@article {pmid41683796,
year = {2026},
author = {Jiang, Y and Chen, Y and Huang, Z and Chen, L and Huang, X},
title = {Tyrosinase-Deficient Skin Melanophore Lineage in Xenopus tropicalis Tadpoles Shows Strong Autofluorescence.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683796},
issn = {1422-0067},
support = {LY20C120003//Zhejiang Provincial Natural Science Foundation/ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/deficiency/metabolism ; *Xenopus/metabolism/genetics ; Larva/metabolism/genetics ; *Melanophores/metabolism ; *Skin/metabolism ; CRISPR-Cas Systems ; Melanins ; Optical Imaging ; Xenopus Proteins/genetics/metabolism ; Skin Pigmentation ; },
abstract = {Tyrosinase, encoded by Tyr, is a key rate-limiting enzyme in melanin biosynthesis. Knockout of Tyr results in a distinct albino phenotype, making it a widely used target for evaluating gene-editing efficiency. Here, we found that the tyrosinase-deficient skin melanophore lineage of Xenopus tropicalis (X. tropicalis) tadpoles shows strong autofluorescence under the GFP filter, which may interfere with in vivo fluorescence imaging. Through spectral scanning analysis, we characterized the emission spectrum of the autofluorescence under commonly used excitation wavelengths for fluorescent proteins. Based on this, we established a reference protocol for identifying and excluding such interference in Tyr-targeted knockin studies. Furthermore, knockout of the GTP cyclohydrolase 2 gene (Gch2) using CRISPR-Cas9 significantly reduced the fluorescence intensity induced by tyrosinase deficiency, indicating an essential role of the enzyme and its mediated pterine biosynthesis in the generation of the autofluorescence. This study systematically characterized these fluorescent mutant melanophores in X. tropicalis tadpoles, providing a practical basis for avoiding fluorescent interference in experimental science and a new perspective on pigment cell development and evolution.},
}

Animals
*Monophenol Monooxygenase/genetics/deficiency/metabolism
*Xenopus/metabolism/genetics
Larva/metabolism/genetics
*Melanophores/metabolism
*Skin/metabolism
CRISPR-Cas Systems
Melanins
Optical Imaging
Xenopus Proteins/genetics/metabolism
Skin Pigmentation

@article {pmid41683697,
year = {2026},
author = {Kowalik, S and Samoń, M and Przyborowski, M},
title = {Molecular Regulators of In Vitro Regeneration in Wheat: Roles of Morphogenic Factors in Transformation, Genome Editing, and Breeding.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683697},
issn = {1422-0067},
support = {Dotacja Celowa task 4.1//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Triticum/genetics/physiology/growth & development ; *Gene Editing/methods ; *Plant Breeding/methods ; *Regeneration/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; *Transformation, Genetic ; Transcription Factors/genetics/metabolism ; },
abstract = {Efficient in vitro regeneration remains a major constraint in the genetic transformation, genome editing, and molecular breeding of wheat (Triticum aestivum L.), largely due to strong genotype-dependent recalcitrance and limited activation of developmental programs required for somatic embryogenesis. Plant regeneration relies on extensive transcriptional reprogramming and epigenetic remodeling orchestrated by morphogenic regulators that modulate meristem identity, as well as cellular pluri- and totipotency. In this review, we synthesize current molecular knowledge on key transcription factors (BBM, WUS/WUS2, GRF-GIF, WOX, LAX1, SERK, WIND1/ERF115) and signaling peptides (CLE/CLV-WUS module, phytosulfokine/PSK) that regulate embryogenic competence in monocot cereals, with emphasis on their orthologs and functional relevance in wheat. We highlight how controlled expression of these morphogenic genes, promoter engineering, and transient or excisable induction systems can significantly enhance regeneration capacity, reduce chimerism in CRISPR-Cas-edited plants, and facilitate genotype-independent transformation. We also discuss epigenetic and metabolic constraints underlying wheat recalcitrance and their potential modulation to improve culture responsiveness. By integrating evidence from wheat, rice, maize, and barley, we outline conserved gene-regulatory networks that reinitiate totipotency and propose strategies to accelerate doubled haploid production and speed-breeding pipelines. Collectively, morphogenic factors emerge as central molecular tools for overcoming regeneration bottlenecks and enabling next-generation wheat improvement. The objective of this review is to synthesize and critically evaluate current molecular knowledge on morphogenic regulators controlling in vitro regeneration in wheat (Triticum aestivum L.), with particular emphasis on their roles in genetic transformation and genome editing.},
}

*Triticum/genetics/physiology/growth & development
*Gene Editing/methods
*Plant Breeding/methods
*Regeneration/genetics
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
*Transformation, Genetic
Transcription Factors/genetics/metabolism

@article {pmid41683605,
year = {2026},
author = {Blazyte, A and Lee, H and Yoon, C and Jeon, S and Lee, J and Bayarsaikhan, D and Kim, J and Park, S and Cho, J and Baek, SA and Byun, G and Lee, B and Bhak, J},
title = {Neurofibromin 1 (NF1) Splicing Mutation c.61-2A>G: From Aberrant mRNA Processing to Therapeutic Implications In Silico.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683605},
issn = {1422-0067},
support = {1.200047.01//Ulsan City Research Fund/ ; RS-2023-00263429//Genome Editing Research Program/ ; KEIT 20018560//Alchemist Project of the Korea Evaluation Institute of Industrial Technology/ ; RS-2024-00435468//Korea Planning & Evaluation Institute of Industrial Technology with support from the Ministry of Trade, Industry and Energy/ ; },
mesh = {*Neurofibromin 1/genetics ; Humans ; *Neurofibromatosis 1/genetics/therapy ; *RNA Splicing/genetics ; *Mutation ; *RNA, Messenger/genetics ; RNA Splice Sites/genetics ; Gene Editing ; Computer Simulation ; DNA Methylation ; CRISPR-Cas Systems ; Male ; },
abstract = {The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein's N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders.},
}

*Neurofibromin 1/genetics
Humans
*Neurofibromatosis 1/genetics/therapy
*RNA Splicing/genetics
*Mutation
*RNA, Messenger/genetics
RNA Splice Sites/genetics
Gene Editing
Computer Simulation
DNA Methylation
CRISPR-Cas Systems
Male

@article {pmid41683574,
year = {2026},
author = {Sopel, J and Sarad, K and Kozinska, A and Mokrzyński, K and Szczygieł, D and Murzyn, A and Drzał, A and Słomiński, A and Szczygieł, M and Elas, M},
title = {Pmel17 Deficiency Affects Melanogenesis and Promotes Tumor Vascularization.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683574},
issn = {1422-0067},
mesh = {Animals ; *Melanins/metabolism/biosynthesis ; Mice ; *Neovascularization, Pathologic/metabolism/genetics/pathology ; *Melanoma, Experimental/metabolism/pathology/genetics/blood supply ; Reactive Oxygen Species/metabolism ; Cell Line, Tumor ; *gp100 Melanoma Antigen/genetics/metabolism/deficiency ; CRISPR-Cas Systems ; Cell Movement ; Melanosomes/metabolism ; Cell Cycle ; Melanogenesis ; },
abstract = {Premelanosomal protein (Pmel, also known as Pmel17) is the major component of melanosomal fibrils and plays a key role in melanin polymerization, making it an important factor in melanogenesis. We investigated how the absence of Pmel affects the properties of B16F10 melanoma cells. Pmel-knockout B16F10 cells were generated using CRISPR/Cas9-mediated genome editing. A viability assay revealed no significant differences between wild-type (WT) and Pmel-knockout (KO) sublines; however, melanosome maturation was impaired. In Pmel KO cells, the cell cycle was disrupted, and higher levels of reactive oxygen species (ROS) were observed compared with WT cells. Moreover, the migration capacity and tube formation of melanoma cells were increased. Tumors derived from Pmel KO cells exhibited unchanged growth kinetics but reduced melanin content, along with enhanced vascularization and oxygenation. Thus, knockout of the Pmel17 gene in melanoma cells alters pigmentation, vascularization, and oxygenation of tumors. These parameters are crucial for both tumor progression and therapeutic response.},
}

Animals
*Melanins/metabolism/biosynthesis
Mice
*Neovascularization, Pathologic/metabolism/genetics/pathology
*Melanoma, Experimental/metabolism/pathology/genetics/blood supply
Reactive Oxygen Species/metabolism
Cell Line, Tumor
*gp100 Melanoma Antigen/genetics/metabolism/deficiency
CRISPR-Cas Systems
Cell Movement
Melanosomes/metabolism
Cell Cycle
Melanogenesis

@article {pmid41638522,
year = {2026},
author = {Niles, A and Kroening, K and Lauer, A and Chakravorty, A},
title = {Identity of protease-based biomarkers for viability and cytotoxicity revealed by CRISPR knockouts.},
journal = {Analytical biochemistry},
volume = {712},
number = {},
pages = {116073},
doi = {10.1016/j.ab.2026.116073},
pmid = {41638522},
issn = {1096-0309},
mesh = {Humans ; Cell Survival/drug effects ; *Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Biomarkers/metabolism/analysis ; *Peptide Hydrolases/metabolism/genetics ; Gene Editing ; },
abstract = {A wide variety of assay chemistries are routinely employed to determine cell health within an in vitro test population. Each method relies on the consistent and robust measurement of specific biological surrogates for cell viability or cytotoxicity. Unfortunately, the precise cellular origin or identity of many of these biomarkers remain poorly characterized or unknown, and thus subject to a host of undetermined biological and chemical interferences. This work details efforts to pinpoint the enzymatic sources of a set of proteolytic activity profiles previously discovered in a phenotypic activity screen and measured in a multiplexed viability ("live cell") and cytotoxicity ("dead cell") assay. First, Clustered Regularly Interspaced Short Palindromic Repeats gene editing (CRISPR) was utilized to knockout (KO) genes encoding candidate enzymes in a human cell background to create clones for identity testing. Next, clones demonstrating discrete reduction of either the live or dead cell signals were further characterized by Western blot analysis for presence of immunogenic protein and by Sanger Sequencing of the targeted edit site. The KO data directed the sourcing of potent and selective inhibitors for orthogonal activity studies of the proteases in a parental population. Last, the utility of the multiplexed assay reagent was further explored in both non-human and human primary cell lines to characterize the universality of the application. Collectively, the positive identification of Cathepsin C (CatC) and Tripeptidyl peptidase II (TPP II) informs assay users about potential modulators of activity leading to possible interferences. Last, the work provides new information about assay performance in previously untested cell types.},
}

Humans
Cell Survival/drug effects
*Gene Knockout Techniques
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Biomarkers/metabolism/analysis
*Peptide Hydrolases/metabolism/genetics
Gene Editing

@article {pmid41581373,
year = {2026},
author = {Akla, N and Boudah, A and Bertomeu, T and Chatr-Aryamontri, A and Desjarlais, M and Annabi, B},
title = {CRISPR-based chemogenomic profiling reveals redox vulnerabilities to epigallocatechin-3-gallate and green tea polyphenol extract.},
journal = {Redox biology},
volume = {90},
number = {},
pages = {104047},
pmid = {41581373},
issn = {2213-2317},
mesh = {*Catechin/analogs & derivatives/pharmacology ; Humans ; Oxidation-Reduction/drug effects ; *Tea/chemistry ; *CRISPR-Cas Systems ; *Polyphenols/pharmacology ; *Plant Extracts/pharmacology/chemistry ; Oxidative Stress/drug effects ; Antioxidants/pharmacology ; Cell Line, Tumor ; },
abstract = {Green tea polyphenols, particularly epigallocatechin-3-gallate (EGCG), are widely recognized for their beneficial preventive effects against chronic diseases including cancer and obesity. These effects are traditionally attributed to EGCG's antioxidant, anti-inflammatory, and metabolic regulatory properties. In conditions characterized by persistent oxidative stress, the disrupted redox signaling further creates a unique vulnerability that EGCG may exploit through a dual redox mechanism. Emerging evidence therefore suggests that EGCG not only mitigates oxidative damage but could also induce selective pro-oxidant stress in cancer cells, enhancing its therapeutic potential. To investigate this duality, we performed a genome-wide CRISPR/Cas9 knockout screen to identify genetic determinants of EGCG sensitivity and resistance. Our chemogenomic analysis revealed that loss of key antioxidant genes, including PRDX1, CAT, GSS, GCLM, and GCLC, significantly heightened cellular susceptibility to EGCG and green tea extract (GTE), underscoring the critical role of glutathione biosynthesis and redox homeostasis in mediating cytotoxicity. In contrast, knockouts of Kelch-like ECH-associated Protein 1 (KEAP1) and peroxisome-associated PEX genes conferred resistance, implicating in part NRF2 (also known as nuclear factor erythroid-derived 2-like 2; NFE2L2) activation and peroxisomal reactive oxygen species clearance in protective responses. Comparative profiling with gallic acid (GA), which lacks EGCG's catechin structure, further highlighted the gallate moiety's contribution to glutathione-dependent antioxidant mechanisms. Altogether, these findings illuminate the complex redox biology of EGCG and identify novel genetic vulnerabilities that may be leveraged to enhance its anticancer efficacy, particularly in obesity-associated cancers. Clinically, this work could support the development of EGCG-based interventions tailored to individual redox profiles, offering a precise chemopreventive strategy for patients at high risk of malignancies driven by metabolic and oxidative dysregulation. Furthermore, the identification of new genetic markers of EGCG sensitivity and resistance may inform future exploration of patient stratification.},
}

*Catechin/analogs & derivatives/pharmacology
Humans
Oxidation-Reduction/drug effects
*Tea/chemistry
*CRISPR-Cas Systems
*Polyphenols/pharmacology
*Plant Extracts/pharmacology/chemistry
Oxidative Stress/drug effects
Antioxidants/pharmacology
Cell Line, Tumor

@article {pmid41526374,
year = {2026},
author = {Xie, N and Pan, Y and Tong, H and Lin, Y and Jiang, Y and Wang, Z and Wan, J and Zhang, W and Wang, X and Sun, X and Yan, S and Yin, P and Sun, Q and Qi, C and Tian, Y and Shen, L and Jiang, H and Liang, D and Tang, B and Li, S and Li, XJ and Liu, Q},
title = {Precise excision of expanded GGC repeats in NOTCH2NLC via CRISPR/Cas9 for treating neuronal intranuclear inclusion disease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1683},
pmid = {41526374},
issn = {2041-1723},
support = {32071037//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Editing/methods ; *Intranuclear Inclusion Bodies/genetics/pathology/metabolism ; *Neurodegenerative Diseases/genetics/therapy/pathology ; *Trinucleotide Repeat Expansion/genetics ; Disease Models, Animal ; Mice, Transgenic ; *Receptor, Notch2/genetics ; Cell Line ; Male ; Nerve Tissue Proteins ; Intercellular Signaling Peptides and Proteins ; },
abstract = {Neuronal intranuclear inclusion disease (NIID) is an adult-onset neurodegenerative disease caused by expanded GGC repeats in the 5' untranslated region of the human-specific NOTCH2NLC gene. The high sequence similarity between NOTCH2NLC and its paralogs poses a significant challenge for precise gene editing. Here, we develop a CRISPR/spCas9-based gene-editing strategy that precisely excises the expanded GGC repeats in NOTCH2NLC without detectable off-target effects on the highly homologous NOTCH2/NOTCH2NL family genes (<2% sequence divergence at this locus). The efficacy, specificity and safety of this approach are rigorously validated across multiple experimental models, including human cell lines, NIID iPSCs, and our previously established transgenic NIID mouse model. Our results demonstrate that precise excision of the expanded GGC repeats effectively alleviates NIID-related neuropathological, molecular and behavioral abnormalities. This study establishes the proof of concept for genome editing as a therapeutic strategy for NIID and other related repeat expansion disorders.},
}

Humans
Animals
*CRISPR-Cas Systems/genetics
Mice
*Gene Editing/methods
*Intranuclear Inclusion Bodies/genetics/pathology/metabolism
*Neurodegenerative Diseases/genetics/therapy/pathology
*Trinucleotide Repeat Expansion/genetics
Disease Models, Animal
Mice, Transgenic
*Receptor, Notch2/genetics
Cell Line
Male
Nerve Tissue Proteins
Intercellular Signaling Peptides and Proteins

@article {pmid41248420,
year = {2026},
author = {Long, SA and Todd, H and Goodhart, G and Chang, WH and Amparo, AM and Bridgens, R and Dilly, J and Park, SJ and Beal, RM and Shehadeh, SM and Satyadi, MA and Trivedi, VK and Ackermann, SE and Mukherjee, R and Goodwin, CM and Edwards, AC and Stalnecker, CA and Greis, KD and Aguirre, AJ and Hobbs, GA and Bryant, KL and Ahmad, SA and Cox, AD and Der, CJ and Waters, AM},
title = {CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer.},
journal = {Cancer research},
volume = {86},
number = {4},
pages = {1035-1053},
doi = {10.1158/0008-5472.CAN-25-1835},
pmid = {41248420},
issn = {1538-7445},
support = {K22CA276632//National Cancer Institute (NCI)/ ; IRG-23-1141524//American Cancer Society (ACS)/ ; PF-23-1072348-01-CDP//American Cancer Society (ACS)/ ; 22-WG_DERB//Pancreatic Cancer Action Network (PCAN)/ ; T32CA244125//National Cancer Institute (NCI)/ ; 23-MF-DILL//Pancreatic Cancer Action Network (PCAN)/ ; R25CA261610//National Cancer Institute (NCI)/ ; R01CA42978//National Cancer Institute (NCI)/ ; P50CA196510//National Cancer Institute (NCI)/ ; U01CA199235//National Cancer Institute (NCI)/ ; P01CA203657//National Cancer Institute (NCI)/ ; R35CA232113//National Cancer Institute (NCI)/ ; P50CA257911//National Cancer Institute (NCI)/ ; R37CA251877//National Cancer Institute (NCI)/ ; 15-90-25-DER//Pancreatic Cancer Action Network (PCAN)/ ; W81XWH2110692//U.S. Department of Defense (DOD)/ ; S10OD026717//National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Pancreatic Neoplasms/genetics/drug therapy/pathology ; *Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors/genetics ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Cell Line, Tumor ; Mutation ; *Protein Kinase Inhibitors/pharmacology ; Erlotinib Hydrochloride/pharmacology ; },
abstract = {UNLABELLED: KRAS inhibitors (KRASi) targeting various KRAS mutations have entered clinical trials for pancreatic cancer. Despite promising preliminary clinical responses, most patients relapse due to intrinsic or acquired resistance. Thus, combination treatments are essential to extend the efficacy of KRAS-targeted therapies. To further determine the genetic mechanisms of KRASi resistance, we performed KRASi-anchored CRISPR-Cas9 loss-of-function screens in KRASG12D-, KRASG12C-, KRASG12R-, and KRASQ61H-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines, using six KRASi, to identify genes that modulate sensitivity to KRAS inhibition. Several hits from the screens, including EGFR, CK2, p110α, p110γ, and YAP, were validated by combining targeted inhibitors with KRASi. KRASQ61H-mutant PDAC cell lines were intrinsically less dependent on KRAS for survival than other KRAS mutational subtypes. Furthermore, the EGFR inhibitor erlotinib synergized with the RAS(ON) multiselective inhibitor RMC-7977 in KRASQ61H-mutant PDAC cell lines and in cell lines with highly active EGFR by mitigating ERK rebound activity. KRASi-resistant cell lines featured sustained ERK/MAPK dependence despite decreased ERK activity. Together, these findings enhance the understanding of intrinsic and acquired resistance to KRASi and identify therapeutic vulnerabilities that can potentially be exploited for KRASi combination therapies in patients with pancreatic cancer.

