@article {pmid41603863,
year = {2026},
author = {Calderón, L and Schäfer, M and Rončević, M and Rauschmeier, R and Jaritz, M and Schwickert, TA and Sun, Q and Pauli, A and Zuber, J and Busslinger, M},
title = {In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
pmid = {41603863},
issn = {1540-9538},
support = {//Boehringer Ingelheim/ ; 740349/ERC_/European Research Council/International ; LT00427/2013//Human Frontier Science Program/ ; },
mesh = {Animals ; *Cell Differentiation/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plasma Cells/immunology/cytology ; *B-Lymphocytes/immunology/cytology ; Mice ; *Lymphocyte Activation/genetics/immunology ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Immune responses to pathogens lead to the generation of plasma cells through a complex interplay of B cells with their microenvironment in lymphoid organs. To identify new regulators of B cell activation and plasmablast differentiation in the context of the splenic microenvironment, we established an in vivo system for pooled sgRNA CRISPR/Cas9 screens in immunized mice. To improve the infection efficiency of naïve B cells, we generated Cd23-Cre Rosa26LSL-EcoR/+ mice exhibiting increased expression of the ecotropic lentivirus receptor EcoR on naïve B cells. Upon adoptive B cell transfer and immunization of recipient mice, 379 sgRNAs, targeting genes with high expression in plasma cells, were analyzed for their effects on plasmablast generation. Gene hits, encoding 23 positive and 18 negative regulators of B cell activation, plasmablast differentiation, or homeostasis, were uniquely identified in these in vivo screens. Validated genes encoded proteins involved in cell adhesion, signal transduction, protein folding, iron transport, and enzymatic processes. Hence, our in vivo screening system identified novel regulators controlling B cell-mediated immune responses.},
}

Animals
*Cell Differentiation/genetics/immunology
*CRISPR-Cas Systems/genetics
*Plasma Cells/immunology/cytology
*B-Lymphocytes/immunology/cytology
Mice
*Lymphocyte Activation/genetics/immunology
Mice, Inbred C57BL
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41603755,
year = {2026},
author = {Yuan, X and Yang, F and Chen, X and Xiao, P and Shen, L},
title = {Single-Nucleotide Variation Analysis in Oral Squamous Cell Carcinoma-Related ctDNA by dCas9/sgRNA Recognition-Mediated Proximity Ligation-Triggered Terminal Hairpin Formation and Self-Priming Amplification.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07087},
pmid = {41603755},
issn = {1520-6882},
abstract = {Circulating tumor DNA (ctDNA) represents a promising noninvasive biomarker for cancer diagnosis, including oral cancer. However, its clinical translation is currently limited by the lack of precise and reliable detection techniques. In this study, we developed a novel fluorescent biosensor for the detection of single-nucleotide variations in ctDNA, which integrates dual dCas9/sgRNA complexes for target recognition, proximity ligation-initiated terminal hairpin formation and self-priming amplification (PS-THSP), and Cas12a/crRNA-mediated signal output. A key innovation of this design is its multilayered specificity strategy, combining mutation-specific recognition by dual dCas9/sgRNA, proximity-dependent ligation, and Cas12a/crRNA-assisted verification of PS-THSP amplicons. This integrated approach offers a significant advance over existing CRISPR/Cas-based methods that rely primarily on signal amplification. Furthermore, the biosensor achieves high sensitivity through the synergistic coupling of PS-THSP amplification and Cas12a trans-cleavage activity, enabling a broad dynamic range spanning 6 orders of magnitude and a detection limit as low as 0.12 fM within 120 min. When applied to serum samples, the biosensor reliably detected ctDNA with high accuracy, demonstrating its strong potential for clinical cancer diagnostics.},
}

@article {pmid41603736,
year = {2026},
author = {Fuglsang, A and Rout, SS and Koutna, EB and Sofos, N and Gallego, AR and Montoya, G},
title = {Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603736},
issn = {1362-4962},
support = {NNF14CC0001//Novo Nordisk Foundation Center for Protein Research (CPR)/ ; NNF14CC0001//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; NNF0024386//Novo Nordisk Foundation/ ; NNF17SA0030214//Novo Nordisk Foundation/ ; NNF18OC0055061//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; *Deoxyribonuclease I/genetics/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Protein Engineering ; Humans ; Cryoelectron Microscopy ; Gene Editing ; Models, Molecular ; DNA, Single-Stranded/metabolism/genetics/chemistry ; DNA/metabolism/chemistry ; Protein Conformation ; },
abstract = {The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.},
}

*CRISPR-Cas Systems/genetics
DNA Cleavage
*Deoxyribonuclease I/genetics/chemistry/metabolism
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
Catalytic Domain
Protein Engineering
Humans
Cryoelectron Microscopy
Gene Editing
Models, Molecular
DNA, Single-Stranded/metabolism/genetics/chemistry
DNA/metabolism/chemistry
Protein Conformation

