@article {pmid40263339,
year = {2025},
author = {Zhao, C and Cao, Y and Ibrahim, N and Wang, Y and Martemyanov, KA},
title = {Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3768},
pmid = {40263339},
issn = {2041-1723},
support = {EY018139//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; EY028033//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; EY030554//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; },
mesh = {Animals ; *Retina/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Synapses/metabolism ; Mice ; },
abstract = {A key application of CRISPR/Cas9-based genomic editing is modification of genes to introduce engineered sequences. However, the editing flexibility is severely constrained by the requirement for targeting sites in proximity to the desired modification site, which makes many modifications intractable. Here, we develop a strategy that overcomes this key limitation to allow CRISPR-based editing at any position with high efficiency. It relies on reconstructing the targeted gene using Silently Mutate And Repair Template (SMART) where we mutate the gap sequence in the repair template to prevent its base pairing with the target DNA while maintaining the same amino acid coding. Using vertebrate retina as a neuronal model system we document the application of SMART editing for labeling endogenous proteins in vivo with high efficiency. We show that SMART editing allows us to access numerous cell types in the retina and address fundamental cell biological questions pertaining to its organization. We propose that this approach will facilitate functional genomic studies in a wide range of systems and increase the precision of corrective gene therapies.},
}

Animals
*Retina/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Synapses/metabolism
Mice

@article {pmid40263274,
year = {2025},
author = {Rengifo-Gonzalez, M and Mazzuoli, MV and Janssen, AB and Rueff, AS and Burnier, J and Liu, X and Veening, JW},
title = {Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3796},
pmid = {40263274},
issn = {2041-1723},
support = {310030_200792//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; NCCR AntiResist 51NF40_180541//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; TMPFP3_210202//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 771534-PneumoCaTChER//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*Streptococcus pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.},
}

*Streptococcus pneumoniae/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
*Genetic Engineering/methods
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid40262634,
year = {2025},
author = {Silverstein, RA and Kim, N and Kroell, AS and Walton, RT and Delano, J and Butcher, RM and Pacesa, M and Smith, BK and Christie, KA and Ha, LL and Meis, RJ and Clark, AB and Spinner, AD and Lazzarotto, CR and Li, Y and Matsubara, A and Urbina, EO and Dahl, GA and Correia, BE and Marks, DS and Tsai, SQ and Pinello, L and De Ravin, SS and Liu, Q and Kleinstiver, BP},
title = {Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-025-09021-y},
pmid = {40262634},
issn = {1476-4687},
abstract = {Engineering and characterizing proteins can be time-consuming and cumbersome, motivating the development of generalist CRISPR-Cas enzymes[1-4] to enable diverse genome editing applications. However, such enzymes have caveats such as an increased risk of off-target editing[3,5,6]. To enable scalable reprogramming of Cas9 enzymes, here we combined high-throughput protein engineering with machine learning (ML) to derive bespoke editors more uniquely suited to specific targets. Via structure/function-informed saturation mutagenesis and bacterial selections, we obtained nearly 1,000 engineered SpCas9 enzymes and characterized their protospacer-adjacent motif[7] (PAM) requirements to train a neural network that relates amino acid sequence to PAM specificity. By utilizing the resulting PAM ML algorithm (PAMmla) to predict the PAMs of 64 million SpCas9 enzymes, we identified efficacious and specific enzymes that outperform evolution-based and engineered SpCas9 enzymes as nucleases and base editors in human cells while reducing off-targets. An in silico directed evolution method enables user-directed Cas9 enzyme design, including for allele-selective targeting of the RHO P23H allele in human cells and mice. Together, PAMmla integrates ML and protein engineering to curate a catalog of SpCas9 enzymes with distinct PAM requirements, and motivates the use of efficient and safe bespoke Cas9 enzymes instead of generalist enzymes for various applications.},
}

@article {pmid40261967,
year = {2025},
author = {Liu, X and Wang, P and Wang, S and Liao, W and Ouyang, M and Lin, S and Lin, R and Sarris, PF and Michalopoulou, V and Feng, X and Zhang, Z and Xu, Z and Chen, G and Zhu, B},
title = {The circular RNA circANK suppresses rice resistance to bacterial blight by inhibiting microRNA398b-mediated defense.},
journal = {The Plant cell},
volume = {37},
number = {4},
pages = {},
doi = {10.1093/plcell/koaf082},
pmid = {40261967},
issn = {1532-298X},
support = {32272479//National Natural Science Foundation of China/ ; CSTB2022NSCQ-MSX0524//Chongqing Natural Science Foundation/ ; 2025YFHZ0043//Science & Technology Department of Sichuan Province/ ; },
mesh = {*Oryza/genetics/microbiology/immunology ; *MicroRNAs/genetics/metabolism ; *RNA, Circular/genetics/metabolism ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Xanthomonas/pathogenicity/physiology ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics/metabolism ; Reactive Oxygen Species/metabolism ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Circular RNAs (circRNAs) are prevalent in eukaryotic cells and have been linked to disease progressions. Their unique circular structure and stability make them potential biomarkers and therapeutic targets. Compared with animal models, plant circRNA research is still in its infancy. The lack of effective tools to specifically knock down circRNAs without affecting host gene expression has slowed the progress of plant circRNA research. Here, we have developed a CRISPR-Cas13d tool that can specifically knock down circRNAs in plant systems, successfully achieving the targeted knockdown of circRNAs in rice (Oryza sativa). We further focused on Os-circANK (a circRNA derived from Ankyrin repeat-containing protein), a circRNA differentially expressed in rice upon pathogen infection. Physiological and biochemical experiments revealed that Os-circANK functions as a sponge for miR398b, suppressing the cleavage of Cu/Zn-superoxidase dismutase (CSD)1/CSD2/copper chaperone for superoxide dismutase/superoxidase dismutaseX through competing endogenous RNA, leading to reduced reactive oxygen species levels following Xanthomonas oryzae pv. oryzae (Xoo) infection and a negative regulation of rice resistance to bacterial blight. Our findings indicate Os-circANK inhibits rice resistance to bacterial blight via the microRNA398b(miR398b)/CSD/SOD pathway.},
}

*Oryza/genetics/microbiology/immunology
*MicroRNAs/genetics/metabolism
*RNA, Circular/genetics/metabolism
*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Xanthomonas/pathogenicity/physiology
Gene Expression Regulation, Plant
*RNA, Plant/genetics/metabolism
Reactive Oxygen Species/metabolism
CRISPR-Cas Systems/genetics
Plant Proteins/genetics/metabolism

@article {pmid40261966,
year = {2025},
author = {Cheng, Y and Li, G and Qi, A and Mandlik, R and Pan, C and Wang, D and Ge, S and Qi, Y},
title = {A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants.},
journal = {The Plant cell},
volume = {37},
number = {4},
pages = {},
doi = {10.1093/plcell/koaf081},
pmid = {40261966},
issn = {1532-298X},
support = {IOS-2029889//NSF Plant Genome Research Program/ ; 2021-67013-34554//USDA-NIFA Agricultural Innovation through Gene Editing/ ; 2020-33522-32274//USDA-NIFA Biotechnology Risk Assessment Research/ ; 21010111//Foundation for Food and Agriculture Research/ ; },
mesh = {Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Solanum lycopersicum/genetics ; Protoplasts/metabolism ; Plants, Genetically Modified ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Plant/genetics ; Acetolactate Synthase/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants.},
}

Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Solanum lycopersicum/genetics
Protoplasts/metabolism
Plants, Genetically Modified
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genome, Plant/genetics
Acetolactate Synthase/genetics

@article {pmid40261268,
year = {2025},
author = {Liu, Y and Wu, Y and Liu, Y and Zhang, Q and Yuan, H and Li, S and Li, Z and Wang, B and Chang, Y and Liu, M},
title = {Arrest of CRISPR-Cas12a by Nonspecific Single-Stranded DNA for Biosensing.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c07081},
pmid = {40261268},
issn = {1520-6882},
abstract = {CRISPR-Cas technologies have emerged as powerful biosensing tools for the sensitive and specific detection of non-nucleic acid targets. However, existing biosensing strategies suffer from poor compatibility across diverse targets due to the complicated engineering of crRNA and DNA activator required for the CRISPR-Cas activity regulation. Herein, we report a novel and straightforward strategy for designing CRISPR-Cas12a-based biosensors that function by switching structures from single-stranded (ss)DNA/CRISPR-Cas12a assembly to DNA activator/CRISPR-Cas12a complex in the presence of target bacterium. The strategy begins with a ssDNA assembly made of a trans-acting RNA-cleaving DNAzyme (tRCD) and an RNA/DNA chimeric substrate (RCS). The ssDNA assembly has the ability to bind Cas12a nonspecifically, thus indeed blocking the CRISPR-Cas12a activity. By exploiting the specific recognition and cleavage capacities of tRCD for RCS in the presence of a target, the target-bound tRCD and the cleaved RCS are released from Cas12a, thus restoring the CRISPR-Cas12a activity. This method has been successfully applied for the sensitive (detection limit: 10[2] CFU/mL) detection of Escherichia coli (E. coli, EC) and Burkholderia gladioli (B. gladioli, BG). For the blind testing of 30 clinical urine samples, it exhibited 100% sensitivity and 100% specificity in identifying E. coli-associated urinary tract infections (UTIs).},
}

@article {pmid40261207,
year = {2025},
author = {Liao, X and Li, Y and Wu, Y and Li, X and Shang, X},
title = {Deep Learning-Based Classification of CRISPR Loci Using Repeat Sequences.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00174},
pmid = {40261207},
issn = {2161-5063},
abstract = {With the widespread application of the CRISPR-Cas system in gene editing and related fields, along with the increasing availability of metagenomic data, the demand for detecting and classifying CRISPR-Cas systems in metagenomic data sets has grown significantly. Traditional classification methods for CRISPR-Cas systems primarily rely on identifying cas genes near CRISPR arrays. However, in cases where cas gene information is absent, such as in metagenomes or fragmented genome assemblies, traditional methods may fail. Here, we present a deep learning-based method, CRISPRclassify-CNN-Att, which classifies CRISPR loci solely based on repeat sequences. CRISPRclassify-CNN-Att utilizes convolutional neural networks (CNNs) and self-attention mechanisms to extract features from repeat sequences. It employs a stacking strategy to address the imbalance of samples across different subtypes and uses transfer learning to improve classification accuracy for subtypes with fewer samples. CRISPRclassify-CNN-Att demonstrates outstanding performance in classifying multiple subtypes, particularly those with larger sample sizes. Although CRISPR loci classification traditionally depends on cas genes, CRISPRclassify-CNN-Att offers a novel approach that serves as a significant complement to cas-based methods, enabling the classification of orphan or distant CRISPR loci. The proposed tool is freely accessible via https://github.com/Xingyu-Liao/CRISPRclassify-CNN-Att.},
}

@article {pmid40259370,
year = {2025},
author = {Wang, T and Brown, C and Doherty, N and Byrne, NM and Islam, R and Doherty, M and Feng, J and Yin, C and Chambers, S and McQuoid, L and Mohamed-Smith, L and Butterworth, KT and Kerr, EM and Coulter, JA},
title = {Mannose and PMI depletion overcomes radiation resistance in HPV-negative head and neck cancer.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {189},
pmid = {40259370},
issn = {1478-811X},
support = {EP/X525625/1//Engineering and Physical Sciences Research Council/ ; MR/X502881/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Radiation Tolerance/drug effects ; *Mannose/metabolism/pharmacology ; *Head and Neck Neoplasms/radiotherapy/pathology/metabolism ; *Mannose-6-Phosphate Isomerase/metabolism/deficiency/genetics ; Animals ; Cell Line, Tumor ; Mice ; Glycolysis ; CRISPR-Cas Systems ; },
abstract = {Radiotherapy is critical component of multidisciplinary cancer care, used as a primary and adjuvant treatment for patients with head and neck squamous cell carcinoma. This study investigates how mannose, a naturally occurring monosaccharide, combined with phosphomannose isomerase (PMI) depletion, enhances the sensitivity of HPV-negative head and neck tumour models to radiation. Isogenic PMI knockout models were generated by CRISPR/Cas9 gene editing, yielding a 20-fold increase in sensitivity to mannose in vitro, and causing significant tumour growth delay in vivo. This effect is driven by metabolic reprogramming, resulting in potent glycolytic suppression coupled with consistent depletion of ATP and glycolytic intermediates in PMI-depleted models. Functionally, these changes impede DNA damage repair following radiation, resulting in a significant increase in radiation sensitivity. Mannose and PMI ablation supressed both oxygen consumption rate and extracellular acidification, pushing cells towards a state of metabolic quiescence, effects contributing to increased radiation sensitivity under both normoxic and hypoxic conditions. In 3D-tumoursphere models, metabolic suppression by mannose and PMI depletion was shown to elevate intra-tumoursphere oxygen levels, contributing to significant in vitro oxygen-mediated radiosensitisation. These findings position PMI as a promising anti-tumour target, highlighting the potential of mannose as a metabolic radiosensitiser enhancing cancer treatment efficacy.},
}

Humans
*Radiation Tolerance/drug effects
*Mannose/metabolism/pharmacology
*Head and Neck Neoplasms/radiotherapy/pathology/metabolism
*Mannose-6-Phosphate Isomerase/metabolism/deficiency/genetics
Animals
Cell Line, Tumor
Mice
Glycolysis
CRISPR-Cas Systems

@article {pmid40259121,
year = {2025},
author = {Potlapalli, BP and Dassau, F and Fuchs, J and Sushmoy, DR and Houben, A},
title = {CRISPR-CISH: an in situ chromogenic DNA repeat detection system for research and life science education.},
journal = {Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology},
volume = {33},
number = {1},
pages = {7},
pmid = {40259121},
issn = {1573-6849},
support = {HO1779/33-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA/genetics ; *In Situ Hybridization/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Repetitive Sequences, Nucleic Acid ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.},
}

*CRISPR-Cas Systems
Humans
*DNA/genetics
*In Situ Hybridization/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Repetitive Sequences, Nucleic Acid
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40259107,
year = {2025},
author = {Yang, J and Song, J and Feng, Z and Ma, Y},
title = {Application of CRISPR-Cas9 in microbial cell factories.},
journal = {Biotechnology letters},
volume = {47},
number = {3},
pages = {46},
pmid = {40259107},
issn = {1573-6776},
support = {BY2021KYQD02//Binzhou Medical University/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Bacteria/genetics/metabolism ; },
abstract = {Metabolically engineered bacterial strains are rapidly emerging as a pivotal focus in the biosynthesis of an array of bio-based ingredients. Presently, CRISPR (clustered regularly interspaced short palindromic repeats) and its associated RNA-guided endonuclease (Cas9) are regarded as the most promising tool, having ushered in a transformative advancement in genome editing. Because of CRISPR-Cas9's accuracy and adaptability, metabolic engineers are now able to create novel regulatory systems, optimize pathways more effectively, and make extensive genome-scale alterations. Nevertheless, there are still obstacles to overcome in the application of CRISPR-Cas9 in novel microorganisms, particularly those industrial microorganism hosts that are resistant to traditional genetic manipulation techniques. How to further extend CRISPR-Cas9 to these microorganisms is an urgent problem to be solved. This article first introduces the mechanism and application of CRISPR-Cas9, and then discusses how to optimize CRISPR-Cas9 as a genome editing tool, including how to reduce off-target effects and how to improve targeting efficiency by optimizing design. Through offering a comprehensive perspective on the revolutionary effects of CRISPR-Cas9 in microbial cell factories, we hope to stimulate additional research and development in this exciting area.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Metabolic Engineering/methods
*Bacteria/genetics/metabolism

@article {pmid40258142,
year = {2025},
author = {Filsinger, GT and Mychack, A and Lyerly, E and Henriksen, C and Bartlett, TM and Kuchwara, H and Eitzinger, S and Bernhardt, TG and Walker, S and Church, GM and Wannier, TM},
title = {A diverse single-stranded DNA-annealing protein library enables efficient genome editing across bacterial phyla.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {17},
pages = {e2414342122},
doi = {10.1073/pnas.2414342122},
pmid = {40258142},
issn = {1091-6490},
mesh = {*Gene Editing/methods ; *DNA, Single-Stranded/genetics/metabolism ; *Genome, Bacterial ; Bacteriophages/genetics ; CRISPR-Cas Systems ; Gene Library ; *Bacteria/genetics/classification ; *Viral Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Phylogeny ; Homologous Recombination ; Staphylococcus aureus/genetics ; },
abstract = {Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA-annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia (Mycobacterium smegmatis and Corynebacterium glutamicum), Alphaproteobacteria (Agrobacterium tumefaciens and Caulobacter crescentus), and Bacilli (Lactococcus lactis and Staphylococcus aureus). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in C. crescentus to introduce single amino acid mutations with >70% efficiency. Second, we adapt SSAPs for dsDNA editing in C. glutamicum and S. aureus, enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in S. aureus to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.},
}

*Gene Editing/methods
*DNA, Single-Stranded/genetics/metabolism
*Genome, Bacterial
Bacteriophages/genetics
CRISPR-Cas Systems
Gene Library
*Bacteria/genetics/classification
*Viral Proteins/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
Phylogeny
Homologous Recombination
Staphylococcus aureus/genetics

@article {pmid40120517,
year = {2025},
author = {Wang, Q and Chen, X and Li, Y and Yang, S and Fan, S and Xia, J and Wu, H},
title = {Protein-inorganic hybrid flowers with a two-stage accelerated strategy for stimulated activation of CRISPR/Cas12a enhance polynucleotide kinase biosensing.},
journal = {Talanta},
volume = {292},
number = {},
pages = {127981},
doi = {10.1016/j.talanta.2025.127981},
pmid = {40120517},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Polynucleotide 5'-Hydroxyl-Kinase/metabolism/analysis ; Humans ; DNA Nucleotidylexotransferase/metabolism/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Polynucleotide kinases (PNK) play a crucial role in DNA damage repair and are closely associated with specific diseases, making them promising targets for therapeutic intervention. In this study, we propose a two-stage accelerated strategy that utilizes protein-inorganic hybrid flowers (PHFs) to enhance the performance of the terminal deoxynucleotidyl transferase (TdT)-combined CRISPR/Cas12a system (TCS) for efficient detection of PNK activity. In TCS, the participation of PHFs confines the substrate probes (SPs) to a limited space, thereby significantly enhancing the local concentration of phosphorylated 3' termini of SPs and effectively promoting the enzymatic reaction kinetics as the first step in the accelerated strategy. Upon encountering the target PNK, the phosphorylated 3' termini were promptly recognized and dephosphorylated to 3'-OH termini. Subsequently, TdT catalyzed the assembly of deoxyadenosine triphosphates (dATPs) without a template, rapidly activating the CRISPR/Cas12a system by forming multiple polyadenine (poly-A) chains. PHF-fixed poly-A chains then substantially boosted the localized concentration of CRISPR/Cas12a systems and vastly enhanced their efficacy in cleaving reporter nucleic acids. Our findings indicated that the spatial confinement effect facilitated by PHFs promoted frequent molecular collisions and accelerated multiple enzymatic reactions. The developed sensing strategy allows for the detection of PNK activity within a linear range of 0.001-1 U/mL, with a detection limit of 1.82 × 10[-4] U/mL. Additionally, this strategy has been successfully applied to detect PNK activity in cell extracts and to screen for PNK inhibitors. Owing to these advantages, PNK can be rapidly and accurately detected with a high sensitivity, specificity, and biostability.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Polynucleotide 5'-Hydroxyl-Kinase/metabolism/analysis
Humans
DNA Nucleotidylexotransferase/metabolism/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39930103,
year = {2025},
author = {Ling, S and Zhang, X and Dai, Y and Jiang, Z and Zhou, X and Lu, S and Qian, X and Liu, J and Selfjord, N and Satir, TM and Lundin, A and Touza, JL and Firth, M and Van Zuydam, N and Bilican, B and Akcakaya, P and Hong, J and Cai, Y},
title = {Customizable virus-like particles deliver CRISPR-Cas9 ribonucleoprotein for effective ocular neovascular and Huntington's disease gene therapy.},
journal = {Nature nanotechnology},
volume = {20},
number = {4},
pages = {543-553},
pmid = {39930103},
issn = {1748-3395},
support = {no. 31971364//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 32370148//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 81970766//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 82171102//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Huntington Disease/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; Induced Pluripotent Stem Cells/metabolism ; Huntingtin Protein/genetics ; HEK293 Cells ; *Virion/genetics ; Mice ; Disease Models, Animal ; },
abstract = {In vivo CRISPR gene editing holds enormous potential for various diseases. Ideally, CRISPR delivery should be cell type-specific and time-restricted for optimal efficacy and safety, but customizable methods are lacking. Here we develop a cell-tropism programmable CRISPR-Cas9 ribonucleoprotein delivery system (RIDE) based on virus-like particles. The efficiency of RIDE was comparable to that of adeno-associated virus and lentiviral vectors and higher than lipid nanoparticles. RIDE could be readily reprogrammed to target dendritic cells, T cells and neurons, and significantly ameliorated the disease symptoms in both ocular neovascular and Huntington's disease models via cell-specific gene editing. In addition, RIDE could efficiently edit the huntingtin gene in patients' induced pluripotent stem cell-derived neurons and was tolerated in non-human primates. This study is expected to facilitate the development of in vivo CRISPR therapeutics.},
}

*Huntington Disease/therapy/genetics
*CRISPR-Cas Systems/genetics
Animals
*Genetic Therapy/methods
Humans
Gene Editing/methods
*Ribonucleoproteins/genetics
Induced Pluripotent Stem Cells/metabolism
Huntingtin Protein/genetics
HEK293 Cells
*Virion/genetics
Mice
Disease Models, Animal

@article {pmid39742661,
year = {2025},
author = {Huang, X and Feng, X and Yan, YH and Xu, D and Wang, K and Zhu, C and Dong, MQ and Huang, X and Guang, S and Chen, X},
title = {Compartmentalized localization of perinuclear proteins within germ granules in C. elegans.},
journal = {Developmental cell},
volume = {60},
number = {8},
pages = {1251-1270.e3},
doi = {10.1016/j.devcel.2024.12.016},
pmid = {39742661},
issn = {1878-1551},
mesh = {Animals ; *Caenorhabditis elegans/metabolism/genetics ; *Caenorhabditis elegans Proteins/metabolism/genetics ; *Germ Cells/metabolism ; *Cytoplasmic Granules/metabolism ; Cell Nucleus/metabolism ; CRISPR-Cas Systems ; },
abstract = {Germ granules, or nuage, are RNA-rich condensates that are often docked on the cytoplasmic surface of germline nuclei. C. elegans perinuclear germ granules are composed of multiple subcompartments, including P granules, Mutator foci, Z granules, SIMR foci, P -bodies, and E granules. Although many perinuclear proteins have been identified, their precise localization within the subcompartments of the germ granule is still unclear. Here, we systematically labeled perinuclear proteins with fluorescent tags via CRISPR-Cas9 technology. Using this nematode strain library, we identified a series of proteins localized in Z or E granules and extended the characterization of the D granule. Finally, we found that the LOTUS domain protein MIP-1/EGGD-1 regulated the multiphase organization of the germ granule. Overall, our work identified the germ-granule architecture and redefined the compartmental localization of perinuclear proteins. Additionally, the library of genetically modified nematode strains will facilitate research on C. elegans germ granules.},
}

Animals
*Caenorhabditis elegans/metabolism/genetics
*Caenorhabditis elegans Proteins/metabolism/genetics
*Germ Cells/metabolism
*Cytoplasmic Granules/metabolism
Cell Nucleus/metabolism
CRISPR-Cas Systems

