@article {pmid41598889,
year = {2025},
author = {Wang, Y and Zhang, C and Li, MJ and Iqbal, A and Ahmed, KS and Idrees, A and Habiba, and Yang, BM and Jiang, L},
title = {Exploring the Role of Pheromones and CRISPR/Cas9 in the Behavioral and Olfactory Mechanisms of Spodoptera frugiperda.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
doi = {10.3390/insects17010035},
pmid = {41598889},
issn = {2075-4450},
support = {2024C014-2//Innovation Capacity Building Project of the Jilin Provincial Development and Reform Commission./ ; },
abstract = {Globally, Spodoptera frugiperda is a major threat to many important crops, including maize, rice, and cotton, causing significant economic damage. To control this invasive pest, environmentally friendly pest control techniques, including pheromone detection and identification of potential molecular targets to disrupt S. frugiperda mating communication, are needed. Female moths biosynthesize pheromones and emit them from the pheromone gland, which significantly depends on the intrinsic factors of the moth. Male S. frugiperda have a sophisticated olfactory circuit on their antennae that recognizes pheromone blends via olfactory receptor neurons (ORNs). With its potential to significantly modify the insect genome, CRISPR/Cas9 offers a revolutionary strategy to control this insect pest. The impairing physiological behaviors and disrupting the S. frugiperda volatile-sensing mechanism are the main potential applications of CRISPR/Ca9 explored in this review. Furthermore, the release of mutant S. frugiperda for their long-term persistence must be integral to the adoption of this technology. Looking forward, CRISPR/Cas9-based gene drive systems have the potential to synergistically target pheromone signaling pathways in S. frugiperda by disrupting pheromone receptors and key biosynthesis genes, thereby effectively blocking intraspecific communication and reproductive success. In conclusion, CRISPR/Cas9 provides an environmentally friendly and revolutionary platform for precise, targeted pest management in S. frugiperda.},
}

@article {pmid41598172,
year = {2025},
author = {Magyar-Tábori, K and Udupa, SM and Hanász, A and Juhász, C and Mendler-Drienyovszki, N},
title = {Rising Demand for Winter Crops Under Climate Change: Breeding for Winter Hardiness in Autumn-Sown Legumes.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/life16010017},
pmid = {41598172},
issn = {2075-1729},
abstract = {Climate change in the Pannonian region is accelerating a shift toward autumn sowing of cool-season grain legumes (pea, faba bean, lentil, chickpea, lupine) to achieve higher yields, greater biomass production, enhanced nitrogen fixation, improved soil cover, and superior resource use efficiency compared with spring sowing. However, successful overwintering depends on the availability of robust winter-hardy cultivars. This review synthesizes recent breeding advances, integrating traditional approaches-such as germplasm screening, hybridization, and field-based selection-with genomics-assisted strategies, including genome-wide association studies (GWAS), quantitative trait locus (QTL) mapping, marker-assisted selection (MAS), and CRISPR/Cas-mediated editing of CBF transcription factors. Key physiological mechanisms-LT50 determination, cold acclimation, osmoprotectant accumulation (sugars, proline), and membrane stability-are assessed using field survival rates, electrolyte leakage assays, and chlorophyll fluorescence measurements. Despite challenges posed by genotype × environment interactions, variable winter severity, and polygenic trait control, the release of cultivars worldwide (e.g., 'NS-Mraz', 'Lavinia F', 'Ghab series', 'Pinklevi', and 'Rézi') and ongoing breeding programs demonstrate substantial progress. Future breeding efforts will increasingly rely on genomic selection (GS), high-throughput phenomics, pangenomics, and G×E modeling to accelerate the development of climate-resilient legume cultivars, ensuring stable and sustainable production under increasingly unpredictable winter conditions.},
}

@article {pmid41597233,
year = {2026},
author = {Bao, C and Channell, CI and Tseng, YH and Bailey, J and Sbaiti, N and Demirkol, A and Tsang, SH},
title = {Chronic In Vivo CRISPR-Cas Genome Editing: Challenges, Long-Term Safety, and Outlook.},
journal = {Cells},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/cells15020156},
pmid = {41597233},
issn = {2073-4409},
support = {the Foundation Fighting Blindness TA-GT-0321-0802-COLU-TRAP, the Lynette and Richard Jaffe Foundation, NYEE Foundation, the Rosenbaum Family Foundation, the Gebroe Family Foundation, the Research to Prevent Blindness (RPB) Physician-Scientist Award, unres//Jonas Children's Vision Care is supported by the National Institute of Health U01EY030580, U01EY034590 R24EY028758, R24EY027285, R01EY033770, R01EY018213, R01EY024698,/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR/Cas systems have transformed molecular medicine, yet the field still lacks principled guidance on when transient editing suffices versus when sustained exposure through in vivo viral delivery is necessary and how to keep prolonged exposure safe. Notably, EDIT-101 was designed for a permanent edit in post-mitotic photoreceptors with lifelong Cas9 persistence. This review addresses this gap by defining the biological and therapeutic conditions that drive benefit from extended Cas activity while minimizing risk. We will (i) examine relationships between expression window and efficacy across Cas9/Cas12/Cas13 modalities, (ii) identify genome-wide off-target liabilities alongside orthogonal assays, and (iii) discuss controllable, self-limiting, and recallable editor platforms. By separating durable edits from persistent nuclease exposure, and by providing validated control levers, this work establishes a generalizable framework for safe, higher-efficacy CRISPR medicines. Furthermore, we highlight key studies in cell lines, murine models, non-human primates, and humans that examine the long-term effects of sustained expression of CRISPR/Cas systems and discuss the safety and efficacy of such approaches. Current evidence demonstrates promising therapeutic outcomes with manageable safety profiles, although there is a need for continued monitoring as CRISPR/Cas therapies are increasingly applied in clinical contexts and therapies are developed for broader clinical applications.},
}

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

@article {pmid41597209,
year = {2026},
author = {Ren, Z and Zhou, J and Yang, D and Guo, Y and Zhang, J and Xu, J and Chen, YE},
title = {Gene Editing Therapies Targeting Lipid Metabolism for Cardiovascular Disease: Tools, Delivery Strategies, and Clinical Progress.},
journal = {Cells},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/cells15020134},
pmid = {41597209},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics/metabolism ; *Lipid Metabolism/genetics ; *Genetic Therapy/methods ; Animals ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; },
abstract = {Gene editing technologies have revolutionized therapeutic development, offering potentially curative and preventative strategies for cardiovascular disease (CVD), which remains a leading global cause of morbidity and mortality. This review provides an introduction to the state-of-the-art gene editing tools-including ZFNs, TALENs, CRISPR/Cas9 systems, base editors, and prime editors-and evaluates their application in lipid metabolic pathways central to CVD pathogenesis. Emphasis is placed on targets such as PCSK9, ANGPTL3, CETP, APOC3, ASGR1, LPA, and IDOL, supported by findings from human genetics, preclinical models, and recent first-in-human trials. Emerging delivery vehicles (AAVs, LNPs, lentivirus, virus-like particles) and their translational implications are discussed. The review highlights ongoing clinical trials employing liver-targeted in vivo editing modalities (LivGETx-CVD) and provides insights into challenges in delivery, off-target effects, genotoxicity, and immunogenicity. Collectively, this review captures the rapid progress of LivGETx-CVD from conceptual innovation to clinical application, and positions gene editing as a transformative, single-dose strategy with the potential to redefine prevention and long-term management of dyslipidemia and atherosclerotic cardiovascular disease.},
}

Humans
*Gene Editing/methods
*Cardiovascular Diseases/therapy/genetics/metabolism
*Lipid Metabolism/genetics
*Genetic Therapy/methods
Animals
*Gene Transfer Techniques
CRISPR-Cas Systems

@article {pmid41596857,
year = {2026},
author = {Tsouggou, N and Korozi, E and Pemaj, V and Drosinos, EH and Kapolos, J and Papadelli, M and Skandamis, PN and Papadimitriou, K},
title = {Advances in Shotgun Metagenomics for Cheese Microbiology: From Microbial Dynamics to Functional Insights.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/foods15020259},
pmid = {41596857},
issn = {2304-8158},
abstract = {The cheese microbiome is a complex ecosystem strongly influenced by both technological practices and the processing environment. Moving beyond traditional cultured-based methods, the integration of shotgun metagenomics into cheese microbiology has enabled in-depth resolution of microbial communities at the species and strain levels. The aim of the present study was to review recent applications of shotgun metagenomics in cheese research, underscoring its role in tracking microbial dynamics during production and in discovering genes of technological importance. In addition, the review highlights how shotgun metagenomics enables the identification of key metabolic pathways, including amino acid catabolism, lipid metabolism, and citrate degradation, among others, which are central to flavor formation and ripening. Results of the discussed literature demonstrate how microbial composition, functional traits, and overall quality of cheese are determined by factors such as raw materials, the cheesemaking environment, and artisanal practices. Moreover, it highlights the analytical potentials of shotgun metagenomics, including metagenome-assembled genomes (MAGs) reconstruction, characterization of various genes contributing to flavor-related biosynthetic pathways, bacteriocin production, antimicrobial resistance, and virulence, as well as the identification of phages and CRISPR-Cas systems. These insights obtained are crucial for ensuring product's authenticity, enabling traceability, and improving the assessment of safety and quality. Despite shotgun metagenomics' advantages, there are still analytical restrictions concerning data handling and interpretation, which need to be addressed by importing standardization steps and moving towards integrating multi-omics approaches. Such strategies will lead to more accurate and reproducible results across studies and improved resolution of active ecosystems. Ultimately, shotgun metagenomics has shifted the field from descriptive surveys to a more detailed understanding of the underlying mechanisms shaping the overall quality and safety of cheese, thus bringing innovation in modern dairy microbiology.},
}

@article {pmid41596715,
year = {2026},
author = {Ansari, RA and Rezaee Danesh, Y and Castello, I and Vitale, A},
title = {Molecular Identification and RNA-Based Management of Fungal Plant Pathogens: From PCR to CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27021073},
pmid = {41596715},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Plant Diseases/microbiology/prevention & control/genetics ; *Fungi/genetics/pathogenicity ; Polymerase Chain Reaction/methods ; *RNA, Fungal/genetics ; Plants/microbiology ; },
abstract = {Fungal diseases continue to limit global crop production and drive major economic losses. Conventional diagnostic and control approaches depend on time-consuming culture-based methods and broad-spectrum chemicals, which offer limited precision. Advances in molecular identification have changed this landscape. PCR, qPCR, LAMP, sequencing and portable platforms enable rapid and species-level detection directly from plant tissue. These tools feed into RNA-based control strategies, where knowledge of pathogen genomes and sRNA exchange enables targeted suppression of essential fungal genes. Host-induced and spray-induced gene silencing provide selective control without the long-term environmental costs associated with chemical use. CRISPR/Cas9 based tools now refine both diagnostics and resistance development, and bioinformatics improves target gene selection. Rising integration of artificial intelligence indicates a future in which disease detection, prediction and management connect in near real time. The major challenge lies in limited field validation and the narrow range of fungal species with complete molecular datasets, yet coordinated multi-site trials and expansion of annotated genomic resources can enable wider implementation. The combined use of molecular diagnostics and RNA-based strategies marks a shift from disease reaction to disease prevention and moves crop protection towards a precise, sustainable and responsive management system. This review synthesizes the information related to current molecular identification tools and RNA-based management strategies, and evaluates how their integration supports precise and sustainable approaches for fungal disease control under diverse environmental settings.},
}

*CRISPR-Cas Systems
*Plant Diseases/microbiology/prevention & control/genetics
*Fungi/genetics/pathogenicity
Polymerase Chain Reaction/methods
*RNA, Fungal/genetics
Plants/microbiology

@article {pmid41596696,
year = {2026},
author = {Boren, C and Kumar, R and Gollahon, L},
title = {In Silico Design and Characterization of a Rationally Engineered Cas12j2 Gene Editing System for the Treatment of HPV-Associated Cancers.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27021054},
pmid = {41596696},
issn = {1422-0067},
support = {NA//Texas Tech University Association of Biologists Grants-in-Aid/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/therapy/virology/genetics ; *Neoplasms/therapy/virology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oncogene Proteins, Viral/genetics ; Computer Simulation ; },
abstract = {CRISPR-Cas9 systems have enabled unprecedented advances in genome engineering, particularly in developing treatments for human diseases, like cancer. Despite potential applications, limitations of Cas9 include its relatively large size and strict targeting requirements. Cas12j2, a variant ofCasΦ-2, shows promise for overcoming these limitations. However, its effectiveness in mammalian cells remains relatively unexplored. This study sought to develop an optimized CRISPR-Cas12j2 system for targeted knockout of the E6 oncogene in HPV-associated cancers. A combination of computational tools (ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and Amber for Molecular Dynamics) was utilized to investigate the impact of engineered modifications on structural integrity and gRNA binding of Cas12j2 fusion constructs, in potential intracellular conditions. Cas12j2_F2, a Cas12j2 variant designed and evaluated in this study, behaves similarly to the wild-type Cas12j2 structure in terms of RMSD/RMSF profiles, compact Rg values, and minimal electrostatic perturbation. The computationally validated Cas12j2 variant was incorporated into a custom expression vector, co-expressing the engineered construct along with a dual gRNA for packaging into a viral vector for targeted knockout of HPV-associated cancers. This study provides a structural and computational foundation for the rational design of Cas12j2 fusion constructs with enhanced stability and functionality, supporting their potential application for precise genome editing in mammalian cells.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
*Papillomavirus Infections/therapy/virology/genetics
*Neoplasms/therapy/virology/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Oncogene Proteins, Viral/genetics
Computer Simulation

@article {pmid41596664,
year = {2026},
author = {Luo, X and Ding, Y and Wang, Z and Liu, J},
title = {MED12 Dictates Epithelial Ovarian Cancer Cell Ferroptosis Sensitivity via YAP-TEAD1 Signaling.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27021020},
pmid = {41596664},
issn = {1422-0067},
support = {82102732//National Natural Science Foundation of China/ ; 82272698//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Ferroptosis/drug effects/genetics ; Female ; *Carcinoma, Ovarian Epithelial/metabolism/genetics/pathology ; *Transcription Factors/metabolism/genetics ; *Signal Transduction ; Cell Line, Tumor ; YAP-Signaling Proteins ; TEA Domain Transcription Factors ; *Ovarian Neoplasms/metabolism/genetics/pathology ; *DNA-Binding Proteins/metabolism/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; *Nuclear Proteins/metabolism/genetics ; Drug Resistance, Neoplasm ; Cysteine-Rich Protein 61/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Epithelial ovarian cancer (EOC) represents the most lethal malignancy arising from the female reproductive tract, largely due to the clinical challenge of chemotherapy resistance. Recent studies indicate that ferroptosis-a distinct form of programmed cell death driven by iron accumulation and lipid peroxidation, could potentially exploit a vulnerability in chemoresistant cancer cells. Here, we identify MED12 as a critical regulator of ferroptosis sensitivity in EOC through modulation of the YAP-TEAD1 signaling pathway. Using CRISPR/Cas9-mediated knockout and rescue experiments in EOC cell lines, we demonstrate that MED12 deficiency significantly enhances sensitivity to ferroptosis inducers (RSL3 and Erastin), as evidenced by reduced IC50 values. Transcriptomic and chromatin accessibility analyses reveal that MED12 loss activates YAP signaling through TEAD1 upregulation, increasing chromatin accessibility at YAP-TEAD1 target loci and elevating the expression of downstream effectors CYR61 and CTGF. Pharmacological inhibition of YAP with verteporfin or siRNA-mediated TEAD1 knockdown reverses ferroptosis sensitivity in MED12-deficient cells, confirming pathway specificity. These findings establish MED12 as a modulator of the YAP-TEAD1-ferroptosis axis and suggest that targeting this pathway could overcome chemoresistance in MED12-deficient EOC. Our work provides a mechanistic foundation for exploiting ferroptosis induction as a therapeutic strategy in ovarian cancer.},
}

Humans
*Ferroptosis/drug effects/genetics
Female
*Carcinoma, Ovarian Epithelial/metabolism/genetics/pathology
*Transcription Factors/metabolism/genetics
*Signal Transduction
Cell Line, Tumor
YAP-Signaling Proteins
TEA Domain Transcription Factors
*Ovarian Neoplasms/metabolism/genetics/pathology
*DNA-Binding Proteins/metabolism/genetics
*Adaptor Proteins, Signal Transducing/metabolism/genetics
Gene Expression Regulation, Neoplastic
*Nuclear Proteins/metabolism/genetics
Drug Resistance, Neoplasm
Cysteine-Rich Protein 61/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41596567,
year = {2026},
author = {Kosmas, CE and Rallidis, LS and Hoursalas, I and Papakonstantinou, EJ and Kostara, CE},
title = {Angiopoietin-like Protein 3 (ANGPTL3) Targeting in the Management of Dyslipidemias.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020921},
pmid = {41596567},
issn = {1422-0067},
mesh = {Humans ; *Angiopoietin-Like Protein 3/antagonists & inhibitors/metabolism/genetics ; *Angiopoietin-like Proteins/antagonists & inhibitors/metabolism/genetics ; *Dyslipidemias/drug therapy/metabolism/genetics ; Animals ; Antibodies, Monoclonal/therapeutic use/pharmacology ; Oligonucleotides, Antisense/therapeutic use ; RNA, Small Interfering/therapeutic use ; },
abstract = {Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, despite advances in pharmacological prevention and treatment. The burden of CVD necessitates implementing the treatment of risk factors including dyslipidemia. Pharmaceutical advancements and in depth understanding of pathophysiology have enabled innovative therapies targeting pathways underlying lipoprotein metabolism disorders. Angiopoietin protein-like 3 (ANGPTL3) plays a crucial role in the regulation of lipoprotein metabolism, therefore being a potential therapeutic target. Inhibition of ANGPTL3 has emerged as a new therapeutic strategy to reduce LDL-cholesterol levels independent of the LDL receptor function. Therapeutic approaches for ANGPTL3 inhibition range from monoclonal antibodies to nucleic acid therapeutics including antisense oligonucleotides and small interfering RNAs. In this review, we briefly explain the structure and mechanism of action of ANGPTL3 and discuss the therapeutic approaches for targeting ANGPTL3 in the clinical setting. We also discuss Evinacumab, a monoclonal antibody, its structure, mechanism of action, safety, tolerability, pharmacokinetics, and pharmacodynamics, as well as its clinical trial-derived results. The antisense oligonucleotides modify ANGPTL3 mRNA to inhibit protein production, and small interfering RNAs induce mRNA degradation; results from clinical trials were reviewed in detail. Finally, we discuss promising gene editing approaches including clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems.},
}

Humans
*Angiopoietin-Like Protein 3/antagonists & inhibitors/metabolism/genetics
*Angiopoietin-like Proteins/antagonists & inhibitors/metabolism/genetics
*Dyslipidemias/drug therapy/metabolism/genetics
Animals
Antibodies, Monoclonal/therapeutic use/pharmacology
Oligonucleotides, Antisense/therapeutic use
RNA, Small Interfering/therapeutic use

@article {pmid41596458,
year = {2026},
author = {Ionas, K and Vukosavljev, M and Bulić, E and Radanović, A and Jocić, S and Kondić-Špika, A and Miladinović, D},
title = {Beyond the Bottleneck: Predicting Regeneration Potential in Sunflower Through Integrated Morphological and Statistical Profiling.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020809},
pmid = {41596458},
issn = {1422-0067},
support = {101081974//European Commission/ ; },
mesh = {*Helianthus/genetics/physiology/growth & development ; *Regeneration/genetics ; Genotype ; Plant Shoots/growth & development/genetics ; Plant Roots/growth & development/genetics ; Plant Breeding ; Gene Expression Regulation, Plant ; Sucrose/pharmacology ; Cluster Analysis ; },
abstract = {This study presents the first integrated analysis of genotype-medium interactions and temporal morphogenesis profiling in sunflower regeneration. It aims to characterize genotype-specific responses, identify predictive morphological markers, and develop a scalable framework for breeding and transformation. Eighteen sunflower genotypes were evaluated to assess organogenic performance. The model genotype Ha-26-PR was used for a complementary experiment, testing varying sucrose concentrations to examine their influence on morphogenic outcomes. Hierarchical Cluster Analysis (HCA), guided by the Elbow method, identified four optimal clusters (K = 4). These aligned with three biologically meaningful categories: High Regenerators (Cluster 1), Moderate/Specific Regenerators (Clusters 2 and 3), and Non-Regenerators (Cluster 4). On S1 medium, NO-SU-12 and AS-1-PR showed superior shoot regeneration, while on R4 medium, HA-26-PR-SU and NO-SU-12 performed best. Genotypes such as NO-SU-12 and AS-1-PR consistently excelled across both media, whereas AB-OR-8 and FE-7 remained non-regenerators. Medium R4 supported superior regeneration, primarily through root formation, while S1 failed to induce roots in any genotype, highlighting the importance of hormonal composition. Although sucrose promoted callus induction, it did not trigger organogenesis. Callus was consistently present across media and time points, but its correlations with shoot and root formation were weak and temporally unstable, limiting its predictive value. Root formation at 14 days (Root 14D) emerged as a robust early predictor of organogenic success. This integration of morphological, temporal, and statistical analyses offers a genotype-tailored regeneration framework with direct applications in molecular breeding and CRISPR/Cas-based genome editing.},
}

*Helianthus/genetics/physiology/growth & development
*Regeneration/genetics
Genotype
Plant Shoots/growth & development/genetics
Plant Roots/growth & development/genetics
Plant Breeding
Gene Expression Regulation, Plant
Sucrose/pharmacology
Cluster Analysis

@article {pmid41596437,
year = {2026},
author = {Luo, H and Zou, H and Lin, S and Liu, J and Zhou, G and Gao, L and Huang, J and Li, J and Gao, J and Ma, C},
title = {Multiplex Editing of OsMads26, OsBsr-d1, OsELF3-2 and OsERF922 with CRISPR/Cas9 Confers Enhanced Resistance to Pathogens and Abiotic Stresses and Boosts Grain Yield in Rice (Oryza sativa).},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020781},
pmid = {41596437},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Proteins/genetics ; *Stress, Physiological/genetics ; Plant Diseases/microbiology/genetics ; Plants, Genetically Modified/genetics ; *Edible Grain/genetics/growth & development ; Gene Expression Regulation, Plant ; Droughts ; },
abstract = {Rice (Oryza sativa) is one of the world's major staple foods. However, stable rice production is constrained by various biotic and abiotic and stresses. Breeding and cultivation of rice varieties with resistance to multiple pathogens and environmental stresses is the most effective strategy to mitigate the adverse effect of pathogen attacks and abiotic stresses. Recently, researchers have focused on the exploitation of CRISPR/Cas9 technology to manipulate some negative defense-regulator genes to generate rice varieties with broad-spectrum resistance against rice pathogens. In this study, four negative regulator genes of rice blast, OsMads26, OsBsr-1, OsELF3-2 and OsERF922, were selected as CRISPR/Cas9 targets. By simultaneously knocking out all four genes via CRISPR/Cas9 technology, we created three mads26/bsr-1/elf3-2/erf922 quadruple knockout mutants. Our results demonstrated that all quadruple mutants exhibited much higher resistance not only to rice blast and bacterial blight but also to drought and salt stresses than the wildtype. Interestingly, grain yield of all three quadruple mutants was also drastically increased by 17.35% to 21.95%. Therefore, this study provides a novel strategy to rapidly improve rice varieties with broad-spectrum resistance to pathogens, elevated tolerance to abiotic stresses and enhanced yield potential.},
}

*Oryza/genetics/microbiology/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
*Disease Resistance/genetics
*Plant Proteins/genetics
*Stress, Physiological/genetics
Plant Diseases/microbiology/genetics
Plants, Genetically Modified/genetics
*Edible Grain/genetics/growth & development
Gene Expression Regulation, Plant
Droughts

@article {pmid41596278,
year = {2026},
author = {Bernacka, KU and Michalski, K and Wojciechowski, M and Sowa, S},
title = {Application of SNV Detection Methods for Market Control of Food Products from New Genomic Techniques.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020626},
pmid = {41596278},
issn = {1422-0067},
support = {101137025//Horizon Europe- European Union/ ; DHR.bz.070.2.2024//Polish Ministry of Agriculture and Rural Development/ ; },
mesh = {*Plants, Genetically Modified/genetics ; *Genomics/methods ; *Polymorphism, Single Nucleotide ; *Food, Genetically Modified ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The detection of single-nucleotide variants (SNVs) is an important challenge in modern genomics, with broad applications in medicine, diagnostics, and agricultural biotechnology. Current detection approaches include PCR-based techniques with high-affinity probes, ligase-based strategies, and sequencing approaches, each with varying degrees of sensitivity, specificity, and practicality. Despite advances in SNV analysis in the medical field, their implementation in the official control and monitoring of genetically modified organisms (GMOs) remains limited. This challenge has gained priority with the advent of new genomic techniques (NGTs), such as CRISPR-Cas nucleases, which allow precise genome editing, including subtle changes at the nucleotide level without introducing foreign DNA. Therefore, traditional methods of GMO detection targeting transgene sequences may not be sufficient to monitor such GMOs. In the European Union, GMO legislation requires distinguishing between conventionally bred and genetically modified plants. The planned introduction of new regulatory categories of NGT plants (NGT1 and NGT2) with different surveillance requirements emphasizes the need for robust, sensitive, and cost-effective SNV detection methods suitable for distinguishing between GMOs, particularly in the context of food and feed safety, traceability, and compliance.},
}

*Plants, Genetically Modified/genetics
*Genomics/methods
*Polymorphism, Single Nucleotide
*Food, Genetically Modified
CRISPR-Cas Systems
Gene Editing

@article {pmid41595497,
year = {2026},
author = {Guan, S and Han, Y and Zhang, J and Du, Y and Chen, Z and Miao, C and Li, J},
title = {Multiplex Gene Editing and Effect Analysis of Yield, Fragrance, and Blast Resistance Genes in Rice.},
journal = {Genes},
volume = {17},
number = {1},
pages = {},
doi = {10.3390/genes17010077},
pmid = {41595497},
issn = {2073-4425},
mesh = {*Oryza/genetics/growth & development/microbiology ; *Gene Editing/methods ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Diseases/genetics/microbiology ; Plant Proteins/genetics ; Genes, Plant ; Plant Breeding ; },
abstract = {BACKGROUND: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement.