@article {pmid41603733,
year = {2026},
author = {Kwon, H and Kim, J and Zhou, L and Dean, A},
title = {LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603733},
issn = {1362-4962},
support = {//Intramural Program of the National Institute of Diabetes/ ; //Digestive and Kidney Diseases/ ; 075033/GF/NIH HHS/United States ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; Kruppel-Like Factor 4 ; *Chromatin/chemistry/metabolism/genetics ; Cell Lineage/genetics ; *DNA-Binding Proteins/genetics/physiology/metabolism ; SOXB1 Transcription Factors/metabolism/genetics ; Embryoid Bodies/metabolism/cytology ; Erythroblasts/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; Kruppel-Like Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Mice, Knockout ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism/cytology ; Enhancer Elements, Genetic ; Embryonic Stem Cells/metabolism/cytology ; *Gene Expression Regulation ; LIM Domain Proteins ; },
abstract = {Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, the role of enhancer looping protein LIM domain-binding 1 (LDB1) in stem cells remains to be fully explored. We generated Ldb1(-/-) embryonic stem cells (ESCs) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Differential gene expression, including of the Lin28-mediated self-renewal pathway genes, was observed between wild-type and Ldb1(-/-) ESC. LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors, and LDB1 loss resulted in loss of Sox2 interactions with the SCR enhancer. Embryoid bodies (EBs) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers. Ldb1(-/-) ESC had impaired ability to undergo terminal differentiation to erythroblasts, and gene dysregulation was very pronounced in Ldb1(-/-) erythroblasts. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important for normal gene expression during differentiation to erythroblasts.},
}

Animals
*Cell Differentiation/genetics
Mice
Kruppel-Like Factor 4
*Chromatin/chemistry/metabolism/genetics
Cell Lineage/genetics
*DNA-Binding Proteins/genetics/physiology/metabolism
SOXB1 Transcription Factors/metabolism/genetics
Embryoid Bodies/metabolism/cytology
Erythroblasts/metabolism/cytology
Mouse Embryonic Stem Cells/metabolism/cytology
Kruppel-Like Transcription Factors/genetics/metabolism
Gene Expression Regulation, Developmental
Mice, Knockout
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism/cytology
Enhancer Elements, Genetic
Embryonic Stem Cells/metabolism/cytology
*Gene Expression Regulation
LIM Domain Proteins

@article {pmid41603729,
year = {2026},
author = {Huang, Y and Xu, H and Zhang, T and Liao, Y and Hu, J and Huang, Z and Hu, H and Li, P and Fan, L and Xie, J},
title = {Mycobacterial non-homologous end joining is required for antiphage defense.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603729},
issn = {1362-4962},
support = {82472325//National Natural Science Foundation/ ; 82072246//National Natural Science Foundation/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; //Chongqing Public Health Key Specialty (Discipline) Construction Project/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; },
mesh = {*DNA End-Joining Repair ; *Mycobacterium smegmatis/virology/genetics ; DNA Breaks, Double-Stranded ; Oxidative Stress ; Homologous Recombination ; },
abstract = {In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos sites, thereby blocking replication. The host modulates the balance between NHEJ and homologous recombination (HR) to control repair fidelity: NHEJ loss shifts the balance toward high-fidelity HR, which in turn promotes phage survival. Furthermore, NHEJ deficiency exacerbates infection-induced oxidative stress, leading to a compromise in bacterial viability. Our findings reveal the multifaceted functions of NHEJ in mycobacterium-phage interactions and provide new insights into how DNA repair systems shape antiphage defense and coevolution.},
}

*DNA End-Joining Repair
*Mycobacterium smegmatis/virology/genetics
DNA Breaks, Double-Stranded
Oxidative Stress
Homologous Recombination

@article {pmid41603018,
year = {2026},
author = {Dolder, RE and Friedman, CE and Loiben, AM and Yang, KC and Glazer, AM},
title = {High-Throughput Methods for Variant Functional Assessment in Cardiac Disease.},
journal = {Circulation. Genomic and precision medicine},
volume = {},
number = {},
pages = {e005239},
doi = {10.1161/CIRCGEN.125.005239},
pmid = {41603018},
issn = {2574-8300},
abstract = {In vitro functional modeling of genetic variants has revolutionized our understanding of which variants can cause cardiac disorders, providing insights into their molecular underpinnings. This review provides an overview of high-throughput methods used for the functional assessment of variants implicated in inherited cardiac diseases. Advances in gene-editing technology now enable the efficient generation of cells expressing individual genetic variants or libraries of variants for robust functional studies. We discuss innovative assays that can evaluate dozens or hundreds of variants sequentially. For example, the electrophysiological properties of numerous cardiac ion channel variants in genes linked to inherited arrhythmias can be characterized using automated patch clamping. The mechanical properties of cardiomyocytes expressing candidate cardiomyopathy variants can be assessed using techniques such as atomic force microscopy, traction force microscopy, and impedance-based methods. Multiplexed assays of variant effect are an emerging family of techniques that use gene-specific or general assays, combined with next-generation sequencing, to characterize hundreds or thousands of pooled genetic variants. We examine the key advantages and limitations of each method and outline future goals for the field. Innovative in vitro studies of cardiac genetic variants will enhance our understanding of variant-disease relationships and improve diagnosis, screening, and treatment options for these disorders.},
}

@article {pmid41601654,
year = {2025},
author = {Yin, L and He, W and Wang, Y and Zhang, H and Huang, M and Yan, Y and Li, S and Feng, X and Saenz, F and Zhang, J and Zhu, D and Yang, C and Ma, T and Fu, J and Chen, J},
title = {FACS-based genome-wide CRISPR screening platform identifies modulators of CD47.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1684539},
pmid = {41601654},
issn = {1664-3224},
mesh = {*CD47 Antigen/genetics/metabolism/immunology ; Animals ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Humans ; Flow Cytometry/methods ; Phagocytosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Macrophages/immunology/metabolism ; Female ; Immunity, Innate ; },
abstract = {BACKGROUND: CD47 is a key innate immune checkpoint that enables tumor cells to evade macrophage-mediated clearance.

METHODS/RESULTS: To systematically identify genetic regulators of CD47 surface expression, we performed FACS-based genome-wide CRISPR screens in three murine cancer cell lines B16 (melanoma), MC38 (colon adenocarcinoma), and EMT6 (breast carcinoma).

