@article {pmid41226739,
year = {2025},
author = {Arana, ÁJ and Veiga-Rua, S and Cora, D and Gónzalez-Gómez, MA and Seijas, A and Carballeda, M and Polo, D and Cuesta, A and Piñeiro, Y and Rivas, J and Novo, M and Al-Soufi, W and Martínez, P and Sánchez, L and Robledo, D},
title = {Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110703},
pmid = {41226739},
issn = {1422-0067},
support = {CRISPR-NANOXEN//Universidade de Santiago de Compostela/ ; Optimizing CRISPR/Cas9 genome editing to improve disease resistance in aquaculture//Fundación Caixa Rural Galega Tomás Notario Vacas/ ; investment line nº 1 and component number 17, which includes the Complementary RTDI Plan for Marine Science//Spain's Recovery and Resilience Plan/ ; No 101076432 (FishTRIM)//European Union ERC Starting Grant programme 2022/ ; (BBS/E/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A)//Oportunius programme of the Axencia Galega the Innovación (GAIN, Xunta de Galicia)/ ; RYC2023-044793-I//Ministerio de Ciencia, Innovación y Universidades/ ; 10.13039/501100011033//Agencia Estatal de Investigación/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line ; Electroporation/methods ; *Fishes/genetics ; Transfection/methods ; *Gene Transfer Techniques ; },
abstract = {Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies-electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)-for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Cell Line
Electroporation/methods
*Fishes/genetics
Transfection/methods
*Gene Transfer Techniques

@article {pmid41226719,
year = {2025},
author = {Amiri, S and Adibzadeh, S and Khazaei Monfared, Y and Kaboli, S and Arashkia, A and Barkhordari, F and Mahmoudian, M and Kheirandish, MH and Trotta, F and Davami, F},
title = {Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110682},
pmid = {41226719},
issn = {1422-0067},
support = {BD-66002183//Pasteur Institute of Iran/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Cyclodextrins/chemistry ; CHO Cells ; Cricetulus ; *Ribonucleoproteins/genetics ; Green Fluorescent Proteins/genetics ; *Nanostructures/chemistry ; },
abstract = {The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
*Cyclodextrins/chemistry
CHO Cells
Cricetulus
*Ribonucleoproteins/genetics
Green Fluorescent Proteins/genetics
*Nanostructures/chemistry

@article {pmid41226716,
year = {2025},
author = {Zhao, R and Zhu, J and Wang, J and Wang, D and Liu, X and Han, L and Li, S},
title = {Functional Characterization of Fp2Cas9, a Cold-Adapted Type II-C CRISPR Nuclease from Flavobacterium psychrophilum.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110681},
pmid = {41226716},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 2024XT01, 2023TD45//the Central Public Interest Scientific Institution Basal Research Fund, CAFS/ ; Identification, Evaluation, and Breeding of Characteristic Aquatic Animal Germplasm Resources in Heilongjiang Province.//Heilongjiang Province Seed Industry Innovation and Development Fund Project/ ; },
mesh = {*Flavobacterium/enzymology/genetics ; Zebrafish/genetics ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Cold Temperature ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5'-SNAAAG-3', and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems.},
}

*Flavobacterium/enzymology/genetics
Zebrafish/genetics
Gene Editing/methods
*CRISPR-Cas Systems
Animals
Cold Temperature
*CRISPR-Associated Protein 9/metabolism/genetics
*Bacterial Proteins/genetics/metabolism

@article {pmid41226505,
year = {2025},
author = {Qian, Y and Liu, X and Wang, B and Li, D and Wu, Z and Tong, J},
title = {Genome-Wide Identification of the LsaPHR1 Gene Family and Preliminary Functional Validation of LsaPHR1.1 in Phosphorus Tolerance in Lactuca sativa.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110466},
pmid = {41226505},
issn = {1422-0067},
support = {KJCX20240508//Special Innovation Ability Construction Fund of Beijing Academy of Agricultural and Forestry Sciences/ ; QNJJ202248//Beijing Academy of Agriculture and Forestry Science Youth Research Fund/ ; KYCX202506//Innovation and Development Program of Beijing Vegetable Research Center/ ; CARS-24-B-02//China Agriculture Research System of the MOF and MARA/ ; KJCX20200205//Project for Scientific and Technological Innovation Capability Building of the Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Lactuca/genetics/metabolism ; *Phosphorus/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; *Multigene Family ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; },
abstract = {Phosphorus (P) is a limiting nutrient for plant growth and productivity. Improving P use efficiency is important for crop production. In Lactuca sativa (lettuce), five phosphate starvation response 1 (PHR1) genes were identified and characterized through a bioinformatics approach. The expression patterns of LsaPHR1s were examined using qRT-PCR under various treatments, including devoid phosphorus (DP), low phosphorus (LP), high phosphorus (HP), darkness, ABA, IAA, and MeJA. The results indicate that LsaPHR1s in lettuce responded to phosphorus stress, hormones, and darkness. Furthermore, we engineered LsaPHR1.1 knock-out mutants via CRISPR/Cas9-mediated genome editing. Then, the mutants were subjected to phosphorus stress (DP, LP, and HP). In contrast to WT, the mutants improved nitrate and ammonium contents, increased antioxidant enzyme activity, and elevated antioxidant and chlorophyll contents. Our results offer a potential strategy for improving phosphorus stress tolerance in lettuce, which holds great significance for maintaining yield and quality.},
}

*Lactuca/genetics/metabolism
*Phosphorus/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Stress, Physiological/genetics
*Multigene Family
CRISPR-Cas Systems
Chlorophyll/metabolism
Gene Editing

@article {pmid41225502,
year = {2025},
author = {Huang, S and Zhou, F and Jiang, Z and Jiang, S and Yang, Q and Yang, L and Huang, J and Shi, J and Ding, Y and Li, E and Li, Y},
title = {Development and validation of PmMAD7 for efficient gene editing in Penaeus monodon.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {125},
pmid = {41225502},
issn = {1472-6750},
support = {2022YFD2401900//National Key R & D Program of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Penaeidae/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing has become a promising approach for enhancing traits in aquaculture species. Nevertheless, traditional CRISPR-Cas systems encounter challenges, including significant off-target effects and strict protospacer adjacent motif (PAM) requirements, which constrain their use in crustaceans such as Penaeus monodon. To address these limitations, this research has developed PmMAD7, a codon-optimized CRISPR system specifically designed for P. monodon, which incorporates nuclear localization signals to improve editing efficiency and precision.

RESULTS: This research successfully synthesized and delivered PmMAD7 mRNA and crRNAs targeting the ECH1 and AQP4 genes into the hemocytes of P. monodon. Quantitative PCR analysis demonstrated that PmMAD7 achieved significant gene silencing, reducing the expression levels of ECH1 and AQP4 by 81.5% and 78.33%, respectively. Next-generation sequencing confirmed targeted insertions and deletions at the gene loci, with knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4, which were significantly higher than those obtained with LbCas12a (7.14% and 12.43%, respectively). Furthermore, functional analysis indicated that ECH1 knockout resulted in increased cell volume and mortality, while AQP4 knockout led to decreased cell volume and reduced viability. These specific results highlight the first successful demonstration of MAD7-based genome editing in shrimp. The broader PAM compatibility and enhanced editing efficiency of PmMAD7 provide a versatile platform for gene editing in shrimp.

CONCLUSION: PmMAD7 constitutes an enhanced CRISPR editing tool specifically designed for P. monodon, exhibiting superior precision, expanded PAM compatibility, and enhanced editing efficacy relative to conventional Cas12a systems. These results lay the groundwork for the advancement of gene editing applications in crustaceans and contribute to sustainable genetic improvements in aquaculture.},
}

Animals
*Gene Editing/methods
*Penaeidae/genetics
*CRISPR-Cas Systems/genetics

@article {pmid41223249,
year = {2025},
author = {Pan, X and Huang, P and Ali, SS and Renslo, B and Greenberg, Z and Erwin, N and Li, Y and Ding, Z and Hutchinson, TE and Warnecke, A and Fernandez, NE and Staecker, H and He, M},
title = {Extracellular vesicle-mediated gene editing for the treatment of nonsyndromic progressive hearing loss in adult mice.},
journal = {Science translational medicine},
volume = {17},
number = {824},
pages = {eadn3993},
doi = {10.1126/scitranslmed.adn3993},
pmid = {41223249},
issn = {1946-6242},
mesh = {Animals ; *Extracellular Vesicles/metabolism ; *Gene Editing/methods ; *Hearing Loss/therapy/genetics ; Mice ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Genetic Therapy/methods ; Ribonucleoproteins/metabolism ; Hair Cells, Auditory/metabolism ; Electroporation ; Humans ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current-controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the Myo7a[WT/Sh1] mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old Myo7a[WT/Sh1] mice resulted in a reduction in Myo7a[Sh1] messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only-injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.},
}

Animals
*Extracellular Vesicles/metabolism
*Gene Editing/methods
*Hearing Loss/therapy/genetics
Mice
CRISPR-Cas Systems/genetics
Disease Models, Animal
Genetic Therapy/methods
Ribonucleoproteins/metabolism
Hair Cells, Auditory/metabolism
Electroporation
Humans
RNA, Guide, CRISPR-Cas Systems/metabolism

@article {pmid41223195,
year = {2025},
author = {Kimata, K and Satou, K},
title = {Improved CRISPR/Cas9 off-target prediction with DNABERT and epigenetic features.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335863},
pmid = {41223195},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Epigenesis, Genetic ; *Gene Editing/methods ; Genome, Human ; Deep Learning ; },
abstract = {CRISPR/Cas9 is a powerful genome editing tool, but its clinical application is hindered by off-target effects. Accurate computational prediction of these unintended edits is crucial for ensuring the safety and efficacy of therapeutic applications. While various deep learning models have been developed, most are trained only on task-specific data, failing to leverage the vast knowledge embedded in entire genomes. To address this limitation, we introduce a novel approach that integrates DNABERT, a deep learning model pre-trained on the human genome, with epigenetic features (H3K4me3, H3K27ac, and ATAC-seq). We conducted a comprehensive benchmark of our model, DNABERT-Epi, against five state-of-the-art methods across seven distinct off-target datasets. Our results demonstrate that the pre-trained DNABERT-based models achieve competitive or even superior performance. Rigorous ablation studies quantitatively confirmed that both genomic pre-training and the integration of epigenetic features are critical factors that significantly enhance predictive accuracy. Furthermore, by applying advanced interpretability techniques (SHAP and Integrated Gradients), we identified the specific epigenetic marks and sequence-level patterns that influence the model's predictions, offering insights into its decision-making process. This study is the first to establish the significant potential of a pre-trained DNA foundation model for CRISPR/Cas9 off-target prediction. Our findings underscore that leveraging both large-scale genomic knowledge and multi-modal data is a key strategy for advancing the development of safer genome editing tools.},
}

*CRISPR-Cas Systems/genetics
Humans
*Epigenesis, Genetic
*Gene Editing/methods
Genome, Human
Deep Learning

@article {pmid41222688,
year = {2025},
author = {Yuan, A and Sun, T and Deng, Z and Ye, L and Shang, Y and Xie, W and Peng, H},
title = {A multifunctional approach: merging CRISPR/Cas technology with DNA nanomachines for advanced biosensing.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {41222688},
issn = {1618-2650},
support = {2023YFA0915102//National Key Research and Development Program of China/ ; 22276199//National Natural Science Foundation of China/ ; 22306195//National Natural Science Foundation of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
abstract = {CRISPR/Cas systems have revolutionized nucleic acid recognition, offering unparalleled programmability for biosensing. Simultaneously, DNA nanomachines have emerged as powerful bioanalytical tools due to their structural precision, biocompatibility, and multifunctional capabilities. The strategic integration of these platforms synergistically enhances sensitivity, specificity, and multiplexing potential, creating next-generation bioanalytical systems. While promising, this convergence presents unique engineering challenges and necessitates critical evaluation. This review systematically analyzes the target recognition mechanisms of CRISPR/Cas systems and critically evaluates prevalent signal readout modalities. A core focus is the critical assessment of innovative biosensing strategies that leverage DNA nanomachines-particularly dynamic walker systems-coupled with CRISPR/Cas activation. We highlight representative integrated platforms, detailing their operational principles and objectively examining their demonstrated advantages against inherent limitations such as signal leakage, complex assembly, and in vivo applicability constraints. By providing a critical analysis of the synergistic potential and current constraints of CRISPR/Cas-DNA nanomachine integration, this review aims to guide rational design towards robust, clinically translatable precision diagnostics and therapeutics.},
}

@article {pmid41221123,
year = {2025},
author = {van Karnebeek, CDM and Gailus-Durner, V and Engelke, UF and Seisenberger, C and Marschall, S and Dragano, NRV and da Silva-Buttkus, P and Leuchtenberger, S and Fuchs, H and Hrabě de Angelis, M and Wevers, RA and Coughlin, CR and Lefeber, DJ},
title = {New treatment for pyridoxine-dependent epilepsy due to ALDH7A1 deficiency: first proof-of-principle of upstream enzyme inhibition in the mouse.},
journal = {Brain communications},
volume = {7},
number = {6},
pages = {fcaf397},
pmid = {41221123},
issn = {2632-1297},
abstract = {Pyridoxine-dependent epilepsy (PDE) due to recessive ALDH7A1 mutations is characterized by intractable epilepsy that is often unresponsive to antiseizure medications. Irrespective of pyridoxine (vitamin B6) supplementation and lysine reduction therapy, patients present severe residual neurocognitive deficits. We evaluated upstream inhibition of 2-aminoadipic semialdehyde synthase (AASS) as a novel therapeutic strategy to reduce the accumulating metabolites (α-aminoadipic semialdehyde, Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) considered neurotoxic. We utilized an existing mouse knockout model of hyperlysinaemia (Aass-knockout) and generated a PDE model, a Aldh7a1 single knockout model via CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) and generated the double-knockout Aass/Aldh7a1 mice. Next-generation metabolomics screening was performed to measure all known biomarkers in brain, liver and plasma of wild-type and mutant mice. Metabolomics confirmed the known metabolite markers for Aldh7a1-knockout and Aass knockout mice in all samples. The potentially neurotoxic metabolites (Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) significantly decreased in double-knockout Aass/Aldh7a1 mice brain and liver tissues compared to Aldh7a1-knockout mice. Plasma analysis revealed a significant reduction of known biomarkers, suggesting a reliable monitoring option in human patients. We demonstrate the first mammalian evidence that AASS inhibition is a viable strategy to rescue abnormal brain metabolism associated with PDE. This may target the intellectual disability and neurologic deficits caused by persistent lysine catabolic-related neurotoxicity despite adequate vitamin B6 supplementation.},
}

@article {pmid41220980,
year = {2025},
author = {Yuan, X and Yu, X and Zhao, W and Sun, J},
title = {A Practical CRISPR-Based Method for Rapid Genome Editing in Caulobacter crescentus.},
journal = {Bio-protocol},
volume = {15},
number = {21},
pages = {e5490},
pmid = {41220980},
issn = {2331-8325},
abstract = {The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example. Key features • CRISPR-SpCas9M-reporting system overcomes the limitation of CRISPR escape and achieves a high apparent editing efficiency. • The method enables multiple kinds of genome editing, generating in-frame and marker-less chromosomal modifications. • The method completes a cycle of genome editing within one week. • The method could be readily applied for genome editing in C. crescentus, A. fabrum, and S. meliloti.},
}

@article {pmid41219964,
year = {2025},
author = {George, NA and Zhou, Z and Anantharaman, K and Hug, LA},
title = {Discarded diversity: novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {370},
pmid = {41219964},
issn = {1743-422X},
support = {R35GM143024/GM/NIGMS NIH HHS/United States ; 2016-03686//Natural Sciences and Engineering Research Council of Canada/ ; Tier II chair//Canada Research Chairs/ ; },
mesh = {*Bacteriophages/genetics/classification/isolation & purification ; *CRISPR-Cas Systems ; *Waste Disposal Facilities ; Metagenomics ; Archaea/virology/genetics ; *Bacteria/virology/genetics ; Genome, Viral ; Host Microbial Interactions ; Computational Biology ; North America ; },
abstract = {BACKGROUND: Viruses are the most abundant microbial entities on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering bacteria and archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of bacteria and archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America.

RESULTS: We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~ 678 kbp), and noted a large diversity of viruses in landfills that has limited overlap across landfills and is distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect hosts across multiple phyla. Auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in the literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector-like proteins appear distinct relative to previously reported Cas effectors and are interesting targets for potential genome editing tools.

CONCLUSIONS: Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.},
}

*Bacteriophages/genetics/classification/isolation & purification
*CRISPR-Cas Systems
*Waste Disposal Facilities
Metagenomics
Archaea/virology/genetics
*Bacteria/virology/genetics
Genome, Viral
Host Microbial Interactions
Computational Biology
North America

@article {pmid41219611,
year = {2025},
author = {Javaid, MM and Ahmed, J and Ahmed, M and Awan, MJA and Waqas, MAB and Ali, Z and Saeed, NA},
title = {CRISPR-Cas9 based editing of the susceptibility allele TaLr34 enhances leaf rust resistance in bread wheat without yield penalty.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {236},
pmid = {41219611},
issn = {1438-7948},
mesh = {*Triticum/genetics/microbiology/growth & development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; *Gene Editing ; Alleles ; *Plant Proteins/genetics/metabolism ; Basidiomycota/pathogenicity ; Plant Leaves/microbiology/genetics ; Plant Breeding ; },
abstract = {Wheat (Triticum aestivum L.) is widely grown and consumed cereal crop around the world, but most wheat-producing regions suffer from rust diseases, especially stripe and leaf rust, which has caused a devastating global pandemic and severely reduced grain yields. The most effective way to control rust problem in wheat is to sow and breed durable, rust resistant wheat varieties. Conventional breeding for disease-resistant crops primarily relies on resistance (R) genes; however, the effectiveness of R gene-mediated resistance is often compromised by mutations in the pathogen. In this study, we employed CRISPR-Cas9-based genome editing as an advanced breeding tool to enhance rust resistance in the bread wheat cultivar Galaxy-13 by knocking out the homologs of the susceptibility allele TaLr34, specifically targeting the conserved regions within exon 11. Out of 21 transformed plants, five carried successful editing and exhibited resistance to moderate resistance against leaf rust. The TaLr34 mutants were evaluated for leaf rust resistance under both glasshouse and field conditions over three consecutive growing seasons at multiple geographical locations. Our results demonstrate that CRISPR-Cas9-mediated knockout of TaLr34 provides a robust strategy for achieving durable leaf rust resistance in the high-yielding elite wheat cultivar Galaxy-13 without compromising grain yield and agronomic performance.},
}

*Triticum/genetics/microbiology/growth & development
*CRISPR-Cas Systems
*Disease Resistance/genetics
*Plant Diseases/genetics/microbiology
*Gene Editing
Alleles
*Plant Proteins/genetics/metabolism
Basidiomycota/pathogenicity
Plant Leaves/microbiology/genetics
Plant Breeding

@article {pmid41218116,
year = {2025},
author = {Wang, X and Cramer, Z and Leu, NA and Monaghan, K and Durning, K and Adams-Tzivelekidis, S and Rhoades, JH and Heintz, J and Tian, Y and Rico, J and Mendez, D and Petroni, R and King, AC and Kim, MS and Matsuda, R and Hanselman, O and Shin, AE and Carrera Rodríguez, MF and Brodsky, IE and Rustgi, A and Li, N and Lengner, CJ and Andrés Blanco, M},
title = {Forward genetic screening in engineered colorectal cancer organoids identifies regulators of metastasis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {46},
pages = {e2510910122},
doi = {10.1073/pnas.2510910122},
pmid = {41218116},
issn = {1091-6490},
support = {P30-DK050306/GF/NIH HHS/United States ; F31CA250267-02/GF/NIH HHS/United States ; F31AI160741-01/GF/NIH HHS/United States ; R01 CA279317-01/GF/NIH HHS/United States ; },
mesh = {*Colorectal Neoplasms/genetics/pathology ; Animals ; *Organoids/pathology/metabolism ; Mice ; Humans ; *Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; *Genetic Testing/methods ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; *Adenocarcinoma/genetics/pathology ; },
abstract = {Metastatic outgrowth requires that cancer cells delaminate from the primary tumor, intravasate, survive in circulation, extravasate, migrate to, and proliferate at a distal site. Recurrent genetic drivers of metastasis remain elusive, suggesting that unlike the early steps of oncogenesis, metastasis drivers may be variable. We develop a framework for identifying metastasis regulators using CRISPR/Cas9-based screening in a genetically defined organoid model of colorectal adenocarcinoma. We conduct in vitro screens for invasion and migration alongside orthotopic, in vivo screens for gain of metastasis in a syngeneic mouse model. We identify CTNNA1 and BCL2L13 as bona fide metastasis-specific suppressors which do not confer any selective advantage in primary tumors. CTNNA1 loss promotes cell invasion and migration, and BCL2L13 loss promotes anchorage-independent survival and non-cell-autonomous changes to macrophage polarization. This study demonstrates proof of principle that large-scale genetic screening can be performed in tumor-organoid models in vivo and identifies novel regulators of metastasis.},
}

*Colorectal Neoplasms/genetics/pathology
Animals
*Organoids/pathology/metabolism
Mice
Humans
*Neoplasm Metastasis/genetics
CRISPR-Cas Systems
*Genetic Testing/methods
Cell Movement/genetics
Cell Line, Tumor
Neoplasm Invasiveness
*Adenocarcinoma/genetics/pathology

@article {pmid41215577,
year = {2025},
author = {Uombe, NPI and Velikkakam, T and Silveira, ACA and Rodrigues, CC and Borges, BC and Teixeira, TL and Pereira, CL and Servato, JPS and Melo, NS and Mortara, RA and Silveira, JFD and Silva, CVD},
title = {Trypanosoma cruzi P21 Is a Pleiotropic Protein That Is Involved in Parasite Host Cell Invasion and Intracellular Parasitism.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70154},
pmid = {41215577},
issn = {2045-8827},
support = {//This study was supported by grants and fellowships from FAPESP, FAPEMIG, CAPES, and CNPq. FAPESP Grant number: 2016/15000-4 and 2019/05049-4 (Postdoctoral fellowship); FAPEMIG - RED-00198-23./ ; },
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/growth & development/physiology/metabolism ; Animals ; *Chagas Disease/parasitology ; Vero Cells ; Chlorocebus aethiops ; Mice ; *Host-Parasite Interactions ; *Protozoan Proteins/genetics/metabolism ; Gene Knockout Techniques ; Virulence ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {We characterized the secreted Trypanosoma cruzi P21 protein and hypothesized its role in parasite invasion and multiplication. To investigate the role of T. cruzi P21 protein in host-parasite interactions, specifically focusing on the low-virulence G strain. P21 knockout parasites were generated using CRISPR/Cas9. Cell invasion, multiplication, egress, and tissue parasitism were assessed in vitro and in vivo, comparing knockout and control parasites. P21 knockout significantly reduced parasite invasion and multiplication in Vero cells. In vivo, knockout parasites also showed reduced heart tissue parasitism in infected mice, despite no observable systemic parasitemia. Accordingly, P21 knockout trypomastigote egress was reduced in Vero cells. P21 plays a pleiotropic role in T. cruzi infection, differentially impacting parasite biology in the low-virulent G strain. In the G strain, P21 promotes invasion and persistence, potentially through mechanisms distinct from its role in the Y strain previously described. This highlights its potential as a therapeutic target for Chagas disease, warranting further investigation into strain-specific functions.},
}