SIGNIFICANCE: A comprehensive assessment of genetic modulators of KRAS inhibitor sensitivity identifies combination approaches to increase the efficacy of KRAS inhibitors and demonstrates the limited response of KRASQ61H-mutant cancer cells to KRAS inhibition.},
}

Humans
*Pancreatic Neoplasms/genetics/drug therapy/pathology
*Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors/genetics
*CRISPR-Cas Systems
*Drug Resistance, Neoplasm/genetics
*Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology
Cell Line, Tumor
Mutation
*Protein Kinase Inhibitors/pharmacology
Erlotinib Hydrochloride/pharmacology

@article {pmid41035413,
year = {2026},
author = {Mazzei, A and Martewicz, S and Amiri, R and Cui, M and Elvassore, N and Luni, C},
title = {Functional CRISPR-Cas9 knockout screening of the genetic determinants of human fibroblast migration propensity.},
journal = {Biotechnology progress},
volume = {42},
number = {1},
pages = {e70076},
doi = {10.1002/btpr.70076},
pmid = {41035413},
issn = {1520-6033},
support = {CUP J45F21002000001//Università di Bologna/ ; F-0301-15-009//ShanghaiTech University/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; *Fibroblasts/cytology/metabolism ; *Gene Knockout Techniques/methods ; MicroRNAs/genetics ; Gene Editing ; },
abstract = {Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. Overall, it is a complex phenomenon at the interface between mechanical forces and biochemical signaling, with cell-specific and context-specific molecular events acting in the process. Here, we focus on human fibroblast migration induced by a biochemical gradient with an approach that connects the identification of molecular players with the actual mechanical function. We show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. Moreover, the screening has been performed after an expansion step aiming at the removal of all the essential genes and miRNAs, so as to identify targets related to the cell migratory ability without affecting other major cellular functions. The results confirm known genes involved in migration, but also highlight new candidates. This work establishes a methodological advancement in the use of CRISPR technology for functional screening and represents a resource for candidate genes and miRNAs playing a role in human fibroblast directional migration under biochemical gradient.},
}

Humans
*Cell Movement/genetics
*CRISPR-Cas Systems/genetics
*Fibroblasts/cytology/metabolism
*Gene Knockout Techniques/methods
MicroRNAs/genetics
Gene Editing

@article {pmid41683064,
year = {2026},
author = {Chen, Z and He, D and Yu, W and Fu, X and Zhang, L and Zhang, M and Yu, X and Ye, Z},
title = {Advancing Bongkrekic Acid Detection: From Conventional Instrumental Analysis to Advanced Biosensing for Cross-Toxin Applications.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41683064},
issn = {2304-8158},
abstract = {Bongkrekic acid (BKA), a highly lethal toxin, has been implicated in frequent poisoning incidents in recent years, posing a serious threat to global food safety and creating an urgent need for rapid and sensitive detection methods. This review provides a systematic analysis of the entire BKA detection technologies, covering sample pretreatment techniques, instrumental analysis, immunoassays, and biosensing methods. It assesses the merits of key methods and also explores the strategic cross-application of detection paradigms developed for analogous toxins. This review delivers a comprehensive and critical evaluation of BKA detection technologies. First, it discusses sample pretreatment strategies, notably solid-phase extraction (SPE) and QuEChERS. Subsequently, it analyzes the principles, performance, and applications of core detection methods, including high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), high-resolution mass spectrometry (HRMS), time-resolved fluorescence immunoassay (TRFIA), dual-mode immunosensors and nanomaterial-based sensors. Instrumental methods (e.g., HRMS) offer unmatched sensitivity [with a limit of detection (LOD) as low as 0.01 μg/kg], yet remain costly and laboratory-dependent. Immunoassay and biosensor approaches (TRFIA and dual-mode sensors) enable rapid on-site detection with high sensitivity (ng/mL to pg/mL), though challenges in stability and specificity remain. Looking forward, the development of next-generation BKA detection could be accelerated by cross-applying cutting-edge strategies proven for toxins-such as Fumonisin B1 (FB1), Ochratoxin A (OTA), and Aflatoxin B1 (AFB1)-including nanobody technology, CRISPR-Cas-mediated signal amplification, and multimodal integrated platforms. To translate this potential into practical tools, future research should prioritize the synthesis of high-specificity recognition elements, innovative signal amplification strategies, and integrated portable devices, aiming to establish end-to-end biosensing systems capable of on-site rapid detection through multitechnology integration.},
}

@article {pmid41680629,
year = {2026},
author = {Selhorst, P and Van Vyve, E and Falconi-Agapito, F and Mariën, J and Ariën, KK},
title = {Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {188},
pmid = {41680629},
issn = {1471-2164},
support = {U01AI151378/NH/NIH HHS/United States ; },
abstract = {UNLABELLED: Metagenomic sequencing for pathogen detection has traditionally suffered from low sensitivity due to the overwhelming presence of host nucleic acids. Commercial host-depletion kits are often prohibitively expensive and limited to specific species, hindering adoption in resource-limited settings, where the burden of zoonotic diseases is highest. To address this, we optimized and combined Sequence-Independent Single Primer Amplification (SISPA) with Depletion of Abundant Sequences by Hybridization (DASH), establishing a low-cost metagenomic protocol on the Oxford Nanopore sequencing platform. Our approach can be adapted to any species to detect microbial RNAs in serum samples at PCR-range sensitivity, outperforming existing methods in the field.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12268-4.},
}

@article {pmid41605411,
year = {2026},
author = {Zhang, Q and Yang, Y and Huang, X and Ma, J and Duan, Y and Ma, G and Lei, H},
title = {Prime editing for ocular gene therapy and disease modeling: a narrative review of advances, delivery, and translational readiness.},
journal = {Experimental eye research},
volume = {265},
number = {},
pages = {110891},
doi = {10.1016/j.exer.2026.110891},
pmid = {41605411},
issn = {1096-0007},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems ; Translational Research, Biomedical ; *Eye Diseases/therapy/genetics ; Disease Models, Animal ; Gene Transfer Techniques ; },
abstract = {Prime editing is a versatile "search-and-replace" genome-editing technology that enables precise and flexible genome correction of genetic sequences by reverse-transcribing an RNA template encoded at the 3' end of a prime editing guide RNA (pegRNA). It supports the introduction of nucleotide substitutions, and insertions or/and deletions (indels) in living cells without requiring double-stranded DNA breaks or exogenous donor templates. Since its introduction in 2019, prime editing has advanced rapidly-from the first-generation prime editor (PE1) to PE7 and other next-generation variants-with editing efficiencies increasing from 0.7 to 5.5 % to more than 50 % in vitro. Optimization strategies including engineering of the Cas9 and reverse transcriptase domains, refinement of pegRNA architecture, recruitment of auxiliary proteins, and modulation of DNA repair pathways have substantially enhanced editing efficiency, product purity, and target scope across diverse cell types and tissues. These developments are particularly relevant to ophthalmology, where many blinding disorders arise from point mutations or small indels ideally suited for prime editing-based correction. Recent work in retinal cells and animal models has demonstrated the growing feasibility of prime editing to treat inherited retinal diseases, modulate pathological angiogenesis, and achieve precise gene repair in post-mitotic photoreceptors and retinal pigment epithelial cells. As delivery vectors and newer PE variants improve, prime editing is a plausible next-generation platform for a wide range of ocular diseases.},
}

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

@article {pmid41485557,
year = {2026},
author = {Li, D},
title = {Rising Star Engineering the Genome for Curative Futures.},
journal = {Journal of molecular biology},
volume = {438},
number = {5},
pages = {169618},
doi = {10.1016/j.jmb.2025.169618},
pmid = {41485557},
issn = {1089-8638},
mesh = {*Gene Editing/methods ; Humans ; Animals ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; *Genetic Engineering/methods ; Mice ; Neoplasms/therapy/genetics ; Disease Models, Animal ; },
abstract = {As a professor of biomedicine in the School of Life Sciences at East China Normal University (ECNU), I am dedicated to developing advanced genome editing technologies for disease modeling and precise gene therapy. My foundational training at Hunan Normal University and Texas A&M University cultivated a deep interest in using engineered cellular and animal models to understand human diseases. Since 2013, my laboratory at ECNU has pioneered the use of TALEN and CRISPR/Cas9 for the rapid generation of knockout mouse and rat models for disease studies. Once stepped in genome editing field, I shifted my focus to advancing editing tools and developing gene therapy strategies for genetic disorders and cancer. My team has developed a suite of high-performance base editors for nuclear DNA, mitochondrial DNA, and RNA, broadening editing capabilities while enhancing precision and safety. Leveraging these technologies, we have designed several therapeutic strategies that have shown efficacy in cellular and animal models of genetic diseases. Through collaborative efforts, we have successfully translated genome editing into clinical applications, contributing to the treatment of patients with β-thalassemia. Additionally, we have developed a non-viral, site-specific CAR-T platform for lymphoma therapy. Looking forward, I aim to develop the next generation of long-fragment, site-specific integration technologies and accelerate clinical translation to bring transformative cures to more patients.},
}

*Gene Editing/methods
Humans
Animals
*Genetic Therapy/methods
CRISPR-Cas Systems
*Genetic Engineering/methods
Mice
Neoplasms/therapy/genetics
Disease Models, Animal

@article {pmid41047302,
year = {2026},
author = {Boultwood, J},
title = {Induced pluripotent stem cell-based modelling of disease evolution in myeloid leukemia: MDS to AML.},
journal = {Advances in biological regulation},
volume = {99},
number = {},
pages = {101119},
doi = {10.1016/j.jbior.2025.101119},
pmid = {41047302},
issn = {2212-4934},
mesh = {Humans ; *Myelodysplastic Syndromes/pathology/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Leukemia, Myeloid, Acute/pathology/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Animals ; },
abstract = {The myelodysplastic syndromes (MDS) are common myeloid malignancies that develop from the successive acquisition of driver mutations in hematopoietic stem cells residing in the bone marrow. Around a third of MDS patients will develop secondary acute myeloid leukemia (sAML) and patients with high-risk MDS or sAML have a dismal prognosis. The study of disease progression in myeloid malignancy has been enhanced in recent years by the use of induced pluripotent stem cells (iPSCs) technology. iPSCs offer the advantage of indefinite expansion and the potential for genetic modification, with reprogramming enabling the capture of the full complement of genetic lesions found in primary patient bone marrow samples. The power of iPSC and CRISPR-Cas9 gene editing technologies have been harnessed to generate a range of iPSC-based cellular models of MDS, reflecting the genetic and biologic heterogeneity of the disease. Stage-specific patient iPSC lines have been produced and sequential gene editing in normal human iPSCs has been performed to map the evolution of MDS to AML. These studies have increased our understanding of the impact of driver mutations, and co-mutations, on disease phenotype and revealed mechanisms underlying disease stage transitions in myeloid malignancy. iPSC-based models of MDS have also proven important tools in high throughput drug screening and have empowered drug testing and drug discovery, offering a new platform to develop personalized therapy.},
}

Humans
*Myelodysplastic Syndromes/pathology/genetics/metabolism
*Induced Pluripotent Stem Cells/metabolism/pathology
*Leukemia, Myeloid, Acute/pathology/genetics/metabolism
Gene Editing
CRISPR-Cas Systems
Animals

@article {pmid40923291,
year = {2026},
author = {Guiltinan, MJ and Landherr, L and Maximova, SN and DelVecchio, D and Sebastian, A and Albert, I},
title = {Reduced Susceptibility to Phytophthora in Non-Transgenic Cacao Progeny Through CRISPR-Cas9 Mediated TcNPR3 Mutagenesis.},
journal = {Plant biotechnology journal},
volume = {24},
number = {2},
pages = {442-454},
doi = {10.1111/pbi.70365},
pmid = {40923291},
issn = {1467-7652},
support = {//Pennsylvania State University College of Agriculture/ ; //Huck Institutes of the Life Sciences/ ; PEN05003//Endowed Program in the Molecular Biology of Cacao and USDA Hatch/ ; PEN4879//Endowed Program in the Molecular Biology of Cacao and USDA Hatch/ ; },
mesh = {*Phytophthora/pathogenicity/physiology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/genetics ; *Cacao/genetics/microbiology ; Gene Editing ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Mutagenesis ; Disease Resistance/genetics ; },
abstract = {Black pod disease, caused by a complex of Phytophthora species, poses a severe threat to global cacao production. This study explores the use of CRISPR-Cas9 genome editing to reduce disease susceptibility in Theobroma cacao L. by targeting the TcNPR3 gene, a known negative regulator of plant defence. Transgenic T0 lines carrying mutations predicted to disrupt TcNPR3 function exhibited reduced susceptibility to Phytophthora infection in in vitro foliar assays. These T0 plants were advanced to maturity and outcrossed with non-transgenic cacao to eliminate T-DNA sequences associated with the CRISPR-Cas9 transgene. Whole-genome sequencing of the T0 parents and 22 progeny revealed single T-DNA insertion sites in each T0 line; seven progeny retained the edited npr3 alleles but lacked T-DNA insertions. Transcriptome analysis of the mutant lines showed upregulation of genes associated with reactive oxygen species (ROS) generation, defence-related transcription factors and pathogenesis-related proteins. Several genes were also downregulated, suggesting that TcNPR3 may function as both a repressor and an activator in regulating basal transcriptional states. Genome-edited plants were phenotypically comparable to wild-type controls and displayed a 42% reduction in lesion size upon Phytophthora challenge. These findings demonstrate the feasibility of generating non-transgenic cacao with reduced susceptibility to Phytophthora through CRISPR-Cas9-mediated genome editing, offering a promising strategy for sustainable cacao cultivation and improved farmer livelihoods. Field trials are underway to evaluate long-term agronomic performance under natural conditions.},
}

*Phytophthora/pathogenicity/physiology
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology/genetics
*Cacao/genetics/microbiology
Gene Editing
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Mutagenesis
Disease Resistance/genetics

@article {pmid41680487,
year = {2026},
author = {Kroell, AS and Hoffmann, KH and Motzkus, NA and Lemmen, N and Happ, N and Wolf, B and von Bachmann, AL and Southern, N and Vogd, F and Aschenbrenner, S and Niopek, D and Mathony, J},
title = {Modular engineering of thermoresponsive allosteric proteins.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41680487},
issn = {1552-4469},
support = {520612620//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 453202693//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Thermogenetics enables noninvasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli, we generated potent, thermoswitchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41 °C). Extending this strategy to mammalian systems, we engineered CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Lastly, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermosensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.},
}

@article {pmid41680202,
year = {2026},
author = {Garmendia-Antoñana, N and Dorado-Morales, P and Gil, C and García, B and Echeverz, M and Solano, C and Penadés, JR and Lasa, I},
title = {Targeted elimination of Staphylococcus aureus mastitis infections with synthetic phage-based CRISPR-Cas delivery systems.},
journal = {NPJ biofilms and microbiomes},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41522-026-00931-x},
pmid = {41680202},
issn = {2055-5008},
support = {PRE2021-097385//Spanish Ministry of Science, Innovation and Universities/ ; PID2020-113494RB-I00/ AEI//Spanish Ministry of Science, Innovation and Universities/ ; BES-2015-07285//the Spanish Ministry of Science, Innovation and Universities/ ; },
abstract = {Treatment options for Staphylococcus aureus infections are increasingly limited, particularly in livestock, where S. aureus causes mastitis requiring prolonged antibiotic therapy. This study engineered Phage Inducible Chromosomal Islands (ePICIs) to deliver CRISPR-Cas9 modules targeting small RNA genes. ePICIs exhibit bactericidal activity without chromosomal integration, an expanded host range compared to their parental phages, and biofilm-dependent efficacy influenced by the extracellular matrix composition. Biofilms mediated by the Bap protein strongly protect bacteria from ePICIs, whereas PIA/PNAG-based biofilms do not. Despite Bap-mediated protection in vitro, ePICIs achieved bactericidal effects comparable to vancomycin in a mouse mastitis model caused by Bap-producing strains. These findings reveal key factors affecting phage-delivered CRISPR-Cas efficacy and highlight that antibiofilm therapies should not be dismissed based solely on in vitro performance. Non-replicative ePICIs thus represent a promising alternative for treating localized infections such as mastitis.},
}

@article {pmid41679307,
year = {2026},
author = {Zhao, J and Wang, Z and Lu, L and Bu, G and Miao, Z and Zhang, Y and Guo, Y and Yang, Z and Ma, J and Jiao, J and Ma, X},
title = {An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair.},
journal = {Cell reports methods},
volume = {},
number = {},
pages = {101299},
doi = {10.1016/j.crmeth.2025.101299},
pmid = {41679307},
issn = {2667-2375},
abstract = {CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: OREC[C] regulated by doxycycline (Dox) and OREC[o] controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC's therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.},
}

@article {pmid41678638,
year = {2026},
author = {El-Brolosy, MA and Oak, A and Hoang, AT and Damergi, Y and Fischer, A and Saunders, RA and Luo, J and Balabaki, A and Guez, J and Whitfield, TW and Goldman, SR and Latifkar, A and Lu, YR and Stainier, DYR and Karczewski, KJ and Corradin, O and Weissman, JS},
title = {Mechanisms linking cytoplasmic decay of translation-defective mRNA to transcriptional adaptation.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6786},
pages = {eaea1272},
doi = {10.1126/science.aea1272},
pmid = {41678638},
issn = {1095-9203},
mesh = {*RNA Stability ; *RNA, Messenger/metabolism/genetics ; *Cytoplasm/metabolism ; Humans ; *Transcription, Genetic ; *Protein Biosynthesis ; *RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Transcriptional adaptation (TA) is a genetic robustness mechanism through which mutant messenger RNA (mRNA) decay induces sequence-dependent up-regulation of so-called adapting genes. How cytoplasmically generated mRNA fragments affect nuclear transcription remains poorly understood. Using genome-wide CRISPR screens, we uncover ILF3 as an RNA binding protein connecting cytoplasmic mRNA decay and transcription during TA and show that it is required for a range of TA substrates. ILF3 is enriched at adapting genes' RNAs, and its artificial recruitment through dCas13 promotes gene expression. Using tiling oligonucleotide screens, we identify trigger RNA fragments that activate adapting genes when introduced into cells. Further functional dissection reveals a critical role for homology between trigger and target sequences. These findings enhance our molecular understanding of TA and inform the design of programmable oligonucleotides for gene expression augmentation.},
}

*RNA Stability
*RNA, Messenger/metabolism/genetics
*Cytoplasm/metabolism
Humans
*Transcription, Genetic
*Protein Biosynthesis
*RNA-Binding Proteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41678468,
year = {2026},
author = {Mendoza-Garcia, P and Keith, B and Nordberg, M and Quist, E and Ferrás, C and Hamza, GM and Elgendy, R and Ashenden, SK and Chi, J and van Zuydam, NR and Hattersley, N and Zhang, X},
title = {Omics-aided design genome editing strategy for challenging human immortalized cell models.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0341124},
pmid = {41678468},
issn = {1932-6203},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; MCF-7 Cells ; Hep G2 Cells ; DNA End-Joining Repair ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Genomics/methods ; Gene Knock-In Techniques ; },
abstract = {CRISPR-Cas9 has become a popular genome editing tool for biomedical research and drug development due to its capability to enable precise correction or integration of genetic mutations in the genome. However, precise genome editing competency varies dramatically between cell types depending on their capabilities for DNA damage. In this proof-of-concept study, we took the example of HepG2 and MCF7 to show that omics profiling identifies bottlenecks that are associated with poor precise knock-in (KI) efficiency in hard-to-engineer cells. These bottlenecks include previously described factors such as the predominance of non-homologous end joining (NHEJ) repair and impaired homologous recombination (HR) capability, but also reveals apoptotic priming status of the cells as a limiting factor. Upon further comparative analysis between HepG2 and MCF7 cells, we pinpointed and validated the proliferating cell nuclear antigen (PCNA) as a target to overexpress to enhance precise KI efficiency in MCF7. Overall, we describe how employing a multi-omics approach to characterize cell models of interest can facilitate an in-depth understanding of their editability molecular signature, empowering us to manipulate the activity of key pathways for precise editing, and therefore increase efficiency of desired editing outcomes.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
MCF-7 Cells
Hep G2 Cells
DNA End-Joining Repair
Proliferating Cell Nuclear Antigen/genetics/metabolism
Genomics/methods
Gene Knock-In Techniques

@article {pmid41678341,
year = {2026},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Bacterial Cofactors for CRISPR Activation.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.5c00738},
pmid = {41678341},
issn = {1520-4995},
abstract = {Anti-CRISPR (Acr) proteins have long exemplified the viral counterattack against CRISPR-Cas immunity. By contrast, comparatively little is known about host proteins that may increase Cas effector activity. Recent work on a compact type V nuclease, Cas12p, demonstrates that this phage-associated effector depends on the bacterial thioredoxin TrxA for efficient DNA cleavage. TrxA binds a dedicated thioredoxin-binding (TB) domain on Cas12p through a redox-sensitive interaction, promoting an active conformation competent for DNA cleavage. This finding adds to a small but growing set of CRISPR activators and highlights that CRISPR-Cas systems are not static defense modules but dynamic networks shaped by auxiliary factors that can fine-tune their activity.},
}

@article {pmid41678334,
year = {2026},
author = {Wang, Y and Su, X and Chen, Y and Chen, Y and Shi, C and Liu, F and Ye, Y and Sun, P and Tan, M and Yu, M and Wang, Y and Xie, S and Liu, J and Yan, Q and Sun, Q and Neculai, D and Liu, W and Shao, J and Liu, Y and Lin, W and Lin, A},
title = {A CRISPR-based mitochondrial gene therapy tool derived by engineering guide RNAs.},
journal = {Cell reports},
volume = {45},
number = {2},
pages = {116958},
doi = {10.1016/j.celrep.2026.116958},
pmid = {41678334},
issn = {2211-1247},
abstract = {Mitochondrial genetic diseases arise from mitochondrial DNA (mtDNA) defects, which gene therapy tools may rectify. However, delivering single-guide RNAs (sgRNAs) into mitochondria remains a challenge limiting CRISPR-mediated mtDNA therapy. Here, through network analysis of mitochondrion-localized long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs), we found that lncRNA RP11-46H11.3 translocates into mitochondria via binding mitochondria-associated RBPs using its key RNA recognition motifs (RRMs); its derived 30 nt ST2-RNA mitochondrial targeting sequence (RMTS) showed the highest mitochondrial localization efficiency. We engineered the RMTS-CRISPR tool by fusing ST2-RMTS to sgRNA, verifying its ability to target and cleave mtDNA. Strikingly, our results demonstrated that RMTS-CRISPR could achieve heteroplasmic mtDNA shifting efficiencies of up to 26.37% in m.3243A>G mutant cell models and 26.79% in vivo, offering a technological approach for the correction of heterogeneous mtDNA mutations. Taken together, our findings reveal a CRISPR-based mitochondrial gene intervention strategy that may have applications in mitochondrial disorders.},
}

@article {pmid41530154,
year = {2026},
author = {Li, H and Liu, F and Li, J and Shi, C and Lin, Z and Qin, Y and Pan, R and Wu, X and Peng, Y and Xing, C and Wang, Y and Qu, Q and Li, G},
title = {Evolutionary dynamics of sex determination in Branchiostoma belcheri driven by repeated transposition of a single novel gene.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1616},
pmid = {41530154},
issn = {2041-1723},
support = {Start-up//Xiamen University (XMU)/ ; 32300346//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32200411//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270439//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32061160471//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022J06004//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Sex Determination Processes/genetics ; Female ; Male ; *Lancelets/genetics ; *Evolution, Molecular ; CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; Sex Chromosomes/genetics ; RNA, Long Noncoding/genetics ; },
abstract = {Sex determination systems display striking evolutionary flexibility, yet the mechanisms underlying their transitions remain poorly understood. Using newly generated genome assemblies, we investigated the evolving sex-determining system in the amphioxus Branchiostoma belcheri. We identified two female-specific sex-determining regions (SDRs) on chromosome 13, both derived from independent transpositions of the autosomal gene tesD, which shows testis-specific expression in amphioxus species. CRISPR/Cas9 knockout experiments in Branchiostoma floridae confirmed that tesD functions as a male-determination gene, with loss of function producing an all-female phenotype. In B. belcheri, the older SDR (tesDwa) inserted into the coding region of twai, while the younger SDR (tesDwb), flanked by active Zator-1 transposons, inserted into the 3' UTR of vps9c and later translocated to autosomes in ~10% of individuals. Transcriptomic analyses revealed that W-linked tesDwa and tesDwb produce antisense long non-coding RNAs that likely suppress tesD transcription in females, whereas autosomal tesDwb is not expressed and appears non-functional. The insertion sites and co-transcription with host genes suggest promoter hijacking. Together, these findings demonstrate that recurrent transpositions can generate new functional SDRs that coexist with older ones, driving dynamic turnover of sex determination in B. belcheri.},
}

Animals
*Sex Determination Processes/genetics
Female
Male
*Lancelets/genetics
*Evolution, Molecular
CRISPR-Cas Systems
*DNA Transposable Elements/genetics
Sex Chromosomes/genetics
RNA, Long Noncoding/genetics

@article {pmid41500180,
year = {2026},
author = {Sheng, C and Wang, J and Tan, M and Zhang, J and Sun, M and Sun, J and Shao, Y and Tu, J and Zhu, L and Song, X},
title = {Establishment of detection method of chicken infectious anemia virus based on CRISPR/Cas12a system.},
journal = {Research in veterinary science},
volume = {201},
number = {},
pages = {106046},
doi = {10.1016/j.rvsc.2025.106046},
pmid = {41500180},
issn = {1532-2661},
mesh = {*Chicken anemia virus/isolation & purification/genetics ; *CRISPR-Cas Systems ; Animals ; Chickens ; *Poultry Diseases/diagnosis/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; Sensitivity and Specificity ; },
abstract = {Chicken Infectious Anemia Virus (CIAV) causes chicken infectious anemia, characterized by anemia and immune dysfunction. The rapid dissemination of this virus is generating substantial economic consequences for poultry producers. The CRISPR/Cas12a system is widely used for virus detection through crRNA-guided target recognition and the paracrine activity of Cas12a. To enable rapid and highly sensitive detection of Chicken Infectious Anemia Virus (CIAV), a CRISPR-Cas12a-based fluorescence assay was refined. Through optimization of the CRISPR/Cas12a system and integration of enzymatic recombinase amplification (ERA), the assay achieved a detection limit of 1 copy/μL, demonstrating its significant utility for CIAV diagnostics. In addition, a CRISPR/Cas12a lateral flow assay was developed and optimized, achieving a sensitivity of 10^3 copies/μL for the rapid and visual detection of target analytes. This technique exhibits high specificity for CIAV, showing no cross-reactivity with other chicken viruses. Overall, the system enables rapid CIAV detection with cost-effective equipment, making it suitable for virus monitoring.},
}

*Chicken anemia virus/isolation & purification/genetics
*CRISPR-Cas Systems
Animals
Chickens
*Poultry Diseases/diagnosis/virology
*Circoviridae Infections/veterinary/diagnosis/virology
Sensitivity and Specificity

@article {pmid41475537,
year = {2026},
author = {Tang, W and Ma, M and Song, W and Kou, M and Wang, X and Yan, H and Li, C and Zhang, A and Gao, T and Gao, R and Zhang, Y and Li, Q},
title = {An efficient CRISPR/Cas9-mediated editing of phytoene desaturase in hexaploid sweetpotato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {364},
number = {},
pages = {112967},
doi = {10.1016/j.plantsci.2025.112967},
pmid = {41475537},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oxidoreductases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Polyploidy ; Chlorophyll/metabolism ; },
abstract = {CRISPR/Cas9-mediated gene editing has emerged as a pivotal tool for functional genomics and crop improvement. For the first time, we applied CRISPR/Cas9-mediated editing to the IbPDS gene in the purple-fleshed cultivar 'XZS-8', achieving mutation efficiencies up to 98.18 %. Loss-of-function mutations in IbPDS induced visually discernible albino phenotypes. Hi-TOM sequencing confirmed deletion mutations within the target locus, with editing efficiencies ranging from 17.77 % to 65.90 % (gRNA1) and 87.87-98.18 % (gRNA2). Knockout lines showed significant reductions in chlorophyll a and b content, confirming functional disruption of IbPDS. Collectively, our results demonstrate efficient CRISPR/Cas9-mediated genome editing for generating mutants in the hexaploid sweetpotato.},
}

*Ipomoea batatas/genetics/enzymology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Oxidoreductases/genetics/metabolism
*Plant Proteins/genetics/metabolism
Polyploidy
Chlorophyll/metabolism

@article {pmid41420106,
year = {2026},
author = {Ge, J and Hirosue, S and Castillon, L and Patel, SA and Wesolowski, L and Dyas, A and Yong, C and de Haan, S and Drost, J and Stewart, GD and Obenauf, AC and Muñoz-Espín, D and Vanharanta, S},
title = {Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities.},
journal = {EMBO molecular medicine},
volume = {18},
number = {2},
pages = {599-619},
pmid = {41420106},
issn = {1757-4684},
support = {C9685/A25177//Cancer Research UK (CRUK)/ ; C62187/A29760//Cancer Research UK (CRUK)/ ; BRC-1215-20014//NIHR | NIHR Cambridge Biomedical Research Centre (NIHR Cambridge BRC)/ ; 955951//European Commission (EC)/ ; MC_UU_12022/7//UKRI | Medical Research Council (MRC)/ ; RP_033_20170303//Kidney Research UK/ ; 338420//Research Council of Finland (AKA)/ ; },
mesh = {*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; CRISPR-Cas Systems ; *Drug Resistance, Neoplasm ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Epithelial Cells ; },
abstract = {The von Hippel-Lindau tumor suppressor (VHL) is a component of a ubiquitin ligase complex that controls cellular responses to hypoxia. Endogenous VHL is also utilized by proteolysis-targeting chimera (PROTAC) protein degraders, a promising class of anti-cancer agents. VHL is broadly essential for cell proliferation, yet it is a key tumor suppressor in renal cell carcinoma. To understand the functional consequences of VHL loss, and to identify targeted approaches for the elimination of VHL null cells, we have used genome-wide CRISPR-Cas9 screening in human renal epithelial cells. We find that, upon VHL loss, the HIF1A/ARNT complex is the central inhibitor of cellular fitness, suppressing mitochondrial respiration, and that VHL null cells show HIF1A-dependent molecular vulnerabilities that can be targeted pharmacologically. Combined VHL/HIF1A inactivation in breast and esophageal cancer cells can also provide resistance to ARV-771, a VHL-based bromodomain degrader that has anti-cancer activity. HIF1A stabilization can thus provide opportunities for early intervention in neoplastic VHL clones, and the VHL-HIF1A axis may be relevant for the development of resistance to the emerging class of PROTAC-based cancer therapies.},
}

*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics
CRISPR-Cas Systems
*Drug Resistance, Neoplasm
Cell Line, Tumor
*Antineoplastic Agents/pharmacology
Epithelial Cells

@article {pmid41201433,
year = {2026},
author = {Jetley, U and Balwani, I and Sharma, P and Miller, IC and Luther, A and Dutta, I and Saravanan, N and Goel, S and Zhang, Q and Zhang, B and Kilic, O and Liu, B and Han, BW and Liu, D and Schultes, B and Prodeus, A and Zhang, Y},
title = {A differentiated and durable allogeneic strategy applicable to cell therapies.},
journal = {Cytotherapy},
volume = {28},
number = {3},
pages = {101991},
doi = {10.1016/j.jcyt.2025.10.001},
pmid = {41201433},
issn = {1477-2566},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Cell- and Tissue-Based Therapy/methods ; Gene Editing/methods ; Killer Cells, Natural/immunology ; *T-Lymphocytes/immunology ; Transplantation, Homologous ; Cell Differentiation ; *Immunotherapy, Adoptive/methods ; },
abstract = {BACKGROUND AIMS: Autologous T-cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily as the result of their individualized manufacturing requirements.

METHODS: To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select human leukocyte antigens (HLA) class I and II alleles (HLA-A, HLA-B and the class II transactivator [CIITA]) while retaining HLA-C to protect from natural killer (NK) cell rejection.

RESULTS AND CONCLUSION: Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus template allowed for a high localized insertion rate while simultaneously removing the endogenous TCR and preventing graft-versus-host disease. Using an optimized T-cell engineering process involving orthogonal CRISPR/Cas9 cleavage and base editors coupled with lipid nanoparticle delivery, we achieved efficient production of Allo-CAR T cells with high editing rates and cell expansion in a scalable manner. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene-editing approach significantly minimized the occurrence of chromosomal aberrations. This promising allogeneic approach also has been applied to induced pluripotent stem cells (iPSCs) with triple edits targeting HLA-A, HLA-B and CIITA (TKO). Pancreatic progenitor cells or cardiomyocytes derived from TKO iPSCs were protected from host peripheral blood mononuclear cell-mediated rejection when matched for HLA-C, suggesting potential applications in regenerative medicine applications.},
}

Humans
CRISPR-Cas Systems/genetics
*Cell- and Tissue-Based Therapy/methods
Gene Editing/methods
Killer Cells, Natural/immunology
*T-Lymphocytes/immunology
Transplantation, Homologous
Cell Differentiation
*Immunotherapy, Adoptive/methods

@article {pmid39973047,
year = {2026},
author = {Ling, X and Zhou, C and Hong, JF and Jiang, YP and Sun, Q and Li, LY and Wang, SY and Xie, XY and Zou, QL and Yang, XL and Xiang, K and Ma, J and Qiao, L and Chen, B and Sun, W},
title = {Increased versatility and convenience: Advances and strategy optimization of Receptor-Mediated Ovary Transduction of Cargo-mediated genetic modification in insects.},
journal = {Insect science},
volume = {33},
number = {1},
pages = {2-12},
doi = {10.1111/1744-7917.70003},
pmid = {39973047},
issn = {1744-7917},
support = {No.KJZD-K202200507;No.KJQN202200533//Scientific and Technological Research Program of Chongqing Municipal Education Commission/ ; No. cx2022052//Venture and Innovation Support Program for Chongqing Overseas Returnees/ ; No.CYS23399;No.CYS240374//Graduate Research and Innovation Foundation of Chongqing under Grant/ ; No. CSTB2022BSXM-JCX0067//Chongqing "Express" Science and Research Program for PhD/ ; No. 202410637012//College Students' Innovation and Entrepreneurship Training Plan Program/ ; Nos.31772527;31872262//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Ovary/metabolism ; *Gene Editing/methods ; Female ; *Insecta/genetics ; CRISPR-Cas Systems ; },
abstract = {Genetic modification via gene editing has become a widely adopted and demonstrably effective method in functional gene research within entomology. However, the optimal efficiency and simplicity of delivering exogenous guide RNA-clustered regularly interspaced short palindromic repeats-associated protein 9 complexes into target tissues are crucial for successful gene editing. The Receptor-Mediated Ovary Transduction of Cargo (ReMOT) strategy, which simplifies the delivery process, target-site selection, technical requirements, and delivery cost compared with embryonic microinjection, enabling efficient editing at the germline level, is gaining increasing attention. Although the feasibility and advantages of this technique have been demonstrated in various insect species, further optimization of operational details and the overcoming of further bottlenecks are still required. This review focuses on advances in developing ReMOT as a valuable technology, exploring its applicability, rationale for selecting the ovary as a delivery target site, factors influencing its efficiency, and improvement recommendations. The versatility and effectiveness of ReMOT make it a promising method for researchers looking to make precise genetic modifications with greater ease and efficiency.},
}

Animals
Ovary/metabolism
*Gene Editing/methods
Female
*Insecta/genetics
CRISPR-Cas Systems

@article {pmid39829059,
year = {2026},
author = {Shi, W and He, L and Li, R and Cao, J},
title = {Role of mitochondrial complex I genes in host plant expansion of Bactrocera tau (Tephritidae: Diptera) by CRISPR/Cas9 system.},
journal = {Insect science},
volume = {33},
number = {1},
pages = {147-158},
doi = {10.1111/1744-7917.13495},
pmid = {39829059},
issn = {1744-7917},
support = {//National Science Foundation of China/ ; //Science and Technology Program of Yunnan Province/ ; },
mesh = {Animals ; *Tephritidae/genetics/growth & development/physiology ; CRISPR-Cas Systems ; *Electron Transport Complex I/genetics/metabolism ; Larva/growth & development/genetics/physiology ; Pupa/growth & development/genetics/physiology ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Host expansion facilitates tephritid flies to expand their ranges. Unraveling the mechanisms of host expansion will help to efficiently control these pests. Our previous works showed mitochondrial complex I genes Ndufs1, Ndufs3, and Ndufa7 being upregulated during host expansion of Bactrocera tau (Walker), one of the highly hazardous species of tephritids. However, their roles in the host expansion of B. tau remain unknown. Here, using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) editing system for the first time, a stable homozygous Ndufa7 strain (Btndufa7[-/-]), heterozygous Ndufs1 (Btndufs1[+/-]), and Ndufs3 strains (Btndufs3[+/-]) were obtained from F3 generation of B. tau, after gene knockout. Reduced sizes of larvae and pupae of the Ndufa7 knockout strain were first observed. Notably, the mean values of fitness estimation (pupal numbers, single-pupal weight and emergence rate) and Ndufa7 gene expression in the Ndufa7 knockout strain were slightly reduced on 2 native hosts (summer squash and cucumber), while it sharply decreased on the novel host banana and the potential host pitaya, compared with those of the wild-type strain. Furthermore, the Ndufa7 knockout strain did not survive on the novel host guava. These results suggested that Ndufa7 disturbs the survival on native hosts, expansion to novel hosts, and further expansion to potential hosts of B. tau. Homozygous lethality occurred after the knockout of Ndufs1 or Ndufs3, suggesting that these 2 genes play a role in the early development of B. tau. This study revealed that Ndufa7 is a target gene for the management of tephritids and opens a new avenue for pest control research.},
}

Animals
*Tephritidae/genetics/growth & development/physiology
CRISPR-Cas Systems
*Electron Transport Complex I/genetics/metabolism
Larva/growth & development/genetics/physiology
Pupa/growth & development/genetics/physiology
*Insect Proteins/genetics/metabolism
Gene Knockout Techniques

@article {pmid41676858,
year = {2026},
author = {Niu, Y and Wu, S and Su, J},
title = {Harnessing CRISPR/Cas systems for food safety detection: biosensor design and emerging applications for food safety detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01791b},
pmid = {41676858},
issn = {1759-9679},
abstract = {Food safety has become a critical global concern, with foodborne diseases affecting approximately 600 million people annually and causing 420 000 deaths each year, posing significant risks to human health and well-being. Rapid, efficient, and reliable detection methods are essential to mitigate these risks. Traditional detection methods, such as PCR and culture-based assays, while widely used, often face challenges related to speed, accuracy, and portability. Over the past 5 years (2020-2025), the (CRISPR)/Cas system has emerged as a powerful tool for food safety detection due to its high sensitivity, specificity, and versatility. This review highlights recent advances in CRISPR/Cas-based biosensors and their applications in food safety. First, we discuss the key challenges in food safety detection and the design principles of CRISPR/Cas biosensors. Next, we comprehensively summarize their applications in detecting foodborne pathogens (viruses and bacteria), food fraud, genetically modified organisms (GMOs), toxins, heavy metals, antibiotic residues, and pesticides. Finally, we address the current limitations and future prospects of CRISPR/Cas biosensors, providing insights into their potential for next-generation food safety solutions.},
}

@article {pmid41674784,
year = {2025},
author = {Yashooa, RK and Nabi, AQ and Smail, SW and Azeez, SS and Nooh, WA and Mustafa, SA and Al-Farha, AA and Capitanio, N and Shekha, MS},
title = {CRISPR-Cas technologies in neurodegenerative disorders: mechanistic insights, therapeutic potential, and translational challenges.},
journal = {Frontiers in neurology},
volume = {16},
number = {},
pages = {1737468},
pmid = {41674784},
issn = {1664-2295},
abstract = {CRISPR-Cas genome-editing technologies have emerged as powerful tools for precise DNA and RNA modulation, offering promising therapeutic strategies for neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). This review critically evaluates current CRISPR/Cas applications in neurodegeneration, with emphasis on mechanistic insights, therapeutic outcomes, and translational feasibility. Preclinical and early translational studies demonstrate that CRISPR-Cas platforms can correct pathogenic mutations, suppress toxic gene expression, and restore neuronal function. Advanced modalities, including base and prime editing, CRISPRi/a, and RNA-targeting Cas systems, improve precision and reduce genomic damage, which is particularly advantageous in post-mitotic neurons. Emerging CRISPR-based diagnostics (e.g., SHERLOCK and DETECTR), AI-assisted sgRNA design, and machine-learning approaches for predicting off-target effects further enhance the safety, stratification, and monitoring of CRISPR therapeutics. In parallel, patient-derived brain organoids and assembloids provide scalable human-relevant platforms for mechanistic studies and preclinical validation. Despite this progress, major challenges remain, including efficient delivery across the blood-brain barrier, immune responses, long-term safety, and ethical and regulatory considerations. Overall, CRISPR-Cas technologies hold strong potential as disease-modifying interventions for neurodegenerative disorders, provided that advances in delivery systems, artificial intelligence integration, and regulatory oversight continue to evolve toward clinical translation.},
}

@article {pmid41674724,
year = {2026},
author = {Baum, R and Telugu, N and Bruyneel, AAN and Kay, M and Nair, P and Perea-Gil, I and Termglinchan, V and Bharucha, N and Lee, E and Mercola, M and Diecke, S and Karakikes, I},
title = {CRISPR-MiX: A pooled single-stranded donor strategy to enhance HDR efficiency in human iPSCs.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102820},
pmid = {41674724},
issn = {2162-2531},
abstract = {CRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in iPSCs remains low and is highly dependent on the target locus. Here, we developed CRISPR-MiX, an improved protocol to enhance HDR efficiency in human iPSCs. Using a GFP-to-BFP reporter system, we identified key single-stranded oligodeoxynucleotide (ssODN) donor design parameters, including homology arm symmetry, CRISPR/Cas-blocking mutations, and strand complementarity, which significantly influence HDR outcomes. We applied this approach to introduce pathogenic variants into five genes related to genetic cardiomyopathies. Quantitative analysis of HDR events showed that both the target locus and ssODN design strongly affect HDR efficiency. To address the locus- and design-specific limitations, we established CRISPR-MiX, a pooled ssODN-based method for scarless genome editing using ribonucleoproteins (RNPs) that does not require selection. CRISPR-MiX consistently improved HDR efficiency across multiple loci. This strategy offers a simple, robust, and versatile approach for precise genome engineering in iPSCs, supporting broad applications in disease modeling and functional genomics.},
}

@article {pmid41674468,
year = {2026},
author = {Takeda, T and Aso, M and Ueda, H and Yuzawa, S},
title = {Direct Pathway Synthesis and Editing (DiPaSE): A One-Pot DNA Assembly Method for Accurate and Efficient Refactoring of High-GC Biosynthetic Gene Clusters.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00934},
pmid = {41674468},
issn = {2161-5063},
abstract = {Natural products (NPs) produced by actinobacteria, particularly Streptomyces species, represent a rich source of bioactive compounds and have yielded many clinically important compounds. Actinobacterial genomes are characterized by high GC content and typically harbor 20-40 biosynthetic gene clusters (BGCs) per genome, which encode diverse NPs such as polyketides, peptides, and glycosides. CRISPR/Cas-based genome editing has emerged as a promising tool to activate silent BGCs and engineer NP biosynthesis. However, the efficiency of multiplex editing drastically decreases as the number of targeted sites increases. Here, we report a novel one-pot DNA assembly method, termed direct pathway synthesis and editing (DiPaSE), for the efficient synthesis and multiplex editing of long, high-GC BGCs. DiPaSE accurately assembles multiple high-GC DNA fragments up to 60 kb and enables simultaneous deletions and insertions within a target BGC without compromising the assembly efficiency. Using this approach, we identified functions of previously uncharacterized genes in the aureothin BGC and significantly enhanced the titer of the corresponding NP. The workflow employs conventional polymerase chain reaction, type IIP restriction enzymes, commercially available DNA assembly reagents, and Escherichia coli, providing a simple, cost-effective, and broadly applicable platform for genome mining, BGC refactoring, and rational design of artificial biosynthetic pathways.},
}

@article {pmid41674382,
year = {2026},
author = {Zhang, Y and Xu, Y and Ding, Z and Cheng, Y and Gao, Y and Ye, J and Zhang, H and Li, P and Wu, H},
title = {Machine learning-optimized long single-stranded DNA synthesis technology empowers high-precision diagnostic-therapeutic integration in living cells.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41674382},
issn = {1362-4962},
support = {2021YFC2100600//National Key Research and Development Program of China/ ; 31872608//National Natural Science Foundation of China/ ; 31872608//National Natural Science Foundation of China/ ; },
mesh = {*DNA, Single-Stranded/chemistry/genetics/chemical synthesis/biosynthesis ; Humans ; *Machine Learning ; Gene Editing/methods ; Nanotechnology/methods ; DNA-Directed DNA Polymerase/metabolism ; CRISPR-Cas Systems ; },
abstract = {While DNA nanotechnology holds transformative potential across biomedical and information storage applications, current technologies face critical limitations in synthesizing long single-stranded DNA (ssDNA) with high purity and homogeneity. To address these challenges, we developed Ouroborosyn-ssDNA, a nicking enzymatic assisted replication (NEAR) platform that synergizes enzymatic engineering with computational optimization. By integrating phi29 DNA polymerase and Nb.BbvCI nickase in formate-based buffers, we achieved extended ssDNA synthesis up to 15 000 nt while preserving sequence fidelity, resulting in a 4.73-fold yield enhancement compared to commercial buffers. Notably, machine learning-guided parameter optimization identified magnesium ion dynamics and thermal modulation as pivotal determinants of enzymatic efficiency. Furthermore, solid-phase synthesis using thiol-gold immobilized templates demonstrated 86.38% purification recovery via automated magnetic bead systems, enabling scalable production. To validate functional utility, we engineered six-helix bundle DNA origami-CRISPR complexes that achieved nucleolin-targeted genome editing in cervical cancer cells, coupling GFP-based diagnostics with therapeutic E7 oncogene disruption. These advancements directly overcome key limitations in enzymatic stochasticity and product heterogeneity through buffer engineering and computational optimization, establishing a scalable pathway for applications in precision nanomedicine, synthetic biology, and molecular data storage. This integrated strategy advances DNA nanotechnology from proof-of-concept studies toward standardized biomanufacturing of sequence-defined macromolecular architectures.},
}

*DNA, Single-Stranded/chemistry/genetics/chemical synthesis/biosynthesis
Humans
*Machine Learning
Gene Editing/methods
Nanotechnology/methods
DNA-Directed DNA Polymerase/metabolism
CRISPR-Cas Systems

@article {pmid41674223,
year = {2026},
author = {Goudar, P and Hall, A},
title = {The influence of GMO media strategies on public perceptions of CRISPR crop technologies in Southern Ontario.},
journal = {GM crops & food},
volume = {17},
number = {1},
pages = {2620131},
doi = {10.1080/21645698.2026.2620131},
pmid = {41674223},
issn = {2164-5701},
mesh = {Ontario ; *Public Opinion ; *Plants, Genetically Modified/genetics ; Humans ; Gene Editing ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Adult ; Surveys and Questionnaires ; Male ; Female ; Middle Aged ; },
abstract = {Genetically modified organisms (GMOs) have often divided public opinion, one factor influencing perceptions of GMO technologies has been misunderstood or poorly communicated scientific messaging. However, advancements in gene editing tools like CRISPR/Cas9 offer new crop modification possibilities, prompting different regulatory frameworks than traditional GMO technologies. This research examines public understanding of GMOs, awareness of CRISPR crops, and how prior experiences with GMOs shape perceptions of new genetic technologies. A mixed-methods approach was employed, combining a public survey of adults in the Greater Toronto-Hamilton area and interviews with science journalists. Results show hesitance toward GMOs and CRISPR crops, with acceptance most impacted by consumer behavior and cost. Key interview themes include journalist's concerns about levels of public education, the role of social media, and the cost of goods. Our findings suggest increased transparency and effective communication could improve public acceptance of GMOs and CRISPR crops. While CRISPR crops do not come under the GMO regulatory framework in Canada, our findings show that the public does not recognize this distinction. Without increased transparency and more effective communication CRISPR crops may become widely associated with the negative media frames that have shaped perceptions of GMOs.},
}

Ontario
*Public Opinion
*Plants, Genetically Modified/genetics
Humans
Gene Editing
*Crops, Agricultural/genetics
*CRISPR-Cas Systems
Adult
Surveys and Questionnaires
Male
Female
Middle Aged

@article {pmid41674084,
year = {2026},
author = {Ghosh, PK and Biswas, S and Malaker, R and Pham, H and Septiningsih, EM and Ravelombola, W},
title = {Optimization of CRISPR/Cas9-mediated CtPDS knockout in guar protoplasts.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70177},
doi = {10.1002/tpg2.70177},
pmid = {41674084},
issn = {1940-3372},
support = {//Texas A&M Institute for Advancing Health through Agriculture/ ; //Texas A&M Advancing Discovery to Market/ ; GSC2024075//Texas Department of Agriculture/ ; 58-6046-2-008//USDA-ARS Germplasm Evaluation/ ; 58-6046-3-005//USDA-ARS Germplasm Evaluation/ ; },
mesh = {*CRISPR-Cas Systems ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Cyamopsis/genetics ; *Plant Proteins/genetics ; Gene Knockout Techniques/methods ; },
abstract = {Guar (Cyamopsis tetragonoloba L. Taub.) is a climate-resilient legume with industrial and agricultural applications. Recently, gene editing has emerged as a key genetic tool for crop improvement. Despite its recent increasing value as a commodity for various uses, there is no documented report of gene editing work in guar to date. In this study, we present the first optimized protocol for protoplast-based clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing in guar. The most intact and viable protoplasts were observed in the cotyledons of 6-day-old seedlings that were isolated using the tape-sandwich method. Enzymatic digestion with 1.5% cellulase RS and 0.3% pectinase in 600 mM mannitol, followed by 10 min of vacuum infiltration, increased protoplast release and viability. Polyethylene glycol (PEG)-mediated transformation had been tailored using 40% PEG for 5 min with green fluorescent protein plasmid, and the cestrum yellow leaf curling virus promoter at room temperature showed the highest transient expression efficiency (∼57%). A multiplexed CRISPR/Cas9 construct was designed to target the Cyamopsis tetragonoloba phytoene desaturase (CtPDS) gene in guar. Polymerase chain reaction amplification and Sanger sequencing of transfected protoplasts confirmed highly efficient editing, with fragment deletions ranging from 714 to 1061 bp in CtPDS. Overall, we achieved 100% editing efficiency, as all successfully transformed samples showed CRISPR/Cas9-induced mutations. These findings establish a reliable, transient protoplast system for functional genomics and targeted trait improvement in guar, providing a key foundation for future crop improvement.},
}

*CRISPR-Cas Systems
*Protoplasts/metabolism
*Gene Editing/methods
*Cyamopsis/genetics
*Plant Proteins/genetics
Gene Knockout Techniques/methods

@article {pmid41673772,
year = {2026},
author = {Gao, H and Li, B and Fengler, K and Yang, M and Schroder, M and Rahe, M and Sanyour-Doyel, N and Qi, J and LIaca, V and Beatty, M and Hu, WN and Barrett, B and Norman, B and Mo, H and Leonard, A and Wilson, B and Meeley, RB and Perugini, L and Krishnamurthy, N and Habben, JE and Tabor, G},
title = {Genome-Edited Maize Expressing Two Native Genes Confers Broad-Spectrum Resistance to Northern Corn Leaf Blight.},
journal = {Molecular plant pathology},
volume = {27},
number = {2},
pages = {e70205},
pmid = {41673772},
issn = {1364-3703},
mesh = {*Zea mays/genetics/microbiology ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Plant Leaves/microbiology/genetics ; *Genes, Plant ; Ascomycota/pathogenicity ; },
abstract = {Northern corn leaf blight (NCLB) can result in yield losses of up to 50% in maize. The most effective strategy for managing NCLB is the deployment of resistant hybrids. Conventional breeding methods typically require 6 or 7 backcross generations to introgress a resistance locus, often bringing along undesirable traits that reduce yield. Recent advances in genome editing offer a precise alternative, enabling the targeted incorporation of resistance genes without linkage drag. In this study, we identified an NCLB resistance gene, NLB18-R, that is allelic to Htn1 and Ht2/Ht3. Using CRISPR-Cas9, we replaced the susceptible allele (NLB18-S) with NLB18-R in an elite inbred, resulting in enhanced resistance to NCLB. In a parallel experiment, we inserted both NLB18-R and the resistance gene Ht1-R into preselected, closely linked sites on chromosome 1. Through genetic crossing, we combined these edits into a stack. The resulting genome-edited plants exhibited resistance to Setosphaeria turcica races 0, 1 and 23N. Field trials under disease-free conditions showed no significant yield differences between hybrids carrying NLB18-R, Ht1-R, or the stack compared to null and wild-type controls. These findings demonstrate that CRISPR-Cas9-mediated genome editing is a powerful tool for rapidly developing commercial-grade maize hybrids with broad-spectrum resistance to NCLB, and potentially other diseases.},
}

*Zea mays/genetics/microbiology
*Gene Editing/methods
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
Plants, Genetically Modified
CRISPR-Cas Systems/genetics
*Plant Leaves/microbiology/genetics
*Genes, Plant
Ascomycota/pathogenicity

@article {pmid41673677,
year = {2026},
author = {Baena, JC and Cabrera-Salcedo, SC and Carrera Suárez, Y and Biancha-Vasco, JM and Rios-Serna, LJ and García-Mantilla, MD and Estrada-Schweineberg, M and Victoria Hincapie, JS and Toro-Pedroza, A and Garcia-Robledo, JE and Cañas, CA and Ortiz-Guzman, J and Loukanov, A},
title = {The avatar principle: exosomal dynamics guiding tumor adaptation and next-generation therapeutic strategies.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {159},
pmid = {41673677},
issn = {1477-3155},
mesh = {*Exosomes/metabolism ; Humans ; *Neoplasms/therapy/immunology ; Tumor Microenvironment ; Immunotherapy/methods ; Animals ; Receptors, Chimeric Antigen ; Avatar ; },
abstract = {Exosomes are nanoscale extracellular vesicles that transfer proteins, nucleic acids, and lipids, reflecting the state of their parent cells. A persistent scientific challenge is that tumor-derived exosomes (TDEs) facilitate immune evasion, remodel the tumor microenvironment, and create premetastatic niches, intensifying tumor aggressiveness and undermining therapeutic efficacy, ultimately narrowing treatment options to palliative strategies in advanced settings. Yet their dual roles as suppressive agents and potential therapeutic tools remain poorly integrated within current cancer immunotherapy frameworks. This review examines the molecular mechanisms underlying TDE-mediated immune suppression and therapeutic resistance, while also highlighting engineering strategies to exploit or counteract exosome biology. Exosomes derived from chimeric antigen receptor (CAR) T cells preserve antigen specificity and cytotoxic components without the risks of uncontrolled proliferation or cytokine release, offering a safer class of cell free immunotherapies. Advances in genetic engineering, hybrid vesicle design, and nanotechnology have extended exosome applications to the delivery of CRISPR/Cas systems, chemotherapeutic agents, immunoregulatory RNAs, and vaccines, with liposome or nanoparticle integration enhancing targeting and efficacy. Remaining obstacles include the lack of standardized protocols, scalability issues in production, and unresolved regulatory frameworks. Drawing on The Art of War, exosomes can be envisioned as avatars of strategy, discreet messengers capable of undermining host defenses while simultaneously carrying the potential to redirect immunity against the tumor. By embodying both deception and counterattack, they illustrate the capacity to penetrate hidden barriers and redefine the therapeutic battlefield, opening new horizons for precision cancer immunotherapy.},
}

*Exosomes/metabolism
Humans
*Neoplasms/therapy/immunology
Tumor Microenvironment
Immunotherapy/methods
Animals
Receptors, Chimeric Antigen
Avatar

@article {pmid41673558,
year = {2026},
author = {Sun, X and Wu, F and Ma, Z and Liang, G and Chen, S and Hu, X and Fan, S and Zhao, Y},
title = {Systematic synthesis of CRISPR/Cas applications for enhancing salt tolerance in crops: a decade of progress and challenges.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08295-2},
pmid = {41673558},
issn = {1471-2229},
support = {42320104006//National Natural Science Foundation of China for International Cooperation/ ; },
abstract = {Soil salinity is a major constraint on global crop productivity, driving the need for salt-tolerant varieties. While CRISPR-Cas genome editing offers targeted solutions for trait improvement, significant biological and technical bottlenecks limit its application in conferring salt stress resilience. This systematic summarizes findings from 83 peer-reviewed studies (2015-2024) employing CRISPR/Cas technologies to improve salt tolerance in five major crops (rice, wheat, maize, sorghum, barley). Our systematic review reveals that early single-gene edits achieved modest gains (30-50% Na[+] exclusion) but often showed limited yield gains in field settings, potentially due to compensatory regulation and environmental variation. The literature suggests that multiplex designs spanning ion homeostasis, osmoprotection, and ROS management can improve salt-tolerance outcomes and help maintain yield under severe salinity; however, the magnitude of benefit varies with crop, genotype, and transformation/regeneration context. Protein-protein interaction networks identified 12 hub genes and three functional modules, highlighting SOS3 and MPK6 as critical bottlenecks whose disruption risks pleiotropic effects. Spatial expression analysis underscored tissue-specific trade-offs, constitutive editing of root-dominant genes in shoots reduced yields by 15-28%, while tissue-optimized promoters minimized physiological conflicts. Persistent challenges include genotype-dependent transformation inefficiencies, epigenetic drift and environmental interactions under salt stress. Collectively, our synthesis consolidates and refines current best practices for salt-tolerance genome editing and highlights major bottlenecks-particularly regeneration/transformability, genotype dependence, and epigenetic constraints-that should be explicitly considered in experimental design and reporting.},
}

@article {pmid41673492,
year = {2026},
author = {Jiang, Y and Hodgson, KJ and Segos, I and Lambie, EJ and Yang, L and Pan, M and Greig, A and Conradt, B},
title = {Tagging of C. elegans apoptosis activator EGL-1 BH3-only reveals CED-9 BCL-2-dependent mitochondrial localization and dynamic control of EGL-1 synthesis and degradation in vivo.},
journal = {Cell death and differentiation},
volume = {},
number = {},
pages = {},
pmid = {41673492},
issn = {1476-5403},
support = {BB/V007572/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V015648/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; C0204/10-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; RSWF\R1\180008//Royal Society/ ; PhD stipend//China Scholarship Council (CSC)/ ; },
abstract = {The BH3-only protein EGL-1 is the key activator of apoptosis during C. elegans development. EGL-1 protein is thought to be synthesized predominantly in cells programmed to die and to localize to mitochondria. We used CRISPR-Cas-mediated modification of the egl-1 locus to add the coding sequence for the monomeric StayGold fluorescent protein or 18 copies of the SunTag peptide to the endogenous open reading frame. We found that tagged EGL-1 protein colocalizes with mitochondria in vivo and that mitochondrial localization is dependent on the anti-apoptotic BCL-2-like protein CED-9. Consistent with the presence of egl-1 mRNA in cells programmed to die as well as their progenitor cells ('mother' cells), EGL-1 protein is detected in both types of cells in vivo. Furthermore, real time imaging reveals that EGL-1 protein rapidly disappears from the mother cell prior to its division and that EGL-1 protein rapidly reappears specifically in the daughter cell programmed to die. Our results demonstrate CED-9 BCL-2-dependent mitochondrial localization of EGL-1 BH3-only protein and dynamic control of EGL-1 protein synthesis and degradation. Furthermore, we have identified additional levels of control of egl-1 BH3-only function that expand our understanding of apoptosis activation in vivo.},
}

@article {pmid41671593,
year = {2026},
author = {Fattahi, M and Pirbadami, F and Motallebirad, T and Beheshti, Z and Azadi, D},
title = {Next-generation gene therapy for infectious disease: Advances, challenges, and future directions.},
journal = {Journal of infection and public health},
volume = {19},
number = {4},
pages = {103164},
doi = {10.1016/j.jiph.2026.103164},
pmid = {41671593},
issn = {1876-035X},
abstract = {Infectious diseases, worsened by antimicrobial resistance and limitations of conventional treatments, demand innovative solutions. This systematic review evaluates recent advances in next-generation gene therapy for HIV, HBV, HPV, and multidrug-resistant(MDR) pathogens, while addressing key challenges and future directions. Following PRISMA guidelines, major databases were searched (2015-2025), yielding 1250 records. the114 peer-reviewed studies were included, assessed using the MMAT, and thematically synthesized for therapeutic strategies, efficacy, challenges, and prospects. Findings show CRISPR-based genome editing achieved HIV remission in preclinical and early clinical trials, engineered bacteriophages demonstrated strong efficacy against MDR bacteria, RNAi effectively silenced HBV, and phage-derived depolymerases reduced MDR biofilms. Adverse events included off-target effects, immunogenicity, and scalability issues. Efficacy varied, with CRISPR offering high specificity and phage therapies showing robust lysis. In conclusion, Gene therapy shows strong potential against resistant pathogens, but faces challenges like study heterogeneity, preclinical reliance, delivery barriers, and unequal access in low-and middle-income countries.},
}

@article {pmid41649152,
year = {2026},
author = {Gallo, D and Bes, M and Mounier, T and Calatayud, C and Meunier, AC and Périn, C},
title = {A Streamlined CRISPRa Architecture with Dual Pol II/Pol III Promoter and Optimized scRNA Enables Robust and Tunable Gene Activation.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {36-48},
doi = {10.1177/25731599251408333},
pmid = {41649152},
issn = {2573-1602},
mesh = {*Promoter Regions, Genetic ; Oryza/genetics ; *RNA Polymerase II/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA Polymerase III/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Gene Expression Regulation, Plant ; },
abstract = {CRISPR activation (CRISPRa) offers a powerful approach to upregulate endogenous genes; yet, existing systems in plants can be complex or difficult to integrate with CRISPR interference (CRISPRi). Here, we present a streamlined and flexible CRISPRa platform that enables robust gene activation. Using a dual-luciferase reporter, we benchmarked a range of guide RNA scaffolds, effector proteins, and promoters. We developed a novel single-guide RNA (sgRNA) architecture, harboring two MS2 aptamers inserted into the tetraloop and driven by a composite Pol II/Pol III promoter, as the most efficient configuration. This scaffold outperformed gR2.0- and SunTag-based constructs, reaching up to 100-fold activation of a minimal 35S promoter and up to 215-fold induction of three endogenous rice genes in protoplast assays. In contrast, scaffold RNAs (scRNAs) with aptamers at the 3' end or in excessive copy numbers were ineffective. Exploratory AlphaFold modeling supports a possible role for aptamer positioning and MCP-VP64 dimerization, although this remains a working hypothesis. This modular design enables tunable gene regulation in rice protoplasts and provides a practical platform for high-throughput screening and synthetic gene circuit prototyping in plants. Given that scRNA geometry and promoter architecture are universal features of CRISPR-based transcriptional modulation, the system is expected to be broadly portable across species. While the architecture is intended to be compatible with CRISPRi, future studies will be needed to establish its practical use in combined CRISPRa/i settings.},
}

*Promoter Regions, Genetic
Oryza/genetics
*RNA Polymerase II/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*RNA Polymerase III/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Transcriptional Activation
Gene Expression Regulation, Plant

@article {pmid41468058,
year = {2026},
author = {Vasileva, A and Abramova, M and Selkova, P and Arseniev, A and Musharova, O and Malysheva, P and Demkina, A and Khodorkovskii, M and Severinov, K},
title = {Streptococcus uberis Cas9-A Compact Type II-A Nuclease Recognizing a Unique PAM and Functional in Human Cells.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {21-35},
doi = {10.1177/25731599251404417},
pmid = {41468058},
issn = {2573-1602},
mesh = {Humans ; *Streptococcus/genetics/enzymology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/metabolism/genetics ; Animals ; *Bacterial Proteins/genetics/metabolism ; Cattle ; },
abstract = {Several type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 RNA-guided nucleases are commonly used for genome engineering. Their relatively large size and requirements for specific protospacer adjacent motif (PAM) sequences flanking their targets prompt continuous searches for additional more compact Cas9 enzymes with new PAM specificities. Here, we present SuCas9, a compact nuclease from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. SuCas9 thus expands the available genome editing toolset and may find biotechnological and medicinal applications in the future.},
}

Humans
*Streptococcus/genetics/enzymology
*CRISPR-Cas Systems
Gene Editing/methods
*CRISPR-Associated Protein 9/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems/genetics
*Endonucleases/metabolism/genetics
Animals
*Bacterial Proteins/genetics/metabolism
Cattle

@article {pmid41338874,
year = {2026},
author = {Villegas, NK and Tran, MH and Keller, A and Plesa, C},
title = {BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {9-20},
doi = {10.1177/25731599251401526},
pmid = {41338874},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; *Genes, Synthetic/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Modern gene synthesis platforms enable investigations of protein function and genome biology at an unprecedented scale. Yet, the proportion of error-free constructs in diverse gene libraries decreases with length due to the propagation of oligo synthesis errors. To rescue these error-free constructs, we developed Barcode-Assisted Retrieval CRISPR-Activated Targeting (BAR-CAT), an in vitro method that uses multiplexed dCas9-single-guide RNA (sgRNA) complexes to extract barcodes corresponding to error-free constructs. After a 15-min incubation and wash regimen, three low-bundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to a 384-gene DropSynth gene library, BAR-CAT enriched 12 targets up to 122-fold and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library
*Genes, Synthetic/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing/methods
Plasmids/genetics

@article {pmid41671414,
year = {2026},
author = {Yu, J and Park, JC and Uhm, H and Kim, YW and Im, HW and Bae, S},
title = {Evolution of Prime Editing: Enhancing Efficiency and Expanding Capacity.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e21015},
doi = {10.1002/advs.202521015},
pmid = {41671414},
issn = {2198-3844},
support = {RS-2024-00332601//Korean Fund for Regenerative Medicine/ ; 25202MFDS003//Ministry of Food and Drug Safety/ ; 25B-001-0700//SNUH Lee Kun-hee Child Cancer and Rare Disease/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2024-00404132//Ministry of Health & Welfare/ ; },
abstract = {Genetic mutations cause approximately 80% of rare human diseases, highlighting the urgent need for precise genome editing. Since clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) nucleases were first used for genome editing in 2012, genome editing technologies have rapidly advanced. Base editors, derived from the CRISPR-Cas system, were developed to introduce specific point mutations without requiring DNA double-strand breaks, and subsequently, prime editing (PE) technology was created to enable insertions, deletions, and all types of point mutations. The precision and versatility of PE make it a promising tool for clinical applications. However, PE has potential limitations, including low editing efficiency and limited capacity for large-scale manipulation. To overcome these limitations, research has been continuously conducted to improve PE efficiency and expand its capabilities. Therefore, this review aims to highlight current efforts and future directions for developing and improving PE-related tools.},
}

@article {pmid41671402,
year = {2026},
author = {Shi, M and Ge, W and Li, C and Liu, B and Deng, X and Liu, C and Zheng, M and Zhang, P and Li, L and Guo, Y and Han, Y and Yang, Y and Yu, YV and Jin, YN},
title = {Versatile CRISPR-Cas Tools for Gene Regulation in Zebrafish via an Enhanced Q Binary System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11485},
doi = {10.1002/advs.202511485},
pmid = {41671402},
issn = {2198-3844},
support = {32070832//National Natural Science Foundation of China/ ; 32150610476//National Natural Science Foundation of China/ ; 2042022dx0003//Fundamental Research Funds for the Central Universities/ ; },
abstract = {CRISPR-Cas systems revolutionize gene regulation across diverse organisms, including zebrafish. However, most zebrafish studies still rely on transient delivery of CRISPR components, with limited use of transgenic models, primarily restricted to Cas9-mediated knockouts. This limitation arises from challenges in achieving sustained, tissue-specific, and efficient expression of transgenic CRISPR effectors. To address these challenges, we introduce CRISPR-Q, a transgenic system that combines the QFvpr/QUAS binary expression platform with CRISPR-Cas technologies. CRISPR-Q overcomes the drawbacks of transient mRNA or protein delivery and circumvents the toxicity and transgene silencing issues associated with other binary systems, such as Gal4/UAS. The system enables robust and spatiotemporal expression of CasRx or dCas9vpr, allowing precise transcript knockdown (CRISPR-QKD) or gene activation (CRISPR-Qa). Using CRISPR-QKD, we achieve effective knockdown of smn1 and simultaneous knockdown of tardbp and tardbpl, modeling spinal muscular atrophy and amyotrophic lateral sclerosis, respectively. CRISPR-Qa activates endogenous lin28a and sox9b, demonstrating its functional versatility. We further validate CRISPR-Q's tissue-specific applicability in heart-specific transgenic zebrafish. Together, CRISPR-Q represents a robust and versatile platform for studying gene function and modeling human diseases in zebrafish, with broad potential for adaptation in other model organisms.},
}

@article {pmid41670257,
year = {2026},
author = {Vollmer, SK and Stetter, MG and Hensel, G},
title = {First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70590},
pmid = {41670257},
issn = {1467-7652},
support = {EXC-2048/1 project ID 390686111//Deutsche Forschungsgemeinschaft/ ; STE 2654/4//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Grain amaranth is a nutritionally rich, stress-tolerant C4 dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.},
}

@article {pmid41669806,
year = {2026},
author = {Wang, ZH and Yang, ZQ},
title = {Advances in site-specific knock-in techniques for gene editing.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {2},
pages = {128-141},
doi = {10.16288/j.yczz.25-076},
pmid = {41669806},
issn = {0253-9772},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Animals ; DNA Nucleotidyltransferases ; },
abstract = {Gene-targeted knock-in technology serves as a cornerstone tool in genetic engineering and gene therapy, designed to circumvent the unpredictability and heterogeneityassociated with conventional random integration methods. However, its practical application has long been constrained by off-target activity and low efficiency during the editing process. Recent advances in site-specific recombinase systems (e.g., Bxb1 integrase) and programmable nuclease systems (e.g., CRISPR/Cas9) have significantly enhanced the precision and efficiency of gene knock-in. Notably, the Cas9-Bxb1 integrase system enables targeted integration of large DNA fragments (5-43 kb) into genomic safe harbor (GSH) sites, offering a transformative platform for disease modeling, functional genomics, and clinical therapeutics. This review systematically summarizes the progress of site-specific recombinase and nuclease systems, discusses GSH screening strategies and the role of multi-omics data in optimizing predictive models, and compares the strengths and limitations of twinPE+Bxb1 and PASTE systems. Future research should focus on developing novel integrases with low off-target activity, refining DSB-free editing technologies, and establishing cross-species GSH databases to advance applications in precision medicine and synthetic biology.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Animals
DNA Nucleotidyltransferases

@article {pmid41669364,
year = {2025},
author = {Elkonin, LA and Gerashchenkov, GA and Borisenko, NV and Sarsenova, SK and Panin, VM},
title = {Study of the progeny of sorghum mutants obtained using the CRISPR/Cas9 genetic construct directed at inducing mutations in the α-kafirin k1C5 gene.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {8},
pages = {1161-1168},
doi = {10.18699/vjgb-25-122},
pmid = {41669364},
issn = {2500-0462},
abstract = {Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T0 generation and their T1 progeny. It was revealed that four of the six studied T0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3' from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T2 and T3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4-15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T2 and T3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84-93 %, p <0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.},
}

@article {pmid41666229,
year = {2026},
author = {Fan, X and Lyu, S and Fan, W and Shu, J and Cheng, X},
title = {Precision targeting: The dawn of artificially customized disease resistance.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013942},
pmid = {41666229},
issn = {1553-7374},
mesh = {*Plant Diseases/immunology/prevention & control/genetics ; *Disease Resistance/genetics/immunology ; *Plant Immunity/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/immunology ; *Crops, Agricultural/genetics/immunology ; },
abstract = {Advanced plant disease management strategies are essential to sustainable agriculture and global food security. Advances in plant immunity have given rise to a variety of innovative disease control strategies, such as NLR gene transfer, RNA silencing technology, and CRISPR/Cas9-based gene disruption, as well as the use of immunity inducers. Recently, several novel resistance strategies, including the bioengineering of immunoreceptors, protease-triggered resistance design, and the sentinel approach, have enabled the customized development of disease resistance traits. These new approaches envisage a new paradigm of precision-targeted, artificially engineered resistance to enhance crop protection.},
}

*Plant Diseases/immunology/prevention & control/genetics
*Disease Resistance/genetics/immunology
*Plant Immunity/genetics
CRISPR-Cas Systems
Plants, Genetically Modified/genetics/immunology
*Crops, Agricultural/genetics/immunology

@article {pmid41665762,
year = {2026},
author = {Farooq, M and Khan, A and Hassan, A and Shah, MM},
title = {Advances in CRISPR/Cas systems for engineering abiotic stress tolerance in plants: mechanisms and future prospects.},
journal = {Planta},
volume = {263},
number = {3},
pages = {72},
pmid = {41665762},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; *Crops, Agricultural/genetics/physiology ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Droughts ; },
abstract = {Abiotic stress factors such as drought, salinity, extreme temperatures, and oxidative stress significantly limit crop productivity and threaten global food security. Traditional breeding and transgenic approaches have been employed to enhance stress tolerance, but they are often time-consuming and face regulatory hurdles. The advent of CRISPR/Cas genome editing technology has revolutionized plant genetic engineering by enabling precise modifications to stress-responsive genes. This review explores recent advancements in CRISPR/Cas-based genome editing for improving abiotic stress resilience in crops. We discuss the mechanisms of CRISPR/Cas systems, their applications in stress tolerance, and emerging approaches such as multiplex genome editing, base editing, and AI-assisted CRISPR. Furthermore, we highlight challenges, ethical considerations, and future directions for integrating CRISPR into agricultural biotechnology. This review underscores the potential of CRISPR-based strategies in developing climate-resilient crops to ensure sustainable food production in the face of global climate change.},
}

*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Stress, Physiological/genetics
*Crops, Agricultural/genetics/physiology
Plants, Genetically Modified/genetics
Genetic Engineering/methods
Droughts

@article {pmid41664621,
year = {2026},
author = {Ullah, Q and Haider, W and Zeshan, M and Waqar, M and Arshad, MT and Parveen, H and Mukhtar, S and Zahir, A},
title = {Advancing climate adaptation in saffron through CRISPR-based modulation of stress tolerance and photoperiodic flowering control.},
journal = {GM crops & food},
volume = {17},
number = {1},
pages = {2626180},
pmid = {41664621},
issn = {2164-5701},
mesh = {*Flowers/genetics/physiology/growth & development ; *Crocus/genetics/physiology/growth & development ; Gene Editing ; *CRISPR-Cas Systems ; Photoperiod ; Stress, Physiological ; Plants, Genetically Modified/genetics ; Adaptation, Physiological ; Droughts ; Climate Change ; },
abstract = {Saffron (Crocus sativus L.) is a high-value crop known for its intricate harvesting process and limited production due to factors like triploid sterility and specific climatic needs. This review discusses biotechnological methods, particularly CRISPR/Cas9 genome editing, aimed at improving heat and drought tolerance and achieving year-round flowering. Such genetic edits as evidenced with experimental CRISPR/Cas9 systems that reach up to 70% callus initiation in saffron. Cultivation efficiency and quality are increased in Hydroponic systems and synthetic bioreactors, which have been proven in trials in non-traditional areas such as North Bengal, India. Nonetheless, internationalization threatens the market value and cultural integrity of saffron, and such measures as fair-trade labels, GI laws, and cooperatives of stakeholders must be implemented fairly and equally.},
}

*Flowers/genetics/physiology/growth & development
*Crocus/genetics/physiology/growth & development
Gene Editing
*CRISPR-Cas Systems
Photoperiod
Stress, Physiological
Plants, Genetically Modified/genetics
Adaptation, Physiological
Droughts
Climate Change

@article {pmid41663678,
year = {2026},
author = {Zhu, Y and Zhang, J and Ruan, Y and Lei, T and Li, S and Cao, L and Zou, X and He, Y and Li, Q and Chen, S and Peng, A},
title = {Application of compact CRISPR/Cas nucleases for citrus genome editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {7},
pmid = {41663678},
issn = {1573-9368},
support = {CSTB2024TIAD-CYKJCXX0021//Sichuan-Chongqing Science and Technology Innovation Cooperation Program Project/ ; CSTB2023TIAD-KPX0044//the Technology Innovation and Application Development Key Project of Chongqing/ ; CARS-26//Earmarked Fund for China Agriculture Research System/ ; CSTB2023NSCQ-MSX1085//Chongqing Natural Science Foundation Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Citrus/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Citrus/genetics
*Genome, Plant
Plants, Genetically Modified/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics

@article {pmid41663312,
year = {2026},
author = {Eraña, H and Vidal, E and Fernández-Borges, N and Charco, JM and Díaz-Domínguez, CM and Sampedro-Torres-Quevedo, C and Galarza-Ahumada, J and Fernández-Muñoz, E and San-Juan-Ansoleaga, M and Pérez-Castro, MÁ and Gonçalves-Anjo, N and Piñeiro, P and Giler, S and González-Martín, N and Lorenzo, NL and Manero-Azua, A and Perez de Nanclares, G and Geijo, M and Sánchez-Martín, MA and Requena, JR and Castilla, J},
title = {The L108I polymorphism in mouse prion protein drives spontaneous disease and enhances transmission of atypical and classical prion strains.},
journal = {Brain pathology (Zurich, Switzerland)},
volume = {},
number = {},
pages = {e70083},
doi = {10.1111/bpa.70083},
pmid = {41663312},
issn = {1750-3639},
support = {PID2024-160022OB-I00//Agencia Estatal de Investigación/ ; PID2021-122201OB-C21//Agencia Estatal de Investigación/ ; PID2020-117465GB-I00//Agencia Estatal de Investigación/ ; PID2021-1222010B-C22//Agencia Estatal de Investigación/ ; CEX2021-001136-S//Ministerio de Ciencia e Innovación/ ; EFA031/01 NEURO-COOP//Interreg/ ; PT23/00123//Instituto de Salud Carlos III/ ; BN661-FTPGB-2023//Fundación Tatiana Pérez de Guzmán el Bueno/ ; //Creutzfeldt-Jakob Disease Foundation-2022/ ; },
abstract = {Prion diseases are fatal neurodegenerative disorders that can be idiopathic, associated with genetic mutations, or acquired by infection with misfolded prion protein. We developed two complementary transgenic mouse models to investigate how the L108I substitution in mouse prion protein (PrP) influences spontaneous prion formation and transmission characteristics. The transgenic mouse model overexpressing the variant at approximately three times wild-type (WT) PrP levels (TgMo(L108I)3x) consistently developed a spontaneous neurodegenerative disorder between 219 and 536 days of age with 100% penetrance. This spontaneous disease exhibited biochemical and neuropathological characteristics of atypical prion disorders, featuring a distinctive 7-10 kDa protease-resistant PrP fragment and pathology comparable to small ruminants' atypical scrapie and certain forms of Gerstmann-Sträussler-Scheinker syndrome (GSS). In contrast, the knock-in model expressing the same variant at physiological levels (TgMo(L108I)1x) showed no spontaneous disease beyond 600 days, demonstrating that both the specific amino acid substitution and elevated expression levels are necessary for spontaneous prion formation. The spontaneously generated prions transmitted efficiently to models expressing the I108 variant and to Tga20 mice overexpressing WT PrP but encountered a robust transmission barrier toward WT mice, indicating strain-specific replication requirements. The TgMo(L108I)3x model demonstrated exceptional versatility as a universal acceptor for heterogeneous prion isolates, demonstrating superior efficiency in propagating atypical variants like GSS A117V (57 ± 0.6 days) and rapid propagation of classical scrapie-derived mouse prion strains, including Rocky Mountains Laboratory mouse prion strain (RML) (85 ± 3.8 days) and 22L (95 ± 1 days). Comparative analysis revealed that the L108I substitution differentially impacts strain propagation, with greater acceleration of RML (~33% shorter incubation) than 22L (~0.5% shorter) compared to WT mice. These complementary systems offer powerful experimental platforms for investigating the molecular determinants of spontaneous prion formation, strain selection and transmission barriers, providing insights into idiopathic prion pathogenesis and developing therapeutic interventions.},
}

@article {pmid41663226,
year = {2026},
author = {Yin, X and Zhang, Z and Luo, H and Qin, X and Chen, Y and Chen, W and Zheng, H},
title = {Amplification-free one-pot RNA detection by pairing CRISPR-Cas13a with cascade amplification circuit-driven DNAzyme (RAPID).},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345138},
doi = {10.1016/j.aca.2026.345138},
pmid = {41663226},
issn = {1873-4324},
mesh = {*DNA, Catalytic/metabolism/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Neisseria gonorrhoeae/genetics/isolation & purification ; *RNA, Bacterial/analysis/genetics ; Humans ; },
abstract = {RNA has become a versatile target for diagnosing a wide range of pathogens. The demand for rapid and accurate diagnostics in point-of-care (POC) or resource-limited settings is growing. However, most RNA-based assays depend on reverse transcription and complex instruments (e.g., RT-qPCR), restricting their use in these settings. Isothermal amplification methods provide a simpler alternative with reduced instrumentation requirements, but their high amplification efficiency raises concerns about nucleic acid carry-over contamination. To address these challenges, we developed RAPID (CRISPR-Cas13a with a cascade amplification circuit-driven DNAzyme), an isothermal, one-pot RNA detection biosensing platform that eliminates the need for sample pre-amplification. RAPID integrates the precise target recognition by CRISPR-Cas13a with robust signal amplification by a toehold-mediated strand-displacement DNA circuit, eliminating the need for reverse transcription and thermal cycling. This platform enables quantitative RNA detection within 30 min at 37 °C. By reprogramming RAPID crRNAs, we successfully detected both bacterial (e.g., Treponema pallidum and Neisseria gonorrhoeae) and viral (e.g., herpes simplex virus) targets. The RAPID platform is designed for versatile detection, being compatible with both fluorescence-based (RAPID-Flu) and lateral flow assay (RAPID-LFA) readouts. The RAPID-Flu and RAPID-LFA both demonstrated a sensitivity of 5 fM per reaction, exhibiting comparable detection limits. Both methods showed excellent specificity and high concordance with clinical diagnoses of Neisseria gonorrhoeae. In summary, the RAPID platform provides rapid, programmable, and visually interpretable solutions with strong potential for POC diagnostics. Its flexibility and portability make it particularly suitable for early diagnosis and on-site monitoring of diverse infectious pathogens.},
}

*DNA, Catalytic/metabolism/chemistry/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
Neisseria gonorrhoeae/genetics/isolation & purification
*RNA, Bacterial/analysis/genetics
Humans

@article {pmid41663224,
year = {2026},
author = {Li, Y and Chen, X and Yang, Z and Wang, Z and Wang, R},
title = {CRISPR/Cas12a empowered electrochemical biosensor for ultrasensitive detection of Vibrio parahaemolyticus in seafood samples.},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345158},
doi = {10.1016/j.aca.2026.345158},
pmid = {41663224},
issn = {1873-4324},
mesh = {*Vibrio parahaemolyticus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Seafood/microbiology/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Contamination/analysis ; Animals ; DNA, Bacterial/genetics/analysis ; },
abstract = {Rapid and ultrasensitive detection of Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is of great significance for the early prevention of foodborne disease. Traditional methods for detecting Vp are time-consuming, exhibiting low sensitivity and specificity. In this study, CRISPR/Cas12a system is integrated with electrochemical sensing and polymerase chain reaction (PCR) to establish a PCR-based E-CRISPR biosensor for Vp detection. The target DNA extracted from Vp is amplified by PCR, then activate CRISPR/Cas12a system to cleave methylene blue (MB)-labeled hairpin DNA probes on electrode, resulting in great changes in current. The employment of hairpin DNA probes reduces the steric hindrance for Cas12a trans-cleavage, acquiring a better cleavage efficiency and sensing performance. Under optimal conditions, the limit of detection reaches 1.17 copies/μL (genomic DNA), 1.23 CFU/mL (standard bacteria), and 12.3 CFU/g (artificially contaminated shrimp samples) respectively. Moreover, the PCR-based E-CRISPR biosensor demonstrates superior reproducibility and specificity. Most importantly, the E-CRISPR biosensor were in 100 % agreement with real time quantitative PCR for the detection of 18 seafood samples, which confirms the biosensor's broad applicability for monitoring Vp in complex food matrix. Our developed E-CRISPR biosensor demonstrates to be a simple, rapid and ultrasensitive method for Vp detection in the food supply chain, and can be extended to other foodborne pathogens.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics
*Biosensing Techniques/methods
*Seafood/microbiology/analysis
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
Limit of Detection
Food Contamination/analysis
Animals
DNA, Bacterial/genetics/analysis

@article {pmid41662274,
year = {2026},
author = {Tek, MI and Budak Tek, K and Sarikaya, P and Ahmed, AR and Fidan, H},
title = {Choosing the best route: Comparative optimization of wheat transformation methods for improving yield by targeting TaARE1-D with CRISPR/Cas9.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0342491},
pmid = {41662274},
issn = {1932-6203},
mesh = {*Triticum/genetics/growth & development ; *CRISPR-Cas Systems ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Proteins/genetics ; Plant Breeding/methods ; },
abstract = {Wheat (Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ~3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D, a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance.},
}

*Triticum/genetics/growth & development
*CRISPR-Cas Systems
Gene Editing/methods
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Plant Proteins/genetics
Plant Breeding/methods

@article {pmid41661420,
year = {2026},
author = {Zhang, H and Feng, G and Feng, Y},
title = {Quinoa as a naturally stress-resistant crop: current status and future promises.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {12},
pmid = {41661420},
issn = {2731-0450},
support = {2022B02010-1//Science and Technology Department of Xinjiang Uygur Autonomous Region/ ; 2022YFF1003403-4//Key Technologies Research and Development Program/ ; 2021YFA1300401//Key Technologies Research and Development Program/ ; 32441015//National Natural Science Foundation of China/ ; },
abstract = {Quinoa (Chenopodium quinoa Willd.), a semi-domesticated halophyte originating in the Andean region, has emerged as a promising crop for exploiting marginal lands, valued for its exceptional nutritional profile and remarkable resilience to high salinity and drought. This review analyzes the current status and future potential of quinoa as a model halophytic crop. We begin by examining the physiological mechanisms that enable quinoa to thrive in marginal environments, which have been the subject of extensive study. Thanks to the advancement in high-throughput sequencing technology, genomic resources - including the recent development of high-quality reference genomes and a Chenopodium pangenome - are rapidly expanding. Sequence-based genetic mapping techniques hold the promise to dissect the molecular basis of complex traits in combination with the utility of functional genomics tools such as virus-induced gene silencing (VIGS) and stable genetic transformation. Ultimately, the application of modern breeding technologies, such as phenomics, genomic selection (GS), and CRISPR/Cas, will expedite the development of locally adapted, climate-resilient quinoa cultivars worldwide.},
}

@article {pmid41661284,
year = {2026},
author = {Li, Z and Chen, L and Luo, J and Lu, Y and Zhang, H and Zhao, P},
title = {One-pot recombinase polymerase amplification and CRISPR/Cas12a assay for Cryptococcus neoformans.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {61},
pmid = {41661284},
issn = {1432-0614},
mesh = {*Cryptococcus neoformans/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Recombinases/genetics/metabolism ; *Cryptococcosis/diagnosis/microbiology ; Humans ; Real-Time Polymerase Chain Reaction/methods ; Point-of-Care Testing ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cryptococcus neoformans, an opportunistic fungal pathogen, can induce central nervous system infections, posing a life-threatening risk and imposing substantial global health challenges and economic burdens. Given the significant reduction in mortality achieved through early monitoring of C. neoformans, there is an urgent demand for a rapid detection method for this pathogen. Herein, we developed a rapid, sensitive, and specific assay for the detection of C. neoformans based on a one-pot recombinase polymerase amplification (RPA) and CRISPR/Cas12a system, which can be read using a real-time fluorescent PCR instrument or lateral flow strips. This assay exhibits high sensitivity, with a detection limit of 1 copy/µL for C. neoformans, and no cross-reactivity was observed across different fungal strains. Notably, the assay can be performed in harsh environments without reliance on complex equipment, making it suitable for point-of-care testing (POCT). Collectively, this method not only provides a robust alternative for C. neoformans detection but also offers valuable insights for the identification of other fungal pathogens. KEY POINTS: • We developed a one-pot RPA and CRISPR/Cas12a assay to detect Cryptococcus neoformans. • This assay exhibits high sensitivity and specificity. • Detection results can be obtained by three ways which is suitable for POCT.},
}

*Cryptococcus neoformans/genetics/isolation & purification
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Recombinases/genetics/metabolism
*Cryptococcosis/diagnosis/microbiology
Humans
Real-Time Polymerase Chain Reaction/methods
Point-of-Care Testing
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41660305,
year = {2026},
author = {Zhang, W and Jiang, A and Jia, BK and Jin, Y and Chen, Y and Li, Z and Liao, Y and Zhang, H and Lin, Z and Fang, X and Wang, L},
title = {Progress in RNA-Targeted Therapeutics for Human Diseases.},
journal = {MedComm},
volume = {7},
number = {2},
pages = {e70607},
pmid = {41660305},
issn = {2688-2663},
abstract = {RNA-targeted therapy is reshaping molecular medicine by shifting the traditional "protein-centric" view toward an "RNA-regulatory network" paradigm. Beyond carrying genetic information, RNA plays essential roles in posttranscriptional regulation, signaling pathways, and epigenetic modulation. Advances in high-throughput sequencing, structural biology, and delivery technologies have accelerated the development of diverse RNA therapeutics, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA) modulators, messenger RNA (mRNA) therapeutics, aptamers, short hairpin RNA, and CRISPR/Cas-guided single-guide RNAs. However, a concise comparison of these major RNA modalities and the translational barriers that limit their broader clinical application is still lacking. This review outlines the mechanisms and representative applications of these RNA-based strategies in gene silencing, editing, protein replacement, immune activation, and targeted drug delivery. Special emphasis is placed on ASOs and siRNAs for neurological, metabolic, and infectious diseases, as well as mRNA therapeutics that are transforming vaccine development. Common challenges-such as in vivo stability, delivery efficiency, and immune activation-are also discussed. Finally, we highlight how chemical modification, nanotechnology, and artificial intelligence-assisted design are enhancing the specificity, stability, and safety of RNA therapeutics, providing a framework for advancing next-generation precision RNA medicine.},
}

