@article {pmid41077083,
year = {2025},
author = {Luo, J and Lu, Y and Zhang, Z and Zhang, Q and Shang, Y and Yuan, Y and Duan, X and Zhang, L and Tang, Y and Hu, H and Tong, X and Dai, F},
title = {A conserved Piezo-mRPL2/Jun axis controls reproduction via mitochondrial bioenergetics.},
journal = {Insect biochemistry and molecular biology},
volume = {185},
number = {},
pages = {104417},
doi = {10.1016/j.ibmb.2025.104417},
pmid = {41077083},
issn = {1879-0240},
mesh = {*Mitochondria/physiology ; *Mechanotransduction, Cellular/physiology ; Bombyx ; CRISPR-Cas Systems ; Oxidative Stress ; Phosphorylation ; Transgenes ; Male ; Animals ; Ion Channels ; *Energy Metabolism ; Mice ; *Ribosomal Proteins/metabolism ; *Mitochondrial Proteins/metabolism ; Insect Proteins/metabolism ; Fertility ; },
abstract = {Piezo channels are essential mechanosensors, but their roles in reproduction remain poorly understood. Here, we identify a critical function for BmPiezo in silkworm fertility and embryonic diapause, mediated through a conserved downstream regulatory axis. CRISPR/Cas9-mediated knockout of BmPiezo resulted in male sterility and loss of embryonic diapause, accompanied by dysregulation of ribosome-associated genes and oxidative phosphorylation, and reduced ATP production. We identified mitochondrial ribosomal protein L2 (BmmRPL2) and transcription factor BmJun as key downstream effectors, required for spermatogenesis and diapause, respectively. Homozygous disruption of either gene caused embryonic lethality. Cross-species expression analysis revealed strong expression of Piezo2 and mRPL2 in early human spermatids. Remarkably, transgenic expression of murine Piezo2 in BmPiezo-/-silkworms reactivated the Piezo-mRPL2/Jun axis, restored ATP levels, and rescued reproductive defects. These findings define a conserved pathway that connects mechanical sensing to mitochondrial bioenergetics in reproductive regulation, advancing our understanding of fertility mechanisms across species.},
}

*Mitochondria/physiology
*Mechanotransduction, Cellular/physiology
Bombyx
CRISPR-Cas Systems
Oxidative Stress
Phosphorylation
Transgenes
Male
Animals
Ion Channels
*Energy Metabolism
Mice
*Ribosomal Proteins/metabolism
*Mitochondrial Proteins/metabolism
Insect Proteins/metabolism
Fertility

@article {pmid41664627,
year = {2026},
author = {Hancock, MB and Ruby, DR and Bieler, RA and Cole, DC and Marsden, KC},
title = {Multi-omic analyses identify molecular targets of Chd7 that contribute to CHARGE syndrome model phenotypes.},
journal = {Disease models & mechanisms},
volume = {19},
number = {3},
pages = {},
doi = {10.1242/dmm.052592},
pmid = {41664627},
issn = {1754-8411},
support = {R21NS120079-01/NS/NINDS NIH HHS/United States ; R01NS116354-01/NS/NINDS NIH HHS/United States ; R21NS120079-01/NS/NINDS NIH HHS/United States ; R01NS116354-01/NS/NINDS NIH HHS/United States ; //North Carolina State University/ ; },
mesh = {*CHARGE Syndrome/genetics/pathology ; Animals ; Zebrafish/genetics/metabolism ; Phenotype ; *Zebrafish Proteins/metabolism/genetics ; Disease Models, Animal ; *DNA Helicases/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; *Proteomics ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Transcriptome/genetics ; CRISPR-Cas Systems/genetics ; Humans ; Mutation/genetics ; Gene Expression Profiling ; Multiomics ; },
abstract = {CHARGE syndrome is a developmental disorder that affects 1 in 10,000 births, and patients exhibit both physical and behavioral characteristics. De novo variants in chromodomain helicase DNA binding protein 7 (CHD7) cause 67% of CHARGE syndrome cases. CHD7 is a DNA-binding chromatin remodeler with thousands of predicted binding sites in the genome, making it challenging to define molecular pathways linking loss of CHD7 to CHARGE syndrome phenotypes. To address this problem, here, we used a previously characterized zebrafish model of CHARGE syndrome to generate transcriptomic and proteomic datasets from larval zebrafish head tissue at two developmental time points. By integrating these datasets with differential expression, pathway and upstream regulator analyses, we identified multiple consistently dysregulated pathways and defined a set of candidate genes that link loss of chd7 with disease-related phenotypes. Finally, we identified that CRISPR/Cas9-mediated knockdown of capgb, nefla or rdh5 phenocopies behavioral defects seen in chd7 mutants, functionally validating the roles of these genes. Our data provide a resource for further investigation of molecular mediators of CHD7 and a template to reveal functionally relevant therapeutic targets to alleviate specific aspects of CHARGE syndrome.},
}

*CHARGE Syndrome/genetics/pathology
Animals
Zebrafish/genetics/metabolism
Phenotype
*Zebrafish Proteins/metabolism/genetics
Disease Models, Animal
*DNA Helicases/metabolism/genetics
*DNA-Binding Proteins/metabolism/genetics
*Proteomics
Gene Expression Regulation, Developmental
Gene Knockdown Techniques
Transcriptome/genetics
CRISPR-Cas Systems/genetics
Humans
Mutation/genetics
Gene Expression Profiling
Multiomics

@article {pmid41859774,
year = {2026},
author = {Fang, Z and Hao, Y and Zuo, X and Wang, S},
title = {Target-Gated Ratiometric pH Sensing via Tetrahedral DNA Framework-Based Dual-CRISPR System.},
journal = {Analytical chemistry},
volume = {98},
number = {12},
pages = {9208-9218},
doi = {10.1021/acs.analchem.5c07666},
pmid = {41859774},
issn = {1520-6882},
mesh = {Hydrogen-Ion Concentration ; *DNA/chemistry/metabolism ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Fluorescent Dyes/chemistry ; Extracellular Vesicles/metabolism/chemistry ; },
abstract = {Extracellular vesicle (EV)-mediated communication is tightly regulated by local pH, which governs vesicle biogenesis, cargo release, and membrane fusion. Accurate and context-specific pH sensing is therefore crucial for elucidating EV function and disease-associated microenvironmental regulation. Here, we present a tetrahedral DNA framework (TDF)-orchestrated dual-CRISPR system that integrates orthogonal Cas12a and Cas13a nucleases for target-activated, ratiometric pH detection at lipid membranes. By exploiting the distinct pH-activity profiles of Cas12a (optimal pH ∼ 8.5) and Cas13a (optimal pH ∼ 7.2), combined with their complete substrate orthogonality, we constructed a self-calibrating nanosensor featuring equimolar coassembly of both nucleases and their corresponding fluorogenic reporters at the four vertices of a TDF. The well-defined tetrahedral geometry ensured reproducible molecular organization and stable fluorescence output, eliminating variability inherent to conventional single-fluorophore probes. The sensor exhibited quantitative assembly fidelity and robust pH responsiveness across physiological ranges. Importantly, the Cas module can be programmed for conditional activation, enabling pH sensing only upon recognition of disease-associated biomarkers. Using miR-146a, a regulatory microRNA enriched in EVs implicated in inflammation and cancer progression, as a model target, we demonstrated target-gated pH monitoring on cell-derived exosomes and during liposome fusion events. This work establishes a versatile and generalizable platform for programmable, ratiometric sensing at biomembrane interfaces, offering new opportunities to probe EV-mediated intercellular communication and dynamic microenvironmental regulation.},
}

Hydrogen-Ion Concentration
*DNA/chemistry/metabolism
Humans
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Fluorescent Dyes/chemistry
Extracellular Vesicles/metabolism/chemistry

@article {pmid41897352,
year = {2026},
author = {Atluri, U and Cvammen, W and Kemp, MG},
title = {The Small Molecule SR8278 Inhibits Cell Proliferation Independent of the REV-ERB Nuclear Receptor Proteins in Human Keratinocytes.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897352},
issn = {2218-273X},
support = {GM103583/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Cell Proliferation/drug effects ; *Keratinocytes/drug effects/metabolism/cytology ; *Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism/genetics/antagonists & inhibitors ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Gene Expression Regulation/drug effects ; *Thiophenes/pharmacology ; },
abstract = {The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play important roles in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of different preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. We therefore carried out an RNA-seq analysis and found that genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, and these effects were confirmed at both the RNA and protein level by RT-qPCR and Western blotting, respectively. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce genotoxic stress or apoptosis. Finally, the use of CRISPR/Cas9 genome editing and siRNA-mediated disruption of REV-ERB gene expression showed that the loss of the REV-ERB proteins did not impact the effect of SR8278 on gene expression and cell proliferation. We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and, thus, care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown, particularly in studies involving cell proliferation.},
}

Humans
*Cell Proliferation/drug effects
*Keratinocytes/drug effects/metabolism/cytology
*Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism/genetics/antagonists & inhibitors
CRISPR-Cas Systems
Apoptosis/drug effects
Gene Expression Regulation/drug effects
*Thiophenes/pharmacology

@article {pmid41897386,
year = {2026},
author = {Richter, PR and Graf, J and Haag, FWM and Scudlo, V and Wiesmeth, S and Hauslage, J and Richter, M and Geißler, D and Lebert, M},
title = {Acceleration and Light-Induced Changes in Cytosolic cAMP Concentration in Euglena gracilis.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897386},
issn = {2218-273X},
support = {50WB1128 and 50WB2218)//Deutsches Zentrum für Luft- und Raumfahrt (DLR) BMWi project/ ; },
mesh = {*Cyclic AMP/metabolism ; *Euglena gracilis/metabolism/radiation effects ; *Light ; Adenylyl Cyclases/metabolism/genetics ; *Cytosol/metabolism/radiation effects ; *Acceleration ; Phototaxis ; },
abstract = {The second messenger cyclic AMP (cAMP) is very likely involved in phototactic as well as gravitactic behavior of the unicellular flagellate Euglena gracilis. A slight but significant increase in cAMP was observed when cells encountered sub-threshold acceleration (0.16 × g) force after microgravity [µg]. No differences in cAMP levels were found between cells on a clinostat and 1x-controls. This observation is consistent with the ones of earlier studies. Illumination of cells resulted in a significant increase in cellular cAMP levels. After RNAi-mediated knockdown or CRISPR-Cas9 knockout of the photoactivated adenylyl cyclases PACα and/or PACβ in the photoreceptor, light-induced changes in cAMP levels were no longer observed. In parallel, phototactic behavior was abolished, supporting the essential role of photoactivated adenylyl cyclases in phototaxis. Cells spin around their length axis during locomotion (1-2 Hz). In order to generate a signal in the light direction, the cells should be capable of synthesizing and degrading cAMP within 0.5-1 s. The rapid fixation of cells upon transition from dark to light or light to dark revealed that detectable changes in cAMP-levels (increase or decrease) occur within a 100-200 ms time window, which is sufficiently fast to account for the proposed theoretical kinetics of cAMP oscillations.},
}

*Cyclic AMP/metabolism
*Euglena gracilis/metabolism/radiation effects
*Light
Adenylyl Cyclases/metabolism/genetics
*Cytosol/metabolism/radiation effects
*Acceleration
Phototaxis

@article {pmid41897402,
year = {2026},
author = {Shahannaz, DC and Sugiura, T},
title = {Next-Generation Metabolic Reprogramming in iPSC-Derived Cardiomyocytes: CRISPR-EV Synergy for Precision Cardiac Regeneration.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897402},
issn = {2218-273X},
mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Regeneration ; Animals ; CRISPR-Cas Systems ; Gene Editing ; Metabolic Reprogramming ; },
abstract = {Cardiovascular disease remains the leading global cause of mortality, largely due to the limited regenerative capacity of adult human myocardium. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a scalable platform for cardiac repair and disease modeling; however, their persistent metabolic immaturity-characterized by reliance on glycolysis, reduced oxidative phosphorylation (OXPHOS), and structurally underdeveloped mitochondria-limits functional integration and long-term therapeutic efficacy. Recent advances indicate that targeted metabolic reprogramming can enhance mitochondrial biogenesis, increase ATP production, and improve stress resilience in iPSC-CMs. This review examines the complementary integration of CRISPR-based metabolic engineering and extracellular vesicle (EV)-mediated metabolic modulation as a systems-level strategy for cardiac maturation. We discuss CRISPR activation, interference, and epigenome-editing approaches targeting regulators such as PGC-1α, TFAM, and PPARs to promote stable enhancement of mitochondrial networks and respiratory capacity. In parallel, engineered EVs delivering miRNAs, metabolic enzymes, and redox modulators provide non-genomic mechanisms to optimize bioenergetic function and mitigate oxidative stress. By synthesizing mechanistic insights, quantitative bioenergetic metrics, and translational considerations, we propose CRISPR-EV synergy as a precision framework for durable metabolic maturation of iPSC-CMs, with implications for regenerative therapy, pharmacologic screening, and myocardial repair.},
}

Humans
*Myocytes, Cardiac/metabolism/cytology
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cellular Reprogramming
*Regeneration
Animals
CRISPR-Cas Systems
Gene Editing
Metabolic Reprogramming

@article {pmid41898355,
year = {2026},
author = {Ye, Y and Huang, L and Fu, H and Wang, J and Jin, Y},
title = {Advances in SRNS Gene Research: From Precision Classification to Precision Diagnosis and Treatment.},
journal = {Biomedicines},
volume = {14},
number = {3},
pages = {},
pmid = {41898355},
issn = {2227-9059},
support = {2024C03211 to Jingjing Wang//Key Research and Development Program of Zhejiang Province/ ; GZY-KJS-ZJ-2026-071//Joint TCM Science &Technology Projects of National Demonstration Zones for Comprehensive TCM Reform/ ; LTGD24H050002//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {To clarify the genetic classification, diagnostic strategies, and precision treatment pathways of steroid-resistant nephrotic syndrome (SRNS), this review systematically reviews the genetic stratification system of SRNS by integrating recent advances in genetic testing technologies and pathogenesis research. It contains the pathogenic mechanisms, diagnostic protocols, and therapeutic correlations of different genetic subtypes, while summarizing current progress and clinical challenges in gene therapy. Results indicate SRNS can be categorized into genetic (38-58%) and non-genetic/immune-mediated (40-60%). A stepwise diagnostic system comprising core proteinuria gene panel testing, whole-genome sequencing (WGS), whole-exome sequencing (WES), and supplementary multi-omics/long-range sequencing is proposed, suited for populations with "typical phenotypes and moderate genetic risk", "atypical phenotypes and high genetic suspicion", and "complex structural/non-coding region variants" respectively. Pathogenic mechanisms directly determine therapeutic strategies: COQ2/PDSS2 mutations respond to coenzyme Q10 suplementation, while NPHS1 mutations necessitate early renal transplantation. Adeno-associated virus (AAV)-mediated gene therapy and CRISPR-Cas editing show preclinical promise but face challenges including incomplete detection coverage and clinical translation difficulties. Genetic technologies are driving SRNS management transformation from "empirical treatment" to "mechanism-oriented precision diagnosis and therapy". Future efforts should focus on overcoming genetic testing limitations and gene therapy translation bottlenecks to enhance diagnostic and therapeutic efficacy.},
}

@article {pmid41898529,
year = {2026},
author = {Thu, KL and Jafari, S and Silvester, J and Cruickshank, J and Soria-Bretones, I and Hodgson, K and Tobin, C and Haight, J and Lau, APY and Bray, T and Wakeham, D and Bray, MR and Mak, TW and Cescon, DW},
title = {Genome-Wide CRISPR Screens Identify ABCG2-Mediated Drug Resistance to the Threonine Tyrosine Kinase (TTK) Inhibitor CFI-402257 in Breast Cancer.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898529},
issn = {1422-0067},
mesh = {Humans ; *ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/metabolism ; *Drug Resistance, Neoplasm/genetics ; Animals ; Female ; Cell Line, Tumor ; Mice ; *Triple Negative Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; *Neoplasm Proteins/genetics/metabolism ; Xenograft Model Antitumor Assays ; *Protein-Tyrosine Kinases ; CRISPR-Cas Systems ; *Protein Kinase Inhibitors/pharmacology ; *Cell Cycle Proteins/antagonists & inhibitors ; *Indoles/pharmacology ; Antineoplastic Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Apoptosis/drug effects ; Protein Serine-Threonine Kinases ; },
abstract = {CRISPR screens are a powerful functional genomics approach for identifying genes that confer sensitivity and resistance to anti-cancer therapies. CFI-402257 (luvixasertib, 2257) is a small molecule inhibitor of threonine tyrosine kinase (TTK), a promising therapeutic target in genomically unstable cancers due to its critical role in establishing the spindle assembly checkpoint (SAC) during mitosis. To inform its ongoing development and evaluation in clinical trials, we sought to use CRISPR activation (i.e., gain of function) screens to identify cellular mechanisms of resistance to 2257 in models of triple-negative breast cancer (TNBC). In vitro screens conducted in two TNBC cell lines nominated ABCG2 as the top resistance-conferring gene in both models. Validation studies assessing clonogenic survival and apoptosis confirmed that ABCG2 overexpression enhanced TNBC resistance to 2257 in vitro, while knockdown enhanced sensitivity. These findings suggest that 2257 is a substrate of ABCG2's drug efflux activity. However, overexpression of ABCG2 failed to confer resistance to 2257 in TNBC xenografts grown in mice and treated with a moderately active dose and schedule. Our results highlight the potential impact of drug transporters in in vitro CRISPR screens and the importance of confirming the relevance of drug response mechanisms identified in cultured cells using in vivo models that recapitulate drug pharmacokinetics and pharmacodynamics.},
}

Humans
*ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/metabolism
*Drug Resistance, Neoplasm/genetics
Animals
Female
Cell Line, Tumor
Mice
*Triple Negative Breast Neoplasms/genetics/drug therapy/pathology/metabolism
*Neoplasm Proteins/genetics/metabolism
Xenograft Model Antitumor Assays
*Protein-Tyrosine Kinases
CRISPR-Cas Systems
*Protein Kinase Inhibitors/pharmacology
*Cell Cycle Proteins/antagonists & inhibitors
*Indoles/pharmacology
Antineoplastic Agents/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
Apoptosis/drug effects
Protein Serine-Threonine Kinases

@article {pmid41898703,
year = {2026},
author = {Seh, BA and Rafiq, K and Legradi, A and Mir, MY},
title = {Targeted Gene and Genome-Editing Strategies for Epilepsy: Experimental Advances and Translational Challenges.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898703},
issn = {1422-0067},
mesh = {Humans ; *Epilepsy/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems ; Translational Research, Biomedical ; },
abstract = {Epilepsy affects more than 50 million individuals worldwide, and approximately one-third of patients remain refractory to existing antiseizure medications. Advances in gene therapy and genome editing have opened new possibilities for disease-modifying interventions that directly target the molecular and circuit-level mechanisms underlying epileptogenesis. Recent progress in central nervous system tropic viral vectors, non-viral delivery systems, and programmable genome-editing technologies has enabled precise manipulation of neuronal and glial function in preclinical epilepsy models. Strategies range from restoration of haploinsufficient genes implicated in monogenic epilepsies, such as SCN1A in Dravet syndrome, to modulation of neuronal excitability through engineered ion channels, neuropeptides, and astrocyte-based approaches. In parallel, CRISPR-derived platforms, including transcriptional activation and repression systems, base editing, and prime editing, offer new avenues for regulating gene expression in post-mitotic neurons without introducing double-strand DNA breaks. Despite these advances, significant translational challenges remain, including efficient and cell-type-specific delivery, long-term safety, and the risk of network-level side effects in the epileptic brain. This review critically examines recent gene therapy and genome-editing approaches for epilepsy, highlights key technological and biological barriers to clinical translation, and discusses emerging strategies that may enable durable and targeted treatments for drug-resistant epilepsies.},
}

Humans
*Epilepsy/genetics/therapy
*Gene Editing/methods
*Genetic Therapy/methods
Animals
CRISPR-Cas Systems
Translational Research, Biomedical

@article {pmid41898716,
year = {2026},
author = {Calbay, O and Hsieh, CL and Lu, C and Ghosh, S and Vijaykumar, V and Watts, I and Sweigard, H and Gandhi, J and den Hollander, AI},
title = {Mapping the Hypoxic Fitness Landscape of Retinal Pigment Epithelial Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898716},
issn = {1422-0067},
mesh = {*Retinal Pigment Epithelium/metabolism/cytology ; Humans ; Cell Hypoxia/genetics ; Gene Expression Profiling ; Cell Line ; Transcriptome ; Macular Degeneration/genetics/metabolism/pathology ; CRISPR-Cas Systems ; *Epithelial Cells/metabolism ; Mitochondria/metabolism ; *Hypoxia/genetics ; Oxygen/metabolism ; },
abstract = {Chronic hypoxia is a hallmark of aging and retinal diseases such as age-related macular degeneration (AMD), yet the molecular mechanisms that enable retinal pigment epithelium (RPE) cells to survive under sustained low-oxygen conditions remain poorly understood. To address this, we conducted transcriptomic profiling and a genome-wide CRISPR-Cas9 loss-of-function screen in ARPE-19 cells exposed to chronic hypoxia (1% and 5% O2), mimicking the retinal disease environment. The CRISPR screen identified genes whose loss compromises RPE viability or fitness under hypoxia, while transcriptomic profiling revealed oxygen-dependent shifts in key functional modules. These findings converged on pathways related to mitochondrial function, extracellular matrix remodeling, vascular signaling, and cell cycle regulation, identifying unique functional nodes specific to RPE cells. These core processes are also implicated in retinal diseases, such as AMD. Together, these complementary approaches provide an integrated view of the molecular networks driving RPE adaptation to hypoxic stress and highlight novel gene candidates that may serve as therapeutic targets in retinal disease.},
}

*Retinal Pigment Epithelium/metabolism/cytology
Humans
Cell Hypoxia/genetics
Gene Expression Profiling
Cell Line
Transcriptome
Macular Degeneration/genetics/metabolism/pathology
CRISPR-Cas Systems
*Epithelial Cells/metabolism
Mitochondria/metabolism
*Hypoxia/genetics
Oxygen/metabolism