*Trypanosoma cruzi/genetics/pathogenicity/growth & development/physiology/metabolism
Animals
*Chagas Disease/parasitology
Vero Cells
Chlorocebus aethiops
Mice
*Host-Parasite Interactions
*Protozoan Proteins/genetics/metabolism
Gene Knockout Techniques
Virulence
Disease Models, Animal
CRISPR-Cas Systems

@article {pmid41185939,
year = {2025},
author = {Kong, H and Liu, X and Xia, K and Guan, Y and Zhang, J and Zhang, Y and Liu, B and Ge, Z and Li, Q and Zuo, X and Wang, L and Li, J and Zhu, Y and Fan, C},
title = {Magnetically Driven High-Speed Rolling Nanoclusters for Enhanced CRISPR/Cas9 Genome Editing.},
journal = {ACS applied materials & interfaces},
volume = {17},
number = {45},
pages = {61707-61717},
doi = {10.1021/acsami.5c15498},
pmid = {41185939},
issn = {1944-8252},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Magnetite Nanoparticles/chemistry ; Plasmids/genetics/chemistry/metabolism ; Jurkat Cells ; Protein Serine-Threonine Kinases/genetics ; Cell Cycle Proteins/genetics ; Viscosity ; },
abstract = {Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe3O4 nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe3O4 nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Magnetite Nanoparticles/chemistry
Plasmids/genetics/chemistry/metabolism
Jurkat Cells
Protein Serine-Threonine Kinases/genetics
Cell Cycle Proteins/genetics
Viscosity

@article {pmid41183022,
year = {2025},
author = {Rajamani, S and Vilchez, L and Cracovia, N and Dule, D and Vata, A and Landaverde, S and Iyengar, A and Dubrovsky, EB},
title = {Mitochondrial-specific perturbation of Drosophila RNase Z in neurons leads to motor impairments, disrupted learning and neurodegeneration.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011938},
doi = {10.1371/journal.pgen.1011938},
pmid = {41183022},
issn = {1553-7404},
mesh = {Animals ; *Mitochondria/genetics/metabolism/pathology ; *Neurons/metabolism/pathology ; *Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; *Endoribonucleases/genetics/metabolism ; Mutation ; Learning/physiology ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology ; Animals, Genetically Modified ; Reactive Oxygen Species/metabolism ; },
abstract = {Clinical studies have linked a rare form of neurological disorder to the highly conserved RNase Z gene, which encodes an endoribonuclease responsible for the processing of nuclear and mitochondrial primary tRNA transcripts. Patients harboring mutant variants of this gene exhibit a spectrum of neurological dysfunction; however, no studies to date have established the causality of RNase Z-linked neuropathology. We employed CRISPR/Cas9 technology to create flies with a neuron-specific knockout of the RNase Z gene, which is rescued with transgenes encoding a wild-type or a mutant copy of RNase Z. Neuronal activity of RNase Z is vital, as mutants display striking morphological abnormalities in central and peripheral neurons, along with attenuated motor circuit function and associative learning performance. Neuron-specific mutations of RNase Z also led to mitochondrial fragmentation and elevated ROS production. By employing the rescue transgene encoding RNase Z devoid of a mitochondrial targeting signal (MTS), we segregated the mitochondrial activity of RNase Z from that in other compartments, allowing us to assess organelle-specific contributions to disease etiology and progression. We found that mutating mitochondrial RNase Z was sufficient to induce the neuropathology in flies, as they recapitulate the salient phenotypes observed in the pan-neuronal mutants. Collectively, our study validates the pathogenicity of mutant RNase Z and establishes mitochondrial-specific contributions to neuropathology.},
}

Animals
*Mitochondria/genetics/metabolism/pathology
*Neurons/metabolism/pathology
*Drosophila Proteins/genetics/metabolism
Drosophila melanogaster/genetics
*Endoribonucleases/genetics/metabolism
Mutation
Learning/physiology
CRISPR-Cas Systems
*Neurodegenerative Diseases/genetics/pathology
Animals, Genetically Modified
Reactive Oxygen Species/metabolism

@article {pmid41170612,
year = {2025},
author = {Pu, J and Zhou, S and Ma, W and Liu, C and Guo, L and Yan, B and Huo, Z},
title = {Light-controlled CRISPR-Cas12a one-pot platform for ultrasensitive cell-free DNA detection in systemic lupus erythematosus diagnosis.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {44},
pages = {8916-8922},
doi = {10.1039/d5ay01286d},
pmid = {41170612},
issn = {1759-9679},
mesh = {*Lupus Erythematosus, Systemic/diagnosis/blood/genetics ; Humans ; *Cell-Free Nucleic Acids/blood/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Systemic lupus erythematosus (SLE), as a complex autoimmune disease with heterogeneous clinical manifestations, presents significant challenges for early diagnosis. Circulating cell-free DNA (cfDNA) has emerged as a promising disease monitoring biomarker due to its correlation with SLE pathological progression in terms of concentration and fragmentation patterns. However, existing detection methods lack sufficient sensitivity and practicality for clinical application. To address this, we developed a spatiotemporally resolved light-controlled biosensor by integrating a photoactivatable CRISPR-Cas12a system with TdT-mediated poly-A tail extension, achieving three major innovations: (1) implementation of NPOM-dt modified crRNA for precise regulation; (2) optimization of 365 nm UV activation protocol to eliminate interference in one-pot reactions; and (3) establishment of a three-phase "Extension-Activation-Detection" workflow. The platform demonstrates outstanding performance with a detection limit of 0.42 pM, excellent linearity (R[2] = 0.9956) in the 0-0.1 nM range, and the novel DNA Integrity Index (DII) as a diagnostic indicator - showing significantly higher values in SLE patients (9.82 × 10[3] nmol g[-1]) versus healthy controls (4.2 × 10[3] nmol g[-1], P < 0.0001) with an AUC of 0.8947. This study provides an innovative detection platform combining high sensitivity with clinical utility for early SLE diagnosis.},
}

*Lupus Erythematosus, Systemic/diagnosis/blood/genetics
Humans
*Cell-Free Nucleic Acids/blood/genetics/analysis
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Limit of Detection

@article {pmid41165493,
year = {2025},
author = {Zhang, J and Li, X and Wang, M and Ren, S and Li, M and Liu, Y and Lu, F and Li, Q and Li, Y},
title = {Rational Construction of a Robust Bacillus amyloliquefaciens Cell Factory for Acid-Stable α Amylase Production.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28883-28895},
doi = {10.1021/acs.jafc.5c04227},
pmid = {41165493},
issn = {1520-5118},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism/enzymology ; *Bacterial Proteins/genetics/metabolism/chemistry ; *alpha-Amylases/genetics/chemistry/metabolism/biosynthesis ; Fermentation ; Gene Editing ; Prophages/genetics/metabolism ; Enzyme Stability ; CRISPR-Cas Systems ; Multigene Family ; },
abstract = {With the development of synthetic biology and biotechnology, chassis engineering has become the main means of industrial protein production, but it has been limited by the lack of efficient gene editing methods and effective engineering strategies. Bacillus amyloliquefaciens shows potential for expressing heterologous proteins, but its cells undergo early autolysis, hindering further application. In this study, an autolysis-related prophage gene cluster was rationally deleted by establishing an efficient CRISPR-nCas9 editing process, and the prophage mutant strain was constructed, which prevented cell lysis. Based on the prophage mutant strain, we screened secondary metabolite biosynthetic gene clusters that hindered the expression of heterologous proteins, and we made reasonable deletions to further improve their efficient expression. Finally, an optimized yield of acid-stable α amylase (2,46,089.21 U/mL) was obtained in a 5-L fed-batch fermentation. Therefore, we successfully constructed an ideal candidate strain for the expression of heterologous proteins, which provides an important research basis for the development of more chassis strains.},
}

*Bacillus amyloliquefaciens/genetics/metabolism/enzymology
*Bacterial Proteins/genetics/metabolism/chemistry
*alpha-Amylases/genetics/chemistry/metabolism/biosynthesis
Fermentation
Gene Editing
Prophages/genetics/metabolism
Enzyme Stability
CRISPR-Cas Systems
Multigene Family

@article {pmid41144643,
year = {2025},
author = {Huang, C and Guo, LJ and Li, JY and Chen, YW and Wu, B and Liu, YW and Zhang, CZ and Lin, WW and Yang, Y and Chen, JY and Liu, ZJ},
title = {A CRISPR-Cas12a Fluorescent Aptasensor for Point-of-Care Drug Concentration Detection: A Synergistic Regulation by DNA Aptamer and RNA Blocker.},
journal = {Analytical chemistry},
volume = {97},
number = {44},
pages = {24415-24424},
doi = {10.1021/acs.analchem.5c03744},
pmid = {41144643},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Vancomycin/analysis/blood ; Limit of Detection ; *RNA/chemistry/antagonists & inhibitors ; Humans ; *Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10-250 nM (R[2] = 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection.},
}

*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Point-of-Care Systems
*Vancomycin/analysis/blood
Limit of Detection
*RNA/chemistry/antagonists & inhibitors
Humans
*Fluorescent Dyes/chemistry
*Anti-Bacterial Agents/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41122778,
year = {2025},
author = {Hasselbeck, S and Wang, J and Bai, Z and Hüfner, T and Hummer, G and Grote, P and Cheng, X},
title = {A Chemically Induced CRISPR/dCas13[FCPF] Platform for Precise and Programmable RNA Regulation.},
journal = {Journal of medicinal chemistry},
volume = {68},
number = {21},
pages = {22633-22649},
doi = {10.1021/acs.jmedchem.5c01609},
pmid = {41122778},
issn = {1520-4804},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *RNA/genetics/metabolism ; Alternative Splicing/drug effects ; Survival of Motor Neuron 2 Protein/genetics ; HEK293 Cells ; Exons ; },
abstract = {Alternative splicing enhances proteomic diversity, yet its dysregulation drives cancer, neurodegeneration, and inherited disease. Small-molecule splicing modulators, while clinically validated, like risdiplam, often lack locus specificity, producing off-target effects. CRISPR/Cas13 enables programmable transcript-level targeting, but dCas13 fusion effectors are bulky and can hamper delivery and RNA homeostasis. Building on our previous Chem-CRISPR/dCas9[FCPF] system for epigenome editing, we now introduce Chem-CRISPR/dCas13[FCPF], a modular platform that covalently tethers a perfluorobiphenyl-tagged small molecule to dCas13 via a four-residue FCPF π-clamp tag. Guided by crRNAs, dRfxCas13d[FCPF] recruits a risdiplam-derived conjugate to the SMN2 exon 7 splice region, inducing exon inclusion at ligand doses ∼500-fold lower than those of free risdiplam and with no detectable effects at known risdiplam-sensitive transcripts in our assays. The approach generalizes to additional transcripts by crRNA redesign. By coupling CRISPR addressability with dose-sparing chemical action, Chem-CRISPR/dCas13[FCPF] establishes a proximity-induced, chemically controllable route to precise RNA modulation suitable for therapeutic exploration.},
}

Humans
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*RNA/genetics/metabolism
Alternative Splicing/drug effects
Survival of Motor Neuron 2 Protein/genetics
HEK293 Cells
Exons

@article {pmid41108054,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {A systematic review of CRISPR applications in demyelinating peripheral nervous system disorders.},
journal = {Regenerative medicine},
volume = {20},
number = {11},
pages = {653-662},
pmid = {41108054},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Demyelinating Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Peripheral Nervous System Diseases/therapy/genetics ; Charcot-Marie-Tooth Disease/therapy/genetics ; Schwann Cells/metabolism/pathology ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {AIM: This review evaluates CRISPR-based strategies for myelin regeneration in peripheral demyelinating disorders, with a focus on Guillain - Barré syndrome (GBS) and Charcot - Marie - Tooth disease type 1A (CMT1A). It aims to identify current therapeutic approaches, delivery systems, and gaps in the literature.

MATERIALS & METHODS: A systematic literature search was conducted across PubMed, Scopus, Web of Science, and Google Scholar for studies published from 2010 onward, following PRISMA guidelines. Study quality was assessed using OHAT and SYRCLE tools, and 14 articles met the inclusion criteria.

RESULTS: In GBS, CRISPR interventions primarily targeted antiviral immune regulation (AXL, IFI6, IFNL2), inhibition of viral entry mechanisms (Integrin αvβ5, SPCS1), and Schwann cell repair. In CMT1A, therapeutic approaches focused on correcting PMP22 overexpression. Lentiviral transduction was the most frequently used delivery method, with no major adverse effects reported. However, most studies were in vitro, and only two were in vivo, highlighting the need for further validation in animal models.

CONCLUSIONS: CRISPR technology shows considerable potential for addressing peripheral nerve demyelination through precise genetic modifications that may enhance Schwann cell function and support myelin repair. Nevertheless, the field remains at an early discovery stage, with no near-term clinical applicability demonstrated.},
}

Humans
Animals
*Demyelinating Diseases/therapy/genetics
*CRISPR-Cas Systems/genetics
Genetic Therapy/methods
*Peripheral Nervous System Diseases/therapy/genetics
Charcot-Marie-Tooth Disease/therapy/genetics
Schwann Cells/metabolism/pathology
Gene Editing
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41061783,
year = {2025},
author = {Ma, Y and Zhao, Q and Yuan, J and Wang, D and Chen, X and Yu, Y and Li, J and Yu, M and Yuan, J and Lou, J and Du, S and Wen, Y and Wang, Y and Wu, R and Yan, QG and Huang, X and Zheng, Y and Zhao, F and Cao, SJ},
title = {RIPK4-p53 interaction drives aflatoxin B1-induced renal mitochondrial apoptosis via Ser15 phosphorylation: A CRISPR-Cas9 mechanistic study.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 3},
pages = {148130},
doi = {10.1016/j.ijbiomac.2025.148130},
pmid = {41061783},
issn = {1879-0003},
mesh = {*Apoptosis/drug effects ; Animals ; *CRISPR-Cas Systems ; *Aflatoxin B1/toxicity ; Phosphorylation/drug effects ; *Mitochondria/metabolism/drug effects ; *Tumor Suppressor Protein p53/metabolism ; *Kidney/metabolism/drug effects/pathology ; Swine ; Cell Line ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Protein Binding ; },
abstract = {Aflatoxin B1 (AFB1), recognized as a highly toxic and carcinogenic mycotoxin, contaminates more than 25 % of the global grain supply, thereby presenting a substantial public health threat and posing significant risks to renal health. However, the host factors that mediate the associated damage remain inadequately understood. This study aimed to identify key host factors in AFB1-induced cytotoxicity using a genome-wide CRISPR/Cas9 screen and elucidate the underlying molecular mechanisms. We developed a porcine kidney epithelial (PK15) cell model, followed by knockout validation, CCK-8 assays, qRT-PCR, western blotting, AO-EB staining, flow cytometry, and co-immunoprecipitation to dissect mechanistic pathways. Receptor-Interacting Protein Kinase 4 (RIPK4) was identified as a critical pro-apoptotic factor. RIPK4 knockout increased PK15 cell viability by ~50 % (P < 0.001) and reduced apoptosis by ~44 %(P < 0.001), accompanied by downregulation of APAF1, Cyt-c, cleaved-Caspase-9/-3, and p53 Ser15 phosphorylation, and upregulation of Bcl-2. Mechanistically, RIPK4 directly interacted with p53 via its N-terminal 1-490 aa region, enhancing its phosphorylation and pro-apoptotic activity. In conclusion, RIPK4 promotes AFB1 nephrotoxicity by activating p53-mediated mitochondrial apoptosis, identifying it as a novel therapeutic target. Future studies should validate these findings in vivo models and explore the potential of RIPK4-specific inhibitors for mitigating nephrotoxicity.},
}

*Apoptosis/drug effects
Animals
*CRISPR-Cas Systems
*Aflatoxin B1/toxicity
Phosphorylation/drug effects
*Mitochondria/metabolism/drug effects
*Tumor Suppressor Protein p53/metabolism
*Kidney/metabolism/drug effects/pathology
Swine
Cell Line
*Protein Serine-Threonine Kinases/metabolism/genetics
Protein Binding

@article {pmid41025776,
year = {2025},
author = {Messina, G and Goerner, A and Bennett, C and Brennan, E and Carruthers, VB and Martorelli Di Genova, B},
title = {Impact of equilibrative nucleoside transporters on Toxoplasma gondii infection and differentiation.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0220725},
pmid = {41025776},
issn = {2150-7511},
support = {P20 GM125498/GM/NIGMS NIH HHS/United States ; },
mesh = {*Toxoplasma/genetics/growth & development/metabolism ; Animals ; Mice ; *Toxoplasmosis/parasitology ; *Protozoan Proteins/genetics/metabolism ; *Nucleoside Transport Proteins/genetics/metabolism ; *Equilibrative Nucleoside Transport Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Toxoplasma gondii cannot synthesize purines de novo and must import them; however, the functional interplay among its four equilibrative nucleoside transporters (ENTs) homologs remains unclear. We systematically deconstructed this network by combining CRISPR-Cas9 knockouts with an auxin-inducible degron. Across all phenotypic assays, tachyzoite replication, nucleoside-analog sensitivity, alkaline-stress-induced differentiation, and murine cyst formation, the ΔTgENT2 strain was indistinguishable from the parental line, indicating that TgENT2 is dispensable under the conditions tested. In contrast, the double mutant ΔTgAT1ΔTgENT3 exhibited delayed bradyzoite differentiation in vitro and produced smaller brain cysts in vivo. This double deletion triggered approximately threefold transcriptional upregulation of TgENT1, whose product we partially localized to the plant-like vacuolar compartment (PLVAC). Conditional depletion of TgENT1 caused complete intracellular growth arrest, PLVAC swelling, and a purine-starvation-like transcriptomic program enriched for nucleoside phosphatases and cyclic-nucleotide phosphodiesterases. These findings reveal a compensatory salvage pathway in which the parasite reroutes purine acquisition through a vacuolar route when plasma-membrane import is compromised. Although this response sustains tachyzoite proliferation, it fails during the energetically demanding transition to bradyzoites, creating a metabolic bottleneck that impairs chronic infection. Our work reveals an adaptable yet ultimately limited purine-import network and identifies TgENT1, along with the vacuolar salvage axis it mediates.

IMPORTANCE: In this manuscript, we demonstrate that Toxoplasma gondii employs a flexible transporter network that redirects to a vacuolar salvage route when primary transporters are compromised. Disrupting this backup pathway disrupts parasite growth, exposing an Achilles' heel in purine homeostasis. Because nucleoside transporters are druggable, these findings suggest that the purine import machinery and TgENT1 may be potential targets for therapies against T. gondii infections.},
}

*Toxoplasma/genetics/growth & development/metabolism
Animals
Mice
*Toxoplasmosis/parasitology
*Protozoan Proteins/genetics/metabolism
*Nucleoside Transport Proteins/genetics/metabolism
*Equilibrative Nucleoside Transport Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40985725,
year = {2025},
author = {Sayson, SG and Ashbaugh, A and Bauer, LC and Smulian, AG},
title = {Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in Pneumocystis murina.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0182525},
pmid = {40985725},
issn = {2150-7511},
support = {R61 AI187097/AI/NIAID NIH HHS/United States ; 1R61AI187097//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Extracellular Vesicles/metabolism/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Pneumocystis/genetics ; Animals ; Mice ; *Transformation, Genetic ; *Gene Transfer Techniques ; Lung/microbiology ; Plasmids/genetics ; },
abstract = {Pneumocystis species are obligate fungal pathogens that cause severe pneumonia, particularly in immunocompromised individuals. The absence of robust genetic manipulation tools has impeded our mechanistic understanding of Pneumocystis biology and the development of novel therapeutic strategies. Herein, we describe a novel method for the stable transformation and CRISPR/Cas9-mediated genetic editing of Pneumocystis murina utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to Pneumocystis, we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful in vitro transformation and subsequent expression of mNeonGreen and Dhps[ARS] in P. murina organisms. Subsequently, we established stable in vivo expression of mNeonGreen in mice infected with transformed P. murina for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the P. murina Dhps gene, confirming DNA cleavage efficiency in vitro. Ultimately, we achieved successful in vivo CRISPR/Cas9-mediated homologous recombination, precisely introducing a Dhps[ARS] mutation into the P. murina genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in Pneumocystis, thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.IMPORTANCEPneumocystis species are obligate fungal pathogens and major causes of pneumonia in immunocompromised individuals. However, their strict dependence on the mammalian lung environment has precluded the development of genetic manipulation systems, limiting our ability to interrogate gene function, study antifungal resistance mechanisms, or validate therapeutic targets. Here, we report the first successful approach for stable transformation and CRISPR/Cas9-based genome editing of Pneumocystis murina, achieved through in vivo delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the dhps locus via homology-directed repair. This modular, scalable platform overcomes a long-standing barrier in the field and establishes a foundation for functional genomics in Pneumocystis and other obligate, host-adapted microbes.},
}

*Extracellular Vesicles/metabolism/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Pneumocystis/genetics
Animals
Mice
*Transformation, Genetic
*Gene Transfer Techniques
Lung/microbiology
Plasmids/genetics

@article {pmid40980888,
year = {2025},
author = {Danskog, K and Petersen, F and Frängsmyr, L and Gonzalez, G and Becker, M and Lenman, A and Arnberg, N},
title = {CD46 is a cellular receptor for species D human adenovirus.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0158725},
pmid = {40980888},
issn = {2150-7511},
support = {101098647//HORIZON EUROPE European Innovation Council/ ; 2023-01831//Vetenskapsrådet/ ; 2019-01472//Vetenskapsrådet/ ; 22 2005 Pj//Cancerfonden/ ; CAN 2018/771//Cancerfonden/ ; },
mesh = {Humans ; *Membrane Cofactor Protein/metabolism/genetics ; *Adenoviruses, Human/physiology/genetics/classification ; *Receptors, Virus/metabolism/genetics ; A549 Cells ; Viral Tropism ; CRISPR-Cas Systems ; Desmoglein 2/genetics/metabolism ; Virus Attachment ; Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism ; },
abstract = {UNLABELLED: Human adenovirus species D (HAdV-D) contains two-thirds of all known HAdV types (116 in total) and is important as a vector in clinical applications. However, the broad tropism exhibited by several HAdV-D types poses challenges for their use as targeted gene delivery vectors. Since adenoviral tropism is largely governed by receptor usage, we aimed to determine the relative importance of known adenovirus receptors in mediating infection by different HAdV-D types. Here, we generated A549 single-cell CRISPR/Cas9 knockout clones of desmoglein 2 (DSG2), CD46, the coxsackievirus and adenovirus receptor (CAR), and cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS; needed for biosynthesis of sialic acid-containing glycans), and assessed their relative importance for infection by 18 different HAdV-D types. We show that CD46 is the most important receptor for a majority of species D HAdVs. Minor changes in infection levels were noted with A549-ΔCAR and A549-ΔDSG2 cells, whereas A549-ΔCMAS cells displayed an increased susceptibility to infection. We proceed to show that HAdV-D types require CD46 for efficient attachment to A549 cells, and surface plasmon resonance analysis demonstrates that their hexon proteins bind CD46 in an avidity-dependent manner. Strategies to retarget HAdV-D vectors should thus consider hexon-CD46 interactions as a critical determinant of tropism, as CD46 is broadly expressed in vivo. These results increase our understanding of adenovirus-host interactions and will guide the development and targeting of vectors based on HAdV-D types.