@article {pmid40255449,
year = {2025},
author = {Harshini, P and Varghese, R and Pachamuthu, K and Ramamoorthy, S},
title = {Enhanced pigment production from plants and microbes: a genome editing approach.},
journal = {3 Biotech},
volume = {15},
number = {5},
pages = {129},
pmid = {40255449},
issn = {2190-572X},
abstract = {Pigments are known for their vital roles in the growth and development of plants and microbes. In addition, they are also an imperative component of several industries, including textiles, foods, and pharmaceuticals, owing to their immense colours and therapeutic potential. Conventionally, pigments are obtained from plant resources, and the advent of in-vitro propagation techniques boosted the massive production. However, it could not meet the booming demand, leading to the incorporation of new genetic engineering tools. This review focuses on the role of various genetic engineering techniques in enhancing pigment production in plants and microorganisms. It also critically analyzes the efficacy and bottlenecks of these techniques in augmenting pigment biosynthesis. Furthermore, the use of microbes as pigment biofactories and the prospects in the field of genome editing to augment pigment synthesis are discussed. The limitations in the existing techniques underline the need for advanced genome editing strategies to broaden the mass production of pigments to meet the surging needs.},
}

@article {pmid40253348,
year = {2025},
author = {Hurtado, JE and Schieferecke, AJ and Halperin, SO and Guan, J and Aidlen, D and Schaffer, DV and Dueber, JE},
title = {Nickase fidelity drives EvolvR-mediated diversification in mammalian cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3723},
pmid = {40253348},
issn = {2041-1723},
support = {T32 GM139780/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Animals ; CRISPR-Cas Systems/genetics ; *DNA-Directed DNA Polymerase/genetics/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; *Directed Molecular Evolution/methods ; Mutation ; Mutagenesis ; },
abstract = {In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistant MAP2K1 variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase's mismatch tolerance limits EvolvR's mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR's performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR's mutation rates.},
}

Humans
Animals
CRISPR-Cas Systems/genetics
*DNA-Directed DNA Polymerase/genetics/metabolism
*Deoxyribonuclease I/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
*Directed Molecular Evolution/methods
Mutation
Mutagenesis

@article {pmid40253208,
year = {2025},
author = {Xin, Y and Guo, T and Qiao, M},
title = {Current application and future prospects of CRISPR-Cas in lactic acid Bacteria: A review.},
journal = {Food research international (Ottawa, Ont.)},
volume = {209},
number = {},
pages = {116315},
doi = {10.1016/j.foodres.2025.116315},
pmid = {40253208},
issn = {1873-7145},
mesh = {*CRISPR-Cas Systems ; *Lactobacillales/genetics/metabolism ; Gene Editing/methods ; *Food Microbiology ; Probiotics ; Fermentation ; Metabolic Engineering ; },
abstract = {Lactic acid bacteria (LABs) have a long history of use in food and beverages fermentation. Recently, several LABs have gained attention as starter or non-starter cultures and probiotics for making functional fermented foods, which have the potential to enhance human health. In addition, certain LABs show great potential as microbial cell factories for producing food-related chemicals. However, enhancing the outcomes of starter and non-starter cultures, exploring the complicated probiotic mechanism of LABs, and engineering strains to enhance the yields of high-value compounds for precision fermentation remains challenging due to the time-consuming and labor-intensive current genome editing tools. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system, originally an adaptive immune system in bacteria, has revolutionized genome editing, metabolic engineering and synthetic biology. Its versatility has resulted in extensive applications across diverse organisms. The widespread distribution of CRISPR-Cas systems and the diversity of CRISPR arrays in LAB genomes highlight their potential for studying the evolution of LABs. This review discusses the current advancement of CRISPR-Cas systems in engineering LABs for food application. Moreover, it outlines future research directions aimed at harnessing CRISPR-Cas systems to advance lactic acid bacterial research and drive innovation in food science.},
}

*CRISPR-Cas Systems
*Lactobacillales/genetics/metabolism
Gene Editing/methods
*Food Microbiology
Probiotics
Fermentation
Metabolic Engineering

@article {pmid40252747,
year = {2025},
author = {Zou, W and Huo, B and Tu, Y and Zhu, Y and Hu, Y and Li, Q and Yu, X and Liu, B and Tang, W and Tan, S and Xiao, H},
title = {Metabolic reprogramming by chemo-gene co-delivery nanoparticles for chemo-immunotherapy in head and neck squamous cell carcinoma.},
journal = {Acta biomaterialia},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.actbio.2025.04.031},
pmid = {40252747},
issn = {1878-7568},
abstract = {The therapeutic effects of platinum-based drugs are closely linked to the dysregulation of tumor metabolic-immune microenvironment, particularly aberrant lactate accumulation. Herein, we engineered multifunctional nanoparticles (PPPt[IV] NPs) through electrostatic self-assembly of poly(β-amino ester) to co-encapsulate the cisplatin prodrug (Pt[IV]) and CRISPR/Cas9-PKM2 plasmids. Mechanistically, PPPt[IV] NPs efficiently entered cells via endocytosis, followed by escape from lysosomal degradation and cargo release. The reduction of Pt[IV] prodrug to the active Pt[II] via GSH depletion induced DNA damage and ROS upregulation, thereby triggering apoptosis. Concurrently, CRISPR/Cas9-mediated PKM2 knockdown suppressed the Warburg effect, resulting in reduced lactate production and downregulated expression of HIF-1α and PD-L1. These alterations drove immune microenvironment remodeling through enhanced dendritic cell maturation, polarized M1 macrophages, and altered cytokine profiles (characterized by upregulation of IFN-γ, TNF-α, and IL-12 alongside suppression of IL-10), ultimately activating T cell-mediated antitumor immunity. Compared to conventional cisplatin, PPPt[IV] NPs demonstrated superior efficacy against both primary and recurrent tumors while reducing nephrotoxicity through synergistic chemo-immunotherapeutic effects, offering a valuable strategy for HNSCC treatment. STATEMENT OF SIGNIFICANCE: This study engineered an innovative nanoplatform (PPPt[IV]) that synergistically integrates a Pt[IV] prodrug with a CRISPR/Cas-PKM2 plasmid for treating head and neck squamous cell carcinoma. By simultaneously enhancing DNA damage and reversing lactate-mediated immunosuppression, PPPt[IV] nanoplatform achieved chemo-immunotherapy that showed greater suppression of primary and recurrent tumors with reduced renal toxicity compared to cisplatin. This nanotechnology-driven strategy provides valuable insights for the combination of platinum-based drugs with immunometabolic interventions.},
}

@article {pmid40251402,
year = {2025},
author = {Gonzalez, E and Anderson, MAE and Ang, JXD and Nevard, K and Shackleford, L and Larrosa-Godall, M and Leftwich, PT and Alphey, L},
title = {Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {13408},
pmid = {40251402},
issn = {2045-2322},
support = {INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Anopheles/genetics ; Animals ; *RNA Polymerase III/genetics ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Mosquito Vectors/genetics ; },
abstract = {Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.},
}

*Anopheles/genetics
Animals
*RNA Polymerase III/genetics
CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Promoter Regions, Genetic
Mosquito Vectors/genetics

@article {pmid40251060,
year = {2025},
author = {Huang, W and Ruyechan, MC and Ralston, KS},
title = {Work with me here: variations in genome content and emerging genetic tools in Entamoeba histolytica.},
journal = {Trends in parasitology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.pt.2025.03.010},
pmid = {40251060},
issn = {1471-5007},
abstract = {Entamoeba histolytica is the causative agent of amoebiasis, a significant source of morbidity and mortality in developing nations. Despite this, E. histolytica is understudied, leading to few treatment options and a poor understanding of pathogenesis. Genetic tools have historically been limited. By applying modern approaches, it was recently revealed that the genome is aneuploid. Interestingly, gene expression levels do not correlate with ploidy, potentially highlighting the importance of RNAi in gene regulation. Characterization of the RNAi pathway has led to potent tools for targeted gene knockdown, and the advent of RNAi-based forward genetics. CRISPR/Cas tools for editing the endogenous genome are an exciting possibility on the horizon. We celebrate the gains that have made E. histolytica tractable and anticipate continued advances.},
}

@article {pmid40250956,
year = {2025},
author = {Eidelman, M and Eisenberg, E and Levanon, EY},
title = {Global quantification of off-target activity by base editors.},
journal = {Methods in enzymology},
volume = {713},
number = {},
pages = {255-270},
doi = {10.1016/bs.mie.2024.11.036},
pmid = {40250956},
issn = {1557-7988},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Adenosine Deaminase/genetics/metabolism

@article {pmid40195937,
year = {2025},
author = {Wu, Q and Li, S and Long, X and Liu, L and Zhao, Q and Cui, Y and Zhang, Y and He, Y},
title = {β-Galactosidase-Mediated, Mn[2+]-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim.},
journal = {Analytical chemistry},
volume = {97},
number = {15},
pages = {8402-8410},
doi = {10.1021/acs.analchem.4c07110},
pmid = {40195937},
issn = {1520-6882},
mesh = {*Benzimidazoles/analysis/metabolism ; *CRISPR-Cas Systems ; *beta-Galactosidase/metabolism/chemistry ; *Carbamates/analysis ; Enzyme-Linked Immunosorbent Assay/methods ; *Manganese/chemistry/metabolism ; Manganese Compounds/chemistry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of p-aminophenyl β-D-galactopyranoside to generate p-aminophenol. This compound subsequently facilitates the decomposition of MnO2 nanosheets, leading to the generation of Mn[2+] ions. The Mn[2+] ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn[2+]-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.},
}

*Benzimidazoles/analysis/metabolism
*CRISPR-Cas Systems
*beta-Galactosidase/metabolism/chemistry
*Carbamates/analysis
Enzyme-Linked Immunosorbent Assay/methods
*Manganese/chemistry/metabolism
Manganese Compounds/chemistry
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40172020,
year = {2025},
author = {Cheng, HJ and Liu, Y and Li, HD and Yin, BC and Ma, PQ and Ye, BC},
title = {Catalytic hairpin assembly-coupled CRISPR/Cas12a biosensor for sensitive detection of melamine in dairy products.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {34},
pages = {6300-6303},
doi = {10.1039/d5cc00114e},
pmid = {40172020},
issn = {1364-548X},
mesh = {*Triazines/analysis ; *Biosensing Techniques/methods ; *Dairy Products/analysis ; *CRISPR-Cas Systems ; Catalysis ; *Food Contamination/analysis ; Limit of Detection ; },
abstract = {We combined catalytic hairpin assembly (CHA) with the Cas12a system for detecting melamine adulteration. This system involved two-step signal conversion and two-level amplification, boosting the sensor's versatility and sensitivity. The sensor showed excellent specificity and applicability for melamine detection in dairy products, and was broadened to viral nucleic acid detection.},
}

*Triazines/analysis
*Biosensing Techniques/methods
*Dairy Products/analysis
*CRISPR-Cas Systems
Catalysis
*Food Contamination/analysis
Limit of Detection

@article {pmid40160100,
year = {2025},
author = {Yang, S and Ren, L and Fan, N and Wang, S and Shen, B and Liu, Z and Li, X and Ding, S},
title = {CRISPR-Cas12a with split crRNA for the direct and sensitive detection of microRNA.},
journal = {The Analyst},
volume = {150},
number = {9},
pages = {1884-1890},
doi = {10.1039/d5an00142k},
pmid = {40160100},
issn = {1364-5528},
mesh = {*MicroRNAs/genetics/blood/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; DNA/genetics/chemistry ; },
abstract = {microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNA detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for the direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.},
}

*MicroRNAs/genetics/blood/analysis
*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
*Endodeoxyribonucleases/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
DNA/genetics/chemistry

@article {pmid40135711,
year = {2025},
author = {He, S and Lin, W and Liu, X and Li, F and Liang, H and Xu, H and Lu, C and Xing, C},
title = {A DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor for sensitive detection of Pb[2+] based on DNAzymes.},
journal = {The Analyst},
volume = {150},
number = {9},
pages = {1778-1784},
doi = {10.1039/d5an00189g},
pmid = {40135711},
issn = {1364-5528},
mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *DNA/chemistry/genetics ; Spectrometry, Fluorescence/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Water Pollutants, Chemical/analysis ; Fluorescence ; Animals ; Milk/chemistry ; Bacterial Proteins ; },
abstract = {Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb[2+] based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb[2+], the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb[2+] concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb[2+], the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb[2+] in milk and lake water samples.},
}

*Lead/analysis
*DNA, Catalytic/chemistry/genetics/metabolism
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
*DNA/chemistry/genetics
Spectrometry, Fluorescence/methods
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/metabolism/genetics
*Water Pollutants, Chemical/analysis
Fluorescence
Animals
Milk/chemistry
Bacterial Proteins

@article {pmid39302534,
year = {2025},
author = {Wiley, L and Cheek, M and LaFar, E and Ma, X and Sekowski, J and Tanguturi, N and Iltis, A},
title = {The Ethics of Human Embryo Editing via CRISPR-Cas9 Technology: A Systematic Review of Ethical Arguments, Reasons, and Concerns.},
journal = {HEC forum : an interdisciplinary journal on hospitals' ethical and legal issues},
volume = {37},
number = {2},
pages = {267-303},
pmid = {39302534},
issn = {1572-8498},
mesh = {Humans ; *Gene Editing/ethics/methods ; *CRISPR-Cas Systems ; },
abstract = {The possibility of editing the genomes of human embryos has generated significant discussion and interest as a matter of science and ethics. While it holds significant promise to prevent or treat disease, research on and potential clinical applications of human embryo editing also raise ethical, regulatory, and safety concerns. This systematic review included 223 publications to identify the ethical arguments, reasons, and concerns that have been offered for and against the editing of human embryos using CRISPR-Cas9 technology. We identified six major themes: risk/harm; potential benefit; oversight; informed consent; justice, equity, and other social considerations; and eugenics. We explore these themes and provide an overview and analysis of the critical points in the current literature.},
}

Humans
*Gene Editing/ethics/methods
*CRISPR-Cas Systems

@article {pmid38858586,
year = {2025},
author = {Pandey, S and Gao, XD and Krasnow, NA and McElroy, A and Tao, YA and Duby, JE and Steinbeck, BJ and McCreary, J and Pierce, SE and Tolar, J and Meissner, TB and Chaikof, EL and Osborn, MJ and Liu, DR},
title = {Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {22-39},
pmid = {38858586},
issn = {2157-846X},
support = {P01 CA065493/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HL160970/HL/NHLBI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *Recombinases/genetics/metabolism ; HEK293 Cells ; Integrases/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; },
abstract = {Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for 'programmable addition via site-specific targeting elements', a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.},
}

Humans
*Gene Editing/methods
*Recombinases/genetics/metabolism
HEK293 Cells
Integrases/genetics/metabolism
Animals
CRISPR-Cas Systems

@article {pmid40250676,
year = {2025},
author = {Gaba, S and Sahu, M and Chauhan, N and Jain, U},
title = {Transforming growth factor alpha: Key insights into physiological role, cancer therapeutics, and biomarker potential (A review).},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {143212},
doi = {10.1016/j.ijbiomac.2025.143212},
pmid = {40250676},
issn = {1879-0003},
abstract = {Transforming Growth Factor Alpha (TGF-α) is a critical member of the epidermal growth factor (EGF) family and a key regulator of various physiological processes, including cellular proliferation, survival, differentiation, wound repair, and tissue regeneration. Deficiencies or mutations in TGF-α have been associated with impaired tissue development and organ growth, underscoring its critical role in maintaining normal and healthy physiology. Alterations in its levels are frequently implicated in the neoplastic transformation of cells, contributing to cancer development. Several strategies for targeting TGF-α in cancer therapy have been explored, such as the use of antibodies, recombinant proteins, oligonucleotide-mediated interference in ligand synthesis, ligand sequestration via binding proteins, and modulation of the signal transduction pathway. Furthermore, there is growing interest in the potential of TGF-α as a diagnostic or prognostic biomarker for cancer. This review delves into the role of TGF-α in normal physiology and its involvement in carcinogenesis. It highlights therapies targeting TGF-α and explores future directions in targeting TGF-α /EGFR signaling using advancing approaches, including nanoparticle-based drug delivery systems, CRISPR-Cas genome editing tool, PROTAC, and combination therapies. By bringing attention to this molecule, we aim to explore its untapped potential in cancer treatment and inspire further research into its promising applications across related fields. While recent studies highlight the promise of TGF-α as a clinical biomarker, further research is needed to validate its specificity and integration into personalized medicine. By providing a comprehensive overview of TGF-α in both normal and pathological contexts, this review aims to offer new insights into its translational applications in cancer therapeutics and biomarker discovery.},
}

@article {pmid40249536,
year = {2025},
author = {Wang, F and Chen, Y and Huang, R and Lu, D and Zhang, J and Yang, Y and Dang, H and Liu, M and Chen, Z and Sun, X and Wang, Z},
title = {Identification of SURF4 and RALGAPA1 as promising therapeutic targets in glioblastoma and pan-cancer through integrative multi-omics, CRISPR-Cas9 screening and prognostic meta-analysis.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {74},
number = {6},
pages = {175},
pmid = {40249536},
issn = {1432-0851},
support = {No. 82171309//National Natural Science Foundation of China/ ; No. 82201445//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Glioblastoma/genetics/pathology/mortality ; Prognosis ; *Brain Neoplasms/genetics/pathology ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; Tumor Microenvironment ; Gene Expression Regulation, Neoplastic ; Multiomics ; },
abstract = {Glioblastoma (GBM) is the most aggressive and malignant type of primary brain tumor, with a median survival time of less than two years and a uniformly poor prognosis, despite multimodal therapeutic approaches, which highlights an urgent need for novel therapeutic targets. In this study, by integrative multi-omics analysis from CPTAC database, DepMap database and seven independent GBM cohorts, four hub genes (CD44, SURF4, IGSF3 and RALGAPA1) were identified as essential genes regulated by cancer driver genes with robust prognostic value. GBM multi-omics data from public and in-house cohorts validated that CD44 and SURF4 might be synthetic lethal partners of loss-of-function tumor suppressor genes. Analysis for immune-related pathway activity revealed complex regulation relationships of the four hub genes in tumor microenvironment (TME). Further investigation on SURF4 in pathway activity, immune therapy response and drug sensitivity proposed that SURF4 emerged as a promising therapeutic target for GBM, even for pan-cancer. Pan-cancer multi-omics exploration suggested that RALGAPA1 may be a tumor suppressor gene. By screening the first-generation and second-generation DepMap database, four genes (CCDC106, GAL3ST1, GDI2 and HSF1) might be considered as synthetic targets after mutation of RALGAPA1 as a tumor suppressor gene.},
}

Humans
*Glioblastoma/genetics/pathology/mortality
Prognosis
*Brain Neoplasms/genetics/pathology
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
*Biomarkers, Tumor/genetics
Tumor Microenvironment
Gene Expression Regulation, Neoplastic
Multiomics

@article {pmid40247031,
year = {2025},
author = {Loughran, AJ and Narina, S and Klein, J and Siwak, JF and Connelly, JP and Pruett-Miller, SM},
title = {Rapid and robust validation of pooled CRISPR knockout screens using CelFi.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {13358},
pmid = {40247031},
issn = {2045-2322},
support = {P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Pooled CRISPR screens are vital in the unbiased interrogation of gene function and are instrumental in uncovering therapeutic targets and biological processes. However, follow-up hit validation is critical to confirm observed results. Researchers need a simple and robust approach to rapidly verify putative hits and test resulting observations. Thus, we developed a CRISPR-based method for hit validation that tests the effect of a genetic perturbation on cell fitness. By editing target loci and monitoring the indel profiles over time, we have created a Cellular Fitness (CelFi) assay that can elucidate cellular vulnerabilities and verify hits from pooled CRISPR knockout screens. Unlike traditional cellular fitness assays that evaluate viability over time, the CelFi assay correlates changes in the indel profile at the target gene with a selective growth advantage or disadvantage in individual cells over time. Moreover, the CelFi assay can be utilized to evaluate gene dependencies and test new hypotheses, regardless of variations in single guide RNA optimization, ribonucleoprotein concentration, and gene copy number.},
}

*CRISPR-Cas Systems
Humans
*Gene Knockout Techniques/methods
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
INDEL Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40246839,
year = {2025},
author = {Nakamura, K and Aoyama-Ishiwatari, S and Nagao, T and Paaran, M and Obara, CJ and Sakurai-Saito, Y and Johnston, J and Du, Y and Suga, S and Tsuboi, M and Nakakido, M and Tsumoto, K and Kishi, Y and Gotoh, Y and Kwak, C and Rhee, HW and Seo, JK and Kosako, H and Potter, C and Carragher, B and Lippincott-Schwartz, J and Polleux, F and Hirabayashi, Y},
title = {Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3401},
pmid = {40246839},
issn = {2041-1723},
support = {R35 NS127232/NS/NINDS NIH HHS/United States ; JP19dm0207082//Japan Agency for Medical Research and Development (AMED)/ ; JP20H04898//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; N/A//Secom Science and Technology Foundation (SSTF)/ ; JP22H05532//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24H01348//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24H01269//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21wm0525015//Japan Agency for Medical Research and Development (AMED)/ ; N/A//Uehara Memorial Foundation/ ; },
mesh = {*Mitochondria/metabolism/ultrastructure ; *Endoplasmic Reticulum/metabolism/ultrastructure ; Humans ; *Tacrolimus Binding Proteins/metabolism/genetics ; *Mitochondrial Membranes/metabolism/ultrastructure ; HeLa Cells ; HEK293 Cells ; Proteomics ; Membrane Proteins/metabolism ; Animals ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; },
abstract = {Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identify the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron tomography, and correlative light-electron microscopy. Single molecule tracking reveals highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and captures at MERCS. Overexpression of FKBP8 is sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrate their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.},
}

*Mitochondria/metabolism/ultrastructure
*Endoplasmic Reticulum/metabolism/ultrastructure
Humans
*Tacrolimus Binding Proteins/metabolism/genetics
*Mitochondrial Membranes/metabolism/ultrastructure
HeLa Cells
HEK293 Cells
Proteomics
Membrane Proteins/metabolism
Animals
Cryoelectron Microscopy
CRISPR-Cas Systems

@article {pmid40244677,
year = {2025},
author = {Wang, X and Yue, Y and Zhai, Y and Wang, F and Zhuang, X and Wu, S and Yang, Y and Tabashnik, BE and Wu, Y},
title = {Functional redundancy in the toxic pathway of Bt protein Cry1Ab, but not Cry1Fa, against the Asian corn borer.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {16},
pages = {e2503674122},
doi = {10.1073/pnas.2503674122},
pmid = {40244677},
issn = {1091-6490},
support = {2022YFD1400901//Ministry of Science and Technology of China, National Key R&D Program/ ; 32372587)//National Natural Science Foundation of China/ ; 2020-67013-31924//USDA National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; },
mesh = {Animals ; *Endotoxins/genetics/toxicity/metabolism ; Bacillus thuringiensis Toxins ; *Bacterial Proteins/toxicity/genetics/metabolism/pharmacology ; *Hemolysin Proteins/genetics/toxicity/metabolism/pharmacology ; *Moths/genetics/drug effects ; Multidrug Resistance-Associated Protein 2 ; Insecticide Resistance/genetics ; Zea mays/parasitology/genetics ; Multidrug Resistance-Associated Proteins/genetics/metabolism ; Cadherins/genetics/metabolism ; Larva/drug effects/genetics ; Bacillus thuringiensis/genetics/metabolism ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Pest Control, Biological ; Mutation ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests, but their benefits decrease when pests evolve resistance. Better understanding of the genetic basis of resistance is needed to effectively monitor, manage, and counter pest resistance to Bt crops. Resistance to Bt proteins in at least 11 species of Lepidoptera, including many important crop pests, is associated with naturally occurring mutations that disrupt one or more of three larval midgut proteins: cadherin and ATP-binding cassette proteins ABCC2 and ABCC3. Here, we determined how CRISPR/Cas9-mediated mutations disrupting cadherin, ABCC2, and ABCC3 singly and in pairs affect resistance to Bt proteins Cry1Ab and Cry1Fa in the Asian corn borer (Ostrinia furnacalis), which is the most damaging pest of corn in Asia and is closely related to the European corn borer (Ostrinia nubilalis), a major pest in Europe and North America. The results from bioassays of six knockout strains and their parent susceptible strain support a model in which Cry1Ab can kill larvae via one path requiring ABCC2 or another path requiring cadherin and ABCC3, whereas Cry1Fa uses only the first path. The model's predictions are generally supported by results from genetic linkage analyses and responses to Cry1Ab and Cry1Fa of Sf9 cells and Xenopus oocytes modified to produce cadherin, ABCC2, and ABCC3 singly or in pairs. The functional redundancy identified here for Cry1Ab could sustain its efficacy against O. furnacalis and may exemplify a widespread natural strategy for delaying resistance.},
}