@article {pmid41898816,
year = {2026},
author = {Abdalla Elsayed, MEA and MacLaren, RE},
title = {Precision Is Not Enough: When Tools Outpace Translation in Ocular Gene Therapy.},
journal = {Genes},
volume = {17},
number = {3},
pages = {},
pmid = {41898816},
issn = {2073-4425},
support = {//Foundation Fighting Blindness Clinical Research Fellowship/ ; //Oxford NIHR Biomedical Research Centre, UK Department of Health/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; Gene Editing/methods ; *Eye Diseases/therapy/genetics ; CRISPR-Cas Systems ; Translational Research, Biomedical ; Animals ; },
abstract = {Advances in molecular biology have positioned the eye as a leading platform for gene therapy, owing to its surgical accessibility, relative immune privilege, and the ability of the contralateral eye to serve as an anatomical control. We trace the historical evolution of gene discovery, synthesize current gene therapy strategies for inherited and acquired ocular disorders, critically evaluating the limitations of CRISPR and related genome-editing technologies, and examine the key scientific and translational challenges that must be addressed for genetic therapies to be integrated into routine ophthalmic practice.},
}

Humans
*Genetic Therapy/methods
Gene Editing/methods
*Eye Diseases/therapy/genetics
CRISPR-Cas Systems
Translational Research, Biomedical
Animals

@article {pmid40148722,
year = {2026},
author = {Mete, R and Das, S and Saha, A and Roy, S and Mondal, S and Bose, A and Basu, B and Elossaily, GM and Prajapati, B},
title = {Transgenesis in Drug Discovery: Enhancing Target Identification and Validation.},
journal = {Molecular biotechnology},
volume = {68},
number = {3},
pages = {998-1022},
pmid = {40148722},
issn = {1559-0305},
mesh = {Animals ; *Drug Discovery/methods ; Humans ; Animals, Genetically Modified/genetics ; *Gene Transfer Techniques ; Gene Editing/methods ; CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; },
abstract = {Transgenesis, the introduction of foreign genetic material into the genome of an organism, has become a crucial and transformative technique in the realm of drug discovery. This review article provides a comprehensive overview of the integral role that transgenesis plays in the drug discovery process, with a specific focus on target identification and target validation. By examining the recent advancements and innovative approaches, this article aims to shed light on the importance of transgenesis in accelerating drug development. In the context of target identification, transgenesis has allowed for the creation of relevant disease models, enabling researchers to study the genetic and molecular basis of various disorders. The use of transgenic animals, such as mice and zebrafish, has facilitated the identification of potential drug targets by mimicking specific human disease conditions. This review also discusses emerging technologies, such as CRISPR-Cas9 and other genome editing tools, which have revolutionized the field of transgenesis. These technologies have enhanced the precision and efficiency of genetic manipulations in transgenic animals, making the creation of disease-relevant models more accessible and cost-effective. Moreover, integration of omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, has provided a holistic view of the molecular changes in transgenic models, further aiding in target identification and validation. This review emphasizes the importance of transgenesis in target identification and validation and underscores its vital role in shaping the future of drug discovery.},
}

Animals
*Drug Discovery/methods
Humans
Animals, Genetically Modified/genetics
*Gene Transfer Techniques
Gene Editing/methods
CRISPR-Cas Systems
Mice
Disease Models, Animal

@article {pmid40493161,
year = {2026},
author = {Kurpejović, E and Sariyar Akbulut, B and Avci, FG},
title = {Tailoring Corynebacterium glutamicum for Sustainable Biomanufacturing: From Traditional to Cutting-Edge Technologies.},
journal = {Molecular biotechnology},
volume = {68},
number = {3},
pages = {1106-1126},
pmid = {40493161},
issn = {1559-0305},
mesh = {*Corynebacterium glutamicum/genetics/metabolism ; *Metabolic Engineering/methods ; Machine Learning ; Genetic Engineering/methods ; CRISPR-Cas Systems ; Artificial Intelligence ; Amino Acids/biosynthesis ; Industrial Microbiology/methods ; Genome, Bacterial ; Biotechnology/methods ; },
abstract = {As the workhorse of industrial amino acid production, Corynebacterium glutamicum is the focus of this review, which provides a comprehensive overview of available techniques employed to engineer strains with desired traits. The review highlights both traditional and cutting-edge approaches with a brief introduction to the bacterium's physiology, serving as a foundation for understanding its metabolic capabilities and potential applications. Genome modulation techniques by contrasting traditional methods with CRISPR-based approaches, as well as transcription modulation strategies that enhance gene expression and metabolic flux, and high-throughput techniques that streamline strain development processes are summarized. Furthermore, the roles of artificial intelligence and machine learning in genetic engineering are explored, emphasizing their growing impact on strain development.},
}

*Corynebacterium glutamicum/genetics/metabolism
*Metabolic Engineering/methods
Machine Learning
Genetic Engineering/methods
CRISPR-Cas Systems
Artificial Intelligence
Amino Acids/biosynthesis
Industrial Microbiology/methods
Genome, Bacterial
Biotechnology/methods

@article {pmid41723140,
year = {2026},
author = {Shen, K and Seow, WY and Keng, CT and Lim, MGL and Lim, DS and Guo, K and Meliani, A and Irfan Bin Hajis, M and Wang, B and Prabhakar, S and Chen, KH and Chew, WL},
title = {Spatial perturb-seq: single-cell functional genomics within intact tissue architecture.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41723140},
issn = {2041-1723},
support = {PREPARE-OC-VT-2024-008//Ministry of Health -Singapore (MOH)/ ; SC18/21-1089UI//Agency for Science, Technology and Research (A*STAR)/ ; OFIRG24jul-0096//MOH | National Medical Research Council (NMRC)/ ; OFYIRG23jul-0050//MOH | National Medical Research Council (NMRC)/ ; NRF-CRP25-2020-0001//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; },
mesh = {Animals ; *Single-Cell Analysis/methods ; Mice ; *Genomics/methods ; Brain/metabolism/cytology ; CRISPR-Cas Systems ; Neurodegenerative Diseases/genetics ; Mice, Knockout ; Neurons/metabolism ; Cell Communication/genetics ; Gene Knockout Techniques ; },
abstract = {We develop Spatial Perturb-Seq, an in vivo CRISPR technology that interrogates multiple genes within single cells of intact tissues, compatible with both sequencing-based and probe-based spatial technologies. We apply Spatial Perturb-Seq to knock out risk genes for neurodegenerative diseases in the mouse brain, uncovering cell autonomous and cell-cell microenvironmental effects within the spatially intact tissue. Spatial Perturb-Seq functionally screens multiple genes in situ and in vivo, bypasses cell processing steps that skew cell type representation, identifies intracellular and intercellular effects of knockouts, and identifies candidate genes underlying dysregulated neuronal intercellular communication pathways.},
}

Animals
*Single-Cell Analysis/methods
Mice
*Genomics/methods
Brain/metabolism/cytology
CRISPR-Cas Systems
Neurodegenerative Diseases/genetics
Mice, Knockout
Neurons/metabolism
Cell Communication/genetics
Gene Knockout Techniques

@article {pmid41840760,
year = {2026},
author = {Karchner, SI and Aluru, N and Franks, DG and Mandl, CA and Goldstone, JV and Burke, T and Champlin, D and La Du, JK and Perone, DM and Stinson, S and Truong, L and Clark, BW and Nacci, D and Tanguay, RL and Hahn, ME},
title = {Using fish models to understand the role of aryl hydrocarbon receptor (AHR)-interacting protein (AIP) in controlling sensitivity and resistance to dioxin-like compounds in vivo.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {209},
number = {3},
pages = {},
doi = {10.1093/toxsci/kfag033},
pmid = {41840760},
issn = {1096-0929},
support = {P42 ES007381/ES/NIEHS NIH HHS/United States ; R01 ES032323/ES/NIEHS NIH HHS/United States ; P30 ES030287/ES/NIEHS NIH HHS/United States ; S10 OD032203/OD/NIH HHS/United States ; R01 ES033888/ES/NIEHS NIH HHS/United States ; P42 ES016465/ES/NIEHS NIH HHS/United States ; R01ES033888//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; R01ES032323//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; P42ES007381//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; P42ES016465//Superfund Research Program at Boston University/ ; P30ES030287//Superfund Research Program at Oregon State University/ ; 1S10OD032203-01//Superfund Research Program at Oregon State University/ ; //Tufts University Core Facility Genomics Core/ ; //NIH/ ; //NIH Public Access Policy/ ; //PubMed Central upon the Official Date of Publication/ ; },
mesh = {Animals ; *Receptors, Aryl Hydrocarbon/metabolism/genetics ; *Fundulidae/genetics/embryology/metabolism ; *Dioxins/toxicity ; Polychlorinated Dibenzodioxins/toxicity ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Polychlorinated Biphenyls/toxicity ; Zebrafish/genetics ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics ; Embryo, Nonmammalian/drug effects ; *Fish Proteins/genetics/metabolism ; },
abstract = {Humans are exposed to chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) that cause toxicity through activation of the aryl hydrocarbon receptor (AHR). There is inter-individual variation in sensitivity to the effects of AHR ligands, but it is not fully explained by variation in the AHR. A clue to the genetic mechanisms underlying differential sensitivity to AHR agonists has emerged from studies of Atlantic killifish (Fundulus heteroclitus) populations with evolved tolerance to PCBs, TCDD, and PAHs. Genomic studies of these populations identified AHR-interacting protein (AIP/Ara9/XAP2) as the strongest candidate resistance gene. However, the precise role of AIP in the mechanism of resistance is unknown. To understand the role of AIP in the toxicity of dioxin-like compounds in vivo, we used CRISPR-Cas9 to generate AIP loss-of-function alleles in killifish and zebrafish (Danio rerio). Homozygous mutant killifish and zebrafish die during larval development-by 30 and 12 d postfertilization, respectively-whereas heterozygous mutants develop, survive, and reproduce normally. During embryonic and early larval stages, homozygous mutant zebrafish exhibit reduced sensitivity to embryotoxic effects of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) and TCDD. Gene expression profiling of aip-deficient larvae revealed hundreds of differentially expressed genes. PCB126 induced similar sets of well-known AHR-regulated genes in mutant and wild-type larvae, although with reduced magnitude overall in AIP mutants. This study highlights the important role of AIP in fish larval development and demonstrates that AIP status can influence the response of vertebrate embryos to dioxin-like compounds in vivo.},
}

Animals
*Receptors, Aryl Hydrocarbon/metabolism/genetics
*Fundulidae/genetics/embryology/metabolism
*Dioxins/toxicity
Polychlorinated Dibenzodioxins/toxicity
*Intracellular Signaling Peptides and Proteins/genetics/metabolism
Polychlorinated Biphenyls/toxicity
Zebrafish/genetics
CRISPR-Cas Systems
Zebrafish Proteins/genetics
Embryo, Nonmammalian/drug effects
*Fish Proteins/genetics/metabolism

@article {pmid41892062,
year = {2026},
author = {Tantai, W and Xu, Q and Zhang, W and Li, Y and Liu, H},
title = {A One-Pot CRISPR/Cas12a-Based Platform for Contamination-Free Nucleic Acid Amplification Detection.},
journal = {Biosensors},
volume = {16},
number = {3},
pages = {},
pmid = {41892062},
issn = {2079-6374},
support = {22074085//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; *Biosensing Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 10[6] copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples.},
}

*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction
*Biosensing Techniques
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41892280,
year = {2026},
author = {Woronkowicz, M and Thomas, MN and Saram, SJ and Carr, AF and Alonso-Carriazo Fernandez, A and Butt, Z and Skopiński, P and Ramsden, CM},
title = {CRISPR and Beyond: Genome-Editing Strategies in Retinal Stem Cell Research.},
journal = {Cells},
volume = {15},
number = {6},
pages = {},
pmid = {41892280},
issn = {2073-4409},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Retina/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Stem Cell Research ; Retinal Degeneration/genetics/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome editing has emerged as a transformative approach for understanding and treating retinal degenerative diseases. Combining this technology with pluripotent stem cells provides an ideal platform for modeling human development and disease, and investigating emerging therapeutic strategies ultimately aimed towards in vivo correction. This approach enables both functional studies to understand retinal degeneration and the early development of targeted therapies for inherited disease. This review offers a comprehensive overview of genome-editing techniques and the ability to create new clinically relevant models to understand human disease in retinal research, focusing on the use of the CRISPR-Cas9 system in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), as well as highlighting recent advancements in base and prime editing. Gene editing in various retinal diseases is discussed in context of studies focusing on disease modeling or developing therapeutic strategies. Continued refinement of these techniques will be essential for advancing translational applications in retinal disease treatment.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
*Retina/cytology
Induced Pluripotent Stem Cells/metabolism/cytology
*Stem Cell Research
Retinal Degeneration/genetics/therapy
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41892424,
year = {2026},
author = {Gomes, E and Mesquita, TG and Serra, P and Araújo, D and Almeida, C and Machado, A and Oliveira, R and Castro, J},
title = {Antimicrobial Resistance in the Food Chain: Bridging Knowledge Gaps for Effective Detection and Control.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41892424},
issn = {2079-6382},
support = {https://doi.org/10.54499/2024.13640.PEX//Fundação para a Ciência e Tecnologia/ ; https://doi.org/10.54499/2022.07654.PTDC//Fundação para a Ciência e Tecnologia/ ; APTA4shiga (number 14840)//Fundação para a Ciência e Tecnologia/ ; },
abstract = {Antimicrobial resistance (AMR) poses a critical global public health threat, with the food chain serving as a significant transmission route connecting animals, environment, and humans. This review adopts a One Health perspective to analyze the key drivers of AMR dissemination across animal agriculture, aquaculture and food processing. We evaluate detection methodologies, contrasting the regulatory gold standard of culture-based phenotypic testing with rapid molecular advancements, including Whole Genome Sequencing (WGS), metagenomics, and emerging CRISPR-Cas diagnostics. While molecular tools offer unprecedented speed and resolution, challenges such as matrix interference, the viable but non-culturable (VBNC) state, and the genotype-phenotype disconnect remain. Finally, integrated mitigation strategies are also described, ranging from on-farm antimicrobial stewardship and innovative biofilm control to consumer hygiene practices. It is essential to bridge the technical and regulatory gaps in AMR surveillance in order to develop effective interventions and ensure a safer food system.},
}

@article {pmid41893903,
year = {2026},
author = {Baliyan, N and Upadhyay, P and Murugan, T and Srivastava, A and Singh, S and Tomar, BS and Mangal, M},
title = {Recent advances in generation of doubled haploid plants for genetic improvement in solanaceous vegetable crops.},
journal = {Planta},
volume = {263},
number = {5},
pages = {},
pmid = {41893903},
issn = {1432-2048},
support = {SR/WOS-A/LS-146/2019//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*Haploidy ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; *Vegetables/genetics ; Genome, Plant ; },
abstract = {Genotype-specific protocols and advancements focusing on CRISPR/Cas9-based haploid induction for doubled haploid (DH) production are poised to revolutionize plant breeding for faster genetic improvement in solanaceous crops. The need for swift development of improved cultivars offering greater resilience to biotic and abiotic stresses stems from the emergence of climate change risks. While conventional methods are effective, novel methods for precise crop genome manipulation are required. The in vivo and in vitro protocols leading to fixation of homozygosity and rapid attainment of homozygous DH (doubled haploids) lines have led to a resurgence in research on haploids and DH. The efforts for haploid production have been primarily concentrated on in vitro androgenesis and gynogenesis. The success of these in vitro techniques depends on various parameters, including culture conditions, developmental stage of the microspore, pretreatment, culture medium, growth regulators, and other media additives. Breakthroughs in genome-editing technologies, such as the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system, have provided a new avenue for exploring in vivo haploid embryogenesis for haploid induction by means of haploid inducer factors, such as DMP (DMP domain of unknown function 679 membrane protein), ECS (egg cell-specific), MTL (matrilineal), and BBM (Baby boom). A successful haploid induction system in vegetable crops will be possible through the development of efficient in vivo and in vitro androgenesis and gynogenesis protocols. For genome-editing to be feasible, protocols must be optimized for commercially significant solanaceous crops like pepper, potatoes, eggplant, and tomatoes. The existing protocols for genome doubling need to be further improved for solanaceous crops. These developments are pivotal for the advancement and harnessing of haploid technology to its full potential in crop breeding.},
}

*Haploidy
*Crops, Agricultural/genetics
*Plant Breeding/methods
Gene Editing/methods
CRISPR-Cas Systems
*Vegetables/genetics
Genome, Plant

@article {pmid41893914,
year = {2026},
author = {Pistone, D and Bevivino, G and Dipaola, MG and Bandi, C and Lombardo, F},
title = {Current and emerging molecular diagnostic approaches in the detection of human parasites.},
journal = {Parasitology research},
volume = {125},
number = {1},
pages = {},
pmid = {41893914},
issn = {1432-1955},
abstract = {Microscopy and morphological identification remain the gold standard for diagnosing most parasitic infections, yet their limited sensitivity in asymptomatic or low-burden cases, along with technical constraints, has accelerated the adoption of molecular diagnostics. Over the past three decades, advances in nucleic acid amplification and sequencing technologies have transformed parasite detection by improving sensitivity, specificity, and reproducibility, enabling earlier intervention and stronger surveillance. PCR remains the foundation of molecular diagnostics, with real-time PCR and digital PCR improving analytical performance and quantification. Multiplex qPCR supports simultaneous detection of multiple pathogens, while dPCR enables absolute quantification and rare variant detection, although broader implementation is limited by instrument cost. Isothermal amplification methods such as tHDA, NASBA, LAMP, and RPA offer rapid, low-cost amplification at constant temperature and are well suited for field diagnostics in resource-limited settings. Next-Generation Sequencing has advanced genotyping and epidemiological surveillance by resolving cryptic species, resistance mutations, and mixed infections through targeted panels, whole-genome sequencing, and metagenomics. CRISPR/Cas-based assays provide rapid and sensitive nucleic acid detection with strong potential for point-of-care deployment due to their simplicity and adaptability. Emerging biomarkers, including circulating cell-free DNA, non-coding RNAs, and microRNAs in extracellular vesicles, offer promising non-invasive diagnostic strategies, though further validation is required. This review offers a concise overview of these molecular approaches, emphasizing recent innovations such as dPCR, NGS, CRISPR/Cas systems, and biomarker-based detection. For each method, core technical principles, representative applications, and comparative strengths and limitations are presented to illustrate their diagnostic potential.},
}

@article {pmid41895443,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {111401},
doi = {10.1016/j.jbc.2026.111401},
pmid = {41895443},
issn = {1083-351X},
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cascade conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}

@article {pmid41896556,
year = {2026},
author = {Walsh, LH and Soni, V and Ancla, J and Somerville, V and Segata, N and Joyce, S and Sinderen, DV and Mahony, J and Shkoporov, AN and Kenny, JG and Cotter, PD and O'Sullivan, O},
title = {Mining of food metagenomes reveals an unexplored diversity of dsDNA bacteriophages.},
journal = {NPJ biofilms and microbiomes},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41522-026-00941-9},
pmid = {41896556},
issn = {2055-5008},
support = {DOMINO-101060218//European Union's Horizon Europe programme/ ; },
abstract = {Bacteriophages are key drivers of microbial ecology, co-existing and co-evolving with bacteria across diverse environments. Limitations in culturing, alongside advances in sequencing and bioinformatics, have driven the use of metagenomics to explore viral diversity. Viral-specific analysis of >3000 food metagenomes from cFMD produced the FVGC, comprising ~3400 metagenome-assembled viruses, most of which belong to novel Caudoviricetes lineages (n = 91), with only ~15% represented in IMG/VR v4. Together, these findings reveal extensive uncharacterized viral diversity in food systems. Beyond serving as a reference, the FVGC facilitates detailed investigation of virus-host interactions. Viral sequences were pervasive across microbial genomes, with several bacterial families exhibiting near-universal associations with viral elements. Bacterial antiviral defence systems were abundant and taxonomically diverse, dominated by restriction-modification systems, while CRISPR-Cas systems showed pronounced lineage-specific distributions; in contrast, viral anti-defence genes were detected at low frequency (<10% of MAVs). Host prediction linked MAVs to clinically relevant taxa, including expanded ESKAPE pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Enterobacter spp., highlighting the ecological connectivity between food-associated viruses and clinically important bacteria. Antimicrobial resistance signals were scarce, suggesting minimal phage-mediated AMR dissemination in food environments. This new publicly available viral database represents a valuable resource for further exploration of viral diversity.},
}

@article {pmid41897294,
year = {2026},
author = {Zhou, Z and Zhu, L},
title = {A Computational Model for Nme1Cas9 HNH Activation Driven by Dynamic Interface Engineering at Residues S593 and W596.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897294},
issn = {2218-273X},
support = {32471296//National Natural Science Foundation of China Projects/ ; },
mesh = {Molecular Dynamics Simulation ; Thermodynamics ; Protein Engineering ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; },
abstract = {Nme1Cas9 is an encouraging genome-editing tool with high fidelity and compactness, but its applications are limited by poor catalytic efficiency compared with SpyCas9. Understanding the dynamic activation mechanism of the HNH nuclease domain is the key to breaking the kinetic bottleneck. Here, we integrated Steered Molecular Dynamics (SMD) with the Traveling-Salesman-based automated Path Searching (TAPS) algorithm to reconstruct the atomic-level activation landscape of the L1-HNH module. The simulations suggest a complex "Lifting-Rearrangement-Sliding" pathway, revealing the critical role of a "Backbone Sliding" conformation; in this step, the HNH domain rotates across the R-loop surface. A thermodynamic analysis using free energy decomposition by MM/PBSA indicates that the intrinsic instability of the wild-type HNH/R-loop interface constitutes the predominant energetic barrier. Hyperactive variants (S593Q/W596K and S593Q/W596R) can overcome this barrier by substantially increasing binding affinity to the R-loop through a "Geometry-Electrostatics Synergism": S593Q improves interfacial proximity, whereas W596K/R acts as an "Electrostatic Anchor." The results of unbiased MD simulations demonstrate that strengthened interfacial interactions effectively promote spontaneous conformational drift toward the activated state. This computational study proposes a novel in silico model for "Dynamic Interface Engineering" in which reinforcing transient interfacial contacts during conformational sliding can be an effective strategy in developing high-efficiency CRISPR-Cas effectors.},
}

