@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation & purification/genetics ; *Bacillus/isolation & purification/genetics ; *Food Microbiology/methods ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; Humans ; Food Contamination/analysis ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}

*Clostridium/isolation & purification/genetics
*Bacillus/isolation & purification/genetics
*Food Microbiology/methods
Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence
Humans
Food Contamination/analysis

@article {pmid41762821,
year = {2026},
author = {Golla, DA and Sun, C and Haugh, L and Straub, N and Gao, X},
title = {Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.},
journal = {Current opinion in biotechnology},
volume = {99},
number = {},
pages = {103470},
doi = {10.1016/j.copbio.2026.103470},
pmid = {41762821},
issn = {1879-0429},
abstract = {Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.},
}

@article {pmid41761908,
year = {2026},
author = {Perry, TN and Mais, CN and Sanchez-Londono, M and Steinchen, W and Plitzko, PA and Randau, L and Pausch, P and Innis, CA and Bange, G},
title = {Structural basis of Cas8-independent Cas3 recruitment in Type I-F2 CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41761908},
issn = {1362-4962},
support = {//Deutsche Forschungsgemeinschaft/ ; 260989694//DFG/ ; 324652314//DFG/ ; 405858511//DFG/ ; 3869//DFG/ ; //Inserm/ ; 5342-2023//Research Council of Lithuania/ ; //Marburg University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism/genetics ; Models, Molecular ; DNA Helicases/chemistry/metabolism/genetics ; Protein Domains ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the Type I-F2 system represents the most compact Type I CRISPR-Cas variant, distinguished by the complete absence of both large (Cas8) and small (Cas11) subunits. In other Type I systems, Cas8 is essential for protospacer adjacent motif (PAM) recognition and for triggering Cas3 recruitment, while Cas11 stabilizes the Cascade backbone and guides the nontarget DNA strand during R-loop formation. To elucidate how I-F2 executes interference in their absence, we determined the cryo-electron microscopy structure of the I-F2 Cascade bound to target DNA and Cas3. Our structure reveals that Cas5 alone mediates PAM sensing, while Cas7 subunits directly recruit Cas3, which adopts a helicase-loaded conformation compatible with DNA engagement. We show how the helicase and C-terminal domains of Cas3 capture the displaced nontarget strand to initiate directional unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference without canonical large and small subunits and emphasize the mechanistic diversity among closely related Type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the hypercompact I-F2 system for genome editing and biotechnological applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
Cryoelectron Microscopy
DNA/chemistry/metabolism/genetics
Models, Molecular
DNA Helicases/chemistry/metabolism/genetics
Protein Domains
Protein Binding
*Bacterial Proteins/chemistry/metabolism/genetics

@article {pmid41759621,
year = {2026},
author = {Yan, X and Chen, M and Yang, S and Guo, Y and Dai, Y and Chen, Y and Zhong, H and Ma, T and Zha, D and He, Y and Li, B and Jia, X and Guo, L and Hu, J and Wei, Y and Chen, X},
title = {Mitochondrial genome editing tools: prospects in animal breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2026.02.018},
pmid = {41759621},
issn = {1673-8527},
abstract = {Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.},
}

@article {pmid41759529,
year = {2026},
author = {Cao, Z and Yu, S and Peng, J and Barrett, DR and Liu, Y and Sussman, JH and Chen, C and Thadi, A and Liu, L and Alikarami, F and Xu, J and Carroll, MP and Tan, K and Bernt, KM and Shi, J},
title = {CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {968-985.e7},
doi = {10.1016/j.molcel.2026.02.003},
pmid = {41759529},
issn = {1097-4164},
support = {R01 CA262260/CA/NCI NIH HHS/United States ; U01 CA243072/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/pathology/drug therapy/therapy ; *CRISPR-Cas Systems ; Animals ; *Genomics/methods ; Mice ; Single-Cell Analysis ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Gene Editing ; Gene Expression Regulation, Leukemic ; Mice, Inbred NOD ; },
abstract = {Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/pathology/drug therapy/therapy
*CRISPR-Cas Systems
Animals
*Genomics/methods
Mice
Single-Cell Analysis
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor
Xenograft Model Antitumor Assays
Gene Editing
Gene Expression Regulation, Leukemic
Mice, Inbred NOD

@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {*Gene Editing/methods ; *Chromatin/metabolism/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CCCTC-Binding Factor/metabolism ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}

*Gene Editing/methods
*Chromatin/metabolism/genetics
Animals
Mice
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
*Neural Stem Cells/metabolism/cytology
Mouse Embryonic Stem Cells/metabolism/cytology
CCCTC-Binding Factor/metabolism

@article {pmid41679300,
year = {2026},
author = {Wang, Y and Hu, W and Xia, R and Yang, X and Gu, Y and Ning, Z and Yang, T and Yu, C and Zhang, L and Li, D and Jin, Y and Li, J and Zhang, F and Xu, Y and Xu, C and Wang, Z and Jing, N and Chen, L and Wang, G},
title = {CLIM-TIME identifies metastatic microenvironment modulators for T cell therapy response.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1555-1572.e23},
doi = {10.1016/j.cell.2025.12.042},
pmid = {41679300},
issn = {1097-4172},
mesh = {*Tumor Microenvironment/immunology/genetics ; Animals ; *T-Lymphocytes/immunology ; Mice ; Humans ; Immunotherapy/methods ; Laser Capture Microdissection/methods ; Mice, Inbred C57BL ; Lung Neoplasms/secondary/immunology/therapy ; Cell Line, Tumor ; Extracellular Matrix/metabolism ; CRISPR-Cas Systems ; Neoplasm Metastasis ; Neoplasms/immunology/therapy/pathology ; },
abstract = {The tumor microenvironment (TME) poses a major barrier to effective immunotherapy, yet high-throughput perturbation-mapping approaches to dissect TME spatial complexity and its contextual immune modulators remain lacking. Here, we introduce CRISPR-laser-captured microdissection (LCM) integration mapping of the tumor-immune microenvironment (CLIM-TIME), a scalable platform that integrates CRISPR screening with LCM of metastatic tumors for transcriptomic, deconvolution, and immunofluorescence analyses. CLIM-TIME enables spatially resolved mapping of how tumor suppressor gene (TSG) loss reshapes the TME and modulates immune responses. We identified seven distinct TME subtypes, revealing that DNA repair and Polycomb repressive complex (PRC) TSG loss is linked to immune-infiltrated TMEs sensitive to T cell therapy. In contrast, knockouts of TSGs in the Hippo pathway promoted immune evasion and therapy resistance by fostering myeloid-enriched but T cell-excluded TMEs with elevated extracellular matrix (ECM). Targeting the ECM-crosslinking enzyme LOXL2 effectively remodeled the metastatic TME, enhancing T cell infiltration and improving therapeutic efficacy in lung metastases across multiple cancers.},
}

*Tumor Microenvironment/immunology/genetics
Animals
*T-Lymphocytes/immunology
Mice
Humans
Immunotherapy/methods
Laser Capture Microdissection/methods
Mice, Inbred C57BL
Lung Neoplasms/secondary/immunology/therapy
Cell Line, Tumor
Extracellular Matrix/metabolism
CRISPR-Cas Systems
Neoplasm Metastasis
Neoplasms/immunology/therapy/pathology

@article {pmid41636073,
year = {2026},
author = {Zhu, X and Gu, G and Shen, Y and Abdurazik, M and Sun, G},
title = {CRISPR/Cas13a-induced self-priming cyclic amplification enables liquid biopsy of exosomal circular RNA in non-small cell lung cancer.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1413-1419},
doi = {10.1039/d5an01345c},
pmid = {41636073},
issn = {1364-5528},
mesh = {*RNA, Circular/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Humans ; *Lung Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Exosomes/genetics/chemistry ; Liquid Biopsy/methods ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; },
abstract = {The precise and reliable identification of circular RNA (circRNA) is essential for both biological studies and clinical diagnostics of non-small cell lung cancer (NSCLC), especially the exosomal circRNA. In this study, we utilize a CRISPR/Cas13a system to specifically recognize the unique back-splice junction of target circRNA and develop a novel detection platform termed CRISPR/Cas13a-induced self-priming cyclic amplification. This method enables highly sensitive and specific circRNA detection. A pair of stem-loop DNA primers was carefully designed, each incorporating complementary single-stranded DNA sequences and five ribouridine (rU) residues at the 3' end serving as an overhang. When Cas13a binds to the target circRNA, its trans-cleavage activity is activated, leading to the cleavage of the rU residues. This cleavage permits the 3' ends of the stem-loop primers to extend along one another, generating multiple double stem-loop DNA structures that initiate successive cycles of self-priming chain elongation. By leveraging the sustained trans-cleavage activity of Cas13a and the high amplification efficiency of the self-priming cyclic reaction, the assay achieves sensitive detection of circRNA at concentrations as low as 564 aM within 90 min. In addition, the proposed method has been successfully applied for the analysis of exosomal hsa_circ_0003026 expression level in normal samples and NSCLC samples and demonstrated the potential of exosomal hsa_circ_0003026 in regulating the pathological progression. Owing to the high specificity of Cas13a, the proposed method can be directly applied to detect circRNA in complex biological samples without prior isolation of corresponding linear RNAs.},
}

*RNA, Circular/genetics/blood
*Carcinoma, Non-Small-Cell Lung/genetics/diagnosis
Humans
*Lung Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Exosomes/genetics/chemistry
Liquid Biopsy/methods
*Nucleic Acid Amplification Techniques/methods
DNA Primers/genetics

@article {pmid41622198,
year = {2026},
author = {Speth, ZJ and Pokhrel, V and Featherston, KM and Rehard, DG and Reid, WR and Franz, AWE},
title = {Monoallelic knockout of r2d2 affects the antiviral RNAi response to Mayaro virus and the reproductive potential in Aedes aegypti.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41622198},
issn = {1756-3305},
support = {R01 AI134661/AI/NIAID NIH HHS/United States ; R56 AI167980/AI/NIAID NIH HHS/United States ; R01-AI134661 (awarded to A.W.E.F), R56-AI167980 (awarded to A.W.E.F), and R56-AI180215 (awarded to Dr. Stefan Rothenburg, UC Davis)//National Institutes of Health - National Institute for Allergy and Infectious Diseases (NIH-NIAID)/ ; },
mesh = {Animals ; *Aedes/virology/genetics/physiology/immunology ; *RNA Interference ; Female ; *Alphavirus/physiology/immunology ; Mosquito Vectors/virology/genetics ; Gene Knockout Techniques ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Reproduction ; Fertility ; Alleles ; Argonaute Proteins/genetics ; RNA, Small Interfering/genetics ; },
abstract = {BACKGROUND: Aedes aegypti is an important vector for several human-pathogenic arboviruses. RNAi is the principal antiviral immune pathway in mosquitoes. Key steps of antiviral RNAi are processing of long dsRNAs into siRNA duplexes by dicer-2; loading of the siRNA duplexes onto Argonaute-2 with the help of R2D2; RISC formation via incorporation of Argonaute-2, which contains an siRNA; RISC-mediated targeting and degradation of homologous viral RNAs. Here, we generated an r2d2 knockout mosquito line to reveal how RNAi impairment during RISC loading complex (RLC) formation would affect arbovirus infection of Ae. aegypti.

METHODS: CRISPR/Cas9 gene editing has been used to knock out r2d2 in Ae. aegypti. Crossing experiments were conducted to reveal the effects of loss of r2d2 function on fecundity and fertility. Mayaro virus (Togaviridae: MAYV) infection and RNAi pathway gene expression levels were monitored using time-course RT-qPCR assays. Small RNA profiling was conducted to determine small RNA abundance in ΔR2D2[(+/-)] mosquitoes.

RESULTS: We show that in Ae. aegypti, the r2d2 allele is linked to the sex determination locus on chromosome 1. It was not possible to generate homozygous ΔR2D2[(-/-)] mosquitoes, indicating that complete loss of r2d2 function is lethal to Ae. aegypti. Our observations suggest that r2d2 function is not limited to RNAi but also affects mosquito fecundity/fertility, likely through follicle development. Monoallelic disruption of r2d2 increased the replication of MAYV, and r2d2 expression was also increased in infected mosquitoes. MAYV infection of ΔR2D2[(+/-)] mosquitoes was associated with an increase in abundance of putative vpiRNAs. However, impairment of r2d2 did not affect the function of dicer-2, as there was no difference in the 21 nt siRNA profiles between the ΔR2D2[(+/-)] mosquitoes and the non-transgenic control.

CONCLUSIONS: The RNAi pathway gene, r2d2, is an essential gene, and it is not possible to generate mosquitoes with biallelic (complete) loss of r2d2 function. Monoallelic impairment of r2d2 compromises the siRNA pathway downstream of dicer-2 function, at the point of RLC formation. In MAYV-infected mosquitoes, this defect in siRNA pathway function is compensated for by an increased piRNA pathway activity, which moderates increases in viral replication over a 10-day period.},
}

Animals
*Aedes/virology/genetics/physiology/immunology
*RNA Interference
Female
*Alphavirus/physiology/immunology
Mosquito Vectors/virology/genetics
Gene Knockout Techniques
*Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Reproduction
Fertility
Alleles
Argonaute Proteins/genetics
RNA, Small Interfering/genetics

@article {pmid41610849,
year = {2026},
author = {Samelson, AJ and Ariqat, N and McKetney, J and Rohanitazangi, G and Parra Bravo, C and Bose, RS and Travaglini, KJ and Lam, VL and Goodness, D and Ta, T and Dixon, G and Marzette, E and Jin, J and Tian, R and Tse, E and Abskharon, R and Pan, HS and Carroll, EC and Lawrence, RE and Gestwicki, JE and Rexach, JE and Eisenberg, DS and Kanaan, NM and Southworth, DR and Gross, JD and Gan, L and Swaney, DL and Kampmann, M},
title = {CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1517-1534.e19},
doi = {10.1016/j.cell.2025.12.038},
pmid = {41610849},
issn = {1097-4172},
support = {U54 NS123746/NS/NINDS NIH HHS/United States ; R01 AG075802/AG/NIA NIH HHS/United States ; K99 AG080116/AG/NIA NIH HHS/United States ; R01 AG085357/AG/NIA NIH HHS/United States ; U19 AG060909/AG/NIA NIH HHS/United States ; R01 AG070895/AG/NIA NIH HHS/United States ; R00 AG080116/AG/NIA NIH HHS/United States ; R01 AG082141/AG/NIA NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; F32 AG063487/AG/NIA NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R01 AG060477/AG/NIA NIH HHS/United States ; U24 AG072458/AG/NIA NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *tau Proteins/metabolism/genetics ; *Neurons/metabolism/cytology ; *Proteostasis ; Tauopathies/metabolism/pathology/genetics ; Ubiquitin-Protein Ligases/metabolism ; *CRISPR-Cas Systems/genetics ; Ubiquitination ; Mitochondria/metabolism ; Proteasome Endopeptidase Complex/metabolism ; },
abstract = {Aggregation of the protein tau defines tauopathies, the most common age-related neurodegenerative diseases, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation, dysfunction, and death. However, molecular mechanisms underlying cell-type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi screen in induced pluripotent stem cell (iPSC)-derived neurons. The screen uncovered both known and unexpected pathways, including UFMylation and GPI anchor biosynthesis, which control tau oligomer levels. We discovered that the E3 ubiquitin ligase CRL5[SOCS4] controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*tau Proteins/metabolism/genetics
*Neurons/metabolism/cytology
*Proteostasis
Tauopathies/metabolism/pathology/genetics
Ubiquitin-Protein Ligases/metabolism
*CRISPR-Cas Systems/genetics
Ubiquitination
Mitochondria/metabolism
Proteasome Endopeptidase Complex/metabolism

@article {pmid41431195,
year = {2026},
author = {Spezzani, E and Capelli, L and Di Lena, D and Chamorro-Garcia, A and Ippodrino, R and Porchetta, A and Bertucci, A},
title = {MARPLE: A Proximity-Triggered CRISPR-Cas13 Platform for Ultrasensitive Antibody Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {13},
pages = {e17799},
pmid = {41431195},
issn = {2198-3844},
support = {//National Recovery and Resilience Plan/ ; MUR 2023-2027//'Departments of Excellence' program of the Italian Ministry for University and Research/ ; 31108//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Antibodies/analysis ; Immunoassay/methods ; },
abstract = {Monitoring clinically relevant antibodies-as biomarkers of disease or therapeutic response-is essential for informed clinical decision-making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point-of-care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR-Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity-triggered Linker Exchange), a modular CRISPR-Cas13-based platform for ultrasensitive antibody detection. MARPLE harnesses antibody-induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13-mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets-including anti-digoxigenin, anti-cholesterol, anti-HA, trastuzumab, and anti-MUC1-highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one-pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR-based sensing beyond nucleic acids to protein biomarker detection.},
}

*CRISPR-Cas Systems/genetics
Humans
*Antibodies/analysis
Immunoassay/methods

@article {pmid40898426,
year = {2026},
author = {Sahin, U},
title = {Cas9 beyond CRISPR - SUMOylation, effector-like potential and pathogenic adaptation.},
journal = {The FEBS journal},
volume = {293},
number = {5},
pages = {1285-1296},
doi = {10.1111/febs.70256},
pmid = {40898426},
issn = {1742-4658},
support = {223Z048//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; 121C230//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; YIN IG3336//European Molecular Biology Organization/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Sumoylation/genetics ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing ; Protein Processing, Post-Translational ; *Bacteria/pathogenicity/genetics ; Host-Pathogen Interactions/genetics ; Animals ; },
abstract = {The CRISPR/Cas9 system has revolutionized molecular biology and gene editing, yet key aspects of its regulation, especially within eukaryotic environments, remain enigmatic. In this Viewpoint article, I will speculate on and explore the provocative hypothesis that Cas9 may possess previously unrecognized effector-like functions when expressed in host cells, potentially shaped by host-mediated post-translational modifications (PTMs). Of particular interest is SUMOylation at lysine 848, a key residue for DNA binding within the catalytic site, raising the possibility that this modification is not incidental, but functionally significant and precisely regulated. SUMOylation, a eukaryotic PTM, is increasingly recognized as a mechanism that also targets bacterial and viral effector proteins and virulence factors during infection, exerting context-dependent effects that may either enhance or hinder pathogen replication. Could Cas9, beyond its canonical role in bacterial CRISPR immunity, act as a host-modulating effector during infection, akin to known bacterial nucleomodulins such as transcription activator-like (TAL) effectors? If so, this would imply that certain pathogenic bacteria may have evolved Cas9 variants capable of exploiting host PTM machinery and targeting the host genome-an adaptation with potential implications for microbial virulence, host-pathogen interactions, and co-evolutionary dynamics. This perspective underscores the importance of systematically mapping Cas9 PTMs and examining their evolutionary conservation, functional significance, and pharmacological tunability, not only for basic biological insight and to deepen our understanding of microbial strategies, but also to refine the precision and safety of Cas9-based therapeutic platforms.},
}

*CRISPR-Cas Systems/genetics
*Sumoylation/genetics
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
Gene Editing
Protein Processing, Post-Translational
*Bacteria/pathogenicity/genetics
Host-Pathogen Interactions/genetics
Animals

@article {pmid41759295,
year = {2026},
author = {Molina, C and Knight, AL and Lisi, GP},
title = {Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a.},
journal = {Physical biology},
volume = {},
number = {},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged in genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and DNA cleavage mechanism. In this review, we examine the biophysical properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.},
}

@article {pmid41758946,
year = {2026},
author = {Saito, R and Umemura, Y and Makino, S and Fukaya, T},
title = {Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila.},
journal = {Science advances},
volume = {12},
number = {9},
pages = {eady7568},
pmid = {41758946},
issn = {2375-2548},
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics/chemistry ; *Zinc Fingers ; *Transcription Factors/metabolism/genetics ; *Evolution, Molecular ; Protein Binding ; *Drosophila/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism/embryology ; DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; CCCTC-Binding Factor ; Microtubule-Associated Proteins ; Nuclear Proteins ; },
abstract = {The zinc finger-associated domain (ZAD)-containing C2H2 zinc finger proteins (ZAD-ZnFs) represent the most abundant class of transcription factors that emerged during insect evolution, yet their molecular diversity and biological functions remain largely unclear. Here, we established a systematic CRISPR-based protein-tagging approach that enables direct, unambiguous comparison of nuclear localization and genome-wide binding profiles of endogenous ZAD-ZnFs in developing Drosophila embryos. Evidence is provided that a subset of ZAD-ZnFs forms nuclear condensates through the stacking of the N-terminal ZAD dimerization surface. Disruption of condensation activity leads to misregulation of genome-wide binding profiles and lethality, underscoring its functional and physiological significance in development. Integrative chromatin immunoprecipitation sequencing and Micro-C analyses reveal that many ZAD-ZnFs colocalize with core insulator proteins such as CCCTC-binding factor and Centrosomal protein 190 kD to control the formation of topological boundaries. We suggest that the diverse molecular functions of ZAD-ZnFs have evolutionarily arisen from their ancestral role as insulator-binding proteins.},
}

Animals
*Drosophila Proteins/metabolism/genetics/chemistry
*Zinc Fingers
*Transcription Factors/metabolism/genetics
*Evolution, Molecular
Protein Binding
*Drosophila/genetics/metabolism
*Drosophila melanogaster/genetics/metabolism/embryology
DNA-Binding Proteins/metabolism/genetics
CRISPR-Cas Systems
CCCTC-Binding Factor
Microtubule-Associated Proteins
Nuclear Proteins

@article {pmid41758452,
year = {2026},
author = {Bharti S, AK and Mukherjee, AG and Gopalakrishnan, AV and Gajendran, B and Vashishth, R and Prince, SE},
title = {From bench to bedside: stem cell therapy as a transformative approach against HIV.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {41758452},
issn = {1573-4978},
mesh = {Humans ; *HIV Infections/therapy/virology/immunology ; *Hematopoietic Stem Cell Transplantation/methods ; Gene Editing/methods ; HIV-1 ; *Stem Cell Transplantation/methods ; Induced Pluripotent Stem Cells ; Translational Research, Biomedical ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Virus Latency ; },
abstract = {Human immunodeficiency virus (HIV) remains a persistent global health burden, as combination antiretroviral therapy (ART) achieves sustained viral suppression but fails to eliminate long-lived latent reservoirs. Stem cell-based therapeutic strategies have emerged as transformative approaches with the potential to induce durable remission and, ultimately, a functional cure. Clinical proof-of-concept has been established through allogeneic hematopoietic stem cell transplantation (HSCT) using CCR5Δ32/Δ32 donor cells, demonstrating that durable resistance to viral entry can result in prolonged HIV remission. Building on these landmark observations, recent advances in autologous gene-edited hematopoietic stem and progenitor cells and induced pluripotent stem cell (iPSC)-derived immune effectors have accelerated the development of scalable, patient-specific interventions. The convergence of stem cell biology with precision genome-editing platforms, including CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), has enabled targeted disruption of viral entry pathways and host dependency factors, while offering new strategies to address viral latency and immune reconstitution. Despite significant challenges related to treatment-associated toxicity, manufacturing complexity, long-term safety, and ethical considerations, rapid progress in cellular engineering and translational immunology continues to advance the field toward curative outcomes. This review critically synthesizes recent progress in stem cell-based HIV therapeutics, elucidates the underlying mechanistic frameworks, evaluates emerging clinical and preclinical evidence, and outlines future directions required to achieve a durable functional cure.},
}

Humans
*HIV Infections/therapy/virology/immunology
*Hematopoietic Stem Cell Transplantation/methods
Gene Editing/methods
HIV-1
*Stem Cell Transplantation/methods
Induced Pluripotent Stem Cells
Translational Research, Biomedical
Receptors, CCR5/genetics
CRISPR-Cas Systems
Virus Latency

@article {pmid41758321,
year = {2026},
author = {Pathak, A and Singh, J and Swati, and Dwibedi, V},
title = {Deciphering microbial biofilm: mechanism, infection, and advanced approaches for control.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41758321},
issn = {1874-9356},
abstract = {Microbial biofilms are densely organised microbial communities that adhere to biotic and abiotic surfaces, encased within an extracellular polymeric substance (EPS). Microorganisms within these biofilm structures gain enhanced protection, versatility, and resistance to external stresses, antibiotics, and host immune systems. The biofilm formation follows a series of steps, including initial microbial adherence, microcolony establishment, EPS production, regulation by quorum sensing (QS), and dispersal. This flexibility enables biofilm survival in multiple environments, such as medical devices and natural systems, posing serious challenges in healthcare, agricultural, and industrial sectors. The review focuses on the mechanisms involved in biofilm formation and discusses the role of EPS in promoting biofilm stability and resistance to antimicrobials. It addresses biofilm-associated infections in medical environments, such as chronic wounds, cystic fibrosis, urinary tract infections (UTIs), and complications with implanted medical devices. The capacity of biofilm-forming microorganisms to evade immune responses and persist through extended antibiotic use highlights the urgent demand for novel therapeutic approaches. The discussion includes emerging strategies for biofilm control, including anti-biofilm agents, QS inhibitors, enzymatic treatments, and innovative combination therapies combining antibiotics with biofilm-disrupting agents. Emerging technologies, like antimicrobial peptides (AMPs), CRISPR-Cas systems, nanotechnology, and bioelectric therapies, present innovative biofilm disruption and removal approaches. This paper discusses the effectiveness of natural products, plant-derived compounds, and bacteriophage therapies for mitigating biofilm-associated infections linked to biofilms. The review examines the dynamic challenges posed by biofilms, particularly their role in chronic and device-related infections, which contribute to significant healthcare complications. The study highlights the significance of adopting new approaches to overcome biofilm-induced antimicrobial resistance (AMR) and improve therapeutic outcomes. Furthermore, this paper discusses the promising potential of emerging technologies, such as nanomaterials, QS interference, and biofilm-specific antimicrobial agents, in enhancing biofilm control and prevention measures across clinical, industrial, and environmental domains.},
}

@article {pmid41757824,
year = {2026},
author = {Chen, J and Davison, CW and Ellis, J and Blevins, B and Presley, W and Myers, MT and Kong, D and Hou, Z and Mian, SI and Prasov, L and Zhang, Y},
title = {CRISPR Base Editing Correction of TGFBI Mutations in Autosomal Dominant Corneal Dystrophies.},
journal = {Investigative ophthalmology & visual science},
volume = {67},
number = {2},
pages = {60},
pmid = {41757824},
issn = {1552-5783},
mesh = {Humans ; *Corneal Dystrophies, Hereditary/genetics/therapy/metabolism ; *Gene Editing/methods ; *Mutation ; *Transforming Growth Factor beta/genetics/metabolism ; *Genetic Therapy/methods ; Epithelium, Corneal/metabolism/pathology ; *Extracellular Matrix Proteins/genetics ; *CRISPR-Cas Systems ; Dependovirus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {PURPOSE: Lattice and granular corneal dystrophy comprise two common TGFBI-associated autosomal dominant corneal disorders. Existing therapies are only temporizing and carry significant morbidity. Here, we develop a novel therapeutic approach using an adenine base editor (ABE) to correct common TGFBI mutations.

METHOD: We generated two human corneal epithelial (HCE) cell models harboring a copy of the most common disease-causing TGBFI mutations, R124C or R555W. These lines were electroporated with an ABE8e-NG-encoding mRNA and guide RNAs targeting the mutations. The resulting A•T-to-G•C editing efficiencies and off-target (OT) effects were assessed by amplicon sequencing. GFP-expressing adeno-associated viruses (AAVs) with different capsid types were transduced into HCE cells and healthy human corneal donor tissues, and GFP fluorescence was evaluated.

RESULTS: Using all-RNA delivery for ABE8e-NG, we achieved 91% and 62% correction of the pathogenic adenines in HCE TGFBIR124C/WT and TGFBIR555W/WT cells, without editing the wild-type allele. Indel formation was negligible (<0.2%), bystander adenine editing was minimal (<0.7%), and editing at top computationally predicted OT sites was modest (<1.2% at all but 1 of the 20 OT sites analyzed), suggesting minimal safety concerns. Correction of TGFBIR124C/WT in HCEs rescued the aberrant lysosomal localization of TGFBI. We further identified AAV1 as the most effective serotype for gene delivery into both human corneal donor tissue and HCE cells.

CONCLUSIONS: Our study demonstrates the feasibility and safety of CRISPR adenine base editing as a new therapeutic strategy for correcting common TGFBI mutations in corneal dystrophies, paving the way for further preclinical testing.},
}

Humans
*Corneal Dystrophies, Hereditary/genetics/therapy/metabolism
*Gene Editing/methods
*Mutation
*Transforming Growth Factor beta/genetics/metabolism
*Genetic Therapy/methods
Epithelium, Corneal/metabolism/pathology
*Extracellular Matrix Proteins/genetics
*CRISPR-Cas Systems
Dependovirus/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41757451,
year = {2026},
author = {Molina-Márquez, A and Kelterborn, S and Hegemann, P and Pérez-Rodríguez, M and Vigara, J and León, R},
title = {Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70811},
pmid = {41757451},
issn = {1399-3054},
support = {2019-110438RB-C22//Agencia Estatal de Investigación-MCIN/AEI/10.13039/501100011033/ ; PID2022-140995OB-C21//Agencia Estatal de Investigación-MCIN/AEI/10.13039/501100011033/ ; 426566805//German Research Foundation (DFG)/ ; //Hertie Foundation/ ; },
mesh = {*Carotenoids/metabolism ; *Oxidoreductases/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Gene Knockout Techniques ; *Microalgae/genetics/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; Mutation/genetics ; Chloroplasts/metabolism/ultrastructure ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.},
}

*Carotenoids/metabolism
*Oxidoreductases/genetics/metabolism
*Ribonucleoproteins/metabolism/genetics
Gene Knockout Techniques
*Microalgae/genetics/metabolism
*Chlamydomonas reinhardtii/genetics/metabolism
Mutation/genetics
Chloroplasts/metabolism/ultrastructure
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41757335,
year = {2026},
author = {Shafiq, T and Khan, N and Kausar, T and Ahmed, W and Zhang, Z and Liang, Y and Duan, L},
title = {Red Blood Cell-Derived Extracellular Vesicles for Gene and RNA Therapeutics: Biological, Engineering, and Translational Challenges.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {579975},
pmid = {41757335},
issn = {1178-2013},
mesh = {Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Genetic Therapy/methods ; *Erythrocytes/cytology/chemistry/metabolism ; CRISPR-Cas Systems ; Animals ; RNA, Messenger/genetics/administration & dosage/therapeutic use ; *RNA/therapeutic use ; Gene Transfer Techniques ; },
abstract = {Gene therapy has great prospects of DNA/RNA manipulations and protein modulations. Its use in clinic is, however, stifled by risks of immunogenicity, low target specificity, and adverse effects. The red blood cell (RBC-EVs) extracellular vesicles can serve as a solution to this issue since they are biocompatible, long-term stable, and with low immunogenicity. RBC-EVs permit the accurate delivery of therapeutic cargo to space and time, thus minimizing systemic toxicity. This review presents the most recent developments on the expansion of the use of RBC-EVs to encapsulate the components of mRNA and CRISPR-Cas. Through the addition of the means to address these deficiencies, including stimulus-sensitive release mechanisms (eg, pH- or light-activated systems) and tissue-selective targeting approaches, RBC-EVs can be applied to enable the precise application in genetic diseases, inflammatory diseases, and cancer. Such innovations have the potential to overcome the clinical need and enable the biological complexity of mRNA- and CRISPR-Cas-based agents to provide a powerful delivery platform. Moreover, the review also demonstrates the unprecedented benefits of red blood cell EVs, which include immune evasion, scalability, and universal loading capacity, which can establish them as the next-generation delivery vehicles. Red blood cell EVs have the potential to increase the efficacy of precision medicine by increasing its feasibility. Lastly, we note the potential and translational issues in the provision of red blood cell EV-based mRNA and CRISPR-Cas therapeutic delivery of gene therapy.},
}

Humans
*Extracellular Vesicles/chemistry/metabolism
*Genetic Therapy/methods
*Erythrocytes/cytology/chemistry/metabolism
CRISPR-Cas Systems
Animals
RNA, Messenger/genetics/administration & dosage/therapeutic use
*RNA/therapeutic use
Gene Transfer Techniques

@article {pmid41755886,
year = {2026},
author = {Kaniganti, S and Saini, H and Chaitanya, AK and Hegde, N and Shah, P and Magar, ND and Rijal, R and Kaushik, JJ and Nanda, D and Sachan, S and Kumar, A and Bhoite, R and Jamedar, HR},
title = {CRISPR/Cas Genome Editing and Its Applications in Cereal Crop Improvement.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {2},
pages = {e70133},
pmid = {41755886},
issn = {2575-6265},
abstract = {CRISPR/Cas-based genome editing has emerged as a transformative tool for precise genetic improvement of cereal crops. Recent advances in CRISPR technologies, including Cas9, Cas12, Cas13, base editing, and prime editing, have enabled targeted modification of genes and regulatory elements controlling yield, stress tolerance, and grain nutritional quality in major cereals such as rice, wheat, maize, and barley. This review summarizes current progress in CRISPR-mediated genome editing systems, delivery strategies, and representative applications in cereal crop improvement. Emphasis is placed on how genome editing reprograms enzymatic activities and biological pathways underlying complex agronomic traits rather than acting through single-gene effects. The review also discusses challenges related to trait complexity, regulatory considerations, and prospects for translating genome-edited cereal crops from laboratory research to field-level application. Collectively, this review highlights the potential of CRISPR/Cas genome editing as a powerful approach for developing high-yielding, resilient, and nutritionally improved cereal crops.},
}

@article {pmid41755634,
year = {2026},
author = {Hu, Z and Liu, Y and Han, Y and Li, M and Deng, K and Lu, X and Huang, Y and Liang, C and Wang, Y and Fu, Y and Xu, A},
title = {CRISPR/Cas9 screening with destabilized bicistronic fluorescent protein reporter revealed PABPN1 as a hub of regulators for alternative polyadenylation.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755634},
issn = {1362-4962},
support = {2022YFA1103900//National Key Research and Development Program of China/ ; 32470586//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 31930084//National Natural Science Foundation of China/ ; 32500472//National Natural Science Foundation of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; 2022YFA1103900//National Key Research and Development Program of China/ ; },
mesh = {*Polyadenylation/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Genes, Reporter ; *Poly(A)-Binding Protein I/genetics/metabolism ; HEK293 Cells ; DEAD-box RNA Helicases/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; Cell Proliferation/genetics ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Alternative polyadenylation (APA) is intricately intertwined with diverse biological processes. Efficient approaches for screening the regulatory factors of specific APA events are essential to elucidate their regulation mechanisms. Here, we first engineered a destabilized bicistronic fluorescent protein reporter (dBFPR) to enhance the sensitivity of APA detection. Then, we developed a robust high-throughput screening platform for APA regulators by integrating CRISPR/Cas9, dBFPR, and fluorescence-activated cell sorting. With this method, we successfully screened the library of RNA binding proteins and found that PTBP1, ELAVL1, and DDX3X play significant roles in regulating APA and promoting cell proliferation through interaction with PABPN1, suggesting that PABPN1 is an important hub for APA regulation.},
}

*Polyadenylation/genetics
Humans
*CRISPR-Cas Systems/genetics
Genes, Reporter
*Poly(A)-Binding Protein I/genetics/metabolism
HEK293 Cells
DEAD-box RNA Helicases/metabolism/genetics
*Luminescent Proteins/genetics/metabolism
Cell Proliferation/genetics
Polypyrimidine Tract-Binding Protein/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics

@article {pmid41755633,
year = {2026},
author = {Aguilar-González, A and Martos-Jamai, I and Ramos-Hernández, I and Molina-Estévez, FJ and Villao, NV and Puig-Serra, P and Rodríguez-Perales, S and Torres, R and Labun, K and Sánchez-Martín, RM and Díaz-Mochón, JJ and Martín, F},
title = {A novel Dual-guide CRISPR-Cas13 strategy improves specificity for single-nucleotide variant detection.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755633},
issn = {1362-4962},
support = {MCIN)/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation/ ; PID2022-141065OB-I00//European Union Next Generation/ ; CV20-77741//Consejería de Economía y Conocimiento/Project/ ; PI21/00298//Instituto de Salud Carlos III/ ; PI24/00888//Instituto de Salud Carlos III/ ; RD21/0017/0004//Instituto de Salud Carlos III/ ; RD24/0014/0005//Instituto de Salud Carlos III/ ; PI-0236-2024//Consejería de Salud y Familias/ ; PIP-0004-2025//Consejería de Salud y Familias/ ; GeneHumdi-CA21113//European Cooperation in Science and Technology/ ; FPU22/03455//Spanish Ministry of Science, Innovation and Universities/ ; RHJ-0053-2025//Consejería de Salud y Familias, Junta de Andalucía/ ; //European Social Fund/ ; //Universidad de Granada/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation & purification ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology/genetics ; RNA, Viral/genetics ; *Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Sensitivity and Specificity ; Leishmania/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13's heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs-dcrRNA and dtracrRNA-which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform's potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.},
}

*CRISPR-Cas Systems/genetics
Humans
*SARS-CoV-2/genetics/isolation & purification
*RNA, Guide, CRISPR-Cas Systems/genetics
*COVID-19/diagnosis/virology/genetics
RNA, Viral/genetics
*Polymorphism, Single Nucleotide
Proto-Oncogene Proteins p21(ras)/genetics
Sensitivity and Specificity
Leishmania/genetics
CRISPR-Associated Proteins/genetics

@article {pmid41740934,
year = {2026},
author = {Sun, W and Zhu, S and Dong, Q and Tang, S and Liu, Q and Sha, Y and Chen, T and Wang, R and Chen, Y and Ying, H},
title = {Engineering Escherichia coli cell Factories for continuous 5'-cytidine monophosphate production via biofilm-anchored dual-enzyme cascade catalysis.},
journal = {Bioresource technology},
volume = {447},
number = {},
pages = {134267},
doi = {10.1016/j.biortech.2026.134267},
pmid = {41740934},
issn = {1873-2976},
mesh = {*Biofilms/growth & development ; *Escherichia coli/metabolism/genetics/enzymology ; *Cytidine Monophosphate/biosynthesis ; Biocatalysis ; CRISPR-Cas Systems/genetics ; Uridine Kinase/metabolism ; },
abstract = {5'-cytidine monophosphate (5'-CMP) serves as a crucial intermediate for diverse nucleotide derivatives and finds extensive applications in the food and pharmaceutical industries. However, existing enzymatic production processes suffer from low catalytic efficiency and poor economic feasibility. In this study, we developed a continuous 5'-CMP production system based on a cell-enzyme co-immobilized biocatalytic platform. First, the pgaABCD gene cluster was integrated into ClearColi BL21(DE3) using CRISPR-Cas9, enhancing its biofilm-forming capacity by 168.93% and enabling robust cell immobilization on the carrier. Second, a dual-anchoring strategy utilizing ice-nucleation protein (INP) and autotransporter (AIDA-I) enabled surface display of uridine kinase (UDK) and acetate kinase (AckA) on the cell surface. This approach successfully addressed the instability and recovery issues of free enzymes by using biofilm engineering to co-immobilize cells and enzymes. The modified strain achieved a 5'-CMP productivity of 1.77 mmol/L/h, 5.98-fold higher than free intracellular enzyme catalysis, and was reused for ten consecutive cycles under the tested conditions while maintaining a cytidine conversion rate above 73.79%, and a 5'-CMP yield above 59.26%. This work demonstrates the first successful realization of continuous 5'-CMP biosynthesis and establishes an efficient route for its industrial production.},
}

*Biofilms/growth & development
*Escherichia coli/metabolism/genetics/enzymology
*Cytidine Monophosphate/biosynthesis
Biocatalysis
CRISPR-Cas Systems/genetics
Uridine Kinase/metabolism

@article {pmid41719905,
year = {2026},
author = {Yu, F and Yue, D and Wang, F and Fu, B and Qin, G and Wei, S and Zang, W and Zhang, Q and Cui, L and Wang, T},
title = {Structure-initiated CHA variant coordinating SDA for cascade amplification in CRISPR/Cas12a-based miRNA analysis.},
journal = {Talanta},
volume = {304},
number = {},
pages = {129558},
doi = {10.1016/j.talanta.2026.129558},
pmid = {41719905},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are well-established biomarkers for tumor diagnosis and monitoring. Herein, we report a novel biosensing platform by engineering a structure-initiated variant of catalytic hairpin assembly (VCHA) that coordinates with strand displacement amplification (SDA) to drive cascade amplification for CRISPR/Cas12a-based detection. This system employs three hairpin probes which, upon recognizing the target miRNA, self-assemble into a key 5'-end dangling three-way conjugate (5'-DTC) structure. This structure serves as the exclusive trigger, simultaneously propagating the VCHA cycle and priming the SDA process through the coordinated action of polymerase and nicking enzyme. Consequently, VCHA and SDA operate synergistically within a unified circuit, generating abundant single-stranded activator DNA (acDNA) products. These acDNA molecules then activate the trans-cleavage activity of CRISPR/Cas12a, yielding a significantly amplified fluorescence readout. The VCHA-SDA/Cas12a platform demonstrated excellent performance for miRNA-155 detection, achieving a broad dynamic range from 1 pmol/L to 10 nmol/L with an ultra-low detection limit of 0.166 pmol/L. Furthermore, the platform successfully quantified miRNA levels in clinical plasma specimens and various cell lines, confirming its considerable potential as a robust tool for molecular diagnostics and clinical translation.},
}

*MicroRNAs/genetics/analysis/blood
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41713574,
year = {2026},
author = {Hu, T and Hou, Z and Zhang, Y and Jing, P and Dai, X and Wang, H},
title = {Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.},
journal = {Journal of invertebrate pathology},
volume = {216},
number = {},
pages = {108569},
doi = {10.1016/j.jip.2026.108569},
pmid = {41713574},
issn = {1096-0805},
mesh = {*White spot syndrome virus 1/isolation & purification ; Animals ; *Penaeidae/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Aquaculture ; Sensitivity and Specificity ; },
abstract = {Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.},
}

*White spot syndrome virus 1/isolation & purification
Animals
*Penaeidae/virology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Aquaculture
Sensitivity and Specificity

@article {pmid41690121,
year = {2026},
author = {Yin, B and Wu, X and Zhou, H and Meng, Y and Liu, J and Ding, L and Zhu, C and Tai, Z and Zhu, Q and Chen, Z},
title = {Topical ionic liquid-mediated GLUT1 gene editing ameliorates psoriasis and prevents recurrence.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124058},
doi = {10.1016/j.biomaterials.2026.124058},
pmid = {41690121},
issn = {1878-5905},
mesh = {Animals ; *Gene Editing/methods ; *Psoriasis/therapy/genetics/pathology ; *Glucose Transporter Type 1/genetics ; Mice ; *Ionic Liquids/chemistry/administration & dosage ; Humans ; CRISPR-Cas Systems/genetics ; Recurrence ; Mice, Inbred C57BL ; Keratinocytes/metabolism ; Disease Models, Animal ; Administration, Topical ; },
abstract = {Psoriasis is a chronic inflammatory skin disorder characterized by immune dysregulation and a high relapse rate. Current therapies seldom achieve lasting remission. Aberrant overexpression of glucose transporter 1 (GLUT1) in keratinocytes enhances glycolysis, fueling inflammation and immune imbalance, thus positioning GLUT1 as a promising therapeutic target. In this work, a composite ionic liquid-mediated transdermal platform was established for the delivery of CRISPR-Cas9 ribonucleoprotein (CIL-RNP), aiming to achieve efficient GLUT1 gene editing in keratinocytes. The CIL-RNP achieved 76.6% editing efficiency, downregulated pyruvate kinase M (PKM) expression, and reduced inflammatory cytokine secretion. In a psoriasis mouse model, topical administration of CIL-RNP decreased lesion severity by 50% PASI (Psoriasis Area and Severity Index) score, alleviating epidermal hyperplasia and immune infiltration. Furthermore, the treatment inhibited M1 macrophage polarization, reduced reactive oxygen species generation, rebalanced Th17/regulatory T cells (Tregs) responses, and diminished the accumulation of tissue-resident memory T cells (TRMs), thereby lowering the risk of relapse. This study establishes ionic liquid-based CRISPR-RNP transdermal editing of GLUT1 as a novel and effective strategy for restoring immune homeostasis in psoriasis, with potential for long-term remission and broader applications in cutaneous immunopathological conditions.},
}

Animals
*Gene Editing/methods
*Psoriasis/therapy/genetics/pathology
*Glucose Transporter Type 1/genetics
Mice
*Ionic Liquids/chemistry/administration & dosage
Humans
CRISPR-Cas Systems/genetics
Recurrence
Mice, Inbred C57BL
Keratinocytes/metabolism
Disease Models, Animal
Administration, Topical

@article {pmid41685943,
year = {2026},
author = {Wang, C and Li, D and Yu, R and Xue, J and Xie, W and Zhang, Q and Gui, X and Wang, L and Guo, S and Xie, Y and Jiang, Y and Liu, G and Wu, J},
title = {A CRISPR/Cas9-regulated dual-ring topological allosteric probe for detection of the EGFR L858R resistance mutation in CTCs.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1815-1825},
doi = {10.1039/d5ay02124c},
pmid = {41685943},
issn = {1759-9679},
mesh = {Humans ; ErbB Receptors/genetics ; *Neoplastic Cells, Circulating/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Lung Neoplasms/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/blood ; Polymorphism, Single Nucleotide ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Fluorescent Dyes/chemistry ; Cell Line, Tumor ; },
abstract = {A single-nucleotide polymorphism (SNP) is a point mutation occurring at a defined genomic locus, and its precise and rapid detection in circulating tumor cells (CTCs) is essential for early diagnosis and therapeutic monitoring of non-small cell lung cancer (NSCLC). In this study, a CRISPR/Cas9-regulated dual-ring topological allosteric probe was developed for ultrasensitive and specific detection of the EGFR L858R mutation. The recognition ring selectively hybridizes with the target sequence and is cleaved by the Cas9-sgRNA complex, triggering the release of the reporter ring. The released reporter ring then serves as a template for rolling circle amplification (RCA), generating products that hybridize with dual-labeled fluorescent probes to yield measurable signals. This assay clearly distinguished L858R from the wild-type sequence and detected mutation frequencies as low as 1.0% with high specificity against other common EGFR variants. Its robustness was further validated using clinical blood samples, enabling sensitive detection of low-abundance L858R mutations. These results demonstrate that the integration of programmable target recognition, efficient signal amplification, and fluorescence readout provides a promising platform for SNP analysis in liquid biopsy, supporting precision diagnosis and treatment monitoring in NSCLC.},
}

Humans
ErbB Receptors/genetics
*Neoplastic Cells, Circulating/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Lung Neoplasms/genetics/blood
*Carcinoma, Non-Small-Cell Lung/genetics/blood
Polymorphism, Single Nucleotide
*Drug Resistance, Neoplasm/genetics
Mutation
Fluorescent Dyes/chemistry
Cell Line, Tumor

@article {pmid41666850,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Jin, M and Liu, K and Liu, Y and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated knockout of the 22 kDa α-prolamin genes orchestrates the regulation of functional amino acid content in foxtail millet.},
journal = {Journal of plant physiology},
volume = {318},
number = {},
pages = {154723},
doi = {10.1016/j.jplph.2026.154723},
pmid = {41666850},
issn = {1618-1328},
mesh = {*CRISPR-Cas Systems/genetics ; *Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; Gene Knockout Techniques ; *Prolamins/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; },
abstract = {Improving the nutritional quality of cereal crops remains a primary objective in modern breeding programs. The composition and content of prolamins directly affect the overall nutritional value. This study elucidates the role of two 22 kDa α-prolamin genes (Seita.9G301400 and Seita.9G406400) in foxtail millet using CRISPR/Cas9-mediated knockout. While simultaneous disruption of both genes in double mutants reduced prolamin content, amino acids, and soluble sugars, single-gene mutants exhibited the opposite effect. These single mutants displayed increased grain size alongside significantly enhanced levels of essential amino acids and sugars. Starch pasting properties were also improved in single mutants but compromised in double mutants. Our findings demonstrate that individual knockout of the two prolamin genes enhances nutritional and sensory quality, providing a potential strategy for developing improved foxtail millet varieties.},
}

*CRISPR-Cas Systems/genetics
*Setaria Plant/genetics/metabolism
*Amino Acids/metabolism
Gene Knockout Techniques
*Prolamins/genetics/metabolism
*Plant Proteins/genetics/metabolism
Genes, Plant

@article {pmid41653514,
year = {2026},
author = {Li, M and He, L and Wang, Z and Wang, L and Pan, Q and Sun, P and Li, D and Zhang, C},
title = {Amplified ferroptosis and immunomodulation triggered by NIR-II photothermal-controllable CRISPR/Cas9 nanoplatform to treat osteosarcoma and prevent postsurgical implant-associated infections.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124043},
doi = {10.1016/j.biomaterials.2026.124043},
pmid = {41653514},
issn = {1878-5905},
mesh = {*Ferroptosis/drug effects ; Humans ; Animals ; *CRISPR-Cas Systems ; *Osteosarcoma/therapy/immunology ; *Immunomodulation ; Mice ; Cell Line, Tumor ; Infrared Rays ; HSP70 Heat-Shock Proteins/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {Ferroptosis has been proven as a promising therapeutic approach with immunomodulatory effect; however, intracellular antioxidant system maintains redox balance and diminishes its efficacy. Nuclear factor erythroid 2-related factor 2 (Nrf2) is identified as a central transcription factor for regulating oxidative homeostasis. Herein, we have developed a thermal-controllable genome-editing nanoplatform BF/pHCN. Specifically, a CRISPR/Cas9 plasmid with an upstream HSP70 promoter sequence (HSP70-Cas9-sgNrf2, named pHCN) was constructed. Subsequent Fe(II) and pHCN were co-loaded into organic small-molecule BTP with near infrared II (NIR-II) absorption and coated with DSPE-mPEG2000. The generated BF/pHCN (BTP@Fe/pHCN) could be internalized within osteosarcoma cells. Subsequent NIR-II laser-triggered hyperthermia at 42 °C activated the HSP70 promoter and facilitated the precise inhibition targeting Nrf2 genomic sequences while promoting Fe(II) release, ultimately disrupting oxidative stress states. Moreover, the amplified ferroptosis fully triggered immunogenic cell death, thus reprogramming macrophages, promoting maturation of dendritic cells, and activating cellular antitumoral immunity. Additionally, BF/pHCN exhibited direct bactericidal activity against planktonic bacteria, and effectively eliminated intracellular bacteria through iron metabolic disorders strategy targeting macrophages, thereby initiating adaptive antimicrobial immunity. Overall, our NIR-II thermal-controllable genome-editing nanoplatform BF/pHCN exhibits remarkable antitumoral properties alongside robust antiinfection and immunomodulation, providing feasible strategies toward effective management of osteosarcoma, and preventing postsurgical implant-associated infections.},
}

*Ferroptosis/drug effects
Humans
Animals
*CRISPR-Cas Systems
*Osteosarcoma/therapy/immunology
*Immunomodulation
Mice
Cell Line, Tumor
Infrared Rays
HSP70 Heat-Shock Proteins/genetics
NF-E2-Related Factor 2/genetics/metabolism

@article {pmid41649281,
year = {2026},
author = {Zhu, Z-J and Cui, M-L and Liu, Y and Yao, X-Q and Lu, M-J and Wang, M-C and Liu, J-H and Li, J-F and Li, E-Z},
title = {CRISPR/Cas14a combined with RPA for visual detection of Marek's disease virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {3},
pages = {e0262525},
pmid = {41649281},
issn = {2165-0497},
support = {22A310017//Key Scientific Research Projects of Universities in Henan/ ; },
mesh = {Animals ; *Marek Disease/diagnosis/virology ; *CRISPR-Cas Systems ; *Herpesvirus 2, Gallid/isolation & purification/genetics ; *Poultry Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Chickens/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Recombinases/metabolism/genetics ; *Mardivirus/isolation & purification/genetics ; },
abstract = {Marek's disease, a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus, infectious bursal disease virus, MDV-1 vaccine strains, or herpesvirus of turkeys. Plasmid DNA standards were used to determine the sensitivity of the assay, and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all Marek's disease virus-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, highly specific nucleic acid detection platform for MDV-1 that enables visual readout without complex instrumentation by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.IMPORTANCEMarek's disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when evaluated using clinical samples. As a cost-effective method that avoids the need for complex detection instruments, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.},
}

Animals
*Marek Disease/diagnosis/virology
*CRISPR-Cas Systems
*Herpesvirus 2, Gallid/isolation & purification/genetics
*Poultry Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
Chickens/virology
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
Recombinases/metabolism/genetics
*Mardivirus/isolation & purification/genetics

@article {pmid41638449,
year = {2026},
author = {Qu, Y and Li, Y and Shao, T and Kuang, J and Qi, Y and Yang, J and Liu, Y and Wang, J and Fu, X and Liu, J and Zhang, X and Peng, T and Yuan, Q and Zhu, L},
title = {Optimizing prime editing: Advances in efficiency enhancement.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108815},
doi = {10.1016/j.biotechadv.2026.108815},
pmid = {41638449},
issn = {1873-1899},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Protein Engineering/methods ; },
abstract = {Prime editing (PE) enables the precise installation of targeted insertions, deletions, and all possible base-to-base conversions without introducing double-strand breaks or donor DNA templates. However, its efficiency remains highly variable across genomic contexts. To address this, multi-faceted optimization strategies have been developed: protein engineering has yielded editor variants with enhanced reverse transcriptase activity and stability; structural refinements to pegRNA design improve its functional integrity and resistance to degradation; regulation of the PE-Flap-mismatch repair (MMR) process favors the retention of desired edits; and the development of protospacer adjacent motif (PAM)-relaxed Cas variants dramatically expands targetable sites. This review systematically consolidates these advances, illustrating how the integration of structural, mechanistic and targeting enhancements is overcoming fundamental bottlenecks. Together, these developments establish PE as a versatile and efficient system for precision genome engineering, paving the way for its reliable application in diverse biological settings.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
Protein Engineering/methods

@article {pmid41506374,
year = {2026},
author = {Marschhofer, M and Chen, S and Molbay, M and Winkeljann, B and Villano, E and Giancaspro, C and Kourou, A and Berninghausen, O and Rieder, S and Ungewickell, C and Beckmann, R and Popper, B and Torres, AM and Vidal, A and Merkel, OM and Carneiro, SP},
title = {Optimized lipid nanoparticles for pulmonary delivery of CRISPR/Cas9 targeting KRAS G12S in lung cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114607},
doi = {10.1016/j.jconrel.2026.114607},
pmid = {41506374},
issn = {1873-4995},
mesh = {Animals ; *Lung Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/administration & dosage/chemistry ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Lipids/chemistry/administration & dosage ; Gene Editing ; A549 Cells ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Mice ; *CRISPR-Associated Protein 9/genetics ; Mice, Nude ; RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics ; Female ; RNA, Messenger/administration & dosage/genetics ; Apoptosis ; Cell Line, Tumor ; Liposomes ; },
abstract = {KRAS G12S mutations in non-small cell lung cancer (NSCLC) remain refractory to current targeted therapies, with few clinical options and frequent resistance. While CRISPR/Cas9 enables mutation-specific gene disruption, its pulmonary application is limited by systemic clearance, hepatic tropism, and airway mucus barriers. Here, we present lipid nanoparticles (LNPs) specifically engineered for pulmonary delivery of Cas9 mRNA and KRAS G12S-targeting sgRNA, optimized through mRNA surrogate screening and orthogonal mixture design to guide lipid composition and Cas9:sgRNA weight-to-weight ratios. Two lead LNP formulations, A6 3:1 and A8 1:1, exhibited robust critical quality attributes, including particle sizes below 120 nm, low polydispersity, near-neutral zeta potential, and over 80 % encapsulation efficiency. Cryo-TEM revealed distinct morphologies correlated with enhanced transfection. In vitro, A8 1:1 achieved up to 90 % on-target gene editing in A549 cells and a 3.6-fold increase in apoptosis, while A6 3:1 induced a 3.7-fold apoptotic response. Both formulations efficiently traversed airway mucus in air-liquid interface cultures and preserved over 80 % cell viability across doses. In vivo, repeated pulmonary administration was well tolerated, with no signs of systemic toxicity or cytokine elevation in healthy or tumor-bearing mice. In an orthotopic A549-luc lung tumor model, intratracheal delivery of A6 3:1 and A8 1:1 modestly suppressed tumor growth, with histological evidence of tumor cell apoptosis for A8 1:1. Quantification confirmed a statistically significant increase of apoptosis in the A8 1:1 group, consistent with effective KRAS disruption in vivo. Overall, lead LNPs, particularly A8 1:1, enabled efficient and localized RNA-based gene editing that induced downstream apoptotic signaling, demonstrating a preliminary, yet promising, proof-of-concept for CRISPR/Cas9 therapy in NSCLC.},
}

Animals
*Lung Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Humans
*Nanoparticles/administration & dosage/chemistry
*Proto-Oncogene Proteins p21(ras)/genetics
*Lipids/chemistry/administration & dosage
Gene Editing
A549 Cells
*Carcinoma, Non-Small-Cell Lung/genetics/therapy
Mice
*CRISPR-Associated Protein 9/genetics
Mice, Nude
RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics
Female
RNA, Messenger/administration & dosage/genetics
Apoptosis
Cell Line, Tumor
Liposomes

@article {pmid41489552,
year = {2026},
author = {Fan, R and Tong, Y and Luo, S and He, Y and Yang, C and Li, W and Liu, J and Pan, J and Zhu, Y and Zhang, X and Zhu, J and Zhu, Y and Guo, Y and Li, L and Situ, B and Yan, X and Ma, W and Chang, L and Zhang, Y},
title = {Integrated Electroporated-Lysis Electrochemical Platform Enables Sensitive and Rapid EV Protein and miRNA Profiling Based on Multiplex-Responsive CRISPR/Cas12a.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {13},
pages = {e13331},
doi = {10.1002/smll.202513331},
pmid = {41489552},
issn = {1613-6829},
support = {2024YFC2419100//National Key R&D Program of China/ ; 82272424//National Natural Science Foundation of China/ ; 82572682//National Natural Science Foundation of China/ ; 82372343//National Natural Science Foundation of China/ ; 82402753//National Natural Science Foundation of China/ ; 22304006//National Natural Science Foundation of China/ ; 62471021//National Natural Science Foundation of China/ ; 2023A1515010909//Guang Dong Basic and Applied Basic Research Foundation of China/ ; JCYJ20230807142204008//Shenzhen Science and Technology Innovation Committee/ ; 2024J004//Southern Medical University/ ; 2024M750606//China Postdoctoral Science Foundation/ ; 2025T180607//China Postdoctoral Science Foundation/ ; T2425021//National Science Fund for Distinguished Young Scholars/ ; },
mesh = {*MicroRNAs/metabolism/genetics ; Humans ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Electroporation/methods ; },
abstract = {Proteins and miRNAs in extracellular vesicles (EVs) have emerged as crucial biomarkers for tumor diagnosis. While CRISPR/Cas12a-based platforms have shown great promise in nucleic acid and protein detection, their susceptibility to off-target activation and structural instability remains a significant limitation. Here, we have developed an electroporation-lysis electrochemical platform integrated with DNA cube-cage-locked CRISPR/Cas12a (DC-Cas12a), termed EL-DC-Cas12a. This platform utilizes an electric field to rapidly lyse EVs, releasing their internal proteins and miRNAs. These released molecules then activate the DC-Cas12a system, thereby triggering the displacement of two distinct crRNA/Cas12a complexes that correspond to EV proteins and miRNAs, respectively. These complexes then specifically recognize and cleave electrochemical probes, generating quantifiable electrochemical signals that enable synchronous and accurate analysis of the two biomarkers. The integrated workflow for EV lysis and detection can be completed within 40 min, greatly simplifying the overall operation. The detection limits (LOD) of this platform for EV PD-L1 protein and miR-1246 were 5.44 × 10[4] particles/mL and 3.59 × 10[3] particles/mL, respectively. Moreover, by applying machine learning algorithms to analyze the EV-associated proteins and miRNAs profiling, the platform demonstrated a diagnostic accuracy of 98.3% in distinguishing healthy donors from early-stage GC patients, and 99% in differentiating early-stage from advanced-stage GC patients in a clinical gastric cancer cohort. Therefore, the proposed platform offers a promising strategy for multiplexed detection of EV biomarkers and precise discrimination of GC.},
}

*MicroRNAs/metabolism/genetics
Humans
*Extracellular Vesicles/metabolism
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*Electroporation/methods

@article {pmid41477961,
year = {2026},
author = {Gong, P and Tao, D and Chen, Q and Yang, Y and Xie, S and Chen, X and Shi, S and Wang, Y and Wang, L and Qian, Y and Ye, S},
title = {A rapid, visual, ultrasensitive and highly specific method for detecting adeno-associated virus 2020 based on the LAMP-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {2},
pages = {106344},
pmid = {41477961},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; *Dependovirus/isolation & purification/genetics ; *Ducks ; *Molecular Diagnostic Techniques/veterinary/methods ; Sensitivity and Specificity ; Phylogeny ; },
abstract = {Avian parvovirus infection would lead to growth retardation, weight loss, physical deformities and increased mortality in poultry, causing substantial economic losses to the poultry industry. Therefore, the development of a rapid, visual, ultrasensitive and highly specific method is essential for timely diagnosis and effective control of the avian parvovirus infection. In this study, we developed a detection platform based on loop-mediated isothermal amplification (LAMP) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system. Firstly, we have identified a novel avian parvovirus strain from diseased Muscovy ducks. Through genome sequencing, sequence assembly and phylogenetic tree analysis, we have identified this novel avian parvovirus as an adeno-associated virus (AAA) belonging to the family Parvoviridae, subfamily Parvovirinae and genus Dependoparvovirus. So, the novel virus strain was named AAV-2020. Next, specific sgRNAs and LAMP primers targeting the 3 capsid proteins (Cap) genes of AAV-2020 were designed and optimized. Moreover, the CRISPR/Cas12a-based system demonstrated a limit of detection as low as 2 copies/μL for AAV-2020. Importantly, the system could effectively distinguish AAV-2020 from 3 closely related AAV strains with high sequence similarity, indicating excellent specificity. In summary, we developed a novel, rapid, visual, ultrasensitive and highly specific detection system for AAV-2020, offering a reliable tool for early diagnosis and on-site detection of avian parvovirus infections, which would aid in the prevention and control of avian parvovirus infection in poultry industry.},
}

Animals
*Poultry Diseases/diagnosis/virology
*Parvoviridae Infections/veterinary/diagnosis/virology
*Nucleic Acid Amplification Techniques/veterinary/methods
*CRISPR-Cas Systems
*Dependovirus/isolation & purification/genetics
*Ducks
*Molecular Diagnostic Techniques/veterinary/methods
Sensitivity and Specificity
Phylogeny

@article {pmid41423250,
year = {2026},
author = {Sharma, A and Pathangey, L and Chirackal, SS and Shim, KG and Fonseca, R and Swaminathan, S},
title = {Ferritin H Knockout Induces Differential Immunomodulatory Drug Responses in Multiple Myeloma Cell Lines.},
journal = {European journal of haematology},
volume = {116},
number = {4},
pages = {391-401},
doi = {10.1111/ejh.70084},
pmid = {41423250},
issn = {1600-0609},
support = {//Paula and Rodger Riney Foundation/ ; },
mesh = {Humans ; *Multiple Myeloma/genetics/metabolism/drug therapy/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; *Ferritins/genetics ; Reactive Oxygen Species/metabolism ; Iron/metabolism ; CRISPR-Cas Systems ; *Immunomodulating Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Oxidative Stress ; Oxidoreductases ; },
abstract = {BACKGROUND: Immunomodulatory agents (IMiDs) are a cornerstone in the successful management of multiple myeloma (MM). However, acquired IMiD resistance leading to disease relapses remains a major barrier. Hydrogen peroxide generation and oxidative stress are key mediators that determine IMiD's effectiveness in MM. Iron plays a key role in the generation of oxidative stress; therefore, cellular iron levels are tightly governed. FTH1 is the major iron storage protein that tightly regulates cellular iron availability. Hence, the present study is targeted to investigate the role of FTH1 in MM and IMiD resistance.

METHODS: IMiD-sensitive and IMiD-resistant MM cells were analyzed for expression of iron-metabolism genes. CRISPR-cas9-mediated knockout of FTH1 was performed and the after-effects were assessed through multiple experiments.

RESULTS: Initial analysis showed a positive correlation between FTH1 expression and IMiD resistance in MM cells. FTH1-KO reduced IMiD sensitivity in the KMS11 cell line but had no effect on the RPMI8226 cell line. RNA-seq data showed downregulation of ER-stress and calcium signaling genes after FTH1-KO. Further, KMS11-FTH1KO cells exhibited lower intracellular ROS, labile-iron, and mitochondrial superoxide levels along with increased CD63, suggesting activation of L-ferritin secretory pathways.

CONCLUSION: Data reveals a link between FTH1, labile iron, ROS, and IMiD resistance in MM cells.},
}

Humans
*Multiple Myeloma/genetics/metabolism/drug therapy/pathology
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
Gene Knockout Techniques
*Ferritins/genetics
Reactive Oxygen Species/metabolism
Iron/metabolism
CRISPR-Cas Systems
*Immunomodulating Agents/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Oxidative Stress
Oxidoreductases

@article {pmid41376159,
year = {2026},
author = {Ha, AS and Kalter, N and Rosenberg, M and Acevedo, LA and Liang, B and Liu, W and Paruthiyil, S and Sinha, M and Vu, A and Nguyen, V and Qi, Z and Krishnappa, N and Shu, J and Yu, J and Catanzaro, J and Bluestone, JA and Tang, Q and Urnov, FD and Marson, A and Hendel, A and Herold, KC and Shy, BR and Esensten, JH},
title = {Gene-corrected regulatory T cell therapy for IL2RA deficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1367-1381},
doi = {10.1016/j.ymthe.2025.12.004},
pmid = {41376159},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/metabolism/transplantation ; *Interleukin-2 Receptor alpha Subunit/genetics/deficiency ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Gene Editing ; Male ; Female ; Interleukin-2 ; Mutation ; },
abstract = {Bi-allelic germline deficiency of IL2RA causes a rare autoimmune disease with impaired regulatory T cell (Treg) function and interleukin-2 (IL-2) signaling. Definitive treatment is currently limited to allogeneic hematopoietic stem cell transplantation, which carries significant morbidity and mortality risks. We previously identified a family with three siblings affected by compound heterozygous mutations in their IL2RA gene, resulting in dysfunctional Tregs. Here, we introduce a novel therapeutic approach involving ex vivo generation of gene-corrected autologous regulatory T cells (gcTregs). One of the two disease-causing mutations in patient-derived Tregs was corrected with CRISPR-Cas9-mediated homology-directed repair, restoring IL2RA expression. The resulting gcTregs demonstrated robust suppressive activity in vitro. Clinical-scale manufacturing from a patient with IL2RA deficiency showed efficient gene correction, restored IL2RA expression, and functional equivalence to healthy donor Tregs. This work establishes a Good Manufacturing Practice-compatible manufacturing process for personalized gcTreg therapies, potentially providing a safer treatment option for patients with IL2RA deficiency as well as a framework for treating other inborn errors of immunity.},
}

Humans
*T-Lymphocytes, Regulatory/immunology/metabolism/transplantation
*Interleukin-2 Receptor alpha Subunit/genetics/deficiency
*Genetic Therapy/methods
CRISPR-Cas Systems
Gene Editing
Male
Female
Interleukin-2
Mutation

@article {pmid41376155,
year = {2026},
author = {Jin, X and Wu, X and Song, J and Luo, M and Ye, Q and Ren, C and Song, L and Li, M and Hu, M and An, Y and Su, J and Fu, J and Xu, Q and Luo, M and Liu, F and Liu, M and Li, Q and Yao, S and Chen, L and Yang, Y},
title = {Comparative evaluation of liver-directed knockin strategies with viral and nonviral vectors in mouse inherited disease models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1775-1793},
doi = {10.1016/j.ymthe.2025.12.012},
pmid = {41376155},
issn = {1525-0024},
mesh = {Animals ; *Genetic Vectors/genetics/administration & dosage ; Mice ; Disease Models, Animal ; Dependovirus/genetics ; *Liver/metabolism ; *Gene Knock-In Techniques/methods ; Genetic Therapy/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Recombinational DNA Repair ; *Hemophilia B/genetics/therapy ; Transgenes ; },
abstract = {CRISPR-Cas9-mediated gene knockin has emerged as a promising strategy for early-onset genetic disease intervention. However, the therapeutic efficacy and editing outcomes of different knockin strategies remain incompletely understood. Here, we systematically evaluated three major liver-directed knockin strategies, namely homology-directed repair (HDR), homology-independent targeted integration (HITI), and homology-mediated end joining (HMEJ), using neonatal mouse models of mucopolysaccharidosis type I and hemophilia B. Although all three approaches effectively rescued disease phenotypes, we observed distinct editing outcomes. Notably, the HMEJ approach, delivered via a combined adeno-associated virus-lipid nanoparticle (AAV-LNP) system, exhibited superior integration efficiency (5.8%-5.9%) and fidelity (97%-98%) compared with HDR and HITI. In contrast, whole-genome sequencing indicated that HITI induced a higher risk of random AAV donor integration than HDR or HMEJ. Furthermore, long-read sequencing analyses revealed that the frequencies of inverted terminal repeat (ITR)-mediated transgene integration differed between the 5' and 3' genomic junctions among the three strategies. Specifically, in HDR- and HMEJ-treated mice, ITR-mediated integration events were 7.7- to 19.7-fold more common at the 3' junctions than at the 5' junctions. These findings highlight the comprehensive advantages of the AAV-LNP-mediated HMEJ approach for liver-directed knockin therapy and suggest its strong potential for clinical translation.},
}

Animals
*Genetic Vectors/genetics/administration & dosage
Mice
Disease Models, Animal
Dependovirus/genetics
*Liver/metabolism
*Gene Knock-In Techniques/methods
Genetic Therapy/methods
Gene Editing/methods
Humans
CRISPR-Cas Systems
Recombinational DNA Repair
*Hemophilia B/genetics/therapy
Transgenes

@article {pmid41370232,
year = {2026},
author = {Alok, A and Raman, V and D'Agostino, L and Kshetry, AO and Rai, KM and Wang, C and Gunapati, S and Stupar, RM and Patil, GB and Zhang, F},
title = {Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiaf640},
pmid = {41370232},
issn = {1532-2548},
support = {IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; #2021-67013-34565//USDA NIFA/ ; //Texas Governor's University Research/ ; },
mesh = {*Glycine max/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Growth Regulators/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Soybean (Glycine max) transformation remains challenging and has not kept pace with rapid advances in genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 (WUS2) and the gene encoding isopentenyltransferase (IPT) achieved higher transformation efficiencies (14.6% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produced heritable transgenic events within 9 to 11 weeks and successfully delivered CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2/IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation frequency, reduces tissue culture requirements, and offers a promising genome-editing platform for soybean improvement.},
}

*Glycine max/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Plant Growth Regulators/metabolism
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism

@article {pmid41332134,
year = {2026},
author = {Inuzuka, T and Mouzannar, K and Zhang, M and Umarova, R and Park, SB and Uchida, T and Ma, CD and Liang, TJ},
title = {A CRISPR-based genome-wide loss-of-function screen defines a role of host metabolism in regulating hepatitis B virus infection.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1616-1632},
pmid = {41332134},
issn = {1525-0024},
support = {Z01 DK054500/ImNIH/Intramural NIH HHS/United States ; Z01 DK054504/ImNIH/Intramural NIH HHS/United States ; Z99 DK999999/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Hepatitis B virus/genetics/physiology ; *Hepatitis B/metabolism/virology/genetics ; *CRISPR-Cas Systems ; Hep G2 Cells ; Virus Replication/genetics ; Hepatocytes/metabolism/virology ; *Host-Pathogen Interactions/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Hepatitis B virus (HBV) co-opts and interacts with an extensive array of host factors for productive infection. Herein, we develop an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host dependency factors. HepG2[NTCP/Cas9] cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR, and NTCP. Among the novel candidate genes, knockout of 12 genes significantly decreased HBV replication markers. Validation using siRNA knockdown in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV proviral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication. Our study demonstrates the value of the HBV reporter system in identifying previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development.},
}

Humans
*Hepatitis B virus/genetics/physiology
*Hepatitis B/metabolism/virology/genetics
*CRISPR-Cas Systems
Hep G2 Cells
Virus Replication/genetics
Hepatocytes/metabolism/virology
*Host-Pathogen Interactions/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41267399,
year = {2026},
author = {Dua, PH and Simon, BMJ and Marley, CBE and Feliciano, CM and Watry, HL and Cowan, QT and Steury, D and Abraham, A and Gilbertson, EN and Ramey, GD and Capra, JA and Conklin, BR and Judge, LM},
title = {Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1633-1651},
doi = {10.1016/j.ymthe.2025.11.026},
pmid = {41267399},
issn = {1525-0024},
support = {R01 NS119678/NS/NINDS NIH HHS/United States ; U01 ES032673/ES/NIEHS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation, Missense ; *Haplotypes ; *Charcot-Marie-Tooth Disease/genetics/therapy ; Motor Neurons/metabolism ; Polymorphism, Single Nucleotide ; Alleles ; Genetic Therapy/methods ; Phenotype ; },
abstract = {Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Induced Pluripotent Stem Cells/metabolism/cytology
*Mutation, Missense
*Haplotypes
*Charcot-Marie-Tooth Disease/genetics/therapy
Motor Neurons/metabolism
Polymorphism, Single Nucleotide
Alleles
Genetic Therapy/methods
Phenotype

@article {pmid41247434,
year = {2026},
author = {Liu, F and Ge, D and Lian, G and Han, Z and Chai, J},
title = {Unraveling plant immunity: from pathogen perception to resistance engineering.},
journal = {Science China. Life sciences},
volume = {69},
number = {3},
pages = {779-793},
pmid = {41247434},
issn = {1869-1889},
mesh = {*Plant Immunity/genetics ; *Disease Resistance/genetics/immunology ; *Plant Diseases/immunology/microbiology ; Receptors, Pattern Recognition/metabolism/immunology ; NLR Proteins/metabolism/genetics ; Signal Transduction ; *Plants/immunology/genetics ; Calcium Signaling ; Plant Proteins/genetics/metabolism ; Host-Pathogen Interactions/immunology ; CRISPR-Cas Systems ; },
abstract = {Unlike animals, which rely on circulatory systems and mobile immune cells, each plant cell must autonomously detect and respond to pathogenic threats. Plant immunity operates through two major layers: pattern-triggered immunity (PTI), initiated by cell-surface pattern recognition receptors (PRRs), and effector-triggered immunity (ETI), primarily mediated by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors. Recent advances have substantially enhanced our understanding of PTI and ETI signaling. Notably, some NLRs following activation by specific recognition of pathogen effectors form higher-order oligomeric complexes termed resistosomes that act as Ca[2+]-permeable channels to trigger immune signaling. Increasing evidence points to extensive crosstalk and mutual potentiation between PTI and ETI, with Ca[2+] functioning as a pivotal second messenger in both pathways. Elucidating the molecular basis of these pathways, combined with emerging tools like CRISPR/Cas9, offers new strategies for engineering durable disease resistance in crops. This review highlights current insights into PTI and ETI, with an emphasis on the central role of Ca[2+] signaling and key challenges in engineering NLR receptors.},
}

*Plant Immunity/genetics
*Disease Resistance/genetics/immunology
*Plant Diseases/immunology/microbiology
Receptors, Pattern Recognition/metabolism/immunology
NLR Proteins/metabolism/genetics
Signal Transduction
*Plants/immunology/genetics
Calcium Signaling
Plant Proteins/genetics/metabolism
Host-Pathogen Interactions/immunology
CRISPR-Cas Systems

@article {pmid41170937,
year = {2026},
author = {Soliman, H and Akram, N and Saleh, M},
title = {CRISPR-Enhanced RAA-SHERLOCK Assay for Point-of-Care Detection of Cyprinid Herpesvirus-3: Development, Validation and Clinical Application.},
journal = {Journal of fish diseases},
volume = {49},
number = {4},
pages = {e70079},
pmid = {41170937},
issn = {1365-2761},
support = {//Veterinary Medicine University/ ; //Academy of Scientific Research and Technology/ ; },
mesh = {*Fish Diseases/diagnosis/virology ; Animals ; *Herpesviridae/isolation & purification/genetics ; *Herpesviridae Infections/veterinary/diagnosis/virology ; *Carps ; CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Point-of-Care Systems ; Recombinases/metabolism ; },
abstract = {Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance.},
}

*Fish Diseases/diagnosis/virology
Animals
*Herpesviridae/isolation & purification/genetics
*Herpesviridae Infections/veterinary/diagnosis/virology
*Carps
CRISPR-Cas Systems
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/veterinary/methods
*Point-of-Care Systems
Recombinases/metabolism

@article {pmid41078118,
year = {2026},
author = {Wang, Y and Yang, J and Hou, H and Song, L and Cheng, X and Liu, YX},
title = {Advancing Plant Microbiome Research Through Host DNA Depletion Techniques.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1189-1203},
pmid = {41078118},
issn = {1467-7652},
support = {32470055//National Natural Science Foundation of China/ ; U23A20148//National Natural Science Foundation of China/ ; CAAS-BRC-CB-2025-01//Basic Research Center for Crop Biosafety Sciences/ ; CAAS-ZDRW202308//Agricultural Science and Technology Innovation Program/ ; },
mesh = {*Microbiota/genetics ; *Plants/microbiology/genetics ; *Metagenomics/methods ; *DNA, Plant/genetics/isolation & purification ; CRISPR-Cas Systems ; },
abstract = {Plants provide ecological habitats for diverse microorganisms, making accurate metagenomic sequencing essential for understanding the complex interactions that support plant growth, development and disease resistance. However, host DNA contamination poses a major challenge in plant microbiome studies, obscuring microbial genetic signatures and complicating the accurate analysis of microbial genomes. This review provides a comprehensive overview of current host DNA depletion strategies, including physical separation (e.g., filtration, gradient centrifugation), selective lysis and enzymatic treatments targeting plant cell walls. Advanced techniques such as targeted sequence capture with magnetic beads, methylation-based enrichment and nanopore selective sequencing offer additional options for host DNA removal. Despite these advances, current methods still face challenges in efficiency, specificity and applicability, emphasising the need for tailored strategies and the exploration of novel approaches for microbial enrichment. Innovations like CRISPR-Cas9 and chromatin immunoprecipitation-based host DNA depletion methods are proposed to provide novel directions for addressing current limitations. The development and refinement of host depletion techniques tailored to plant systems are crucial for enabling high-resolution, cost-effective metagenomic studies. These efforts promise to deepen our understanding of microbial diversity and functionality, ultimately accelerating microbiome-based innovations in crop improvement, sustainable agriculture and ecosystem resilience.},
}

*Microbiota/genetics
*Plants/microbiology/genetics
*Metagenomics/methods
*DNA, Plant/genetics/isolation & purification
CRISPR-Cas Systems

@article {pmid40744834,
year = {2026},
author = {Qin, Z and Deng, Z and Li, C and Wang, D and Ji, X and Chen, Z},
title = {AI sheds new light on genome editing.},
journal = {Trends in biotechnology},
volume = {44},
number = {3},
pages = {696-709},
doi = {10.1016/j.tibtech.2025.07.011},
pmid = {40744834},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Artificial Intelligence ; CRISPR-Cas Systems/genetics ; Humans ; Biotechnology ; },
abstract = {Artificial intelligence (AI) has revolutionized life sciences, driving transformative advances in engineering clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based genome editors for therapeutic and agricultural applications. Recent breakthroughs demonstrate how deep learning accelerates the discovery, engineering, and design of next-generation genome editing tools. In this review, we explore how AI-driven approaches are supercharging genome editing in three aspects: (i) structure-based methods for discovering novel genome editors neglected by conventional methods, (ii) engineering genome editors with enhanced properties, and (iii) the de novo design of entirely new genome editors endowed with bespoke functions. Finally, we discuss the current challenges and envision the future potential of data-driven AI to unlock new possibilities in genome editing, catalyzing innovations across biology and biotechnology.},
}

*Gene Editing/methods
*Artificial Intelligence
CRISPR-Cas Systems/genetics
Humans
Biotechnology

@article {pmid41754561,
year = {2026},
author = {Wupori, K and Garnett, L and Bello, A and Strong, JE},
title = {CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754561},
issn = {1999-4915},
support = {8//Genomics Research and Development Initiative/ ; CSSP-2022-CP-2546//Canadian Safety and Security Program/ ; },
mesh = {Humans ; *Hemorrhagic Fevers, Viral/diagnosis/virology ; *Point-of-Care Systems ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Point-of-Care Testing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.},
}

Humans
*Hemorrhagic Fevers, Viral/diagnosis/virology
*Point-of-Care Systems
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Point-of-Care Testing
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity

@article {pmid41754535,
year = {2026},
author = {Li, H and Wang, R and Li, J and Duan, W and Liang, Y and Sun, Q and Zhou, J and Zhang, Y},
title = {SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754535},
issn = {1999-4915},
support = {GJJKJ-2024-ZY//National Disease Control and Prevention Administration Public Health Talent Training Support Project/ ; ZDGWNLJS25-36//National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID)/ ; 2021YFC2302003//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Mice ; Virus Replication ; *Coxsackievirus Infections/virology ; *Enterovirus A, Human/genetics/physiology ; Humans ; *Enterovirus/genetics/physiology ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hand, Foot and Mouth Disease/virology ; Cell Line, Tumor ; Viral Load ; Interferon-beta/pharmacology ; },
abstract = {Coxsackievirus A16 (CVA16), a major etiological agent of hand, foot, and mouth disease, is increasingly contributing to neurological complications, with no vaccines or virus-specific antivirals currently available. To identify CVA16-restricting host factors, we investigated the role of the interferon-stimulated gene shiftless (SHFL), previously implicated in the control of other RNA viruses. Using CRISPR-Cas 9, we generated SHFL knockout rhabdomyosarcoma cells and assessed viral replication, cytopathic effects, and replication stage dynamics. We evaluated disease progression and tissue injury in neonatal mice infected with a mouse-adapted CVA16 strain. SHFL expression was strongly induced during CVA16 infection and was inducible by exogenous interferon-β treatment, and its loss markedly increased infectious virus production, accelerated early replication, and exerted severe cytopathic effects. In vivo, SHFL deficiency led to rapid weight loss, pronounced neurological signs, increased viral burden across multiple tissues, and uniform mortality, together with high viral loads and extensive pathological damage in the central nervous system, lungs, and skeletal muscle. Transcriptomic analyses revealed SHFL-dependent modulation of adhesion- and mitogen-activated protein kinase-related pathways. Overall, our results suggest SHFL as a key determinant of host resistance to CVA16, acting mainly at the post-transcriptional stage to limit viral spread and tissue injury, and highlight SHFL-linked pathways as promising host-directed antiviral targets.},
}

Animals
Mice
Virus Replication
*Coxsackievirus Infections/virology
*Enterovirus A, Human/genetics/physiology
Humans
*Enterovirus/genetics/physiology
Host-Pathogen Interactions
CRISPR-Cas Systems
Hand, Foot and Mouth Disease/virology
Cell Line, Tumor
Viral Load
Interferon-beta/pharmacology

@article {pmid41753780,
year = {2026},
author = {Palanisamy, V and Bosilevac, JM and Barkhouse, DA and Velez, SE and Dass, SC},
title = {Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants.},
journal = {Microorganisms},
volume = {14},
number = {2},
pages = {},
pmid = {41753780},
issn = {2076-2607},
support = {2020-67017-30776//USDA-NIFA/ ; },
abstract = {Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage-bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction-modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria-phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms.},
}

@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Brain Neoplasms/therapy/genetics ; Animals ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}

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

@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*CRISPR-Cas Systems ; *Hordeum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Ascomycota/pathogenicity ; *Microbiota/genetics ; Alleles ; Plant Roots/microbiology/genetics ; *Plant Proteins/genetics ; Mutation ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}

*CRISPR-Cas Systems
*Hordeum/genetics/microbiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
*Ascomycota/pathogenicity
*Microbiota/genetics
Alleles
Plant Roots/microbiology/genetics
*Plant Proteins/genetics
Mutation

@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genomics/methods ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems
*Genomics/methods
Phenotype
Genotype
Animals

@article {pmid41751799,
year = {2026},
author = {Cai, X and Liang, X and Zou, P and Xiao, R and Wang, Y},
title = {CRISPRi Screening Identifies Essential E. coli Virulence Factors for Placental Barrier Breach in a Maternal-Fetal Infection Model.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751799},
issn = {1422-0067},
support = {82574169//National Natural Science Foundation of China/ ; 7232009, 7244289//Natural Science Foundation of Beijing Municipality/ ; 20240484724//Cross-cooperation project of Beijing Science and Technology New Star Program/ ; Subject leaders-03-02//High Level Public Health Technical Personnel Construction Project/ ; //Research Foundation of Capital Institute of Pediatrics/ ; //Beijing Chaoyang District Postdoctoral Research Foundation 2024/ ; },
mesh = {Female ; Pregnancy ; Animals ; *Virulence Factors/genetics/metabolism ; *Escherichia coli Infections/microbiology/genetics ; *Escherichia coli/pathogenicity/genetics ; Rats ; *Placenta/microbiology/metabolism ; Humans ; Disease Models, Animal ; Escherichia coli Proteins/genetics/metabolism ; Host-Pathogen Interactions ; Trophoblasts/microbiology/metabolism ; CRISPR-Cas Systems ; *Pregnancy Complications, Infectious/microbiology ; Neonatal Sepsis/microbiology ; },
abstract = {Early-onset neonatal sepsis caused by Escherichia coli (E. coli) threatens neonates' lives due to the pathogen's high virulence and multidrug resistance. The mechanisms that enable its placental barrier breach are poorly understood. Using a clinically isolated ST95 ExPEC strain from a neonatal sepsis case, along with a pregnant rat model and an in vitro placental barrier model, we performed CRISPR interference screening. This screen targeted 264 virulence factor genes and identified virulence factors for motility, iron acquisition, hemolysin secretion, and adherence/invasion as critical. We demonstrated that hlyB is essential for uterine infection, and we elucidated a mechanism for ibeA that facilitates syncytial trophoblast cell layer penetration by interacting with the host receptor(s) PSF/VIM to enhance bacterial internalization. Host cells countered ibeA+ E. coli infection via a novel host defense pathway involving upregulation of ASPHD1. This study systematically mapped the virulence factors required for E. coli placental translocation and delineated key host-pathogen interactions.},
}

Female
Pregnancy
Animals
*Virulence Factors/genetics/metabolism
*Escherichia coli Infections/microbiology/genetics
*Escherichia coli/pathogenicity/genetics
Rats
*Placenta/microbiology/metabolism
Humans
Disease Models, Animal
Escherichia coli Proteins/genetics/metabolism
Host-Pathogen Interactions
Trophoblasts/microbiology/metabolism
CRISPR-Cas Systems
*Pregnancy Complications, Infectious/microbiology
Neonatal Sepsis/microbiology

@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {*Bombyx/genetics/growth & development ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Agriculture/methods ; Silk/genetics ; Genome, Insect ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}

*Bombyx/genetics/growth & development
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Agriculture/methods
Silk/genetics
Genome, Insect
Genomics/methods

@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Cell Proliferation/genetics ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; HEK293 Cells ; Apoptosis/genetics ; CRISPR-Cas Systems ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; Gene Knockout Techniques ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}

Humans
*Cell Proliferation/genetics
*Vesicular Transport Proteins/genetics/deficiency/metabolism
HEK293 Cells
Apoptosis/genetics
CRISPR-Cas Systems
Mitochondrial Dynamics/genetics
Mitochondria/metabolism/genetics
Gene Knockout Techniques

@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Disease Models, Animal ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; Sheep/genetics ; Male ; Female ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.

METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.

RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}

Animals
*Fragile X Syndrome/genetics/pathology
*Disease Models, Animal
*Fragile X Messenger Ribonucleoprotein 1/genetics
Sheep/genetics
Male
Female
CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
Humans

@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Candida/genetics/pathogenicity ; Humans ; Virulence/genetics ; Genome, Fungal ; Candidiasis/microbiology/genetics ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Candida/genetics/pathogenicity
Humans
Virulence/genetics
Genome, Fungal
Candidiasis/microbiology/genetics

@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Listeria monocytogenes/drug effects ; *Probiotics ; *CRISPR-Cas Systems ; *Endopeptidases/genetics/pharmacology/metabolism ; *Saccharomyces boulardii/genetics/metabolism ; Humans ; Bacteriophages/enzymology ; *Anti-Bacterial Agents/pharmacology/metabolism ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}

*Listeria monocytogenes/drug effects
*Probiotics
*CRISPR-Cas Systems
*Endopeptidases/genetics/pharmacology/metabolism
*Saccharomyces boulardii/genetics/metabolism
Humans
Bacteriophages/enzymology
*Anti-Bacterial Agents/pharmacology/metabolism
Saccharomyces cerevisiae/genetics

@article {pmid41719925,
year = {2026},
author = {Park, YJ and Song, DY and Jeon, HB and Kim, DM},
title = {Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.},
journal = {Biosensors & bioelectronics},
volume = {301},
number = {},
pages = {118514},
doi = {10.1016/j.bios.2026.118514},
pmid = {41719925},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Cell-Free System ; *Protein Biosynthesis/genetics ; *Escherichia coli O157/genetics ; Bacillus anthracis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Ribosomal, 16S/genetics/analysis ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins/genetics ; },
abstract = {We present a modular platform that converts CRISPR target recognition into programmable protein outputs for nucleic acid detection. This system couples Cas-mediated collateral cleavage with cell-free protein synthesis. In the presence of a target, Cas-mediated collateral cleavage releases an extension-competent trigger DNA that gates reporter expression. Although collateral cleavage is inherently indiscriminate, we achieve deterministic fragment generation by employing chemically programmed precursors bearing backbone modifications-exemplified here by phosphorothioate linkages-that constrain cleavage to predefined sites. Using Bacillus anthracis and E. coli O157:H7 as a model, the developed CRIVER assay enables concurrent readouts of 16S rRNA together with the species-specific capB or ecf1 loci by integrating Cas13a-mediated RNA recognition and Cas12a-mediated DNA recognition into a dual-channel workflow. Taken together, proposed method establishes addressable signaling at the protein layer, supports protein-based outputs in a modular workflow, and provides a general route to sensitive, dual-channel nucleic acid detection.},
}

*Biosensing Techniques/methods
Cell-Free System
*Protein Biosynthesis/genetics
*Escherichia coli O157/genetics
Bacillus anthracis/genetics
*CRISPR-Cas Systems/genetics
*RNA, Ribosomal, 16S/genetics/analysis
*Nucleic Acids/analysis/genetics
Bacterial Proteins/genetics

@article {pmid41700332,
year = {2026},
author = {Huang, Y and Zhao, Z and Li, J and Wang, X and Qu, H and Zheng, Y},
title = {An aptamer-CRISPR/Cas12a biosensor for rapid and sensitive detection of florfenicol.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1910-1916},
doi = {10.1039/d5ay01903f},
pmid = {41700332},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *Thiamphenicol/analogs & derivatives/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Animals ; Food Contamination/analysis ; },
abstract = {Florfenicol (FF), a broad-spectrum antibacterial agent widely used in livestock and poultry farming, has raised significant food safety concerns due to the accumulation of its residues in animal-derived products (e.g., eggs), posing potential threats to human health. Herein, we developed a novel aptamer-CRISPR/Cas12a biosensor for the rapid and sensitive detection of FF. The biosensor employs streptavidin-modified magnetic beads (SA-MBs) as a solid carrier to achieve efficient enrichment of FF-specific aptamers (APT), while integrating the dual advantages of the APT's high-specificity target recognition and the CRISPR/Cas12a system's powerful signal amplification capability. The detection mechanism is based on a competitive displacement: APT pre-hybridizes with its complementary strand (APT-c) to form stable duplexes. The presence of FF triggers the release of APT-c from APT, and the liberated APT-c then activates the trans-cleavage activity of the CRISPR/Cas12a system. This process converts the small-molecule FF into a CRISPR/Cas12a-detectable nucleic acid signal and enables quantitative FF detection. Under optimized conditions, the biosensor demonstrated a linear detection range of 10 nM to 100 µM for FF (R[2] = 0.9907) and a limit of detection (LOD) of 1.41 nM. The accuracy and practicality were confirmed through spiked recovery experiments in egg samples, yielding recoveries between 97.1% and 100.8%. Furthermore, the modular design of this platform allows its easy adaptation for detecting other antibiotics simply by replacing the specific APT and its corresponding APT-c, highlighting its considerable potential for broad applications in food safety monitoring.},
}

*Biosensing Techniques/methods
*Thiamphenicol/analogs & derivatives/analysis
*Aptamers, Nucleotide/chemistry/genetics
*CRISPR-Cas Systems
*Anti-Bacterial Agents/analysis
Limit of Detection
Animals
Food Contamination/analysis

@article {pmid41693124,
year = {2026},
author = {Xin, W and Tang, Z and Wang, S and Song, ZL and Luo, X},
title = {Scaffold-Proximal DNA Extensions Enhance Cas12a Trans-cleavage for Direct and Broad-Scope Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {8},
pages = {6149-6162},
doi = {10.1021/acs.analchem.5c07125},
pmid = {41693124},
issn = {1520-6882},
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Polymorphism, Single Nucleotide ; *RNA, Messenger/analysis ; *Bacterial Proteins/metabolism ; },
abstract = {The CRISPR/Cas12a system has revolutionized nucleic acid diagnostics, but its direct application for RNA detection remains constrained by the requisite reverse transcription step, insufficient sensitivity, and poor compatibility across diverse RNA targets such as microRNAs (miRNAs), long mRNAs, and single nucleotide polymorphisms (SNPs). Here, we introduce a rationally engineered DNA activator architecture that markedly enhances Cas12a trans-cleavage activity and expands its analytical utility. A key mechanistic finding is the strong positional dependence of activator extensions: appending an overhang specifically at the scaffold-proximal end of the DNA activator (termed Proximal-Extended Activator, PEA) potently boosts Cas12a activation through ribonucleoprotein (RNP) stabilization, whereas distal extensions are inhibitory due to steric hindrance of essential interdomain motions. This optimized PEA system facilitates direct, amplification-free RNA detection, achieving exceptional sensitivity with detection limits of 1.3 fM for miRNA and 93 fM for mRNA, all without reverse transcription. Furthermore, a Split-PEA format confers exceptional discriminatory power for SNPs, enabling robust identification of the EGFR T790 M mutation at a 0.1% allelic frequency. This work establishes a facile and versatile platform where simple sequence modification enables highly sensitive and specific analysis of a broad range of nucleic acid targets, effectively overcoming a significant hurdle in CRISPR-based diagnostics.},
}

*CRISPR-Associated Proteins/metabolism/chemistry
CRISPR-Cas Systems
*DNA/chemistry/metabolism/genetics
*MicroRNAs/analysis/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
Polymorphism, Single Nucleotide
*RNA, Messenger/analysis
*Bacterial Proteins/metabolism

@article {pmid41671611,
year = {2026},
author = {Raban, R and James, AA and Akbari, OS},
title = {Advances in CRISPR gene drives for mosquito population control.},
journal = {Current opinion in microbiology},
volume = {90},
number = {},
pages = {102712},
doi = {10.1016/j.mib.2026.102712},
pmid = {41671611},
issn = {1879-0364},
mesh = {Animals ; *Gene Drive Technology/methods ; *Culicidae/genetics ; *Mosquito Control/methods ; *CRISPR-Cas Systems ; *Mosquito Vectors/genetics ; },
abstract = {CRISPR-based gene drive (GD) systems bias allele inheritance during meiosis, enabling transgenes to spread at rates exceeding Mendel's law of segregation. This capability underlies their potential as powerful tools for controlling mosquito-borne diseases. GDs can be engineered either to suppress mosquito populations or to modify them by introducing traits that block pathogen transmission. Recent advances have focused on improving evolutionary stability, with modeling studies providing insights into expected population dynamics. With a focus on the most current population modification GDs, we discuss advances in GD architectures - including integral and allelic drives, combined modification-suppression systems, and both homing and non-homing toxin-antidote designs - that expand the range of possible strategies and address limitations of early homing drives. Numerous antipathogen effectors with strong pathogen-blocking activity can now be coupled to these systems, with current efforts assessing their durability against genetically diverse pathogens. Key challenges remain, including resistance evolution, ecological impacts, and long-term stability. Nonetheless, GDs offer a promising approach for reducing disease transmission, especially in regions where conventional interventions are difficult to sustain.},
}

Animals
*Gene Drive Technology/methods
*Culicidae/genetics
*Mosquito Control/methods
*CRISPR-Cas Systems
*Mosquito Vectors/genetics

@article {pmid41671237,
year = {2026},
author = {Bagheri, N and Bertucci, A and Merlo, R and Porchetta, A},
title = {Synthetic DNA Transducers Integrate DNA Repair to CRISPR Signal Transduction.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1634-1644},
doi = {10.1021/acssensors.5c04118},
pmid = {41671237},
issn = {2379-3694},
mesh = {*DNA Repair ; Humans ; *CRISPR-Cas Systems/genetics ; DNA Glycosylases/metabolism ; Uracil-DNA Glycosidase/metabolism ; *Signal Transduction ; *DNA/genetics/metabolism/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-based molecular diagnostics have revolutionized nucleic acid detection, yet the integration of upstream enzyme activity into programmable CRISPR output remains largely unexplored. Here, we present a synthetic transduction platform that directly couples endogenous DNA repair activity with CRISPR-Cas12a activation. By linking base excision repair (BER) events to the structural switching of a programmable DNA transducer, we convert the activity of DNA glycosylases, such as uracil DNA glycosylase (UDG) and human 8-oxoguanine glycosylase (hOGG1), into a robust fluorescence signal via Cas12a-mediated collateral (trans-) cleavage. This one-step assay allows rapid and sensitive lysate-based detection of repair activity with high specificity. In addition, it can also be easily adapted to achieve rapid throughput screening of small molecule inhibitors. The rational modular design supports the adaptation to various glycosylase activities, establishing a general framework for transducing DNA repair activity into programmable CRISPR output. Beyond bioanalytical applications, this approach paves the way for the development of synthetic gene circuits that respond to DNA repair activity and CRISPR-based drug screening platforms.},
}

*DNA Repair
Humans
*CRISPR-Cas Systems/genetics
DNA Glycosylases/metabolism
Uracil-DNA Glycosidase/metabolism
*Signal Transduction
*DNA/genetics/metabolism/chemistry
CRISPR-Associated Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/metabolism/genetics

@article {pmid41670012,
year = {2026},
author = {Miao, Y and Li, C and Su, Y and Peng, T and Wang, J and Liu, S and Ma, C and Li, L and Wang, Y},
title = {The application of CRISPR-Cas9 system in brain diseases.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {6},
pages = {899-920},
pmid = {41670012},
issn = {1748-6963},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Brain Diseases/therapy/genetics ; *Gene Editing/methods ; Blood-Brain Barrier/metabolism ; *Genetic Therapy/methods ; Animals ; Genetic Vectors/genetics ; },
abstract = {As an efficient genome-editing technology, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated protein9 (Cas9) system is increasingly being recognized as a significant therapeutic strategy for brain diseases. In recent years, researchers have continuously tried to regulate the expression of genes related to the nervous system through CRISPR-Cas9 system, which provides a new and efficient strategy for the treatment of brain diseases. At the same time, various delivery vectors of CRISPR-Cas9 system have been reported. Although some delivery vectors have not been applied to the research of brain diseases, they still provide valuable ideas for the brain delivery of CRISPR-Cas9 system. In this review, we summarized the principle of CRISPR-Cas9 system and its application in the nervous system, discussed the barrier of blood-brain barrier (BBB) to the treatment of brain diseases, overviewed various delivery vectors of CRISPR-Cas9 system and their applications, and highlighted advanced of CRISPR-Cas9 system applied to various brain diseases. Furthermore, we also discussed the existing obstacles and promising avenues for future investigation regarding CRISPR-Cas9-based therapeutic approaches. This article, through retrieving keyword combinations[PubMed,from Jan. 2018 to Dec. 2025], aims to elucidate the CRISPR-Cas9 system's potential for extensive future research and application as a therapeutic strategy for brain disorders.},
}

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

@article {pmid41663776,
year = {2026},
author = {Pearce, JC and Campbell, JS and Prior, JL and Titball, RW and Wakefield, JG},
title = {PiggyBac-mediated transgenesis and CRISPR-Cas9 knockout in the greater wax moth, Galleria mellonella.},
journal = {Lab animal},
volume = {55},
number = {3},
pages = {95-102},
pmid = {41663776},
issn = {1548-4475},
support = {NC/W002388/1//National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)/ ; NC/T001518/1//National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Moths/genetics ; *DNA Transposable Elements/genetics ; *Gene Knockout Techniques/methods ; Larva/genetics ; Animals, Genetically Modified/genetics ; *Gene Transfer Techniques ; },
abstract = {The larvae of the greater wax moth, Galleria mellonella, are gaining prominence as a versatile nonmammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37 °C, coupled with a broad susceptibility to human pathogens and a distinct melanization response that serves as a visual indicator for larval health, positions G. mellonella as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and Drosophila melanogaster, has hindered development of the full potential of G. mellonella as a model organism. Here we describe a robust methodology for generating transgenic G. mellonella using the PiggyBac transposon system and for precise gene knockouts via CRISPR-Cas9 technology. These advances significantly enhance the utility of G. mellonella in molecular research, paving the way for its widespread use as an inexpensive and ethically compatible animal model in infection biology and beyond.},
}

Animals
*CRISPR-Cas Systems
*Moths/genetics
*DNA Transposable Elements/genetics
*Gene Knockout Techniques/methods
Larva/genetics
Animals, Genetically Modified/genetics
*Gene Transfer Techniques

@article {pmid41649900,
year = {2026},
author = {Zakiyyah, SN and Irkham, and Wulandari, DA and Zein, MIHL and Gaffar, S and Ozsoz, M and Ogata, G and Einaga, Y and Hartati, YW},
title = {CRISPR-Cas13a-Powered Electrochemical Biosensor for Label-Free Detection of Chikungunya Virus RNA Using a Ceria-Modified Screen-Printed Carbon Electrode.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {923-938},
doi = {10.1021/acssensors.5c01924},
pmid = {41649900},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Chikungunya virus/genetics/isolation & purification ; *RNA, Viral/analysis/genetics ; *Cerium/chemistry ; Electrodes ; *Electrochemical Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Carbon/chemistry ; Limit of Detection ; Humans ; },
abstract = {This study aims to develop a label-free electrochemical biosensor for the detection of chikungunya virus (CHIKV) RNA, based on CRISPR-Cas13a integrated with a cerium oxide (ceria)-modified screen-printed carbon electrode (SPCE). The ceria film was deposited through cathodic electrodeposition, forming a uniform, needle-like film, as observed by SEM, and a crystalline fluorite structure was confirmed by XRD with characteristic (111), (200), and (220) reflections. The results showed that Raman spectroscopy demonstrated a dominant F2g band at ∼463 cm[-1], indicative of cubic ceria, while XPS analysis displayed the presence of 13.65% Ce[3+], contributing to favorable surface reactivity for biomolecule immobilization. This interface enhanced the attachment of a biotinylated RNA probe through streptavidin binding. Furthermore, a guide RNA (gRNA) was rationally designed to target the conserved region of the CHIKV E1 gene, with high specificity confirmed through in silico arrangement against related viruses. Upon target recognition, the activated Cas13a enzyme triggered collateral cleavage of the immobilized probe, leading to a measurable reduction in the guanine oxidation signal, detected by differential pulse voltammetry (DPV). This detection strategy was entirely label-free and amplification-free, simplifying both sensor fabrication and operation. The biosensor achieved a detection limit of 1.325 ppt, had a linear response in the range of 1-10,000 ppt, and showed excellent selectivity against DENV and SARS-CoV-2. It also retained signal stability over 45 days and yielded a recovery rate of 94.98% in spiked human serum. In conclusion, this study represents a modular and programmable sensing platform for direct RNA detection that integrates RNA-guided molecular recognition and signal transduction without the need for labeled substrates or amplification, simplifying CRISPR-based diagnostics supporting good health and well-being through field-deployable applications.},
}

*Biosensing Techniques/methods/instrumentation
*Chikungunya virus/genetics/isolation & purification
*RNA, Viral/analysis/genetics
*Cerium/chemistry
Electrodes
*Electrochemical Techniques/methods/instrumentation
*CRISPR-Cas Systems
Carbon/chemistry
Limit of Detection
Humans

@article {pmid41643803,
year = {2026},
author = {Kim, C and Zhu, Z and Tagmount, A and Barbazuk, WB and Bacher, R and Vulpe, CD},
title = {Physiologically relevant 3D CRISPR screening enhances mechanistic insight into chemical toxicity compared to 2D screening.},
journal = {Toxicology},
volume = {522},
number = {},
pages = {154422},
doi = {10.1016/j.tox.2026.154422},
pmid = {41643803},
issn = {1879-3185},
mesh = {Humans ; Hep G2 Cells ; *Toxicogenetics/methods ; *CRISPR-Cas Systems ; *Doxorubicin/toxicity ; *Toxicity Tests/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Spheroids, Cellular/drug effects ; Bioreactors ; *Cell Culture Techniques, Three Dimensional ; },
abstract = {CRISPR-based approaches can complement other genomics-based toxicology studies by enabling causal interrogation of gene function modulating chemical-induced toxicity. Moreover, CRISPR screens enable scalable and systematic identification of functional pathways involved in cellular response to chemical exposure. Cell-based functional toxicogenomics approaches using CRISPR provide a potential powerful tool for the development of mechanism-driven new approach methodologies (NAMs) for toxicodynamic and toxicokinetic hazard screening to enable more effective risk assessment. To improve the physiological relevance of in vitro functional toxicogenomics, we developed a three-dimensional (3D) CRISPR screening platform using HepG2/C3A spheroids cultured in a continuously rotating bioreactor (ClinoStar). We evaluated the potential utility of a 3D CRISPR screen as compared to conventional 2D screen using a custom CRISPR sgRNA library representing common loss-of-function genetic variants in the human population and exposure to the well characterized DNA damaging toxicant, doxorubicin. The 3D platform identified more genes and pathways in which variants have previously been associated with doxorubicin toxicity in clinical studies than the 2D system. These results support the utility of 3D CRISPR screening to identify physiologically relevant genetic determinants underlying chemical toxicity.},
}

Humans
Hep G2 Cells
*Toxicogenetics/methods
*CRISPR-Cas Systems
*Doxorubicin/toxicity
*Toxicity Tests/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Spheroids, Cellular/drug effects
Bioreactors
*Cell Culture Techniques, Three Dimensional

@article {pmid41637835,
year = {2026},
author = {Xie 谢飞, F and Liu 刘晓宙, X and Wang 王露露, L and Zhang 张聪, C and Liu 刘传宏, C and Huo 霍振庆, Z and Zhao 赵正东, Z and Zhao 赵清远, Q and He 贺秋月, Q and Guo 郭科男, K and Sun 孙宇, Y and Wang 王勇, Y},
title = {A porcine congenital deafness model with unconditional knockout of GJB2 generated by CRISPR/Cas9 genomic editing.},
journal = {Hearing research},
volume = {472},
number = {},
pages = {109552},
doi = {10.1016/j.heares.2026.109552},
pmid = {41637835},
issn = {1878-5891},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Connexin 26/genetics ; *Gene Editing ; *Deafness/genetics/physiopathology/congenital/pathology ; *Connexins/genetics/deficiency ; Phenotype ; *Hearing/genetics ; Gene Knockout Techniques ; Sus scrofa ; Homozygote ; Female ; Organ of Corti/physiopathology/pathology ; Mutation ; Animals, Genetically Modified ; Hair Cells, Auditory/pathology/metabolism ; Genetic Predisposition to Disease ; Male ; },
abstract = {GJB2, the primary gene responsible for DFNB1, the most prevalent non-syndromic hearing loss (NSHL), has variants that account for over 50% of all prelingual hearing loss (HL). Mice are the main model for congenital hearing loss (CHL) research, but they have delayed auditory maturation postnatally, and unconditional Gjb2 knockout in mice causes embryonic lethality. Pigs have similar inner-ear anatomy to humans and, like humans, have matured auditory function and fully differentiated cochlea at birth. Currently, there is no GJB2 unconditional knockout animal model for GJB2-related CHLs research, and whether unconditional GJB2 deletion causes embryonic lethality in pigs or if GJB2-deficient pigs can recapitulate typical clinical pathological characteristics remains unclear. In this study, we employed CRISPR/Cas9 to establish the first unconditional GJB2 knockout pig model. The mutant GJB2 alleles in the founder pig were stably germline-transmitted to subsequent generations. Homozygous GJB2 knockout pigs exhibited no embryonic lethality and showed profound hearing loss, cochlear hair cell depletion and impaired Organ of Corti's development. This GJB2 unconditional knockout pig model has not been reported before and demonstrates GJB2 mutation pathological characteristics consistent with clinical patients, validating its potential in investigating the pathogenic mechanisms and therapeutic interventions of GJB2-deficient CHLs.},
}