RESULTS: Comparative analysis of cells with high or low CD47 surface expression using DrugZ revealed CD47 itself as the top hit, validating the screens. Notably, DNAJC13 emerged as a consistent and robust regulator of CD47 expression across all three cell lines. Functional validation using DNAJC13-knockout cells confirmed a significant reduction in CD47 surface levels. Furthermore, in co-culture assays with macrophages, DNAJC13-deficient tumor cells exhibited increased susceptibility to phagocytosis, supporting a functional role for DNAJC13 in innate immune evasion. Finally, we verify that DNAJC13-knockout decrease tumor burden when treated with CD47 blockade.

CONCLUSIONS: Overall, this study highlights a previously unrecognized regulator of CD47 and demonstrates the utility of high-throughput FACS-based CRISPR screening to uncover modulators of key immune checkpoint pathways.},
}

*CD47 Antigen/genetics/metabolism/immunology
Animals
Mice
Cell Line, Tumor
*CRISPR-Cas Systems
Humans
Flow Cytometry/methods
Phagocytosis
*Clustered Regularly Interspaced Short Palindromic Repeats
Macrophages/immunology/metabolism
Female
Immunity, Innate

@article {pmid41601145,
year = {2026},
author = {Jiao, Y and Liu, Y and Sun, F and Wang, W and Li, H and Gao, Q and Li, Y and Lu, N and Tian, X and Ding, X and Du, J},
title = {Genetically Modified Plant Beneficial Microorganisms: A Sustainable Solution or a New Challenge for Agriculture.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c14342},
pmid = {41601145},
issn = {1520-5118},
abstract = {Plant diseases significantly impact crop yield and quality, while conventional pesticide treatments often disrupt beneficial plant microbiota essential for pathogen prevention and immune regulation. Although plant beneficial microorganisms (PBMs) show promise as disease control agents, their effectiveness is constrained by strain-dependent variations, survival challenges, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems combined with complementary technologies like RecE/T, enable precise modifications of PBMs to enhance their protective potential. Enhanced PBMs improve functionality via multiple mechanisms: targeted gene-expression-mediated colonization, specific antimicrobial activity, and immune regulation. Studies demonstrate that genetically modified PBMs can prevent and control plant diseases through competitive exclusion, antibiotic production, barrier reinforcement, and immune modulation. We analyzed the considerations for the environmental release of engineered PBMs to reduce risks. Future research should focus on optimizing PBMs for specific applications while addressing biosafety concerns, thereby unlocking their full potential in safeguarding plant health.},
}

@article {pmid41600812,
year = {2025},
author = {Tian, C and Feng, L and Zhou, X and Huang, K and Wang, F and Luo, R and Meng, F and Yang, H and Qiao, C and Wang, X and Shi, J and Chen, Y},
title = {A Portable One-Tube Assay Integrating RT-RPA and CRISPR/Cas12a for Rapid Visual Detection of Eurasian Avian-like H1N1 Swine Influenza Virus in the Field.},
journal = {Viruses},
volume = {18},
number = {1},
pages = {},
pmid = {41600812},
issn = {1999-4915},
support = {2021YFD1800200//Nnational Key Research and Development Program of China/ ; },
mesh = {*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; Animals ; *CRISPR-Cas Systems ; Swine ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/virology/veterinary ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The widespread circulation of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus poses significant zoonotic and pandemic risks worldwide. However, current diagnostic methods are difficult to deploy in the field, as they generally require specialized laboratory infrastructure and trained personnel. Here, we present a novel dual-signal detection platform that combines reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology for rapid, on-site EA H1N1 detection. We established an integrated one-tube assay by designing and optimizing RT-RPA primers targeting a conserved region of the hemagglutinin (HA) gene, together with engineered CRISPR/Cas12a guide RNAs exhibiting high specificity. The platform incorporates two complementary readout modes: real-time fluorescence monitoring and visual colorimetric detection using a smartphone. The assay shows excellent analytical specificity, with no cross-reactivity observed against other swine influenza virus subtypes or common swine pathogens, (including CSFV, PRRSV, PEDV, PCV, TGEV, and RV). The detection limit is 2 copies/μL, and the entire procedure can be completed within 30 mins using simple portable equipment. When evaluated on 86 clinical samples, the assay demonstrated 94.18% concordance with RT-qPCR. Compared with conventional diagnostic methods, this RT-RPA-CRISPR/Cas12a assay offers greater convenience and cost-effectiveness. Its strong potential for field-based rapid testing underscores promising application prospects in swine influenza surveillance and control programs.},
}

*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Animals
*CRISPR-Cas Systems
Swine
Sensitivity and Specificity
*Orthomyxoviridae Infections/diagnosis/virology/veterinary
*Swine Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41599334,
year = {2026},
author = {Wang, X and Chen, X and Zhou, Y and Zhao, Y and Shi, C and Li, R and Liu, L and Mu, C and Song, W and Wang, C},
title = {Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {2},
pages = {},
pmid = {41599334},
issn = {1420-3049},
support = {2022YFD2400104//the National Key R&D Program of China/ ; 32341058 and 31972783//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Brachyura/genetics ; Electroporation ; Myostatin/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {Portunus trituberculatus is an economically important marine crustacean in East Asia's aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems
*Brachyura/genetics
Electroporation
Myostatin/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
INDEL Mutation

@article {pmid41599202,
year = {2026},
author = {Yoon, B and Kim, JA and Kang, YK},
title = {CRISPR-Cas-Mediated Reprogramming Strategies to Overcome Antimicrobial Resistance.},
journal = {Pharmaceutics},
volume = {18},
number = {1},
pages = {},
pmid = {41599202},
issn = {1999-4923},
support = {grant number RS-2024-00417430//National Research Foundation of Korea (NRF)/ ; grant number RS-2024-00399808//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET)/ ; 2025-RISE-16-001//Regional Innovation System & Education (RISE) program/ ; A26234, CTRQQR-2021\100009//CRUK Convergence Science Centre at The Institute of Cancer Research, London, and Imperial Col-lege London/ ; },
abstract = {Antimicrobial resistance (AMR) is escalating worldwide, posing a serious threat to global public health by driving infections that are no longer treatable with conventional antibiotics. CRISPR-Cas technology offers a programmable and highly specific therapeutic alternative by directly targeting the genetic determinants responsible for resistance. Various CRISPR systems can restore antibiotic susceptibility and induce selective bactericidal effects by eliminating resistance genes, disrupting biofilm formation, and inhibiting virulence pathways. Moreover, CRISPR can suppress horizontal gene transfer (HGT) by removing mobile genetic elements such as plasmids, thereby limiting the ecological spread of AMR across humans, animals, and the environment. Advances in delivery platforms-including conjugative plasmids, phagemids, and nanoparticle-based carriers-are expanding the translational potential of CRISPR-based antimicrobial strategies. Concurrent progress in Cas protein engineering, spatiotemporal activity regulation, and AI-driven optimization is expected to overcome current technical barriers. Collectively, these developments position CRISPR-based antimicrobials as next-generation precision therapeutics capable of treating refractory bacterial infections while simultaneously suppressing the dissemination of antibiotic resistance.},
}

@article {pmid41599009,
year = {2025},
author = {Lv, YR and Liu, YY and Zhang, R and Yang, B and Xue, SY and Ding, YL and Jia, JT and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Mycobacterium avium subsp. paratuberculosis Using a Lateral Flow Assay Based on CRISPR-Cas12a Combined with Recombinase Polymerase Amplification or Nested PCR.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {1},
pages = {},
pmid = {41599009},
issn = {2076-0817},
support = {1.(YF20240007) 2.(BR251303) 3.(YLXKZX-NND-012)//1.Ordos Science & Technology Plan 2.Special Project for the Construction of Scientific Research and Innovation Teams, Class B Team 3.Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*CRISPR-Cas Systems ; *Mycobacterium avium subsp. paratuberculosis/genetics/isolation & purification ; *Paratuberculosis/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; Animals ; Sensitivity and Specificity ; Feces/microbiology ; Recombinases/genetics ; DNA, Bacterial/genetics ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA-CRISPR-Cas12a and nested PCR-CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10[-10] μg/μL for RPA-CRISPR-Cas12a and 1 × 10[-14] μg/μL for nested PCR-CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10[-4] μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control.},
}

*CRISPR-Cas Systems
*Mycobacterium avium subsp. paratuberculosis/genetics/isolation & purification
*Paratuberculosis/diagnosis/microbiology
*Polymerase Chain Reaction/methods
Animals
Sensitivity and Specificity
Feces/microbiology
Recombinases/genetics
DNA, Bacterial/genetics
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41598917,
year = {2026},
author = {Pei, X and Xie, J and Liang, C and Utkina, AO},
title = {Next-Generation Precision Breeding in Peanut (Arachis hypogaea L.) for Disease and Pest Resistance: From Multi-Omics to AI-Driven Innovations.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
pmid = {41598917},
issn = {2075-4450},
abstract = {Peanut (Arachis hypogaea L.) is a globally important oilseed and food legume, yet its productivity is persistently constrained by devastating diseases and insect pests that thrive under changing climates. This review aims to provide a comprehensive synthesis of advances in precision breeding and molecular approaches for enhancing disease and pest resistance in peanut. Traditional control measures ranging from crop rotation and cultural practices to chemical protection have delivered only partial and often unsustainable relief. The narrow genetic base of cultivated peanut and its complex allotetraploid genome further hinder the introgression of durable resistance. Recent advances in precision breeding are redefining the possibilities for resilient peanut improvement. Multi-omics platforms genomics, transcriptomics, proteomics, and metabolomics have accelerated the identification of resistance loci, effector-triggered immune components, and molecular cross-talk between pathogen, pest, and host responses. Genome editing tools such as CRISPR-Cas systems now enable the precise modification of susceptibility genes and defense regulators, overcoming barriers of conventional breeding. Integration of these molecular innovations with phenomics, machine learning, and remote sensing has transformed resistance screening from manual assessment to real-time, data-driven prediction. Such AI-assisted breeding pipelines promise enhanced selection accuracy and faster deployment of multi-stress-tolerant cultivars. This review outlines current progress, technological frontiers, and persisting gaps in leveraging precision breeding for disease and pest resistance in peanut, outlining a roadmap toward climate-resilient, sustainable production systems.},
}

@article {pmid41552883,
year = {2026},
author = {Xu, Q and Ji, M},
title = {A Bst-driven Cas12a cascade amplification strategy for microRNA detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {4},
pages = {899-906},
doi = {10.1039/d5ay01957e},
pmid = {41552883},
issn = {1759-9679},
mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Quantification of trace microRNAs is crucial for early disease diagnosis but remains technically challenging. Herein, we developed an ultrasensitive fluorescence platform for microRNA-21 (miR-21) detection by integrating Bst DNA polymerase - assisted target recycling with CRISPR/Cas12a-mediated signal amplification. In this design, the target miRNA triggers toehold-mediated opening of a hairpin probe, followed by Bst-driven primer extension that enables efficient target recycling and the generation of abundant DNA duplex activators. Subsequently, these activators induce strong trans-cleavage activity of Cas12a, producing markedly enhanced fluorescence responses. Benefiting from the dual amplification of enzymatic recycling and Cas12a activation, the proposed assay exhibits high sensitivity toward miR-21 with a detection limit down to 9.25 × 10[-12] M. Furthermore, the platform exhibited excellent sequence selectivity and was successfully applied to monitor miR-21 in both cell lysates and clinical serum samples. Considering its convenient operation, strong analytical performance, and simple readout mode, this method holds great potential for trace biomarker analysis in clinical diagnostics.},
}

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

@article {pmid41534496,
year = {2026},
author = {Gogate, A and Chahrour, MH},
title = {Recent advances in the neurogenomics of autism spectrum disorder.},
journal = {Current opinion in genetics & development},
volume = {96},
number = {},
pages = {102431},
doi = {10.1016/j.gde.2025.102431},
pmid = {41534496},
issn = {1879-0380},
mesh = {Humans ; *Autism Spectrum Disorder/genetics/pathology ; *Gene Regulatory Networks/genetics ; *Genomics ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Neurogenomics has provided exceptional insights into the genetic architecture underlying autism spectrum disorder (ASD), which is increasingly understood as a collection of individually rare disorders. This review synthesizes current advancements in the field, examining how both rare and common genetic variants contribute to ASD etiology. To functionally interpret the convergence on biological pathways that has emerged despite this genetic heterogeneity, multiomic approaches have been applied to identify gene regulatory networks disrupted in ASD. High-throughput technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR) editing and massively parallel reporter assays, have been employed in human induced pluripotent stem cells and organoids to bridge the gap between genetic association and biological function. Finally, machine learning methods play a pivotal role in integrating and leveraging these complex datasets to inform personalized interventions.},
}

Humans
*Autism Spectrum Disorder/genetics/pathology
*Gene Regulatory Networks/genetics
*Genomics
Induced Pluripotent Stem Cells/metabolism
*Genetic Predisposition to Disease
CRISPR-Cas Systems/genetics
Gene Editing

@article {pmid41468840,
year = {2026},
author = {Shi, T and Jin, X},
title = {Probing neuropsychiatric disorders through in vivo CRISPR screening.},
journal = {Current opinion in genetics & development},
volume = {96},
number = {},
pages = {102424},
pmid = {41468840},
issn = {1879-0380},
support = {R01 HG012819/HG/NHGRI NIH HHS/United States ; R01 MH137042/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Mental Disorders/genetics/pathology ; Mice ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis/methods ; Genetic Predisposition to Disease ; Schizophrenia/genetics ; Brain/pathology/metabolism ; },
abstract = {Although there are many known risk alleles associated with adult-onset psychiatric disorders such as schizophrenia [1-4], bipolar disorder [5-7], and major depressive disorder [8-10], the mechanistic links between these risk alleles and disease pathology, especially on a circuit-level, remain unclear. In vivo pooled CRISPR screening with single‑cell readout (in vivo Perturb‑seq) has begun to fill this gap by mapping causal genes to defined cell states directly in animal tissues [11-14]. Here, we review recent developments and applications of in vivo Perturb-seq in the mouse brain and highlight the potential of utilizing human cellular systems to extend these approaches. Additionally, we discuss how in vivo Perturb-seq can couple genetic perturbation with physiological or environmental perturbations to better model psychiatric diseases with environmental triggers.},
}

Humans
Animals
*Mental Disorders/genetics/pathology
Mice
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Single-Cell Analysis/methods
Genetic Predisposition to Disease
Schizophrenia/genetics
Brain/pathology/metabolism

@article {pmid41456843,
year = {2026},
author = {Chang, C and Yang, J and Liu, Z and Chen, J and Wang, B and Li, J and Liu, H},
title = {Layer-by-layer coated chitosan-CRISPR/Cas9 mTOR nanoparticles: A novel approach to inhibit lens epithelial cell proliferation and migration for preventing posterior capsule opacification.},
journal = {Experimental eye research},
volume = {264},
number = {},
pages = {110828},
doi = {10.1016/j.exer.2025.110828},
pmid = {41456843},
issn = {1096-0007},
mesh = {*Capsule Opacification/prevention & control/pathology/metabolism ; *Chitosan/pharmacology/chemistry ; Cell Proliferation ; *TOR Serine-Threonine Kinases/antagonists & inhibitors/genetics ; *Epithelial Cells/pathology/metabolism ; *Nanoparticles/chemistry ; Cell Movement ; *CRISPR-Cas Systems ; Animals ; Humans ; Rabbits ; *Lens, Crystalline/cytology ; Posterior Capsule of the Lens/pathology ; Epithelial-Mesenchymal Transition ; Coated Materials, Biocompatible ; Lenses, Intraocular ; },
abstract = {Posterior capsular opacification (PCO) is the most common complication following cataract surgery and a significant cause of vision impairment. PCO arises from the proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs), driven by an activated mTOR signalling pathway. Previous research has demonstrated that inhibiting mTOR activity effectively reduces LEC proliferation and EMT in rabbit models. However, achieving sustained mTOR inhibition remains a challenge. In this study, we encapsulated the CRISPR/Cas9 system targeting mTOR into chitosan nanoparticles (Chi-gRNA) with an average size of 135 nm. These nanoparticles exhibited resistance to DNase I digestion. To prolong release duration, we incorporated these Chi-gRNA nanoparticles onto the surface of intraocular lenses (IOLs) via layer-by-layer (LbL) assembly. The LbL coatings consisted of alternating layers of positively charged polyethyleneimine (PEI) and negatively charged heparin, interspersed with Chi-gRNA nanoparticles over five consecutive cycles. Spectral analysis confirmed the successful integration and coating of nanoparticles, with characteristic peaks validating the electrostatic assembly of the layers. In vitro assays demonstrated that Chi-gRNA-coated IOLs significantly inhibited the proliferation, migration, and adhesion of human lens epithelial cells (hLECs). These findings highlight the potential of LbL-coated IOLs to deliver CRISPR/Cas9 system-targeting mTOR nanoparticles as a novel and effective strategy to prevent PCO in patients undergoing cataract surgery. This approach offers a promising avenue for the long-term management of this prevalent postoperative complication.},
}

