@article {pmid41588790,
year = {2026},
author = {Favaratto, L and da Silva, ML and Buss, DS and Quadros, OF and Tapia-Tussell, R and Ventura, JA and Fernandes, AAR and Fernandes, PMB},
title = {The regulatory frameworks surrounding CRISPR-edited papaya and their impact on international commerce.},
journal = {Journal of the science of food and agriculture},
volume = {106},
number = {11},
pages = {6262-6270},
pmid = {41588790},
issn = {1097-0010},
support = {# 404972/2021-7 and 441499/2023-6//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; # 1132/2024//Fundação de Amparo à Pesquisa do Espírito Santo/ ; #301052/25-5 and #307905/2020-9//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; },
mesh = {*Carica/genetics/virology/immunology/growth & development ; *Plant Diseases/virology/economics/genetics ; *Gene Editing/economics/legislation & jurisprudence ; *Commerce/legislation & jurisprudence/economics ; CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics/virology/immunology ; Plant Proteins/genetics/metabolism/immunology ; Fruit/genetics/virology/economics ; },
abstract = {The papaya tree (Carica papaya L.), native to the Americas, is cultivated in tropical regions and holds substantial economic importance, with an estimated export volume of 365 000 t in 2023. However, diseases caused by viruses, fungi, bacteria, and nematodes can lead to severe losses. Among the more than 38 known viral diseases affecting papaya, only a few poses serious threats to cultivation, notably Papaya Ringspot, Papaya Mosaic, and Papaya Sticky Disease (PSD). Emerging technologies, particularly CRISPR/Cas9 gene editing, offer promising avenues to enhance plant resistance. This study examines regulatory paradigms in key papaya-producing and importing countries, highlighting the need for international regulatory harmonization to reduce trade barriers and improve market access for CRISPR-edited cultivars. We demonstrate the feasibility of CRISPR-based genome editing in papaya (Carica papaya L.) by targeting phytoene desaturase as a proof-of-concept marker gene and β-1,3-glucanase, a resistance gene identified through proteomic profiling of host-pathogen interactions during infection by the papaya meleira virus (PMeV and PMeV2) complex. This virus complex causes PSD, a major threat to papaya production, rendering the fruit commercially unviable due to negative effects on texture and flavor as well as inhibiting the formation of benzyl isothiocyanate (BITC), and the fruits become susceptible to fruit flies, which are quarantine pests. Despite extensive traditional breeding efforts, resistant papaya genotypes have yet to be identified, underscoring the need for innovative approaches. However, translating advancements into commercial applications remains challenging due to the diverse and often inconsistent regulatory frameworks governing genome-edited crops across different jurisdictions. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.},
}

*Carica/genetics/virology/immunology/growth & development
*Plant Diseases/virology/economics/genetics
*Gene Editing/economics/legislation & jurisprudence
*Commerce/legislation & jurisprudence/economics
CRISPR-Cas Systems
*Plants, Genetically Modified/genetics/virology/immunology
Plant Proteins/genetics/metabolism/immunology
Fruit/genetics/virology/economics

@article {pmid42116141,
year = {2026},
author = {Ford, H and Dalvie, NC and Lorgeree, TR and Barry, RM and Sibel, AN and Ahlmark, RA and Narayanan, H and Sunday, BC and Whittaker, CA and Acharya, R and Elenberger, CM and Love, JC},
title = {Deletion of low-essentiality, secretion-associated genes enhances recombinant protein production in Komagataella phaffii.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {42116141},
issn = {1475-2859},
mesh = {*Recombinant Proteins/biosynthesis/genetics ; *Saccharomycetales/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Deletion ; Antibodies, Monoclonal/biosynthesis/genetics ; *Genes, Essential ; },
abstract = {BACKGROUND: Komagataella phaffii (K. phaffii) is used to manufacture biologic medicines, food proteins, reagents, and materials. Despite its increasing prevalence, further improvements to its productivity would enhance its economic and operational benefits. Genomic engineering represents one approach to increase its cell-specific productivity. We hypothesized that combining the metrics for the relative essentiality of genes with biological inference for relevance to protein secretion could identify genes that, when disrupted, would improve specific productivity in the resulting strains.

RESULTS: The essentiality of genes in K. phaffii (NRRL Y-11430) were predicted through a genome-wide knockout screen using CRISPR-Cas9. Based on the results from this screen, we selected and subsequently disrupted the least essential genes from two gene groups heavily associated with secretion, namely those relating to the cell wall and vacuolar transport. Strains of K. phaffii with single gene disruptions from these gene sets showed significantly improved production of a monoclonal antibody (mAb). These strains exhibited no discernible differences in growth or apparent profiles of host cell proteins when compared to the parental strain. The best-performing strains consistently showed 2-3x enhancements in specific productivity and titers across scales (3-150 mL), culture formats (plates, flasks, bioreactors), and processing operations (batch and fed-batch).

CONCLUSIONS: This study demonstrates how combining data on gene essentiality and prior knowledge of biological pathways related to a phenotypic trait of interest (here protein secretion) can inform strain engineering to enhance the trait. This study expands the catalog of genetically engineered strains of K. phaffii with improved productivity. These strains support the long-term goal of achieving low-cost, high-volume production of recombinant proteins using this host. Further engineering of these strains and optimization of fermentation processes could enable volumetric productivities comparable to those of other established hosts used to produce mAbs and other complex recombinant proteins.},
}

*Recombinant Proteins/biosynthesis/genetics
*Saccharomycetales/genetics/metabolism
CRISPR-Cas Systems
*Gene Deletion
Antibodies, Monoclonal/biosynthesis/genetics
*Genes, Essential

@article {pmid42148575,
year = {2026},
author = {Kang, B and Kim, J-Y and Oh, S and Kweon, J and Park, H and Kim, M and Choi, I-G and Chang, IS},
title = {A CRISPR interference system for tunable gene expression integrated with a promoter library for Eubacterium callanderi KIST612, an acetogen of functional diversity and versatility.},
journal = {Microbiology spectrum},
volume = {14},
number = {7},
pages = {e0377925},
pmid = {42148575},
issn = {2165-0497},
support = {RS-2021-NR060081//National Research Foundation of Korea/ ; RS-2023-00249146//National Research Foundation of Korea/ ; },
mesh = {*Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Bacterial ; Gene Library ; *CRISPR-Cas Systems ; Metabolic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; },
abstract = {UNLABELLED: Acetogens are key biocatalysts for carbon-neutral biorefineries, yet their metabolic engineering is limited by the lack of tunable transcriptional regulation systems. Here, we developed a synthetic promoter library for Eubacterium callanderi KIST612 and integrated it with a CRISPR interference (CRISPRi) system to establish precise and scalable gene regulation. Motif analysis of 3,109 putative native promoters revealed conserved and semi-conserved -35 and -10 elements, which were used to construct a promoter library spanning a >20-fold dynamic range of transcriptional strengths. The system was validated by knockdown of pyrF, where promoter strength directly determined repression efficiency (R[2] = 0.92), with high-strength promoters achieving near-complete gene silencing. Application to lactate dehydrogenase (ldh) revealed that increasing promoter strength progressively reduced lactate production from 93.3% to 0.0% of control. This study establishes a versatile synthetic promoter-CRISPRi platform tailored for acetogens, enabling precise control of gene expression and mechanistic dissection of redox metabolism.

IMPORTANCE: Transitioning to a carbon-neutral economy requires biocatalysts that can efficiently convert waste-derived substrates into valuable products. Acetogens are industrially relevant organisms for gas fermentation, but the lack of genetic toolkits tailored to their physiology has constrained metabolic engineering. We present the first synthetic promoter-CRISPRi platform specifically optimized for Eubacterium callanderi KIST612, a model acetogen with high industrial potential. This system provides tunable and predictable regulation of gene expression, extending from mild repression to a near-complete knockdown that could alternate gene deletion systems. This system could be used for not only advancing fundamental understanding of acetogen physiology but also providing a broadly applicable genetic toolbox for precision engineering of sustainable microbial biorefineries.},
}

*Promoter Regions, Genetic/genetics
Gene Expression Regulation, Bacterial
Gene Library
*CRISPR-Cas Systems
Metabolic Engineering/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism

@article {pmid42148800,
year = {2026},
author = {Zheng, J and Wen, Z and Li, Y and Zou, W and Ge, P and Peng, H},
title = {Rapid detection of Enterococcus faecalis using RPA-CRISPR/Cas12a-assisted technology.},
journal = {Microbiology spectrum},
volume = {14},
number = {7},
pages = {e0016826},
pmid = {42148800},
issn = {2165-0497},
support = {2023A1515011733//Natural Science Foundation of Guangdong Province/ ; 82272364, 82330072//National Natural Science Foundation of China/ ; },
mesh = {*Enterococcus faecalis/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Gram-Positive Bacterial Infections/microbiology/diagnosis ; Rapid Diagnostic Tests ; Recombinases ; CRISPR-Associated Proteins/genetics ; },
abstract = {Enterococcus faecalis (E. faecalis) is an opportunistic pathogen capable of causing various life-threatening infections, including urinary tract infections, bloodstream infections, infective endocarditis, and meningitis. As a major etiological agent of healthcare-associated infections (HAIs), its global prevalence continues to rise, a trend closely linked to the increasing problem of multidrug resistance driven by overuse of antibiotics. Therefore, rapid and accurate detection is essential for timely treatment and improved prognosis. In this study, the pheS gene of E. faecalis was rapidly amplified using recombinase polymerase amplification (RPA), and detection was achieved via a CRISPR/Cas12a system. The Cas12a-crRNA complex specifically recognized the amplification product and triggered nonspecific cleavage of a single-stranded DNA (ssDNA) reporter, generating a fluorescent signal that could be quantified in a real-time PCR system or visualized directly under ultraviolet (UV) light. After optimization of key parameters-including RPA primers, reaction conditions, crRNA sequence, and the crRNA/Cas12a combination-the assay achieved a limit of detection (LOD) of 10[-2] ng/μL within a short turnaround time, and showed no cross-reactivity with other common pathogen detection results from clinical isolates and spiked samples were fully consistent with those obtained through PCR/qPCR, confirming high reliability. In summary, the RPA-CRISPR/Cas12a detection method established in this study is sensitive, specific, and reliable. Its simplicity, minimal equipment requirements, and cost-effectiveness make it a promising tool for rapid clinical detection of E. faecalis.IMPORTANCEEnterococcus faecalis is a major opportunistic pathogen responsible for severe healthcare-associated infections, with rising prevalence linked to antibiotic resistance. Rapid and accurate detection is critical for timely treatment and infection control. Conventional methods are often time-consuming or require complex laboratory infrastructure, limiting their use at the point of care. This study developed a rapid detection assay by integrating recombinase polymerase amplification with the CRISPR/Cas12a system, targeting the pheS gene of E. faecalis. The method is sensitive and specific, providing visual results under UV light within a short turnaround time. It offers a simple, cost-effective, and requires minimal equipment, suitable for clinical and resource-limited settings, potentially improving diagnostic efficiency and supporting antimicrobial stewardship.},
}

*Enterococcus faecalis/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
Bacterial Proteins/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Gram-Positive Bacterial Infections/microbiology/diagnosis
Rapid Diagnostic Tests
Recombinases
CRISPR-Associated Proteins/genetics

@article {pmid42155443,
year = {2026},
author = {Liang, WW and Mueller, SJ and Hart, SK and Wessels, HH and Méndez-Mancilla, A and Sookdeo, A and Choi, O and Caragine, CM and Corman, A and Lu, L and Kolumba, O and Williams, B and Sanjana, NE},
title = {Essential lncRNAs in the human transcriptome.},
journal = {Cell genomics},
volume = {6},
number = {7},
pages = {101253},
doi = {10.1016/j.xgen.2026.101253},
pmid = {42155443},
issn = {2666-979X},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Transcriptome/genetics ; Gene Expression Profiling/methods ; CRISPR-Cas Systems/genetics ; Neoplasms/genetics ; Apoptosis/genetics ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Cell Cycle/genetics ; },
abstract = {Mammalian genomes host a diverse array of RNAs, including protein-coding and noncoding transcripts. However, the functional roles of most long noncoding RNAs (lncRNAs) remain elusive. Using RNA-targeting CRISPR-Cas13 screens, we probed how the loss of ∼5,500 lncRNAs impacts cell fitness across five human cell lines and identified 788 lncRNAs with context-specific or broad essentiality. We confirm their essentiality through individual perturbations and find that the majority of essential lncRNAs operate independently of their nearest protein-coding genes. Using transcriptome profiling in single cells, we discover that loss of essential lncRNAs impairs cell cycle progression and drives apoptosis. Many essential lncRNAs demonstrate dynamic expression across tissues during development. Using ∼9,000 primary tumors, we pinpoint those lncRNAs whose expression in tumors correlates with survival, yielding new biomarkers and potential therapeutic targets. This transcriptome-wide survey of functional lncRNAs advances our understanding of noncoding transcripts and demonstrates the potential of transcriptome-scale noncoding screens with Cas13.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Transcriptome/genetics
Gene Expression Profiling/methods
CRISPR-Cas Systems/genetics
Neoplasms/genetics
Apoptosis/genetics
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Cell Cycle/genetics

@article {pmid42214793,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {CRISPR/Cas9 system-mediated p21 knockout impairs the MITF signaling pathway.},
journal = {Journal of biotechnology},
volume = {417},
number = {},
pages = {81-90},
doi = {10.1016/j.jbiotec.2026.05.014},
pmid = {42214793},
issn = {1873-4863},
mesh = {*Microphthalmia-Associated Transcription Factor/metabolism/genetics ; Animals ; *Signal Transduction/genetics ; *Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Melanogenesis ; Gene Knockout Techniques/methods ; Melanins/metabolism ; Cell Line, Tumor ; },
abstract = {The CRISPR/Cas9 method facilitates targeted disruption of gene sequences, providing a reliable means to analyze gene-dependent regulatory pathways. This study aims to investigate melanogenesis in p21-knockout B16F1 cells generated by the CRISPR/Cas9 system. The mutation was confirmed by DNA Sanger sequencing, which identified frameshift-inducing indels in the p21 locus. The protein structure of p21 in KO cells was predicted by the α-Fold2 and ChimeraX models. The expression level of the p21 gene was completely reduced in RT-PCR and qPCR assays. Notably, while p21-knockout cells exhibited significantly reduced SA-β-galactosidase activity, this was not indicative of cellular rejuvenation. Instead, it correlated with a loss of melanocytic functionality, as evidenced by the concurrent decrease in melanin synthesis and collagen production. Western blotting and immunofluorescence analyses were performed to examine cell cycle and melanogenesis-associated proteins in p21-deficient cells. Loss of p21 resulted in reduced expression of p21, phosphorylated p21, p53, acetylated p53, CDK2, Cyclin D, Cyclin E, MITF, TRP-1, TRP-2, TYR, and p-ERK. Collectively, these findings indicate that p21 is essential for maintaining MITF-driven melanogenic signaling.},
}

*Microphthalmia-Associated Transcription Factor/metabolism/genetics
Animals
*Signal Transduction/genetics
*Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism
*CRISPR-Cas Systems/genetics
Mice
Melanogenesis
Gene Knockout Techniques/methods
Melanins/metabolism
Cell Line, Tumor

@article {pmid42284907,
year = {2026},
author = {Xiao, S and Zhou, L and Tian, Z and Zhou, S and Kong, J and Zhang, X},
title = {CRISPR-Cas12a-regulated photo-ATRP for ultrasensitive detection of lipopolysaccharide.},
journal = {Food chemistry},
volume = {522},
number = {},
pages = {149992},
doi = {10.1016/j.foodchem.2026.149992},
pmid = {42284907},
issn = {1873-7072},
mesh = {*Lipopolysaccharides/analysis ; *Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Limit of Detection ; *Bacterial Proteins/genetics/chemistry ; Food Contamination/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Beverages/analysis ; },
abstract = {Lipopolysaccharide (LPS), a primary virulence factor produced by Gram-negative bacteria, demands rapid and ultrasensitive detection for safeguarding food safety and supporting clinical diagnosis. This study developed a novel electrochemical biosensing platform by integrating the programmable recognition of CRISPR-Cas12a with the high-gain signal amplification of photoinduced ATRP. In this mechanism, the binding of LPS to its aptamer regulated Cas12a activity, which controlled an N3-DNA initiator probe. This probe, in turn, guided in-situ photopolymerization via click chemistry, generating a dramatically enhanced signal. The sensor exhibited a linear range from 10 fg/mL to 1 ng/mL with a detection limit of 2.48 fg/mL, along with high selectivity, reproducibility, and stability. Tests in spiked beverage samples showed high recovery rates and strong anti-interference capability. This work not only achieves ultrasensitive LPS detection but also provides a new approach for extending CRISPR-based biosensing to non-nucleic acid targets.},
}

*Lipopolysaccharides/analysis
*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems
Limit of Detection
*Bacterial Proteins/genetics/chemistry
Food Contamination/analysis
*Electrochemical Techniques/methods
*CRISPR-Associated Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
Beverages/analysis

@article {pmid42297231,
year = {2026},
author = {Fatima, SW},
title = {Cellular plasticity and epigenetic instability in cancer: Mechanistic insights and functional dissection with CRISPR-based epigenome editing.},
journal = {Cancer letters},
volume = {656},
number = {},
pages = {218679},
doi = {10.1016/j.canlet.2026.218679},
pmid = {42297231},
issn = {1872-7980},
mesh = {Humans ; *Epigenome Editing ; *Cell Plasticity/genetics ; *Neoplasms/genetics/pathology/therapy ; *Epigenesis, Genetic ; Tumor Microenvironment/genetics ; *CRISPR-Cas Systems ; Animals ; Gene Expression Regulation, Neoplastic ; DNA Methylation ; },
abstract = {Cellular plasticity is a fundamental driver of tumor heterogeneity, cancer stemness, immune evasion, therapeutic resistance, and disease progression. In malignancies such as breast cancer and glioblastoma, tumor cells undergo reversible phenotypic transitions between proliferative, stem-like, invasive, and drug-tolerant states in response to intrinsic regulatory programs and extrinsic signals from the tumor microenvironment. These adaptive dynamics are governed by complex interactions among signaling pathways, transcriptional networks, chromatin remodeling, DNA methylation, histone modifications, non-coding RNAs, and immune-mediated microenvironmental cues. Such epigenetic instability enables stochastic and therapy-induced shifts between alternative cellular states, thereby contributing to tumor evolution, metastasis, resistance to targeted therapies, and variable responses to immunotherapy. Understanding the mechanisms that govern epigenetic plasticity remains a central challenge in cancer biology. Recent advances in CRISPR/dCas9-based epigenome editing have provided powerful experimental tools for investigating the functional consequences of locus-specific chromatin modifications without altering the underlying DNA sequence. Catalytically inactive Cas9 (dCas9) fused to epigenetic effector domains, including DNMT3A, TET1, KRAB, and p300, enables targeted modulation of gene expression programs implicated in cell-state transitions, lineage specification, and adaptive resistance. These technologies offer a versatile platform for interrogating causal relationships between chromatin states and cellular phenotypes and for modeling mechanisms of tumor adaptation. This review examines the molecular basis of epigenetic plasticity in cancer, evaluates current CRISPR-based epigenome editing strategies, and discusses their application in studying tumor heterogeneity, microenvironment-driven adaptation, immune escape, and therapy resistance. This study highlights emerging opportunities and persistent challenges associated with epigenome editing, including delivery barriers, durability of epigenetic modifications, context-dependent biological responses, and translational limitations. Collectively, these approaches provide valuable experimental frameworks for dissecting the regulatory logic of cancer cell plasticity while informing future therapeutic development.},
}

Humans
*Epigenome Editing
*Cell Plasticity/genetics
*Neoplasms/genetics/pathology/therapy
*Epigenesis, Genetic
Tumor Microenvironment/genetics
*CRISPR-Cas Systems
Animals
Gene Expression Regulation, Neoplastic
DNA Methylation

@article {pmid42303162,
year = {2026},
author = {Durairaj, S and Durairaj, S and Krishnan, S and Raju, A},
title = {CRISPR Cas9 revolutionizing genetic engineering and therapeutic applications.},
journal = {Journal of biotechnology},
volume = {417},
number = {},
pages = {246-264},
doi = {10.1016/j.jbiotec.2026.06.012},
pmid = {42303162},
issn = {1873-4863},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; *Genetic Engineering/methods ; },
abstract = {Genetic engineering has been transformed by CRISPR-Cas9 technology, offering high precision and adaptability in biological research and therapeutic innovation. Originating from a bacterial defense system, CRISPR-Cas9 enables targeted DNA editing through guide RNA-directed Cas9 nuclease activity, allowing gene modification, mutation correction, and disease mechanism analysis. This has opened new avenues in personalized medicine and gene therapy, particularly for cancer and inherited disorders, alongside applications in agriculture. In oncology, CRISPR-Cas9 demonstrates strong potential in oncogene targeting, immune cell engineering, and CAR-T-based immunotherapy, supported by substantial preclinical success. Delivery efficiency is enhanced through systems such as exosomes, liposomes, and nanoparticles, improving stability and tumor targeting. However, clinical translation remains constrained by off-target effects, delivery limitations, and ethical concerns in human genome editing, particularly germline modification. CRISPR shows therapeutic promise for muscular dystrophy, sickle cell disease, and cystic fibrosis. Emerging platforms including base editing, prime editing, and dCas9-based epigenome editing enable precise genome and gene regulation without double-strand breaks, reducing toxicity and expanding therapeutic scope in cancer and genetic diseases. Regulatory frameworks remain heterogeneous, affecting translation. The United States leads in approvals and clinical progress, the European Union emphasizes safety and ethics, China shows rapid expansion in clinical trials, and India remains in early stages due to regulatory and infrastructure constraints. Public perception influences adoption, shaped by misinformation and limited awareness. Persistent gaps in long-term safety, clinical efficacy, and population diversity remain challenges. Overall, CRISPR-Cas9 represents a transformative but carefully regulated platform for advancing biotechnology and medicine.},
}

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

@article {pmid42398757,
year = {2026},
author = {Wang, X and Dong, W and Shen, R and Yu, X and Zhang, Y and Wang, W and Yin, X and Hu, Y and Peng, X and Yang, G and Rao, Q and Deng, X and Wang, R and Tang, F and Huang, Y and Jin, Z and Cai, Q and Xu, H and Tang, Y and Du, D},
title = {Development of optimized fluorogenic DNA aptamers for a portable one-pot CRISPR-Cas12a platform for rapid and sensitive detection of monkeypox virus and chikungunya virus.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.07.003},
pmid = {42398757},
issn = {2090-1224},
abstract = {INTRODUCTION: The recent global outbreaks of monkeypox virus (MPXV) and chikungunya virus (CHIKV) underscore the urgent need for rapid, accessible, and cost-effective diagnostic methods. Conventional CRISPR/Cas fluorescence assays rely on trans-cleavage of ssDNA/RNA reporters labeled with expensive fluorophores and quenchers, which limits widespread application.

OBJECTIVES: This study aims to develop and optimize a label-free, fluorogenic DNA aptamer-based reporter for a portable, one-pot Cas12a detection system capable of highly sensitive detection of MPXV and CHIKV directly from clinical specimens.

METHODS: We evaluated commonly used ssDNA aptamers for their fluorescence emission upon Thioflavin T (ThT) binding and their cleavage efficiency by Cas12a. Through systematic mutagenesis targeting G-rich regions, we enhanced fluorescence emission. Additionally, poly-A linkers were introduced between G-rich motifs to promote Cas12a cleavage efficiency. Circular dichroism (CD) spectroscopy confirmed G-quadruplex (G4) formation in the aptamers. The assay's sensitivity and specificity were assessed using simulated clinical samples, followed by validation with actual clinical specimens. The performance of direct detection from simulated clinical samples was compared to qRT-PCR. A battery-powered heating-pad, a mini-centrifuge, and a flashlight were used to validate its POCT applicability.

RESULTS: We designed and optimized a cost-effective, stable fluorogenic ssDNA aptamer that specifically binds to ThT. The aptamer ThT-3-5.1 exhibited the highest fluorescence enhancement and cleavage efficiency by Cas12a. Leveraging this aptamer, we developed a rapid, portable, one-pot detection platform (ROD-ThT) capable of detecting as few as 1 copy/reaction of MPXV and CHIKV nucleic acids within 35 min. Validation with clinical samples confirmed the assay's reliability without the need for nucleic acid purification.

CONCLUSION: Our simple, efficient, portable, and affordable ROD-ThT platform holds great promise for disease diagnostics and management, particularly in resource-limited settings.},
}

@article {pmid42399052,
year = {2026},
author = {Kim, J and Kovacs, H and Wisnovsky, S},
title = {CRISPR screens to identify and characterize ligands for glycan-binding proteins.},
journal = {Methods in enzymology},
volume = {732},
number = {},
pages = {219-263},
doi = {10.1016/bs.mie.2026.03.005},
pmid = {42399052},
issn = {1557-7988},
mesh = {Ligands ; *Polysaccharides/metabolism ; Humans ; *Lectins/metabolism/genetics ; Flow Cytometry/methods ; *CRISPR-Cas Systems ; Protein Binding ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cell surface glycans regulate key biological processes including immune signaling, cell communication, and pathogen recognition. Glycan-driven signaling is primarily mediated by glycan-binding proteins (lectins), whose functions depend on the identity and presentation of their glycoprotein ligands. However, identifying ligands for glycan-binding proteins remains challenging due to the structural complexity of carbohydrates and the importance of cellular context in determining binding specificity. Here, we describe a fluorescence-activated cell sorting (FACS)-based pooled CRISPR screening workflow for systematic identification of genetic factors that regulate lectin binding in living cells. The protocol covers lentiviral transduction of pooled sgRNA libraries and phenotypic selection of high- and low-lectin-binding populations by flow cytometry. Genomic DNA extraction, sequencing, and computational sgRNA enrichment analysis enable identification of genes influencing ligand biosynthesis and presentation. Subsequent analysis of these genetic factors can provide a comprehensive view of the structural determinants that govern lectin-glycan binding. The approach is compatible with CRISPR knockout, interference, and activation strategies, allowing broad interrogation of both loss- and gain-of-function effects. Key considerations for maintaining library coverage, optimizing sorting parameters, and performing robust statistical analysis are highlighted to maximize screening performance. Overall, this workflow offers a scalable framework for mapping glycan ligand landscapes in health and disease.},
}

Ligands
*Polysaccharides/metabolism
Humans
*Lectins/metabolism/genetics
Flow Cytometry/methods
*CRISPR-Cas Systems
Protein Binding
HEK293 Cells
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42399613,
year = {2026},
author = {Wei, R and Wang, S and Li, Y and Li, N and Pan, W and Tang, B},
title = {DNAzyme-Enhanced CRISPR/Cas12a Cascade Enables Isothermal, One-Pot RNA Diagnostics.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.6c05456},
pmid = {42399613},
issn = {1944-8252},
abstract = {While integrating DNAzymes with Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems offers a promising route to enhance CRISPR/Cas12a-based molecular diagnosis via enzyme-coupled cascade amplification, their implementation in simple, specific, and sensitive nucleic acid detection remains challenging, largely due to reliance on complex, multistep workflows. Here, we report an RNA-triggered DNAzyme circuit integrated with CRISPR/Cas12a that serves as a universal nucleic acid preamplifier, enabling one-pot and homogeneous detection. The catalytic activity of DNAzyme, initially suppressed by a complementary blocker strand, was restored upon the recognition of the target analyte. The activated DNAzyme then cleaved a hairpin-shaped substrate, liberating multiple activators that triggered a secondary CRISPR/Cas amplification reaction. This cascade generated a visible red band signal on a lateral flow assay via the collateral cleavage of a reporter. By employing the DNAzyme as a signal amplifier, the system efficiently converted a single RNA molecule into numerous initiators, breaking the one-to-one activation relationship between the target and Cas12a ribonucleoprotein and thereby greatly enhancing the detection sensitivity. Additionally, the system exhibited high programmability and universality, as a biosensor for a given target could be easily constructed by simply customizing the corresponding region of the blocker strand that is complementary to the target sequence. This integrated cascade system enables efficient signal amplification within a simple one-pot format and holds significant promise for practical applications.},
}

@article {pmid42400295,
year = {2026},
author = {Liang, D and Guo, H and Wei, J and Zhu, F and Zhang, Y and Green, J and Ji, Y and Jin, H and Zhang, X and Gui, H and Dan, H and Liu, Y and Zhang, Y and Wang, H and Jiang, Y and Geng, L and Lv, J and Cai, W and Song, W and Kelliher, T and Chen, X and Egger, R},
title = {A Robust Framework for Maize Elite Line Genome Editing Through Enhanced HI-Edit via LbCas12a Activity Optimization.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70715},
pmid = {42400295},
issn = {1467-7652},
abstract = {Haploid induction coupled with genome editing (HI-Edit) enables direct modification of commercial crop varieties, bypassing the need for trait introgression or direct transformation of elite lines with CRISPR machinery. However, its widespread application has been constrained by low haploid editing rates (HER), the proportion of haploids carrying edits within the short window between double fertilization and uniparental chromosome elimination. Here, we report substantial improvements in maize HI-Edit efficiency through three complementary strategies: (1) driving an optimized LbCas12a variant (LbCas12aV) using promoters that are highly active in sperm cells and early zygotes; (2) applying a post-pollination heat treatment; and (3) fusing LbCas12aV with the UBA2 domain (ubiquitin-associated domain-2 of Arabidopsis thaliana RAD23) to enhance protein stability during haploid induction. Post-pollination heat treatment alone increased HER to 19.1% (up to 12-fold improvement depending on the target site), providing a simple and effective method to boost the yield of edited doubled haploid (DH) plants. UBA2 fusion improved HER by 6-fold at the Waxy1 (Wx1) locus and 4.5-fold at the Glossy2 (Gl2) locus under normal conditions. Strikingly, combining UBA2 fusion with heat treatment raised the average HER to 25% across multiple events targeting Wx1, with the highest HER reaching 33%. Collectively, these findings demonstrate that increasing CRISPR-Cas protein abundance and modulating environmental conditions can overcome key bottlenecks in HI-Edit. We establish a robust, scalable framework that is readily transferable to other crops for elite-line genome editing.},
}

@article {pmid42400407,
year = {2026},
author = {Wan, L and Zhou, J and Yu, L and Huang, X and Fu, J and Xiao, F and Jia, N and Zhang, Y and Chen, M and Feng, Z and Wang, Y},
title = {A One-Pot CRISPR-Cas12b Assay for Rapid Detection of Human Adenovirus Serotypes 3 and 7.},
journal = {Journal of medical virology},
volume = {98},
number = {7},
pages = {e71032},
doi = {10.1002/jmv.71032},
pmid = {42400407},
issn = {1096-9071},
support = {7262007//Beijing Natural Science Foundation/ ; L234047//Beijing Natural Science Foundation-Changping Innovation Joint Fund Project/ ; XZDX-2025-002//Research Foundation of Capital Institute of Pediatrics/ ; BJRID2025-009//Beijing research center for respiratory infectious diseases project/ ; BJRID2026-011//Beijing research center for respiratory infectious diseases project/ ; 2024-0040//Pathogen spectrum and host marker analysis in respiratory tract infection of children/ ; MEKLCEPP/SXMU-202412//Key Laboratory of Coal Environmental Pathogenicity and Prevention (Shanxi Medical University), Ministry of Educatio/ ; },
mesh = {Humans ; *Adenoviruses, Human/isolation & purification/genetics/classification ; *CRISPR-Cas Systems ; *Adenovirus Infections, Human/diagnosis/virology ; Serogroup ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; China ; *Molecular Diagnostic Techniques/methods ; Respiratory Tract Infections/virology/diagnosis ; },
abstract = {Human adenovirus (HAdV) is a leading cause of acute respiratory tract infections (ARTIs) in children. The high prevalence of HAdV serotypes 3 and 7 in regions such as China presents a significant public health challenge. Here, we propose a one-pot assay that integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b for the detection of HAdV-3 and HAdV-7, termed HAdV-MCDA-One. In this system, MCDA provides exponential target amplification, while the collateral cleavage activity of Cas12b enables secondary signal amplification. The entire reaction is performed isothermally at 60°C in a single tube, providing a fluorescent readout within 50 min, making the assay suitable for point-of-care testing (POCT). Leveraging the single-base recognition capability of CRISPR-Cas12b, the assay demonstrates high specificity, with no cross-reactivity observed against the other 13 identified pathogens. The limit of detection was determined to be 1.59 copies per reaction using target plasmids. Moreover, when evaluated with 96 clinical pharyngeal swabs, the assay showed 100% concordance with quantitative PCR (qPCR), confirming its clinical reliability. These results demonstrate HAdV-MCDA-One as a rapid and robust tool for HAdV-3 and HAdV-7 detection, with significant potential for clinical diagnosis and public health surveillance.},
}

Humans
*Adenoviruses, Human/isolation & purification/genetics/classification
*CRISPR-Cas Systems
*Adenovirus Infections, Human/diagnosis/virology
Serogroup
Sensitivity and Specificity
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
China
*Molecular Diagnostic Techniques/methods
Respiratory Tract Infections/virology/diagnosis

@article {pmid42400607,
year = {2026},
author = {Liu, F and Jiang, T and Tanwir, SE and Ardi, WH and Huo, H},
title = {CRISPR/Cas9-mediated DFR disruption suggests coordinated changes in flavonoid flux and development in Petunia × hybrida.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42400607},
issn = {1432-203X},
support = {2019-67013-29236//National Institute of Food and Agriculture/ ; FLA-MFC-006387//USDA HATCH/ ; },
mesh = {*Petunia/genetics/metabolism/growth & development/enzymology ; *Flavonoids/metabolism ; *CRISPR-Cas Systems/genetics ; *Alcohol Oxidoreductases/genetics/metabolism ; Gene Expression Regulation, Plant ; Anthocyanins/metabolism ; *Plant Proteins/genetics/metabolism ; Flowers/metabolism/genetics ; Pigmentation/genetics ; Phenotype ; Plant Leaves/metabolism/genetics ; Plants, Genetically Modified ; Chlorophyll/metabolism ; Carotenoids/metabolism ; Oxidoreductases/metabolism ; },
abstract = {Loss of DFR function in petunia alters pigment metabolism and reduces organ size, suggesting previously underexplored associations among flavonoid biosynthesis, plastidial pigments, and development. Dihydroflavonol 4-reductase (DFR) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase. While DFR's role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated DFR-edited lines in the commercial Petunia × hybrida cultivar 'Carmine Velour'. The edited lines showed a spectrum of floral pigmentation loss that was broadly consistent with the representative editing patterns inferred from Sanger sequencing, supporting the major contribution of DFR-A to corolla anthocyanin accumulation. Beyond pigmentation, dfr mutants exhibited unexpected reductions in floral dimensions (20-40%), leaf biomass (30-50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35-60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, indicating organ-specific associations between DFR disruption and growth-related traits. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: chalcone synthase A (CHSA) and chalcone isomerase A (CHIA) were downregulated while the competing branch enzyme flavonol synthase (FLS) was upregulated almost twofold, consistent with the possibility of altered flux partitioning toward flavonol biosynthesis. Strikingly, protochlorophyllide oxidoreductase A (PORA), encoding a key chlorophyll biosynthetic enzyme, was severely suppressed by 60-75%, suggesting a possible connection between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses suggested coordinated variation, with floral anthocyanin content positively associated with leaf chlorophyll and carotenoid levels across genotypes. These findings support the view that DFR acts as a functionally important metabolic node whose disruption is associated with effects across pigment classes and organ types, with implications for precision trait engineering in floriculture.},
}

*Petunia/genetics/metabolism/growth & development/enzymology
*Flavonoids/metabolism
*CRISPR-Cas Systems/genetics
*Alcohol Oxidoreductases/genetics/metabolism
Gene Expression Regulation, Plant
Anthocyanins/metabolism
*Plant Proteins/genetics/metabolism
Flowers/metabolism/genetics
Pigmentation/genetics
Phenotype
Plant Leaves/metabolism/genetics
Plants, Genetically Modified
Chlorophyll/metabolism
Carotenoids/metabolism
Oxidoreductases/metabolism

@article {pmid42400669,
year = {2026},
author = {Tamrakar, VK and Sharma, K and Singh, P and Bhargava, A and Thakur, P and Negi, SS},
title = {Development of a CRISPR/dCas9-based membrane-assisted colorimetric assay for detection of high-risk HPV16 and HPV18: a proof-of-concept study.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42400669},
issn = {1573-4978},
support = {Extramural Grant IIPR-2030-3960/F1.//Indian Council of Medical Research/ ; },
mesh = {*Colorimetry/methods ; *Human papillomavirus 18/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Human papillomavirus 16/genetics/isolation & purification ; Humans ; *Papillomavirus Infections/diagnosis/virology ; Proof of Concept Study ; Genotype ; Female ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; Genotyping Techniques/methods ; },
abstract = {BACKGROUND AND AIMS: Persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, is the leading cause of cervical cancer. While molecular diagnostics offer high sensitivity, their deployment in decentralized settings remains limited. This study presents a proof-of-concept CRISPR/dCas9-based membrane-assisted detection platform for HR-HPV genotyping.

METHODS: A membrane-based assay integrating recombinase polymerase amplification (RPA) with CRISPR/dCas9-mediated sequence-specific recognition was developed. FAM-labeled amplicons were captured by immobilized dCas9-sgRNA ribonucleoprotein complexes and detected via antibody-mediated colorimetric readout.

RESULTS: The assay enabled specific detection of HPV16 and HPV18 using genotype-specific sgRNAs, producing visually interpretable signals on a nitrocellulose membrane. No signal was observed in negative controls, demonstrating high analytical specificity. Semi-quantitative signal assessment confirmed clear differentiation between positive and negative samples.

CONCLUSION: This study demonstrates the feasibility of a CRISPR/dCas9-based membrane-assisted detection system for HR-HPV genotyping. While not yet configured as a fully integrated lateral flow device, the platform provides a foundation for future development of simplified, point-of-care molecular diagnostics.},
}

*Colorimetry/methods
*Human papillomavirus 18/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Human papillomavirus 16/genetics/isolation & purification
Humans
*Papillomavirus Infections/diagnosis/virology
Proof of Concept Study
Genotype
Female
Sensitivity and Specificity
Rapid Diagnostic Tests
Genotyping Techniques/methods

@article {pmid41981786,
year = {2026},
author = {Munir, F and Zaheer, U and Asad, M and Yang, G},
title = {Knockout of Ku70 and Ku80 elevates homology-directed repair efficiency in Plutella xylostella.},
journal = {Insect molecular biology},
volume = {35},
number = {4},
pages = {434-447},
doi = {10.1111/imb.70039},
pmid = {41981786},
issn = {1365-2583},
support = {2024L3004//Fujian Province Science and Technology Project/ ; 2024-P-002//Fuzhou-Xiamen-Quanzhou National Independent Innovation Demonstration Zone RNAi Microbial Pesticide Collaborative Innovation Platform Project/ ; 2024I0007//the External Cooperation Project of Fujian Province/ ; KRA16001A//fund of the "111" program/ ; 2023J02009//Fujian Province Natural Science Foundation/ ; W2533068//Overseas Young Researcher Project of National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Moths/genetics/metabolism ; *Ku Autoantigen/genetics/metabolism ; Female ; Male ; Phylogeny ; Amino Acid Sequence ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Sequence Alignment ; },
abstract = {The suppression of Ku70 and Ku80 has been verified to increase homology-directed repair (HDR) efficiency in fruit fly, silkworm and mosquito, but not in other insects. In this study, PxKu70 and PxKu80 were identified from the Plutella xylostella genome. Domain analysis revealed that PxKu70 contained three conserved domains: Ku N, Ku and Ku C, while PxKu80 comprised the Ku N, Ku and Ku PK bind domains. Phylogenetic analysis and multiple-sequence alignment indicated strong conservation of both proteins among lepidopteran insects. RT-qPCR analysis showed that PxKu70 and PxKu80 were highly expressed in adult stages, particularly in reproductive tissues such as the ovary and testis, suggesting their role in maintaining genomic stability during gametogenesis. Two homozygous knockout lines (ΔPxKu70 and ΔPxKu80) were successfully generated through CRISPR/Cas9-mediated genome editing. These knockout lines remained viable and fertile without observable fitness effects. A donor construct carrying an EGFP cassette designed for insertion at the PxKmo locus was generated to assess HDR-mediated integration. The HDR insertion rate was significantly elevated in both knockout lines compared with the wild-type. These findings demonstrate that suppression of either PxKu70 or PxKu80 can enhance HDR in P. xylostella, offering an effective approach for precise genome editing in lepidopteran species.},
}

Animals
*Moths/genetics/metabolism
*Ku Autoantigen/genetics/metabolism
Female
Male
Phylogeny
Amino Acid Sequence
*Insect Proteins/genetics/metabolism
Gene Knockout Techniques
*Recombinational DNA Repair
CRISPR-Cas Systems
Sequence Alignment

@article {pmid42062284,
year = {2026},
author = {Sun, J and Noss, S and Smolen, C and Bhavana, VH and Banerjee, D and Das, M and Giardine, B and Prabhu, A and Amor, DJ and Pope, K and Lockhart, PJ and Girirajan, S},
title = {Functional impact of genetic background on variable expressivity in neurodevelopmental disorders.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42062284},
issn = {2041-1723},
support = {R01 GM121907/GM/NIGMS NIH HHS/United States ; R21 NS122398/NS/NINDS NIH HHS/United States ; GM121907//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; NS122398//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {*Neurodevelopmental Disorders/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Forkhead Transcription Factors/genetics/metabolism ; Phenotype ; Neurons/metabolism ; Chromatin/metabolism ; Signal Transduction/genetics ; Neural Stem Cells/metabolism ; Neurodevelopment ; Cell Proliferation ; CRISPR-Cas Systems ; Animals ; },
abstract = {Disease-associated variants can lead to variable phenotypic outcomes in neurodevelopmental disorders, but the biological mechanisms underlying this variability remain poorly understood. Here, we develop a framework to investigate this phenomenon using the 16p12.1 deletion as a paradigm of variable expressivity. Using induced pluripotent stem cell models from affected families and CRISPR-edited lines with the 16p12.1 deletion, we find that the deletion and rare variants in the genetic background jointly influence chromatin accessibility and expression of neurodevelopmental genes. Cellular analyses identify family-specific phenotypes, including altered inhibitory neuron production and neural progenitor cell proliferation, which correlate with head-size variation. CRISPR activation of individual 16p12.1 genes variably rescue these defects by modulating key developmental signaling pathways. Integrative analyses further identify regulatory hubs, including transcription factors FOXG1 and JUN, as mediators of these effects. Our study provides a functional framework for investigating how individual genetic architectures contribute to phenotypic variability in neurodevelopmental disorders.},
}

*Neurodevelopmental Disorders/genetics
Humans
Induced Pluripotent Stem Cells/metabolism
Forkhead Transcription Factors/genetics/metabolism
Phenotype
Neurons/metabolism
Chromatin/metabolism
Signal Transduction/genetics
Neural Stem Cells/metabolism
Neurodevelopment
Cell Proliferation
CRISPR-Cas Systems
Animals

@article {pmid42091648,
year = {2026},
author = {Chandrasekaran, J and Suthanthiram, B and Selvaraj, EP and Swaminathan, S and Chandran, SA and Ramasamy, S},
title = {Targeting a conserved functional motif in the PDS gene enables efficient CRISPR/Cas9 editing in banana.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42091648},
issn = {2045-2322},
support = {DST/WOS-A/LS-202/2021(G)//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Musa/genetics ; *Oxidoreductases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Base Sequence ; *Gene Editing ; Conserved Sequence ; },
abstract = {Incomplete editing and chimeric phenotypes are major challenges in CRISPR/Cas9-mediated genome editing of polyploid crops. In this study, a single guide RNA (gRNA) was designed to target a conserved dinucleotide-binding motif within exon 3 of the phytoene desaturase (PDS) gene in 'Grand Naine' banana. The gRNA was carefully selected for GC content, guanine residues near the PAM, and predicted secondary structure to enhance Cas9 cleavage efficiency. Agrobacterium-mediated transformation of embryonic cell suspensions produced 102 putative transgenic plants, all exhibiting altered phenotypes, with 91% displaying albino and 9% pale green coloration, indicating efficient PDS gene knockout and absence of chimerism. Sequencing confirmed tri-allelic editing, with all edited plants consistently showing two identical and one distinct mutation. Notably, small in-frame deletions of two to six amino acids within the conserved motif were sufficient to abolish PDS function, confirming its critical role in carotenoid biosynthesis. This strategy is adaptable to clonally propagated polyploid crops, providing a practical framework for achieving high-efficiency, uniform genome edits and supporting the development of precise, non-chimeric CRISPR/Cas9 editing approaches.},
}

*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Musa/genetics
*Oxidoreductases/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Base Sequence
*Gene Editing
Conserved Sequence

@article {pmid42229614,
year = {2026},
author = {Kalmotia, V},
title = {Enhancing statistical accuracy in gene perturbation studies.},
journal = {Bio Systems},
volume = {266},
number = {},
pages = {105819},
doi = {10.1016/j.biosystems.2026.105819},
pmid = {42229614},
issn = {1872-8324},
mesh = {Humans ; *Gene Expression Profiling/methods ; *Single-Cell Analysis/methods ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems/genetics ; Genomics/methods ; },
abstract = {Accurately analysing gene expression changes in high-throughput perturbation studies remains a challenge due to confounding technical factors. This paper evaluates and extends the SCEPTRE (Single-Cell PerTurbation screens via Conditional REsampling) framework, originally introduced by Barry et al. (2021), demonstrating its applicability to high-multiplicity-of-infection (MOI) CRISPR screens. By leveraging a resampling-based methodology, our approach effectively adjusts for sequencing biases, reducing false discoveries while maintaining statistical power.},
}

Humans
*Gene Expression Profiling/methods
*Single-Cell Analysis/methods
High-Throughput Nucleotide Sequencing/methods
CRISPR-Cas Systems/genetics
Genomics/methods

@article {pmid42335458,
year = {2026},
author = {Yang, L and Zhou, Y and Li, H and Chen, X and Zhang, S and Zhu, L},
title = {An Entropy-Driven Autocatalysis-Regulated Signal-On CRISPR/Cas12a Biosensor Supported by DNA Triangular Prism Scaffold.},
journal = {Analytical chemistry},
volume = {98},
number = {26},
pages = {19636-19651},
doi = {10.1021/acs.analchem.6c01471},
pmid = {42335458},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *DNA/chemistry ; *Entropy ; *MicroRNAs/blood/analysis ; *CRISPR-Cas Systems/genetics ; *alpha-Fetoproteins/analysis ; Humans ; Electrochemical Techniques ; Limit of Detection ; DNA Nanostructures ; Carcinoma, Hepatocellular/diagnosis/blood ; },
abstract = {Accurate and sensitive detection of low-abundance biomarkers in complex matrices remains challenging due to the inherent trade-off between amplification efficiency and background suppression in conventional biosensing strategies. Herein, a synergistic amplification strategy was constructed by integrating an entropy-driven autocatalysis (EDAC), a signal-on CRISPR/Cas12a assay, and a DNA triangular prism (DTP) interface. In this strategy, EDAC achieved exponential signal amplification through the recycling of target molecules and reaction byproducts, and its output strands simultaneously served as specific inhibitors of CRISPR/Cas12a. Based on this mechanism, the signal-on CRISPR/Cas12a assay strictly coupled signal generation to the presence of the target, thereby fundamentally circumventing the high background interference inherent to conventional signal-off modes. As a rigid three-dimensional interfacial scaffold, DTP provided high-density and well-ordered nucleic acid assembly sites, reduced steric hindrance through a solution-like microenvironment, suppressed nonspecific adsorption, and efficiently initiated downstream hybridization chain reaction for robust electrochemical readout via methylene blue intercalation. With hepatocellular carcinoma-associated biomarkers alpha-fetoprotein and microRNA-122 as model targets, the biosensor achieved detection limits as low as 11.37 fg/mL and 18.13 aM, respectively. In clinical serum sample assays, the biosensor showed strong agreement with the classical ELISA method, with an area under the curve value of 1.00, demonstrating its promising potential for the diagnosis of hepatocellular carcinoma. With its modular architecture and adaptable recognition elements, this strategy establishes a versatile framework for ultrasensitive biosensing and holds promise for clinical translation in early disease diagnosis.},
}

*Biosensing Techniques/methods
*DNA/chemistry
*Entropy
*MicroRNAs/blood/analysis
*CRISPR-Cas Systems/genetics
*alpha-Fetoproteins/analysis
Humans
Electrochemical Techniques
Limit of Detection
DNA Nanostructures
Carcinoma, Hepatocellular/diagnosis/blood

@article {pmid42342147,
year = {2026},
author = {Yuan, J and Ma, Y and Li, J and Chen, X and Wen, Y and Wu, R and Hang, X and Huang, H and Du, S and Wang, Y and Yan, QG and Huang, X and Zhao, F and Yi, Z and Cao, SJ and Zhao, Q},
title = {CXCL8 is associated with aflatoxin B1-triggered injury and caspase-3 activation in porcine kidney epithelial PK15 cells: integrated transcriptomics and CRISPR/Cas9 knockout.},
journal = {Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association},
volume = {215},
number = {},
pages = {116236},
doi = {10.1016/j.fct.2026.116236},
pmid = {42342147},
issn = {1873-6351},
mesh = {Animals ; *Aflatoxin B1/toxicity ; Swine ; *Caspase 3/metabolism/genetics ; *Interleukin-8/genetics/metabolism ; *Epithelial Cells/drug effects/metabolism ; Cell Line ; *Kidney/drug effects/cytology/metabolism ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Gene Knockout Techniques ; *Transcriptome/drug effects ; Reactive Oxygen Species/metabolism ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent food- and feed-borne mycotoxin, and growing evidence indicates that the renal epithelium is a vulnerable target. However, host determinants that modify epithelial susceptibility remain poorly defined. Here, we investigated AFB1-triggered injury in porcine kidney epithelial PK15 cells and examined the contribution of CXCL8. PK15 cells were exposed to AFB1 (0-32 μM) to define dose-dependent cytotoxicity, and sub-IC50 conditions (4 and 8 μM for 24 h) were used for mechanistic analyses; RNA sequencing at 4 μM for 24 h was used as an exploratory screen to prioritize candidate susceptibility factors. CXCL8 emerged as the most strongly induced transcript and was subsequently evaluated using CRISPR/Cas9-mediated knockout. CXCL8 deficiency attenuated AFB1-induced loss of viability, reduced Annexin V/PI-positive cells, and alleviated mitochondrial ultrastructural injury. In parallel, CXCL8 knockout decreased ROS accumulation, partially restored intracellular GSH and the BCL2/BAX transcriptional ratio, and reduced caspase-3 induction and cleavage. Collectively, these data support CXCL8 as an AFB1-inducible susceptibility factor associated with oxidative stress amplification and caspase-3 activation in PK15 cells, while indicating that the upstream regulatory axis and the precise downstream signaling route require further validation in physiologically relevant renal models.},
}

Animals
*Aflatoxin B1/toxicity
Swine
*Caspase 3/metabolism/genetics
*Interleukin-8/genetics/metabolism
*Epithelial Cells/drug effects/metabolism
Cell Line
*Kidney/drug effects/cytology/metabolism
CRISPR-Cas Systems
Apoptosis/drug effects
Gene Knockout Techniques
*Transcriptome/drug effects
Reactive Oxygen Species/metabolism

@article {pmid42387926,
year = {2026},
author = {Van Heurck, R and Hammar, E and Marconi, C and Korff, C and Abramowicz, M},
title = {[Targeted therapy for ultra-rare diseases].},
journal = {Revue medicale suisse},
volume = {22},
number = {969},
pages = {1216-1220},
doi = {10.53738/REVMED.2026.22.969.48835},
pmid = {42387926},
issn = {1660-9379},
mesh = {Humans ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Oligonucleotides, Antisense/administration & dosage ; *Molecular Targeted Therapy/methods ; Gene Therapy Agents ; CRISPR-Cas Systems ; },
abstract = {Nano-rare diseases, affecting fewer than 30 individuals worldwide, are mostly genetic and severe, with no effective treatments available. Nucleic acid-based therapies, such as antisense oligonucleotides, allow for the targeted modulation of gene expression. Successes like nusinersen and ultrapersonalized treatments (for example, Milasen) highlight their potential. Other approaches, including viral gene therapy and CRISPR-Cas9, enable the addition or correction of genes. However, major challenges remain, including high costs, difficulties in conducting clinical trials, and inadequate regulatory frameworks, especially for "N-of-1" therapies. International initiatives are emerging to facilitate access to these innovative treatments in Europe and Switzerland.},
}

Humans
*Rare Diseases/therapy/genetics
*Genetic Therapy/methods
Oligonucleotides, Antisense/administration & dosage
*Molecular Targeted Therapy/methods
Gene Therapy Agents
CRISPR-Cas Systems

@article {pmid42388033,
year = {2026},
author = {Sultana, H and Mohanty, S and Solomon, AD and Iqbal, MY and Wani, AK and Kumar, V and Khattri, A},
title = {AI in Genomics: From Variant Calling to Multi-Omics Integration.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {48},
number = {7},
pages = {e70160},
doi = {10.1002/bies.70160},
pmid = {42388033},
issn = {1521-1878},
mesh = {Multiomics ; *Genomics/methods ; *Artificial Intelligence ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Machine Learning ; },
abstract = {Artificial intelligence (AI) strategies are revolutionizing genomics by extracting complex patterns that traditional statistical pipelines are likely to miss. This mini-review aims to provide a concise overview of how AI is transforming major genomic technologies including variant calling, gene expression analysis, single-cell transcriptomics, CRISPR-Cas9 optimization, and multi-omics integration. In genome sequencing, machine learning variant callers greatly improve the accuracy and the rate at which single nucleotide and structural variants are called. In bulk RNA-Seq, AI augmented quantification, denoising, and differential expression modules complement the highly established STAR-featureCounts-DESeq2 pipeline, revealing subtle signals in big data sets. In single cell transcriptomics, deep learning approaches enhance batch correction, automate cell type annotation, and track developmental trajectories, hence clarifying cellular heterogeneity. AI-assisted guide RNA design, outcome prediction, and nuclease engineering enable more efficient CRISPR-Cas9 editing, reducing experimental cycles, and off-target effects. Finally, integrated platforms that combine genomic, transcriptomic, epigenomic, proteomic, and metabolomic layers provide an integrative view of cellular regulation and disease mechanisms. The review also covers current limitations, sparsity of data, model bias, privacy, and the need for standardized benchmarks and offers future directions in the form of interpretable models, collaborative learning, and open science practices. Together, these developments render AI an indispensable partner to unravel genomic complexity and accelerate precision medicine applications.},
}

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

@article {pmid42389571,
year = {2026},
author = {Kohabir, KAV and Rietveld, AWJ and Nooi, LO and Beijer, RE and van Dongen, JE and Linthorst, J and Wolthuis, RMF and Jonges, M and Welkers, MRA and Segerink, LI and Sistermans, EA},
title = {Toward point-of-care and amplification-free detection of human cytomegalovirus using CRISPR-Cas12a.},
journal = {iScience},
volume = {29},
number = {7},
pages = {116494},
pmid = {42389571},
issn = {2589-0042},
abstract = {Human cytomegalovirus (hCMV) is a herpesvirus that establishes lifelong latency in myeloid cells, posing health concerns particularly in fetal development and in immunocompromised individuals. Point-of-care (PoC) detection of hCMV DNA in liquid biopsies supports timely diagnosis and proper mitigation. However, ultra-low concentrations and high fragmentation rates, challenge primer-based preamplification methods. We present a proof-of-concept amplification-free CRISPR-based assay, exploiting the inherent specificity and signal-amplification of Cas12a and improving signal using a combinatorial approach. Optimizing Cas12a's trans-cleavage activity and multiplexing hCMV loci, significantly increased detection sensitivity in-bulk. Additionally, we found that AsCas12a trans-cleaves cytosine-rich reporters 4× more efficiently than conventional probes, further improving assay kinetics to reach a femtomolar limit of detection. Translating these optimizations to a microfluidic assay enables sensitive detection even if additional measures may be needed for quantitative, single molecule measurements. Our assay opens avenues toward PoC detection in low-resource settings, supporting effective and affordable infection management.},
}

@article {pmid42390428,
year = {2026},
author = {Shinoda, H and Makino, A and Yoshimura, M and Iida, T and Yamazaki, D and Minagawa, N and Kogo, Y and Sano, T and Jinnai, M and Hishiki, T and Ohya, H and Nishimasu, H and Watanabe, R},
title = {Single-Molecule Characterization of CRISPR-Cas12a for Amplification-Free Genetic Testing.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c00936},
pmid = {42390428},
issn = {1520-6882},
abstract = {CRISPR-Cas-based genetic testing has gained considerable attention for rapid and accurate diagnosis of infectious and genetic diseases. Cas12a, an RNA-guided nuclease that targets DNA, has been widely applied for DNA detection; however, its detailed enzymatic properties have not been fully elucidated. Here, we performed a systematic single-molecule analysis of Cas12a using microchamber arrays to biophysically characterize its catalytic behavior including protospacer adjacent motif (PAM) specificity, mismatch tolerance, and reaction kinetics and to evaluate the feasibility and inherent challenges of amplification-free DNA detection. Cas12a exhibited high activation efficiency toward target DNA, demonstrating the feasibility of amplification-free DNA detection with a detection limit of 104 aM within 15 min. However, Cas12a also exhibited substantial nonspecific cross-reactivity with genome-length targets, particularly mammalian genomic DNA, identifying off-target activation as a major challenge for reliable diagnostic application. Together, these findings provide a quantitative biophysical characterization of Cas12a-based amplification-free DNA detection and highlight the need to improve its target specificity for future reliable application of Cas12a in amplification-free molecular diagnostics.},
}

@article {pmid42390463,
year = {2026},
author = {Mir, YB and Manzoor, T and Mushtaq, D and Najar, AH and Kawoosa, F and Bhatia, D and Qadri, SM and Siraj, F and Ahmad, SM},
title = {Diagnostic challenges in re-emerging rickettsioses: why current tools fall short.},
journal = {Clinical microbiology reviews},
volume = {},
number = {},
pages = {e0002026},
doi = {10.1128/cmr.00020-26},
pmid = {42390463},
issn = {1098-6618},
abstract = {SUMMARYRickettsial diseases, encompassing scrub typhus, spotted fever group rickettsioses, and typhus group rickettsioses, represent a significant and escalating public health threat worldwide, particularly in the Asia-Pacific and sub-Saharan African regions. Despite their high morbidity and potential for fatal outcomes if left untreated, these infections remain notoriously underdiagnosed due to their nonspecific clinical presentation, which frequently overlaps with other acute undifferentiated febrile illnesses (AUFIs) such as dengue, malaria, and leptospirosis. This review evaluates the evolving diagnostic landscape, highlighting the severe limitations of conventional methods: the Weil-Felix test lacks necessary specificity, while the gold standard indirect immunofluorescence assay (IFA) is primarily retrospective due to delayed seroconversion. Molecular diagnostics, particularly multiplex polymerase chain reaction (mPCR), have emerged as a critical advancement, enabling early, species-specific identification during the acute phase of infection when doxycycline therapy is most effective. We further explore the paradigm shift toward syndromic molecular panels, such as the TaqMan Array Card (TAC), which facilitate simultaneous screening for multiple AUFI pathogens. Emerging platforms, including digital PCR (dPCR) for absolute quantification and CRISPR-Cas-based point-of-care (POC) systems (SHERLOCK and DETECTR), offer promising solutions for low-resource settings. Finally, this review underscores the necessity of integrating molecular surveillance within a One Health framework and utilizing artificial intelligence (AI) to address technical and implementation barriers. Overcoming these challenges is essential for transforming rickettsial diagnosis from a reactive to a proactive strategy, ultimately reducing the global burden of these neglected zoonoses.},
}

@article {pmid42391043,
year = {2026},
author = {Lin, S and Jiang, S and Tang, L and Kumbhakonam, SK and Dingman, JC and Lee, JY and Yang, R and Frudakis, T and Kirchner, M and Xu, S and Tao, C and Wang, X and Russo, JJ and Zhang, X and Chen, Q and Zhang, S},
title = {De novo direct sequencing of small therapeutic RNAs by layer-by-layer intensity-resolved mass spectrometry.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42391043},
issn = {1362-4962},
support = {R01 HG012853/HG/NHGRI NIH HHS/United States ; R41 HG013624/HG/NHGRI NIH HHS/United States ; R41 HG014125/HG/NHGRI NIH HHS/United States ; U24 HG011735/HG/NHGRI NIH HHS/United States ; RM1 HG011563/HG/NHGRI NIH HHS/United States ; R01 HD092431/HD/NICHD NIH HHS/United States ; R01 ES032024/ES/NIEHS NIH HHS/United States ; },
mesh = {*Sequence Analysis, RNA/methods ; *High-Throughput Nucleotide Sequencing/methods ; *RNA, Small Interfering/genetics/chemistry/therapeutic use ; MicroRNAs/genetics/chemistry ; Algorithms ; *Mass Spectrometry/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; },
abstract = {The rapid growth of RNA-based therapeutics demands accurate sequencing of all RNA species, including minor and modified variants. Conventional LC-MS/MS typically confirms only a predefined target sequence rather than determining RNA sequences de novo from the analyzed sample, thereby overlooking coexisting impurities and modifications. Here, we present 3D NGMS-Seq, a three-dimensional next-generation mass spectrometry-based sequencing platform for de novo direct sequencing of mixed RNA samples with essentially 100% sequence accuracy. This method incorporates MS intensity into traditional 2D mass-retention time (tR) analysis and introduces a nested algorithm that aligns ladder fragment intensities with parent RNA abundances for computational separation. Controlled acid hydrolysis produces RNA ladder fragments, which are segregated into mass-intensity-tR layers. Within each layer, short reads are generated de novo by sequentially base-calling each nucleotide, canonical or modified, from mass differences between adjacent ladder fragments and subsequently assembled into full-length RNA sequences. Guided by hydrolysis kinetics and statistical modeling, 3D NGMS-Seq accurately sequences synthetic siRNA, miRNA, and CRISPR/Cas9 sgRNAs, reveals unexpected low-abundance RNA impurities, and resolves subtle methylation ambiguities (Um versus mU; Am versus mA), while providing a quantitative profile of each RNA's relative abundance and site-specific modifications. By enabling direct, unbiased sequencing of heterogeneous RNAs without prior sequence knowledge, 3D NGMS-Seq addresses key limitations of current RNA analysis and provides a powerful tool to aid small RNA drug development, quality control, and regulatory validation.},
}

*Sequence Analysis, RNA/methods
*High-Throughput Nucleotide Sequencing/methods
*RNA, Small Interfering/genetics/chemistry/therapeutic use
MicroRNAs/genetics/chemistry
Algorithms
*Mass Spectrometry/methods
Humans
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry

@article {pmid42392238,
year = {2026},
author = {Chen, S and Pi, C and Zhang, B and Wu, X and Gao, L and Chen, X},
title = {Technology-driven revolution in CO2 fixation: From natural pathways to programmable Biosystems.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108964},
doi = {10.1016/j.biotechadv.2026.108964},
pmid = {42392238},
issn = {1873-1899},
abstract = {The escalating atmospheric CO2 concentration, exceeding 430 ppm since the pre-industrial era, presents a critical threat to global climate stability. Moving beyond mere carbon capture, this review synthesizes cutting-edge advancements in technology-driven CO2 fixation, focusing on microbial conversion systems. It begins by examining inherent limitations of natural pathways like the Calvin-Benson-Bassham cycle, constrained by low energy efficiency (<1%) and enzymatic inefficiencies of RuBisCO. The discussion then progresses to engineering native pathways and de novo design of synthetic routes (e.g., rGly, CETCH, THETA cycles), which demonstrate superior thermodynamic and kinetic properties for efficient carbon conversion. CRISPR-Cas systems' revolutionary impact, overcoming genetic barriers in carbon-fixing microorganisms. These tools enable precise metabolic rewiring and conversion of heterotrophic chassis into synthetic autotrophs. Furthermore, the convergence of microbiology with electrochemistry and materials science is detailed, highlighting innovative platforms like microbial electrosynthesis and semi-artificial photosynthetic systems. These biohybrid technologies create synergistic interfaces where microbes utilize electrons from electrodes or artificial materials to drive efficient CO2 reduction into multicarbon compounds, addressing critical energy supply challenges. The review analyzes the transition from natural pathway optimization to custom artificial system construction, underscoring a paradigm shift from isolated improvements to deeply integrated approaches. This new paradigm fuses metabolic engineering, synthetic biology, electrochemistry, and nanomaterials, guided by AI-aided design and modeling. The conclusion emphasizes that seamless integration of microbial capabilities, advanced materials, and artificial intelligence is pivotal for advancing CO2 fixation toward precision, high efficiency, and carbon negativity, laying the essential foundation for sustainable carbon-negative biomanufacturing and contributing meaningfully to global carbon neutrality goals.},
}

@article {pmid42392979,
year = {2026},
author = {Pant, DC and Lone, MA and Parameswaran, J and Ma, F and Ziak, N and Dutta, P and Wang, Z and Pun, D and Verma, S and Hornemann, T and Jiang, J},
title = {Deletion of exon 2 in ALS-linked Sptlc1 causes lethality in homozygous mice but not in heterozygotes.},
journal = {Life science alliance},
volume = {9},
number = {9},
pages = {},
pmid = {42392979},
issn = {2575-1077},
mesh = {Animals ; *Amyotrophic Lateral Sclerosis/genetics ; *Exons/genetics ; Mice ; Heterozygote ; Homozygote ; Disease Models, Animal ; Humans ; *Sequence Deletion/genetics ; Mutation ; Genes, Lethal ; CRISPR-Cas Systems ; Male ; Gene Knock-In Techniques ; },
abstract = {Mutations in the human SPTLC1 gene have recently been linked to early-onset amyotrophic lateral sclerosis (ALS), characterized by global atrophy, motor impairments, and symptoms such as tongue fasciculations. All known ALS-linked SPTLC1 mutations cluster within exon 2, and a specific variant, c.58G>T, results in exon 2 skipping. However, it is unclear how the exon 2 deletion affects SPTLC1 function in vivo and contributes to ALS pathogenesis. Leveraging the high genomic sequence similarity between mouse and human SPTLC1, we created a novel knock-in mouse model with a CRISPR/Cas9-mediated deletion of exon 2 in the endogenous murine Sptlc1 locus. Although heterozygous mice did not develop motor defects or ALS-like neuropathology, homozygous mutants died prematurely. These findings provide valuable insights into SPTLC1 exon 2 biology and serve as a useful resource for future mechanistic studies.},
}

Animals
*Amyotrophic Lateral Sclerosis/genetics
*Exons/genetics
Mice
Heterozygote
Homozygote
Disease Models, Animal
Humans
*Sequence Deletion/genetics
Mutation
Genes, Lethal
CRISPR-Cas Systems
Male
Gene Knock-In Techniques

@article {pmid42394824,
year = {2026},
author = {Wu, Y and Cai, H and Wu, Q and Wu, J and Hu, J and Huang, E and Li, Z and Liang, S and Hu, X and Dai, J and Liao, R},
title = {The CRISPR-Cas toolkit for mosquito-borne virus surveillance: detection, tracing, and discovery.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1873187},
pmid = {42394824},
issn = {2235-2988},
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; *Mosquito-Borne Diseases/virology ; *Epidemiological Monitoring ; *Culicidae/virology ; *Mosquito Vectors/virology ; *Viruses/genetics/isolation & purification/classification ; *Virus Diseases/virology/transmission/diagnosis ; },
abstract = {Mosquito-borne virus surveillance increasingly requires rapid, distributed detection of co-circulating pathogens, serotypes, and lineages across clinical and vector-sampling sites. CRISPR-Cas platforms offer a programmable toolkit for this purpose, but their readiness differs substantially across surveillance functions. Here, we review CRISPR-Cas methods for mosquito-borne virus surveillance across detection, tracing, and discovery-supporting targeted screening. Detection is the most advanced application: selected Cas12- and Cas13-based assays for dengue, Zika, chikungunya, West Nile, Japanese encephalitis, and related mosquito-associated viruses report sub-hour workflows, portable readouts, and targeted serotype- or lineage-marker discrimination. However, performance remains assay-, target-, and sample-matrix-dependent, and validation in pooled mosquito samples and field settings is still limited. Tracing currently relies mainly on validated portable amplicon-sequencing workflows, whereas CRISPR-aided sample-preparation methods such as DASH, FLASH, RAPID-DASH, and Cas9-targeted enrichment remain transferable opportunities for host depletion or target enrichment rather than established mosquito-borne virus genomic-surveillance workflows. For discovery-oriented surveillance, multiplex CRISPR-Cas systems such as CARMEN can support targeted screening of known or near-neighbor viruses represented by predesigned crRNAs, while metagenomic next-generation sequencing remains necessary for divergent or previously unknown viruses. Across these functions, CRISPR-Cas programmability may accelerate parts of assay redesign, but practical retargeting still requires compatible amplification primers, effector-specific target constraints, cross-reactivity assessment, and analytical revalidation. Routine surveillance use will require integrated demonstrations with clinical and pooled-vector samples, comparison against established molecular and sequencing methods, cost validation, and regulatory evidence.},
}

Animals
*CRISPR-Cas Systems
Humans
*Mosquito-Borne Diseases/virology
*Epidemiological Monitoring
*Culicidae/virology
*Mosquito Vectors/virology
*Viruses/genetics/isolation & purification/classification
*Virus Diseases/virology/transmission/diagnosis

@article {pmid42395789,
year = {2026},
author = {Abdallah, NM and Mohammed, MR and Al Haideri, H and Alahmari, AS and Alwutayd, KM and Zarah, RK and Hamdi, H and Al Masoudi, LM and Al Thagafi, NT and Althobaiti, AT and Khormi, MA and Binshaya, AS and Abalkhail, A and Soliman, MKY},
title = {Green synthesis of ZnO/Fe3O4 nanocomposites from Citrus reticulata peel: antibacterial activity against MDR Acinetobacter baumannii, CRISPR-Cas gene modulation, and anticancer potential.},
journal = {RSC advances},
volume = {},
number = {},
pages = {},
pmid = {42395789},
issn = {2046-2069},
abstract = {The emergence of multidrug-resistant (MDR) Acinetobacter baumannii necessitates the development of alternative antimicrobial strategies. In this study, ZnO/Fe3O4 nanocomposites (NCs) were green-synthesized using Citrus reticulata peel extract and evaluated for antibacterial, CRISPR-Cas gene modulation, anticancer, and antioxidant activities. Phytochemical profiling by GC-MS and HPLC confirmed a terpene- and polyphenol-rich composition supporting nanoparticle formation and stabilization. The synthesized NCs were characterized by UV-vis, FTIR, and XRD analyses, supporting the formation of crystalline ZnO and Fe3O4 phases, while TEM revealed nanoscale morphology (55.64 ± 24.2 nm) with hydrodynamic size of ∼181.3 nm and a zeta potential of +2.64 mV. Fifteen clinical A. baumannii isolates were identified, among which nine exhibited multidrug-resistant (MDR) profiles. CRISPR-associated genes were screened in the MDR isolates, and four isolates harboring the target genes were selected for further molecular analyses. The ZnO/Fe3O4 NCs exhibited antibacterial activity with inhibition zones ranging from 17 to 24 mm and MIC values of 250-500 µg mL[-1]. TEM analysis of treated bacteria demonstrated severe structural damage, including membrane disruption and cytoplasmic leakage. Furthermore, sub-MIC exposure resulted in downregulation of CRISPR-associated genes (Cas1: 0.61-0.98; Csy1: 0.54-0.95; Csy3: 0.70-0.83). Cytotoxicity assays revealed selective antiproliferative effects against Caco-2 colorectal cancer cells (IC50 = 60.7 µg mL[-1]) compared to normal Vero cells (IC50 = 254.66 µg mL[-1]), accompanied by increased apoptosis (15.8%) and G2/M cell cycle arrest (39.7%). Additionally, the NCs exhibited concentration-dependent antioxidant activity, reaching up to 69.44% (DPPH), 76.12% (ABTS), 62.24% (H2O2 scavenging), 56.13% (metal chelation), and 63.52% (reducing power). Overall, these findings demonstrate that green-synthesized ZnO/Fe3O4 NCs are multifunctional nanomaterials with promising antibacterial, anticancer, and antioxidant properties.},
}

@article {pmid42140622,
year = {2026},
author = {Lin, YC and Lee, YJ and Li, CX and Chang, HC and Kowalczyk, M and Haque, M and Dragoiescu, A and Losier, TT and Taylor, JA and Rousseaux, MWC and Huang, WH},
title = {CRISPR screening identifies TRIM27 as a destabilizer of the Smith-Magenis syndrome protein RAI1.},
journal = {Genetics},
volume = {233},
number = {3},
pages = {},
doi = {10.1093/genetics/iyag121},
pmid = {42140622},
issn = {1943-2631},
support = {RGPIN-2020-04094//Natural Sciences and Engineering Research Council of Canada Discovery/ ; //Canada First Research Excellence Fund/ ; //Fonds de recherche du Québec awarded/ ; //McGill University/ ; },
mesh = {Humans ; Animals ; *Smith-Magenis Syndrome/genetics/metabolism/pathology ; Mice ; Ubiquitination ; *Trans-Activators/metabolism/genetics ; Protein Stability ; *Transcription Factors/metabolism/genetics ; HEK293 Cells ; Neurons/metabolism ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Proteolysis ; *DNA-Binding Proteins/metabolism/genetics ; },
abstract = {The nervous system is highly sensitive to alterations in the dosage of genes crucial for neurodevelopment, as exemplified by retinoic acid-induced 1 (RAI1). A 50% change in RAI1 gene copy number, resulting in either reduced or increased protein levels, leads to distinct neurodevelopmental disorders. RAI1 haploinsufficiency causes Smith-Magenis syndrome (SMS), whereas RAI1 duplication underlies Potocki-Lupski syndrome. We recently demonstrated that restoring Rai1 levels can improve SMS-related disease phenotypes in mice. However, despite protein stability being a major determinant of protein abundance, there are currently no therapeutic approaches to modulate RAI1 protein stability. Here, we performed a forward CRISPR screen in human cells to identify post-translational regulators of RAI1 steady-state levels and identified tripartite motif-containing 27 (TRIM27) as a destabilizer of RAI1. We show that RAI1 degradation occurs primarily through the ubiquitin-proteasome system, with TRIM27 interacting with RAI1 and enabling TRIM27-dependent lysine(K)48- and K63-linked RAI1 ubiquitination. Finally, in SMS mouse primary neurons, we demonstrate that knocking down TRIM27 partially rescues SMS-associated morphological phenotypes. Our findings provide the first mechanistic insight into RAI1 proteostasis and highlight TRIM27 as a potential therapeutic target for SMS, highlighting the potential of manipulating ubiquitin-mediated proteostasis to restore gene dosage altered by copy number variations.},
}

Humans
Animals
*Smith-Magenis Syndrome/genetics/metabolism/pathology
Mice
Ubiquitination
*Trans-Activators/metabolism/genetics
Protein Stability
*Transcription Factors/metabolism/genetics
HEK293 Cells
Neurons/metabolism
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Proteolysis
*DNA-Binding Proteins/metabolism/genetics

@article {pmid42382580,
year = {2026},
author = {Pan, S and Wang, X and He, J and Peng, N},
title = {RecN: A tunable switch for DNA repair choice and stress tolerance in Zymomonas mobilis.},
journal = {Biodesign research},
volume = {8},
number = {2},
pages = {100088},
pmid = {42382580},
issn = {2693-1257},
abstract = {Engineering polyploid industrial microorganisms is hindered by their intrinsic capacity to repair induced mutations, limiting the efficiency of genome editing and directed evolution. Using the ethanologenic bacterium Zymomonas mobilis- a polyploid alphaproteobacterium that exhibits exceptionally efficient microhomology-mediated end joining (MMEJ)- we demonstrate that RecN is essential for MMEJ and homologous recombination (HR) in vivo. Strikingly, a specialized mutant RecN-K35A, with strongly impaired ATP hydrolysis, specifically blocks MMEJ while leaving HR fully intact. The physiological importance of RecN-mediated MMEJ is highlighted by the cell elongation phenotype and increased stress sensitivity observed in the RecN-K35A mutant. Based on this connection, we developed a high-phosphorus cultivation strategy that increases cellular DNA content and significantly enhanced ethanol fermentation efficiency under industrial stress conditions. In summary, this work defines RecN as a key ATP-dependent effector of MMEJ and positions it as a potential engineering target for modulating DNA repair pathway choice and stress tolerance in Z. mobilis. Moreover, the essential role of RecN in both HR and MMEJ suggests that RecN-deficient polyploid strains could facilitate directed evolution by preventing repair of newly introduced mutations, offering a new strategy for strain improvement.},
}

@article {pmid42385699,
year = {2026},
author = {Pandey, S and Burman, N and Henriques, WS and Wiegand, T and Zahl, T and Nyquist, H and Spreeuw, T and Buyukyoruk, M and Wiedenheft, B},
title = {Identification and structure determination of a type III-Bv CRISPR complex that post-translationally modifies an associated toxin.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2026.06.002},
pmid = {42385699},
issn = {1878-4186},
abstract = {Cas7-family proteins form the scaffolds of multi-subunit CRISPR RNA-guided surveillance complexes. To explore how Cas7 diversification expands CRISPR function, we identified Cas7 fusion proteins linked to diverse accessory domains, including a type III-B variant (III-Bv) in which a Cas7 homolog (Cmr1) is fused to the MntA antitoxin and encoded adjacent to a HEPN-family toxin. Structures reveal that the core Cas proteins assemble into a stable surveillance complex in the absence of crRNA, whereas incorporation of the Cmr1-MntA fusion is crRNA-dependent. Target RNA recognition triggers conformational changes that expose the Cas10 cyclase active site and promote cyclic oligoadenylate synthesis. Biochemical analyses show that the CRISPR-associated MntA is enzymatically active and AMPylates the associated HEPN protein. Together, these findings establish the structural basis for assembly of a type III-Bv surveillance complex containing an enzymatically active toxin-antitoxin module.},
}

@article {pmid42387043,
year = {2026},
author = {Dalal, B and Reena, R and Baloda, A and Saini, S and Sharma, P and Sharma, A},
title = {Nitric oxide and abscisic acid: two intimate collaborators regulating plant defense against drought.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {42387043},
issn = {1615-6102},
abstract = {Drought represents one of the most pervasive and intensifying abiotic stresses under changing climate regimes severely constraining agricultural productivity, ecosystem stability, and global food security. Water deficit disrupts cellular homeostasis, reduces photosynthetic efficiency, and induces excessive accumulation of reactive oxygen species (ROS), resulting in oxidative damage. To survive under such conditions, plants employ a diverse array of adaptive responses, including osmotic adjustment, antioxidant defense, hormonal signalling, and stress-responsive gene regulation. Among the key signalling molecules involved in drought tolerance, nitric oxide (NO) and abscisic acid (ABA) have emerged as pivotal signalling molecules orchestrating a wide spectrum of physiological and molecular responses under drought. NO functions as a versatile signalling molecule that regulates redox homeostasis, enhances antioxidant activity, and promotes the accumulation of osmoprotectant. ABA maintains drought perception by inducing stomatal closure, and activating stress-responsive pathways. Co-application of NO and ABA regulates seed germination, root-shoot growth, and stomatal movement, thereby improving relative water content (RWC), membrane stability index (MSI), and photosynthetic efficiency while reducing oxidative stress markers such as malondialdehyde (MDA) and hydrogen peroxide (H2O2). This comprehensive review navigates through a clear and integrative overview of the mechanistic role of NO and ABA, and at the molecular level, NO and ABA modulate drought tolerance through transcriptional regulation, mRNA-level control, and translational modification of stress-responsive genes. Additionally, emerging strategies, including plant-growth promoting rhizobacteria (PGPR), marker-assisted selection (MAS) with QTL mapping, and genome editing tools such as CRISPR/Cas systems, offer promising approaches for enhancing drought tolerance and developing climate-resilient crop varieties.},
}

@article {pmid42387193,
year = {2026},
author = {Xu, H and Hu, X and Chen, R and Huang, S and Wang, L and Yu, L and Li, X and Zhu, X},
title = {Triple-amplification electrochemiluminescence aptasensor integrating single-atom nanozyme catalysis with CRISPR-Cas12a/HCR cascade for zearalenone detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {7},
pages = {},
pmid = {42387193},
issn = {1436-5073},
support = {81773894//NSFC/ ; 2023J01152//Natural Sciences Foundation of Fujian Province/ ; },
mesh = {*Zearalenone/analysis/chemistry/blood ; *Electrochemical Techniques/methods ; Luminescent Measurements/methods ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; *CRISPR-Cas Systems ; Catalysis ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; DNA/chemistry ; Electrodes ; },
abstract = {Single-atom nanozymes (SANs) are emerging as interfacial catalysts that can modulate surface-confined reactive oxygen species (ROS) generation at the electrode/solution boundary. Herein, an interface-driven electrochemiluminescence (ECL) aptasensor was constructed for the ultrasensitive detection of zearalenone (ZEN), a mycotoxin of significant concern. Oxidase-mimetic Co-N/C SANs (Co-SAC@NC) immobilized on a glassy carbon electrode catalyze the reduction of dissolved O2 to ·OH and O2·[-], followed by in-situ generation of ROS, producing a 13-fold enhancement of luminol ECL without the addition of external H2O2. A ferrocene-labelled reporter DNA (Fc-DNA) tethered to the Co-SAC@NC surface quenches luminol ECL by trapping holes at the electrode interface; upon target binding, a magnetic-bead-supported HCR-CRISPR-Cas12a cascade is triggered, trans-cleaving the Fc-DNA and thus restoring the native ECL intensity. The concentration-dependent interfacial cleavage affords a linear range of 0.3-200 ng/mL and a LOD of 0.087 ng/mL (S/N = 3) for the determination of ZEN. This work establishes a modular interfacial amplification platform with potential for generalization by marrying SANs catalysis with a CRISPR-Cas12a/HCR nucleic acid cascade for advanced ECL bioanalysis.},
}

*Zearalenone/analysis/chemistry/blood
*Electrochemical Techniques/methods
Luminescent Measurements/methods
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry/genetics
Limit of Detection
*CRISPR-Cas Systems
Catalysis
Metallocenes/chemistry
Ferrous Compounds/chemistry
DNA/chemistry
Electrodes

@article {pmid42387220,
year = {2026},
author = {Yaseen, Z and M, M},
title = {Overcoming Immunological Barriers in MSC-Derived Insulin-Producing Cells through CRISPR-Based Hypoimmunogenic Engineering and Translational Perspectives for Type 1 Diabetes.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {42387220},
issn = {2629-3277},
abstract = {Mesenchymal stromal cell (MSC)-derived insulin-producing cells (IPCs) represent an emerging strategy for β-cell replacement in type 1 diabetes mellitus (T1DM) owing to their differentiation potential, intrinsic immunomodulatory properties, and lower tumorigenic risk compared with pluripotent stem cell-derived platforms. However, accumulating evidence indicates that differentiation-associated immunogenicity, context-dependent immune recognition, and recurrent autoimmune responses may substantially limit long-term graft survival and therapeutic durability following transplantation. This review critically examines the immunological barriers associated with MSC-derived IPCs, including altered MHC expression, susceptibility to alloimmune and autoimmune-mediated rejection, and potential reactivation of autoreactive immune memory. We discuss the application of CRISPR-based hypoimmunogenic engineering strategies targeting antigen presentation pathways, NK-cell activation, and immune checkpoint modulation to generate more immune-evasive MSC-derived IPCs while preserving β-cell functionality. By integrating insights from T1DM immunopathogenesis, MSC biology, genome editing, and translational immunology, we propose a framework linking immune engineering with controlled differentiation, functional maturation, and long-term safety evaluation. In parallel, we comparatively position MSC-derived IPCs alongside clinically advancing iPSC-derived β-cell platforms to highlight their distinct translational niche, including potential advantages related to safety, immunomodulatory capacity, manufacturing accessibility, and scalability, while acknowledging the superior functional maturity and clinical progression currently demonstrated by iPSC-derived systems. Finally, we discuss key translational challenges, including genomic stability, immune-evasion durability, GMP-compliant manufacturing, and the need for rigorous functional and immunological benchmarking prior to clinical application of hypoimmunogenic MSC-derived IPC therapies in T1DM.},
}

@article {pmid42387274,
year = {2026},
author = {Yang, H and Zhu, L and Zhi, J and Zhong, Q},
title = {Targeting Bacterial Quorum Sensing: Insights into Quorum Sensing Inhibitors and Innovative Antimicrobial Strategies for Enhancing Food Safety and Combating Antibiotic Resistance.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c17992},
pmid = {42387274},
issn = {1520-5118},
abstract = {Antibiotic resistance transmission and the increasing diversity of antibiotic resistance phenotypes pose growing threats to food safety and public health. Foodborne bacteria employ quorum sensing (QS) to regulate virulence expression and biofilm formation, enhancing pathogenicity and drug resistance. Therefore, targeting QS is regarded as a promising strategy to control bacteria. Quorum sensing inhibitors (QSIs) are highly promising for addressing bacterial resistance, as they do not rely on the direct killing of bacteria but rather on attenuation of bacterial spoilage effects in food by disrupting their group behavior. This review focuses on natural and synthetic compounds with QSI activity, elaborating their mechanisms and potential as antimicrobial agents. Additionally, the review proposes innovative antimicrobial strategies, including nanotechnology-based delivery systems, combination with phage, CRISPR-Cas technology, and multitargeted approaches cooperated with existing QSIs. These integrated strategies are designed to overcome challenges, providing novel methodologies for controlling bacterial contamination and infections while holding broad application prospects.},
}

@article {pmid42049733,
year = {2026},
author = {Nguyen, JT and Huang, L and Levine, H and Lu, M and Bleris, L},
title = {Rewiring miR-22/SNAI1 via CRISPR-based edge editing destabilizes the epithelial phenotype.},
journal = {NPJ systems biology and applications},
volume = {12},
number = {1},
pages = {},
pmid = {42049733},
issn = {2056-7189},
support = {2114192//National Science Foundation (NSF)/ ; 2029121//National Science Foundation (NSF)/ ; CA283330//National Institute of Health/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *Snail Family Transcription Factors/genetics/metabolism ; *Epithelial-Mesenchymal Transition/genetics ; Humans ; Phenotype ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Signal Transduction/genetics ; Transforming Growth Factor beta/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Epithelial-to-Mesenchymal Transition (EMT) is a critical biological process by which cells acquire enhanced migratory and invasive properties. A key signaling pathway involved in EMT phenotypes includes transforming growth factor β (TGFβ) and transcription factors (TFs) such as SNAIL, ZEB, and TWIST. Additionally, microRNAs (miRNAs) - small, non-coding molecules that regulate gene expression by targeting mRNA transcripts - directly regulate genes central to the EMT process. Notably, miR-22 has been identified as a significant regulator of EMT through direct inhibition of EMT drivers like SNAI1 and indirect regulation of upstream genes. In this study, we performed CRISPR-based network rewiring by selectively removing an edge-the connection between two nodes-to investigate its impact on EMT dynamics. Specifically, we disrupted the connection between miR-22 and SNAI1 without affecting other interactions involving miR-22 or SNAI1 and examined the resulting effects on EMT. We demonstrate that the removal of the miR-22 target site from the SNAI1 gene renders cells more sensitive to TGFβ-mediated EMT. This finding highlights the unique advantage of edge-specific perturbation by ablating the direct regulatory connection between miR-22 and SNAI1. We demonstrate that all measured downstream effects on EMT can be attributed to this single interaction, independent of miR-22's influence on other targets or indirect pathways. More generally, our results underscore the importance of CRISPR-mediated edge ablation for exploring the interactions that govern biological networks and highlight an underexplored opportunity to develop edge-based therapeutic modalities.},
}

*MicroRNAs/genetics/metabolism
*Snail Family Transcription Factors/genetics/metabolism
*Epithelial-Mesenchymal Transition/genetics
Humans
Phenotype
CRISPR-Cas Systems/genetics
*Gene Editing/methods
Signal Transduction/genetics
Transforming Growth Factor beta/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42106859,
year = {2026},
author = {Su, N and Xu, C and Liu, W and Xue, M and Huang, Z and Jiang, N and Meng, Y and Zhou, Y and Fan, Y and Zheng, Y},
title = {Establishment and application of a detection method for chinese rice-field eels rhabdovirus (CrERV) using the RPA-CRISPR/Cas12a System.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {42106859},
issn = {1743-422X},
support = {No. 2024XT0602//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; No. 2023TD46//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; No.YFICG2025001//Chinese Academy of Fishery Sciences Yangtze River Fisheries Research Institute/ ; grant numbers 2023YFD2402800//National Key Research and Development Program/ ; },
mesh = {Animals ; *Eels/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Fish Diseases/diagnosis/virology ; Reproducibility of Results ; *Rhabdoviridae/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; China ; Rapid Diagnostic Tests ; },
abstract = {The Chinese rice-field eel rhabdovirus (CrERV) is an emerging pathogen that causes hemorrhagic disease in Chinese rice-field eels (Monopterus albus), leading to epidemic outbreaks, mass mortality, and considerable economic losses in aquaculture. Thus, the development of rapid and reliable diagnostic tools for on-site detection is urgently needed to address this issue. In this study, we established an RPA-CRISPR/Cas12a-based assay for CrERV detection, which exhibited superior sensitivity, specificity, and stability. The assay achieved a detection limit of 10[1] copies/µL. Specificity testing confirmed the absence of cross-reactivity with five other major aquatic viruses, including Grass carp reovirus (GCRV-II), Spring viraemia of carp virus (SVCV), Largemouth bass virus (LMBV), Cyprinid herpesvirus 2 (CyHV-2), and White spot syndrome virus (WSSV). Reproducibility analysis showed intra- and inter-assay coefficients of variation below 10%. Analysis of the 26 clinical samples showed that the RPA‑CRISPR/Cas12a assay achieved a higher positivity rate (23.08%, 6/26) compared to qRT‑PCR (15.38%, 4/26), providing preliminary evidence for its diagnostic potential in detecting CrERV. Collectively, these findings indicate that the RPA-CRISPR/Cas12a platform is a highly sensitive, specific, and user-friendly tool for rapid CrERV surveillance in aquaculture settings.},
}

Animals
*Eels/virology
Sensitivity and Specificity
*CRISPR-Cas Systems
*Fish Diseases/diagnosis/virology
Reproducibility of Results
*Rhabdoviridae/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
China
Rapid Diagnostic Tests

@article {pmid42381030,
year = {2026},
author = {Senu, E and Ikechukwu, SC and Aboagye, C and Agyen, GK and Kamara, S and Tamakloe, VCKT and Nouwati, DD and Diawuo, HY and Doku, EE and Akpobi, S and Asori, M and Effah, A},
title = {Knowledge and perception as determinants of CRISPR application for infectious diseases in Sub-Saharan Africa: a multi-country study in Ghana, Nigeria, and Sierra Leone using regression and structural equation models.},
journal = {Journal of health, population, and nutrition},
volume = {},
number = {},
pages = {},
doi = {10.1186/s41043-026-01380-0},
pmid = {42381030},
issn = {2072-1315},
abstract = {BACKGROUND AND AIM: Infectious diseases, particularly acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV), remain a major global health burden, especially in low- and middle-income countries, despite advances in antiretroviral therapy. Emerging genome-editing technologies, such as CRISPR/Cas systems, hold promise for transforming the diagnosis and treatment of infectious diseases. However, little is known about how these technologies are understood and perceived by individuals in high-burden regions, where significant implementation challenges may limit their adoption. This study assessed knowledge, perceptions, and the potential application of CRISPR technology for infectious disease control in Ghana, Nigeria, and Sierra Leone.

METHODS: This multi-center cross-sectional study was conducted among 300 participants, including students, healthcare professionals, and researchers across the three countries. Data was collected using a well-structured questionnaire and analyzed using logistic regression and structural equation modeling (SEM) to identify the predictors of potential CRISPR applications.

RESULTS: The study revealed a significant gap, such that the majority (87.0%) of the participants demonstrated inadequate knowledge, with 70.7% exhibiting poor perception of CRISPR technology. Major barriers to implementation included funding constraints (81.7%) and inadequate infrastructure (62.3%). Adjusted multivariable logistic regression identified inadequate knowledge (aOR: 3.90; p < 0.0001) and poor perception (aOR: 1.96; p = 0.0060) as independent predictors of low CRISPR application potential. Structural equation modeling confirmed that knowledge significantly enhances perception (β = 0.55), and both constructs jointly influence the potential application of CRISPR gene editing technology.

CONCLUSION: There is a critical deficit in knowledge and perception regarding CRISPR gene editing in Sub-Saharan Africa, which significantly hinders its potential application for infectious diseases. Comprehensive educational strategies and capacity building are essential to foster the adoption of CRISPR technology, especially in resource-limited settings.},
}

@article {pmid42381438,
year = {2026},
author = {Cheng, L and Liu, D and Zhang, H and Lin, H and Zhang, P and Dong, J and Li, C and Ren, F and Ge, X and An, S and Yang, F and Liu, J and Fu, J and Tang, F and Wang, F and Liu, T and Pan, H and Zou, L and Zhou, J and Ma, H and Rong, S},
title = {Synergistic CRISPR-Cas and Nanozyme-Based Fluorescent Sensors for Ultrasensitive Heavy Metal Detection.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/10408347.2026.2684551},
pmid = {42381438},
issn = {1547-6510},
abstract = {Heavy metal pollution poses a serious threat to ecological balance and human health. Traditional detection methods, such as atomic absorption spectrometry and inductively coupled plasma mass spectrometry, suffer from complex procedures, high costs and susceptibility to interference, making them unable to meet the requirements of on-site rapid detection. To address this bottleneck, this review focuses on the innovative solution of the synergistic detection strategy combining nanozymes and the CRISPR-Cas systems. The core advantages of this strategy are as follows: it leverages the excellent specific recognition and signal amplification capabilities of the CRISPR-Cas system to achieve a precise response to heavy metal trigger signals; meanwhile, it utilizes the high stability and enzyme-mimicking catalytic activity of nanozymes to convert signals into readable outputs, thereby constructing an efficient detection platform. We elaborate on how this synergistic mechanism enables high-sensitivity detection without sample pretreatment and remarkably improves analytical performance, with a wider linear range and a lower detection limit. Numerous studies have demonstrated that this combined strategy lays a solid foundation for developing portable, high-sensitivity heavy metal detection devices suitable for complex matrices such as food, water and blood samples, holding great promise for field on-site detection applications.},
}

@article {pmid42108505,
year = {2026},
author = {Pratumkaew, P and Wattanapanitch, M and Viprakasit, V and Kheolamai, P and Issaragrisil, S},
title = {Induced pluripotent stem cell-based modeling of hemolytic anemia in patients with compound heterozygous KLF1 mutations reveals defective erythroid differentiation.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {},
pmid = {42108505},
issn = {1757-6512},
mesh = {Humans ; *Kruppel-Like Transcription Factors/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology/cytology ; *Cell Differentiation ; *Mutation/genetics ; *Anemia, Hemolytic/genetics/pathology/metabolism ; *Erythroid Cells/metabolism/pathology ; Female ; Heterozygote ; CRISPR-Cas Systems ; Male ; },
abstract = {BACKGROUND: Transfusion-dependent hemolytic anemia caused by compound heterozygosity due to mutations in the erythroid Krüppel-like factor 1 (KLF1) gene is a rare and severe blood disorder. The clinical manifestations of the patient are mainly related to erythroid cells. Moreover, the roles of the identified KLF1 mutations in the pathophysiology of this disease remain unclear due to the lack of an appropriate study model. The advent of genome editing technology combined with the generation of patient-specific induced pluripotent stem cells (iPSCs) may provide a better understanding of the molecular mechanisms underlying this disease in an in vitro system and offer a novel therapeutic approach in the future.

METHODS: KLF1-mutant iPSCs were generated from patients with compound heterozygosity of KLF1 mutations, and the mutation was corrected through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system together with a single-stranded oligodeoxynucleotide donor template (ssODN). The obtained iPSC lines were differentiated towards erythroid cells, and the disease-related phenotypes were examined.

RESULTS: Erythroid cells derived from KLF1-mutated iPSCs had lower proliferative capacity, showed delayed maturation, and expressed lower level of the KLF1-related gene, CD44. These results were consistent with some of the phenotypes observed in the patients. After CRISPR/Cas9 gene editing, the corrected iPSCs retained pluripotency, exhibited a normal karyotype, and had undetectable off-target mutations. Importantly, some of the defects were partially restored after genetic correction of the KLF1 gene.

CONCLUSIONS: KLF1-iPSCs presented disease-related phenotypes of compound heterozygous KLF1 mutations, which could be mediated by gene editing through CRISPR/Cas9 and ssODN. This study offers a useful strategy for studying the underlying disease mechanisms of rare diseases, which could be applied to the development of novel treatments for inherited blood disorders in the future.},
}

Humans
*Kruppel-Like Transcription Factors/genetics/metabolism
*Induced Pluripotent Stem Cells/metabolism/pathology/cytology
*Cell Differentiation
*Mutation/genetics
*Anemia, Hemolytic/genetics/pathology/metabolism
*Erythroid Cells/metabolism/pathology
Female
Heterozygote
CRISPR-Cas Systems
Male

@article {pmid42371202,
year = {2026},
author = {Bayramoğlu, Z},
title = {Effects of CRISPR technology on agricultural sustainability: global applications and turkish perspective.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {42371202},
issn = {1573-9368},
mesh = {*Plants, Genetically Modified/genetics/growth & development ; *Agriculture/methods ; *Crops, Agricultural/genetics/growth & development ; *Gene Editing/methods ; Turkey ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions-environmental, economic, social and policy, and scientific and technological-we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms-particularly through producer cooperatives-that allow smallholder farmers to benefit from edited varieties.},
}

*Plants, Genetically Modified/genetics/growth & development
*Agriculture/methods
*Crops, Agricultural/genetics/growth & development
*Gene Editing/methods
Turkey
*CRISPR-Cas Systems/genetics
Humans

@article {pmid42371672,
year = {2026},
author = {Yuan, G and Gao, Z and Qi, Y and Zhang, Y and Tian, X and Zhao, P and Feng, X and She, Q},
title = {CREAT: A CRISPR-Based Genome Trimming Strategy for Systematic Identification of Dispensable Regions and Rapid Genome Reduction.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e76042},
doi = {10.1002/advs.76042},
pmid = {42371672},
issn = {2198-3844},
support = {2023YFC3402003//National Key R & D Program of China/ ; FX//National Key R & D Program of China/ ; 2020YFA0906800//National Key R & D Program of China/ ; QS//National Key R & D Program of China/ ; ZR2024QC306//Department of Science and Technology of Shandong Province/ ; GY//Department of Science and Technology of Shandong Province/ ; 2023KJ009//Department of Education of Shandong Province/ ; XF//Department of Education of Shandong Province/ ; 25-1-1-250-zyyd-jch//Qingdao Natural Science Foundation/ ; SKLMTFCP-2023-05//SKLMT Frontiers and Challenges Project/ ; },
abstract = {The construction of minimal-genome microbes offers an ideal platform for understanding fundamental biological processes and synthetic biology, yet the research is hindered by incomplete lists of essential genes in microbes and by multiple rounds of genome trimming with a trial-and-error nature. To address this, we introduce CREAT (CRISPR-based genome trimming with a multi-homology-arm template)-a streamlined approach that integrates CRISPR-targeted genome cleavage and homology arm walking to classify essential from non-essential genomic subregions, thus providing the basis for predicting essential genes in a given organism. These essential genes were then assembled into synthetic gene cassettes for one-step replacement of the targeted non-deletable genomic regions for further genome trimming. Eight consecutive rounds of CREAT genome trimming achieved a 20.8% reduction in genome size in Saccharolobus islandicus. Furthermore, Cas9-based CREAT genome trimming was developed for Bacillus subtilis and Escherichia coli, with efficiency greatly enhanced by the λ-Red recombinase in the latter. Together, this iterative application of CREAT provides a scalable and generally applicable strategy for rapidly constructing minimal genomes across diverse microorganisms.},
}

@article {pmid42378111,
year = {2026},
author = {Liu, X and Chen, T and Li, X and Li, Q and Zou, G and Fan, W and Liu, C},
title = {Engineering Guide RNAs for CRISPR-Based Biosensors.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01763},
pmid = {42378111},
issn = {2379-3694},
abstract = {CRISPR-Cas systems, with their programmable nucleic acid-targeting capabilities, represent an ideal platform for constructing next-generation, highly sensitive biosensors. However, the clinical translation of these platforms is hindered by key limitations inherent to native single-guide RNAs (sgRNAs), including insufficient stability, potential immunogenicity, and off-target effects. To address these challenges, engineering sgRNAs has emerged as a central strategy to overcome such barriers and enhance overall biosensor performance. In this review, we provide a systematic overview of the field, beginning with the classification, molecular mechanisms, and structural features of representative CRISPR-Cas effector proteins to establish their foundational role as sensing elements. We then examine the specific limitations of native sgRNAs in biosensing applications. Building on this analysis, we highlight recent advances in sgRNA engineering strategies, which encompass three major approaches, including chemical modifications, structural remodeling, and modular functional integration. Furthermore, we review the integration of these engineered sgRNAs into advanced biosensor platforms, including microfluidic paper-based devices, centrifugal platforms, wearable patches, microneedles, and point-of-care testing (POCT) systems, and present a comparative table summarizing their performance in terms of detection signals, limits of detection, and other key metrics. Finally, we discuss persistent challenges such as the fine control of off-target effects, in vivo delivery bottlenecks, and system robustness in complex environments, and outline future directions toward amplification-free, multiplexed, and clinically translatable CRISPR-based biosensors. Overall, the engineering of sgRNAs offers a powerful means to systematically enhance the stability, specificity, and reliability of CRISPR-based biosensors, thereby accelerating their practical deployment in clinical diagnostics.},
}

@article {pmid42379562,
year = {2026},
author = {Del Giovane, S and Migliorelli, D and Paoletti, S and Bagheri, N and Altug, H and Burr, L},
title = {CRISPR-Cas-Based Platform for Single-Step Quantification of Monoclonal Antibodies at Point-of-Care.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c04532},
pmid = {42379562},
issn = {2379-3694},
abstract = {We report a streamlined, cost-effective point-of-care platform for the quantitative detection of monoclonal antibodies for therapeutic drug monitoring, addressing the limitations of bulky, complex, and expensive instrumentation required for standard analytical techniques. In contrast to conventional immunoassays that rely on primary/secondary antibodies for quantification, our method leverages a DNA circuit for the recognition of the target monoclonal antibody coupled with CRISPR-Cas12a signal amplification. This assay is integrated into a microfluidic chip that enables a single-step workflow, eliminating the multiple incubations and reagent addition steps typical of laboratory methods. The platform demonstrates quantitative detection of a model anti-hemagglutinin antibody within a 60-min sample-to-answer timeframe, in the nanomolar range, with a chip manufacturing cost below 1€ and reagent stability confirmed at -20 °C for over one month. This proof-of-concept illustrates the potential of simplified CRISPR-based assays for decentralized rapid protein quantification in clinical settings.},
}

@article {pmid42379944,
year = {2026},
author = {Cui, T and Li, B and Cai, B and Wang, H and Li, W},
title = {Precision gene editing: From proof-of-concept to curative therapies.},
journal = {Trends in molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molmed.2026.06.004},
pmid = {42379944},
issn = {1471-499X},
abstract = {Gene therapy is evolving from gene addition to precise genome editing, enabling the direct correction of disease-causing mutations. Breakthrough technologies, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) nucleases, base editors, prime editors, and CRISPR-associated transposases are reshaping the therapeutic landscape. This review covers the progression of precision editing technologies and their clinical applications, spanning from ex vivo therapies to in vivo treatments targeting vital organs. The rise of personalized medicine, highlighted by therapies, such as carbamoyl phosphate synthetase 1 editing, underscores the shift toward N-of-1 medicine for rare diseases. Clinical trial progress, delivery and accessibility challenges, and the role of AI in optimizing editing tools and predicting outcomes are also discussed. These innovations are transforming genetic medicine, offering the promise of safer, more durable, and personalized cures.},
}

@article {pmid42380749,
year = {2026},
author = {Kurt, IC and Guner, H and Erdem, ZA and Can, O and Gumustop, I and Sirin, A and Erol, I and Kotil, ES and Ortakci, F},
title = {Genomic evidence of ecological flexibility and cross-niche CRISPR spacerome targeting phage-plasmid hybrids in Latilactobacillus curvatus.},
journal = {BMC genomics},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12864-026-13098-8},
pmid = {42380749},
issn = {1471-2164},
support = {MGA-2024-45355//Bilimsel Araştırma Projeleri Birimi, İstanbul Teknik Üniversitesi/ ; },
abstract = {BACKGROUND: Latilactobacillus curvatus is a lactic acid bacterium with a remarkable ability to persist in diverse niches, including fermented foods and gut. Despite its industrial and potential probiotic relevance, the genomic underpinnings of its cross-niche adaptability remain poorly characterized.

METHODS: We conducted a species-contextualized comparative genomic analysis of 53 L. curvatus strains from food and gut isolates. This analysis integrated pangenome structure, metabolic repertoire, CRISPR-Cas immunity profiles, and mobilome analysis. Additionally, binding mode predictions and dynamics simulations were used to evaluate the theoretical binding energies of bacteriocins to the BamA target.

RESULTS: Phylogenomics revealed a polyphyletic population structure, indicating that long-term evolution is not strictly niche-specific. In contrast, genome-wide similarity showed clustering by isolation source, highlighting horizontal gene transfer (HGT) as a plausible contributor to niche adaptation. We identified a highly active mobilome, encompassing diverse plasmids, IS elements, and multiple intact prophages, reflecting high genomic plasticity characteristic of a multihabitat lifestyle. CRISPR-Cas systems were widespread, and analysis of 2,029 spacers revealed a broad immune repertoire targeting mobile genetic elements represented in fermented food, gut, and environmental datasets. We also identified spacer matches to phage-plasmid hybrid-like elements, highlighting the diversity of mobile genetic elements associated with the L. curvatus spacerome.

CONCLUSION: Our study reveals genomic features consistent with ecological flexibility in L. curvatus, including high genomic plasticity and a broad CRISPR spacer repertoire. Rather than demonstrating strict niche-specific evolution or a causal mechanism for cross-niche persistence, these findings support the hypothesis that this species has experienced diverse interactions with mobile genetic elements across multiple ecological contexts.},
}

@article {pmid42210583,
year = {2026},
author = {Slaufova, M and Karakaya, T and Di Filippo, M and Kündig, T and Beer, HD},
title = {Self-Assembled Skin Equivalents with Monoclonal CRISPR/Cas9-Modified N/TERT-1 Keratinocytes: A Cutting-Edge Model for Human Skin and its Diseases.},
journal = {Advanced healthcare materials},
volume = {15},
number = {25},
pages = {e71283},
doi = {10.1002/adhm.71283},
pmid = {42210583},
issn = {2192-2659},
support = {2360/2024//Vontobel Foundation/ ; 2025/09//Wolfermann-Nägeli Foundation/ ; #23C201//Jubiläumsstiftung Swiss Life, Novartis Foundation for medical-biological Research/ ; //Monique Dornonville de la Cour Foundation/ ; LF-OC-23-001156//LEO Foundation/ ; 310030_197426/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Humans ; *Keratinocytes/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Skin/metabolism ; Cell Line ; *Skin Diseases/pathology/metabolism ; CARD Signaling Adaptor Proteins/genetics/metabolism ; Inflammasomes/metabolism ; Interleukin-1beta/metabolism ; },
abstract = {Human skin is a complex organ consisting of multiple cell types and serves as an essential barrier against environmental stressors. Due to ethical considerations and interspecies differences, in vitro human skin equivalents (SEs) are increasingly used to complement or replace animal models in mechanistic, pharmacological, and disease-modeling studies. Scaffold-free full-thickness SEs, in which fibroblasts generate their own extracellular matrix, are particularly attractive because they provide high structural stability even during extended culture. However, the use of genetically defined keratinocyte populations in these SEs has remained limited. Here, scaffold-free full-thickness SEs incorporating wild-type, polyclonal or monoclonal CRISPR/Cas9-modified N/TERT-1 keratinocytes, generated via electroporation, are established. Monoclonal N/TERT-1 keratinocytes with targeted knockout (KO) of the crucial inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) form a differentiated epidermis but fail to secrete the proinflammatory cytokines interleukin (IL)-1β and IL-18 upon inflammasome activation, indicating complete functional ablation of inflammasome signaling in the 3D model. Moreover, SEs generated with gasdermin A (GSDMA)-KO N/TERT-1 keratinocytes illustrate the feasibility of analyzing genes induced during keratinocyte differentiation under physiological conditions. These results establish scaffold-free full-thickness SEs with monoclonal genetically modified N/TERT-1 keratinocytes as a robust and reproducible human skin model for mechanistic studies and future disease-modeling applications.},
}

Humans
*Keratinocytes/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Skin/metabolism
Cell Line
*Skin Diseases/pathology/metabolism
CARD Signaling Adaptor Proteins/genetics/metabolism
Inflammasomes/metabolism
Interleukin-1beta/metabolism

@article {pmid42230849,
year = {2026},
author = {Gao, P and Feng, W and Zhao, X and Shen, J and Wang, X and Wu, X and Zhou, W and Wang, Y and Chen, J and An, W},
title = {Constructing EGF mRNA-Enriched Extracellular Vesicles Based on the AAVS1 Safe Harbor Site to Promote Skin Wound Healing.},
journal = {Advanced healthcare materials},
volume = {15},
number = {25},
pages = {e04092},
doi = {10.1002/adhm.202504092},
pmid = {42230849},
issn = {2192-2659},
support = {//National Vaccine and Serum Institute/ ; },
mesh = {*Wound Healing ; *Epidermal Growth Factor/genetics/metabolism ; Animals ; *Extracellular Vesicles/metabolism ; Humans ; *RNA, Messenger/metabolism/genetics ; Rats ; *Skin/metabolism/pathology/injuries ; Cell Proliferation ; Cell Movement ; Fibroblasts/metabolism ; Cell Line ; *Dependovirus/genetics ; CRISPR-Cas Systems/genetics ; Rats, Sprague-Dawley ; },
abstract = {Skin wound healing is a complex biological process that requires the coordinated regulation of cell proliferation, migration, and extracellular matrix (ECM) remodeling. Epidermal growth factor (EGF) plays a key role in this process, but its clinical application is limited by its rapid degradation at the wound site. Extracellular vesicles (EVs), as natural nanocarriers, can protect nucleic acids from degradation and enhance their bioavailability. In this study, using CRISPR/Cas9 technology, we site-specifically integrated the EGF gene carrying the TPA signal peptide into the AAVS1 safe harbor site of 293F cells, generating a cell line that stably secretes EVs enriched in EGF mRNA. Characterization and in vitro and in vivo functional evaluation of these engineered EVs (293F-EGF-EV) demonstrated that they significantly promoted fibroblast proliferation and migration and inhibited excessive collagen production. In a rat skin defect model, 293F-EGF-EV promoted wound recovery. High-concentration 293F-EGF-EV focused on "high-quality repair," such as promoting angiogenesis, hair follicle regeneration, and epidermal structural remodeling. Low-concentration 293F-EGF-EV favored "high-efficiency closure", such as reducing scar area. This study offers new insights into skin wound treatment.},
}

*Wound Healing
*Epidermal Growth Factor/genetics/metabolism
Animals
*Extracellular Vesicles/metabolism
Humans
*RNA, Messenger/metabolism/genetics
Rats
*Skin/metabolism/pathology/injuries
Cell Proliferation
Cell Movement
Fibroblasts/metabolism
Cell Line
*Dependovirus/genetics
CRISPR-Cas Systems/genetics
Rats, Sprague-Dawley

@article {pmid42304391,
year = {2026},
author = {Gorbenko, F and Sala, I and Lee, YY and van de Venn, L and Yeh, CD and Tálas, A and Karvelis, T and Druteika, G and Bechter, LV and Vykhlyantseva, I and Schröder, MS and Gvozdenovic, A and Schwank, G and Siksnys, V and Corn, JE},
title = {Directed evolution of compact RNA-guided nucleases for enhanced activity in mammalian cells.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42304391},
issn = {1474-760X},
mesh = {Humans ; *Directed Molecular Evolution ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: RNA-guided nucleases enable DNA editing and offer promise for treating genetic diseases, particularly when used for precise sequence replacement. However, many of the most effective enzymes, such as Streptococcus pyogenes Cas9, are too large for delivery using vectors like adeno-associated virus. This has prompted interest in smaller alternatives from the Cas12f and TnpB families. Yet, these nucleases often show low activity in mammalian cells, limiting their utility.

RESULTS: We use directed evolution in human cells to select variants with greatly improved activity. The resulting variants, Cas12f1Super and TnpBSuper, exhibit up to 11-fold increase in editing efficiency without increased off-target effects. When tested as a base editor, Cas12f1Super shows up to tenfold improvement relative to the previously engineered CasMINI, suggesting utility beyond nuclease-related activities.

CONCLUSIONS: These compact and efficient genome editors expand the current toolkit and hold promise for both research and therapeutic use in mammalian systems.},
}

Humans
*Directed Molecular Evolution
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*Endonucleases/genetics/metabolism
HEK293 Cells
CRISPR-Cas Systems

@article {pmid42366709,
year = {2026},
author = {Naqvi, RF and Ali, M and Zuberi, SA and Aamir, N and Rafique, S},
title = {Nanocarrier-Based Gene Delivery Systems: Mechanisms, Clinical Translation, and Future Perspectives.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70289},
pmid = {42366709},
issn = {1097-0290},
abstract = {Gene therapy holds revolutionary potential for managing genetic disorders, cancers and infectious illnesses. However, one of the biggest challenges is delivering DNA or RNA into targeted cells and in the safe and effective way. In this review, nano carrier-based approaches for gene delivery are critically examined, focusing on both viral and non-viral systems. The advancement of CRISPR-Cas genome editing, machine learning-assisted nanocarrier optimization, and biologically inspired delivery systems is being quickly pushed forward in this area. In this review, a comparative analysis of gene delivery systems is being provided, and the key challenges to clinical translation are being pointed out. In addition, expert opinions on future research directions are being offered, with a heavy focus on the development of multifunctional, precisely targeted, and easily scalable delivery systems that can be integrated with next-generation therapeutic technologies.},
}

@article {pmid42366824,
year = {2026},
author = {Tang, H and Xing, Y and Lu, G and Shen, J and Zhou, Q},
title = {CRISPR/Cas12a Technology Combined with Immunochromatographic Strips for the Portable Detection of SFTS Bunyavirus.},
journal = {Journal of microbiology and biotechnology},
volume = {36},
number = {},
pages = {e2603014},
doi = {10.4014/jmb.2603.03014},
pmid = {42366824},
issn = {1738-8872},
mesh = {Sensitivity and Specificity ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/methods ; *Chromatography, Affinity/methods ; *CRISPR-Cas Systems ; Humans ; *Phlebovirus/isolation & purification/genetics ; RNA, Viral/genetics ; Molecular Diagnostic Techniques/methods ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; },
abstract = {Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by SFTS virus (SFTSV), is a widely distributed infection with significant mortality. Diagnosis in resource-limited settings remains challenging. For rapid and convenient diagnosis, we developed a portable rapid diagnosis method that combines Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a technology with immunochromatographic test strips. The SFTSV RNA was amplified by reverse transcription loop-mediated isothermal amplification (RT-LAMP). The homologous target sequence and single stranded DNA (ssDNA) reporter gene were cleaved by CRISPR/Cas12a in parallel, and ssDNA probes labeled with FAM fluorescein and biotin were captured by an immunochromatographic strip. Finally, the signal on the immunochromatographic strips became visible to the naked eye. Based on CRISPR/Cas12a, a rapid SFTSV detection method was developed, featuring simplicity, rapidity, low cost, and ease of use. The method was applied for the nucleic acid detection of SFTSV in 40 clinical serum samples and compared with RT-polymerase chain reaction (PCR). The new method showed 100% sensitivity and 100% specificity with a detection agreement rate of 100%. The minimum detection limit of the method was 2.5 copies/μL, and no cross-reactivity with nucleic acids from other common pathogens was observed. Detection can be completed within 80 min, and results are observable with the naked eye. For the analysis of clinical samples, the method exhibits good detection performance and thus provides an attractive option for the nucleic acid detection of SFTSV in point-of-care and resource-limited medical settings.},
}

Sensitivity and Specificity
Rapid Diagnostic Tests
Nucleic Acid Amplification Techniques/methods
*Chromatography, Affinity/methods
*CRISPR-Cas Systems
Humans
*Phlebovirus/isolation & purification/genetics
RNA, Viral/genetics
Molecular Diagnostic Techniques/methods
*Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology

@article {pmid42369443,
year = {2025},
author = {Meerman, JJ and de Bruijn, VMP and Luechtefeld, T and van Engelen, JGM and Kienhuis, AS and Heusinkveld, HJ},
title = {Including genetic susceptibility towards Parkinson's disease in NAM-based hazard and risk assessment of pesticides: a semi-systematic review.},
journal = {NAM journal},
volume = {1},
number = {},
pages = {100036},
pmid = {42369443},
issn = {3050-6204},
abstract = {Parkinson's disease (PD) prevalence has been steeply increasing over the last years. Environmental pollution, including certain pesticides, is considered a significant contributor to this rise. Certain genetic factors may pose an individual or subpopulation at increased risk to develop pesticide-induced PD. To support New Approach Methodologies (NAMs)-based hazard and risk assessment while considering known sources of interindividual variability, the aim of the current work was to identify biological factors related to increased susceptibility towards pesticide-induced PD onset and progression. To this end, a semi-systematic literature search was performed using Sysrev. We identified 61 relevant records. Potential gene-environment interactions were identified for several genes related to toxicokinetics, mitochondrial functioning, neurotransmission and proteostasis. These genes, except those related to toxicokinetics, have been described in Adverse Outcome Pathways (AOPs) leading to parkinsonian motor symptoms. Untargeted (epi)genetic analyses identified potential new targets. NAMs offer the opportunity to include human-relevant genetic interindividual variability in toxicological hazard and risk assessment. For example, an additional test condition with mutations of interest for each Key Event (KE) could be included in a test strategy. Whole-organism models combined with CrispR-CAS techniques are suitable to study the effects of specific mutations on PD risk. Induced pluripotent stem cells (iPSCs) are promising to study interindividual variability in disease susceptibility based on material from genetically diverse donors. Since additional experiments are resource-intensive, further research is required to establish the need and way to include genetic susceptibilities in a regulatory context.},
}

@article {pmid42370783,
year = {2026},
author = {Wang, R and Liu, X and Li, H and Wang, Y and Zhang, J and Jin, B and Zhang, Y and Yin, H and Li, Y},
title = {Effect of BRD0539 on Gene Editing and Mosaicism Rate in Porcine Gene Editing Embryos by CRISPR/Cas9.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {61},
number = {7},
pages = {e70255},
doi = {10.1111/rda.70255},
pmid = {42370783},
issn = {1439-0531},
support = {32272881//National Natural Science Foundation of China/ ; 2024AH040078//Anhui Province Higher Education Institutions Scientific Research Project/ ; 202513b10050016//Anhui Provincial Science and Technology Commissioner Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Myostatin/genetics ; *Gene Editing/veterinary/methods ; *Mosaicism/veterinary ; Swine/embryology ; Embryo, Mammalian ; Parthenogenesis ; Microinjections/veterinary ; Female ; *Sus scrofa/genetics/embryology ; },
abstract = {Microinjection is a common method for generating gene-edited animals; however, persistent Cas9 activity post-cleavage often results in mosaic embryos due to editing occurring in different blastomeres. This study investigated whether co-injecting the CRISPR/Cas9 system with the small-molecule Cas9 inhibitor BRD0539, or supplementing it in the culture medium, could reduce mosaicism while maintaining editing efficiency in porcine parthenogenetic activation embryos targeting the myostatin (MSTN) gene. The findings are as follows: Co-injection of 10 or 100 μM BRD0539 with Cas9 mRNA: sgRNA significantly reduced gene editing efficiency (28.5% ± 11.6% and 33.8% ± 4.1%, respectively, vs. 86.9% ± 4.5% in control, p < 0.05). Supplementing the culture medium with 10 or 50 μM BRD0539 also reduced both editing efficiency (20.8% ± 12.4% and 47.7% ± 14.6%, respectively, vs. 85.6% ± 4.8%) and mosaicism rate (25.0% ± 15.9% and 12.5% ± 12.5%, respectively, vs. 87.1% ± 7.8%, p < 0.05). Immunofluorescence revealed sustained Cas9 protein expression up to 48 h post-injection. Crucially, short-term addition of 10 μM BRD0539 to the culture medium between 24 and 48 h post-activation significantly reduced mosaicism (32.6% ± 7.5% vs. 78.7% ± 9.6%, p < 0.05) without compromising editing efficiency. Furthermore, this treatment did not adversely affect cleavage rates, blastocyst development, total cell number. These results demonstrate that transient inhibition of Cas9 activity with 10 μM BRD0539 during a critical window effectively reduces mosaicism in microinjected porcine embryos, offering a promising strategy to enhance the efficiency of generating non-mosaic gene-edited livestock.},
}

Animals
*CRISPR-Cas Systems
Myostatin/genetics
*Gene Editing/veterinary/methods
*Mosaicism/veterinary
Swine/embryology
Embryo, Mammalian
Parthenogenesis
Microinjections/veterinary
Female
*Sus scrofa/genetics/embryology

@article {pmid42371108,
year = {2026},
author = {Das, A and Rajput, VD and Pal, S and Banerjee, S and Adak, MK},
title = {Integrative transcriptional regulatory networks governing cereal root responses to heavy metals and drought.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42371108},
issn = {1432-203X},
support = {NTA Ref. No.: 211610058334//University Grants Commission/ ; },
mesh = {*Plant Roots/genetics/physiology/drug effects/metabolism ; *Edible Grain/genetics/physiology/drug effects/metabolism ; *Metals, Heavy/toxicity ; *Gene Regulatory Networks ; Gene Expression Regulation, Plant/drug effects ; Droughts ; Stress, Physiological/genetics ; Drought Resistance ; Transcription Factors/metabolism/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Cereal root systems perceive the onset of drought and heavy metal toxicity, which rapidly triggers signal transduction and extensive transcriptional reprogramming that underpins plant stress tolerance. This review discusses the transcriptional basis of root responses to abiotic stresses, emphasizing key regulatory genes and networks that orchestrate hormone signaling, redox dynamics, ion homeostasis, and structural modifications. Key transcription factor families-NAC, WRKY, bZIP, DREB, ARF, and MYB-serve as the nexus between the early perception and adaptive outputs such as controlled root growth, suberization, aerenchyma formation, and metal sequestration. Integrative transcriptomic, proteomic, metabolomic, and chromatin data in rice, wheat, maize, and millets highlight cell-type- and zone-specific regulatory programs, with single-cell and spatial omics uncovering modules obscured in bulk datasets. We discuss how CRISPR/Cas editing, cis-element engineering, and root-specific promoters refine core regulators, and how quantitative trait loci (QTL)/genome-wide association study (GWAS) advance breeding for drought and metal tolerance. Emerging concepts encompass multi-stress omics, high-throughput root phenomics, and artificial intelligence-driven network modeling, and collectively enable targeting of core transcriptional regulatory nodes. Overall, emerging knowledge supports targeted engineering of transcriptional regulators to develop resilient cereal root systems, contributing to sustainable yields and improved stress tolerance in real-world agroecosystems.},
}

*Plant Roots/genetics/physiology/drug effects/metabolism
*Edible Grain/genetics/physiology/drug effects/metabolism
*Metals, Heavy/toxicity
*Gene Regulatory Networks
Gene Expression Regulation, Plant/drug effects
Droughts
Stress, Physiological/genetics
Drought Resistance
Transcription Factors/metabolism/genetics
Plant Proteins/genetics/metabolism

@article {pmid41319305,
year = {2026},
author = {Point, V and Achache, W and Laudouze, J and Sepulveda Ramos, E and Maziero, M and Crauste, C and Canaan, S and Santucci, P},
title = {Mycobacterial cell division arrest and smooth-to-rough envelope transition using CRISPRi-mediated genetic repression systems.},
journal = {FEBS open bio},
volume = {16},
number = {7},
pages = {1271-1292},
pmid = {41319305},
issn = {2211-5463},
support = {//Institut des sciences biologiques/ ; ANR-24-CE15-2633//Agence Nationale de la Recherche/ ; AMX-19-IET-006//Initiative d'Excellence d'Aix-Marseille Université - A*MIDEX - IM2B/ ; //FEBS Excellence Award/ ; Project n°ANRS0358//Agence Nationale de Recherches sur le Sida et les Hépatites Virales/ ; //Aix-Marseille Université/ ; },
mesh = {Mycobacterium smegmatis/genetics ; Cell Division/genetics ; *CRISPR-Cas Systems/genetics ; Gene Silencing ; *Nontuberculous Mycobacteria/genetics ; Mycobacterium abscessus/genetics ; Bacterial Proteins/genetics ; },
abstract = {The genetic basis underlying nontuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9Sth1-mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use Mycobacterium smegmatis (M. smeg) and Mycobacterium abscessus (M. abs) as NTM model species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.},
}

Mycobacterium smegmatis/genetics
Cell Division/genetics
*CRISPR-Cas Systems/genetics
Gene Silencing
*Nontuberculous Mycobacteria/genetics
Mycobacterium abscessus/genetics
Bacterial Proteins/genetics

@article {pmid42082826,
year = {2026},
author = {Kazerani, M and Cagiral, U and Tabatabaei, SZ and Akalper, RN and Bora, U and Babashah, S and Ozhan, G and Totonchi, M},
title = {A Novel BIRC6 Variant Impairs Apoptotic Regulation in Familial Premature Ovarian Insufficiency: Functional Validation in a CRISPR/Cas9 Zebrafish Model.},
journal = {Reproductive sciences (Thousand Oaks, Calif.)},
volume = {33},
number = {6},
pages = {1170-1188},
pmid = {42082826},
issn = {1933-7205},
mesh = {Animals ; Female ; Zebrafish ; *Primary Ovarian Insufficiency/genetics/metabolism/pathology ; Humans ; *Apoptosis/genetics ; Disease Models, Animal ; *CRISPR-Cas Systems ; Pedigree ; *Inhibitor of Apoptosis Proteins/genetics/metabolism ; Adult ; *Zebrafish Proteins/genetics/metabolism ; Mutation, Missense ; },
abstract = {Premature ovarian insufficiency (POI), characterized by ovarian dysfunction before age 40, remains idiopathic in over 50% of cases, underscoring the urgent need to elucidate its genetic underpinnings. Here, we report a consanguineous Iranian family with five females diagnosed with POI, exhibiting elevated gonadotropins, undetectable anti-Müllerian hormone, and bilateral ovarian atrophy. Whole Exome Sequencing identified a novel homozygous missense variant in BIRC6 (NM_016252.4: c.11266C > T; p.Arg3756Cys), a gene encoding an apoptosis regulator. Segregation analysis confirmed an autosomal recessive inheritance pattern, with homozygosity exclusively in affected individuals. Functional studies in a CRISPR/Cas9-generated birc6[del12] zebrafish model recapitulated POI phenotypes: homozygous females exhibited reduced fecundity, aberrant oocyte morphology, and elevated embryonic death. Transcriptional analysis revealed dysregulation of apoptotic (badb, sortilin, apc) and fertility-related (nanos1, vtg1) genes, alongside unaltered estradiol levels, implicating apoptosis-driven follicular atresia rather than endocrine dysfunction. Compensatory increase in egg production in mutants mirrored human POI progression, where initial irregular cycles culminate in follicular depletion. This study establishes BIRC6 as a novel POI candidate, links its anti-apoptotic function to ovarian homeostasis, and highlights zebrafish as a tractable model for dissecting POI mechanisms. Our findings expand the genetic landscape of infertility and suggest therapeutic potential for apoptosis modulation in fertility preservation.},
}

Animals
Female
Zebrafish
*Primary Ovarian Insufficiency/genetics/metabolism/pathology
Humans
*Apoptosis/genetics
Disease Models, Animal
*CRISPR-Cas Systems
Pedigree
*Inhibitor of Apoptosis Proteins/genetics/metabolism
Adult
*Zebrafish Proteins/genetics/metabolism
Mutation, Missense

@article {pmid42092133,
year = {2026},
author = {Scholz, P and Thompson, J and Crosby, KT and Fauth, T and Krah, NM and Schlauderaff, G and Back, R and Berkheimer, ZA and Jolley, A and Sombroek, D and Medert, R and Zurek, C and Dmytrenko, O and Wilson, E and Schut, FT and Rutter, J and Zhang, X and Krohn, M and Jackson, RN and Beisel, CL and Liu, Y},
title = {RNA-triggered cell killing with CRISPR-Cas12a2.},
journal = {Nature},
volume = {655},
number = {8121},
pages = {230-239},
pmid = {42092133},
issn = {1476-4687},
mesh = {Humans ; *Cell Death/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; DNA/metabolism/genetics/chemistry ; DNA Breaks, Double-Stranded ; *Endodeoxyribonucleases/metabolism/genetics ; *Gene Editing ; RNA/metabolism/genetics ; Saccharomyces cerevisiae/genetics/cytology ; Bacterial Proteins ; },
abstract = {Selectively eradicating target cells on the basis of their genetic or transcriptional identity remains important in basic research, medicine, biotechnology and agriculture[1-3]. For applications involving bacteria, CRISPR nucleases offer promising options due to their ability to enact RNA-guided counterselection[4-7]; however, using these same nucleases for counterselection in eukaryotes has proven much more restrictive[8-14]. Here we show that Cas12a2, a recently discovered type V CRISPR nuclease, exhibits RNA-triggered DNA shredding[15,16], and enables programmable and sequence-specific elimination of yeast and human cells expressing a target transcript. Triggering Cas12a2 elicits rampant double-stranded DNA breaks in trans, leading to cell death. Cell killing can be activated by a wide range of target transcripts, with no observed off-target activation. Leveraging this approach, we selectively eliminate cells that harbour human papillomavirus, cells that failed to undergo gene editing, or cells that encode a prevalent oncogenic point mutation in KRAS. These findings expand the CRISPR toolbox to allow the selective elimination of eukaryotic cells on the basis of their transcriptional profile.},
}

Humans
*Cell Death/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*CRISPR-Cas Systems
DNA/metabolism/genetics/chemistry
DNA Breaks, Double-Stranded
*Endodeoxyribonucleases/metabolism/genetics
*Gene Editing
RNA/metabolism/genetics
Saccharomyces cerevisiae/genetics/cytology
Bacterial Proteins

@article {pmid42353483,
year = {2026},
author = {Xu, J and Cheng, F and Fang, J and Cao, K and Li, G and Luo, W and Hu, D and Zhang, J and Hu, Q},
title = {Advances in Fish Gene Editing.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {12},
pages = {},
pmid = {42353483},
issn = {2076-2615},
support = {CARS-47//China Agriculture Research System/ ; 2025LZGC010, 2024CXPT071-1//Department of Science and Technology of Shandong Province/ ; B24YQ0010, B25H1QC08//Hainan Seed Industry Laboratory/ ; BRESG-JB202502//Basic Research Project of State Key Laboratory of Mariculture Biobreeding and Sustainable Goods/ ; QDLYY-2024012//Blue Seed Industry Innovation Project of Qingdao Institute of Blue Seed Industry/ ; TSQNZ20240843//Taishan Scholars Program/ ; },
abstract = {Fish represent the most species-rich group within the phylum Chordata, possessing exceptional nutritional and ornamental value. Global aquaculture, particularly finfish farming, is experiencing rapid expansion worldwide, and fish serve as crucial model organisms for vertebrate developmental biology and functional genomics research. However, traditional breeding methods are plagued by limitations such as low precision and lengthy breeding cycles. Currently, gene editing technologies represented by the CRISPR/Cas system, base editing, and prime editing have provided revolutionary tools for dissecting gene function, modeling human diseases, targeted trait improvement, and ecological adaptation studies. This review describes the evolutionary history of gene editing technology, compares gene delivery strategies in fish embryos, and highlights landmark applications in key areas, including gene function research, aquaculture breeding, ornamental fish coloration regulation, and human disease model construction. Finally, we propose that innovation should be pursued while ensuring biosafety and regulatory compliance, to promote the transformation of fish gene editing toward large-scale and safe application.},
}

@article {pmid42353704,
year = {2026},
author = {Boyanova, L and Boyanova, LY and Medeiros, J and Dimitrov, G and Hadzhiyski, P and Gergova, R and Markovska, R},
title = {Some Newer Antibiotics Active Against Helicobacter pylori and Anaerobic Bacteria and the Potential Benefits of Their Wider Availability in More Countries: A Narrative Review.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42353704},
issn = {2079-6382},
abstract = {It is crucial to consider newer antibiotics with activity against anaerobes and Helicobacter pylori, given their healthcare importance, and the constantly growing antibiotic resistance/multidrug resistance, which complicates the therapy. The aim of this review was to emphasize certain recently approved or still-under-investigation antibiotics with potential benefits for treating Clostridioides difficile infections (CDIs), other anaerobic infections, and those caused by H. pylori, covering recent data from articles published primarily in 2020-2026. Given the limited number of antibiotics for treating CDI and fidaxomicin nonavailability in many countries, it is necessary to conduct more extensive laboratory and clinical studies of promising antibiotics such as ibezapolstat, delafloxacin, lascufloxacin, omadacycline, eravacycline, ridinilazole, and CRS3123. Against Bacteroides fragilis group species, delafloxacin and eravacycline showed good activity. Research on rifasutenizol for bacterial vaginosis, sarecycline and nadifloxacin for acne vulgaris and amixicile for periodontal diseases needs to be expanded. For H. pylori infection, delafloxacin, sitafloxacin, nemonoxacin, zoliflodacin, and rifasutenizol may improve the suboptimal success of most eradication regimens. However, more efforts, in coordination between medical, scientific, manufacturing, and government representatives, should ensure wider access to and research on the newer antibacterials. Establishing more research groups, careful examination of market issues, and additional approaches, such as nanomaterials, efflux pump inhibitors, phage therapy, and CRISPR-Cas systems, should be beneficial. Notwithstanding the difficulties, there are many opportunities to promote research on and potential use of newer antibiotics which show advantages over the older antibacterials, and to make them available to numerous countries and patients worldwide.},
}

@article {pmid42353780,
year = {2026},
author = {Zhu, C and Wang, Y and Zhu, M and Chen, G and Wang, F and Li, B and Xu, Z and Wang, G and Xu, J and Lu, X and Wang, Y and Jin, S},
title = {High-Density CRISPR/Cas12a-Mediated Multiplex Genome Editing Reveals Genome Instability in Allotetraploid Cotton.},
journal = {Genes},
volume = {17},
number = {6},
pages = {},
pmid = {42353780},
issn = {2073-4425},
support = {XL202401//Xinjiang Talent Development Fund/ ; 2025SNGGGCC-BT06//Tianshan Talents Program for Rural Development Leaders/ ; },
mesh = {*Gossypium/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genomic Instability ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Tetraploidy ; },
abstract = {BACKGROUND: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing.

METHODS: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA-crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon.

RESULTS: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65-24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations.

CONCLUSIONS: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes.},
}

*Gossypium/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genomic Instability
*Genome, Plant
Plants, Genetically Modified/genetics
Tetraploidy

@article {pmid42353789,
year = {2026},
author = {Wu, Q and Sun, J and Yang, S and Zhang, M and Yang, D and Xue, H and Wu, H and Guo, Y and Li, S and An, Y},
title = {TaKMT-7A Gene Positively Regulates Spike Number in Wheat.},
journal = {Genes},
volume = {17},
number = {6},
pages = {},
pmid = {42353789},
issn = {2073-4425},
support = {2024CXPT072//Department of Science and Technology of Shandong Province/ ; },
mesh = {*Triticum/genetics/growth & development ; *Quantitative Trait Loci/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Chromosome Mapping ; Phenotype ; Edible Grain/genetics/growth & development ; CRISPR-Cas Systems ; Genes, Plant ; Frameshift Mutation ; },
abstract = {Wheat (Triticum aestivum L.) is a crucial global food crop that plays a central role in agricultural production and food security. The spike number per unit area (SN) is one of the three component factors of grain yield. In this study, we combined the UG-Map with 27 environments of a recombinant inbred line (RIL) population, and mapped a quantitative trait locus (QTL) for SN, QSn-7A-9048, in which the meta-QTL interval contains only one candidate gene, TraesCS7A02G-364700 (TaKMT-7A). Using the CRISPR/Cas9 system, we generated two homozygous mutant lines, aa-1 and aa-2 of TaKMT-7A, which resulted in frameshift mutations, leading to the premature termination of the translation process. The SN values for the wild type (WT), aa-1, and aa-2 were 4.48, 3.43, and 3.48, respectively. Compared with the WT, the SN of the two mutant lines significantly decreased, and no significant differences for grain number per spike (GNS) and thousand-grain weight (TGW) were detected. We also obtained two overexpression (OE) lines of TaKMT-7A, OE-1 and OE-2. The SN values for the negative control (NC), OE-1, and OE-2 were 2.31, 3.33, and 3.00, respectively. Compared with NC, the SN values in the OE lines significantly increased. The phenotypes of the knockout (KO) lines and OE lines demonstrate that TaKMT-7A acts as a positive regulator of SN in wheat. We performed RNA-Seq analysis using young tiller buds from the WT and aa-1 mutant lines at the tillering stage, and a total of 2315 differentially expressed genes (DEGs) were identified. We screened 22 wheat genes, of which 18 orthologous genes have previously been cloned and are associated with branching in rice and Arabidopsis. These genes included nitrogen transporter, amino metabolism, auxin transporter, auxin homeostasis, auxin response, auxin biosynthesis, strigolactone biosynthesis, and repress gibberellin responses. These genes may represent potential downstream targets of TaKMT-7A.},
}

*Triticum/genetics/growth & development
*Quantitative Trait Loci/genetics
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Chromosome Mapping
Phenotype
Edible Grain/genetics/growth & development
CRISPR-Cas Systems
Genes, Plant
Frameshift Mutation

@article {pmid42353791,
year = {2026},
author = {Rodriguez, SH and Yokota, T},
title = {Building CRISPR-Based Gene-Editing Platforms for Personalized Medicine: The Next Step in Interventional Genetics.},
journal = {Genes},
volume = {17},
number = {6},
pages = {},
pmid = {42353791},
issn = {2073-4425},
mesh = {Humans ; *Precision Medicine/methods/trends ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; },
abstract = {Recent advances in CRISPR technology have expanded beyond traditional double-strand break-based genome editing to include base editors and prime editors, enabling precise and programmable sequence modifications. This evolution marks a shift from conventional mutation correction toward platform-based therapeutic systems capable of targeting a broad spectrum of pathogenic variants. Such versatility holds promise for addressing a substantial proportion of known disease-causing mutations in rare monogenic disorders. This review discusses the technological progression of CRISPR systems, highlighting the principles, applications, and limitations of emerging editing modalities. We will explore their translation into personalized gene therapies, emphasizing delivery challenges, off-target safety, and the need for regulatory innovation. The paper will also introduce the concept of interventional genetics, an emerging medical framework linking genomic diagnosis directly to therapeutic intervention through adaptive gene-editing platforms. Finally, we will outline strategies for establishing unified, scalable, and regulatory-ready editing platforms that can accelerate the clinical implementation of individualized therapies for rare diseases.},
}

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

@article {pmid42354828,
year = {2026},
author = {Flores, AI and Morales-Cedeño, LR and Loeza-Lara, PD and Schoebitz, M and Orozco-Mosqueda, MDC and Santoyo, G},
title = {Engineering Plant-Associated Microorganisms for Bioremediation and Sustainable Agriculture.},
journal = {Microorganisms},
volume = {14},
number = {6},
pages = {},
pmid = {42354828},
issn = {2076-2607},
abstract = {As food demand increases, agricultural practices have evolved, prompting increased exploration of sustainable ecological techniques and utilization of plant-associated microorganisms. In this context, plant fitness has been enhanced by plant growth-promoting microorganisms (PGPM), which stimulate growth through direct mechanisms, such as improved nutrient availability and phytohormone production, as well as indirect mechanisms, including protection against phytopathogens and suppression of soil-borne diseases. However, these innate capabilities of PGPM can be further improved through genomic modification or editing. This article reviews advances in the genomic engineering of plant-beneficial microorganisms as tools to enhance their positive effects on crop performance and environmental remediation. The genetic modification strategies analyzed here include random mutagenesis, targeted genome editing (such as CRISPR-Cas), gene over-expression, genome shuffling, RNA interference, metabolic pathway engineering, and synthetic biology approaches. These tools have enabled the optimization of functions, such as nitrogen fixation, phosphate solubilization, secondary metabolite production, biocontrol, stress tolerance, and bioremediation. However, we propose expanding the discussion of their regulation and use in various countries. Additionally, these modifications must be efficient and safe for the beneficial microbiota associated with the target crop, as well as for humans, animals, and the environment, all of which depend on sustainable agricultural practices.},
}

@article {pmid42359401,
year = {2026},
author = {Gain, H and Banerjee, J},
title = {Cis-regulatory elements in CAMTA-mediated stress signalling: mechanisms and prospects for CRISPR-based crop improvement.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1864496},
pmid = {42359401},
issn = {1664-462X},
abstract = {Enhancements in crop resilience strategies that maintain production are essential to address the challenges posed by climate change and increasing food consumption. Calcium-dependent signaling networks are essential for plant responses to abiotic and biotic stressors, with calmodulin-binding transcription activator (CAMTA) transcription factors serving as crucial regulators within this framework, as these factors govern gene expression through specific cis-regulatory elements located in promoter regions. Recent investigations have expanded to include CAMTA-binding motifs as the stress-responsive cis-regulatory modules across several plant species under examination. These findings indicate that CAMTA-associated cis-elements, comprising CGCG motifs and ABA-responsive regions, facilitate the integration of environmental signals that influence transcription. Cis-regulatory elements (CREs), such as promoters, enhancers, silencers, and insulators, control the exact timing and location of stress-responsive gene expression in plants. Recent breakthroughs in genome editing have enabled the direct manipulation of these cis-regulatory areas, facilitating precise control over gene expression. This work presents an overview of CAMTA structures, their interaction with promoter cis-regulatory regions, and the potential for promoter cis-element engineering to enhance agricultural performance under diverse settings. It emphasizes CRISPR-based strategies for precise CRE modifications and highlights the role of CAMTA in identifying stress-responsive regions. This establishes the foundation for the advancement of next-generation stress-resilient crops, which will ensure food security.},
}

@article {pmid42360551,
year = {2026},
author = {Pramanik, S and Debnath, B and Chakraborty, A and Islam, A and Mullick, S and Chaudhary, P and Nath, R and Chellappan, DK and Mondal, M and Ashique, S},
title = {Targeting mtDNA to Modulate Mitochondrial Dysfunction in Neurodegenerative Diseases.},
journal = {Molecular neurobiology},
volume = {63},
number = {1},
pages = {},
pmid = {42360551},
issn = {1559-1182},
mesh = {Humans ; *Neurodegenerative Diseases/genetics/therapy/pathology ; *DNA, Mitochondrial/genetics/metabolism ; Animals ; *Mitochondria/genetics/metabolism/pathology ; Gene Editing ; },
abstract = {Mitochondrial dysfunction is a common pathological feature of neurodegenerative diseases namely Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Although these disorders are primarily driven by disease-specific genetic and proteopathic mechanisms, increasing evidence suggests that secondary mitochondrial DNA (mtDNA) damage and heteroplasmy shifts may exacerbate bioenergetic failure and neuronal vulnerability. Distinguishing primary disease mechanisms from downstream mtDNA alterations is critical to accurately evaluate emerging therapeutic strategies. Recent advances in mtDNA-targeted genome editing have enabled the direct manipulation of mitochondrial genomes. Mitochondrially targeted zinc finger nucleases and TALENs can selectively alter mutant mtDNA to induce heteroplasmy shifts, whereas DddA-derived cytosine base editors allow precise base editing without double-strand breaks. However, each platform has distinct limitations related to the target scope, off-target risk, design complexity, and delivery efficiency. The application of CRISPR/Cas-based systems to mammalian mtDNA remains constrained by the unresolved challenges in guiding RNA import. This review critically examines mitochondrial dysfunction and mutant mtDNA accumulation in neurodegenerative diseases. It also evaluates current and emerging mtDNA-editing techniques, and highlights key translational barriers. We highlighted that mtDNA-targeted interventions can be a promising approach for disease-modifying or adjunctive strategies, rather than curative approaches.},
}

Humans
*Neurodegenerative Diseases/genetics/therapy/pathology
*DNA, Mitochondrial/genetics/metabolism
Animals
*Mitochondria/genetics/metabolism/pathology
Gene Editing

@article {pmid42361119,
year = {2026},
author = {Dupzyk, AJ and Waldman, BS and Zengel, J and Zanini, F and Carette, JE},
title = {Dissecting the host determinants of orthoflavivirus infection using QIC-seq.},
journal = {PLoS pathogens},
volume = {22},
number = {6},
pages = {e1014279},
pmid = {42361119},
issn = {1553-7374},
mesh = {Animals ; Humans ; *Host-Pathogen Interactions ; *Flavivirus/genetics ; CRISPR-Cas Systems ; *Flavivirus Infections/virology/genetics ; Dengue Virus/genetics ; Membrane Proteins/genetics/metabolism ; West Nile virus/genetics ; Hexosyltransferases ; },
abstract = {Orthoflaviviruses are genetically related, yet cause distinct disease patterns ranging from hepatitis and vascular shock syndrome to encephalitis and congenital abnormalities. There is an incomplete understanding of the cellular pathways co-opted by orthoflaviviruses, and differences in host response to infection may underlie the diverse pathologies caused. We present a single-cell approach (Quantification of Infection and CRISPR guide sequencing; QIC-seq) that combines CRISPR/Cas9 knockout with virus-inclusive transcriptomics to systematically compare host factor requirements and host transcriptional response to orthoflaviviral challenge. Using a CRISPR library focused on select ER-proteostasis genes, we show that dengue and yellow fever viruses are strictly dependent on subunits of the oligosaccharyltransferase complex, while the more distantly related West Nile and Langat viruses are dependent on components of the ER-associated degradation machinery. Our data further shows virus-induced upregulation of interferon-stimulated genes, and activation of the unfolded protein response. QIC-seq enables quantitative comparisons of viral host factor utilization, which may inform development of host-directed antiviral therapies.},
}

Animals
Humans
*Host-Pathogen Interactions
*Flavivirus/genetics
CRISPR-Cas Systems
*Flavivirus Infections/virology/genetics
Dengue Virus/genetics
Membrane Proteins/genetics/metabolism
West Nile virus/genetics
Hexosyltransferases

@article {pmid42362581,
year = {2026},
author = {Chen, J and Huang, L and Chen, H and Li, X and Lin, X and Guo, C and Liu, X and Fu, G and Chen, Y and Liu, L},
title = {Potential role of a CRISPR-Cas-activated toxin-antitoxin system in bacterial immunity.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74930-z},
pmid = {42362581},
issn = {2041-1723},
support = {32371346//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32301007//National Natural Science Foundation of China (National Science Foundation of China)/ ; 324B2056//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023J01023//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; 2024J011007//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; 2023J05008//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; },
abstract = {CRISPR-Cas and toxin-antitoxin systems can serve as antiviral defense mechanisms in prokaryotes. In typical toxin-antitoxin systems, toxin activation can limit phage propagation by inducing growth arrest or reduced cellular fitness, while the antitoxin neutralizes toxin activity. Here, we study potential functional synergy between a CRISPR-Cas13a system and a type II toxin-antitoxin module (HicAB) from a Leptotrichia bacterium, when heterologously expressed in E. coli, as well as in biochemical and structural analyses. We show that the antitoxin HicB exhibits toxic properties, and Cas13a directly activates HicB, triggering growth inhibition and conferring protection against bacteriophages. Structural analyses reveal that Cas13a binding promotes the spatial proximity of HicB tetramers, likely enabling its activation. The toxin HicA competitively binds to HicB, thereby inhibiting Cas13a-mediated HicB activation. Importantly, both CRISPR RNA and HicB independently suppress HicA toxicity. Structural evidence indicates that CRISPR RNA forms a hetero-tetradecameric complex with HicAB, occluding HicA's active site and neutralizing its toxic function. Thus, our findings indicate functional synergy between distinct bacterial immune strategies.},
}

@article {pmid41057262,
year = {2026},
author = {Char, SN and Liu, H and Birchler, JA and Wang, K and Yang, B},
title = {CRISPR-Cas9 Toolkit for Maize: Vector Design, Construction, and Analysis of Edited Plants.},
journal = {Cold Spring Harbor protocols},
volume = {2026},
number = {7},
pages = {pdb.prot108659},
doi = {10.1101/pdb.prot108659},
pmid = {41057262},
issn = {1559-6095},
mesh = {*Zea mays/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.},
}

*Zea mays/genetics
*CRISPR-Cas Systems
*Genetic Vectors/genetics
Plants, Genetically Modified/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41318237,
year = {2026},
author = {Shin, SW and Lee, GM and Lee, JS},
title = {CRISPR screen-based mammalian cell engineering for complex biotherapeutics.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1817-1820},
doi = {10.1016/j.tibtech.2025.11.010},
pmid = {41318237},
issn = {1879-3096},
mesh = {Animals ; Humans ; *Cell Engineering/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mammals ; },
abstract = {The rise of complex biotherapeutics has introduced bottlenecks in production using mammalian cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-based screens enable unbiased discovery of engineering targets that mitigate biomanufacturing-relevant constraints. This forum gives an overview of recent advances and remaining challenges in applying CRISPR screening to build robust, modality-specific cell factories.},
}

Animals
Humans
*Cell Engineering/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mammals

@article {pmid41421891,
year = {2026},
author = {Durán-Vinet, B and Stanton, JL and Jeunen, GJ and Pochon, X and Zaiko, A and Gemmell, NJ},
title = {CRISPR as a next-generation environmental biosurveillance tool for air, land, and water.},
journal = {Trends in biotechnology},
volume = {44},
number = {7},
pages = {1872-1891},
doi = {10.1016/j.tibtech.2025.11.014},
pmid = {41421891},
issn = {1879-3096},
mesh = {*Environmental Monitoring/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Air Microbiology ; Water Microbiology ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based environmental biosurveillance (CRISPR-eBx) offers a portable, specific, sensitive, and cost-effective platform for detecting organisms from environmental nucleic acids. Applications are broad, ranging from pathogen detection to monitoring invasive and endangered species across a range of environmental sources, including water, soil, and air. However, if CRISPR-eBx is to be deployed for novel biological/gene targets and environmental sources, key challenges must be addressed. This review synthesizes recent developments at the intersection of CRISPR technology, computational science, synthetic biology, and biosurveillance. We highlight promising innovations and identify knowledge gaps to present a strategic road map for establishing CRISPR-eBx as a next-generation, frontline biosurveillance solution.},
}

*Environmental Monitoring/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems
Air Microbiology
Water Microbiology

@article {pmid41513118,
year = {2026},
author = {Amieva, R and Román, LR and Coronado, M and Powell, J and Hassan, MA and Hemphill, A and Boubaker, G and Pfarrer, C and Ortega-Mora, LM and Collantes-Fernández, E and Horcajo, P},
title = {NcROP24 loss attenuates Neospora caninum virulence and alters rhoptry organization.},
journal = {International journal for parasitology},
volume = {56},
number = {7},
pages = {104770},
doi = {10.1016/j.ijpara.2026.104770},
pmid = {41513118},
issn = {1879-0135},
mesh = {*Neospora/pathogenicity/genetics ; Animals ; Female ; Cattle ; *Coccidiosis/parasitology/veterinary ; Virulence ; *Protozoan Proteins/genetics/metabolism ; Pregnancy ; Mice ; Macrophages/parasitology ; Host-Parasite Interactions ; Cattle Diseases/parasitology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a leading cause of abortion and economic loss in cattle worldwide. Despite its veterinary significance, the molecular mechanisms underlying parasite virulence and host-pathogen interaction remain poorly understood. In particular, the contribution of rhoptry proteins, key secretory effectors involved in host cell invasion and immune modulation, has yet to be fully elucidated. Here, we investigate NcROP24, a previously understudied rhoptry protein whose expression correlates with isolate virulence. Using CRISPR/Cas9, we generated NcROP24 knock-out mutants (NcΔROP24) by deleting all three genomic copies and confirmed loss of expression with a single-copy insertion of a selectable marker DHFR-TS. In a pregnant mouse model, NcΔROP24 parasites displayed markedly reduced congenital transmission, higher neonatal survival, and lower maternal brain parasite burdens compared to wild-type controls, demonstrating significant attenuation of systemic and vertical infection. Also, in bovine monocyte-derived macrophages, NcΔROP24 tachyzoites showed impaired intracellular growth. Dual RNA-seq of infected macrophages revealed that NcΔROP24 loss prevents the parasite from reprogramming key host metabolic and degradative pathways, instead promoting a stress-induced, lipogenic state that favours clearance. Concurrently, parasites lacking NcROP24 upregulated stress-associated transcripts and downregulated additional secreted effectors, indicating a shift away from aggressive proliferation. Together, these findings establish NcROP24 as a key factor of N. caninum pathogenicity. By defining its role in host-pathogen interactions, our work highlights NcROP24 as a promising target for next-generation vaccines or therapeutics against bovine neosporosis.},
}

*Neospora/pathogenicity/genetics
Animals
Female
Cattle
*Coccidiosis/parasitology/veterinary
Virulence
*Protozoan Proteins/genetics/metabolism
Pregnancy
Mice
Macrophages/parasitology
Host-Parasite Interactions
Cattle Diseases/parasitology
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid41593356,
year = {2026},
author = {Wan, Y and He, Y and Chen, X and Wang, S and Zhou, G and Ying, X and Zhang, H},
title = {Effective delivery of genome editor to cervical cancer targeting Mcl1 for cancer therapy.},
journal = {Cancer gene therapy},
volume = {33},
number = {4},
pages = {378-389},
pmid = {41593356},
issn = {1476-5500},
mesh = {Humans ; Female ; *Uterine Cervical Neoplasms/genetics/therapy/pathology ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists & inhibitors ; Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Editing/methods ; Apoptosis ; Genetic Therapy/methods ; Cell Line, Tumor ; Extracellular Vesicles/metabolism/genetics ; },
abstract = {CRISPR/Cas9 represents a transformative advancement in precision therapies, offering the promise of more effective and targeted treatment options. However, there are still limitations (including off-target editing as well as unsatisfied delivery tool) which obstruct the wide application of CRISPR/Cas9. Here, an endogenic artificial extracellular vesicles (EVs) system is engineered for effective delivery of Cas9 ribonucleoprotein (RNP). We demonstrated that the endogenic Cas9 RNP were sorted by the Lamp2b and delivered by the artificial EVs, which could markedly inhibit the growth of cervical cancer cells by inducing cell apoptosis. Moreover, artificial endogenic EVs[RNP] (Cas9-Mcl1) could result in remarkable antitumor effects in animal models of cervical cancer through suppressing Mcl1 expression. Our findings indicate that the artificial EVs delivery strategy could deliver Cas9 RNP effectively to inhibit cancer progression, which might be a promising treatment.},
}

Humans
Female
*Uterine Cervical Neoplasms/genetics/therapy/pathology
*Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists & inhibitors
Animals
*CRISPR-Cas Systems
Mice
*Gene Editing/methods
Apoptosis
Genetic Therapy/methods
Cell Line, Tumor
Extracellular Vesicles/metabolism/genetics

@article {pmid41662723,
year = {2026},
author = {De Silva Weligodage, H and Goenaga, R and Gutierrez, OA and Brown, JK},
title = {Development of a Recombinase Polymerase Amplification-CRISPR/Cas12a Detection System for Cacao Mild Mosaic Virus.},
journal = {Phytopathology},
volume = {116},
number = {7},
pages = {1173-1184},
doi = {10.1094/PHYTO-10-25-0319-R},
pmid = {41662723},
issn = {0031-949X},
mesh = {*Cacao/virology ; *Plant Diseases/virology ; Recombinases/metabolism/genetics ; *Caulimovirus/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; DNA Primers/genetics ; Plant Leaves/virology ; Sensitivity and Specificity ; },
abstract = {Plant viruses that cause minimal to no disease symptoms may not support readily detectable virus levels. Such viruses are of concern when they persist in plant germplasm collections or in breeding populations because they can provide an inoculum that can be spread and potentially cause outbreaks in susceptible plant species. The mealybug-transmitted cacao mild mosaic virus (CaMMV) causes symptomatic and asymptomatic infection of cacao trees that varies seasonally. The virus accumulates to low levels in leaves and petioles of at least some cacao genetic groups, which has confounded reliable CaMMV detection. Here, a multiplex recombinase polymerase amplification (RPA) assay was developed to increase the reliability of CaMMV detection. Three RPA primers were designed to amplify two regions of the movement protein (mp) gene of CaMMV, yielding fragments of 362 and 284 bp. To increase the detection sensitivity and specificity of CaMMV, two guide RNAs (20 bp) targeting both the CaMMV RPA amplicons were designed to activate Cas12a-mediated collateral cleavage of a fluorescent reporter. An RPA detection efficiency of 100% was achieved with respect to six known CaMMV mp variants, and the analytical sensitivity ranged from approximately 3 to 40 detectable CaMMV genomes. No signal was observed when cloned cacao-infecting badnavirus sequences or virus-free cacao were used as the template, indicating that this assay is highly specific for CaMMV.},
}

*Cacao/virology
*Plant Diseases/virology
Recombinases/metabolism/genetics
*Caulimovirus/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
DNA Primers/genetics
Plant Leaves/virology
Sensitivity and Specificity

@article {pmid41792998,
year = {2026},
author = {Shin, EJ and Choi, Y and Jeon, EJ and Lee, KI and Lee, KJ and Son, YJ and Son, MJ and Kim, S and Cho, SW and Lee, JY},
title = {Targeted KEAP1 disruption enhances antioxidant defense and mesenchymal stromal cell therapy for chronic limb-threatening ischemia.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3949-3961},
doi = {10.1016/j.ymthe.2026.03.005},
pmid = {41792998},
issn = {1525-0024},
mesh = {*Kelch-Like ECH-Associated Protein 1/genetics/metabolism ; Animals ; *Mesenchymal Stem Cells/metabolism/cytology ; Humans ; Mice ; *Mesenchymal Stem Cell Transplantation/methods ; NF-E2-Related Factor 2/metabolism/genetics ; Oxidative Stress ; *Antioxidants/metabolism ; Disease Models, Animal ; Gene Editing ; *Chronic Limb-Threatening Ischemia/therapy/metabolism/genetics ; *Ischemia/therapy/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Reactive Oxygen Species/metabolism ; },
abstract = {Chronic limb-threatening ischemia (CLTI) is a severe vascular disorder characterized by tissue hypoxia and oxidative stress that limit the efficacy of regenerative therapies. Mesenchymal stem/stromal cells (MSCs) hold promise for CLTI treatment through paracrine angiogenic and immunomodulatory signaling, yet their survival and function are compromised in the reactive oxygen species-rich ischemic microenvironment. Here, we utilized CRISPR-Cas9 to generate a targeted knockout of Kelch-like ECH-associated protein 1 (KEAP1), the negative regulator of the antioxidant transcription factor NRF2, in human bone marrow-derived MSCs. KEAP1 editing activated the NRF2 pathway, reduced intracellular oxidative stress, and reprogrammed redox and paracrine gene networks. Edited MSCs exhibited enhanced viability, sustained secretion of proangiogenic cytokines, and improved tissue perfusion and arteriogenesis in a murine model of CLTI. These findings establish KEAP1 gene editing as a permanent, integration-free strategy to augment MSC resistance and therapeutic efficacy in oxidative ischemic environments.},
}

*Kelch-Like ECH-Associated Protein 1/genetics/metabolism
Animals
*Mesenchymal Stem Cells/metabolism/cytology
Humans
Mice
*Mesenchymal Stem Cell Transplantation/methods
NF-E2-Related Factor 2/metabolism/genetics
Oxidative Stress
*Antioxidants/metabolism
Disease Models, Animal
Gene Editing
*Chronic Limb-Threatening Ischemia/therapy/metabolism/genetics
*Ischemia/therapy/metabolism
CRISPR-Cas Systems
Signal Transduction
Reactive Oxygen Species/metabolism

@article {pmid41814651,
year = {2026},
author = {Tao, D and Xu, B and Li, S and Liu, H and Wei, Y and Cao, X and Shi, S and Wang, Y and Jiang, R and Zhang, Y and Zhao, C and Ruan, J and Fu, L and Huang, X and Li, X and Zhao, S and Xie, S},
title = {Structural mining and engineering of metagenome-derived Cas12a orthologs expands the CRISPR genome editing and multiplex diagnostics toolkit.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {4104-4120},
doi = {10.1016/j.ymthe.2026.03.011},
pmid = {41814651},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Associated Proteins/genetics/chemistry ; *Metagenome ; Mice ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Swine ; Pathology, Molecular/methods ; },
abstract = {CRISPR-Cas12a is a compact, RNA-guided nuclease widely deployed in genome editing and molecular diagnostics, yet its broader utility is limited by suboptimal cis-cleavage efficiency and incompletely defined trans-cleavage behavior. To overcome these constraints, we developed an artificial intelligence-guided structural discovery pipeline powered by AlphaFold2, which identified 1,261 previously uncharacterized Cas12a orthologs. From this set, 21 structurally conserved but sequence-divergent candidates were selected for biochemical characterization. Using structure-informed engineering, we generated PcuCas12a MAX, a high-fidelity variant that achieves genome-editing efficiencies in human cells comparable to the benchmark AsCas12a Ultra while retaining robust activity in murine and porcine systems. In addition, four orthologs (LcoCas12a, FcaCas12a, EsoCas12a, and Mac2Cas12a), when paired with specifically engineered CRISPR RNAs, exhibited distinct single-stranded DNA trans-cleavage signatures. These properties enabled construction of a multiplex CRISPR sensor capable of simultaneously detecting multiple nucleic acid targets. Together, these findings expand the Cas12a endonuclease repertoire and enhance its utility in genome engineering and next-generation diagnostics.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
*CRISPR-Associated Proteins/genetics/chemistry
*Metagenome
Mice
*Bacterial Proteins/genetics/chemistry/metabolism
*Endodeoxyribonucleases/genetics/chemistry/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems/genetics
Swine
Pathology, Molecular/methods

@article {pmid41814652,
year = {2026},
author = {Iyer, S and Daman, K and Sun, Y and Tutto, A and Holbrook, SE and Joynt, AT and Yan, J and Ambegaokar, P and Guo, D and Liu, P and Stauffer, JE and Maitland, SA and Lee, SM and Xiao, Y and Huang, HC and Zhu, LJ and Gallagher, TL and Cox, GA and Keeler, AM and Siegwart, DJ and Emerson, CP and Wolfe, SA},
title = {SORT LNPs encapsulating Cas9 mRNA achieve efficient editing in skeletal muscle in a dystrophic mouse model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3885-3902},
pmid = {41814652},
issn = {1525-0024},
support = {UH3 TR002668/TR/NCATS NIH HHS/United States ; R01 HL150669/HL/NHLBI NIH HHS/United States ; R01 HL170629/HL/NHLBI NIH HHS/United States ; R01 CA269787/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; R37 AI147868/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Muscle, Skeletal/metabolism ; Disease Models, Animal ; *RNA, Messenger/genetics/administration & dosage ; *Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; Genetic Therapy/methods ; *Muscular Dystrophies, Limb-Girdle/genetics/therapy ; Liposomes ; },
abstract = {Limb girdle muscular dystrophy (LGMD) is the fourth most common type of muscular dystrophy. Gene editing holds promise for treating neuromuscular disorders such as LGMD, but clinical translation remains challenging due to lack of complementary delivery tools for skeletal muscle. Lipid nanoparticles (LNPs) offer a promising platform for transient delivery of gene-editing reagents as mRNA or ribonucleoprotein complexes (RNPs) to skeletal muscle, but editing efficiencies remain modest. While lipid compositions have been optimized to improve delivery to muscle, the impact of cargo type on editing efficiency, biodistribution, and immune response has not been evaluated. Here, we demonstrate that selective organ targeting (SORT) LNPs encapsulating optimized Cas9 cargo facilitate efficient, local delivery to skeletal muscle. Using an LGMDR7 mouse model harboring a mutation in TCAP as a proof-of-concept target, we show that LNP cargo type impacts LNP size, delivery to neighboring muscle groups, and editing efficiency. RNP and mRNA LNPs also provoked distinct innate and adaptive immune responses upon repeated dosing. The optimized SORT LNP platform resulted in 40% restoration of Telethonin expression in treated muscle. Overall, these findings offer valuable insights for the continued development of LNP-based gene-editing reagents to facilitate disease-modifying interventions for neuromuscular diseases.},
}

Animals
Mice
*Muscle, Skeletal/metabolism
Disease Models, Animal
*RNA, Messenger/genetics/administration & dosage
*Nanoparticles/chemistry
*Gene Editing/methods
*CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems/genetics
*Lipids/chemistry
Genetic Therapy/methods
*Muscular Dystrophies, Limb-Girdle/genetics/therapy
Liposomes

@article {pmid41877484,
year = {2026},
author = {Ralu, M and Guiraud, S and Dastidar, S and Galbiati, P and Sadaoui, E and Mazed, F and Amor, F and de Cian, A and Richard, I and Mamchaoui, K and Ronzitti, G and Tedesco, FS and Amendola, M},
title = {CRISPR-Cas9-mediated upregulation of utrophin ameliorates Duchenne muscular dystrophy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3903-3923},
doi = {10.1016/j.ymthe.2026.03.025},
pmid = {41877484},
issn = {1525-0024},
mesh = {*Muscular Dystrophy, Duchenne/genetics/therapy/metabolism/pathology ; *Utrophin/genetics/metabolism ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; MicroRNAs/genetics ; Genetic Therapy/methods ; Disease Models, Animal ; Up-Regulation ; Dependovirus/genetics ; Gene Editing ; Muscle, Skeletal/metabolism/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Gene Therapy Agents ; Gene Expression Regulation ; Mice, Inbred mdx ; Binding Sites ; },
abstract = {Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulating utrophin, a dystrophin paralog, is a promising gene therapy approach. Here, we present a CRISPR-Cas9-based strategy to enhance utrophin expression by disrupting repressor binding sites. Using a Cas9/guide RNA (gRNA) ribonucleoprotein complex, we disrupted several such sites in DMD myoblasts and identified microRNA Let-7c binding site as effective in relieving repression of the UTRN gene. Interestingly, Cas9-generated insertions or deletions (indels) were as effective as the complete removal of Let-7c binding site in upregulating UTRN expression, with minimal off-target effects. In a three-dimensional tissue-engineered human skeletal muscle model of DMD, this editing strategy resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, in mdx mice, local or systemic delivery of recombinant adeno-associated viruses encoding Cas9 and gRNA targeting the Let-7c binding site resulted in utrophin upregulation and amelioration of muscle histopathology and function. These findings provide the foundations for a mutation-independent, potentially universal gene-editing therapeutic strategy for DMD.},
}

*Muscular Dystrophy, Duchenne/genetics/therapy/metabolism/pathology
*Utrophin/genetics/metabolism
Animals
Humans
Mice
*CRISPR-Cas Systems
MicroRNAs/genetics
Genetic Therapy/methods
Disease Models, Animal
Up-Regulation
Dependovirus/genetics
Gene Editing
Muscle, Skeletal/metabolism/pathology
RNA, Guide, CRISPR-Cas Systems/genetics
Myoblasts/metabolism
Gene Therapy Agents
Gene Expression Regulation
Mice, Inbred mdx
Binding Sites

@article {pmid41935953,
year = {2026},
author = {Fumagalli, M and An, D and Simula, L and Combe, C and Aziez, L and Simoni, Y and Alves-Guerra, MC and Valentini, A and Marchais, M and Vermare, A and Moraly, J and Manni, S and Quadraccia, MC and Quintarelli, C and De Angelis, B and Bercovici, N and Donnadieu, E and Pendino, F},
title = {An in vivo CRISPR screen unveils promising target genes to improve CAR-T cell efficacy in a solid tumor model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3976-4000},
doi = {10.1016/j.ymthe.2026.04.009},
pmid = {41935953},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; Disease Models, Animal ; *Neoplasms/therapy/genetics/immunology ; Xenograft Model Antitumor Assays ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *T-Lymphocytes/immunology/metabolism ; },
abstract = {CAR-T cell therapies are revolutionizing the treatment of refractory or relapsed hematological malignancies, but many patients do not achieve durable responses, and these therapies remain ineffective against solid tumors. Therapeutic failure is closely associated with a poor persistence of CAR-T cells in patients, highlighting the need to identify strategies promoting in vivo expansion. Although numerous gene-editing strategies have been proposed, comparative studies to identify the most effective ones are still lacking. Here, using a focused CRISPR-knockout library targeting 50 selected gene candidates, we developed a competitive screening that revealed ZC3H12A, SOCS1, PTPN2, and CDKN2A as the most robust targets to improve persistence of EGFR CAR-T cells in human lung tumor-bearing mice. Surprisingly, disruption of other genes previously reported to improve CAR-T cell efficacy in other preclinical models-MED12, PRDM1, and BATF-had a detrimental effect in this context. These results suggest that some gene-editing strategies can yield beneficial, neutral, or even deleterious effects on CAR-T cell persistence, depending on specific conditions. Altogether, these findings highlight the importance of performing context-specific evaluations of genetic modifications to accelerate the clinical translation of the most promising editing strategies for optimizing CAR-T cell therapies.},
}

Animals
Humans
Mice
*Immunotherapy, Adoptive/methods
*CRISPR-Cas Systems
*Gene Editing/methods
Cell Line, Tumor
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
Disease Models, Animal
*Neoplasms/therapy/genetics/immunology
Xenograft Model Antitumor Assays
*Clustered Regularly Interspaced Short Palindromic Repeats
*T-Lymphocytes/immunology/metabolism

@article {pmid41981915,
year = {2026},
author = {Truong, LB and Li, S and Domkofski, C and Lin, J and Co, C and Halwasia, S and Kuefner, M and Hu, Y and Mao, HQ and Wang, TH and Zhu, S},
title = {Messenger RNA and guide RNA distributions in lipid nanoparticles impact gene-editing efficiency in vivo.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3962-3975},
pmid = {41981915},
issn = {1525-0024},
support = {R01 AI183336/AI/NIAID NIH HHS/United States ; R01 CA260628/CA/NCI NIH HHS/United States ; R01 CA293906/CA/NCI NIH HHS/United States ; },
mesh = {*Nanoparticles/chemistry ; *RNA, Messenger/genetics/chemistry ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Humans ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are among the most advanced non-viral vectors for CRISPR-based gene-editing therapeutics. Co-packaging of messenger RNA (mRNA) and guide RNA (gRNA) inherently produces heterogeneous payload distributions. The impact of this heterogeneity on editing performance remains unclear. Here, we utilize cylindrical illumination confocal spectroscopy (CICS) for single-particle interrogation of ALC-0315 and DLin-MC3-DMA LNPs prepared by three different mixing methods. CICS resolves four distinct subpopulations: co-encapsulated (50.7%-60.4%), gRNA only (30.0%-36.5%), mRNA only (2.0%-3.4%), and empty (4.2%-13.8%), and it uncovers broad, particle-to-particle variability in RNA copy number within each class. Structure-function analysis reveals that LNP formulation and mixing processes influence payload distribution, resulting in a negative correlation between the fraction of empty LNPs and RNA loading per particle. We further investigated the correlation between these quality attributes and therapeutic performance. In mice, ALC-0315 LNPs carrying higher cargo loads (9.8 vs. 8.0 mRNA copies and 25.4 vs. 20.3 gRNA copies per co-encapsulated particle) yielded 1.5-fold higher in vivo editing activity (55.4% vs. 36.3% insertions and deletions [indels]) despite nearly identical biophysical characteristics including LNP size and RNA encapsulation. These results establish payload distribution as a potential determinant of gene-editing potency and demonstrate single-particle CICS as a powerful tool for rational design of multi-component nucleic acid-delivery systems.},
}

*Nanoparticles/chemistry
*RNA, Messenger/genetics/chemistry
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*Lipids/chemistry
*Gene Editing/methods
Mice
Humans
CRISPR-Cas Systems
Liposomes

@article {pmid42015511,
year = {2026},
author = {He, L and Fu, Y and Wang, Z and Zhou, Q and Sun, H and Wang, H},
title = {In vivo systematic detection of the outcomes of CRISPR-Cas9-mediated DNA repair in skeletal muscle stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {7},
pages = {3924-3948},
doi = {10.1016/j.ymthe.2026.04.032},
pmid = {42015511},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscle, Skeletal/cytology/metabolism ; *Stem Cells/metabolism/cytology ; *DNA Repair ; Dependovirus/genetics ; Genetic Vectors/genetics ; DNA End-Joining Repair ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRSIPR-associated protein 9 (Cas9) has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profile CRISPR-Cas9 editing outcomes at 95 loci using our established CRISPR-Cas9/adeno-associated virus (AAV)9-single guide RNA (sgRNA) system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR-Cas9-mediated editing in vivo largely align with those observed in vitro. In addition to the anticipated small editing insertions or deletions (indels), such as microhomology-mediated end joining (MMEJ)-mediated deletions and non-homologous end joining (NHEJ)-mediated templated insertions, we uncover a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by 3D chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo, we identify their distinct roles in regulating the large on-target modifications. Together, our work systematically profiles CRISPR-Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR-Cas9-based gene therapies.},
}

*CRISPR-Cas Systems
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Muscle, Skeletal/cytology/metabolism
*Stem Cells/metabolism/cytology
*DNA Repair
Dependovirus/genetics
Genetic Vectors/genetics
DNA End-Joining Repair
Humans

@article {pmid42031729,
year = {2026},
author = {Moon, HC and Herschl, MH and Sclip, A and Tran, VQ and Pawluk, A and Konermann, S and Hsu, PD},
title = {A combinatorial domain screening platform reveals epigenetic effector interactions for transcriptional perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42031729},
issn = {2041-1723},
mesh = {Humans ; *Epigenesis, Genetic ; DNA Methylation ; Epigenome Editing ; *Transcription, Genetic ; Protein Domains/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetic regulation involves the coordinated interplay of diverse proteins. To systematically explore these combinations, we present COMBINE (combinatorial interaction exploration), a high-throughput platform that tests over 50,000 pairs of epigenetic effector domains up to 2,094 amino acids in length for their ability to modulate endogenous human gene transcription. COMBINE reveals diverse synergistic interactions between epigenetic protein domains, including a potent KRAB-L3MBTL3 fusion that increases the effective targeting window, enhances gene silencing in dose-limited conditions, and enables robust dual-directional CRISPR perturbation. Inducible screening shows DNA methylation modifiers are essential for epigenetic memory, with distinct combinations driving long-term repression and activation. This systematic analysis of pairwise domain interactions advances our understanding of epigenetic crosstalks and the development of next-generation epigenome editing tools. More broadly, COMBINE offers a generalizable platform to functionally characterize combinatorial biological processes at scale.},
}

Humans
*Epigenesis, Genetic
DNA Methylation
Epigenome Editing
*Transcription, Genetic
Protein Domains/genetics
CRISPR-Cas Systems

@article {pmid42049824,
year = {2026},
author = {Galindo-González, L and Dupras, AA},
title = {CRISPR-Cas12a fluorescence assays identify weedy Amaranthus species.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42049824},
issn = {2045-2322},
support = {N-000470//Canadian Food Inspection Agency/ ; },
mesh = {*Amaranthus/genetics/classification ; *CRISPR-Cas Systems ; Fluorescence ; *Plant Weeds/genetics/classification ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {Tall Waterhemp (Amaranthus tuberculatus) and Palmer Amaranth (Amaranthus palmeri) populations have developed multiple-herbicide resistance and impact the production of important crops, including corn, soybean and cotton. Morphological plasticity of these species, their persistence in the soil seed bank and their presence as contaminants during trade require efficient and sensitive methodologies to support their identification. Cas12 enzymes can be directed to a specific genomic region by a crRNA, cutting the DNA double strand and exhibiting collateral enzymatic activity on free-floating single-stranded DNA. This characteristic can be used to generate single-stranded oligonucleotide reporters bearing a fluorophore and a quencher, which will produce fluorescence when cut by Cas12. We designed CRISPR-Cas12 fluorescent assays to differentiate A. tuberculatus, A. palmeri and A. palmeri`s sister species (A. watsonii) from other Amaranthus species. Our assays for identifying the A. palmeri + A. watsonii clade and A. tuberculatus were 100% accurate when presented with blind samples of 14 Amaranthus species. Fluorescence could be detected using a blue light filter on a transilluminator within minutes. A preliminary recombinase amplification step to increase the limit of detection, and the Cas12 reaction, can be performed at room temperature and within an hour once DNA has been isolated.},
}

*Amaranthus/genetics/classification
*CRISPR-Cas Systems
Fluorescence
*Plant Weeds/genetics/classification
*Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases

@article {pmid42055327,
year = {2026},
author = {Mazur, CM and Kotsalidis, PE and George, M and Whalley, T and Sato, T and Doench, JG and Surface, LE and Wein, MN},
title = {Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {6},
pages = {113075},
pmid = {42055327},
issn = {1083-351X},
mesh = {*Osteocytes/cytology/metabolism ; Animals ; Mice ; *Microtubule-Associated Proteins/genetics/metabolism ; Cell Differentiation/genetics ; Cell Line ; Adaptor Proteins, Signal Transducing/genetics ; Extracellular Matrix Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Osteocytes play critical roles in bone, making them attractive targets for therapeutics aimed at improving bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here, we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61[high] cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule-binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify the role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.},
}

*Osteocytes/cytology/metabolism
Animals
Mice
*Microtubule-Associated Proteins/genetics/metabolism
Cell Differentiation/genetics
Cell Line
Adaptor Proteins, Signal Transducing/genetics
Extracellular Matrix Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42134180,
year = {2026},
author = {Lee, H and Jeon, BJ and Jang, HS and Choi, JY and Jang, G},
title = {Non-surgical deep uterine embryo transfer combined with electroporation-based genome editing enables scalable production of CD163-Knockout pigs.},
journal = {Theriogenology},
volume = {263},
number = {},
pages = {117977},
doi = {10.1016/j.theriogenology.2026.117977},
pmid = {42134180},
issn = {1879-3231},
mesh = {Animals ; *Embryo Transfer/veterinary/methods ; *Electroporation/veterinary/methods ; *Gene Editing/veterinary/methods ; Female ; Swine/genetics ; *Antigens, CD/genetics/metabolism ; Animals, Genetically Modified ; *Antigens, Differentiation, Myelomonocytic/genetics/metabolism ; CD163 Antigen ; *Receptors, Cell Surface/genetics ; CRISPR-Cas Systems ; *Gene Knockout Techniques/veterinary ; Pregnancy ; Electroporation Therapies ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) remains the most economically devastating viral disease in the global swine industry, causing annual losses exceeding $600 million in the United States alone. While CD163-knockout (KO) pigs have demonstrated complete resistance to the PRRS virus (PRRSV) infection, conventional production methods relying on microinjection-based genome editing and surgical embryo transfer present significant bottlenecks for industrial-scale application. Here, we demonstrate that the integration of electroporation-based CRISPR-Cas9 delivery with non-surgical deep intrauterine embryo transfer enables scalable production of CD163-KO pigs. Our approach achieved successful pregnancies and live births of genome-edited piglets without requiring surgical intervention or specialized microinjection expertise. Genotyping analysis revealed complete biallelic KOs in a subset of offspring, while others exhibited mosaic patterns reflecting the stochastic nature of electroporation-mediated editing. These findings establish a technically accessible and scalable pipeline for producing gene-edited pigs. By eliminating the need for complex surgical facilities and specialized microinjection equipment, this approach provides a practical and field-applicable strategy that can be readily implemented in standard commercial swine farms, representing a critical step toward the widespread deployment of genome-edited livestock in global agriculture.},
}

Animals
*Embryo Transfer/veterinary/methods
*Electroporation/veterinary/methods
*Gene Editing/veterinary/methods
Female
Swine/genetics
*Antigens, CD/genetics/metabolism
Animals, Genetically Modified
*Antigens, Differentiation, Myelomonocytic/genetics/metabolism
CD163 Antigen
*Receptors, Cell Surface/genetics
CRISPR-Cas Systems
*Gene Knockout Techniques/veterinary
Pregnancy
Electroporation Therapies

@article {pmid42319782,
year = {2026},
author = {Liu, Z and Li, M and Wang, J and Chen, B},
title = {Copper-only Superoxide dismutase 6 contributes to reactive oxygen species regulation, genotoxic stress tolerance, and virulence in Candida albicans.},
journal = {Medical mycology},
volume = {64},
number = {7},
pages = {},
doi = {10.1093/mmy/myag063},
pmid = {42319782},
issn = {1460-2709},
support = {2023YXNS187//Development Plan of Jining/ ; 202411000427//Medical and Health Science and Technology Development Project of Shandong, China/ ; SDYWZGKCJHLH2023079//Shandong medical staff science and technology innovation plan project/ ; },
mesh = {*Candida albicans/pathogenicity/genetics/enzymology ; *Reactive Oxygen Species/metabolism ; Virulence ; Animals ; Oxidative Stress ; *Superoxide Dismutase/genetics/metabolism ; Mice ; Candidiasis/microbiology ; *DNA Damage ; Disease Models, Animal ; Gene Expression Profiling ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; },
abstract = {Superoxide dismutase (SOD) is a major antioxidant enzyme that protects cells against reactive oxygen species (ROS)-mediated oxidative stress. The identification of a fungal-specific copper-only SOD family in Candida albicans (C. albicans) has revealed a previously unrecognized component of fungal oxidative stress defence, yet the role of SOD6 remains unclear. Here, we generated a sod6Δ/Δ mutant in C. albicans using a transient CRISPR/Cas9 approach and examined the contribution of SOD6 to virulence, ROS homeostasis, oxidative stress resistance, and genome maintenance. Compared with the wild-type (WT) strain, the sod6Δ/Δ mutant showed attenuated virulence in both the Galleria mellonella (G. mellonell) infection model and a murine systemic candidiasis model. This virulence defect occurred without detectable alterations in hyphal formation or other major pathogenicity-associated traits. Instead, the mutant accumulated higher intracellular and extracellular ROS levels and displayed increased sensitivity to hydrogen peroxide, zeocin, and camptothecin (CPT), consistent with impaired oxidative stress adaptation and genome maintenance. Transcriptomic analysis further revealed downregulation of genes involved in DNA replication and repair, with dpb4, which encodes a subunit of DNA polymerase epsilon required for DNA replication and genome stability, showing the greatest reduction. Together, these findings identify SOD6 as an important determinant of oxidative stress adaptation, genome maintenance, and pathogenic fitness in C. albicans.},
}

*Candida albicans/pathogenicity/genetics/enzymology
*Reactive Oxygen Species/metabolism
Virulence
Animals
Oxidative Stress
*Superoxide Dismutase/genetics/metabolism
Mice
Candidiasis/microbiology
*DNA Damage
Disease Models, Animal
Gene Expression Profiling
CRISPR-Cas Systems
Gene Expression Regulation, Fungal

@article {pmid42349956,
year = {2026},
author = {Dutta, S and Pal, A and Srivatsan, SG},
title = {Chemoenzymatic labeling of RNA using terminal uridylyl transferase and bioorthogonal click chemistry.},
journal = {Methods in enzymology},
volume = {731},
number = {},
pages = {223-252},
doi = {10.1016/bs.mie.2026.05.007},
pmid = {42349956},
issn = {1557-7988},
mesh = {*Click Chemistry/methods ; *RNA Nucleotidyltransferases/metabolism/chemistry ; Azides/chemistry ; Uridine Triphosphate/chemistry/analogs & derivatives ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics ; *RNA/chemistry ; Staining and Labeling/methods ; *Nucleotidyltransferases/metabolism ; CRISPR-Cas Systems ; Schizosaccharomyces pombe Proteins ; },
abstract = {The ability of native and engineered nucleic acid-processing enzymes to incorporate clickable nucleotide substrates has greatly advanced bioorthogonal labeling of nucleic acids, overcoming the limitations of conventional solid-phase oligonucleotide (ON) synthesis. In this chemoenzymatic approach, template-dependent polymerases routinely enable the incorporation of nucleotides bearing small reactive handles. The resulting nucleic acids undergo chemoselective reactions, such as azide-alkyne cycloaddition, inverse-electron-demand Diels-Alder, or Staudinger ligation, to install desired functionalities. Alternatively, the promiscuity of template-independent transferases, such as terminal uridylyl transferase (TUTase), provides access to site-specific labeling of RNA ONs at the 3'-end. In this methods chapter, we detail protocols for incorporating azide-modified UTP analogs into short RNA ONs and highly structured CRISPR guide RNAs (sgRNAs) using the terminal uridylyl transferase SpCID1. We describe methods to control the enzyme's incessant incorporation behavior and enable subsequent click functionalization of the RNAs. Finally, we demonstrate remodeling of the CRISPR system via synthesis of azide-modified sgRNAs, which when complexed with dCas9, recruit azide groups to specific gene targets for post-hybridization functionalization.},
}

*Click Chemistry/methods
*RNA Nucleotidyltransferases/metabolism/chemistry
Azides/chemistry
Uridine Triphosphate/chemistry/analogs & derivatives
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics
*RNA/chemistry
Staining and Labeling/methods
*Nucleotidyltransferases/metabolism
CRISPR-Cas Systems
Schizosaccharomyces pombe Proteins

@article {pmid42351104,
year = {2026},
author = {Zhu, Y and Bi, Z and Zhang, Z and Zhang, M and Du, Q and Hu, M and Zhou, T and Fan, Y and Zhang, S and Wang, G and Liu, G},
title = {Virus-like particles in cancer immunotherapy: bridging human and veterinary medicine through one health.},
journal = {Journal of nanobiotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12951-026-04703-9},
pmid = {42351104},
issn = {1477-3155},
support = {2024M753586//the China Postdoctoral Science Foundation/ ; 2026JB09//the Central Public-interest Scientific Institution Basal Research Fund/ ; 32473001//the National Natural Science Foundation of China/ ; 2023YFD1800700//the National Key Research and Development Program of China/ ; },
abstract = {Virus-like particles (VLPs) are engineered nanoplatforms that mimic viral structures, offering high immunogenicity, biocompatibility, and functional versatility for cancer immunotherapy. While widely explored in human oncology as nanovaccines and targeted delivery systems for chemo-/immuno-therapeutics and genetic payloads (e.g., mRNA, siRNA, and CRISPR/Cas systems), their potential in veterinary oncology remains underexploited. This review synthesizes recent advances in VLP design, including scaffold engineering, antigen display, cargo encapsulation, and surface functionalization, and discusses the mechanistic basis of VLP-induced antitumor immunity, encompassing dendritic cell activation, adaptive immune amplification, and tumor microenvironment remodeling. Importantly, we highlight the emerging role of companion animals with spontaneous tumors-such as lymphoma, melanoma, and mammary carcinoma-as immunocompetent translational models within the One Health framework. Comparative oncology reveals striking parallels in oncogenic pathways, immune landscapes, and therapeutic responses, supporting the use of canine and feline cancers as biologically relevant intermediates between murine studies and human clinical trials. We provide an evidence-based assessment of representative VLP platforms, evaluate their translational readiness, and examine cross-species opportunities for shared target development, biomarker discovery, and regulatory convergence, while also addressing species-specific biological and technical limitations. Finally, we propose a forward-looking roadmap that prioritizes manufacturing standardization, biomarker development, comparative validation, precision engineering, and emerging technologies such as AI-guided design and tumor-on-chip systems. Collectively, we position One Health as an operational strategy to accelerate the bidirectional translation of VLP-based immunotherapies for both human and veterinary cancer patients.},
}

@article {pmid42352260,
year = {2026},
author = {Kuroda, T and Yokota, T},
title = {RNA Therapeutics Targeting Skeletal Muscle: Emerging Antisense and Gene-Modifying Strategies.},
journal = {Biomolecules},
volume = {16},
number = {6},
pages = {},
pmid = {42352260},
issn = {2218-273X},
mesh = {Humans ; *Oligonucleotides, Antisense/therapeutic use/genetics ; *Muscle, Skeletal/metabolism/drug effects ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Animals ; *Genetic Therapy/methods ; RNA, Small Interfering/therapeutic use/genetics ; Morpholinos/therapeutic use ; },
abstract = {RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides-eteplirsen, golodirsen, viltolarsen, and casimersen-have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies-including base and prime editing-offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships.},
}

Humans
*Oligonucleotides, Antisense/therapeutic use/genetics
*Muscle, Skeletal/metabolism/drug effects
*Muscular Dystrophy, Duchenne/genetics/therapy
Animals
*Genetic Therapy/methods
RNA, Small Interfering/therapeutic use/genetics
Morpholinos/therapeutic use

@article {pmid42352295,
year = {2026},
author = {McGill, LP and Banas, KH and Tiesi, G and Kmiec, EB},
title = {Genotypic and Phenotypic Diversity as a Function of CRISPR-Directed Gene Knock-Out of NRF2 in Pancreatic Adenocarcinoma Cells, a Feasibility Study.},
journal = {Biomolecules},
volume = {16},
number = {6},
pages = {},
pmid = {42352295},
issn = {2218-273X},
mesh = {Humans ; *NF-E2-Related Factor 2/genetics/metabolism ; *Pancreatic Neoplasms/genetics/pathology ; Cell Line, Tumor ; Phenotype ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Drug Resistance, Neoplasm/genetics ; *Adenocarcinoma/genetics/pathology ; Genotype ; Feasibility Studies ; Gemcitabine ; Deoxycytidine/analogs & derivatives/pharmacology ; *Carcinoma, Pancreatic Ductal/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Survival/drug effects ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) presents unique treatment challenges, often due to the development of anti-cancer drug resistance. Previously, we demonstrated that CRISPR-directed gene ablation disabled the master regulator gene NRF2, a transcription factor known to control drug resistance in squamous cell carcinoma tumor cells, and restored chemosensitivity. In this short study, we evaluated a broad range of CRISPR/Cas9 molecules for their capacity to elicit similar responses in PDAC cells. Synthetic single guide RNAs (sgRNAs) were designed to target multiple functional domains encoded by NRF2. These molecules were delivered to cells via nucleofection, with outcomes analyzed by genotypic, phenotypic, and functional assays. We observed targeting efficiencies ranging from 25% to 100% with a high level of random insertions and deletions (indels). sgRNAs targeting exons 2, 3 and 4 demonstrated a high degree of genotypic, phenotypic and functional outcomes. Targeted disruption of exons 3 and 4 reveals significant loss of cell viability while overcoming drug resistance through the restoration of sensitivity to gemcitabine (>1.75 μM). Our study identifies domain-specific sites within NRF2 that, when disabled, restore sensitivity to gemcitabine, potentiating a more in-depth analysis of this novel augmentative therapeutic approach.},
}

Humans
*NF-E2-Related Factor 2/genetics/metabolism
*Pancreatic Neoplasms/genetics/pathology
Cell Line, Tumor
Phenotype
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
Drug Resistance, Neoplasm/genetics
*Adenocarcinoma/genetics/pathology
Genotype
Feasibility Studies
Gemcitabine
Deoxycytidine/analogs & derivatives/pharmacology
*Carcinoma, Pancreatic Ductal/genetics/pathology
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Survival/drug effects

@article {pmid42353201,
year = {2026},
author = {Walflor, HSM and Medeiros, LCS},
title = {Machine Learning for CRISPR-Based Diagnostics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353201},
issn = {1422-0067},
mesh = {Humans ; *Machine Learning ; *CRISPR-Cas Systems ; Deep Learning ; Predictive Learning Models ; Classification Algorithms ; },
abstract = {CRISPR-based diagnostics now detect viral, bacterial, and cancer-associated nucleic acids with sensitivities approaching quantitative PCR; however, their translation to decentralized care rests on computational design and interpretation that current datasets cannot sustain. Pandemic-era Cas12a assays reached 95% positive predictive agreement against reverse transcription quantitative PCR (RT-qPCR) at 10 copies/μL, and deep neural networks now design Cas13 detection assays spanning 1933 vertebrate-infecting viruses, ranking candidate guides at Spearman correlations of 0.69 to 0.84 across internal and external validation. Generative deep-learning systems improve single-nucleotide discrimination two- to three-fold, computer vision classifies lateral flow outputs at 96.5% accuracy, and multi-biomarker fusion reaches an area under the receiver operating characteristic curve (AUC) of 0.998 in lung cancer detection. These results mask a narrow data foundation. Cas13a guide prediction still draws from a single screening library of 19,209 guide-target pairs, Cas12a has one published diagnostic model, and signal classifiers almost uniformly validate on single-site cohorts. This review synthesizes mechanistic constraints, predictive and generative models, and point-of-care classifiers, and maps the path beyond this data ceiling. Evolutionary pretraining on RNA corpora and lab-in-the-loop agents that convert model failure into targeted data acquisition define the route forward.},
}

Humans
*Machine Learning
*CRISPR-Cas Systems
Deep Learning
Predictive Learning Models
Classification Algorithms

@article {pmid42353287,
year = {2026},
author = {Kim, SJ and Nam, YH and Joo, EY and Park, J and Park, S and Jung, SC and Jin, DK},
title = {Systemic AAV-hGCDH Gene Therapy Alleviates Glutaric Acid Accumulation and Attenuates Chronic Brain Vacuolation in a Novel Mouse Model of Glutaric Aciduria Type I.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353287},
issn = {1422-0067},
support = {75440-1//Inha University/ ; },
mesh = {Animals ; *Dependovirus/genetics ; *Genetic Therapy/methods ; *Glutaryl-CoA Dehydrogenase/deficiency/genetics/metabolism ; Mice ; Disease Models, Animal ; *Amino Acid Metabolism, Inborn Errors/therapy/genetics/metabolism/pathology ; *Brain Diseases, Metabolic/therapy/genetics/metabolism/pathology ; *Brain/pathology/metabolism ; *Glutarates/metabolism ; Genetic Vectors/genetics/administration & dosage ; Gene Therapy Agents ; Male ; CRISPR-Cas Systems ; Liver/metabolism ; },
abstract = {Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder caused by glutaryl-CoA dehydrogenase (GCDH) deficiency, leading to the accumulation of neurotoxic metabolites that can cause both acute encephalopathic crises and progressive, insidious brain injury. Current management primarily relies on a protein-restricted diet, which remains therapeutically insufficient and burdensome for patients, highlighting the need for disease-modifying therapies. In this study, we established a novel GA1 mouse model using CRISPR/Cas9 technology and evaluated the preclinical efficacy of systemic recombinant adeno-associated virus (rAAV)-mediated gene therapy. Under standard dietary conditions without high-lysine challenge, our GA1 model exhibited sustained cerebral and hepatic glutaric acid (GA) accumulation and distinct chronic vacuolation in the hippocampus and cerebellum, mirroring the insidious-onset GA1 phenotype. Five-week-old mice received a single intravenous injection of rAAV-hGCDH using either rAAV2/8 or rAAV2/9 serotypes. Systemic rAAV-mediated gene therapy significantly reduced GA accumulation and attenuated chronic neuropathological changes in this GA1 mouse model for both serotypes. Our findings support the hypothesis that peripheral metabolic correction may play an important role in preventing the chronic neuropathological changes associated with GCDH deficiency. However, further investigation using tissue-specific expression systems is required to definitively delineate the relative contributions of hepatic versus central GCDH restoration to the observed neuroprotection.},
}

Animals
*Dependovirus/genetics
*Genetic Therapy/methods
*Glutaryl-CoA Dehydrogenase/deficiency/genetics/metabolism
Mice
Disease Models, Animal
*Amino Acid Metabolism, Inborn Errors/therapy/genetics/metabolism/pathology
*Brain Diseases, Metabolic/therapy/genetics/metabolism/pathology
*Brain/pathology/metabolism
*Glutarates/metabolism
Genetic Vectors/genetics/administration & dosage
Gene Therapy Agents
Male
CRISPR-Cas Systems
Liver/metabolism

@article {pmid42353300,
year = {2026},
author = {Afifi, N and Colussi, D and Perez-Leal, O},
title = {CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353300},
issn = {1422-0067},
support = {1R21HG012241-03/NH/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Gene Editing/methods ; *Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology.},
}

Humans
*CRISPR-Cas Systems
Animals
*Gene Editing/methods
*Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42353328,
year = {2026},
author = {Jan, R and Iqbal, S and Ali, S and Almalki, MA and Alfredan, M and Ibrahim, RIH and Asaf, S and Kim, KM},
title = {Early Flowering (ELF) Gene Integrates Vegetative Growth, Flowering Regulation, and Reproductive Development in Arabidopsis thaliana.},
journal = {International journal of molecular sciences},
volume = {27},
number = {12},
pages = {},
pmid = {42353328},
issn = {1422-0067},
mesh = {*Arabidopsis/genetics/growth & development ; *Flowers/genetics/growth & development ; *Arabidopsis Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Reproduction/genetics ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Germination/genetics ; Gene Editing ; },
abstract = {Early flowering-related factors play pivotal roles in coordinating plant growth and reproductive development. In this study, we investigated the biological function of early flowering gene (ELF) in Arabidopsis thaliana using CRISPR/Cas9-mediated genome editing and construction of overexpression approaches. Two independent ELF overexpression (OE-ELF) and genome-edited (ge-elf) lines were generated and systemically analyzed. ELF overexpression significantly enhanced early seedling performance, increasing germination rate and seedling fresh weight by up to 8.7%, while genome-edited lines exhibited a marked reduction. Root growth was strongly promoted in OE-ELF plants, with root length increase of 85% and 75%, whereas ge-elf lines showed a reduction of up to 48%. At later developmental stages, OE-ELF plants displayed enhanced vegetative growth, including increased leaf length (32%), leaf area (91%), and accelerated flowering (21% earlier than wild type). In contrast, ge-elf delayed flowering by up to 25% and resulted in compact plant architecture. Reproductive development was severely compromised in ge-elf plants, which exhibited malformed inflorescences, reduced pollen germination, shortened silique (45%), and a drastic decrease in seed number per silique (70%). Conversely, OE-ELF plants showed increased silique number and seed per silique. Molecular analysis revealed that ELF positively regulates key flowering-related genes, including FLC, SOC1, AP1, and LFY, which correlated strongly with growth and reproductive traits. Our results demonstrate that ELF functions as a central regulator integrating vegetative growth, floral development, male fertility, and seed production in Arabidopsis thaliana.},
}

*Arabidopsis/genetics/growth & development
*Flowers/genetics/growth & development
*Arabidopsis Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified
Reproduction/genetics
*Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Germination/genetics
Gene Editing

@article {pmid42087135,
year = {2026},
author = {Ouyang, M and Wang, J and Luo, X and Tian, R},
title = {CaRPOOL: a pooled calcium‑recording CRISPR screening platform identifies CCR7 as a modulator of cellular osmomechanosensing.},
journal = {Cell communication and signaling : CCS},
volume = {24},
number = {1},
pages = {},
pmid = {42087135},
issn = {1478-811X},
support = {2024YFA0919800//National Key Research and Development Program of China/ ; A2303039//Shenzhen Medical Research Fund/ ; 2023B151502007//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *Receptors, CCR7/metabolism/genetics ; *Calcium/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Calcium Signaling ; *CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Cells must continuously sense and respond to environmental changes by translating physical and chemical cues into intracellular signals. However, systematic discovery of genes governing these sensory processes has been limited by the transient nature of signaling events and the low throughput of measurement assays. Here, we present CaRPOOL, a pooled, high‑throughput genetic screening platform that integrates the calcium‑activity recorder CaMPARI2 with CRISPR interference (CRISPRi), enabling stable capture of transient calcium signals for genome‑scale functional screening. Using osmomechanical stimulation as a model, we demonstrate that CaMPARI2 photoconversion faithfully reports stimulus‑dependent calcium responses and supports pooled fluorescence‑activated cell sorting (FACS)-based screening. A CRISPRi library targeting membrane‑associated genes identified both known and previously uncharacterized regulators of osmomechanosensing, including the chemokine receptor CCR7. Mechanistic analyses revealed that CCR7 promotes osmomechanical calcium signaling through a PIEZO1‑dependent Gαs-cAMP-PKA pathway, establishing it as a GPCR regulator of osmomechanical response. Notably, osmotic stress upregulated CCR7 expression in immune cell lines and enhanced osmomechanical responsiveness, suggesting a role in osmomechanical adaptation. Together, these findings introduce a broadly applicable platform for high‑throughput discovery of genes controlling dynamic signaling responses and reveal a GPCR-ion channel crosstalk mechanism in osmomechanotransduction with potential implications for immune cell mechanoadaptation.},
}

Humans
*Receptors, CCR7/metabolism/genetics
*Calcium/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Calcium Signaling
*CRISPR-Cas Systems
HEK293 Cells

@article {pmid42258637,
year = {2026},
author = {Wei, G and Huang, Z and Wang, S and Zheng, F and Yao, J and Jin, Q and Zhang, Y and Luo, H and Zhang, X and He, Y and Zhou, M and Gu, M and Mao, C and Wang, Z},
title = {Improving low-phosphate tolerance via tissue-specific CRISPR/Cas9 knockout to balance growth and stress responses in rice.},
journal = {The Plant cell},
volume = {38},
number = {6},
pages = {},
doi = {10.1093/plcell/koag170},
pmid = {42258637},
issn = {1532-298X},
support = {2021YFF1000402//National Key Research and Development Program of China/ ; LR24C150001//Natural Science Foundation of Zhejiang Province, China/ ; 92581113//National Natural Science Foundation of China/ ; 32170262//National Natural Science Foundation of China/ ; 226-2024-00102//Fundamental Research Funds for the Central University/ ; },
mesh = {*Oryza/genetics/physiology/growth & development/metabolism ; *Phosphates/metabolism/deficiency ; *CRISPR-Cas Systems/genetics ; Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Gene Knockout Techniques ; Plant Proteins/genetics/metabolism ; Plant Roots/genetics/metabolism ; },
abstract = {Balancing growth and stress responses is critical for improving crop stress tolerance. Inorganic phosphate (Pi) deficiency reduces agricultural yields. Plants have evolved a Pi-starvation response (PSR) network that coordinates growth and responds to fluctuating environmental Pi levels. Null mutations or whole-plant knockdown of PSR repressor genes, such as PHOSPHATE2 (OsPHO2) and OsSPX (Syg1, Pho81, XPR1) family genes, enhance Pi absorption and transfer but disrupt Pi homeostasis, inhibiting growth and reducing yields. To overcome this, we developed a CRISPR/Cas9 tissue-specific knockout (TSKO) system for efficient, vascular-specific somatic knockout of OsPHO2 in rice (Oryza sativa) cv. "Nipponbare" across several generations. The plants showed moderately increased Pi concentrations, maintained Pi homeostasis in hydroponic culture, and increased effective tiller number and grain yield in a Pi-deficient paddy. Vascular-specific OsPHO2 knockout moderately increased OsPHO2-repressed, vascular-expressed, Pi-starvation-induced signaling in roots and alleviated disordered PSR in roots and leaves. Vascular-specific knockout of OsPHO2 or OsSPX1/2 in the Zhonghua 11 background gave similar results. Field trials confirmed the enhanced low-Pi tolerance of TSKO plants in a Pi-deficient paddy and these plants showed normal growth in a Pi-sufficient paddy. This highlights the utility of improving rice low-Pi tolerance via a tissue-specific CRISPR/Cas9 knockout, provides insights into the role of vascular tissues in PSR, and offers a promising spatial-targeting strategy for crop improvement.},
}

*Oryza/genetics/physiology/growth & development/metabolism
*Phosphates/metabolism/deficiency
*CRISPR-Cas Systems/genetics
Stress, Physiological/genetics
Gene Expression Regulation, Plant
Plants, Genetically Modified
Gene Knockout Techniques
Plant Proteins/genetics/metabolism
Plant Roots/genetics/metabolism

@article {pmid42347182,
year = {2026},
author = {Feng, C and Yin, J},
title = {Advances in CRISPR-Cas for Diagnosis and Treatment of Klebsiella pneumoniae.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {42347182},
issn = {2076-0817},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Klebsiella Infections/diagnosis/therapy/drug therapy/microbiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Gene Editing/methods ; Drug Resistance, Bacterial ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant pathogen for both hospital-acquired and community-acquired infections, characterized by its strong epidemic potential and high mortality rate, posing a severe threat to global public health. CRKP spreads widely across the globe through the horizontal transfer of plasmid-mediated resistance genes such as *blaKPC*, *blaNDM*, and *blaOXA-48*. The clinical treatment options for this bacterium are limited, and its resistance has been increasing year by year, urgently necessitating the development of new antimicrobial drugs or alternative strategies. In recent years, the CRISPR-Cas system has shown great potential in the diagnosis and treatment of CRKP, including rapid detection and identification, gene editing, antimicrobial strategies, and resistance inhibition. For instance, CRISPR-Cas12a/13a can be used for the rapid detection and identification of CRKP, while CRISPR-Cas9/Cas3 can target resistance genes to reverse the resistance of strains. With the advancement of delivery and biotechnologies, the CRISPR-Cas system is expected to become an important tool against drug-resistant CRKP. This review focuses on the application of the CRISPR-Cas system in the detection and treatment of CRKP, analyzing its technical advantages, limitations, and future development directions.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification
Humans
*CRISPR-Cas Systems
*Klebsiella Infections/diagnosis/therapy/drug therapy/microbiology
Anti-Bacterial Agents/pharmacology/therapeutic use
Gene Editing/methods
Drug Resistance, Bacterial