IMPORTANCE: Several human adenovirus species D (HAdV-D) types are currently used, or under development, as viral vectors for vaccines and gene delivery. However, the unusually broad tropism observed in many HAdV-D types limits their specificity and effectiveness as targeted vectors. Since tropism is largely governed by receptor usage, and previous studies have reported conflicting findings on receptor preferences within this species, clarifying receptor usage is essential. In this study, we systematically investigated receptor usage in 18 different HAdV-D types and identified CD46 as the primary receptor. Since CD46 is widely expressed across human tissues, our findings explain the broad cellular tropism of these viruses and provide valuable insight for the rational design and refinement of HAdV-D-based vectors.},
}

Humans
*Membrane Cofactor Protein/metabolism/genetics
*Adenoviruses, Human/physiology/genetics/classification
*Receptors, Virus/metabolism/genetics
A549 Cells
Viral Tropism
CRISPR-Cas Systems
Desmoglein 2/genetics/metabolism
Virus Attachment
Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism

@article {pmid40973826,
year = {2025},
author = {Petri, K and D'Ippolito, E and Künkele, A and Köhl, U and Busch, DH and Einsele, H and Hudecek, M},
title = {Next-generation T cell immunotherapies engineered with CRISPR base and prime editing: challenges and opportunities.},
journal = {Nature reviews. Clinical oncology},
volume = {22},
number = {12},
pages = {902-923},
pmid = {40973826},
issn = {1759-4782},
mesh = {Humans ; *Gene Editing/methods ; *T-Lymphocytes/immunology/transplantation ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {T cells can be reprogrammed with transgenic antigen recognition receptors, including chimeric antigen receptors and T cell receptors, to selectively recognize and kill cancer cells. Such adoptive T cell therapies are effective in patients with certain haematological cancers but challenges persist, including primary and secondary resistance, a lack of efficacy in patients with solid tumours, a narrow range of targetable antigens, and time-consuming and complex manufacturing processes. CRISPR-based genome editing is a potent strategy to enhance cellular immunotherapies. Conventional CRISPR-Cas9 systems are useful for gene editing, transgene knock-in or gene knockout but can result in undesired editing outcomes, including translocations and chromosomal truncations. Base editing and prime editing technologies constitute a new generation of CRISPR platforms and enable highly precise and programmable installation of defined nucleotide variants in primary T cells. Owing to their high precision and versatility, base editing and prime editing systems, hereafter collectively referred to as CRISPR 2.0, are advancing to become the new standard for precision-engineering of cellular immunotherapies. CRISPR 2.0 can be used to augment immune cell function, broaden the spectrum of targetable antigens and facilitate streamlined production of T cell therapies. Notably, CRISPR 2.0 is reaching clinical maturity, with multiple clinical trials of CRISPR 2.0-modified cellular therapies currently ongoing. In this Review, we discuss emerging CRISPR 2.0 technologies and their progress towards clinical translation, highlighting challenges and opportunities, and describe strategies for the use of CRISPR 2.0 to advance cellular immunotherapy for haematological malignancies and solid tumours in the future.},
}

Humans
*Gene Editing/methods
*T-Lymphocytes/immunology/transplantation
*CRISPR-Cas Systems/genetics
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology/genetics
Receptors, Antigen, T-Cell/genetics

@article {pmid40973646,
year = {2025},
author = {Dinneen, E and Dasgupta, P and Sharma, A and Nisaa, K and Silva-García, CG},
title = {A single-copy knock-in system: one plasmid to target all chromosomes in C. elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {11},
pages = {},
pmid = {40973646},
issn = {2160-1836},
support = {//NIA/ ; R00AG065508/GF/NIH HHS/United States ; A24058//American Federation for Aging Research/ ; //Brown University Division of Research Seed Award/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Plasmids/genetics ; CRISPR-Cas Systems ; *Chromosomes/genetics ; *Gene Knock-In Techniques/methods ; Transgenes ; },
abstract = {Successful transgenesis in model organisms has significantly helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce a single-copy knock-in system that uses 1 plasmid to target all chromosomes in Caenorhabditis elegans (SKI PLACE), designed for inserting a transgene by CRISPR/Cas9. The SKI PLACE system uses the pSKI plasmid to insert a desired transgene at specific harbor loci on each chromosome. The pSKI plasmid contains multiple restriction sites for cloning and serves as a CRISPR/Cas9-based insertion repair template because it has 2 synthetic and long homology arms that recombine with the SKI PLACE cassettes. This system also uses a single crRNA guide, which acts as a Co-CRISPR enrichment marker. Overall, the SKI PLACE system is flexible; with the same SKI PLACE cassette on each chromosome, researchers can select the insertion site, work with 1 plasmid, and streamline tracking using standard primers.},
}

Animals
*Caenorhabditis elegans/genetics
*Plasmids/genetics
CRISPR-Cas Systems
*Chromosomes/genetics
*Gene Knock-In Techniques/methods
Transgenes

@article {pmid40972573,
year = {2025},
author = {Pfefferle, A and Malmberg, KJ},
title = {Rewiring natural killer cells for next-generation cancer therapies.},
journal = {Cancer cell},
volume = {43},
number = {11},
pages = {1980-1982},
doi = {10.1016/j.ccell.2025.08.007},
pmid = {40972573},
issn = {1878-3686},
mesh = {*Killer Cells, Natural/immunology ; Humans ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy/methods ; CRISPR-Cas Systems ; Animals ; },
abstract = {Harnessing the cytotoxic potential of natural killer (NK) cells for cancer immunotherapy has proven challenging. In this issue of Cancer Cell, Biederstädt et al. and Nikolic et al. utilize genome-wide CRISPR screening to uncover novel regulators of NK cell function, paving the way for developing next-generation NK cell therapies.},
}

*Killer Cells, Natural/immunology
Humans
*Neoplasms/therapy/immunology/genetics
*Immunotherapy/methods
CRISPR-Cas Systems
Animals

@article {pmid40902837,
year = {2025},
author = {Huang, M and Ewadi, A and Servatian, N and Noormohamadi, H and Aminov, Z and Muzammil, K and Esfahani, MA and Abak, N and Soleimani Samarkhazan, H},
title = {Epigenetic mechanisms and next-gen editing platforms in hematology: From molecular basis to therapeutic frontiers.},
journal = {Critical reviews in oncology/hematology},
volume = {215},
number = {},
pages = {104916},
doi = {10.1016/j.critrevonc.2025.104916},
pmid = {40902837},
issn = {1879-0461},
mesh = {Humans ; *Gene Editing/methods ; *Epigenesis, Genetic ; *Hematologic Diseases/therapy/genetics ; CRISPR-Cas Systems ; Animals ; *Hematology/methods ; Genetic Therapy/methods ; },
abstract = {Epigenetic regulation is fundamental to hematopoiesis, influencing stem cell fate, lineage commitment, and the development of hematologic diseases. Recent technological innovations have transitioned from traditional genetic editing towards programmable, reversible epigenetic modulation without altering the DNA sequence. This review explores the evolution of epigenetic editing platforms, from zinc finger proteins and TALEs to the transformative CRISPR-dCas9 system, and introduces next-generation technologies leveraging dCas12, dCas13, and modular RNA-guided systems. By fusing catalytically inactive CRISPR variants with chromatin or RNA-modifying enzymes, these tools enable precise control of gene expression and epitranscriptomic landscapes. In hematology, these advances offer novel strategies to modulate oncogenes, reactivate silenced tumor suppressors, and correct epigenetic dysregulation in malignancies such as leukemia, lymphoma, and myelodysplastic syndromes, as well as in inherited disorders like β-thalassemia and sickle cell disease. The integration of epigenetic editing into immune engineering, particularly in enhancing CAR-T and NK cell therapies, underscores its growing clinical impact. Together, these next-generation approaches herald a paradigm shift, enabling safer, more dynamic, and tunable interventions for blood disorders. This review highlights the current landscape and future directions of epigenetic editing, positioning it as a cornerstone of precision hematologic therapy.},
}

Humans
*Gene Editing/methods
*Epigenesis, Genetic
*Hematologic Diseases/therapy/genetics
CRISPR-Cas Systems
Animals
*Hematology/methods
Genetic Therapy/methods

@article {pmid40845844,
year = {2025},
author = {Biederstädt, A and Basar, R and Park, JM and Uprety, N and Shrestha, R and Reyes Silva, F and Dede, M and Watts, J and Acharya, S and Xiong, D and Liu, B and Daher, M and Rafei, H and Banerjee, P and Li, P and Islam, S and Fan, H and Shanley, M and Jin, J and Kumar, B and Woods, V and Lin, P and Tiberti, S and Nunez Cortes, AK and Jiang, XR and Biederstädt, I and Zhang, P and Li, Y and Rawal, S and Liu, E and Muniz-Feliciano, L and Deyter, GM and Shpall, EJ and Fowlkes, NW and Chen, K and Rezvani, K},
title = {Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency.},
journal = {Cancer cell},
volume = {43},
number = {11},
pages = {2069-2088.e11},
pmid = {40845844},
issn = {1878-3686},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA280827/CA/NCI NIH HHS/United States ; P50 CA281701/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; P01 CA148600/CA/NCI NIH HHS/United States ; P50 CA127001/CA/NCI NIH HHS/United States ; S10 OD024977/OD/NIH HHS/United States ; R01 CA288617/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation/metabolism ; *Immunotherapy, Adoptive/methods ; Animals ; Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics/immunology ; *Neoplasms/therapy/immunology/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Tumor Microenvironment/immunology ; },
abstract = {Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer.},
}

Humans
*Killer Cells, Natural/immunology/transplantation/metabolism
*Immunotherapy, Adoptive/methods
Animals
Gene Editing/methods
Mice
*CRISPR-Cas Systems
*Receptors, Chimeric Antigen/genetics/immunology
*Neoplasms/therapy/immunology/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Xenograft Model Antitumor Assays
Cell Line, Tumor
Tumor Microenvironment/immunology

@article {pmid40845150,
year = {2025},
author = {Carballar-Lejarazú, R and Pham, TB and Tushar, T and James, AA},
title = {Mutant allele formation and inheritance during Cas9/guide RNA-mediated gene drive in a population modification mosquito strain for human malaria control.},
journal = {Genetics},
volume = {231},
number = {3},
pages = {},
pmid = {40845150},
issn = {1943-2631},
support = {//University of California Irvine Malaria Initiative/ ; INV-043645/GATES/Gates Foundation/United States ; AI170692/NH/NIH HHS/United States ; //AAJ/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; *Gene Drive Technology/methods ; Alleles ; *Malaria/prevention & control/parasitology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Mutation ; Mosquito Vectors/genetics ; *CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Gene-drive systems are under development for the population modification of anopheline vectors of human malaria parasites. The key to their success is the fixation of genes in target mosquito populations that encode molecules preventing parasite transmission. High-efficiency Cas9/guide RNA (gRNA)-based gene-drive systems can facilitate this objective. A potential challenge to these systems is the presence of naturally-occurring or drive system-induced sequence polymorphisms in the genomic target site that could impede Cas9/gRNA-mediated cleavage and negatively impact gene-drive dynamics and fixation. Careful choice of the target site can mitigate the impact of natural variation, and here we analyze drive system-mediated, target-site mutagenesis in the outcross and testcross progeny of an Anopheles gambiae strain homo- and hemizygous for the TP13-based gene-drive system. The resulting data allow for estimation of the rates at which drive-system activity generates mutant target-site alleles in the germline and the impact of inherited paternal- and maternal-effect mutations. Functional and nonfunctional mutant alleles are recovered from the germlines at average rates per target gene/generation of 0.08% for paternal and 0.33% for maternal testcross lineages, with an overall average rate of 0.21%. Clustering effects amplify the potential inheritance frequencies of the mutant alleles. Mutations originating in the germlines represent 47% of the total inherited in testcross progeny, with the balance coming from mutant alleles generated by paternal and maternal effects inherited through the respective parental lineages. This approach allows the estimation of potential cleavage-resistant allele formation and inheritance for this drive system in this species and provides empirically derived values to inform more realistic data-driven gene-drive modeling.},
}

Animals
*Anopheles/genetics/parasitology
*Gene Drive Technology/methods
Alleles
*Malaria/prevention & control/parasitology
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Mutation
Mosquito Vectors/genetics
*CRISPR-Cas Systems
Female
Male

@article {pmid40827355,
year = {2025},
author = {Spilsbury, K and Wu, J and Reidy, M and Kropp, PA},
title = {The mitochondrial trans-2-enoyl-CoA reductase is necessary for mitochondrial homeostasis in C. elegans.},
journal = {Genetics},
volume = {231},
number = {3},
pages = {},
doi = {10.1093/genetics/iyaf166},
pmid = {40827355},
issn = {1943-2631},
support = {//Intramural Research Program/ ; //Digestive and Kidney Diseases/ ; //National Heart, Blood, and Lung Institute/ ; //Kenyon College/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Mitochondria/metabolism/genetics/enzymology ; *Homeostasis ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Fatty Acids/metabolism/biosynthesis ; CRISPR-Cas Systems ; *Acyl-CoA Dehydrogenases/genetics/metabolism ; Oxidoreductases Acting on CH-CH Group Donors ; },
abstract = {Fatty acids function not only as signaling molecules and for energy storage but also as essential cofactors for mitochondrial enzymes. These fatty acid cofactors are produced by the mitochondrial fatty acid synthesis (mtFAS) pathway, the terminal enzyme of which is mitochondrial trans-2-enoyl-CoA reductase (MECR). Dysfunction of MECR prevents the synthesis of fatty acids and is the monogenic cause of Mitochondrial Enoyl-CoA Protein Associated Neurodegeneration (MEPAN) syndrome, a rare mitochondrial disease characterized by dystonia, basal ganglia degeneration, and optic nerve atrophy. Given the necessity of mtFAS products for mitochondrial function, MECR should be essential. Yet, evidence from MEPAN individuals and model organisms with MECR loss of function indicate that mitochondrial function is not as severely impaired as would be expected. However, many of these studies have been limited to single cells or cell types. To better understand the role of MECR and its products in a multicellular system, we used CRISPR/Cas9 to knock out its 2 orthologs in Caenorhabditis elegans, MECR-1 and MECR-2. We found that only MECR-1 is necessary for normal mitochondrial function, germline development, and neuromuscular function. We thus establish a model in which further studies of MECR/MECR-1 can clarify its biochemical, developmental, and physiological roles.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
*Mitochondria/metabolism/genetics/enzymology
*Homeostasis
*Caenorhabditis elegans Proteins/genetics/metabolism
Fatty Acids/metabolism/biosynthesis
CRISPR-Cas Systems
*Acyl-CoA Dehydrogenases/genetics/metabolism
Oxidoreductases Acting on CH-CH Group Donors

@article {pmid40784261,
year = {2026},
author = {Wang, Y and Fu, L and Li, S and Tao, D and Gong, P and Yang, Y and Ruan, J and Xie, S and Wang, C and He, D},
title = {A rapid and highly sensitive CRISPR assay utilizing Cas12a orthologs for the detection of Novel Duck Reovirus.},
journal = {Talanta},
volume = {297},
number = {Pt B},
pages = {128680},
doi = {10.1016/j.talanta.2025.128680},
pmid = {40784261},
issn = {1873-3573},
mesh = {Nucleic Acid Amplification Techniques/methods ; Animals ; *Orthoreovirus, Avian/genetics/isolation & purification ; Ducks/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Reoviridae Infections/diagnosis/veterinary/virology ; Poultry Diseases/virology/diagnosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; },
abstract = {The Novel Duck Reovirus (NDRV) seriously threatens the global poultry industry due to the lack of effective therapies. Preventive measures for NDRV heavily depend on early disease detection, highlighting the need for rapid and sensitive diagnostic methods. This study used Cas12a orthologs Gs12-18 to develop a visual CRISPR-based detection assay targeting the NDRV S3 gene. Comparative analysis of candidate Cas12a proteins, Gs12-16 and Gs12-18, showed that Gs12-18 has significantly better trans-cleavage activity, making it especially suitable for highly sensitive nucleic acid detection. We integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to create a LAMP-CRISPR/Gs12-18 detection platform. This method enables visual detection of the NDRV S3 gene with high specificity and sensitivity, with a detection limit of 38 copies per reaction. It does not require complex equipment and is suitable for point-of-care testing. This research provides a reliable diagnostic tool for the early prevention and control of NDRV.},
}

Nucleic Acid Amplification Techniques/methods
Animals
*Orthoreovirus, Avian/genetics/isolation & purification
Ducks/virology
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Limit of Detection
*Reoviridae Infections/diagnosis/veterinary/virology
Poultry Diseases/virology/diagnosis
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins

@article {pmid40591237,
year = {2025},
author = {Sharma, J and Biswas, R and Khare, P},
title = {Design principle of successful genome editing applications using CRISPR-based toolkits.},
journal = {Journal of applied genetics},
volume = {66},
number = {4},
pages = {1065-1077},
pmid = {40591237},
issn = {2190-3883},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Streptococcus pyogenes/genetics/enzymology ; Plasmids/genetics ; Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are the most promising toolkit of synthetic biology for genetic engineering applications across species. Essentially, the Type II CRISPR system, featuring Cas9 nuclease from Streptococcus pyogenes complexed with sgRNA, introduces targeted DNA cleavage, enabling modifications with exceptional precision. This technology can be utilized for not only editing but also modulating gene expressions, thereby finding widespread utility in various biotechnological applications. Here we discuss strategies to construct a consolidated platform aiming at developing a CRISPR-based gene editing system in microbial hosts such as yeast. Employing the well-known gene editing enzymes, i.e., Cpf1 and dCas9, two independent strategies to develop a one-pot plasmid system have been proposed. Furthermore, approaches to reduce off-target cleavages introduced by non-specific targeting of CRISPR complex have been discussed. Finally, an overarching discussion on advanced strategies to design robust CRISPR components is provided for streamlining future genome editing applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Streptococcus pyogenes/genetics/enzymology
Plasmids/genetics
Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Synthetic Biology/methods

@article {pmid40579953,
year = {2025},
author = {Ye, Z and Fan, G and Wei, Y and Li, L and Liu, F},
title = {CRISPR/Cas9-mediated germline mutagenesis in the subsocial parasitoid wasp, Sclerodermus guani.},
journal = {Insect molecular biology},
volume = {34},
number = {6},
pages = {939-947},
doi = {10.1111/imb.13014},
pmid = {40579953},
issn = {1365-2583},
support = {Qian Ke He Ji Chu ZK [2024] General 423//Guizhou Provincial Science and Technology Department/ ; 32401587//National Natural Science Foundation of China/ ; QSXM [2022] B09//Guizhou Normal University/ ; Qian Jiao Ji [2024] 54//Department of Education of Guizhou Province/ ; },
mesh = {Animals ; *Wasps/genetics/growth & development ; *CRISPR-Cas Systems ; Female ; Male ; *Mutagenesis ; *Kynurenine 3-Monooxygenase/genetics/metabolism ; Insect Proteins/genetics/metabolism ; Phylogeny ; Gene Editing ; },
abstract = {The ectoparasitoid wasp Sclerodermus guani (Hymenoptera: Bethylidae), as a subsocial insect, is widely applied in biological control against beetle vectors of pine wood nematodes. Despite significant advances in behavioural research, functional genetics in S. guani remains underdeveloped due to the absence of efficient gene manipulation tools. In this study, we employed CRISPR-mediated mutagenesis to achieve germline gene knockout targeting the eye pigment-associated gene kynurenine 3-monooxygenase (KMO). Phylogenetic analysis revealed that S. guani KMO shares a close relationship with its homologue in Prorops nasuta (Hymenoptera: Bethylidae). Two single-guide RNAs (sgRNAs), coupled with Cas9 protein with and without nuclear localisation signal (NLS) were tested. Both sgRNAs induced specific in vitro DNA cleavage and in vivo heritable indels at the target genomic loci. Homozygous null mutant females and males exhibit a white-eye phenotype, which was identified during pupal stage. Optimal editing efficiency in vivo was achieved using the Cas9-NLS variant. Given the complication of germline gene editing in eusocial Hymenopterans, the application of CRISPR in the subsocial parasitoid wasp S. guani provides an accessible research platform for the molecular evolution of insect sociality.},
}

Animals
*Wasps/genetics/growth & development
*CRISPR-Cas Systems
Female
Male
*Mutagenesis
*Kynurenine 3-Monooxygenase/genetics/metabolism
Insect Proteins/genetics/metabolism
Phylogeny
Gene Editing