Molecular Dynamics Simulation
Thermodynamics
Protein Engineering
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism

@article {pmid41864147,
year = {2026},
author = {Zhang, L and Tang, J and Feng, M and Chen, S},
title = {Development of a plasmid-free Escherichia coli strain for high-yield production of ergothioneine.},
journal = {Enzyme and microbial technology},
volume = {197},
number = {},
pages = {110850},
doi = {10.1016/j.enzmictec.2026.110850},
pmid = {41864147},
issn = {1879-0909},
mesh = {*Ergothioneine/biosynthesis ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; Plasmids/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; },
abstract = {Ergothioneine (ERG), a sulfur-containing amino acid derivative known for its antioxidant activity, has a wide range of applications in healthcare and nutrition. Escherichia coli has been extensively studied as a platform for ERG production due to its rapid growth and well-established genetic tools. However, most engineered strains rely on plasmid-based expression system, which are genetically unstable. Additionally, the requirement for antibiotics to maintain plasmid stability further limits the feasibility of plasmid-based systems for industrial-scale production. Here, we established a plasmid-free E. coli platform for ERG biosynthesis using a multi-copy chromosomal integration CRISPR-associated transposase (MUCICAT) system. We first integrated a three-gene ERG biosynthetic pathway into the E. coli genome at varying copy numbers, resulting in a five-copy strain (P5) that exhibited the highest ERG titer of 222.5 ± 5.0 mg/L. Subsequently, we reinforced the two key catalytic modules-histidine methylation and SAM biosynthesis-through iterative genomic integration of the corresponding genes, yielding a plasmid-free strain P18 that produced 370.0 ± 7.0 mg/L ERG. The engineered strain P18 exhibited excellent genetic stability, as confirmed by serial passaging. When scaled up in a 5-L bioreactor under fed-batch condition, an ERG titer of 10.1 g/L was achieved. This study demonstrates a plasmid-free ERG production strategy based on stable, multi-copy chromosomal integration of the ERG biosynthetic pathway in E. coli, highlighting its potential as an efficient platform for scalable ERG production.},
}

*Ergothioneine/biosynthesis
*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
Plasmids/genetics
Biosynthetic Pathways/genetics
CRISPR-Cas Systems

@article {pmid41889958,
year = {2026},
author = {Ocampo, RF and Orosco, C and Huang, B and West, MS and Jain, PK and Taylor, DW},
title = {Architecture of a DNA-guided Cas12a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.03.19.712971},
pmid = {41889958},
issn = {2692-8205},
abstract = {CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The ΨDNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering.},
}

@article {pmid41891780,
year = {2026},
author = {Gu, C and Gu, J and Li, J and Zou, D and Qi, Y and Xia, R and Zhou, Z and Li, M},
title = {Insights into the Arms Race between Prokaryotic Hosts and Their Viruses in Mangrove Ecosystem.},
journal = {Environmental science & technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.5c14802},
pmid = {41891780},
issn = {1520-5851},
abstract = {The coevolutionary arms race between prokaryotes and viruses has driven the diversification of various microbial immune mechanisms including restriction-modification (RM) and clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems. While recent efforts have expanded the catalog of antiviral systems, their ecological dynamics within complex microbial communities remain underexplored. Here, we analyzed prokaryotic communities, viruses, and defense systems in mangrove habitats from Futian, China, by integrating DNA and RNA sequencing of sediment cores collected across multiple depths and seasons at two sites. Prokaryotic genomes harbored 65 distinct defense system types, representing ∼43% of known systems, with transcriptional activity dominated by Pseudomonadota, Planctomycetota, and Chloroflexiota. The key systems, including abortive infection (AbiD, AbiE, AbiU), Eleos, CRISPR-Cas, MazEF, Retron, and Wadjet, exhibited high transcriptional activity across samples. Strikingly, viruses encoded highly expressed defense systems such as AbiE and RM, despite their co-occurring prokaryotic hosts lacking detectable antiviral systems. The abundance ratio of prokaryotes and viruses with defense systems shows an opposite trend as the depth variation. Heterologous validation confirmed the antiviral efficacy of the selected systems. Our findings suggest that viral-encoded defense systems may functionally augment host immunity in mangrove habitats, revealing a nuanced coevolutionary interplay within these ecosystems. This study advances our understanding of host-virus interactions at the community level in mangrove wetland microbiomes.},
}

@article {pmid41891877,
year = {2026},
author = {Wan, Y and Zhao, X and Lin, X and Wang, L and Ai, X and Jiang, J and Han, L and Huang, D and Du, H and Huang, L},
title = {Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41891877},
issn = {1362-4962},
support = {2024ZDZ07//Guangdong Province Drug Administration Science and Technology Innovation/ ; 202206010073//Science and Technology Program of Guangzhou/ ; 2023A03J0542//Science and Technology Program of Guangzhou/ ; 2025A1515010459//Natural Science Foundation of Guangdong Provinc/ ; GZYZH2023001//Open Project of the China NMPA Key Laboratory for Animal Alternative Testing Technology of Cosmetics/ ; 2022A1515011733//Guangdong Basic and Applied Basic Research Foundation/ ; 2024ZDZ07//Guangdong Province Drug Administration Science and Technology Innovation Project/ ; 202206010073//Science and Technology Program of Guangzhou/ ; 2023A03J0542//Science and Technology Program of Guangzhou/ ; 2025A1515010459//Natural Science Foundation of Guangdong Provinc/ ; GZYZH2023001//Open Project of the China NMPA Key Laboratory for Animal Alternative Testing Technology of Cosmetics/ ; 2022A1515011733//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *INDEL Mutation ; DNA Breaks, Double-Stranded ; *Gene Editing/methods ; *DNA Repair/genetics ; Humans ; DNA End-Joining Repair ; Gene Regulatory Networks ; },
abstract = {CRISPR/Cas9-induced DNA double-strand breaks (DSBs) trigger diverse repair outcomes, yet the dynamic regulatory networks governing these outcomes remain incompletely understood. Here, we develop indel pattern-guided repair mapping, an integrative framework that deciphers DSB repair mechanisms by integrating repair outcome spectra, kinetic dynamics, and functional gene regulation. Our analysis categorizes Cas9-mediated repair outcomes into seven distinct patterns based on their frequency and sequence characteristics, revealing differential repair kinetics among these subtypes. Functional clustering identifies three regulatory pillars: (i) microhomology-mediated end joining (MMEJ)-driven MH deletions form a cohesive module defined by a shared regulatory network of protein-coding genes and miRNAs, rather than by the core repair enzymes themselves; (ii) non-homologous end joining coordinates 1 bp insertions and non-MH deletions, with RFC4/5 stabilizing repair templates to suppress large deletions; (iii) Atypical repair outcomes show distinct genetic signatures: large insertions are associated with polymerase-related regulators, whereas mutations are associated with a signature enriched for chromatin-associated regulators. Strikingly, S100A8 emerges as a potent MMEJ suppressor via direct interaction with PARP1, revealing unappreciated cross-talk between inflammatory signaling and DSB repair pathway choice. By linking repair outcome patterns to molecular determinants, our work provides a transformative platform to interrogate DNA repair mechanisms for precise genome editing optimization and therapeutic genome stabilization.},
}

*CRISPR-Cas Systems/genetics
*INDEL Mutation
DNA Breaks, Double-Stranded
*Gene Editing/methods
*DNA Repair/genetics
Humans
DNA End-Joining Repair
Gene Regulatory Networks

@article {pmid41581881,
year = {2026},
author = {Zhao, AH and Koganti, PP and Qian, M and Garcia, A and O'Day, P and Auchus, RJ and Covey, DF and Selvaraj, V},
title = {Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {3},
pages = {111191},
pmid = {41581881},
issn = {1083-351X},
mesh = {*Receptors, GABA/metabolism/genetics ; Animals ; *Leydig Cells/metabolism/drug effects/cytology ; Mice ; Male ; *Androstenes/pharmacology ; Progesterone/biosynthesis ; *3-Hydroxysteroid Dehydrogenases/metabolism/antagonists & inhibitors/genetics ; Pregnenolone ; Mitochondria/metabolism ; Cell Line ; *Steroids/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3β,17β,19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo-deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt2-cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3β-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3β-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.},
}

*Receptors, GABA/metabolism/genetics
Animals
*Leydig Cells/metabolism/drug effects/cytology
Mice
Male
*Androstenes/pharmacology
Progesterone/biosynthesis
*3-Hydroxysteroid Dehydrogenases/metabolism/antagonists & inhibitors/genetics
Pregnenolone
Mitochondria/metabolism
Cell Line
*Steroids/biosynthesis
CRISPR-Cas Systems

@article {pmid41708610,
year = {2026},
author = {Klann, M and Miura, S and Lee, SH and Vianello, SD and Ross, R and Watanabe, M and Gairin, E and Liang, Y and Hutto, HW and McCluskey, BM and Herrera, M and Solnica-Krezel, L and Besseau, L and Pigolotti, S and Parichy, DM and Kinoshita, M and Laudet, V},
title = {Cell-cell communication as underlying principle governing color pattern formation in teleost fishes.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708610},
issn = {2041-1723},
mesh = {Animals ; *Cell Communication/genetics/physiology ; Zebrafish/genetics ; Gap Junctions/metabolism ; *Pigmentation/genetics/physiology ; Mutation, Missense ; Connexins/genetics/metabolism ; Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Phenotype ; Gene Editing ; *Perciformes/genetics ; },
abstract = {The diverse pigmentation patterns of animals are crucial for predation avoidance and behavioral display. This diversity arises from interactions among distinct pigment cell types, yet mechanisms generating pattern variation across teleost fishes remain incompletely understood. In zebrafish, Turing models have been proposed to explain stripe patterns, but it is unclear if they apply to other fishes. Here, we investigate the Snowflake mutant of the anemonefish Amphiprion ocellaris, which displays enlarged white bars with irregular boundaries. Using genome-wide association mapping and targeted sequencing, we identify a missense mutation (E42K) in gja5b, encoding the gap junction protein Connexin 41.8. CRISPR/Cas9-mediated genome editing recapitulates the Snowflake phenotype, while pharmacological inhibition of gap junctions phenocopies the boundary defects, supporting a causal role for impaired intercellular communication. Expression analyses reveal that, unlike zebrafish, anemonefish gja5b is predominantly expressed in iridophores. With functional in vitro assays we demonstrate that the E42K mutation acts as a dominant negative, strongly reducing gap junctional coupling. Introducing the same mutation in zebrafish reveals context-dependent effects on pigment patterning. Taken together our findings highlighting gap junction-mediated communication as a conserved but flexible mechanism controlling pigment boundary positioning and pattern diversification.},
}

Animals
*Cell Communication/genetics/physiology
Zebrafish/genetics
Gap Junctions/metabolism
*Pigmentation/genetics/physiology
Mutation, Missense
Connexins/genetics/metabolism
Fish Proteins/genetics/metabolism
CRISPR-Cas Systems
Genome-Wide Association Study
Phenotype
Gene Editing
*Perciformes/genetics

@article {pmid41708664,
year = {2026},
author = {Huo, Y and Mei, J and Zhang, D and Yan, B and Zhang, D and Dong, C and Yin, S and Liu, M and Wang, X and Chen, D and Guan, Y and Song, G and Du, B and Wang, Y and Zheng, Z and Liu, H and Li, D and Yang, L and Wang, L},
title = {Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708664},
issn = {2041-1723},
support = {U24A20677//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230064//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32311530111//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24J22800400//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Genome, Human ; Mutation ; Genetic Therapy/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Humans
Mice
HEK293 Cells
Genome, Human
Mutation
Genetic Therapy/methods
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid41874403,
year = {2026},
author = {Zhang, H and Liu, L and Wang, D and Yang, X and Kang, Y and Huang, J and Ouyang, Y and Yu, H and Zhang, Y},
title = {CRISPR-Cas gene editing technology in biomanufacturing to enhance stress tolerance of microbial strains.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnag030},
pmid = {41874403},
issn = {1574-6968},
abstract = {In response to the loss of microbial efficiency caused by environmental stress in biomanufacturing, CRISPR-Cas gene editing technology has become a core tool for enhancing stress tolerance by accurately targeting genomic loci. This article systematically reviews the progress of its application. By optimizing engineered nucleases, gRNA design, and innovative delivery strategies, this technology successfully regulates key pathways in oxidative stress responses. It integrates functional genome screening with dynamic regulation to examine the networks of multi-gene collaborative tolerance. In the construction of high-stress-tolerant industrial chassis cells, the stress survival rate (>90% in Bacillus subtilis under thermal stress) and product synthesis ability (such as cellulose producing ethanol up to 4.5 g/L) of strains such as Escherichia coli and Corynebacterium glutamicum were significantly improved. Current challenges focus on delivery efficiency, off-target risks, and complex regulatory bottlenecks. In the future, the development of new editing tools and intelligent circuits will promote their industrial application in sustainable bio-manufacturing.},
}

@article {pmid41875153,
year = {2026},
author = {Magarditchian, G and Berest, I and Ziogou, A and Matsushita, M and Reid, M and Othman, A and Kopf, M},
title = {The phospholipid profile of T cells shapes ACSL4 dependency and ferroptosis sensitivity of naive, effector, and memory T cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {13},
pages = {e2528153123},
doi = {10.1073/pnas.2528153123},
pmid = {41875153},
issn = {1091-6490},
support = {ETH-30-20-1//ETH grant/ ; },
mesh = {*Ferroptosis/immunology ; *Coenzyme A Ligases/metabolism/genetics ; *Phospholipids/metabolism ; Animals ; Mice ; *CD8-Positive T-Lymphocytes/metabolism/immunology ; *Memory T Cells/metabolism/immunology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Humans ; Fatty Acids, Unsaturated/metabolism ; Mice, Knockout ; Iron/metabolism ; },
abstract = {Iron-dependent phospholipid (PL) peroxidation, which is reduced by glutathione peroxidase 4, is recognized as the hallmark of cells undergoing ferroptosis. Although studies have attempted to elucidate the molecular mechanisms underlying ferroptosis in cancer cells, the regulation of ferroptosis in effector and memory T cells remains largely unknown. Here, using genome-wide CRISPR-Cas9 knockout screens, we demonstrate that acyl-CoA synthetase long-chain family member 4 (ACSL4) is the predominant ferroptosis inducer in primary T cells cultured in vitro, while other identified iron- and lipid metabolism-related genes only slightly modulate their sensitivity to ferroptosis. However, ACSL4 dependency relies on the PL composition of the cells. In vitro cultured T cells treated with polyunsaturated fatty acids (PUFAs), as well as effector CD8[+] T cells that are enriched in PUFA-containing PLs (PUFA-PLs), undergo ferroptosis in the absence of ACSL4. In contrast to effector T cells, naive and memory T cells share a similar PL profile, characterized by a scarcity of PUFA-PLs, and are resistant to ferroptosis. Overall, the PL composition is a central feature and determines the differential susceptibility of effector and memory T cells to ferroptosis and its molecular mechanism.},
}

*Ferroptosis/immunology
*Coenzyme A Ligases/metabolism/genetics
*Phospholipids/metabolism
Animals
Mice
*CD8-Positive T-Lymphocytes/metabolism/immunology
*Memory T Cells/metabolism/immunology
Mice, Inbred C57BL
CRISPR-Cas Systems
Humans
Fatty Acids, Unsaturated/metabolism
Mice, Knockout
Iron/metabolism

@article {pmid41876528,
year = {2026},
author = {Lin, YH and Kompa, J and Sun, DE and Mao, R and Koch, B and Hinnah, K and Wilhelm, J and Franz, N and Kühn, S and Menche, T and Adow, A and Breuer, P and Hiblot, J and Johnsson, K},
title = {A high-affinity split-HaloTag for live-cell protein labeling.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41876528},
issn = {2041-1723},
mesh = {Humans ; Microscopy, Fluorescence/methods ; *Staining and Labeling/methods ; Fluorescent Dyes/chemistry ; HEK293 Cells ; CRISPR-Cas Systems ; Peptides/chemistry/metabolism ; *Proteins/metabolism ; },
abstract = {We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHaloΔ3). Hpep binds to cpHaloΔ3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHaloΔ3 and in fixed cells through incubation with cpHaloΔ3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.},
}

Humans
Microscopy, Fluorescence/methods
*Staining and Labeling/methods
Fluorescent Dyes/chemistry
HEK293 Cells
CRISPR-Cas Systems
Peptides/chemistry/metabolism
*Proteins/metabolism

@article {pmid41876645,
year = {2026},
author = {Ren, F and Liu, D and Ren, H and Zhang, H and Zhang, P and Lin, H and Li, C and Dong, J and An, S and Ge, X and Cheng, L and Yang, F and Liu, J and Fu, J and Tang, F and Wang, F and Liu, T and Pan, H and Rong, S and Ma, H and Zou, L},
title = {Synergistic integration of CRISPR/Cas and nanozymes in next-generation biosensors for ultrasensitive bacterial detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41876645},
issn = {1436-5073},
support = {82574099//National Natural Science Foundation of China/ ; LH2023H054//Natural Science Foundation of Heilongjiang Province/ ; 2022-MYHJ-014//Foundation for Huoju Plan Research of Mudanjiang Medical University/ ; YJSZX2022137//Foundation for Special Program of Supervisor Scientific Research of Mudanjiang Medical University/ ; 2024-KYYWFMY-0455//The Fundamental Research Funds for the Universities of Heilongjiang Province/ ; CYQN24028//Youth the Science and Technology Talents Team Project of Chunyan Plan of Heilongjiang Province/ ; },
}

@article {pmid41876887,
year = {2026},
author = {Xu, R and Cong, T and Yuan, J and Chen, X and Li, Y and Lan, X and Zhu, M},
title = {Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {41876887},
issn = {1750-2799},
abstract = {Tracking-seq is a highly sensitive method for genome-wide detection of off-target effects in cells edited with diverse genome editing modalities, including Cas9, cytosine base editors, adenine base editors and prime editors. Since most genome editors induce DNA repair pathways and generate single-stranded DNA (ssDNA) intermediates, Tracking-seq leverages this process by tracking replication protein A-a key protein that binds and protects ssDNA-to identify on-target and off-target events. Here we provide a detailed protocol for Tracking-seq, covering genome editing of cells, extraction of replication protein A-bound ssDNA, sequencing library construction and data analysis using our custom computational tool Offtracker. Tracking-seq is applicable to various genome editing scenarios with low cell input, delivering high-performance results. The entire workflow, from genome editing to data analysis, can be completed within 1-2 weeks, making it a rapid solution for assessing genome-wide off-target activity.},
}

@article {pmid41877594,
year = {2026},
author = {Cucuy, A and Ben-Tov, D and Melamed-Bessudo, C and Honig, A and Cohen, BA and Levy, AA},
title = {Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70809},
pmid = {41877594},
issn = {1365-313X},
support = {//Israel Innovation Authority/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; Promoter Regions, Genetic/genetics ; DNA Breaks, Double-Stranded ; CRISPR-Associated Protein 9 ; DNA Repair ; Introns/genetics ; Exons/genetics ; },
abstract = {CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Plant/genetics
Promoter Regions, Genetic/genetics
DNA Breaks, Double-Stranded
CRISPR-Associated Protein 9
DNA Repair
Introns/genetics
Exons/genetics

@article {pmid41877893,
year = {2025},
author = {Srinivasa, MA and Escobar, M},
title = {CRISPR-based Transcriptional Regulation: Technologies, Applications, and Future Directions.},
journal = {DNA},
volume = {5},
number = {4},
pages = {57},
pmid = {41877893},
issn = {2673-8856},
support = {25TPA1463933/AHA/American Heart Association-American Stroke Association/United States ; },
abstract = {CRISPR-based transcriptional regulation technologies, including CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), offer precise and programmable control over gene expression, representing a major advance in gene and epigenetic therapy. CRISPRa uses nuclease-inactive Cas proteins fused to transcriptional activators to upregulate target genes, while CRISPRi employs repressor domains for gene silencing. Preclinical studies have demonstrated the efficacy of CRISPRa/i in models of metabolic, neurological, muscular, and oncological diseases. Notably, CRISPRi-based therapies have entered clinical trials for conditions like hepatitis B and muscular dystrophy, showing encouraging safety and efficacy profiles. Despite ongoing challenges related to delivery efficiency, immunogenicity, and off-target activity, innovations in protein engineering and guide RNA design are rapidly enhancing the precision and safety of these technologies. Overall, CRISPRa and CRISPRi are poised to transform the treatment of genetic and epigenetic disorders, with continued optimization expected to accelerate their clinical adoption and broaden their therapeutic impact.},
}

@article {pmid41879319,
year = {2026},
author = {Cheng, W and Li, J and Lei, L and Zhu, Y and Luo, S and Wang, X and Zhang, Q and Cao, M and Zheng, Y and Peng, W},
title = {Unlocking genome engineering in Alcaligenes faecalis by exploiting its native type I-F CRISPR-Cas.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0278625},
doi = {10.1128/spectrum.02786-25},
pmid = {41879319},
issn = {2165-0497},
abstract = {Alcaligenes faecalis is an environmentally significant bacterium for pollutant biodegradation and aerobic denitrification, yet its genetic engineering has been hindered by a lack of high-throughput tools. Conventional methods like homologous recombination are time-consuming and cannot achieve large genomic deletions, while technologies based on heterologous CRISPR-Cas systems failed due to cytotoxicity. This study resolves these limitations by developing a genome editing toolkit based on the endogenous type I-F CRISPR-Cas of A. faecalis J481. The toolkit enables efficient single-gene knockout and accomplishes the previously unattainable precise deletion of large genomic fragments. By engineering a PheS-mutant counterselection marker, we achieved rapid plasmid curing, allowing two rounds of large-fragment removal (~47 kb total) within 5 days. This breakthrough provides the first CRISPR-based platform for complex genome engineering in A. faecalis, overcoming intrinsic constraints of heterologous systems. The work establishes a scalable genetic toolbox to enhance A. faecalis' capabilities in bioremediation and eutrophication control. Moreover, the strategy of harnessing endogenous CRISPR-Cas systems offers a blueprint for developing advanced genome editing tools in other prokaryotes.IMPORTANCEThis study breaks through the longstanding genetic engineering bottleneck in an environmentally crucial bacterium, Alcaligenes faecalis, by creating a fast, efficient, and versatile toolkit using its native CRISPR-Cas system. This enables complex edits, such as large genomic deletions previously impossible, unlocking new potential for bioremediation and eutrophication control, providing a blueprint for other prokaryotes, and setting a precedent for genetic tool development in other hard-to-engineer microbes.},
}