*Capsule Opacification/prevention & control/pathology/metabolism
*Chitosan/pharmacology/chemistry
Cell Proliferation
*TOR Serine-Threonine Kinases/antagonists & inhibitors/genetics
*Epithelial Cells/pathology/metabolism
*Nanoparticles/chemistry
Cell Movement
*CRISPR-Cas Systems
Animals
Humans
Rabbits
*Lens, Crystalline/cytology
Posterior Capsule of the Lens/pathology
Epithelial-Mesenchymal Transition
Coated Materials, Biocompatible
Lenses, Intraocular

@article {pmid41456781,
year = {2026},
author = {Puri, B and Gaikwad, AB},
title = {Targeting LncRNAs with CRISPR/Cas9 for Kidney Therapeutics: A Review.},
journal = {International journal of biological macromolecules},
volume = {339},
number = {Pt 1},
pages = {149932},
doi = {10.1016/j.ijbiomac.2025.149932},
pmid = {41456781},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Long Noncoding/genetics ; Gene Editing/methods ; Animals ; *Kidney Diseases/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Long noncoding RNAs (lncRNAs) have emerged as key players in the pathogenesis of kidney diseases, including acute kidney injury (AKI), AKI-to-chronic kidney disease (CKD) transition, CKD, diabetic kidney disease (DKD), renal cell carcinoma (RCC), polycystic kidney diseases (PKD), and lupus nephritis (LN). Although the roles of lncRNAs in disease progression have been investigated in preclinical models, their underlying mechanisms remain poorly understood. The therapeutic potential of lncRNA-based therapies remains largely unexplored in clinical settings. Recently, an advancement in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology offers a novel strategy for treating sickle cell anemia and β-thalassemia. Additionally, CRISPR/Cas9 is currently being evaluated in clinical trials for various diseases, including kidney diseases like RCC. However, the application of CRISPR/Cas9 to target lncRNAs is still in the early stages. Preclinical experiments have revealed that CRISPR/Cas9 could effectively target lncRNAs in kidney disorders. However, its clinical translation in AKI and CKD conditions remains unclear, and various biological challenges remain to be addressed. This review aims to investigate advancements in CRISPR/Cas9 that target lncRNAs in the kidney, highlighting the limitations and future directions for advancing CRISPR/Cas9-based lncRNA therapy and translating these findings into clinical applications.},
}

Humans
*CRISPR-Cas Systems/genetics
*RNA, Long Noncoding/genetics
Gene Editing/methods
Animals
*Kidney Diseases/therapy/genetics
Genetic Therapy/methods

@article {pmid41397613,
year = {2026},
author = {Yin, C and Chen, B and Zheng, X and Wang, N and Wang, J and Li, R and Li, J and Yao, S and Zhai, Y and Song, X},
title = {Portable visual platform integrates polymerase spiral amplification and CRISPR/Cas12a for foodborne bacteria point-of-care testing.},
journal = {Journal of dairy science},
volume = {109},
number = {2},
pages = {1036-1051},
doi = {10.3168/jds.2025-27493},
pmid = {41397613},
issn = {1525-3198},
mesh = {*Staphylococcus aureus/isolation & purification/genetics ; *Point-of-Care Testing ; CRISPR-Cas Systems ; Animals ; Nucleic Acid Amplification Techniques ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Staphylococcus aureus, a prominent global foodborne pathogen, frequently triggers epidemics with severe public health impacts. Timely and reliable detection of S. aureus is crucial for mitigating the disease burden in low- and middle-income countries. However, conventional laboratory-based detection methods remain impractical in resource-limited settings, highlighting the urgent need for accessible point-of-care solutions. Here, we present an inner-outer-tube (IOT) assay that synergistically integrates the polymerase spiral amplification (PSR) technology for enhanced sensitivity with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a (Cas12a) system for sequence-specific identification. Additionally, we have created a portable all-in-one mobile detection (PAMD) device that combines all the steps needed for testing in the field, allowing for quick visual detection of S. aureus in just 60 min. The PSR-CRISPR/Cas12a-IOT method implemented with the PAMD device achieves a detection limit of 10 cfu/mL without needing extra preparation or costly equipment. The detection platform developed in this work has advantages of ease of operation, manageable costs, and robust performance, making it highly ideal for low-resource contexts and on-site detection scenarios. Furthermore, the PSR-CRISPR/Cas12a-IOT-PAMD detection platform provides global versatility through the interchangeable use of primer sets, hence broadening its applicability to various infections.},
}