@article {pmid39681701,
year = {2025},
author = {Uijttewaal, ECH and Lee, J and Sell, AC and Botay, N and Vainorius, G and Novatchkova, M and Baar, J and Yang, J and Potzler, T and van der Leij, S and Lowden, C and Sinner, J and Elewaut, A and Gavrilovic, M and Obenauf, A and Schramek, D and Elling, U},
title = {CRISPR-StAR enables high-resolution genetic screening in complex in vivo models.},
journal = {Nature biotechnology},
volume = {43},
number = {11},
pages = {1848-1860},
pmid = {39681701},
issn = {1546-1696},
support = {TFRI Project #1107//Terry Fox Research Institute (Institut de Recherche Terry Fox)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; },
abstract = {Pooled genetic screening with CRISPR-Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
*Genetic Testing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Gene Editing/methods

@article {pmid39604565,
year = {2025},
author = {Cullot, G and Aird, EJ and Schlapansky, MF and Yeh, CD and van de Venn, L and Vykhlyantseva, I and Kreutzer, S and Mailänder, D and Lewków, B and Klermund, J and Montellese, C and Biserni, M and Aeschimann, F and Vonarburg, C and Gehart, H and Cathomen, T and Corn, JE},
title = {Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations.},
journal = {Nature biotechnology},
volume = {43},
number = {11},
pages = {1778-1782},
pmid = {39604565},
issn = {1546-1696},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; DFG CA 311/4-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *DNA-Activated Protein Kinase/antagonists & inhibitors ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems/genetics ; *Recombinational DNA Repair/drug effects/genetics ; Protein Kinase Inhibitors/pharmacology ; Pyrimidines ; },
abstract = {The DNA-PKcs inhibitor AZD7648 enhances CRISPR-Cas9-directed homology-directed repair efficiencies, with potential for clinical utility, but its possible on-target consequences are unknown. We found that genome editing with AZD7648 causes frequent kilobase-scale and megabase-scale deletions, chromosome arm loss and translocations. These large-scale chromosomal alterations evade detection through typical genome editing assays, prompting caution in deploying AZD7648 and reinforcing the need to investigate multiple types of potential editing outcomes.},
}

*Gene Editing/methods
Humans
*DNA-Activated Protein Kinase/antagonists & inhibitors
*Pyrazoles/pharmacology
CRISPR-Cas Systems/genetics
*Recombinational DNA Repair/drug effects/genetics
Protein Kinase Inhibitors/pharmacology
Pyrimidines

@article {pmid41219913,
year = {2025},
author = {Muttappagol, M and Sharma, M and Adhikari, DN and Suma, R and Borgohain, T and Saikia, B and Keithellakpam, A and Dutt, A and Laskar, A and Hiremath, S and Chikkaputtaiah, C},
title = {Towards Climate-Resilient Crops: Integrative Strategies for Reproductive Stage Abiotic Stress Tolerance.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70635},
doi = {10.1111/ppl.70635},
pmid = {41219913},
issn = {1399-3054},
support = {MMP025301//Council of Scientific and Industrial Research, India/ ; CRG/2022/007073 TSA//Anusandhan National Research Foundation (ANRF), Government of India/ ; BT/PR38410/GET/119/310/2020//Department of Biotechnology, Government of India (DBT)/ ; },
abstract = {Abiotic stresses such as drought, heat, cold, and salinity, threaten global crop productivity, particularly during the highly sensitive reproductive phase. These stresses disrupt female gametophyte formation, ovule development, fertilization, and seed maturation, resulting in reduced fertility and yield losses. While the mechanisms of vegetative stress tolerance are relatively well studied, reproductive-stage resilience, especially the resilience of the female gametophyte, remains insufficiently understood, despite its decisive role in determining yield stability. Recent advances provide an opportunity to close this gap through an integrative perspective. Physiological assessments and high-throughput phenotyping offer reliable tools to evaluate gametophyte and embryo performance under stress. Multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, are covering regulatory pathways underlying reproductive success. Emerging technologies such as CRISPR/Cas genome editing, biomarker discovery and machine learning further accelerate trait identification and their application in breeding pipelines. The novelty of this review lies in its focus on the female gametophyte as an overlooked determinant of stress resilience and in presenting a systems-level framework that integrates physiology, omics, breeding, and AI. This integration does more than catalogue stress effects; it connects mechanistic insights with breeding strategies and scales them with computational tools. The ultimate outcome is climate-resilient varieties that sustain productivity under stress, which support farmer livelihoods and contribute to global food security.},
}

@article {pmid41219189,
year = {2025},
author = {Uchigashima, M and Iguchi, R and Fujii, K and Shiku, K and Kumar, P and Liu, X and Isogai, M and Hoshino, C and Abe, M and Nozumi, M and Okamura, Y and Igarashi, M and Sakimura, K and Bise, R and Lavis, LD and Mikuni, T},
title = {Single-cell synaptome mapping of endogenous protein subpopulations in mammalian brain.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9705},
pmid = {41219189},
issn = {2041-1723},
support = {JP19dm0207080, JP21wm0525014, and JP24wm0625117//Japan Agency for Medical Research and Development (AMED)/ ; JPMJPR16F9//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; CDA00043/2019-C//Human Frontier Science Program (HFSP)/ ; N/A//Uehara Memorial Foundation/ ; JP20K21461, JP20H03349, JP20H05918, JP23K18160, and JP24K02130//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJFR231M//MEXT | Japan Science and Technology Agency (JST)/ ; N/A//Kowa Life Science Foundation/ ; },
mesh = {Animals ; *Synapses/metabolism ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Mice ; *Neurons/metabolism ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {Different spatial or temporal protein populations, such as cell-surface/intracellular or pre-existing/nascent subpopulations, determine the basal and activity-induced functions of individual synapses within a neuron in vivo. Here, we developed a simple and generalizable platform to image different spatial and temporal subpopulations of endogenous proteins at thousands of synapses in single neurons in the mammalian brain. The platform is based on the development, improvement and integration of CRISPR-Cas9-mediated protein labeling methods, chemical tag labeling techniques, and a semi-automatic analytical pipeline. The combined platform enables whole-cell mapping of total, cell-surface, intracellular, pre-existing, nascent or nascent-and-surface populations of endogenous proteins, such as receptor, scaffold and signaling proteins, at thousands of synapses in individual neurons in living or fixed mouse brain. Our single-cell "synaptome" mapping of endogenous protein subpopulations comprehensively visualizes the spatial representation of synapse diversity in protein localization, trafficking and turnover, providing valuable insights into single-cell organization and computations in the brain.},
}

Animals
*Synapses/metabolism
*Brain/metabolism/cytology
*Single-Cell Analysis/methods
Mice
*Neurons/metabolism
CRISPR-Cas Systems
Mice, Inbred C57BL

@article {pmid41218598,
year = {2025},
author = {Nazarudeen, A and Aswathy, VA and Rauf, AA and Aswathy, UV and Mahesh Chandran, VR and Abhirami, N and Sudhina, S and Chandran, A and Ayyappan, JP},
title = {Targeting Circular RNAs (circRNAs) in Atherosclerosis Using CRISPR Technology.},
journal = {The journal of gene medicine},
volume = {27},
number = {11},
pages = {e70048},
doi = {10.1002/jgm.70048},
pmid = {41218598},
issn = {1521-2254},
support = {//University of Kerala/ ; //Start-up Research Grant from the Science and Engineering Research Board (SERB), India/ ; //Start-up grant for new faculty from the University Grants Commission (UGC), India/ ; //University of Kerala research support in the form of Plan and Nonplan funds/ ; },
mesh = {*RNA, Circular/genetics ; Humans ; *Atherosclerosis/genetics/therapy/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Gene Editing/methods ; Genetic Therapy/methods ; Lipid Metabolism/genetics ; },
abstract = {Atherosclerosis is a chronic inflammatory condition that remains a major global cause of cardiovascular morbidity and death. Circular RNAs (circRNAs), emerging as key regulators of biological processes, have been linked to atherosclerosis because of their functions in inflammation, lipid metabolism, and plaque stability. This review explores the biogenesis and cellular functions of circRNAs, highlighting specific circRNAs, such as circANRIL, circHIPK, and circRSF1, which influence atherosclerosis progressions and development. CRISPR-Cas technology, specifically Cas9 and Cas13, has transformed the way atherosclerosis is studied and potentially treated. Targeting PCSK9, LDLR, and APOB to modify lipid metabolism, including lowering LDL cholesterol and repairing mutations in familial hypercholesterolemia, has been made possible using CRISPR-Cas9 in atherosclerosis models. In parallel, CRISPR-Cas13 offers a novel approach for RNA-level intervention by selectively editing circRNAs, providing a dynamic approach to regulate atherosclerosis-related pathways. In order to convert these findings into therapeutic treatments, future research should focus on elucidating the mechanics of circRNA, which in turn determines CRISPR-Cas13, and designing specific delivery systems. This review paper demonstrates the revolutionary promise of circRNA research and CRISPR innovation in the treatment of atherosclerosis and underscores the need for extensive preclinical validation to bridge the gap towards clinical use.},
}

*RNA, Circular/genetics
Humans
*Atherosclerosis/genetics/therapy/metabolism
*CRISPR-Cas Systems/genetics
Animals
Gene Editing/methods
Genetic Therapy/methods
Lipid Metabolism/genetics

@article {pmid41217051,
year = {2025},
author = {Alam, SS and Mehdi, A and Zafar, A and Ali, S and Rehman, AU and Liaqat, I and Peng, L and Kanwal, F and Afzal, S and Haq, IU and Aftab, MN},
title = {Advances in microbial biofuel production by metabolic and enzyme engineering, synthetic biology, metagenomics, and genome editing applications.},
journal = {Emerging topics in life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1042/ETLS20240002},
pmid = {41217051},
issn = {2397-8554},
abstract = {Microorganisms are the primary source of genetic diversity on earth due to their unparalleled metabolic and functional variability. With the depletion of fossil fuels, a sustainable alternative approach is the use of biofuels, where plant biomass as feedstock is essentially degraded to sugars with the aid of microbe-derived enzymes, followed by the conversion of those sugars to biofuels. Several cellulolytic and non-cellulolytic enzymes are involved in biofuel synthesis. Molecular cloning, along with the advancements in genetic and metabolic engineering in microbial cells, plays a significant contribution to biofuel overproduction. Advanced molecular technologies such as metagenomics and synthetic biology approaches are also being used to construct effective microorganisms for biofuel manufacturing. Obtaining novel enzymes from undiscovered microbial consortia and functional gene analysis is possible through a metagenomics approach. While synthetic biology provides engineered biological systems to generate required biofuel productivity, the CRISPR-Cas genome editing tool is another revolutionary tool being utilized for efficient biofuel production. This article provides a brief overview of different methods of biofuel production using microorganisms.},
}

@article {pmid41216030,
year = {2025},
author = {Naki, D and Gophna, U},
title = {Evolutionary insights into provirus-encoded CRISPR-Cas systems in halophilic archaea.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf033},
pmid = {41216030},
issn = {2633-6693},
abstract = {Prokaryotic microorganisms coexist with mobile genetic elements (MGEs), which can be both genetic threats and evolutionary catalysts. In Haloferax lucentense, a halophilic archaeon, we have recently identified an unusual genomic arrangement: a complete type I-B CRISPR-Cas system encoded on a megaplasmid and an incomplete type I-B system within an integrated provirus in the main chromosome. The provirus-encoded system lacks the adaptation genes (cas1, cas2, and cas4), suggesting its potential reliance on the megaplasmid-encoded CRISPR-Cas module for the acquisition of new spacers. This arrangement suggests a potential instance of "adaptive outsourcing," where a provirus might leverage a co-resident MGE for a key function. Through comparative genomics, we show that similar proviral CRISPR-Cas systems are found in distantly related haloarchaea (e.g. Natrinema and Halobacterium), indicating probable virus-mediated horizontal transfer and suggesting they may function as mobile defense modules. Phylogenetic analysis highlights distinct evolutionary origins of the two systems: the plasmid system clusters with other Haloferax CRISPR-Cas systems, while the proviral system clusters with those from other genera, consistent with horizontal acquisition. Interestingly, spacer analysis reveals that the proviral systems predominantly target viral sequences, while the plasmid system appears to target both plasmids and viral sequences, a distribution mirroring broader trends observed in other plasmid- and chromosome-encoded CRISPR systems. This observed targeting preference suggests a potential for complementarity that could support a model of cooperative immunity, where each system may protect its genetic "owner" from competition and, indirectly, the host.},
}

@article {pmid41214117,
year = {2025},
author = {Bakthavachalam, V and Sanborn, MA and Mathayan, M and Salunkhe, SS and Wood, JM and Maienschein-Cline, M and Setty, S and Kabeer, BSA and Rehman, J and Prabhakar, BS},
title = {CRISPR/Cas9-mediated deletion of MADD induces cell cycle arrest and apoptosis in anaplastic thyroid cancer cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {39264},
pmid = {41214117},
issn = {2045-2322},
support = {VA Merit Review, Award # 5 I01 BX004697//United States (U.S.) Department of Veteran's Affairs Biomedical Laboratory Research and Development Services (BLRD)/ ; },
mesh = {*Apoptosis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Thyroid Carcinoma, Anaplastic/genetics/pathology/metabolism ; Animals ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; Mice ; *Thyroid Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; *Death Domain Receptor Signaling Adaptor Proteins/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics ; Gene Deletion ; Guanine Nucleotide Exchange Factors ; },
abstract = {Anaplastic thyroid cancer (ATC) is an aggressive malignancy with a poor prognosis and limited treatment options. Previous studies have shown that selective downregulation of the MADD (MAP-kinase-activating death domain-containing protein) gene isoform increases ATC cell susceptibility to TRAIL-induced apoptosis. However, the existence of multiple MADD gene isoforms raised the possibility of functional compensation. This study aimed to definitively evaluate the role of MADD in ATC by employing CRISPR-Cas9 to Cas9 to target exon 3, a conserved exon expressed in all known MADD isoforms, resulting in functional knockout of MADD expression. CRISPR-Cas9-mediated MADD knockout, performed in three ATC cell lines (8505 C, C643, and HTH7) with distinct mutational backgrounds, significantly impaired ATC cell function in vitro, as indicated by reduced viability, increased apoptosis, decreased migration, and G0/G1 cell cycle arrest. RNA-seq analysis revealed alterations in genes related to cell survival, proliferation, and metastasis. In the orthotopic ATC mouse model, MADD deletion dramatically suppressed tumor growth, reduced lung metastases, and prolonged survival. Our findings demonstrate that MADD plays a crucial role in ATC cell survival, proliferation, and metastasis. The consistent effects observed across multiple cell lines and in vivo models suggest that MADD may represent a promising therapeutic target for this aggressive malignancy.},
}

*Apoptosis/genetics
*CRISPR-Cas Systems/genetics
Humans
*Thyroid Carcinoma, Anaplastic/genetics/pathology/metabolism
Animals
Cell Line, Tumor
*Cell Cycle Checkpoints/genetics
Mice
*Thyroid Neoplasms/genetics/pathology
Cell Proliferation/genetics
*Death Domain Receptor Signaling Adaptor Proteins/genetics
Gene Expression Regulation, Neoplastic
Cell Movement/genetics
Gene Deletion
Guanine Nucleotide Exchange Factors

@article {pmid41213972,
year = {2025},
author = {Chisholm, CG and Bartlett, R and Brown, ML and Proctor, EJ and Farrawell, NE and Gorman, J and Delerue, F and Ittner, LM and Vine-Perrow, KL and Ecroyd, H and Cashman, NR and Saunders, DN and McAlary, L and Lum, JS and Yerbury, JJ},
title = {Development of a targeted BioPROTAC degrader selective for misfolded SOD1.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9713},
pmid = {41213972},
issn = {2041-1723},
mesh = {Animals ; *Superoxide Dismutase-1/metabolism/genetics/chemistry ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology/drug therapy ; Mice, Transgenic ; Mice ; Protein Folding ; Humans ; Disease Models, Animal ; Proteolysis ; Motor Neurons/metabolism/pathology ; Ubiquitin-Protein Ligases/metabolism/genetics ; CRISPR-Cas Systems ; Disease Progression ; HEK293 Cells ; },
abstract = {The accumulation of misfolded proteins underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein superoxide dismutase 1 (SOD1), using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction reveals a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we demonstrate that the presence of the BioPROTAC delays disease progression in the SOD1[G93A] mouse model of ALS. Delayed disease progression is associated with protection of motor neurons, a reduction of insoluble SOD1 accumulation and preservation of innervated neuromuscular junctions. These findings provide proof-of-concept evidence and a platform for developing BioPROTACs as a therapeutic strategy for the targeted degradation of neurotoxic misfolded species in the context of neurodegenerative diseases.},
}

Animals
*Superoxide Dismutase-1/metabolism/genetics/chemistry
*Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology/drug therapy
Mice, Transgenic
Mice
Protein Folding
Humans
Disease Models, Animal
Proteolysis
Motor Neurons/metabolism/pathology
Ubiquitin-Protein Ligases/metabolism/genetics
CRISPR-Cas Systems
Disease Progression
HEK293 Cells

@article {pmid41212595,
year = {2025},
author = {Wu, Y and Su, J and Sun, J and Li, Z and Yang, J and Wu, C and Liu, Z and Wang, J and Wang, Y and Liu, J and Duan, L and Wang, C and Zhu, G and Wen, X},
title = {Rapid detection of Seneca Valley virus based on reverse transcription recombinase polymerase amplification and CRISPR-Cas13a.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2585719},
pmid = {41212595},
issn = {2150-5608},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Picornaviridae/isolation & purification/genetics ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/genetics ; *Nucleic Acid Amplification Techniques/methods ; Reverse Transcription ; },
abstract = {The Seneca Valley virus (SVV) is an emerging novel etiological agent that triggers vesicular stomatitis in pigs. SVV leads to considerable financial detriment to the global swine industry. Availability of expeditious, simple, and precise detection techniques would aid in the diagnosis and control of Seneca Valley disease. In this study, we developed a novel SVV detection assay based on reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR-Cas13a. This technique can specifically identify SVV with no cross-reactivity with other viruses, such as the porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus, and foot-and-mouth disease virus. It boasts a detection threshold as sensitive as 2.43 copies/μL, matching the proficiency of reverse transcriptase quantitative PCR in clinical diagnostics. The streamlined RT-RPA/CRISPR-Cas13a assay for SVV minimizes procedural complexity and the likelihood of contamination. Integrating this method with nucleic acid acquisition via nucleic acid releaser enables a turnaround time of only 40 min from sample acquisition to result determination, underscoring its suitability for on-site diagnostics and affirming its role as a robust instrument for the monitoring and detection of SVV.},
}

Animals
Swine
*CRISPR-Cas Systems
*Swine Diseases/diagnosis/virology
Sensitivity and Specificity
*Picornaviridae/isolation & purification/genetics
*Picornaviridae Infections/diagnosis/veterinary/virology
Recombinases/genetics
*Nucleic Acid Amplification Techniques/methods
Reverse Transcription

@article {pmid41208113,
year = {2025},
author = {Hermann, MD and Fernandez Perez, N and Yeroslaviz, A and Murray, PJ},
title = {Normal macrophage signaling and gene expression in Rosa26 Cas9-expressing mice.},
journal = {ImmunoHorizons},
volume = {9},
number = {11},
pages = {},
pmid = {41208113},
issn = {2573-7732},
support = {//Deutsche Forschungsgemeinschaft/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {Animals ; Mice ; *Macrophages/metabolism/immunology ; *Signal Transduction/genetics ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *RNA, Untranslated/genetics ; Mice, Inbred C57BL ; Toll-Like Receptor 3/metabolism ; Mice, Transgenic ; Toll-Like Receptor 4/metabolism ; },
abstract = {Cas9-expression from the Rosa26 "safe harbor" locus are widely used for gene manipulation and Crispr-based screening. Recently, experimental evidence suggested that macrophages isolated from Rosa26-Cas9 mice may have signaling differences compared to control mice in terms of TRIF signaling downstream of TLR3 and TLR4. As we frequently use the Rosa26-Cas9 mice made by Feng Zhang (Cas9-FZ, Jackson Laboratory stock No. 026179), arguably the strain with the widest distribution and utilization, we were motivated to test macrophage signaling in these mice under our conventional conditions. We used different macrophage polarization and signaling conditions combined with RNA sequencing and measurement of TLR signaling by immunoblotting. Our results suggest that the Cas9-FZ mice bear no obvious defects in any commonly used macrophage signaling pathway. We document the differences in our macrophage culture techniques compared to Raychowdhury et al., which may aid in how individual laboratories use Cas9-expressing macrophages, especially for focused or genome-wide screening.},
}

Animals
Mice
*Macrophages/metabolism/immunology
*Signal Transduction/genetics
CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
*RNA, Untranslated/genetics
Mice, Inbred C57BL
Toll-Like Receptor 3/metabolism
Mice, Transgenic
Toll-Like Receptor 4/metabolism

@article {pmid41206041,
year = {2025},
author = {Liu, Q and Pan, TT and Wang, LJ and Zhang, CY},
title = {Mirror-synchronized asymmetric CRISPR nanoswitch for single-molecule profiling of multiple circRNAs in different stages of breast cancer.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206041},
issn = {1362-4962},
support = {22474019//National Natural Science Foundation of China/ ; },
mesh = {*RNA, Circular/genetics ; *Breast Neoplasms/genetics/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; Female ; *RNA/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Single Molecule Imaging/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs characterized by their covalently closed circular structures. They have been implicated in significant transcriptional and post-transcriptional regulation of gene expression. Here, we present a one-pot method for the detection of circRNAs based on engineered DNA hairpins and CRISPR-Cas12a signal amplification, which involves signal pre-amplification via coupled probe-mediated hairpin amplification of two palindromic hairpins and Cas12a signal generation via trans-cleavage. We demonstrate that this platform is sensitive (detection limit of 1.07 aM), specific (capable of single-mismatch discrimination), and fast (reaction time of 25 min) and can be used to detect different circRNAs from RNase R-treated RNA (both in vitro and in clinically relevant samples, including correct classification of disease progression). This method enables single-molecule profiling and can be extended to detect other types of nucleic acids.},
}

*RNA, Circular/genetics
*Breast Neoplasms/genetics/pathology
Humans
*CRISPR-Cas Systems/genetics
Female
*RNA/genetics
CRISPR-Associated Proteins/genetics/metabolism
Single Molecule Imaging/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41205588,
year = {2025},
author = {Kumar, KRR},
title = {Plant genome editing goes viral: balancing innovation and biosafety.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2684-2685},
doi = {10.1016/j.tibtech.2025.09.006},
pmid = {41205588},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Genome, Plant/genetics ; *Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Containment of Biohazards ; },
abstract = {Weiss and colleagues demonstrated a breakthrough in transgene-free heritable genome editing using viral vectors. While promising for controlled laboratory applications, the open-field use envisaged by Sajjad and colleagues raises ecological and biosafety concerns. Rigorous risk assessment is essential to harness innovation responsibly while safeguarding biodiversity and public trust.},
}

*Gene Editing/methods
*Genome, Plant/genetics
*Plants, Genetically Modified/genetics
Genetic Vectors/genetics
CRISPR-Cas Systems
Containment of Biohazards

@article {pmid41190788,
year = {2025},
author = {Orzechowski, M and Hoikkala, V and Chi, H and McMahon, S and Gloster, T and White, MF},
title = {A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4.},
journal = {The Biochemical journal},
volume = {482},
number = {22},
pages = {},
doi = {10.1042/BCJ20253271},
pmid = {41190788},
issn = {1470-8728},
mesh = {*Viral Proteins/metabolism/genetics/chemistry ; *Adenine Nucleotides/metabolism ; *CRISPR-Cas Systems ; *Second Messenger Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oligoribonucleotides ; },
abstract = {Type III CRISPR systems typically generate cyclic oligoadenylate second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The Calp (CRISPR associated Lon protease) system elicits a transcriptional response to infection when CalpL (Calp Lon protease) binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS (Calp Sigma factor) for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2 (second anti-CRISPR of type III systems), which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.},
}

*Viral Proteins/metabolism/genetics/chemistry
*Adenine Nucleotides/metabolism
*CRISPR-Cas Systems
*Second Messenger Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Oligoribonucleotides

@article {pmid41187216,
year = {2025},
author = {Treaster, MJ and McCann, J and Solovei, KS and Palmieri, RJ and White, MA},
title = {A Y-linked duplication of anti-Mullerian hormone is the sex determination gene in threespine stickleback.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011932},
pmid = {41187216},
issn = {1553-7404},
mesh = {Animals ; *Smegmamorpha/genetics ; Male ; *Anti-Mullerian Hormone/genetics ; *Sex Determination Processes/genetics ; Female ; *Y Chromosome/genetics ; *Gene Duplication ; CRISPR-Cas Systems ; Evolution, Molecular ; Sex Differentiation/genetics ; },
abstract = {Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. We find that amhy contributes to a higher total dosage of amh early in development and likely contributes to differential germ cell proliferation key to sex determination. The creation of sex-reversed lines also allowed us to investigate the genetic basis of secondary sex characteristics. Threespine stickleback have striking differences in behavior and morphology between sexes. Here we show one of the classic traits important for reproductive success, blue male nuptial coloration, is controlled by both sex-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the "Four Core Genotypes" model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.},
}

Animals
*Smegmamorpha/genetics
Male
*Anti-Mullerian Hormone/genetics
*Sex Determination Processes/genetics
Female
*Y Chromosome/genetics
*Gene Duplication
CRISPR-Cas Systems
Evolution, Molecular
Sex Differentiation/genetics

@article {pmid41170863,
year = {2025},
author = {Liu, J and Feng, G and Guo, C and Liu, D and Li, Y},
title = {Opportunities and Challenges of CRISPR-Cas9 in the Sustainable Development of Animal Husbandry.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28575-28587},
doi = {10.1021/acs.jafc.5c09076},
pmid = {41170863},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Animals ; Gene Editing ; Sustainable Development ; *Animal Husbandry/methods ; *Livestock/genetics/metabolism/growth & development ; Animals, Genetically Modified/genetics/metabolism ; },
abstract = {Food security is of vital importance to the destiny of the entire globe. However, the realization of the United Nations' Sustainable Development Goals, which advocate for the eradication of hunger and the improvement of nutrition, appears somewhat bleak. The advent of gene editing has made precise modification of biological genomes a reality. Among these technologies, CRISPR-Cas9, due to its convenience and high efficiency, has been widely applied in fields such as agriculture. The application of CRISPR-Cas9 in genetically engineered animals can rapidly improve animal traits, reduce resource consumption, and has great potential in addressing food security issues. Therefore, this review focuses on the editing principles of CRISPR-Cas9, its developmental trajectory, and the opportunities and challenges it presents in animal husbandry, aiming to provide a theoretical reference for the further application of CRISPR-Cas9 to promote the sustainable development of animal husbandry.},
}

*CRISPR-Cas Systems
Animals
Gene Editing
Sustainable Development
*Animal Husbandry/methods
*Livestock/genetics/metabolism/growth & development
Animals, Genetically Modified/genetics/metabolism

@article {pmid41162785,
year = {2025},
author = {Coda, DM and Watt, L and Glauser, L and Batiuk, MY and Burns, AM and Stahl, CL and Wong, LY and Gräff, J},
title = {Cell-type- and locus-specific epigenetic editing of memory expression.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2661-2668},
pmid = {41162785},
issn = {1546-1718},
support = {101043457//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 310030_219342//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 310030_197752//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Epigenesis, Genetic/genetics ; *Memory/physiology ; Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Nerve Tissue Proteins/genetics ; Neuronal Plasticity/genetics ; Male ; Cytoskeletal Proteins/genetics ; Neurons/metabolism/physiology ; Epigenome Editing ; },
abstract = {Epigenetic mechanisms have long been proposed to act as molecular mnemonics[1-3], but whether the epigenetic makeup of a single genomic site can guide learnt behaviors remains unknown. Here we combined CRISPR-based epigenetic editing tools[4,5] with c-Fos-driven engram technologies[6,7] to address this question in memory-bearing neuronal ensembles. Focusing on the promoter of Arc, which encodes a master regulator of synaptic plasticity[8], we found that its locus-specific and temporally controlled epigenetic editing is necessary and sufficient to regulate memory expression. Such effects occurred irrespective of the memory phase-during the initially labile period after learning and for fully consolidated memories-and were reversible within subject, testifying to their inherent plasticity. These findings provide a proof-of-principle that site-specific epigenetic dynamics are causally implicated in memory expression.},
}

*Epigenesis, Genetic/genetics
*Memory/physiology
Animals
*Gene Editing/methods
Mice
CRISPR-Cas Systems/genetics
Promoter Regions, Genetic
Nerve Tissue Proteins/genetics
Neuronal Plasticity/genetics
Male
Cytoskeletal Proteins/genetics
Neurons/metabolism/physiology
Epigenome Editing

@article {pmid41087678,
year = {2025},
author = {Kaplan, EG and Steger, RJ and Shah, ST and Drepanos, LM and Griffith, AL and Reint, G and Doench, JG},
title = {Activity-based selection for enhanced base editor mutational scanning.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2920-2929},
pmid = {41087678},
issn = {1546-1718},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome that is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
Tumor Suppressor Protein p53/genetics

@article {pmid40996032,
year = {2025},
author = {Mofed, D and Gowripalan, A and Berrigan, J and Das, PK and Pujari, N and Ajasin, D and Haldar, S and McCullough, J and Zhang, Y and Kalpana, GV and Bresnick, A and Kielian, M and Wilson, DW and Zhang, J and Chandran, K and Prasad, VR},
title = {Influence of CCL2-mediated modulation of ALIX in the budding and replication of viruses from multiple families.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0224125},
doi = {10.1128/mbio.02241-25},
pmid = {40996032},
issn = {2150-7511},
support = {R01 AI125244/AI/NIAID NIH HHS/United States ; R01 AI153008/NH/NIH HHS/United States ; R01 AI125244/NH/NIH HHS/United States ; R01 AI075647/NH/NIH HHS/United States ; R01 AI185073/NH/NIH HHS/United States ; P30CA013330/NH/NIH HHS/United States ; S10OD026833-01/NH/NIH HHS/United States ; S10OD032169-01/NH/NIH HHS/United States ; },
mesh = {Humans ; HeLa Cells ; *Virus Replication ; *Endosomal Sorting Complexes Required for Transport/metabolism/genetics ; *Calcium-Binding Proteins/metabolism/genetics ; *Chemokine CCL2/genetics/metabolism ; *Virus Release ; HIV-1/physiology/genetics ; *Cell Cycle Proteins/metabolism/genetics ; Gene Knockout Techniques ; Signal Transduction ; CRISPR-Cas Systems ; Animals ; },
abstract = {UNLABELLED: Signaling by C-C motif ligand 2 (CCL2), a β-chemokine, modulates HIV-1 budding and release by mobilizing ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the cytosol. Immunodepleting CCL2 in the medium sequesters ALIX to F-actin. We developed a novel tool to study HIV budding and release without mutating viral late domains or silencing ESCRT genes, but by blocking CCL2 signaling using CRISPR-Cas9 knockout (KO) of the CCL2 or CCR2 genes. We knocked out CCL2 (CCL2KO) and CCR2 (CCR2KO) singly or together (double knockout) in HeLa cells and confirmed that knockout was associated with the absence of CCL2 or CCR2 expression. In KO cells, ALIX was associated with the F-actin cytoskeleton, while in control cells, it was associated with the cytosolic soluble fraction. In KO cells, HIV-1 production was profoundly reduced (10-fold). Strikingly, for CCL2KO cells, the addition of CCL2 mobilized ALIX to the soluble fraction, and virus production was stimulated to levels higher than those of untreated HeLa cells. We utilized these cells to test the involvement of ALIX in the budding and/or replication of several viruses, including Simian Immunodeficiency Virus (SIV), Equine Infectious Anemia Virus (EIAV), Herpes Simplex Virus type 1 (HSV-1), Dengue virus (DENV), and Hazara virus (HAZV). Budding and release of SIV and EIAV were both inhibited in CCL2KO cells and rescued by CCL2 addition. Replication of HSV-1 and DENV was unaffected in CCL2KO cells, confirming that ALIX is not involved in their replication. Finally, HAZV replication was affected by CCL2 signaling. Our studies indicate that CCL2 signaling and ALIX mobilization are important for several viral families.

IMPORTANCE: C-C motif ligand 2 (CCL2) plays a regulatory role in the budding and release of HIV-1 in macrophages and HeLa cells. CCL2 signaling mobilizes ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the soluble cytosol, where it is accessible for recruitment by the HIV-1 Gag polyprotein in the assembling virions at the plasma membrane. In previous studies, CCL2 immunodepletion, which blocks CCL2 signaling, resulted in ALIX sequestration to the F-actin cytoskeleton and inhibited virus production. Here, we developed a HeLa CCL2 gene knockout cell line and found that abrogation of CCL2 signaling can be restored by CCL2 addition, as evidenced by the restoration of ALIX to the cytosolic fraction and rescue of HIV-1 release. Employing such a system, we tested Simian Immunodeficiency Virus, Equine Infectious Anemia Virus, Herpes Simplex Virus type 1, Dengue, and Hazara virus for their dependence on ALIX for virus replication. The results indicate that CCL2 signaling and ALIX release from F-actin may play a role in the replication of several viruses.},
}

Humans
HeLa Cells
*Virus Replication
*Endosomal Sorting Complexes Required for Transport/metabolism/genetics
*Calcium-Binding Proteins/metabolism/genetics
*Chemokine CCL2/genetics/metabolism
*Virus Release
HIV-1/physiology/genetics
*Cell Cycle Proteins/metabolism/genetics
Gene Knockout Techniques
Signal Transduction
CRISPR-Cas Systems
Animals

@article {pmid40980903,
year = {2025},
author = {Khurram, I and Choudhery, MS and Ghani, MU and Arif, T and Naeem, A and Mahmood, R and Niaz, A and Khan, MU},
title = {Gene Editing for Cystic Fibrosis: Advances and Prospects of CRISPR-Cas9 Therapy.},
journal = {Cell biology international},
volume = {49},
number = {12},
pages = {1564-1578},
doi = {10.1002/cbin.70082},
pmid = {40980903},
issn = {1095-8355},
mesh = {*Cystic Fibrosis/genetics/therapy/diagnosis ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Genetic Therapy/methods ; Mutation/genetics ; Animals ; },
abstract = {Cystic fibrosis (CF) is an inherited, autosomal recessive disorder that is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). CFTR maintains the balance between water and salts by transporting chloride ions along various epithelial surfaces. CFTR impairment affects the function of several organs, including the lungs. Newborn screening, prenatal diagnosis, and pharmacological interventions have altered the prevalence and incidence of cystic fibrosis. Although CFTR modulators are a promising treatment option, their ability to target and correct only one mutation at a time restricts their therapeutic potential. The development of genome editing technologies such as Clustered Regularly Interspaced Short Palindromic Repeats-Cas(CRISPR-Cas9) has the potential to correct genetic mutations, including those associated with CF, thereby offering a permanent treatment by fixing the root cause of CF. This article summarizes cystic fibrosis development, prognosis, and diagnosis, as well as possibilities for correcting various types of CFTR gene mutations. The review focuses on the potential of gene editing technologies to repair CFTR mutations and their applications in the advancement of CF treatment.},
}

*Cystic Fibrosis/genetics/therapy/diagnosis
Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
*Genetic Therapy/methods
Mutation/genetics
Animals

@article {pmid40975652,
year = {2025},
author = {Sajjad, MW and Naqvi, RZ and Amin, I},
title = {Viral genome editing: striking a balance between promises and precautions.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2686-2687},
doi = {10.1016/j.tibtech.2025.09.010},
pmid = {40975652},
issn = {1879-3096},
mesh = {*Genome, Viral/genetics ; *Genetic Vectors/genetics ; *Genetic Engineering/methods ; Humans ; *Viruses/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Agriculture ; },
abstract = {Viral vectors can provide transgene-free genome editing. The forward-looking frameworks for their use must be biosafety and freedom from ecological risk. The responsible innovations, showing a balance between promise and caution following regulatory frameworks, can guarantee that viral genome editing develops into a revolutionary but environmentally conscious strategy for sustainable agricultural improvement.},
}

*Genome, Viral/genetics
*Genetic Vectors/genetics
*Genetic Engineering/methods
Humans
*Viruses/genetics
*Gene Editing
CRISPR-Cas Systems
Agriculture

@article {pmid40962831,
year = {2025},
author = {Su, X and Zhang, H and Hong, Y and Yang, Q and Wang, L and Le, T and Liu, J and Cheruvu, L and Labour, E and Zhang, S and Mendez-Maldonado, K and Kreimer, A and Song, H and Ming, GL and Duan, J and Pang, ZP},
title = {Mutations of schizophrenia risk gene SETD1A dysregulate synaptic function in human neurons.},
journal = {Molecular psychiatry},
volume = {30},
number = {12},
pages = {5680-5693},
pmid = {40962831},
issn = {1476-5578},
support = {ASF_23-004/ASF/Autism Science Foundation/United States ; R01MH125528//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01 MH106575/MH/NIMH NIH HHS/United States ; 74260//Robert Wood Johnson Foundation (RWJF)/ ; R01 AG081374/AG/NIA NIH HHS/United States ; R01 AG063175/AG/NIA NIH HHS/United States ; RM1 MH133065/MH/NIMH NIH HHS/United States ; ASF_23-004/ASF/Autism Science Foundation/United States ; },
mesh = {Humans ; *Schizophrenia/genetics/metabolism ; *Histone-Lysine N-Methyltransferase/genetics/metabolism ; Neurons/metabolism/physiology ; Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Synapses/genetics/metabolism ; Genetic Predisposition to Disease ; Haploinsufficiency/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Schizophrenia (SCZ) is a complex neuropsychiatric disorder associated with both common risk variants of small effect sizes and rare risk variants of high penetrance. Rare premature open reading frame (ORF) termination variants in SETD1A (SET Domain Containing 1A) show a strong association with SCZ; however, it remains largely unclear how rare premature ORF termination variants in SETD1A contribute to the pathophysiology of SCZ. To understand the impact of SETD1A rare premature ORF termination variants in human neurons, we CRISPR/Cas9-engineered five isogenic pairs of human induced pluripotent stem cells (iPSCs), with a recurrent heterozygous patient-specific premature ORF termination mutation c.4582-2delAG in two donor lines and a heterozygous frameshift mutation c.4596_4597insG (p. Leu1533fs) in three donor lines. These two mutations are predicted to cause a premature stop codon in exon 16 of SETD1A, leading to SETD1A haploinsufficiency. We found that these presumably loss-of-function (LoF) mutations caused the SETD1A mRNAs to be degraded by nonsense-mediated decay (NMD), accompanied by a reduction of full-length SETD1A protein level in iPSCs. We then characterized the morphological, electrophysiological, and transcriptomic impacts of SETD1A[+/-] LoF mutations in iPSC-derived human excitatory neurons induced by NGN2. We found that the SETD1A[+/-] exon-16 LoF mutations altered dendrite complexity, dysregulated synaptic transmission, and synaptic plasticity, likely by dysregulating genes involved in synaptic function. These results provide mechanistic insights into how SETD1A[+/-] exon-16 patient-specific LoF mutations affect neuron phenotypes that may be relevant to the pathophysiology of SCZ.},
}

Humans
*Schizophrenia/genetics/metabolism
*Histone-Lysine N-Methyltransferase/genetics/metabolism
Neurons/metabolism/physiology
Induced Pluripotent Stem Cells/metabolism
Mutation/genetics
Synapses/genetics/metabolism
Genetic Predisposition to Disease
Haploinsufficiency/genetics
CRISPR-Cas Systems/genetics

@article {pmid40817029,
year = {2025},
author = {Vidal, P and Giménez-Dejoz, J and Fernandez-Lopez, L and Romero, S and Nazemi, SA and Luengo, M and Gonzalez-Alfonso, JL and Martinez-Sugrañes, M and Robles-Martín, A and Almendral, D and Roda, S and Pérez-García, P and Kruse, L and Jaeger, KE and Streit, WR and Plou, FJ and Floor, M and Shahgaldian, P and Bargiela, R and Guallar, V and Ferrer, M},
title = {Computationally guided genome rewiring of Escherichia coli and its application for nanopolyethylene terephthalate (PET) biodegradation and upcycling.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2874-2903},
doi = {10.1016/j.tibtech.2025.07.008},
pmid = {40817029},
issn = {1879-3096},
mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Escherichia coli/genetics/metabolism ; Biodegradation, Environmental ; *Metabolic Engineering/methods ; *Genome, Bacterial ; *Nanoparticles/chemistry/metabolism ; CRISPR-Cas Systems ; Escherichia coli Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Numerous strategies for the biodegradation and upcycling of polyethylene terephthalate (PET) are under investigation. Here, we present a proof-of-concept study for reprogramming the Escherichia coli BL21(DE3) strain to degrade PET nanoparticles (nPET) without introducing foreign DNA and compromising native cellular fitness. In brief, native proteins selected in silico from the genome were repurposed to acquire artificial PETase activity without compromising their function and were subsequently replaced via CRISPR/Cas9 editing. A variant of the transport protein LsrB, selected for its ability to bind PET, was engineered to degrade PET powder (at 37-60°C). Building on LsrB periplasmic localization, we engineered a strain that degrades nPET at 37°C. The strain was further engineered to grow on nPET degradation products and produce valuable compounds. Our method, which is applicable across diverse genomes and microbial chassis, expands the potential of metabolic engineering to address plastic biodegradation and upcycling while reducing reliance on foreign DNA.},
}

*Polyethylene Terephthalates/metabolism/chemistry
*Escherichia coli/genetics/metabolism
Biodegradation, Environmental
*Metabolic Engineering/methods
*Genome, Bacterial
*Nanoparticles/chemistry/metabolism
CRISPR-Cas Systems
Escherichia coli Proteins/genetics/metabolism
Gene Editing/methods

@article {pmid40473489,
year = {2025},
author = {Sen, MK and Sellamuthu, G and Mondal, SK and Varshney, RK and Roy, A},
title = {Epigenome editing for herbicide-resistant crops.},
journal = {Trends in plant science},
volume = {30},
number = {11},
pages = {1201-1210},
doi = {10.1016/j.tplants.2025.05.003},
pmid = {40473489},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/drug effects ; *Herbicide Resistance/genetics ; CRISPR-Cas Systems/genetics ; *Epigenome/genetics ; *Herbicides/pharmacology ; Plants, Genetically Modified/genetics ; Epigenome Editing ; },
abstract = {Herbicide resistance (HR) is fundamental for sustainable agriculture as global food security increasingly relies on efficient and eco-friendly weed management. Recent advances in CRISPR/dCas9-based epigenome editing offer a promising, non-genetic approach by precisely targeting regulatory regions of genes involved in herbicide sensitivity and detoxification. While CRISPR/Cas9 has successfully been used to develop HR crops, CRISPR/dCas9 remains underexplored in this field. We propose that CRISPR/dCas9-driven epigenome editing could enable time- and tissue-specific control of gene expression, allowing for the introduction of heritable HR traits without altering DNA sequences. This innovative approach could transform sustainable HR development, offering a powerful solution to enhance agricultural resilience and food security while aligning with eco-friendly weed management strategies.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics/drug effects
*Herbicide Resistance/genetics
CRISPR-Cas Systems/genetics
*Epigenome/genetics
*Herbicides/pharmacology
Plants, Genetically Modified/genetics
Epigenome Editing

@article {pmid41215733,
year = {2025},
author = {Claisse, O and Mosterd, C and Marrec, CL and Samot, J},
title = {Defense Systems and Prophage Detection in Streptococcus mutans Strains.},
journal = {Molecular oral microbiology},
volume = {},
number = {},
pages = {e70014},
doi = {10.1111/omi.70014},
pmid = {41215733},
issn = {2041-1014},
support = {//Association France Parkinson/ ; },
abstract = {Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans. The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty-four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR-Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti-CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction-modification systems being the most prevalent, followed by the MazEF toxin-antitoxin system and CRISPR-Cas systems. Type II-C CRISPR-Cas systems were not identified here in S. mutans. Novel variations in type II-A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting "dark matter". A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.},
}

@article {pmid41215480,
year = {2025},
author = {Ali, RM and Arshad, H and Zafar, AQ and Gull, S and Gul, A and Mansoor, F and Zahid, R},
title = {A Comprehensive Review on Engineering Lactic Acid Bacteria: Emerging Genetic Tools and Synthetic Biology Strategies.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.70079},
pmid = {41215480},
issn = {1470-8744},
abstract = {Lactic acid bacteria (LAB) are pivotal in food, pharmaceutical, and environmental applications due to their metabolic versatility and probiotic potential. This review explores the advancements in genetic engineering and synthetic biology strategies to enhance LAB functionality. We examine the genomic architecture of key LAB species, such as Lactobacillus and Lactococcus, highlighting their natural genetic traits and metabolic constraints. Emerging genetic tools, including electroporation, conjugation, and CRISPR-Cas systems, have revolutionized LAB modification, enabling precise gene editing and expression control. Synthetic biology approaches, such as genetic circuits, riboswitches, and biosensor development, offer novel pathways for optimizing LAB for functional foods, mucosal therapeutics, and industrial biotechnology. We discuss applications in probiotic delivery, bioremediation, and agricultural enhancement, emphasizing LAB's role in producing bioactive metabolites and combating pathogens. Challenges, including plasmid instability, metabolic burden, and regulatory hurdles, are addressed alongside socio-ethical considerations for genetically modified LAB. The integration of genome-scale engineering and CRISPR-based technologies holds promise for overcoming these barriers, paving the way for next-generation LAB with enhanced stress tolerance and tailored functionalities. This review synthesizes current knowledge and future prospects, underscoring the transformative potential of engineered LAB in addressing global health, environmental, and industrial needs while navigating biosafety and public perception challenges.},
}