@article {pmid41616513,
year = {2026},
author = {Xu, L and Jin, J and Lyu, W and Liang, X and Wang, Q and Zhang, J and Luo, Y and Chen, J and Lu, H and Li, X and Shen, F},
title = {Point-of-care profiling of H. pylori virulence and antibiotic resistance from endoscopic biopsies using an integrated restriction enzyme-CRISPR microfluidic platform.},
journal = {Biosensors & bioelectronics},
volume = {299},
number = {},
pages = {118438},
doi = {10.1016/j.bios.2026.118438},
pmid = {41616513},
issn = {1873-4235},
mesh = {*Helicobacter pylori/genetics/pathogenicity/drug effects/isolation & purification ; Humans ; *Helicobacter Infections/microbiology/diagnosis/drug therapy ; *Drug Resistance, Bacterial/genetics ; Point-of-Care Systems ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; Biopsy ; Biosensing Techniques ; Clarithromycin/pharmacology ; Levofloxacin/pharmacology ; Polymorphism, Single Nucleotide ; Gastric Mucosa/microbiology/pathology ; },
abstract = {Endoscopically obtained gastric mucosal biopsies contain abundant molecular information that could inform Helicobacter pylori (H. pylori) eradication therapy. However, the lack of point-of-care tools for processing solid tissue limits rapid, on-site genotyping, leading to empirical therapy and higher eradication failure rates. Here, we present the SlipChip-based On-site and User-friendly Testing (SCOUT) together with the restriction enzyme-assisted CRISPR/Cas12a (RCut) method for point-of-care genotyping from raw endoscopic gastric biopsies. This fully automated platform enables analysis of key virulence genes and resistance-associated single-nucleotide variants (SNVs) at mutant allele frequencies down to 0.1 %. Results were available within 1 h, enabling actionable molecular profiling during the same endoscopic encounter. In 159 clinical validations, SCOUT showed concordant virulence profiling with quantitative PCR (κ = 0.978) and accurately detected clarithromycin and levofloxacin resistance mutations with up to 100 % sensitivity and specificity. These results demonstrate that SCOUT bridges the gap between diagnostic sampling and informed eradication strategies, providing a scalable framework for point-of-care molecular genotyping.},
}

*Helicobacter pylori/genetics/pathogenicity/drug effects/isolation & purification
Humans
*Helicobacter Infections/microbiology/diagnosis/drug therapy
*Drug Resistance, Bacterial/genetics
Point-of-Care Systems
Anti-Bacterial Agents/pharmacology
Virulence/genetics
CRISPR-Cas Systems
Lab-On-A-Chip Devices
Biopsy
Biosensing Techniques
Clarithromycin/pharmacology
Levofloxacin/pharmacology
Polymorphism, Single Nucleotide
Gastric Mucosa/microbiology/pathology

@article {pmid41607132,
year = {2026},
author = {Yu, K and Li, H and Hu, Y and Yu, Y and Deng, S and Yang, Y and Guo, M and Li, M and Zhe, M and He, H and Fan, C},
title = {Integrating transcriptomics and high-throughput gene editing uncovers shoot apical meristem regulators in Brassica napus.},
journal = {Plant physiology},
volume = {200},
number = {2},
pages = {},
doi = {10.1093/plphys/kiag032},
pmid = {41607132},
issn = {1532-2548},
support = {2022YFD1200400//National Key Research and Development Program of China/ ; 2023YFF1000700//National Key Research and Development Program of China/ ; 31671279//National Natural Science Foundation of China/ ; 2024BBA001//Agricultural Gene Editing Platform Technology and Breeding Research & Development/ ; },
mesh = {*Brassica napus/genetics/growth & development ; *Meristem/genetics/growth & development ; *Gene Editing/methods ; *Transcriptome ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plant Shoots/genetics/growth & development ; Plant Proteins/genetics/metabolism ; Mutation ; },
abstract = {The shoot apical meristem (SAM) determines plant architecture, but the key components of its regulatory network remain elusive in rapeseed (Brassica napus L.). Here, we integrated transcriptomic profiling of 3 multilocular silique mutants (Bnaclv1, Bnaclv2, and Bnaclv3) across key SAM development stages (IM, stage6, and stage8) with large-scale CRISPR/Cas9 functional screening to identify regulators of SAM maintenance. Differential gene expression and GO enrichment highlighted genes significantly associated with meristem development processes. Weighted gene co-expression network analysis of stage-specific transcriptomes identified 42 candidate genes potentially related to SAM development. To enable systematic functional screening, we established a high-throughput multiplex CRISPR/Cas9 pipeline, simultaneously targeting 198 sites across 42 candidate genes through optimized sgRNA design and pooled transformation. We successfully obtained mutants for 25 genes with homozygous mutants for 9 genes. Phenotypic analysis demonstrated that mutants of BnaSCPL family genes (SCPL29, SCPL44, and SCPL45) exhibited a multi-stem phenotype and disrupted SAM organization. Mechanistic studies revealed that BnaSCPL mutations disrupt the canonical CLV3/WUS feedback loop, uncovering their roles in SAM homeostasis. Additionally, knockout of BnaLFY homologs caused permanent vegetative state and sterility, demonstrating their conserved role in floral meristem identity in Brassica napus. Collectively, our study not only elucidates the critical function of BnaSCPLs in SAM maintenance but also establishes a regulatory framework for understanding meristem phase transitions in B. napus, providing potential targets for crop architecture improvement.},
}

*Brassica napus/genetics/growth & development
*Meristem/genetics/growth & development
*Gene Editing/methods
*Transcriptome
Gene Expression Profiling
Gene Expression Regulation, Plant
CRISPR-Cas Systems
Plant Shoots/genetics/growth & development
Plant Proteins/genetics/metabolism
Mutation

@article {pmid41591771,
year = {2026},
author = {Chen, D and Zhu, B and Zhou, Y and Fang, Z and Zhu, Z and Chen, C and Shen, T},
title = {Dual-mode CRISPR/Cas12a-mediated alkaline phosphatase detection (CAD) biosensor.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1239-1247},
doi = {10.1039/d5ay01195g},
pmid = {41591771},
issn = {1759-9679},
mesh = {*Alkaline Phosphatase/analysis/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Immunoassay/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Alkaline phosphatase (ALP), a crucial biomarker for hepatobiliary disorders, bone diseases, and cancer progression, requires ultrasensitive detection methods to meet clinical diagnostic requirements. Current methodologies predominantly depend on single-readout mechanisms that fail to address the growing requirements of sensitivity, operational simplicity, and adaptability to resource-limited settings. Herein, we present a CRISPR/Cas12a-mediated ALP detection (CAD) isothermal amplification system that overcomes these challenges through a novel dual-signal (fluorescence and lateral flow immunoassay (LFIA)) readout mechanism. The system features a rationally engineered hairpin DNA probe (HPP) that initiates Klenow (exo-)-driven polymerase elongation upon ALP recognition, subsequently activating Cas12a's trans-cleavage activity for exponential signal amplification. With fluorescence readout, this cascade amplification strategy achieves unprecedented sensitivity with a detection limit of 0.1 U L[-1] and a wide linear range (0.1-10 U L[-1]), outperforming conventional colorimetric methods by one order of magnitude while maintaining exceptional specificity against biological interferents. Furthermore, the LFIA adaptation of the readout bridges the gap between laboratory-based detection and point-of-care applications. This user-friendly adaptation enables instrument-free visual detection with a clear cut-off value of about 7 U L[-1], offering the potential to effectively differentiate pathological samples from normal physiological levels with an appropriate dilution factor for clinical samples. Our dual-mode biosensing strategy not only enables high-precision quantitative analysis in clinical settings but also facilitates rapid qualitative detection under resource-limited conditions, thus offering significant potential for early disease diagnosis and long-term therapeutic monitoring.},
}

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

@article {pmid41590404,
year = {2026},
author = {Li, W and Wang, M and Wang, S},
title = {A label-free and universal CRISPR/Cas12a platform for the detection of hazardous substances in food.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1216-1223},
doi = {10.1039/d5ay01887k},
pmid = {41590404},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; *Food Contamination/analysis ; *Hazardous Substances/analysis ; *Food Analysis/methods ; Limit of Detection ; },
abstract = {The present study proposes a label-free and universal CRISPR/Cas12a-based platform for the detection of hazardous substances in food. A label-free reporter, termed the G4 reporter, was carefully engineered. In the absence of the target analyte, the catalytic hairpin assembly (CHA) remains inactive, thereby preventing activation of the CRISPR/Cas12a system and preserving the cleavage of the G4 reporter, which emits a label-free fluorescence signal. Conversely, in the presence of the target analyte, the CHA process is triggered, activating the CRISPR/Cas12a system, which cleaves the G4 reporter into fragments, leading to a decrease in the label-free fluorescence signal. This detection strategy follows a negative response model, wherein quantification is based on the net fluorescence difference between positive and negative controls. The developed label-free signal output modality for the CRISPR/Cas12a system offers advantages of simplicity and cost-effectiveness. Moreover, the proposed method incorporates a CHA process to facilitate signal transduction and activate the subsequent CRISPR system. The system upon integration of CHA with CRISPR functions in a dual-amplification mode, resulting in enhanced signal amplification efficiency and superior sensitivity compared to the standalone CRISPR system. Thus, the platform achieves highly sensitive detection of hazardous substances, with limits of detection (LODs) of 3.0 fg mL[-1] for aflatoxin B1 (AFB1) and 0.02 pg mL[-1] for acetamiprid (ACE). Moreover, trace amounts of AFB1 and ACE were successfully identified in real food samples. By altering the sequences of the detection components, this platform can be readily adapted for the detection of other hazardous substances in food matrices. Therefore, this work introduces a novel, label-free, universal, and sensitive CRISPR/Cas12a-based detection platform, demonstrating considerable potential for applications in food safety surveillance and public health protection.},
}

*CRISPR-Cas Systems/genetics
*Food Contamination/analysis
*Hazardous Substances/analysis
*Food Analysis/methods
Limit of Detection

@article {pmid41554880,
year = {2026},
author = {Bot, JF and Zhao, Z and Li, M and Kammeron, D and Shang, P and Geijsen, N},
title = {Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {233},
pmid = {41554880},
issn = {2399-3642},
support = {NNF21CC0073729//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; W.OR18-11//Prinses Beatrix Spierfonds/ ; 15804//Netherlands Organisation for Scientific Research | Stichting voor de Technische Wetenschappen (Technology Foundation STW)/ ; 201706890022//China Scholarship Council (CSC)/ ; 202206300033//China Scholarship Council (CSC)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics ; *RNA/metabolism/genetics ; HEK293 Cells ; Ribonucleoproteins/metabolism/genetics ; Apoptosis/genetics ; },
abstract = {CRISPR-Cas13 exclusively targets RNA. In prokaryotes, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. Here we report that LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage starts within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulate genes associated with the stress and innate immune response, ultimately leading to apoptotic cell death. This enables us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, using both total RNA sequencing and Nanopore sequencing, we find that LbuCas13a activation leads to rapid and near-global depletion of cytoplasmic RNAs, and that cleavage occurs at specific nucleotide positions.},
}

Humans
*CRISPR-Cas Systems
*RNA Cleavage
*CRISPR-Associated Proteins/metabolism/genetics
*RNA/metabolism/genetics
HEK293 Cells
Ribonucleoproteins/metabolism/genetics
Apoptosis/genetics

@article {pmid41459814,
year = {2026},
author = {Celle, M and Aniorte, S and Issa, AR and Falabregue, M and Jin, H and Sanchez-Mirasierra, I and Ding, S and Soukup, SF and Seugnet, L and Liao, L and Lesca, G and Walter, L and Mollereau, B},
title = {A dwdr45 knock-out drosophila model to decipher the role of autophagy in BPAN.},
journal = {Human molecular genetics},
volume = {35},
number = {3},
pages = {},
doi = {10.1093/hmg/ddaf198},
pmid = {41459814},
issn = {1460-2083},
support = {//French Ministry of Higher Education and Research/ ; 101067877//Marie Sklodowska-Curie Action fellowship/ ; //China Scholarship Council/ ; },
mesh = {Animals ; *Autophagy/genetics ; Disease Models, Animal ; *Drosophila Proteins/genetics/metabolism ; Gene Knockout Techniques ; Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology/metabolism ; *Carrier Proteins/genetics/metabolism ; Phenotype ; Drosophila/genetics ; Humans ; },
abstract = {Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurological disorder characterized by severe cognitive and motor impairments. BPAN is caused by de novo pathogenic variants in the WDR45 gene on the X chromosome. WDR45 gene encodes the protein WDR45/WIPI4, a known regulator of autophagy. A defective autophagy has been observed in cellular models of BPAN disease and is associated with neurological dysfunctions in wdr45 knockout (KO) mice. However, it remains unclear whether the autophagic defect directly contributes to all WDR45 loss-induced phenotypes or whether other WDR45-dependent cellular functions are involved. To investigate this, we generated a CRISPR/Cas9-mediated KO of CG11975 (dwdr45 KO), the Drosophila homolog of WDR45. Our analysis revealed that dwdr45 KO flies display BPAN-like phenotypes, including impaired locomotor function, seizure-like behavior, autophagy dysregulation and iron dyshomeostasis. Additionally, dwdr45 KO flies exhibit shortened lifespan compared to control flies. These findings demonstrate that dwdr45 KO fly is a relevant in-vivo model for investigating the key cellular and molecular mechanisms underlying BPAN-associated phenotypes. Here we showed that induction of autophagy in dwdr45 KO flies improved both the shortened lifespan and the seizure-like behavior, but did not restore locomotor function. This suggests that defective autophagy contributes to some, but not all, aspects of the phenotypes resulting from loss of dWdr45 function.},
}

Animals
*Autophagy/genetics
Disease Models, Animal
*Drosophila Proteins/genetics/metabolism
Gene Knockout Techniques
Drosophila melanogaster/genetics
CRISPR-Cas Systems
*Neurodegenerative Diseases/genetics/pathology/metabolism
*Carrier Proteins/genetics/metabolism
Phenotype
Drosophila/genetics
Humans

@article {pmid41330380,
year = {2026},
author = {Feng, C and Peets, EM and Zhou, Y and Crepaldi, L and Usluer, S and Dunham, A and Braunger, JM and Su, J and Strauss, ME and Muraro, D and Xian Cheam, KA and Bonder, MJ and Nogales, EG and Cooper, S and Bassett, A and Leonard, S and Gu, Y and Fussing, B and Burke, D and Parts, L and Stegle, O and Velten, B},
title = {A genome-scale single-cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines.},
journal = {Cell genomics},
volume = {6},
number = {2},
pages = {101076},
doi = {10.1016/j.xgen.2025.101076},
pmid = {41330380},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation/genetics ; *Genome, Human/genetics ; Quantitative Trait Loci/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Population-scale resources of genetic, molecular, and cellular information form the basis for understanding human genomes, charting the heritable basis of disease and tracing the effects of mutations. Pooled perturbation assays, probing the effect of many perturbations coupled with single-cell RNA sequencing (scRNA-seq) readout, are especially potent references for interpreting disease-linked mutations or gene-expression changes. However, the utility of existing maps has been limited by the comprehensiveness of perturbations conducted and the relevance of their cell-line context. Here, we present a genome-scale CRISPR interference perturbation map with scRNA-seq readout across many genetic backgrounds in human pluripotent cells. We map trans expression changes induced by knockdowns and characterize their variation across donors, with expression quantitative trait loci linked to higher genetic modulation of perturbation effects. This study pioneers population-scale CRISPR perturbations with high-dimensional readouts, which will fuel the future of effective modulation of cellular disease phenotypes.},
}

Humans
*Single-Cell Analysis/methods
*Pluripotent Stem Cells/metabolism
*CRISPR-Cas Systems/genetics
*Gene Expression Regulation/genetics
*Genome, Human/genetics
Quantitative Trait Loci/genetics
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41659011,
year = {2025},
author = {Wang, B and Lu, J and Zhang, X and Hu, R and Ma, H},
title = {Advances in nanomaterial-mediated CRISPR/Cas delivery: from lipid nanoparticles to vesicle-derived systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1669104},
pmid = {41659011},
issn = {2296-4185},
abstract = {Gene and genome editing therapies are increasingly connected with nanomaterials, which protect and transport fragile nucleic acids and CRISPR/Cas systems through biological barriers safely and accurately. This review discusses how different nanocarriers, including lipid-based, polymeric, inorganic, and vesicle-derived systems, can improve delivery efficiency, cell targeting, endosomal escape, and intracellular movement for gene and genome editing. It summarizes findings from early clinical and preclinical studies, comparing several carrier types such as ionizable lipid nanoparticles, polymeric nanoparticles, micelles, gold and silica nanostructures, and engineered extracellular vesicles. The review also explains how specific design factors, such as surface ligands, charge modification, PEGylation, and stimulus-responsive behaviors, influence biodistribution, and improve on-target efficiency while lowering immune responses and off-target effects. Ethical and regulatory concerns for in vivo editing are highlighted, along with current methods used to study nano-bio interactions. Among these carriers, ionizable lipid nanoparticles show the most advanced performance for delivering nucleic acids and CRISPR systems. However, new polymer-based and exosome-inspired carriers are progressing rapidly for repeated and targeted applications. Hybrid and responsive systems may also enable better spatial and temporal control of editing. Future research should focus on stronger in vivo potency testing, improved biocompatibility evaluation, and standardized manufacturing to ensure clinical safety and reliability.},
}

@article {pmid41658853,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {In silico exploration of the genomic repertoire of Iranian aquatic bacteria: Prophage carriage, bioactive compound potential, CRISPR-Cas immunity, and integrated defensive-metabolic islands.},
journal = {Biochemistry and biophysics reports},
volume = {45},
number = {},
pages = {102452},
pmid = {41658853},
issn = {2405-5808},
abstract = {The unique and underexplored aquatic ecosystems of Iran represent a significant reservoir of microbial diversity. This study presents the first comprehensive genomic survey of 38 native Iranian bacterial strains from hypersaline lakes and wetlands, integrating in silico analyses of their secondary metabolome, bacteriocin potential, resident prophages, and genomic architecture. Our genome mining revealed a prolific capacity for secondary metabolite production, identifying dozens of biosynthetic gene clusters (BGCs). Ectoine biosynthesis was ubiquitous, underscoring its role as a key osmoprotectant, while diverse BGCs for terpenes, polyketides, and hybrid metabolites were also prevalent. Concurrently, we identified a wide array of ribosomally synthesized and post-translationally modified peptides (RiPPs), including known bacteriocins. Furthermore, we characterized eight high-quality prophages integrated within these genomes, encoding auxiliary genes such as carbohydrate-active enzymes (CAZymes) and putative anti-CRISPR (ACR) proteins. The bacterial hosts themselves were equipped with robust defense systems, with CRISPR-Cas loci, predominantly Type I, detected in most strains. Crucially, we identified multi-functional genomic islands that physically link BGCs with defense systems (e.g., CRISPR-Cas, restriction-modification) and prophage regions. We propose the "Fortress Hypothesis" to explain this architecture, wherein the co-localization of metabolite production and defense machinery protects metabolic investment against phage predation and genetic loss. This integrative genomic arrangement highlights a sophisticated co-evolutionary strategy for survival in extreme environments. Our findings position these indigenous bacteria as a promising genetic repository for discovering novel bioactive compounds, enzymes, and biotechnological tools, with implications for antibiotic discovery, CRISPR modulation, and understanding adaptive microbial genomics in extreme niches.},
}

@article {pmid41658434,
year = {2026},
author = {Han, Y and Chen, R and Shentu, X},
title = {Advances and challenges of CRISPR/Cas gene editing for corneal diseases.},
journal = {Advances in ophthalmology practice and research},
volume = {6},
number = {1},
pages = {68-79},
pmid = {41658434},
issn = {2667-3762},
abstract = {BACKGROUND: Corneal diseases are a major cause of global visual impairment, and current treatments remain inadequate for severe or refractory cases. The CRISPR/Cas system offers robust and precise gene-editing capabilities, yet its therapeutic potential for corneal disorders remains largely unexplored.

MAIN TEXT: This narrative review introduces the CRISPR/Cas system and summarizes its recent advances in treating various corneal diseases, including inherited corneal dystrophies, infectious keratitis, corneal injury, and pathological neovascularization. We outline emerging preclinical and clinical studies, and analyze key issues that should be addressed for translation, including administration strategies, vector platform optimization and the mitigation of off-target toxicity.