*Staphylococcus aureus/isolation & purification/genetics
*Point-of-Care Testing
CRISPR-Cas Systems
Animals
Nucleic Acid Amplification Techniques
Food Microbiology
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40522814,
year = {2026},
author = {Blumberg, LC and Bakker, GM and van der Kaaij, A and Gariépy, É and van de Geest, H and Slootweg, EJ and Los, FCO and Westerhof, LB and Wilbers, RHP},
title = {Highly efficient transgene-free ErCas12a RNP-protoplast genome editing and single-cell regeneration in Nicotiana benthamiana for glyco-engineering.},
journal = {Plant biotechnology journal},
volume = {24},
number = {1},
pages = {239-255},
doi = {10.1111/pbi.70141},
pmid = {40522814},
issn = {1467-7652},
support = {101080784//Horizon Europe/ ; 16740//The Netherlands Organization for Scientific Research/ ; //Hudson River Biotechnology B.V./ ; },
mesh = {*Nicotiana/genetics/metabolism ; *Gene Editing/methods ; *Protoplasts/metabolism ; CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Plants, Genetically Modified ; Polysaccharides/metabolism ; Regeneration ; Transgenes ; },
abstract = {Nicotiana benthamiana serves as a unique platform for biopharmaceutical production, offering advantages such as efficient and scalable protein synthesis. In addition, custom N-glycans can be engineered on biopharmaceutical glycoproteins. Yet, plant-native glycosyltransferases and glycoside hydrolases need to be removed to prevent undesired modifications of tailored N-glycans. CRISPR-based systems offer tremendous potential; however, the ploidy of the allotetraploid N. benthamiana can make genome editing challenging when attempting to knock out multiple undesired enzymes using transgenes. Here, we report a highly efficient CRISPR ribonucleoprotein (RNP)-protoplast genome editing strategy for rapid, single-generation platform engineering. We delineate the editing characteristics of ErCas12a RNPs and apply hydrogel protoplast immobilization to characterize true single-cell regeneration. We target three β-hexosaminidases responsible for removing terminal GlcNAc and/or GalNAc residues from N-glycans and verify their inactivity via MALDI-TOF-MS N-glycan analysis. We achieve up to 89.6%, 95.3% and 86.5% on-target editing in the absence of off-target editing. We demonstrate the feasibility of low cell density (10[4] ml[-1]) regeneration of individual CRISPR-edited protoplasts in 12-14 weeks, carrying intended tetra-allelic and/or deca-allelic mutations while maintaining monoclonality. Despite the occurrence of genome duplications during the single-cell regeneration of N. benthamiana protoplasts, high-efficiency genome editing paired with shoot induction frequencies exceeding 89% facilitated the ubiquitous identification of desired β-hexosaminidase mutants. We anticipate that this genome-editing method will rapidly advance glyco-engineering in polyploids such as N. benthamiana.},
}

*Nicotiana/genetics/metabolism
*Gene Editing/methods
*Protoplasts/metabolism
CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
Plants, Genetically Modified
Polysaccharides/metabolism
Regeneration
Transgenes

@article {pmid40379239,
year = {2026},
author = {Wu, J and Wang, X and Xu, J and Li, T and Shan, G and Zhang, L and Yan, T and Song, X and Sun, Y and Guo, H and Zeng, F},
title = {Overexpression of soybean flavonoid 3'-hydroxylase enhances plant salt tolerance by promoting ascorbic acid biosynthesis.},
journal = {Journal of advanced research},
volume = {80},
number = {},
pages = {111-123},
doi = {10.1016/j.jare.2025.05.009},
pmid = {40379239},
issn = {2090-1224},
mesh = {*Glycine max/genetics/metabolism/enzymology ; *Salt Tolerance/genetics ; Plants, Genetically Modified/genetics ; *Ascorbic Acid/biosynthesis ; Gene Expression Regulation, Plant ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Salt Stress ; },
abstract = {INTRODUCTION: Salt stress is a major cause of crop loss. Soybean (Glycine max), a globally vital legume crop, faces mounting yield constraints due to soil salinization. It is known that the flavonoid biosynthesis pathway involving flavonoid 3'-hydroxylase (F3'H) plays an important role in salt tolerance. However, the precise molecular basis of F3'H-mediated salt tolerance remains inadequately characterized.

OBJECTIVES: This study aimed to elucidate the function and explore the pleiotropic molecular basis of F3'H protein in soybean salt tolerance. Innovation on elite new crop varieties facilitates breeding and production applications on salt tolerance.

METHODS: We employed CRISPR/Cas9-mediated knockout and Agrobacterium-based overexpression to generate GmF3'H allelic variants and ectopic expression in soybeans. Sanger sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to confirm the specificity of gene editing and quantify expression levels in overexpression transgenic plants, respectively. As well as Subcellular localization analysis, Yeast two-hybrid (Y2H) assay, LUC activity assay and plant physiological measurements were carried out to elucidate the F3'H-mediated salt tolerance molecular basis in plants.

RESULTS: In this study, we identified the flavonoid 3' hydroxylase gene (GmF3'H) in soybeans, which as a master regulator of salt stress adaptation during seed germination and seedling stages in both soybean and Arabidopsis thaliana. Furthermore, our study revealed that the evolutionarily conserved F3'H protein competitively binds to photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) and disrupts its interaction with GDP-mannose pyrophosphorylase 1 (VTC1), a key enzyme in ascorbate biosynthesis. This competitive inhibition redirects metabolic flux toward the L-galactose pathway, leading to an increase in ascorbic acid (AsA) biosynthesis. The enhanced AsA production subsequently improves seedling salt stress tolerance in plants by maintaining redox homeostasis through ROS scavenging.