Animals
*CRISPR-Cas Systems
Disease Models, Animal
*Connexin 26/genetics
*Gene Editing
*Deafness/genetics/physiopathology/congenital/pathology
*Connexins/genetics/deficiency
Phenotype
*Hearing/genetics
Gene Knockout Techniques
Sus scrofa
Homozygote
Female
Organ of Corti/physiopathology/pathology
Mutation
Animals, Genetically Modified
Hair Cells, Auditory/pathology/metabolism
Genetic Predisposition to Disease
Male

@article {pmid41636718,
year = {2026},
author = {Xu, ZH and Hu, X and Weng, X and Lin, RM and Xu, W and Yu, LS and Gao, H},
title = {An "off-on" electrochemiluminescence biosensor based on CRISPR-Cas12a for ultrasensitive determination of aflatoxin B1.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1325-1331},
doi = {10.1039/d5an01369k},
pmid = {41636718},
issn = {1364-5528},
mesh = {*Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; Quantum Dots/chemistry ; *CRISPR-Associated Proteins/genetics ; Food Contamination/analysis ; Electrodes ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Accurate and sensitive determination of aflatoxin B1 (AFB1) is of vital importance for food safety. Herein, an electrochemiluminescence (ECL) "off-on" switch sensor combined with CRISPR-Cas12a is fabricated for the quantitative analysis of AFB1. Initially, the DNA activator is effectively locked by two AFB1 aptamers. Once the target AFB1 is introduced, the activator is released in an open state and thus is recognized by the Cas12a-crRNA duplex. Black hole quencher (BHQ)-marked DNA strands are digested by the activated CRISPR-Cas12a system. Owing to the resonance energy transfer (RET) between aggregation-induced ECL active (AIECL-active) polymer dots (Pdots) and BHQ, the ECL signal of Pdots switches from signal "off" to "on" along with the detachment of BHQ from the electrode surface. The proposed ECL sensor thus achieves sensitive quantification of AFB1 with a detection limit of 0.06 pg mL[-1]. This work provides an effective strategy for mycotoxin determination.},
}

*Aflatoxin B1/analysis
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Luminescent Measurements/methods
*CRISPR-Cas Systems
Aptamers, Nucleotide/chemistry/genetics
Limit of Detection
Quantum Dots/chemistry
*CRISPR-Associated Proteins/genetics
Food Contamination/analysis
Electrodes
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41620000,
year = {2026},
author = {Shah, W and Hussain, M and Serwat, A and Bilal, M and Raza, Y and Mansoor, A and Faraz, A},
title = {CRISPR/Cas9 and reproductive failure: applications, ethical challenges, and future perspectives in human germline genome editing.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {584},
number = {},
pages = {120875},
doi = {10.1016/j.cca.2026.120875},
pmid = {41620000},
issn = {1873-3492},
mesh = {Humans ; *Gene Editing/ethics ; *CRISPR-Cas Systems/genetics ; *Germ Cells/metabolism ; Animals ; *Infertility/genetics/therapy ; },
abstract = {Reproductive failure affects millions of couples worldwide and frequently arises from genetic defects that impair gametogenesis, fertilization, or early embryonic development. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome-editing technology has emerged as a powerful experimental platform for dissecting infertility-associated genes and, in principle, correcting pathogenic variants in germline cells or preimplantation embryos. This review critically examines current applications of CRISPR/Cas9 in reproductive biology, including disease modeling in animal systems, editing of spermatogonial stem cells (SSCs), manipulation of oocytes and zygotes, and proof-of-concept studies in human embryos. Particular emphasis is placed on the major technical barriers that currently preclude clinical translation, including off-target mutagenesis, embryo mosaicism, and the low efficiency of homology-directed repair relative to non-homologous end joining. Limitations related to delivery strategies, DNA damage responses, chromosomal rearrangements, and the genetic heterogeneity of infertility are also evaluated. Comparative discussion highlights how germline editing differs fundamentally from somatic CRISPR therapies that have already reached clinical application in hematologic disorders. The review further analyzes ethical and regulatory challenges associated with heritable genome modification, including long-term safety, consent across generations, international governance disparities, and the continued reliance on assisted reproductive technologies combined with preimplantation genetic testing as safer clinical alternatives. Collectively, current evidence indicates that CRISPR/Cas9 remains primarily a research tool for elucidating reproductive biology rather than an imminent therapeutic option for human infertility. Continued technological refinement, rigorous preclinical validation, and globally harmonized oversight will be essential before germline applications can be ethically or clinically justified.},
}

Humans
*Gene Editing/ethics
*CRISPR-Cas Systems/genetics
*Germ Cells/metabolism
Animals
*Infertility/genetics/therapy

@article {pmid41616988,
year = {2026},
author = {Yao, B and Öktem, M and Yang, G and Wang, Q and Daniels, MA and Dokter, I and Lefferts, JW and Gonçalves, MAFV and Doevendans, PA and van Mil, A and Sluijter, JPG and Schiffelers, R and Mastrobattista, E and Lei, Z},
title = {LAH5-mediated delivery of prime editor ribonucleoprotein complexes for genome editing.},
journal = {International journal of pharmaceutics},
volume = {692},
number = {},
pages = {126622},
doi = {10.1016/j.ijpharm.2026.126622},
pmid = {41616988},
issn = {1873-3476},
mesh = {Humans ; *Gene Editing/methods ; *Ribonucleoproteins/administration & dosage/genetics ; HEK293 Cells ; *Cell-Penetrating Peptides/administration & dosage/chemistry ; Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a precise gene-editing technology with potential for treating genetic disorders, but efficient delivery systems remain a challenge. Viral vectors offer high efficiency but pose safety concerns related with their immunogenicity, while non-viral methods struggle with stability and scalability. Cell-penetrating peptides (CPPs) present a promising alternative due to their low immunogenicity. In this study, we explored LAH5, a histidine-rich CPP, for delivering PE ribonucleoproteins (RNPs) into PLN R14del mutant cell lines. We purified engineered SpGPEmax protein, evaluating its intracellular uptake and editing frequency in HEK293T.PLN R14del reporter cells and human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our results demonstrate that LAH5 effectively delivers intracellularly SpGPEmax RNP components, resulting in correction of the R14del mutation, thereby offering a viable non-viral strategy for direct cellular precise genome editing.},
}

Humans
*Gene Editing/methods
*Ribonucleoproteins/administration & dosage/genetics
HEK293 Cells
*Cell-Penetrating Peptides/administration & dosage/chemistry
Induced Pluripotent Stem Cells/metabolism
Myocytes, Cardiac/metabolism
CRISPR-Cas Systems

@article {pmid41615337,
year = {2026},
author = {Liu, X and Wang, X and He, S and Li, M and Lu, C and Xing, C},
title = {An oxidative cleavage-based HCR-CRISPR/Cas12a biosensor for highly sensitive detection of hypochlorous acid.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1317-1324},
doi = {10.1039/d5an01299f},
pmid = {41615337},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Hypochlorous Acid/analysis ; *CRISPR-Cas Systems ; Oxidation-Reduction ; Limit of Detection ; DNA/chemistry/genetics ; Nucleic Acid Hybridization ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Water Pollutants, Chemical/analysis ; Bacterial Proteins ; },
abstract = {Hypochlorous acid (HClO) is a critical disinfectant in public health and water treatment, yet its excessive presence in environmental matrices poses significant risks to human health (e.g., respiratory irritation, tissue damage) and aquatic ecosystems (e.g., biodiversity loss). Herein, we developed an oxidative cleavage-based hybridization chain reaction (HCR)-CRISPR/Cas12a biosensor for highly sensitive detection of HClO. The sensor utilizes a dual-lock switch mechanism: HClO selectively cleaves a phosphorothioated (PS) DNA hairpin (Lock 1), releasing an initiator strand to trigger the HCR and generate PAM-containing long dsDNA. Simultaneously, the locked crRNA (L-crRNA), which is caged by a PS-modified silent chain (Lock 2), can be released when the PS-modified silent chain is degraded under HClO incubation, allowing the activated crRNA to dynamically regulate Cas12a-crRNA complex formation. Then, HCR-dsDNA activates Cas12a's trans-cleavage activity, enabling fluorescence signal readout via reporter DNA cleavage. The dual-lock strategy minimizes nonspecific activation in CRISPR/Cas12a systems, significantly enhancing sensitivity and specificity. Our work establishes a robust platform for environmental pollutant monitoring, with applications in water safety assessment and food quality control.},
}

*Biosensing Techniques/methods
*Hypochlorous Acid/analysis
*CRISPR-Cas Systems
Oxidation-Reduction
Limit of Detection
DNA/chemistry/genetics
Nucleic Acid Hybridization
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/genetics/metabolism
*Water Pollutants, Chemical/analysis
Bacterial Proteins

@article {pmid41605928,
year = {2026},
author = {Ganguly, C and Aribam, SD and Dos Santos, AM and Martin, L and Thomas, LM and Shao, Y and Rajan, R},
title = {Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41605928},
issn = {2041-1723},
support = {MCB-1716423 and MCB-2424888//National Science Foundation (NSF)/ ; R35GM153297 and R44GM133270//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Francisella/genetics/enzymology ; *R-Loop Structures ; DNA Cleavage ; Gene Editing ; DNA/metabolism ; },
abstract = {CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a[KD2P]) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12a[KD2P] that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a[WT]) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.},
}

*CRISPR-Associated Proteins/metabolism/genetics/chemistry
CRISPR-Cas Systems/genetics
Allosteric Regulation
*Endodeoxyribonucleases/metabolism/genetics/chemistry
*Bacterial Proteins/metabolism/genetics/chemistry
*Francisella/genetics/enzymology
*R-Loop Structures
DNA Cleavage
Gene Editing
DNA/metabolism

@article {pmid41603430,
year = {2026},
author = {Li, W and Yang, Y and Xu, W and Zhu, Y and Li, Y and Cao, L and Lyu, S and Liu, J and Xie, Y and Li, X and Hu, X and Huang, L},
title = {An Extraction-free One-Pot Assay for Rapid Field Discrimination of African Swine Fever Virus Variants by a Single-Step RPA-CRISPR/Cas12a Strategy.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1201-1213},
doi = {10.1021/acssensors.5c03287},
pmid = {41603430},
issn = {2379-3694},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *African Swine Fever/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {African swine fever virus (ASFV) causes devastating outbreaks in swine populations worldwide. The co-existence of wild-type and emerging gene-deleted variants (e.g., ASFV-ΔI177L) necessitates rapid on-site diagnostic tools for strain identification, which is critical for timely disease control and tailored farm management. Here, we developed a field-deployable, extraction-free one-pot assay (CORDSv2) that combines multiplex RPA and CRISPR/Cas12a to target conserved ASFV sequences and specific fluorescent markers (eGFP/mCherry) of gene-deleted variants. The assay achieved ultrasensitive detection (LOD: 6 copies/μL) and demonstrated 97.9% accuracy with 96 field samples. To streamline field operations, we integrated an extraction-free protocol (for serum/saliva) with freeze-dried reagent microspheres, enabling stable storage and direct use with minimal manual handling. When paired with a portable fluorometer (hippoCORDS), the system completes the entire sample-to-answer workflow within 1 h with a single step: addition of lysate to the microspheres. This robust, portable system addresses the urgent need for simple, on-site ASFV variant surveillance and is adaptable for other high-threat pathogens.},
}

*African Swine Fever Virus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Animals
Swine
*African Swine Fever/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
Limit of Detection

@article {pmid41591278,
year = {2026},
author = {Li, L and Wang, Y and Liu, L and Lou, Y and Lin, K and Li, T and Yu, C and Han, Y and Wei, H and Wang, D and Wang, S and Rong, Z},
title = {Miniaturized Single-Step Duplex CRISPR Diagnostic Platform for At-Home Molecular Testing of HPV16 and HPV18.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1117-1128},
doi = {10.1021/acssensors.5c03020},
pmid = {41591278},
issn = {2379-3694},
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; *Human papillomavirus 18/genetics/isolation & purification ; Humans ; Female ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Uterine Cervical Neoplasms/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Miniaturization ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The early screening, precise diagnosis, and effective treatment of invasive cervical cancer necessitate at-home molecular testing of human papillomaviruses (HPVs). However, current HPV DNA tests cannot meet the need for an affordable, rapid, and accurate diagnosis using a streamlined workflow. Here, we present a miniaturized single-step duplex CRISPR diagnostic platform, termed SCOPEv2 (Streamlined CRISPR On Pod Evaluation platform, version 2), for rapid and highly sensitive at-home molecular testing of high-risk HPV16 and HPV18 for population screening of cervical cancer. Dual-target recombinase polymerase amplification (RPA) was initially incorporated with Cas12a/Cas13a cleavage reactions in a single-step reaction system. A miniaturized and low-cost dual-color wireless analysis device was further developed to execute the analysis workflow. SCOPEv2 can detect HPV16 and HPV18 with limits of detection of 2.5 copies/μL (5 copies/reaction) and 5 copies/μL (10 copies/reaction) in 30 min, respectively. The analysis results for 128 clinical cervicovaginal swab samples revealed 94.7% sensitivity and 100% specificity. SCOPEv2 demonstrates an easy-to-use workflow, low cost, high analytical performance, and superior clinical feasibility, which enable accurate and simultaneous point-of-care testing of HPV16 and HPV18.},
}

*Human papillomavirus 16/genetics/isolation & purification
*Human papillomavirus 18/genetics/isolation & purification
Humans
Female
*Papillomavirus Infections/diagnosis/virology
*CRISPR-Cas Systems
Uterine Cervical Neoplasms/diagnosis/virology
Nucleic Acid Amplification Techniques
Miniaturization
Limit of Detection
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41582136,
year = {2026},
author = {Guo, S and Zhao, S and Tang, S and Leng, H and Wu, Y and Li, W and Xing, S and Feng, Y and Zhang, Y},
title = {Establishment of a CRISPR/Cas12a/13a-driven dual-detection platform for rapid diagnosis of swine influenza virus and porcine reproductive and respiratory syndrome virus infection.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {41582136},
issn = {1743-422X},
support = {2021YFD1800200//National Key Research and Development Program of China/ ; 32170539//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Swine ; *Porcine respiratory and reproductive syndrome virus/isolation & purification/genetics ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Porcine Reproductive and Respiratory Syndrome/diagnosis/virology ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/veterinary/virology ; *Swine Diseases/diagnosis/virology ; Coinfection/diagnosis/virology/veterinary ; },
abstract = {BACKGROUND: Swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) are leading pathogens in pigs, whose co-infections exacerbate disease severity. Current diagnostics like RT-PCR lack suitability for rapid, on-site use, while CRISPR-based systems face challenges in convenient multiplex detection.

RESULTS: We developed an RT-LAMP-CRISPR-Cas12a/13a-LFD dual-detection platform that integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the orthogonal trans-cleavage activities of CRISPR-Cas12a and Cas13a, followed by lateral flow dipstick (LFD) visualization. This assay achieved detection limits of 5 copies/µL for SIV and 2 copies/µL for PRRSV, and exhibited high specificity against other common swine pathogens. The entire process, including a 20-minute amplification at 40 °C and 5-minute LFD readout, enables rapid and visual diagnosis. A preliminary validation was conducted using respiratory infection samples, demonstrating high concordance with reference methods and specificity against non-target pathogens.

CONCLUSIONS: The RT-LAMP-CRISPR-Cas12a/13a-LFD assay provides a sensitive, specific, and potentially field-adaptable tool for the simultaneous detection of SIV and PRRSV. It is ideally suited for early screening and precise control of these pathogens in resource-limited settings.},
}

Animals
Swine
*Porcine respiratory and reproductive syndrome virus/isolation & purification/genetics
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Porcine Reproductive and Respiratory Syndrome/diagnosis/virology
Sensitivity and Specificity
*Orthomyxoviridae Infections/diagnosis/veterinary/virology
*Swine Diseases/diagnosis/virology
Coinfection/diagnosis/virology/veterinary

@article {pmid41577687,
year = {2026},
author = {Montagud-Martínez, R and Ruiz, R and Baldanta, S and Delicado-Mateo, R and Rodrigo, G},
title = {CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41577687},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *R-Loop Structures ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Protein Domains ; DNA, Single-Stranded/metabolism/genetics ; Models, Molecular ; DNA/metabolism ; },
abstract = {Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5' side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
*R-Loop Structures
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
Protein Domains
DNA, Single-Stranded/metabolism/genetics
Models, Molecular
DNA/metabolism

@article {pmid41572361,
year = {2026},
author = {Mulero-Sánchez, A and Bosma, A and Visuvasam, B and Pouliopoulou, N and van de Ven, M and Proost, N and Boeije, M and Lieftink, C and Beijersbergen, R and Bernards, R and Mainardi, S},
title = {CRISPR knockout screens reveal JUN as the master mediator of resistance to MAPK inhibition in KRAS-mutant pancreatic cancer.},
journal = {Journal of experimental & clinical cancer research : CR},
volume = {45},
number = {1},
pages = {},
pmid = {41572361},
issn = {1756-9966},
support = {SU2C-AACR-PCC-01-18//Stand Up To Cancer/ ; KWF 12539//KWF Kankerbestrijding/ ; },
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Pancreatic Neoplasms/genetics/drug therapy/pathology/metabolism ; Animals ; Mice ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Cell Line, Tumor ; *Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Xenograft Model Antitumor Assays ; Gene Knockout Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is often driven by KRAS mutations, but inhibitors targeting the most frequent KRAS substitutions in PDAC are not yet approved in the clinic. We previously discovered that KRAS-mutant PDAC is sensitive to the combination of SHP2 and ERK inhibitors, recently investigated in the Phase I/Ib clinical trial NCT04916236. Lately, RAS(ON) multi-selective inhibitors have entered clinical development, representing a promise for mono or combination therapies in PDAC. However, resistance may arise even for combination therapies. Here, we aimed at anticipating mechanisms of resistance to SHP2 plus ERK or RAS(ON) multi-selective inhibitors.

METHODS: We performed a genome-wide CRISPR-KO screening, followed by four follow-up focused screenings, leading to the identification of resistance mediators, which were further validated through functional genetic and pharmacological experiments, both in vitro and in vivo.

RESULTS: Through unbiased CRISPR-based screenings, we identified mTOR and JUN hyperactivation as interconnected mechanisms that overcome MAPK suppression. Further investigation pointed at JUN as the most downstream resistance mediator, and indirect therapeutic target, using MAP2K4 inhibitors.

CONCLUSIONS: Alterations in the PI3K/AKT/mTOR and JUN pathways can induce resistance to multiple combinations of MAPK pathway inhibitors, and may serve as biomarkers for sensitivity/resistance in clinical trials exploring such combinations in KRAS-mutant PDAC.},
}

Humans
*Proto-Oncogene Proteins p21(ras)/genetics
*Pancreatic Neoplasms/genetics/drug therapy/pathology/metabolism
Animals
Mice
*Drug Resistance, Neoplasm/genetics
Mutation
Cell Line, Tumor
*Protein Kinase Inhibitors/pharmacology
CRISPR-Cas Systems
*Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology
Xenograft Model Antitumor Assays
Gene Knockout Techniques
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41512567,
year = {2026},
author = {Sushmita, and Srivastava, A and Jain, G and Singh, M and Verma, PC},
title = {Targeted disruption of a cell wall-modifying gene α-Mannosidase using CRISPR-Cas9 enhances post-harvest shelf life in tomato through ABA accumulation.},
journal = {Plant physiology and biochemistry : PPB},
volume = {231},
number = {},
pages = {111017},
doi = {10.1016/j.plaphy.2026.111017},
pmid = {41512567},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism/enzymology ; *Abscisic Acid/metabolism ; *Cell Wall/metabolism/genetics ; *CRISPR-Cas Systems ; *alpha-Mannosidase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Fruit/genetics/metabolism ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; },
abstract = {Tomato ripening is a complex process regulated by transcription factors, hormones, and physiological changes. While this intricate regulation ensures desirable traits such as color, texture, and flavor, it is often accompanied by rapid post-harvest deterioration and poor shelf life, leading to significant economic losses and limiting market potential. Various cell wall-modifying enzymes, including N-glycan-processing enzyme α-Mannosidase, play crucial roles in the softening and senescence of tomato fruits. Our study shows that α-Mannosidase knockout through CRISPR/Cas9 results in fruits with enhanced firmness, longer shelf life, and improved moisture retention. Additionally, the expression of SlRIN (Ripening Inhibitor), a key regulator of ripening and several downstream genes, including those involved in cell wall degradation, ethylene biosynthesis, and signaling, was downregulated. Interestingly, in the later stages of storage, they also exhibited higher accumulation of abscisic acid (ABA) and lower accumulation of reactive oxygen species, along with better antioxidant capacity, compared to the control fruits, which may confer delayed softening and increased shelf life. Our findings highlight α-Mannosidase as a ripening-specific regulator and a promising genetic target for extending tomato shelf life, offering a sustainable strategy to minimize post-harvest losses without compromising plant development.},
}

*Solanum lycopersicum/genetics/metabolism/enzymology
*Abscisic Acid/metabolism
*Cell Wall/metabolism/genetics
*CRISPR-Cas Systems
*alpha-Mannosidase/genetics/metabolism
Plant Proteins/genetics/metabolism
Fruit/genetics/metabolism
Gene Expression Regulation, Plant
Reactive Oxygen Species/metabolism

@article {pmid41505207,
year = {2026},
author = {Gao, JL and Li, Z and Calderon-Perez, R and Pavek, A and Kim, L and McDermott, DH and Murphy, PM},
title = {Gene therapy via CRISPR/Cas9-mediated Cxcr4 disease allele inactivation reverses leukopenia in WHIM mice.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {5},
pages = {},
pmid = {41505207},
issn = {1558-8238},
mesh = {Animals ; *Receptors, CXCR4/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy ; *Leukopenia/therapy/genetics/pathology ; *Warts/therapy/genetics/pathology ; *Alleles ; *Immunologic Deficiency Syndromes/therapy/genetics/pathology ; Primary Immunodeficiency Diseases/therapy/genetics ; Hematopoietic Stem Cell Transplantation ; Humans ; Gene Editing ; },
abstract = {Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an immunodeficiency caused by autosomal dominant hyperfunctional mutations in chemokine receptor CXCR4 that promote panleukopenia due to BM retention. We previously reported a preclinical gene therapy protocol involving allele-nonspecific Cxcr4 CRISPR/Cas9 inactivation, leveraging the known in vivo dominance of Cxcr4+/o (+, WT; o, inactivated) hematopoietic stem cells (HSCs) for autologous BM engraftment and leukocyte reconstitution over HSCs with other Cxcr4 genotypes. Here, we show that without BM conditioning, this approach is not able to correct leukopenia in WHIM mice. We therefore modified the protocol by adding conditioning with a nongenotoxic CD117-targeted immunotoxin, CD117-antibody-saporin-conjugate. With this change, donor-derived blood cells rapidly reached ~95% chimerism after transplantation, which was stable without adverse events. Mice receiving edited HSCs showed rapid normalization of absolute myeloid cell counts, the key blood subset responsible for WHIM syndrome. In competitive transplants using equal numbers of edited and unedited donor HSCs, over 80% of blood cells originated from the edited population, predominantly with the Cxcr4+/o genotype. These results provide proof of principle that CRISPR/Cas9-mediated inactivation of the Cxcr4 disease allele, combined with nongenotoxic HSC-targeted conditioning, may offer a safe and effective gene therapy strategy generalizable to all WHIM-causing mutations.},
}

Animals
*Receptors, CXCR4/genetics
Mice
*CRISPR-Cas Systems
*Genetic Therapy
*Leukopenia/therapy/genetics/pathology
*Warts/therapy/genetics/pathology
*Alleles
*Immunologic Deficiency Syndromes/therapy/genetics/pathology
Primary Immunodeficiency Diseases/therapy/genetics
Hematopoietic Stem Cell Transplantation
Humans
Gene Editing

@article {pmid41485979,
year = {2026},
author = {Shimizu, T and Okamoto, M and Kawamoto, K},
title = {Evaluation of a d-octaarginine-linked polymer for CRISPR-Cas9 ribonucleoprotein (RNP) delivery and genome editing in murine dendritic cells.},
journal = {The Journal of veterinary medical science},
volume = {88},
number = {3},
pages = {374-383},
doi = {10.1292/jvms.25-0334},
pmid = {41485979},
issn = {1347-7439},
mesh = {Animals ; *Dendritic Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mice ; *Ribonucleoproteins/genetics ; *Oligopeptides/chemistry ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; *Polymers/chemistry ; },
abstract = {We previously reported that poly (N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) efficiently introduces proteins and nucleic acids into cells. Based on these results, we hypothesized that VP-R8 can introduce a complex composed of guide RNA and Cas9 (RNP complex) into cells and induce genome editing mediated by the CRISPR-Cas9 system. We tested this hypothesis using a mouse dendritic cell line and mouse primary dendritic cells. The RNP complexes formed by guide RNA consisting of CRISPR RNA (crRNA), fluorescently labeled trans-activating crRNA (tracrRNA), and GFP-fused Cas9 were introduced into a mouse dendritic cell line using VP-R8 or control transfection reagents. Cells transfected using VP-R8 exhibited higher fluorescence than those transfected with other transfection reagents, indicating that VP-R8 efficiently introduced the RNP complex into the mouse dendritic cell line. Genome editing of the target DNA was detected in cells transfected with the RNP complex using VP-R8 and dominant relative to other transfection reagents. We also observed that VP-R8 effectively delivered RNP complexes consisting of single-guide RNA and Cas9 and induced genome editing in the dendritic cell line. Additionally, VP-R8 efficiently delivered RNP complexes into mouse primary dendritic cells and induced genome editing of the functional gene without producing early inflammatory cytokines. Thus, VP-R8 shows potential as a transfection tool to generate dendritic cells with specific gene regions deleted by genome editing via the CRISPR-Cas9 system. This approach aims to elucidate the detailed molecular mechanisms of dendritic cell function for its application to vaccines.},
}

Animals
*Dendritic Cells/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Mice
*Ribonucleoproteins/genetics
*Oligopeptides/chemistry
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Transfection
*Polymers/chemistry

@article {pmid41432359,
year = {2026},
author = {Fruitet, E and de Fouchier, A and Heckel, DG and Groot, AT},
title = {Multiple CRISPR/Cas9 modifications of an esterase reveal its role in influencing acetate esters in the pheromone blend of a moth.},
journal = {Insect molecular biology},
volume = {35},
number = {2},
pages = {166-176},
doi = {10.1111/imb.70016},
pmid = {41432359},
issn = {1365-2583},
support = {//Instituut voor Biodiversiteit en Ecosysteem Dynamica, Universiteit van Amsterdam/ ; //Universiteit van Amsterdam/ ; //International Max Plank Research School, Max-Planck-Instituts für chemische Ökologie/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {*Moths/genetics/metabolism/enzymology ; Animals ; CRISPR-Cas Systems ; Female ; Male ; *Esterases/genetics/metabolism ; *Acetates/metabolism ; Esters/metabolism ; *Insect Proteins/genetics/metabolism ; *Sex Attractants/metabolism ; Quantitative Trait Loci ; },
abstract = {Sexual signalling by pheromones is essential for mate finding and mate choice in moths and plays an important role in reproductive isolation. Acetates (i.e., acetate esters) produced by females of Heliothis (Chloridea) subflexa Fabricius, 1777 (Lepidoptera: Noctuidae) attract conspecific males but repel Heliothis virescens Fabricius, 1777 (Lepidoptera: Noctuidae) males. A QTL (quantitative trait locus) harbouring carboxylesterases and lipases was previously shown to affect acetates, and CRISPR/Cas9-induced knockouts increased acetate amounts by blocking hydrolysis of the esters as expected. A second, unlinked QTL, containing a cluster of three different carboxylesterases (CXEs), unexpectedly yielded decreased acetate amounts. In one of these genes, esterase CXE24, we found a naturally occurring transposable element insertion in exon 8. A CRISPR/Cas9-induced frameshift at the same position yielded the same results. The paradox was resolved by a CRISPR/Cas9-induced frameshift in exon 2 of CXE24 which increased acetate amounts. The frameshift in exon 2 produced a truncated protein lacking the substrate binding site and the catalytic triad, while the frameshift in exon 8 removed only the third residue of the catalytic triad. In silico modelling showed that the exon-8-truncated protein could not hydrolyse the esters by itself, which likely explains the decreased acetate amounts. To place our findings in an evolutionary context, we explored variation in the esterase cluster in 16 species of Lepidoptera with completely sequenced genomes. Geographic and temporal variation in acetates has been observed in H. subflexa, and variation in the frequency of the transposable element could be a possible explanation.},
}

*Moths/genetics/metabolism/enzymology
Animals
CRISPR-Cas Systems
Female
Male
*Esterases/genetics/metabolism
*Acetates/metabolism
Esters/metabolism
*Insect Proteins/genetics/metabolism
*Sex Attractants/metabolism
Quantitative Trait Loci

@article {pmid41411488,
year = {2026},
author = {Bär, I and Groten, SA and Barraclough, A and Bürgisser, PE and van Kwawegen, C and Lenting, PJ and van Moort, I and Eikenboom, JCJ and Leebeek, FWG and Voorberg, J and van den Biggelaar, M and Bierings, R},
title = {Allele-selective disruption of pathogenic VWF variants in type 2 von Willebrand disease using CRISPR/Cas9.},
journal = {Blood advances},
volume = {10},
number = {5},
pages = {1429-1443},
pmid = {41411488},
issn = {2473-9537},
mesh = {Humans ; *von Willebrand Factor/genetics ; *Alleles ; *CRISPR-Cas Systems ; *von Willebrand Disease, Type 2/genetics/therapy ; Polymorphism, Single Nucleotide ; *Gene Editing/methods ; Genetic Therapy/methods ; Mutation ; },
abstract = {In contrast to major innovations in treating severe hemophilia, the treatment of severe von Willebrand disease (VWD) remains limited to intravenous infusion of von Willebrand factor (VWF) concentrates. To date, no gene therapy-based approaches for the treatment of VWD have been developed, largely owing to the disease's heterogeneous mutational landscape and the challenge of specifically targeting VWF production in endothelial cells. In this study, we developed a novel gene therapy strategy for patients with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF. We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted allele. Variant mapping mass spectrometry that discriminates between wild-type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs. This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.},
}

Humans
*von Willebrand Factor/genetics
*Alleles
*CRISPR-Cas Systems
*von Willebrand Disease, Type 2/genetics/therapy
Polymorphism, Single Nucleotide
*Gene Editing/methods
Genetic Therapy/methods
Mutation

@article {pmid41370881,
year = {2026},
author = {Li, SR and Li, Y and Yang, KB and Wang, SW and Sun, ML and Liu, Z and Zhang, XP and Zhong, Y and Yao, J},
title = {CRISPR/Cas12a coupled with MIRA: A specific and rapid assay for human DNA in challenging forensic matrices.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103393},
doi = {10.1016/j.fsigen.2025.103393},
pmid = {41370881},
issn = {1878-0326},
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Cytochromes b/genetics ; Mice ; Swine ; Chickens ; *DNA Fingerprinting/methods ; DNA ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human DNA detection is crucial in forensic medicine, particularly for trace, degraded, or mixed samples, which demand high sensitivity, specificity, and rapid processing. Traditional methods, such as immunological assays and PCR-based techniques, often suffer from operational complexity, limited sensitivity, or high equipment dependency. To address these challenges, we developed a novel detection system combining multienzyme isothermal rapid amplification (MIRA) with CRISPR-Cas12a for the rapid, specific, and portable human DNA identification. By targeting the human mitochondrial cytochrome b (CYTB) gene and sex-determining Region Y(SRY) gene, we designed MIRA primers and CRISPR-Cas12a crRNA to enable dual recognition and signal amplification. The method involves isothermal amplification at 37°C followed by CRISPR-Cas12a-mediated cleavage, producing detectable fluorescence or lateral flow chromatographic signals. Our system achieves ultra-sensitive detection and high specificity, distinguishing human DNA from non-human sources (e.g., pig, chicken, mouse), and also enables accurate gender identification, further enhancing its utility in forensic and genetic studies. Compared to traditional qPCR, this approach demonstrates superior sensitivity, faster turnaround (≤ 45 min), and minimal equipment requirements, making it ideal for forensic applications. Moreover, the blood, mixed, and degraded samples were used to confirm its robustness, with results interpretable via blue-light fluorescence or colloidal gold test strips. In summary, the MIRA-CRISPR/Cas12a system overcomes the limitations of conventional techniques, offering a rapid, cost-effective, and reliable solution for forensic human DNA identification, with potential extensions to wildlife conservation and food safety testing.},
}

Humans
*CRISPR-Cas Systems
Animals
*Nucleic Acid Amplification Techniques/methods
Cytochromes b/genetics
Mice
Swine
Chickens
*DNA Fingerprinting/methods
DNA
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41211856,
year = {2026},
author = {Kelly, AA and Fulda, M and Aden, M and Abreu, IN and Feussner, K and Feussner, I},
title = {Reducing the Sinapine Levels of Camelina sativa Seeds Through Targeted Genome Editing of REF1.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1839-1865},
pmid = {41211856},
issn = {1467-7652},
support = {INST 186/1434-1//Deutsche Forschungsgemeinschaft/ ; ZUK 45/2010//Deutsche Forschungsgemeinschaft/ ; 031B0343A//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Seeds/metabolism/genetics ; *Gene Editing/methods ; *Choline/analogs & derivatives/metabolism ; *Brassicaceae/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Sinapine (O-sinapoyl choline) is the major phenolic metabolite typically found in the oil-rich seeds of Brassicaceae such as Camelina sativa and Brassica napus. It imparts a bitter taste to the seeds as a defence mechanism against herbivores, but it also renders them less palatable to livestock. To improve Camelina flour for human consumption or as animal feed, we reduced sinapine content through CRISPR/Cas9-based genome editing of REF1 (REDUCED EPIDERMAL FLUORESCENCE1), which encodes the NADP[+]-dependent coniferaldehyde/sinapaldehyde dehydrogenase (CALDH/SALDH), a key enzyme for sinapine biosynthesis in Arabidopsis thaliana and B. napus. Inactivation of all three homoeologues found in C. sativa lowered the sinapine content in seeds by an overall 56% in two cultivars indicating the presence of a REF1-independent pathway for sinapine biosynthesis. Most importantly however, crucial agronomic seed traits such as total lipid or protein content of the seeds, seed weight or germination were not affected. Hence, the ref1 mutant lines produced here provide a valuable trait, that can be combined with other traits through gene stacking to obtain crops with significantly improved product quality. Furthermore, metabolite fingerprinting by ultra-performance liquid chromatography-electrospray ionisation-quadrupole time-of-flight mass spectrometry of ref1 mutant lines revealed a contrasting phenylpropanoid profile in seeds and leaves, indicating that REF1 oxidises sinapaldehyde to sinapate in seeds and coniferyl aldehyde to ferulate in leaves. In contrast to Arabidopsis however, Camelina accumulates no comparable levels of sinapoyl malate, but substantial amounts of chlorogenic acid, of which an additional chlorogenic acid isomer distinguishes the two different Camelina cultivars as a metabolite marker.},
}

*Seeds/metabolism/genetics
*Gene Editing/methods
*Choline/analogs & derivatives/metabolism
*Brassicaceae/genetics/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
CRISPR-Cas Systems

@article {pmid41195477,
year = {2026},
author = {Lee, SK and Hong, WJ and Kim, EJ and Lee, C and Moon, S and Ha, SH and Jung, KH},
title = {Developing an Efficient System for Hybrid Rice Seed Production Using Partial Male Sterility.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1740-1749},
pmid = {41195477},
issn = {1467-7652},
support = {RS-2024-00322278//Rural Development Administration/ ; RS-2021-NR059380//National Research Foundation of Korea/ ; RS-2021-NR060084//National Research Foundation of Korea/ ; },
mesh = {*Oryza/genetics/physiology/growth & development ; *Seeds/genetics/growth & development ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Pollination/genetics ; Hybridization, Genetic ; CRISPR-Cas Systems ; Pollen/genetics ; Mutation ; },
abstract = {Efficient production of hybrid rice seeds requires male sterility systems to overcome the challenges of self-pollination. In this study, we identified male gametic transfer defect (GTrD)5 and GTrD9 as essential genes for the process of male gamete transmission in rice. Mutations in these genes cause partial male sterility by impairing pollen tube elongation, thereby reducing the fertilisation rate. Using CRISPR/Cas9 technology, we developed gtrd5flo5 and gtrd9flo5 double mutants that combined the gtrd5 and gtrd9 genes responsible for partial male sterility with the floury endosperm (FLO)5 gene, whose defects result in seeds with an opaque endosperm, allowing us to easily differentiate between hybrid and self-pollinated seeds. This two-line hybrid system demonstrated a high rate of hybrid seed production with significantly reduced self-pollination ratios. The hybrid seeds resulted in plants with increased height, panicle size and grain yield compared with those obtained from the parental lines and also displayed heterosis. Unlike the current two-line hybrid systems based on photoperiod- and thermosensitive genic male-sterile lines, our approach is independent of environmental factors, ensuring a stable and reliable hybrid seed production. This novel method simplifies seed production, enhances efficiency and offers a cost-effective and environmentally sustainable solution for hybrid rice breeding.},
}

*Oryza/genetics/physiology/growth & development
*Seeds/genetics/growth & development
*Plant Infertility/genetics
*Plant Breeding/methods
Pollination/genetics
Hybridization, Genetic
CRISPR-Cas Systems
Pollen/genetics
Mutation

@article {pmid41190715,
year = {2026},
author = {Simon, MK and Yuan, L and Che, P and Day, K and Jones, T and Godwin, ID and Koltunow, AMG and Albertsen, MC},
title = {Induction of Synthetic Apomixis in Two Sorghum Hybrids Enables Seed Yield and Genotype Preservation Over Multiple Generations.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1712-1724},
pmid = {41190715},
issn = {1467-7652},
support = {INV-002955/GATES/Gates Foundation/United States ; OPP1076280//Bill and Melinda Gates Foundation/ ; //Corteva Agriscience/ ; INV-002955/GATES/Gates Foundation/United States ; },
mesh = {*Sorghum/genetics/physiology ; *Apomixis/genetics ; *Seeds/genetics/growth & development/physiology ; Genotype ; Hybridization, Genetic ; CRISPR-Cas Systems ; Plant Breeding/methods ; Meiosis/genetics ; Parthenogenesis/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Induction of apomixis, or clonal reproduction through seed, could economise commercial hybrid seed production and enable smallholder farmers to save and sow hybrid seed. Here, we demonstrate the synthetic induction of apomixis in two sorghum hybrids and show that the clonal hybrid seed can be maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Meiotic avoidance was generated by CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell by the expression of the Cenchrus ASGR-BBML2 gene coding sequence. Two different strategies were used to combine these components to induce synthetic apomixis in two sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in both apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other components, but this led to a reduction in seed set. By contrast, a single in-frame edited allele of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimising fertility; however, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.},
}

*Sorghum/genetics/physiology
*Apomixis/genetics
*Seeds/genetics/growth & development/physiology
Genotype
Hybridization, Genetic
CRISPR-Cas Systems
Plant Breeding/methods
Meiosis/genetics
Parthenogenesis/genetics
Plant Proteins/genetics/metabolism

@article {pmid41186962,
year = {2026},
author = {Misceo, D and Terkelsen, T and Bøen Keim, SM and Bjørnstad, PM and Strand, ME and Orszagh, VC and Jensen, UB and Frengen, E},
title = {CRISPR Activation Reveals the Spliceogenicity of an Intronic NEB Variant in Fetuses With Arthrogryposis Multiplex Congenita 6.},
journal = {Clinical genetics},
volume = {109},
number = {4},
pages = {772-777},
pmid = {41186962},
issn = {1399-0004},
support = {//Norwegian National Advisory Unit on Rare Disorders/ ; //Legatet til Henrik Homans Minde/ ; },
mesh = {Humans ; *Arthrogryposis/genetics/diagnosis/pathology ; *Introns/genetics ; Female ; Fetus/pathology ; Pregnancy ; *RNA Splicing/genetics ; *CRISPR-Cas Systems/genetics ; Pedigree ; Male ; Frameshift Mutation ; RNA Splice Sites/genetics ; },
abstract = {Whole-genome sequencing identifies intronic variants whose pathogenicity can be predicted with tools like SpliceAI. However, an actionable classification of such variants may require RNA-based validation, which can be limited by low expression in clinically accessible tissues. We report two fetuses from one family with Arthrogryposis multiplex congenita 6 (AMC6 [OMIM # 619334]) and biallelic NEB variants: a paternally inherited likely pathogenic frameshift variant, Chr2(GRCh38):g.151579391del; NM_001164508.2:c.16653del; NP_001157980.2:p.(Asp5552ThrfsTer5), and a maternally inherited intronic variant of uncertain clinical significance, Chr2(GRCh38):g.151496267G>A; NM_001164508.2:c.24486+9C>T; NP_001157980.2:p.(?). Because NEB is poorly expressed in fibroblasts, we used CRISPR activation to induce its expression in fibroblasts from the heterozygous mother. RNA-sequencing subsequently confirmed that the intronic variant generated a novel splice donor site associated with inferred loss of splicing at the canonical donor site. After NMD-inhibition, we could thus identify 45.5% of NEB transcripts with a 7 bp exon extension, predicted to result in a protein-coding frameshift. The intronic variant was classified as likely pathogenic, allowing a genetic diagnosis.},
}

Humans
*Arthrogryposis/genetics/diagnosis/pathology
*Introns/genetics
Female
Fetus/pathology
Pregnancy
*RNA Splicing/genetics
*CRISPR-Cas Systems/genetics
Pedigree
Male
Frameshift Mutation
RNA Splice Sites/genetics

@article {pmid41159426,
year = {2026},
author = {Kato, M and Kleidon, J and Phillips, A and Tabrett, A and Harding, R and Dale, J and Paul, JY},
title = {Transgene-Free, Gene-Edited Cavendish Bananas (Musa acuminata, AAA).},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1620-1634},
pmid = {41159426},
issn = {1467-7652},
support = {//Cooperative Research Centre Projects/ ; //Australian Banana Research Pty Ltd/ ; },
mesh = {*Gene Editing/methods ; *Musa/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Transgenes/genetics ; DNA, Bacterial/genetics ; Oxidoreductases/genetics ; Intramolecular Lyases/genetics ; Carotenoids/metabolism ; },
abstract = {Global consumer acceptance of gene-edited food crops is increasing with new breeding technologies that can modify the genome without foreign DNA integration. Here, we report an Agrobacterium-based system for transgene-free, gene editing of the banana cultivar, Cavendish. The protocol uses a three-tiered approach whereby cells containing T-DNA are enriched by positive antibiotic selection over a 48-h period, CRISPR/Cas9-mediated gene editing occurs over a short transient window and negative selection of cells containing T-DNA is achieved by the addition of 5-FC, which is converted to cytotoxic 5-FU by the co-expressed CODA enzyme. Two key enzymes in the carotenoid biosynthesis pathway were targeted as visual markers of editing: phytoene desaturase (pds) and lycopene β-cyclase (LCYb). Disruptive edits of the LCYb gene were easily identifiable at the calli stage of the regeneration process with cells appearing pink due to lycopene accumulation. Eight of 32 plants for pds and 34 of 125 plants for LCYb contained edits and were likely free of integrated vector DNA as determined by targeted genome sequencing and T-DNA-specific PCR, respectively. Plants containing disruptive tri-allelic edits in either gene displayed an albino phenotype. A subset of potentially transgene-free, edited plants was verified by whole genome sequencing. The transient editing protocol has an estimated 17.6% to 21.9% efficiency in generating plants containing disruptive tri-allelic modifications that are free of novel DNA. The protocol overcomes the regulatory limitations associated with the release of gene-edited, vegetatively propagated crops and provides an effective means of creating new disease-resistant and agronomically superior Cavendish cultivars.},
}

*Gene Editing/methods
*Musa/genetics
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Transgenes/genetics
DNA, Bacterial/genetics
Oxidoreductases/genetics
Intramolecular Lyases/genetics
Carotenoids/metabolism

@article {pmid41123354,
year = {2026},
author = {Choi, NY and Kim, MH and Jang, HA and Pyo, SW and Park, KY and Lee, H and Bae, EK and Ko, JH},
title = {Elucidating the Genetic Basis of Columnar Upright Architecture in Populus Through CRISPR Disruption of TILLER ANGLE CONTROL1.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1377-1397},
pmid = {41123354},
issn = {1467-7652},
support = {RS-2023-NR076519//National Research Foundation of Korea/ ; FG0702-2023-01-2025//Korea Forest Service/ ; 2023489B10-2325-AA01//R&D Program for Forest Science Technology/ ; },
mesh = {*Populus/genetics/growth & development/anatomy & histology ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Gene Editing ; Plant Leaves/genetics/growth & development/anatomy & histology ; Gene Expression Regulation, Plant ; },
abstract = {Narrow or upright branch angles in shoots and leaves lead to columnar, upright-growing tree architectures, as observed in various tree species such as Lombardy poplar (Populus nigra var. italica). However, the genetic mechanism underlying this unique growth habit in Lombardy poplar has not yet been elucidated. In this study, we identified a nonsense mutation in the PnTAC1-1 gene of Lombardy poplar, an ortholog of the rice TILLER ANGLE CONTROL 1 (TAC1) gene known to regulate branch angles. To confirm the functional role of TAC1 in regulating tree architecture, we generated transgenic hybrid poplar (Populus alba × Populus glandulosa, clone BH) with targeted mutations in TAC1 homologues using CRISPR/Cas9 gene editing. The resulting TAC1-CRISPR hybrid poplars exhibited a stable upright branching phenotype closely resembling that of Lombardy poplar, as confirmed by two consecutive years of living modified organism (LMO) field trials. Anatomical analysis revealed increased cell elongation specifically in the lower petiole region and significantly enhanced gravitropic responses in TAC1-CRISPR hybrid poplars compared to wild-type BH clones. RNA sequencing analysis further demonstrated that TAC1 disruption triggered extensive transcriptomic reprogramming of axillary meristem, notably altering hormonal and photomorphogenic signalling pathways, which redirected auxin accumulation toward the abaxial region and increased gibberellin biosynthesis, ultimately promoting upright growth. This research uncovers the genetic and molecular mechanisms behind columnar growth in poplar and provides a promising approach for engineering tree architecture to enhance planting density, harvest efficiency and woody biomass productivity.},
}

*Populus/genetics/growth & development/anatomy & histology
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Gene Editing
Plant Leaves/genetics/growth & development/anatomy & histology
Gene Expression Regulation, Plant

@article {pmid41108596,
year = {2026},
author = {Liu, Y and Merino, I and Gutensohn, M and Johansson, AI and Johansson, K and Andersson, M and Hofvander, P and Sitbon, F},
title = {Glycoalkaloid-Free Starch Potatoes Generated by CRISPR/Cas9-Mediated Mutations of Genes in the Glycoalkaloid Biosynthesis Pathway Enable More Sustainable Uses of By-Products From Starch Production.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1344-1358},
pmid = {41108596},
issn = {1467-7652},
support = {//SLU Grogrund/ ; },
mesh = {*Solanum tuberosum/genetics/metabolism ; *Starch/metabolism/biosynthesis ; *CRISPR-Cas Systems/genetics ; Plant Tubers/metabolism/genetics ; Mutation ; Gene Editing ; Plants, Genetically Modified ; Biosynthetic Pathways/genetics ; },
abstract = {Steroidal glycoalkaloids (SGAs) are toxic cholesterol-derived secondary metabolites present in several Solanaceae species. In potato, tuber SGA levels are for reasons of toxicity of concern in both table and starch cultivars. In the latter, SGAs bind to proteins and fibres in starch production side-streams and prevent their further uses as food and feed. To enable more sustainable uses of starch by-products, we have here reduced SGA biosynthesis in a starch potato cultivar using DNA-free CRISPR/Cas9. Six SGA genes were targeted, encoding enzymes acting either before cholesterol (SMO1-L, DWF1-L, DWF7-L), or after (16DOX, CYP88B1, TAMiso2). Editing efficiencies varied between 20% and 49%, and generated mutants were investigated under greenhouse and field conditions. Target mass-spectrometric analyses confirmed reduced SGA levels and alterations of sterol metabolism in mutated events. Plant height and tuber yield were reduced in several events, although this was not correlated to low SGA levels. Several knockout mutants had almost SGA-free leaves and tubers, the latter also under two SGA-inducing conditions. Similarly, both fibre and protein fractions isolated from side-streams in the starch production process from mutant tubers had very low SGA levels. By contrast, the corresponding wild-type SGA levels were almost 10-fold and, respectively, 40-fold higher than the recommended upper safe limit. The results demonstrate that glycoalkaloid-free mutants can be generated and grown with moderate yield reductions under both greenhouse and field conditions. This suggests a potential for sustainable production of high-value products, e.g., food-grade protein and fibre, from starch production side-streams of SGA knockout tubers.},
}

*Solanum tuberosum/genetics/metabolism
*Starch/metabolism/biosynthesis
*CRISPR-Cas Systems/genetics
Plant Tubers/metabolism/genetics
Mutation
Gene Editing
Plants, Genetically Modified
Biosynthetic Pathways/genetics

@article {pmid41063698,
year = {2026},
author = {Roman, G and Lauritzen, KH and Harrison, SP and Bhattacharya, A and Andresen, MS and Mowinckel, MC and Smolkova, B and Henriksson, CE and Glosli, H and Iversen, N and Thiede, B and Sullivan, GJ and Almaas, R and Sandset, PM and Stavik, B and Chollet, ME},
title = {Ex vivo correction of severe coagulation Factor VII deficiency in patient-derived 3D liver organoids.},
journal = {Haematologica},
volume = {111},
number = {3},
pages = {941-954},
pmid = {41063698},
issn = {1592-8721},
mesh = {Humans ; *Factor VII Deficiency/therapy/genetics/pathology/metabolism ; *Organoids/metabolism/cytology ; *Liver/metabolism/pathology/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Factor VII/genetics/metabolism ; Mutation, Missense ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Coagulation factor (F) VII deficiency is the most frequent among the rare, inherited bleeding disorders and is predominantly caused by missense mutations in the F7 gene. The disease phenotype ranges from asymptomatic cases to extremely severe hemorrhagic forms, requiring prophylactic injections with plasma-derived or recombinant FVII concentrates. In response, we have developed an autologous cell-based approach that corrects the disease-causing mutation in patient- derived induced pluripotent stem cells (iPSC) and generates therapeutic, three-dimensional hepatic organoids (HO). We report the CRISPR-mediated correction of homozygous c.718G>C (p.G240R), a missense mutation associated with a severe, life-threatening bleeding phenotype. The HO contain all liver cell types and exhibit key liver functions, including coagulation factor production. After correction, our data indicate that the patient-derived HO secrete consistent amounts of functional FVII protein, resulting in improved thrombin generation times. These results represent a significant milestone toward the establishment of an autologous cell-based therapy for patients with FVII- and other coagulation factor deficiencies.},
}

Humans
*Factor VII Deficiency/therapy/genetics/pathology/metabolism
*Organoids/metabolism/cytology
*Liver/metabolism/pathology/cytology
Induced Pluripotent Stem Cells/metabolism/cytology
*Factor VII/genetics/metabolism
Mutation, Missense
CRISPR-Cas Systems
Gene Editing

@article {pmid40954224,
year = {2026},
author = {Hu, X and Atwal, RS and Xiao, S and Ahmed, SU and Wang, Z and Zhao, C and Wang, H and Kelley, SO},
title = {ELOVL6 activity attenuation induces mutant KRAS degradation.},
journal = {Nature chemical biology},
volume = {22},
number = {3},
pages = {424-434},
pmid = {40954224},
issn = {1552-4469},
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; Mutation ; Fatty Acid Elongases/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Proteolysis/drug effects ; *Acetyltransferases/metabolism/genetics/antagonists & inhibitors ; },
abstract = {KRAS is one of the most frequently mutated oncogenes in cancer. Targeting mutant KRAS directly has been challenging because of minor structural changes caused by mutations. Despite recent success in targeting KRAS-G12C, targeted therapy for another hotspot mutant, KRAS-G12V, has not been described. We used CRISPR-Cas9 genome-wide knockout screens to identify genes that specifically modulate mutant KRAS harboring the G12V substitution. Our top hit, a fatty acid elongase (ELOVL6), showed remarkable selectivity in diminishing KRAS-G12V protein expression and aberrant oncogenic signaling associated with mutant KRAS. Our studies reveal that ELOVL6 can be targeted to control the production of phospholipids exploited by KRAS mutants for function-targeted and trigger-targeted degradation of the protein. Our results demonstrate the basis for a first-in-class small-molecule inhibitor to selectively clear KRAS-G12V from cancer cells.},
}

Humans
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism
Mutation
Fatty Acid Elongases/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
Proteolysis/drug effects
*Acetyltransferases/metabolism/genetics/antagonists & inhibitors

@article {pmid40789664,
year = {2026},
author = {Li, H and Lu, Q and Yu, Z and Wu, Z and Zhu, Z and Li, J and Zhang, Z and Wang, Z and Yang, N and Chen, Y and Lu, H and Niu, T and Tong, A},
title = {CRISPR/Cas9-engineered universal CD123/B7-H3 tandem CAR-T cell for the treatment of acute myeloid leukemia.},
journal = {Chinese medical journal},
volume = {139},
number = {5},
pages = {728-740},
pmid = {40789664},
issn = {2542-5641},
mesh = {*Leukemia, Myeloid, Acute/therapy/immunology/genetics ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Interleukin-3 Receptor alpha Subunit/metabolism/genetics ; Mice ; *B7 Antigens/metabolism/genetics ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; Cell Line, Tumor ; Gene Editing/methods ; },
abstract = {BACKGROUND: Autologous chimeric antigen receptor T (CAR-T) cell therapy has demonstrated efficacy in the treatment of acute myeloid leukemia (AML). Nevertheless, the intrinsic characteristics of autologous therapy, such as extended manufacturing timelines and patient-specific limitations, contribute to delays in treatment availability. More critically, relapse due to antigen escape following single-targeted CAR-T therapy constitutes a significant clinical obstacle. To address the dual challenges of delayed treatment accessibility and antigen escape relapse, this study proposes the development of universal tandem CAR-T cells. These cells, engineered to target CD123 and B7-H3 through clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology, represent an innovative therapeutic strategy for AML.

METHODS: In this study, an immune phage display nanobody library was developed for the purpose of screening CD123-specific nanobodies. The CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system was utilized to disrupt the T-cell receptor alpha chain (TRAC) and B2M genes present in T cells, resulting in the generation of universal CD123/B7-H3 bispecific universal CAR-T (UCAR-T) cells. The efficacy of these dual-specific UCAR-T cells in combating tumors was subsequently assessed through in vitro and in vivo experiments.

RESULTS: Through four rounds of panning against CD123 from an immunized camelid VHH library, we identified 21 antigen-specific nanobodies. Tandem bispecific UCAR-T engineered with these binders demonstrated CAR transduction efficiencies ranging from 82% to 87%. In vitro functional profiling revealed a significantly enhanced cytotoxicity of bispecific UCAR-Ts against CD123 + /B7-H3 + AML cell lines when compared to single-target constructs, while effectively regulating the secretion of effector cytokines (IL-2, IFN-γ, TNF-α). In AML xenograft models, treatment with bispecific UCAR-T notably inhibited tumor progression, extended the survival of tumor-bearing mice with recurrence-free persistence throughout the observation period, and did not result in significant body weight loss or cytokine release syndrome.

CONCLUSIONS: The findings of the study address the issue of tumor antigen evasion in the treatment of AML, circumvent certain constraints associated with autologous CAR-T cell therapy, and offer novel insights and strategies for managing AML.},
}

*Leukemia, Myeloid, Acute/therapy/immunology/genetics
Animals
Humans
*CRISPR-Cas Systems/genetics
*Interleukin-3 Receptor alpha Subunit/metabolism/genetics
Mice
*B7 Antigens/metabolism/genetics
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/metabolism
T-Lymphocytes/metabolism
Cell Line, Tumor
Gene Editing/methods

@article {pmid41752921,
year = {2026},
author = {Panov, J and Elbert, A and Rosenthal, DS and Levi, M and Chumakov, K and Andino, R and Brodsky, L and Kaphzan, H},
title = {Spacio-Linear Screening for Ligand-Docking Cavities in Protein Structures: SLAM Algorithm.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {2},
pages = {},
pmid = {41752921},
issn = {2075-1729},
support = {N/A//Tauber Foundation/ ; },
abstract = {Identifying structurally similar ligand-binding sites in unrelated proteins can facilitate drug repurposing, reveal off-target effects, and deepen our understanding of protein function. A number of tools were developed for structural screening, but many of them suffer from limited sensitivity and scalability. Using a data bank of crystallized protein structures, we aimed to discover novel protein targets for a ligand by leveraging a known ligand-binding query protein with a resolved structure. Here, we present SLAM (Spacio-Linear Alignment of Macromolecules), a novel alignment-based algorithm that detects local 3D similarities between ligand-binding cavities or protein-exposed surfaces of query and target proteins. SLAM encodes spatial substructure neighborhoods into short linear sequences of physicochemically annotated atoms, then applies pairwise sequence alignment combined with distance-correlation scoring to identify high-fidelity structural matches. Benchmarking using the Kahraman-36 dataset demonstrated that SLAM outperforms the state-of-the-art ProBiS algorithm in true-positive rate for predicting ligand-docking compatibility. Furthermore, SLAM identifies candidate ligands that may inhibit functionally critical domains of CRISPR-Cas proteins and predicts novel binding partners of toxic per- and polyfluoroalkyl Substance (PFAS) compounds (PFOA, PFOS) with plausible mechanistic links to toxicity. In conclusion, SLAM is a robust computationally efficient and flexible structural screening tool capable of detecting subtle physicochemical compatibilities between protein surfaces, promising to accelerate target discovery in pharmacology and elucidate protein-ligand interactions in environmental toxicology.},
}

@article {pmid40954296,
year = {2025},
author = {Zhang, R and Mirdita, M and Söding, J},
title = {De novo discovery of conserved gene clusters in microbial genomes with Spacedust.},
journal = {Nature methods},
volume = {22},
number = {10},
pages = {2065-2073},
pmid = {40954296},
issn = {1548-7105},
support = {CompLifeSci project horizontal4meta//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; RS-2023- 00250470//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Metagenomics/methods ; Metagenome/genetics/physiology ; *Multigene Family ; *Genome, Microbial/genetics ; Conserved Sequence ; *Software ; *Computational Biology/methods ; Algorithms ; Genome, Bacterial ; CRISPR-Cas Systems ; },
abstract = {Metagenomics has revolutionized environmental and human-associated microbiome studies. However, the limited fraction of proteins with known biological processes and molecular functions presents a major bottleneck. In prokaryotes and viruses, evolution favors keeping genes participating in the same biological processes colocalized as conserved gene clusters. Conversely, conservation of gene neighborhood indicates functional association. Here we present Spacedust, a tool for systematic, de novo discovery of conserved gene clusters. To find homologous protein matches, Spacedust uses fast and sensitive structure comparison with Foldseek. Partially conserved clusters are detected using novel clustering and order conservation P values. We demonstrate Spacedust's sensitivity with an all-versus-all analysis of 1,308 bacterial genomes, identifying 72,843 conserved gene clusters containing 58% of the 4.2 million genes. It recovered 95% of antiviral defense system clusters annotated by the specialized tool PADLOC. Spacedust's high sensitivity and speed will facilitate the annotation of large numbers of sequenced bacterial, archaeal and viral genomes.},
}

*Metagenomics/methods
Metagenome/genetics/physiology
*Multigene Family
*Genome, Microbial/genetics
Conserved Sequence
*Software
*Computational Biology/methods
Algorithms
Genome, Bacterial
CRISPR-Cas Systems

@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}

@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}

@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}

@article {pmid41741764,
year = {2026},
author = {Shivgotra, R and Soni, B and Bakrey, H and Kaur, P and Jain, SK},
title = {Bridging Gaps in Fungal Keratitis Management: Novel Diagnostics, Drug Delivery Systems, and Gene Therapies.},
journal = {AAPS PharmSciTech},
volume = {27},
number = {3},
pages = {},
pmid = {41741764},
issn = {1530-9932},
abstract = {Globally, fungal keratitis has become a major health concern, particularly in tropical and humid regions. The disease burden is exacerbated because of the underlying complexity of fungal pathogens, delayed or insufficient diagnosis, and limitations in existing therapeutic strategies, often leading to progressive corneal damage and impaired vision. Additional challenges include insufficient therapy optimization and variable efficacy of topical and systemic antifungal treatments, which may be influenced by factors such as rapid ocular drug clearance, ineffective ocular penetration, and the increasing prevalence of antifungal resistance. Conventional diagnostic techniques, such as microscopy and culture, continue to serve as reference standards but have limitations due to comparatively modest sensitivity and the prolonged turnaround times. In comparison, recent advances in diagnostic techniques, including CRISPR-based assays, PCR, MALDI-ToF MS, and in vivo confocal microscopy, as well as the novel drug-delivery nanocarriers, have been reported to show noticeable improvements in diagnostic accuracy and therapeutic outcomes. Furthermore, emerging DNA-based gene therapies and RNA-based therapeutics, along with advanced ocular drug carriers, have shown promising outcomes in preclinical research and early-phase clinical trials, suggesting potential advantages in enhanced tissue targeting and reduced therapeutic resistance. However, before widespread clinical acceptability, extensive clinical validation, long-term safety evaluations, and cost assessments are required, as existing findings are mainly limited to short-term and experimental research. This review focuses on the pathophysiology of fungal keratitis while underscoring the unmet diagnostic and therapeutic needs. It further explores the potential for developing translatable technologies aimed at predictive diagnosis and the effective management of this sight-threatening condition.},
}

@article {pmid41741653,
year = {2026},
author = {Saxton, DS and DeWeirdt, PC and Doering, CR and Roney, IJ and Laub, MT},
title = {A membrane-bound nuclease directly cleaves phage DNA during genome injection.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41741653},
issn = {1476-4687},
abstract = {From mammals to bacteria, the direct recognition and cleavage of viral nucleic acids is a potent defence strategy against viral infection, but it requires mechanisms for distinguishing self from non-self[1,2]. In bacteria, CRISPR-Cas and restriction-modification systems achieve this discrimination by recognizing specific DNA sequences or DNA modifications, respectively. Alternative mechanisms probably remain to be discovered. Here, we characterize SNIPE, an anti-bacteriophage defence system that constitutively localizes to the bacterial cell membrane in Escherichia coli to block phage λ infection. Using radiolabelled phage DNA and time-lapse microscopy to track phage genomes, we demonstrate that SNIPE directly cleaves phage DNA during genome injection. Based on proximity labelling, we find that SNIPE associates with host proteins essential for λ genome entry and with the λ tape measure protein, which facilitates λ genome injection across the inner membrane. SNIPE also defends against diverse siphoviruses, probably through direct interactions with their tape measure proteins. Our findings establish SNIPE as a widespread bacterial defence system that exploits the spatial organization of phage genome injection to specifically target viral DNA, representing a previously unknown strategy for distinguishing self from non-self in prokaryotic immune systems.},
}

@article {pmid41740122,
year = {2026},
author = {Yıldırım, K and Kavas, M},
title = {The Role of CRISPR in Modern Plant Breeding: Overcoming Breeding Barriers and Legislative Challenges Through Transgene-Free Genome Editing.},
journal = {Genome},
volume = {},
number = {},
pages = {},
doi = {10.1139/gen-2025-0063},
pmid = {41740122},
issn = {1480-3321},
abstract = {Traditional plant breeding techniques-such as crossbreeding, mutation breeding, and marker-assisted selection-have significantly contributed to crop improvement over the past century. However, these methods are often limited by long breeding cycles, low precision, and the unintended transfer of undesirable traits. To address these challenges, transgenic breeding emerged as a powerful tool, enabling the introduction of specific foreign genes to confer desirable traits such as pest resistance or herbicide tolerance. While highly effective and precise, transgenic approaches face considerable regulatory and public acceptance barriers, particularly in regions with strict GMO legislation.The advent of CRISPR/Cas genome editing has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved crops. Among CRISPR-based methods, transgene-free genome editing has gained prominence for its ability to produce enhanced plant varieties without integrating foreign DNA, thus avoiding many regulatory constraints associated with GMOs. This review aims to provide a comprehensive overview of transgenefree CRISPR-mediated genome editing technologies, emphasizing their use in modern plant breeding and their transformative potential to overcome the limitations of conventional methods while providing a regulation-friendly pathway for crop improvement.},
}