@article {pmid41880574,
year = {2026},
author = {Katsumura, T and Sato, S and Yamashita, K and Oda, S and Gakuhari, T and Tanaka, S and Fujitani, K and Nishimaki, T and Imai, T and Yoshiura, Y and Takeshima, H and Hashiguchi, Y and Sekita, Y and Mitani, H and Ogawa, M and Takeuchi, H and Oota, H},
title = {DNA methylation site loss for plasticity-led novel trait genetic fixation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {13},
pages = {e2534817123},
doi = {10.1073/pnas.2534817123},
pmid = {41880574},
issn = {1091-6490},
support = {JP16K21352//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP19K16201//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP19H05737//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24K02078//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17H01453//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17H03738//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16J07227//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; *Oryzias/genetics ; *DNA Methylation/genetics ; Epigenesis, Genetic ; CpG Islands/genetics ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {Phenotypic plasticity allows organisms to adapt traits in response to environmental changes, yet the molecular basis by which such plastic traits become genetically fixed remains unclear. Here, we investigated gut-length plasticity in medaka fish (Oryzias latipes) through genome-wide methylation profiling, CRISPR/Cas9-mediated deletion, and population genomic analyses. We found that seasonal methylation of CpG sites upstream of the Plxnb3 is correlated with gut-length plasticity, and deletion of this region abolishes plasticity. Additionally, standing variation in Ppp3r1 is associated with genetically fixed longer gut length in populations lacking plasticity. These results suggest that loss of epigenetic regulation via CpG site reduction triggers the genetic fixation of novel traits. Our findings provide molecular evidence linking epigenetic plasticity and genetic assimilation, advancing understanding of plasticity-led evolution in natural populations.},
}

Animals
*Oryzias/genetics
*DNA Methylation/genetics
Epigenesis, Genetic
CpG Islands/genetics
Phenotype
CRISPR-Cas Systems

@article {pmid41882914,
year = {2026},
author = {Kambakam, S and Thomas, J and Robbe-Austerman, S and Shanmuganatham, K and Palinski, R},
title = {Rapid identification of African swine fever virus in diagnostic samples using CRISPR-Cas.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {},
number = {},
pages = {10406387261432985},
pmid = {41882914},
issn = {1943-4936},
abstract = {African swine fever virus (ASFV) is a highly transmissible pathogen affecting swine, causing a devastating disease with high mortality rates in naive populations. Given the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten the U.S. swine industry, given their proximity and involvement in movement to and from North America. Although surveillance programs are ongoing, limited point-of-care (POC) tests are available during outbreaks with the sensitivity and specificity standards of laboratory testing (e.g., real-time PCR [rtPCR]). However, the recently developed CRISPR-Cas-based testing systems may offer comparable high-quality results. We sought to develop a low-cost visual detection method for ASFV by employing a recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a POC assay. Our CRISPR-Cas12a assay had comparable sensitivity and specificity to rtPCR, both visually and when quantified using a fluorescence reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, our CRISPR assay achieved a sensitivity of 98.3% (10[2] DNA copies) and a specificity of 100%. Test results of our RPA-CRISPR assay can be visualized in as few as 7 min, with peak fluorescence at 40 min (RPA and CRISPR steps). Our results lay the groundwork for a large-scale POC assay assessment for ASFV detection and offer a robust workflow that works with commonly submitted diagnostic samples.},
}

@article {pmid41883584,
year = {2026},
author = {Kobel, L and Van de Venn, L and Schröder, M and Bechter, LV and Huang, D and Abdolazimi, Y and Pertel, T and Gopalakrishnan, S and Corn, JE and Kontarakis, Z},
title = {DisTAL-Seq: A TALEN-specific adaptation of DISCOVER-Seq for off-target profiling.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {2},
pages = {102883},
pmid = {41883584},
issn = {2162-2531},
abstract = {Programmable guided nucleases have revolutionized genome editing and biomedical research, with transformative potential for gene and cell therapy. Although the widespread adoption of the CRISPR-Cas system has provided deep insights into target recognition and specificity, the behavior of clinically relevant tools like transcription activator-like effector nucleases (TALENs) remains poorly characterized in human cells. To address this gap, we implemented DisTAL-Seq, a TALEN-specific adaptation of the DISCOVER-Seq pipeline, which detects MRE11 recruitment to double-strand breaks (DSBs). Based on the DISCOVER-Seq principle, DisTAL-Seq incorporates alignment logic tailored to TALEN-binding properties, including variable RVD specificity, cleavage offset, and dimerization behavior. Using DisTAL-Seq, we identified and validated on- and off-target sites across diverse TALENs and T cell donors. This unbiased approach revealed key features of TALEN activity in human cells, including number of tolerated mismatches to a target site and relative location of the induced DSB. DisTAL-Seq thus extends DISCOVER-Seq to the TALEN family and provides a robust platform for assessing modifications in enzyme architecture and application contexts on a genome-wide scale, supporting the development of safer and more effective genome editing tools.},
}

@article {pmid41885207,
year = {2026},
author = {Yin, W and Jin, Z and Jiang, Q and Jin, S and Wang, X and He, R and Qiao, B and Qiao, J and Zhang, X and Liu, Y},
title = {Elimination of cis-cleavage in CRISPR diagnostics for one-pot rapid nucleic acid detection.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41885207},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Associated Proteins/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Endodeoxyribonucleases/genetics/metabolism ; COVID-19/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics/metabolism ; Tumor Suppressor Protein p53/genetics ; *COVID-19 Nucleic Acid Testing/methods ; },
abstract = {Current one-pot clustered regularly interspaced short palindromic repeats diagnostics are limited by the cis-cleavage activity of Cas nucleases, which leads to amplicon degradation during amplification. Here, we report a streamlined strategy that overcomes this limitation. By integrating a bipartite split-crRNA into Cas12a (SCas12a), we separate target recognition from PAM dependency and completely eliminate cis-cleavage while preserving robust trans-cleavage. This strategy is broadly applicable for one-pot testing, compatible with recombinase polymerase amplification, RT-RPA, and loop-mediated isothermal amplification, as well as multiple Cas12a orthologs, including As, Lb, and Ct Cas12a. Moreover, the SCas12a accelerates one-pot testing with 100-1000-fold improved sensitivity and achieves >10-fold reduction in time-to-signal, enabling detection of targets at attomolar levels within 30 min. Additionally, it provides single-base resolution with up to 91-fold selectivity. The system has been successfully applied to detect HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples. Together, we have developed a PAM-independent and cis-cleavage-free one-pot Cas12a assay, which holds strong potential for point-of-care diagnostics.},
}

Humans
*CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics/isolation & purification
*CRISPR-Associated Proteins/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
*Endodeoxyribonucleases/genetics/metabolism
COVID-19/diagnosis/virology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacterial Proteins/genetics/metabolism
Tumor Suppressor Protein p53/genetics
*COVID-19 Nucleic Acid Testing/methods

@article {pmid41885929,
year = {2026},
author = {Idrees, J and Shabbir, AQ and Alvi, IA and Sana, S and Rehman, SU and Asif, M},
title = {CRISPR-anti-CRISPR dynamics: evolutionary, ecological and biotechnological perspectives.},
journal = {Archives of microbiology},
volume = {208},
number = {6},
pages = {},
pmid = {41885929},
issn = {1432-072X},
mesh = {*CRISPR-Cas Systems ; *Bacteriophages/genetics/physiology ; *Bacteria/genetics/virology ; Biotechnology ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ecosystem ; Biological Evolution ; Viral Proteins/metabolism/genetics ; Evolution, Molecular ; },
abstract = {Phages and bacteria are engaged in an evolutionary arms race. CRISPR-Cas systems allow bacteria to resist phage predation, contributing to competitive advantages for certain bacterial strains. However, phages generate a diverse anti-CRISPR (Acr) proteins to effectively neutralise the CRISPR-Cas system. Acr proteins restore phage infectivity, which may contribute in limiting the dominance of CRISPR-armed bacterial strains, potentially influencing the microbial diversity in certain environments. Acrs can influence microbial community dynamics which may indirectly affect ecosystem functions such as nutrient cycling in certain marine and soil ecosystems. The potential of these Acrs proteins in controlled and reversible genome editing, highlights their potential as regulatory components for genome editing systems. This review discusses the molecular mechanism of CRISPR and anti-CRISPRs, highlights the diversity and limitations of known inhibitory mechanisms, ecological role of anti-CRISPRs and highlights their expanding application in microbial evolution and biotechnology.},
}

*CRISPR-Cas Systems
*Bacteriophages/genetics/physiology
*Bacteria/genetics/virology
Biotechnology
Gene Editing
*Clustered Regularly Interspaced Short Palindromic Repeats
Ecosystem
Biological Evolution
Viral Proteins/metabolism/genetics
Evolution, Molecular

@article {pmid41886492,
year = {2026},
author = {Tu, KJ and Roy, SK and Kingsbury, TJ and Shukla, HD},
title = {HIF1α mediates resistance to radiation and to KRAS inhibitors in pancreatic adenocarcinoma.},
journal = {PloS one},
volume = {21},
number = {3},
pages = {e0341912},
pmid = {41886492},
issn = {1932-6203},
mesh = {*Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Humans ; *Pancreatic Neoplasms/metabolism/genetics/radiotherapy/pathology/drug therapy ; *Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors/genetics/metabolism ; Cell Line, Tumor ; Animals ; *Radiation Tolerance/genetics/drug effects ; Mice ; *Drug Resistance, Neoplasm ; *Carcinoma, Pancreatic Ductal/genetics/radiotherapy/metabolism/pathology/drug therapy ; Tumor Suppressor Protein p53/metabolism ; Cell Proliferation/drug effects ; Apoptosis/drug effects/radiation effects ; *Adenocarcinoma/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is highly treatment resistant and characterized by a hypoxic microenvironment. Here, we investigated the role of hypoxia-inducible factor 1α (HIF1α) in regulating resistance to radiation and KRAS-inhibitor. We employed CRISPR/Cas9 to knock out (KO) HIF1α from the murine KRASG12D/+; p53R172H/+ KPC and the KRASG12D/+; p53R273H; CDK2NA-/- Panc-1 human pancreatic cell lines. Compared to WT, the HIF1α KO cell lines demonstrated a shift toward an epithelial phenotype and had decreased proliferation and migration under hypoxia. HIF1α KO cell lines were less likely to survive after radiotherapy, and neutral comet assays demonstrated DNA damage four hours after treatment, suggesting that HIF1α promotes radioresistance through non-homologous end joining. When treated with a KRASG12D inhibitor, HIF1α KO cells exhibited significantly increased apoptosis due to decreased p53 degradation, likely mediated through Mdm2. Confirming this, enrichment of hypoxic signaling was associated with KRAS inhibitor resistance in a cohort of 31 KRASG12D cell lines. Our results thus suggest that inhibiting HIF1α may sensitize PDAC to radiation and KRAS inhibitors. To explore this, we conducted a drug repurposing screen and identified three HIF1α inhibitors (bakuchiol, BAY-87-2243, 2-methoxyestradiol) whose sensitivities were correlated with sensitivity to Deltarasin, a KRAS inhibitor. Our findings suggest that HIF1α inhibitors could be used to sensitize PDAC to radiotherapy and KRAS inhibitors.},
}

*Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism
Humans
*Pancreatic Neoplasms/metabolism/genetics/radiotherapy/pathology/drug therapy
*Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors/genetics/metabolism
Cell Line, Tumor
Animals
*Radiation Tolerance/genetics/drug effects
Mice
*Drug Resistance, Neoplasm
*Carcinoma, Pancreatic Ductal/genetics/radiotherapy/metabolism/pathology/drug therapy
Tumor Suppressor Protein p53/metabolism
Cell Proliferation/drug effects
Apoptosis/drug effects/radiation effects
*Adenocarcinoma/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41886504,
year = {2026},
author = {Maestas, MM and Bradley, K and Shunkarova, M and Mukherjee, N and Ishahak, M and Lu, J and Millman, JR},
title = {Whole-genome CRISPR screening identifies genetic modifiers of stem cell-derived islet transplantation.},
journal = {Stem cells translational medicine},
volume = {15},
number = {4},
pages = {},
doi = {10.1093/stcltm/szag012},
pmid = {41886504},
issn = {2157-6580},
support = {UG3DK142188//National Institutes of Health (NIH)/ ; 2022-001//Beatson Foundation/ ; //Edward J. Mallinckrodt Foundation/ ; //Washington University School of Medicine Department of Medicine/ ; //Anita Palmer Corbin Trust/ ; T32GM139774//Cellular and Molecular Biology Training/ ; //Bill & Melinda Gates Foundation through the Gates Millennium Scholars Program/ ; //Rita Levi-Montalcini Postdoctoral Fellowship in Regenerative Medicine/ ; //Rita Levi-Montalcini Postdoctoral Fellowship in Regenerative -Medicine/ ; T32DK007120/GF/NIH HHS/United States ; },
mesh = {Animals ; *Islets of Langerhans Transplantation/methods ; Humans ; Mice ; Mice, Inbred NOD ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Islets of Langerhans/metabolism/cytology ; },
abstract = {INTRODUCTION: Genetically engineering human pluripotent stem cell (hPSC)-derived islets is a promising strategy for improving transplantation for diabetes cell therapy; however, genetic perturbations that modulate transplantation outcomes have yet to be systematically explored.

METHODS: To identify potential targets, we performed an unbiased whole-genome CRISPR-activation screen in transplanted stem cell-derived islets (SC-islets). Specifically, we created a stem cell line with CRISPR-activation components (HUES8-VPR) and then transduced these stem cells with a lentiviral guide RNA library targeting the whole human genome. Following transduction, the stem cells were differentiated into SC-islets, which were subsequently transplanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) immunodeficient mice. After transplantation, SC-islets were extracted for next-generation sequencing.

RESULTS: The screen identified multiple candidates, including the Fc alpha/mu receptor (FCAMR). In vitro characterization revealed that FCAMR overexpression did not negatively affect SC-islet function or transcriptomic identity. Mice subcutaneously transplanted with SC-islets overexpressing FCAMR had reduced blood glucose levels and increased C-peptide compared to controls. Additionally, mice receiving FCAMR-modified grafts into the kidney capsule or hindleg muscle maintained a higher body weight compared to controls in a diabetic setting.

CONCLUSIONS: In conclusion, this study demonstrats improved glucose regulation at a subcutaneous transplant site. In addition, we show that FCAMR SC-islets could play a role in systemic metabolism when transplanted into the kidney capsule or hindleg muscle. Overall, our study establishes a functional screening approach to identify gene candidates to improve SC-islet transplantation.},
}

Animals
*Islets of Langerhans Transplantation/methods
Humans
Mice
Mice, Inbred NOD
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
Cell Differentiation
Islets of Langerhans/metabolism/cytology

@article {pmid41888353,
year = {2026},
author = {Wang, J and Bobrik, M and Pankaew, N and Gratacap, R and Digard, P and Bean, TP and Jin, Y and Robledo, D},
title = {Efficient genome editing in a Mozambique tilapia cell line using CAS ribonucleoprotein complexes.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-42702-w},
pmid = {41888353},
issn = {2045-2322},
support = {STG5443//Ministry of Science and Technology of Thailand/ ; BBS/E/RL/230002A/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Genome editing using the CRISPR/Cas system makes it possible to rapidly characterise gene function in vitro and in vivo, and provides a powerful platform through which the genetics of farmed fish can be altered to improve traits such as resistance to important pathogens. Tilapia is one of the most important farmed fish globally; however, its farming is heavily impacted by Tilapia lake virus (TiLV). The Mozambique tilapia (Oreochromis mossambicus) brain (OmB) cell line is susceptible to TiLV, making it an ideal in vitro model for studying host-pathogen interactions and mechanisms of disease resistance. To establish OmB cells as a model for gene editing in Tilapia, it is essential to optimise genome editing protocols. In this study, we optimized a CRISPR/Cas9-based genome editing system for OmB cells using ribonucleoprotein complexes. With the optimized protocol, we successfully edited two endogenous genes with efficiencies ranging from 67% to 70%. In conclusion, we establish a highly efficient CRISPR/Cas9 RNP based gene editing workflow optimized for OmB tilapia cell lines. This optimized platform will facilitate future functional genomic studies in tilapia and support the development of TiLV-resistant tilapia.},
}

@article {pmid40966091,
year = {2025},
author = {Wang, Y and Hu, M and Zou, Y and Wang, X},
title = {In vivo CRISPR screening protocol to identify metastasis mediators using iteratively selected mouse models.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104042},
pmid = {40966091},
issn = {2666-1667},
mesh = {Animals ; Mice ; Female ; *CRISPR-Cas Systems/genetics ; Humans ; Disease Models, Animal ; *Neoplasm Metastasis/genetics ; *Ovarian Neoplasms/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {In vivo CRISPR screens uncover metastasis genes in native contexts, surpassing in vitro model limitations. Here, we present a protocol to identify metastasis-driving genes in ovarian cancer using an in vivo CRISPR screening technique. Key steps include single-guide RNA (sgRNA) library design and validation, lentiviral transduction, establishment of metastatic mouse models, tissue collection, sgRNA amplification for sequencing, bioinformatics-based candidate gene identification, and functional validation. For complete details on the use and execution of this protocol, please refer to Wang et al.[1].},
}

Animals
Mice
Female
*CRISPR-Cas Systems/genetics
Humans
Disease Models, Animal
*Neoplasm Metastasis/genetics
*Ovarian Neoplasms/genetics/pathology
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40975871,
year = {2025},
author = {Washer, SJ and Navarro-Guerrero, E and Cowley, SA and Ebner, DV and Bassett, AR},
title = {Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104111},
pmid = {40975871},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Microglia/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Flow Cytometry/methods ; Lentivirus/genetics ; Phagocytosis/genetics ; },
abstract = {Here, we present a protocol for CRISPR knockout screening in human induced pluripotent stem cell (hiPSC)-derived microglia (iMGL) using lentiviral delivery of CRISPR-Cas9 and co-transduction of VPX virus-like particles (VPX-VLPs). We first describe large-scale production of iMGL from hiPSCs, production of the lentiviral and VPX-VLP libraries, and titration. Next, we describe how to perform a pooled CRISPR screen for phagocytosis including the computational analysis pipeline of CRISPR screening data. For complete details on the use and execution of this protocol, please refer to Perez-Alcantara et al.[1].},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Microglia/cytology/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*Flow Cytometry/methods
Lentivirus/genetics
Phagocytosis/genetics

@article {pmid41014566,
year = {2025},
author = {Hazan, JM and Lahoud-Jeries, N and Bester, AC},
title = {Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104110},
pmid = {41014566},
issn = {2666-1667},
mesh = {*RNA, Long Noncoding/genetics ; *Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing multiple parallel perturbations tailored to the research question, followed by gRNA amplification and data analysis.},
}

*RNA, Long Noncoding/genetics
*Gene Library
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid41108681,
year = {2025},
author = {Espinoza, A and Ingersoll, S and Ren, X},
title = {Protocol for quantifying phase-separated condensates in living cells using HILO microscopy imaging and genetic engineering.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104151},
pmid = {41108681},
issn = {2666-1667},
mesh = {Humans ; *Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; *Biomolecular Condensates ; Microscopy, Fluorescence/methods ; *Microscopy/methods ; },
abstract = {Spatial clustering of epigenetic factors through phase separation is an emerging concept in transcriptional regulation and genome organization. Here, we present a protocol to quantify the phase separation capacity of epigenetic factors in living cells via HILO (highly inclined and laminated optical sheet) microscopy. We describe steps for using CRISPR-Cas9 to generate a cell line with two distinct fusion proteins containing fluorophores and either CRE-LoxP- or dTAG-inducible degradation systems. We then detail the procedures for imaging fusion proteins to analyze and quantify phase separation capacity. For complete details on the use and execution of this protocol, please refer to Brown et al.[1].},
}

Humans
*Genetic Engineering/methods
CRISPR-Cas Systems/genetics
*Biomolecular Condensates
Microscopy, Fluorescence/methods
*Microscopy/methods

@article {pmid41201092,
year = {2025},
author = {Wu, Y and Zhong, A and Ramirez, B and Wai, SS and Zhou, T},
title = {Protocol to rapidly verify silent gene reporter in human pluripotent stem cells using CRISPR activation.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104164},
pmid = {41201092},
issn = {2666-1667},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; UM1 HG012654/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Pluripotent Stem Cells/metabolism/cytology ; *Genes, Reporter/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation/genetics ; },
abstract = {Validating human pluripotent stem cell (hPSC) reporters targeting silent genes typically requires inducing gene expression through cell state transitions, which can be time consuming and complex. Here, we present a rapid workflow to verify reporter knockins at unexpressed loci in hPSCs using CRISPR-mediated transcriptional activation (CRISPRa). We detail steps for designing and cloning single-guide RNA (sgRNA), delivery of CRISPRa into reporter cells, and detection of reporter gene. In this protocol, we illustrate this process using KLF17-GFP reporter hPSCs. For complete details on the use and execution of this protocol, please refer to Wu et al.[1].},
}

Humans
*Pluripotent Stem Cells/metabolism/cytology
*Genes, Reporter/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcriptional Activation/genetics