Animals
*Endotoxins/genetics/toxicity/metabolism
Bacillus thuringiensis Toxins
*Bacterial Proteins/toxicity/genetics/metabolism/pharmacology
*Hemolysin Proteins/genetics/toxicity/metabolism/pharmacology
*Moths/genetics/drug effects
Multidrug Resistance-Associated Protein 2
Insecticide Resistance/genetics
Zea mays/parasitology/genetics
Multidrug Resistance-Associated Proteins/genetics/metabolism
Cadherins/genetics/metabolism
Larva/drug effects/genetics
Bacillus thuringiensis/genetics/metabolism
Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Pest Control, Biological
Mutation

@article {pmid40215828,
year = {2025},
author = {Miskel, D and Kurzella, J and Rings, F and Tholen, E and Tesfaye, D and Schellander, K and Salilew-Wondim, D and Held-Hoelker, E and Große-Brinkhaus, C and Hoelker, M},
title = {Functional COPA is indispensable for early embryo development beyond major genome activation in bovines.},
journal = {Theriogenology},
volume = {241},
number = {},
pages = {117415},
doi = {10.1016/j.theriogenology.2025.117415},
pmid = {40215828},
issn = {1879-3231},
mesh = {Animals ; *Embryonic Development/genetics/physiology ; Cattle/embryology/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental/physiology ; *Vesicular Transport Proteins/genetics/metabolism ; Blastocyst ; Female ; },
abstract = {Embryonic genome activation is divided into a minor and a major wave of transition to endogenous transcription. In bovines, minor genome activation begins early in the 2-cell stage and is completed by the 8-cell stage when major genome activation becomes dominant. While the activation of genes known to regulate early development have been studied extensively, genes involved in more central cellular functions have not been examined. Taking advantage of the CRISPR Cas9 system, the present study investigated the effect of knocking out the Golgi retrograde protein transporter COPA on early bovine development. After the electroporation of presumptive zygotes with Cas9 ribonucleoproteins targeting COPA exon 6, sequences of 2 (11 %) and 4-cell (16 %) embryos showed knockouts of COPA whereas 8-cell embryos and blastocysts did not, demonstrating that COPA is necessary for development to the 8-cell stage and beyond. Using a repair template containing silent mutations along the target site, COPA loss of wildtype was observed in 5 blastocysts, with successful knock-in of the template on at least one allele. This shows that an edited yet functional copy of COPA can save the developmental capacity of the embryo and demonstrates that Cas9 activity at the target region itself is not responsible for the loss of function. Together, the present study revealed that COPA is necessary for embryonic development, and that the timing of this necessity is before major genome activation onset. More generally, this study further demonstrates the utility of genome editing within reproductive biotechnology for the interrogation of gene function and early embryonic development.},
}

Animals
*Embryonic Development/genetics/physiology
Cattle/embryology/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Developmental/physiology
*Vesicular Transport Proteins/genetics/metabolism
Blastocyst
Female

@article {pmid38379101,
year = {2025},
author = {Zhang, X and Van Treeck, B and Horton, CA and McIntyre, JJR and Palm, SM and Shumate, JL and Collins, K},
title = {Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.},
journal = {Nature biotechnology},
volume = {43},
number = {1},
pages = {42-51},
pmid = {38379101},
issn = {1546-1696},
support = {DP1 HL156819/HL/NHLBI NIH HHS/United States ; F32 GM139306/GM/NIGMS NIH HHS/United States ; T32 GM007232/GM/NIGMS NIH HHS/United States ; GM139306//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *Transgenes/genetics ; *Retroelements/genetics ; CRISPR-Cas Systems/genetics ; *Mutagenesis, Insertional/methods ; Animals ; Gene Editing/methods ; },
abstract = {Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.},
}

Humans
*Transgenes/genetics
*Retroelements/genetics
CRISPR-Cas Systems/genetics
*Mutagenesis, Insertional/methods
Animals
Gene Editing/methods

@article {pmid40244417,
year = {2025},
author = {Bono, H},
title = {Recent Advances in Genome Editing and Bioinformatics: Addressing Challenges in Genome Editing Implementation and Genome Sequencing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244417},
issn = {1422-0067},
support = {JPMJPF2010//Japan Science and Technology Agency/ ; },
mesh = {*Gene Editing/methods ; *Computational Biology/methods ; Humans ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; Animals ; Genome ; },
abstract = {Genome-editing technology has advanced significantly since the 2020 Nobel Prize in Chemistry was awarded for the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). While CRISPR-Cas9 has become widely used in academic research, its social implementation has lagged due to unresolved patent disputes and slower progress in gene function analysis. To address this, new approaches bypassing direct gene function analysis are needed, with bioinformatics and next-generation sequencing (NGS) playing crucial roles. NGS is essential for sequencing the genome of target species, but challenges such as data quality, genome heterogeneity, ploidy, and small individual sizes persist. Despite these issues, advancements in sequencing technologies, like PacBio high-fidelity (HiFi) long reads and high-throughput chromosome conformation capture (Hi-C), have improved genome sequencing. Bioinformatics contributes to genome editing through off-target prediction and target gene selection, both of which require accurate genome sequence information. In this review, I will give updates on the development of genome editing and bioinformatics technologies with a focus on the rapid progress in genome sequencing.},
}

*Gene Editing/methods
*Computational Biology/methods
Humans
CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing/methods
Animals
Genome

@article {pmid40244221,
year = {2025},
author = {Fan, S and Jia, L and Wu, J and Zhao, Y},
title = {Harnessing the Potential of CRISPR/Cas in Targeted Alfalfa Improvement for Stress Resilience.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244221},
issn = {1422-0067},
support = {42320104006//National Natural Science Foundation of China/ ; },
mesh = {*Medicago sativa/genetics/physiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Climate Change ; Genome, Plant ; },
abstract = {Alfalfa (Medicago sativa), recognized as the most valuable legume feed crop, faces significant challenges in enhancing both qualitative and quantitative production amidst the pressures of climate change. This review highlights these challenges, including the underutilization of genomic and genetic resources, while proposing potential solutions through genome editing. Our focus is on leveraging CRISPR/Cas technology in conjunction with decades of advancements in conventional breeding to expedite the improvement of alfalfa. By adopting this approach, we aim to overcome the limitations of traditional alfalfa improvement approaches and accelerate the development of improved cultivars capable of thriving in changing climates. Key candidate traits for CRISPR/Cas genome editing, as reviewed in the latest literature, include nutrient use efficiency, freezing tolerance, and resistance to pests and diseases. We dissect literature on potential gene pathways associated with these traits, providing molecular breeders with valuable insights for utilizing CRISPR/Cas genome editing. Furthermore, we propose editing modalities to expedite the development of stress-resilient, genome-edited alfalfa that can effectively cope with climate change.},
}

*Medicago sativa/genetics/physiology
*CRISPR-Cas Systems
*Gene Editing/methods
*Stress, Physiological/genetics
Plant Breeding/methods
Climate Change
Genome, Plant

@article {pmid40244157,
year = {2025},
author = {Zanatta, CB and Narendja, F and El Jawhary, H and Abou-Sleymane, G and Subburaj, S and Nodari, RO and Agapito-Tenfen, SZ},
title = {Suitability of Real-Time PCR Methods for New Genomic Technique Detection in the Context of the European Regulations: A Case Study in Arabidopsis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244157},
issn = {1422-0067},
support = {301911//The Research Council of Norway/ ; },
mesh = {*Arabidopsis/genetics ; *Real-Time Polymerase Chain Reaction/methods ; *Plants, Genetically Modified/genetics ; Europe ; Polymorphism, Single Nucleotide ; *Genomics/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; DNA, Plant/genetics ; Genome, Plant ; },
abstract = {PCR methods are widely applied for the detection of genetically modified organisms (GMOs) in Europe, facilitating compliance with stringent regulatory requirements and enabling the accurate identification and quantification of genetically modified traits in various crops and foodstuffs. This manuscript investigates the suitability of real-time PCR methods for detecting organisms generated through new genomic techniques (NGTs), specifically focusing on a case study using Arabidopsis thaliana as a model gene-edited plant. Given the complexities of European regulations regarding genetically modified organisms (GMOs) and the classification of gene-edited plants, there is a pressing need for robust detection methods. Our study highlights the development and validation of a novel single-plex real-time PCR method targeting a specific single nucleotide polymorphism (SNP) in the grf1-3 gene modified using CRISPR-Cas9 technology. We emphasize the effectiveness of locked nucleic acid (LNA)-modified primers in improving specificity. The results demonstrate that while the grf1-3 LNA method successfully detected and quantified gene-edited Arabidopsis DNA, achieving absolute specificity remains a challenge. This study also addresses the significance of the cross-laboratory method for validation, demonstrating that the method developed for an SNP-modified allele can be performed in accordance with the precision and trueness criteria established by the European Network of GMO Laboratories (ENGL). Furthermore, we call for continued collaboration among regulatory agencies, academia, and industry stakeholders to refine detection strategies. This proactive approach is essential not only for regulatory compliance but also for maintaining public trust in the safe integration of gene-edited organisms into food products.},
}

*Arabidopsis/genetics
*Real-Time Polymerase Chain Reaction/methods
*Plants, Genetically Modified/genetics
Europe
Polymorphism, Single Nucleotide
*Genomics/methods
CRISPR-Cas Systems
Gene Editing/methods
DNA, Plant/genetics
Genome, Plant

@article {pmid40215680,
year = {2025},
author = {Ito, R and Nakano, T and Sugawara, A and Yokoyama, A},
title = {A CRISPR-based high-throughput screening system identifies bromodomain inhibitors as transcriptional suppressors of CYP11B1.},
journal = {Biochemical and biophysical research communications},
volume = {762},
number = {},
pages = {151779},
doi = {10.1016/j.bbrc.2025.151779},
pmid = {40215680},
issn = {1090-2104},
mesh = {*Steroid 11-beta-Hydroxylase/genetics/antagonists & inhibitors/metabolism ; *High-Throughput Screening Assays/methods ; Humans ; *Triazoles/pharmacology ; *Azepines/pharmacology ; *CRISPR-Cas Systems ; *Transcription, Genetic/drug effects ; Cell Line ; Promoter Regions, Genetic/drug effects ; *Enzyme Inhibitors/pharmacology ; Epigenesis, Genetic/drug effects ; },
abstract = {CYP11B1 encodes steroid 11β-hydroxylase, the final rate-limiting enzyme for cortisol biosynthesis in the adrenal cortex. Excessive cortisol production is a hallmark of Cushing's disease (CD). While direct enzymatic inhibitors have been explored, achieving specificity remains a challenge due to the high homology between CYP11B1 and CYP11B2, highlighting transcriptional suppression of CYP11B1 as an alternative therapeutic strategy. To identify transcriptional regulators of CYP11B1, we generated genome-edited H295R adrenal cells carrying a luciferase reporter knocked into the endogenous CYP11B1 locus. Using this reporter cell line, we established a high-throughput screening (HTS) platform and screened a focused chemical library targeting epigenetic-related factors, given the importance of epigenetic mechanisms in gene regulation. Among eight candidate compounds identified, we focused on JQ1, a bromodomain inhibitor. JQ1 significantly suppressed Forskolin-induced CYP11B1 promoter activity and mRNA expression without causing cytotoxicity, suggesting the involvement of epigenetic readers in the transcriptional regulation of steroidogenic genes. Furthermore, the reporter-based HTS platform developed here, when combined with our previously established CYP11B2-luciferase system, may facilitate the identification of compounds that selectively modulate adrenal steroidogenic pathways. These findings provide a foundation for the development of novel transcription-targeted therapies for CD.},
}

*Steroid 11-beta-Hydroxylase/genetics/antagonists & inhibitors/metabolism
*High-Throughput Screening Assays/methods
Humans
*Triazoles/pharmacology
*Azepines/pharmacology
*CRISPR-Cas Systems
*Transcription, Genetic/drug effects
Cell Line
Promoter Regions, Genetic/drug effects
*Enzyme Inhibitors/pharmacology
Epigenesis, Genetic/drug effects

@article {pmid40196691,
year = {2025},
author = {Tang, S and Žedaveinytė, R and Burman, N and Pandey, S and Ramirez, JL and Kulber, LM and Wiegand, T and Wilkinson, RA and Ma, Y and Zhang, DJ and Lampe, GD and Berisa, M and Jovanovic, M and Wiedenheft, B and Sternberg, SH},
title = {Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40196691},
issn = {2692-8205},
abstract = {Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo. It remains unknown whether similar mechanisms of defense are employed by other DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.},
}

@article {pmid40054045,
year = {2025},
author = {Li, X and Wang, Z and Man, X and Dai, X and Zhou, Q and Zhang, S},
title = {Research advances CRISPR gene editing technology generated models in the study of epithelial ovarian carcinoma.},
journal = {Gynecologic oncology},
volume = {195},
number = {},
pages = {34-44},
doi = {10.1016/j.ygyno.2025.02.022},
pmid = {40054045},
issn = {1095-6859},
mesh = {Female ; *Carcinoma, Ovarian Epithelial/genetics/therapy ; *Gene Editing/methods ; Animals ; Humans ; *Ovarian Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Mice ; *Disease Models, Animal ; Genetic Therapy/methods ; },
abstract = {Epithelial ovarian carcinoma (EOC), the most lethal gynecologic cancer, is often diagnosed at advanced stages, which urge us to explore the novel therapeutic strategies. Mouse models have played a crucial role in elucidating the molecular mechanisms for the development ovarian cancer and its therapeutic strategies. However, there are still various challenges in modeling the genetic drivers of ovarian cancer in animal models. Here, we provided an overview of the research advances for the molecular mechanisms underlying EOC development, therapeutic strategies, the CRISPR genome editing technology and its generated EOC models. The review also comprehensively discussed the advantages and obstacles of CRISPR in generating EOC mouse models and the promising therapeutic approach by correcting the oncogenes of EOC through in vivo delivery of gene-edited components. The development of more precise animal models, along with a deeper understanding of EOC molecular mechanisms, will dramatically benefit the investigation and treatment of EOC.},
}

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

@article {pmid40244092,
year = {2025},
author = {Ciapaite, J and Albersen, M and Savelberg, SMC and Bosma, M and Meijer, NWF and Tessadori, F and Bakkers, JPW and van Haaften, G and Jans, JJ and Verhoeven-Duif, NM},
title = {Broad Vitamin B6-Related Metabolic Disturbances in a Zebrafish Model of Hypophosphatasia (TNSALP-Deficiency).},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244092},
issn = {1422-0067},
support = {OZF 2014-2015//Wilhelmina Children's Hospital Research Fund/ ; },
mesh = {Animals ; *Zebrafish/metabolism/genetics ; *Vitamin B 6/metabolism ; *Hypophosphatasia/metabolism/genetics ; Disease Models, Animal ; *Alkaline Phosphatase/genetics/metabolism/deficiency ; Pyridoxal Phosphate/metabolism ; Pyridoxine ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B6-responsive seizures. To study HPP, we generated alpl[-/-] zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl[-/-] compared to alpl[+/+] embryos. The survival of alpl[-/-] zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl[-/-] embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl[-/-] embryos. Accumulation of d3-pyridoxal 5'-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl[-/-] embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl[-/-] embryos. Metabolic profiling of alpl[-/-] larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.},
}

Animals
*Zebrafish/metabolism/genetics
*Vitamin B 6/metabolism
*Hypophosphatasia/metabolism/genetics
Disease Models, Animal
*Alkaline Phosphatase/genetics/metabolism/deficiency
Pyridoxal Phosphate/metabolism
Pyridoxine
Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40243700,
year = {2025},
author = {He, J and Liu, J and Yue, Y and Wang, L and Liu, Z and Xi, G and An, L and Tian, J and Wang, Y},
title = {Genome Editing in Mouse Embryo Using the CRISPR/Cas12i3 System.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40243700},
issn = {1422-0067},
support = {2022YFD1302202//National Key R&D Program of China/ ; No.2023TC019//Chinese Universities Scientific Fund/ ; 2022TC114//Chinese Universities Scientific Fund/ ; 2022TC123//Chinese Universities Scientific Fund/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Embryo, Mammalian/metabolism ; Nanog Homeobox Protein/genetics ; Female ; Embryonic Development/genetics ; Blastocyst/metabolism ; },
abstract = {The CRISPR/Cas system is a sizable family that is currently a popular and efficient gene editing tool. Cas12i3, as a member of the Type V-I family, has the characteristics of recognizing T-rich PAM sequences and being guided by shorter crRNA and has higher gene editing efficiency than Cas9 in rice. However, as a potential tool in accelerating the breeding process, the application of Cas12i3 in mammalian embryos has not yet been reported. Our study systematically evaluated the feasibility of applying CRISPR/Cas12i3 to gene editing in mouse embryos, with the core pluripotency regulator gene Nanog as the target. We successfully constructed a Nanog loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted Nanog locus, its editing efficiency exceeded that of the Cas9 system under matched experimental conditions; no off-target phenomenon was detected. Moreover, the Cas12i3 system exhibited no side effect on mouse embryo development and proliferation of blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring by optimizing the concentration of the Cas12i3 mixture. These results confirm the feasibility and safety of CRISPR/Cas12i3 for gene editing in mammals, which provides a reliable tool for one-step generation of gene-edited animals for applications in biology, medical research, and large livestock breeding.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
*Embryo, Mammalian/metabolism
Nanog Homeobox Protein/genetics
Female
Embryonic Development/genetics
Blastocyst/metabolism

@article {pmid40243657,
year = {2025},
author = {Daliri, K and Hescheler, J and Newby, GA and Clement, K and Liu, DR and Pfannkuche, K},
title = {Modulating Collagen I Expression in Fibroblasts by CRISPR-Cas9 Base Editing of the Collagen 1A1 Promoter.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40243657},
issn = {1422-0067},
support = {4346564//Imhoff foundation/ ; },
mesh = {Humans ; *Fibroblasts/metabolism ; *Promoter Regions, Genetic ; *Gene Editing/methods ; *Collagen Type I/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Collagen Type I, alpha 1 Chain ; MCF-7 Cells ; Fibrosis/genetics ; },
abstract = {Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to-CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.},
}

Humans
*Fibroblasts/metabolism
*Promoter Regions, Genetic
*Gene Editing/methods
*Collagen Type I/genetics/metabolism
*CRISPR-Cas Systems/genetics
Collagen Type I, alpha 1 Chain
MCF-7 Cells
Fibrosis/genetics

@article {pmid40243589,
year = {2025},
author = {Kim, T and Park, BS and Heo, S and Jeon, H and Kim, J and Kim, D and Kook Lee, S and Jung, SY and Kong, SY and Lu, T},
title = {Combinatorial CRISPR screen reveals FYN and KDM4 as targets for synergistic drug combination for treating triple negative breast cancer.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40243589},
issn = {2050-084X},
support = {2021R1A2C1093499//National Research Foundation of Korea/ ; 2E32331//Korea Institute of Science and Technology/ ; 2020M3A9A5036362//National Research Foundation of Korea/ ; },
mesh = {*Triple Negative Breast Neoplasms/drug therapy/genetics ; Humans ; Animals ; *Proto-Oncogene Proteins c-fyn/genetics/metabolism/antagonists & inhibitors ; Female ; Cell Line, Tumor ; Mice ; *Jumonji Domain-Containing Histone Demethylases/genetics/metabolism/antagonists & inhibitors ; Drug Synergism ; *Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Drug Resistance, Neoplasm ; },
abstract = {Tyrosine kinases play a crucial role in cell proliferation and survival and are extensively investigated as targets for cancer treatment. However, the efficacy of most tyrosine kinase inhibitors (TKIs) in cancer therapy is limited due to resistance. In this study, we identify a synergistic combination therapy involving TKIs for the treatment of triple negative breast cancer. By employing pairwise tyrosine kinase knockout CRISPR screens, we identify FYN and KDM4 as critical targets whose inhibition enhances the effectiveness of TKIs, such as NVP-ADW742 (IGF-1R inhibitor), gefitinib (EGFR inhibitor), and imatinib (ABL inhibitor) both in vitro and in vivo. Mechanistically, treatment with TKIs upregulates the transcription of KDM4, which in turn demethylates H3K9me3 at FYN enhancer for FYN transcription. This compensatory activation of FYN and KDM4 contributes to the resistance against TKIs. FYN expression is associated with therapy resistance and persistence by demonstrating its upregulation in various experimental models of drug-tolerant persisters and residual disease following targeted therapy, chemotherapy, and radiotherapy. Collectively, our study provides novel targets and mechanistic insights that can guide the development of effective combinatorial targeted therapies, thus maximizing the therapeutic benefits of TKIs.},
}

*Triple Negative Breast Neoplasms/drug therapy/genetics
Humans
Animals
*Proto-Oncogene Proteins c-fyn/genetics/metabolism/antagonists & inhibitors
Female
Cell Line, Tumor
Mice
*Jumonji Domain-Containing Histone Demethylases/genetics/metabolism/antagonists & inhibitors
Drug Synergism
*Protein Kinase Inhibitors/pharmacology
CRISPR-Cas Systems
*Antineoplastic Agents/pharmacology
Drug Resistance, Neoplasm

@article {pmid40243584,
year = {2025},
author = {Zhao, X and Mai, C and Xia, L and Jia, G and Li, X and Lu, Y and Li, Z and Yang, H and Wang, L},
title = {Molecular Insights into the Positive Role of Soybean Nodulation by GmWRKY17.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40243584},
issn = {1422-0067},
support = {32241046, 32472158//National Natural Science Foundation of China/ ; 2025xczx03//Shanxi Breeding Innovation Joint research and development projects/ ; 2023ZD040350103//Scientific and Technological Innovation 2030-Major Projects/ ; 202204051001020//Science and Technology Innovation Young Talent Team of Shanxi Province/ ; 2021xG003, 2022xG0014//Scientific research fund for talents of Shanxi Agricultural University/ ; },
mesh = {*Glycine max/genetics/microbiology/metabolism ; *Plant Root Nodulation/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Root Nodules, Plant/genetics/metabolism/microbiology ; CRISPR-Cas Systems ; Symbiosis ; *Transcription Factors/genetics/metabolism ; Cytokinins/metabolism ; Plants, Genetically Modified ; Abscisic Acid/metabolism ; RNA Interference ; Nitrogen Fixation ; },
abstract = {Soybean is an important economic oilseed crop, being rich in protein and plant oil, it is widely cultivated around the world. Soybeans have been shown to establish a symbiotic nitrogen fixation (SNF) with their compatible rhizobia, resulting in the formation of nodules. Previous studies have demonstrated the critical roles of phytohormones, such as abscisic acid and cytokinin, in the process of legume nodulation. The present study investigated the role of GmWRKY17, a homolog of Rosa hybrida (Rh)WRKY13 in regulating plant immunity through cytokinin content and abscisic acid signaling in soybean nodulation. Utilizing real-time PCR and histochemical staining, we demonstrated that GmWRKY17 is predominantly expressed in soybean root nodules. Subsequently, we analyzed the function of GmWRKY17-overexpression, RNA interference (RNAi), and the CRISPR/Cas9 system. Overexpression of GmWRKY17 significantly increases soybean nodule number, while RNAi or CRISPR/Cas9-mediated knockout of GmWRKY17 resulted in a dramatic repression of nodule formation in soybeans. These results highlight that GmWRKY17 functions as a positive regulator involved in soybean nodulation. Furthermore, manipulation of GmWRKY17 expression impacts the expression of genes associated with the nod factor (NF) signaling pathway, thereby influencing soybean nodulation. This study demonstrated that WRKY-type transcription factors are involved in the regulation of legume nodulation, offering new light on the molecular basis of the symbiotic interaction between legumes and rhizobia.},
}