@article {pmid41213945,
year = {2025},
author = {Hoetzel, J and Wang, T and Suess, B},
title = {Beyond the niche - unlocking the full potential of synthetic riboswitches.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9897},
pmid = {41213945},
issn = {2041-1723},
support = {SU402/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; WA 5722/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Synthetic riboswitches have undergone great development in the past decade, evolving into valuable regulatory tools. Operating entirely at the RNA level and independently of auxiliary proteins, they offer a promising alternative to protein-based systems such as TetON/OFF or CRISPR-Cas. As compact, modular RNA elements they unite sensing and regulatory functions within a single molecule, giving them the advantages of high modularity, portability and low metabolic burden. Here, we explore the unique features of synthetic riboswitches, highlight key applications, assess current bottlenecks and limitations and put them in context with emerging solutions, to emphasise the potential of synthetic riboswitches.},
}

@article {pmid41212235,
year = {2025},
author = {Yilmaz-Çolak, Ç},
title = {The CRISPR-cas repertoire of Kluyvera ascorbata: insights from genomic data.},
journal = {Current genetics},
volume = {71},
number = {1},
pages = {28},
pmid = {41212235},
issn = {1432-0983},
abstract = {The genus Kluyvera has gained increasing attention due to their emerging role as opportunistic pathogens and their antibiotic resistance determinants. Various approaches have been employed to reveal genomic insights into the evolution and pathogenicity of Kluyvera species. However, detailed knowledge about Kluyvera-specific clustered regularly interspaced short palindromic repeats (CRISPR) is still missing. In this study, a genome-mining approach was employed for the characterization of CRISPR-Cas loci in a total of 13 complete Kluyvera genomes using CRISPRCasFinder and related tools. Out of 13 species, only K. ascorbata displayed multiple CRISPR-Cas arrays and a complete set of cas genes characteristics of a type I-E system. Spacer analysis revealed potential targets within phage and plasmid sequences, indicating historical exposure to mobile genetic elements. Furthermore, a phylogenetic tree constructed using the Cas3 protein sequence positioned K. ascorbata closely with other enteric bacteria, including Salmonella spp. and Citrobacter spp. This study provides the first detailed insight into the CRISPR-Cas architecture of K. ascorbata. Although there is no significant diversity of the CRISPR-Cas system identified in this species, it can emphasize a role as active immune defenses against invaders and offer a foundation for future functional and evolutionary investigations. Moreover, difficulties in identification of the genus Kluyvera can be overcome through the CRISPR-Cas system using next-generation diagnostics tools.},
}

@article {pmid41211267,
year = {2025},
author = {Ramesh, B and Fakoya, AO},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Unleashed: Transforming Gene Editing With Breakthroughs, Applications, and Ethical Dilemmas.},
journal = {Cureus},
volume = {17},
number = {11},
pages = {e95908},
pmid = {41211267},
issn = {2168-8184},
abstract = {The most significant breakthrough in gene editing is the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. This innovative technology enables scientists to insert or delete genes using specific enzymes, facilitating modifications to genomes that can influence an organism's phenotype. The Cas9 enzyme is the most widely used within the CRISPR framework and has already received approval for treating sickle cell disease, with many other applications likely to follow. As this rapidly evolving field continues to advance, it holds great promise for addressing genetic disorders and diseases. This article will explore the various enzymes available in the CRISPR system, the range of diseases and conditions that could be treated using this technology, alternative gene therapy methods, and the ethical considerations surrounding its use.},
}

@article {pmid41211185,
year = {2025},
author = {Allmang, S and Witzel, HR and Hausen, A and Marquard, S and Eckert, C and Marnet, N and Hörner, N and Mayer, P and Heinrich, S and Dang, H and Roth, W and Gaida, MM},
title = {Aberrant Expression of A Disintegrin and Metalloproteinase With Thrombospondin Motifs 13 (ADAMTS13) in Pancreatic Cancer Leads to Dichotomic Functions.},
journal = {MedComm},
volume = {6},
number = {11},
pages = {e70462},
pmid = {41211185},
issn = {2688-2663},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers characterized by highly invasive growth into the surrounding peripancreatic fat tissue, where tumor cells can directly interact with adipocytes. Due to poor response to the currently available (radio)chemotherapies, there is an urgent need for advanced therapy concepts. The present study shows that ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a key factor in blood coagulation, is significantly overexpressed in human PDAC. Immunohistochemical analysis revealed that ADAMTS13 expression is associated with prolonged survival and negatively correlated with vascular density. In vitro and in vivo experiments demonstrate its partial induction by leptin. Mechanistically, CRISPR/Cas-mediated ADAMTS13 knockout in PDAC cells resulted in reduced migration and invasion. In an avian xenograft tumor model, ADAMTS13 loss led to increased vascularization, decreased vascular length, and diminished tumor growth, accompanied by reduced expression of multiple key angiogenic and angioplastic factors. Furthermore, loss of ADAMTS13 was associated with decreased expression of mesenchymal markers. In conclusion, we identified an aberrant expression and alternative function of ADAMTS13 in PDAC linked to tumor progression, plasticity, and angiogenesis, partly induced by the peripancreatic fat tissue, making this metalloproteinase an interesting target for personalized therapies.},
}

@article {pmid41210586,
year = {2025},
author = {Joo, JH and Lee, S and Kim, KP},
title = {Precision gene editing: The power of CRISPR-Cas in modern genetics.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102733},
pmid = {41210586},
issn = {2162-2531},
abstract = {Gene editing has transformed molecular biology by enabling precise modifications to genomic DNA across a wide variety of organisms. Gene editing technologies make it possible to add, remove, or modify specific DNA sequences, with a range of applications including gene knockouts, therapeutic gene correction, and the design of targeted genetic traits. These techniques depend on two main DNA repair mechanisms: homology-directed repair (HDR), which facilitates precise changes to the genome, and non-homologous end joining (NHEJ), which often results in mutations such as deletions or frameshift errors. Among the diverse gene-editing platforms, the CRISPR-Cas system has emerged as the most extensively employed, owing to its simplicity, low cost, and efficiency. This review presents the evolution of gene-editing technologies, with a particular emphasis on the CRISPR-Cas system and its expanding applications in genetics, biotechnology, agriculture, and medicine. Furthermore, advanced gene editing approaches are discussed, offering an overview of emerging trends.},
}

@article {pmid41209346,
year = {2025},
author = {Wei, Y and Sun, J and Zhu, R},
title = {CRISPR-epigenetic crosstalk: From bidirectional regulation to therapeutic potential.},
journal = {Computational and structural biotechnology journal},
volume = {27},
number = {},
pages = {4496-4504},
pmid = {41209346},
issn = {2001-0370},
abstract = {Recent advances in epigenetics have elucidated the pivotal roles of epigenetic modifications in genomic regulation and disease pathogenesis. Concurrently, CRISPR-based technologies have transcended conventional gene-editing applications and have emerged as powerful tools for target gene screening, chromatin imaging, and epigenetic modulation. Notably, epigenetic landscapes substantially influence the CRISPR editing efficiency, whereas CRISPR itself can reshape epigenetic states, forming a dynamic CRISPR-Epigenetics Regulatory Circuit. This review systematically examines the bidirectional interplay between CRISPR systems and epigenetic modifications, emphasizing their collective impact on genome-editing precision, disease progression, and therapeutic development. Existing studies have predominantly focused on the application of CRISPR in epigenetic modifications or the impact of epigenetic landscapes on CRISPR, exhibiting unidirectional characteristics. However, accumulating evidence suggests a bidirectional interaction between the two. Here, a transformative "CRISPR-Epigenetics Regulatory Circuit" model is synthesized and presented, supported by three pivotal breakthroughs: demonstrating CRISPR as an active epigenetic programmer, synthesizing the epigenetic preconditioning therapeutic paradigm, and elucidating the first predictive mathematical model (EPIGuide). Further exploration of this circuit is expected to enhance CRISPR performance, optimize sgRNA design via epigenetic predictive models, and pioneer sequential epigenetic or gene editing therapies.},
}

@article {pmid41206864,
year = {2025},
author = {Docherty, JAD and Cook, R and Kiu, R and Dyball, X and Brown, TL and Kujawska, M and Smith, RL and Phillips, S and Watt, R and Telatin, A and Tiwari, SK and Hall, LJ and Adriaenssens, EM},
title = {Diverse defense systems and prophages in human-associated Bifidobacterium species reveal coevolutionary "arms race" dynamics.},
journal = {Cell reports},
volume = {44},
number = {11},
pages = {116542},
doi = {10.1016/j.celrep.2025.116542},
pmid = {41206864},
issn = {2211-1247},
abstract = {Bacteria of the genus Bifidobacterium are pivotal for human health, especially in early life, where they dominate the gut microbiome in healthy infants. Bacteriophages, as drivers of gut bacterial composition, can affect bifidobacterial abundance. Here, we use a bioinformatics approach to explore direct interactions between human-associated Bifidobacterium spp. and prophages, as evidenced by their genomes. Analysis of 1,086 bifidobacterial genomes reveals the presence of complex systems that prevent viral invasion, with 34 defense systems and 56 subtypes detected, including several different CRISPR-Cas systems. CRISPR spacers target almost three-quarters of bifidobacteria-derived prophages, indicating dynamic interactions. At least one prophage is present in ∼67% of strains, with phages exhibiting high genomic diversity and evidence of historical recombination. These prophages encode various defense and anti-defense systems, such as anti-CRISPR genes and restriction-modification mechanisms. Overall, this investigation reveals that coevolutionary "arms race" dynamics drive genomic diversity in both bifidobacteria and their phages.},
}

@article {pmid41206491,
year = {2025},
author = {Landesman, WJ and Hudson, TR and Bedore, SE and Suarez, MC and Hayden, MS},
title = {A CRISPR/LbCas12a system for Borrelia burgdorferi sensu stricto detection in blacklegged ticks.},
journal = {Journal of medical entomology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jme/tjaf163},
pmid = {41206491},
issn = {1938-2928},
support = {P20GM103449//Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health/ ; U01CK000661/ACL/ACL HHS/United States ; /CC/CDC HHS/United States ; },
abstract = {CRISPR/Cas systems have the potential to revolutionize DNA detection of vector-borne pathogens with highly specific and user-friendly assays. One such system, named DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), uses a guide RNA (gRNA) and Cas enzyme to bind to and cut DNA targets. Following cutting, Cas12a exhibits non-specific collateral cleavage of single-stranded DNA (ssDNA). A ssDNA reporter in the reaction allows the trans-cleavage activity to be harnessed as an amplified output signal upon recognition of the target by the Cas12a/gRNA complex. We developed a DETECTR assay to detect Borrelia burgdorferi sensu stricto, the primary Lyme disease pathogen in the United States, in blacklegged ticks (Ixodes scapularis) collected from forests in southern Vermont. We compared DETECTR to gel electrophoresis of PCR-amplified products and used quantitative real-time PCR (qPCR) of a different B. burgdorferi primer set for independent confirmation. We found that 123/125 of the samples had identical results for DETECTR and gel electrophoresis. Both assays identified the same 33 B. burgdorferi-positive samples and the same 90 B. burgdorferi-negative samples. On a subset of eight samples, we tested DETECTR using lateral flow test strips and obtained identical results to those obtained with the fluorescence-based DETECTR. The sensitivity of DETECTR was lower than qPCR, which detected nine additional B. burgdorferi-positive samples. When qPCR is not available, the DETECTR assay offers a robust alternative to gel electrophoresis that is more user-friendly and requires less time. Due to the highly specific nature of the assay, DETECTR provides additional confidence that a B. burgdorferi target is present.},
}

@article {pmid41206049,
year = {2025},
author = {Wu, JN and Li, C and Liu, Z and Li, X and Wang, J and Lou, X and Xia, F and Dai, J and Wu, T},
title = {Photocontrolled dissociation and toehold-mediated strand displacement-based synergistic regulation of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206049},
issn = {1362-4962},
support = {22 474 045//National Natural Science Foundation of China/ ; 82 172 372//National Natural Science Foundation of China/ ; HUST: 2024JYCXJ010//Central Universities/ ; S202410487577//Undergraduates of Hubei Province/ ; },
abstract = {The trans-cleavage activity of the CRISPR-Cas system holds broad potential across diverse fields, yet precise spatiotemporal regulation remains challenging due to the predominantly single-direction control strategies available. Here, we present a bidirectional, multi-round modulation strategy for CRISPR-Cas12a trans-cleavage activity, utilizing toehold-mediated strand displacement and photocontrolled dissociation. This approach enables dynamic transitions between on and off states: Cas12a activity is activated by an activator, inhibited by a photosensitive blocker, and reactivated via UV light. We further integrated this system with DNA cryptography, establishing a hierarchical temporal authorization system that enhanced cryptographic security. The method supported multi-round modulation, achieving restoration of 95.4% activity after multiple cycles in the on state while maintaining suppression to 12.4% in the off state. This precise control strategy provides a versatile tool for spatiotemporal regulation in CRISPR-based applications, with significant implications for advanced gene editing, diagnostics, and bioengineering.},
}

@article {pmid41206045,
year = {2025},
author = {Nakamura, J and Shiraishi, M and Yamamoto, J and Suzuki, K},
title = {Development of programmable RNA imaging with RNA-guided GFP via click chemistry.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206045},
issn = {1362-4962},
support = {25K21779//The Uehara Memorial Foundation and the Japan Society/ ; JPMJSP2138//JST/ ; //Japan Society for the Promotion of Science/ ; },
abstract = {The CRISPR-Cas system revolutionized molecular biology by guiding Cas proteins to target nucleic acid sequences using customizable guide RNAs, offering unparalleled precision and versatility. Inspired by this innovation, we developed RNA-guided green fluorescent protein (RGG), a simple and programmable platform for targeting nucleic acid. Using a streamlined click chemistry approach, known for its high efficiency and specificity, we conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids, designed to complement target sequences, with azide-exposed proteins to construct RGG. Systematic optimization identified 30-nt RNA with 3'-DBCO modifications as the most effective configuration for RGG, enabling precise visualization of nuclear-localized RNAs, including NEAT1 and Satellite III RNA, in living cells. This establishes RGG as a customizable and efficient system for RNA imaging and molecular analysis, underscoring the potential of direct conjugation between guide nucleic acids and proteins to enable precise nucleic acid recognition and dynamic molecular modification in living cells.},
}

@article {pmid41205654,
year = {2025},
author = {Yu, L and Niu, M and Dong, Z and Dong, X and Han, Y and An, J and Jiang, T and Chen, Y and Feng, Y and Sun, Y and Li, H},
title = {High-efficiency inhibition of human adenovirus type 55 replication by CRISPR-Cas12a.},
journal = {The Journal of infection},
volume = {},
number = {},
pages = {106650},
doi = {10.1016/j.jinf.2025.106650},
pmid = {41205654},
issn = {1532-2742},
abstract = {OBJECTIVES: Human adenovirus 55 (HAdV-55), a highly pathogenic double-stranded DNA virus, presents a serious global public health challenge due to its rapid transmission and complex pathogenesis. Current antiviral treatment options for HAdV-55 are limited, with no specific antiviral drugs available. The CRISPR-Cas system, capable of precisely targeting viral genomes, has emerged as a novel approach for antiviral therapy. This study aimed to leverage targeted DNA cleavage activity of the CRISPR-Cas12a system to develop a therapeutic strategy for effectively inhibiting HAdV-55 replication.

METHODS: We developed a rapid and efficient screening platform for identifying antiviral targets by integrating CRISPR-Cas12a fluorescence detection technology with bioinformatics analysis. Using this platform, we systematically screened 194 candidate targets against HAdV-55.

RESULTS: The E1B-crRNA6-Cas12a system was identified, demonstrating a highly potent antiviral activity with 99.17% inhibitory efficiency and a selectivity index (SI) of 2482.80. This target significantly outperformed the clinical broad-spectrum anti-adenovirus drug cidofovir in both inhibitory efficacy and duration.

CONCLUSIONS: This study not only holds promise for providing safe and highly effective antiviral candidate targets for HAdV-55 therapy but also, through the construction of an interdisciplinary technical platform, is expected to enhance the translational potential of CRISPR antiviral technology for preclinical applications.},
}

@article {pmid41203870,
year = {2025},
author = {Chen, X and Xiao, L and Wang, Q and Zhou, L and Xu, Y and Zhou, C and Dai, M and Wang, F and Xu, H and Luo, D and Sirois, P and Li, K and Liao, D and Zhang, J},
title = {Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays.},
journal = {Biotechnology letters},
volume = {47},
number = {6},
pages = {128},
pmid = {41203870},
issn = {1573-6776},
support = {SKY2021047//the Suzhou Science and Technology Planning Project/ ; ND2022B04//Program of Clinical Research Center of Neurological Disease/ ; NS2021016//Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul/ ; },
mesh = {*Gene Editing/methods ; Escherichia coli/genetics ; *CRISPR-Cas Systems ; *Endonucleases/genetics/metabolism ; Humans ; beta-Galactosidase/genetics/metabolism ; },
abstract = {More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.},
}

*Gene Editing/methods
Escherichia coli/genetics
*CRISPR-Cas Systems
*Endonucleases/genetics/metabolism
Humans
beta-Galactosidase/genetics/metabolism

@article {pmid41203686,
year = {2025},
author = {Sun, Y and Qu, K and Corsi, GI and Anthon, C and Pan, X and Xiang, X and Jensen, LJ and Lin, L and Luo, Y and Gorodkin, J},
title = {Deep learning models simultaneously trained on multiple datasets improve base-editing activity prediction.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9821},
pmid = {41203686},
issn = {2041-1723},
support = {NNF21OC0068988//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {*Deep Learning ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Humans ; Software ; },
abstract = {CRISPR-derived base editors (BE) enable precise single nucleotide substitution without introducing double-stranded DNA breaks. Apart from the base editing enzymes, efficient base editing strongly depends on both the CRISPR guide RNA (gRNA) efficiency and the edited position. Here, we show that the accuracy of BE gRNA design can be significantly improved by generating more data and by introducing deep neural networks trained on multiple different datasets simultaneously. Generating ~20,000 gRNAs for A•T to G•C and C•G to T•A conversions, we present such deep learning models, which also allow users to do dataset-aware predictions. The methods are available online and as stand-alone software.},
}

*Deep Learning
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
Neural Networks, Computer
Humans
Software

@article {pmid41203361,
year = {2025},
author = {Xia, N and Gupta, N},
title = {Function and therapeutic potential of transketolase enzymes in parasitic protists.},
journal = {Methods in enzymology},
volume = {722},
number = {},
pages = {97-115},
doi = {10.1016/bs.mie.2025.07.010},
pmid = {41203361},
issn = {1557-7988},
mesh = {*Toxoplasma/enzymology/genetics/pathogenicity ; *Transketolase/genetics/metabolism ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Toxoplasmosis/parasitology/therapy ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Humans ; },
abstract = {Transketolase (TKT), the rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), catalyzes the reversible transfer of glycolaldehyde moieties between sugar phosphates. Here, we present wide-ranging methods to study TKT function in a clinically-relevant and globally-prevalent model intracellular pathogen, Toxoplasma gondii. The experimental framework utilizes heterologous expression of recombinant TgTKT proteins in a bacterial system, anti-TgTKT antibody production, CRISPR/Cas9-assisted genome editing to generate TgTKT-null mutants, and in-depth phenotyping, including [13]C-based metabolomic analysis to determine the carbon flux in the acutely-infectious tachyzoite stage of T. gondii. The phenotypic studies comprise the parasite proliferation in vitro and virulence assessment in a murine model of acute toxoplasmosis. The data suggest TgTKT as a metabolic rheostat regulating nucleotide biosynthesis and enabling adaptation of this highly promiscuous parasite in different nutritional environments. Last but not least, the work presents the therapeutic potential of the TgTKT-null mutant as a metabolically-attenuated vaccine, highlights the evolutionary divergence of TgTKT proteins, and provides a robust foundation to investigate TgTKT orthologs in other pathogenic protozoan organisms.},
}

*Toxoplasma/enzymology/genetics/pathogenicity
*Transketolase/genetics/metabolism
Animals
Mice
*Protozoan Proteins/genetics/metabolism
*Toxoplasmosis/parasitology/therapy
CRISPR-Cas Systems
Recombinant Proteins/genetics/metabolism
Gene Editing
Humans

@article {pmid41203292,
year = {2025},
author = {Yu, D and Duan, C},
title = {[Epigenetics and precise crop breeding for resistance].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3918-3938},
doi = {10.13345/j.cjb.250513},
pmid = {41203292},
issn = {1872-2075},
mesh = {*Epigenesis, Genetic/genetics ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; DNA Methylation/genetics ; Gene Editing ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetics refers to a heritable phenomenon that dynamically modulates gene expression without altering the DNA sequence, through molecular mechanisms such as DNA methylation, histone modification, non-coding RNA, chromatin remodeling, and RNA modifications. In plants, these modifications are extensively involved in key biological processes, including flowering time, gametogenesis, stress responses, and immune defenses. Over the past few decades, the research on epigenetics has gradually shifted from fundamental studies primarily conducted in Arabidopsis thaliana to investigations in various crop species such as rice and tomato. This transition has revealed the multifaceted roles of epigenetic regulation in shaping agronomic traits. This review integrates current knowledge of epigenetic regulatory mechanisms and their functions in plant responses to both biotic and abiotic stresses. Epigenetic editing tools such as CRISPR-dCas9 enable targeted DNA methylation or histone acetylation. Emerging transformation technologies, including magnetic nanoparticles and virus-based delivery systems, have the potential to overcome the bottlenecks of plant regeneration, offering new possibilities for precise epigenetic editing. In future agriculture, it is essential to further elucidate multi-layered epigenetic regulatory mechanisms at the single-cell level, develop efficient delivery systems, and leverage artificial intelligence to advance the application of epigenetic breeding for sustainable agricultural development.},
}

*Epigenesis, Genetic/genetics
*Crops, Agricultural/genetics
*Plant Breeding/methods
DNA Methylation/genetics
Gene Editing
*Disease Resistance/genetics
CRISPR-Cas Systems

@article {pmid41203276,
year = {2025},
author = {Ding, Y and Guo, W},
title = {[Advances in the application of genome editing technologies in plant pathogenic fungi].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3683-3700},
doi = {10.13345/j.cjb.250539},
pmid = {41203276},
issn = {1872-2075},
mesh = {*Gene Editing/methods ; *Fungi/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Plants/microbiology ; *Genome, Fungal/genetics ; },
abstract = {Filamentous fungi represent an important group of eukaryotic microorganisms with diverse ecological functions and ubiquitous distribution in various ecosystems. Among them, many species are closely associated with agriculture, functioning as major plant pathogens that cause yield losses and produce mycotoxins to compromise both the quality and safety of agricultural products. In recent years, the CRISPR/Cas system has emerged as a powerful and programmable genome editing tool, and it has been extensively applied to the genetic study of plant pathogenic fungi. This technology has greatly facilitated the investigation of pathogenic mechanisms, mycotoxin biosynthetic pathways and key gene functions, antifungal resistance, and rapid pathogen detection. This review summarizes the development of CRISPR/Cas systems and the key strategies for their application in plant pathogenic fungi and makes an outlook on the practical deployment. With the continuous advancement of gene editing technologies, emerging fungal-adapted editing systems hold great promise for advancing functional genomics and enabling innovations in disease-resistant breeding and sustainable crop protection.},
}

*Gene Editing/methods
*Fungi/genetics/pathogenicity
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology
*Plants/microbiology
*Genome, Fungal/genetics

@article {pmid40178656,
year = {2025},
author = {Gao, C and Cai, Y and Wu, X and Song, J and Zheng, Q and Wang, M and Luo, Y and Luo, Y and Fei, X and Zhang, Y and Yang, Y and Kuai, L and Ru, Y and Hong, S and Tian, N and Li, B and Zhang, Z},
title = {CRISPR/Cas9-Mediated Knockout and Overexpression Studies Unveil the Role of PD-L1 in Immune Modulation in a Psoriasis-like Mouse Model.},
journal = {Inflammation},
volume = {48},
number = {5},
pages = {3519-3528},
pmid = {40178656},
issn = {1573-2576},
support = {20224Y0373, 20234Y0269, 20234Y0075//Shanghai Municipal Health Commission Health Industry Clinical Research Special Project/ ; No. 82174383, 82204954, 82304819, 82305232, 82374458, W2433194//Shanghai Municipal Health Commission Health Industry Clinical Research Special Project/ ; 22YF1441300//Shanghai Science and Technology Development Funds (Sailing Program)/ ; lczh2021-05, lcfy2022-04, lczh2023-01//Clinical Transformation Incubation Program in Hospital/ ; zyyzdxk-2023065//Evidence-based dermatology base sponsored by State Administration of Traditional Chinese Medicine, High-level Chinese Medicine Key Discipline Construction Project (Integrative Chinese and Western Medicine Clinic) of National Administration of TCM/ ; 2023ZZ02017//Shanghai Dermatology Research Center/ ; SHDC2023CRW009//Shanghai Dermatology Hospital Demonstration Research Ward Project/ ; shzyyzdxk-2024104//Shanghai Key Discipline Construction Project of Traditional Chinese Medicine/ ; 2024-QNRC2-B04//Youth Medical Talents-Specialist Program of Shanghai "Rising Stars of Medical Talents" Youth Development Program, and Youth Talent Promotion Project of China Association of Traditional Chinese Medicine (2024-2026) Category B/ ; },
mesh = {Animals ; *B7-H1 Antigen/genetics/immunology ; *Psoriasis/immunology/chemically induced/genetics/pathology/metabolism ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; Mice, Knockout ; Imiquimod ; },
abstract = {The role of programmed death-ligand 1 (PD-L1), an essential immune checkpoint protein, has garnered considerable interest in recent years due to its influence on immune responses, particularly inhibiting immature Th cells into Th17 cells. This study aims to examine the effect of PD-L1 on psoriasis progress, which is the condition characterized by an immune response dominated by Th17 cells. We constructed the PD-L1 knockout (PD-L1[KO]) and overexpression (PD-L1[OE]) mice through CRISPR/Cas9 technology to assess the impact of PD-L1 in an imiquimod (IMQ)-induced psoriasis-like mouse model. In comparison to IMQ, the ear thickness exhibited a reduction, the PASI score decreased, and HE sections revealed a thinning of the epidermal spines in PD-L1[OE] mice. PD-L1[KO] mice, however, showed opposite results. Moreover, immunohistochemical assessments of the skin lesion tissues demonstrated heightened epidermal proliferation and inflammatory infiltration in the PD-L1[KO] group, accompanied by elevated tissue expression of proliferating cell nuclear antigen (PCNA), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50, and F4/80 in comparison to IMQ-treated and WT mice. The absence of PD-L1 in IMQ-induced mice was found to intensify the immune response, as evidenced by heightened expression of phosphorylated signal transducers and activators of transcription 3 (pSTAT3) and CD3 in the affected tissues compared to both IMQ-treated and WT mice. According to our findings, PD-L1 plays important roles in inhibiting inflammation, proliferation, and regulating immune responses. Targeting PD-L1 may present a promising therapeutic strategy for the management of psoriasis.},
}

Animals
*B7-H1 Antigen/genetics/immunology
*Psoriasis/immunology/chemically induced/genetics/pathology/metabolism
Mice
*CRISPR-Cas Systems
Disease Models, Animal
Mice, Knockout
Imiquimod

@article {pmid41202215,
year = {2025},
author = {Wang, T and Brogan, DJ and Zawalick, NM and Chen, F and Mkrtchian, Y and Komor, AC and Akbari, OS},
title = {CTRL Enables Gene-Specific RNA Regulation Using CRISPR-Cas7-11.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00658},
pmid = {41202215},
issn = {2161-5063},
abstract = {Advancements in synthetic biology have enabled the development of precision gene expression technologies for comprehensive investigations of biological and biochemical networks. Here, we describe the development of a refined and innovative tool, CRISPR-Cas Transgenic Repressible eLement (CTRL), which utilizes the direct repeat processing activity of the recently discovered CRISPR-Cas7-11 effector to site-specifically target synthetic mRNA molecules. We demonstrate that CTRL exhibits high efficiency, tunable regulation of expression, and gene-specific repression of mRNA and protein expression. We engineered multiple permutations of the Cas7-11 effector that differ in their ability to reduce gene expression, suggesting flexibility for the application of choice. CTRL is a novel variation on gene repression technology that exhibits broad applicability across multiple model systems.},
}

@article {pmid41202131,
year = {2025},
author = {Liu, Y and Ban, Y and Gao, D},
title = {Oligo-CALL: A next-generation barcoding platform for studying resistance to targeted therapy.},
journal = {Science advances},
volume = {11},
number = {45},
pages = {eadw9990},
doi = {10.1126/sciadv.adw9990},
pmid = {41202131},
issn = {2375-2548},
mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Lung Neoplasms/genetics/drug therapy/pathology ; Single-Cell Analysis ; Molecular Targeted Therapy ; Proto-Oncogene Proteins p21(ras)/genetics/antagonists & inhibitors ; },
abstract = {Understanding therapy resistance requires deconvolving heterogeneous cell populations and tracking clonal trajectories. While CRISPR-based cellular barcoding is powerful for lineage tracing, many platforms suffer from low efficiency and limited compatibility with single-cell transcriptomics. We developed Oligo-CALL (Oligonucleotide-inducible CRISPR transcriptional activator-Assisted Lineage Labeling), an advanced barcoding system enabling precise lineage tracing, live clone isolation, and seamless integration with single-cell RNA sequencing. Applied to lung cancer cells treated with a KRAS[G12C] inhibitor, Oligo-CALL identified clones consistently enriched posttreatment, supporting a model of predestined resistance. Oligo-CALL achieved >95% efficiency in linking lineage identity to transcriptomes, uncovering diverse clone-specific pathways with underlying resistance. Paired analysis of barcode-matched clones from naïve and resistant populations revealed transient and fixed resistance phenotypes. Notably, DNA repair pathways are recurrently altered in resistant clones, and inhibition of poly(adenosine 5'-diphosphate-ribose) polymerase synergizes with KRAS G12C inhibition to overcome resistance. Together, Oligo-CALL provides a versatile platform for dissecting lineage evolution and molecular dynamics of targeted therapy resistance.},
}

Humans
*Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
CRISPR-Cas Systems
*Lung Neoplasms/genetics/drug therapy/pathology
Single-Cell Analysis
Molecular Targeted Therapy
Proto-Oncogene Proteins p21(ras)/genetics/antagonists & inhibitors

@article {pmid41202069,
year = {2025},
author = {Verkuyl, C and Belotserkovsky, A and Zerbes, T and Williams, D and Krishnan, MR and Zhu, S and Grunnesjӧ, S and Eid, S and Zhang, C and Zhao, W and Xu, L and Lin, E and O'Shea, T and Draper, B and Jungman, A and Most, P and Schmitt-Ulms, G},
title = {Toward an all-in-one recombinant adeno-associated virus vector for functionally ablating the prion gene using CRISPR-Cas technology.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0336578},
doi = {10.1371/journal.pone.0336578},
pmid = {41202069},
issn = {1932-6203},
mesh = {*Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Vectors/genetics ; Mice ; Humans ; Gene Editing/methods ; Genetic Therapy/methods ; Prion Diseases/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Any strategy that can selectively and persistently lower the brain levels of the cellular prion protein (PrPC) is expected to extend survival in prion diseases. Recent advances in the virus-mediated delivery of gene therapies prompted us to explore if a recombinant adeno-associated virus (rAAV) vector delivering a CRISPR-Cas-based gene editor can be devised that induces a functional knockout of the prion gene. Whereas the eventual objective is to assess the therapeutic potency of an optimized vector in prion-infected mice, in this proof-of-concept study, we evaluated tools and methods that are suited to achieve this goal. The result of these efforts is a first-generation all-in-one rAAV vector that codes for a prion gene-specific guide RNA and a small Cas9 endonuclease, whose expression is controlled by a truncated neural cell adhesion molecule 1 (NCAM1) promoter that is active in PrPC expressing cells. We also constructed a second rAAV vector coding for a prion gene-specific 'traffic light reporter' (TLR). The TLR can be used to monitor prion gene-editing efficacy by coding for red and green fluorescent proteins separated by a segment of the prion gene that is targeted by the gene editor. For the purification of AAVs, we adopted a robust and scalable rAAV vector assembly pipeline and undertook proof-of-concept prion gene editing experiments in human cells and mice, which to date yielded prion gene editing rates of approximately 20% and 5%, respectively. Finally, we compared brain distributions of rAAV vectors following intrathalamic versus retro-orbital injection, and selected the 9P31 capsid for future studies based on a 7.5-fold higher heterologous gene expression level as compared to the PHP.eB capsid.},
}

*Dependovirus/genetics
*CRISPR-Cas Systems/genetics
Animals
*Genetic Vectors/genetics
Mice
Humans
Gene Editing/methods
Genetic Therapy/methods
Prion Diseases/genetics/therapy
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid41201953,
year = {2025},
author = {Fan, J and Huang, L and Chen, J and Jiang, Y and Du, X and Wang, L and Ding, K and Huang, J and Qiu, S and Liu, H and Song, H},
title = {Highly Sensitive Field Detection Technology for Anthrax Based on the CRISPR/Cas13a System.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70240},
doi = {10.1111/1751-7915.70240},
pmid = {41201953},
issn = {1751-7915},
support = {32141003//National Science Foundation of China/ ; 32300080//National Science Foundation of China/ ; },
mesh = {*Bacillus anthracis/genetics/isolation & purification ; *Anthrax/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Humans ; Point-of-Care Testing ; },
abstract = {In this study, we established a highly sensitive on-site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point-of-care testing technology. By developing a CRISPR point-of-care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on-site detection of B. anthracis. It holds significant value for on-site detection of emerging infectious diseases.},
}

*Bacillus anthracis/genetics/isolation & purification
*Anthrax/diagnosis/microbiology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Humans
Point-of-Care Testing

@article {pmid41200999,
year = {2025},
author = {Moreira, R and Nascimento, H and Martins, TMDM and Barbieri, G and Pires, P and Carvalho, LN and Rosa, LR and Almeida, A and Pessuti, CL and Ferrer, H and Gomes, JÁP and Goulart, E and Raia, S and Belfort, R},
title = {Porcine Corneal Tissue and Xenozoonotic Risks: A Review of the Current Evidence.},
journal = {Xenotransplantation},
volume = {32},
number = {6},
pages = {e70068},
doi = {10.1111/xen.70068},
pmid = {41200999},
issn = {1399-3089},
support = {//by FAPESP/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods/adverse effects ; Swine ; Humans ; *Corneal Transplantation/methods/adverse effects ; *Cornea/microbiology/virology ; Heterografts/microbiology ; },
abstract = {Corneal opacities affect millions worldwide, with corneal transplantation as the primary treatment. However, donor shortages remain a challenge, leaving thousands waiting for transplants. Xenotransplantation using porcine corneas has emerged as a promising alternative due to anatomical and physiological similarities with human corneas. Advances in CRISPR/Cas technology enable genetic modifications to address immune rejection and zoonotic risks. A key concern is xenozoonosis, the transmission of infectious agents from pigs to humans. Pathogens such as porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), and bacteria pose potential risks. While PERVs can infect human cells in vitro, no transmission has been documented in vivo. Regulatory bodies, including the WHO and IXA, have established guidelines for monitoring and clinical trials. The first human corneal xenotransplantation trials in South Korea and China are underway. Enhanced biosecurity measures in tissue banks have reduced microbial contamination, improving safety. Corneal xenotransplantation presents lower risks compared to solid organ xenotransplants. With ongoing research, stringent regulations, and improved pathogen-free animal models, this technique could become a viable clinical option. Future human trials will provide crucial insights into its long-term safety and effectiveness.},
}

Animals
*Transplantation, Heterologous/methods/adverse effects
Swine
Humans
*Corneal Transplantation/methods/adverse effects
*Cornea/microbiology/virology
Heterografts/microbiology

@article {pmid41199512,
year = {2025},
author = {Friess, L and van Sinderen, D and Lee, C},
title = {A CRISPRi Gene Regulation System for Bifidobacteria.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70260},
doi = {10.1111/1751-7915.70260},
pmid = {41199512},
issn = {1751-7915},
support = {883766//Horizon 2020 Framework Programme/ ; SFI/12/RC/2273-412 P1/SFI_/Science Foundation Ireland/Ireland ; SFI/12/RC/2273 P2/SFI_/Science Foundation Ireland/Ireland ; //APC Microbiome Institute/ ; },
mesh = {*CRISPR-Cas Systems ; Streptococcus thermophilus/genetics/enzymology ; *Gene Expression Regulation, Bacterial ; *Bifidobacterium/genetics ; Plasmids ; *Bifidobacterium breve/genetics ; },
abstract = {This work describes the development of a CRISPR interference (CRISPRi) system for targeted gene repression in bifidobacteria. We first validated the CRISPRi-based approach using Bifidobacterium breve strains engineered to express nuclease-dead orthologs of Cas9 and demonstrated that the CRISPR-Cas system from Streptococcus thermophilus is efficient at targeting both reporter and endogenous genes through the use of single guide RNAs corresponding to the gene of interest. We also developed a one-plasmid system for targeted gene repression in bifidobacteria and demonstrated its utility by targeting genes involved in nucleotide metabolism and carbohydrate metabolism in several species of bifidobacteria. Efficient gene repression was achieved across all tested bifidobacterial species without the requirement for extensive optimization of transformation parameters or sequence optimization to avoid restriction modification systems thus removing the key barriers to genetic manipulation in this genus. This CRISPRi system provides a novel approach to functional genomics in bifidobacteria which facilitates future mechanistic studies in these commercially important microbes.},
}

*CRISPR-Cas Systems
Streptococcus thermophilus/genetics/enzymology
*Gene Expression Regulation, Bacterial
*Bifidobacterium/genetics
Plasmids
*Bifidobacterium breve/genetics

@article {pmid41199069,
year = {2025},
author = {Singh, S and Chaudhary, R and Tiwari, S},
title = {CRISPR/Cas9-mediated gene targeting at BBM2 locus demonstrates HDR-assisted precise knock-in in banana cv. Grand Naine.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {265},
pmid = {41199069},
issn = {1432-203X},
support = {BRIC-NABI Core Research Grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*Musa/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Gene Targeting/methods ; *Plant Proteins/genetics/metabolism ; *Recombinational DNA Repair/genetics ; },
abstract = {The present study demonstrates the first CRISPR/Cas-mediated precise knock-in of the eGFP gene at the BABYBOOM2 (GN-BBM2) locus in banana cv. Grand Naine, facilitating the detection of editing events in early embryogenic developmental stages. Genome editing has accelerated crop improvement programs by introducing targeted and precise genetic modifications. Among different tools, CRISPR/Cas-based genome editing has been widely used for enabling mutations through double-stranded breaks (DSBs), repaired either by non-homologous end joining (NHEJ) for gene knockouts or homology-directed repair (HDR) to generate knock-in events. While gene knockouts are well established in banana, efficient knock-in remains a major challenge due to low HDR activity, sterility, and the vegetatively propagated nature of banana. In the present study, we report the first successful CRISPR/Cas-based gene knock-in editing in banana by targeting the BABYBOOM2 (BBM2) gene, which encodes a transcription factor involved in somatic embryogenesis. The enhanced green fluorescent protein (eGFP) gene was precisely inserted at the BBM2 locus in banana cv. Grand Naine to enable visual detection during embryogenesis. In vitro validation showed ~ 95% target cleavage efficiency of the selected gRNA. The PCR-based screening and shift-in amplicon size analyses confirmed three edited lines (#3, #11, and #14) harboring eGFP knock-in at the targeted locus. Sequencing of the amplicon from these lines further confirmed the precise knock-in events. Hence, this study establishes a foundation for precise knock-in-based genome modification in banana and opens new avenues for targeted trait improvement in this important clonally propagated crop.},
}

*Musa/genetics
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Gene Editing/methods
Green Fluorescent Proteins/genetics/metabolism
Plants, Genetically Modified
*Gene Targeting/methods
*Plant Proteins/genetics/metabolism
*Recombinational DNA Repair/genetics

@article {pmid41198952,
year = {2025},
author = {Makarova, KS and Shmakov, SA and Wolf, YI and Mutz, P and Altae-Tran, H and Beisel, CL and Brouns, SJJ and Charpentier, E and Cheng, D and Doudna, J and Haft, DH and Horvath, P and Moineau, S and Mojica, FJM and Pausch, P and Pinilla-Redondo, R and Shah, SA and Siksnys, V and Terns, MP and Tordoff, J and Venclovas, Č and White, MF and Yakunin, AF and Zhang, F and Garrett, RA and Backofen, R and van der Oost, J and Barrangou, R and Koonin, EV},
title = {An updated evolutionary classification of CRISPR-Cas systems including rare variants.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41198952},
issn = {2058-5276},
support = {Intramural funds//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {The known diversity of CRISPR-Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR-Cas systems. The updated CRISPR-Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR-Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR-Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR-Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.},
}

@article {pmid41198678,
year = {2025},
author = {Nukaga, S and Shiraishi, K and Hamabe, K and Mochizuki, A and Hamaguchi, Y and Ogawa, E and Le, NT and Shimada, Y and Ono, H and Nishinakamura, H and Kobayashi, Y and Hamamoto, J and Ui, A and Araki, M and Sagae, Y and Ohgino, K and Sugihara, K and Endo, S and Miyakoshi, J and Shiraishi, Y and Yasuda, H and Okuno, Y and Yoshida, T and Goto, Y and Ohe, Y and Watanabe, SI and Yatabe, Y and Nishikawa, H and Hamamoto, R and Kohno, T and Nakaoku, T},
title = {Mutation of CMTR2 in Lung Adenocarcinoma Alters RNA Alternative Splicing and Reveals Therapeutic Vulnerabilities.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9754},
pmid = {41198678},
issn = {2041-1723},
support = {JP23ak0101205//Japan Agency for Medical Research and Development (AMED)/ ; JP24ama221233//Japan Agency for Medical Research and Development (AMED)/ ; JP24ck0106905//Japan Agency for Medical Research and Development (AMED)/ ; 25K02539//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 22KJ3158//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 20H00545//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJCR1689//MEXT | Japan Science and Technology Agency (JST)/ ; JPMJCR18Y4//MEXT | Japan Science and Technology Agency (JST)/ ; },
mesh = {Humans ; *Adenocarcinoma of Lung/genetics/drug therapy ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Alternative Splicing/genetics ; *Mutation ; Cell Line, Tumor ; *Methyltransferases/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {RNA splicing dysregulation has emerged as a hallmark of cancer and a promising therapeutic target; however, its full landscape in human solid cancer remains poorly characterized. To address this, we perform alternative splicing analyses using RNA-sequencing data from 751 lung adenocarcinoma samples from our cohort integrated with 519 samples from The Cancer Genome Atlas. Visualization of splicing patterns using t-distributed stochastic neighbor embedding reveals substantial inter-tumor heterogeneity driven by distinct molecular subtypes and histological differentiation. We identify a unique molecular subtype associated with inactivating mutations in CMTR2, which encodes Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2. CMTR2 mutations are observed in 3.8% of cases and are predominantly truncating mutations, which form an isolated cluster within the splicing landscape. Intrinsic and CRISPR-Cas9-engineered CMTR2 mutations disrupt alternative splicing and sensitize cancer cells to sulfonamide-based RNA splicing modulators and immune checkpoint blockade therapy. Retrospective patient data confirm the increased sensitivity of CMTR2-deficient tumors to immune checkpoint blockade therapy. These findings uncover a previously unrecognized RNA splicing deficiency in human cancers and define a molecular subtype of lung adenocarcinoma driven by RNA splicing dysregulation, suggesting targets for therapeutic intervention in lung cancer.},
}

Humans
*Adenocarcinoma of Lung/genetics/drug therapy
*Lung Neoplasms/genetics/drug therapy/pathology
*Alternative Splicing/genetics
*Mutation
Cell Line, Tumor
*Methyltransferases/genetics/metabolism
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems

@article {pmid41197724,
year = {2025},
author = {Ito, C and Yamamoto, M and Yokota, N and Nakamura, N and Shiomi, M and Kizu, M and Ichinose, M and Fujii, S and Fujii, T and Fukunaga, R},
title = {Dissecting stress-activated protein kinase (SAPK)-signaling pathways using multiplex gene knockout HeLa cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110901},
doi = {10.1016/j.jbc.2025.110901},
pmid = {41197724},
issn = {1083-351X},
abstract = {The stress-activated protein kinase (SAPK) family consists of three JNK and four p38 members. To explore the isotype-specific or overlapping roles of SAPK members, HeLa-derived multiplex SAPK-knockout (KO) cells, such as JNK1/2/3-triple KO, p38α/β/γ/δ-quadruple KO, and JNK1/2/3/p38α/β/γ/δ-septuple KO cells were generated using the CRISPR/Cas9 method. Also, 'sole survivor' hextuple KO (ss-HKO) cells, in which only one of seven SAPK genes remains intact, were generated. Western blot analyses using phospho-specific antibodies for SAPK substrates showed that serum or anisomycin-induced phosphorylation of MAPKAPK2, MSK1, Mnk1, and CREB/ATF-1 largely depended on p38, while anisomycin-induced phosphorylation of c-Jun/JunD depended on JNK. Similar analyses using the ss-HKO cell lines revealed that JNK1 rather than JNK2 contributed to the c-Jun/JunD phosphorylation, while p38α was the primary species phosphorylating the examined p38 substrates. Expression analyses of three typical immediate-early genes, c-Jun, EGR1, and c-Fos, demonstrated that JNK1 and 2 are responsible for c-Jun expression induced by interleukin-1β, tumor necrosis factor-α, ultraviolet-C (UV), and heat shock (HS), whereas p38 is predominant in EGR1 expression induced by UV and HS, and in c-Fos expression induced by the cytokines, UV, and HS. On the other hand, neither JNK nor p38 contributed significantly to the cytokine-induced EGR1 expression, suggesting complicated SAPK-signaling mechanisms that regulate immediate-early gene expression. Together, these results demonstrate the utility of the comprehensive multigene KO and sole-survivor KO strategy in dissecting intracellular signaling pathways consisting of multiple family members.},
}

@article {pmid41193960,
year = {2025},
author = {Van Goethem, MW and Vikram, S and Cowan, DA and Makhalanyane, TP},
title = {Comparative genomics reveals adaptive traits in novel Antarctic lithic cyanobacteria.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {994},
pmid = {41193960},
issn = {1471-2164},
abstract = {BACKGROUND: Terrestrial polar cyanobacteria persist at the cold limits of life, enduring a suite of extreme stressors including sub-zero temperatures, frequent freeze–thaw cycles, oligotrophic soils, variable light regimes with long periods of darkness, and desiccation. To survive, cyanobacteria have evolved diverse physiological strategies. A key adaptation among Antarctic terrestrial cyanobacteria is niche colonization: inhabiting the undersides of translucent quartz rocks (hypoliths) and the interior spaces of porous rocks (endoliths), which buffer environmental extremes and sustains the potential for photoautotrophic carbon fixation. However, the full genomic repertoire facilitating their resilience is incomplete.