METHODS: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2) and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed using the Agrobacterium-mediated method with the japonica rice variety Xin Dao 53 as the recipient. Mutation editing efficiency was detected in T0 transgenic plants. Grain length, grain number per panicle, thousand-grain weight, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T3 generations.

RESULTS: Effectively edited plants were obtained in the T0 generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T2 generation. In the T3 generation, compared with the wild-type (WT), the edited homozygous lines showed increased grain number per panicle (14.60-25.61%), increased grain length (7.39-11.16%), increased grain length-width ratio (8.37-13.02%), increased thousand-grain weight (3.79-9.15%), a 42-64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits.

CONCLUSIONS: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance.},
}

*Oryza/genetics/growth & development/microbiology
*Gene Editing/methods
*Disease Resistance/genetics
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
*Plant Diseases/genetics/microbiology
Plant Proteins/genetics
Genes, Plant
Plant Breeding

@article {pmid41595237,
year = {2026},
author = {Karnik, M and Tulimilli, SV and Anantharaju, PG and Bettadapura, ADS and Natraj, SM and Mohideen, HS and Dovat, S and Sharma, A and Madhunapantula, SV},
title = {An Overview of the Mechanisms of HPV-Induced Cervical Cancer: The Role of Kinase Targets in Pathogenesis and Drug Resistance.},
journal = {Cancers},
volume = {18},
number = {2},
pages = {},
doi = {10.3390/cancers18020318},
pmid = {41595237},
issn = {2072-6694},
support = {No. JSSAHER/REG/RES/URG/54/2023-24//Intramural grant sanctioned by JSS AHER/ ; No. JSSAHER/REG/RES/URG/54/2025-26//JSS AHER/ ; },
abstract = {Despite a thorough understanding of the structure of human papillomavirus (HPV) and its genotypic variations (high-risk and low-risk variants), the mechanisms underlying HPV-induced cervical cancer (CC) pathogenesis and the molecular signatures of drug resistance remain to be fully understood. Accumulating evidence has shown the involvement of kinase targets in the induction of drug resistance in high-risk (HR) HPV-CC. Molecularly, the genome of high-risk HPV is reported to control the expression of host kinases. In particular, Aurora kinases A, B, and C (ARKA, ARKB, and ARKC), phosphotidylinositol-trisphosphate kinase (PI3K)-Akt, and Glycogen synthase kinase3-α/β (GSK3 α/β) promote the transformation of infected cells, and also enhance the resistance of cells to various chemotherapeutic agents such as nelfinavir and cisplatin. However, the precise mechanisms through which HPV activates these kinases are yet to be fully elucidated. Furthermore, there is still ambiguity surrounding whether targeting HPV-induced kinases along with HPV-targeted therapies (such as phytopharmaceuticals and PROTAC/CRISPR-CAS-based systems) synergistically inhibit cervical tumor growth. Given the critical role of kinases in the pathogenesis and treatment of CC, a comprehensive review of current evidence is warranted. This review aims to provide key insights into the mechanisms of HPV-induced CC development, the involvement of kinases in drug resistance induction, and the rationale for combination therapies to improve clinical outcomes.},
}

@article {pmid41594570,
year = {2025},
author = {Anschuetz, A and Robinson, L and Mondesir, M and Melis, V and Platt, B and Harrington, CR and Riedel, G and Schwab, K},
title = {Effect of the Icelandic Mutation APP[A673T] in the Murine APP Gene on Phenotype of Line 66 Tau Mice.},
journal = {Biomolecules},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/biom16010028},
pmid = {41594570},
issn = {2218-273X},
support = {PAR1577 and PAR2074//TauRx Therapeutics Ltd Singapore/ ; },
mesh = {Animals ; *tau Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Male ; Female ; *Amyloid beta-Protein Precursor/genetics/metabolism ; *Alzheimer Disease/genetics/pathology/metabolism ; Phenotype ; Iceland ; *Mutation ; Humans ; Mice, Inbred C57BL ; Brain/metabolism/pathology ; Disease Models, Animal ; },
abstract = {The Icelandic mutation in the amyloid precursor protein (APP), APP[A673T], has been identified in Icelandic and Scandinavian populations and is associated with a significantly lower risk of developing Alzheimer's disease (AD). The introduction of the human APP[A673T] form led to a reduction in amyloid β-protein (Aβ) production and tau pathology, but the effect of mouse APP[A673T] on tau and Aβ pathology is not well studied. We have crossed line 66 (L66) tau transgenic mice that overexpress the P301S aggregation-prone form of tau with C57Bl6/J mice expressing a single-point mutation edited into the murine APP gene via CRISPR-Cas gene editing, known as mAPP[A673T]. We have performed ELISA, histopathological, and behavioural analyses of heterozygous male/female L66 and L66 xmAPP[A673T] crosses at the age of 6 months to investigate the effect of the murine A673T mutation on tau brain pathology and behavioural deficits in these mice. Using immunohistochemistry, we found only a moderate, yet significant, reduction in mAb 7/51-reactive tau for female L66 x mAPP[A673T] compared to L66 mice. Quantification of tau in soluble/insoluble brain homogenate fractions by ELISA confirmed the lack of overt differences between genotypes, as did our extensive behavioural phenotyping using six different paradigms assessing motor function, olfaction, depression/apathy-like behaviour, as well as exploration and sociability. Therefore, the mAPP[A673T] mutation has a moderate impact on tau pathology but does not appear to impact motor and neuropsychiatric behaviour in L66 tau transgenic mice.},
}

Animals
*tau Proteins/genetics/metabolism
Mice
Mice, Transgenic
Male
Female
*Amyloid beta-Protein Precursor/genetics/metabolism
*Alzheimer Disease/genetics/pathology/metabolism
Phenotype
Iceland
*Mutation
Humans
Mice, Inbred C57BL
Brain/metabolism/pathology
Disease Models, Animal

@article {pmid41594550,
year = {2025},
author = {Wang, R and Guo, C and Zhang, J and Wang, Z and Jin, W and Liu, W},
title = {Evaluation of the Unintended Effects of fad2-1-Gene-Edited Soybean Line AE15 Seeds.},
journal = {Biomolecules},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/biom16010008},
pmid = {41594550},
issn = {2218-273X},
support = {2023YFF1001603//the National Key Research and Development Program/ ; ZDYF2025GXJS151//the Hainan Provincial Sanya Yazhou Bay Science and Technology Innovation Joint Project/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Seeds/genetics/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Proteomics ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; },
abstract = {A data-independent acquisition (DIA)-based proteomic analysis was performed to evaluate the unintended effects of fad2-1-gene-edited soybean line AE15 seeds. A total of 561, 269, and 227 differentially expressed proteins (DEPs) were identified in seeds from three consecutive generations of AE15 soybean, respectively, and were primarily enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to carbon metabolism, protein processing in the endoplasmic reticulum, and proteasome function. Furthermore, eight commonly differentially expressed proteins (co-DEPs) were detected across all three generations of AE15 soybean seeds, among which two-beta-amylase and endoplasmic reticulum (ER) lumen protein-retaining receptor-exhibited consistently upregulated expression. In the wild-type soybean control groups, 1063, 989, and 671 DEPs were identified across the three comparisons (ZhH302E3/ZhH10, ZhH10/ZhH42, and ZhH42/ZhH302E3), among which 71 co-DEPs were observed. These findings indicate that the protein expression profile alterations resulting from fad2-1 gene editing are considerably less pronounced compared to those caused by natural genetic variation among soybean seeds.},
}

*Glycine max/genetics/metabolism
*Seeds/genetics/metabolism
*Gene Editing
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
Proteomics
Plants, Genetically Modified/genetics
CRISPR-Cas Systems

@article {pmid41593344,
year = {2026},
author = {Munir, S and Wan, S and Gao, X and Lai, M and Mu, Z and Wang, H and Liu, Z and Li, F and Xia, L and Tan, Y},
title = {Elucidating the roles of essential genes in autotrophic metabolism and cell morphology of Clostridium ljungdahlii by CRISPRi.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {44},
pmid = {41593344},
issn = {1432-0614},
mesh = {*Clostridium/genetics/metabolism/cytology/growth & development ; *Autotrophic Processes/genetics ; *Genes, Essential ; Ethanol/metabolism ; Fermentation ; Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Understanding the function of essential genes in Clostridium ljungdahlii is critical for unraveling its autotrophic metabolism and optimizing its potential as a platform for syngas fermentation. However, study on essential genes of this species remains insufficient. Here, we employed an inducible CRISPR interference (CRISPRi) system to investigate the roles of key metabolic and cell division genes in C. ljungdahlii. Targeted repression of genes encoding pyruvate:ferredoxin oxidoreductase (PFOR1, PFOR2), acetaldehyde:ferredoxin oxidoreductase (AOR1, AOR2), and glyceraldehyde phosphate hydrogenase type I (GAP-I) revealed their essential contributions to autotrophic growth, as knockdown strains exhibited impaired growth and reduced ethanol production. Furthermore, downregulation of the cell division gene ftsZ resulted in elongated cell morphology, highlighting its critical role in cell shape regulation. These findings provide new insights into the functional importance of essential genes in C. ljungdahlii and demonstrate how targeted gene repression can advance our understanding of autotrophic metabolism and cellular processes.},
}

*Clostridium/genetics/metabolism/cytology/growth & development
*Autotrophic Processes/genetics
*Genes, Essential
Ethanol/metabolism
Fermentation
Gene Expression Regulation, Bacterial
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41592905,
year = {2026},
author = {Shabbir, R and Javed, T and Sun, SR and Wang, ZQ and Zhang, W and Gao, SJ and Wang, QN},
title = {Functional genomics in sugarcane breeding: key challenges and strategies.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/07388551.2026.2614075},
pmid = {41592905},
issn = {1549-7801},
abstract = {Sugarcane, a leading source of sugar and bio-energy around the globe stands at the cross-road of genome complexity and agricultural innovation, offering the immense potential to fuel a sustainable future. Functional genomics with its precise identification and manipulation of genes could enable researchers unlock this potential and accelerate the breeding efforts. However, the polyploid genome of sugarcane with: high heterozygosity, high-repetitive DNA content, multiple copies of homo(eo)logous gene, epistatic interaction of alleles, etc., challenges the gene annotation, genome sequencing, genome editing, and phenotypic characterization. Similarly long breeding cycle, low transformation efficiency, time-consuming, and labor-intensive transformation methods further complicates the genome editing. Recent advances of functional genomics are transforming this scenario, such as current availability of reference genome "R570," has provided a significant insight of genome architect and function. Genome wide association studies (GWAS)/genome selection (GS) are enhancing trait-mapping and prediction of breeding values to accelerate the breeding cycles. The current era of smart breeding with integrative bio-informatics, advance genome editing tools, i.e., CRISPR/Cas-systems (Cas-proteins, Cas-RNPs, d-Cas-RNPs, and CRISPRa/i), and high-throughput phenomics offers a significant approach to: overcome transformation bottlenecks, explore complex trait architect and address polyploidy challenges. Therefore, this review summarizes the key challenges and focuses on elaborating recent advances and suggests optimized strategies for future improvement in functional genomics of sugarcane breeding.},
}

@article {pmid41592109,
year = {2026},
author = {Saglam, M and Tsakirpaloglou, N and Bridgeland, A and Miller, R and Thomson, MJ and Septiningsih, EM},
title = {Carbon nanotube and carbon dot mediated plasmid DNA delivery in cowpea leaves.},
journal = {PloS one},
volume = {21},
number = {1},
pages = {e0340716},
doi = {10.1371/journal.pone.0340716},
pmid = {41592109},
issn = {1932-6203},
mesh = {*Nanotubes, Carbon/chemistry ; *Plant Leaves/genetics/metabolism ; *Vigna/genetics/metabolism ; *Plasmids/genetics ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Glucuronidase/genetics/metabolism ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; *Quantum Dots/chemistry ; Gene Editing ; Carbon/chemistry ; },
abstract = {CRISPR-Cas9 technology has been widely used as a key molecular biology tool for crop improvement. However, the advance of this technology has been hindered by host species- or genotype-dependent tissue culture protocols and poor transformation efficiencies. Recent research has shown that plasmid DNA delivered by single-walled carbon nanotubes (SWCNTs) and carbon dots (CDs) can diffuse through plant cell walls, enabling the transient expression of genetic material in plant tissues. However, such an experiment has not been performed in legumes, most of which are considered recalcitrant species for transformation. In this study, we aim to investigate the capability of a SWCNT or CD-based plasmid delivery system in expressing a target gene in cowpea (Vigna unguiculata) leaves via infiltration using the β-glucuronidase (GUS) reporter gene. Further, we aim to see the potential of SWCNTs and CDs for a CRISPR-Cas9 gene construct delivery system, with phytoene desaturase (PDS) as the target gene. Our results showed that SWCNTs and CDs can deliver the GUS reporter gene construct in the surrounding area near the site of the infiltration, which results in the temporary expression of GUS by observing the blue color in this area. Likewise, infiltration of the CRISPR-Cas9 vectors targeting the PDS gene for the knockout resulted in multiplex editing and large deletions within the target gene. Overall, our findings pave the way for overcoming conventional DNA delivery challenges. However, further research is needed to explore optimal germline targets for plant tissues to avoid chimerism and to allow for more efficient CRISPR-Cas9 editing resulting in heritable mutations.},
}

*Nanotubes, Carbon/chemistry
*Plant Leaves/genetics/metabolism
*Vigna/genetics/metabolism
*Plasmids/genetics
*Gene Transfer Techniques
CRISPR-Cas Systems
Glucuronidase/genetics/metabolism
Plants, Genetically Modified/genetics
Oxidoreductases/genetics
*Quantum Dots/chemistry
Gene Editing
Carbon/chemistry

@article {pmid41591594,
year = {2026},
author = {Singhal, P and Saini, S and Saini, O and Bishnoi, A and E R, R and Meena, BR and Singh, J and Yogendra, K},
title = {Molecular gatekeepers: eukaryotic translation factors decoding plant-virus dynamics for resistance engineering.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {9},
pmid = {41591594},
issn = {2731-0450},
abstract = {Plant viruses are among the most significant biotic stressors, posing a severe threat to crop productivity and global food security. Their success largely depends on the exploitation of host eukaryotic translation factors (eTFs), including initiation factors (eIFs) and elongation factors (eEFs), which act as molecular gatekeepers of the viral life cycle. Key members such as eIF4E, eIF(iso)4E, eIF4G, eEF1A, and eEF1B have been identified as susceptibility factors that mediate viral translation, replication, and systemic movement. Viruses have co-evolved specialized proteins and RNA elements, including VPg and IRES structures, to hijack these host factors and circumvent plant defense barriers. This review synthesizes current understanding of the mechanistic roles of eTFs in virus-host dynamics and highlights strategies to mitigate viral stress. Approaches such as natural allele mining, induced mutagenesis, TILLING/EcoTILLING, RNA interference, and precise genome editing with CRISPR/Cas systems are explored as practical tools for reducing susceptibility. Targeted manipulation of eTFs offers a promising avenue to reprogram plants for resistance while maintaining essential cellular functions. By integrating molecular biology with applied strategies, we propose an eTF-centered framework for resistance breeding within a broader stress biology perspective. Future research combining functional genomics, synthetic biology, and breeding innovation will be pivotal in delivering broad-spectrum, durable, and environmentally sustainable resistance to plant viral stress.},
}

@article {pmid41590698,
year = {2025},
author = {Antonacci, A and Masi, A and Vedi, V and Colella, S and Musella, F and Fiorentino, G and Scognamiglio, V},
title = {CRISPR-Cas Technology Turns Chlamydomonas reinhardtii into a Flagship for Algal Biotechnology.},
journal = {Marine drugs},
volume = {24},
number = {1},
pages = {},
doi = {10.3390/md24010001},
pmid = {41590698},
issn = {1660-3397},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Biotechnology/methods ; Gene Editing/methods ; *Microalgae/genetics/metabolism ; Biofuels ; Metabolic Engineering/methods ; },
abstract = {Microalgae represent some of the most promising eukaryotic platforms in biotechnology due to their rapid growth, simple cultivation requirements, reliance on sunlight as a primary energy source, and ability to synthesize high-value bioactive compounds. These characteristics have made microalgae attractive candidates in various fields, including biofuel production, carbon capture, and pharmaceutical development. However, several technical limitations have limited their large-scale use as sustainable biofactories. A paradigm shift is currently occurring thanks to the genetic manipulation of microalgae, driven by CRISPR-Cas technology. Significant progress has been made in the model species Chlamydomonas reinhardtii, particularly in the targeted and efficient insertion of foreign DNA. Despite this progress, key challenges remain, and further optimization of CRISPR-Cas methodologies is needed to fully unleash the genetic potential of this organism. This review provides an overview of the convergence of CRISPR-Cas technologies in microalgae research, highlighting their impact on genetic studies, metabolic engineering, and industrial applications. It summarizes recent advances in microalgal genome editing through CRISPR systems, outlines current technical challenges, and highlights future directions for improving the implementation of this innovative technology in microalgal biotechnology.},
}

*Chlamydomonas reinhardtii/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Biotechnology/methods
Gene Editing/methods
*Microalgae/genetics/metabolism
Biofuels
Metabolic Engineering/methods

@article {pmid41590485,
year = {2026},
author = {Gupta, DR and Kasfy, SH and Ali, J and Hia, FT and Hoque, MN and Rahman, M and Islam, T},
title = {Validation and Improvement of a Rapid, CRISPR-Cas-Free RPA-PCRD Strip Assay for On-Site Genomic Surveillance and Quarantine of Wheat Blast.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/jof12010073},
pmid = {41590485},
issn = {2309-608X},
support = {Grant Code: V0156.01//Bill and Melinda Gates Foundation and the Foreign, Commonwealth & Development Office (FCDO), UK/ ; },
abstract = {As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve a Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum, and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI), and in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in a wheat blast hotspot district, Meherpur, Bangladesh. Training 52 diverse stakeholders validated the kit's field readiness, with 88% of trainees endorsing its user-friendly design. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site genomic surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as an early warning modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.},
}

@article {pmid41590284,
year = {2026},
author = {Zhang, Z and Fu, Q and Wen, T and Zheng, Y and Ma, Y and Liu, S and Liu, G},
title = {Integrated Colorimetric CRISPR/Cas12a Detection of Double-Stranded DNA on Microfluidic Paper-Based Analytical Devices.},
journal = {Biosensors},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/bios16010032},
pmid = {41590284},
issn = {2079-6374},
support = {22174121, 22211530067, T2250710180//National Natural Science Foundation of China/ ; },
mesh = {*Colorimetry ; Paper ; CRISPR-Cas Systems ; *Biosensing Techniques ; Humans ; Human papillomavirus 16 ; Lab-On-A-Chip Devices ; Gold/chemistry ; *DNA/analysis ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; },
abstract = {Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a's trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV.},
}

*Colorimetry
Paper
CRISPR-Cas Systems
*Biosensing Techniques
Humans
Human papillomavirus 16
Lab-On-A-Chip Devices
Gold/chemistry
*DNA/analysis
Metal Nanoparticles/chemistry
Nucleic Acid Amplification Techniques

@article {pmid41590269,
year = {2025},
author = {Liang, Z and Zhang, J and Zhang, S},
title = {Engineering a CRISPR-Mediated Dual Signal Amplification-Based Biosensor for miRNA Determination.},
journal = {Biosensors},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/bios16010017},
pmid = {41590269},
issn = {2079-6374},
support = {2023A1515110638//Guangdong Basic and Applied Basic Research Foundation/ ; 2025A1515011683//Guangdong Basic and Applied Basic Research Foundation/ ; 2025A04J4037//Guangzhou Science and Technology Planning Project/ ; 2022GDASZH-2022010110//GDAS' Project of Science and Technology Development/ ; },
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electrochemical Techniques ; },
abstract = {MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO4[3-] modified single-stranded DNAs (ssDNAs); the resulting 3' hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 10[8] fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy.},
}

*MicroRNAs/analysis
*Biosensing Techniques/methods
Humans
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Electrochemical Techniques

@article {pmid41588195,
year = {2026},
author = {Taveneau, C and Chai, HX and D'Silva, J and Bamert, RS and Chen, H and Hayes, BK and Calvert, RW and Purcell, J and Curwen, DJ and Munder, F and Martin, LL and Barr, JJ and Rosenbluh, J and Fareh, M and Grinter, R and Knott, GJ},
title = {De novo design of potent CRISPR-Cas13 inhibitors.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41588195},
issn = {1552-4469},
abstract = {CRISPR-Cas systems are transformative tools for gene editing that can be tuned or controlled by anti-CRISPRs (Acrs)-phage-derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that can inhibit biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful and rapid approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Here, using Leptotrichia buccalis CRISPR-Cas13a as a representative example, we demonstrate that Acrs designed using artificial intelligence (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13a nuclease activity. We present a comprehensive workflow for design validation and demonstrate AIcr functionality in controlling CRISPR-Cas13 activity in bacterial and human cells. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture and microbiology.},
}