CONCLUSION: The discovery and characterization of F3'H-mediated salt tolerance provide a crucial framework for the genetic improvement of crops. This work provides new insights into plant salt stress tolerance and develops innovative strategies to enhance broad-spectrum salt tolerance, a crucial aspect for ensuring food security in crops.},
}

*Glycine max/genetics/metabolism/enzymology
*Salt Tolerance/genetics
Plants, Genetically Modified/genetics
*Ascorbic Acid/biosynthesis
Gene Expression Regulation, Plant
*Cytochrome P-450 Enzyme System/genetics/metabolism
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Salt Stress

@article {pmid40274228,
year = {2026},
author = {Song, Y and Guan, C and Zhang, Y and Xu, Y and Li, P and Luo, L and Feng, C and Chen, G},
title = {A novel CRISPR-Cas9 nickase-mediated rolling circle amplification (CRIRCA) technique for gene identification and quantitative analysis of extrachromosomal DNA.},
journal = {Journal of advanced research},
volume = {80},
number = {},
pages = {239-248},
doi = {10.1016/j.jare.2025.04.031},
pmid = {40274228},
issn = {2090-1224},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Circular/genetics ; *Deoxyribonuclease I/metabolism/genetics ; Cell Line, Tumor ; In Situ Hybridization, Fluorescence ; Microscopy, Atomic Force ; },
abstract = {INTRODUCTION: Extrachromosomal DNA (ecDNA) plays an important role in the initiation and progression of cancerous tumors. Although Circle-seq and other genetic technologies can be utilized for ecDNA analysis, they fail to provide multi-dimensional information from ecDNA, which is time-consuming and laborious.

OBJECTIVES: Herein, by combining the netlike rolling circle amplification (NRCA) with CRISPR, we developed a novel CRISPR-Cas9 nickase-mediated RCA (CRIRCA) technology that can meet the clinical analysis needs of ecDNA.

METHODS: Atomic force microscope (AFM) was applied to confirm the circular structure of the ecDNA. Agarose gel electrophoresis was performed to analyze the CRIRCA products. Fluorescent detection was applied to characterize the fluorescence signal of amplified products. qPCR and FISH techniques were applied to verify the CRIRCA results of gene identification of ecDNA.

RESULTS: Our data revealed that CRIRCA achieved more efficient signal amplification compared to traditional RCA methods, allowing it to sensitively analyze small amounts of ecDNA in single tumor cells. Utilizing computer-aided design, we successfully constructed the primer library and sgRNA library of oncogene in ecDNA, and adopted CRIRCA technology to identify the oncogenes of ecDNA in breast cancer cells.

CONCLUSION: Therefore, CRIRCA can simultaneously obtain the information from structure, sequence and quantitation of ecDNA. This work will fill the gap in the current research on the early monitoring of cancer targeting ecDNA, and provide support for the accurate diagnosis and treatment of cancer.},
}

Humans
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*DNA, Circular/genetics
*Deoxyribonuclease I/metabolism/genetics
Cell Line, Tumor
In Situ Hybridization, Fluorescence
Microscopy, Atomic Force

@article {pmid40274227,
year = {2026},
author = {Wang, X and Zhang, X and Liu, Y and Ru, L and Yan, G and Xu, Y and Yu, Y and Zhu, Z and He, Y},
title = {miR398-SlCSD1 module participates in the SA-H2O2 amplifying feedback loop in Solanum lycopersicum.},
journal = {Journal of advanced research},
volume = {80},
number = {},
pages = {19-30},
doi = {10.1016/j.jare.2025.04.035},
pmid = {40274227},
issn = {2090-1224},
mesh = {*Solanum lycopersicum/metabolism/genetics ; *MicroRNAs/genetics/metabolism ; *Hydrogen Peroxide/metabolism ; *Salicylic Acid/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; Signal Transduction ; Reactive Oxygen Species/metabolism ; Feedback, Physiological ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {INTRODUCTION: Salicylic acid (SA) is essential for immune response signal transduction in higher plants, with its signaling thought to be enhanced through interactions with reactive oxygen species (ROS). However, the exact mechanisms behind this SA self-amplifying signaling are still not well understood.

OBJECTIVES: In this study, we report the involvement of the miR398b-SlCSD1 module in the SA-H2O2 amplifying feedback loop in tomato (Solanum lycopersicum).

METHODS: Experiments were conducted using various concentrations of SA to assess its impact on ROS metabolism and the expression of SlCSD1 and sly-miR398. CRISPR/Cas9 was employed to knock out sly-miR398 and SlCSD1. Bioinformatics analyses, dual-luciferase reporter assays (Dual-Luc), and electrophoretic mobility shift assays (EMSA) were used to identify SA-responsive transcription factors and validate their regulation of sly-miR398b. The role of miR398 in endogenous SA synthesis was examined using overexpression and knockout tomato lines.

RESULTS: Low SA concentrations stimulated H2O2 accumulation, increased superoxide dismutase (SOD) activity, and suppressed sly-miR398 expression, effects absent in NahG plants with reduced SA levels. Knockout of SlCSD1 via CRISPR/Cas9 partially inhibited SA-induced H2O2 accumulation, confirming SlCSD1's role in SA-dependent H2O2 signaling. Furthermore, Dual-Luc and EMSA results revealed that TGACG-sequence-specific binding protein 2 (TGA2) mediated the regulation of miR398-SlCSD1 module by SA in tomato. Additionally, overexpression and mutation of sly-miR398b promoted SA synthesis via the phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) pathways, highlighting its regulatory role in SA biosynthesis.

CONCLUSION: Taken together, our results shed light on the involvement of the miR398-SlCSD1 module in the SA-H2O2 amplifying feedback loop, providing new insights into SA signaling in tomato. These findings contribute to understanding SA-ROS interactions and offer a potential strategy for enhancing stress tolerance in crops by targeting microRNA-regulated pathways.},
}

*Solanum lycopersicum/metabolism/genetics
*MicroRNAs/genetics/metabolism
*Hydrogen Peroxide/metabolism
*Salicylic Acid/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/metabolism/genetics
Signal Transduction
Reactive Oxygen Species/metabolism
Feedback, Physiological
CRISPR-Cas Systems
Plants, Genetically Modified

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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