CONCLUSIONS: This review provides a comprehensive and integrated overview of the current translational directions and challenges of CRISPR/Cas technology in corneal diseases from a novel perspective. It offers valuable guidance for future research and may accelerate the development of gene-editing therapies toward clinical application.},
}

@article {pmid41656481,
year = {2026},
author = {Laxmi, V and Verma, S and Kumar, M and Venkatesh, V and Mohit, and Maury, J and Mohd, S and Tripathi, S},
title = {Lactic Acid Bacteria-derived Bacteriocins: A Promising Antimicrobial Strategy against Multidrug-resistant for Neonatal Sepsis Pathogens.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41656481},
issn = {1867-1314},
abstract = {Neonatal sepsis continues to pose rising critical challenges to global health, particularly in low and middle-income countries, where it accounts for a substantial proportion of neonatal morbidity and mortality. The increasing prevalence of multidrug-resistant (MDR) pathogens in neonatal sepsis has significantly weakened the efficacy of conventional antibiotics, necessitating urgent exploration of alternative antimicrobial therapies for better clinical outcomes. Emerging research interest is growing to develop the microbial-derived peptides as novel antimicrobial agents, particularly "bacteriocins." In comparison to traditional antibiotics, many bacteriocins exhibit narrow-spectrum action, enabling them to inhibit specific pathogens without disrupting the host microbiota. Recent studies have highlighted the remarkable potential of lactic acid bacteria (LAB) derived bacteriocins in combating MDR pathogens responsible for neonatal sepsis. In this review, we compile current literature on the in vitro antimicrobial activity of LAB-derived bacteriocins, molecular diversity, mechanisms of action and clinical potential. Special attention is given to bacteriocins produced by LAB associated with the milk-derived microbiota, whose natural protective functions could be particularly beneficial for immunocompromised newborns. Additionally, we discuss the physicochemical properties of microbial peptides, including thermal stability, enzymatic resistance, and pH tolerance, which support their suitability for pharmaceutical applications. Overall, LAB-derived bacteriocins represent a novel, biocompatible, and complementary antimicrobial strategy integrated with conventional antibiotics to fight against MDR in neonatal sepsis. Further research and multicentric clinical trials are necessary to fully explore its compliance and efficacy as a future antimicrobial agent in neonatal medicine to underscore their potential as adjunct or preventive biotherapeutics in NICU settings.},
}

@article {pmid41656299,
year = {2026},
author = {Fang, T and Bogensperger, L and Feer, L and Allam, A and Bezshapkin, V and Balázs, Z and von Mering, C and Sunagawa, S and Krauthammer, M and Schwank, G},
title = {Uncovering Cas9 PAM diversity through metagenomic mining and machine learning.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-69098-5},
pmid = {41656299},
issn = {2041-1723},
abstract = {Recognition of protospacer adjacent motifs (PAMs) is crucial for target site recognition by CRISPR-Cas systems. In genome editing applications, the requirement for specific PAM sequences at the target locus imposes substantial constraints, driving efforts to search for novel Cas9 orthologs with extended or alternative PAM compatibilities. Here, we present CRISPR-PAMdb, a comprehensive and publicly accessible database compiling Cas9 protein sequences from 3.8 million bacterial and archaeal genomes and PAM profiles from 7.4 million phage and plasmid sequences. Through spacer-protospacer alignment, we infer consensus PAM preferences for 8003 unique Cas9 clusters. To extend PAM discovery beyond traditional alignment-based approaches, we develop CICERO, a machine learning model predicting PAM preferences directly from Cas9 protein sequences. Built on the ESM2 protein language model and trained on the CRISPR-PAMdb database, CICERO achieves an average cosine similarity of 0.69 on test data and 0.75 on experimentally validated Cas9 orthologs. For Cas9 clusters where alignment-based predictions are infeasible, CICERO generates PAM profiles for an additional 50,308 Cas9 proteins, including 17,453 high-confidence predictions with CICERO confidence scores above 0.8. Together, CRISPR-PAMdb and CICERO enable large-scale exploration of PAM diversity across Cas9 proteins, accelerating design of next-generation CRISPR-Cas9 tools for precise genome engineering.},
}

@article {pmid41656257,
year = {2026},
author = {Ruis, BL and Ward, H and Myers, CL and Bielinsky, AK and Hendrickson, EA},
title = {SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes.},
journal = {Epigenetics & chromatin},
volume = {19},
number = {1},
pages = {10},
pmid = {41656257},
issn = {1756-8935},
support = {AG077174//National Institute of Aging/ ; AG077174//National Institute of Aging/ ; R35GM141805//National Institutes of General Medical Sciences/ ; GM088351/GM/NIGMS NIH HHS/United States ; CA154461/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {BACKGROUND: The use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) was developed such that targeted genomic lesions [usually DNA double-stranded breaks (DSBs)] could be introduced in vivo with ease and precision. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. Because DSBs can lead to genomic instability, however, a large amount of effort has been invested in methodologies (e.g., base editors) that only require nicking the chromosomal DNA on one strand. Indeed, we have demonstrated in human cells that oligodeoxynucleotide (ODN)-mediated PGE using nickase variants of Cas9 can proceed by at least two HDR subpathways termed synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI). Which pathway is utilized is determined by which chromosomal strand (sense or anti-sense/Watson or Crick) is nicked and by the strandedness (sense or anti-sense/Watson or Crick) of the donor ODN.

RESULTS: While the mechanism of mammalian SDSA is moderately well understood, that of ssDI is not. To gain genetic insight into ssDI, we carried out a genome-wide CRISPR knockout screen to identify those genes which, when absent, enable increased ssDI. This screen identified the protein lysine methyl transferase (PKMT) Su(var)3-9, enhancer-of-zeste and trithorax (SET) domain bifurcated histone lysine methyltransferase 1 (SETDB1):activating transcription factor 7-interacting protein (ATF7IP) heterodimer and the downstream human silencing hub (HUSH) complex as strong negative regulators of ssDI. Consistent with their well-known biological effects, the negative regulation of ssDI by SETDB1/ATF7IP and HUSH was specific for transgenic reporters and for a HUSH-regulated single-copy gene, but was not observed at other (non-HUSH regulated) single-copy endogenous loci.

CONCLUSIONS: In toto, these experiments underscore the profound impact that chromatin modifiers - and by extension, chromatin structure - have on PGE outcomes. Specifically, we have identified SETDB1/ATF7IP and the HUSH complex as major negative regulators of the HDR subpathway, ssDI, when the target is a transgene. These experiments are a proof-of-principle that chromatin can act as a potent barrier to genetic recombination and they strongly support the feasibility of extending similar chromatin modulating strategies to enhance PGE efficiency at endogenous single-copy loci.},
}

Humans
*Histone-Lysine N-Methyltransferase/metabolism/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Recombinational DNA Repair
DNA Breaks, Double-Stranded
HEK293 Cells

@article {pmid41655694,
year = {2026},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {Cytomegalovirus (CMV)-encoded immediate early 1 (IE1) protein perturbs neural progenitor proliferation via interfering with host PML-DISC1 interaction.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {111269},
doi = {10.1016/j.jbc.2026.111269},
pmid = {41655694},
issn = {1083-351X},
abstract = {Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation in congenital CMV infection. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. We further observed that CMV-encoded IE1 protein interferes with host PML-DISC1 interaction, resulting in disturbance of the Notch pathway in vitro and in embryonic brains. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}

@article {pmid41654278,
year = {2026},
author = {Wang, X and You, J and Li, X and Xu, Y and Li, Z and Wang, L},
title = {Limitations of traditional mycotoxin control and biotechnological advances toward sustainable solutions.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108836},
doi = {10.1016/j.biotechadv.2026.108836},
pmid = {41654278},
issn = {1873-1899},
abstract = {Mycotoxins are harmful fungal metabolites that contaminate food and feed, posing serious health and economic risks worldwide. Traditional control methods often fall short due to inefficiency and safety concerns, prompting the development of innovative biotechnological approaches. This review explores recent advances in mycotoxin management, focusing on engineered microbes for targeted degradation, nanotechnology-based detection and removal systems, phage therapy targeting toxin-producing fungi, CRISPR-Cas gene editing of mycotoxin biosynthesis pathways, and plant-microbe interactions that suppress fungal growth. Additionally, enzyme immobilization strategies are highlighted for improving enzyme stability and reusability in detoxification processes. These integrated biotechnological tools offer promising, sustainable solutions to mitigate mycotoxin contamination, enhancing food safety and agricultural productivity. The review also discusses current challenges and future directions for translating these advances into practical applications. Highlighting that biotechnological tools are technically feasible and increasingly close to industrial deployment in food and feed chains.},
}

@article {pmid41621146,
year = {2026},
author = {Kundu, A and Jerala, R},
title = {Small RNAs, big potential: Engineering microRNA-based synthetic gene circuits.},
journal = {Current opinion in chemical biology},
volume = {90},
number = {},
pages = {102652},
doi = {10.1016/j.cbpa.2026.102652},
pmid = {41621146},
issn = {1879-0402},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Gene Regulatory Networks ; *Synthetic Biology/methods ; Animals ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression. Their dysregulation is closely associated with various diseases, positioning them as biomarkers of cellular state. Synthetic biology has leveraged these properties to engineer miR-based genetic circuits capable of sensing and interpreting endogenous miR levels. Early miR-OFF systems relied on reporter gene repression but were limited by ambiguous signal interpretation. Subsequent advances introduced miR-ON architectures, logic-based classifiers integrating multiple miRs, and layered regulatory strategies combining transcriptional, translational, and cleavage-based modules to enhance sensitivity and specificity. Recent innovations include CRISPR-associated miR-responsive systems and incoherent feed-forward loop (iFFL) architectures that stabilize gene expression amid cellular variability, shifting applications from passive sensing to therapeutic intervention. Despite challenges such as leakage, cellular resource resources, and delivery, progress in orthogonal miR toolkits, computational modeling, and RNA-based delivery platforms is rapidly driving miR-based circuits toward diagnostic and therapeutic applications.},
}

*MicroRNAs/genetics/metabolism
Humans
*Gene Regulatory Networks
*Synthetic Biology/methods
Animals
*Genetic Engineering/methods
CRISPR-Cas Systems

@article {pmid41577003,
year = {2026},
author = {Zade, NH and Jain, M and Garg, M and Checker, R and Ghosh, A and Khattar, E},
title = {Proteomics method for identifying POT1-associated complexes at telomeres using ChIP-Mass spectrometry.},
journal = {Methods (San Diego, Calif.)},
volume = {247},
number = {},
pages = {161-174},
doi = {10.1016/j.ymeth.2026.01.007},
pmid = {41577003},
issn = {1095-9130},
mesh = {Shelterin Complex ; *Telomere/metabolism/genetics/chemistry ; *Proteomics/methods ; Humans ; *Telomere-Binding Proteins/metabolism/genetics ; Mass Spectrometry/methods ; CRISPR-Cas Systems ; *Chromatin Immunoprecipitation/methods ; Protein Binding ; Gene Editing ; Tripeptidyl-Peptidase 1 ; },
abstract = {POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1's role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3' overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.},
}

Shelterin Complex
*Telomere/metabolism/genetics/chemistry
*Proteomics/methods
Humans
*Telomere-Binding Proteins/metabolism/genetics
Mass Spectrometry/methods
CRISPR-Cas Systems
*Chromatin Immunoprecipitation/methods
Protein Binding
Gene Editing
Tripeptidyl-Peptidase 1

@article {pmid41576935,
year = {2026},
author = {Hommersom, MP and Puvogel, S and Scheefhals, N and Carpentiero, E and Bouma, M and van Beusekom, E and Dillen, L and van de Warrenburg, BPC and Nadif Kasri, N and van Bokhoven, H},
title = {Human neuronal networks on micro-electrode arrays as a tool to assess genotype-phenotype correlation in CACNA1A-related disorders.},
journal = {Stem cell reports},
volume = {21},
number = {2},
pages = {102783},
doi = {10.1016/j.stemcr.2025.102783},
pmid = {41576935},
issn = {2213-6711},
mesh = {Humans ; *Genetic Association Studies/methods ; *Nerve Net/metabolism ; *Neurons/metabolism ; *Calcium Channels/genetics ; Phenotype ; CRISPR-Cas Systems/genetics ; Electrodes ; Genotype ; Calcium Channels, N-Type ; },
abstract = {CACNA1A-related disorders constitute a diverse group of neurological conditions, including ataxia, migraine, and epilepsy. Despite extensive genetic studies, clear genotype-phenotype correlations remain elusive. Moreover, next-generation sequencing has identified many variants of uncertain significance (VUS). Here, we leveraged patient-derived and CRISPR-Cas9-engineered human neuronal networks to explore relationships between CACNA1A variants and neurophysiological activity. CACNA1A haploinsufficiency induced subtle alterations in glutamatergic network activity, whereas missense variants had a more pronounced effect on overall network function. Network fingerprints were most affected from patients where ataxia co-occurred with migraine or epilepsy. Furthermore, we analyzed the impact of CRISPR-Cas9-induced VUS on network developmental trajectories. Although functional changes could not be directly linked to clinical phenotypes, all tested variants induced measurable alterations in neuronal network function, supporting their classification as likely pathogenic. These findings highlight the potential of human neuronal networks as a translational model for evaluating CACNA1A variant effects and improving clinical variant interpretation.},
}

Humans
*Genetic Association Studies/methods
*Nerve Net/metabolism
*Neurons/metabolism
*Calcium Channels/genetics
Phenotype
CRISPR-Cas Systems/genetics
Electrodes
Genotype
Calcium Channels, N-Type

@article {pmid41565761,
year = {2026},
author = {Beauchemin, KS and Supattapone, S},
title = {Genome-wide screens identify core regulators of cell surface prion protein expression.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5895},
pmid = {41565761},
issn = {2045-2322},
support = {1R37NS125431/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; },
mesh = {*Neurons/metabolism/cytology ; Humans ; CRISPR-Cas Systems ; *Prion Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Gene Expression Regulation ; *PrPC Proteins/genetics/metabolism ; Animals ; Cell Line ; Cell Membrane/metabolism ; },
abstract = {Expression of the cellular prion protein, PrP[C], on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrP[C] expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrP[C] expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrP[C]. We also sought to determine whether the same or different genes regulate cell surface PrP[C] in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrP[C] expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrP[C] expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrP[C] expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.},
}

*Neurons/metabolism/cytology
Humans
CRISPR-Cas Systems
*Prion Proteins/genetics/metabolism
Cell Differentiation/genetics
*Gene Expression Regulation
*PrPC Proteins/genetics/metabolism
Animals
Cell Line
Cell Membrane/metabolism

@article {pmid41524400,
year = {2026},
author = {Clarke, JE and Faulkner, TR and Seipke, RF},
title = {A platform for CRISPRi-seq in Streptomyces albidoflavus.},
journal = {mBio},
volume = {17},
number = {2},
pages = {e0306525},
doi = {10.1128/mbio.03065-25},
pmid = {41524400},
issn = {2150-7511},
support = {BB/T014962/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Streptomyces/genetics ; Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; *CRISPR-Cas Systems ; Genomics/methods ; },
abstract = {UNLABELLED: Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.

IMPORTANCE: Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.},
}

*Streptomyces/genetics
Operon
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Bacterial
*CRISPR-Cas Systems
Genomics/methods

@article {pmid41467797,
year = {2026},
author = {Ji, HJ and Jang, A-Y and Han, SH and Kim, M-K and Lamien, CE and Wijewardana, V and Ahn, KB and Kim, K-H and Song, JY and Seo, HS},
title = {Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).},
journal = {Journal of clinical microbiology},
volume = {64},
number = {2},
pages = {e0098425},
doi = {10.1128/jcm.00984-25},
pmid = {41467797},
issn = {1098-660X},
support = {22202MFDS171//Ministry of Food and Drug Safety/ ; 2018E240600//Korea Disease Control and Prevention Agency/ ; 523140-26//Korea Atomic Energy Research Institute/ ; RS-2022-00164721//National Research Foundation of Korea/ ; CRP D32039//International Atomic Energy Agency/ ; },
mesh = {*Streptococcus pneumoniae/classification/genetics/isolation & purification ; Humans ; *Serotyping/methods ; *Pneumococcal Infections/microbiology ; Serogroup ; *Nanopore Sequencing/methods ; Republic of Korea ; CRISPR-Cas Systems ; Pneumococcal Vaccines ; },
abstract = {Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.},
}

*Streptococcus pneumoniae/classification/genetics/isolation & purification
Humans
*Serotyping/methods
*Pneumococcal Infections/microbiology
Serogroup
*Nanopore Sequencing/methods
Republic of Korea
CRISPR-Cas Systems
Pneumococcal Vaccines

@article {pmid41654255,
year = {2026},
author = {Saini, H and Yadav, J and Pandey, S and Kumar, A and Nanda, D and Sachan, S and Kaushik, JJ},
title = {Field-deployable CRISPR-cas variants for rapid on-site detection of plant pathogens.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {365},
number = {},
pages = {113028},
doi = {10.1016/j.plantsci.2026.113028},
pmid = {41654255},
issn = {1873-2259},
abstract = {Rapid, field-deployable diagnostics are essential for effective plant disease management. Although CRISPR-Cas systems offer high sensitivity and programmability, their use in on-site plant pathogen detection has been hindered by the lack of standardized, practical workflows. Here we present implementable CRISPR-Cas diagnostic protocols using Cas12a, Cas13a, and miniature Cas variants for rapid detection of major plant pathogens. Three field-ready assays are described: (i) an RPA-Cas12a lateral-flow test for DNA pathogens, (ii) a Cas13a RT-RPA assay for RNA viruses, and (iii) an amplification-free Cas12a electrochemical biosensor suited for portable laboratories. Each protocol includes sample preparation steps, reagent formulations, incubation conditions, and troubleshooting guidance. Across platforms, detection limits of 1-100 copies µL[-1] were achieved within 20-45 min, demonstrating analytical sensitivity comparable to conventional PCR-based diagnostics while offering substantially reduced assay time and improved field deployability. We also address practical constraints including sample inhibitors, reagent stability, and biosafety and propose solutions for field implementation. These standardized workflows translate recent advances in CRISPR diagnostics into reproducible, field-deployable tools for plant health surveillance and rapid disease detection.},
}

@article {pmid41653672,
year = {2026},
author = {Bai, T and Qu, X and Pan, J and Tang, Y and Wang, L and Zhou, P and Hu, Z and Guo, Z and Zhu, Y and Zhang, Y},
title = {CRISPR-initiated exponential amplification on fluorescently-barcoded microspheres for deep learning-assisted multiplexed HPV detection.},
journal = {Biosensors & bioelectronics},
volume = {300},
number = {},
pages = {118488},
doi = {10.1016/j.bios.2026.118488},
pmid = {41653672},
issn = {1873-4235},
abstract = {Rapid, low-cost, and multiplexed nucleic acid testing is essential for human health but remains challenging. Although CRISPR-Cas systems offer high specificity, their integration into Multiplexed platforms suitable for near-patient testing has been limited. Here, we introduce a biosensing platform that combines CRISPR-initiated enzymatic amplification with quantum-dot encoded microbeads and deep-learning based image analysis for Multiplexed detection of human papillomavirus (HPV) DNA. The high specificity of CRISPR/Cas9 first triggers an exponential amplification. The products are then specifically captured on the microbead surface for localized fluorescent readout. A custom deep-learning algorithm automatically quantifies the bead fluorescence, enabling robust and automated analysis. The integrated approach achieves simultaneous detection of HPV16, HPV18, and HPV33 with detection limits as low as 0.2 pM. By using recognition-triggered amplification and a simple deep-learning assisted fluorescence readout, the workflow is significantly simplified. The platform establishes a universal and practical strategy for molecular diagnostics, demonstrating strong potential for near-patient testing.},
}

@article {pmid41617656,
year = {2026},
author = {He, M and Chen, J and Chao, M and Yin, F and Pan, H and Wang, Q and Cheng, W and Wang, Z and Xiang, Y},
title = {Novel One-Pot Detection of Tumor mRNA Vaccine by the Target Serving as crRNA of the Split Cas12a System and Synergistically Triggering an Entropy-Driven Reaction.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4134-4142},
doi = {10.1021/acs.analchem.5c06932},
pmid = {41617656},
issn = {1520-6882},
mesh = {*Entropy ; *Cancer Vaccines/analysis/genetics ; *RNA, Messenger/genetics/analysis ; Humans ; *mRNA Vaccines/analysis ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {mRNA vaccines, as a new form of vaccine, are gradually revealing their potential in the field of tumor treatment. With the development of mRNA vaccines, monitoring their levels has become particularly important. In this work, a novel and innovative strategy was proposed for the first time, which directly used the target as crRNA and simultaneously triggered an entropy-driven split Cas12a system for triple signal amplification for the high-sensitivity detection of mRNA vaccines. This study broke the traditional full-length crRNA and innovatively designed a split crRNA structure, directly using the target mRNA as the variable sequence of the crRNA. This design avoided the need for additional introduction of variable sequences, thereby eliminating nonspecific signal leakage and multicomponent interference from the source. At the same time, the target molecule served as the initiating chain for the EDC reaction, allowing the split Cas12a system to be coupled with the entire entropy-driven reaction process, achieving efficient collaboration and triple signal amplification and significantly improving the detection sensitivity and specificity. Moreover, the entire detection process required only one reaction step, which was simple, fast, and efficient. The aim was to provide strategies for screening and pharmacokinetic studies of mRNA vaccines.},
}

*Entropy
*Cancer Vaccines/analysis/genetics
*RNA, Messenger/genetics/analysis
Humans
*mRNA Vaccines/analysis
*Endodeoxyribonucleases/metabolism/genetics
CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Nucleic Acid Amplification Techniques/methods

@article {pmid41587498,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO},
title = {Toehold-mediated strand displacement in CRISPR/Cas12a reactions: Advances in programmable and universal biosensing strategies.},
journal = {Talanta},
volume = {302},
number = {},
pages = {129442},
doi = {10.1016/j.talanta.2026.129442},
pmid = {41587498},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based biosensors have emerged as powerful tools for nucleic acid detection due to their programmability, high sensitivity, and versatility. However, challenges such as PAM dependence, limited mismatch discrimination, and difficulty in detecting non-nucleic acid analytes constrain their universality. Toehold-mediated strand displacement (TMSD) offers a programmable mechanism to overcome these limitations by dynamically regulating hybridization kinetics and molecular interactions. This review systematically summarizes recent advances integrating TMSD into CRISPR/Cas12a systems, including crRNA release, crRNA-DNA activation, activator generation, and reporter signal modulation. By coupling TMSD's precise strand exchange capabilities with Cas12a's trans-cleavage activity, these hybrid biosensors achieve improved specificity, tunable kinetics, and multi-analyte adaptability. The review further discusses design principles, thermodynamic foundations, and application examples across biomedical, environmental, and food diagnostics. Collectively, TMSD-assisted CRISPR/Cas12a biosensing provides a universal, programmable framework for next-generation molecular diagnostics with enhanced control, sensitivity, and functional diversity.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
DNA/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41586479,
year = {2026},
author = {Huang, C and Yang, Y and Yin, H and Chang, H and Gong, R and Jiang, Z and Zhang, XE and Chen, Y},
title = {Ultrasensitive CRISPR Platform Enables Rapid and Extraction-Free Detection of Viral Nucleic Acid.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4028-4035},
doi = {10.1021/acs.analchem.5c06518},
pmid = {41586479},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Nasopharynx/virology ; },
abstract = {CRISPR-based molecular diagnostics offers great potential for the rapid identification of pathogens. However, existing one-pot detection systems remain constrained by their restricted versatility and operational complexity. Herein, we report a novel strategy termed interspaced phosphorothioate primer-mediated one-pot detection (iPSOT), which substitutes conventional phosphodiester primers with interspaced phosphorothioate (iPS) primers. The iPS primers demonstrate strong compatibility with AapCas12b during loop-mediated isothermal amplification, enabling robust fluorescence signal generation within 15 min. The iPSOT system achieves highly specific nucleic acid detection and reliably identifies low-copy ribonucleic acid targets at concentrations of as low as 0.5 aM (0.3 copies/μL). Clinical validation further demonstrated that iPSOT enables the direct detection of SARS-CoV-2 from nasopharyngeal swabs without RNA extraction. This method reduces reagent cost and shortens assay time, achieving sample-to-result under 20 min. Overall, iPSOT enhances both sensitivity and specificity in one-pot detection and offers a promising platform for rapid, reliable point-of-care testing and large-scale pathogen surveillance.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*RNA, Viral/analysis/genetics
Nucleic Acid Amplification Techniques/methods
*COVID-19/diagnosis/virology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Limit of Detection
*Clustered Regularly Interspaced Short Palindromic Repeats
*COVID-19 Nucleic Acid Testing/methods
Nasopharynx/virology