@article {pmid41240344,
year = {2025},
author = {Banerjee, N and Walsh, B and Patel, R and Castelletto, ML and Hallem, EA},
title = {Protocol to generate stable knockout lines in the human-parasitic nematode Strongyloides stercoralis.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104201},
pmid = {41240344},
issn = {2666-1667},
mesh = {Animals ; *Strongyloides stercoralis/genetics ; *Gene Knockout Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Gerbillinae ; },
abstract = {A major limitation to the study of gene function in parasitic nematodes was the inability to make stable mutant lines. Here, we present a protocol for generating stable knockout lines in the human-parasitic nematode Strongyloides stercoralis. We describe steps for generating CRISPR components and microinjecting them into worms. We also detail procedures for identifying potential gene disruptions and propagating mutants by host passage in gerbils to generate stable homozygous knockout lines. This protocol enables studies of gene function in S. stercoralis. For complete details on the use and execution of this protocol, please refer to Banerjee et al.[1].},
}

Animals
*Strongyloides stercoralis/genetics
*Gene Knockout Techniques/methods
Humans
CRISPR-Cas Systems/genetics
Gerbillinae

@article {pmid41277604,
year = {2026},
author = {Xu, M and Yan, L and Zhu, M and Zhan, Z and Chen, H and Wang, D and Zheng, Z and Zhang, Y and Xiong, L and He, Y},
title = {TKC-MC: An Effective Strategy for Generating Heritable Heterozygous Mutations in Essential Genes in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2092-2104},
doi = {10.1111/pbi.70472},
pmid = {41277604},
issn = {1467-7652},
support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32200335//National Natural Science Foundation of China/ ; U21A20207//National Natural Science Foundation of China/ ; Y2023QC39//Youth innovation Program of Chinese Academy of Agricultural Sciences/ ; YBXM2504//Nanfan special project of CAAS/ ; YBXM2505//Nanfan special project of CAAS/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Plants, Genetically Modified/genetics ; *Mutation/genetics ; *Genes, Essential/genetics ; *Genes, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing technology has been widely used in defining gene functions and crop improvement. However, some genes are essential for plant growth and development. Loss-of-function homozygous mutations in essential genes lead to plant death or sterility. Mutations in essential genes need to be maintained and propagated in heterozygous plants. CRISPR/Cas9 technology is highly efficient in generating homozygous or bi-allelic mutations at T0 generation in rice, making it difficult to generate useful genetic materials for essential genes using traditional gene editing technology. In this study, we designed Transgene-Killer CRISPR (TKC)-mediated mismatch-spacer targeting (TKC-M) to efficiently generate heritable heterozygous mutations in essential genes in rice. Leveraging our earlier transgenic offspring self-elimination TKC platform, TKC-M relied on timely self-elimination of Cas9 and engineered gRNA-target mismatches to enrich heritable heterozygous or mosaic incomplete-edited T0 mutants and heterozygous progeny. We found that the sensitivity of targets to spacer mismatch(es) varies. A single-base mismatch at gRNA positions 11 or 17 yielded abundant heritable heterozygotes in sensitive targets. For insensitive targets, dual mismatches at positions 8 and 15 maximised heritable heterozygotes. Co-transformation of rice with TKC vectors carrying gRNA without mismatches (G1), gRNA with a mismatch at position 11 (M11) and M8 + M15 spacers, termed TKC-M Cocktail (TKC-MC) significantly increased the incomplete-edited mutant ratio compared with using G1 alone. This work establishes a technical foundation for generating mutant libraries that cover every single gene in a plant genome and for in-depth research on essential genes.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Heterozygote
Plants, Genetically Modified/genetics
*Mutation/genetics
*Genes, Essential/genetics
*Genes, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41432570,
year = {2026},
author = {Hu, J and Li, X and Gao, Y and Guo, Y and Liu, Y and Wang, C and Xu, G and Du, C and Liu, S and Zhao, Z and Wang, Y and Wu, Y and Dong, X and Li, C and Wan, J},
title = {Cas9-Embedding Hyperactive TadA8e Confers Efficient and Highly Specific A-To-G Base Editing in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2576-2591},
doi = {10.1111/pbi.70511},
pmid = {41432570},
issn = {1467-7652},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; //Ministry of Education of the People's Republic of China/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Adenosine Deaminase/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Adenine base editors (ABEs) produce precise A-to-G conversion in the genomic target sites without causing double-strand breaks. However, the hyperactive adenosine deaminase TadA8e raises safety concerns on genome-wide off-target edits. We engineered 11 chimeric proteins for ABEs (CP-ABEs) by embedding hyperactive TadA8e within Cas9 nickase to minimise the sgRNA-independent off-target effects. Four CP-ABEs exhibited robust on-target activity with minimal sgRNA-independent off-target edits. Then we developed four chimeric high-fidelity ABEs (CH-ABEs) to minimise both sgRNA-dependent and sgRNA-independent off-target effects by employing high-fidelity Cas9 variants. The CH-ABEs achieved reductions of up to 7.0-fold and 79.4-fold in the respective off-target edits, while generating 22.0%-72.4% homozygous and biallelic rice mutants. Whole-genome and whole-transcriptome sequencing (WGS/WTS) confirmed the specificity of CH-ABEs. Incorporating Sniper2L into CH-ABEs further enhanced both specificity and on-target activity. Two PAM-less SpRY variants (SpRY-K2, SpRY-KK) expanded the targeting scope of CP-ABEs and boosted activity by 80.0%. Furthermore, we demonstrated that CP-ABE8e-RY[KK] could discriminate paralogous targets in rice and successfully applied it to create herbicide-resistant rice by precisely installing the OsALS-K591E mutation.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Associated Protein 9/metabolism/genetics
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
*Adenosine Deaminase/genetics/metabolism
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41433155,
year = {2026},
author = {Hemani, D and Grissom, JH and Chi, RJ},
title = {Protocol for marker-free genome editing in Saccharomyces cerevisiae using universal donor templates and multiplexed CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104280},
pmid = {41433155},
issn = {2666-1667},
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal/genetics ; },
abstract = {Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers. For complete details on the use and execution of this protocol, please refer to Grissom et al.[1].},
}

*Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Fungal/genetics

@article {pmid41575844,
year = {2026},
author = {Bourner, LA and Acken, KA and Long, H and Chung, LA and Roth, KD and Dorsey, FC},
title = {Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104304},
pmid = {41575844},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Succinates/metabolism/analysis ; THP-1 Cells ; Macrophages/metabolism ; Chromatography, Liquid/methods ; Mass Spectrometry ; Carboxy-Lyases ; },
abstract = {Itaconate mimetics inadequately represent endogenous itaconate, a negative regulator of innate immune-driven pro-inflammatory cytokines. We present a CRISPR-Cas9 protocol to delete a 4-nucleotide region in the immunoresponsive gene 1 (IRG1), ablating ACOD1 (the itaconate-producing enzyme) in THP-1 cells. We describe the functional validation of ACOD1 deletion using immunoblotting, ELISA, and liquid chromatography-mass spectrometry (LC-MS) quantification of itaconate, enabling the study of endogenous itaconate in THP-1 macrophages. For complete details on the use and execution of this protocol, please refer to Bourner et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
*Succinates/metabolism/analysis
THP-1 Cells
Macrophages/metabolism
Chromatography, Liquid/methods
Mass Spectrometry
Carboxy-Lyases

@article {pmid41575845,
year = {2026},
author = {Priesmeier, L and Tiburcy, M and Zelarayán, LC},
title = {Protocol for differentiation of vascular smooth muscle cells from human iPSCs and their application in CRISPRa-mediated gene regulation.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104345},
pmid = {41575845},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Differentiation/genetics ; *Muscle, Smooth, Vascular/cytology ; *Myocytes, Smooth Muscle/cytology/metabolism ; *Cell Culture Techniques/methods ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; Gene Expression Regulation ; },
abstract = {Directed differentiation of human induced pluripotent stem cells (hiPSCs) holds major promise for the development of disease models, drug screening platforms, and regenerative medicine. Here, we provide a step-by-step highly reproducible protocol for differentiating vascular smooth muscle cells (vSMCs) from hiPSCs, including hiPSC culture, hiPSC differentiation, and vSMC passaging under chemically defined conditions. We also detail molecular and functional analysis procedures for hiPSC-derived contractile vSMCs along with endogenous transcriptional activation modulation ready for any downstream application.},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Cell Differentiation/genetics
*Muscle, Smooth, Vascular/cytology
*Myocytes, Smooth Muscle/cytology/metabolism
*Cell Culture Techniques/methods
*CRISPR-Cas Systems/genetics
Cells, Cultured
Gene Expression Regulation

@article {pmid41626796,
year = {2026},
author = {Jones, LE and Kellermeyer, R and Anand, R and Watson, J and Smith, L and Huang, X and Wang, Z and Yan, W and Zhang, H and Mastick, CC and Kidd, T and Mastick, GS},
title = {SLIT2 repellent is cleaved by TLL1 protease and promotes sensory axon fasciculation.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {16},
pages = {},
doi = {10.1242/dev.205124},
pmid = {41626796},
issn = {1477-9129},
support = {P20 GM103650/GM/NIGMS NIH HHS/United States ; P30 GM145646/GM/NIGMS NIH HHS/United States ; P30 GM145646; P20 GM103650/NH/NIH HHS/United States ; RO1 NS114219/NH/NIH HHS/United States ; RO1 NS114219/NH/NIH HHS/United States ; },
mesh = {Animals ; Ganglia, Spinal/metabolism/cytology/embryology ; *Axons/metabolism ; *Nerve Tissue Proteins/metabolism/genetics ; Mice ; *Intercellular Signaling Peptides and Proteins/metabolism/genetics ; Slit Homolog 2 Protein ; Mice, Knockout ; *Sensory Receptor Cells/metabolism ; CRISPR-Cas Systems ; Proteolysis ; *Tolloid-Like Metalloproteinases/metabolism/genetics ; },
abstract = {SLIT2 is a secreted protein that repels axons from the CNS midline. Full-length SLIT2 (SLIT2-FL) is proteolytically cleaved into two fragments, SLIT2-N and SLIT2-C. SLIT2-FL and SLIT2-N have opposing biological effects on cultured dorsal root ganglion (DRG) axons. This study identified SLIT2 cleavage mechanisms and functional significance for DRG axon guidance. The Tolloid-related protease TLL1 cleaved SLIT2 in cultured cells, with TLL1 requiring activation by furin/prohormone convertases. We used CRISPR editing in mice to produce a Slit2ΔTLS allele lacking the TLL1 cleavage site. Slit2ΔTLS embryos retained dorsal repulsion of DRG axons, in contrast to DRG midline invasion in Slit2 knockouts. However, DRG axons in Slit2 knockouts and Slit2ΔTLS mutants showed reduced fasciculation of rootlets and longitudinal DRG projections. In vitro, SLIT2-N promoted fasciculation of DRG axons. These results suggest that proteolytic cleavage generates additional SLIT2 biological functions for organizing DRG central axon projections.},
}

Animals
Ganglia, Spinal/metabolism/cytology/embryology
*Axons/metabolism
*Nerve Tissue Proteins/metabolism/genetics
Mice
*Intercellular Signaling Peptides and Proteins/metabolism/genetics
Slit Homolog 2 Protein
Mice, Knockout
*Sensory Receptor Cells/metabolism
CRISPR-Cas Systems
Proteolysis
*Tolloid-Like Metalloproteinases/metabolism/genetics

@article {pmid41649928,
year = {2026},
author = {Yoon, DE and Yang, J and Jeong, TY and Park, J and Lee, H and Seong, JK and Kim, K},
title = {Protocol for non-invasive delivery of CRISPR RNPs via virus-like particles for mouse model generation.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104352},
pmid = {41649928},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems/genetics ; Female ; Models, Animal ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-virus-like particle (VLP)-induced targeted mutagenesis (CRISPR-VIM) enables genome editing in mouse embryos through non-invasive delivery of CRISPR ribonucleoproteins (RNPs) via VLPs, eliminating the need for physical manipulation and specialized expertise. We detail protocols for VLP production, titration, and treatment for diverse genome edits. This protocol is compatible with zygotes and in vitro fertilization (IVF)-derived embryos via simple co-culture, facilitating high-efficiency and heritable mutations with minimized off-target effects, independent of specialized equipment and conducive to reduced animal use. For complete details on the use and execution of this protocol, please refer to Jeong et al.[1].},
}

Animals
Mice
*Gene Editing/methods
*Ribonucleoproteins/genetics
*CRISPR-Cas Systems/genetics
Female
Models, Animal
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41665608,
year = {2026},
author = {Hanson, E and Gold, R and Lee, DH and Yigit, MV},
title = {Cas12a-Programmed Modular CRISPR Cascade Reaction on Paper Supports for Dual-Mode Detection of Pathogenic Genomes.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2774-2783},
doi = {10.1021/acssensors.5c04848},
pmid = {41665608},
issn = {2379-3694},
mesh = {*Paper ; *CRISPR-Cas Systems/genetics ; *Listeria monocytogenes/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Campylobacter jejuni/genetics/isolation & purification ; *Endodeoxyribonucleases/genetics/metabolism ; *Genome, Bacterial ; *Bacterial Proteins/genetics ; Escherichia coli/genetics/isolation & purification ; Metal Nanoparticles/chemistry ; Silver/chemistry ; DNA, Bacterial/genetics ; },
abstract = {We developed an optical biosensing platform using cost-efficient and scalable paper support for the detection and identification of three major bacterial pathogens using fluorescent DNA-templated silver nanoclusters (FNPs) and an innovative CRISPR-Cas12a cascade reaction. The sensors were fabricated as ∼5 mm letter-shaped paper cutouts, with each letter representing a specific pathogen: "C" for Campylobacter jejuni, "E" for Shiga toxin-producing Escherichia coli, and "L" for Listeria monocytogenes. Detection was initially achieved via an ON-to-OFF mechanism, wherein target recognition by Cas12a led to FNP degradation and fluorescence loss using target strands identified from the conserved genomic regions from each pathogen. This platform successfully detected individual and multiple targets in all possible seven combinations. To enhance diagnostic clarity, we developed a two-step CRISPR-Cas12a cascade reaction enabling an ON signal output when the target is present, a more intuitive and desirable reporting format. In this design, the first Cas12a reaction detects the target and cleaves an activator strand, preventing activation of a second Cas12a reaction that would otherwise degrade FNPs. Consequently, fluorescence is retained in the presence of the target (ON-retention) and lost in its absence, providing a clear ON signal when the target is detected, and an OFF signal when it is not. Finally, we demonstrated both ON-to-OFF and ON-retention detection modes using the whole Listeria genome amplified by isothermal recombinase polymerase amplification, with reliable detection of as few as 40 full genomic copies using fluorescent images on paper substrates. This work represents a significant advancement in Cas12a-based biosensing, uniquely demonstrating multistep biochemical reactions directly on paper support, and offers a promising platform for low-cost, scalable pathogen detection in resource-limited settings.},
}

*Paper
*CRISPR-Cas Systems/genetics
*Listeria monocytogenes/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
Campylobacter jejuni/genetics/isolation & purification
*Endodeoxyribonucleases/genetics/metabolism
*Genome, Bacterial
*Bacterial Proteins/genetics
Escherichia coli/genetics/isolation & purification
Metal Nanoparticles/chemistry
Silver/chemistry
DNA, Bacterial/genetics

@article {pmid41686171,
year = {2026},
author = {Su, M and Lv, MM and Pan, MX and Zha, CJ and Nie, YG and Ying, ZM},
title = {From Self-Processing to Responsive Assembly Enabling an Autocatalytic Cas13a Circuit for Enhanced Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2697-2706},
doi = {10.1021/acssensors.5c04673},
pmid = {41686171},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems ; Survivin/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; MicroRNAs/analysis/genetics ; DNA, Viral/analysis/genetics ; RNA, Messenger/analysis/genetics ; DNA-Directed RNA Polymerases ; Viral Proteins ; },
abstract = {Despite the success of non-classical crRNA designs in Cas12a-based biosensing, application to Cas13a systems faces fundamental challenges. Our research discovers that site-specific splitting within the crRNA seed region enables effective activation of Cas13a trans-cleavage. Here, we developed an autocatalytic Cas13a circuit that transitions from self-processing to responsive assembly for enhanced biosensing (PRA-Cas13a). The system employs engineered pre-crRNA as a molecular switch, which undergoes self-processing upon target binding to assemble an active Cas13a complex and activate its trans-cleavage activity. By integrating a dual-UUU site DNA switch template and a T7 RNA polymerase-mediated signal amplification module, a "processing-assembly-amplification" cycle is constructed to enhance the detection signal. Through validation using various targets including miRNA, mRNA, and viral DNA, the PRA-Cas13a system not only achieves an attomolar (aM) level detection limit but also enables visual field detection within 10 min using a lateral flow test strip. Analysis of single-base mutations demonstrated that its sensitivity is significantly superior to conventional CRISPR-based methods. Moreover, the system successfully enabled accurate detection of survivin mRNA in different cell lines and HPV16 in clinical cervical swab samples, showing strong concordance with qPCR gold standard methods. The PRA-Cas13a strategy leverages intrinsic self-processing assembly and autocatalytic signal amplification, addresses the critical issue of off-target cleavage inherent in conventional Cas13a systems while expanding the range of applicable targets, and demonstrates high specificity and point-of-care testing potential in cancer and viral diagnostics.},
}

*Biosensing Techniques/methods
Humans
CRISPR-Cas Systems
Survivin/genetics
*CRISPR-Associated Proteins/genetics/metabolism
MicroRNAs/analysis/genetics
DNA, Viral/analysis/genetics
RNA, Messenger/analysis/genetics
DNA-Directed RNA Polymerases
Viral Proteins

@article {pmid41699288,
year = {2026},
author = {Hu, C and Tai, H and Zhu, R and Shu, Z and Guo, G and Ma, D and Zuo, S and Tang, L and Zeng, Z},
title = {CRISPR-Cas9 screening identifies ATOX1-driven cisplatin resistance mechanisms in liver cancer and evaluates targeted inhibitor efficacy.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41699288},
issn = {2399-3642},
mesh = {*Cisplatin/pharmacology ; Humans ; *Drug Resistance, Neoplasm/genetics ; *Liver Neoplasms/drug therapy/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Animals ; Cell Line, Tumor ; Mice ; *Copper Transport Proteins/genetics/antagonists & inhibitors/metabolism ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Male ; Receptor, Notch1/genetics/metabolism ; },
abstract = {Liver cancer treatment with cisplatin is often hindered by drug resistance. This study aimed to identify key genes associated with cisplatin resistance in liver cancer and develop targeted inhibitors. Using genome-wide CRISPR-Cas9 screening, ATOX1 was identified as a critical gene for cisplatin resistance. ATOX1 was highly expressed in liver cancer tissues and associated with poor prognosis. Knockdown of ATOX1 in liver cancer cells enhanced cisplatin sensitivity in vitro and in vivo. Molecular dynamics simulation and virtual screening identified compound 8 as a potent ATOX1 inhibitor with high affinity (Kd = 12.5 μM) and exhibited synergistic effects with cisplatin on liver cancer cell growth. Mechanistically, compound 8 inhibits the activity of ATOX1, leading to intracellular copper accumulation. The elevated copper levels subsequently promote increased DNA methylation at the NOTCH1 promoter, resulting in suppression of the NOTCH1/HES1 signaling pathway and enhancing the sensitivity of liver cancer cells to cisplatin. In conclusion, ATOX1 is crucial for cisplatin resistance in liver cancer and linked to poor prognosis. Targeting ATOX1 with compound 8 may be a novel therapeutic strategy for overcoming cisplatin resistance.},
}

*Cisplatin/pharmacology
Humans
*Drug Resistance, Neoplasm/genetics
*Liver Neoplasms/drug therapy/genetics/metabolism/pathology
*CRISPR-Cas Systems
*Antineoplastic Agents/pharmacology
Animals
Cell Line, Tumor
Mice
*Copper Transport Proteins/genetics/antagonists & inhibitors/metabolism
Female
Gene Expression Regulation, Neoplastic/drug effects
Male
Receptor, Notch1/genetics/metabolism

@article {pmid41702943,
year = {2026},
author = {de Menezes, MN and Chen, AXY and Kulkarni, N and Sampurno, S and Saw, NYL and Yap, KM and Pérez-Núñez, I and Roth, S and Deguit, CDT and Haugen, B and Ramsbottom, KM and Munoz, I and Beavis, PA and Parish, IA},
title = {High efficiency CRISPR knock-in demonstrates that TCF1 is insufficient to reverse T cell exhaustion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41702943},
issn = {2041-1723},
support = {MCRF21019//Victorian Cancer Agency (VCA)/ ; MCRF20011//Victorian Cancer Agency (VCA)/ ; Lloyd J. Old STAR Grant CRI5578//Cancer Research Institute (CRI)/ ; },
mesh = {Animals ; *Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; *CD8-Positive T-Lymphocytes/immunology/metabolism/cytology ; Cell Differentiation ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; *T Cell Transcription Factor 1/genetics/metabolism ; Mice, Transgenic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; T-Cell Exhaustion ; },
abstract = {CD8[+] T cell exhaustion is a regulatory state triggered by chronic antigen stimulation in both cancer and persistent infection. The less differentiated stem-like sub-populations of exhausted T cells have been heavily studied given their importance to the efficacy of current immunotherapies. While the transcription factor TCF1 is both necessary and sufficient for formation and maintenance of these stem-like populations, it remains unclear whether TCF1 can actively de-differentiate more terminally exhausted subsets back into a stem-like state. To address this question, here we utilize and optimize a high efficiency CRISPR knock-in methodology, compatible with mouse in vivo exhaustion models, to engineer T cells that either constitutively over-express TCF1, or conditionally over-express TCF1 following differentiation of the cells into a CX3CR1[+] intermediate-exhausted state. Strikingly, we find that only constitutive, and not conditional, TCF1 over-expression can increase the size of the stem-like T cell pool. Thus, while TCF1 can slow stem-like T cell differentiation, it is insufficient to revert more differentiated cells back into a stem-like state.},
}

Animals
*Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism
*Gene Knock-In Techniques/methods
Mice
*CD8-Positive T-Lymphocytes/immunology/metabolism/cytology
Cell Differentiation
Mice, Inbred C57BL
CRISPR-Cas Systems
*T Cell Transcription Factor 1/genetics/metabolism
Mice, Transgenic
*Clustered Regularly Interspaced Short Palindromic Repeats
T-Cell Exhaustion

@article {pmid41715004,
year = {2026},
author = {Metz, P and Alves-Vasconcelos, S and Wallbank, R and Riepsaame, J and Brown, S and Hassan, AB},
title = {Variation in guide RNA library representation results in gene effect score bias in genome-wide CRISPR screens.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41715004},
issn = {1471-2164},
support = {OxPOS//F. Hoffmann-La Roche/ ; GEO//John Fell Fund, University of Oxford/ ; MPNST//Grenfell Shaw Charity/ ; Oxford Clinical Fellowships/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enAsCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells, as well as external datasets containing Cas9-based CRISPR library screens, including from DepMap. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A potential contributor to this effect is gRNA representation within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also propose strategies for addressing gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation dependent bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Gene Library
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Human

@article {pmid41715150,
year = {2026},
author = {Zhao, Y and Li, X and Du, Y},
title = {AI-driven CRISPR screening: optimizing gene editing through automation and intelligent decision support.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {41715150},
issn = {1479-5876},
support = {No. Z04J2024E107-B-12//Bethune Charitable Foundation/ ; },
mesh = {Humans ; *Artificial Intelligence ; *Gene Editing ; *Automation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Decision Support Techniques ; *CRISPR-Cas Systems/genetics ; Deep Learning ; *Genetic Testing/methods ; },
abstract = {BACKGROUND: CRISPR-based genetic screening has become a central methodology in functional genomics, enabling systematic interrogation of gene function, genetic interactions and context-dependent vulnerabilities at scale. However, the rapid expansion of screening modalities-including multi-condition designs, combinatorial perturbations, in vivo applications and single-cell readouts-has exposed fundamental limitations of heuristic-driven experimental design and post hoc statistical analysis.

MAIN BODY: This Review synthesizes how artificial intelligence is reshaping CRISPR screening by introducing predictive, adaptive and system-level intelligence across the experimental lifecycle. We organize recent advances into two tightly coupled modules. First, machine learning and deep learning (ML/DL) methods optimize experimental design by learning context-dependent perturbation behavior, anticipating confounding effects and enabling iterative, information-efficient screening strategies. Second, large language model-agent (LLM-agent) systems complement these advances by externalizing scientific reasoning, integrating biological knowledge at scale and coordinating analysis and decision-making in human-in-the-loop workflows.

CONCLUSIONS: Together, ML/DL and LLM-agent approaches reframe CRISPR screening from a static analytical pipeline into an intelligent experimental system, with important implications for robustness, scalability and biological discovery.},
}

Humans
*Artificial Intelligence
*Gene Editing
*Automation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Decision Support Techniques
*CRISPR-Cas Systems/genetics
Deep Learning
*Genetic Testing/methods

@article {pmid41718462,
year = {2026},
author = {Ren, Y and Wu, R and Yang, X and Yao, B and Wang, Y},
title = {Membrane-Confined CRISPR-Cas12a Activation via Split Activator for Wash-Free Detection of Tumor-Specific Extracellular Vesicles.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2356-2365},
doi = {10.1021/acssensors.5c04166},
pmid = {41718462},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *Neoplasms/diagnosis ; },
abstract = {Current CRISPR-Cas12a biosensing systems for protein analysis rely on the release of a blocked activator from an aptamer through a competitive mechanism. However, the design of the complementary length between the activator and the aptamer involves a critical trade-off: excessively long sequences impede aptamer-target binding, whereas overly short sequences fail to activate Cas12a effectively. To overcome this limitation, we split the full-length Cas12a activator into two short single-stranded DNA fragments (S1 and S2; 10 nt each). S1 was specifically sequestered within the hairpin structure of an allosteric aptamer. The binding of the target protein to the aptamer triggered a conformational change, exposing S1 and thereby enabling its assembly with S2 to activate Cas12a. The strategy successfully preserved the binding affinity of the aptamer without compromising the efficiency of Cas12a activation. Furthermore, we anchored the Cas12a substrate to the membrane surface of extracellular vesicles (EVs) via hydrophobic interactions, localizing the cleavage reaction to the EVs interface and thereby significantly enhancing local catalytic efficiency. Finally, the strategy provided highly sensitive detection of tumor-specific EVs, not only accurately distinguishing cancer patients from healthy individuals (77.5% accuracy for early-stage and 100% for advanced-stage) but also holding potential for monitoring dynamic postoperative changes. Overall, our study provided a wash-free, one-pot detection platform for EVs analysis and established a new paradigm for CRISPR-based clinical diagnostics.},
}

*Extracellular Vesicles/metabolism/chemistry
*CRISPR-Cas Systems
Humans
Aptamers, Nucleotide/chemistry/metabolism
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism
*Neoplasms/diagnosis

@article {pmid41736562,
year = {2026},
author = {Purvis, IJ and Ochoa Olmos, OE and Park, KU and Kaufman, ML and Henry, CM and Schaaf, C and Clise, OJ and Tesdahl, CD and Haas, A and Brzezinski, JA},
title = {A robust cis-regulatory network ensures Otx2 expression during retinal development.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {6},
pages = {},
doi = {10.1242/dev.204881},
pmid = {41736562},
issn = {1477-9129},
support = {R01-EY024272/NH/NIH HHS/United States ; //Research to Prevent Blindness/ ; R01 EY024272/EY/NEI NIH HHS/United States ; F31-EY034794/NH/NIH HHS/United States ; F31 EY034794/EY/NEI NIH HHS/United States ; R01-EY024272/NH/NIH HHS/United States ; F31EY034794/NH/NIH HHS/United States ; //Gates Institute/ ; //Anschutz Medical Campus, University of Colorado/ ; //University of Colorado/ ; },
mesh = {*Otx Transcription Factors/genetics/metabolism ; Animals ; *Retina/metabolism/embryology ; *Gene Expression Regulation, Developmental ; Enhancer Elements, Genetic/genetics ; Mice ; *Gene Regulatory Networks/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand how Otx2 expression is regulated, we explored the activity and function of three of its enhancers (DHS2, DHS4 and DHS15). Enhancer reporter assays and lineage tracing show that DHS4 initiates Otx2 expression while DHS2 and DHS15 maintain expression in photoreceptors. Matched CRISPR/Cas9 and CRISPR interference systems were used to mutate or epigenetically silence enhancers, respectively. CRISPR reduced OTX2 expression acutely, but failed to significantly alter cell fate choice over the long term. In contrast, CRISPR interference of these enhancers caused permanent OTX2 loss and corresponding cell fate changes. While these data suggest that each enhancer is needed for normal Otx2 expression, it also highlights that the enhancers can interact and substitute for each other during development. This cis-regulatory element flexibility likely promotes Otx2 expression robustness. Such robustness may enable complex genes, like Otx2, to resist environmental stressors and regulatory disruptions to promote reproducible developmental outcomes.},
}

*Otx Transcription Factors/genetics/metabolism
Animals
*Retina/metabolism/embryology
*Gene Expression Regulation, Developmental
Enhancer Elements, Genetic/genetics
Mice
*Gene Regulatory Networks/genetics
CRISPR-Cas Systems/genetics

@article {pmid41758644,
year = {2026},
author = {Adami, A and Garza, R and Dorazehi, F and Douse, CH and Jakobsson, J},
title = {Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104398},
pmid = {41758644},
issn = {2666-1667},
mesh = {Humans ; *Gene Silencing ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retroelements/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al.[1].},
}

Humans
*Gene Silencing
*Induced Pluripotent Stem Cells/metabolism/cytology
*Retroelements/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Pluripotent Stem Cells/metabolism

@article {pmid41800529,
year = {2026},
author = {Yan, J and Wang, S and Xiong, S and Luo, X and Li, Y and Deng, X},
title = {Cas12a Trans-Cleavage of Hairpins Triggers a CHA Cascade for an Ultrasensitive SERS Aptasensor.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2828-2839},
doi = {10.1021/acssensors.6c00041},
pmid = {41800529},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; Limit of Detection ; DNA/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; CRISPR-Cas Systems ; Humans ; Gold/chemistry ; Bacterial Proteins ; },
abstract = {Ultrasensitive detection of non-nucleic acid biomarkers using CRISPR/Cas12a remains a major challenge due to the lack of intrinsic signal amplification. Moreover, linear DNA reporters fail to maintain efficient downstream signal amplification after trans-cleavage, while pre-amplification procedures often lead to nonspecific signals, thereby compromising assay accuracy, particularly in complex biological matrices. Here, a highly SERS aptasensor is developed by harnessing CRISPR/Cas12a-driven trans-cleavage of hairpin substrates to trigger catalytic hairpin assembly (Cas12a-CHA), achieving robust cascade signal amplification. Target recognition is converted into customizable DNA triggers that precisely activate Cas12a, while a thymine-rich DNA/RNA reporter with dT5 motifs facilitates high enhances trans-cleavage efficiency, sustaining continuous CHA cycles. The integration of AuNF@4-MBA@Ag@H2 SERS nanotags that generate abundant plasmonic hotspots, the system provides significantly enhanced Raman readout. Benefiting from synergistic molecular amplification and nanostructure engineering, the aptasensor achieves an ultralow detection limit of 1.97 × 10[-17] g/mL, nearly 20,000-fold higher sensitivity than traditional sandwich assays, along with a broad dynamic range and high specificity. Furthermore, it exhibits excellent uniformity, reproducibility, stability, and recovery in spiked serum samples, using FGF2 used as a representative biomarker to validate its performance, highlighting great potential for clinical diagnostics and real-world applications.},
}

*Spectrum Analysis, Raman/methods
*Aptamers, Nucleotide/chemistry/metabolism
*Biosensing Techniques/methods
Limit of Detection
DNA/chemistry
*CRISPR-Associated Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/chemistry
CRISPR-Cas Systems
Humans
Gold/chemistry
Bacterial Proteins

@article {pmid41805294,
year = {2026},
author = {Anfang, M and Yahya, RH and Caldararu, O and Ben Yaakov, S and Landau, U and Berman, A and Hu, Y and Belew, ZM and Crocoll, C and Xu, D and Nour-Eldin, HH and Mayrose, I and Shani, E},
title = {Targeting redundant gene families: A multiplexed, tissue-specific CRISPR toolbox for Arabidopsis genetic screens.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117055},
pmid = {41805294},
issn = {2211-1247},
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genetic Testing/methods ; *Multigene Family ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Organ Specificity/genetics ; Genome, Plant ; },
abstract = {Genome-scale targeted CRISPR libraries for forward genetic screens in plants are powerful tools for functional analysis, but they suffer from limited spatial control, single sgRNA design, and poor handling of genetic redundancy. We develop multiplexed CRISPR libraries in which each construct contains two sgRNAs that simultaneously target multiple members of a gene family. The libraries can also function at the cell-type-specific and tissue levels. A double-barcoding strategy enables efficient tracking and identification of sgRNA combinations at the plant level without individually sequencing each line. Using this platform, we generate over 1,000 Arabidopsis lines that express sgRNAs targeting 707 transporter genes across 114 gene families involved in nutrient uptake. The multiplexed design increases gene coverage and editing efficiency, underscoring its improved targeting capability to reveal hidden phenotypes. This toolbox provides a scalable resource for multi-targeted genome editing and spatially precise forward genetic screens in plants.},
}

*Arabidopsis/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Genetic Testing/methods
*Multigene Family
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Organ Specificity/genetics
Genome, Plant

@article {pmid41816845,
year = {2026},
author = {Chen, WD and Liu, L and Cheng, L},
title = {Nitroreductase-Responsive Oligomeric crRNAs for Enzyme-Triggered Regulation of CRISPR Activity.},
journal = {The Journal of organic chemistry},
volume = {91},
number = {12},
pages = {4494-4501},
doi = {10.1021/acs.joc.5c02287},
pmid = {41816845},
issn = {1520-6904},
mesh = {*Nitroreductases/metabolism/chemistry ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism ; },
abstract = {Hypoxic tumors overexpress nitroreductase (NTR), providing an endogenous trigger for selective biomolecular activation. Here, we describe the synthesis of NTR-responsive clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs via the site-specific incorporation of a p-nitrobenzyl (p-NB) phosphoramidite at the 5' terminus of crRNAs. Click-mediated oligomerization into trimeric and tetrameric constructs effectively suppressed Cas nuclease activity. Enzymatic reduction by NTR induced linker cleavage, releasing active crRNAs and restoring DNA cleavage in vitro, establishing a strategy for enzyme-regulated CRISPR control.},
}

*Nitroreductases/metabolism/chemistry
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism

@article {pmid41837829,
year = {2026},
author = {Bao, J and Ju, X and Zhang, H and Tang, D and Yan, J and Cui, C and Gao, SS},
title = {Synergistic CRISPR-Cas9 Host Engineering and Enzyme Evolution for Enantioselective Synthesis of a Vibegron Pyrrolidine Intermediate.},
journal = {Organic letters},
volume = {28},
number = {12},
pages = {3695-3700},
doi = {10.1021/acs.orglett.6c00065},
pmid = {41837829},
issn = {1523-7052},
mesh = {Stereoisomerism ; *Pyrrolidines/chemistry/chemical synthesis/metabolism ; Molecular Structure ; *CRISPR-Cas Systems ; Biocatalysis ; *Oxidoreductases/metabolism/chemistry ; Directed Molecular Evolution ; },
abstract = {The stereoselective synthesis of chiral pyrrolidine motifs is essential to vibegron production but remains challenging using conventional chemical routes. Here we report an imine reductase (IRED) catalyzed asymmetric imine reduction to access a key vibegron intermediate. Directed evolution afforded a highly efficient variant delivering 94% conversion and >99% d.e. Combined enzyme and host engineering enabled clean whole cell catalysis, establishing a robust and scalable biocatalytic platform.},
}

Stereoisomerism
*Pyrrolidines/chemistry/chemical synthesis/metabolism
Molecular Structure
*CRISPR-Cas Systems
Biocatalysis
*Oxidoreductases/metabolism/chemistry
Directed Molecular Evolution

@article {pmid41865271,
year = {2026},
author = {Kahr, J and Diaz-Peregrino, R and Sandalcioglu, IE and John, P and Mawrin, C},
title = {RagC and Map4K3 deficiency in high-grade gliomas drives proliferation and modulates mTORC1-dependent cellular functions.},
journal = {Journal of neuropathology and experimental neurology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jnen/nlag010},
pmid = {41865271},
issn = {1554-6578},
abstract = {Cellular growth and homeostasis via amino acid-responsive pathways are mediated by the mTOR signaling pathway. Rag GTPases and Map4K3 modify mTOR signaling as amino acid sensors. Altered mTOR signaling in relation to amino acid sensors might represent factors that modify proliferation and treatment responses in astrocytic tumors. To investigate this hypothesis, RagC and Map4K3 expression was studied in human gliomas, glioma cells (U87MG/U138MG), and nonglial cells (MCF-7, IOMM-Lee). RagC and Map4K3 knockout in glioma cells was generated using CRISPR-Cas and shRNA. High-grade astrocytomas had significantly reduced immunoreactivity for RagC and Map4K3 compared to low-grade astrocytomas. RagC- and Map4K3-deficient glioma cells had significantly increased proliferation and showed altered morphology and motility. Induced amino acid deficiency (leucine deprivation) reduced proliferation in Map4K3- but not in RagC-deficient cells. mTOR signaling in RagC- and Map4K3-deficient U87 cells was altered with increased phosphorylation of p70S6K and increased expression of RagD and transcription factor EB. In this context, uncoupled, exaggerated autophagy occurred in Map4K3-deficient U87 cells. In contrast, RagC-deficient U87 cells showed increased senescence but no autophagy induction. These data show that losses of RagC and Map4K3 in malignant gliomas have proliferation-inducing effects and differentially modulate key mTOR signaling-dependent cellular mechanisms.},
}

@article {pmid41865648,
year = {2026},
author = {Marková, K},
title = {Affinity-based nanostructured platforms for the selective pretreatment, enrichment and detection of miRNA biomarkers.},
journal = {Journal of chromatography. B, Analytical technologies in the biomedical and life sciences},
volume = {1276},
number = {},
pages = {125017},
doi = {10.1016/j.jchromb.2026.125017},
pmid = {41865648},
issn = {1873-376X},
abstract = {MicroRNAs (miRNAs) are clinically relevant liquid-biopsy biomarkers, yet their reliable quantification is still limited by low abundance, matrix complexity, and frequent association with protective carriers such as Argonaute-2 and exosomes. This review focuses on affinity-based nanostructured platforms as superior tools for the selective pretreatment and enrichment of miRNAs, bridging the gap between raw clinical samples and high-performance analysis. We summarize key nanomaterial architectures, including functional nanoparticles, electrospun nanofibers, 2D nanomaterial platforms, and hybrid nanocomposites, together with surface functionalization strategies that enable the liberation of miRNAs from protein complexes and their subsequent sequence-specific capture. Emphasis is placed on how these enrichment workflows address target accessibility in protein-rich biofluids and mitigate matrix-induced interference. Beyond sample preparation, we evaluate the integration of these platforms with advanced detection modalities, including Surface-Enhanced Raman Spectroscopy (SERS) using inverse molecular sentinel (iMS) nanoprobes, enzyme-free hybridization chain reactions (HCR) and CRISPR/Cas-based assays. These innovative strategies circumvent the limitations of enzymatic amplification, offering high sensitivity and specificity. Finally, we address challenges in automation and standardization, highlighting the need for integrated enrichment-to-detection workflows that accelerate the translation of nanomaterial innovation and next-generation, point-of-care miRNA diagnostics.},
}

@article {pmid41867779,
year = {2026},
author = {Wester, M and Lim, J and Van, AB and Koprowski, K and Valera, E and Bashir, R},
title = {Kinetic Modeling of Target-Amplification-Free CRISPR-Cas-Based Autocatalysis Reactions.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.03.03.709462},
pmid = {41867779},
issn = {2692-8205},
abstract = {CRISPR-Cas-based diagnostics utilize the Cas enzyme's trans-cleavage activity to generate signal and have become popular platforms for sensitive nucleic acid detection. Recently, autocatalytic systems have been demonstrated to improve the time to response and sensitivity in some cases. However, mechanistic description of these assays is limited and optimization relies on simple trial-and-error. In this work, we present the first comprehensive kinetic model that integrates all major biochemical processes involved in these assays, including cleavage reactions, nucleic acid equilibrium kinetics, inhibition of trans-cleavage by single-stranded DNA, and degradation of single-stranded reaction components. We discuss the biochemical foundations and implementation of the ordinary differential equation model, which is built for adaptation to different reaction schemes. We use the full model to investigate the role of nucleic acid stability in assay performance for a typical nucleic acid design and show that our model demonstrates inhibition effects consistent with experimental data. We describe the reaction behavior, derive a simplified analytical model and compare its performance to the full analytical model. Finally, we demonstrate tools developed for rapid in silico optimization to guide the rational design of future target-amplification-free CRISPR-Cas-based autocatalysis assays.},
}

@article {pmid41869310,
year = {2026},
author = {Petri, K and Ferrari, S and Cianciotti, BC},
title = {Editorial: Safety and efficacy of CRISPR/Cas-based genome editing tools: applications and considerations in cell and gene therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1809809},
doi = {10.3389/fimmu.2026.1809809},
pmid = {41869310},
issn = {1664-3224},
}

@article {pmid41870078,
year = {2026},
author = {Bayurova, E and Kostyushev, D and Tikhonov, A and Chulanov, V and Gordeychuk, I},
title = {Broad-acting antivirals: the pursuit of pan-viral therapeutics in the era of pandemics.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0007726},
doi = {10.1128/jvi.00077-26},
pmid = {41870078},
issn = {1098-5514},
abstract = {The ever-present threat of new viral epidemics makes the scientific community relentlessly work on the development of universal methods of antiviral therapy. The development of broad-spectrum antivirals (BSAs) focuses either on substances acting directly on viral proteins (direct-acting antivirals [DAA]) or on substances directed at the cell's own proteins (host-targeting antivirals [HTA]). Decades of development have led to the market entry of a number of DAAs with a wide range of antiviral activities; however, their clinical approval has been obtained for individual infections. HTAs have a number of advantages over DAAs, such as a wider range of antiviral activities and a high genetic barrier to viral resistance, which is undoubtedly important when preparing for a battle with an unknown pathogen. The COVID-19 pandemic has allowed for multiple clinical trials for repurposed HTAs, previously licensed for the treatment of other diseases, including cancer. Despite the enormous work done, the arsenal of BSAs capable of protecting against future pandemics caused by pathogen X is very limited. In this review, we described data on the most studied DAAs and HTAs, effective against at least two unrelated viral pathogens, focusing on those that have been studied in late preclinical and clinical trials. In the end, we highlighted alternative new approaches such as CRISPR-Cas therapy.},
}

@article {pmid41870756,
year = {2026},
author = {Gowtham, K and Shanmugaraj, B and Thangavel, LS and Srinivasan, A and Malla, A},
title = {Advancing the frontier of plant-based therapeutics: critical innovations in molecular farming and bioprocess Integration.},
journal = {Biotechnology letters},
volume = {48},
number = {2},
pages = {},
pmid = {41870756},
issn = {1573-6776},
support = {PSGCAS/IRSG/2024-2025/Biotechnology/004//PSG/ ; },
mesh = {*Molecular Farming/methods ; Gene Editing ; CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics/metabolism ; *Plants/genetics/metabolism ; Synthetic Biology ; Biotechnology/methods ; },
abstract = {Plant molecular farming (PMF) has emerged as a promising strategy for producing biopharmaceuticals and high-value biomolecules in plant systems. In this review, we present a comprehensive synthesis of current methodologies while introducing novel approaches to genetic transformation, protein expression, glycan engineering, and downstream processing. We offer in-depth analyses of recent advancements such as CRISPR/Cas9-mediated pathway editing, synthetic biology frameworks for optimizing protein yield and quality, and integrated bioprocessing solutions that enhance purification efficiency. Further, detailed case studies are discussed to illustrate actionable strategies, and future research directions are proposed to bridge current gaps. By focusing on transformative techniques and critical problem-solving perspectives, this review aims to guide researchers toward more effective and scalable PMF applications.},
}

*Molecular Farming/methods
Gene Editing
CRISPR-Cas Systems
*Plants, Genetically Modified/genetics/metabolism
*Plants/genetics/metabolism
Synthetic Biology
Biotechnology/methods

@article {pmid41871065,
year = {2026},
author = {Huang, Y and Liang, W and Huang, M and Deng, Y and Huang, Z and Ai, C and Tan, W and Jiang, L},
title = {Application of CRISPR/Cas13a system on the rapid detection of Salmonella spp.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {3},
pages = {e0014150},
doi = {10.1371/journal.pntd.0014150},
pmid = {41871065},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Salmonella/genetics/isolation & purification ; Sensitivity and Specificity ; *Salmonella Infections/diagnosis/microbiology ; Prospective Studies ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Diarrhea/microbiology/diagnosis ; Female ; Male ; Adult ; Nucleic Acid Amplification Techniques/methods ; Middle Aged ; },
abstract = {BACKGROUND: Salmonella spp. infections can manifest in various clinical symptoms, from asymptomatic carriage to gastroenteritis, and even severe sepsis. Given the rapid progression of the disease and its potential to cause severe outcomes or trigger cluster outbreaks, making the detection of Salmonella spp. critically important. Although broth enrichment culture is considered the gold standard, it is time-consuming and involves multiple steps, making it difficult to meet urgent diagnostic needs. Hence, prompt and precise detection of Salmonella spp. is crucial not only for early diagnosis and effective treatment, but also for preventing transmission, controlling outbreaks, and screening asymptomatic Salmonella carrier.