RESULTS: We cultivated cyanobacteria from endolithic and hypolithic niches in Victoria Valley, Eastern Antarctica, and recovered four near-complete genomes (100% completeness, < 1% contamination). Three hypolithic genomes showed near-identical sequence similarity (whole genome average nucleotide identity = 99.98%) and phylogenomic proximity to the genus Coleofasciculus, yet represent a novel species, Coleofasciculus caryii H7-2. The fourth genome, derived from an endolith, showed moderate similarity to Aliterella antarctica (whole genome average nucleotide identity = 79.1%), and is proposed as a new species, Aliterella bergstromii E5.1. C. caryii H7-2 possessed a larger genome (~ 6.1 Mbp) than A. bergstromii E5.1 (~ 5.4 Mbp). Both genomes encoded complete pathways for carbon fixation via oxygenic photosynthesis (RuBisCO and phosphoribulokinase), extensive phycobilisomes, and multiple photoprotective mechanisms. Predicted optimal growth temperatures were 21.7 °C and 23.2 °C, respectively. Shared stress-mitigation genes included those for osmotic, thermal and oxidative (superoxide dismutase) stress response. All genomes contained biosynthetic gene clusters associated with stress-adaptive secondary metabolites, including heterocyst glycolipids, siderophores, phenazines, compounds related to nostopeptolide and merocyclophane. The C. caryii H7-2 genome encoded multiple CRISPR-Cas systems, suggesting adaptive immunity and historical phage exposure. In contrast, A. bergstromii E5.1 harboured a single prophage and an array of 24 plasmids.

CONCLUSIONS: These finding reveal that the newly-described cyanobacteria possess a rich genomic repertoire of adaptations to withstand Antarctic extremes, emphasizing the resilience and ecological importance of lithobiontic cyanobacteria in polar deserts.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12203-7.},
}

@article {pmid41191214,
year = {2025},
author = {Choi, S and Lee, JM and Kim, KK},
title = {Biomolecular condensates: molecular structure, biological functions, diseases, and therapeutic targets.},
journal = {Molecular biomedicine},
volume = {6},
number = {1},
pages = {99},
pmid = {41191214},
issn = {2662-8651},
support = {RS-2025-00513647//National Research Foundation of Korea/ ; 20240678//Bisa Research Grant of Keimyung University/ ; },
mesh = {Humans ; *Biomolecular Condensates/chemistry/metabolism ; Animals ; Neurodegenerative Diseases/metabolism ; },
abstract = {Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.},
}

Humans
*Biomolecular Condensates/chemistry/metabolism
Animals
Neurodegenerative Diseases/metabolism

@article {pmid41058551,
year = {2025},
author = {Dear, HS and Franks, NE and Markley, EE and Holtz, AM and Song, JY and Johnson, CN and Medina-Cabrera, PI and Hernandez, D and Joel, P and Pasca di Magliano, M and Wellik, DM and Allen, BL},
title = {GliFHV mice: a tool to investigate GLI processing and localization.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {21},
pages = {},
doi = {10.1242/dev.204251},
pmid = {41058551},
issn = {1477-9129},
support = {R01 CA275182/CA/NCI NIH HHS/United States ; R01 CA275182/CA/NCI NIH HHS/United States ; //Rogel Cancer Center, University of Michigan/ ; },
mesh = {Animals ; Mice ; *Zinc Finger Protein GLI1/metabolism/genetics ; Cilia/metabolism ; Fibroblasts/metabolism ; Hedgehog Proteins/metabolism ; Zinc Finger Protein Gli3/metabolism/genetics ; Gene Editing ; Signal Transduction ; Alleles ; CRISPR-Cas Systems ; Female ; Mice, Transgenic ; Nerve Tissue Proteins ; },
abstract = {GLI proteins (GLI1-3) are the transcriptional effectors of mammalian hedgehog (HH) signaling. However, studies of GLI function have been hampered by the lack of robust GLI antibodies. To address this, we utilized CRISPR-based gene editing to generate endogenous epitope-tagged Gli alleles for each Gli gene (Gli1FLAG, Gli2HA, Gli3V5). Through breeding, we established a novel mouse model, Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5, referred to as GliFHV. Importantly, GliFHV animals are viable and fertile with no overt phenotypes. Sanger and long-range DNA sequencing confirmed proper editing of each Gli allele, while qPCR and western blot analysis confirmed similar gene expression and protein levels, respectively, between wild-type and GliFHV animals. We utilized these mice to assess GLI localization in the developing limb, finding that all three GLIs localize to primary cilia with distinct distributions. Finally, we generated immortalized GliFHV mouse embryonic fibroblasts, demonstrating that these cells are HH responsive and that GLIs localize to primary cilia and nuclei in a HH-dependent fashion. These animals and cell lines provide a valuable resource for analyses of GLI processing, localization and function throughout embryogenesis, postnatal development, and in adults.},
}

Animals
Mice
*Zinc Finger Protein GLI1/metabolism/genetics
Cilia/metabolism
Fibroblasts/metabolism
Hedgehog Proteins/metabolism
Zinc Finger Protein Gli3/metabolism/genetics
Gene Editing
Signal Transduction
Alleles
CRISPR-Cas Systems
Female
Mice, Transgenic
Nerve Tissue Proteins

@article {pmid40944362,
year = {2025},
author = {Hillebrand, GH and Carlin, SC and Giacobe, EJ and Stephenson, HA and Collins, J and Hooven, TA},
title = {A Cas12a Toolbox for Rapid and Flexible Group B Streptococcus Genomic Editing and CRISPRi.},
journal = {Molecular microbiology},
volume = {124},
number = {5},
pages = {449-461},
pmid = {40944362},
issn = {1365-2958},
support = {P20GM125504//Centers of Biomedical Research Excellence/ ; P20 GM125504/GM/NIGMS NIH HHS/United States ; R01 AI182835/AI/NIAID NIH HHS/United States ; //UPMC Children's Hospital of Pittsburgh Research Advisory Council/ ; R01AI177991/NH/NIH HHS/United States ; R01 AI177991/AI/NIAID NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; R01AI182835/NH/NIH HHS/United States ; R21 AI178067/AI/NIAID NIH HHS/United States ; R01AI177991/NH/NIH HHS/United States ; R01AI182835/NH/NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; },
mesh = {*Streptococcus agalactiae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Genome, Bacterial ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Mutagenesis ; Enterococcus faecium/genetics ; },
abstract = {Streptococcus agalactiae (group B Streptococcus; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids-pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference-derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. In benchmarking, template-less Cas12a mutagenesis yielded sequence-confirmed mutants in ~7 days and homology-directed edits in ~7-14 days; aTC-resistant colonies arose at ~10[-4] of uninduced CFU, and 27%-65% of resistant clones carried the intended homology-directed edit depending on locus and homology arm length (e.g., ~27% markerless deletion; ~35% insertion; 65% with 1 kb arms). These workflows provide a rapid alternative to temperature-sensitive plasmid mutagenesis protocols that typically require ≥ 4 weeks. This Cas12a-based platform offers an efficient, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.},
}

*Streptococcus agalactiae/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plasmids/genetics
Genome, Bacterial
*Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
Mutagenesis
Enterococcus faecium/genetics

@article {pmid40675807,
year = {2025},
author = {Bykonya, AG and Guschin, DY and Barlev, NA},
title = {Modern approaches to engineering human reporter cell lines using CRISPR within Safe Harbor loci and endogenous genes.},
journal = {Critical reviews in biotechnology},
volume = {45},
number = {8},
pages = {1816-1832},
doi = {10.1080/07388551.2025.2515834},
pmid = {40675807},
issn = {1549-7801},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Cell Line ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Reporter systems are gaining increasing popularity in modern molecular biology as they provide reliable and clear readouts for various types of assays, both in cellulo and in vivo. The generation of reporter cell lines is instrumental for screening activators and inhibitors of signaling pathways to develop new therapeutic approaches. Reporter cell lines are those with stably integrated reporter constructs containing signaling genes (often luciferase or fluorescent proteins), enabling the visualization and tracking of protein expression. Although seemingly harmless and straightforward, untargeted genomic integration of reporter genes may severely affect the expression of neighboring genes, causing unwanted and unpredictable effects. Unlike the untargeted approach, the CRISPR/Cas9 system provides a more precise method of reporter integration, especially when reporters are integrated into Safe Harbor loci. This ensures minimal influence on neighboring genomic regions. This review discusses recent advancements in creating reporter lines using the CRISPR/Cas9 system and experimental approaches for identifying suitable Safe Harbor loci.},
}

Humans
*CRISPR-Cas Systems/genetics
*Genes, Reporter/genetics
Cell Line
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41191093,
year = {2025},
author = {Sun, Y and Han, H and Zhao, R and Liu, X and Liu, Z and Liang, J and Wang, B and Zhao, B and Pang, Y and Xiao, R},
title = {CRISPR-assisted MoS2@TQD nanosheets-based lateral flow assay for dual-mode and sensitive diagnosis of SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {792},
pmid = {41191093},
issn = {1436-5073},
mesh = {Humans ; *SARS-CoV-2/isolation & purification/genetics ; *COVID-19/diagnosis ; *Molybdenum/chemistry ; *Quantum Dots/chemistry ; Limit of Detection ; *Disulfides/chemistry ; Colorimetry/methods ; CRISPR-Cas Systems ; Nanostructures/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; COVID-19 Testing/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) assembled lateral flow assay (LFA) have been developed for rapid virus diagnosis, however, the single-mode visual half-quantitative characteristics limit their applications in accurate quantitative diagnosis. Herein, a CRISPR assisted MoS2@TQD (MoS2 nanosheet assembled ternary quantum dot) based colorimetric and fluorescent dual-mode LFA was fabricated for the ultrasensitive and flexible detection of the SARS-CoV-2. Compared with single QDs, MoS2@TQD can provide stable and superior colorimetric/fluorescent signals in various bio-samples. RT-RAA/CRISPR-Cas13 was adopted to couple with MoS2@TQD so that the detection sensitivity can be further enhanced. Based on the dual-mode LFA, the colorimetric visual detection limit was 400 copies/mL, and for fluorescence quantification mode, as low as 250 copies/mL can be obtained. For clinic diagnosis, 35 SARS-CoV-2 positive throat swab samples and 18 negative samples were detected by this LFA, 100% accuracy and 100% consistency with quantitative polymerase chain reaction (qPCR) results can be obtained. It indicated that MoS2@TQD-based dual-mode LFA can be a rapid, sensitive, and accurate detection platform for SARS-CoV-2 and other infectious pathogens in the point-of-care diagnosis.},
}

Humans
*SARS-CoV-2/isolation & purification/genetics
*COVID-19/diagnosis
*Molybdenum/chemistry
*Quantum Dots/chemistry
Limit of Detection
*Disulfides/chemistry
Colorimetry/methods
CRISPR-Cas Systems
Nanostructures/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats
COVID-19 Testing/methods

@article {pmid41188812,
year = {2025},
author = {Hussain, T and Awasthi, S and Shahid, F and Yi, SS and Sahni, N and Aldaz, CM},
title = {Therapeutic potential of PRMT1 as a critical survival dependency target in multiple myeloma.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1704},
pmid = {41188812},
issn = {1471-2407},
support = {R35GM133658/GM/NIGMS NIH HHS/United States ; R35GM137836/NH/NIH HHS/United States ; 7016-18//Leukemia and Lymphoma Society/ ; },
mesh = {*Protein-Arginine N-Methyltransferases/antagonists & inhibitors/genetics/metabolism ; Humans ; *Multiple Myeloma/drug therapy/genetics/pathology/metabolism ; Cell Survival/drug effects ; Cell Line, Tumor ; *Repressor Proteins/antagonists & inhibitors/genetics/metabolism ; Arginine/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; DNA Damage ; },
abstract = {Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.},
}

*Protein-Arginine N-Methyltransferases/antagonists & inhibitors/genetics/metabolism
Humans
*Multiple Myeloma/drug therapy/genetics/pathology/metabolism
Cell Survival/drug effects
Cell Line, Tumor
*Repressor Proteins/antagonists & inhibitors/genetics/metabolism
Arginine/metabolism
Gene Expression Regulation, Neoplastic/drug effects
CRISPR-Cas Systems
Cell Cycle/drug effects
DNA Damage

@article {pmid41188277,
year = {2025},
author = {Yeh, TY and Chiu, IM and Huang, YJ and Wu, SC and Hsieh, CC and Sung, HH and Hwu, WL and Wang, GJ and Ju, JC},
title = {Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {38648},
pmid = {41188277},
issn = {2045-2322},
support = {108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Blastocyst/metabolism/cytology ; *Kidney/metabolism/embryology ; Mice, Knockout ; Female ; Gene Targeting ; RNA, Guide, CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Blastocyst complementation has been reported to produce exogenous mouse organs including the pancreas, lungs, and kidneys, but the complemented kidneys still failed to rescue the host animals. In the present study, we generated mouse complemented kidneys through a two-step procedure: using CRISPR/Cas9 ribonucleoproteins (RNP) to knockout Osr1 alleles, followed by injecting mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP). When two different sgRNAs targeting the exon 2 of Osr1 were microinjected into the pronucleus of a mouse zygote, 34% of the embryos had deletions on both alleles, and these Osr1-knockouts died with no mesonephric duct development shown by histochemical staining. With three sgRNA injections, the knockout efficiencies increased, and mesonephric duct development with EGFP-positive cells was observed in ES cell-injected E12.5 embryos. Most of the ES cell-injected Osr1-knockout embryos degenerated from E13.5 to E15.5. Four of the 264 ES cell-injected embryos were born alive and survived to the second day, with strong EGFP signals observed in both the kidneys and the heart. Therefore, complementation of the Osr1-knockout blastocyst is a potential method to produce exogenous kidneys, although further modification is still needed to increase the efficiency.},
}

Animals
*CRISPR-Cas Systems
Mice
*Blastocyst/metabolism/cytology
*Kidney/metabolism/embryology
Mice, Knockout
Female
Gene Targeting
RNA, Guide, CRISPR-Cas Systems/genetics
Green Fluorescent Proteins/genetics/metabolism
Gene Editing

@article {pmid41187082,
year = {2025},
author = {Avaro, AS and Mirjalili, S and Griffiths, AD and Santiago, JG},
title = {Microfluidic networks using isotachophoresis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {45},
pages = {e2511724122},
doi = {10.1073/pnas.2511724122},
pmid = {41187082},
issn = {1091-6490},
support = {//Swedish e-Science Research Centre (SeRC)/ ; //Kungliga Tekniska Högskolan (KTH)/ ; },
abstract = {The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.},
}

@article {pmid41171899,
year = {2025},
author = {Gil-Campillo, C and Mignolet, J and Domínguez-San Pedro, A and Rapún-Araiz, B and Janssen, AB and de Bakker, V and Veening, JW and Garmendia, J},
title = {CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants.},
journal = {PLoS pathogens},
volume = {21},
number = {10},
pages = {e1013650},
pmid = {41171899},
issn = {1553-7374},
mesh = {*Haemophilus influenzae/genetics/growth & development ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Genomics/methods ; },
abstract = {Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and we refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae.},
}

*Haemophilus influenzae/genetics/growth & development
*Genome, Bacterial
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation, Bacterial
Genomics/methods

@article {pmid41109141,
year = {2026},
author = {Song, L and Bu, S and Ding, Y and Chai, Y and Fu, Y and Zhang, P and Yuan, R},
title = {CRISPR-enhanced low-potential ECL sensor based on AuPt-functionalized porphyrin AlOG for ultrasensitive aflatoxin B1 detection.},
journal = {Biosensors & bioelectronics},
volume = {292},
number = {},
pages = {118089},
doi = {10.1016/j.bios.2025.118089},
pmid = {41109141},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/isolation & purification/chemistry ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; *Porphyrins/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Aluminum/chemistry ; Food Contamination/analysis ; },
abstract = {Herein, an ultrasensitive low-potential electrochemiluminescence (ECL) sensor was developed by integrating an AuPt nanoparticle-functionalized porphyrin aluminum-organic gel (P-AlOG@AuPt) emitter with an enhanced CRISPR/Cas12a amplification module for the detection of aflatoxin B1 (AFB1) related to food safety. Impressively, the P-AlOG@AuPt emitter exhibited strong ECL emission and high signal-to-noise ratio at a low triggering potential (-0.8 V), addressing key limitations of previous high-potential aluminum-organic gels. Meanwhile, the in situ enriched AuPt as a coreaction accelerator catalyzed the reduction of coreactant S2O8[2-] to produce abundant SO4[•-] for facilitating its interactions with P-AlOG@AuPt[•-], resulting in a significant ECL enhancement. Furthermore, a synergistic catalytic hairpin assembly (CHA) pre-amplification and tetrahedral DNA nanostructure (TDN)-anchored ssDNA reporters significantly improved the cleavage efficiency of CRISPR/Cas12a, boosting the detection sensitivity of the ECL sensor for AFB1. The established ECL sensor exhibited ultrasensitive detection of AFB1 with a linear range of 0.1 pg mL[-1] - 500 ng mL[-1] and detection limit of 0.044 pg mL[-1], outperforming most reported ECL- and CRISPR-based AFB1 sensors. The practical usability of the proposed ECL sensor was verified by applying it for the quantification of AFB1 in rice samples. This work offers a promising low-potential ECL sensing platform for practical mycotoxin monitoring and broader biosensing applications.},
}

*Aflatoxin B1/analysis/isolation & purification/chemistry
*Biosensing Techniques/methods
CRISPR-Cas Systems
Limit of Detection
Luminescent Measurements/methods
Electrochemical Techniques/methods
*Porphyrins/chemistry
Gold/chemistry
Metal Nanoparticles/chemistry
Aluminum/chemistry
Food Contamination/analysis

@article {pmid41052497,
year = {2026},
author = {Huang, X and Wang, A and Lin, Z and Xu, Y and Zheng, J},
title = {Novel electrochemiluminescence resonance energy transfer biosensor driven by CRISPR-Cas12a system for ctDNA detection.},
journal = {Biosensors & bioelectronics},
volume = {292},
number = {},
pages = {118067},
doi = {10.1016/j.bios.2025.118067},
pmid = {41052497},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Circulating Tumor DNA/genetics/blood/isolation & purification ; *Lung Neoplasms/genetics/blood/diagnosis ; *Luminescent Measurements/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Gold/chemistry ; *Carcinoma, Non-Small-Cell Lung/genetics/blood/diagnosis ; Energy Transfer ; Mutation ; },
abstract = {Efficient energy donor-acceptor pairing and distance control are critical in resonance energy transfer-based electrochemiluminescence (ECL-RET) biosensor. In this work, a novel ECL-RET system with Ru(phen)3[2+] as ECL energy donor and Au nanocages as energy acceptor was constructed based on the characteristic that Ru(phen)3[2+] can be stably embedded into double-stranded DNA. On this basis, a homogeneous ECL biosensor based on target activated CRISPR-Cas12a system and driving ECL-RET effect was developed. The ECL biosensor has been successfully applied to the detection of circulating tumor DNA (ctDNA) and demonstrated efficient recognition of L858R mutation in ctDNA of non-small cell lung cancer patients. The presented ECL-RET biosensor achieves a wide linear detection range from 10 fM to 1 nM and an excellent detection limit as low as 3.0 fM for ctDNA due to its combination of the highly efficient and specific cleavage of the CRISPR-Cas12a system.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Circulating Tumor DNA/genetics/blood/isolation & purification
*Lung Neoplasms/genetics/blood/diagnosis
*Luminescent Measurements/methods
Limit of Detection
Electrochemical Techniques/methods
Gold/chemistry
*Carcinoma, Non-Small-Cell Lung/genetics/blood/diagnosis
Energy Transfer
Mutation