@article {pmid41582525,
year = {2026},
author = {Ding, S and Li, Y and Wang, F and Liu, Q and Liu, L and Li, J and Wu, Y and Liu, X and Jiang, D and Xu, X and Gao, X and Huang, C},
title = {DNA Flap-Controlled CRISPR/Cas12a Trans-Cleavage Enables Mix-and-Read FEN1 Activity Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {3998-4007},
doi = {10.1021/acs.analchem.5c06447},
pmid = {41582525},
issn = {1520-6882},
mesh = {*Flap Endonucleases/metabolism/analysis ; Humans ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as a powerful biosensing tool, owing to its exceptional specificity and trans-cleavage-mediated signal amplification capability, for detecting flap endonuclease 1 (FEN1) activity. However, its reliance on additional preligation or replication steps increases operational complexity and raises the risk of false signals. Herein, we report a DNA flap-controlled CRISPR/Cas12a trans-cleavage (FCT-CRISPR) strategy that enables sensitive, accurate, and mix-and-read detection for FEN1 activity. In FCT-CRISPR, a flap-structured dumbbell DNA probe was ingeniously designed, in which the flap domain serves as a split activator, and the dumbbell scaffold acts as a steric hindrance unit. Upon FEN1 recognition and cleavage, the split activator is liberated from its constrained configuration in the dumbbell scaffold, thereby activating CRISPR/Cas12a trans-cleavage activity. FCT-CRISPR strategy avoids the reliance on exogenous DNA ligation or replication processes, allowing mix-and-read detection of FEN1 activity with a detection limit as low as 0.2 mU and excellent specificity against nontarget enzymes. In addition, the successful detection application in lysates of cancer cells demonstrates the potential of FCT-CRISPR for clinical use. This work establishes a sensitive, accurate, and mix-and-read platform for monitoring FEN1 activity and offers a promising tool for the early diagnosis of FEN1-related diseases.},
}

*Flap Endonucleases/metabolism/analysis
Humans
*CRISPR-Cas Systems
*DNA/chemistry/metabolism/genetics
*Biosensing Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41496452,
year = {2026},
author = {Liu, J and Liu, Z and Qin, D and Qi, X and Chen, M and Cheng, D and Guo, S and Zhang, X and Zhang, L and Yan, T and Li, F and He, W and Lu, Z and Wang, H and Li, J and Yang, X and Shi, Y and Zhang, S and Zhao, H and Liu, C and Chen, S and Zhong, Y},
title = {An all-in-one visual selection system for male-sterile line production in maize and rice.},
journal = {Plant communications},
volume = {7},
number = {2},
pages = {101693},
doi = {10.1016/j.xplc.2026.101693},
pmid = {41496452},
issn = {2590-3462},
mesh = {*Zea mays/genetics/physiology ; *Oryza/genetics/physiology ; *Plant Infertility/genetics ; Seeds/genetics/growth & development ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Hybrid Vigor/genetics ; Plants, Genetically Modified ; },
abstract = {Heterosis has significantly improved crop yields, yet hybrid seed production remains hindered by labor-intensive manual emasculation. Although current male-sterility systems, such as cytoplasmic male sterility and environment-sensitive genic male sterility, have improved the efficiency of hybrid seed production, their limited genetic adaptability and high environmental dependence remain major challenges. Here, we report an all-in-one seed production technology (ASPT) that integrates CRISPR-Cas9, RUBY, and key seed production technology (SPT) components into a single vector, enabling efficient generation and propagation of male-sterile lines in both maize and rice. The engineered RUBY marker enables visual identification of male-sterile and maintainer lines, with an accuracy of 99.81% in automated seed sorting and 100% in secondary field screening. Notably, ASPT was successfully introduced into 21 genetically diverse elite maize inbred lines, demonstrating broad compatibility. ASPT enables scalable and precise propagation of male-sterile lines in both maize and rice, providing a broadly applicable strategy to advance hybrid seed production in crops.},
}

*Zea mays/genetics/physiology
*Oryza/genetics/physiology
*Plant Infertility/genetics
Seeds/genetics/growth & development
*Plant Breeding/methods
CRISPR-Cas Systems
Hybrid Vigor/genetics
Plants, Genetically Modified

@article {pmid40579542,
year = {2026},
author = {Ullah, HMA and Huang, Q and Chiola, S and Wang, Y and Shcheglovitov, A},
title = {Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes.},
journal = {Nature protocols},
volume = {21},
number = {2},
pages = {718-748},
pmid = {40579542},
issn = {1750-2799},
support = {R01 MH113670/MH/NIMH NIH HHS/United States ; T32HG008962//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01NS123849//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01MH113670//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01 NS123849/NS/NINDS NIH HHS/United States ; R21NS104963//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R21 CA279773/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Organoids/cytology ; *Telencephalon/cytology ; *Neural Stem Cells/cytology ; Nerve Tissue Proteins/genetics ; CRISPR-Cas Systems ; Neurons/cytology ; *Cell Culture Techniques/methods ; },
abstract = {We have developed a method for generating human telencephalic organoids from stem cell-derived isolated single neural rosettes. The use of single neural rosettes for generating organoids offers several important advantages. First, it mimics the development of neural tissue from a singular neural tube in vivo. Second, single neural rosette-derived organoids exhibit a relatively consistent and reproducible composition of telencephalic neural cells. Finally, single neural rosette-derived organoids demonstrate predictable organization of the identified neural cells around a single neural rosette-derived lumen and contain a large proportion of functionally mature neurons that generate action potentials and receive both excitatory and inhibitory synaptic inputs. These unique features of our protocol enable the study of the specification and organization of different neural cells in the developing human telencephalon, as well as modeling of neurodevelopmental disorders associated with disrupted neural networks. Here, we describe our protocols for generating CRISPR-Cas9-engineered human stem cells with a hemizygous SHANK3 deletion, stem cell-derived single neural rosettes and telencephalic brain organoids. We also offer insights on how to conduct single-cell RNA sequencing, immunohistochemistry and slice patch-clamp electrophysiology on these organoids. Completion of the protocols takes 5-6 months and requires experience working with cultured cells. We expect this protocol will prove useful for studies of human brain development and disease, as well as for advancing the development of new organoid-based biocomputers.},
}

Humans
*Organoids/cytology
*Telencephalon/cytology
*Neural Stem Cells/cytology
Nerve Tissue Proteins/genetics
CRISPR-Cas Systems
Neurons/cytology
*Cell Culture Techniques/methods

@article {pmid41652748,
year = {2026},
author = {Lei, Y and Ma, W and Wang, L and Chen, Y and Chen, X},
title = {Genomic insights into prophage and CRISPR-Cas system present in Lactobacillus delbrueckii subsp. bulgaricus strains.},
journal = {Food research international (Ottawa, Ont.)},
volume = {227},
number = {},
pages = {118213},
doi = {10.1016/j.foodres.2025.118213},
pmid = {41652748},
issn = {1873-7145},
mesh = {*Prophages/genetics ; *Lactobacillus delbrueckii/virology/genetics ; *CRISPR-Cas Systems/genetics ; Genomics ; Yogurt/microbiology ; Base Composition ; Genome, Bacterial ; Genome, Viral ; },
abstract = {As a key starter culture for yogurt fermentation, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) was subjected to bioinformatics analysis to investigate the distribution of prophages and the structure of the CRISPR-Cas system in 119 L. bulgaricus strains, and explored the targeting relationship between them and annotated the functional genes of targeted prophages. A total of 1704 prophage fragments were identified, of which 8.74 % (149/1704) were classified as complete prophages, none of them carried virulence factor genes and antibiotic resistance genes. Among all complete prophage genomes, the GC content was significantly affected by geographical sources (Asia, Europe, and North America) of host bacteria (P < 0.01). The 123 CRISPR-Cas systems detected in L. bulgaricus, type I-E (69) and type II-A (40) systems were predominant. Spacers-prophages targeting analysis revealed a negative correlation (P < 0.05), with 23.43 % (1055/4503) spacers showing homology to prophages. Functional gene annotation revealed that the diversity of prophage functional genes showed significant differences in under different grouping conditions (P < 0.05). Nevertheless, in-depth analysis of targeted prophage genomes revealed a conserved functional gene--ABC transporter. This study provides data support for exploring the genomic diversity and adaptive evolution mechanism of prophages in L. bulgaricus, and providing insights for screening phage-resistant strains and developing novel anti-phage strategies in dairy industry.},
}

*Prophages/genetics
*Lactobacillus delbrueckii/virology/genetics
*CRISPR-Cas Systems/genetics
Genomics
Yogurt/microbiology
Base Composition
Genome, Bacterial
Genome, Viral

@article {pmid41652223,
year = {2026},
author = {Zhang, J and Zhang, J and Tang, S and Liu, C and Cai, Y and Zeng, H and Meng, X and Liu, B and Zhang, Y and Wang, Y},
title = {Single-round evolution of RNA aptamers with GRAPE-LM.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41652223},
issn = {1546-1696},
support = {62302311//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82273967//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M742397//China Postdoctoral Science Foundation/ ; 2024T170585//China Postdoctoral Science Foundation/ ; },
abstract = {The directed evolution of biomolecules is an iterative process. Although advancements in language models have expedited protein evolution, effectively evolving RNA remains a challenge. RNA aptamers, selected for their binding properties, provide an ideal system to address this challenge, yet traditional aptamer discovery still relies on labor-intensive, multi-round screening. Here we introduce GRAPE-LM (generator of RNA aptamers powered by activity-guided evolution and language model), a generative artificial intelligence framework designed for the one-round evolution of RNA aptamers. GRAPE-LM integrates a transformer-based conditional autoencoder with nucleic acid language models and is guided by CRISPR-Cas-based aptamer screening data derived from intracellular environments. We validate GRAPE-LM on three disparate targets: the human T cell receptor CD3ε, the receptor-binding domain of the SARS-CoV-2 spike protein and the human oncogenic transcription factor c-Myc (an intracellular disordered protein). GRAPE-LM, informed with only a single round of CRISPR-Cas-based screening, successfully obtains RNA aptamers that outperform those driven from multiple rounds of human selection and optimization.},
}

@article {pmid41652115,
year = {2026},
author = {Omelchenko, DO and Barkovskaya, AM and Omelchenko, LV and Klepikova, AV and Penin, AA and Logacheva, MD},
title = {Optimization of functional genetics tools for a model tetraploid Capsella bursa-pastoris, with focus on homoeolog-aware gene editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {6},
pmid = {41652115},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Capsella/genetics/growth & development ; Tetraploidy ; *Plants, Genetically Modified/genetics/growth & development ; Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; },
abstract = {Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.},
}

*Gene Editing/methods
*Capsella/genetics/growth & development
Tetraploidy
*Plants, Genetically Modified/genetics/growth & development
Genome, Plant/genetics
Agrobacterium tumefaciens/genetics
CRISPR-Cas Systems

@article {pmid41652090,
year = {2026},
author = {Pan, X and Yildiz, U and Armstrong, SK and Bissonnette, K},
title = {Status and advancement of root-knot nematode management strategies and the emerging CRISPR/Cas biotechnology application.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {38},
pmid = {41652090},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Animals ; *Plant Diseases/parasitology/genetics/prevention & control ; Gene Editing ; *Tylenchoidea/pathogenicity/genetics ; Crops, Agricultural/genetics/parasitology ; Disease Resistance/genetics ; Plant Roots/parasitology/genetics ; Gossypium/parasitology/genetics ; },
abstract = {Root-knot nematodes (RKNs), Meloidogyne spp., exhibit a broad host range, threatening more than 3000 species of plants, including agriculturally important crops such as cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) and rice (Oryza sativa). Among the over 90 RKN species, the four most prevalent are M. incognita, M. arenaria, M. javanica, and M. hapla, with M. incognita being the most damaging. This paper reviewed the current RKN management strategies, including chemical nematicides, biological control, crop rotation, and resistant varieties, with a focus on the application of the revolutionary CRISPR/Cas genome editing tool in developing RKN resistance in plants. CRISPR/Cas has been widely utilized for improving crop traits due to its specificity, streamline, and inheritability. Recent progress has demonstrated the simplicity and robustness of CRISPR/Cas technology in improving plant traits. Among these, the development of nematode resistance by CRISPR/Cas knocking out of plant compatibility factors in model and commercial plants, has achieved significant progress. This review summarizes the RKN parasitism mechanisms and plant compatibility factors that would be promising CRISPR/Cas targets. The fundamentals and key aspects of CRISPR/Cas genome editing technology are addressed and discussed, and an example experimental pipeline for developing nematode resistance in cotton is described.},
}

*CRISPR-Cas Systems
Animals
*Plant Diseases/parasitology/genetics/prevention & control
Gene Editing
*Tylenchoidea/pathogenicity/genetics
Crops, Agricultural/genetics/parasitology
Disease Resistance/genetics
Plant Roots/parasitology/genetics
Gossypium/parasitology/genetics

@article {pmid41651998,
year = {2026},
author = {Natsagdorj, ME and Hara, H and Uosaki, H and Nakahara, F and Inoue, M and Hanazono, Y},
title = {ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {177},
pmid = {41651998},
issn = {2399-3642},
support = {JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP22bm0804018//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP18am0301002//Japan Agency for Medical Research and Development (AMED)/ ; JP22ae0201007//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics/metabolism ; *Dependovirus/genetics ; Animals ; Mice ; *Apoptosis/genetics ; *Gene Knock-In Techniques/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Morpholines/pharmacology ; Gene Editing/methods ; Mouse Embryonic Stem Cells/metabolism ; Thioxanthenes ; },
abstract = {CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.},
}

*Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics/metabolism
*Dependovirus/genetics
Animals
Mice
*Apoptosis/genetics
*Gene Knock-In Techniques/methods
DNA Breaks, Double-Stranded
CRISPR-Cas Systems
Morpholines/pharmacology
Gene Editing/methods
Mouse Embryonic Stem Cells/metabolism
Thioxanthenes

@article {pmid41651087,
year = {2026},
author = {Cao, W and Lu, J and Jiang, Y and Yuan, M and Wang, M and Mu, X and Gao, Z and Yang, S and Li, T and Wei, X and Guo, L},
title = {Recent advances in metabolic engineering of purple non‑sulfur photosynthetic bacteria for enhanced biohydrogen production.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108832},
doi = {10.1016/j.biotechadv.2026.108832},
pmid = {41651087},
issn = {1873-1899},
abstract = {Photo-fermentative hydrogen production from lignocellulosic biomass offers a sustainable and carbon-neutral route for bioenergy conversion, providing an effective strategy to mitigate fossil resource depletion and greenhouse gas emissions. Among diverse microbial candidates, purple non‑sulfur bacteria (PNSB) have emerged as promising photosynthetic platforms owing to their broad substrate utilization, intrinsic tolerance to lignocellulose-derived inhibitors, and high hydrogen yields under light-anaerobic conditions. Their metabolic versatility allows dynamic redistribution of carbon and electron fluxes, facilitating efficient energy conversion. Recent progress in metabolic engineering has substantially advanced the hydrogen-producing capacity of PNSB through targeted manipulation of photosynthetic metabolism, redox balance and stress response pathways. Engineering strategies have focused on optimizing pigment biosynthesis to enhance light harvesting, reinforcing redox homeostasis and adenosine triphosphate (ATP) generation, and improving tolerance to environmental stresses such as ammonia, pH, and temperature fluctuations. These efforts have led to engineered strains exhibiting extraordinary improvement in hydrogen yield, stability and robustness. This review provides an overview of the fundamental mechanisms underlying photo-fermentative hydrogen metabolism in PNSB, summarizes recent advances in the metabolic and systems-level engineering strategies, and outlines the prospects of developing strains capable of approaching the theoretical limit of hydrogen yield through integrated engineering strategies, advanced tools such as CRISPR-Cas, and adaptive laboratory evolution methods.},
}

@article {pmid41648912,
year = {2026},
author = {Wang, R and Li, J and Zheng, Y and Wei, N and Li, W},
title = {CRISPR/Cas12a platform activated by a protospacer adjacent motif-engineered DNA circuit for specific target sensing.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay02079d},
pmid = {41648912},
issn = {1759-9679},
abstract = {CRISPR/Cas systems have found extensive applications in nucleic acid diagnostics. However, the generalizability of this approach, particularly for the sensing of non-nucleic acid targets, remains a challenge. This study presents the development of a CRISPR/Cas12a platform activated by a protospacer adjacent motif (PAM)-engineered DNA circuit. Initially, the influence of the presence or absence of the PAM on the DNA circuit and the subsequent CRISPR/Cas12a system was investigated, demonstrating that a PAM-engineered DNA circuit functions as an effective activator of Cas12a, whereas a DNA circuit lacking the PAM does not induce activation. Subsequently, through the strategic design of recognition elements, sensitive and selective detection of specific targets is achieved, with limits of detection (LODs) of 0.023 fM for circulating tumor DNA (ctDNA), 0.00004 U mL[-1] for uracil-DNA glycosylase (UDG), and 0.12 fM for acetamiprid (ACE). This approach exemplifies a two-stage signal amplification mechanism, achieving improved sensitivity relative to either the CRISPR/Cas12a system or the DNA circuits alone. Moreover, quantitative assays for these targets were successfully conducted in real samples, suggesting the practical applicability of the proposed method. This research establishes a versatile sensing platform for various targets, which holds significant promise for advancements in molecular diagnostics, food safety assessment, and environmental monitoring.},
}

@article {pmid41648761,
year = {2025},
author = {Lu, Z and Bukhari, STH and Azeem, M and Tariq, N and Shabbir, MAB},
title = {A narrative review of precision and ethical considerations in cardiovascular health: CRISPR-Cas9, telemedicine, and lifestyle interventions.},
journal = {Frontiers in public health},
volume = {13},
number = {},
pages = {1737251},
pmid = {41648761},
issn = {2296-2565},
mesh = {Humans ; *Telemedicine/ethics ; *Cardiovascular Diseases/prevention & control/therapy ; *CRISPR-Cas Systems ; *Life Style ; *Precision Medicine ; Gene Editing ; },
abstract = {BACKGROUND: Cardiovascular diseases (CVDs) remain the leading cause of global morbidity and mortality, influenced by lifestyle, socioeconomic status, and genetic factors. Emerging innovations, including wearable health technologies, telemedicine, and CRISPR-Cas9 gene editing, provide new possibilities for rapid prevention and personalized management.

METHODS: This narrative review collected evidence from Scopus, PubMed, and Google Scholar, using keywords such as cardiovascular (CV) prevention, lifestyle determinants, digital health, telemedicine, CRISPR-Cas9, and public health ethics. Eligible peer-reviewed studies, clinical guidelines, and policy documents were included to assess behavioral, technological, and genomic strategies for CVD care.

RESULTS: Modifications in lifestyle, such as quitting smoking, regular physical activity, following a heart-healthy diet plan, and getting adequate sleep, can significantly reduce the risk of CVD. Additionally, telemedicine and wearable devices facilitate early detection, better self-management, and treatment adherence, especially in underserved communities. CRISPR-Cas9 holds a significant potential for correcting genetic variants related to lipid disorders and inherited cardiomyopathies, but its clinical translation remains in early stages. However, existing evidence is limited by heterogeneity in study design, brief follow-up, particularly for digital health and CRISPR applications. Additional challenges, such as health inequities, digital access, data privacy, and ethical oversight, further influence their real-world implementation.

CONCLUSION: Effective integration of behavioral, digital and genomic innovations requires policy frameworks that ensure equity, ethical governance, and long-term sustainability. Combining precision medicine with efforts to address social determinants of health will be crucial in reducing the global burden of CVD and shaping the future of CV care.},
}

Humans
*Telemedicine/ethics
*Cardiovascular Diseases/prevention & control/therapy
*CRISPR-Cas Systems
*Life Style
*Precision Medicine
Gene Editing

@article {pmid41648339,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.01.24.701482},
pmid = {41648339},
issn = {2692-8205},
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cas8 conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}

@article {pmid41637875,
year = {2026},
author = {Wu, H and Merényi, Z and Virágh, M and Liu, XB and Hegedüs, B and Hou, Z and Ábrahám, E and Fürtön, A and Kristóffy, Z and Lipinszki, Z and Nagy, LG},
title = {Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles.},
journal = {Microbiological research},
volume = {306},
number = {},
pages = {128462},
doi = {10.1016/j.micres.2026.128462},
pmid = {41637875},
issn = {1618-0623},
mesh = {*Transcription Factors/genetics/metabolism ; Fruiting Bodies, Fungal/growth & development/genetics ; *Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; *Agaricales/genetics/growth & development ; Morphogenesis/genetics ; CRISPR-Cas Systems ; Phylogeny ; Gene Expression Profiling ; Gene Deletion ; Evolution, Molecular ; Zinc Fingers ; },
abstract = {Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but its role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species' genome. Transcriptome analyses identified several misregulated genes in the Δgcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.},
}

*Transcription Factors/genetics/metabolism
Fruiting Bodies, Fungal/growth & development/genetics
*Fungal Proteins/genetics/metabolism
Gene Expression Regulation, Fungal
*Agaricales/genetics/growth & development
Morphogenesis/genetics
CRISPR-Cas Systems
Phylogeny
Gene Expression Profiling
Gene Deletion
Evolution, Molecular
Zinc Fingers

@article {pmid41553775,
year = {2026},
author = {Zheng, I and Learn, B and Bailey, S},
title = {Structural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.},
journal = {The Biochemical journal},
volume = {483},
number = {3},
pages = {289-300},
doi = {10.1042/BCJ20250364},
pmid = {41553775},
issn = {1470-8728},
support = {GM097330//HHS | National Institutes of Health (NIH)/ ; },
mesh = {*Streptococcus pyogenes/genetics/enzymology/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism/antagonists & inhibitors/genetics ; *CRISPR-Cas Systems ; *Viral Proteins/chemistry/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; Cryoelectron Microscopy ; *Bacteriophages/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Models, Molecular ; },
abstract = {CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-single-guide RNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the protospacer adjacent motif (PAM) duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple anti-CRISPRs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.},
}

*Streptococcus pyogenes/genetics/enzymology/metabolism
*CRISPR-Associated Protein 9/chemistry/metabolism/antagonists & inhibitors/genetics
*CRISPR-Cas Systems
*Viral Proteins/chemistry/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
Cryoelectron Microscopy
*Bacteriophages/metabolism/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Models, Molecular