@article {pmid41588051,
year = {2026},
author = {Molina, MC and Quiroga, C},
title = {Functional characterization of a type I-F1 CRISPR-cas system from the clinical isolate Shewanella xiamenensis Sh95 reveals constitutive activity and plasmid-curing capability.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-025-34486-2},
pmid = {41588051},
issn = {2045-2322},
support = {ANPCyT 2020-03222//Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación/ ; IP-PUE 0085//Consejo Nacional de Investigaciones Científicas y Técnicas/ ; PIDAE-UBA 2025 EX-2024-04115022-UBA-DME#REC//Universidad de Buenos Aires/ ; },
}

@article {pmid41587008,
year = {2026},
author = {Cai, M and Song, K and Yao, C and Wang, S and Wang, R and Wang, Q and Chen, H and Wang, H},
title = {Global spread and evolution of KPC-2 and NDM-1-producing Gram-negative bacteria.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {41587008},
issn = {1869-1889},
abstract = {The co-occurrence of KPC and NDM carbapenemases in Gram-negative bacteria presents a serious and expanding global health threat. This study characterized 338 KPC-2/NDM-1 dual-positive isolates from 23 countries, including 41 clinical strains sequenced through hybrid second- and third-generation platforms from China's national surveillance network. These isolates spanned six genera, 58 species, and 138 sequence types, reflecting substantial taxonomic and geographic diversity. Molecular analysis identified IncFII(p14) plasmids as the principal vectors for cross-genus dissemination of KPC-2, while IncX3, IncN, and IncFIB(pB171)/IncFII(Yp) plasmids were dominant carriers of NDM-1 among the studied strains. Codon usage analysis indicated stronger bias in KPC-2 plasmids (effective codon number: 39.17, optimal codons: 17) compared to NDM-1 plasmids (effective codon number: 41.25, optimal codons: 12), indicating differential evolutionary pressures. Dual-positive strains exhibited significantly higher virulence scores and broader resistance profiles than reference strains (P<0.001). Notably, only 14.6% of isolates harbored Type I-E CRISPR-Cas systems, all of which encoded the anti-CRISPR protein AcrIE10. Furthermore, Type II methyltransferase numbers were significantly enriched in dual-positive strains (P<0.005), suggesting a potential role in modulating host defense evasion. We propose that in Klebsiella spp., KPC-2 plasmids are typically acquired prior to NDM-1 plasmids and can form hybrid plasmids. In non-Klebsiella genera, dual resistance is primarily driven by independent acquisition of high-risk plasmids such as IncFII(p14) and IncX3, without a fixed temporal order. These findings highlight the convergence of global plasmid-mediated resistance, host-pathogen immune interplay, and pan-resistance evolution. Targeting high-risk plasmid lineages and host defense-modulating elements may be key to forecasting resistance emergence and guiding early interventions against dual-carbapenemase-producing pathogens.},
}

@article {pmid41586841,
year = {2026},
author = {Graça, M and Virgolini, N and Correia, R and Escandell, J and Roldão, A},
title = {An improved CRISPR-Cas9 protein-based method for knocking out insect Sf9 cell genes.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {42},
pmid = {41586841},
issn = {1432-0614},
mesh = {Animals ; *CRISPR-Cas Systems ; Sf9 Cells ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; Apoptosis ; Baculoviridae/genetics ; Spodoptera/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production. KEY POINTS: • Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells. • Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection. • Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.},
}

Animals
*CRISPR-Cas Systems
Sf9 Cells
*Gene Knockout Techniques/methods
*Gene Editing/methods
Apoptosis
Baculoviridae/genetics
Spodoptera/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41586305,
year = {2025},
author = {Pérez-Rodríguez, M and Serrano-Pertierra, E and Blanco-López, MC},
title = {Advances in biosensor technologies for the detection of antimicrobial resistance in Staphylococcus aureus.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1741845},
pmid = {41586305},
issn = {2235-2988},
mesh = {*Biosensing Techniques/methods ; Humans ; *Staphylococcal Infections/diagnosis/microbiology ; *Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/isolation & purification ; *Staphylococcus aureus/drug effects/genetics/isolation & purification ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests/methods ; },
abstract = {The rise of methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent need for rapid, sensitive, and portable diagnostics. In this paper, we have critically reviewed recent advances in biosensor technologies, integrating nanomaterials, aptamers, CRISPR/Cas systems, and microfluidic lab-on-a-chip platforms, that enable sub-hour and ultrasensitive detection of S. aureus and its resistance genes. These innovations offer powerful alternatives to conventional culture and PCR assays, forming the way for real-time, point-of-care antimicrobial resistance testing. Remaining challenges include matrix interference, lack of standardization, and limited clinical validation, yet continued integration with artificial intelligence and digital systems promises transformative diagnostic capabilities.},
}

*Biosensing Techniques/methods
Humans
*Staphylococcal Infections/diagnosis/microbiology
*Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/isolation & purification
*Staphylococcus aureus/drug effects/genetics/isolation & purification
CRISPR-Cas Systems
*Drug Resistance, Bacterial
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests/methods

@article {pmid41584446,
year = {2026},
author = {Yang, F and Ran, Q and Chen, J and Bao, G and Xian, Y and Zhang, C},
title = {Spatiotemporally regulated mitochondrial genome editing via enzyme and NIR-activated CRISPR/Cas9 nanoplatform.},
journal = {Chemical science},
volume = {},
number = {},
pages = {},
pmid = {41584446},
issn = {2041-6520},
abstract = {Mitochondrial DNA (mtDNA) mutations play critical roles in tumor progression and metabolic reprogramming. Controllable gene editing within tumor cell mitochondria remains a challenge due to the double-membrane barrier and the lack of tumor-selective activation. Herein, we report a dual-responsive CRISPR/Cas delivery platform (UCRP-TPP) that enables spatiotemporally regulated mtDNA editing for targeted tumor therapy. This nanoplatform integrates near infrared light-responsive upconversion nanoparticle (UCNP), an apurinic endonuclease 1 (APE-1)-responsive DNA complex, and a mitochondrial-targeting ligand (TPP), ensuring selective activation and mitochondrial release of Cas9/sgRNA complexes. Upon activation by endogenous APE-1 enzyme and exogenous NIR light, UCRP-TPP induces mtDNA editing by CRISPR/Cas, which leads to mtDNA copy number reduction, mitochondrial membrane depolarization, reactive oxygen species generation, and tumor cell apoptosis. In vivo studies further confirm the robust antitumor efficacy of the UCRP-TPP-based nanoplatform. This work presents a versatile and controllable mitochondrial gene-editing strategy.},
}

@article {pmid41581986,
year = {2026},
author = {Dhariwal, R and Jain, M},
title = {Cell-free systems for low-cost diagnostics.},
journal = {Progress in molecular biology and translational science},
volume = {218},
number = {},
pages = {157-185},
doi = {10.1016/bs.pmbts.2025.08.005},
pmid = {41581986},
issn = {1878-0814},
mesh = {Humans ; Biosensing Techniques ; *Cell-Free System ; Point-of-Care Systems ; Diagnosis ; },
abstract = {Cell-free systems have also become a revolutionary platform for low-cost diagnostics, providing fast, flexible, and scalable solutions to the conventional cell-based assays. Such systems, which utilize the fundamental biochemical machinery of cells without the intricacies of living organisms, have been of great use in point-of-care (POC) diagnostics, particularly in resource-poor environments. This chapter offers a broad overview of the basic principles, design approaches, and technological breakthroughs behind cell-free diagnostic development. It discusses the biochemical underpinnings of cell-free expression, such as ribosomal function, transcriptional control, and energy regeneration, with emphases on the leading platforms including E. coli lysates, wheat germ extracts, and PURE systems. The application of synthetic biology in the form of gene circuits, CRISPR-Cas tools, and RNA aptamers is presented here in the framework of improving the sensitivity and specificity of diagnostics. The chapter further discusses recent innovations in paper-based assays, microfluidic biosensors, and wearable biosensors, which are capable of offering real-time and field-deployable diagnostics. Major challenges in the form of reagent stability, scalability, and regulatory implications are analyzed carefully along with recent trends such as AI-based system design and personalization of diagnostics. In extensive case studies, the chapter highlights the promise of cell-free systems in filling diagnostic gaps, enhancing access to healthcare, and revolutionizing global health. This book strives to offer an encyclopedic sourcebook for researchers, clinicians, and innovators interested in bringing cell-free diagnostics forward.},
}

Humans
Biosensing Techniques
*Cell-Free System
Point-of-Care Systems
Diagnosis

@article {pmid41578173,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {Genomic landscape of biosynthetic gene clusters in Iranian extremophiles reveals prolific metabolite potential, prophage associations, and integrated defensive-metabolic islands.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-025-04690-1},
pmid = {41578173},
issn = {1471-2180},
abstract = {The extreme and underexplored ecosystems of Iran represent a significant reservoir of microbial diversity with profound biosynthetic potential. To systematically investigate this resource, we employed a comprehensive genome mining approach on 16 bacterial isolates from hypersaline, desert, and petroleum-contaminated soils. Our analysis revealed an extraordinary density and complexity of biosynthetic gene clusters (BGCs), identifying 229 BGCs in total. A substantial majority (56.8%) showed no significant similarity to known clusters, underscoring the extensive novelty encoded within these extremophiles. Notably, we discovered highly intricate "trio" and "quartet" hybrid BGCs, which encode the machinery for three or four distinct classes of secondary metabolites, pushing the boundaries of known biosynthetic complexity. Parallel analysis identified six novel, high-quality prophages, largely uncharacterized in public databases. These prophages were found to carry a putative bacteriocin cluster (UviB) indicating a direct role in enhancing host fitness. Furthermore, we uncovered a dynamic co-evolutionary arms race, with bacterial genomes fortified by diverse defense systems, including abundant CRISPR-Cas arrays, and prophages encoding a repertoire of counter-defense anti-CRISPR proteins. Genomic architecture analysis revealed widespread co-localization of BGCs, prophages, and defense systems into functional genomic islands, suggesting a synergistic linkage between secondary metabolism and phage resistance. This study illuminates the remarkable biosynthetic and defensive landscape of Iranian extremophiles, highlighting them as a premier resource for discovering novel natural products and understanding virus-host evolutionary dynamics.},
}

@article {pmid41578091,
year = {2026},
author = {Torres-Higuera, LD and Rojas-Tapias, DF and Jiménez-Velásquez, S and Renjifo-Ibáñez, C},
title = {Comprehensive genotyping and taxonomic analysis uncovers extensive distribution of intermediate Leptospira species in Colombia.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {2},
pages = {57},
pmid = {41578091},
issn = {1573-0972},
mesh = {Colombia/epidemiology ; *Leptospira/genetics/classification/isolation & purification ; Phylogeny ; *Leptospirosis/microbiology/epidemiology/veterinary ; Humans ; Animals ; RNA, Ribosomal, 16S/genetics ; Genotype ; Genetic Variation ; DNA, Bacterial/genetics ; Multilocus Sequence Typing ; Whole Genome Sequencing ; Genome, Bacterial ; Ribotyping ; DNA-Directed RNA Polymerases/genetics ; Virulence Factors/genetics ; Minisatellite Repeats ; },
abstract = {Leptospirosis, a globally prevalent zoonosis caused by pathogenic and intermediate Leptospira species, poses significant threats to public health and livestock industries. Despite its substantial impact, knowledge gaps persist regarding the prevalence and genetic diversity of Leptospira strains in many regions, including South America. This study aimed to characterize a diverse collection of Leptospira strains isolated from various sources in Colombia to enhance our understanding of the genetic diversity within this genus. Using a tiered approach combining conventional and genomic methods, we genotyped 55 isolates from various sources using 16S rRNA and rpoB gene sequencing, DNA ribotyping, and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). Most isolates were classified into phylogenetic groups containing pathogenic and intermediate strains of L. interrogans and L. wolffii, respectively, which was corroborated by ribotyping and MLVA. Whole-genome sequencing of selected strains revealed distinct genomic characteristics compared to related strains. Pan-genome analysis identified strain-specific genes, primarily hypothetical, while virulence factor analysis distinguished species-specific patterns. Furthermore, CRISPR-Cas system analysis uncovered genetic variations among the isolates. This study provides a framework for understanding Leptospira genetic diversity in Colombia and its potential implications on human and animal health. Our findings highlight the need for improved diagnostic methods and surveillance strategies that encompass both pathogenic and intermediate Leptospira species, which could significantly impact public health policies and veterinary practices in the region.},
}

Colombia/epidemiology
*Leptospira/genetics/classification/isolation & purification
Phylogeny
*Leptospirosis/microbiology/epidemiology/veterinary
Humans
Animals
RNA, Ribosomal, 16S/genetics
Genotype
Genetic Variation
DNA, Bacterial/genetics
Multilocus Sequence Typing
Whole Genome Sequencing
Genome, Bacterial
Ribotyping
DNA-Directed RNA Polymerases/genetics
Virulence Factors/genetics
Minisatellite Repeats

@article {pmid41578087,
year = {2026},
author = {Gautam, B and Jarvis, BA and Esfahanian, M and McGinn, M and Williams, D and Liu, S and Phippen, ME and Heller, NJ and Wesley, TL and Phippen, WB and Ulmasov, T and Marks, MD and Chopra, R and Sedbrook, JC},
title = {Creating a new oilseed crop, pennycress, by combining key domestication traits using CRISPR genome editing.},
journal = {Nature plants},
volume = {12},
number = {1},
pages = {74-87},
pmid = {41578087},
issn = {2055-0278},
support = {2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; },
mesh = {*Gene Editing ; *Domestication ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Seeds/genetics ; *Brassica/genetics ; },
abstract = {Considerable off-season farmland lies fallow because few crops can profitably fit between primary crops. As a remedy, we performed de novo domestication of the freeze-tolerant, rapid-cycling wild brassica Thlaspi arvense L. (field pennycress), identifying and stacking CRISPR-Cas9-induced mutations that have minimal impacts on seed yields. High-yielding varieties were created with seed compositions such as 'double-low' canola (low erucic acid and reduced glucosinolate) and reduced seed fibre content. Seed glucosinolate content was reduced by 75% by combining mutations in R2R3-MYB (MYB28/HAG1) and basic helix-loop-helix MYC (MYC3) transcription factors. Pennycress weediness was greatly reduced by knockout of the basic helix-loop-helix transcription factor TRANSPARENT TESTA8 (TT8), which lowered seed dormancy and seed coat protections, thereby mitigating re-emergence in fields. Domesticated pennycress offers farmers a low-carbon-intensity intermediate crop that fits between two full-season summer crops, resulting in three cash crops in 2 years, conferring cover-crop-like ecosystem benefits while producing grain for renewable fuels and enhanced food security.},
}

*Gene Editing
*Domestication
*Crops, Agricultural/genetics
CRISPR-Cas Systems
Seeds/genetics
*Brassica/genetics

@article {pmid41577144,
year = {2026},
author = {Mocchetti, A and Steelant, P and Hosseinkhani, M and De Rouck, S and Khajehali, J and Van Leeuwen, T},
title = {Knockout of nAChR subunits in spider mites and their phytoseiid predators confers spinosyn cross-resistance and reveals a conserved mode of action in mites.},
journal = {Insect biochemistry and molecular biology},
volume = {},
number = {},
pages = {104498},
doi = {10.1016/j.ibmb.2026.104498},
pmid = {41577144},
issn = {1879-0240},
abstract = {Spinosyns are allosteric modulators of nicotinic acetylcholine receptors (nAChRs) which in insects specifically target subunit α6. However, their mode of action in mites and compatibility with phytoseiid predators remain unclear. We combined phylogenetics with CRISPR/Cas-based reverse genetics to test whether α6-like subunits mediate spinosyn toxicity in mites and to assess prospects for resistance breeding in phytoseiids. The phylogenetic analysis identified seven α and three β subunits in multiple phytoseiids and in Tetranychus urticae. A single phytoseiid subunit clustered within the insect α6/α7 clade, whereas T. urticae possessed three (Tuα5/α6/α7) without strict one-to-one insect orthology. Using SYNCAS maternal delivery of CRISPR RNPs, we disrupted the putative α6 ortholog in Amblyseius swirskii (Asα6) and each of the three α6/α7-clade genes in T. urticae. In A. swirskii, all survivors of a discriminating spinosad dose carried Asα6 indels, and three independently edited lines exhibited insensitivity to both spinosad and spinetoram (no significant mortality at 10,000 mg a.i./L), whereas the wild type showed LC50 = 163 mg/L (spinosad) and 54 mg/L (spinetoram). In T. urticae, Tuα6 knockouts conferred high cross-resistance to both compounds, while Tuα5 knockouts slightly increased susceptibility and Tuα7 knockouts produced modest resistance. Our data demonstrate that α6-mediated spinosyn action is conserved in mites, with α6 loss conferring strong cross-resistance in a key phytoseiid predator and in a model tetranychid. These findings enable marker-assisted editing/selection of spinosyn-resistant phytoseiid strains to improve pesticide-biocontrol compatibility and establish α6 as a practical universal marker gene for genome editing in acarine systems.},
}

@article {pmid41576918,
year = {2026},
author = {Pinglay, S and Atwater, JT and Brosh, R and Shendure, J and Maurano, MT and Boeke, JD},
title = {Mammalian genome writing: Unlocking new length scales for genome engineering.},
journal = {Cell},
volume = {189},
number = {2},
pages = {356-374},
pmid = {41576918},
issn = {1097-4172},
support = {DP5 OD036167/OD/NIH HHS/United States ; R01 HG012743/HG/NHGRI NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Genome ; *Genetic Engineering/methods ; *Mammals/genetics ; CRISPR-Cas Systems ; },
abstract = {The ability to design and engineer mammalian genomes across arbitrary length scales would transform biology and medicine. Such capabilities would enable the systematic dissection of mechanisms governing gene regulation and the influence of complex haplotypes on human traits and disease. They would also facilitate the engineering of disease models that more faithfully recapitulate human physiology and of next-generation cell therapies harboring sophisticated genetic circuits. Over the past decade, advances in genome editing have made small, targeted modifications at single sites routine. However, achieving multiple coordinated alterations across long sequence windows (>10 kb) or installing large synthetic DNA segments in mammalian cells remains a major challenge. Recent advances in mammalian genome writing-the bottom-up design, assembly, and targeted integration of large custom DNA sequences, independent of any natural template-offer a potential solution. Here, we review key technological developments, highlight emerging applications, and discuss current bottlenecks and strategies for overcoming them.},
}

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

@article {pmid41574295,
year = {2025},
author = {Zhang, R and Zheng, Y and Ding, C and Wu, J and Zhu, W and Zhu, X and Xu, G and Chen, L},
title = {OxyR contributes to the oxidative stress capacity and virulence of hypervirulent Klebsiella pneumoniae ATCC 43816.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1661384},
pmid = {41574295},
issn = {2235-2988},
mesh = {*Oxidative Stress ; *Klebsiella pneumoniae/pathogenicity/genetics/growth & development/drug effects ; Animals ; Virulence ; Klebsiella Infections/microbiology/pathology ; Biofilms/growth & development ; Mice ; Hydrogen Peroxide/toxicity ; Disease Models, Animal ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Gene Deletion ; Moths/microbiology ; *Repressor Proteins/genetics/metabolism ; Genetic Complementation Test ; Virulence Factors/genetics ; Gene Expression Profiling ; CRISPR-Cas Systems ; Lepidoptera/microbiology ; },
abstract = {INTRODUCTION: Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathogen associated with severe invasive infections and high mortality, in which resistance to host-derived reactive oxygen species (ROS) is critical for immune evasion and persistence. However, the mechanisms underlying oxidative stress resistance in hvKP remain poorly understood, and the role of the global regulator OxyR in this species has not been fully elucidated.

METHODS: In this study, VK055_RS16305 was first identified as an OxyR homologue in K. pneumoniae ATCC 43816 by sequence alignment. The oxyR deletion mutant was generated using a CRISPR/Cas9-based genome editing system, whereas the complemented strain was obtained using the pSTV28 plasmid carrying oxyR. We then compared their growth characteristics, susceptibility to H₂O₂, biofilm formation, and virulence in Galleria mellonella and mouse infection models, and performed RNA sequencing followed by qRT-PCR to characterize the OxyR regulon under oxidative stress.

RESULTS: Deletion of oxyR did not alter bacterial growth or colony morphology under non-stress conditions, but markedly increased susceptibility to H₂O₂ and impaired biofilm formation. In vivo, the oxyR mutant exhibited attenuated virulence, with improved survival of Galleria mellonella and mice and significantly reduced bacterial burdens in blood, liver, lung, and spleen, all of which were restored by genetic complementation. Transcriptomic analysis revealed that OxyR positively regulates multiple oxidative stress-associated genes, including hemH, grxA, gsk, katG, and ahpC, in response to H₂O₂.