*Glycine max/genetics/microbiology/metabolism
*Plant Root Nodulation/genetics
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Root Nodules, Plant/genetics/metabolism/microbiology
CRISPR-Cas Systems
Symbiosis
*Transcription Factors/genetics/metabolism
Cytokinins/metabolism
Plants, Genetically Modified
Abscisic Acid/metabolism
RNA Interference
Nitrogen Fixation

@article {pmid40199056,
year = {2025},
author = {Li, M and Pan, L and Ma, C and Wu, H and Xiang, G and Li, LF and Wang, T and Luo, R and Li, Y and Liu, D and Zhai, H and Assad, M and Song, X and Wang, Y and Gallardo, F and Qiu, HJ and Sun, Y},
title = {Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the N2a cells.},
journal = {Veterinary microbiology},
volume = {304},
number = {},
pages = {110503},
doi = {10.1016/j.vetmic.2025.110503},
pmid = {40199056},
issn = {1873-2542},
mesh = {*Herpesvirus 1, Suid/physiology/genetics ; Animals ; *Virus Replication ; Mice ; *Virus Internalization ; Genome, Viral ; Staining and Labeling/methods ; Cell Line ; Pseudorabies/virology ; Cell Line, Tumor ; Luminescent Proteins/genetics ; CRISPR-Cas Systems ; Red Fluorescent Protein ; },
abstract = {Pseudorabies virus (PRV) is a neurotropic herpesvirus. It is not easy to be track the whole replication progress of PRV, especially the nascent viral genome in the host cells. In this study, we developed a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with the viral genome and the envelope protein gM labeled by ANCHOR DNA labeling system and mCherry, respectively. Through single-virus tracking of rPRV-Anchor3-mCherry, we observed that PRV invaded mouse neuroblastoma Neuro-2a cells via both endocytosis and plasma membrane fusion pathway. During the replication stage, parental and progeny viral genome of rPRV-Anchor3-mCherry in the cell nuclei could be visible, and viral nucleocapsid appeared more specifically than traditional capsid protein labeled PRV particles (rPRV-VP26-EGFP). We found that numerous progeny viral particles were produced in the nuclear, causing the nucleus membrane to break using three-dimensional (3D) live-cell imaging and electron microscopy. Moreover, our findings confirmed that simultaneously targeting of the UL9 and UL54 genes using a CRISPR-Cas9 system led to the complete inhibition PRV replication. rPRV-Anchor3-mCherry can be used to research multiple steps of the viral cycle.},
}

*Herpesvirus 1, Suid/physiology/genetics
Animals
*Virus Replication
Mice
*Virus Internalization
Genome, Viral
Staining and Labeling/methods
Cell Line
Pseudorabies/virology
Cell Line, Tumor
Luminescent Proteins/genetics
CRISPR-Cas Systems
Red Fluorescent Protein

@article {pmid40185099,
year = {2025},
author = {Lee, JS and Dan, T and Zhang, H and Cheng, Y and Rehfeld, F and Brugarolas, J and Mendell, JT},
title = {An ultraconserved snoRNA-like element in long noncoding RNA CRNDE promotes ribosome biogenesis and cell proliferation.},
journal = {Molecular cell},
volume = {85},
number = {8},
pages = {1543-1560.e10},
doi = {10.1016/j.molcel.2025.03.006},
pmid = {40185099},
issn = {1097-4164},
mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Cell Proliferation/genetics ; *RNA, Small Nucleolar/genetics/metabolism ; *Ribosomes/metabolism/genetics ; *Carcinoma, Renal Cell/genetics/pathology/metabolism ; *Kidney Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Cell Nucleolus/metabolism/genetics ; Alternative Splicing ; HEK293 Cells ; RNA, Ribosomal/genetics/metabolism ; Conserved Sequence ; CRISPR-Cas Systems ; },
abstract = {Cancer cells frequently upregulate ribosome production to support tumorigenesis. While small nucleolar RNAs (snoRNAs) are critical for ribosome biogenesis, the roles of other classes of noncoding RNAs in this process remain largely unknown. Here, we performed CRISPR interference (CRISPRi) screens to identify essential long noncoding RNAs (lncRNAs) in renal cell carcinoma (RCC) cells. This revealed that an alternatively spliced isoform of lncRNA colorectal neoplasia differentially expressed (CRNDE) containing an ultraconserved element (UCE), referred to as CRNDE[UCE], is required for RCC cell proliferation. CRNDE[UCE] localizes to the nucleolus and promotes 60S ribosomal subunit biogenesis. The UCE of CRNDE functions as an unprocessed C/D box snoRNA that directly interacts with ribosomal RNA precursors. This facilitates delivery of eukaryotic initiation factor 6 (eIF6), a key 60S biogenesis factor, which binds to CRNDE[UCE] through a sequence element adjacent to the UCE. These findings highlight the functional versatility of snoRNA sequences and expand the known mechanisms through which noncoding RNAs orchestrate ribosome biogenesis.},
}

*RNA, Long Noncoding/genetics/metabolism
Humans
*Cell Proliferation/genetics
*RNA, Small Nucleolar/genetics/metabolism
*Ribosomes/metabolism/genetics
*Carcinoma, Renal Cell/genetics/pathology/metabolism
*Kidney Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Cell Nucleolus/metabolism/genetics
Alternative Splicing
HEK293 Cells
RNA, Ribosomal/genetics/metabolism
Conserved Sequence
CRISPR-Cas Systems

@article {pmid40174266,
year = {2025},
author = {Khan, TG and Bragazzi Cunha, J and Raut, C and Burroughs, M and Vyas, HS and Leix, K and Goonewardena, SN and Smrcka, AV and Speliotes, EK and Emmer, BT},
title = {Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening.},
journal = {Atherosclerosis},
volume = {403},
number = {},
pages = {119174},
doi = {10.1016/j.atherosclerosis.2025.119174},
pmid = {40174266},
issn = {1879-1484},
mesh = {Humans ; *Lipoprotein(a)/metabolism/blood/genetics ; *Receptors, LDL/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; United Kingdom ; *Hepatocytes/metabolism ; },
abstract = {BACKGROUND AND AIMS: An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial.

METHODS: We performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Screen validation was performed by single gene disruption and overexpression. Direct binding between purified lipoproteins and recombinant protein was tested using biolayer interferometry. An association between human genetic variants and circulating Lp(a) levels was analyzed in the UK Biobank cohort.

RESULTS: The top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort.

CONCLUSIONS: Our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.},
}

Humans
*Lipoprotein(a)/metabolism/blood/genetics
*Receptors, LDL/genetics/metabolism
Ubiquitin-Protein Ligases/genetics/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
United Kingdom
*Hepatocytes/metabolism

@article {pmid40173337,
year = {2025},
author = {Kandlbinder, A and Peter-Spiess, MH and Leeners, B and Mollaysa, A and Cavazza, T and Meier, A and Braunschweig, M and Ioannidi, E and Schwank, G and Krauthammer, M},
title = {Strategies for Interdisciplinary Human Gene Editing Research: Insights from a Swiss Project.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {79-88},
doi = {10.1089/crispr.2024.0041},
pmid = {40173337},
issn = {2573-1602},
mesh = {*Gene Editing/methods/ethics ; Humans ; CRISPR-Cas Systems ; Genetic Therapy/methods ; *Interdisciplinary Research ; Switzerland ; },
abstract = {CRISPR gene editing is a cutting-edge technology that has advanced tremendously in recent years. The first clinical CRISPR applications have been approved, and more gene editing therapies are to be expected in human medicine. Consequently, continuous basic research is needed to assess possibilities and prime future clinical applications. Because this technology not only offers new possibilities for treating diseases but also raises important ethical and societal questions, collaboration between human, life, biomedical, and medical sciences is needed. In this article, we discuss the practical challenges of such interdisciplinary projects and present strategies for addressing them based on our experience of conducting an interdisciplinary project on CRISPR. This work aims to help and encourage interdisciplinary collaborations and discussions on modern scientific endeavors that, such as gene editing, tend to blur the lines between traditional disciplines. The strategies suggested include realistic expectations, shared goals, space setting, and expert and lay dialogue.},
}

*Gene Editing/methods/ethics
Humans
CRISPR-Cas Systems
Genetic Therapy/methods
*Interdisciplinary Research
Switzerland

@article {pmid40163416,
year = {2025},
author = {Tordoff, J and Alfonse, LE and Makarova, KS and Ornstein, A and Garrity, AJ and Yan, WX and Scott, DA and Koonin, EV and Cheng, DR},
title = {Initial Characterization of 12 New Subtypes and Variants of Type V CRISPR Systems.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {149-154},
doi = {10.1089/crispr.2024.0100},
pmid = {40163416},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteriophages/genetics ; *CRISPR-Associated Proteins/genetics ; Gene Editing ; Prophages/genetics ; Genetic Variation ; },
abstract = {Type V CRISPR systems are highly diverse in sequence, mechanism, and function. Although recent efforts have greatly expanded our understanding of their evolution, the diversity of type V systems remains to be completely explored, and many clades have not been experimentally characterized. In this work, we mined metagenomic databases to identify three new subtypes and nine new variants of Cas12, the effector of Type V systems, and provide experimental and computational characterization of their Protospacer-Adjacent Motif (PAM), interference activity, loci architecture, and tracrRNA dependence. Half of the new Cas12s are found in phages or prophages. New subtypes Cas12o and Cas12p lack the canonical RuvC catalytic residues, suggesting they interfere with the target without cleavage, possibly by blocking transcription or replication. One variant, Cas12f10, displays substantial activity on PAM-less targets. Our work expands the diversity of the functionally characterized Cas12 effectors and provides some promising candidates for genome engineering tools.},
}

*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacteriophages/genetics
*CRISPR-Associated Proteins/genetics
Gene Editing
Prophages/genetics
Genetic Variation

@article {pmid40163415,
year = {2025},
author = {Noel, EA and Sahu, SU and Wyman, SK and Krishnappa, N and Jeans, C and Wilson, RC},
title = {Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance Editing in Primary Human Lymphocytes.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {105-119},
doi = {10.1089/crispr.2024.0080},
pmid = {40163415},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; *Nuclear Localization Signals/genetics ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Lymphocytes/metabolism ; T-Lymphocytes/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Nucleus ; },
abstract = {The incorporation of nuclear localization signal (NLS) sequences at one or both termini of CRISPR enzymes is a widely adopted strategy to facilitate genome editing. Engineered variants of CRISPR enzymes with diverse NLS sequences have demonstrated superior performance, promoting nuclear localization and efficient DNA editing. However, limiting NLS fusion to the CRISPR protein's termini can negatively impact protein yield via recombinant expression. Here we present a distinct strategy involving the installation of hairpin internal NLS sequences (hiNLS) at rationally selected sites within the backbone of CRISPR-Cas9. We evaluated the performance of these hiNLS Cas9 variants by editing genes in human primary T cells following the delivery of ribonucleoprotein enzymes via either electroporation or co-incubation with amphiphilic peptides. We show that hiNLS Cas9 variants can improve editing efficiency in T cells compared with constructs with terminally fused NLS sequences. Furthermore, many hiNLS Cas9 constructs can be produced with high purity and yield, even when these constructs contain as many as nine NLS. These hiNLS Cas9 constructs represent a key advance in optimizing CRISPR effector design and may contribute to improved editing outcomes in research and therapeutic applications.},
}

Humans
*Gene Editing/methods
*Nuclear Localization Signals/genetics
*CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism
*Lymphocytes/metabolism
T-Lymphocytes/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Nucleus

@article {pmid40151952,
year = {2025},
author = {Yuan, H and Song, C and Xu, H and Sun, Y and Anthon, C and Bolund, L and Lin, L and Benabdellah, K and Lee, C and Hou, Y and Gorodkin, J and Luo, Y},
title = {An Overview and Comparative Analysis of CRISPR-SpCas9 gRNA Activity Prediction Tools.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {89-104},
doi = {10.1089/crispr.2024.0058},
pmid = {40151952},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Machine Learning ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Deep Learning ; Computational Biology/methods ; Computer Simulation ; Software ; Animals ; },
abstract = {Design of guide RNA (gRNA) with high efficiency and specificity is vital for successful application of the CRISPR gene editing technology. Although many machine learning (ML) and deep learning (DL)-based tools have been developed to predict gRNA activities, a systematic and unbiased evaluation of their predictive performance is still needed. Here, we provide a brief overview of in silico tools for CRISPR design and assess the CRISPR datasets and statistical metrics used for evaluating model performance. We benchmark seven ML and DL-based CRISPR-Cas9 editing efficiency prediction tools across nine CRISPR datasets covering six cell types and three species. The DL models CRISPRon and DeepHF outperform the other models exhibiting greater accuracy and higher Spearman correlation coefficient across multiple datasets. We compile all CRISPR datasets and in silico prediction tools into a GuideNet resource web portal, aiming to facilitate and streamline the sharing of CRISPR datasets. Furthermore, we summarize features affecting CRISPR gene editing activity, providing important insights into model performance and the further development of more accurate CRISPR prediction models.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Machine Learning
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
Deep Learning
Computational Biology/methods
Computer Simulation
Software
Animals

@article {pmid40147800,
year = {2025},
author = {Yu, WB and Ye, ZH and Shi, JJ and Deng, WQ and Chen, J and Lu, JJ},
title = {Dual blockade of GSTK1 and CD47 improves macrophage-mediated phagocytosis on cancer cells.},
journal = {Biochemical pharmacology},
volume = {236},
number = {},
pages = {116898},
doi = {10.1016/j.bcp.2025.116898},
pmid = {40147800},
issn = {1873-2968},
mesh = {*CD47 Antigen/antagonists & inhibitors/metabolism/immunology/genetics ; *Phagocytosis/physiology/drug effects ; Humans ; *Macrophages/metabolism/drug effects/immunology ; *Glutathione Transferase/antagonists & inhibitors/metabolism/genetics ; Cell Line, Tumor ; Animals ; Mice ; RAW 264.7 Cells ; CRISPR-Cas Systems ; *Neoplasms/metabolism ; },
abstract = {CD47 is a crucial anti-phagocytic signal in regulating macrophage responses and its manipulation offers the therapeutic potential in cancer treatment. However, in many cases, blockade of CD47 by itself is insufficient to activate macrophage effectively, indicating other unidentified phagocytosis-regulating factors to resist the macrophage activity. In this study, a genome-wide human CRISPR-Cas9 library was developed for comprehensive screening of phagocytosis-regulating factors in the context of CD47 blockade. The screening results identified GSTK1 as a potential anti-phagocytic signal counteracting the efficacy of CD47-based phagocytosis. The disruption of GSTK1 significantly increased the phagocytosis rate of cancer cells by macrophages in combination with anti-CD47 antibody. Further mechanism investigation unveiled that GSTK1 blockade increased the membrane exposure of calreticulin in different cancer cells, which might be the primary mechanism driving enhanced macrophage-mediated phagocytosis. To this end, siGSTK1-loaded nanoparticles (siGSTK1-LNPs) were designed to suppress the GSTK1 expression efficiently. The comparable phagocytosis efficacy was also observed when combining siGSTK1-LNPs with anti-CD47 antibody. Above all, GSTK1 blockade was identified as a promising and feasible stimulus for enhancing the effectiveness of anti-CD47 antibody, introducing a novel and effective combination approach in cancer immunotherapy.},
}

*CD47 Antigen/antagonists & inhibitors/metabolism/immunology/genetics
*Phagocytosis/physiology/drug effects
Humans
*Macrophages/metabolism/drug effects/immunology
*Glutathione Transferase/antagonists & inhibitors/metabolism/genetics
Cell Line, Tumor
Animals
Mice
RAW 264.7 Cells
CRISPR-Cas Systems
*Neoplasms/metabolism

@article {pmid40118290,
year = {2025},
author = {Cros-Perrial, E and Beaumel, S and Gimbert, M and Camus, N and Vicente, C and Sekiou, I and Figuet, L and Duruisseaux, M and Dumontet, C and Jordheim, LP},
title = {SLX4 and XPF are involved in cell migration and EMT in a cell-specific manner.},
journal = {Biochemical pharmacology},
volume = {236},
number = {},
pages = {116885},
doi = {10.1016/j.bcp.2025.116885},
pmid = {40118290},
issn = {1873-2968},
mesh = {Humans ; *Epithelial-Mesenchymal Transition/physiology/drug effects ; *Cell Movement/physiology/drug effects ; Animals ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; Mice ; HEK293 Cells ; Cell Line, Tumor ; A549 Cells ; HT29 Cells ; CRISPR-Cas Systems ; Cell Proliferation ; },
abstract = {SLX4 and XPF are two proteins involved in DNA repair, but very little is known about their potential roles in other processes of cancer cell biology. We developed original cell models with CRISPR-Cas9-mediated knock-out of SLX4 and/or XPF using five different cell lines (A549, NCI-H1703, COLO-357, HT-29 and HEK-293 T), and performed characterization with cell biology experiments including migration assays, drug sensitivity testing, cell proliferation assessment and Western blots for relevant proteins. Results showed decreased migration of all models in HT-29 cells, of XPF-deficient COLO-357 cells and of SLX4-deficient HEK-293 T cells. Modified cell models had overall increased sensitivity to cisplatin and mitomycine C, and some models showed an increased frequency of double-stranded DNA damages. One NCI-H1703 cell model showed major karyotypic modifications, and epithelial to mesenchymal transition (EMT)-related proteins were modified in several models. Finally, knocking out one or both proteins in A549 cells had not the same impact on in vivo growth in mice. These original cell models allowed us to identify new and DNA repair-unrelated cellular roles of SLX4 and XPF in cancer cell biology. Our results should be considered within work on Nucleotide Excision Repair (NER) inhibition targeting SLX, XPF or other related proteins.},
}

Humans
*Epithelial-Mesenchymal Transition/physiology/drug effects
*Cell Movement/physiology/drug effects
Animals
*DNA-Binding Proteins/genetics/metabolism/deficiency
Mice
HEK293 Cells
Cell Line, Tumor
A549 Cells
HT29 Cells
CRISPR-Cas Systems
Cell Proliferation

@article {pmid40099789,
year = {2025},
author = {Huang, L and Zhou, Y and Feng, Y and Jia, S and Wang, S and Zhong, C},
title = {Tailoring Bacterial Cellulose through the CRISPR/Cas9-Mediated Gene Editing Tool in Komagataeibacter xylinus.},
journal = {ACS synthetic biology},
volume = {14},
number = {4},
pages = {1161-1172},
doi = {10.1021/acssynbio.4c00785},
pmid = {40099789},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; *Cellulose/genetics/biosynthesis/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *Gluconacetobacter xylinus/genetics/metabolism ; Metabolic Engineering/methods ; *Acetobacteraceae/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. Komagataeibacter xylinus was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in K. xylinus has not been reported. To facilitate metabolic pathway engineering in K. xylinus, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in K. xylinus, the strain's ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (bcsH, bcsX, bcsY, and bcsZ) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in K. xylinus is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.},
}

*Gene Editing/methods
*Cellulose/genetics/biosynthesis/metabolism/chemistry
*CRISPR-Cas Systems/genetics
*Gluconacetobacter xylinus/genetics/metabolism
Metabolic Engineering/methods
*Acetobacteraceae/genetics/metabolism
Bacterial Proteins/genetics/metabolism

@article {pmid40081369,
year = {2025},
author = {Binan, L and Jiang, A and Danquah, SA and Valakh, V and Simonton, B and Bezney, J and Manguso, RT and Yates, KB and Nehme, R and Cleary, B and Farhi, SL},
title = {Simultaneous CRISPR screening and spatial transcriptomics reveal intracellular, intercellular, and functional transcriptional circuits.},
journal = {Cell},
volume = {188},
number = {8},
pages = {2141-2158.e18},
doi = {10.1016/j.cell.2025.02.012},
pmid = {40081369},
issn = {1097-4172},
mesh = {Humans ; Animals ; *Transcriptome/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; Single-Cell Analysis/methods ; Astrocytes/metabolism/cytology ; Mice ; *CRISPR-Cas Systems/genetics ; Monocytes/metabolism ; *Gene Regulatory Networks ; Autism Spectrum Disorder/genetics ; Immunity, Innate ; Gene Expression Profiling/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipopolysaccharides/pharmacology ; },
abstract = {Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but they have not yet leveraged the power of highly plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present a combination of imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular effects that are consistent with single-cell RNA-sequencing-based readouts of perturbation effects (Perturb-seq) in a screen of lipopolysaccharide response in cultured monocytes, and it uncovers intercellular and density-dependent regulation of the innate immune response. Similarly, in three-dimensional xenograft models, Perturb-FISH identifies tumor-immune interactions altered by genetic knockout. When paired with a functional readout in a separate screen of autism spectrum disorder risk genes in human-induced pluripotent stem cell (hIPSC) astrocytes, Perturb-FISH shows common calcium activity phenotypes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a general method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.},
}

Humans
Animals
*Transcriptome/genetics
Induced Pluripotent Stem Cells/metabolism/cytology
Single-Cell Analysis/methods
Astrocytes/metabolism/cytology
Mice
*CRISPR-Cas Systems/genetics
Monocytes/metabolism
*Gene Regulatory Networks
Autism Spectrum Disorder/genetics
Immunity, Innate
Gene Expression Profiling/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Lipopolysaccharides/pharmacology

@article {pmid40073612,
year = {2025},
author = {Durazo-Martínez, K and Chaudhari, J and Sherry, LM and Webster, DA and Martins, K and Bostrom, JR and Carlson, DF and Sonstegard, TS and Vu, HLX},
title = {Modification of the splice acceptor in CD163 exon 7 of pigs is insufficient to confer resistance to PRRSV.},
journal = {Veterinary microbiology},
volume = {304},
number = {},
pages = {110450},
doi = {10.1016/j.vetmic.2025.110450},
pmid = {40073612},
issn = {1873-2542},
mesh = {Animals ; CD163 Antigen ; Swine ; *Receptors, Cell Surface/genetics ; *Porcine respiratory and reproductive syndrome virus ; *Porcine Reproductive and Respiratory Syndrome/genetics/immunology/virology ; *Antigens, CD/genetics ; *Antigens, Differentiation, Myelomonocytic/genetics ; *Exons/genetics ; *RNA Splice Sites/genetics ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; RNA, Messenger/genetics ; Gene Editing ; },
abstract = {CD163 is the primary receptor for PRRSV, and its SRCR5 domain, encoded by exon 7, is crucial for supporting PRRSV infection. Previous studies have used CRISPR/Cas9 technology to remove exon 7 from the host genome, and the edited pigs were completely resistant to PRRSV infection. In this study, we used CRISPR/Cas9 technology mimicking an adenine base editor (ABE) to edit the splice acceptor site of exon 7, rendering it nonfunctional. This alteration was intended to cause exon 6 to join directly to exon 8 during mRNA processing, resulting in a mature mRNA transcript that lacks exon 7, which encodes the SRCR5 domain. Piglets carrying the exon 7 splice site modification (CD163Ex7-ABE) were successfully generated. However, these pigs remained fully susceptible to infection with a PRRSV-2 isolate. Analysis of CD163 mRNA from the CD163Ex7-ABE pigs revealed that they predominantly expressed a mature CD163 mRNA lacking exon 7. However, due to cryptic splice sites, two additional mRNA isoforms were expressed, including an in-frame variant containing all of exon 7 and an extra 48 base pairs. This likely resulted in the expression of a full-length CD163 with a 16-amino-acid insertion upstream of the SRCR5 domain, which was sufficient to render the animals susceptible to PRRSV. Overall, our results demonstrate that merely modifying the splice acceptor site of CD163 exon 7 is not sufficient to generate PRRSV-resistant pigs.},
}

Animals
CD163 Antigen
Swine
*Receptors, Cell Surface/genetics
*Porcine respiratory and reproductive syndrome virus
*Porcine Reproductive and Respiratory Syndrome/genetics/immunology/virology
*Antigens, CD/genetics
*Antigens, Differentiation, Myelomonocytic/genetics
*Exons/genetics
*RNA Splice Sites/genetics
*Disease Resistance/genetics
CRISPR-Cas Systems
RNA, Messenger/genetics
Gene Editing

@article {pmid40019800,
year = {2025},
author = {Major, RM and Mills, CA and Xing, L and Krantz, JL and Wolter, JM and Zylka, MJ},
title = {Exploring the Cytoplasmic Retention of CRISPR-Cas9 in Eukaryotic Cells: The Role of Nuclear Localization Signals and Ribosomal Interactions.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {120-136},
doi = {10.1089/crispr.2024.0074},
pmid = {40019800},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Ribosomes/metabolism/genetics ; *Cytoplasm/metabolism/genetics ; Cell Nucleus/metabolism/genetics ; *Nuclear Localization Signals/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Eukaryotic Cells/metabolism ; Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; Gene Editing/methods ; Neurons/metabolism ; },
abstract = {Cas9 must be localized to the nucleus to access the genome of mammalian cells. For most proteins, adding a single nuclear localization signal (NLS) is sufficient to promote nuclear entry. However, Cas9 nuclear entry appears to be inefficient as multiple NLSs are typically added to Cas9. Here, we found that three different Cas9 variants interact with the ribosome in HEK293T cells, and that this interaction is RNA mediated. Following immunoprecipitation-mass spectrometry of cytoplasmic-localized Cas9-0NLS and nuclear-localized Cas9-4NLS constructs, we identified novel Cas9 interactors in postmitotic neurons, including KEAP1 and additional ribosomal subunits, the latter were enriched in Cas9-0NLS samples. Collectively, our results suggest that Cas9 is sequestered in the cytoplasm of mammalian cells, in part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants.},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
*Ribosomes/metabolism/genetics
*Cytoplasm/metabolism/genetics
Cell Nucleus/metabolism/genetics
*Nuclear Localization Signals/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
*Eukaryotic Cells/metabolism
Kelch-Like ECH-Associated Protein 1/metabolism/genetics
Gene Editing/methods
Neurons/metabolism

@article {pmid39792480,
year = {2025},
author = {Buyukyoruk, M and Krishna, P and Santiago-Frangos, A and Wiedenheft, B},
title = {Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {137-148},
doi = {10.1089/crispr.2024.0093},
pmid = {39792480},
issn = {2573-1602},
support = {K99 GM147842/GM/NIGMS NIH HHS/United States ; R00 GM147842/GM/NIGMS NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleotide Motifs ; *Archaea/genetics ; CRISPR-Cas Systems ; *Bacteria/genetics ; Genome, Archaeal ; Genome, Bacterial ; Conserved Sequence ; },
abstract = {Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.},
}

*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleotide Motifs
*Archaea/genetics
CRISPR-Cas Systems
*Bacteria/genetics
Genome, Archaeal
Genome, Bacterial
Conserved Sequence

@article {pmid40243059,
year = {2025},
author = {Liu, Q and Jiang, Z and Li, S and Li, Y and Wan, Y and Hu, Z and Ma, S and Zou, Z and Yang, R},
title = {Nonequilibrium hybridization-driven CRISPR/Cas adapter with extended energetic penalty for discrimination of single-nucleotide variants.},
journal = {Nucleic acids research},
volume = {53},
number = {7},
pages = {},
doi = {10.1093/nar/gkaf287},
pmid = {40243059},
issn = {1362-4962},
support = {22334005//National Natural Science Foundation of China/ ; 2022JJ20038//Natural Science Foundation of Hunan Province/ ; 23A0059//Scientific Research Fund of Hunan Provincial Education Department/ ; 2022XKQ0205//Hunan Normal University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; Humans ; Nucleic Acid Hybridization ; Proto-Oncogene Proteins B-raf/genetics ; ErbB Receptors/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Mutation ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Base Pair Mismatch ; },
abstract = {Accurate identification of single-nucleotide variants (SNVs) is critical in clinical diagnostics but remains challenging due to subtle free energy variations, particularly for hard-to-detect SNVs such as wobble base pairs and those in high guanine-cytosine (GC) regions. Here we report a high-energetic-penalty SNV detection (HEPSD) platform that redesigns the hybridization regions of CRISPR RNA (crRNA) in the CRISPR/Cas12a system. This system employs a binary crRNA architecture design that enables the activation of the cleavage activity of Cas12a while amplifying the energetic penalty for single-nucleotide mismatches through nonequilibrium hybridization-driven regulation. Consequently, the entire targeting region of CRISPR/Cas exhibits a marked preference for mutations in genomic DNA, while preventing false activation induced by sequences containing a single mismatched nucleotide. Moreover, HEPSD exhibits exceptional differentiation performance for hard-to-detect SNVs including wobble mutations at extreme GC contents. As proof of principle, profiling of BRAF V600E and EGFR L858R tumor mutations down to a 0.01% variant allele frequency was achieved, enabling accurate discrimination of 132 clinical sample pairs, which showed high consistency with quantitative polymerase chain reaction-based techniques and next-generation sequencing. The proven effectiveness of this platform showcases its potential for clinical molecular diagnostics and expands the fundamental scope of hybridization-based protocols.},
}

*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide
Humans
Nucleic Acid Hybridization
Proto-Oncogene Proteins B-raf/genetics
ErbB Receptors/genetics
CRISPR-Associated Proteins/metabolism/genetics
Mutation
Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Base Pair Mismatch

@article {pmid40242952,
year = {2025},
author = {Aloliqi, AA and Alnuqaydan, AM and Albutti, A and Alharbi, BF and Rahmani, AH and Khan, AA},
title = {Current updates regarding biogenesis, functions and dysregulation of microRNAs in cancer: Innovative approaches for detection using CRISPR/Cas13‑based platforms (Review).},
journal = {International journal of molecular medicine},
volume = {55},
number = {6},
pages = {},
doi = {10.3892/ijmm.2025.5531},
pmid = {40242952},
issn = {1791-244X},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Neoplastic ; Animals ; Biomarkers, Tumor/genetics ; },
abstract = {MicroRNAs (miRNAs) are short non‑coding RNAs, which perform a key role in cellular differentiation and development. Most human diseases, particularly cancer, are linked to miRNA functional dysregulation implicated in the expression of tumor‑suppressive or oncogenic targets. Cancer hallmarks such as continued proliferative signaling, dodging growth suppressors, invasion and metastasis, triggering angiogenesis, and avoiding cell death have all been demonstrated to be affected by dysregulated miRNAs. Thus, for the treatment of different cancer types, the detection and quantification of this type of RNA is significant. The classical and current methods of RNA detection, including northern blotting, reverse transcription‑quantitative PCR, rolling circle amplification and next‑generation sequencing, may be effective but differ in efficiency and accuracy. Furthermore, these approaches are expensive, and require special instrumentation and expertise. Thus, researchers are constantly looking for more innovative approaches for miRNA detection, which can be advantageous in all aspects. In this regard, an RNA manipulation tool known as the CRISPR and CRISPR‑associated sequence 13 (CRISPR/Cas13) system has been found to be more advantageous in miRNA detection. The Cas13‑based miRNA detection approach is cost effective and requires no special instrumentation or expertise. However, more research and validation are required to confirm the growing body of CRISPR/Cas13‑based research that has identified miRNAs as possible cancer biomarkers for diagnosis and prognosis, and as targets for treatment. In the present review, current updates regarding miRNA biogenesis, structural and functional aspects, and miRNA dysregulation during cancer are described. In addition, novel approaches using the CRISPR/Cas13 system as a next‑generation tool for miRNA detection are discussed. Furthermore, challenges and prospects of CRISPR/Cas13‑based miRNA detection approaches are described.},
}

Humans
*MicroRNAs/genetics/metabolism
*Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Gene Expression Regulation, Neoplastic
Animals
Biomarkers, Tumor/genetics

@article {pmid40241992,
year = {2025},
author = {Haque, US and Yokota, T},
title = {Gene Editing for Duchenne Muscular Dystrophy: From Experimental Models to Emerging Therapies.},
journal = {Degenerative neurological and neuromuscular disease},
volume = {15},
number = {},
pages = {17-40},
pmid = {40241992},
issn = {1179-9900},
abstract = {The CRISPR system has emerged as a ground-breaking gene-editing tool, offering promising therapeutic potential for Duchenne muscular dystrophy (DMD), a severe genetic disorder affecting approximately 1 in 5000 male births globally. DMD is caused by mutations in the dystrophin gene, which encodes a critical membrane-associated protein essential for maintaining muscle structure, function and repair. Patients with DMD experience progressive muscle degeneration, loss of ambulation, respiratory insufficiency, and cardiac failure, with most succumbing to the disease by their third decade of life. Despite the well-characterized genetic basis of DMD, curative treatments- such as exon skipping therapies, micro-dystrophin, and steroids- remain elusive. Recent preclinical studies have demonstrated the promise of CRISPR-based approaches in restoring dystrophin expression across various models, including human cells, murine systems, and large animal models. These advancements highlight the potential of gene editing to fundamentally alter the trajectory of the disease. However, significant challenges persist, including immunogenicity, off-target effects, and limited editing efficiency, which hinder clinical translation. This review provides a comprehensive analysis of the latest developments in CRISPR-based therapeutic strategies for DMD. It emphasizes the need for further innovation in gene-editing technologies, delivery systems, and rigorous safety evaluations to overcome current barriers and harness the full potential of CRISPR/Cas as a durable and effective treatment for DMD.},
}

@article {pmid40240328,
year = {2025},
author = {Torelli, F and Butterworth, S and Lockyer, E and Matias, AN and Hildebrandt, F and Song, OR and Pearson-Farr, J and Treeck, M},
title = {GRA12 is a common virulence factor across Toxoplasma gondii strains and mouse subspecies.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3570},
pmid = {40240328},
issn = {2041-1723},
support = {223192/Z/21/Z//Wellcome Trust (Wellcome)/ ; 223192/Z/21/Z//Wellcome Trust (Wellcome)/ ; 223192/Z/21/Z//Wellcome Trust (Wellcome)/ ; CR2023/030/2132//Francis Crick Institute (Francis Crick Institute Limited)/ ; CR2132//Francis Crick Institute (Francis Crick Institute Limited)/ ; CR2023/030/2132//Francis Crick Institute (Francis Crick Institute Limited)/ ; CC2132/WT_/Wellcome Trust/United Kingdom ; CC2132/WT_/Wellcome Trust/United Kingdom ; CR2023/030/2132//Francis Crick Institute (Francis Crick Institute Limited)/ ; CC0199/WT_/Wellcome Trust/United Kingdom ; CC0199/WT_/Wellcome Trust/United Kingdom ; 2023.06167.CEECIND//Ministry of Education and Science | Fundação para a Ciência e a Tecnologia (Portuguese Science and Technology Foundation)/ ; TO 1349/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; WGF2024-0027//Wenner-Gren Foundation (Wenner-Gren Foundation for Anthropological Research, Inc.)/ ; },
mesh = {Animals ; *Toxoplasma/pathogenicity/genetics/metabolism ; Mice ; *Virulence Factors/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; Macrophages/parasitology/immunology ; CRISPR-Cas Systems ; *Toxoplasmosis, Animal/parasitology/immunology ; Female ; Mice, Inbred C57BL ; Toxoplasmosis/parasitology ; Host-Parasite Interactions ; Interferon-gamma ; },
abstract = {Toxoplasma gondii parasites exhibit extraordinary host promiscuity owing to over 250 putative secreted proteins that disrupt host cell functions, enabling parasite persistence. However, most of the known effector proteins are specific to Toxoplasma genotypes or hosts. To identify virulence factors that function across different parasite isolates and mouse strains that differ in susceptibility to infection, we performed systematic pooled in vivo CRISPR-Cas9 screens targeting the Toxoplasma secretome. We identified several proteins required for infection across parasite strains and mouse species, of which the dense granule protein 12 (GRA12) emerged as the most important effector protein during acute infection. GRA12 deletion in IFNγ-activated macrophages results in collapsed parasitophorous vacuoles and increased host cell necrosis, which is partially rescued by inhibiting early parasite egress. GRA12 orthologues from related coccidian parasites, including Neospora caninum and Hammondia hammondi, complement TgΔGRA12 in vitro, suggesting a common mechanism of protection from immune clearance by their hosts.},
}

Animals
*Toxoplasma/pathogenicity/genetics/metabolism
Mice
*Virulence Factors/genetics/metabolism
*Protozoan Proteins/genetics/metabolism
Macrophages/parasitology/immunology
CRISPR-Cas Systems
*Toxoplasmosis, Animal/parasitology/immunology
Female
Mice, Inbred C57BL
Toxoplasmosis/parasitology
Host-Parasite Interactions
Interferon-gamma

@article {pmid40239991,
year = {2025},
author = {Nam, HJ and Han, JH and Yu, J and Cho, CS and Kim, D and Kim, YE and Kim, MJ and Kim, JH and Jo, DH and Bae, S},
title = {Autophagy induction enhances homologous recombination-associated CRISPR-Cas9 gene editing.},
journal = {Nucleic acids research},
volume = {53},
number = {7},
pages = {},
doi = {10.1093/nar/gkaf258},
pmid = {40239991},
issn = {1362-4962},
support = {2020M3A9I4036072//NRF/ ; KK2431-10//Korea Research Institute of Chemical Technology/ ; //National Research Council of Science & Technology/ ; GTL24021-300//MSIT/ ; 18-2023-0010//Seoul National University Hospital/ ; RS-2023-00260351//Genome Editing Research Program/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Autophagy/genetics ; Mice ; *Homologous Recombination ; Humans ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9)-based gene editing via homologous recombination (HR) enables precise gene correction and insertion. However, its low efficiency poses a challenge due to the predominance of nonhomologous end-joining during DNA repair processes. Although numerous efforts have been made to boost HR efficiency, there remains a critical need to devise a novel method that can be universally applied across cell types and in vivo animals, which could ultimately facilitate therapeutic treatments. This study demonstrated that autophagy induction using different protocols, including nutrient deprivation or chemical treatment, significantly improved HR-associated gene editing at diverse genomic loci in mammalian cells. Notably, interacting cofactor proteins that bind to Cas9 under the autophagic condition have been identified, and autophagy induction could also enhance in vivo HR-associated gene editing in mice. These findings pave the way for effective gene correction or insertion for in vivo therapeutic treatments.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Autophagy/genetics
Mice
*Homologous Recombination
Humans
HEK293 Cells
CRISPR-Associated Protein 9/metabolism

@article {pmid40239628,
year = {2025},
author = {Chen, J and Tan, J and Wang, N and Li, H and Cheng, W and Li, J and Wang, B and Sedgwick, AC and Chen, Z and Chen, G and Zhang, P and Zheng, W and Liu, C and Chen, J},
title = {Specific macrophage RhoA targeting CRISPR-Cas9 for mitigating osteoclastogenesis-induced joint damage in inflammatory arthritis.},
journal = {Cell reports. Medicine},
volume = {6},
number = {4},
pages = {102046},
doi = {10.1016/j.xcrm.2025.102046},
pmid = {40239628},
issn = {2666-3791},
mesh = {*rhoA GTP-Binding Protein/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Macrophages/metabolism/pathology ; *Osteogenesis/genetics ; Animals ; *Osteoclasts/metabolism/pathology ; *Arthritis, Rheumatoid/pathology/genetics/metabolism ; Mice ; Humans ; Gene Editing ; *Joints/pathology ; Inflammation/pathology ; RAW 264.7 Cells ; Mice, Inbred C57BL ; },
abstract = {Rheumatoid arthritis (RA) is the most prevalent inflammatory arthritis with unknown etiology, characterized by synovial inflammation and articular bone erosion. Studies have highlighted that inhibiting macrophage-induced osteoclastogenesis holds promise in mitigating bone destruction. However, specifically halting this pathological cascade remains a challenge for the management of RA. Here, initially, we identify that Ras homolog gene family member A (RhoA) is a pivotal target in inducing osteoclastogenesis of macrophages. Subsequently, we develop a strategy termed specific macrophages RhoA targeting (SMART), in which phosphatidylserine (PS)-enriched macrophage membranes are engineered to deliver macrophage-specific promoter-containing CRISPR-Cas9 plasmids (SMART-Cas9), enabling targeted editing of RhoA in RA joint macrophages. Multiscale imaging techniques confirm the highly specific targeted effect of SMART-Cas9 on the macrophages of inflamed joints. SMART-Cas9 successfully reduces osteoclastogenesis by macrophages, thus mitigating bone erosion by modulating cytoskeletal dynamics and immune balance in inflammatory arthritis, representing a therapeutic avenue for RA and other inflammatory bone diseases.},
}

*rhoA GTP-Binding Protein/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Macrophages/metabolism/pathology
*Osteogenesis/genetics
Animals
*Osteoclasts/metabolism/pathology
*Arthritis, Rheumatoid/pathology/genetics/metabolism
Mice
Humans
Gene Editing
*Joints/pathology
Inflammation/pathology
RAW 264.7 Cells
Mice, Inbred C57BL

@article {pmid40238250,
year = {2025},
author = {Zhao, J and Yang, W and Cai, H and Cao, G and Li, Z},
title = {Current Progress and Future Trends of Genomics-Based Techniques for Food Adulteration Identification.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/foods14071116},
pmid = {40238250},
issn = {2304-8158},
support = {Grant No. ZDYF2024GXJS316//Key Research and Development Projects in Hainan Province/ ; },
abstract = {Addressing the pervasive issue of food adulteration and fraud driven by economic interests has long presented a complex challenge. Such adulteration not only compromises the safety of the food supply chain and destabilizes the market economy but also poses significant risks to public health. Food adulteration encompasses practices such as substitution, process manipulation, mislabeling, the introduction of undeclared ingredients, and the adulteration of genetically modified foods. Given the diverse range of deceptive methods employed, genomics-based identification techniques have increasingly been utilized for detecting food adulteration. Compared to traditional detection methods, technologies such as polymerase chain reaction (PCR), next-generation sequencing (NGS), high-resolution melt (HRM) analysis, DNA barcoding, and the CRISPR-Cas system have demonstrated efficacy in accurately and sensitively detecting even trace amounts of adulterants. This paper provides an overview of genomics-based approaches for identifying food adulteration, summarizes the latest applications in certification procedures, discusses current limitations, and explores potential future trends, thereby offering new insights to enhance the control of food quality and contributing to the development of more robust regulatory frameworks and food safety policies.},
}

@article {pmid40234780,
year = {2025},
author = {Tian, L and Gao, Y and Zi, L and Zhe, R and Yang, J},
title = {Dual Cas12a and multiplex crRNA CRISPR strategy ultrasensitive detection novel circRNA biomarker for the diagnosis of ovarian cancer.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {695},
pmid = {40234780},
issn = {1471-2407},
support = {2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; Female ; *RNA, Circular/genetics ; *Ovarian Neoplasms/diagnosis/genetics/blood ; *Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; Limit of Detection ; Bacterial Proteins ; },
abstract = {BACKGROUND: Ovarian cancer (OC), as a malignant tumor, currently lacks effective screening early diagnosis measures. Clinical biomarkers CA-125 and HE4 are limited by false positives and insufficient sensitivity. Therefore, it's of great significance to search for new biomarker and construct sensitive detection method.

METHODS: We found a novel circRNA biomarker (hsa_circ_0049101) by RNA sequencing, and simultaneously propose a strategy, which integrates reverse transcription rolling circle amplification (RT-RCA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a to amplify and detect novel circRNA biomarker. This strategy use Dual Cas12a protein (FnCas12a and LbCas12a) and Multiplex CrRNA (DCMC-CRISPR) to enhance detection sensitivity. The sensitivity mechanism of CRISPR to detect circRNA was verified in detail.

RESULTS: The DCMC-CRISPR assay exhibited a broad detection range of 2000 pM to 0.5 fM and the limit of detection (LOD) as low as 0.5 fM. The DCMC-CRISPR system has 4-11 times higher sensitivity than single-crRNA CRISPR/Cas12a system. Clinical assessment of RNA extracts from patient's peripheral blood of 22 clinical OC patients and 28 controls demonstrates the DCMC-CRISPR strategy outperformed CA-125, HE4, and the ROMA index. The assay demonstrated comparable performance to RT-qPCR, exhibiting favorable sensitivity and specificity in this pilot cohort.

CONCLUSIONS: The DCMC-CRISPR platform offers a promising solution for circRNA biomarker screening and circRNA diagnostic. It highlights the possibility of expanding its applicability to address other cancer diseases.},
}

Humans
Female
*RNA, Circular/genetics
*Ovarian Neoplasms/diagnosis/genetics/blood
*Biomarkers, Tumor/genetics
*CRISPR-Cas Systems
Sensitivity and Specificity
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics
Limit of Detection
Bacterial Proteins

@article {pmid40234414,
year = {2025},
author = {Kusumawardhani, H and Zoppi, F and Avendaño, R and Schaerli, Y},
title = {Engineering intercellular communication using M13 phagemid and CRISPR-based gene regulation for multicellular computing in Escherichia coli.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3569},
pmid = {40234414},
issn = {2041-1723},
support = {310030_200532//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Escherichia coli/genetics/physiology ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Cell Communication/genetics ; *Gene Expression Regulation, Bacterial ; Synthetic Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computers, Molecular ; *Genetic Engineering/methods ; },
abstract = {Engineering multicellular consortia, where information processing is distributed across specialized cell types, offers a promising strategy for implementing sophisticated biocomputing systems. However, a major challenge remains in establishing orthogonal intercellular communication, or "wires," within synthetic bacterial consortia. In this study, we address this bottleneck by integrating phagemid-mediated intercellular communication with CRISPR-based gene regulation for multicellular computing in synthetic E. coli consortia. We achieve intercellular communication with high sensitivity by regulating the transfer of single guide RNAs (sgRNAs) encoded on M13 phagemids from sender to receiver cells. Once inside the receiver cells, the transferred sgRNAs mediate gene regulation via CRISPR interference. Leveraging this approach, we successfully constructed one-, two-, and four-input logic gates. Our work expands the toolkit for intercellular communication and paves the way for complex information processing in synthetic microbial consortia, with diverse potential applications, including biocomputing, biosensing, and biomanufacturing.},
}

*Escherichia coli/genetics/physiology
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Cell Communication/genetics
*Gene Expression Regulation, Bacterial
Synthetic Biology/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Computers, Molecular
*Genetic Engineering/methods

@article {pmid40232991,
year = {2025},
author = {Sahin, GN and Seli, E},
title = {Gene editing using CRISPR-Cas9 technology: potential implications in assisted reproduction.},
journal = {Current opinion in obstetrics & gynecology},
volume = {},
number = {},
pages = {},
doi = {10.1097/GCO.0000000000001022},
pmid = {40232991},
issn = {1473-656X},
abstract = {PURPOSE OF REVIEW: This article reviews the mechanisms, advancements, and potential implications of clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) gene editing technology, with a specific focus on its applications in reproductive biology and assisted reproduction. It aims to explore the benefits and challenges of integrating this revolutionary technology into clinical and research settings.

RECENT FINDINGS: CRISPR-Cas9 is a transformative tool for precise genome editing, enabling targeted modifications through mechanisms like nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Innovations such as Cas9 nickase and dCas9 systems have improved specificity and expanded applications, including gene activation, repression, and epigenetic modifications. In reproductive research, CRISPR has facilitated gene function studies, corrected genetic mutations in animal models, and demonstrated potential in addressing human infertility and hereditary disorders. Emerging applications include mitochondrial genome editing, population control of disease vectors via gene drives, and detailed analyses of epigenetic mechanisms.

SUMMARY: CRISPR-Cas9 technology has revolutionized genetic engineering by enabling precise genome modifications. This article discusses its mechanisms, focusing on the repair pathways (NHEJ and HDR) and methods to mitigate off-target effects. In reproductive biology, CRISPR has advanced our understanding of fertility genes, allowed corrections of hereditary mutations, and opened avenues for novel therapeutic strategies. While its clinical application in human-assisted reproduction faces ethical and safety challenges, ongoing innovations hold promise for broader biomedical applications.},
}

@article {pmid40231967,
year = {2025},
author = {Zhang, X and Chen, S and Li, J and Liu, DA and Lai, J and Song, X and Hu, R and Qiu, Y and Chen, K and Xu, Y and Li, X},
title = {One-Step RAA and CRISPR-Cas13a Method for Detecting Influenza B Virus.},
journal = {Microbial biotechnology},
volume = {18},
number = {4},
pages = {e70144},
pmid = {40231967},
issn = {1751-7915},
support = {2024XZ011//Zhejiang Shuren University Basic Scientific Research Special Funds/ ; },
mesh = {*Influenza B virus/isolation & purification/genetics ; Sensitivity and Specificity ; Humans ; *Influenza, Human/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *Recombinases/metabolism ; CRISPR-Cas Systems ; },
abstract = {We developed a sensitive and specific method based on recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13a (Cas13a). This method, named CRISPR-based Rapid and Efficient Test (CRISPRET), is designed for the early diagnosis of Influenza B (FluB) with the aim of shortening its transmission chain. We identified conserved regions in the Influenza B Virus (IBV) NS gene and designed forward and reverse primers along with crRNAs. We then established and optimised the reaction system, and Nucleic Acid Positive Reference Materials of IBV were used to evaluate the detection limit (DL) of CRISPRET. Additionally, we collected 257 clinical samples, comprising 127 samples from patients with IBV infection and 130 samples from healthy individuals, and subjected them to dual detection using CRISPRET and qPCR to evaluate the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of CRISPRET. We designed one forward primer, two reverse primers, and two crRNAs to establish and optimise the CRISPR ET. The method demonstrated the DL of 500 copies·μL[-1] when assisted by appropriate equipment. Despite requiring auxiliary equipment and a 30-min reaction, the CRISPR ET method enables the detection of IBV nucleic acid within approximately the first 5 min, achieving high sensitivity (100%), specificity (97.69%), PPV (97.69%) and NPV (100%), with a concordance rate of 98.83% to qPCR. CRISPRET offers a simple, field-applicable, one-step method for the rapid detection of IBV. It has strong potential for field-testing applications and intelligent integration into existing diagnostic systems.},
}

*Influenza B virus/isolation & purification/genetics
Sensitivity and Specificity
Humans
*Influenza, Human/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
*Recombinases/metabolism
CRISPR-Cas Systems

@article {pmid40231554,
year = {2025},
author = {Halat, M and Klimek-Chodacka, M and Domagała, A and Zając, G and Oleszkiewicz, T and Kapitán, J and Baranski, R},
title = {Chiral sensing combined with nuclease activity assay to track Cas9 dynamics in solution: ROA and CPL study.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc00971e},
pmid = {40231554},
issn = {1364-548X},
abstract = {Chiroptical studies of the SpyCas9 protein are extremely rare. Nondestructive methods are needed to characterize its active ribonucleoprotein form. Using Raman optical activity (ROA) and circularly polarized luminescence (CPL), we present a new approach to detect key biomolecules involved in CRISPR-Cas technology while preserving their original nucleolytic activity.},
}

@article {pmid40230958,
year = {2025},
author = {Rust, S and Randau, L},
title = {Real-time imaging of bacterial colony growth dynamics for cells with Type IV-A1 CRISPR-Cas activity.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf006},
pmid = {40230958},
issn = {2633-6693},
abstract = {The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans provides defense against mobile genetic elements in the absence of target DNA degradation. In recent studies, Escherichia coli BL21-AI cells with Type IV-A1 CRISPR-Cas activity displayed a heterogeneous colony growth phenotype. Here, we developed a convenient smartphone-mediated automatic remote-controlled time-lapse imaging system (SMARTIS), that enables monitoring of growing bacteria over time. The system's design includes a custom-built imaging box equipped with LED lights, an adjustable heating system and a smartphone that can be remotely controlled using freely available, user-friendly applications. SMARTIS allowed long-term observation of growing colonies and was utilized to analyze different growth behaviors of E. coli cells expressing Type IV-A1 CRISPR ribonucleoproteins. Our findings reveal that heterogeneity in colonies can emerge within hours of initial growth. We further examined the influence of different expression systems on bacterial growth and CRISPR interference activity and demonstrated that the observed heterogeneity of colony-forming units is strongly influenced by plasmid design and backbone identity. This study highlights the importance of careful assessment of heterogenous colony growth dynamics and describes a real-time imaging system with wide applications beyond the study of CRISPR-Cas activity in bacterial hosts.},
}

@article {pmid40229905,
year = {2025},
author = {Chen, RD and Yang, Y and Liu, KM and Hu, JZ and Feng, YL and Yang, CY and Jiang, RR and Liu, SC and Wang, Y and Han, PA and Tian, RG and Wang, YL and Xu, SM and Xie, AY},
title = {Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {96},
pmid = {40229905},
issn = {1474-760X},
support = {32071439//National Natural Science Foundation of China/ ; 32371348//National Natural Science Foundation of China/ ; 2023ZD04048//National Science and Technology Major Project of China/ ; 2023ZD0500501//National Major Science and Technology Projects of China/ ; },
mesh = {*DNA End-Joining Repair ; *DNA Breaks, Double-Stranded ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Bacterial Proteins ; },
abstract = {BACKGROUND: After Cas12a cleaves its DNA target, it generates a DNA double strand break (DSB) with two compatible 5'-staggered ends. The Cas12a-gRNA complex remains at the protospacer adjacent motif (PAM)-proximal end (PPE) while releasing the PAM-distal end (PDE). The effects of this asymmetric retention on DSB repair are currently unknown.

RESULTS: Post-cleavage retention of LbCas12a at PPEs suppresses the recruitment of classical non-homologous end joining (c-NHEJ) core factors, leading to longer deletions at PPEs compared to PDEs. This asymmetry in c-NHEJ engagement results in approximately tenfold more accurate ligation between two compatible PDEs induced by paired LbCas12a than ligation involving a compatible PPE. Moreover, ligation to a given end of SpCas9-induced DSBs demonstrates more efficient ligation with a PDE from Cas12a-induced DSBs than with a PPE. In LbCas12a-induced NHEJ-mediated targeted integration, only two compatible PDEs from LbCas12a-induced DSBs-one from donor templates and the other from target sites-promote accurate and directional ligation. Based on these findings, we developed a strategy called Cas12a-induced PDE ligation (CIPDEL) for NHEJ-mediated efficient and precise gene correction and insertion.

CONCLUSIONS: The asymmetric retention of CRISPR-LbCas12a at DSB ends suppresses c-NHEJ at PPEs, not at PDEs. This unique repair mechanism can be utilized in the CIPDEL strategy, offering a potentially better alternative for homology-directed targeted integration.},
}

*DNA End-Joining Repair
*DNA Breaks, Double-Stranded
*CRISPR-Associated Proteins/metabolism/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Bacterial Proteins

@article {pmid40198713,
year = {2025},
author = {Ashokkumar, M and Hafer, TL and Felton, A and Archin, NM and Margolis, DM and Emerman, M and Browne, EP},
title = {A targeted CRISPR screen identifies ETS1 as a regulator of HIV-1 latency.},
journal = {PLoS pathogens},
volume = {21},
number = {4},
pages = {e1012467},
doi = {10.1371/journal.ppat.1012467},
pmid = {40198713},
issn = {1553-7374},
mesh = {*Proto-Oncogene Protein c-ets-1/genetics/metabolism ; *HIV-1/physiology/genetics ; Humans ; *Virus Latency/physiology/genetics ; *CD4-Positive T-Lymphocytes/virology/metabolism ; *HIV Infections/virology/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Viral ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Human Immunodeficiency virus (HIV) infection is regulated by a wide array of host cell factors that combine to influence viral transcription and latency. To understand the complex relationship between the host cell and HIV-1 latency, we performed a lentiviral CRISPR screen that targeted a set of host cell genes whose expression or activity correlates with HIV-1 expression. We further investigated one of the identified factors - the transcription factor ETS1, and found that it is required for maintenance of HIV-1 latency in both latently infected cell lines and in a primary CD4 T cell latency model. Interestingly, ETS1 played divergent roles in actively infected and latently infected CD4 T cells, with knockout of ETS1 leading to reduced HIV-1 expression in actively infected cells, but increased HIV-1 expression in latently infected cells, indicating that ETS1 can play both a positive and negative role in HIV-1 expression. CRISPR/Cas9 knockout of ETS1 in CD4 T cells from ART-suppressed people with HIV-1 (PWH) confirmed that ETS1 maintains transcriptional repression of the clinical HIV-1 reservoir. Transcriptomic profiling of ETS1-depleted cells from PWH identified a set of host cell pathways involved in viral transcription that are controlled by ETS1 in resting CD4 T cells. In particular, we observed that ETS1 knockout increased expression of the long non-coding RNA MALAT1 that has been previously identified as a positive regulator of HIV-1 expression. Furthermore, the impact of ETS1 depletion on HIV-1 expression in latently infected cells was partially dependent on MALAT1. Additionally, we demonstrate that ETS1 knockout resulted in enhanced abundance of activating modifications (H3K9Ac, H3K27Ac, H3K4me3) on histones located at the HIV-1 long terminal repeat (LTR), indicating that ETS1 regulates the activity of chromatin-targeting complexes at the HIV-1 LTR. Overall, these data demonstrate that ETS1 is an important regulator of HIV-1 latency that impacts HIV-1 expression through repressing MALAT1 expression and by regulating modification of proviral histones.},
}

*Proto-Oncogene Protein c-ets-1/genetics/metabolism
*HIV-1/physiology/genetics
Humans
*Virus Latency/physiology/genetics
*CD4-Positive T-Lymphocytes/virology/metabolism
*HIV Infections/virology/genetics/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Viral
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39957248,
year = {2025},
author = {Yin, X and Wang, R and Thackeray, A and Baehrecke, EH and Alkema, MJ},
title = {VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {4},
pages = {},
doi = {10.1093/g3journal/jkaf023},
pmid = {39957248},
issn = {2160-1836},
support = {R01 GM140480/NH/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/physiology ; *Mitochondria/metabolism/genetics ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *Locomotion/genetics ; *Homeostasis/genetics ; *Mutation ; Unfolded Protein Response/genetics ; CRISPR-Cas Systems ; Humans ; },
abstract = {Mitochondria control cellular metabolism, serve as hubs for signaling and organelle communication, and are important for the health and survival of cells. VPS13D encodes a cytoplasmic lipid transfer protein that regulates mitochondrial morphology, mitochondria and endoplasmic reticulum contact, and quality control of mitochondria. VPS13D mutations have been reported in patients displaying ataxic and spastic gait disorders with variable age of onset. Here, we used CRISPR/Cas9 gene editing to create VPS13D-related spinocerebellar ataxia-4 missense mutations and C-terminal deletion in VPS13D's ortholog vps-13D in Caenorhabditis elegans. Consistent with SCAR4 patient movement disorders and mitochondrial dysfunction, vps-13D mutant worms exhibit locomotion defects and abnormal mitochondrial morphology. Importantly, animals with a vps-13D deletion or a N3017I missense mutation exhibited an increase in mitochondrial unfolded protein response. The cellular and behavioral changes caused by VPS13D mutations in C. elegans advance the development of animal models that are needed to study SCAR4 pathogenesis.},
}

Animals
*Caenorhabditis elegans/genetics/physiology
*Mitochondria/metabolism/genetics
*Caenorhabditis elegans Proteins/genetics/metabolism
*Locomotion/genetics
*Homeostasis/genetics
*Mutation
Unfolded Protein Response/genetics
CRISPR-Cas Systems
Humans

@article {pmid39849901,
year = {2025},
author = {Tobias, IC and Moorthy, SD and Shchuka, VM and Langroudi, L and Cherednychenko, M and Gillespie, ZE and Duncan, AG and Tian, R and Gajewska, NA and Di Roberto, RB and Mitchell, JA},
title = {A Sox2 enhancer cluster regulates region-specific neural fates from mouse embryonic stem cells.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {4},
pages = {},
doi = {10.1093/g3journal/jkaf012},
pmid = {39849901},
issn = {2160-1836},
support = {FRN PJT153186/CAPMC/CIHR/Canada ; //Canada Foundation for Innovation/ ; //Ontario Ministry of Research and Innovation/ ; //University of Toronto/ ; //Canadian Institutes of Health Research fellowship/ ; //Natural Science and Engineering Research Council of Canada/ ; },
mesh = {Animals ; Mice ; *SOXB1 Transcription Factors/genetics/metabolism ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism/cytology ; *Neural Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; *Multigene Family ; Neurogenesis/genetics ; CRISPR-Cas Systems ; },
abstract = {Sex-determining region Y box 2 (Sox2) is a critical transcription factor for embryogenesis and neural stem and progenitor cell (NSPC) maintenance. While distal enhancers control Sox2 in embryonic stem cells (ESCs), enhancers closer to the gene are implicated in Sox2 transcriptional regulation in neural development. We hypothesize that a downstream enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), regulates Sox2 transcription in neural stem cells and we investigate this in NSPCs derived from mouse ESCs. Using functional genomics and CRISPR-Cas9-mediated deletion analyses, we investigate the role of SRR2-18 in Sox2 regulation during neural differentiation. Transcriptome analyses demonstrate that the loss of even 1 copy of SRR2-18 disrupts the region-specific identity of NSPCs, reducing the expression of genes associated with more anterior regions of the embryonic nervous system. Homozygous deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which causes differentiation into posterior neural fates, specifically the caudal neural tube. This study highlights the importance of precise Sox2 regulation by SRR2-18 in neural differentiation.},
}

Animals
Mice
*SOXB1 Transcription Factors/genetics/metabolism
*Enhancer Elements, Genetic
*Mouse Embryonic Stem Cells/metabolism/cytology
*Neural Stem Cells/metabolism/cytology
Cell Differentiation/genetics
Gene Expression Regulation, Developmental
*Multigene Family
Neurogenesis/genetics
CRISPR-Cas Systems

@article {pmid40227971,
year = {2025},
author = {Chen, WD and Liu, L and Cheng, L},
title = {Functionally Tunable Star-Shaped Multivalent crRNAs for Photocontrol CRISPR/Cas Editing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202506527},
doi = {10.1002/anie.202506527},
pmid = {40227971},
issn = {1521-3773},
abstract = {Clustered regularly interspaced shortpalindromic repeats/CRISPR-associated (CRISPR/Cas)-based genome editing has significantly advanced genetic engineering due to its precision, simplicity, and versatility. However, achieving precise spatial and temporal control remains challenging, restricting therapeutic and research applications. Herein, we introduce a novel class of star-shaped, multivalent crRNAs engineered for precise spatiotemporal control of CRISPR/Cas9 and Cas12a editing systems. These crRNAs are synthesized via single-site chemical modification and can be efficiently purified. By integrating distinct photo-responsive chemical linkages, we achieved selective activation of crRNA activity upon irradiation with specific wavelengths, enabling orthogonal regulation of multiple genetic targets simultaneously. This method demonstrated robust OFF-ON switching capabilities in vitro, characterized by minimal leakage and rapid activation. Importantly, the approach also proved highly effective for temporally controlled gene editing in mammalian cells in vivo, achieving considerable editing efficiency following brief photoactivation. Due to its target sequence-independent, single-site modification design, this strategy may serve as a universal solution for diverse CRISPR/Cas systems, eliminating cumbersome optimization processes. Future advancements incorporating long-wavelength responsive and reversible linkers promise further enhancement of tissue penetration and control, significantly broadening the applicability and impact of this approach in biological research and therapeutic interventions.},
}

@article {pmid40226913,
year = {2025},
author = {Lin, CP and Li, H and Brogan, DJ and Wang, T and Akbari, OS and Komives, EA},
title = {CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.},
journal = {Nucleic acids research},
volume = {53},
number = {7},
pages = {},
pmid = {40226913},
issn = {1362-4962},
support = {T32 GM008326/GF/NIH HHS/United States ; R01AI151004/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Protein Binding ; *CRISPR-Cas Systems ; Catalytic Domain ; Models, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Hydrogen Deuterium Exchange-Mass Spectrometry ; *RNA/metabolism/chemistry ; RNA Recognition Motif ; },
abstract = {Type III-E CRISPR-Cas effectors, referred to as Cas7-11 or giant Repeat-Associated Mysterious Protein, are single proteins that cleave target RNAs (tgRNAs) without nonspecific collateral cleavage, opening new possibilities for RNA editing. Here, biochemical assays combined with amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments reveal the dynamics of apo Cas7-11. The HDX-MS results suggest a mechanism by which CRISPR RNA (crRNA) stabilizes the folded state of the protein and subsequent tgRNA binding remodels it to the active form. HDX-MS shows that the four Cas7 RNA recognition motif (RRM) folds are well-folded, but insertion sequences, including disordered catalytic loops and β-hairpins of the Cas7.2/Cas7.3 active sites, fold upon binding crRNA leading to stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. TgRNA binding causes conformational changes around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot independently process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces tgRNA cleavage. The insertion domain interacts most stably with mature crRNA. Finally, we show a crRNA-induced conformational change in one of the tetratricopeptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding.},
}

*CRISPR-Associated Proteins/chemistry/metabolism/genetics
Protein Binding
*CRISPR-Cas Systems
Catalytic Domain
Models, Molecular
*Clustered Regularly Interspaced Short Palindromic Repeats
Hydrogen Deuterium Exchange-Mass Spectrometry
*RNA/metabolism/chemistry
RNA Recognition Motif

@article {pmid40225577,
year = {2025},
author = {Karapurkar, JK and Rajkumar, S and Jung, JH and Kim, JY and Birappa, G and Gowda, DAA and Colaco, JC and Suresh, B and Choi, JY and Woo, SH and Jo, WJ and Lee, JH and Kim, KS and Hong, SH and Ramakrishna, S},
title = {Targeting USP11 counteracts SFTPC[I73T] -associated interstitial lung disease in hiPSCs-derived alveolar organoids and in vivo models.},
journal = {Theranostics},
volume = {15},
number = {10},
pages = {4526-4549},
pmid = {40225577},
issn = {1838-7640},
mesh = {Animals ; Mice ; Humans ; *Organoids/metabolism ; *Lung Diseases, Interstitial/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems ; *Pulmonary Surfactant-Associated Protein C/genetics/metabolism ; Bleomycin ; Alveolar Epithelial Cells/metabolism ; Ubiquitination ; Mutation ; Pulmonary Alveoli/metabolism/pathology ; },
abstract = {Background: Interstitial lung disease (ILD) is a pulmonary disorder characterized by a combination of inflammation and fibrosis in the lung parenchyma, which initiates with the dysfunction of alveolar epithelial cells (AECs). The alveolar cells secrete surfactant proteins that lowers the surface tension of fluids in the lungs and maintains the stability of pulmonary tissue. Mutations on surfactant protein C (SFTPC), particularly I73T, are associated with a toxic gain of function that causes misfolding and the accumulation of immature SFTPC proteins, triggering pulmonary fibrosis (PF). Therefore, it is crucial to block the accumulation of the SFTPC [I73T] protein during ILD progression. Methods: We used a loss-of-function-based CRISPR/Cas9 library kit to screen genome-wide for deubiquitinating enzymes that regulate the SFTPC protein. The interaction between USP11 and SFTPC and its ubiquitination status was validated by immunoprecipitation and the TUBEs assay. HDR-directed knock-in of the I73T mutation into the SFTPC locus in human induced pluripotent stem cells (hiPSCs) was performed using the CRISPR/Cas9 system, and then those cells were differentiated into alveolar organoids (AOs) using a forced aggregation protocol. The clinical relevance of the USP11 inhibitor and its effect on preventing PF were investigated in a TGF-β-induced fibrosis in AOs and bleomycin (BLM)-induced mouse model. Results: We identified USP11 as a novel deubiquitinase that interacts with, stabilizes, deubiquitinates, and extends the half-life of SFTPC. Remarkably, USP11 stabilized and prolonged the half-life of the SFTPC [I73T] mutant protein significantly more than the wild type. In vitro functional studies revealed that USP11 exacerbates SFTPC [I73T] -induced fibrosis and enhances the epithelial-to-mesenchymal transition. Furthermore, we present a human in vitro model for investigating SFTPC[I73T] -induced fibrosis: hiPSCs-derived-AOs carrying the pathogenic SFTPC[I73T] variant. Interestingly, USP11 depletion in the organoids mitigated SFTPC [I73T] -induced fibrosis. Finally, pharmacological inhibition of USP11 prevented PF caused by TGF-β in hiPSCs-SFTPC[I73T]-AOs and BLM-induced mouse model, underscoring its therapeutic potential. Conclusions: Altogether, USP11 is a major protein stabilizer of SFTPC, and the clinical inhibition of USP11 during PF could be a novel therapeutic approach for ILD patients.},
}

Animals
Mice
Humans
*Organoids/metabolism
*Lung Diseases, Interstitial/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism
Disease Models, Animal
CRISPR-Cas Systems
*Pulmonary Surfactant-Associated Protein C/genetics/metabolism
Bleomycin
Alveolar Epithelial Cells/metabolism
Ubiquitination
Mutation
Pulmonary Alveoli/metabolism/pathology

@article {pmid40223602,
year = {2025},
author = {Mukhare, R and Gandhi, KA and Kadam, A and Raja, A and Singh, A and Madhav, M and Chaubal, R and Pandey, S and Gupta, S},
title = {Integration of Organoids With CRISPR Screens: A Narrative Review.},
journal = {Biology of the cell},
volume = {117},
number = {4},
pages = {e70006},
pmid = {40223602},
issn = {1768-322X},
mesh = {*Organoids/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Neoplasms/genetics/pathology ; Gene Editing/methods ; },
abstract = {Organoids represent a significant advancement in disease modeling, demonstrated by their capacity to mimic the physiological/pathological structure and functional characteristics of the native tissue. Recently CRISPR/Cas9 technology has emerged as a powerful tool in combination with organoids for the development of novel therapies in preclinical settings. This review explores the current literature on applications of pooled CRISPR screening in organoids and the emerging role of these models in understanding cancer. We highlight the evolution of genome-wide CRISPR gRNA library screens in organoids, noting their increasing adoption in the field over the past decade. Noteworthy studies utilizing these screens to investigate oncogenic vulnerabilities and developmental pathways in various organoid systems are discussed. Despite the promise organoids hold, challenges such as standardization, reproducibility, and the complexity of data interpretation remain. The review also addresses the ideas of assessing tumor organoids (tumoroids) against established cancer hallmarks and the potential of studying intercellular cooperation within these models. Ultimately, we propose that organoids, particularly when personalized for patient-specific applications, could revolutionize drug screening and therapeutic approaches, minimizing the reliance on traditional animal models and enhancing the precision of clinical interventions.},
}

*Organoids/metabolism
Humans
*CRISPR-Cas Systems/genetics
Animals
*Neoplasms/genetics/pathology
Gene Editing/methods

@article {pmid40223236,
year = {2025},
author = {Santiago Rivera, E and Scheibel, T},
title = {Spider Eye Development Editing and Silk Fiber Engineering Using CRISPR-Cas.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202502068},
doi = {10.1002/anie.202502068},
pmid = {40223236},
issn = {1521-3773},
abstract = {CRISPR-Cas9 gene editing represents an effective and precise technology to induce mutations in the genome, and it has been applied to a wide range of organisms for diverse purposes. However, CRISPR-based gene editing in spiders has not been reported to date. In this study, we demonstrate CRISPR-mediated microinjection in parental spiders leading to both knock-out (KO) and knock-in (KI) mutations within the spider's offspring. The KO of the gene sine oculis causes total eye loss, confirming the role of the gene in the development of all spider eyes. The knock-in of a monomeric red fluorescent protein (mRFP-KI) within a spider silk gene encoding one compound of the major ampullate silk of the spider Parasteatoda tepidariorum yields red fluorescent silk fibers. This finding demonstrates the feasibility of functionalizing silk proteins in spiders using CRISPR-based gene editing without influencing silk assembly. Our study expands the application of CRISPR to spiders and provides insights in the fields of developmental genetics as well as material sciences.},
}

@article {pmid40180619,
year = {2025},
author = {Zhang, D and Wang, W and Tang, M and Qu, C and Jiang, Z and Li, X and Luan, Y},
title = {In Situ Gene Engineering Approach to Overcome Tumor Resistance and Enhance T Cell-Mediated Cancer Immunotherapy.},
journal = {Nano letters},
volume = {25},
number = {15},
pages = {6200-6208},
doi = {10.1021/acs.nanolett.5c00488},
pmid = {40180619},
issn = {1530-6992},
mesh = {Animals ; Interleukin-12/genetics/immunology ; CRISPR-Cas Systems ; *T-Lymphocytes, Cytotoxic/immunology ; *Immunotherapy/methods ; Mice ; Humans ; *Gene Editing ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics/pathology ; Tumor Microenvironment/immunology ; Dendrimers/chemistry ; Endosomal Sorting Complexes Required for Transport/genetics ; Genetic Engineering ; },
abstract = {T cell-mediated cancer immunotherapy harnesses the power of cytotoxic T lymphocytes (CTLs) to target and eradicate tumor cells. However, tumor cells often evade immune attack through membrane repair mechanisms involving endosomal sorting complexes required for transport (ESCRT) and immune suppression within the tumor microenvironment. Here, we developed a robust TMV@PpCHIL nanomedicine to address these issues by reprogramming tumor cells via in situ gene editing. Using CRISPR/Cas9, we disrupted the Chmp4b gene, a key component of the ESCRT machinery, preventing tumor cells from repairing CTL-induced membrane damage. Simultaneously, we genetically engineered tumor cells to produce interleukin-12 (IL-12), a cytokine that enhances CTL activation. The TMV@PpCHIL nanomedicine, designed by coating tumor membrane vesicles (TMVs) onto polyamidoamine (PAMAM) dendrimer-condensed plasmid complexes, ensured efficient CRISPR/Cas9-based gene editing and sustained IL-12 production. This approach significantly enhanced CTL-mediated tumor cell cytotoxicity, suppressed tumor growth, reduced metastasis, and prolonged survival, providing a promising strategy for durable cancer treatment.},
}

Animals
Interleukin-12/genetics/immunology
CRISPR-Cas Systems
*T-Lymphocytes, Cytotoxic/immunology
*Immunotherapy/methods
Mice
Humans
*Gene Editing
Cell Line, Tumor
*Neoplasms/therapy/immunology/genetics/pathology
Tumor Microenvironment/immunology
Dendrimers/chemistry
Endosomal Sorting Complexes Required for Transport/genetics
Genetic Engineering

@article {pmid40177728,
year = {2025},
author = {Liu, S and Lun, J and Zhan, Y and Li, Z and Tian, J and Zhang, C and Pan, L},
title = {CRISPR/Cas12a Combined with RAA for On-Site Detection of ALS W574L Mutation in Three Alopecurus Species: A Visual Approach for Herbicide Resistance Monitoring.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {15},
pages = {8907-8914},
doi = {10.1021/acs.jafc.5c02188},
pmid = {40177728},
issn = {1520-5118},
mesh = {*Herbicide Resistance ; CRISPR-Cas Systems ; *Acetolactate Synthase/genetics/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism ; Mutation ; *Poaceae/genetics/drug effects/enzymology ; Plant Weeds/genetics/drug effects/enzymology ; },
abstract = {The genus Alopecurus encompasses several weed species, including Alopecurus japonicus, Alopecurus aequalis, and Alopecurus myosuroides, which represent significant threats to agricultural productivity, particularly in wheat and oilseed rape fields. ALS-inhibiting herbicides have been extensively used for controlling Alopecurus weeds. However, the widespread use of these herbicides has led to the rapid emergence of resistance in Alopecurus populations with the Trp-574-Leu (W574L) mutation in the ALS gene being one of the most common resistance mechanisms. This study aims to develop a novel molecular detection method combining recombinase-aided amplification (RAA) with CRISPR/Cas12a technology to detect the W574L mutation in Alopecurus species. The method was optimized for key parameters, balancing efficiency with experimental costs, and was evaluated for specificity, sensitivity, and field applicability. This approach offers a rapid, accurate, and visual tool for identifying W574L target-site resistance in A. japonicus, A. aequalis, and A. myosuroides, with significant potential for monitoring resistance and enhancing weed management strategies.},
}

*Herbicide Resistance
CRISPR-Cas Systems
*Acetolactate Synthase/genetics/metabolism
*Herbicides/pharmacology
*Plant Proteins/genetics/metabolism
Mutation
*Poaceae/genetics/drug effects/enzymology
Plant Weeds/genetics/drug effects/enzymology

@article {pmid40033995,
year = {2025},
author = {Hanson, E and Kalla, N and Tharu, RJ and Demir, MM and Tok, BH and Canbaz, MA and Yigit, MV},
title = {CRISPR-Responsive Reprogrammable Label-Free Fluorescent Nanoclusters for ML-Assisted Pathogenic Genome Detection on Solid Substrates.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {15},
pages = {e2500784},
doi = {10.1002/smll.202500784},
pmid = {40033995},
issn = {1613-6829},
support = {1R35GM156250-01//National Institute of General Medical Sciences of the National Institutes of Health/ ; 2022-08596//USDA National Institute of Food and Agriculture (NIFA)/ ; 2022-08596//National Institute of Food and Agriculture/ ; 1R35GM156250-01/GM/NIGMS NIH HHS/United States ; 1R35GM156250-01/GM/NIGMS NIH HHS/United States ; },
mesh = {*Salmonella/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Metal Nanoparticles/chemistry ; Silver/chemistry ; Fluorescence ; },
abstract = {The development of a paper-based genome detection assay using target-responsive DNA-templated silver nanoclusters (DFN-1) is presented. The reported nanoclusters exhibit intrinsic fluorescence, which is regulated by the cleavage of the DNA template surrounding the silver core. To enable the nanoclusters to respond to a specific genome, CRISPR-Cas12a is employed for highly specific and programmable digestion of the nanoclusters. Upon detection of the target, the DNA template is cleaved by the CRISPR-Cas12a complex, leading to a reduction in fluorescence. This assay successfully demonstrates for the detection of the Salmonella genome in the liquid phase and on 2 mm solid filter paper discs. By altering only the crRNA in the CRISPR complex, the assay is programmed to detect two different Salmonella serotypes. The selectivity of the assay is evaluated in DNA mixtures with and without the target genomic fragments. The assay also demonstrates the detection of as little as 33 copies of the full Salmonella genome by incorporating an isothermal amplification step. Furthermore, 60 unknown samples with different target content in standard 344 well plates are evaluated. The results are analyzed using custom-developed machine-learning algorithms, successfully detecting the presence of the target with 100% prediction accuracy.},
}

*Salmonella/genetics
*CRISPR-Cas Systems/genetics
*Genome, Bacterial/genetics
*Metal Nanoparticles/chemistry
Silver/chemistry
Fluorescence

@article {pmid39786576,
year = {2025},
author = {Zafeer, MF and Ramzan, M and Duman, D and Mutlu, A and Seyhan, S and Kalcioglu, MT and Fitoz, S and DeRosa, BA and Guo, S and Dykxhoorn, DM and Tekin, M},
title = {Human organoids for rapid validation of gene variants linked to cochlear malformations.},
journal = {Human genetics},
volume = {144},
number = {4},
pages = {375-389},
pmid = {39786576},
issn = {1432-1203},
support = {R01 DC009645/DC/NIDCD NIH HHS/United States ; R01 DC012836/DC/NIDCD NIH HHS/United States ; NIH R01DC009645/DC/NIDCD NIH HHS/United States ; NIH R01DC009645/DC/NIDCD NIH HHS/United States ; },
mesh = {Humans ; *Organoids/metabolism/pathology ; *Cochlea/abnormalities/pathology/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Male ; Female ; Polymorphism, Single Nucleotide ; Exome Sequencing ; CRISPR-Cas Systems ; Fibroblast Growth Factor 3 ; },
abstract = {Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital. The majority of patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for the genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families deafness. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a potential tool to validate the pathogenicity of DNA variants associated with cochlear malformations.},
}

Humans
*Organoids/metabolism/pathology
*Cochlea/abnormalities/pathology/metabolism
Induced Pluripotent Stem Cells/metabolism
Male
Female
Polymorphism, Single Nucleotide
Exome Sequencing
CRISPR-Cas Systems
Fibroblast Growth Factor 3

@article {pmid39761726,
year = {2025},
author = {Vázquez Salgado, AM and Cai, C and Lee, M and Yin, D and Chrystostome, ML and Gefre, AF and He, S and Kieckhaefer, JE and Wangensteen, KJ},
title = {In Vivo CRISPR Activation Screening Reveals Chromosome 1q Genes VPS72, GBA1, and MRPL9 Drive Hepatocellular Carcinoma.},
journal = {Cellular and molecular gastroenterology and hepatology},
volume = {19},
number = {5},
pages = {101460},
pmid = {39761726},
issn = {2352-345X},
mesh = {Animals ; *Carcinoma, Hepatocellular/genetics/pathology ; Humans ; *Liver Neoplasms/genetics/pathology ; Mice ; *Chromosomes, Human, Pair 1/genetics ; Gene Expression Regulation, Neoplastic ; *Vesicular Transport Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) frequently undergoes regional chromosomal amplification, resulting in elevated gene expression levels. We aimed to elucidate the role of these poorly understood genetic changes by using CRISPR activation (CRISPRa) screening in mouse livers to identify which genes within these amplified loci are cancer driver genes.

METHODS: We used data from The Cancer Genome Atlas to identify that frequently copy number-amplified and up-regulated genes all reside on human chromosomes 1q and 8q. We generated CRISPRa screening transposons that contain oncogenic Myc to drive tumor formation. We conducted CRISPRa screens in vivo in the liver to identify tumor driver genes. We extensively validated the findings in separate mice and performed RNA sequencing analysis to explore mechanisms driving tumorigenesis.

RESULTS: We targeted genes that frequently undergo amplification in human HCC using an in vivo CRISPRa screening system in mice, which induced extensive liver tumorigenesis. Human chromosome 1q genes Zbtb7b, Vps72, Gba1, and Mrpl9 emerged as drivers of liver tumorigenesis. In human HCC there is a trend in correlation between levels of MRPL9, VPS72, or GBA1 and poor survival. In validation assays, activation of Vps72, Gba1, or Mrpl9 resulted in extensive liver tumorigenesis and decreased survival in mice. RNA sequencing revealed different mechanisms driving HCC, with Mrpl9 activation altering genes functionally related to mitochondrial function, Vps72 levels altering phospholipid metabolism, and Gba1 activation enhancing endosomal-lysosomal activity, all leading to promotion of cellular proliferation. Analysis of human tumor tissues with high levels of MRPL9, VPS72, or GBA1 revealed congruent results, indicating conserved mechanisms driving HCC.

CONCLUSIONS: This study reveals chromosome 1q genes Vps72, Gba1, and Mrpl9 as drivers of HCC. Future efforts to prevent or treat HCC can focus on these new driver genes.},
}

Animals
*Carcinoma, Hepatocellular/genetics/pathology
Humans
*Liver Neoplasms/genetics/pathology
Mice
*Chromosomes, Human, Pair 1/genetics
Gene Expression Regulation, Neoplastic
*Vesicular Transport Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems

@article {pmid39760438,
year = {2025},
author = {Montilla-Rojo, J and Eleveld, TF and van Soest, M and Hillenius, S and Timmerman, DM and Gillis, AJM and Roelen, BAJ and Mummery, CL and Looijenga, LHJ and Salvatori, DCF},
title = {Depletion of TP53 in Human Pluripotent Stem Cells Triggers Malignant-Like Behavior.},
journal = {Advanced biology},
volume = {9},
number = {4},
pages = {e2400538},
doi = {10.1002/adbi.202400538},
pmid = {39760438},
issn = {2701-0198},
support = {184.034.019//Stichting Kinderen Kankervrij/ ; NNF21CC0073729//Novo Nordisk Foundation Center for Stem Cell Medicine reNEW/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/genetics/metabolism ; *Pluripotent Stem Cells/metabolism/pathology ; CRISPR-Cas Systems ; Mutation ; Cell Proliferation/genetics ; },
abstract = {Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, TP53 is knocked out in hPSCs using CRISPR-Cas9 and compared them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency, and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. The results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, can impact their behavior and safety in a clinical setting.},
}

Humans
*Tumor Suppressor Protein p53/genetics/metabolism
*Pluripotent Stem Cells/metabolism/pathology
CRISPR-Cas Systems
Mutation
Cell Proliferation/genetics

@article {pmid39478215,
year = {2024},
author = {Huynh, P and Hoffmann, JD and Gerhardt, T and Kiss, MG and Zuraikat, FM and Cohen, O and Wolfram, C and Yates, AG and Leunig, A and Heiser, M and Gaebel, L and Gianeselli, M and Goswami, S and Khamhoung, A and Downey, J and Yoon, S and Chen, Z and Roudko, V and Dawson, T and Ferreira da Silva, J and Ameral, NJ and Morgenroth-Rebin, J and D'Souza, D and Koekkoek, LL and Jacob, W and Munitz, J and Lee, D and Fullard, JF and van Leent, MMT and Roussos, P and Kim-Schulze, S and Shah, N and Kleinstiver, BP and Swirski, FK and Leistner, D and St-Onge, MP and McAlpine, CS},
title = {Myocardial infarction augments sleep to limit cardiac inflammation and damage.},
journal = {Nature},
volume = {635},
number = {8037},
pages = {168-177},
pmid = {39478215},
issn = {1476-4687},
support = {R00 HL151750/HL/NHLBI NIH HHS/United States ; R01 AG082185/AG/NIA NIH HHS/United States ; R01 HL158534/HL/NHLBI NIH HHS/United States ; T32 AI078892/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Chemotaxis, Leukocyte ; Choroid Plexus/metabolism ; Glutamic Acid/metabolism ; Heart/physiopathology ; Heart Rate ; *Inflammation/pathology/prevention & control ; Lateral Thalamic Nuclei/metabolism ; Macrophages/cytology/metabolism ; Mice, Inbred C57BL ; Microglia/cytology/metabolism ; Monocytes/cytology/metabolism ; *Myocardial Infarction/physiopathology/complications/pathology/metabolism ; *Myocardium/pathology/metabolism ; Neurons/metabolism ; Receptors, Adrenergic, beta-2/metabolism ; Receptors, Tumor Necrosis Factor, Type I/metabolism ; *Sleep/physiology ; Sleep, Slow-Wave/physiology ; Sympathetic Nervous System/physiopathology ; Tachycardia, Ventricular/physiopathology/etiology/metabolism ; Tumor Necrosis Factors/metabolism ; },
abstract = {Sleep is integral to cardiovascular health[1,2]. Yet, the circuits that connect cardiovascular pathology and sleep are incompletely understood. It remains unclear whether cardiac injury influences sleep and whether sleep-mediated neural outputs contribute to heart healing and inflammation. Here we report that in humans and mice, monocytes are actively recruited to the brain after myocardial infarction (MI) to augment sleep, which suppresses sympathetic outflow to the heart, limiting inflammation and promoting healing. After MI, microglia rapidly recruit circulating monocytes to the brain's thalamic lateral posterior nucleus (LPN) via the choroid plexus, where they are reprogrammed to generate tumour necrosis factor (TNF). In the thalamic LPN, monocytic TNF engages Tnfrsf1a-expressing glutamatergic neurons to increase slow wave sleep pressure and abundance. Disrupting sleep after MI worsens cardiac function, decreases heart rate variability and causes spontaneous ventricular tachycardia. After MI, disrupting or curtailing sleep by manipulating glutamatergic TNF signalling in the thalamic LPN increases cardiac sympathetic input which signals through the β2-adrenergic receptor of macrophages to promote a chemotactic signature that increases monocyte influx. Poor sleep in the weeks following acute coronary syndrome increases susceptibility to secondary cardiovascular events and reduces the heart's functional recovery. In parallel, insufficient sleep in humans reprogrammes β2-adrenergic receptor-expressing monocytes towards a chemotactic phenotype, enhancing their migratory capacity. Collectively, our data uncover cardiogenic regulation of sleep after heart injury, which restricts cardiac sympathetic input, limiting inflammation and damage.},
}

Animals
Female
Humans
Male
Mice
Chemotaxis, Leukocyte
Choroid Plexus/metabolism
Glutamic Acid/metabolism
Heart/physiopathology
Heart Rate
*Inflammation/pathology/prevention & control
Lateral Thalamic Nuclei/metabolism
Macrophages/cytology/metabolism
Mice, Inbred C57BL
Microglia/cytology/metabolism
Monocytes/cytology/metabolism
*Myocardial Infarction/physiopathology/complications/pathology/metabolism
*Myocardium/pathology/metabolism
Neurons/metabolism
Receptors, Adrenergic, beta-2/metabolism
Receptors, Tumor Necrosis Factor, Type I/metabolism
*Sleep/physiology
Sleep, Slow-Wave/physiology
Sympathetic Nervous System/physiopathology
Tachycardia, Ventricular/physiopathology/etiology/metabolism
Tumor Necrosis Factors/metabolism

@article {pmid40221789,
year = {2025},
author = {Davydova, S and Liu, J and Liu, Y and Prince, K and Mann, J and Kandul, NP and Braswell, WE and Champer, J and Akbari, OS and Meccariello, A},
title = {A self-limiting sterile insect technique alternative for Ceratitis capitata.},
journal = {BMC biology},
volume = {23},
number = {1},
pages = {97},
pmid = {40221789},
issn = {1741-7007},
support = {AP23PPQS&T00C108//U.S. Department of Agriculture/ ; AP23PPQS&T00C108//U.S. Department of Agriculture/ ; 23-8130-1007-IA//U.S. Department of Agriculture/ ; 101059523//Horizon 2020/ ; },
mesh = {*Ceratitis capitata/genetics/physiology ; Animals ; *CRISPR-Cas Systems ; *Pest Control, Biological/methods ; Female ; Male ; },
abstract = {BACKGROUND: Genetic biocontrol systems have broad applications in population control of insects implicated in both disease spread and food security. Ceratitis capitata (the Mediterranean fruit fly), a major agricultural pest with a global distribution, is one of the appealing targets for such genetic control.

RESULTS: In this study, we establish and characterise a novel split-CRISPR/Cas9 system we term Sex Conversion Induced by CRISPR (SCIC) in C. capitata. Using the white eye gene for toolkit selection we achieved up to 100% CRISPR/Cas9 efficiency, displaying the feasibility of C. capitata split-CRISPR/Cas9 systems using constitutive promoters. We then induce sex conversion by targeting the transformer gene in a SCIC approach aimed for SIT-mediated releases upon radiation-based sterilisation. Knock-out of transformer induced partial to full female-to-male sex conversion, with the remaining individuals all being intersex and sterile. SCIC population modelling shows a strong potential to outcompete traditional SIT, allowing for faster population elimination with fewer released sterile males.

CONCLUSION: Overall, we construct an appropriate CRISPR/Cas9 toolkit for the use in C capitata. Our results build the foundation for further genetic pest control methods in the species and related tephritid agricultural pests.},
}

*Ceratitis capitata/genetics/physiology
Animals
*CRISPR-Cas Systems
*Pest Control, Biological/methods
Female
Male

@article {pmid40221460,
year = {2025},
author = {Myers, G and Friedman, A and Yu, L and Pourmandi, N and Kerpet, C and Ito, MA and Saba, R and Tang, V and Ozel, AB and Bergin, IL and Johnson, CN and Ku, CJ and Wang, Y and Balbin-Cuesta, G and Lim, KC and Lin, Z and Drysdale, C and McGee, B and Kurita, R and Nakamura, Y and Liu, X and Siemieniak, D and Singh, SA and Lyssiotis, CA and Maillard, I and Weisman, LS and Engel, JD and Khoriaty, R},
title = {A genome-wide screen identifies genes required for erythroid differentiation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3488},
pmid = {40221460},
issn = {2041-1723},
support = {R01 AI091627/AI/NIAID NIH HHS/United States ; U2C DK129445/DK/NIDDK NIH HHS/United States ; AI091627//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; F31 HL162544/HL/NHLBI NIH HHS/United States ; K08 DK127013/DK/NIDDK NIH HHS/United States ; P01 HL146372/HL/NHLBI NIH HHS/United States ; R01 HL157062/HL/NHLBI NIH HHS/United States ; U01 HL117658/HL/NHLBI NIH HHS/United States ; R01 HL148333/HL/NHLBI NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; TL1 DK136046/DK/NIDDK NIH HHS/United States ; T32-GM007315//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *Erythropoiesis/genetics ; *Cell Differentiation/genetics ; CRISPR-Cas Systems ; Animals ; Genome-Wide Association Study ; *Erythroid Precursor Cells/cytology/metabolism ; Erythroblasts/metabolism/cytology ; Mice ; Cell Line ; Gene Knockout Techniques ; Erythroid Cells/cytology ; Mutation ; },
abstract = {The complete array of genes required for terminal erythroid differentiation remains unknown. To address this knowledge gap, we perform a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2 and validate candidate regulators of erythroid differentiation in a custom secondary screen. Comparison of sgRNA abundance in the CRISPR library, proerythroblasts, and orthochromatic erythroblasts, resulted in the identification of genes that are essential for proerythroblast survival and genes that are required for terminal erythroid differentiation. Among the top genes identified are known regulators of erythropoiesis, underscoring the validity of this screen. Notably, using a Log2 fold change of <-1 and false discovery rate of <0.01, the screen identified 277 genes that are required for terminal erythroid differentiation, including multiple genes not previously nominated through GWAS. NHLRC2, which was previously implicated in hemolytic anemia, was a highly ranked gene. We suggest that anemia due to NHLRC2 mutation results at least in part from a defect in erythroid differentiation. Another highly ranked gene in the screen is VAC14, which we validated for its requirement in erythropoiesis in vitro and in vivo. Thus, data from this CRISPR screen may help classify the underlying mechanisms that contribute to erythroid disorders.},
}

Humans
*Erythropoiesis/genetics
*Cell Differentiation/genetics
CRISPR-Cas Systems
Animals
Genome-Wide Association Study
*Erythroid Precursor Cells/cytology/metabolism
Erythroblasts/metabolism/cytology
Mice
Cell Line
Gene Knockout Techniques
Erythroid Cells/cytology
Mutation

@article {pmid40220102,
year = {2025},
author = {Romanowski, AJ and Richardson, RR and Plachez, C and Erzurumlu, RS and Poulopoulos, A},
title = {Gene Knockout in the Developing Brain of Wild-Type Rodents by CRISPR In Utero Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2910},
number = {},
pages = {221-238},
pmid = {40220102},
issn = {1940-6029},
mesh = {Animals ; *Electroporation/methods ; *CRISPR-Cas Systems ; Mice ; *Gene Knockout Techniques/methods ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Brain/metabolism/embryology/growth & development ; Pregnancy ; Hippocampus/metabolism ; Plasmids/genetics ; Neurons/metabolism ; },
abstract = {CRISPR/Cas9 constructs can be delivered by in utero electroporation to knock out a gene of interest in neurons of the developing brain in wild-type rodents. This approach allows for high-throughput genetic screening, circuit-specific gene knockout, and knockout cell phenotyping using sparse labeling within a wild-type in vivo context. Here we outline the methods and steps of designing guide RNAs in silico, cloning guide RNAs into plasmid backbones, and introducing these plasmids into the developing mouse cortex and hippocampus.},
}

Animals
*Electroporation/methods
*CRISPR-Cas Systems
Mice
*Gene Knockout Techniques/methods
Female
RNA, Guide, CRISPR-Cas Systems/genetics
*Brain/metabolism/embryology/growth & development
Pregnancy
Hippocampus/metabolism
Plasmids/genetics
Neurons/metabolism

@article {pmid40220094,
year = {2025},
author = {Robertson, CD and Richardson, RR and Steyert, M and Martin, CA and Flynn, C and Poulopoulos, A},
title = {Prime Editing of Mouse Primary Neurons.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2910},
number = {},
pages = {69-84},
pmid = {40220094},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *Neurons/metabolism/cytology ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plasmids/genetics ; High-Throughput Nucleotide Sequencing ; Cells, Cultured ; },
abstract = {Prime editing is a hybrid genome editing technology that introduces small edits on the genome with high precision. It combines nickase Cas9 with reverse transcriptase to prime and synthesizes edited DNA from RNA, reducing unintended insertions and deletions on the genome. This protocol describes the design of prime editing guide RNAs (pegRNAs), cloning of plasmids, nucleofection of mouse primary neurons, and preparation for next-generation sequencing. Directions are given for pegRNA and PE3b gRNA design and construction using PegAssist, a publicly available webtool and plasmid set. Prime editing in neurons allows genome manipulation while maintaining endogenous gene expression, making it ideal for studying protein structure/function relationships and pathogenic variants in a native neuronal context.},
}

Animals
*Gene Editing/methods
Mice
*Neurons/metabolism/cytology
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Plasmids/genetics
High-Throughput Nucleotide Sequencing
Cells, Cultured

@article {pmid40219967,
year = {2025},
author = {Liu, X and Qi, Q and Xiong, W and Zhang, Y and Shen, W and Xu, X and Zhao, Y and Li, M and Zhou, E and Tian, T and Zhou, X},
title = {Tailoring and reversing m6A editing with sequential RNA bioorthogonal chemistry.},
journal = {Nucleic acids research},
volume = {53},
number = {7},
pages = {},
pmid = {40219967},
issn = {1362-4962},
support = {22037004//National Natural Science Foundation of China/ ; 2042023kf0204//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *RNA/chemistry/genetics/metabolism ; *Adenosine/analogs & derivatives/chemistry/metabolism ; CRISPR-Cas Systems ; Methylation ; *RNA Editing ; Azides/chemistry ; HEK293 Cells ; },
abstract = {Many existing methods for post-transcriptional RNA modification rely on a single-step approach, limiting the ability to reversibly control m6A methylation at specific sites. Here, we address this challenge by developing a multi-step system that builds on the concept of sequential RNA bioorthogonal chemistry. Our strategy uses an azide-based reagent (NAI-N3) capable of both cleavage and ligation reactions, thereby allowing iterative and reversible modifications of RNA in living cells. By applying this approach in CRISPR (clustered regularly interspaced short palindromic repeats)-based frameworks, we demonstrate tailored editing of m6A marks at targeted RNA sites, overcoming the one-way restriction of conventional bioorthogonal methods. This sequential protocol not only broadens the scope for fine-tuned RNA regulation but also provides a versatile platform for exploring dynamic m6A function in genetic and epigenetic research.},
}

Humans
*RNA/chemistry/genetics/metabolism
*Adenosine/analogs & derivatives/chemistry/metabolism
CRISPR-Cas Systems
Methylation
*RNA Editing
Azides/chemistry
HEK293 Cells

@article {pmid40219064,
year = {2025},
author = {Marques, BM and Sulis, DB and Suarez, B and Yang, C and Cofre-Vega, C and Thomas, RD and Whitehill, JGA and Whetten, RW and Barrangou, R and Wang, JP},
title = {A Protoplast System for CRISPR-Cas Ribonucleoprotein Delivery in Pinus taeda and Abies fraseri.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
pmid = {40219064},
issn = {2223-7747},
support = {20-070-4013//North Carolina Specialty Crop Block Grants/ ; NCZ04214//Cooperative State Research Service of the U.S. Department of Agriculture/ ; 2044721//U.S. National Science Foundation Small Business Technology Transfer Program/ ; 190549MA//North Carolina State University Chancellor's Innovation Fund/ ; },
abstract = {Climate change profoundly impacts the health, productivity, and resilience of forest ecosystems and threatens the sustainability of forest products and wood-based industries. Innovations to enhance tree growth, development, and adaptation offer unprecedented opportunities to strengthen ecosystem resilience and mitigate the effects of climate change. Here, we established a method for protoplast isolation, purification, and CRISPR-Cas ribonucleoprotein (RNP) delivery in Pinus taeda and Abies fraseri as a step towards accelerating the genetic improvement of these coniferous tree species. In this system, purified protoplasts could be isolated from somatic embryos with up to 2 × 10[6] protoplasts/g of tissue and transfected with proteins and nucleotides, achieving delivery efficiencies up to 13.5%. The delivery of functional RNPs targeting phenylalanine ammonia lyase in P. taeda and phytoene desaturase in A. fraseri yielded gene editing efficiencies that reached 2.1% and 0.3%, respectively. This demonstration of RNP delivery for DNA-free genome editing in the protoplasts of P. taeda and A. fraseri illustrates the potential of CRISPR-Cas to enhance the traits of value in ecologically and economically important tree species. The editing system provides a foundation for future efforts to regenerate genome-edited forest trees to improve ecosystem health and natural resource sustainability.},
}

@article {pmid40183470,
year = {2025},
author = {Haley, RM and Padilla, MS and El-Mayta, RD and Joseph, RA and Weber, JA and Figueroa-Espada, CG and Mukalel, AJ and Ricciardi, AS and Palanki, R and Geisler, HC and Jester, MT and Davidson, BL and Mitchell, MJ},
title = {Lipid Nanoparticles for In Vivo Lung Delivery of CRISPR-Cas9 Ribonucleoproteins Allow Gene Editing of Clinical Targets.},
journal = {ACS nano},
volume = {19},
number = {14},
pages = {13790-13804},
doi = {10.1021/acsnano.4c16617},
pmid = {40183470},
issn = {1936-086X},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/chemistry/administration & dosage/metabolism ; *Lung/metabolism ; Animals ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; Mice ; *CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {In the past 10 years, CRISPR-Cas9 has revolutionized the gene-editing field due to its modularity, simplicity, and efficacy. It has been applied for the creation of in vivo models, to further understand human biology, and toward the curing of genetic diseases. However, there remain significant delivery barriers for CRISPR-Cas9 application in the clinic, especially for in vivo and extrahepatic applications. In this work, high-throughput molecular barcoding techniques were used alongside traditional screening methodologies to simultaneously evaluate LNP formulations encapsulating ribonucleoproteins (RNPs) for in vitro gene-editing efficiency and in vivo biodistribution. This resulted in the identification of a lung-tropic LNP formulation, which shows efficient gene editing in endothelial and epithelial cells within the lung, targeting both model reporter and clinically relevant genomic targets. Further, this LNP shows no off-target indel formation in the liver, making it a highly specific extrahepatic delivery system for lung-editing applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/chemistry/administration & dosage/metabolism
*Lung/metabolism
Animals
*Nanoparticles/chemistry
Humans
*Lipids/chemistry
Mice
*CRISPR-Associated Protein 9
Liposomes

@article {pmid40173419,
year = {2025},
author = {Towell, SE and Jareczek, MJ and Cooke, LS and Godfrey, DR and Zhukhovitskiy, AV},
title = {Skeletal Editing of Polymer Backbones and Its Impact Across the Polymer Lifecycle.},
journal = {Accounts of chemical research},
volume = {58},
number = {8},
pages = {1275-1283},
doi = {10.1021/acs.accounts.5c00054},
pmid = {40173419},
issn = {1520-4898},
mesh = {*Polymers/chemistry ; CRISPR-Cas Systems ; },
abstract = {ConspectusIn the last five years, interest in the precise modification of molecular cores─termed skeletal editing─has rapidly expanded in the Chemistry community. Beyond the intrinsic value of these transformations, skeletal editing also has value in the attention it brings to under-explored chemical challenges, whose solutions could transform the practice of Chemistry at large. In few contexts does this perspective ring as true as in the realm of polymers. Inspired by the revolutionary power of biologically derived machinery called CRISPR-Cas9 to edit nucleic acid polymers and, consequently, the genetic meaning encoded in them, we envisioned that skeletal editing of synthetic polymer backbones may also enable control over the structure and "meaning"─i.e., properties and function─of plastics. However, the idea of editing polymer backbones brings about numerous fundamental chemical questions that must be answered to make the vision a reality: for instance, how to constructively activate carbon-carbon and carbon-heteroatom bonds that make up typical polymer backbones and how to do so in a site-selective manner? While many fundamental questions have begun to be answered by the small molecule community, they are yet to be applied to the realm of polymers, and such adaptation often begets new scientific challenges. Moreover, as we begin to tackle these questions, we must always consider how advances in skeletal editing of polymer backbones impact the broader contexts of applications and sustainability of plastics.In this Account, we summarize our efforts to advance the skeletal editing of polymer backbones, focusing on how such methods can affect each stage of the polymer lifecycle: (1) provide an entry to previously challenging-to-access functional polymers or to existing ones but from new feedstocks, (2) evolve one type of polymer into another with associated changes in material properties, and (3) enable the breakdown of otherwise intractable polymer backbones. Along the way, we describe our rationale behind the selection and development of reactions utilized for skeletal editing. We explain how small molecule reactions often need to be adapted to suit polymeric substrates and the methodology optimizations we needed to do to accomplish our edits. We also discuss the considerations involved in the selection or design of polymeric substrates for editing with an eye toward what edits can add to polymer function and how to advance the field. We conclude with an outlook on outstanding challenges that we aim to address in future work establishing areas for future exploration within each of our topic areas.},
}

*Polymers/chemistry
CRISPR-Cas Systems

@article {pmid40173086,
year = {2025},
author = {Liu, X and Zhou, E and Qi, Q and Xiong, W and Tian, T and Zhou, X},
title = {Innovative Chemical Strategies for Advanced CRISPR Modulation.},
journal = {Accounts of chemical research},
volume = {58},
number = {8},
pages = {1262-1274},
doi = {10.1021/acs.accounts.5c00052},
pmid = {40173086},
issn = {1520-4898},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Metal-Organic Frameworks/chemistry ; Humans ; },
abstract = {ConspectusOver the past decade, RNA-guided gene editing technologies, particularly those derived from CRISPR systems, have revolutionized life sciences and opened unprecedented opportunities for therapeutic innovation. Despite their transformative potential, achieving precise control over the activity and specificity of these molecular tools remains a formidable challenge, requiring advanced and innovative regulatory strategies. We and others have developed new approaches that integrate chemical ingenuity with bioorthogonal techniques to achieve remarkable precision in CRISPR regulation. One key innovation lies in the chemical modulation of guide RNA (gRNA), significantly expanding the CRISPR toolkit. Strategies such as CRISPR-ON and CRISPR-OFF switches rely on selective chemical masking and demasking of gRNA. These approaches use either bulky chemical groups to preemptively mask RNA or minor, less obstructive groups to fine-tune its function, followed by bioorthogonal reactions to restore or suppress activity. These methodologies have proven to be pivotal for controlled gene editing and expression, addressing the challenges of precision, reversibility, and dynamic regulation.Parallel to these advances, the development of mesoporous metal-organic frameworks (MOFs) has emerged as a promising solution for RNA deprotection and activation. By serving as catalytic tools, MOFs enhance the versatility and efficiency of CRISPR systems, pushing their applications beyond the conventional boundaries. In addition, the synthesis of novel small molecules for regulating CRISPR-Cas9 activity marks a critical milestone in the evolution of gene therapy protocols. Innovative RNA structural control strategies have also emerged, particularly through the engineering of G-quadruplex (G4) motifs and G-G mismatches. These methods exploit the structural propensities of engineered gRNAs, employing small-molecule ligands to induce specific conformational changes that modulate the CRISPR activity. Whether stabilizing G4 formation or promoting G-G mismatches, these strategies demonstrate the precision and sophistication required for the molecular-level control of gene editing.Further enhancing these innovations, techniques like host-guest chemistry and conditional diacylation cross-linking have been developed to directly alter gRNA structure and function. These approaches provide nuanced, reversible, and safe control over CRISPR systems, advancing both the precision and reliability of gene editing technologies. In conclusion, this body of work highlights the convergence of chemistry, materials science, and molecular biology to create integrative solutions for gene editing. By combination of bioorthogonal chemistry, RNA engineering, and advanced materials, these advancements offer unprecedented accuracy and control for both fundamental research and therapeutic applications. These innovations not only advance genetic research but also contribute to developing safer and more effective gene editing strategies, moving us closer to realizing the full potential of these technologies.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
Metal-Organic Frameworks/chemistry
Humans

@article {pmid40062839,
year = {2025},
author = {Khasa, R and Ogden, SC and Wang, Y and Mou, Z and Metzler, AD and Xie, X and Dai, X and Tang, H},
title = {A single mutation in the PrM gene of Zika virus determines AXL dependency for infection of human neural cells.},
journal = {Journal of virology},
volume = {99},
number = {4},
pages = {e0187324},
doi = {10.1128/jvi.01873-24},
pmid = {40062839},
issn = {1098-5514},
support = {R01AI146342, U19 AI131130//National Institute of Allergy and Infectious Diseases/ ; R35GM151043/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Zika Virus/genetics/physiology ; *Receptor Protein-Tyrosine Kinases/genetics/metabolism ; *Proto-Oncogene Proteins/metabolism/genetics ; Axl Receptor Tyrosine Kinase ; Virus Internalization ; *Zika Virus Infection/virology/metabolism/genetics ; Mutation ; *Neurons/virology/metabolism ; *Viral Envelope Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Cell Line, Tumor ; Gene Knockout Techniques ; },
abstract = {Zika virus (ZIKV) is spread by mosquito bites and is unique among known flaviviruses for being able to cause microcephaly. Entry factors for ZIKV are incompletely understood, but phosphatidylserine (PS) receptors, including the TAM (Tyro3, AXL, and Mer) and TIM (T-cell Ig mucin) families, can serve as cofactors for flavivirus entry in a cell type-specific manner. We identify AXL as the top hit in a CRISPR/Cas9 genome-wide screen in human glioblastoma cells and establish a definitive role of AXL, but not TYRO3 or MerTK, for ZIKV infection. Additionally, Spondweni virus also shows AXL dependency, while dengue virus infection is not affected by AXL knockout. Passage of ZIKV in AXL knockout (KO) cells generated a mutant virus capable of infection via AXL-independent mechanisms, and multiple independent selections identified a common mutation, H83R, in the prM coding region of the ZIKV genome. The mutant virus exhibits an increased infectivity rate in AXL KO cells as compared to wild-type ZIKV and is dependent upon the single H83R mutation. The mutant virus' ability to infect cells in an AXL-independent manner is unrelated to interferon signaling antagonism but likely pertains to a change in virus maturation that leads to a structural disturbance of the ZIKV virion. Our study provides evidence for a potential mechanism linking the viral structural proteins and host PS receptor usage during flavivirus infection.IMPORTANCEA major challenge in elucidating the mechanism of Zika virus (ZIKV) pathogenesis is the multitude of cell types it infects with distinct requirements. The role of phosphatidylserine (PS) receptors in ZIKV infection is cell type-specific, and the controversy surrounds their function in flavivirus entry. Here, we establish a definitive requirement of AXL for infection of human glioblastoma cells by both Zika and Spondweni virus. We then identified a single amino acid mutation (H83R) in the prM protein of ZIKV that allowed AXL-independent infection of these cells. The H83R-mediated escape of AXL requirement is independent of interferon (IFN) signaling suppression by AXL; instead, the mutation has the potential to disrupt the virus assembly and virion structure. This study reveals a previously unknown connection between the PS receptor usage and the flavivirus prM gene, which can guide detailed molecular mechanism studies of the interplay between virion assembly and virus entry.},
}

Humans
*Zika Virus/genetics/physiology
*Receptor Protein-Tyrosine Kinases/genetics/metabolism
*Proto-Oncogene Proteins/metabolism/genetics
Axl Receptor Tyrosine Kinase
Virus Internalization
*Zika Virus Infection/virology/metabolism/genetics
Mutation
*Neurons/virology/metabolism
*Viral Envelope Proteins/genetics/metabolism
CRISPR-Cas Systems
Animals
Cell Line, Tumor
Gene Knockout Techniques

@article {pmid40029713,
year = {2025},
author = {Youngquist, BM and Mnguni, AT and Pungan, D and Lai, RP and Dai, G and Ng, CF and Samson, A and Abdelgaliel, Y and Lyon, CJ and Ning, B and Husain, S and Wasserman, S and Kolls, JK and Hu, TY},
title = {CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis.},
journal = {The Journal of clinical investigation},
volume = {135},
number = {8},
pages = {},
doi = {10.1172/JCI177241},
pmid = {40029713},
issn = {1558-8238},
mesh = {*Pneumonia, Pneumocystis/diagnosis/microbiology/genetics/blood ; Animals ; Humans ; Mice ; *Bronchoalveolar Lavage Fluid/microbiology ; *Oropharynx/microbiology ; *Pneumocystis carinii/genetics ; Female ; Male ; Infant ; *CRISPR-Cas Systems ; Adult ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA, Fungal/genetics/blood ; *Pneumocystis/genetics ; Mice, Knockout ; },
abstract = {BACKGROUNDPneumocystis jirovecii pneumonia (PCP) is a leading cause of fungal pneumonia, but its diagnosis primarily relies on invasive bronchoalveolar lavage (BAL) specimens that are difficult to obtain. Oropharyngeal swabs and serum could improve the PCP diagnostic workflow, and we hypothesized that CRISPR could enhance assay sensitivity to allow robust P. jirovecii diagnosis using swabs and serum. Herein, we describe the development of an ultrasensitive RT-PCR-coupled CRISPR assay with high active-infection specificity in infant swabs and adult BAL and serum.METHODSMouse analyses employed an RT-PCR CRISPR assay to analyze P. murina transcripts in WT and Rag2-/- mouse lung RNA, BAL, and serum at 2-, 4-, and 6-weeks after infection. Human studies used an optimized RT-PCR CRISPR assay to detect P. jirovecii transcripts in infant oropharyngeal swab samples, adult serum, and adult BAL specimens from patients who were infected with P. jirovecii and those who were not.RESULTSThe P. murina assays sensitively detected Pneumocystis RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay results identified infants infected with P. jirovecii with greater sensitivity (96.3% versus 66.7%) and specificity (100% versus 90.6%) than RT-qPCR compared with mitochondrial large subunit rRNA gene (mtLSU) standard marker, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% versus 26.7%) in adult serum specimens.CONCLUSIONSince swabs are routinely collected in pediatric patients with pneumonia and serum is easier to obtain than BAL, this assay approach could improve the accuracy and timing of pediatric and adult Pneumocystis diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.FUNDINGThe work was supported by the NIH (R01AI120033), NHLBI (R35HL139930), the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program, and by research funding provided by National Institute of Allergy and Infectious Diseases (NIAID) (R01AI144168, R01AI175618, R01AI173021). This research was also funded by the NIHR (project 134342) using UK aid from the UK government to support global health research.},
}

*Pneumonia, Pneumocystis/diagnosis/microbiology/genetics/blood
Animals
Humans
Mice
*Bronchoalveolar Lavage Fluid/microbiology
*Oropharynx/microbiology
*Pneumocystis carinii/genetics
Female
Male
Infant
*CRISPR-Cas Systems
Adult
*Clustered Regularly Interspaced Short Palindromic Repeats
*RNA, Fungal/genetics/blood
*Pneumocystis/genetics
Mice, Knockout

@article {pmid40217300,
year = {2025},
author = {Khalili-Tanha, G and Radisky, ES and Radisky, DC and Shoari, A},
title = {Matrix metalloproteinase-driven epithelial-mesenchymal transition: implications in health and disease.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {436},
pmid = {40217300},
issn = {1479-5876},
support = {R01CA237406/NH/NIH HHS/United States ; R01CA258274/NH/NIH HHS/United States ; R01 CA258274/CA/NCI NIH HHS/United States ; R01GM132100/NH/NIH HHS/United States ; R01HL157424/NH/NIH HHS/United States ; },
mesh = {*Epithelial-Mesenchymal Transition ; Humans ; *Matrix Metalloproteinases/metabolism ; Animals ; *Health ; *Disease ; Extracellular Matrix/metabolism ; Neoplasms/pathology ; },
abstract = {Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells, defined by apical-basal polarity and tight intercellular junctions, acquire migratory and invasive properties characteristic of mesenchymal cells. Under normal conditions, EMT directs essential morphogenetic events in embryogenesis and supports tissue repair. When dysregulated, EMT contributes to pathological processes such as organ fibrosis, chronic inflammation, and cancer progression and metastasis. Matrix metalloproteinases (MMPs)-a family of zinc-dependent proteases that degrade structural components of the extracellular matrix-sit at the nexus of this transition by dismantling basement membranes, activating pro-EMT signaling pathways, and cleaving adhesion molecules. When normally regulated, MMPs promote balanced ECM turnover and support the cyclical remodeling necessary for proper development, wound healing, and tissue homeostasis. When abnormally regulated, MMPs drive excessive ECM turnover, thereby promoting EMT-related pathologies, including tumor progression and fibrotic disease. This review provides an integrated overview of the molecular mechanisms by which MMPs both initiate and sustain EMT under physiological and disease conditions. It discusses how MMPs can potentiate EMT through TGF-β and Wnt/β-catenin signaling, disrupt cell-cell junction proteins, and potentiate the action of hypoxia-inducible factors in the tumor microenvironment. It discusses how these pathologic processes remodel tissues during fibrosis, and fuel cancer cell invasion, metastasis, and resistance to therapy. Finally, the review explores emerging therapeutic strategies that selectively target MMPs and EMT, ranging from CRISPR/Cas-mediated interventions to engineered tissue inhibitors of metalloproteinases (TIMPs), and demonstrates how such approaches may suppress pathological EMT without compromising its indispensable roles in normal biology.},
}

*Epithelial-Mesenchymal Transition
Humans
*Matrix Metalloproteinases/metabolism
Animals
*Health
*Disease
Extracellular Matrix/metabolism
Neoplasms/pathology