METHODS: This study developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) -SE assay that integrated the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas13a system for detecting Salmonella spp. The clinical performance of CRISPR/Cas13a-SE assay was evaluated by a cohort of 94 inpatients with diarrhea and three prospective studies.

RESULTS: The CRISPR/Cas13a-SE assay can be completed within 60 minutes, and its limit of detection was 100 fg/μL. Compared to the broth enrichment culture, the CRISPR/Cas13a-SE assay demonstrated a sensitivity of 87.5% and a specificity of 98.8% in a cohort of 94 inpatients with diarrhea. In our prospective studies involved three distinct cohorts: 1,662 food handlers, 211 outpatients with diarrhea, and 154 inpatients with Gram-negative bacteremia. Compared with broth enrichment culture, CRISPR/Cas13a-SE assay had a high concordance rate of 98.79% (1,642/1,662), 99.52% (210/211), and 100.00% (154/154) respectively.

CONCLUSIONS: We demonstrated that the CRISPR/Cas13a-SE system showed excellent detection performance for infectious diarrhea caused by Salmonella spp. The combined use of CRISPR/Cas13a-SE with the blood culture method enhances the rapid diagnosis of invasive salmonellosis, which is crucial for early target-based therapy. Additionally, screening of asymptomatic Salmonella carrier will be benefit for disease prevention and control.},
}

Humans
*CRISPR-Cas Systems
*Salmonella/genetics/isolation & purification
Sensitivity and Specificity
*Salmonella Infections/diagnosis/microbiology
Prospective Studies
Clustered Regularly Interspaced Short Palindromic Repeats
*Molecular Diagnostic Techniques/methods
Diarrhea/microbiology/diagnosis
Female
Male
Adult
Nucleic Acid Amplification Techniques/methods
Middle Aged

@article {pmid41872458,
year = {2026},
author = {Lkhagvadorj, K and Okamura, E and Taki, T and Suzuki, H and Kuno, A and Itoh, Y and Mizuno, S and Woltjen, K and Ema, M},
title = {Optimizing CRISPR precision in mouse embryos via microhomology-mediated end joining-dominant targeting.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41872458},
issn = {2399-3642},
support = {25K02195//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; 24K18045//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP223fa627008//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA End-Joining Repair ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells/metabolism ; *Embryo, Mammalian/metabolism ; Female ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblast Growth Factor 10/genetics ; },
abstract = {CRISPR/Cas9 technology enables efficient gene editing in mice, but its reliance on non-homologous end joining often leads to unpredictable and mosaic mutations in founder (F0) animals. Here, we present a hybrid genome editing strategy that combines in silico prediction software with in vitro validation using mouse embryonic stem cells (mESCs). Although the software was trained on mESC datasets, actual editing outcomes in mESCs more accurately reflected mutation patterns observed in blastocysts and post-implantation embryos. Using this information to develop an integrated pipeline, we pre-selected guide RNAs (gRNAs) predicted to promote microhomology-mediated end joining (MMEJ)-dominant repair and validated them in mESCs prior to embryo injection. Applied to the Tyr and Fgf10 genes, this approach enabled efficient generation of F0 mice with highly uniform genotypes. Our strategy enhances the predictability and reproducibility of CRISPR-based genome editing in mice and may help reduce animal usage in gene editing studies.},
}

Animals
Mice
*Gene Editing/methods
*CRISPR-Cas Systems
*DNA End-Joining Repair
RNA, Guide, CRISPR-Cas Systems/genetics
Mouse Embryonic Stem Cells/metabolism
*Embryo, Mammalian/metabolism
Female
*Clustered Regularly Interspaced Short Palindromic Repeats
Fibroblast Growth Factor 10/genetics

@article {pmid41872465,
year = {2026},
author = {Ahmed, MF and Sarkar, MMH and Mehzabin, K and Hossain, MI and Bhim, M and Chowdhury, SF and Naser, SR and Mumtaz, T and Faruk, MO},
title = {Genomic convergence of multidrug resistance, virulence-associated loci, and phage defense systems in Klebsiella pneumoniae from pharmaceutical wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-45102-2},
pmid = {41872465},
issn = {2045-2322},
abstract = {Klebsiella pneumoniae strains that combine multidrug resistance and enhanced virulence pose a growing global public health threat. Understanding the genetic basis of these high-risk lineages is critical for surveillance and mitigation. We isolated K. pneumoniae JU-BAEC-01 from treated effluent of antibiotic-manufacturing pharmaceutical facilities in Bangladesh and performed whole-genome sequencing with comparative genomic analyses to characterize its phylogeny, resistome, virulence-associated loci, mobile genetic elements, and predicted antiviral defense systems. JU-BAEC-01 belongs to a phylogenetically distinct lineage, serotype O3b: KL150 with resistance to nearly all clinically relevant antibiotic classes except carbapenems and colistin, mediated by an extensive acquired resistome, including tmexCD3-toprJ3 (tigecycline), armA, aac(6')-Ib-cr, qnrB4, oqxAB, blaDHA-1, blaSHV-182, and blaTEM-1B, mostly carried on conjugative IncC, IncFIB, IncHI1B, and IncR plasmids. Classical hypervirulence markers are present: complete aerobactin (iucABCD-iutA) and salmochelin (iroBCDEN) clusters, rmpA2, type 1 and type 3 fimbriae, T6SS, and pgaABCD. Notably, the strain encodes one of the most elaborate anti-phage defense arsenals reported in Klebsiella to date, comprising functional Type I-E, III-A, and IV-A CRISPR-Cas systems, multiple restriction-modification systems, BREX Type I, abortive infection systems (AbiE, AbiU), and additional novel defenses that coexist with phage-derived anti-CRISPR (AcrIE9) and anti-restriction (ArdA) proteins. K. pneumoniae JU-BAEC-01 is a "perfect storm" pathogen that combines multi-drug resistance (MDR), hypervirulence, and a multilayered, highly developed defense against bacteriophages. Together, these findings highlight the environmental emergence of a genetically distinct, multidrug-resistant K. pneumoniae with substantial virulence potential and complex phage-host interaction capacity, underscoring the need for genomic surveillance of pharmaceutical wastewater systems.},
}

@article {pmid41873079,
year = {2026},
author = {Luan, M and Liu, S and Yang, Q and Zhang, Y and Wang, M and Liu, R and Niu, G},
title = {[Metabolic engineering of Streptomyces for production of hyaluronic acid].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {3},
pages = {1242-1260},
doi = {10.13345/j.cjb.250734},
pmid = {41873079},
issn = {1872-2075},
support = {2023YFD1700700//the National Key Research and Development Program of China/ ; },
mesh = {*Metabolic Engineering/methods ; *Streptomyces/metabolism/genetics ; *Hyaluronic Acid/biosynthesis ; Streptomyces coelicolor/metabolism/genetics ; Hyaluronan Synthases ; },
abstract = {Hyaluronic acid (HA) is a glycosaminoglycan with significant biological activities, which render it widely applicable in the cosmetics and pharmaceutical industries. The development of safe and efficient chassis cells to enhance HA synthesis efficiency has thus emerged as a key factor in HA production. Our study aims to construct a high-performance HA biosynthesis system using Streptomyces as the chassis cell, thereby providing technical support for the efficient microbial production of HA. Thus, our study focused on the metabolic engineering of Streptomyces for strengthening the HA synthesis pathway and then optimized the culture conditions for efficient HA synthesis. First, the HA-synthesizing capabilities of four hyaluronate synthases from different sources were evaluated in two host strains: Streptomyces coelicolor M1146 and Streptomyces albus J1074. The results indicated that the hyaluronate synthases derived from Streptococcus pyogenes exhibited the strongest HA synthesis capability. Notably, the HA yield in S. albus J1074 was higher. Building on this finding, S. albus J1074 was selected as the starting strain to construct a chassis strain tailored for HA synthesis: key genes in the competitive metabolic pathway of HA synthesis were knocked out, while the expression levels of genes associated with the bypass pathway were down-regulated. Furthermore, different combinations of key genes involved in the HA precursor synthesis pathway were designed, and their expression levels were enhanced via a constitutive strong promoter. The recombinant strain obtained therefrom achieved a maximum HA yield of 2.62 g/L. Finally, the synthetic capacity of this high-yield engineered strain was further unleased through the optimization of culture conditions, leading to a final HA yield of 4.63 g/L. The recombinant strain constructed in this study not only lays a foundation for the development of engineered Streptomyces but also provides an excellent chassis strain for the microbial production of HA and other related bioproducts.},
}

*Metabolic Engineering/methods
*Streptomyces/metabolism/genetics
*Hyaluronic Acid/biosynthesis
Streptomyces coelicolor/metabolism/genetics
Hyaluronan Synthases

@article {pmid41873757,
year = {2026},
author = {Yu, Z and Bao, Y and Alcalá-Lalinde, A and Andrade Dos Ramos, Z and Koroglu, M and Berkhout, B and Herrera-Carrillo, E},
title = {Elucidating the kinetics of CRISPR-SaCas9 action to obtain effective HIV DNA excision with two gRNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41873757},
issn = {1362-4962},
support = {1R01AI145045IH//NIH RO1/ ; DGP_EMEC_2023_00154//Junta de Andalucía/ ; PID2024-162385OB-I00//Spanish State Research Agency/ ; },
mesh = {*HIV-1/genetics ; *CRISPR-Cas Systems ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; Kinetics ; *DNA, Viral/genetics/metabolism ; Proviruses/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Genome, Viral ; HIV Infections/virology ; },
abstract = {The persistence of integrated human immunodeficiency virus (HIV) proviral DNA poses a major barrier to viral eradication, as the viral reservoir enables rapid rebound upon treatment interruption, despite effective virus inhibition. CRISPR-Cas-based editing strategies, especially those using double-site cleavage, show promise in excising proviral DNA, yet the rate and determinants of excision efficiency remain poorly understood. In this study, we systematically evaluated both single- and dual-SaCas9/gRNA approaches for HIV-1 inactivation. Sequence analysis revealed that SaCas9 can eliminate all wild-type HIV-1 genomes with a single gRNA, unlike other CRISPR-Cas systems. Dual-gRNA strategies improved antiviral efficacy, with the Gag3 + Pol5 combination achieving 97% excision efficiency. Kinetic analysis showed that excision efficiency correlates with the kinetic compatibility of paired gRNAs. Pairs of gRNAs with fast and similar kinetics achieved the highest excision efficiency. In contrast, the Gag3 + Env4 pair exhibited discordant kinetic characteristics (fast and slow), resulting in the failure to induce excision as the cut DNA will be repaired before the second cut is realized. Consequently, no excision but regular editing occurred at the two target sites. These findings provide a mechanistic framework for optimizing CRISPR-Cas-mediated excision, highlighting the critical role of both antiviral activity and kinetic synergy in guiding gRNA selection.},
}

*HIV-1/genetics
*CRISPR-Cas Systems
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Gene Editing/methods
Kinetics
*DNA, Viral/genetics/metabolism
Proviruses/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells
Genome, Viral
HIV Infections/virology

@article {pmid39508585,
year = {2024},
author = {Androsiuk, L and Maane, S and Tal, S},
title = {CRISPR spacers acquired from plasmids primarily target backbone genes, making them valuable for predicting potential hosts and host range.},
journal = {Microbiology spectrum},
volume = {12},
number = {12},
pages = {e0010424},
pmid = {39508585},
issn = {2165-0497},
support = {3-17700//Ministry of Health, State of Israel (Ministry of Health)/ ; //BGU | Kreitman School of Advanced Graduate Studies, Ben-Gurion University of the Negev (Kreitman school of Advanced Graduate Studies)/ ; },
mesh = {*Plasmids/genetics ; *Bacteria/genetics/classification ; *Host Specificity/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Metagenomics/methods ; Gene Transfer, Horizontal ; CRISPR-Cas Systems ; },
abstract = {In recent years, there has been a surge in metagenomic studies focused on identifying plasmids in environmental samples. Although these studies have unearthed numerous novel plasmids, enriching our understanding of their environmental roles, a significant gap remains: the scarcity of information regarding the bacterial hosts of these newly discovered plasmids. Furthermore, even when plasmids are identified within bacterial isolates, the reported host is typically limited to the original isolate, with no insights into alternative hosts or the plasmid's potential host range. Given that plasmids depend on hosts for their existence, investigating plasmids without the knowledge of potential hosts offers only a partial perspective. This study introduces a method for identifying potential hosts and host ranges for plasmids through alignment with CRISPR spacers. To validate the method, we compared the PLSDB plasmids database with the CRISPR spacers database, yielding host predictions for 46% of the plasmids. When compared with reported hosts, our predictions achieved 84% concordance at the family level and 99% concordance at the phylum level. Moreover, the method frequently identified multiple potential hosts for a plasmid, thereby enabling predictions of alternative hosts and the host range. Notably, we found that CRISPR spacers predominantly target plasmid backbone genes while sparing functional genes, such as those linked to antibiotic resistance, aligning with our hypothesis that CRISPR spacers are acquired from plasmid-specific regions rather than insertion elements from diverse sources. Finally, we illustrate the network of connections among different bacterial taxa through plasmids, revealing potential pathways for horizontal gene transfer.IMPORTANCEPlasmids are notorious for their role in distributing antibiotic resistance genes, but they may also carry and distribute other environmentally important genes. Since plasmids are not free-living entities and rely on host bacteria for survival and propagation, predicting their hosts is essential. This study presents a method for predicting potential hosts for plasmids and offers insights into the potential paths for spreading functional genes between different bacteria. Understanding plasmid-host relationships is crucial for comprehending the ecological and clinical impact of plasmids and implications for various biological processes.},
}

*Plasmids/genetics
*Bacteria/genetics/classification
*Host Specificity/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Metagenomics/methods
Gene Transfer, Horizontal
CRISPR-Cas Systems

@article {pmid41191525,
year = {2026},
author = {Wang, K and Wang, J and Feng, R and Dudnyk, K and Kim, YJ and Lim, JYS and Lee, M and Zhang, Y and Gao, X and Cheng, Y and Orkin, SH and Zhou, J and Weiss, MJ and Yue, F and Xu, J},
title = {Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation.},
journal = {Blood},
volume = {147},
number = {13},
pages = {1470-1484},
doi = {10.1182/blood.2025030211},
pmid = {41191525},
issn = {1528-0020},
mesh = {Humans ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; *Gene Silencing ; CRISPR-Cas Systems ; Erythroid Cells/metabolism ; Repressor Proteins ; *Nuclear Proteins/genetics/metabolism ; *Carrier Proteins/genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Chromatin/genetics/metabolism ; },
abstract = {The transcription factor BCL11A is a genetically and clinically validated regulator of the fetal-to-adult hemoglobin switch in human erythroid cells. CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as the first CRISPR-based therapy for β-hemoglobinopathies. However, the molecular basis for the remarkable efficacy of CRISPR-mediated enhancer ablation remains elusive. Here, we describe a new genome architecture, an enhancer-dependent chromatin rosette, that is essential for epigenetic insulation and the developmentally regulated, hematopoietic lineage-specific expression of BCL11A. CRISPR-mediated disruption of the BCL11A erythroid enhancer impairs transcription of enhancer-driven RNAs and NIPBL-dependent cohesin loading, leading to the destabilization of the rosette structure, loss of chromatin insulation, and epigenetic silencing of BCL11A. Moreover, targeted depletion of enhancer RNAs using antisense oligonucleotides silences BCL11A by disrupting epigenetic insulation, causing HbF reactivation in adult erythroid cells. These findings uncover an essential role for enhancer-driven epigenetic insulation in transcriptional control, presenting a new strategy for the therapeutic targeting of BCL11A.},
}

Humans
*Enhancer Elements, Genetic
*Epigenesis, Genetic
*Gene Silencing
CRISPR-Cas Systems
Erythroid Cells/metabolism
Repressor Proteins
*Nuclear Proteins/genetics/metabolism
*Carrier Proteins/genetics/metabolism
Fetal Hemoglobin/genetics/metabolism
Chromatin/genetics/metabolism

@article {pmid41717698,
year = {2026},
author = {Tadokoro, T and Li, H and Gan, P and Xu, Z and Tan, W and Alzhanov, D and Sánchez-Ortiz, E and McAnally, JR and Guo, L and Xu, L and Ruan, P and Liu, N and Olson, EN},
title = {Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure.},
journal = {Circulation research},
volume = {138},
number = {7},
pages = {e326738},
pmid = {41717698},
issn = {1524-4571},
support = {P01 HL160488/HL/NHLBI NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Protein Kinase C-alpha/genetics/metabolism ; *Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism ; Phosphorylation ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Humans ; Myocytes, Cardiac/metabolism/enzymology ; Mice, Inbred C57BL ; Male ; Disease Models, Animal ; Induced Pluripotent Stem Cells ; },
abstract = {BACKGROUND: The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα.

METHODS: We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (Prkca[T497A]) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of Prkca[T497A] was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCA[T497A] mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca[2+] homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCA[T497A] under chronic AngII (angiotensin II) stimulation.

RESULTS: The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca[2+] transients.

CONCLUSIONS: The editing of PRKCA[T497A] through adenine base editing represents a potential therapeutic approach for human cardiac diseases.},
}

Animals
*Protein Kinase C-alpha/genetics/metabolism
*Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism
Phosphorylation
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
Humans
Myocytes, Cardiac/metabolism/enzymology
Mice, Inbred C57BL
Male
Disease Models, Animal
Induced Pluripotent Stem Cells

@article {pmid41834687,
year = {2026},
author = {Yang, J and Huang, Q},
title = {Cas13a/crRNA trans-cleavage triggered primer exchange reaction based self-priming chain extension for sensitive and label-free infantile pneumonia related microRNA analysis.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {12},
pages = {2524-2531},
doi = {10.1039/d6ay00090h},
pmid = {41834687},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Pneumonia/genetics/diagnosis ; Limit of Detection ; *RNA, Circular/genetics ; },
abstract = {Accurate and sensitive detection of microRNAs (miRNAs) is crucial for both pathophysiological studies and clinical diagnostics. Conventional amplification methods often face limitations such as dependence on thermal cycling, susceptibility to contamination, and insufficient specificity to discriminate among closely related miRNA family members. To address these challenges, we developed a label-free isothermal detection platform that integrates the precise RNA-targeting ability of the CRISPR/Cas13a system with a self-priming amplification cascade driven by the primer exchange reaction (PER). In this assay, target miRNA binding directly activates the trans-cleavage activity of the Cas13a/crRNA complex, which subsequently cleaves a uracil-rich toehold region on a stem-loop DNA primer (H1). Following dephosphorylation, the cleaved primer initiates a PER-mediated self-priming amplification process, generating long tandem double-stranded DNA products that can be sensitively detected using the fluorescent dye SYBR Green I. The proposed method demonstrates several key advantages: (i) high specificity enabled by the programmable Cas13a/crRNA complex, allowing clear distinction between the target miRNA and sequences with single-base mismatches or high homology; (ii) exceptional sensitivity, achieving a detection limit of 406 aM and a dynamic range spanning six orders of magnitude, through coupling Cas13a collateral cleavage with exponential isothermal amplification; (iii) excellent reproducibility, reflected by low relative standard deviations and a coefficient of variation of 3.65% in spiked serum samples; and (iv) strong concordance with the reference RT-qPCR method in mock clinical specimens, highlighting its reliability for potential clinical use. In summary, this CRISPR/Cas13a-coupled self-priming amplification strategy provides a robust, accurate, and highly sensitive means for miRNA quantification, offering a promising alternative for point-of-care molecular diagnostic applications.},
}

*MicroRNAs/genetics/analysis
Humans
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Pneumonia/genetics/diagnosis
Limit of Detection
*RNA, Circular/genetics

@article {pmid41860589,
year = {2026},
author = {Walsh, DJ and Hynes, R and Guo, W and Surgenor, C and Prodöhl, PA and Parle-McDermott, A},
title = {Development and Laboratory Validation of a Field-Deployable CRISPR-Cas12a eDNA Assay for Phylogeographic Lineage Detection in Arctic Char (Salvelinus alpinus).},
journal = {Molecular ecology resources},
volume = {26},
number = {3},
pages = {e70125},
pmid = {41860589},
issn = {1755-0998},
support = {//Higher Education Authority/ ; },
mesh = {Animals ; *Trout/genetics/classification ; Phylogeography/methods ; *CRISPR-Cas Systems ; *DNA, Environmental/genetics/isolation & purification ; Ireland ; Sensitivity and Specificity ; },
abstract = {Environmental DNA (eDNA) tools are increasingly used for biodiversity monitoring, with most existing assays targeting species-level identification. However, the use of eDNA to resolve intraspecific genetic variation remains rare and methodologically underdeveloped. This study presents the development and laboratory validation of a novel molecular assay capable of detecting specific phylogeographic lineages, advancing eDNA applications by enabling resolution below the species level. The assay combines Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a technologies with a lateral flow platform for field-ready, on-site detection. Irish Arctic char (Salvelinus alpinus) was selected as the model due to its conservation relevance and post-glacial lineage diversity in Ireland. Mitochondrial genome sequencing of known Irish lineages identified a Protospacer Adjacent Motif (PAM) site unique to the Atlantic Subclade 1 lineage, allowing clear discrimination from co-occurring lineages. Two assays were optimised: a species-specific assay detecting all Arctic char lineages and a lineage-specific assay targeting Lineage 1. Both showed high sensitivity and specificity under laboratory conditions, with LbCas12a outperforming AsCas12a at optimised buffer concentrations. The lateral flow adaptation, utilising a dual-labelled FAM-Biotin probe, enabled portable and rapid detection with minimal equipment. Field validation using eDNA from Irish lakes highlighted the need for improved sampling protocols, as lake-edge surface samples failed to yield detections. This assay represents the first reported example of a CRISPR-based eDNA tool for phylogeographic lineage detection in the field. It offers a novel, non-invasive, and scalable approach to fine-scale ecological monitoring and establishes a foundation for future conservation tools targeting intraspecific diversity.},
}

Animals
*Trout/genetics/classification
Phylogeography/methods
*CRISPR-Cas Systems
*DNA, Environmental/genetics/isolation & purification
Ireland
Sensitivity and Specificity

@article {pmid41860948,
year = {2026},
author = {Hong, Y and Si, X and Liu, W and Mai, X and Zhang, Y},
title = {Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41860948},
issn = {2050-084X},
support = {2021YFA1101002//National Key Research and Development Program of China/ ; 81773304//National Natural Science Foundation of China/ ; 81572795//National Natural Science Foundation of China/ ; 2019A39//the "Hundred, Thousand and Ten Thousand Talent Project" by Beijing municipal government/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Glycosylation ; Mice ; *Galactosyltransferases/metabolism/genetics ; *CD8-Positive T-Lymphocytes/immunology ; *Lymphocyte Activation ; Mice, Inbred C57BL ; Humans ; Receptors, Antigen, T-Cell/metabolism ; },
abstract = {Cytotoxic CD8[+] T-cells play central roles in tumor immunotherapy. Understanding the mechanisms that regulate development, differentiation, and functions of cytotoxic CD8[+] T-cells leads to the development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8[+] T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1,4-galactosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8[+] T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8[+] T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8[+] T-cells. Finally, the expression levels of B4GALT1 normalized to tumor-infiltrated CD8[+] T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8[+] T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.},
}

Animals
*CRISPR-Cas Systems
Glycosylation
Mice
*Galactosyltransferases/metabolism/genetics
*CD8-Positive T-Lymphocytes/immunology
*Lymphocyte Activation
Mice, Inbred C57BL
Humans
Receptors, Antigen, T-Cell/metabolism

@article {pmid41861390,
year = {2026},
author = {Huang, Y and Yi, X and Yang, X and Li, C and Li, Y and Ye, Z and He, J},
title = {Characterization of CRISPR-Cas systems in the Haemophilus genus CRISPR-Cas in Haemophilus spp.},
journal = {Genetics and molecular biology},
volume = {49},
number = {1},
pages = {e20250166},
pmid = {41861390},
issn = {1415-4757},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system constitutes a crucial adaptive defense mechanism in prokaryotes against foreign genetic elements. Although CRISPR-Cas systems have been characterized in numerous bacteria, the architecture and function of these systems in the Haemophilus genus remain poorly understood. This study aims to analyze CRISPR-Cas systems in 138Haemophilusstrains and investigate their function, particularly in relation to virulence factors. Results revealed that CRISPR-Cas systems were identified in 31.88% of the Haemophilusstrains. Subtype I-C was the most prevalent, followed by subtypes II-C and III-A. Repeat sequences and thecas1gene were highly conserved within the same subtype. 29.62% of spacer sequences exhibited homology to plasmids or bacteriophages. phiMHaA1 was an important target of the CRISPR-Cas system in Haemophilusgenus. The protospacer adjacent motif sequences (PAM) were determined to be 5'-TTC-3' for subtype I-C and 5'-TTT-3' for subtype II-C. Comparative analysis of virulence genes showed that CRISPR-positive strains carried more ompP2 than CRISPR-negative strains, while the distribution of hmw2C and hmw1C exhibited an opposite trend. These findings provide novel insights into the diversity and function of CRISPR-Cas systems inHaemophilusgenus and propose potential strategies for attenuating the impact ofHaemophilusvirulence factors.},
}

@article {pmid41863808,
year = {2026},
author = {Louis, EM and Fu, L and Luu, N and Sachs, LM and Shi, YB},
title = {Protocol for streamlining genotyping of germline-transmissible mutants from genome editing by using a parallel qPCR-based index and R analysis.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104454},
doi = {10.1016/j.xpro.2026.104454},
pmid = {41863808},
issn = {2666-1667},
abstract = {Targeted genome editing using CRISPR-Cas, ZFNs, or TALENs enables precise gene function studies but often produces point mutations or insertions or deletions (indels) that are difficult to detect by conventional PCR. We developed a parallel qPCR assay with an iGenotype index for simple, reliable genotyping. iGenotype values (1, 0, -1) remained constant across allele-specific primers. qPCR data can be analyzed via an R program, enabling large-scale or automated genotyping. For complete details on the use and execution of this protocol, please refer to Fu et al.[1].},
}

@article {pmid41865126,
year = {2026},
author = {Marei, HE},
title = {Recent Advances in the Non-viral Delivery of Genes to Central Nervous System Disorders.},
journal = {Cellular and molecular neurobiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s10571-026-01703-z},
pmid = {41865126},
issn = {1573-6830},
abstract = {Disorders of the central nervous system (CNS), neurological disorders, neurodegenerative disorders, genetic disorders) constitute a significant burden on global health, and current treatment options remain challenging. As treatment for CNS disorders is primarily palliative, the underlying causes of disease progression are not addressed through conventional pharmacologic therapies. Gene therapy has the potential to address these root causes of disease progression; however, many of the vectors used in gene therapy (e.g., adeno-associated viruses (AAVs)) have limitations such as immunogenicity, low cargo capacity, and crossing the blood-brain barrier (BBB). These limitations have led to significant progress in the development of non-viral gene delivery systems. Compared with viral vectors, non-viral platforms offer improved safety profiles, greater design flexibility, lower production costs, and superior suitability for repeated administration. This review reports recent advancements in the development of non-viral platforms for CNS gene delivery and focuses on lipid-based nanoparticles, polymeric nanoparticles, exosome-based techniques, and new hybrid technologies. Particular emphasis is placed on nanoparticle modification approaches to enhance BBB penetration and enable delivery of genome-editing technologies (CRISPR/Cas systems). The review provides explanations of clinical trials, regulatory considerations, and manufacturing issues that result from the recent developments noted above. It also explores the emerging role of artificial intelligence in supporting carrier design and enhancing delivery efficiency. Both artificial intelligence and non-viral platforms have the potential to facilitate the advancement of safe, effective, and repeatably administered gene therapies for patients with CNS disorders.},
}

@article {pmid40162765,
year = {2025},
author = {Zeng, X and Jiang, Q and Yang, F and Wu, Q and Lyu, T and Zhang, Q and Wang, J and Li, F and Xu, D},
title = {Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system.},
journal = {Microbiology spectrum},
volume = {13},
number = {5},
pages = {e0026825},
pmid = {40162765},
issn = {2165-0497},
mesh = {*Candida albicans/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Candidiasis/diagnosis/microbiology ; *Recombinases/metabolism/genetics ; Temperature ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Invasive candidiasis is a fungal infection caused by various pathogenic yeasts, with Candida albicans as the predominant pathogen. Traditional culturing and identification methods for C. albicans are slow, requiring several days to weeks to produce results, which hampers rapid diagnosis. In this study, we proposed three amplification methods to combine with CRISPR/Cas12a and selected the enzymatic recombinase amplification (ERA) and CRISPR/Cas12a two-step method for the detection of C. albicans in terms of sensitivity, and then the two-step method was optimized to a temperature-controlled one-step method for the detection of C. albicans by enzymatic recombinase amplification (ERA)-CRISPR/Cas12a. The temperature-controlled system employs a combination of liquid and solid paraffin wax to maintain the desired melting point, thus facilitating spatial separation of the ERA amplification system from the CRISPR/Cas12a detection system within a single tube. After a reaction at 37°C, the temperature is raised to 45°C, melting the wax and allowing the amplification system to merge with the detection system, initiating the reaction. This one-step detection platform simplifies and expedites the procedure, achieving a sensitivity level on par with that of two-step methods. The reaction completes in about 30 minutes, detecting as little as 100 ag/µL of genomic DNA from C. albicans pure cultures. It shows high specificity and resistance to clinical nucleic acid interference, without cross-reactivity. Additionally, the method eliminates the need to open the reaction tube, effectively preventing aerosol contamination and providing a stable, thus offering a new tool for the rapid clinical diagnosis of C. albicans.

IMPORTANCE: This study established a two-step method through optimization, compared its sensitivity, and then combined the specific detection capabilities of ERA and CRISPR/Cas12a. Furthermore, a one-step method was developed based on the two-step method, creating a one-step system for the detection of Candida albicans. This system does not require the lid to be opened during the reaction process, reducing aerosol contamination and minimizing the risk of false positives. This method does not require advanced instruments or equipment and shows strong specificity without being affected by other pathogens. It can serve as a new method for the detection of Candida albicans and has significant practical application prospects.},
}

*Candida albicans/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Candidiasis/diagnosis/microbiology
*Recombinases/metabolism/genetics
Temperature
*Molecular Diagnostic Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41691189,
year = {2026},
author = {Lv, J and Geng, L and Shi, W and Li, C and Wang, S and Zhang, B and Yu, K and Cui, R and Liu, Y and Xiong, F and Lv, J and Dong, S and Barco, B and Xu, J},
title = {Enhancing heritable genome editing in soybean by optimizing promoter combinations for the LbCas12a system.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41691189},
issn = {1471-2229},
abstract = {UNLABELLED: The CRISPR-Cas system, adapted from prokaryotic immune mechanisms, enables programmable DNA targeting and editing. Recent advancements include base editors and prime editors, expanding genetic research applications. The LbCas12a (Cpf1) system offers unique advantages, including simplified operation and multiplex editing, yet achieving stable heritable edits in soybean remains challenging due to its complex paleo-tetraploid genome and transformation constraints. Here we optimized promoter combinations for the LbCas12a editing system to maximize both editing efficiency in the initial (E0) generation and the frequency of homozygous or biallelic mutants in the subsequent (E1) generation. We identified prAtHSP70-1 (At5G02500), prAtEF1αA4 (At5G60390), and prGmUbi1 (Glyma.10G251900) as an effective promoter set, and demonstrated that LbCas12a–crRNA accumulation needs to exceed a defined threshold to achieve efficient target cleavage. These findings provide a practical strategy for enhancing heritable genome editing in soybean and may be broadly applicable to other crops.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08336-w.},
}

@article {pmid41733889,
year = {2026},
author = {Son, SH and Woo, S and Choi, A and Ahn, S and Yoo, HC},
title = {Advances in Engineered Virus-Like Particles for Genome Editing and Therapy.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {40},
number = {2},
pages = {317-333},
pmid = {41733889},
issn = {1179-190X},
support = {RS202400403169//Korea Basic Science Institute/ ; 2021R1C1C2006283//National Research Foundation/ ; RS-2024-00412879//National Research Foundation/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; Genetic Engineering/methods ; Genetic Vectors ; CRISPR-Cas Systems ; Gene Transfer Techniques ; },
abstract = {Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents. By combining the efficient cellular entry of viral vectors with the safety advantages of nonviral platforms, eVLPs enable transient delivery of ribonucleoproteins such as Cas9, base editors, and prime editors. Successive design strategies, from rational engineering to directed optimization, have progressively improved particle assembly, cargo stability, and editing efficiency. In parallel, pseudotyping approaches have expanded the versatility of eVLPs for cell-type-specific targeting. Recent preclinical studies highlight their potential across diverse applications, ranging from monogenic disease models to complex disorders, and support their advancement toward clinical translation. This review summarizes the structural and production principles of eVLPs, surveys key advances in their development, and discusses therapeutic opportunities and future challenges for their application in genome editing.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
Animals
Genetic Engineering/methods
Genetic Vectors
CRISPR-Cas Systems
Gene Transfer Techniques

@article {pmid41741470,
year = {2026},
author = {Aird, EJ and Serrano-Benitez, A and Siegner, SM and Cannavo, E and Belotserkovskaya, R and Gueorguieva, N and Fielden, J and Cullot, G and Ammann, S and Bader, AS and Gupta, V and Andrieux, G and Raab, R and Del Rey González, M and Cathomen, T and Cejka, P and Corn, JE and Jackson, SP},
title = {ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741470},
issn = {2041-1723},
support = {855741-DDREAMM-ERC-2019-SyG//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *DNA Helicases/metabolism/genetics ; *DNA Repair ; DNA-Binding Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; MRE11 Homologue Protein/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Cell Cycle Proteins/metabolism/genetics ; DNA Repair Enzymes/metabolism/genetics ; Poly-ADP-Ribose Binding Proteins/metabolism ; HEK293 Cells ; Nuclear Proteins/metabolism ; Bacterial Proteins ; Acid Anhydride Hydrolases ; CRISPR-Associated Proteins ; },
abstract = {DNA double-strand breaks (DSBs) both pose threats to genome integrity and are commonly used for genome editing applications. Structural features of DSB ends play key roles in determining DNA repair pathway usage and outcomes during genome editing, but the cellular factors involved in these processes are only partially known. Through genome-wide CRISPRi screening, we identify ERCC6L2 as critical for repairing Cas12a-induced staggered DSBs but irrelevant for Cas9-induced blunt DSBs. We show that ERCC6L2 acts as a protection factor for staggered DSBs with either 5' or 3' polarity, preventing large deletions and translocations stemming from DNA damage induced by Cas12a, TALENs, or dual Cas9 nicks. Furthermore, ERCC6L2 loss hyper-sensitizes cells to multiple staggered DSBs induced by promiscuous Cas12a activity or etoposide-induced TOP2 trapping. By combining genetics and biochemical reconstitution, we find that ERCC6L2 counteracts MRE11-RAD50-NBS1 (MRN)-mediated resection by binding and melting staggered DNA ends, thereby promoting accurate end joining. Our data reveal a protective role of ERCC6L2 in staggered-end DSB repair, which suggests the molecular underpinnings of pathology in patients with ERCC6L2 mutations and cautions against using overhang-inducing genome editing tools for their treatment.},
}

*DNA Breaks, Double-Stranded
Humans
*DNA Helicases/metabolism/genetics
*DNA Repair
DNA-Binding Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
MRE11 Homologue Protein/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Cell Cycle Proteins/metabolism/genetics
DNA Repair Enzymes/metabolism/genetics
Poly-ADP-Ribose Binding Proteins/metabolism
HEK293 Cells
Nuclear Proteins/metabolism
Bacterial Proteins
Acid Anhydride Hydrolases
CRISPR-Associated Proteins

@article {pmid41812941,
year = {2026},
author = {Wang, M and Niu, D and Zhang, Q and Tang, Y and Zhao, Y and Chen, F},
title = {CRISPR-based correction of apolipoprotein E4 in Alzheimer's disease: Therapeutic strategies and macromolecular delivery innovations.},
journal = {International journal of biological macromolecules},
volume = {354},
number = {},
pages = {151352},
doi = {10.1016/j.ijbiomac.2026.151352},
pmid = {41812941},
issn = {1879-0003},
mesh = {*Alzheimer Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Apolipoprotein E4/genetics ; Blood-Brain Barrier/metabolism ; Exosomes/metabolism ; Genetic Therapy/methods ; Nanoparticles/chemistry ; },
abstract = {Alzheimer's disease (AD) is the leading cause of dementia worldwide, with substantial unmet clinical needs. The apolipoprotein E4 (APOE4) allele is the strongest genetic risk factor for late onset AD, with each copy increasing risk approximately two- to three-fold, and homozygous carriers facing up to a 10- to 15-fold higher risk compared to APOE3 carriers. APOE4 contributes to diverse pathogenic mechanisms including lipid dysregulation, neuroinflammation, synaptic dysfunction, and vascular compromise. The precise, allele-specific correction of APOE4 therefore holds transformative therapeutic potential. CRISPR-based genome editing technologies, including nuclease disruption, base editing, and prime editing, offer unprecedented opportunities to directly modify APOE4 at its genomic source. Here, we review mechanistic underpinnings of APOE4 pathology, summarize current gene editing platforms for APOE4 correction, evaluate relevant in vitro and in vivo model systems, and assess delivery strategies with an emphasis on nanoparticle and exosome based approaches. We highlight recent breakthroughs in exosome mediated APOE4 editing while addressing ongoing technical hurdles in allele specificity and translational barriers such as Cas nuclease immunogenicity, limited delivery efficiency across the blood brain barrier (BBB), and concerns over long term genomic safety. This review concludes that overcoming BBB constraints remains the most significant challenge for clinical translation, and that innovations in exosome and nanoparticle based delivery platforms represent the most promising strategies for advancing CRISPR therapeutics for AD.},
}

*Alzheimer Disease/genetics/therapy
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Apolipoprotein E4/genetics
Blood-Brain Barrier/metabolism
Exosomes/metabolism
Genetic Therapy/methods
Nanoparticles/chemistry

@article {pmid41854526,
year = {2026},
author = {Gao, Z and Liu, G},
title = {Advancing Point-of-Care Testing for Helicobacter pylori toward CRISPR-Cas-Enabled Diagnostics.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00089},
pmid = {41854526},
issn = {2379-3694},
abstract = {Helicobacter pylori (H. pylori) chronically infects nearly half of the global population and is a major risk factor for gastric cancer. Timely and accurate diagnosis is critical to enable targeted eradication therapy and prevent disease progression. However, current gold-standard methods, such as invasive endoscopy and laboratory-based polymerase chain reaction, are costly, time-consuming, and logistically impractical for large-scale screening, particularly in resource-limited settings. Point-of-care testing (POCT) emerges as a transformative solution, offering rapid, user-friendly, and minimally invasive detection at the point of need. In this review, we systematically trace the evolution of H. pylori POCT, with a focus on revolutionary CRISPR-Cas-based diagnostic systems, cutting-edge advancements in substrate engineering (e.g., paper, polymer, hydrogels) and multi-modal signal transduction (e.g., optical, electrochemical). We further outline key design principles for next-generation POCT platforms that strictly align with the World Health Organization's ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Deliverable), aiming to accelerate early detection, reduce healthcare disparities, and improve global clinical management of H. pylori infection.},
}

@article {pmid41855975,
year = {2026},
author = {Yong, Q and Ou, X and Zhao, Y and Kang, X and Gao, H and Liu, H and Li, K and Guo, Y},
title = {CRISPR/Cas and isothermal amplification in Pathogen Detection: Applications and future perspectives.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129662},
doi = {10.1016/j.talanta.2026.129662},
pmid = {41855975},
issn = {1873-3573},
abstract = {Conventional pathogen detection methods are often limited by prolonged turnaround times and laboratory dependency. The integration of CRISPR/Cas systems with isothermal amplification (IA) has emerged as a promising approach to enable rapid, accurate, and field-deployable molecular diagnostics. This review systematically outlines the principles, optimization strategies, and recent advances in CRISPR-Cas and IA-integrated platforms. It highlights how synergistic mechanisms enhance detection sensitivity and examines innovative integration strategies-such as physical compartmentalization, chemical regulation, and intelligent system design-that address key compatibility challenges. The role of nanomaterials in enhancing signal amplification and facilitating system integration is thoroughly discussed. Furthermore, the suitability of various readout modalities-including fluorescence, lateral flow assays, electrochemical sensing, and digital detection-is critically evaluated. While challenges remain in terms of stability, cost, and standardization, future advances in intelligent design, portable device development, and quantitative methodologies are expected to establish this technology as a versatile platform for public health control, food safety monitoring, and related fields. This review provides a comprehensive perspective and methodological reference for researchers engaged in point-of-care testing and diagnostic technology development.},
}

@article {pmid41856106,
year = {2026},
author = {Yuan, S and Zhu, H and Yu, M and Jia, H and Peng, S and Ma, Y},
title = {Discovery of human gut phage-encoded anti-CRISPR proteins unveils diverse mechanisms for phages to evade type II CRISPR immunity.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2026.02.017},
pmid = {41856106},
issn = {1934-6069},
abstract = {Phages encode diverse anti-CRISPR (Acr) proteins to counteract bacterial CRISPR-Cas systems. However, gut phage Acrs remain poorly characterized. Using an integrated bioinformatics and high-throughput functional screening approach, we identify 651 phage-encoded positive Acr candidates that target type II CRISPR systems, which predominate in the human gut. Among these, a subset of Acrs is verified through plasmid interference assays, with plaque assays confirming CRISPR-Cas inhibitory activity for 36 Acr candidates. Mechanistic characterization of five Acrs, including the Acr against subtype II-B systems (AcrIIB-1), reveals distinct inhibition strategies. Remarkably, 213 positive Acr candidates, designated here as GutAcraca, exhibit structural convergence by adopting similar folds and exhibit dual functionality: transcription regulation to support their production and inhibition of CRISPR-Cas systems. These GutAcraca are widely distributed across microbial species (detected in 26% of species). Our work uncovers the extensive diversity of phage-encoded Acrs in the human gut and highlights their potential as biotechnology tools.},
}