DISCUSSION: Together, these findings demonstrate that OxyR is a key regulator of oxidative stress defense, biofilm formation, and systemic virulence in hvKP, providing new insight into OxyR-mediated pathogenic mechanisms in K. pneumoniae.},
}

*Oxidative Stress
*Klebsiella pneumoniae/pathogenicity/genetics/growth & development/drug effects
Animals
Virulence
Klebsiella Infections/microbiology/pathology
Biofilms/growth & development
Mice
Hydrogen Peroxide/toxicity
Disease Models, Animal
Gene Expression Regulation, Bacterial
*Bacterial Proteins/genetics/metabolism
Gene Deletion
Moths/microbiology
*Repressor Proteins/genetics/metabolism
Genetic Complementation Test
Virulence Factors/genetics
Gene Expression Profiling
CRISPR-Cas Systems
Lepidoptera/microbiology

@article {pmid41570748,
year = {2026},
author = {Gholami, S and Aghbash, PS and Ravanlo, ZZ and Rahimi, SB and Baghi, HB},
title = {Therapeutic targeting of the HPV E7 oncoprotein: Current advances and emerging strategies.},
journal = {International immunopharmacology},
volume = {172},
number = {},
pages = {116193},
doi = {10.1016/j.intimp.2026.116193},
pmid = {41570748},
issn = {1878-1705},
abstract = {Cervical cancer is one of the most common malignancies among women, with persistent infection by high-risk human papillomavirus (HPV) types, particularly HPV16 and HPV18, being the primary etiological factor. The viral oncoproteins E6 and E7 play pivotal roles in carcinogenesis by inactivating the tumor suppressor proteins p53 and pRb, respectively. E7 has emerged as a promising therapeutic target due to its continuous expression in transformed cells and its essential role in maintaining the malignant phenotype. Recent advances in molecular biology and nanotechnology have led to the development of novel therapeutic strategies aimed at silencing or inhibiting E7, such as immunotherapy, RNA interference (RNAi), CRISPR/Cas9-based genome editing, and the use of natural bioactive compounds. Immunotherapeutic approaches aim to elicit specific cytotoxic T-cell responses against E7, whereas RNAi and CRISPR/Cas systems enable precise suppression or disruption of the E7 oncogene. As a result, it leads to the reactivate of p53 and pRb pathways, cell cycle arrest, and apoptosis. Additionally, the design of innovative delivery systems, such as liposomal nanoparticles, polymeric carriers, and viral vectors, has improved the efficiency and safety of therapeutic gene delivery. Collectively, these targeted approaches offer promising prospects for the treatment of HPV-related cancers. However, further optimization of delivery platforms and minimization of off-target effects are essential for the successful clinical translation of E7-targeted therapies in cervical cancer.},
}

@article {pmid41570230,
year = {2025},
author = {Demidova, NA and Klimova, RR and Kushch, AA and Karpov, DS},
title = {CRISPR-Cas genome editing system in the diagnosis and therapy of infection caused by herpes simplex virus type 1 (Orthoherpesviridae: Alphaherpesviridae: Simplexvirus: Simplexvirus humanalpha1).},
journal = {Voprosy virusologii},
volume = {70},
number = {6},
pages = {493-507},
doi = {10.36233/0507-4088-307},
pmid = {41570230},
issn = {2411-2097},
mesh = {Humans ; *Herpesvirus 1, Human/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Herpes Simplex/therapy/diagnosis/genetics/virology ; Animals ; Genome, Viral ; *Genetic Therapy/methods ; },
abstract = {Herpes simplex virus type 1 (HSV-1), newly named as Simplexvirus humanalpha1 is one of the most common pathogens in the human population, which can cause severe disease, often with fatal outcomes. Diagnostic methods currently in use are specific and sensitive, but time-consuming, require expensive laboratory equipment and highly qualified personnel. Existing therapeutic agents have a number of significant drawbacks. To successfully treat and prevent the spread of the infection, new rapid, easy-to-use, and highly sensitive diagnostic tools and effective therapeutic agents are required. One approach to achieve this goal is CRISPR-based technology. This review analyzes information obtained from a literature search in the Scopus, Web of Science and MedLine databases on the topics «HSV-1, structure, distribution, life cycle», «new methods for molecular diagnosis of HSV-1-infection», «classification of CRISPR-Cas systems», «nucleic acid amplification methods», «CRISPR-Cas effector proteins», «application of CRISPR-Cas systems in molecular diagnostics of HSV-1-infection», «application of CRISPR-Cas systems in therapy of HSV-1-infection». New approaches of CRISPR using effector proteins Cas12 and Cas13 in the diagnosis of HSV-1 infections are reviewed. The article discusses the progress in the development of CRISPR-Cas-based therapies against HSV-1-infection in vitro and in vivo. CRISPR gene therapy in vivo has a great clinical potential, but its safety and efficacy require further investigation. An analysis of the available data suggests that CRISPR-based technologies offer promising prospects for expanding the arsenal of diagnostic tools and antiviral drugs in the context of current and future outbreaks of viral diseases.},
}

Humans
*Herpesvirus 1, Human/genetics/pathogenicity
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Herpes Simplex/therapy/diagnosis/genetics/virology
Animals
Genome, Viral
*Genetic Therapy/methods

@article {pmid41569521,
year = {2026},
author = {Yang, X and Wu, H and Zeng, Z and Chen, WN and Luan, GX and Zhang, QL and Chen, JM},
title = {Recent advances in highly sensitive and specific functional nucleic acid sensors for environmental pollutant detection: from mechanism to application.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5an01139f},
pmid = {41569521},
issn = {1364-5528},
abstract = {Functional nucleic acids (FNAs) have emerged as a cutting-edge tool in environmental pollutant detection, attributed to their exceptional stability, robust specificity, and remarkable capacity for signal transduction and amplification. This review elaborates comprehensively on four pivotal categories of FNAs-aptamers, RNA-cleaving DNAzymes, G-quadruplex/hemin DNAzymes, and gRNAs-alongside their applications in monitoring a spectrum of pollutants. These encompass organic contaminants (e.g., pesticides and bisphenols), heavy metals (such as Pb[2+] and Hg[2+]), biotoxins, and pathogenic microorganisms. It also underscores the integration of FNAs with sophisticated technologies like nanomaterials and CRISPR/Cas systems to augment detection sensitivity and efficacy. Despite prevailing challenges, including susceptibility to environmental variables (pH and temperature) and intricate synthesis procedures, FNAs hold immense potential for advancing environmental monitoring and pollution control.},
}

@article {pmid41569163,
year = {2026},
author = {Luo, Y and Jiang, Q and Qu, Y and Li, W and Liu, R and Zhu, Y and Xie, Y and Jiang, C and Chen, C and Cong, L and Han, F and Bao, J and Wang, C},
title = {Compact bacterial recombination complexes drive efficient kilobase-scale knock-in in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41569163},
issn = {1362-4962},
support = {2024YFC3408100//National Key Research and Development Program of China/ ; JSSCTD202450//Jiangsu Shuangchuang Project/ ; TJ-2023-005//Jiangsu Science and Technology Association Youth Science and Technology/ ; QYPY20230032//Center for Advanced Interdisciplinary Science and Biomedicine of IHM/ ; BK20240529//Natural Science Foundation of Jiangsu Province/ ; //Nanjing Medical University/ ; 82403421//National Natural Science Foundation of China/ ; 2408085J016//Anhui Provincial Natural Science Foundation/ ; },
mesh = {Animals ; Humans ; Mice ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Recombinational DNA Repair ; Escherichia coli/genetics/enzymology ; *Escherichia coli Proteins/genetics/metabolism ; HEK293 Cells ; Neurons/metabolism ; *Rec A Recombinases/genetics/metabolism ; },
abstract = {Efficient homologous recombination, homology-directed repair (HDR), remains a major hurdle for precise genome editing in mammalian cells, particularly for kilobase-scale insertions. Bacterial recombineering proteins, such as RecE and RecT, offer potential solutions, but their activity in eukaryotic systems has been largely uncharacterized. Here, we identify Escherichia coli RecE (EcRecE) as a potent enhancer of HDR in mammalian cells. Targeted recruitment of EcRecE via CRISPR/Cas9 significantly increased HDR efficiency at multiple genomic loci across different cellular contexts, including human embryonic stem cells, achieving a 3-6-fold enhancement in the integration efficiency of kilobase-scale sequences. Furthermore, in combination with RecT and a catalytically inactive Cas9 (dCas9), applying functional domain engineering, we developed a dCas9-miniRecTE editor that enhances large-fragment integration without introducing double-strand breaks in human cells and primary mouse neurons, achieving ∼20% kilobase-scale knock-in efficiency. These results establish EcRecE as a versatile tool for improving precision genome engineering, with potential applications in therapeutic gene editing.},
}

Animals
Humans
Mice
*Gene Knock-In Techniques/methods
CRISPR-Cas Systems
*Gene Editing/methods
*Recombinational DNA Repair
Escherichia coli/genetics/enzymology
*Escherichia coli Proteins/genetics/metabolism
HEK293 Cells
Neurons/metabolism
*Rec A Recombinases/genetics/metabolism

@article {pmid41569151,
year = {2026},
author = {Zhang, W and Kong, J and Zeng, Y and Su, Y and Zhang, S and Li, Y and Hu, C and Chen, Q and Xiao, Y and Lu, M},
title = {Structural plasticity enables broad cAn binding and dual activation of CRISPR-associated ribonuclease Cdn1.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41569151},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//STI2030-Major Projects/ ; 31970547//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Binding ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Adenine Nucleotides/metabolism/chemistry ; Catalytic Domain ; *Ribonucleases/metabolism/chemistry/genetics ; Oligoribonucleotides ; },
abstract = {Prokaryotes have naturally evolved diverse RNA-guided defense systems against viral infections, with the type III CRISPR-Cas systems representing the most intricate. These systems feature accessory proteins activated by cyclic oligoadenylates (cOAs) produced upon target RNA recognition, synergizing with the CRISPR-Cas machinery to defend against exogenous invaders. Typically, each accessory protein is activated by only one specific cOA type. Here, we characterize Cdn1, a type III-B CRISPR accessory protein from Psychrobacter lutiphocae, which binds to cA3, cA4, and cA6, but activated by cA4 and cA6 with different efficacies to catalyze ssRNA cleavage. Combined structural and biochemical analyses reveal that cOA binding triggers dramatic conformational reorganization, including the formation of a dimerization interface of nuclease domains, the emergence of substrate binding cleft, and the reconstruction of a metal-dependent catalytic center essential for RNA cleavage. This dual activation mechanism illustrates evolutionary innovation within CRISPR-associated Rossman-fold nucleases. We propose that such structural plasticity evolved to maximize defensive resilience during microbial competition and horizontal gene transfer, while preserving broad-spectrum antiviral ability. These findings not only elucidate the activation mechanisms of Cdn1 within the type III systems but also underscore the functional complexity and adaptability of CRISPR-Cas ancillary proteins.},
}

*CRISPR-Cas Systems/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Protein Binding
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Models, Molecular
Adenine Nucleotides/metabolism/chemistry
Catalytic Domain
*Ribonucleases/metabolism/chemistry/genetics
Oligoribonucleotides

@article {pmid41568169,
year = {2026},
author = {Zhang, W and Wang, H and Liu, D and Mao, X and Zhang, Y and Yang, Y and Liu, Z and Pan, T and Liu, Y and Zhang, Q},
title = {Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102811},
pmid = {41568169},
issn = {2162-2531},
abstract = {The CRISPR-Cas13 system exhibits potent RNA cleavage activity and has been widely utilized for RNA-targeting applications. However, its collateral cleavage of bystander RNAs limits in vivo therapeutic applications. In this study, we generated a series of LwaCas13a mutants through structure-based design and site-directed mutagenesis strategies. A triple mutant enCas13a (Q521R/E796A/E810A) was obtained with significantly enhanced target RNA cleavage activity along with only slightly increased collateral activity. To reduce the collateral activity, we optimized crRNA terminal extensions and obtained M1crRNA and M3crRNA variants that, in combination with enCas13a, maintained or reduced collateral activity while preserving enhanced targeted cleavage activity. Thus, by optimizing the Cas protein and crRNA, we have created an improved CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity. This system demonstrated superior performance in targeting endogenous genes and antiviral applications. Mechanistic studies revealed that enhanced protein-crRNA interactions and altered complex conformations underlie the improved cleavage activity. This engineering approach provides a generalizable strategy for developing CRISPR-Cas systems with enhanced therapeutic potential.},
}

@article {pmid41564866,
year = {2026},
author = {Watterson, A and Picco, G and Veninga, V and Samarakoon, Y and Cattaneo, CM and Vieira, SF and Karakoc, E and Bhosle, S and Battaglia, TW and Consonni, S and Halim, TYF and Voest, EE and Garnett, MJ and Coelho, MA},
title = {CRISPR screens in the context of immune selection identify CHD1 and MAP3K7 as mediators of cancer immunotherapy resistance.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102565},
doi = {10.1016/j.xcrm.2025.102565},
pmid = {41564866},
issn = {2666-3791},
mesh = {Humans ; Animals ; *Immunotherapy/methods ; Mice ; *MAP Kinase Kinase Kinases/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; CD8-Positive T-Lymphocytes/immunology ; *Drug Resistance, Neoplasm/genetics ; *Neoplasms/immunology/therapy/genetics ; Interferon-gamma ; Melanoma/immunology/genetics ; Mice, Inbred C57BL ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here, we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR-Cas9 knockout screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 (encoding TAK1) potentiates the transcriptional response to IFN-γ, thereby creating an acquired vulnerability by sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade is more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and is associated with elevated intra-tumoral CD8[+] T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer, and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.},
}

Humans
Animals
*Immunotherapy/methods
Mice
*MAP Kinase Kinase Kinases/genetics/metabolism
*DNA Helicases/genetics/metabolism
*CRISPR-Cas Systems/genetics
*DNA-Binding Proteins/genetics/metabolism
Cell Line, Tumor
Immune Checkpoint Inhibitors/pharmacology/therapeutic use
CD8-Positive T-Lymphocytes/immunology
*Drug Resistance, Neoplasm/genetics
*Neoplasms/immunology/therapy/genetics
Interferon-gamma
Melanoma/immunology/genetics
Mice, Inbred C57BL
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41564857,
year = {2026},
author = {Balke-Want, H and Keerthi, V and Del Carmen Arenas, M and Chen, Y and Malipatlolla, M and Klysz, DD and Xu, P and Ho, K and Asano, K and Stahl, D and Huang, J and Retherford, A and Patel, S and Fowler, C and Maas, L and Gkitsas-Long, N and Jiang, Q and Liu, X and Ullrich, R and George, J and Heitzeneder, S and Tunuguntla, R and Sage, J and Sotillo, E and Mackall, CL and Feldman, SA},
title = {c-JUN enhances CRISPR knockin anti-B7-H3 CAR T cell function in small cell lung cancer and thoracic SMARCA4-deficient undifferentiated tumors.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102549},
doi = {10.1016/j.xcrm.2025.102549},
pmid = {41564857},
issn = {2666-3791},
mesh = {Humans ; *DNA Helicases/deficiency/genetics/metabolism ; *Lung Neoplasms/immunology/pathology/genetics/therapy/metabolism ; *Nuclear Proteins/deficiency/genetics/metabolism ; *Transcription Factors/deficiency/genetics/metabolism ; *Small Cell Lung Carcinoma/immunology/pathology/therapy/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-jun/metabolism/genetics ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive/methods ; Animals ; },
abstract = {Small cell lung cancer (SCLC), a highly lethal disease, limits T cell responses by downregulating major histocompatibility (MHC) class I molecules. Because chimeric antigen receptor (CAR) T cells are not MHC restricted, they may provide a powerful strategy against SCLC. However, few CAR targets for SCLC are known. Here, we show that B7-H3/CD276 is expressed in SCLC and thoracic SMARCA4-deficient undifferentiated tumors (UTs) that can clinicopathologically mimic SCLC. Thoracic SMARCA4-deficient UTs limit killing by B7-H3 CAR T cells via secretion of transforming growth factor β1 (TGF-β1). To overcome tumor-driven CAR T cell suppression, we knock in c-JUN alongside a B7-H3 CAR into the TRAC locus of primary human T cells utilizing CRISPR-Cas9. Non-viral c-JUN+B7-H3 CAR T cells show enhanced killing of both SCLC cells with low antigen density and thoracic SMARCA4-deficient UTs, providing a platform to address these highly aggressive entities. We also provide evidence that good manufacturing practice (GMP) clinical-scale manufacturing is feasible for c-JUN+B7-H3 CAR T cells.},
}

Humans
*DNA Helicases/deficiency/genetics/metabolism
*Lung Neoplasms/immunology/pathology/genetics/therapy/metabolism
*Nuclear Proteins/deficiency/genetics/metabolism
*Transcription Factors/deficiency/genetics/metabolism
*Small Cell Lung Carcinoma/immunology/pathology/therapy/genetics/metabolism
CRISPR-Cas Systems/genetics
*T-Lymphocytes/immunology/metabolism
Cell Line, Tumor
*Proto-Oncogene Proteins c-jun/metabolism/genetics
*Receptors, Chimeric Antigen/metabolism/immunology
Immunotherapy, Adoptive/methods
Animals

@article {pmid41535129,
year = {2026},
author = {Du, J and Pu, X and Yuan, T and Peng, F and Hu, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Zhu, X and Chen, W and Xie, Q and Jiang, L and Xiong, E and Yang, R},
title = {Plug-and-Play Photo-Initiated CRISPR-Cas12a One-Pot Nucleic Acid Detection via Universal Repeat RNA Acylation Strategy.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2136-2145},
doi = {10.1021/acs.analchem.5c05769},
pmid = {41535129},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Acylation ; *RNA/genetics/analysis/chemistry ; Humans ; Gene Editing ; CRISPR-Associated Proteins/metabolism ; Photochemical Processes ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Precise spatiotemporal control of CRISPR activity is central to both accurate gene editing and sensitive molecular diagnostics. However, current regulatory strategies are often sequence-specific, labor-intensive, and difficult to generalize. Here, we report a minimalist plug-and-play tactic: acylation of the repeat region (rRNA) of a split crRNA with photolabile groups. Because the modification is introduced post-synthesis and is independent of the spacer region (sRNA), every rRNA, regardless of its target sequence, can be activated by light irradiation alone, entirely eliminating the need for redesign or reoptimization. Integrating the photo-initiated CRISPR-Cas12a system with recombinase polymerase amplification into a one-pot format yields an upgraded platform, named POIROTv2 (PhotO-Initiated CRISPR-Cas12a system for Robust One-pot Testing, version 2). POIROTv2 achieves a 100-fold sensitivity gain over conventional always-on Cas12a-based one-pot assays and matches the analytical performance of a two-step assay while remaining a more streamlined and potentially faster detection process and avoiding the risk of aerosol contamination. In clinical validation with HCMV- and EBV-suspected samples, POIROTv2 delivered diagnostic accuracy statistically indistinguishable from that of gold-standard qPCR, highlighting its potential for robust and sensitive molecular diagnostics. Overall, the strategy opens up exciting possibilities for applications in infectious virus diagnostics and has broad prospects in the field of spatiotemporally controllable gene editing.},
}

*CRISPR-Cas Systems/genetics
Acylation
*RNA/genetics/analysis/chemistry
Humans
Gene Editing
CRISPR-Associated Proteins/metabolism
Photochemical Processes
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41533833,
year = {2026},
author = {Chen, X and Mao, C and Gao, Y and Shi, C and Wang, Y and Jin, Z and Xia, B and Zhou, Y},
title = {Ultrasensitive Detection of Cardiac Troponin I via CRISPR/Cas12a-Mediated Liposomal Amplification Coupled with Electrospray Ionization Mass Spectrometry.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2183-2190},
doi = {10.1021/acs.analchem.5c05804},
pmid = {41533833},
issn = {1520-6882},
mesh = {*Troponin I/blood/analysis ; Humans ; *Spectrometry, Mass, Electrospray Ionization/methods ; *CRISPR-Cas Systems ; *Liposomes/chemistry ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Direct quantitative analysis of low-abundance protein biomarkers by electrospray ionization mass spectrometry (ESI-MS) remains challenging due to poor ionization efficiency and matrix interferences. Herein, we report an ultrasensitive analytical platform, termed CRISPR/Cas12a-mediated liposomal amplification coupled with electrospray ionization mass spectrometry (CMLA-MS), that overcomes this limitation by integrating CRISPR/Cas12a-mediated dual-cascade signal amplification with an ESI-MS readout. The strategy converts the detection of poorly ionizable protein molecules into the quantification of numerous, highly ionizable small-molecule reporters: proteins trigger Cas12a trans-cleavage (first amplification), which subsequently cleaves single-stranded DNA (ssDNA) probes anchored to signal-loaded liposomes, causing the burst release of thousands of MS-detectable reporters (second, physical amplification). This dual-amplification strategy enabled an exceptionally low limit of detection (LOD) of 10.8 fg/mL, and the method successfully quantified cardiac troponin I (cTnI) in clinical serum samples with high recoveries (90.3-101.6%).},
}

*Troponin I/blood/analysis
Humans
*Spectrometry, Mass, Electrospray Ionization/methods
*CRISPR-Cas Systems
*Liposomes/chemistry
Limit of Detection
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41512333,
year = {2026},
author = {Zhao, J and Sui, Z and Chen, B and Wu, R and Xu, J and Dong, H},
title = {Customizable NAND Logic-Gate Biosensing System Enabled by an Engineered Methylation-CRISPR/Cas12a Consensus Sequence for Ultrasensitive DNA Methyltransferase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2368-2378},
doi = {10.1021/acs.analchem.5c06772},
pmid = {41512333},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism/analysis ; Limit of Detection ; Humans ; Logic ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methyltransferases (MTases) play crucial roles in epigenetic regulation, and their abnormal activity is closely associated with various human diseases. Here, we report a customizable NAND logic-gate biosensing platform for highly sensitive and intelligent detection of DNA adenine methyltransferase (Dam MTase). An engineered methylation-CRISPR/Cas12a consensus sequence (MCCS, 5'-TTTGATC-3') was rationally designed to integrate the Cas12a PAM site, Dam methylation site, and DpnI recognition sequence into a unified functional motif. Coupled with a primer-triggered hybridization chain reaction (HCR), multiple tandem MCCS units were generated to amplify the fluorescence signal output. In this logic circuit, Dam, SAM, and DpnI serve as three biochemical inputs, and their combined presence ("1,1,1") yields a low-fluorescence "OFF" output according to the NAND logic rule. The system exhibited a broad linear detection range with an ultralow detection limit of 0.00032 U mL[-1], outstanding selectivity toward nontarget MTases, and satisfactory recoveries (98.16-100.03%) in human serum samples. Furthermore, it enabled quantitative evaluation of Dam inhibitors, revealing IC50 values of 1.75 μM for 5-fluorouracil and 11.9 μM for penicillin G. This strategy provides a universal molecular computation-driven biosensing framework for enzyme activity analysis and inhibitor screening in complex biological systems.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism/analysis
Limit of Detection
Humans
Logic
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41511442,
year = {2026},
author = {Li, Y and Li, X and Chen, Y and Wang, Y and Zuo, Z},
title = {Differential Allosteric Modulation of Cas9 Specificity.},
journal = {Journal of chemical theory and computation},
volume = {22},
number = {2},
pages = {806-817},
doi = {10.1021/acs.jctc.5c01919},
pmid = {41511442},
issn = {1549-9626},
mesh = {Allosteric Regulation ; Molecular Dynamics Simulation ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Both RNA- and protein-based strategies have been developed to mitigate off-target cleavage by CRISPR-Cas9, yielding noncanonical guide RNAs (gRNAs) and Cas9 variants with enhanced gene-editing precision. However, the molecular mechanisms by which such PAM-distal alterations─remote from the nuclease centers─modulate Cas9 activity and specificity remain incompletely understood. Here, we performed near-millisecond all-atom molecular dynamics simulations to elucidate how diverse PAM-distal perturbations─including gRNA truncation, base mismatching, and evolved mutations─reshape the conformational dynamics and allosteric regulation of Cas9. Despite their distinct origins, all perturbations ultimately modulate Cas9 function by altering HNH dynamics that impede the transition from the checkpoint to the catalytically active state, yet they do so through distinct allosteric routes. The 16-nt gRNA induces a pronounced REC3 reorientation toward the L2 linker and HNH domain, while PAM-distal mismatches with the 18-nt gRNA promote engagement of the unwound target DNA strand with L2─both effectively restraining HNH rotation. In contrast, evolved mutations remodel the global motional modes so that REC2 swivels inward, constraining the HNH flexibility. These perturbations delineate multiple structural paths converging on a shared allosteric outcome─HNH immobilization and catalytic suppression─thereby unifying RNA-, DNA-, and protein-level effects within a single dynamic framework linking distal structural perturbations to activity control. This work provides mechanistic insight into the regulation of Cas9 fidelity and offers principles for the design of next-generation genome editors.},
}

Allosteric Regulation
Molecular Dynamics Simulation
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
CRISPR-Cas Systems
Mutation

@article {pmid41475353,
year = {2026},
author = {Marks, D and Garcia, E and Kumar, S and Tyson, K and Koch, C and Ivanov, AP and Edel, JB and Mirza, HB and Flanagan, W and Dunsby, C and French, PMW and McNeish, IA},
title = {Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101270},
doi = {10.1016/j.crmeth.2025.101270},
pmid = {41475353},
issn = {2667-2375},
mesh = {Female ; Humans ; *Ovarian Neoplasms/drug therapy/pathology/metabolism/genetics ; *Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *Poly(ADP-ribose) Polymerases/metabolism ; *Poly (ADP-Ribose) Polymerase-1/metabolism/genetics ; },
abstract = {Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood, as current experimental methodologies lack resolution and throughput. To address this, we develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells. This biosensor enables real-time, single-cell analysis of PARP trapping dynamics. Using fluorescence lifetime imaging microscopy (FLIM), we reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe reduced trapping in PARPi-resistant models in vitro and in vivo. This biosensor provides critical insights into PARPi resistance mechanisms, with implications for developing more effective therapies and advancing personalized treatment for ovarian cancer patients.},
}

Female
Humans
*Ovarian Neoplasms/drug therapy/pathology/metabolism/genetics
*Fluorescence Resonance Energy Transfer/methods
*Biosensing Techniques/methods
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
Cell Line, Tumor
*CRISPR-Cas Systems/genetics
Animals
Mice
*Poly(ADP-ribose) Polymerases/metabolism
*Poly (ADP-Ribose) Polymerase-1/metabolism/genetics

@article {pmid41436498,
year = {2025},
author = {Jiang, J and Jiang, Z and Luo, Q and Chen, X and Zhuang, J and Chen, J and Mu, Q and Qiu, J and Li, Y and Chen, S and Zhang, P and Yu, K and Chen, S and Liu, GS and Zhuang, J},
title = {Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening.},
journal = {Cell death & disease},
volume = {17},
number = {1},
pages = {128},
pmid = {41436498},
issn = {2041-4889},
support = {82472143//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82372131//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024A1515012562//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; GNT2029648//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {*Topotecan/pharmacology/therapeutic use ; Humans ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Retinoblastoma/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; Cell Line, Tumor ; Topoisomerase I Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Apoptosis/drug effects ; *Retinal Neoplasms/genetics/drug therapy/pathology ; Female ; },
abstract = {The topoisomerase I inhibitor topotecan is an effective chemotherapeutic agent for retinoblastoma; however, treatment resistance remains a major clinical challenge, and its mechanisms remain elusive. Using genome-wide CRISPR-Cas9 knockout screening, we identified ELF2 as a key gene involved in topotecan resistance. Here, we show that surviving retinoblastoma cells exposed to topotecan showed progressively decreased ELF2 expression, accompanied by reduced apoptosis. In a mouse xenograft model, ELF2 disruption diminished the antitumor efficacy of topotecan, with ELF2-knockout cells exhibiting reduced topotecan-induced apoptosis. RNA sequencing further revealed that the MT-CYB pathway, associated with ATP synthesis, contributes to ELF2-mediated resistance. Importantly, clinical analysis demonstrated a correlation between ELF2 expression and tumor volume in retinoblastoma patients treated with topotecan. Together, these findings interrogate the mechanisms underlying topotecan resistance in retinoblastoma and suggest ELF2 as a potential therapeutic target to overcome drug resistance.},
}

*Topotecan/pharmacology/therapeutic use
Humans
*Drug Resistance, Neoplasm/genetics/drug effects
*Retinoblastoma/genetics/drug therapy/pathology/metabolism
*CRISPR-Cas Systems/genetics
Animals
Mice
Cell Line, Tumor
Topoisomerase I Inhibitors/pharmacology
Xenograft Model Antitumor Assays
Apoptosis/drug effects
*Retinal Neoplasms/genetics/drug therapy/pathology
Female

@article {pmid41427723,
year = {2026},
author = {Ortiz-Severin, J and Geoffroy, P and Aravena, P and Hodar, C and Palma, DE and González, M and Cambiazo, V},
title = {Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0156025},
pmid = {41427723},
issn = {1098-5336},
support = {1211893//ANID-Fondecyt/ ; 2024T2DID//Doctoral Fellowship/ ; ICN2021_044//Millennium Science Initiative Program/ ; },
mesh = {*Piscirickettsia/genetics ; Fish Diseases/microbiology ; Piscirickettsiaceae Infections/microbiology/veterinary ; Animals ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics ; Gene Silencing ; },
abstract = {UNLABELLED: Piscirickettsia salmonis is the causative agent of salmonid rickettsial septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression of dCas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains when the expression of the fur gRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Fe[2+] and Fe[3+] acquisition systems and iron mobilization in the fur1 knockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system in P. salmonis has been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.

IMPORTANCE: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.},
}

*Piscirickettsia/genetics
Fish Diseases/microbiology
Piscirickettsiaceae Infections/microbiology/veterinary
Animals
*CRISPR-Cas Systems
Bacterial Proteins/genetics
Gene Silencing

@article {pmid41423621,
year = {2025},
author = {Goswami, SG and Gupta, P and Arvinden, VR and Bhargava, N and Iyer, AR and Saravanakumar, V and Yadav, P and Jha, SK and Singh, S and Kumar, A and Singh, P and Gunda, P and Jain, S and Mehta, P and Nakamura, Y and Kurita, R and Bajaj, A and Ramalingam, S},
title = {CRISPR editing of HPFH3 genotype induces γ-globin expression and reverses sickle cell disease and β-thalassemia phenotypes.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {46},
pmid = {41423621},
issn = {1757-6512},
mesh = {*beta-Thalassemia/genetics/therapy/pathology/metabolism ; *Anemia, Sickle Cell/genetics/therapy/pathology/metabolism ; Humans ; *gamma-Globins/genetics/metabolism ; Animals ; *Gene Editing/methods ; *Fetal Hemoglobin/genetics/metabolism ; CRISPR-Cas Systems ; Genotype ; Mice ; Phenotype ; Hematopoietic Stem Cells/metabolism ; },
abstract = {BACKGROUND: Hereditary persistence of Fetal Hemoglobin (HPFH) is a benign condition known to mitigate symptoms in individuals with co-inherited β-hemoglobinopathies, such as β-thalassemia (BT) and sickle cell disease (SCD), through the reactivation of fetal hemoglobin (HbF). HPFH typically arises from deletions of varying sizes affecting the β-globin gene cluster or point mutations in the promoters of the γ-globin genes. While the therapeutic benefits of point mutations have been extensively studied, the potential of deletional forms of HPFH remains underexplored in preclinical settings.

METHOD: In this study, we generated benign deletional HPFH3 genotype in SCD and BT patient-derived HSPCs using CRISPR/Cas9 and showed that therapeutically relevant levels of HbF reactivation result in the alleviation of the pathological phenotypes.

RESULTS: In edited cells derived from SCD patients, we observed reduced sickling and oxidative stress, while in edited from BT cells, restoration of the α-globin/β-globin ratio improved erythroid lineage maturation and reduced ROS levels. Importantly, HPFH3-edited HSPCs retained their genome integrity and showed no detrimental effect on their regeneration or differentiation into erythroid, myeloid, T, and B cell lineages in immunodeficient NBSGW mice post-xenotransplantation. Additionally, we showed a reduced interaction between the LCR and HBB, suggesting that the HPFH3 deletion specifically promoted LCR interactions with HBG1/2, likely due to the absence of the HBB locus.

CONCLUSIONS: Collectively, our preclinical findings suggest that the generation of the HPFH3 genotype has the potential to significantly enhance HbF levels, offering a promising universal therapeutic strategy for treating both SCD and β-thalassemia.},
}

*beta-Thalassemia/genetics/therapy/pathology/metabolism
*Anemia, Sickle Cell/genetics/therapy/pathology/metabolism
Humans
*gamma-Globins/genetics/metabolism
Animals
*Gene Editing/methods
*Fetal Hemoglobin/genetics/metabolism
CRISPR-Cas Systems
Genotype
Mice
Phenotype
Hematopoietic Stem Cells/metabolism

@article {pmid41344324,
year = {2026},
author = {Djamshidi, M and Hill, A and Heshmatzad, K and Langley, J and Krowicki, H and Ali, M and Yang, Y and Tanida, R and Abdul-Careem, MF and Billon, P and Riabowol, K},
title = {FAME-CRISPR improves CRISPR-Cas9 genome editing via HDAC inhibition and engineered virus-like particle delivery.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101248},
doi = {10.1016/j.crmeth.2025.101248},
pmid = {41344324},
issn = {2667-2375},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Histone Deacetylase Inhibitors/pharmacology ; *Virion/genetics ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {CRISPR-mediated gene editing using engineered virus-like particles (eVLPs) can achieve high efficiency, but performance varies with reduced effectiveness often seen in primary cells or when generating polyclonal models at scale. We developed a faster, accurate and 4-fold more efficient CRISPR-Cas9 (FAME-CRISPR) method using pan-histone deacetylase inhibitors with eVLP transduction compared to previous reports using other histone deacetylase inhibitors. Combined optimization of pan-HDACi treatment with eVLP enhanced double-strand break (DSB)-mediated CRISPR and base editing gave significantly edited populations within 2- to 3-cell mean population doublings, reducing the need for post-editing selection in immortalized cancer cells and in primary diploid fibroblasts that have limited replicative lifespans.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
*Histone Deacetylase Inhibitors/pharmacology
*Virion/genetics
DNA Breaks, Double-Stranded
HEK293 Cells

@article {pmid41208252,
year = {2026},
author = {Patterson, FM and Nguyen Tran, MT and Guinan, T and Mohd Khalid, MKN and Kc, R and Fairfax, KA and Liu, GS and Cook, AL and Hung, SS and Hewitt, AW},
title = {Generalizable features of pegRNA design for prime editing of inherited retinal diseases.},
journal = {Ophthalmic genetics},
volume = {47},
number = {1},
pages = {59-66},
doi = {10.1080/13816810.2025.2576786},
pmid = {41208252},
issn = {1744-5094},
mesh = {*Gene Editing/methods ; Humans ; *Retinal Diseases/genetics/therapy ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND AND OBJECTIVES: The variety of ocular cell types involved in inherited retinal disease (IRD) necessitates the use of gene editing therapeutics which have generalizable components. In our study, we investigate the generalizable characteristics of non-engineered pegRNA design (PE2) for efficient, proof-in-principle gene correction of over 21 genes implicated in IRDs and associated syndromes. We use a single-transgene oligopool approach, comprising approximately 12,000 uniquely barcoded pegRNAs that target a synthetically integrated, 50 bp sequence motif, which faithfully recapitulate the disease context of their various counterpart IRDs. Using this approach, we perform a high throughput, pooled analysis of pegRNA characteristics across non- and ocular cell types to propose a cell-line agnostic set of pegRNA design guidelines.

RESULTS: Briefly, we find that non-engineered pegRNA 3' extensions should mediate substitution-type edits and that the desired edit should be placed within five nucleotides upstream of the nick site induced by the Cas-endonuclease. Further, PBS and RTT lengths of at least 12 and 14 nucleotides, respectively, should be used and each non-engineered pegRNA 3' extension should obviate an initial templating cytosine nucleotide.

CONCLUSION: We establish a set of recommendations for the generalizable design of the non-engineered pegRNA 3' extension for the correction of several IRDs, enabling overall simplification of design parameters for PE2-based systems.},
}

*Gene Editing/methods
Humans
*Retinal Diseases/genetics/therapy
*Genetic Therapy/methods
CRISPR-Cas Systems

@article {pmid41084330,
year = {2026},
author = {Huppertz, F and Caturelli, MS and Lehmann, LS and Kurth, F and Maier, AG and Matuschewski, K},
title = {Plasmodium falciparum gametogenesis essential protein 1 (GEP1) is a transmission-blocking target.},
journal = {FEBS letters},
volume = {600},
number = {2},
pages = {239-250},
pmid = {41084330},
issn = {1873-3468},
support = {IRTG2290//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects/metabolism/growth & development ; Animals ; *Protozoan Proteins/genetics/metabolism ; *Gametogenesis/genetics/drug effects ; Mice ; Humans ; *Malaria, Falciparum/transmission/parasitology ; Female ; Male ; Xanthurenates/pharmacology ; Anopheles/parasitology ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; },
abstract = {Transmission of Plasmodium parasites to Anopheles mosquitoes relies on rapid activation of mature gametocytes in the midgut, triggered by a temperature drop and xanthurenic acid. In Plasmodium yoelii, the gametogenesis essential protein 1 (GEP1) was linked to xanthurenic acid (XA)-dependent gamete activation. We characterized GEP1 in Plasmodium falciparum using CRISPR-Cas9 to create PfGEP1 loss-of-function lines. These lines failed to undergo male or female gametogenesis, even when stimulated by XA or a temperature drop. The defect persisted despite treatment with the phosphodiesterase inhibitor Zaprinast. Analysis of field samples revealed two GEP1 single-nucleotide polymorphisms (V241L and S263P) in 12% and 20% of 49 cases. Our findings confirm GEP1's essential role in gamete activation, highlight an XA-independent function, and support its potential as a transmission-blocking target. Impact statement For sustainable malaria control, transmission-blocking drug targets are urgently needed. Work in murine models showed that GEP1 is a candidate. We show complete block of life cycle progression of the human malarial parasite Plasmodium falciparum when GEP1 is deleted, warranting targeted drug development to achieve gamete-free mosquito blood meals.},
}

*Plasmodium falciparum/genetics/drug effects/metabolism/growth & development
Animals
*Protozoan Proteins/genetics/metabolism
*Gametogenesis/genetics/drug effects
Mice
Humans
*Malaria, Falciparum/transmission/parasitology
Female
Male
Xanthurenates/pharmacology
Anopheles/parasitology
CRISPR-Cas Systems
Polymorphism, Single Nucleotide

@article {pmid41039747,
year = {2026},
author = {Chen, L and Huang, Q and Liu, Y and Chen, K and Yang, Q and Tang, H and Wang, D and Tang, Z},
title = {A Galactose-Engineered Dual-Responsive Nanocarrier for ASO/CRISPR-Cas9 Delivery to Inhibit HBV Replication.},
journal = {Advanced healthcare materials},
volume = {15},
number = {4},
pages = {e02835},
doi = {10.1002/adhm.202502835},
pmid = {41039747},
issn = {2192-2659},
support = {2023MD734126//China Postdoctoral Science Foundation/ ; BJRC202426//Chongqing Medical University Top Talent Cultivation Program/ ; CSTB2024NSCQ-MSX0202//Natural Science Foundation of Chongqing/ ; 2022COBSHTB3003//Chongqing Municipal Key Research and Development Program of China/ ; },
mesh = {*Hepatitis B virus/physiology/drug effects ; Animals ; *CRISPR-Cas Systems/genetics ; *Virus Replication/drug effects ; Humans ; Mice ; *Oligonucleotides, Antisense/pharmacology/chemistry ; *Galactose/chemistry ; Hepatitis B/virology ; Gene Editing ; *Nanoparticles/chemistry ; *Drug Carriers/chemistry ; Hep G2 Cells ; },
abstract = {Complete hepatitis B virus (HBV) cure is hindered primarily by the stable persistence of covalently closed circular DNA (cccDNA). Gene editing approaches to eradicate HBV by targeting cccDNA face challenges and limitations due to suboptimal editing efficiency and substantial off-target effects. Herein, a combinatorial therapeutic strategy is developed that integrates CRISPR/Cas9-mediated cccDNA disruption with an antisense oligonucleotide (ASO)-targeted degradation of pregenomic RNA (pgRNA). To overcome delivery challenges, a hepatocyte-targeting nanocarrier (UACPG) is engineered, featuring low immunogenicity, high payload capacity, and dual-stimuli responsiveness. The UACPG platform enabled liver-specific delivery through surface-conjugated targeting ligands, followed by on-demand release of Cas9 ribonucleoprotein complexes and ASO via RNase H-dependent degradation and near-infrared (NIR) light activation. The results demonstrated that UACPG can effectively reduce HBV replication and viral antigen levels, while significantly lowering cccDNA in hydrodynamic HBV-infected mouse models, with no significant off-target effects observed. This nanocarrier achieved the spatiotemporally controlled release of gene-editing systems in vitro and in vivo, significantly inhibiting the replication of HBV, thereby establishing an innovative technological platform for developing curative HBV therapies.},
}

*Hepatitis B virus/physiology/drug effects
Animals
*CRISPR-Cas Systems/genetics
*Virus Replication/drug effects
Humans
Mice
*Oligonucleotides, Antisense/pharmacology/chemistry
*Galactose/chemistry
Hepatitis B/virology
Gene Editing
*Nanoparticles/chemistry
*Drug Carriers/chemistry
Hep G2 Cells

@article {pmid40372972,
year = {2025},
author = {Sobral, LM and Walker, FM and Madhavan, K and Janko, E and Donthula, S and Danis, E and Bompada, P and Balakrishnan, I and Wang, D and Pierce, A and Haag, MM and Carstens, BJ and Serkova, NJ and Foreman, NK and Venkataraman, S and Veo, B and Vibhakar, R and Dahl, NA},
title = {Targeting processive transcription for Myc-driven circuitry in medulloblastoma.},
journal = {Neuro-oncology},
volume = {27},
number = {10},
pages = {2697-2710},
doi = {10.1093/neuonc/noaf121},
pmid = {40372972},
issn = {1523-5866},
support = {K08 NS121592/NS/NINDS NIH HHS/United States ; P30 CA046934/CA/NCI NIH HHS/United States ; R01 NS091219/NS/NINDS NIH HHS/United States ; S10 OD023485/OD/NIH HHS/United States ; K08NS121592/NS/NINDS NIH HHS/United States ; R01NS091219/NS/NINDS NIH HHS/United States ; MUJP.2021.003//University of Colorado Cancer Center/Molecular, Cellular, and Developmental Biology/ ; P30 CA046934/CA/NCI NIH HHS/United States ; S10 OD023485/CD/ODCDC CDC HHS/United States ; },
mesh = {*Medulloblastoma/genetics/pathology/drug therapy ; Humans ; Animals ; Mice ; *Proto-Oncogene Proteins c-myc/genetics ; *Cerebellar Neoplasms/genetics/pathology/drug therapy ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; *Gene Expression Regulation, Neoplastic/drug effects ; *Transcription, Genetic ; Tumor Cells, Cultured ; Cyclin-Dependent Kinase 9/antagonists & inhibitors ; Cell Proliferation ; },
abstract = {BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB.

METHODS: Independent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate the effect on Myc-driven programs and tumor growth.

RESULTS: Eight CRISPR-Cas9 screens performed across 3 independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting a potent antitumor effect.

CONCLUSIONS: Our findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB.},
}

*Medulloblastoma/genetics/pathology/drug therapy
Humans
Animals
Mice
*Proto-Oncogene Proteins c-myc/genetics
*Cerebellar Neoplasms/genetics/pathology/drug therapy
CRISPR-Cas Systems
Xenograft Model Antitumor Assays
*Gene Expression Regulation, Neoplastic/drug effects
*Transcription, Genetic
Tumor Cells, Cultured
Cyclin-Dependent Kinase 9/antagonists & inhibitors
Cell Proliferation

@article {pmid41564380,
year = {2026},
author = {Myojin, Y and Kodama, T and Takahashi, R and Nagasawa, H and Kondo, Y and Yusa, K and Yoshida-Hashidate, T and Shindou, H and Furuta, K and Murai, K and Saito, Y and Hikita, H and Takehara, T},
title = {Genome-wide CRISPR screen identifies ACSL3 as a regulator of lipotoxicity and progression of MASLD.},
journal = {Hepatology communications},
volume = {10},
number = {2},
pages = {},
pmid = {41564380},
issn = {2471-254X},
mesh = {Humans ; *Coenzyme A Ligases/genetics/metabolism ; Long-Chain-Fatty-Acid-CoA Ligase ; Hepatocytes/metabolism ; Disease Progression ; Endoplasmic Reticulum Stress/genetics ; Apoptosis/genetics ; *Fatty Liver/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Animals ; Oxidative Stress ; Mice ; Male ; Liver/pathology/metabolism ; Lipid Metabolism ; Lipogenesis ; },
abstract = {BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, metabolic dysfunction-associated steatohepatitis, are highly prevalent and lack effective pharmacotherapies. Hepatocellular lipotoxicity-driven by the accumulation of saturated fatty acids (eg, palmitate)-promotes disease progression; however, the determinants of hepatocyte susceptibility remain incompletely defined.

METHODS: We performed a genome-wide CRISPR-Cas9 loss-of-function screening to identify the regulators of palmitate-induced lipotoxicity. The top candidates were validated using genetic perturbation and pharmacological inhibition. Lipid handling, endoplasmic reticulum/oxidative stress, apoptosis, and lipogenic transcriptional programs were also quantified. Human MASLD liver tissues were analyzed for ACSL3 expression in relation to histology and aminotransferases. Single-cell and spatial transcriptomics were used to localize ACSL3 expression and the associated pathway signatures in metabolic dysfunction-associated steatohepatitis.

RESULTS: The screen recovered established mediators (CASPASE-8, AGPAT9, RNF213) and identified ACSL3 as a novel determinant of hepatocyte survival under lipotoxic stress. Genetic deletion or pharmacological inhibition of ACSL3 renders hepatocytes resistant to palmitate-induced apoptosis and endoplasmic reticulum stress, accompanied by reduced lipid-droplet accumulation, decreased incorporation of saturated fatty acids into neutral lipids and phospholipids, and blunted induction of lipogenic programs. In human MASLD, hepatic ACSL3 expression positively correlated with histological severity and aminotransferase levels. Single-cell transcriptomics localized ACSL3 predominantly to hepatocytes in advanced metabolic dysfunction-associated steatohepatitis displaying oxidative and endoplasmic reticulum stress signatures, whereas spatial transcriptomics showed ACSL3-high hepatocyte regions enriched for apoptotic and inflammatory pathways and spatially coupled to macrophage-rich and plasma cell-rich niches.

CONCLUSIONS: ACSL3 is a central regulator of lipotoxic hepatocyte injury and MASLD progression, mechanistically linking lipid-droplet biogenesis to apoptosis and inflammatory niche formation. These data suggest that ACSL3 is a promising therapeutic target and support further translational studies to evaluate ACSL3 modulation in steatotic liver disease.},
}

Humans
*Coenzyme A Ligases/genetics/metabolism
Long-Chain-Fatty-Acid-CoA Ligase
Hepatocytes/metabolism
Disease Progression
Endoplasmic Reticulum Stress/genetics
Apoptosis/genetics
*Fatty Liver/genetics/metabolism/pathology
CRISPR-Cas Systems
Animals
Oxidative Stress
Mice
Male
Liver/pathology/metabolism
Lipid Metabolism
Lipogenesis

@article {pmid41563484,
year = {2026},
author = {Pardy, F and Reblova, K and Svozilova, H and Tichy, B and Pospisilova, S and Kotaskova, J and Navrkalova, V},
title = {Assessment of long-read strategies for the enrichment of clinically relevant breakpoints in lymphomas: towards a diagnostic implementation.},
journal = {Annals of hematology},
volume = {105},
number = {2},
pages = {47},
pmid = {41563484},
issn = {1432-0584},
mesh = {Humans ; *Translocation, Genetic ; *Lymphoma/genetics/diagnosis ; *Chromosome Breakpoints ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; },
abstract = {Recurrent chromosomal translocations are hallmarks of many hematological malignancies, including lymphomas and leukemias. Accurate breakpoint detection is essential for diagnostics, treatment optimization, and disease monitoring. Long-read sequencing (Oxford Nanopore Technologies) enables unambiguous mapping and translocation identification. We designed a Cas9-based enrichment panel targeting common translocations in lymphoid malignancies. To accommodate both well-defined and promiscuous translocation partners, we employed single-side and dual-side sequencing strategies. Using well-established lymphoid cell lines, we benchmarked three enrichment approaches: (i) Cas9 read-out, (ii) Cas9 excision with multiplexing, and (iii) adaptive sampling. Cas9-mediated enrichment achieved superior on-target coverage, particularly in densely targeted regions (such as the IGH locus), while single-probe targets showed lower coverage depth. Adaptive sampling offered higher throughput, flexibility, and better pore occupancy, however with limited breakpoint detection. Cas9 excision has been demonstrated as a fast and reliable method to detect canonical translocation partners in clinical lymphoma samples. Our findings indicate that long-read enrichment strategies are suitable for targeting breakpoint hotspots, although the choice of approach depends heavily on the laboratory's specific goal. We propose a decision algorithm for selecting the optimal method based on experimental and clinical needs: Cas9-mediated enrichment suits focused diagnostic intent, while adaptive sampling is preferable for broader research use.},
}

Humans
*Translocation, Genetic
*Lymphoma/genetics/diagnosis
*Chromosome Breakpoints
Cell Line, Tumor
High-Throughput Nucleotide Sequencing/methods
CRISPR-Cas Systems

@article {pmid41491310,
year = {2026},
author = {Arena, KA and Kearns, CA and Ahmed, M and O'Rourke, R and Sagerström, CG and Franco, SJ and Appel, B},
title = {Gsx2 regulates oligodendrocyte precursor formation in the zebrafish spinal cord.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {30-44},
doi = {10.1016/j.ydbio.2026.01.001},
pmid = {41491310},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Spinal Cord/embryology/cytology/metabolism ; *Zebrafish Proteins/metabolism/genetics ; *Oligodendroglia/metabolism/cytology ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; *Oligodendrocyte Precursor Cells/metabolism/cytology ; Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; *Homeodomain Proteins/genetics/metabolism ; Gene Regulatory Networks ; Cell Lineage ; },
abstract = {Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Spinal Cord/embryology/cytology/metabolism
*Zebrafish Proteins/metabolism/genetics
*Oligodendroglia/metabolism/cytology
Cell Differentiation/genetics
Gene Expression Regulation, Developmental
*Oligodendrocyte Precursor Cells/metabolism/cytology
Neural Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
*Homeodomain Proteins/genetics/metabolism
Gene Regulatory Networks
Cell Lineage

@article {pmid41443566,
year = {2026},
author = {Ghosh, P and Wadsworth, BC and Terry, L and Evans, TA},
title = {Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {1-9},
doi = {10.1016/j.ydbio.2025.12.015},
pmid = {41443566},
issn = {1095-564X},
mesh = {Animals ; *Axon Guidance/physiology/genetics ; *Tribolium/embryology/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Axons/metabolism ; Signal Transduction ; Netrin Receptors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Biological Evolution ; CRISPR-Cas Systems ; },
abstract = {The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance are evolutionarily conserved within insects.},
}

Animals
*Axon Guidance/physiology/genetics
*Tribolium/embryology/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
*Drosophila melanogaster/embryology/genetics/metabolism
Axons/metabolism
Signal Transduction
Netrin Receptors/genetics/metabolism
Gene Expression Regulation, Developmental
Biological Evolution
CRISPR-Cas Systems

@article {pmid41435996,
year = {2026},
author = {Gopalakrishnan, R and Kannan, K and Gunasekaran, R and Ramachandran, P and Ganapathy, D and Pitchiah, S},
title = {A comparative review of vector insertion techniques in Saccharomyces cerevisiae.},
journal = {Journal of microbiological methods},
volume = {241},
number = {},
pages = {107378},
doi = {10.1016/j.mimet.2025.107378},
pmid = {41435996},
issn = {1872-8359},
mesh = {*Saccharomyces cerevisiae/genetics ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; Homologous Recombination ; *Genetic Engineering/methods ; *Mutagenesis, Insertional/methods ; Synthetic Biology/methods ; Gene Editing/methods ; Genome, Fungal ; },
abstract = {Saccharomyces cerevisiae, a model organism in genetics and molecular biology has been extensively engineered using various vector insertion techniques. This review compares and contrasts three prominent techniques: In vivo homologous recombination (HR), Cre-lox recombination and CRISPR/Cas9. In vivo HR leverages the organism's innate DNA repair machinery for easy vector integration and targeted genome modifications. Cre-lox recombination offers high specificity and efficiency at loxP sites, making it ideal for targeted gene excision or integration. CRISPR/Cas9 has revolutionized genome engineering with its precision and ability to target multiple loci simultaneously. Each technique has its strengths and limitations, including site dependency, off-target effects, and strain-specific variability. This review provides a comprehensive overview of these vector insertion techniques, highlighting their applications, advantages, and limitations in S. cerevisiae genome engineering and synthetic biology.},
}

*Saccharomyces cerevisiae/genetics
*Genetic Vectors/genetics
CRISPR-Cas Systems
Homologous Recombination
*Genetic Engineering/methods
*Mutagenesis, Insertional/methods
Synthetic Biology/methods
Gene Editing/methods
Genome, Fungal

@article {pmid41420208,
year = {2025},
author = {Holmlund, H and Yamauchi, Y and Tekayev, M and Jakobs, S and Robin, A and Fujii, W and Ward, MA},
title = {CRISPR/Cas9-mediated knock-in of the murine Y chromosomal genes Zfy1 and Zfy2.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {88},
pmid = {41420208},
issn = {1471-2164},
support = {NIH F31HD111279/GF/NIH HHS/United States ; HD114645/GF/NIH HHS/United States ; 17CON-86294//Hawai'i Community Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Male ; Mice ; *Gene Knock-In Techniques ; Spermatogenesis/genetics ; *Y Chromosome/genetics ; Testis/metabolism ; Gene Editing ; Zinc Fingers/genetics ; },
abstract = {BACKGROUND: The Y-linked mouse zinc finger genes Zfy1 and Zfy2 are critical fertility factors in mice but the mechanisms by which they regulate spermatogenesis remain unclear. We recently produced Zfy1/2 double knock-out mice and observed a complete loss in fertility. However, the biochemical mechanism by which Zfy regulates spermatogenesis is unknown, and ZFY expression has not yet been confirmed at the protein level. As both Zfy homologues share ~ 95% sequence similarity, it is difficult to produce an anti-ZFY antibody specific to either homologue.

RESULTS: To overcome this technical challenge, we used CRISPR/Cas9 genome editing to develop tagged Zfy1 knock-in (XY[Zfy1-HA]), Zfy2 knock-in (XY[Zfy2-FLAG], XY[Zfy2-3xFLAG], and XY[Zfy2-HA]), and Zfy1/2 double knock-in (XY[Zfy1-HA,Zfy2-MYC]) mice. Successful targeting was confirmed by genotyping and sequencing. The knock-in lines were fertile with normal sperm parameters. Using Western blot on testes, knock-in specific bands were detected matching the predicted ZFY expression patterns. Using immunofluorescence on testis sections from knock-in males, ZFY1 and ZFY2 expression was detected in zygotene spermatocytes, and ZFY2 expression was also detected in spermatids step 7-8 and 9.

CONCLUSIONS: These novel knock-in mice can be used in future investigations to determine how ZFY controls spermatogenesis.},
}

Animals
*CRISPR-Cas Systems
Male
Mice
*Gene Knock-In Techniques
Spermatogenesis/genetics
*Y Chromosome/genetics
Testis/metabolism
Gene Editing
Zinc Fingers/genetics

@article {pmid41160201,
year = {2026},
author = {Wang, YY and Lin, YH and Ke, CC and Lai, TH and Yu, IS and Au, CF and Singh, R and Lin, YH},
title = {Human teratozoospermia-related AGTPBP1 R791H mutation is associated with sperm head and tail defects in a CRISPR-engineered murine model.},
journal = {Journal of assisted reproduction and genetics},
volume = {43},
number = {1},
pages = {133-142},
pmid = {41160201},
issn = {1573-7330},
support = {NSTC 111-2320-B-030 -007-MY3//National Science and Technology Council/ ; CTH-109A-2215//Cardinal Tien Hospital/ ; },
mesh = {Animals ; Male ; *Teratozoospermia/genetics/pathology ; Mice ; *Sperm Head/pathology ; Humans ; *Sperm Tail/pathology ; Disease Models, Animal ; Spermatozoa/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; *Infertility, Male/genetics/pathology ; *Poly-ADP-Ribose Binding Proteins/genetics ; },
abstract = {BACKGROUND: Infertility is a pervasive global health concern affecting millions of couples worldwide. Approximately 7% of the male population is infertile. Teratozoospermia, defined by > 96% abnormal sperm morphology, is a major cause of infertility often linked to genetic defects. In our previous study, we identified three AGTPBP1 mutations (p.Glu423Asp, p.Pro631Leu, and p.Arg811His) in teratozoospermia cases. AGTPBP1 is a key enzyme involved in regulating tubulin polyglutamylation and generating Δ-2 tubulin, a major structural component of the sperm tail and an essential structure for sperm head differentiation. However, functional proof of the impact of AGTPBP1 Arg811His on sperm head and tail impairment remained unestablished.

METHODS: Knock-in mice carrying the equivalent mutation, Arg791His (R791H) corresponding to the human mutation (R811H), in the Agtpbp1 gene were generated and analyzed for sperm morphological abnormalities.

RESULTS: Sperm morphological evaluation revealed a significant increase in the proportion of morphologically abnormal sperm in the Agtpbp1[R791H/R791H] mice. Detailed morphological analysis revealed a significantly higher incidence of sperm head abnormalities and abnormal attachment of the head to the midpiece in the Agtpbp1[R791H/R791H] mice relative to wild-type controls. Further, sperm with head defects from Agtpbp1[R791H/R791H] mice exhibited abnormal accumulation of polyglutamylated tubulin within the sperm head. The mutant mice showed exactly the same morphological defects as seen in human patients and those displayed by mice lacking the complete carboxypeptidase A domain of AGTPBP1 but at a relatively lesser frequency.

CONCLUSIONS: We conclude that the R791H mutation in the Agtpbp1 gene impairs sperm head and tail differentiation, resulting in sperm morphological defects.},
}

Animals
Male
*Teratozoospermia/genetics/pathology
Mice
*Sperm Head/pathology
Humans
*Sperm Tail/pathology
Disease Models, Animal
Spermatozoa/pathology
CRISPR-Cas Systems/genetics
Mutation
*Infertility, Male/genetics/pathology
*Poly-ADP-Ribose Binding Proteins/genetics

@article {pmid41128695,
year = {2026},
author = {Plugge, SF and Ma, H and van der Vaart, JY and Sprangers, J and Morsink, FHM and Xanthakis, D and Jamieson, C and Stroot, KBW and Keijzer, AR and Margaritis, T and Candelli, T and Straver, R and de Ridder, J and Holstege, FCP and de Leng, WWJ and Offerhaus, GJA and Merenda, A and Maurice, MM},
title = {Intestinal LKB1 Loss Drives a Premalignant Program Along the Serrated Cancer Pathway.},
journal = {Gastroenterology},
volume = {170},
number = {2},
pages = {298-314},
doi = {10.1053/j.gastro.2025.07.041},
pmid = {41128695},
issn = {1528-0012},
mesh = {Humans ; *Protein Serine-Threonine Kinases/genetics/deficiency/metabolism ; Animals ; AMP-Activated Protein Kinase Kinases ; *Peutz-Jeghers Syndrome/genetics/pathology/enzymology ; Organoids/pathology ; *Colorectal Neoplasms/genetics/pathology/enzymology ; Mice ; *Precancerous Conditions/genetics/pathology/enzymology ; Signal Transduction ; ErbB Receptors/metabolism ; Mice, Knockout ; Colon/pathology/enzymology ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Disease Models, Animal ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Cell Transformation, Neoplastic/genetics/pathology ; Intestinal Mucosa/pathology ; CRISPR-Cas Systems ; AMP-Activated Protein Kinases ; },
abstract = {BACKGROUND & AIMS: Heterozygous inactivating mutations of Serine Threonine Kinase 11/Liver Kinase B1 (LKB1) are causative to the Peutz-Jeghers syndrome (PJS), a hereditary disease characterized by gastrointestinal hamartomatous polyposis and increased cancer susceptibility. Although LKB1 loss-induced polyp formation has been ascribed to nonepithelial tissues, how LKB1 deficiency increases cancer risk of patients by altering the phenotypical landscape and hierarchical organization of epithelial tissues remains poorly understood.

METHODS: Using CRISPR/Cas9, we generated heterozygous and homozygous Lkb1-deficient mouse small intestinal and human colon organoids. These organoids were characterized by an integrated approach that combines imaging, bulk and single-cell RNA sequencing, and growth factor dependency assays. Our findings were validated in human PJS-derived tissues using immunohistochemistry and linked to colorectal cancer profiles using the Cancer Genome Atlas (TCGA) cancer database.

RESULTS: Our results reveal that heterozygous Lkb1 loss is sufficient to push intestinal cells into a premalignant transcriptional program associated with serrated colorectal cancer, which is further amplified by loss of heterozygosity. This altered epithelial growth state associates with persistent features of regeneration and enhanced EGFR ligand and receptor expression, conferring niche-independent growth properties to Lkb1-deficient organoids. Moreover, our newly generated LKB1-mutant signature is enriched in sporadic serrated colorectal cancer, and synergistic cooperation of Lkb1 deficiency with mutant Kras was experimentally confirmed by assessing organoid growth properties and transcriptomes.

CONCLUSIONS: Heterozygous loss of LKB1 pushes intestinal cells into a chronic regenerative state, which is amplified on loss of heterozygosity. Lkb1 deficiency thereby generates fertile ground for serrated colorectal cancer formation in the intestine, potentially explaining the increased cancer risk observed in PJS.},
}

Humans
*Protein Serine-Threonine Kinases/genetics/deficiency/metabolism
Animals
AMP-Activated Protein Kinase Kinases
*Peutz-Jeghers Syndrome/genetics/pathology/enzymology
Organoids/pathology
*Colorectal Neoplasms/genetics/pathology/enzymology
Mice
*Precancerous Conditions/genetics/pathology/enzymology
Signal Transduction
ErbB Receptors/metabolism
Mice, Knockout
Colon/pathology/enzymology
Cell Proliferation
Gene Expression Regulation, Neoplastic
Disease Models, Animal
Proto-Oncogene Proteins p21(ras)/genetics/metabolism
*Cell Transformation, Neoplastic/genetics/pathology
Intestinal Mucosa/pathology
CRISPR-Cas Systems
AMP-Activated Protein Kinases

@article {pmid39894889,
year = {2026},
author = {Shahid, A and Zahra, A and Aslam, S and Shamim, A and Ali, WR and Aslam, B and Khan, SH and Arshad, MI},
title = {Appraisal of CRISPR Technology as an Innovative Screening to Therapeutic Toolkit for Genetic Disorders.},
journal = {Molecular biotechnology},
volume = {68},
number = {1},
pages = {71-94},
pmid = {39894889},
issn = {1559-0305},
support = {DDWP-2021//Ministry of Science and Technology, Pakistan/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Diseases, Inborn/diagnosis/therapy/genetics ; *Genetic Therapy/methods ; },
abstract = {The high frequency of genetic diseases compels the development of refined diagnostic and therapeutic systems. CRISPR is a precise genome editing tool that offers detection of genetic mutation with high sensitivity, specificity and flexibility for point-of-care testing in low resource environment. Advancements in CRISPR ushered new hope for the detection of genetic diseases. This review aims to explore the recent advances in CRISPR for the detection and treatment of genetic disorders. It delves into the advances like next-generation CRISPR diagnostics like nano-biosensors, digitalized CRISPR, and omics-integrated CRISPR technologies to enhance the detection limits and to facilitate the "lab-on-chip" technologies. Additionally, therapeutic potential of CRISPR technologies is reviewed to evaluate the implementation potential of CRISPR technologies for the treatment of hematological diseases, (sickle cell anemia and β-thalassemia), HIV, cancer, cardiovascular diseases, and neurological disorders, etc. Emerging CRISPR therapeutic approaches such as base/epigenetic editing and stem cells for the development of foreseen CRIPSR drugs are explored for the development of point-of-care testing. A combination of predictive models of artificial intelligence and machine learning with growing knowledge of genetic disorders has also been discussed to understand their role in acceleration of genetic detection. Ethical consideration are briefly discussed towards to end of review. This review provides the comprehensive insights into advances in the CRISPR diagnostics/therapeutics which are believed to pave the way for reliable, effective, and low-cost genetic testing.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Genetic Diseases, Inborn/diagnosis/therapy/genetics
*Genetic Therapy/methods

@article {pmid41562469,
year = {2026},
author = {Wan, Z and Xu, C and Wang, Y and Song, L and Yuan, W and Chen, M and Gong, R and Zhang, XE},
title = {An AND-Logic Gate-Based Biosensor for Simultaneous Detection of SARS-CoV-2 Nucleic Acids and Nucleocapsid Proteins.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07321},
pmid = {41562469},
issn = {1520-6882},
abstract = {Nucleic acids and proteins are recognized as gold standard biomarkers for disease diagnosis and pathogen detection. However, conventional single-analyte detection methods remain susceptible to false positives caused by manual operational errors or sample contamination, thereby undermining diagnostic reliability and increasing the burden on healthcare systems. To address this limitation, we developed a one-pot isothermal amplification and CRISPR-Cas cooperative system (OIACS) that functions as an AND-logic gate biosensor for the simultaneous detection of SARS-CoV-2 RNA and nucleocapsid protein. Unlike conventional methods relying solely on CRISPR RNA (crRNA) recognition, the OIACS employs antibody-mediated target binding with blocker release for target recognition, offering increased flexibility in assay design for different targets. A universal Cas12a-targetable DNA barcode is generated via strand displacement isothermal amplification, enabling signal amplification upon dual-target recognition. The OIACS assay exhibited practical utility by reliably detecting SARS-CoV-2 transcription- and replication-competent virus-like-particles at 5000 copies/mL, and the limit of detection was determined to be as low as 1698 copies/mL, highlighting its robustness and potential for clinical diagnosis.},
}

@article {pmid41562237,
year = {2026},
author = {Mattivi, C and Wang, S and Ji, L and Xiao, Q and Cao, J},
title = {HLA-Knockout: Enabling Allele-Specific Knockout of HLA Class I Genes for Immunogenic Engineering.},
journal = {HLA},
volume = {107},
number = {1},
pages = {e70548},
pmid = {41562237},
issn = {2059-2310},
support = {P30CA072720//Rutgers Cancer Institute of New Jersey/ ; //Nanjing University/ ; //Science Fund Program for Distinguished Young Scholars (Overseas)/ ; 82473308//National Natural Science Foundation of China Grant/ ; 2025300355//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *Alleles ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Histocompatibility Antigens Class I/genetics/immunology ; Receptors, Antigen, T-Cell/genetics/immunology ; },
abstract = {The interaction between T-cell receptors (TCRs) and antigenic peptides presented by HLA molecules is fundamental to adaptive immunity. However, the extreme polymorphism of HLA genes poses major challenges for transplantation, antigen discovery, immunotherapy and studies of allele-specific function. Although CRISPR/Cas9 has transformed gene editing, existing sgRNA design tools are not optimised for knockout of HLA Class I genes due to their high rates of polymorphism. To address this, we developed HLA-Knockout (https://hlaknockout.rutgers.edu), a novel web-based tool that enables precise, allele-specific targeting of HLA Class I genes. HLA-Knockout retrieves user-defined HLA sequences from the IPD-IMGT/HLA database and applies stringent design criteria, including mismatch filtering and PAM disruption analysis, to ensure high specificity and minimal off-target effects on non-target HLA Class I alleles. Using HLA-Knockout, we achieved efficient single- and double-allele HLA Class I knockouts in human cells without disrupting non-target HLA Class I alleles. Functional assays confirmed allele-specific loss of antigen-specific TCR activation, validating the platform's utility. HLA-Knockout provides a unique resource for dissecting HLA-restricted immune interactions and has broad applications in transplantation biology, autoimmunity and cancer immunotherapy.},
}

Humans
*Alleles
*Gene Knockout Techniques/methods
CRISPR-Cas Systems
*Gene Editing/methods
*Histocompatibility Antigens Class I/genetics/immunology
Receptors, Antigen, T-Cell/genetics/immunology

@article {pmid41562083,
year = {2025},
author = {Liu, J and Hong, W and Sun, Z and Zhang, S and Xue, C and Dong, N},
title = {The gut-lung axis: effects and mechanisms of gut microbiota on pulmonary diseases.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1693964},
pmid = {41562083},
issn = {1664-3224},
mesh = {Humans ; *Gastrointestinal Microbiome/immunology ; Animals ; *Lung/immunology/metabolism/microbiology ; Dysbiosis/immunology ; *Lung Diseases/microbiology/immunology/metabolism/therapy/etiology ; Fatty Acids, Volatile/metabolism ; Fecal Microbiota Transplantation ; },
abstract = {The proposal of the gut-lung axis has profoundly reshaped our understanding of the mechanisms underlying respiratory diseases. As a crucial component of this axis, the gut microbiota plays a central role in pulmonary immune regulation through inter-organ communication mediated by metabolic products. However, a systematic integration of mechanisms explaining how gut microbes achieve precise cross-organ immune regulation remains elusive. Existing research predominantly focuses on descriptive observations, such as the association between early-life microbiota dysbiosis and an increased risk of asthma and chronic obstructive pulmonary disease (COPD), as well as the frequent occurrence of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF), often accompanied by gut microbiome disruption. This paper focuses on three key gut microbial metabolites-short-chain fatty acids (SCFAs), tryptophan metabolites, and polyamines (PAs)-to examine their roles in immune regulation, maintenance of barrier function, and modulation of metabolic signaling networks. Based on the latest experimental and clinical evidence, this study systematically elucidates how dysbiosis of the gut microbiota, a key component of the gut-lung axis, crosses physiological barriers to exacerbate pulmonary inflammation. Regarding intervention strategies, probiotics, fecal microbiota transplantation (FMT), and CRISPR-Cas systems have demonstrated significant therapeutic potential in restoring gut microbial balance. Finally, this paper outlines future research directions, emphasizing the need to further explore non-invasive microbial sampling techniques, molecular interaction mechanisms of the gut-lung axis, and personalized microbiome-based diagnostic and therapeutic strategies to provide new insights for the prevention and treatment of respiratory diseases involving gut microbiota.},
}

Humans
*Gastrointestinal Microbiome/immunology
Animals
*Lung/immunology/metabolism/microbiology
Dysbiosis/immunology
*Lung Diseases/microbiology/immunology/metabolism/therapy/etiology
Fatty Acids, Volatile/metabolism
Fecal Microbiota Transplantation

@article {pmid41561392,
year = {2026},
author = {Choudhary, DK and Turgeman-Grott, I and Robinzon, S and Gophna, U},
title = {CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf047},
pmid = {41561392},
issn = {2633-6693},
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11-Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.},
}

@article {pmid41561390,
year = {2026},
author = {Fehrenbach, A and Mitrofanov, A and Backofen, R and Baumdicker, F},
title = {The complexity of multiple CRISPR arrays in strains with (co-occurring) CRISPR systems.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf042},
pmid = {41561390},
issn = {2633-6693},
abstract = {CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas-type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.},
}

@article {pmid41560876,
year = {2026},
author = {More, R and Yadav, V and Vadakedath, N},
title = {Calyptranema fuscum gen. sp. nov.: a novel cyanobacterial genus within Oculatellaceae based on polyphasic and genomic characterization.},
journal = {Current research in microbial sciences},
volume = {10},
number = {},
pages = {100542},
pmid = {41560876},
issn = {2666-5174},
abstract = {This study presents a comprehensive characterization and classification of a novel cyanobacterial isolate, strain S582, proposed as Calyptranema fuscum gen. sp. nov. within the family Oculatellaceae using an integrated polyphasic approach. Strain S582 was isolated from a lake in the Botanical Garden, Sarangpur, Chandigarh, India. Initial molecular characterization with the 16S rRNA gene revealed ≤ 94.90% of similarity with related genera and showed unique 16S-23S ITS secondary structures, indicating its delineation as a novel genus. Morphological assessment highlighted the presence of a cap-like structure called calyptra at the terminal cells, further distinguishing it from related genera. Furthermore, whole genome sequencing yielded an assembly of 7962,515 bp with GC content of 48.27%. Genome-based analysis encompassing average nucleotide identity (ANI), average amino acid identity (AAI), percentage of conserved proteins (POCP) was subsequently performed. The observed values for ANI (71.15% to 73.00%) and AAI (63.30% to 69.62%) were below the established genus-level thresholds. Phylogenetic analysis using maximum-likelihood and Bayesian inference along with phylogenomic reconstruction based on 1434 single copy core genes supported its taxonomic novelty. Functional classification revealed the presence of biosynthetic gene clusters (BGCs), tRNAs, insertion elements, CRISPR/Cas systems, and genes associated with metabolism, carbon fixation and photosynthesis. Additionally, the pangenome was constructed to study the genomic diversity of the studied isolate and related genera among the Oculatellaceae family and identified core, accessory, and singleton gene clusters. Collectively, these findings establish Calyptranema fuscum gen. sp. nov. as a novel genus within Oculatellaceae while expanding our understanding of cyanobacterial diversity and genomic potential.},
}

@article {pmid41556308,
year = {2026},
author = {Xie, Z and Jin, YS and Miller, MJ},
title = {Exploiting the Endogenous Type II-A CRISPR-Cas System for Functional Engineering of Probiotic Lacticaseibacillus rhamnosus GG.},
journal = {Microbial biotechnology},
volume = {19},
number = {1},
pages = {e70303},
pmid = {41556308},
issn = {1751-7915},
support = {//University of Illinois at Urbana-Champaign/ ; },
mesh = {*CRISPR-Cas Systems ; *Lacticaseibacillus rhamnosus/genetics ; *Gene Editing/methods ; *Probiotics ; Glucuronidase/genetics/metabolism ; },
abstract = {Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable targeted deletions and insertions across multiple loci, achieving modest but practically relevant editing efficiencies (11.1-25.0% of recovered transformants) that support routine strain construction. Using this optimised and precise genome engineering method, we generated a β-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics.},
}

*CRISPR-Cas Systems
*Lacticaseibacillus rhamnosus/genetics
*Gene Editing/methods
*Probiotics
Glucuronidase/genetics/metabolism

@article {pmid41556260,
year = {2026},
author = {Neherin, K and Holloway, K and Song, Y and Houston, A and Chen, F and Ding, L and Zhang, H},
title = {Introducing Cellular Senescence in Human Induced Pluripotent Stem Cells and Differentiated Neural Lineage for Modeling of Age-Associated Diseases.},
journal = {Advanced biology},
volume = {10},
number = {1},
pages = {e00468},
doi = {10.1002/adbi.202500468},
pmid = {41556260},
issn = {2701-0198},
support = {RF1AG056302/AG/NIA NIH HHS/United States ; R21AG086894/AG/NIA NIH HHS/United States ; U54AG075934//NIH Common Fund/ ; },
mesh = {Humans ; *Cellular Senescence/genetics/physiology ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Differentiation ; *Neural Stem Cells/cytology/metabolism ; *Aging/genetics ; CRISPR-Cas Systems ; Cell Lineage ; },
abstract = {Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenetic landscapes that mark the aging clock, and consequently cells differentiated from iPSCs resemble fetal cells rather than adult or aged cells. The lack of proper cellular aging in cells differentiated from iPSCs presents a unique challenge in iPSC-based modeling of age-associated diseases such as neurodegeneration. To address this challenge, we seek to introduce cellular senescence, a hallmark of aging, into iPSC-based models in a robust and temporally controlled manner. An inducible CRISPR interference (CRISPRi) is used to suppress the expression of TERF2, a key component of the telomere protecting Shelterin complex. We demonstrate that suppression of TERF2 robustly activates the DNA damage response, p53/p21 signaling, and cellular senescence in iPSCs in a highly homogeneous and synchronous manner. Applying this inducible CRISPRi-TERF2 system to differentiation of iPSCs to neural progenitor cells (NPCs), we show efficient activation of senescence-associated phenotypes in NPCs. This inducible cell model allows isogenic comparisons of the same cell populations over the course of differentiation with or without the activation of cellular senescence in a synchronous and homogeneous manner, and has broad applications in investigating the role of cellular senescence in the progression of age-related diseases.},
}

Humans
*Cellular Senescence/genetics/physiology
*Induced Pluripotent Stem Cells/cytology/metabolism
*Cell Differentiation
*Neural Stem Cells/cytology/metabolism
*Aging/genetics
CRISPR-Cas Systems
Cell Lineage

@article {pmid41555514,
year = {2026},
author = {Xiao, B and Zhang, J},
title = {A Simple Programmable Cas12a/crRNA Induced Walking System for Sensitive Methicillin-Resistant Staphylococcus aureus Detection via Integrated cis- and trans-Cleavage Activity.},
journal = {Journal of microbiology and biotechnology},
volume = {36},
number = {},
pages = {e2511026},
doi = {10.4014/jmb.2511.11026},
pmid = {41555514},
issn = {1738-8872},
mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; Aptamers, Nucleotide/genetics ; *Staphylococcal Infections/diagnosis/microbiology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Biosensing Techniques/methods ; Humans ; },
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat to public health due to its strong antibiotic resistance, wide dissemination, and high infection rates. Rapid identification of MRSA strains is essential for accurate diagnosis and timely treatment of related infections. In this study, we propose an analytical method for MRSA that employs a hairpin-structured locker-probe to directly regulate the trans-cleavage activity of Cas12a. This designed locker-probe connects a target-specific aptamer to an inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to the specific target, the probe undergoes a conformational change that abolishes its inhibitory effect on Cas12a. As a result, the structure-switchable probe modulates Cas12a activity in a target-dependent manner. Additionally, the sensing substrate combines a "cis-cleavage trigger" and a "trans-cleavage trigger" to integrate both cis- and trans-cleavage activities of Cas12a/crRNA within a single probe. This design significantly simplifies the probe architecture while maintaining high signal amplification efficiency. The proposed method was successfully applied to detect MRSA, achieving a detection limit as low as 2.5 CFU/ml with high specificity. By exploiting the inhibitory aptamer of Cas12a as a regulatory element for MRSA analysis, this work expands the toolbox of CRISPR/Cas12a-based methodologies and offers a promising strategy for bacterial detection.},
}

*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Bacterial Proteins/genetics/metabolism
Aptamers, Nucleotide/genetics
*Staphylococcal Infections/diagnosis/microbiology
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*Biosensing Techniques/methods
Humans

@article {pmid41555077,
year = {2026},
author = {Yu, L and Yin, M and Zhu, Y and Lu, Z and Xiao, B and Zhou, F and Yu, Y and Huang, Z},
title = {An anti-CRISPR targets the sgRNA to block Cas9 and guides the design of enhanced genome editors.},
journal = {Nature structural & molecular biology},
volume = {},
number = {},
pages = {},
pmid = {41555077},
issn = {1545-9985},
abstract = {Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.},
}

@article {pmid41554027,
year = {2025},
author = {Ireri, SW and Cao, M},
title = {CRISPR-Cas9-based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {226},
pages = {},
doi = {10.3791/68932},
pmid = {41554027},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Mutagenesis ; *Rhabditida/genetics ; Microinjections/methods ; Gene Editing/methods ; },
abstract = {Entomopathogenic nematodes (EPNs) in the genus Steinernema and Heterorhabditis maintain mutualistic interactions with Xenorhabdus and Photorhabdus symbiotic bacteria, respectively. Together, these nematode-bacterium pairs infect and kill insect hosts that are primarily larvae from the orders of Lepidoptera and Coleoptera, forming a tractable tripartite system for dissecting the molecular basis of mutualism and parasitism. A key step towards fully utilizing this model is the development of stable and transgenerational genetic tools in EPNs. Here, we demonstrate a reliable CRISPR-Cas9 genome editing platform in the emerging model Steinernema hermaphroditum, a species that is readily maintained in vivo and in vitro, and is highly amenable to gonadal microinjection. Importantly, its hermaphroditic reproduction greatly streamlines the generation and maintenance of homozygous mutant lines. We provide a detailed protocol for efficient, targeted gene disruption using microinjection-based delivery of Cas9 ribonucleoprotein complexes. As a proof of concept, we modified the conserved muscle-associated gene unc-22, generating a characteristic twitching phenotype that validates targeted mutagenesis in this system. This CRISPR-Cas9 platform opens the door to stable genetic manipulation in S. hermaphroditum, such as transgene expression, and provides a framework that can be extended to additional EPN species of agricultural and ecological importance.},
}

Animals
*CRISPR-Cas Systems
Mutagenesis
*Rhabditida/genetics
Microinjections/methods
Gene Editing/methods

@article {pmid41540063,
year = {2026},
author = {Port, F and Buhmann, MA and Zhou, J and Stricker, M and Vaughan-Brown, A and Michalsen, AC and Roßmanith, E and Pöltl, A and Großkurth, L and Huber, J and Menendez Kury, LB and Weberbauer, B and Hübl, M and Puscher, E and Heigwer, F and Boutros, M},
title = {Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {877},
pmid = {41540063},
issn = {2041-1723},
support = {ERC-DECODE//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; SFB1324//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; Drosophila melanogaster/genetics ; Gene Targeting/methods ; *Bacterial Proteins/genetics/metabolism ; Drosophila/genetics ; },
abstract = {CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (<1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.},
}

Animals
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
Gene Editing/methods
Drosophila melanogaster/genetics
Gene Targeting/methods
*Bacterial Proteins/genetics/metabolism
Drosophila/genetics

@article {pmid41506457,
year = {2026},
author = {Jiménez Lancho, V and Leitner, K and Agarwal, K and Krishnakumar, A and Khetan, A and Borth, N and Marx, N},
title = {CRISPR-based precise methylation of specific FUT8 promoter regions allows isolation of CHO cells with a fine-tuned glycoprofile.},
journal = {Journal of biotechnology},
volume = {410},
number = {},
pages = {341-352},
doi = {10.1016/j.jbiotec.2026.01.001},
pmid = {41506457},
issn = {1873-4863},
mesh = {CHO Cells ; Cricetulus ; Animals ; *DNA Methylation/genetics ; *Promoter Regions, Genetic/genetics ; *Fucosyltransferases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Glycosylation ; Gene Editing/methods ; },
abstract = {A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve. While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach. By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35-70 % fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and repressed promoters was found to potentially define clones with a fine-tuned expression. Combined, the data demonstrates that using targeted DNA methylation in a manner confined to specific promoter regions opens new engineering strategies to fine-tune gene expression in mammalian cells.},
}

CHO Cells
Cricetulus
Animals
*DNA Methylation/genetics
*Promoter Regions, Genetic/genetics
*Fucosyltransferases/genetics/metabolism
*CRISPR-Cas Systems/genetics
Glycosylation
Gene Editing/methods

@article {pmid41496287,
year = {2026},
author = {Chatrousse, L and Poullion, T and Mahiou, H and El-Kassar, L and Giraud-Triboult, K and Boissart, C and Benchoua, A},
title = {Knock-out of specific DMD gene isoforms in the parental hESC line SA001 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103899},
doi = {10.1016/j.scr.2025.103899},
pmid = {41496287},
issn = {1876-7753},
mesh = {Humans ; *Dystrophin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Protein Isoforms/genetics/metabolism ; Cell Line ; Male ; Muscular Dystrophy, Duchenne/genetics ; *Gene Knockout Techniques ; },
abstract = {The DMD gene, which encodes the protein dystrophin, is involved in a group of diseases known as dystrophinopathies, which includes Duchenne Muscular Dystrophy (DMD). DMD is a progressive and lethal muscular disorder mainly affecting boys that results from the loss of function of the longer dystrophin isoform DP427 in skeletal muscles. Dystrophinopathies are also associated with poorly understood neurocognitive and neurodevelopmental disorders. To investigate the role of dystrophin isoforms in neural development, we specifically disrupted three dystrophin isoforms expressed in the brain, namely DP427, DP140 and DP71, in the male human embryonic stem cell line SA001 using the CRISPR/Cas9 system. (100 / 100 words).},
}

Humans
*Dystrophin/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
Protein Isoforms/genetics/metabolism
Cell Line
Male
Muscular Dystrophy, Duchenne/genetics
*Gene Knockout Techniques

@article {pmid41496285,
year = {2026},
author = {Wang, J and Gao, Y and Jin, K and Jin, ZB and Xu, J},
title = {Generation of RB1 knockout human embryonic stem cell lines derived from H9 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103897},
doi = {10.1016/j.scr.2025.103897},
pmid = {41496285},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques ; *Retinoblastoma Binding Proteins/genetics/metabolism/deficiency ; Cell Line ; *Ubiquitin-Protein Ligases/genetics/deficiency/metabolism ; Cell Differentiation ; Animals ; Mice ; },
abstract = {RB1 is a tumor suppressor gene which plays a crucial role in cell cycle and cellular differentiation. Mutations or loss of RB1 are associated with retinoblastoma and a variety of other cancers. We generated RB1 knockout human embryonic stem cell (hESC) lines (H9) using CRISPR/Cas9-mediated gene targeting. These RB1-deficient cell lines maintain typical stem cell morphology, normal karyotype and expression of pluripotent marker genes. Furthermore, they retain their in vivo differentiation capacity, enabling the generation of multiple cell lineages. These RB1 knockout hESC lines provide valuable models for investigating the role of RB1 in tumorigenesis, neurodevelopment, and cell cycle regulation.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Knockout Techniques
*Retinoblastoma Binding Proteins/genetics/metabolism/deficiency
Cell Line
*Ubiquitin-Protein Ligases/genetics/deficiency/metabolism
Cell Differentiation
Animals
Mice

@article {pmid41496284,
year = {2026},
author = {Fan, M and Zhao, M and Su, W and Tang, Z and Sun, W and Zhou, T and Liu, P},
title = {Generation of a PLIN2-GFP2-P2A-Puro human induced pluripotent stem cell line (SEUi001-A) via CRISPR/Cas9-mediated gene editing technology.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103896},
doi = {10.1016/j.scr.2025.103896},
pmid = {41496284},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Perilipin-2/genetics/metabolism ; Cell Line ; Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; },
abstract = {Perilipin 2 (PLIN2) dysregulation drives metabolic pathologies including non-alcoholic fatty liver disease (NAFLD). To enable real-time tracking of PLIN2 dynamics, we established a human induced pluripotent stem cell (hiPSC) line with endogenous GFP2 knock-in at the PLIN2 locus via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). This PLIN2-GFP2 reporter line demonstrated synchronous fluorescence and transcriptional expression validated by flow cytometry. Genomic integrity was confirmed by normal diploid karyotype (46, XY). Pluripotency markers (POU5F1, SOX2, NANOG) were stably expressed. Furthermore, the cells possessed the ability to differentiate into three germ layers. As the first reported endogenous PLIN2 reporter in human stem cells, this model overcomes limitations of antibody-based detection and transgenic overexpression systems, preserving native regulatory mechanisms. The model provides a physiologically relevant platform for: (1) live monitoring of LD-mitochondria interactions, (2) high-throughput compound screening for metabolic disorders, and (3) modeling NAFLD pathogenesis in vitro, advancing precision therapeutics and mechanistic disease modeling.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Perilipin-2/genetics/metabolism
Cell Line
Green Fluorescent Proteins/metabolism/genetics
Cell Differentiation

@article {pmid41496283,
year = {2026},
author = {Low, YC and McKnight, CL and Stojanovski, D and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent human AGK knockout embryonic stem cell model (WAe009-A-3C) of Sengers syndrome.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103895},
doi = {10.1016/j.scr.2025.103895},
pmid = {41496283},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism/deficiency ; Cell Line ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Gene Knockout Techniques ; *Mitochondrial Diseases/genetics/pathology ; Gene Editing ; Cell Differentiation ; },
abstract = {Sengers syndrome is a rare mitochondrial disorder caused by the loss of a nuclear encoded mitochondrial protein, acylglycerol kinase (AGK). Here, we describe the generation of a novel in vitro stem cell model of Sengers syndrome (AGK[KO] C10) using CRISPR/Cas9 gene editing. This cell line displayed normal characteristics of pluripotent stem cells, including colony morphology, expression of pluripotency markers, trilineage potential, and no karyotypic abnormalities. Together with the parental H9 hESC control line, the AGK[KO] C10 line can ultimately be used for investigation of disease mechanisms and drug testing.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism/deficiency
Cell Line
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism
Gene Knockout Techniques
*Mitochondrial Diseases/genetics/pathology
Gene Editing
Cell Differentiation

@article {pmid41496278,
year = {2026},
author = {Bozaoglu, K and Massie, S and Irion, FE and Davies, KC and Kantor, I and Raabus, M and Haebich, KM and Vlahos, K and Howden, SE and Wright, J and Payne, JM and Lockhart, PJ},
title = {Simultaneous reprogramming and gene correction to generate six iPSC lines and isogenic controls from individuals with neurofibromatosis type 1.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103904},
doi = {10.1016/j.scr.2025.103904},
pmid = {41496278},
issn = {1876-7753},
mesh = {Humans ; *Neurofibromatosis 1/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Gene Editing ; CRISPR-Cas Systems ; Cell Line ; Male ; Female ; },
abstract = {Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder that affects ∼ 1: 2700 individuals (Lee et al., 2023) however the underlying pathogenic mechanisms are poorly understood. In this study, we performed simultaneous reprogramming and CRISPR-Cas9 genome editing to generate pluripotent stem cell (iPSCs) lines and their respective isogenic controls from six individuals with different pathogenic NF1 variants. All iPSC lines had a normal karyotype, were pluripotent and able to differentiate into the three embryonic germ layers. These iPSC lines are valuable pre-clinical models to investigate the pathomechanisms of NF1 and can be used for future screening to identify new therapeutic treatments for NF1.},
}

Humans
*Neurofibromatosis 1/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cellular Reprogramming
*Gene Editing
CRISPR-Cas Systems
Cell Line
Male
Female

@article {pmid41483428,
year = {2026},
author = {Deng, X and Gao, Q and Shen, K and Mu, W and Ge, T and Gu, J and Yang, X and Cheng, J and Wang, J and Zhang, W and Li, D and Zhou, J and Xiao, M},
title = {TNFRSF13B Variant-Induced TACI Dysregulation Underlies CAEBV Pathogenesis.},
journal = {Journal of clinical immunology},
volume = {46},
number = {1},
pages = {12},
pmid = {41483428},
issn = {1573-2592},
support = {82200259//National Natural Science Foundation of China/ ; 81830008//National Natural Science Foundation of China/ ; 82270203//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Transmembrane Activator and CAML Interactor Protein/genetics/metabolism ; *Epstein-Barr Virus Infections/genetics/immunology/metabolism ; *Herpesvirus 4, Human/physiology/immunology ; Frameshift Mutation ; Signal Transduction ; Exons/genetics ; CRISPR-Cas Systems ; },
abstract = {The tumor necrosis factor (TNF) receptor superfamily member, transmembrane activator and CAML interactor (TACI) encoded by TNFRSF13B, are extensively involved in immune responses. In our previous work, TNFRSF13B exon 2 variants were recurrently identified in chronic active Epstein-Barr virus disease (CAEBV). Here we aim to reveal the roles of TNFRSF13B variants in CAEBV, and investigate the feasibility of targeting TNFRSF13B/TACI as a new approach to control EBV infection. The lymphoblastoid cell lines (LCL) models carrying homozygous TNFRSF13B exon 2 frameshift mutations were constructed using CRISPR/Cas9. Immunological assays, transcriptomic analysis, and gene silencing experiments were performed on LCL models to measure the effect of TNFRSF13B exon 2 variants and explore the underlying mechanisms. TACI ligands and a TLR9 agonist were applied to modulate TACI signaling and EBV activities. Frameshift mutations in exon 2 of TNFRSF13B significantly up-regulated the short isoforms of TACI (TACI-S) at the expense of its long isoforms (TACI-L) in LCLs. The up-regulated TACI-S induced more intense activation of NF-κB, MAPK, and Rho signaling pathways, leading to the switch of EBV activities to lytic reactivation. The subsequent increased viral load and viral IL-10 provide a rational for the susceptibility of variant carriers to CAEBV. The BAFF trimer, an indirect TACI-signaling inhibitor, also significantly suppressed the EBV lytic program. Gene silencing experiments indicated that XBP-1 might be involved in the TACI-mediated regulation of EBV lytic activities in EBV-immortalized B cells. This study underscores the impact of TNFRSF13B variants on EBV infection and host immune responses, offering insights into CAEBV pathogenesis and potential therapeutic strategies.},
}

Humans
*Transmembrane Activator and CAML Interactor Protein/genetics/metabolism
*Epstein-Barr Virus Infections/genetics/immunology/metabolism
*Herpesvirus 4, Human/physiology/immunology
Frameshift Mutation
Signal Transduction
Exons/genetics
CRISPR-Cas Systems

@article {pmid41478438,
year = {2026},
author = {Ma, Q and Wang, N and Qiao, K and Luo, K and Zhao, C and Yan, J and Fan, S and Rong, J and Ma, Q},
title = {The R2R3-MYB transcription factor GhMYB35 governs anther development and pollen viability in upland cotton.},
journal = {Gene},
volume = {984},
number = {},
pages = {149990},
doi = {10.1016/j.gene.2025.149990},
pmid = {41478438},
issn = {1879-0038},
mesh = {*Gossypium/genetics/growth & development/metabolism ; *Pollen/genetics/growth & development ; *Transcription Factors/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Flowers/genetics/growth & development ; Plant Infertility/genetics ; CRISPR-Cas Systems ; },
abstract = {Male reproductive development is fundamental to the life cycle of flowering plants, culminating in seed production. Aberrations in anther development frequently lead to male sterility, yet the underlying molecular mechanisms in upland cotton (Gossypium hirsutum) remain largely uncharacterized. The R2R3-MYB family of transcription factors are known key regulators of diverse developmental processes, including male fertility in several model species. Here, we identify and functionally characterize GhMYB35, an R2R3-MYB transcription factor that plays an essential role in cotton anther development. CRISPR/Cas9-mediated knockout of GhMYB35 resulted in complete male sterility, with mutants (ghmyb35) exhibiting indehiscent anthers, shorter filaments, and a total absence of viable pollen. Expression analyses reveal that GhMYB35 is predominantly expressed in anthers, with peak expression of its A- and D-subgenome homoeologs occurring at developmental stage 7. Subcellular localization results show that bothGhMYB35_AandGhMYB35_Dare nuclear-localized transcription factors. Furthermore, the total absence of GhMYB35 leads to pollen abortion and subsequent anther collapse without dehiscence. Collectively, our findings establish GhMYB35 as a critical regulator of anther maturation, thereby elucidating a key component of the molecular network governing male fertility in cotton.},
}

*Gossypium/genetics/growth & development/metabolism
*Pollen/genetics/growth & development
*Transcription Factors/genetics/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Flowers/genetics/growth & development
Plant Infertility/genetics
CRISPR-Cas Systems

@article {pmid41476041,
year = {2025},
author = {Xi, W and Xu, Y and Bao, W and Ding, Z and Xiao, H and Yang, J and Yan, X and Ping, Y},
title = {In vivo chemogenetic RNA editing of macrophages by bioengineered viruses for sepsis treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {927},
pmid = {41476041},
issn = {2041-1723},
support = {82425055//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32261143727//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82504685//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M762890//China Postdoctoral Science Foundation/ ; 2025T180980//China Postdoctoral Science Foundation/ ; GZB20240672//China Postdoctoral Science Foundation/ ; LMS25H300002//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {*Sepsis/therapy/genetics/immunology ; Animals ; *Macrophages/metabolism ; Mice ; *RNA Editing/genetics ; *Lentivirus/genetics ; Disease Models, Animal ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; Mice, Inbred C57BL ; Genetic Vectors/genetics ; Humans ; Genetic Therapy/methods ; Bioengineering ; CRISPR-Cas Systems ; Male ; Chemogenetics ; },
abstract = {Sepsis, a life-threatening condition arising from a dysregulated host response to infection, remains a significant clinical challenge with limited therapeutic options. RNA editing presents a promising avenue for modulating gene expression to attenuate the inflammatory cascade characteristic of sepsis. Here, we introduce an approach utilizing chemogenetic activation of CasRx-based RNA editing via bioengineered lentiviruses for the treatment of sepsis. Our strategy involves the targeted delivery of biomineralized lentiviral vectors encoding RNA-editing enzymes and chemogenetic switches to M1 macrophage populations implicated in sepsis pathogenesis. Upon the administration of a small molecule ligand, the chemogenetic switches activate the RNA-editing tool, CasRx, thereby enabling precise and repeated downregulation of NLRP3 mRNA. We demonstrate the efficacy and repeatability of this viral-based approach in mouse models of sepsis, highlighting its potential as a versatile therapeutic strategy for mitigating sepsis-induced inflammation. This study underscores the utility of chemogenetic technologies in harnessing the power of RNA editing for the treatment of sepsis and other inflammatory disorders.},
}

*Sepsis/therapy/genetics/immunology
Animals
*Macrophages/metabolism
Mice
*RNA Editing/genetics
*Lentivirus/genetics
Disease Models, Animal
NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism
Mice, Inbred C57BL
Genetic Vectors/genetics
Humans
Genetic Therapy/methods
Bioengineering
CRISPR-Cas Systems
Male
Chemogenetics

@article {pmid41422102,
year = {2025},
author = {Zhong, C and Yu, L and Zhao, T and Shen, X and Li, Z and Zhu, W and Hu, Z and Tian, R and Han, Z and He, D},
title = {A Plug-in system for reprogramming the editing patterns of base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {910},
pmid = {41422102},
issn = {2041-1723},
support = {32171465//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32500460//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M734091//China Postdoctoral Science Foundation/ ; },
mesh = {*Gene Editing/methods ; Zebrafish/genetics/embryology ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase's spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE's versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.},
}

*Gene Editing/methods
Zebrafish/genetics/embryology
Animals
Humans
CRISPR-Cas Systems/genetics
Genetic Therapy/methods
DNA/genetics/metabolism
HEK293 Cells

@article {pmid41421670,
year = {2026},
author = {Fan, X and Li, B and Xu, X and Long, B and Jia, Z and Wang, R and Gao, J and Chen, Y and Peng, M and Zhou, M},
title = {Deciphering the regulatory role of the pfs gene on biofilm formation in Lactobacillus plantarum R: Insights from transcriptome and metabolome.},
journal = {Bioresource technology},
volume = {443},
number = {},
pages = {133833},
doi = {10.1016/j.biortech.2025.133833},
pmid = {41421670},
issn = {1873-2976},
mesh = {*Lactiplantibacillus plantarum/genetics/physiology/metabolism ; *Biofilms/growth & development ; *Transcriptome/genetics ; *Metabolome/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Quorum Sensing/genetics ; *Genes, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Lactobacillus plantarum is a widely recognized probiotic that forms biofilms to enhance environmental tolerance and probiotic properties, but the mechanisms regulating its biofilm formation remain unclear. This study successfully used CRISPR-Cas9 to delete the pfs gene in the high biofilm-producing strain L. plantarum R, and first investigated its role by integrated transcriptomic and metabolomic analyses. The pfs gene participates in the activated methyl cycle and AI-2 synthesis, which is involved in quorum sensing and biofilm formation. Deletion of pfs increased biofilm biomass by 91% and markedly enhanced matrix accumulation, including exopolysaccharides, extracellular proteins and extracellular DNA (eDNA). Transcriptomic analysis revealed significant perturbation of cysteine and methionine metabolism and altered expression of key genes involved in exopolysaccharide synthesis. Metabolomic profiling identified 223 differentially expressed metabolites, primarily associated with carbon flux and EPS precursor pathways. In summary, pfs deletion enhances biofilm formation via metabolic reprogramming rather than classical AI-2 dependent QS pathways. This study provides new insights into the pfs gene-mediated regulation of biofilm formation in L. plantarum and establishes a foundation for future strategies to manipulate biofilm formation in industrial applications.},
}

*Lactiplantibacillus plantarum/genetics/physiology/metabolism
*Biofilms/growth & development
*Transcriptome/genetics
*Metabolome/genetics
Gene Expression Regulation, Bacterial
*Bacterial Proteins/genetics/metabolism
Quorum Sensing/genetics
*Genes, Bacterial/genetics
CRISPR-Cas Systems/genetics

@article {pmid41419745,
year = {2025},
author = {Ying, Q and Chen, Y and Shen, L and Xu, Y and Tian, R},
title = {SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {911},
pmid = {41419745},
issn = {2041-1723},
support = {82171416//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*X-Box Binding Protein 1/metabolism/genetics ; *Endoribonucleases/metabolism/genetics ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *RNA Splicing/genetics ; Humans ; Signal Transduction/genetics ; Animals ; *Endoplasmic Reticulum Stress/genetics ; Protein Phosphatase 1/metabolism/genetics/antagonists & inhibitors ; Unfolded Protein Response/genetics ; Mice ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq (SPLicing regulator identification through CRISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 (PPP1R15A) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway.},
}

*X-Box Binding Protein 1/metabolism/genetics
*Endoribonucleases/metabolism/genetics
*Protein Serine-Threonine Kinases/metabolism/genetics
*RNA Splicing/genetics
Humans
Signal Transduction/genetics
Animals
*Endoplasmic Reticulum Stress/genetics
Protein Phosphatase 1/metabolism/genetics/antagonists & inhibitors
Unfolded Protein Response/genetics
Mice
CRISPR-Cas Systems
HEK293 Cells
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41418729,
year = {2026},
author = {Wang, Y and Zheng, W and Qiu, B and Chen, Q and Yang, T and Zhou, S and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-3F) with Cas9 expression cassette integrated at the AAVS1 locus via CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103882},
doi = {10.1016/j.scr.2025.103882},
pmid = {41418729},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Homologous Recombination/genetics ; Cell Line ; Gene Editing ; Genetic Loci ; Cell Differentiation ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {Cas9, an RNA-guided nuclease, enables precise genome editing by recognizing sgRNA-complementary sequences and cleaving target DNA. In this study, we used CRISPR/Cas9-mediated homologous recombination to integrate a loxP-flanked STOP cassette-controlled Cas9 expression framework (LSL-Cas9) into the AAVS1 safe-harbor locus of human embryonic stem cells. The resulting cell line, SMUDHe010-A-3F, allows Cre-dependent activation of Cas9 but remains inactive in the absence of Cre recombinase. Karyotype and tri-lineage differentiation confirmed genomic stability and pluripotency. This line provides a valuable platform for organoid gene editing and studies of human development and disease.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Homologous Recombination/genetics
Cell Line
Gene Editing
Genetic Loci
Cell Differentiation
CRISPR-Associated Protein 9/metabolism

@article {pmid41406514,
year = {2026},
author = {Ghodrat, R and Ramachandran, H and Hildebrandt, B and Binder, S and Rossi, A and Reichert, AS},
title = {CRISPR/Cpf1-mediated editing of PINK1 in induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103887},
doi = {10.1016/j.scr.2025.103887},
pmid = {41406514},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Protein Kinases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; },
abstract = {The PTEN induced kinase 1 (PINK1) gene is crucial for mitophagy and mitochondrial quality control. Mutations in the PINK1 gene are associated with several neurological disorders. To decipher the role of PINK1-mediated mitophagy in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with homozygous out-of-frame deletions by targeting exon 6 of the PINK1 gene. The generated homozygous PINK1 mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Protein Kinases/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Cell Differentiation
Cell Line

@article {pmid41398410,
year = {2025},
author = {Chi, H and Hoikkala, V and McMahon, S and Graham, S and Gloster, T and White, MF},
title = {Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {889},
pmid = {41398410},
issn = {2041-1723},
support = {101018608//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Crystallography, X-Ray ; *CRISPR-Cas Systems ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Bacteriophages/genetics ; Nucleotides, Cyclic/metabolism ; Oligoribonucleotides/metabolism ; },
abstract = {Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.},
}

Crystallography, X-Ray
*CRISPR-Cas Systems
Adenine Nucleotides/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Models, Molecular
*Endonucleases/metabolism/chemistry/genetics
Bacteriophages/genetics
Nucleotides, Cyclic/metabolism
Oligoribonucleotides/metabolism

@article {pmid41385899,
year = {2026},
author = {Fathy, K and Bharti, J and Khan Sony, S and Nehra, M and Kaul, R and Rawat, B and Sopory, SK and Agrawal, PK and Prakash, A and Kaul, T},
title = {Triumphing over hidden hunger: Redesigning rice (Oryza sativa L.) for enhanced nutraceutical grain composition utilizing multiplexed genome editing.},
journal = {Journal of plant physiology},
volume = {316},
number = {},
pages = {154667},
doi = {10.1016/j.jplph.2025.154667},
pmid = {41385899},
issn = {1618-1328},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Gene Editing/methods ; *Edible Grain/chemistry/genetics ; Cadmium/metabolism/analysis ; *Dietary Supplements ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Zinc/metabolism ; Iron/metabolism ; },
abstract = {Rice, a staple food crop, is consumed by most of the world's population. Micronutrient malnutrition is a severe health issue, leading to diseases such as cancer, anemia, diabetes, heart disease, and disorders in physical and psychological development. We aimed to create rice with low cadmium in the grain but having high cadmium in shoots, safe biofortified protein, high iron, and zinc using CRISPR/Cas9 and breeding technologies instead of adding drugs. The triple gene Knockout rice lines for two iron sensors and one negative regulator gene for cadmium were created to offer high Fe/Zn and low Cd content for breeders. Multiplexed gene editing mediated biolistic transformation of rice callus, and genotyping was used to check the genetic stability of the edited rice lines. Rice lines were found to have enhanced iron, zinc, and protein content, with concentrations varying based on growth conditions. These lines can be used as phytoremediators for cadmium by storing Cd on plant shoots. The rice-edited plants possessed excellent agro-morphological traits, photosynthetic, and physiological performance. The developed edited indica rice lines have crucial agronomic traits with more nutritional value. Compared to the other lines and the wild wildtype, the genome-edited free Cas9 line 2 showed better traits: 13.48 μg/g (iron), 22.9 μg/g (zinc), and a high protein content, which depends on how bioavailable metals and nutrients are in the soil. The line also had 20.60 g of seeds per 1000 g of plant, a total plant yield of 102.76 g, and 101 days of 50 % flowering. This work offers efficient and precise multiple gene-editing in rice with an effective, sustainable strategy for multi-trait enhancement. The developed lines could be used in breeding programs for sustainable solutions for malnutrition worldwide. The experimental results can provide reference and support for the safe use of edited crops as a diet.},
}

*Oryza/genetics/metabolism/chemistry
*Gene Editing/methods
*Edible Grain/chemistry/genetics
Cadmium/metabolism/analysis
*Dietary Supplements
Plants, Genetically Modified/genetics
CRISPR-Cas Systems
Plant Breeding
Zinc/metabolism
Iron/metabolism

@article {pmid41285723,
year = {2025},
author = {Morshedi Rad, D and Richards, C and Zhand, S and de Alwis, N and Hannan, NJ and Faiz, A and McClements, L and Ebrahimi Warkiani, M},
title = {CRISPR/Cas9-mediated gene editing in trophoblast cells via mechanoporation for preeclampsia insight.},
journal = {Cell death & disease},
volume = {17},
number = {1},
pages = {61},
pmid = {41285723},
issn = {2041-4889},
support = {106628//National Heart Foundation of Australia (Heart Foundation)/ ; DP200101860//Department of Education and Training | Australian Research Council (ARC)/ ; 2021CDF1148//Cancer Institute NSW (Cancer Institute New South Wales)/ ; },
mesh = {*Trophoblasts/metabolism/pathology ; Female ; *Pre-Eclampsia/genetics/pathology/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Pregnancy ; Cell Movement/genetics ; Cell Line ; },
abstract = {Preeclampsia is a severe pregnancy complication marked by impaired trophoblast function and abnormal placental development, leading to significant maternal and fetal morbidity. FK506-binding protein-like (FKBPL) has been identified as a potential biomarker as it is significantly downregulated in early pregnancy stages of women who progress to develop preeclampsia. However, editing the Fkbpl gene in trophoblast cells to create a model of preeclampsia using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is challenging due to inefficient delivery, leading to low editing efficiency and reduced cell viability. To address these challenges, we developed a cost-effective and minimally invasive mechanoporation system using micro-engineered filters to deliver CRISPR/Cas9 plasmid DNA (pDNA) targeting the Fkbpl gene into trophoblast cells. This approach successfully generated cell lines with a 38% knockout (K/O) of Fkbpl expression, significantly reducing cell migration (wildtype (WT): 28.77% ± 4.7 vs. 38% K/O: 4.95% ± 0.8, wound closure, **p < 0.01) and proliferation (WT: 1.26 ± 0.06 vs. 38% K/O: 0.81 ± 0.01, ****p < 0.0001). Lower Fkbpl-K/O efficiency of 17% showed a similar reduction in cell proliferation as the 38% K/O clone. Although a full Fkbpl-K/O in the ACH-3P first-trimester trophoblast cell line was not achieved, the partial K/O provided valuable insights into Fkbpl's role in trophoblast function relevant to preeclampsia pathogenesis. Moreover, treatment with mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) or MSC-sEVs did not restore migratory capacity in Fkbpl-deficient cells (p = 0.14). MSC-sEVs increased proliferation in WT ACH-3P cells at 1 µg (p < 0.05) and 2 µg (p < 0.01) doses, however, were not effective in either 17% or 38% Fkbpl-K/O clones, suggesting that FKBPL is an important mechanism of MSC-sEV-mediated therapeutic effect in trophoblasts in the context of preeclampsia. This study advances gene-editing techniques in placental biology and proposes new therapeutic strategies and mechanisms for pregnancy-related complications. A Schematic overview of CRISPR/Cas9 plasmid delivery using microfiltroporation compared to gold standard electroporation and lipofection technologies in trophoblast cells. A CRISPR/Cas9 plasmid targeting Fkbpl was delivered to the first trimester trophoblast cell line, ACH-3P. Cells were sorted according to green fluorescence protein (GFP) expression, expanded and assessed for changes in cell function using proliferation and migration assays. B Actual images of the isopore silicon nitride (SiN) microfilters used in this study and diagram of cell membrane dynamics in response to mechanoporation. This figure was created with Biorender.com. CRISPR clustered regularly interspaced short palindromic repeats, EP electroporation, MFP microfiltroporation.},
}

*Trophoblasts/metabolism/pathology
Female
*Pre-Eclampsia/genetics/pathology/metabolism
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Pregnancy
Cell Movement/genetics
Cell Line

@article {pmid41194485,
year = {2026},
author = {Yang, TT and Zhang, JR and Xie, ZH and Ren, ZL and Zhao, MY and Hu, WJ and Yan, JW and Ni, M},
title = {CRISPR-Cas9-Targeted Nanopore Sequencing for STR Typing.},
journal = {Electrophoresis},
volume = {47},
number = {1},
pages = {57-67},
doi = {10.1002/elps.70051},
pmid = {41194485},
issn = {1522-2683},
support = {2021QY2004//We thank the National Key Research and Development Program of China/ ; 82030058//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanopore Sequencing/methods ; *Microsatellite Repeats/genetics ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *DNA Fingerprinting/methods ; Pilot Projects ; *Sequence Analysis, DNA/methods ; DNA/genetics ; },
abstract = {CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3 µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.},
}

Humans
*Nanopore Sequencing/methods
*Microsatellite Repeats/genetics
*CRISPR-Cas Systems/genetics
HEK293 Cells
*DNA Fingerprinting/methods
Pilot Projects
*Sequence Analysis, DNA/methods
DNA/genetics

@article {pmid40522172,
year = {2025},
author = {Xue, B and Zhang, MX and Bi, XC and Lai, SP and Bie, XT and Dong, Y and Li, JF and Gao, F and Zhang, X and Wang, Y},
title = {An Amygdala-hippocampus Circuit for Endocannabinoid Modulation of Anxiety Avoidance.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {34},
pages = {e05121},
pmid = {40522172},
issn = {2198-3844},
support = {2021ZD0203000 (2021ZD0203002//STI2030-Major/ ; 82090033//National Natural Science Foundation of China/ ; 81830035//National Natural Science Foundation of China/ ; 82003729//National Natural Science Foundation of China/ ; ZR2019ZD35//Natural Science Foundation of Shandong Province/ ; ZR2020QH357//Natural Science Foundation of Shandong Province/ ; 202205AF150068//Zhang Xia Expert Workstation of the Yunnan Province/ ; ZYGD23034//West China Hospital, Sichuan University/ ; },
mesh = {*Amygdala/physiology ; *Hippocampus/physiology ; *Endocannabinoids/metabolism ; *Anxiety/metabolism/physiopathology ; Male ; Animals ; Mice ; Receptor, Cannabinoid, CB1/metabolism ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/physiology ; Neurons/physiology ; Avoidance Learning/physiology ; CRISPR-Cas Systems ; Brain/physiology ; },
abstract = {Recent studies indicate a therapeutic potential of increased brain endocannabinoids (eCBs) in anxiety disorders, but the underlying brain circuits are still elusive. Here, it is observed that optogenetic inhibition and activation of anterior basolateral amygdala (aBLA) - ventral hippocampus (vHPC) glutamatergic projections respectively decrease and increase anxiety avoidance behaviors. Then, the contributions of eCBs in aBLA-vHPC projections to anxiety avoidance are investigated by employing three newly developed synapse- and circuit-specific eCB-targeted viral strategies to achieve real-time monitoring of eCB release, in vivo optogenetic activation of CB1 receptors, and CRISPR-Cas9 gene knockdown of eCB biosynthesis enzymes. Prominent eCB release are surprisingly found at aBLA-vHPC glutamatergic synapses during anxiety avoidance, suggesting inhibitory effects of increased eCBs in aBLA-vHPC projections on anxiety avoidance. This idea is further supported by findings that specific activation of CB1 receptors at aBLA-vHPC synapses inhibit presynaptic glutamate release and reduce anxiety avoidance. In contrast, specific knockdown of eCB biosynthesis enzymes at aBLA-vHPC synapses reduce eCB levels at aBLA-vHPC glutamatergic synapses and increase anxiety avoidance. Additionally, inhibition of aBLA-innervated vHPC glutamatergic neurons alleviates anxiety avoidance. Together, these findings reveal counteracting effects of increased eCB signaling in aBLA-vHPC circuits on anxiety avoidance.},
}

*Amygdala/physiology
*Hippocampus/physiology
*Endocannabinoids/metabolism
*Anxiety/metabolism/physiopathology
Male
Animals
Mice
Receptor, Cannabinoid, CB1/metabolism
Mice, Inbred C57BL
Basolateral Nuclear Complex/physiology
Neurons/physiology
Avoidance Learning/physiology
CRISPR-Cas Systems
Brain/physiology

@article {pmid40434188,
year = {2026},
author = {Wu, F and Li, N and Xiao, Y and Palanki, R and Yamagata, H and Mitchell, MJ and Han, X},
title = {Lipid Nanoparticles for Delivery of CRISPR Gene Editing Components.},
journal = {Small methods},
volume = {10},
number = {2},
pages = {e2401632},
pmid = {40434188},
issn = {2366-9608},
support = {XDB0570000//Strategic Priority Research Program of the Chinese Academy of Science/ ; 32471401//National Natural Science Foundation of China/ ; ssIII-2024B01//Shanghai Sci-Tech Inno Center for Infection & Immunity/ ; },
mesh = {*Gene Editing/methods ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Liposomes ; },
abstract = {Gene editing has emerged as a promising therapeutic option for treating genetic diseases. However, a central challenge in the field is the safe and efficient delivery of these large editing tools, especially in vivo. Lipid nanoparticles (LNPs) are attractive nonviral vectors due to their low immunogenicity and high delivery efficiency. To maximize editing efficiency, LNPs should efficiently protect gene editing components against multiple biological barriers and release them into the cytoplasm of target cells. In this review, the widely used CRISPR gene editing systems are first overviewed. Then, each component of LNPs, as well as their effects on delivery, are systematically discussed. Following this, the current LNP engineering strategies to achieve non-liver targeting are summarized. Finally, preclinical and clinical applications of LNPs for in vivo genome editing are highlighted, and perspectives for the future development of LNPs are provided.},
}

*Gene Editing/methods
*Nanoparticles/chemistry
Humans
*Lipids/chemistry
Animals
*CRISPR-Cas Systems/genetics
*Gene Transfer Techniques
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Liposomes

@article {pmid41553913,
year = {2026},
author = {Garnica, M and San Martin-Uriz, P and Rodriguez-Marquez, P and Calleja-Cervantes, ME and Rodriguez-Diaz, S and Martinez-Turrillas, R and Hernaez, M and Prosper, F and Rodriguez-Madoz, JR},
title = {Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {227},
pages = {},
doi = {10.3791/69721},
pmid = {41553913},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Gene Library ; Immunotherapy, Adoptive/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not been fully replicated in solid tumors. Moreover, even in hematological cancers, relapse after CAR T cell infusion continues to compromise long-term outcomes. These challenges highlight the urgent need to develop strategies that enhance CAR T cell efficacy, persistence, overcoming tumor and microenvironment-mediated resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening platforms provide a powerful approach to systematically identify genes that regulate CAR T cell function. By linking genetic perturbations to phenotypic outcomes, these assays enable the discovery of pathways controlling activation, proliferation, memory formation, and cytotoxicity. Standard workflows involve transduction of substantial numbers of cells with a single guide RNA (sgRNA) library, Cas9-mediated editing, selection of edited cells, and PCR amplification of sgRNA cassettes from genomic DNA (gDNA) prior to sequencing. However, PCR amplification using large amounts of gDNA poses significant challenges and often fails to selectively amplify and retrieve sgRNAs. Here, we describe an optimized CRISPR-Cas9 knockout screening protocol, which we have tested on primary human CAR T cells. The method here incorporates an intermediate step during sgRNA library preparation that reduces gDNA carryover through enzymatic digestion and selective pulldown of the sgRNA cassette, thereby increasing the efficiency of the first PCR amplification. This modification allowed us to retrieve sgRNA information across our CAR T cell screens, which had remained elusive in our previous attempts using traditional 1 and 2-step PCR amplification protocols. In conclusion, this optimized workflow facilitates CRISPR screening library preparation in challenging samples and enables the identification of key genetic determinants that can be targeted to improve therapeutic efficacy.},
}

Humans
*CRISPR-Cas Systems
*T-Lymphocytes/immunology
*Receptors, Chimeric Antigen/genetics/immunology
*Gene Library
Immunotherapy, Adoptive/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods