable CD25-KO cell lines thus generated was illustrated by setting up proliferation assays, and characterizing a panel of IL-2-derived recombinant muteins, including agonists, super-agonists and antagonists. Problems and pitfalls of such assays, as well as the ways to mitigate them, were depicted. Stat5 phosphorylation assays based on KO cell lines were also performed. These cellular models, and the experience accumulated during their use, could contribute to the evaluation of the output of IL-2 engineering strategies developed across different laboratories.},
}

*Interleukin-2 Receptor alpha Subunit/genetics/metabolism
Animals
Humans
*Interleukin-2 Receptor beta Subunit/metabolism/genetics
Mice
Interleukin-2/metabolism
*Interleukin Receptor Common gamma Subunit/metabolism/genetics
Cell Line
Signal Transduction
Cell Proliferation
CRISPR-Cas Systems
STAT5 Transcription Factor/metabolism
Gene Knockout Techniques

@article {pmid42497864,
year = {2026},
author = {Zhang, C and Li, T and Hao, H and Wang, G and Zhang, Y and Huang, Y and Chen, L and Wang, J and Yang, XJ and Bi, X},
title = {CRISPR-Cas9-producing probiotic bacteria for editing NOX2/gp91[phox] and treating inflammatory bowel disease.},
journal = {Cell reports. Medicine},
volume = {7},
number = {8},
pages = {102940},
doi = {10.1016/j.xcrm.2026.102940},
pmid = {42497864},
issn = {2666-3791},
mesh = {Animals ; *NADPH Oxidase 2/genetics/metabolism ; Mice ; *Probiotics/metabolism ; *Inflammatory Bowel Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; Reactive Oxygen Species/metabolism ; RAW 264.7 Cells ; *Gene Editing/methods ; Colitis/chemically induced ; Dextran Sulfate ; Humans ; Oxidative Stress ; Disease Models, Animal ; Intestinal Barrier Function ; Mice, Inbred C57BL ; },
abstract = {The primary pathogenic mechanism of inflammatory bowel disease (IBD) involves elevated levels of reactive oxide species (ROS) in the gut, leading to oxidative stress and damage to the intestinal barrier function. We engineered a non-pathogenic bacterial strain, Escherichia coli Nissle 1917 (EcN), for oral CRISPR-Cas9 delivery to edit NOX2 (encoding the gp91[phox] subunit of NADPH oxidase 2), thereby alleviating IBD symptoms by reducing ROS. EcN expressing the Cas9/sgRNA ribonucleoprotein (RNP) was encapsulated in a hydrogel (composed of hyaluronic acid, chitosan, and MgCl2), which protected EcN-RNP from degradation and increased survival from 0.07% to 12%. EcN-RNP hydrogel reduced NOX2 expression by 47% and ROS by 88% in lipopolysaccharide-induced RAW264.7 cells. Moreover, oral delivery of EcN-RNP hydrogel mitigated inflammation in dextran sodium sulfate-induced colitis mouse models. Mechanistically, the hydrogel could activate the NRF2-HO1/GPX4 pathway by inhibiting NOX2 expression, enhancing oxidative defense, and promoting glutathione accumulation. Thus, the EcN-RNP hydrogel offers a promising therapeutic strategy for IBD.},
}

Animals
*NADPH Oxidase 2/genetics/metabolism
Mice
*Probiotics/metabolism
*Inflammatory Bowel Diseases/therapy/genetics
*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/metabolism
Reactive Oxygen Species/metabolism
RAW 264.7 Cells
*Gene Editing/methods
Colitis/chemically induced
Dextran Sulfate
Humans
Oxidative Stress
Disease Models, Animal
Intestinal Barrier Function
Mice, Inbred C57BL

@article {pmid42585941,
year = {2026},
author = {Wang, R and Pang, W and Yu, M and Dong, W and Wang, R},
title = {Evolution of CRISPR-cas detection systems: From nucleic acid recognition to omnidirectional point-of-care diagnostic applications.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {4},
pages = {117589},
doi = {10.1016/j.diagmicrobio.2026.117589},
pmid = {42585941},
issn = {1879-0070},
abstract = {Molecular diagnostic technologies play an indispensable role in modern medicine and public health. However, traditional diagnostic platforms frequently face an inherent trade-off between laboratory-grade analytical precision and the speed and operational simplicity required for point-of-care testing. In recent years, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein system has precipitated disruptive technological changes to this field. CRISPR-Cas system possesses high-fidelity target recognition capability and exhibits a distinctive trans-cleavage activity upon activation, which functions as signal amplification. This technology alleviates the inherent trade-off between sensitivity and portability. This review systematically summarizes the core molecular mechanisms of CRISPR-Cas detection platforms, addressing the differences in substrate preference and cleavage behavior among mainstream effector proteins (e.g., Cas9, Cas12, Cas13, and Cas14) and prokaryotic Argonaute (pAgo) proteins. Furthermore,this review sorts out the technological iteration path of detection platforms and presents the applications of this technology in fields such as infectious disease surveillance, cancer liquid biopsy, preliminary screening of genetic diseases, food and environmental safety, and veterinary port quarantine. Despite the challenges in quantitative accuracy and anti-interference ability, CRISPR biosensors are powerfully driving precision medicine towards decentralized, on-site, and accessible Point-of-Care Testing (POCT).},
}

@article {pmid42586052,
year = {2026},
author = {Wang, MR and Sánchez-Rivera, FJ},
title = {eVLP compound delivery breaks the prime editing efficiency ceiling.},
journal = {Cell genomics},
volume = {6},
number = {8},
pages = {101333},
pmid = {42586052},
issn = {2666-979X},
support = {P01 CA291694/CA/NCI NIH HHS/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; T32 GM136540/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {Prime editing could resolve gene variant function at scale, but the editing machinery needs to be delivered efficiently and reproducibly. Langley, Baudrier, et al.[1] show that timing of editor delivery is rate limiting and introduce PRIME-VLP, a repeated dosing strategy with engineered virus-like particles that improves editing efficiency and screening performance.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems

@article {pmid42586245,
year = {2026},
author = {Rathore, S and Gupta, A and Shah, K and Chauhan, NS and Gupta, SK},
title = {Targeting the hallmarks of ageing: Pharmacological challenges and breakthroughs in CRISPR delivery systems and future prospects.},
journal = {Ageing research reviews},
volume = {121},
number = {},
pages = {103301},
doi = {10.1016/j.arr.2026.103301},
pmid = {42586245},
issn = {1872-9649},
abstract = {CRISPR has emerged as a next-generation gene-editing tool with the potential to target the molecular pathways associated with ageing and related disorders. It functions through RNA-guided Cas nucleases, directing DNA cleavage and utilizing the native DNA repair machinery for genetic manipulations. Advances in CRISPR technology have significantly enhanced the precision and flexibility of techniques for genome editing. The enzyme Cas9's ability to cut DNA at exact site has revolutionized genome editing by enabling accurate modifications within living eukaryotic cells. This review critically examines recent developments in CRISPR-based technologies, including Cas9, Cas12, base editing, prime editing, and CRISPR-mediated gene regulation. It highlights their rising applications in ageing research, with more emphasis on neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The review also discusses the major pharmacological and translational challenges that currently limit clinical applications, including inefficient tissue-specific delivery, off-target genome editing, immunogenicity, manufacturing complexity, and long-term safety concerns. Also, recent progress in both, viral and non-viral delivery methods are critically evaluated, including adeno-associated viruses, lentivirus vectors, lipid nanoparticles, gold nanoparticles, exosomes, electroporation, and microinjection, is thoroughly discussed to highlight their therapeutic potential and translational limitations. Current studies indicate that CRISPR-based approaches have preclinical potential for targeting important hallmarks of ageing, particularly genomic instability, telomere attrition, and mitochondrial dysfunction. Other hallmarks of ageing, such as stem cell exhaustion, epigenetic modifications, and microbiome changes, are at earlier stages of development. Overall, this review describes future strategies for developing safe, precise, and clinically translatable CRISPR-based treatments to promote healthy ageing.},
}

@article {pmid42586508,
year = {2026},
author = {Hasan, MU and Azhar, MW and Noor-Ul-Saba, and Khurshid, M and Riaz, R},
title = {Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.},
journal = {Journal of virological methods},
volume = {346},
number = {},
pages = {115447},
doi = {10.1016/j.jviromet.2026.115447},
pmid = {42586508},
issn = {1879-0984},
abstract = {The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.},
}

@article {pmid42587136,
year = {2026},
author = {Hsu, A and Chen, PJ and Li, AH and Hemez, CF and Gao, XD and Terrey, M and Nelson, C and Selvam, V and Cristian, A and McElroy, AN and Steinbeck, BJ and Mahadeshwar, GK and Pandey, S and Barsdale, Z and Chen, PZ and Sousa, AA and Sakai, HA and Silverstein, RA and Morad, I and Krueger, RK and Shen, MW and Kleinstiver, BP and Lutz, CM and Tolar, J and Blazar, BR and Osborn, MJ and Liu, DR},
title = {Mechanistic machine learning for prediction of prime editing outcomes.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42587136},
issn = {1546-1696},
support = {U01AI142756//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01EB022376//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R35GM118062//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {Prime editing (PE) can make specific local changes to genomic DNA in living systems but its efficient application currently requires extensive optimization of PE guide RNA (pegRNA) sequences. Here we present OptiPrime, a machine learning model of PE efficiency based on current understanding of PE mechanisms. OptiPrime achieves state-of-the-art accuracy on PE efficiency prediction and enables prediction of nicking guide RNA (PE3) and dual pegRNA (twinPE) outcomes. We validate that OptiPrime has learned the determinants of mammalian mismatch repair (MMR) and is well suited for nominating MMR-evasive silent edits that improve PE efficiency. We demonstrate the use of OptiPrime in a variety of prospective therapeutic contexts in primary human and mouse cells. Lastly, we show that OptiPrime can be used to achieve streamlined and efficient in vivo correction of a pathogenic mutation in the brain of a mouse model of KIF1A-associated neurological disorder. We provide a webserver for OptiPrime (https://optipri.me/) as a community resource.},
}

@article {pmid42589580,
year = {2026},
author = {Siddika, A and Husseiny, FE and Rousseau, J and Tremblay, JP},
title = {Prime Editing Mediated Generation and Correction of the mdx5cv Mutation Restores Dystrophin Expression in Myoblasts.},
journal = {International journal of molecular sciences},
volume = {27},
number = {15},
pages = {},
pmid = {42589580},
issn = {1422-0067},
support = {53320215//Defeat Duchenne Foundation, Canada/ ; },
mesh = {Animals ; *Dystrophin/genetics/metabolism ; *Myoblasts/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/genetics/metabolism/therapy ; *Gene Editing/methods ; Cell Line ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; },
abstract = {Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish dystrophin expression. Prime editing enables precise genome modification without generating double-strand DNA breaks or requiring donor DNA templates. We established an in vitro prime editing workflow to generate and subsequently correct the mdx5cv mutation in mouse C2C12 myoblasts. Following optimization of engineered prime editing guide RNAs (epegRNAs) and PAM-flexible prime editors, wild-type cells were edited, clonally isolated, and genotyped. Mutation correction was then evaluated using optimized epegRNA designs. Two rounds of prime editing introduced the mdx5cv mutation into approximately 20% of alleles in C2C12 cells creating the mdx5cv C2C12 cell line. Clonal isolation yielded five homozygous mutant clones among 59 expanded colonies. Optimization studies identified an epegRNA containing a 16 nucleotide reverse transcription template and a 10 nucleotide primer binding site (RTT16/PBS10) as the most efficient design. Correction of the pathogenic allele reached approximately 26%, whereas longer PBS lengths reduced editing efficiency. In silico off-target analysis using Cas-OFFinder identified no candidate genomic loci with fewer than three mismatches for the spacer sequences used in either mutation generation or correction, suggesting a favorable predicted specificity profile. Corrected mdx5cv C2C12 myoblasts retained their capacity to differentiate into multinucleated myotubes. Representative Western blot analysis detected dystrophin protein expression in differentiated corrected mdx5cv myotubes, consistent with successful correction of the pathogenic mutation. These findings establish a robust prime editing platform for both the generation and correction of the mdx5cv mutation and provide proof of concept that precise correction of the pathogenic mutation is associated with restoration of dystrophin expression following myogenic differentiation.},
}

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

@article {pmid42589608,
year = {2026},
author = {Steiman, S and Kinsella, C and Zhang, Y and Omer, S and Brown, C and Forguson, G and Elbakr, L and Pastore, S and Ivakine, EA},
title = {Restoring the Balance: CRISPRa-Driven β-Tubulin Compensation as a Strategy for Tubulinopathy Treatment.},
journal = {International journal of molecular sciences},
volume = {27},
number = {15},
pages = {},
pmid = {42589608},
issn = {1422-0067},
support = {//Nicol Family Foundation/ ; },
mesh = {*Tubulin/genetics/metabolism ; Humans ; Cilia/metabolism/genetics ; *CRISPR-Cas Systems ; Microtubules/metabolism/genetics ; *Gene Editing/methods ; Animals ; Mutation ; *Ciliopathies/genetics/therapy/metabolism ; },
abstract = {Microtubules are essential cytoskeletal components comprising alpha- and beta-tubulin proteins that facilitate organelle positioning, cell migration, division, and intracellular trafficking. Mutations in tubulin genes can lead to tubulinopathies, a class of rare genetic neurodevelopmental disorders characterized by a range of brain malformations and other clinical features. Recent studies have shown that pathogenic variants in beta-tubulin genes such as TUBB[G308S] have been found to underlie the development of ciliopathies, disorders impacting cilia, important organelles for development and cell motility. Thus, mutations in distinct tubulin genes, which present a significant hurdle for the development of therapeutic gene editing strategies and targeted therapeutics. Here, we describe the development of a mutation-independent treatment strategy based on the upregulation of non-mutated beta-tubulin isotype protein using CRISPR-Cas9 activation. By increasing the expression of various beta-tubulin proteins, we demonstrate a restoration of the microtubule network and primary cilia formation.},
}

*Tubulin/genetics/metabolism
Humans
Cilia/metabolism/genetics
*CRISPR-Cas Systems
Microtubules/metabolism/genetics
*Gene Editing/methods
Animals
Mutation
*Ciliopathies/genetics/therapy/metabolism

@article {pmid42589635,
year = {2026},
author = {Kong, L and Iwabuchi, S and Li, YY and Murai, K and Korai, R and Kawaguchi, K and Nio, K and Imafuku, T and Shimakami, T and Yamashita, T and Tajima, A and Suzuki, Y and Honda, M and Hashimoto, S},
title = {Chromatin Accessibility-Guided Targeting Identifies Structurally Constrained Regions in HBV cccDNA and Suppresses Viral Replication.},
journal = {International journal of molecular sciences},
volume = {27},
number = {15},
pages = {},
pmid = {42589635},
issn = {1422-0067},
support = {17fk0310110h0001, 20fk0310110s8003, 21fk0310110s8005, 22fk0310514s0101, 23fk0310514s0102.//AMED/ ; },
mesh = {*Hepatitis B virus/genetics/physiology ; *DNA, Circular/genetics ; Humans ; *Virus Replication/genetics ; *DNA, Viral/genetics ; *Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; Hepatitis B/virology/genetics ; Hepatocytes/virology ; Genome, Viral ; },
abstract = {Covalently closed circular DNA (cccDNA) is a stable episomal form of the hepatitis B virus (HBV) genome that serves as the template for viral transcription and replication and represents a major barrier to HBV cure. Here, we investigated chromatin accessibility patterns of cccDNA in HBV-infected hepatocyte cells at single-molecule resolution. We found that most cccDNA copies exhibited limited accessibility around nucleotides 800-1000, a region overlapping the polymerase open reading frame and the pregenomic RNA transcriptional region. Notably, a small subset of cccDNA showed detectable accessibility at this site, suggesting the presence of heterogeneous chromatin states. Based on this observation, we targeted this accessibility-associated region using a CRISPR/Cas9-based approach and observed reductions in HBV DNA-related signals, including cccDNA-enriched fractions and total HBV DNA levels across complementary experimental systems. These findings suggest that chromatin accessibility profiling may provide an additional framework for identifying candidate cccDNA target regions. Our study provides a proof-of-concept for accessibility-informed HBV targeting and supports further investigation of chromatin-associated vulnerability within HBV cccDNA.},
}

*Hepatitis B virus/genetics/physiology
*DNA, Circular/genetics
Humans
*Virus Replication/genetics
*DNA, Viral/genetics
*Chromatin/genetics/metabolism
CRISPR-Cas Systems
Hepatitis B/virology/genetics
Hepatocytes/virology
Genome, Viral

@article {pmid42592160,
year = {2026},
author = {Christopher, CW and Zhou, X},
title = {In vivo CRISPR editing for cancer immunotherapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1872510},
pmid = {42592160},
issn = {1664-3224},
support = {R00 CA282989/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; *Immunotherapy/methods ; Animals ; Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; },
abstract = {Cancer immunotherapy has shown significant promise in certain patient populations, but further advancements are needed to extend its benefits to a wider range of patients. Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing has rapidly evolved in recent years, enabling its transition into direct therapeutic applications. This review summarizes recent progress in applying CRISPR systems in vivo for cancer immunotherapy, focusing on approaches that target cancer cells and the tumor microenvironment, as well as those that directly engineer immune cell populations themselves. Novel CRISPR editing platforms and strategies enabling multiplexed editing have also recently demonstrated promising impacts on driving antitumor immunity, however, the platforms investigated are still in the early stages and further investigation will be needed to robustly assess the potential for clinical translation. Future work can expand the array of therapeutic targets by incorporating data from functional genomics and must also carefully evaluate both editing modalities and delivery systems to optimize efficacy, safety, and scalability.},
}

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

@article {pmid42593176,
year = {2026},
author = {Kilani, H and Hamzaoui, Z and Ferjani, S and Lengliz, S and Tayh, G and Ben Zedira, A and Mosbahi, M and Kanzari, L and Ben Chehida, F and Abbassi, MS and Boutiba-Ben Boubaker, I},
title = {Whole-Genome Sequencing of Feline Uropathogens Reveals Multidrug Resistance and Zoonotic Potential in Domestic Cats in Tunisia.},
journal = {Vector borne and zoonotic diseases (Larchmont, N.Y.)},
volume = {},
number = {},
pages = {15303667261477573},
doi = {10.1177/15303667261477573},
pmid = {42593176},
issn = {1557-7759},
abstract = {BACKGROUND: Urinary tract infections (UTIs) in cats are increasingly recognized as clinically relevant conditions frequently associated with multidrug-resistant (MDR) bacteria of potential zoonotic origin, yet genomic data on feline uropathogens remain scarce in Tunisia.

METHODS: We used whole-genome sequencing to characterize seven bacterial isolates recovered from six cats with clinical signs of UTI: Mammaliicoccus lentus (n = 2), Staphylococcus schleiferi (n = 1), Mammaliicoccus sciuri (n = 1), Enterococcus faecalis (n = 1), Enterococcus casseliflavus (n = 1), and Klebsiella aerogenes (n = 1).

RESULTS: Resistome analysis revealed determinants conferring resistance to β-lactams (blaZ, blaCMY-132), methicillin (mecC-type), macrolides (erm(43), ermB), tetracyclines (tet(M), tet(45), tetB), fosfomycins (fosI, fosB, fosA5), and aminoglycosides (aac(6'), aph(3')-IIIa, aph(6)-Id), alongside efflux pump genes (efrA, sepA, sdrM, oqxA, KpnE/F/G), vancomycin-operon genes (vanT, vanY, vanC, vanG), and biofilm/biocide-tolerance genes (salB, qacG). Notably, M. lentus S104 carried mecC-type elements, the first such report in Tunisia, while K. aerogenes displayed an extensive MDR profile, including blaCMY-132 and fosA5. Multilocus sequence typing/ribosomal multilocus sequence typing (MLST/rMLST) identified diverse lineages, including the internationally distributed E. faecalis ST19 and the rarely reported K. aerogenes ST242. Plasmids were absent in all isolates; a Tn916/1545-type transposon occurred in E. casseliflavus, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were unevenly distributed.

CONCLUSIONS: These findings highlight companion animals as reservoirs of clinically important resistance genes, reinforcing the need for One Health AMR surveillance.},
}

@article {pmid42593847,
year = {2026},
author = {Lozano-Montalbá, L and Denis, Z and Courgnaud, V and Moreno-García, J and Majzoub, K and Sanjuán, R and Dufloo, J},
title = {Glycosphingolipids are essential entry factors for non-influenza orthomyxoviruses.},
journal = {The Journal of general virology},
volume = {107},
number = {8},
pages = {},
pmid = {42593847},
issn = {1465-2099},
mesh = {*Virus Internalization ; *Glycosphingolipids/metabolism ; Animals ; *Thogotovirus/physiology/genetics ; Glucosyltransferases/metabolism/genetics ; Virus Replication ; Cell Line ; *Orthomyxoviridae/physiology ; Humans ; CRISPR-Cas Systems ; },
abstract = {Thogotoviruses and quaranjaviruses are arthropod-borne orthomyxoviruses that circulate widely in wildlife and domestic animals and include several zoonotic members. Despite their close phylogenetic relationship to influenza viruses, the mechanisms underlying their replication remain poorly understood, and the host factors mediating viral entry are unknown. Here, we performed genome-wide loss-of-function CRISPR-Cas9 screens using replication-competent recombinant vesicular stomatitis viruses expressing thogoto- and quaranjavirus glycoproteins to identify cellular determinants of viral entry. These screens identified the glycosphingolipid (GSL) biosynthesis pathway as a key regulator of viral entry, with the upstream enzyme uridine diphosphate (UDP)-glucose ceramide glucosyltransferase (UGCG) emerging as a central host entry factor. Pharmacological inhibition of UGCG impaired thogoto- and quaranjavirus entry. The importance of GSL biosynthesis for thogotovirus replication was further validated using multiple thogotovirus isolates. Together, our findings establish GSLs as critical host entry factors for non-influenza orthomyxoviruses and identify UGCG as a potential target for antiviral intervention.},
}

*Virus Internalization
*Glycosphingolipids/metabolism
Animals
*Thogotovirus/physiology/genetics
Glucosyltransferases/metabolism/genetics
Virus Replication
Cell Line
*Orthomyxoviridae/physiology
Humans
CRISPR-Cas Systems

@article {pmid42595700,
year = {2026},
author = {Mani, M and Poonguzhali, S and Baghyalakshmi, K},
title = {CRISPR/Cas-based genome editing for enhancing grain quality traits in rice (Oryza sativa L.).},
journal = {Plant signaling & behavior},
volume = {21},
number = {1},
pages = {2713868},
pmid = {42595700},
issn = {1559-2324},
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Gene Editing/methods ; Plants, Genetically Modified ; },
abstract = {Rice is one of the most important food crops and feeds more than half of the world's population. Enhancing grain quality is currently a highly important issue since consumers are now more concerned with the taste, appearance, and nutritional value of the grain. The quality of grain in rice is complex and regulated by a multitude of genes that influence qualities such as amylase content, grain size and shape, chalkiness, aroma and nutrient content. The traditional forms of breeding, such as hybridization and marker-assisted selection, are slow and less effective since such characteristics are regulated by many genes and are influenced by environmental conditions. CRISPR/Cas genome editing has become a potent tool that enables scientists to directly and specifically edit grain quality-related genes. Important genes such as Wx (amylose), GS3, GW8, and TGW3 (grain size), Chalk5 (chalkiness), BADH2 (aroma), and nutrient-related grain size genes such as OsAAP6, OsAAP10, and OsVIT1/2, have been successfully edited to enhance the quality of rice. Newer methods, such as base editing and prime editing, enable this process to become even more precise by modifying the specific bases of DNA without cutting the DNA. CRISPR has assisted in the improvement of rice by controlling the amylose content, reducing chalkiness, enhancing aroma, and improving nutritional quality. It is more accurate and quicker than traditional breeding, and it can enhance various traits simultaneously. Nonetheless, other challenges, such as off-target effects, regulatory concerns, and acceptance by the people, still have to be overcome. Combining CRISPR with artificial intelligence and genomic selection in the future will aid in creating superior versions of rice in shorter periods of time. Overall, CRISPR/Cas genome editing is a potential method to enhance the quality of rice and secure food security in the whole world.},
}

*Oryza/genetics
*CRISPR-Cas Systems/genetics
*Edible Grain/genetics
*Gene Editing/methods
Plants, Genetically Modified

@article {pmid42595703,
year = {2026},
author = {Mukherjee, A and Thakur, D and Ahuja, M and Nayan, P and Kumar, L},
title = {Enzyme-driven antimicrobial resistance and advancements in sustainable anti-infective strategies.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-39},
doi = {10.1080/07388551.2026.2693159},
pmid = {42595703},
issn = {1549-7801},
abstract = {Antibiotic resistance is an emerging global issue that has reduced the efficacy of antibiotics for treating life-threatening bacterial infections. Bacterial adaptive enzymatic defense mechanisms allow cells to activate or modify specific enzymes that inactivate antibiotics and support survival under antimicrobial stress. Antibiotics are predominantly inactivated through enzymatic degradation or chemical modification. Most of the β-lactamases, macrolide esterases, tetracycline-modifying enzymes, fosfomycin degrading enzymes, aminoglycoside-modifying enzymes, and other bacterial enzymes chemically modify or degrade the antibiotics making them inactive. This review covers classification and mechanisms of enzyme-mediated resistance and emphasizes the significant enzymes involved in inactivation of various antibiotic classes. It also summarizes recent biotechnological advances to combat antibiotic resistance, including β-lactamase inhibitors, phage therapy, antimicrobial peptides, and CRISPR-Cas9 systems, along with emerging therapeutic approaches and current trends in antibiotic research. A deeper understanding of enzyme-mediated resistance and the cellular intelligence driving bacterial adaptation is crucial for designing effective therapeutic strategies, preserving antibiotic efficacy, and reducing the global burden of resistant infections.},
}

@article {pmid42341757,
year = {2026},
author = {Kim, J and Kovacs, H and Wisnovsky, S},
title = {Rapid discovery of cell-surface glycosylation regulators using a lectin-based magnetic CRISPR screen.},
journal = {Cell reports methods},
volume = {6},
number = {8},
pages = {101507},
doi = {10.1016/j.crmeth.2026.101507},
pmid = {42341757},
issn = {2667-2375},
mesh = {Humans ; Glycosylation ; *Lectins/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Polysaccharides/metabolism ; *Cell Membrane/metabolism ; Flow Cytometry/methods ; Cell Line, Tumor ; *CRISPR-Cas Systems ; },
abstract = {FACS-based CRISPR screening has emerged as a potent tool for dissecting the genetic networks that regulate cell-surface glycosylation. However, existing protocols can be tedious and are not compatible with many cell models. We developed a lectin-based magnetic-activated cell sorting platform (Lec-MACS) that enables rapid identification of genes controlling expression of specific cell-surface glycans. Lec-MACS offers superior speed and multiplexability compared to FACS, while also being well-suited to studying cell models that are not amenable to flow-based sorting. We subsequently applied Lec-MACS to map genes that regulate hypersialylation in an adherent breast cancer cell line. Subsequent hit validation confirmed an unexpected link between DNA damage response signaling and cell-surface sialylation. The Lec-MACS method will expand the scope and throughput of genetic screens targeted at cell-surface glycans.},
}

Humans
Glycosylation
*Lectins/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Polysaccharides/metabolism
*Cell Membrane/metabolism
Flow Cytometry/methods
Cell Line, Tumor
*CRISPR-Cas Systems

@article {pmid42420316,
year = {2026},
author = {Favoino, G and Pšenka, D and Frideres, L and Volke, DC and Nikel, PI},
title = {A portable Cas6f-based system for multiplex translational repression in bacteria.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42420316},
issn = {2041-1723},
support = {NNF20CC0035580//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF18OC0034818//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF21OC0067996//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF24OC0091501//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF23OC0083631//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF24SA0100980//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF20CC0035596//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; 101082049//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*Pseudomonas putida/genetics/metabolism ; *Escherichia coli/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; *Protein Biosynthesis ; Metabolic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockdown Techniques/methods ; Synthetic Biology/methods ; },
abstract = {Engineered small RNAs (sRNAs) enable programmable gene knockdowns and support metabolic engineering and multiplex regulation in model bacteria. Still, precise, tunable, and multiplex gene repression remains a challenge in synthetic biology. Common tools can impose genetic burden, depend on host RNA factors, or do not transfer well across species. Here we present MORTISE (Multiplex, ORthogonal Translation Interference SystEm), a compact Cas6f-based platform for programmable translational repression in Gram-negative bacteria. The system functions without host Hfq or RNases and operates robustly in Escherichia coli and Pseudomonas putida. We demonstrate repression in both species using chromosomal reporter assays, with performance improving when guide and target transcription are matched and when the translation initiation region is targeted. Single-promoter multiplexing enables simultaneous knockdowns and a cloning toolbox facilitates assembly of up to nine guides in a single step. Finally, MORTISE is leveraged to boost malonyl-coenzyme A-dependent production in P. putida, supporting pathway balancing.},
}

*Pseudomonas putida/genetics/metabolism
*Escherichia coli/genetics/metabolism
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic
*Protein Biosynthesis
Metabolic Engineering/methods
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockdown Techniques/methods
Synthetic Biology/methods

@article {pmid42585319,
year = {2026},
author = {Dinh, DT and Smith, KM and McPhee, T and Foot, NJ and Bersten, DC and White, MA and Thomas, PQ and Robker, RL and Russell, DL},
title = {Reassessing the role of progesterone receptor isoforms PGR-A and PGR-B in female fertility.},
journal = {Science advances},
volume = {12},
number = {33},
pages = {eadz7757},
pmid = {42585319},
issn = {2375-2548},
support = {INV-001616//Bill & Melinda Gates Foundation/United States ; INV-024199//Bill & Melinda Gates Foundation/United States ; },
mesh = {Female ; *Receptors, Progesterone/genetics/metabolism ; Animals ; *Fertility/genetics ; Protein Isoforms/genetics/metabolism ; Mice ; CRISPR-Cas Systems ; Mice, Knockout ; },
abstract = {Progesterone is a critical reproductive hormone that acts via progesterone receptor transcriptional regulators. The short PGR-A isoform lacks a 164-amino acid amino-terminal region present in PGR-B that markedly enhances transcriptional activity. Isoform-specific mutants inactivating either PGR-A (PRAKO) or PGR-B (PRBKO) indicated that PGR-A is specifically essential for female fertility. This study revises that interpretation by showing that an inadvertent frameshift mutation in the PRAKO caused ablation of both isoforms, not the intended PGR-A isoform-specific mutation. A true PGR-A-specific mutant generated through CRISPR-Cas9 editing with a complete lack of PGR-A but retained PGR-B expression showed a phenotype indistinguishable from wild type. Thus, while the short PGR-A isoform has distinct function, it is not independently essential for female reproduction in mice, and the physiological role of these highly conserved isoforms must be reconsidered in light of this new information.},
}

Female
*Receptors, Progesterone/genetics/metabolism
Animals
*Fertility/genetics
Protein Isoforms/genetics/metabolism
Mice
CRISPR-Cas Systems
Mice, Knockout

@article {pmid42584997,
year = {2026},
author = {Wu, P and Zhou, X},
title = {Canon enables causal inference of downstream genes in single-cell CRISPR studies via instrumental variable analysis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {33},
pages = {e2525359123},
doi = {10.1073/pnas.2525359123},
pmid = {42584997},
issn = {1091-6490},
support = {R01HG009124//HHS | NIH (NIH)/ ; R01GM144960//HHS | NIH (NIH)/ ; R01GM126553//HHS | NIH (NIH)/ ; R01HG011883//HHS | NIH (NIH)/ ; },
mesh = {Humans ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; *Gene Regulatory Networks/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A critical analytical task in sc-CRISPR screening is identifying downstream genes influenced by perturbed target genes. Existing methods for this task primarily rely on traditional association-based analyses, which not only fall short in establishing causal relationships between genes but also suffer from high false positive rates and limited statistical power. To overcome these limitations, we introduce a causal inference-based framework that leverages the perturbation status of gRNAs in single cells as instrumental variables (IVs) to infer causal gene relationship via IV analysis. Building upon this framework, we further present Canon, a one-sample IV analysis method specifically tailored to systematically identify genes that are potentially causally influenced by perturbed target genes across diverse sc-CRISPR platforms. Canon ensures robust type I error control while maintaining high statistical power. We evaluated its performance through comprehensive simulations and real data applications. The gene-gene relationships identified by Canon provide valuable insights into the causal gene regulatory network, uncovering candidate therapeutic targets with potential relevance for cancer biology and demonstrating the transformative potential of sc-CRISPR screening to resolve causal regulatory networks at an unprecedented scale.},
}

Humans
*Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
*Gene Regulatory Networks/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42582274,
year = {2026},
author = {Liu, W and Gu, J and Xie, C and Du, B and Liu, M and Zhang, J},
title = {Rewriting CAR-T cell fate: CRISPR/Cas gene editing for solid tumor therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1910092},
pmid = {42582274},
issn = {1664-3224},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *Receptors, Chimeric Antigen/immunology/genetics ; Animals ; Tumor Microenvironment/immunology ; *T-Lymphocytes/immunology/metabolism ; T-Cell Exhaustion ; },
abstract = {Although chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematological malignancies, its therapeutic efficacy in solid tumors remains limited by several challenges, including insufficient tumor infiltration, T cell exhaustion and the immunosuppressive tumor microenvironment (TME). CRISPR/Cas, a third-generation gene editing technology developed in recent years, is characterized by its simplicity and high efficiency. This technology has demonstrated broad application potential across multiple fields and has emerged as a powerful tool for improving CAR-T cell therapy. In this review, we summarize recent advances in the application of CRISPR/Cas gene editing technology to enhance the antitumor activity of CAR-T cells against solid tumors. We also discuss the key challenges currently faced and systematically propose potential strategies for overcoming the limitations.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology/genetics
*Receptors, Chimeric Antigen/immunology/genetics
Animals
Tumor Microenvironment/immunology
*T-Lymphocytes/immunology/metabolism
T-Cell Exhaustion

@article {pmid42582990,
year = {2026},
author = {Ma, AJ and Brown, BH and Kim, S and Hilton, IB},
title = {Clinical translation of epigenome editing technologies.},
journal = {Current opinion in biomedical engineering},
volume = {38},
number = {},
pages = {},
pmid = {42582990},
issn = {2468-4511},
support = {R01 EB036003/EB/NIBIB NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR/Cas-based epigenome editing technologies hold great promise for identifying novel therapeutic targets, improving gene and cell therapies, and directly addressing the underlying issues in many diseases, all while minimizing risks of genotoxicity often associated with conventional genome editing. Exciting recent advances in CRISPR/Cas-based epigenome editing technologies have drastically enhanced the ability to precisely control the timing, levels, and durations of endogenous gene expression and reprogram epigenetic states in human cells. As a result, epigenome editing is now poised to unlock new biomedical discoveries and treatments for diseases driven by transcriptional and epigenetic dysregulation as well as those stemming from aberrantly repetitive genomic regions or complex genomic arrangements that are difficult to target using conventional genome editing. Additionally, the power of epigenome editors is generating new strategies to control cell fate and function, which has direct and important implications for cell therapies and regenerative medicines. Here, as the first wave of CRISPR/Cas-based epigenome editors move into clinical trials, we cover recent advances as the field looks to address pressing hurdles facing widespread clinical deployment of epigenome editing technologies including delivery, performance, and safety. For instance, the discovery of compact Cas chassis, engineering efforts to reduce effector sizes for efficient delivery, and campaigns to tailor the targeting discrimination of epigenome editors are rapidly progressing, as is research into the development of new effector domains with high specificity, robust performance, and a lack of immunogenicity and cytotoxicity. This exciting progress is quickly moving the community closer to fulfilling the promise of CRISPR/Cas-based epigenome editing as a powerful class of platform technologies for biological discoveries, biotechnological innovations, and medicines.},
}

@article {pmid42584708,
year = {2026},
author = {Skoczek, D and Hohendorff, J and Malecki, MT and Roig-Merino, A and Bak, RO and Kachamakova-Trojanowska, N},
title = {CRISPR/Cas9-based repair of a heterozygous HNF1A mutation in patient-derived hiPSCs.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {42584708},
issn = {1432-1203},
support = {2020/38/E/NZ3/00516//Narodowe Centrum Nauki/ ; 2020/38/E/NZ3/00516//Narodowe Centrum Nauki/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Hepatocyte Nuclear Factor 1-alpha/genetics ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Frameshift Mutation ; Mutation ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor's genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Hepatocyte Nuclear Factor 1-alpha/genetics
*CRISPR-Cas Systems/genetics
Heterozygote
Frameshift Mutation
Mutation

@article {pmid41820594,
year = {2026},
author = {Lee, SJ and Nam, BG and Hong, SA and Jo, DH and Lee, SM and Bae, S and Kim, JH},
title = {Topical application of Cas9 ribonucleoproteins inhibits corneal neovascularization in a mouse model of alkali burn injury.},
journal = {Gene therapy},
volume = {33},
number = {4},
pages = {435-447},
pmid = {41820594},
issn = {1476-5462},
support = {2022M3A9E4017127//National Research Foundation of Korea (NRF)/ ; RS-2023-00260351//National Research Foundation of Korea (NRF)/ ; RS-2024-00467177//National Research Foundation of Korea (NRF)/ ; 18-2023-0010//Seoul National University Hospital (SNUH)/ ; GTL24021-000//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; },
mesh = {Animals ; *Corneal Neovascularization/therapy/genetics ; Mice ; *Burns, Chemical/therapy ; *Genetic Therapy/methods ; Disease Models, Animal ; *Eye Burns/therapy/chemically induced ; Vascular Endothelial Growth Factor A/genetics/metabolism ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/administration & dosage ; Gene Therapy Agents ; Gene Editing ; Mice, Inbred C57BL ; Administration, Topical ; *CRISPR-Associated Protein 9/genetics ; },
abstract = {Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.},
}

Animals
*Corneal Neovascularization/therapy/genetics
Mice
*Burns, Chemical/therapy
*Genetic Therapy/methods
Disease Models, Animal
*Eye Burns/therapy/chemically induced
Vascular Endothelial Growth Factor A/genetics/metabolism
*CRISPR-Cas Systems
*Ribonucleoproteins/genetics/administration & dosage
Gene Therapy Agents
Gene Editing
Mice, Inbred C57BL
Administration, Topical
*CRISPR-Associated Protein 9/genetics

@article {pmid42174157,
year = {2026},
author = {Li, JX and Zhang, SM and Ma, XY and Deng, DH and Bai, PD and Zhao, JJ and Gong, A and Pang, SC and Dong, F and Wang, SD and Zhang, JP and Zhang, XB},
title = {AviTag-seq unifies nucleotide-resolution maps of CRISPR off-targets and AAV vector integrations.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42174157},
issn = {2399-3642},
support = {82402188//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Genetic Vectors/genetics ; *Dependovirus/genetics ; Mice ; *Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Liver/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Virus Integration ; },
abstract = {Comprehensive safety assessment of gene-editing therapies requires quantifying both off-target cleavage and vector integration. However, current double-strand break (DSB)-dependent assays are fundamentally limited when evaluating nickase-based editors and are hindered by tag polarity constraints. Here, we present AviTag-seq, a platform repurposing AAV Inverted Terminal Repeats (ITRs) as universal capture tags. By exploiting the ITRs' single-stranded hairpin structure, AviTag-seq overcomes polarity issues, enabling high-sensitivity detection with a single primer pair, particularly in iPSCs. Crucially, it captures off-target events from prime and base editors that evade conventional detection. In vivo, AviTag-seq outperformed DISCOVER-Seq+ in profiling Pcsk9 off-targets in mouse liver while simultaneously mapping AAV integration sites. This dual profiling revealed that, unlike in vitro, AAV vectors in vivo preferentially integrate into active gene promoters, highlighting a specific genotoxic risk for liver-directed therapies. AviTag-seq thus offers a unified, regulatory-grade solution for evaluating diverse genome-editing modalities.},
}

Animals
*Genetic Vectors/genetics
*Dependovirus/genetics
Mice
*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Liver/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*Virus Integration

@article {pmid42243947,
year = {2026},
author = {Wei, Z and Xu, X and Qiao, Q and Guo, J and Wu, T and Rong, H and Ning, S and Zhu, X and Zhao, K and Ke, J and He, L and Chi, Y and Ge, Y and Cui, L and Min, X},
title = {A temporally controlled isothermal amplification-CRISPR/Cas12a platform for the rapid detection of monkeypox virus.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42243947},
issn = {1743-422X},
support = {JSJK2026D00033//Science and Technology Program Project of Jiangsu Provincial Bureau of Disease Prevention and Control/ ; MQ2025039//Scientific Research Project of Jiangsu Commission of Health/ ; M2024024//Scientific Research Project of Jiangsu Commission of Health/ ; BK20231374//Natural Science Foundation of Jiangsu Province/ ; 2023YFC2605100, 2023YFC2605104//National Key Research and Development Program of China/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Monkeypox virus/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Mpox, Monkeypox/diagnosis/virology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Humans ; Animals ; },
abstract = {BACKGROUND: Given the continuous threat posed by emerging and re-emerging infectious diseases worldwide, a rapid, sensitive, and practical molecular detection technology is urgently required for timely point-of-care (POC) diagnosis. Although the quantitative real-time PCR (qPCR) based technologies exhibit high sensitivity and specificity compared with traditional pathogen detection methods, they are not applicable for POC detection scenarios.

METHODS: Based on the photocontrolled principle, this study established a universal photoactivation strategy for LbCas12a by modifying four sites in the repeat region of LbCas12a crRNA with the photocleavable protecting group 6-nitropiperonyloxymethyl (NPOM). This strategy was integrated with multienzyme isothermal rapid amplification (MIRA) to develop a photocontrolled one-pot rapid detection method for monkeypox virus (MPXV), termed the temporally controlled MIRA-CRISPR/Cas12a (TC-MIRA-CRISPR/Cas12a) assay.

RESULTS: The TC-MIRA-CRISPR/Cas12a assay exhibited a 100-fold improvement in detection sensitivity over the conventional one-step assay, achieving a limit of detection (LOD) of 3.9 copies per reaction, which was comparable to stepwise detection assay. The entire assay can be completed within 40 min, faster than the widely used qPCR. In clinical sample detection, this method showed good consistency with qPCR, with a Kappa coefficient of 0.980 (P < 0.001), a sensitivity of 98.4%, and a specificity of 100%.

CONCLUSIONS: This method effectively avoids amplicon contamination while maintaining high sensitivity, and exhibits excellent universality and expandability. It enables detection of other pathogens simply by modifying the spacer sequence of crRNA, providing a novel technical approach and research perspective for POC detection of MPXV and other pathogens.},
}

*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Monkeypox virus/isolation & purification/genetics
*CRISPR-Cas Systems
*Mpox, Monkeypox/diagnosis/virology
Rapid Diagnostic Tests
*Molecular Diagnostic Techniques/methods
Humans
Animals

@article {pmid42265600,
year = {2026},
author = {Jiang, S and Chen, F and Ma, H and Wu, S and Tang, X and Pan, X and Li, Q and Tao, A and Xu, J and Qi, J and Fang, P and Chen, J and Zhang, L},
title = {Cloning and functional verification of endogenous U6 promoters for developing an efficient CRISPR/Cas9-mediated genome editing system in kenaf (Hibiscus cannabinus L.).},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {42265600},
issn = {1471-2229},
support = {32472219//the National Natural Science Foundation of China/ ; 2023J01443//Fujian Provincial Natural Science Foundation of China/ ; CARS-16//China Agricultural Research System of MOF and MARA/ ; KFB23001//Science and Technology Innovation Project of Fujian Agriculture and Forestry University/ ; ASTIP-IBFC-01//Agriculture Science and Technology Innovation Program/ ; },
mesh = {*Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems ; *Hibiscus/genetics ; *Gene Editing/methods ; Cloning, Molecular ; *RNA, Small Nuclear/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {BACKGROUND: The U6 promoter is a critical component of the CRISPR/Cas9 system, as it drives the transcription of single-guide RNAs (sgRNAs) to enable precise genome editing. Endogenous promoters typically exhibit higher transcriptional activity than their exogenous counterparts, which can significantly enhance editing efficiency. However, the endogenous U6 promoter in kenaf (Hibiscus cannabinus L.), an important fiber crop, has not yet been characterized.

METHODS: Using the Arabidopsis U6-26 (AtU6-26) promoter as a reference, we performed a homologous sequence search and identified two candidate U6 promoters in kenaf, designated HcU6-1 and HcU6-14. Promoter fragments were amplified from the kenaf cultivar 'Fuhong 952' and cloned into a β-glucuronidase (GUS) reporter vector. Histochemical GUS staining assays revealed that both HcU6 promoters were transcriptionally active, with HcU6-14 showing significantly stronger expression levels compared to HcU6-1.

RESULTS: To further evaluate the utility of these promoters for genome editing, we constructed CRISPR/Cas9 vectors targeting the kenaf acetolactate synthase (ALS) gene, driven by either HcU6-14P or the exogenous cotton GbU6-9P promoter. Agrobacterium rhizogenes K599-mediated transformation was used to induce hairy roots, and mutation analysis of the ALS gene was performed via Sanger sequencing. Notably, targeted mutations in the ALS gene were detected in hairy roots transformed with the HcU6-14P-driven CRISPR/Cas9 vector, whereas no mutations were observed in roots transformed with the exogenous GbU6-9P promoter. These results demonstrate that the endogenous HcU6-14 promoter confers superior genome editing efficiency compared to the heterologous promoter, which facilitates the development of improved varieties with enhanced agronomic traits.},
}

*Promoter Regions, Genetic/genetics
*CRISPR-Cas Systems
*Hibiscus/genetics
*Gene Editing/methods
Cloning, Molecular
*RNA, Small Nuclear/genetics
Plants, Genetically Modified
Genome, Plant

@article {pmid42409798,
year = {2026},
author = {Zhao, C and Shih, M and Ahmed, S and Song, S and Lennon, A and Mayes, JM and Fan, MJ and Lo, PY and Perera, J and Tutar, A and Kang, A and Warren, E and McClure, R and Khan, AA and Kelley, SO and Abdrabou, AM},
title = {AI-guided CRISPR screening reveals therapeutic targets in psoriasis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42409798},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Psoriasis/genetics/drug therapy/chemically induced ; Keratinocytes/metabolism/drug effects ; Mice ; *Receptors, Interleukin-17/metabolism/genetics ; Arachidonate 5-Lipoxygenase/genetics/metabolism ; Imiquimod ; Receptors, Oxytocin/antagonists & inhibitors/genetics/metabolism ; CRISPR-Cas Systems ; Hydroxyurea/pharmacology/analogs & derivatives ; Clustered Regularly Interspaced Short Palindromic Repeats ; Interleukin-17 ; },
abstract = {Psoriasis affects over 125 million people globally. Biologics targeting the IL-17/IL-17RA axis are effective but require systemic administration, are costly, and are unsuitable for some patients. Developing topical small-molecule alternatives requires a better understanding of how IL-17 receptor A (IL17RA) is regulated in keratinocytes, the principal effector cells of psoriatic lesions. Here, we report a genome-wide CRISPR knockout screen for regulators of surface IL17RA in primary human epidermal keratinocytes. We prioritize hits using experimental enrichment together with VirtualCRISPR, a language-model framework trained on functional-genomics data, and validate two regulators with minimal prior connection to IL17RA: 5-lipoxygenase (ALOX5) and the oxytocin receptor (OXTR), which act through distinct cell-intrinsic mechanisms. Topical zileuton, an ALOX5 inhibitor, and cligosiban, an OXTR antagonist, suppress imiquimod-induced psoriasiform dermatitis in mice, mirroring systemic anti-IL17RA antibody efficacy. By linking AI- guided selection to genetic perturbation screening, this study provides an efficient route from candidate gene nomination to biological validation and therapeutic discovery.},
}

Humans
Animals
*Psoriasis/genetics/drug therapy/chemically induced
Keratinocytes/metabolism/drug effects
Mice
*Receptors, Interleukin-17/metabolism/genetics
Arachidonate 5-Lipoxygenase/genetics/metabolism
Imiquimod
Receptors, Oxytocin/antagonists & inhibitors/genetics/metabolism
CRISPR-Cas Systems
Hydroxyurea/pharmacology/analogs & derivatives
Clustered Regularly Interspaced Short Palindromic Repeats
Interleukin-17

@article {pmid42414323,
year = {2026},
author = {Wu, X and Li, Y and Cao, Y and Zhao, Z and Lu, H and Liang, S and Lui, GCY and Chan, DPC and Hsing, IM},
title = {Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42414323},
issn = {2041-1723},
support = {16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*Polymorphism, Single Nucleotide/genetics ; Thermodynamics ; *Point-of-Care Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genotyping Techniques/methods ; DNA Primers/genetics ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Genotype ; },
abstract = {One-pot CRISPR diagnostics face a fundamental incompatibility: isothermal nucleic acid amplification enables rapid target accumulation, whereas CRISPR activation irreversibly consumes those substrates, destabilizing reaction kinetics. Here we show that reaction order can be programmed into DNA primers through thermodynamic design. Differences in primer-binding strength create two sequential amplification stages, delaying CRISPR activation until enough amplicons have accumulated without physical separation or external control. The design also introduces the protospacer adjacent motif (PAM), a short sequence required for CRISPR recognition, through the primer rather than relying on its presence in the native target, expanding target accessibility while retaining single-nucleotide discrimination. An ordinary differential equation model captures the threshold behavior and establishes a predictable framework for primer design. Building on this principle, we develop Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), which achieves attomolar sensitivity within 30 min and enables sequencing-concordant SNP genotyping and pathogen detection in a single-step microfluidic format.},
}

*Polymorphism, Single Nucleotide/genetics
Thermodynamics
*Point-of-Care Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Genotyping Techniques/methods
DNA Primers/genetics
Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Genotype

@article {pmid42574383,
year = {2026},
author = {Xu, A and Cates, K and Kerr, C and Wysong, K and Barna, M},
title = {Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/66881},
pmid = {42574383},
issn = {1940-087X},
mesh = {*Biotinylation/methods ; *Ribosomes/chemistry/metabolism ; Carbon-Nitrogen Ligases/chemistry/metabolism/genetics ; Biotin/chemistry ; Humans ; Ribosomal Proteins/chemistry/genetics ; CRISPR-Cas Systems ; },
abstract = {Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of specific mRNAs is key to ensuring that proteins with compartment-specific functions are produced in the ideal milieu and in appropriate quantities. However, techniques for subcellular isolation and characterization of the ribosomes that translate these mRNAs have been limited to date, such that much remains unknown about how the composition of translational machinery contributes to regulation of localized mRNA translation. Here, we demonstrate AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi), a method that combines epitope tagging, a newly developed optogenetically activated split-biotin ligase, and affinity purification to rapidly and specifically label and isolate ribosomes localized to any subcellular compartment of interest. First, CRISPR/Cas9 editing is used to fuse an AviTag peptide, a tobacco etch virus (TEV) protease cleavage site, and a FLAG epitope tag to a ribosomal protein. The split biotin ligase, fused to an organelle-targeting domain, is expressed in this cell line and is inactive under normal biotin concentrations. Upon activation by supplemental biotin and blue light illumination, the ligase biotinylates AviTagged ribosomes in the immediate vicinity, allowing for affinity purification of biotinylated ribosomes and associated proteins and mRNAs on streptavidin-coated beads. Non-denaturing elution via TEV protease cleavage yields samples suitable for downstream characterization of core ribosomal proteins, ribosome-associated proteins, and ribosome-bound mRNAs via RNA sequencing or mass spectrometry proteomics. In this protocol, we review design principles for fusing AviTag to a ribosomal protein and targeting the split biotin ligase enzyme to the organelle of interest. We demonstrate activation of the ALIBi system, cell lysis, affinity purification, and sample elution. Finally, we discuss typical results and troubleshooting.},
}

*Biotinylation/methods
*Ribosomes/chemistry/metabolism
Carbon-Nitrogen Ligases/chemistry/metabolism/genetics
Biotin/chemistry
Humans
Ribosomal Proteins/chemistry/genetics
CRISPR-Cas Systems

@article {pmid42574397,
year = {2026},
author = {Langille, ER and Al-Zahrani, KN and Nurtanto, J and Lee, Y and Chiu, CH and Schramek, D},
title = {Parallel In Vivo Screening of Gene Knockout and Activation in the Mouse Mammary Gland.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71115},
pmid = {42574397},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Mammary Glands, Animal/physiology/metabolism ; Female ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Forward genetics screens are routinely employed to perturb thousands of genetic elements in a pooled fashion with the goal of producing large-scale genotype-to-phenotype maps. While often carried out in cell culture systems, accumulating evidence supports that in vivo screens have the power to unveil new biology that cannot be recapitulated in vitro. However, the widespread application of this approach has been limited by two major challenges: a predominant focus on loss-of-function perturbations rather than gene activation and the significant technical hurdles of delivering complex genetic libraries to specific tissues in vivo. To overcome these challenges, we describe a simple and versatile intraductal injection strategy that enables efficient and rapid functional genomic screening in the mouse mammary gland, by generating tens of thousands of discrete epithelial clones. Furthermore, we provide all the details necessary for library generation, intraductal injection, screen deconvolution, and analysis of CRISPR-Knockout and Activation libraries for comprehensive in vivo screens. Using these tools, which we termed CRISPR-KOALA (Knockout and Activation Linked Assay), we have identified new tumor suppressors and oncogenes within the coding and non-coding genome in pooled libraries ranging from 46 loci to one-fifth of the genome. Importantly, this approach and analysis can be applied to other organs to study the biological function of any gene during homeostasis or disease.},
}

Animals
Mice
*Mammary Glands, Animal/physiology/metabolism
Female
*Gene Knockout Techniques/methods
CRISPR-Cas Systems

@article {pmid42574536,
year = {2026},
author = {Yedier-Bayram, O and Guvener, EA and Bagci-Onder, T},
title = {Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/71175},
pmid = {42574536},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Paclitaxel/pharmacology ; *Gene Knockout Techniques/methods ; *Epigenome/genetics ; *Triple Negative Breast Neoplasms/genetics/drug therapy ; Female ; },
abstract = {Chemotherapy resistance remains a major challenge in cancer treatment, driven by cancer cells' ability to acquire adaptive properties, rewire signaling pathways, and alter chromatin structure to evade drug-induced cytotoxicity. Because these processes rely heavily on epigenetic mechanisms that regulate chromatin organization and transcriptional plasticity, epigenetic regulators have emerged as key contributors to chemotherapy resistance. To investigate resistance to paclitaxel, one of the most widely used chemotherapeutic agents in triple-negative breast cancer (TNBC), we employed an epigenome-focused knockout library (EPIKOL), a CRISPR-Cas9-based library, designed to systematically disrupt genes involved in chromatin regulation. Chemoresistant cell lines were generated through a stepwise dose-escalation protocol that recapitulates clinically relevant drug adaptation. However, these resistant cells exhibit a multidrug-resistant (MDR) phenotype, posing significant challenges for efficient viral transduction and the selection of stable cell populations. In this study, we describe key methodological steps for achieving high-efficiency lentiviral transduction and selection, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models. Following the described protocol, an epigenome-wide CRISPR screen was conducted on chemoresistant TNBC cells, and novel epigenetic regulators of chemoresistance were identified. This protocol provides a robust framework for identifying epigenetic regulators that contribute to acquired paclitaxel resistance using a CRISPR-based loss-of-function approach.},
}

Humans
*CRISPR-Cas Systems
*Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
Paclitaxel/pharmacology
*Gene Knockout Techniques/methods
*Epigenome/genetics
*Triple Negative Breast Neoplasms/genetics/drug therapy
Female

@article {pmid42577382,
year = {2026},
author = {Lin, W and Shi, J and Chen, H and Chai, R and Zhu, S and Chen, L and Mo, S and Wang, Z and Li, H and Feng, Y and Zhao, L and Chen, J and Yu, G and Lu, T and Wang, J},
title = {Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1878099},
pmid = {42577382},
issn = {1664-3224},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Genetic Vectors/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; HEK293 Cells ; Animals ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Cell Engineering/methods ; CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; Cell Line ; },
abstract = {INTRODUCTION: CRISPR-Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches.

METHODS: We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9-gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules-Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope-into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days.

RESULTS: Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci.

DISCUSSION: It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*Genetic Vectors/genetics
*Receptors, Chimeric Antigen/genetics/immunology
HEK293 Cells
Animals
*Gene Editing/methods
*Immunotherapy, Adoptive/methods
*Cell Engineering/methods
CRISPR-Cas Systems
*T-Lymphocytes/immunology
Cell Line

@article {pmid42577913,
year = {2026},
author = {Tenkolu, LA and Balcha, F},
title = {Molecular Diagnosis of Bacterial Meningitis in Ethiopia: A Narrative Review of Current Evidence and Implementation Gaps.},
journal = {The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale},
volume = {2026},
number = {},
pages = {4447180},
pmid = {42577913},
issn = {1712-9532},
abstract = {BACKGROUND: Bacterial meningitis (BM) remains a significant public health concern in Ethiopia, particularly due to diagnostic challenges posed by limited laboratory infrastructure, late patient presentation, and technical skills required for cerebrospinal fluid (CSF) sampling. Molecular diagnostic methods offer rapid, sensitive, and specific alternatives to conventional culture.

OBJECTIVE: This narrative review synthesizes available evidence on molecular diagnostics for BM in Ethiopia, evaluates their performance compared to conventional methods, and identifies barriers to implementation.

METHODS: A structured literature search was conducted in PubMed, Google Scholar, Scopus, and Web of Science (August 1977-September 2024) using the terms: Meningitis OR Bacterial meningitis AND Diagnostics OR Molecular diagnostics, including PCR, CRISPR, next-generation sequencing (NGS), and MALDI-TOF. The review used a repeatable research selection procedure, well-defined inclusion/exclusion criteria, and PRISMA reporting requirements. Studies from Ethiopia and other low-resource settings were included. After screening titles/abstracts and full texts, 66 articles were selected.

RESULTS: In Ethiopia, multiplex PCR detected bacterial DNA in 10%-22% of the CSF samples, whereas culture was positive in only 0.5%-1% of the cases, primarily due to pre-admission antibiotic use. Globally, molecular panels (e.g., BioFire FilmArray ME) show > 90% sensitivity and specificity. Advanced techniques (CRISPR-Cas, NGS, and MALDI-TOF) have not yet been implemented in Ethiopian routine diagnostics.

CONCLUSION: Molecular methods vastly improve the detection of BM in Ethiopia, but high costs, infrastructure gaps, and shortage of trained personnel prevent widespread adoption. Molecular assays have outstanding analytical sensitivity, but culture remains the gold standard for confirmation of the viability of live pathogens. Implementation gaps require urgent targeted investment in point-of-care diagnostics and specialized transport networks.},
}

@article {pmid42578365,
year = {2026},
author = {Lv, S and Wang, Q and Gao, S and Wang, L and Zheng, L},
title = {Specific cleavage of 5' overhangs of non-target strand by Cas9 activated by target DNA binding.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42578365},
issn = {1362-4962},
support = {U23A20265//National Natural Science Foundation of China/ ; 32530084//National Natural Science Foundation of China/ ; 2408085MC072//Anhui Provincial Natural Science Foundation/ ; PA2025GDGP0026//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*DNA/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; DNA Cleavage ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; SARS-CoV-2/genetics ; *CRISPR-Associated Protein 9/metabolism ; Humans ; Base Sequence ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become a powerful genome-editing tool that uses RNA-DNA pairing to cleave target DNA with protospacer adjacent motif (PAM) sequences. While its primary function is well-studied, secondary activities remain poorly understood, causing unintended off-target effects. This study reports for the first time that Cas9 specifically cleaves the 5' overhang of the non-target strand (NTS) in target double-stranded DNA. This specific cleavage requires an additional PAM element at the NTS 5' region, is mediated by Cas9's RuvC domain, and is regulated by the HNH domain. It depends on the exact positioning of the NTS 5' end, but not on the overhang homopolymer sequence or overhang length. Adequate single-guide RNA-DNA complementarity is also essential. This discovery potentially advances our understanding of Cas9's enzymatic versatility to enhance genome-editing precision and efficacy and offers new nucleic acid detection strategies. Based on this cleavage, we developed a sensitive assay for Severe Acute Respiratory Syndrome Coronavirus 2 pseudovirus down to 2.4 copies μL-1, demonstrated extremely high sensitivity in diagnostic applications.},
}

*DNA/metabolism/genetics/chemistry
*CRISPR-Cas Systems
DNA Cleavage
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
SARS-CoV-2/genetics
*CRISPR-Associated Protein 9/metabolism
Humans
Base Sequence

@article {pmid42578374,
year = {2026},
author = {Zou, S and Ye, T and Fu, L and Zhou, JQ},
title = {A CRISPR/Cas9-induced blunt-end telomere system in S. pombe reveals RNase H2-dependent RNA primer removal at the terminal Okazaki fragment of lagging telomeres.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42578374},
issn = {1362-4962},
support = {2023YFA0913400//National Key Research and Development Program of China/ ; //Shanghai Academy of Natural Sciences/ ; },
mesh = {*Telomere/genetics/metabolism/chemistry ; *Schizosaccharomyces/genetics/metabolism ; *Ribonuclease H/metabolism/genetics ; *CRISPR-Cas Systems ; *RNA/metabolism/genetics ; DNA Replication/genetics ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; *DNA/genetics/metabolism ; },
abstract = {Studying the fine-scale dynamics of telomere replication has been hindered by the heterogeneity of native telomeres and the limitations of existing tools. Here, we report a highly efficient and inducible CRISPR/Cas9-mediated system for generating de novo telomeres with defined blunt ends in S. pombe. This fine setting allows for the precise dissection of post-replicative telomere end structures at near single-nucleotide resolution. Using this system, we show that the replicated leading-strand telomere is blunt-ended, while the lagging-strand counterpart contains an ∼10-nt 3' overhang resulting from RNA primer removal. By analyzing mutants deficient in ribonucleases, we found that the removal of this terminal RNA primer is specifically dependent on RNase H2, but not RNase H1. This RNase H2-dependent mechanism is essential for defining the mature structure of the lagging-strand telomere with authentic telomeric sequences. Our findings reveal a fundamental asymmetry in telomere end processing after replication and establish RNase H2 as the key enzyme responsible for resolving the terminal RNA primer in lagging-strand telomeres. This mechanism, conserved from budding to fission yeast, underscores the critical and evolutionarily ancient role of RNase H2 in defining eukaryotic telomere architecture.},
}

*Telomere/genetics/metabolism/chemistry
*Schizosaccharomyces/genetics/metabolism
*Ribonuclease H/metabolism/genetics
*CRISPR-Cas Systems
*RNA/metabolism/genetics
DNA Replication/genetics
*Schizosaccharomyces pombe Proteins/genetics/metabolism
*DNA/genetics/metabolism

@article {pmid42579777,
year = {2026},
author = {Ma, Y and Deng, Y and Xu, J and Wang, S and Shi, B and Liang, K and Deng, R and Yang, H},
title = {Engineering CRISPR for Point-of-Care Tests.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01485},
pmid = {42579777},
issn = {2379-3694},
support = {2026YFHZ0080//International S and T Cooperation Program of Sichuan Province/ ; 22504094//National Natural Science Foundation of China/ ; 32271392//National Natural Science Foundation of China/ ; 32571536//National Natural Science Foundation of China/ ; 82270970//National Natural Science Foundation of China/ ; 82470967//National Natural Science Foundation of China/ ; //Sichuan University/ ; 2023NSFSC0333//Natural Science Foundation of Sichuan Province/ ; 2024NSFSC0676//Natural Science Foundation of Sichuan Province/ ; 2025ZNSFSC1041//Natural Science Foundation of Sichuan Province/ ; },
abstract = {CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.},
}

@article {pmid42580522,
year = {2026},
author = {Reis, BCC and Georget, C and Meunier, AC and de Andrade Silva, EM and Perin, C and Herbert, L and Dos Santos Lopes, N and Micheli, F and de Oliveira Mendes, TA},
title = {Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software.},
journal = {New biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.nbt.2026.07.005},
pmid = {42580522},
issn = {1876-4347},
abstract = {Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. High-resolution melting (HRM) analysis is an established approach for screening CRISPR/Cas-induced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wild-type melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.},
}

@article {pmid42580703,
year = {2026},
author = {Yamaji, M and Tabata, H and Nakamura, M and Fuse, R and Okada, T and Ohba, SI and Ohishi, T and Kawada, M and Saito, I and Sato, S and Nakanishi, T},
title = {Adenovirus Vector-Mediated In Vivo Knock-in Treatment of Neonatal Phenylketonuria Mice Using Terminally Cleaved Donor DNA.},
journal = {The journal of gene medicine},
volume = {28},
number = {8},
pages = {e70104},
pmid = {42580703},
issn = {1521-2254},
support = {JP21fk0108560//Japan Agency for Medical Research and Development/ ; JP24fk0310523//Japan Agency for Medical Research and Development/ ; JP19K06476//Japan Society for the Promotion of Science/ ; JP23K27095//Japan Society for the Promotion of Science/ ; JP24K21938//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Genetic Vectors/genetics/administration & dosage ; *Gene Knock-In Techniques/methods ; Mice ; *Adenoviridae/genetics ; *Phenylalanine Hydroxylase/genetics ; *Phenylketonurias/therapy/genetics ; CRISPR-Cas Systems ; Genetic Therapy ; Disease Models, Animal ; Animals, Newborn ; Humans ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA/genetics ; },
abstract = {BACKGROUND: Adenovirus vectors (AdVs) are widely used and have an advantage of large insert capacity compared with adeno-associated virus vectors. However, AdVs have scarcely been used in genome-editing knock-in strategies because of low efficiency.

METHODS: Novel AdVs possessing a very large, 3.7 kb donor DNA fragment and six or eight multiplex gRNA expression units were developed for CRISPR/Cas9-mediated knock-in to correct a phenylalanine hydroxylase (Pah) gene in a Pah[enu2] phenylketonuria mouse model. These AdVs were co-infected to Hepa1-6 cells or liver cells in vivo together with an AdV expressing either native Cas9 or Cas9 nickase (Cas9n) for double-nicking cleavage.

RESULTS: In vitro knock-in of the AdVs carrying 3.7 kb donor DNA and six gRNA units targeting the cell genome was observed in both cases using Cas9 and Cas9n, though their efficiencies were low. Therefore, we generated AdVs carrying an additional two gRNA units that cleave the donor DNA terminus in the AdV genome via native Cas9 or Cas9 nickase. The knock-in efficiency increased approximately twofold for both vectors and reached a maximum of 8% for native Cas9 without selection. Newborn phenylketonuria model mice were intravenously administered the knock-in AdV together with the native-Cas9 AdV. Although the knock-in efficiency by homologous recombination occurred in only approximately 1% of hepatocytes, blood phenylalanine levels were reduced by up to 30%. Also, unintended fragments produced by nonhomologous end-joining were observed between the cleavage site at the terminus of the donor DNA in the AdV genome and the target site in the cell genome.

CONCLUSIONS: The knock-in efficiency of AdVs can be increased by cleaving the terminus of the donor DNA, although it would be desirable to avoid nonhomologous end-joining between double-strand break termini.},
}

Animals
*Genetic Vectors/genetics/administration & dosage
*Gene Knock-In Techniques/methods
Mice
*Adenoviridae/genetics
*Phenylalanine Hydroxylase/genetics
*Phenylketonurias/therapy/genetics
CRISPR-Cas Systems
Genetic Therapy
Disease Models, Animal
Animals, Newborn
Humans
Gene Editing
RNA, Guide, CRISPR-Cas Systems/genetics
*DNA/genetics

@article {pmid42581041,
year = {2026},
author = {Kinney, KJ and Jia, K and Zhang, H and Schmaljohn, E and Osborne, T and Thommandru, B and Murugan, K and Sánchez-Peña, A and West, S and Chen, S and Codipilly, R and Sturgeon, M and Turk, R and McNeill, MS and Behlke, M and Jacobi, A and Cromer, MK and Rettig, G and Kurgan, GL},
title = {UNCOVERseq enables sensitive and controlled gene editing off-target nomination across CRISPR-Cas modalities and systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42581041},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Hematopoietic Stem Cells/metabolism ; DNA Breaks, Double-Stranded ; },
abstract = {The rapid expansion of CRISPR-Cas gene editing enables new therapeutic strategies but complicates assessment of unintended editing risks due to emerging modalities and unclear analytical standards. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow that sensitively identifies rare off-target events using defined inputs and analytical process controls. Using an inter-method off-target confirmation benchmarking dataset, UNCOVERseq demonstrates high analytical sensitivity (97.6%) and precision (78%), outperforming published nomination methods. We apply UNCOVERseq across 192 guide RNAs and identify six guides spanning a broad specificity range, enabling relative risk assessment across S. pyogenes Cas9, high-fidelity variants, and base editors in hematopoietic stem and progenitor cells. We further show that double-strand break nomination sites retain strong rank-order concordance with single-strand break-mediated base editing. Together, these results establish UNCOVERseq as a robust framework for informed off-target risk assessment in translational gene-editing systems.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Hematopoietic Stem Cells/metabolism
DNA Breaks, Double-Stranded

@article {pmid42086878,
year = {2026},
author = {Wang, MM and Li, Y and Ho, CEH and Yu, WSS and Coupland, SE and Park, G and Chan, ASY and Figueiredo, CR},
title = {Novel CRISPR-Cas9 BAP1 knockout pre-clinical tumor model recapitulates human melanoma tumorigenesis and immune evolution.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42086878},
issn = {2399-3642},
mesh = {Animals ; *Ubiquitin Thiolesterase/genetics ; Humans ; *Tumor Suppressor Proteins/genetics ; *Melanoma/genetics/immunology/pathology/metabolism ; *CRISPR-Cas Systems ; Mice ; *Carcinogenesis/genetics ; Mice, Knockout ; Disease Models, Animal ; Cell Line, Tumor ; Uveal Melanoma ; *Skin Neoplasms/genetics/pathology/immunology ; Uveal Neoplasms/genetics/immunology/pathology ; Female ; },
abstract = {BAP1-deficient melanocytic tumors exhibit strong immunosuppressive features and poor prognosis. Currently, no immune-competent preclinical models exist to study their tumor-immune interactions or test new immunotherapies. This limitation hinders progress in understanding how BAP1 loss drives tumor aggressiveness and immune evasion. To address this, we generate a syngeneic BAP1 knockout melanocyte tumor line using CRISPR-Cas9. We then evaluate its functional and immunological impact in immune-competent mice, including its ability to recapitulate metabolic and immunosuppressive features of human BAP1-deficient melanomas. The selected knockout clone exhibits hallmarks of aggressive skin and intraocular melanomas, including epithelioid morphology, in vivo tumorigenic potential, rapid growth, and key immunosuppressive features, mirroring those observed in human BAP1-deficient melanomas. Cross-species single-cell transcriptome analysis demonstrates strong molecular overlap between BAP1 knockout mouse tumors and high-risk (class 2) human uveal melanomas, highlighting shared pathways in lipid metabolism, transmembrane receptor signaling, and immune modulation. Gene Set Enrichment Analysis confirms that lipid metabolic reprogramming, previously described in human tumors, is also a key feature of our model, validating its ability to recapitulate human disease biology. This study introduces a syngeneic preclinical model that mimics the immunosuppressive landscape of BAP1-deficient melanocytic tumors, enabling the development and optimization of new combination immunotherapies.},
}

Animals
*Ubiquitin Thiolesterase/genetics
Humans
*Tumor Suppressor Proteins/genetics
*Melanoma/genetics/immunology/pathology/metabolism
*CRISPR-Cas Systems
Mice
*Carcinogenesis/genetics
Mice, Knockout
Disease Models, Animal
Cell Line, Tumor
Uveal Melanoma
*Skin Neoplasms/genetics/pathology/immunology
Uveal Neoplasms/genetics/immunology/pathology
Female

@article {pmid42120907,
year = {2026},
author = {Jajarmi, J and Guest, MR and Ma, LJ and Thu, KL and Chari, R and Lockwood, WW},
title = {Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA libraries.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42120907},
issn = {2399-3642},
support = {N/A//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; HHSN261201500003I/CA/NCI NIH HHS/United States ; HHSN261201500003I//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Promoter Regions, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Library ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Mice ; Gene Editing ; Plasmids/genetics ; },
abstract = {CRISPR-Cas9 screening relies on uniform representation of single-guide RNA (sgRNA) libraries to enable accurate gene discovery. However, technical biases during library preparation can compromise screen performance. Here we show that commonly used "all-in-one" CRISPR vectors expressing both Cas9 and sgRNAs drive unintended Cas9 protein expression in Escherichia coli during plasmid amplification. This bacterial Cas9 expression causes guide-specific toxicity, leading to selective loss of sgRNAs and highly skewed library representation. We demonstrate that this effect occurs across multiple bacterial strains and affects both targeted and genome-wide libraries, including widely used human CRISPR libraries. Mechanistically, toxicity is driven by Cas9 expression rather than plasmid size and is only partially alleviated by catalytically inactive Cas9. Importantly, replacing the EF-1α promoter with a mouse phosphoglycerate kinase promoter suppresses Cas9 expression in bacteria while preserving genome editing efficiency in mammalian cells, restoring sgRNA uniformity. These findings identify a previously unrecognized source of bias in CRISPR library preparation and provide a practical solution to improve screening fidelity.},
}

*Escherichia coli/genetics/metabolism
*Promoter Regions, Genetic
*RNA, Guide, CRISPR-Cas Systems/genetics
Animals
*CRISPR-Cas Systems
*Gene Library
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
Mice
Gene Editing
Plasmids/genetics

@article {pmid42213648,
year = {2026},
author = {Kaminski, PJ and Min, K and Traxler, EA and Khandros, E and Abdulmalik, O and Godfrey, B and Keller, CA and Giardine, BM and Hardison, RC and Shi, J and Blobel, GA},
title = {Dissecting polycomb complexes for enhanced fetal hemoglobin production.},
journal = {Blood},
volume = {148},
number = {7},
pages = {882-895},
doi = {10.1182/blood.2026033804},
pmid = {42213648},
issn = {1528-0020},
mesh = {*Fetal Hemoglobin/genetics/biosynthesis/metabolism ; Animals ; Humans ; Mice ; *Enhancer of Zeste Homolog 2 Protein/genetics/metabolism ; Erythroid Cells/metabolism ; CRISPR-Cas Systems ; Polycomb Repressive Complex 2/metabolism/genetics ; Histones/metabolism ; *Polycomb-Group Proteins/metabolism/genetics ; },
abstract = {Polycomb repressive complex 1 (PRC1) and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin (Hb) production, a process whose reversal is a goal for the treatment of sickle cell disease and β-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal Hb (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-CRISPR-associated protein 9 (Cas9) mutagenesis screen identified a region in EZH2 in which Cas9 induced exon 14 skipping (EZH2Δ14). EZH2Δ14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2Δ14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human β-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting nonenzymatic domains within chromatin-modifying complexes.},
}

*Fetal Hemoglobin/genetics/biosynthesis/metabolism
Animals
Humans
Mice
*Enhancer of Zeste Homolog 2 Protein/genetics/metabolism
Erythroid Cells/metabolism
CRISPR-Cas Systems
Polycomb Repressive Complex 2/metabolism/genetics
Histones/metabolism
*Polycomb-Group Proteins/metabolism/genetics

@article {pmid42486984,
year = {2026},
author = {Meng, H and Lei, Z and Yan, Y and Wang, L and Zhang, S and Rao, X and Shao, C and Zhang, X and Chen, K and Yang, L and Liu, R and Yang, G and Shen, R and Gu, R and Wang, X and Wang, Y and Lu, S and Lv, Z and He, B and Wen, H and Li, D and Yi, C},
title = {Precise DNA base editing using AlphaFold3-based contact modelling.},
journal = {Nature},
volume = {656},
number = {8127},
pages = {463-473},
pmid = {42486984},
issn = {1476-4687},
mesh = {*DNA/genetics/chemistry/metabolism ; *Gene Editing/methods ; *Models, Molecular ; CRISPR-Cas Systems/genetics ; Adenine/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Substrate Specificity ; CRISPR-Associated Proteins/metabolism/chemistry ; Base Sequence ; Adenosine Deaminase ; Escherichia coli Proteins ; },
abstract = {Achieving high specificity in biochemical transformations is crucial for research and therapeutics. This is particularly important for genome editing, where enhancing tool specificity ensures effective and precise editing outcomes[1,2]. Current strategies are constrained by activity-specificity trade-offs, high labour intensity and low success rates[3,4]. Here we present ContactSeek, an artificial-intelligence-driven framework that uses AlphaFold3 (AF3)-predicted contact probability[5] to improve the specificity of genome editors. Using Cas9-TadA adenine base editors[6-8] as a demonstration, we mapped their genome-wide off-targets and fed the off-target DNA sequences to AF3. Among AF3 outputs, we found that contact probability was more sensitive than predicted three-dimensional structures for detecting differential interactions between on- and off-target complexes. Correlating contact probability with sequencing-based off-target signals, ContactSeek identified and ranked consensus contact regions, which are neighbouring Cas residues with consistent contact changes to DNA/guide RNA, and pinpointed specificity-determining residues within them. ContactSeek can also be applied modularly and identified key residues in the TadA8e deaminase. Targeted amplicon sequencing, genome-wide profiling, R-loop assay and RNA-sequencing together confirmed the greatly enhanced specificity; our best variant, combining two mutations of Cas9 and TadA8e, outperformed several known high-fidelity adenine base editors. ContactSeek is also generalized to Cas12a-based cytosine base editors. Collectively, our framework represents an AF3-driven model tailored for specificity improvement, establishing a paradigm for improving the precision of genome editing tools through the integration of structural and functional dimensions.},
}

*DNA/genetics/chemistry/metabolism
*Gene Editing/methods
*Models, Molecular
CRISPR-Cas Systems/genetics
Adenine/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
Substrate Specificity
CRISPR-Associated Proteins/metabolism/chemistry
Base Sequence
Adenosine Deaminase
Escherichia coli Proteins

@article {pmid42570142,
year = {2026},
author = {Ahad, A and Hullon, D and Singh, T and Dabiry, SM and Sakthivel, L and Padaria, J and Rekhraj, AS and Bhattacharjee, A},
title = {The Vascular Genome as a Therapeutic Target: A Systematic Review of CRISPR-based Gene Editing In Vascular Disease.},
journal = {Cardiovascular toxicology},
volume = {26},
number = {8},
pages = {},
pmid = {42570142},
issn = {1559-0259},
mesh = {Humans ; Animals ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; *Vascular Diseases/genetics/therapy/metabolism/physiopathology ; *CRISPR-Cas Systems ; Vascular Remodeling/genetics ; Genetic Predisposition to Disease ; Phenotype ; },
abstract = {Despite advances in therapy, arterial, venous, and pulmonary vascular diseases remain leading causes of morbidity and mortality. Persistent endothelial dysfunction, inflammation, oxidative stress, and maladaptive vascular remodeling continue to drive disease progression and residual risk. CRISPR/Cas9 technology offers a unique opportunity to modify the molecular pathways underlying vascular pathophysiology directly. The PRISMA 2020 guidelines guided the systematic review. The databases PubMed/MEDLINE, Embase, Web of Science, Cochrane Library, ClinicalTrials.gov, and Google Scholar were searched from their inception until September 2025 for experimental and/or clinical studies evaluating the application of CRISPR/Cas9 on vascular disease. Included were in vitro studies, animal model studies, and early-phase human studies aimed at targeting the endothelial cell regulatory pathways, inflammatory pathways, metabolic remodeling processes, and hereditary causes of vasculopathy. Seventeen studies met the inclusion criteria. CRISPR technologies targeting PCSK9, NOS3, HIF1A, NLRP3, METTL4, BMPR2, and ACTA2 were identified to enhance repair mechanisms in endothelial cells, regulate inflammation, modulate lipid metabolism, and remodel the vascular system. The human studies demonstrated sustained gene silencing effects following a single dose of CRISPR-induced in vivo editing. The use of CRISPR technology to edit cell genomes offers potential to alter disease progression in vascular medicine, with a growing body of translational evidence supporting the feasibility and durability of the approach.},
}

Humans
Animals
*Gene Editing
*Genetic Therapy/adverse effects/methods
*Vascular Diseases/genetics/therapy/metabolism/physiopathology
*CRISPR-Cas Systems
Vascular Remodeling/genetics
Genetic Predisposition to Disease
Phenotype

@article {pmid42570236,
year = {2026},
author = {Jiang, S and Tian, X and Wang, F and Han, W and She, Q and Feng, X},
title = {Recruitment of Cas3 enables DNA cleavage by the type I-A CRISPR-Cas system of Saccharolobus islandicus.},
journal = {Cell reports},
volume = {45},
number = {8},
pages = {117820},
doi = {10.1016/j.celrep.2026.117820},
pmid = {42570236},
issn = {2211-1247},
abstract = {Type I CRISPR-Cas systems constitute the most prevalent prokaryotic adaptive immune pathways and are classified into seven subtypes (I-A to I-G). These antiviral systems typically exploit a Cascade complex for RNA-guided DNA recognition and a Cas3 helicase-nuclease effector for DNA degradation, yet their diverse activation mechanisms remain not fully understood. In this study, we isolate the I-A Cascade from Saccharolobus islandicus, revealing a minimal form of Cascade lacking both Cas3 and the CRISPR-RNA maturase Cas6. Cas3 is recruited to the R-loop structure formed after Cascade binding to target DNA, which activates the effector for both cis- and trans-DNA cleavage. Strikingly, ATP not only enables the processive target degradation by Cas3 but also suppresses the trans-cleavage of the same enzyme. Together, the Sa. islandicus I-A system operates via target-dependent Cas3 recruitment-a mechanism distinct from other characterized I-A systems, thus underscoring the mechanistic diversity within type I CRISPR-Cas immunity.},
}

@article {pmid42571080,
year = {2026},
author = {Ullah, O},
title = {Breaking the growth-defense trade-off in cereal crops: CRISPR/Cas and moonlighting proteins in biotic stress resistance.},
journal = {Molecular breeding : new strategies in plant improvement},
volume = {46},
number = {8},
pages = {80},
pmid = {42571080},
issn = {1572-9788},
abstract = {Recurrent crop disease outbreaks linked to global warming pose challenges to sustainable food production. Conventional plant breeding techniques may become less effective at addressing these threats, as improving disease resistance often causes yield reduction. Amid these challenges, CRISPR/Cas-based gene editing offers targeted and tractable solutions. This review synthesizes recent approaches to uncoupling immunity from productivity in cereals. We show that susceptibility (S) gene disruption can provide resistance without activating costly defense mechanisms. We also discuss the generation of new alleles through targeted modifications that mitigate autoimmunity-associated fitness costs. Here, we propose CRISPR-mediated de-moonlighting, an approach for decoupling multifunctional protein activities. Multiplex editing of minor resistance loci, especially in polyploids such as wheat, offers long-lasting, broad-spectrum protection. These strategies converge on the manipulation of canonical moonlighting proteins, multifunctional signaling hubs, and pleiotropic regulators, which serve as regulatory nodes that link development and immunity. CRISPR-mediated precision modification of these regulators can fine-tune the growth-defense balance. Combining these approaches with systems biology, AI-driven design and advanced breeding pipelines can help develop high-yielding, disease-resistant cereals for sustainable agriculture.},
}

@article {pmid42571242,
year = {2026},
author = {Eken, JA and Havenaar, FRM and de Groen, RAL and Quinten, E and Mei, H and Sepúlveda-Yáñez, JH and Navarrete, MA and Drexler, HG and Vermaat, JSP and van Bergen, CAM and Veelken, H},
title = {Alternative oncogenic drivers and sensitivity to BTK inhibition in CRISPR/Cas gene-edited human DLBCL cell models.},
journal = {Blood neoplasia},
volume = {3},
number = {3},
pages = {100261},
pmid = {42571242},
issn = {2950-3280},
abstract = {Diffuse large B-cell lymphoma (DLBCL) can be subclassified by phenotype into germinal center B-cell-like and activated B-cell-like (ABC) subtypes and by recurrent potentially oncogenic mutations into 5 to 7 genetic clusters. In ABC-DLBCL, potentially oncogenic mutations frequently occur in genes involved in B-cell receptor (BCR) signaling and NF-κB activation. Autonomous BCR signaling acts as an alternative immunologic driver predominantly in ABC-type DLBCL that cannot be captured by either subclassification system. The relative functional contribution and interdependence of these mechanistically diverse oncogenic drivers have not been completely defined. To directly compare the effects of autonomously signaling BCR and signalosome-activating CARD11 mutations on NF-κB activation and survival of ABC-DLBCL, we reciprocally exchanged these driver mechanisms in the MYD88[L265P]-mutated ABC-DLBCL cell lines TMD8 and OCI-Ly3. Only CARD11[L251P] (not CARD11[K215N], CARD11[D230N], and CARD11[R337Q]) compensated TMD8 cells for the loss of autonomous BCR signaling, as indicated by survival of BCR knockout and conversion to complete resistance to acalabrutinib. Transduction of the TMD8 BCR rescued OCI-Ly3 cells from replacing the CARD11[L215P] variant with CARD11[WT]. The autonomous TMD8 BCR signal provided a slight growth advantage over CARD11[L251P]-driven cells in both reciprocal systems. Unsupervised clustering of genetically engineered TMD8 and OCI-Ly3 clones demonstrated tight clustering with their parental cells and only minor alterations of cellular pathways. Only the strongest signalosome-activating mutation has functional near-equivalency to an autonomously signaling BCR for NF-κB activation and growth and survival in ABC-DLBCL. Quantifying the effects of co-occurring potential NF-κB-activating mechanisms is essential to predict Bruton tyrosine kinase (BTK) inhibition sensitivity in individual ABC-DLBCL cases.},
}

@article {pmid42572397,
year = {2026},
author = {Baoling, L and Lina, S and Tong, X and Qinrui, J and Ling, Z and Zhiwen, X},
title = {A Rapid, Field-Deployable Diagnostic Platform for Getah Virus Based on RT-RAA and CRISPR EsCas13d.},
journal = {Microbial biotechnology},
volume = {19},
number = {8},
pages = {e70429},
pmid = {42572397},
issn = {1751-7915},
support = {2024YFD1800102//National Key Research and Development Program of China during the 14th Five-Year Plan: Research and Development of Key Technologies for the Prevention and Control of Important Diseases in Wild Animals/ ; 2024YFD1800500//Research and Application of Integrated Prevention, Control and Purification Technologies for Major Swine Epidemic Diseases/ ; 2021ZDZX0010-3//Sichuan Province Major Science and Technology Project for Sichuan Pigs during the 14th Five-Year Plan Period/ ; sccxtd-2024-08//Sichuan Pig Innovation Team of the National Modern Agricultural Industry Technology System/ ; },
mesh = {Animals ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; RNA, Viral/genetics ; Reverse Transcription ; *CRISPR-Cas Systems ; Temperature ; },
abstract = {The emerging zoonotic Getah virus (GETV) poses an increasing threat to both animal and human health, underscoring the need for rapid, sensitive and field-deployable diagnostic tools. In this study, we developed and optimized a rapid, one-step, visual detection (ROSVD) platform for GETV by integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-EsCas13d-mediated collateral RNA cleavage. Notably, the ROSVD assay uses a simplified sample-preparation strategy based on rapid nucleic acid release, eliminating the need for conventional nucleic acid extraction and purification. The entire workflow, including amplification and detection, is completed within 30 min at 37°C or ambient temperature (25°C) without specialized instrumentation. Detection results can be visualized directly under ultraviolet light or with a lateral flow assay. At 37°C, the ROSVD assay achieved sensitivity comparable to RT-qPCR, and evaluation of clinical specimens showed 100% concordance with RT-qPCR results. Collectively, these findings demonstrate that ROSVD is a rapid, sensitive, cost-effective and instrument-independent diagnostic platform, providing a practical solution for on-site surveillance of GETV and a versatile framework for the detection of other emerging RNA pathogens.},
}

Animals
Rapid Diagnostic Tests
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Humans
RNA, Viral/genetics
Reverse Transcription
*CRISPR-Cas Systems
Temperature

@article {pmid42573069,
year = {2026},
author = {Fan, X and Liang, L and Wang, H and Liu, G and Tan, H and Zhang, F and Liu, L and Liu, R},
title = {A self-iterative orthogonal base-editing platform enables multiplex N-to-N diversification and genome-scale functional screening in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {15},
pages = {},
pmid = {42573069},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 22278058//National Natural Science Foundation of China/ ; 22578048//National Natural Science Foundation of China/ ; 22208044//National Natural Science Foundation of China/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project for Young Faculty/ ; XLYC2203075//Xingliao Talent Plan/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Gene Editing/methods ; *Genome, Bacterial ; Butanols/metabolism ; Escherichia coli Proteins/genetics/metabolism ; DNA Repair/genetics ; Codon/genetics ; Gene Library ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Base editing enables precise genome modification without double-strand breaks but remains limited by narrow editing windows, DNA repair pathway biases, and restricted nucleotide diversity. Here, we report MUTATOR, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli. MUTATOR combines CWBE and ABE with iterative editing on two complementary DNA strands, thereby overcoming endogenous DNA repair constraints and expanding A-to-N and C-to-N editing outcomes across both strands. This strategy substantially expands accessible nucleotide outcomes, codon variants, and amino-acid diversity within existing editing windows relative to conventional editors. Using four gRNAs, MUTATOR facilitated four-site editing of ompR, generating 84 distinct amino-acid combinations and 252 codon combinations, with the synonymous OmpR_P160P variant increasing isobutanol production by up to 56.2%. We further applied MUTATOR to a 151-gene library encompassing transcriptional regulators, translation factors, DNA repair proteins, ribosomal components, and NAD(P)H-associated metabolic genes, identifying single and combinatorial mutations that markedly enhanced cell growth and ethanol utilization when ethanol was used as the sole carbon source. Together, these results establish MUTATOR as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.},
}

*Escherichia coli/genetics/metabolism
*Gene Editing/methods
*Genome, Bacterial
Butanols/metabolism
Escherichia coli Proteins/genetics/metabolism
DNA Repair/genetics
Codon/genetics
Gene Library
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42573087,
year = {2026},
author = {Guy, J and Hein, E and Alexander-Howden, B and von Bock Und Polach, T and Mathieson, T and Kleinstiver, BP and Zoghbi, HY and Bird, A},
title = {Translational reading frame predicts the pathogenicity of C-terminal frameshift deletions in MeCP2.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42573087},
issn = {2050-084X},
support = {10.35802/222507//Wellcome/ ; 13669477_13669480//Rett Syndrome Research Trust/ ; DP2CA281401/NH/NIH HHS/United States ; 13800235_1380239//Simons Initiative for the Developing Brain/ ; },
mesh = {*Methyl-CpG-Binding Protein 2/genetics/metabolism ; Animals ; Humans ; *Frameshift Mutation ; Mice ; *Rett Syndrome/genetics/pathology ; Disease Models, Animal ; *Sequence Deletion ; },
abstract = {Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) removes ~100 amino acids and accounts for approximately 10% of RTT-causing mutations. Their pathogenicity is unexpected because this C-terminal domain is dispensable in mice. Analysis of pathogenic and benign human MECP2 variants reveals that some individuals with apparently typical CTDs do not develop Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human sequence data and mouse models we show that pathogenicity results from a marked reduction in MeCP2 levels and depends on the presence of a proline proline stop motif (-PPX) generated by a shift to the +2 reading frame. CTDs that shift to the +1 frame avoid this motif and are benign. Replacing the stop codon of the PPX motif with tryptophan restores MeCP2 expression and rescues RTT-like phenotypes in a CTD mouse model. An adenine base editor efficiently introduces this substitution in cultured cells. These findings define a reliable prognostic distinction between benign and pathogenic CTDs and establish a potential editing strategy for correcting disease-causing CTD mutations.},
}

*Methyl-CpG-Binding Protein 2/genetics/metabolism
Animals
Humans
*Frameshift Mutation
Mice
*Rett Syndrome/genetics/pathology
Disease Models, Animal
*Sequence Deletion

@article {pmid42573653,
year = {2026},
author = {Nguyen, BD and Kolluri, SK},
title = {Genome-wide CRISPR screen reveals CGS-15943 induced heme-dependent cell death mediated by aryl hydrocarbon receptor in lung cancer cells.},
journal = {Apoptosis : an international journal on programmed cell death},
volume = {31},
number = {8},
pages = {},
pmid = {42573653},
issn = {1573-675X},
mesh = {Humans ; *Receptors, Aryl Hydrocarbon/genetics/metabolism ; *Heme/metabolism ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; Cell Line, Tumor ; *Apoptosis/drug effects ; Cell Death/drug effects ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic/drug effects ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Oxidative Stress/drug effects ; },
abstract = {Induction of programmed cancer cell death by selective aryl hydrocarbon receptor (AHR) ligands represents a promising strategy for developing novel anticancer therapeutics. In this study, we characterized the anticancer activity and underlying mechanism of the selective AHR ligand CGS-15943 in lung cancer cells. CGS-15943 potently inhibited the growth of lung cancer cell lines expressing high levels of AHR, whereas CRISPR-mediated knockout of AHR in H460 and H69AR cells markedly rescued cells from CGS-15943-induced cell death, demonstrating an essential role for AHR. To identify additional mediators of this response, we performed a genome-wide CRISPR knockout screen, which revealed eight enzymes involved in the heme biosynthesis pathway, three heme-containing enzymes, as well as AHR and its transcriptional partner ARNT, as critical determinants of CGS-15943-induced cell death. Transcriptomic analyses further showed that CGS-15943 induced AHR-dependent transcriptional programs enriched for oxidative stress and oxidized phospholipid response pathways. Together, these findings identify key components of the AHR signaling network that regulate a programmed heme-dependent cell death pathway and establish CGS-15943 as a promising lead compound for targeting AHR-positive lung cancers.},
}

Humans
*Receptors, Aryl Hydrocarbon/genetics/metabolism
*Heme/metabolism
*Lung Neoplasms/genetics/drug therapy/metabolism/pathology
Cell Line, Tumor
*Apoptosis/drug effects
Cell Death/drug effects
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic/drug effects
*Basic Helix-Loop-Helix Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Oxidative Stress/drug effects

@article {pmid42573697,
year = {2026},
author = {Ward, JD},
title = {Protein Depletion in Caenorhabditis elegans Using the Auxin-Inducible Degradation System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3069},
number = {},
pages = {169-185},
pmid = {42573697},
issn = {1940-6029},
mesh = {Animals ; *Indoleacetic Acids/pharmacology/metabolism ; *Caenorhabditis elegans/genetics/metabolism ; F-Box Proteins/genetics/metabolism ; Degrons ; *Proteolysis/drug effects ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; Receptors, Cell Surface/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knock-In Techniques/methods ; Arabidopsis/genetics ; },
abstract = {The auxin-inducible degradation (AID) system is a powerful tool in modern molecular genetics that allows for conditional protein depletion. In Caenorhabditis elegans, tools exist to allow for rapid, tissue-specific depletion of target proteins. The system requires tagging a gene of interest with an AID degron and a transgene expressing the Arabidopsis thaliana TIR1 F-box protein, which can form a functional SCF ubiquitin ligase with endogenous Skp and Cullin proteins. Here, I describe how to generate degron knock-ins by CRISPR, cross them to TIR1-expressing strains, and perform depletion experiments. I provide a description of our current methods and highlight alternative approaches.},
}

Animals
*Indoleacetic Acids/pharmacology/metabolism
*Caenorhabditis elegans/genetics/metabolism
F-Box Proteins/genetics/metabolism
Degrons
*Proteolysis/drug effects
*Caenorhabditis elegans Proteins/genetics/metabolism
Arabidopsis Proteins/genetics/metabolism
Receptors, Cell Surface/genetics/metabolism
CRISPR-Cas Systems
Gene Knock-In Techniques/methods
Arabidopsis/genetics

@article {pmid42323652,
year = {2026},
author = {Liu, X and Wang, Y and Wong, JCY and Zhang, X and Wu, C and Chen, P and Yang, Y and Liu, P and Lau, CS and Cook, M and Li, PH},
title = {Modeling Hereditary Angioedema With Personalized Expanded Potential Stem Cell-Derived Hepatocytes: A CRISPR-Validated Platform for Mutation-Specific Mechanisms and Therapeutic Innovation.},
journal = {Allergy},
volume = {81},
number = {8},
pages = {2858-2873},
pmid = {42323652},
issn = {1398-9995},
support = {//the InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government/ ; },
mesh = {Humans ; *Hepatocytes/metabolism/cytology ; *Mutation ; *Angioedemas, Hereditary/genetics/therapy/metabolism ; Cell Differentiation ; *Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; Complement C1 Inhibitor Protein/genetics/metabolism ; Models, Biological ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Precision Medicine ; },
abstract = {Hereditary angioedema (HAE) with C1 esterase inhibitor (C1INH) deficiency is caused by pathogenic SERPING1 mutations that disrupt production of the plasma protease inhibitor C1INH. However, the molecular mechanisms and consequences of patient-specific mutations remain poorly understood due to the lack of physiologically relevant human models. Here, we established a personalized, isogenic, stem-cell-derived hepatocyte platform to investigate the underlying mutation-specific mechanisms of HAE. Specifically, peripheral blood mononuclear cell (PBMC)-expanded erythroblasts from four representative HAE-C1INH-Type1 patients containing distinct point, insertion, deletion, or large fragment SERPING1 mutations were reprogrammed into expanded potential stem cells (EPSCs) and further differentiated into hepatocyte-like cells (HLCs). These HLCs exhibited appropriate transcriptional transitions, mature hepatic features, and C1INH secretion comparable to that observed in human plasma. All patient-derived HLCs demonstrated impaired C1INH secretion with mutation-specific differences in both SERPING1 transcription and intracellular accumulation. Moreover, to verify that the mutations directly drive the phenotype, we performed CRISPR/Cas9-mediated genome repair, which restored SERPING1 mRNA expression and C1INH secretion. Conversely, identical patient mutations installed into healthy EPSCs showed the same transcriptional and secretory defects, confirming sufficiency. Collectively, we have established a robust human hepatocyte model that accurately recapitulates key hepatocyte-specific aspects of HAE pathophysiology and provides a scalable foundation for investigation of future precision therapies.},
}

Humans
*Hepatocytes/metabolism/cytology
*Mutation
*Angioedemas, Hereditary/genetics/therapy/metabolism
Cell Differentiation
*Stem Cells/cytology/metabolism
*CRISPR-Cas Systems
Complement C1 Inhibitor Protein/genetics/metabolism
Models, Biological
*Clustered Regularly Interspaced Short Palindromic Repeats
Precision Medicine

@article {pmid42389905,
year = {2026},
author = {Minaiyan, G and Aussel, C and Ammann, S and Cathomen, T},
title = {Genome Editing for Familial Hemophagocytic Lymphohistiocytosis: Design Principles, Challenges, and Translational Perspectives.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {702-712},
doi = {10.1177/10430342261465555},
pmid = {42389905},
issn = {1557-7422},
mesh = {Humans ; *Lymphohistiocytosis, Hemophagocytic/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; Translational Research, Biomedical ; Perforin/genetics ; Membrane Proteins/genetics ; Mutation ; CRISPR-Cas Systems ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by genetic defects in cytotoxic lymphocyte function. Current therapies can control disease activity, but transplantation of allogeneic hematopoietic stem and progenitor cells (HSPCs) remains the only curative option and is associated with substantial risks. These limitations have accelerated development of genome editing approaches enabling precise correction of disease-causing mutations in autologous cells. Familial HLH (FHL) represents a compelling target for genome editing, but successful and safe clinical translation has remained challenging. Preclinical studies demonstrate that targeted editing of key genes, such as PRF1 and UNC13D, can restore cytotoxic function in HSPCs and T cells. Translation to the clinic, however, depends on multiple factors, including the choice of target cell population, the level of functional correction required, and gene-specific constraints such as locus complexity and regulation of gene expression. In this review, we synthesize current progress in genome editing for FHL and highlight critical biological and technical barriers to clinical implementation. We propose a conceptual framework for designing genome editing strategies tailored to FHL, emphasizing the alignment of editing platform, gene architecture, and cellular context to enable effective and clinically translatable therapies.},
}

Humans
*Lymphohistiocytosis, Hemophagocytic/genetics/therapy
*Gene Editing/methods
*Genetic Therapy/methods
Animals
Translational Research, Biomedical
Perforin/genetics
Membrane Proteins/genetics
Mutation
CRISPR-Cas Systems
Hematopoietic Stem Cell Transplantation

@article {pmid42393087,
year = {2026},
author = {Weickert, P and Liu, Y and Strecker, J},
title = {Prokaryotic Schlafen proteins cleave tRNAs during type III CRISPR immunity.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42393087},
issn = {2041-1723},
support = {DP2HL185100//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {*RNA, Transfer/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Schlafen nucleases restrict viral infection in mammals by cleaving self RNAs, however, their function and mechanism in prokaryotic immunity is unknown. Here, we uncover CRISPR-associated Schlafen (Cash) proteins containing a Schlafen domain fused to Csx15, an uncharacterized member of Rossmann-like nucleotide-binding sensors. Cash is activated by cyclic tetra-adenylate (cA4) produced during type III CRISPR interference and induces cell toxicity by cleaving tRNAs, primarily in the T-loop. Cryo-electron microscopy structures of Chloroflexi bacterium Cash reveal an inactive dodecamer, the formation of a filament upon cA4 binding to align catalytic interfaces, and the molecular basis of substrate recognition and cleavage in a tRNA-bound complex. We identify numerous families of prokaryotic Schlafen proteins associated with diverse antiviral defense systems and characterized by unique sensor domains. This work highlights tRNA depletion by Schlafen nucleases as an evolutionary recurring antiviral strategy and reveals mechanistic differences between Cash and human Schlafen members.},
}

*RNA, Transfer/metabolism/genetics
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42424999,
year = {2026},
author = {Dong, J and Li, X and Gu, T and Zhang, Y and Deng, L and Luo, X and Hou, C and Huo, D},
title = {Compartmentalization-inspired dual-chamber CRISPR sensing coupled with single-atom electrocatalysis for crosstalk-free multiplex microRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119016},
doi = {10.1016/j.bios.2026.119016},
pmid = {42424999},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/analysis/blood ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Electrochemical Techniques ; Nucleic Acid Amplification Techniques ; Catalysis ; Equipment Design ; },
abstract = {Compartmentalization is a hallmark of cells, enabling parallel biochemical processes to proceed with high fidelity and minimal interference. Drawing inspiration from this spatial isolation principle, we developed a compartmentalization-inspired dual-chamber sensing platform for crosstalk-minimized multiplex miRNA analysis. In each physically isolated compartment, the target miRNA directs padlock-probe ligation to form a circular template, followed by rolling-circle-extension-driven loop-mediated isothermal amplification (R-LAMP). The resulting amplicons specifically activate the corresponding CRISPR/Cas12a-crRNA complex, triggering trans-cleavage of a hairpin-DNA biogate that seals Fe-MOF nanocontainers. Gate opening releases distinct electroactive reporters (3,3',5,5'-tetramethylbenzidine, TMB; or methylene blue, MB) from their respective chambers. After the compartmentalized reactions finish, the two supernatants are combined and read out on a screen-printed electrode modified with a Co-N-C single-atom catalyst, producing two well-resolved DPV peaks for simultaneous quantification. The platform achieves femtomolar detection limits in simultaneous assays (0.87 fM for miRNA-21 and 0.72 fM for miRNA-155), a broad linear range (1 fM-100 pM), and high discrimination against non-cognate or mismatched sequences. Accurate recoveries in diluted human serum and consistent trends in cell lysates (validated by RT-qPCR) confirm practical applicability. By integrating bioinspired compartmentalization with CRISPR precision and single-atom electrocatalysis, this platform provides a generalizable route to multiplex nucleic acid diagnostics with enhanced fidelity and sensitivity.},
}

*MicroRNAs/genetics/isolation & purification/analysis/blood
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
Limit of Detection
Electrochemical Techniques
Nucleic Acid Amplification Techniques
Catalysis
Equipment Design

@article {pmid42431049,
year = {2026},
author = {Ji, S and Wang, B and Yan, Y and Zhang, C and Ren, H and Yang, J and Wei, X and Hou, W and Huang, M and Song, J and Wang, S and Qiu, L and Wang, H},
title = {TOPS-CRISPR: Thermally-regulated and oligonucleotide-mediated one-pot CRISPR-Cas12a assay for ultra-sensitive and rapid on-site diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {118998},
doi = {10.1016/j.bios.2026.118998},
pmid = {42431049},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; Oligonucleotides/chemistry/genetics ; *Brucella/isolation & purification/genetics/pathogenicity ; *Brucellosis/diagnosis/microbiology ; Rapid Diagnostic Tests ; Temperature ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; },
abstract = {CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.},
}

*CRISPR-Cas Systems/genetics
Humans
*Biosensing Techniques/methods
Oligonucleotides/chemistry/genetics
*Brucella/isolation & purification/genetics/pathogenicity
*Brucellosis/diagnosis/microbiology
Rapid Diagnostic Tests
Temperature
CRISPR-Associated Proteins/genetics
Limit of Detection

@article {pmid42447864,
year = {2026},
author = {Langley, J and Baudrier, L and Curry, J and Narta, K and Todesco, HM and Potts, K and Morrissy, S and Mahoney, DJ and Billon, P},
title = {Compound delivery of eVLPs enhances prime editing for targeted genome engineering and high-throughput screening.},
journal = {Cell genomics},
volume = {6},
number = {8},
pages = {101302},
doi = {10.1016/j.xgen.2026.101302},
pmid = {42447864},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; *Virion/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Engineered virus-like particles (eVLPs) enable transgene-free ribonucleoprotein delivery for genome editing, yet optimized strategies for high-throughput applications remain unexplored. Prime editing enables precise genomic modifications but suffers from limited efficiency. Here, we present PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), which exploits eVLP-mediated editing kinetics through multiple sequential sub-saturating transductions at optimal intervals. PRIME-VLP achieves 1.5- to 2.9-fold improvements in prime editing efficiency across diverse genomic targets and cell types without increasing off-target editing, compromising cellular viability or causing transcriptional perturbations. By decoupling pegRNA and editor delivery through pegRNA-free eVLPs, PRIME-VLP enables pooled prime editing screens and circumvents transgene silencing limitations. Using a 6,000-pegRNA library targeting TP53, PRIME-VLP achieved 2.8-fold higher editing and improved reproducibility compared to conventional lentiviral delivery, identifying TP53 loss-of-function variants conferring Nutlin-3 resistance. This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics.},
}

Humans
*Gene Editing/methods
*High-Throughput Screening Assays/methods
HEK293 Cells
CRISPR-Cas Systems/genetics
Lentivirus/genetics
*Virion/genetics
Tumor Suppressor Protein p53/genetics

@article {pmid42470832,
year = {2026},
author = {Azhar, M and Malviya, R and Chandra, P and Sridhar, SB and Shareef, J and Wadhwa, T},
title = {Precision prime editing of TP53 mutations for functional tumor suppression in colorectal cancer.},
journal = {Biochemical and biophysical research communications},
volume = {831},
number = {},
pages = {154311},
doi = {10.1016/j.bbrc.2026.154311},
pmid = {42470832},
issn = {1090-2104},
mesh = {*Colorectal Neoplasms/genetics/therapy ; Humans ; *Mutation ; *Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; Precision Medicine/methods ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is a major global health concern, with high mortality due to genetic heterogeneity and resistance to treatment. Tumor Protein p53 (TP53) mutations are also among the most important molecular changes that can disrupt genomic stability and facilitate tumor progression, so it is a critical target for precision-based interventions.

AIM: This review aims to discuss the future potential of prime editing as a new generation of genome engineering to identify precise approaches to correct TP53 mutations in colorectal cancer.

METHOD: A focused literature review was conducted on PubMed, Scopus, Web of Science, and Google Scholar for articles published between the years of 2010 and 2026. The keywords used in the search were CRC, TP53 mutation, prime editing, Prime Editing Guide RNA (pegRNA), CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9), and precision oncology. Studies were screened for experimental, mechanistic, and translational, and the focus was on mutation-specific editing, delivery platforms, organoid validation, clinically relevant barriers, etc. RESULTS/DISCUSSION: Prime editing is a programmable search-and-replace method that does not involve two single-stranded Deoxyribonucleic Acid (DNA) breaks, resulting in fewer Insertions/deletions (indels) and greater precision compared with traditional CRISPR-Cas9 approaches. Recent systems like Prime Editor Max (PEmax), PE5/PE5max, engineered pegRNAs, twin prime editors, PrimeDel, and PASTE have enhanced the efficiency, range, and flexibility. Hotspot and organoid studies suggest that variants of TP53, particularly R175H, R248Q/W, R273 H/C, and R282W, can be repaired. But cargo size, delivery specificity, tumor heterogeneity, varying cargo editing efficiency, cargo recognition by the immune system, and off-target risk are all barriers to clinical translation.

CONCLUSION: Precision oncology with prime editing has the potential to be a useful tool for CRC, though optimized delivery, thorough preclinical testing, and safety monitoring will be required for therapeutic adoption.

ORIGINALITY: This review combines TP53 hotspot biology, recent breakthroughs in prime editing technology, and CRC-specific translational challenges, and provides a step-by-step approach to its clinical application in a unique way.},
}

*Colorectal Neoplasms/genetics/therapy
Humans
*Mutation
*Tumor Suppressor Protein p53/genetics
CRISPR-Cas Systems
Animals
Genetic Therapy/methods
Precision Medicine/methods
*Gene Editing/methods

@article {pmid42472526,
year = {2026},
author = {Liu, Z and Wang, J and Yang, Z and Huang, H and Zhang, Z and Wu, T},
title = {High-specificity gene point mutation detection by PAM-free Cas12a system with double-stranded substrate positioning-unwinding.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119043},
doi = {10.1016/j.bios.2026.119043},
pmid = {42472526},
issn = {1873-4235},
mesh = {*Point Mutation/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/genetics/chemistry ; *CRISPR-Associated Proteins/genetics ; DNA Mutational Analysis/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR/Cas12a system holds great promise for nucleic acid detection, but its strict dependence on the protospacer adjacent motif (PAM) severely limits its application in gene point mutation analysis, with fewer than 2% of known mutation sites naturally harboring adjacent PAM sequences. Herein, we developed a PAM-free Cas12a system with double-stranded substrate positioning-unwinding (dsPU-Cas12a), wherein "bubble" structures formed by unpaired base pairs release partial single-stranded target strand as a toehold, and excess auxiliary strands induce local unwinding of double-stranded DNA to facilitate R-loop formation. After optimization, the dsPU-Cas12a system achieved an ultra-low limit of detection of 0.013% for gene point mutations, with excellent linearity over the mutation abundance range of 0-10%. Furthermore, it exhibited robust feasibility and accuracy in detecting the JAK2 V617F mutation in blood samples from patients with myeloproliferative neoplasms. This simple and universal strategy overcomes the sequence limitation of Cas12a, providing a high-performance tool for clinical gene point mutation detection.},
}

*Point Mutation/genetics
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*DNA/genetics/chemistry
*CRISPR-Associated Proteins/genetics
DNA Mutational Analysis/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases