@article {pmid40298457,
year = {2025},
author = {Zhang, Y and Liu, J and Zhou, Y and Hu, X and Geng, B and Yang, S},
title = {Establishment of a RecET-Assisted CRISPR-Cas12a System for Large Deoxyribonucleic Acid-Fragment Manipulation in Zymomonas mobilis.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00863},
pmid = {40298457},
issn = {2161-5063},
abstract = {The lack of effective and efficient genome-editing toolkits for large DNA-fragment manipulation impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. Zymomonas mobilis is an important nonmodel polyploid industrial strain with excellent industrial characteristics. Although diverse CRISPR-Cas systems have been established in Z. mobilis for gene deletion, replacement, and ssDNA recombination, it is still challenging to achieve large DNA-fragment manipulation due to its low recombination and repair efficiencies for double-strand DNA breaks. In this study, a RecET-assisted CRISPR-Cas12a genome editing system was developed using a chromosome-borne cas12a and recET-encoded recombinase, as well as an all-in-one editing plasmid. Different promoters were used for recET and cas12a to determine optimal expression. The combination of PB-cas12a_Pt-recET had the highest efficiency of 97.92 ± 2.95% for 9-kb DNA-fragment deletion, which also had efficiencies about 100%, 80%, and 5%, respectively, for the deletion of 9-16, 20-25, and 30 kb DNA fragments. The RecET-assisted CRISPR-Cas12a was further applied for deletions of different large gene clusters and had the potential for efficient pathway knock-in. This study highlights the importance of the Cas12a nuclease expression levels and the combination of the RecET system in improving the double-strand DNA repair capability for large DNA-fragment manipulation in Z. mobilis. The RecET-assisted CRISPR-Cas12a system established in this study provides a versatile and powerful tool for large DNA-fragment manipulation in Z. mobilis, which is beneficial for functional genomic research, strain improvement, as well as the development of synthetic microbial chassis.},
}

@article {pmid40298107,
year = {2025},
author = {Sun, J and Yu, X and Tang, G and Chen, M and Zheng, Y and Hu, Y and Li, Q and Li, X and Li, N and Li, Z and Li, Y and Lu, N and Tan, W and Yang, Y and Lyu, X and Zhao, G and Wang, H and Dai, L and Zhao, GP and Ai, L and Zhao, W},
title = {A CRISPR-SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
pmid = {40298107},
issn = {1362-4962},
support = {2024YFA0917100//National Key R&D Program of China/ ; XDB0480000//Chinese Academy of Sciences/ ; 32025029//National Natural Science Foundation of China/ ; 2023A1515030069//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*Caulobacter crescentus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Bacterial ; Bacterial Proteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are wildly studied for their unique asymmetric life cycle and versatile applications in industry, agriculture, and biomedicine. However, genetic manipulation in these bacteria remains challenging, typically requiring time-consuming and labor-intensive procedures. Here, we report a practical CRISPR-SpCas9M-reporting system that overcomes the limitations of SpCas9 expression and CRISPR escape, enabling efficient, markerless, and rapid genome editing in C. crescentus. Two genes encoding for a pair of scaffold proteins were knocked out individually or iteratively, demonstrating their direct involvements in cellular signaling asymmetry. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized in the system, finally achieving the apparent editing efficiency up to 80% in C. crescentus. Furthermore, we applied the CRISPR-SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and general editing strategy. We anticipate that this system could be applied to other CRISPR-Cas-recalcitrant organisms, accelerating both basic and applied research in α-proteobacteria.},
}

*Caulobacter crescentus/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Genome, Bacterial
Bacterial Proteins/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40295218,
year = {2025},
author = {Jeong, SO and Kim, HJ and Lee, SJ},
title = {Adaptive Evolution of GatC, a Component of the Galactitol Phosphotransferase System, for Glucose Transport in Escherichia coli.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2502021},
doi = {10.4014/jmb.2502.02021},
pmid = {40295218},
issn = {1738-8872},
mesh = {*Escherichia coli/genetics/metabolism/enzymology ; *Glucose/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Phosphoenolpyruvate Sugar Phosphotransferase System/genetics/metabolism ; Directed Molecular Evolution ; Fermentation ; CRISPR-Cas Systems ; Biological Transport ; Mutation, Missense ; Metabolic Engineering ; Mutation ; Succinic Acid/metabolism ; },
abstract = {Microbial adaptive laboratory evolution is a powerful approach for uncovering novel gene functions within metabolic pathways. Building on our previous discovery of ExuT as a glucose transporter in ptsG-deficient Escherichia coli, this study investigates strains lacking recognized glucose transporters (ptsG, manX, and exuT). Successive rounds of experimental evolution revealed key genetic adaptations, including loss-of-function mutations in malI and nagC, which encode repressors of the maltose and N-acetylglucosamine phosphotransferase systems (PTS), respectively. Additionally, a gain-of-function mutation in gatC, a component of the galactitol PTS EIIC, was identified. The functional significance of these mutations was validated through transcript analysis, genetic knockouts, and CRISPR-Cas9-mediated site-specific genome mutagenesis, with a particular focus on the gatC missense mutation (F340C). The resulting modifications were examined for their effects on sugar specificity and metabolic flux. Furthermore, our findings identified succinate as the predominant fermentation product in engineered strains utilizing alternative glucose transport pathways, including the maltose, N-acetylglucosamine, and galactitol PTS. This study advances our understanding of sugar transport mechanisms in E. coli and offers insights into regulatory networks, fermentative metabolism, and substrate specificity, which can be leveraged for evolutionary engineering in biotechnological applications.},
}

*Escherichia coli/genetics/metabolism/enzymology
*Glucose/metabolism
*Escherichia coli Proteins/genetics/metabolism
*Phosphoenolpyruvate Sugar Phosphotransferase System/genetics/metabolism
Directed Molecular Evolution
Fermentation
CRISPR-Cas Systems
Biological Transport
Mutation, Missense
Metabolic Engineering
Mutation
Succinic Acid/metabolism

@article {pmid40295092,
year = {2025},
author = {Dipalo, LL and Mikkelsen, JG and Gijsbers, R and Carlon, MS},
title = {Trojan Horse-Like Vehicles for CRISPR-Cas Delivery: Engineering Extracellular Vesicles and Virus-Like Particles for Precision Gene Editing in Cystic Fibrosis.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2024.258},
pmid = {40295092},
issn = {1557-7422},
abstract = {The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.},
}

@article {pmid40293975,
year = {2025},
author = {Deragon, MA and Sharifi, HJ and LaRocca, TJ},
title = {Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {218},
pages = {},
doi = {10.3791/66077},
pmid = {40293975},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; U937 Cells ; *Gene Knockout Techniques/methods ; *Receptor-Interacting Protein Serine-Threonine Kinases/genetics ; *Nuclear Pore Complex Proteins/genetics ; },
abstract = {This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated guide RNA plasmids and lentiviral packaging plasmids to achieve RIP1 gene knockout. The protocol addresses challenges and improvements to traditional CRISPR methods, enabling its replication for future cell death studies. The resulting mutant cells can be used to investigate mechanistic changes in cell death, where functional RIP1 proteins would otherwise play a role. Viability assays demonstrated a significant reduction in cell death in knockout cells following necroptosis induction. Fluorescence microscopy revealed a marked decrease in mitochondrial reactive oxygen species (ROS) in knockout cells under the same conditions. Together, these functional assays confirm the loss of RIP1 protein. Optimized for use with U937 human monocytes, this procedure may also be adapted to target other key cell death regulators, yielding functional, non-lethal mutants. Potential pitfalls are addressed throughout to provide insights into challenges that may arise during mutant generation.},
}

Humans
*CRISPR-Cas Systems
U937 Cells
*Gene Knockout Techniques/methods
*Receptor-Interacting Protein Serine-Threonine Kinases/genetics
*Nuclear Pore Complex Proteins/genetics

@article {pmid40160040,
year = {2025},
author = {Cetin, B and Erendor, F and Eksi, YE and Sanlioglu, AD and Sanlioglu, S},
title = {Advancing CRISPR genome editing into gene therapy clinical trials: progress and future prospects.},
journal = {Expert reviews in molecular medicine},
volume = {27},
number = {},
pages = {e16},
doi = {10.1017/erm.2025.10},
pmid = {40160040},
issn = {1462-3994},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods/trends ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Anemia, Sickle Cell/therapy/genetics ; Animals ; beta-Thalassemia/therapy/genetics ; },
abstract = {Genome editing has recently evolved from a theoretical concept to a powerful and versatile set of tools. The discovery and implementation of CRISPR-Cas9 technology have propelled the field further into a new era. This RNA-guided system allows for specific modification of target genes, offering high accuracy and efficiency. Encouraging results are being announced in clinical trials employed in conditions like sickle cell disease (SCD) and transfusion-dependent beta-thalassaemia (TDT). The path finally led the way to the recent FDA approval of the first gene therapy drug utilising the CRISPR/Cas9 system to edit autologous CD34+ haematopoietic stem cells in SCD patients (Casgevy). Ongoing research explores the potential of CRISPR technology for cancer therapies, HIV treatment and other complex diseases. Despite its remarkable potential, CRISPR technology faces challenges such as off-target effects, suboptimal delivery systems, long-term safety concerns, scalability, ethical dilemmas and potential repercussions of genetic alterations, particularly in the case of germline editing. Here, we examine the transformative role of CRISPR technologies, including base editing and prime editing approaches, in modifying the genetic and epigenetic codes in the human genome and provide a comprehensive focus, particularly on relevant clinical applications, to unlock the full potential and challenges of gene editing.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods/trends
*CRISPR-Cas Systems
Clinical Trials as Topic
Anemia, Sickle Cell/therapy/genetics
Animals
beta-Thalassemia/therapy/genetics

@article {pmid40139379,
year = {2025},
author = {Shuo, Y and Feng, X and Zhao, C and Zhang, J and Wang, Y and Wang, J and Ding, Y and Shi, X and Li, J and Song, X and Wang, J},
title = {A dual enhancement strategy for ultrasensitive detection of norovirus based on catalytic hairpin assembly-assisted CRISPR/Cas12a sensing system.},
journal = {Journal of dairy science},
volume = {108},
number = {5},
pages = {4726-4733},
doi = {10.3168/jds.2024-26150},
pmid = {40139379},
issn = {1525-3198},
mesh = {*Norovirus/isolation & purification ; Animals ; Milk/virology ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Noroviruses are a leading cause of food-borne illnesses, responsible for over 50% of global gastroenteritis outbreaks. Whereas reverse transcriptase PCR (RT-PCR) and other nucleic acid amplification methods are crucial for norovirus detection, their reliance on specialized equipment highlights the urgent need for more accessible detection methods. Herein, we propose an isothermal cascade signal amplification assay that integrates catalytic hairpin assembly (CHA) and cluster regularly interspaced short palindromic repeats (CRISPR)/Cas12a for rapid and accurate detection of norovirus in food samples. By incorporating the advantages of CHA and CRISPR/Cas12a, the dual signal enhancement sensing strategy can achieve high sensitivity low to 14 fM within 70 min, and good specificity in adenovirus, human enterovirus, rotavirus, and other interfering agents. The proposed dual enhancement strategy for norovirus detection has satisfactory accuracy and acceptable recoveries in milk samples compared with RT-PCR assay, and holds promise for improving food safety monitoring, particularly in dairy products.},
}

*Norovirus/isolation & purification
Animals
Milk/virology
CRISPR-Cas Systems
Nucleic Acid Amplification Techniques/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40043602,
year = {2025},
author = {Liu, G and Gong, H and Tang, H and Meng, Z and Wang, Z and Cui, W and Zhang, K and Chen, Y and Yang, Y},
title = {Enhanced lignocellulose degradation in Bacillus subtilis RLI2019 through CRISPR/Cas9-mediated chromosomal integration of ternary cellulase genes.},
journal = {International journal of biological macromolecules},
volume = {306},
number = {Pt 3},
pages = {141727},
doi = {10.1016/j.ijbiomac.2025.141727},
pmid = {40043602},
issn = {1879-0003},
mesh = {*Bacillus subtilis/genetics/metabolism/enzymology ; *Lignin/metabolism ; *CRISPR-Cas Systems/genetics ; *Cellulase/genetics/metabolism ; Gene Editing ; Biomass ; *Chromosomes, Bacterial/genetics ; },
abstract = {Bacillus subtilis (B. subtilis) is a crucial industrial microorganism for lignocellulose biomass degradation. However, wild-type strains from natural environments have inherent deficiencies in the composition of cellulase genes, so constructing recombinant strains through genome engineering is a generalizable strategy to overcome these shortcomings. Herein, eglS, cel48S, and bglS were integrated into the aprE, epr, and amyE loci of the B. subtilis RLI2019 chromosome, respectively, through CRISPR/Cas9-mediated genome editing, deriving the engineered strain B. subtilis AEA3. The activities of endoglucanase, exoglucanase, β-glucosidase, xylanase, and total cellulase in B. subtilis AEA3 were enhanced by 3.1-fold, 6.6-fold, 3.0-fold, 1.2-fold, and 1.8-fold, respectively, reaching 26.31 U/mL, 9.77 U/mL, 3.91 U/mL, 19.63 U/mL, and 2.42 U/mL. Notably, the engineered strain improved the saccharification efficiency of crop straws, effectively disrupting fiber structure, and significantly reducing the content of neutral and acid detergent fibers, lignocellulose and hemicellulose. In summary, this study provides a general strategy for enhancing the cellulose degradation capabilities of B. subtilis through comprehensive and systematic multi-module genetic engineering, broadening its potential application in lignocellulose biomass conversion.},
}

*Bacillus subtilis/genetics/metabolism/enzymology
*Lignin/metabolism
*CRISPR-Cas Systems/genetics
*Cellulase/genetics/metabolism
Gene Editing
Biomass
*Chromosomes, Bacterial/genetics

@article {pmid39846250,
year = {2025},
author = {Adjumain, S and Daniel, P and Sun, CX and Bradshaw, G and Chew, NJ and Tsui, V and Lee, H and Loi, M and Zhukova, N and Habarakada, D and Yoel, A and Vaghjiani, VG and Game, S and Ludlow, LE and Neeman, N and Sweet-Cordero, EA and Eisenstat, DD and Cain, JE and Firestein, R},
title = {Multidimensional, integrative profiling identifies BCL2L1 methylation as a predictor of MCL1 dependency in pediatric malignancies.},
journal = {JCI insight},
volume = {10},
number = {2},
pages = {},
pmid = {39846250},
issn = {2379-3708},
mesh = {Humans ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists & inhibitors ; *DNA Methylation ; Cell Line, Tumor ; Child ; *Glioma/genetics/pathology/drug therapy/metabolism ; *Brain Neoplasms/genetics/pathology/drug therapy/metabolism ; *bcl-X Protein/genetics/metabolism ; Mice ; Gene Expression Regulation, Neoplastic ; Animals ; CRISPR-Cas Systems ; Apoptosis/genetics ; Female ; },
abstract = {Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 (MCL1) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.},
}

Humans
*Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists & inhibitors
*DNA Methylation
Cell Line, Tumor
Child
*Glioma/genetics/pathology/drug therapy/metabolism
*Brain Neoplasms/genetics/pathology/drug therapy/metabolism
*bcl-X Protein/genetics/metabolism
Mice
Gene Expression Regulation, Neoplastic
Animals
CRISPR-Cas Systems
Apoptosis/genetics
Female

@article {pmid39846200,
year = {2025},
author = {Zheng, X and Huang, CH and Yan, S and Rong, MD},
title = {Advances and applications of genome-edited animal models for severe combined immunodeficiency.},
journal = {Zoological research},
volume = {46},
number = {1},
pages = {249-260},
pmid = {39846200},
issn = {2095-8137},
mesh = {Animals ; *Severe Combined Immunodeficiency/genetics/therapy ; *Gene Editing/methods ; *Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Severe combined immunodeficiency disease (SCID), characterized by profound immune system dysfunction, can lead to life-threatening infections and death. Animal models play a pivotal role in elucidating biological processes and advancing therapeutic strategies. Recent advances in gene-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editing, have significantly enhanced the generation of SCID models. These models have not only deepened our understanding of disease pathophysiology but have also driven progress in cancer therapy, stem cell transplantation, organ transplantation, and infectious disease management. This review provides a comprehensive overview of current SCID models generated using novel gene-editing approaches, highlighting their potential applications in translational medicine and their role in advancing biomedical research.},
}

Animals
*Severe Combined Immunodeficiency/genetics/therapy
*Gene Editing/methods
*Disease Models, Animal
CRISPR-Cas Systems

@article {pmid39837195,
year = {2025},
author = {Boismier, EC and Aboulnaga, EA and TerAvest, MA},
title = {Zymomonas mobilis: bringing an ancient human tool into the genomic era.},
journal = {Current opinion in biotechnology},
volume = {92},
number = {},
pages = {103257},
doi = {10.1016/j.copbio.2025.103257},
pmid = {39837195},
issn = {1879-0429},
mesh = {*Zymomonas/genetics/metabolism ; *Metabolic Engineering/methods ; Gene Editing ; Biofuels ; Ethanol/metabolism ; Humans ; Genomics ; CRISPR-Cas Systems ; Genome, Bacterial ; },
abstract = {Zymomonas mobilis is an ethanologenic bacterium that has been used for over 1500 years to produce alcoholic beverages. Recently, this microbe has become a top candidate for biofuel production due to its efficient metabolism. Z. mobilis is being developed to utilize lignocellulosic biomass as a feedstock and synthesize a range of valuable chemicals and fuels. Genetic and metabolic engineering strategies are crucial to reach these goals. Recent advances include genome engineering, CRISPR editing, and CRISPRi knockdown of genes. Metabolic engineering has enabled redirection of carbon from the natural product ethanol to chemicals such as 2,3-butanediol and polyhydroxybutyrate. The approaches summarized here will streamline the development of Z. mobilis as an industrial chassis for sustainable liquid fuels and chemicals.},
}

*Zymomonas/genetics/metabolism
*Metabolic Engineering/methods
Gene Editing
Biofuels
Ethanol/metabolism
Humans
Genomics
CRISPR-Cas Systems
Genome, Bacterial

@article {pmid39692298,
year = {2025},
author = {Walker, RL and Ferguson, Z and Mitchell, L and Waltz, M},
title = {Enhancing Animals is "Still Genetics": Perspectives of Genome Scientists and Policymakers on Animal and Human Enhancement.},
journal = {AJOB empirical bioethics},
volume = {16},
number = {2},
pages = {94-102},
doi = {10.1080/23294515.2024.2441688},
pmid = {39692298},
issn = {2329-4523},
mesh = {Humans ; *Gene Editing/ethics ; Animals ; Animal Welfare/ethics ; Policy Making ; CRISPR-Cas Systems ; Research Personnel ; Animals, Genetically Modified ; Genome ; },
abstract = {BACKGROUND: Nonhuman animals are regularly enhanced genomically with CRISPR and other gene editing tools as scientists aim at better models for biomedical research, more tractable agricultural animals, or animals that are otherwise well suited to a defined purpose. This study investigated how genome editors and policymakers perceived ethical or policy benefits and drawbacks for animal enhancement and how perceived benefits and drawbacks are alike, or differ from, those for human genome editing.

METHODS: We identified scientists through relevant literature searches as well as conference presentations. Policymakers were identified through rosters of genome editing oversight groups (e.g., International Commission on the Clinical Use of Human Germline Genome Editing, World Health Organization) or efforts aimed at influencing policy (e.g., deliberative democracy groups). Interviews covered participants' views on ethical differences between interventions affecting somatic or germline cells and distinctions between using gene editing for disease treatment, prevention, and enhancement purposes.

RESULTS: Of the 92 participants interviewed, 81 were genome editing scientists, and 33 were policymakers, with 22 interviewees being both scientists and policymakers. Multiple areas were identified in which the ethical implications of genomic enhancements for nonhuman animals differ from those for human animals including with respect to experiential welfare; germline edits; environmental sustainability; and justice.

CONCLUSIONS: Overall, respondents viewed that animal enhancement is unburdened by the ethical complexities of human enhancement. These views may be related to participant perceptions of animals' lesser moral status and because germline editing in animals is common practice.},
}

Humans
*Gene Editing/ethics
Animals
Animal Welfare/ethics
Policy Making
CRISPR-Cas Systems
Research Personnel
Animals, Genetically Modified
Genome

@article {pmid39363579,
year = {2025},
author = {Ying, X and Ying, Z and Gao, X and Wang, Y and Lv, X},
title = {CRISPR-mediated WNK4 point mutation aggravates tumor progression and weakens chemotherapy sensitivity in gastric cancer.},
journal = {Histology and histopathology},
volume = {40},
number = {5},
pages = {711-720},
pmid = {39363579},
issn = {1699-5848},
mesh = {*Stomach Neoplasms/genetics/pathology/drug therapy/enzymology ; Animals ; Humans ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; Mice ; *Point Mutation ; *Drug Resistance, Neoplasm/genetics ; Apoptosis/drug effects ; CRISPR-Cas Systems ; STAT3 Transcription Factor/metabolism ; Cisplatin/pharmacology ; Disease Progression ; Mice, Nude ; Fluorouracil/pharmacology ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology ; Xenograft Model Antitumor Assays ; Male ; },
abstract = {OBJECTIVE: Gastric cancer (GC) is the fifth most common malignancy, the molecular targets of which have been increasingly explored in recent years. As a serine/threonine protein kinase, the role of WNK lysine deficient protein kinase 4 (WNK4) in GC was clarified in this study.

METHODS: Human GC lines AGS and MKN45 were stably transfected with a WNK4 mutant constructed by the CRISPR/Cas9 method and treated with cis-dichlorodiammine platinum (CDDP, 2 μg/mL) and 5-fluorouracil (5-FU, 5 μg/mL) for 48h. Tumor-bearing mice were established with 5×10[6] mutant-type AGS cells, and injected with 40 mg/kg WP1066, the inhibitor of signal transducer and activator of transcription 3 (STAT3), for 21 days. Cell malignant potential and tumor growth were assessed. STAT3 activation was identified by western blot and immunohistochemistry. The interaction between WNK4 and STAT3 was determined using co-immunoprecipitation and immunofluorescence co-localization.

RESULTS: WNK4 mutation promoted proliferation and invasion, and upregulated the p-STAT3/STAT3 value in GC cells with/without 5-FU and CDDP treatments, while inhibiting apoptosis of GC cells without drug treatment. In tumor-bearing mice, WNK4 mutation accelerated tumor growth, increased levels of p-STAT3, STAT3, and p-STAT3/STAT3, and strengthened the co-immunoprecipitation and co-localizing with STAT3; however, these effects were reversed by WP1066 treatment.

CONCLUSION: Through activating STAT3, WNK4 mutation impacts both the natural and drug-treated growth of GC cells or tumors, suggesting a new avenue for preclinical research.},
}

*Stomach Neoplasms/genetics/pathology/drug therapy/enzymology
Animals
Humans
Cell Line, Tumor
*Protein Serine-Threonine Kinases/genetics/metabolism
Cell Proliferation/drug effects/genetics
Mice
*Point Mutation
*Drug Resistance, Neoplasm/genetics
Apoptosis/drug effects
CRISPR-Cas Systems
STAT3 Transcription Factor/metabolism
Cisplatin/pharmacology
Disease Progression
Mice, Nude
Fluorouracil/pharmacology
Mice, Inbred BALB C
Antineoplastic Agents/pharmacology
Xenograft Model Antitumor Assays
Male

@article {pmid40292247,
year = {2025},
author = {Suthar, MK and Mittal, MK},
title = {Genome-wide identification and characterization of dicer-like genes in Andrographis paniculata (Burm. f.) Wall. ex Nees and their expression response to methyl jasmonate elicitation.},
journal = {3 Biotech},
volume = {15},
number = {5},
pages = {141},
pmid = {40292247},
issn = {2190-572X},
abstract = {Andrographis paniculata, commonly known as the "King of Bitters," is a medicinal plant valued for its bioactive diterpenoids, particularly andrographolides. Dicer-like (DCL) proteins are central to the miRNA pathway, processing precursor miRNAs into mature miRNAs that regulate gene expression. While miRNAs influence plant metabolism, their role in secondary metabolite biosynthesis in A. paniculata remains unexplored. This study identified and characterized five ApDCL genes, mapped to distinct chromosomes in A. paniculata. The structural analysis revealed that ApDCL3 contained the highest number of introns (24), whereas ApDCL2 had the fewest (10). Conserved RNA-processing domains were confirmed, and phylogenetic analysis revealed evolutionary conservation, showing that ApDCLs are closely related to the DCLs of Sesamum indicum and Salvia miltiorrhiza. Expression analysis showed ApDCL1 and ApDCL2 were predominantly expressed in roots, whereas ApDCL3, ApDCL4a, and ApDCL4b were more abundant in leaves. Methyl jasmonate treatment upregulated ApDCL3 (~ 3.5-fold) and ApDCL4a (~ 1.5-fold), but further research is needed to determine whether this response directly influences secondary metabolism or results from MeJ-induced stress. These findings provide a foundation for functional validation through gene knockdown, overexpression, and CRISPR-Cas-based genome editing to elucidate DCL-mediated regulatory mechanisms. Future research leveraging these insights could aid in modulating RNA silencing pathways to enhance the biosynthesis of pharmacologically significant metabolites in A. paniculata through biotechnological interventions.},
}

@article {pmid40288310,
year = {2025},
author = {Qian, J and Zhang, B and Liu, C and Xue, Y and Zhou, H and Huang, L and Zheng, S and Chen, M and Fu, YQ},
title = {Reconfigurable acoustic tweezer for precise tracking and in-situ sensing of trace miRNAs in tumor cells.},
journal = {Biosensors & bioelectronics},
volume = {282},
number = {},
pages = {117505},
doi = {10.1016/j.bios.2025.117505},
pmid = {40288310},
issn = {1873-4235},
abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis and monitoring. However, their isolation from clinical samples typically yields only trace amounts, significantly limiting the sensitivity and efficiency of cancer detection. To address this challenge, we present a octangular reconfigurable acoustic tweezer (ORAT) as an integrated platform for precise tumor cell tracking and in-situ detection of trace miRNAs. By simultaneously modulating multidirectional acoustic signals and parameters, the ORAT dynamically reshapes the acoustic field, enabling precise control over manipulation areas, particle spacing, array angles, distribution patterns, and node rotation. This device allows selective particle manipulation across entire regions or specific areas through adaptive adjustments of the microchamber boundary. Notably, the ORAT achieves rapid and accurate localization and labeling of rare tumor cells within a large population of normal cells. Furthermore, it enhances the sensitivity of CRISPR/Cas-based miRNA detection in digital microdroplets by three orders of magnitude, if compared to that of the conventional tube-based method. With its versatile capabilities, the ORAT holds remarkable promise for advancing nucleic acid analysis in a wide range of cancers and related diseases.},
}

@article {pmid40014690,
year = {2025},
author = {Faure, G and Saito, M and Wilkinson, ME and Quinones-Olvera, N and Xu, P and Flam-Shepherd, D and Kim, S and Reddy, N and Zhu, S and Evgeniou, L and Koonin, EV and Macrae, RK and Zhang, F},
title = {TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6746},
pages = {eadv9789},
doi = {10.1126/science.adv9789},
pmid = {40014690},
issn = {1095-9203},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *Bacteriophages/genetics ; Protein Domains ; *DNA/metabolism ; *CRISPR-Cas Systems ; Bacterial Proteins/chemistry/genetics/metabolism ; },
abstract = {RNA-guided systems provide remarkable versatility, enabling diverse biological functions. Through iterative structural and sequence homology-based mining starting with a guide RNA-interaction domain of Cas9, we identified a family of RNA-guided DNA-targeting proteins in phage and parasitic bacteria. Each system consists of a tandem interspaced guide RNA (TIGR) array and a TIGR-associated (Tas) protein containing a nucleolar protein (Nop) domain, sometimes fused to HNH (TasH)- or RuvC (TasR)-nuclease domains. We show that TIGR arrays are processed into 36-nucleotide RNAs (tigRNAs) that direct sequence-specific DNA binding through a tandem-spacer targeting mechanism. TasR can be reprogrammed for precise DNA cleavage, including in human cells. The structure of TasR reveals striking similarities to box C/D small nucleolar ribonucleoproteins and IS110 RNA-guided transposases, providing insights into the evolution of diverse RNA-guided systems.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism
*Bacteriophages/genetics
Protein Domains
*DNA/metabolism
*CRISPR-Cas Systems
Bacterial Proteins/chemistry/genetics/metabolism

@article {pmid39824180,
year = {2025},
author = {Gui, L and Chen, K and Yan, J and Chen, P and Gao, WQ and Ma, B},
title = {Targeting the mevalonate pathway potentiates NUAK1 inhibition-induced immunogenic cell death and antitumor immunity.},
journal = {Cell reports. Medicine},
volume = {6},
number = {2},
pages = {101913},
pmid = {39824180},
issn = {2666-3791},
mesh = {*Mevalonic Acid/metabolism ; Humans ; Animals ; Reactive Oxygen Species/metabolism ; *Immunogenic Cell Death/drug effects ; Mice ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/antagonists & inhibitors/metabolism/genetics ; Endoplasmic Reticulum Stress/drug effects ; Cholesterol/metabolism ; Simvastatin/pharmacology ; *Neoplasms/immunology/drug therapy ; CRISPR-Cas Systems ; Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology ; },
abstract = {The induction of immunogenic cell death (ICD) impedes tumor progression via both tumor cell-intrinsic and -extrinsic mechanisms, representing a robust therapeutic strategy. However, ICD-targeted therapy remains to be explored and optimized. Through kinome-wide CRISPR-Cas9 screen, NUAK family SNF1-like kinase 1 (NUAK1) is identified as a potential target. The ICD-provoking effect of NUAK1 inhibition depends on the production of reactive oxygen species (ROS), consequent to the downregulation of nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant gene expression. Moreover, the mevalonate pathway/cholesterol biosynthesis, activated by spliced form of X-box binding protein 1 (XBP1s) downstream of ICD-induced endoplasmic reticulum (ER) stress, functions as a negative feedback mechanism. Targeting the mevalonate pathway with CRISPR knockout or the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor simvastatin amplifies NUAK1 inhibition-mediated ICD and antitumor activity, while cholesterol dampens ROS and ICD, and therefore also dampens tumor suppression. The combination of NUAK1 inhibitor and statin enhances the efficacy of anti-PD-1 therapy. Collectively, our study unveils the promise of blocking the mevalonate-cholesterol pathway in conjunction with ICD-targeted immunotherapy.},
}

*Mevalonic Acid/metabolism
Humans
Animals
Reactive Oxygen Species/metabolism
*Immunogenic Cell Death/drug effects
Mice
Cell Line, Tumor
*Protein Serine-Threonine Kinases/antagonists & inhibitors/metabolism/genetics
Endoplasmic Reticulum Stress/drug effects
Cholesterol/metabolism
Simvastatin/pharmacology
*Neoplasms/immunology/drug therapy
CRISPR-Cas Systems
Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology

@article {pmid39814710,
year = {2025},
author = {Neugebauer, E and Walter, S and Tan, J and Drayman, N and Franke, V and van Gent, M and Pennisi, S and Veratti, P and Stein, KS and Welker, I and Tay, S and Verjans, GMGM and Timmers, HTM and Akalin, A and Landthaler, M and Ensser, A and Wyler, E and Full, F},
title = {Herpesviruses mimic zygotic genome activation to promote viral replication.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {710},
pmid = {39814710},
issn = {2041-1723},
support = {En 423/5-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; DP2 AI154437/AI/NIAID NIH HHS/United States ; 01KI2017//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; FOR5200 DEEP-DV, 443644894//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; AI154437//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 GM127527/GM/NIGMS NIH HHS/United States ; },
mesh = {*Virus Replication/genetics ; Humans ; Genome, Viral/genetics ; *Herpesvirus 1, Human/physiology/genetics ; *Homeodomain Proteins/metabolism/genetics ; *Zygote/metabolism/virology ; Gene Expression Regulation, Viral ; *Herpesviridae/physiology/genetics ; Animals ; CRISPR-Cas Systems ; Transcription, Genetic ; HEK293 Cells ; },
abstract = {Zygotic genome activation (ZGA) is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In humans, ZGA is induced by DUX4, a pioneer factor that drives expression of downstream germline-specific genes and retroelements. Here we show that herpesviruses from all subfamilies, papillomaviruses and Merkel cell polyomavirus actively induce DUX4 expression to promote viral transcription and replication. Analysis of single-cell sequencing data sets from patients shows that viral DUX4 activation is of relevance in vivo. Herpes-simplex virus 1 (HSV-1) immediate early proteins directly induce expression of DUX4 and its target genes, which mimics zygotic genome activation. Upon HSV-1 infection, DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for infection, since genetic depletion by CRISPR/Cas9 as well as degradation of DUX4 by nanobody constructs abrogates HSV-1 replication. Our results show that DNA viruses including herpesviruses mimic an embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.},
}

*Virus Replication/genetics
Humans
Genome, Viral/genetics
*Herpesvirus 1, Human/physiology/genetics
*Homeodomain Proteins/metabolism/genetics
*Zygote/metabolism/virology
Gene Expression Regulation, Viral
*Herpesviridae/physiology/genetics
Animals
CRISPR-Cas Systems
Transcription, Genetic
HEK293 Cells

@article {pmid40285901,
year = {2025},
author = {Lin, W and Huang, M and Fu, H and Yu, L and Chen, Y and Chen, L and Lin, Y and Wen, T and Luo, X and Cong, Y},
title = {An EXPAR-CRISPR/Cas12a Assay for Rapid Detection of Salmonella.},
journal = {Current microbiology},
volume = {82},
number = {6},
pages = {262},
pmid = {40285901},
issn = {1432-0991},
support = {20231800500552//Dongguan Science and Technology Commissioner Project/ ; 81974299//National Natural Science Foundation of China/ ; 2023A1515010938//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
mesh = {*Salmonella/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; Salmonella Infections/diagnosis/microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Salmonella is considered as one of the primary pathogens associated with foodborne diseases globally. The effective treatment of these illnesses depends on the rapid and accurate identification of this organism. Traditional culture methods, however, necessitate extended testing periods, while many alternative techniques often lack precision. This research presents an innovative detection system that employs CRISPR-Cas12a for the detection of Salmonella. The detection system specifically targets the yfiR gene, which is amplified through isothermal exponential amplification (EXPAR). Target DNA hybridizes with the hairpin probe to form the DNA strand. The DNA strand was nicked to generate a nick by nicking endonuclease owing to its recognition sequence toward the hairpin probe. DNA polymerase can extend the 3'-end of the nicked site, which simultaneously displaces the newly synthesized strand. Thus, a large number of single-stranded DNA (ssDNA) were produced in the circle of nicking, polymerization, and strand displacement to achieve exponential amplification. The resultant amplified ssDNA products are subsequently recognized by CRISPR/Cas12a, resulting in the emission of a fluorescence signal. The detection system demonstrates a limit of detection of 10 fM for synthetic DNA and exhibits a strong linear relationship between 10 fM and 100 nM. Furthermore, the EXPAR-CRISPR/Cas12a detection system successfully identifies extracted genomic DNA samples containing Salmonella strains less than one hour, achieving a detection threshold of 1 pg/μL. This assay not only offers rapid results, requiring less than one hour for sample-to-answer outcomes, but is also cost-effective, minimizes aerosol risks, and provides exceptional specificity and sensitivity for the detection of Salmonella.},
}

*Salmonella/genetics/isolation & purification
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Bacterial Proteins/genetics
Sensitivity and Specificity
DNA, Bacterial/genetics
Salmonella Infections/diagnosis/microbiology
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40285354,
year = {2025},
author = {John, T and Czechowicz, A},
title = {Clinical Hematopoietic Stem Cell-Based Gene Therapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.04.029},
pmid = {40285354},
issn = {1525-0024},
abstract = {Hematopoietic stem cell (HSC)-based gene therapies have seen extraordinary progress since their initial conception, now fundamentally transforming the treatment paradigms for various inherited hematologic, immunologic and metabolic conditions - with additional use cases under exploration. Decades worth of work with advances in viral vector technologies and cell manufacturing have paved the way for HSC gene therapy with marked improvement in the safety and efficiency of gene delivery into HSCs. These have been augmented by the recent rise of innovative genome editing techniques, particularly using clustered regularly interspaced short palindromic repeats - CRISPR-associated proteins (CRISPR-Cas)-based technologies, which have enabled more precise and reproducible genome alterations in HSCs and fostered opportunities for targeted gene modification or gene correction. These breakthroughs have led to the development of many active clinical trials and culminated in the recent federal regulatory agency approvals of multiple clinical HSC gene therapies for various indications that are now becoming available across different geographies. These treatments aim to offer significant, long-lasting benefits to patients world-wide without the toxicities of alternative treatment approaches. This review explores the history and advancements in HSC gene therapies and provides a comprehensive overview of the latest clinical innovations and cell therapy products. Further, it concludes with a discussion of the persistent challenges that have limited adoption and potential future opportunities that aspire to enable curative treatment of many different patients through such personalized medicines.},
}

@article {pmid40284926,
year = {2025},
author = {Yin, J and Cui, J and Zheng, H and Guo, T and Wei, R and Sha, Z and Gu, S and Ni, B},
title = {Implementation of RT-RAA and CRISPR/Cas13a for an NiV Point-of-Care Test: A Promising Tool for Disease Control.},
journal = {Viruses},
volume = {17},
number = {4},
pages = {},
pmid = {40284926},
issn = {1999-4915},
support = {2021YFD1800301-04//National Key R&D Program for the 14th Five-Year Plan/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Nipah Virus/genetics/isolation & purification ; *Point-of-Care Testing ; *Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; },
abstract = {Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks.},
}

Animals
Swine
*CRISPR-Cas Systems
*Swine Diseases/diagnosis/virology
Sensitivity and Specificity
*Nipah Virus/genetics/isolation & purification
*Point-of-Care Testing
*Nucleic Acid Amplification Techniques/methods
Point-of-Care Systems

@article {pmid40284749,
year = {2025},
author = {Hu, M and Chua, SL},
title = {Antibiotic-Resistant Pseudomonas aeruginosa: Current Challenges and Emerging Alternative Therapies.},
journal = {Microorganisms},
volume = {13},
number = {4},
pages = {},
pmid = {40284749},
issn = {2076-2607},
abstract = {Antibiotic-resistant Pseudomonas aeruginosa is a pathogen notorious for its resilience in clinical settings due to biofilm formation, efflux pumps, and the rapid acquisition of resistance genes. With traditional antibiotic therapy rendered ineffective against Pseudomonas aeruginosa infections, we explore alternative therapies that have shown promise, including antimicrobial peptides, nanoparticles and quorum sensing inhibitors. While these approaches offer potential, they each face challenges, such as specificity, stability, and delivery, which require careful consideration and further study. We also delve into emerging alternative strategies, such as bacteriophage therapy and CRISPR-Cas gene editing that could enhance targeted treatment for personalized medicine. As most of them are currently in experimental stages, we highlight the need for clinical trials and additional research to confirm their feasibility. Hence, we offer insights into new therapeutic avenues that could help address the pressing issue of antibiotic-resistant Pseudomonas aeruginosa, with an eye toward practical applications in future healthcare.},
}

@article {pmid40282900,
year = {2025},
author = {Ganesh, I and Karthiga, I and Murugan, M and Rangarajalu, K and Ballambattu, VB and Ravikumar, S},
title = {CRISPR/Cas-Based Prenatal Screening for Aneuploidy: Challenges and Opportunities for Early Diagnosis.},
journal = {Medicina (Kaunas, Lithuania)},
volume = {61},
number = {4},
pages = {},
pmid = {40282900},
issn = {1648-9144},
mesh = {Humans ; Female ; Pregnancy ; *Aneuploidy ; *Prenatal Diagnosis/methods/trends ; *CRISPR-Cas Systems/genetics ; Early Diagnosis ; Pregnancy Trimester, First ; Genetic Testing/methods ; },
abstract = {Aneuploidy is increasingly recognized globally as a common cause of miscarriage among expectant mothers. The existing prenatal screening techniques for detecting aneuploidy have several limitations. The ability to diagnose aneuploidy early in a non-invasive manner is not feasible with the current screening methods, as they may produce false positive or false negative results. Recently, the widely used gene editing tool CRISPR/Cas has shown great promise in diagnostics. This review summarizes the prenatal screening tests used in the first trimester to assess aneuploidy conditions. Additionally, we discuss the advantages and disadvantages of molecular diagnostic tests, including the benefits and challenges of CRISPR/Cas-based trisomy detection. Thus, the proposed prenatal screening using CRISPR/Cas could provide significant benefits to expectant mothers by potentially enabling the early diagnosis of trisomy, helping to prevent miscarriage and birth defects. Furthermore, it opens new avenues for research, allowing clinicians and researchers to develop, optimize, and implement CRISPR/Cas-based prenatal screening assays in the future.},
}

Humans
Female
Pregnancy
*Aneuploidy
*Prenatal Diagnosis/methods/trends
*CRISPR-Cas Systems/genetics
Early Diagnosis
Pregnancy Trimester, First
Genetic Testing/methods

@article {pmid40282347,
year = {2025},
author = {Zheng, Y and Zou, Q and Li, J and Yang, Y},
title = {CRISPR-MFH: A Lightweight Hybrid Deep Learning Framework with Multi-Feature Encoding for Improved CRISPR-Cas9 Off-Target Prediction.},
journal = {Genes},
volume = {16},
number = {4},
pages = {},
pmid = {40282347},
issn = {2073-4425},
support = {32471860//National Natural Science Foundation of China/ ; Y202352570//scientific research project of the education department of Zhejiang province/ ; },
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {BACKGROUND: The CRISPR-Cas9 system has emerged as one of the most promising gene-editing technologies in biology. However, off-target effects remain a significant challenge. While recent advances in deep learning have led to the development of models for off-target prediction, these models often fail to fully leverage sequence pair information. Furthermore, as the models' parameter sizes increase, so do their complexities, limiting their practical applicability.

METHODS: In this study, we introduce a novel multi-feature independent encoding method, which encodes the gRNA-DNA sequence pair into three distinct feature matrices to minimize information loss. Additionally, we propose a lightweight hybrid deep learning framework, CRISPR-MFH, that integrates multi-scale separable convolutions and hybrid attention mechanisms for efficient and accurate off-target prediction.

RESULTS: Extensive experiments across multiple benchmark datasets demonstrate that the proposed encoding method effectively captures critical features and that CRISPR-MFH outperforms or matches state-of-the-art models with significantly fewer parameters across multiple evaluation metrics.

CONCLUSIONS: This study offers a novel perspective for advancing deep learning technology in the realm of CRISPR-Cas9 off-target detection.},
}

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

@article {pmid40282329,
year = {2025},
author = {Rathe, SK and Marko, TA and Edwards, EN and Ridder, PH and Varshney, J and Williams, KB and Johnson, JE and Moriarity, BS and Largaespada, DA},
title = {Techniques for Validating CRISPR Changes Using RNA-Sequencing Data.},
journal = {Genes},
volume = {16},
number = {4},
pages = {},
pmid = {40282329},
issn = {2073-4425},
support = {MSTP grant T32 GM008224/NH/NIH HHS/United States ; R01-CA113636/NH/NIH HHS/United States ; #123939//ACS Research Professorship/ ; NA//Zach Sobiech Osteosarcoma Fund of the Children's Cancer Research Fund, Minneapolis MN/ ; NA//Children's Cancer Research Fund Emerging Scientist Award/ ; NA//Children's Tumor Foundation Young Investigator Award/ ; 5T32CA009138-40//Children's Tumor Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Sequence Analysis, RNA/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptome/genetics ; *RNA-Seq/methods ; Gene Editing/methods ; Gene Knockout Techniques/methods ; },
abstract = {UNLABELLED: The use of CRISPR to knockdown or knockout genes is a powerful tool for understanding the specific role of a gene in disease development. However, it can cause many unanticipated changes to the transcriptome that are not detected by DNA amplification and Sanger sequencing of the target site. Various RNA-sequencing techniques can be used to identify these changes and effectively gauge the full impact of the CRISPR knockout, thereby providing a means of selecting appropriate clones for further experimentation.

BACKGROUND/OBJECTIVES: RNA-seq data from 4 CRISPR knockout experiments were analyzed and techniques developed to both confirm the success of the CRISPR modifications and identify potential issues.

METHODS: A broad-based analysis of RNA-sequencing data identified many CRISPR-based changes not identified by PCR amplification of DNA around the CRISPR target site. These changes included an inter-chromosomal fusion event, exon skipping, chromosomal truncation, and the unintentional transcriptional modification and amplification of a neighboring gene.

CONCLUSIONS: The inadvertent modifications identified by the evaluation of 4 CRISPR experiments highlight the value of using RNA-seq to identify transcriptional changes to cells altered by CRISPR, many of which cannot be recognized by evaluating DNA alone. Specific guidelines are presented for designing and analyzing CRISPR experiments using RNA-seq data.},
}

*CRISPR-Cas Systems/genetics
Humans
*Sequence Analysis, RNA/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcriptome/genetics
*RNA-Seq/methods
Gene Editing/methods
Gene Knockout Techniques/methods

@article {pmid40280947,
year = {2025},
author = {Chen, H and Song, F and Wang, B and Huang, H and Luo, Y and Han, X and He, H and Lin, S and Wan, L and Huang, Z and Fu, Z and Ledesma-Amaro, R and Yin, D and Mao, H and He, L and Yang, T and Chen, Z and Ma, Y and Xue, EY and Wan, Y and Mao, C},
title = {Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3929},
pmid = {40280947},
issn = {2041-1723},
support = {42376184//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bacteria/isolation & purification/genetics ; DNA, Bacterial/genetics ; },
abstract = {Clinical pathogen diagnostics detect targets by qPCR (but with low sensitivity) or blood culturing (but time-consuming). Here we leverage a dual-stem-loop DNA amplifier to enhance non-specific collateral enzymatic cleavage of an oligonucleotide linker between a fluophore and its quencher by CRISPR-CasΦ, achieving ultrasensitive target detection. Specifically, the target pathogens are lysed to release DNA, which binds its complementary gRNA in CRISPR-CasΦ to activate the collateral DNA-cleavage capability of CasΦ, enabling CasΦ to cleave the stem-loops in the amplifier. The cleavage product binds its complementary gRNA in another CRISPR-CasΦ to activate more CasΦ. The activated CasΦ collaterally cleaves the linker, releasing the fluophore to recover its fluorescent signal. The cycle of stem-loop-cleavage/CasΦ-activation/fluorescence-recovery amplifies the detection signal. Our target amplification-free collateral-cleavage-enhancing CRISPR-CasΦ method (TCC), with a detection limit of 0.11 copies/μL, demonstrates enhanced sensitivity compared to qPCR. It can detect pathogenic bacteria as low as 1.2 CFU/mL in serum within 40 min.},
}

*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
RNA, Guide, CRISPR-Cas Systems/genetics
*Bacteria/isolation & purification/genetics
DNA, Bacterial/genetics

@article {pmid40280080,
year = {2025},
author = {Xiong, Q and Zhu, C and Yin, X and Zhu, L},
title = {CRISPR/Cas and Argonaute-based biosensors for nucleic acid detection.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128210},
doi = {10.1016/j.talanta.2025.128210},
pmid = {40280080},
issn = {1873-3573},
abstract = {Nowadays, nucleic acid detection technology has been applied to disease diagnosis, prevention, food safety, environmental testing and many other aspects. However, traditional methods still have shortcomings. Therefore, there is an urgent need for a simple, rapid, sensitive, and specific new method to supersede traditional nucleic acid detection technology. CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) system and Argonaute (Ago) system play an important role in microbial immune defense. Their targeting specificity, programmability and special trans-cleavage activity make it possible to develop some new platforms for nucleic acid detection in combination with a variety of biosensors. We introduce the origins of these two systems and the biosensors developed based on CRISPR/Cas system and Ago system, respectively, especially the prospects for the future development of Cascade Amplification biosensors. This review is expected to provide useful guidance for researchers in related fields and provide inspiration for the development of Cascade Amplification biosensors in the future.},
}

@article {pmid40278504,
year = {2025},
author = {Lv, J and Jin, J and Ding, L and Xiang, L and Xie, B and Wu, K and Chen, Q},
title = {Directed Evolution of OgeuIscB With Enhanced Activity in Human Cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {8},
pages = {e70570},
doi = {10.1096/fj.202500082R},
pmid = {40278504},
issn = {1530-6860},
support = {Y20220148//Science and Technology Project of Wenzhou City/ ; Y2023784//Science and Technology Project of Wenzhou City/ ; ZCLTGD24H1202//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; LTGD23H120001//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; LTGC23H120001//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; 82101169//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Directed Molecular Evolution/methods ; HEK293 Cells ; Protein Engineering/methods ; Mutation ; CRISPR-Cas Systems ; },
abstract = {The miniature RNA-guided endonuclease IscB, as the evolutionary progenitor of Cas9, is attracting increased attention for genome editing due to its compact size and suitability for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells presents a significant challenge to its widespread application in precise site-specific human genome editing. In this study, we employed structure-guided rational design and protein engineering to optimize OgeuIscB, resulting in the identification of enIscB-F138R, which further enhanced editing activity up to 3.49-fold in mammalian cells compared to the high-activity OgeuIscB variant enIscB. Furthermore, we engineered an enIscB-F138R nickase-based adenine base editor, termed miABE-F138R, exhibiting enhanced base editing efficiency relative to miABE. To illustrate the practical applications of miABE-F138R, we applied it to rectify the prevalent R560C mutation in Pde6β associated with autosomal recessive retinitis pigmentosa, resulting in a significant improvement in activity compared to miABE. In conclusion, enIscB-F138R and miABE-F138R offer adaptable platforms for genome editing with potential significance in future biomedical applications.},
}

Humans
*Gene Editing/methods
*Directed Molecular Evolution/methods
HEK293 Cells
Protein Engineering/methods
Mutation
CRISPR-Cas Systems

@article {pmid40277901,
year = {2025},
author = {Plümers, R and Jelinek, S and Lindenkamp, C and Osterhage, MR and Knabbe, C and Hendig, D},
title = {Investigation on ABCC6-Deficient Human Hepatocytes Generated by CRISPR-Cas9 Genome Editing.},
journal = {Cells},
volume = {14},
number = {8},
pages = {},
pmid = {40277901},
issn = {2073-4409},
mesh = {Humans ; *Hepatocytes/metabolism ; *CRISPR-Cas Systems/genetics ; *Multidrug Resistance-Associated Proteins/deficiency/genetics/metabolism ; *Gene Editing/methods ; Pseudoxanthoma Elasticum/genetics/pathology/metabolism ; },
abstract = {Patients affected by the rare disease pseudoxanthoma elasticum (PXE) exhibit the calcification of elastic fibers in ocular, dermal, and vascular tissues. These symptoms are triggered by mutations in the ATP-binding cassette transporter subfamily C member 6 (ABCC6), whose substrate remains unknown. Interestingly, ABCC6 is predominantly expressed in the liver tissue, leading to the hypothesis that PXE is a metabolic disorder. We developed a genome-editing system targeting ABCC6 in human immortalized hepatocytes (HepIms) for further investigations. The HepIms were transfected with an ABCC6-specific clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) genome-editing plasmid, resulting in the identification of a heterozygous (ht[ABCC6]HepIm) and a compound heterozygous (cht[ABCC6]HepIm) clone. These clones were analyzed for key markers associated with the PXE pathobiochemistry. Hints of impaired lipid trafficking, defects in the extracellular matrix remodeling, the induction of calcification inhibitor expression, and the down regulation of senescence and inflammatory markers in ABCC6-deficienct HepIms were found. Our ABCC6 knock-out model of HepIms provides a valuable tool for studying the metabolic characteristics of PXE in vitro. The initial analysis of the clones mirrors various features of the PXE pathobiochemistry and provides an outlook on future research approaches.},
}

Humans
*Hepatocytes/metabolism
*CRISPR-Cas Systems/genetics
*Multidrug Resistance-Associated Proteins/deficiency/genetics/metabolism
*Gene Editing/methods
Pseudoxanthoma Elasticum/genetics/pathology/metabolism

@article {pmid40277547,
year = {2025},
author = {Xue, B and Qiao, B and Jia, L and Chi, J and Su, M and Song, Y and Du, J},
title = {A Sensitive and Fast microRNA Detection Platform Based on CRlSPR-Cas12a Coupled with Hybridization Chain Reaction and Photonic Crystal Microarray.},
journal = {Biosensors},
volume = {15},
number = {4},
pages = {},
pmid = {40277547},
issn = {2079-6374},
support = {2021YFA0805100//National Key Research and Development Program of China/ ; 82070407//National Natural Science Foundation of China/ ; 82200459//National Natural Science Foundation of China/ ; },
mesh = {*MicroRNAs/analysis ; Nucleic Acid Hybridization ; CRISPR-Cas Systems ; *Biosensing Techniques ; Oligonucleotide Array Sequence Analysis ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Changes in microRNA (miRNA) levels are closely associated with the pathological processes of many diseases. The sensitive and fast detection of miRNAs is critical for diagnosis and prognosis. Here, we report a platform employing CRISPR/Cas12a to recognize and report changes in miRNA levels while avoiding complex multi-thermal cycling procedures. A non-enzyme-dependent hybridization chain reaction (HCR) was used to convert the miRNA signal into double-stranded DNA, which contained a Cas12a activation sequence. The target sequence was amplified simply and isothermally, enabling the test to be executed at a constant temperature of 37 °C. The detection platform had the capacity to measure concentrations down to the picomolar level, and the target miRNA could be distinguished at the nanomolar level. By using photonic crystal microarrays with a stopband-matched emission spectrum of the fluorescent-quencher modified reporter, the fluorescence signal was moderately enhanced to increase the sensitivity. With this enhancement, analyzable fluorescence results were obtained in 15 min. The HCR and Cas12a cleavage processes could be conducted in a single tube by separating the two procedures into the bottom and the cap. We verified the sensitivity and specificity of this one-pot system, and both were comparable to those of the two-step method. Overall, our study produced a fast and sensitive miRNA detection platform based on a CRISPR/Cas12a system and enzyme-free HCR amplification. This platform may serve as a potential solution for miRNA detection in clinical practice.},
}

*MicroRNAs/analysis
Nucleic Acid Hybridization
CRISPR-Cas Systems
*Biosensing Techniques
Oligonucleotide Array Sequence Analysis
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40277544,
year = {2025},
author = {Chao, A and Hu, Q and Yin, K},
title = {A Label-Free CRISPR/Cas12a-G4 Biosensor Integrated with FTA Card for Detection of Foodborne Pathogens.},
journal = {Biosensors},
volume = {15},
number = {4},
pages = {},
pmid = {40277544},
issn = {2079-6374},
support = {ZDYF2022SHFZ321//Hainan Province Science and Technology Special Fund/ ; X2020-02-08-00-12-F01110//Shanghai Agriculture Applied Technology Development Program/ ; 202340069//Project of Shanghai Municipal Health Commission/ ; 22104090//National Natural Science Foundation of China/ ; 22ZR1436200//Natural Science Foundation of Shanghai/ ; NA//Shanghai Oriental Talent Program (Youth Project)/ ; 22474076//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques ; *Escherichia coli O157/isolation & purification ; *CRISPR-Cas Systems ; *Food Microbiology ; G-Quadruplexes ; Limit of Detection ; },
abstract = {CRISPR/Cas-based diagnostics offer unparalleled specificity, but their reliance on fluorescently labeled probes and complex nucleic acid extraction limits field applicability. To tackle this problem, we have developed a label-free, equipment-free platform integrating FTA card-based extraction, CRISPR/Cas12a, and pre-folded G-quadruplex (G4)-Thioflavin T (ThT) signal reporter. This system eliminates costly fluorescent labeling by leveraging G4-ThT structural binding for visible fluorescence output, while FTA cards streamline nucleic acid isolation without centrifugation. Achieving a limit of detection (LOD) to 10[1] CFU/mL for Escherichia coli O157:H7 in spiked food samples, the platform demonstrated 100% concordance with qPCR and standard fluorescent probe-based CRISPR/Cas12a system. Its simplicity, minimal equipment (portable heating/imaging), and cost-effectiveness make it a revolutionary tool for detecting foodborne pathogens in resource-limited environments.},
}

*Biosensing Techniques
*Escherichia coli O157/isolation & purification
*CRISPR-Cas Systems
*Food Microbiology
G-Quadruplexes
Limit of Detection

@article {pmid40277382,
year = {2025},
author = {Xu, D and Wu, Q and Yang, F and Zhang, Q and Jiang, Q and Zeng, X and Zhang, Y and Lv, T and Wang, J and Li, F},
title = {Fast-Flu: RT-RPA-CRISPR/Cas12a assisted one-step platform for rapid influenza B virus detection.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0036525},
doi = {10.1128/spectrum.00365-25},
pmid = {40277382},
issn = {2165-0497},
abstract = {UNLABELLED: Influenza B virus (Flu B) is a prevalent respiratory pathogen responsible for seasonal influenza epidemics. Despite its clinical significance, there remains a lack of rapid and accurate diagnostic methods for Flu B detection. In this study, we developed a novel Flu B detection system, named Fast-Flu, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (CRISPR/Cas). Through optimization of reaction temperature and adjustment of Cas12a concentrations, we successfully balanced RPA amplification and CRISPR/Cas12a trans-cleavage activity, enabling the establishment of a one-step detection system. The one-step Fast-Flu system demonstrated the ability to specifically identify Flu B within 45 min, with a limit of detection of 58 copies per test. It eliminates the need for uncapping operations and minimizes the risk of cross-contamination, without cross-reactivity with other pathogens. When evaluated using 101 clinical throat swab samples, the one-step Fast-Flu system achieved a sensitivity of 56.25% and a specificity of 100% compared to the PCR-based method, with an overall concordance rate of 93.06% (94/101). The development of this one-step RT-RPA-CRISPR/Cas12a system represents a significant advancement in the rapid, convenient, and accurate detection of Flu B, highlighting its potential for clinical diagnosis. Furthermore, with future technical improvements to enhance sensitivity, this one-step RT-RPA-CRISPR assay holds promise as a versatile tool for the rapid nucleic acid detection of other RNA viruses.

IMPORTANCE: Influenza B virus (Flu B) is a significant global health concern, and rapid, accurate pathogen diagnosis is crucial for effective influenza prevention and control. The integration of isothermal amplification methods with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has achieved high sensitivity and specificity for nucleic acid detection. Although CRISPR/Cas-based systems have been developed for influenza detection, existing platforms require the transfer of amplified products into the CRISPR/Cas12a detection system through uncapping operations, which increases the risk of cross-contamination. In this study, we developed a one-step reverse transcription recombinase polymerase amplification-CRISPR/Cas12a Flu B detection method using a one-pot detection system. By optimizing the reaction temperature and Cas12a concentration, we achieved a streamlined and contamination-free workflow. This innovative approach not only improves Flu B detection but also serves as a valuable reference for constructing CRISPR/Cas systems for the detection of other pathogens and targets, paving the way for broader applications in molecular diagnostics.},
}

@article {pmid40252041,
year = {2025},
author = {Wen, TT and Yang, YM and Zhang, YX and Liu, MQ and Qian, ZY and Zhang, ZY and Dong, CH and Sun, L and Xu, L and Sun, WJ and Cui, FJ},
title = {CRISPR-Cas9/Safe Harbor-Targeted Overexpression of Glucan Synthase Gene CmGls in Edible Mushroom Cordyceps militaris.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {17},
pages = {10456-10469},
doi = {10.1021/acs.jafc.5c01310},
pmid = {40252041},
issn = {1520-5118},
mesh = {*Glucosyltransferases/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Cordyceps/genetics/enzymology/metabolism/growth & development ; Gene Expression Regulation, Fungal ; Cell Wall/metabolism/genetics ; Fermentation ; },
abstract = {The membrane-integrated β-1,3-glucan synthase is the key enzyme involved in the biosynthesis of the core component β-1,3-glucan of the fungal cell wall. To date, the precise and targeted insertion of the β-1,3-glucan synthase gene into the genomes of edible fungi for safe and predictable overexpression has been extremely difficult due to the large DNA sequences (>5.0 kb) encoding the multitransmembrane domains and large molecular weights. In the present study, a large 5.9 kb DNA sequence of the membrane-bound β-1,3-glucan synthase gene CmGls was successfully and precisely inserted at a genomic safe harbor site CmSh1 of the C. militaris genome for the first time. By comparing mycelial and fermentation performance, overexpression of the β-1,3-glucan synthase gene CmGls resulted in rapid radial growth with a more pronounced yellowish color and increased resistance to cell wall stresses. Overexpression of CmGls significantly improved exopolysaccharide production with higher molecular weights, accompanied by an increase in the transcription levels of genes associated with polysaccharide/glucan synthesis, such as CmPgm, CmPgi, and CmUgp. Our findings provide convincing proof for the elucidation of glucan biosynthetic pathways and a basis for developing safe strains with highly efficient production of polysaccharides/glucans by edible fungi.},
}

*Glucosyltransferases/genetics/metabolism
*Fungal Proteins/genetics/metabolism
CRISPR-Cas Systems
*Cordyceps/genetics/enzymology/metabolism/growth & development
Gene Expression Regulation, Fungal
Cell Wall/metabolism/genetics
Fermentation

@article {pmid40023418,
year = {2025},
author = {Helalat, SH and Kristinsdóttir, HT and Petersen, AD and Téllez, RC and Boye, MN and Sun, Y},
title = {Enzymatic assembly for CRISPR split-Cas9 system: The emergence of a Sortase-based split-Cas9 technology.},
journal = {International journal of biological macromolecules},
volume = {306},
number = {Pt 2},
pages = {141583},
doi = {10.1016/j.ijbiomac.2025.141583},
pmid = {40023418},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *Aminoacyltransferases/metabolism/genetics ; *Cysteine Endopeptidases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Jurkat Cells ; Escherichia coli/genetics ; },
abstract = {CRISPR-Cas9 has been widely used in research and medical investigations as a pioneering technology. However, challenges such as the large size of the Cas9 sequence and the need for precise control over its activity in specific cell types have impeded its widespread adoption. Various alternatives, such as split-Cas9 technology, have emerged. Split-Cas9 systems allow the large Cas9 sequence to be divided into two segments to aid in the delivery of the enzyme. Nevertheless, challenges persist in achieving precise control over the timing and location of Cas9 reassembly and activity to ensure targeted action. This study presents an enzymatic-based split-Cas9 system, introducing a new approach utilizing the Sortase enzyme for the reconstitution of the full Cas9 protein. The developed method eliminates the need for chemical or physical induction and allows for precise genome editing in specific cells through the utilization of various specific promoters or targeted drug delivery. Experimental validation of the enzymatic system was conducted in E. coli, HEK cells, and Jurkat cells, demonstrating successful assembly and activity of the assembled Cas9 enzyme. In addition, this study explored the incorporation of nuclear localization signals, the evaluation of inducible promoters, and the delivery of the system's components in mRNA or protein form. Furthermore, we investigated the potential of S/MAR minicircle technology instead of viral vectors within the system. Overall, we highlighted the feasibility and utility of the Sortase-based split-Cas9 system to enhance control and efficiency compared to traditional CRISPR-Cas9 approaches. Additionally, this study revealed the potential of using the Sortase enzyme for posttranslational modifications and protein assembly in human cells.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
HEK293 Cells
*Aminoacyltransferases/metabolism/genetics
*Cysteine Endopeptidases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
Jurkat Cells
Escherichia coli/genetics

@article {pmid39804565,
year = {2025},
author = {Gu, B and Li, M and Li, D and Huang, K},
title = {CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis.},
journal = {Journal of cardiovascular translational research},
volume = {18},
number = {2},
pages = {424-441},
pmid = {39804565},
issn = {1937-5395},
mesh = {Humans ; *Proprotein Convertase 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Atherosclerosis/genetics/therapy/enzymology/blood/diagnosis ; *Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; Animals ; Biomarkers/blood ; *Cholesterol, LDL/blood ; Genetic Predisposition to Disease ; Treatment Outcome ; },
abstract = {CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.},
}

Humans
*Proprotein Convertase 9/genetics/metabolism
*CRISPR-Cas Systems
*Atherosclerosis/genetics/therapy/enzymology/blood/diagnosis
*Gene Editing/methods
*Genetic Therapy/methods/adverse effects
Animals
Biomarkers/blood
*Cholesterol, LDL/blood
Genetic Predisposition to Disease
Treatment Outcome

@article {pmid39799394,
year = {2025},
author = {Dragon, AC and Bonifacius, A and Lienenklaus, S and Verboom, M and Gerhards, JP and Ius, F and Hinze, C and Hudecek, M and Figueiredo, C and Blasczyk, R and Eiz-Vesper, B},
title = {Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {3},
pages = {1031-1047},
pmid = {39799394},
issn = {1525-0024},
mesh = {*Graft Rejection/prevention & control/immunology ; Animals ; Mice ; *B-Lymphocytes/immunology/metabolism ; Humans ; *T-Lymphocytes/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Isoantigens/immunology ; *Receptors, Antigen, T-Cell/genetics/metabolism/immunology ; Lymphocyte Depletion ; CRISPR-Cas Systems ; },
abstract = {Antibody-mediated rejection (AMR) remains a major complication after solid organ transplantation (SOT). Current treatment options are inefficient and result in drastic impairment of the general immunity. To selectively eliminate responsible alloreactive B cells characterized by anti-donor-HLA B cell receptors (BCRs), we generated T cells overcoming rejection by antibodies (CORA-Ts) engineered with a novel chimeric receptor comprising a truncated donor-HLA molecule as antigen recognition domain. As proof-of-concept, CORA receptors based on HLA-A∗02 were developed. In co-cultures with anti-HLA-A∗02 B cell lines, CORA-Ts were specifically activated, released pro-inflammatory mediators, and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A∗02 antibody release. Significant reduction of growth of an anti-HLA-A∗02 B cell line could be confirmed using an in vivo mouse model. Modification of the CORA receptor effectively abrogated T cell binding, thereby avoiding T cell sensitization. Additionally, using CRISPR-Cas9-mediated knockout of the FKBP12 gene, CORA-Ts were able to resist immunosuppressive treatment with tacrolimus, thereby allowing high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically eliminate alloreactive, anti-HLA B cells, thus selectively preventing anti-HLA antibody release even under immunosuppressive conditions. This suggests CORA-Ts as potent approach to combat AMR and improve long-term graft survival in SOT patients while preserving their overall B cell immunity.},
}

*Graft Rejection/prevention & control/immunology
Animals
Mice
*B-Lymphocytes/immunology/metabolism
Humans
*T-Lymphocytes/immunology/metabolism
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
Isoantigens/immunology
*Receptors, Antigen, T-Cell/genetics/metabolism/immunology
Lymphocyte Depletion
CRISPR-Cas Systems

@article {pmid39761351,
year = {2025},
author = {Ansari, MA and Verma, D and Hamizan, MA and Mukherjee, MD and Mohd-Naim, NF and Ahmed, MU},
title = {Trends in Aptasensing and the Enhancement of Diagnostic Efficiency and Accuracy.},
journal = {ACS synthetic biology},
volume = {14},
number = {1},
pages = {21-40},
doi = {10.1021/acssynbio.4c00591},
pmid = {39761351},
issn = {2161-5063},
mesh = {Humans ; *Biosensing Techniques/methods/trends ; *Aptamers, Nucleotide ; Machine Learning ; Point-of-Care Systems ; Precision Medicine/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; },
abstract = {The field of healthcare diagnostics is navigating complex challenges driven by evolving patient demographics and the rapid advancement of new technologies worldwide. In response to these challenges, these biosensors offer distinctive advantages over traditional diagnostic methods, such as cost-effectiveness, enhanced specificity, and adaptability, making their integration with point-of-care (POC) platforms more feasible. In recent years, aptasensors have significantly evolved in diagnostic capabilities through the integration of emerging technologies such as microfluidics, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, wearable devices, and machine learning (ML), driving progress in precision medicine and global healthcare solutions. Moreover, these advancements not only improve diagnostic accuracy but also hold the potential to revolutionize early detection, reduce healthcare costs, and improve patient outcomes, especially in resource-limited settings. This Account examines key advancements, focusing on how scientific breakthroughs, including artificial intelligence (AI), have improved sensitivity and precision. Additionally, the integration of aptasensors with these technologies has enabled real-time monitoring and data analysis, fostering advances in personalized healthcare. Furthermore, the potential commercialization of aptasensor technologies could increase their availability in clinical settings and support their use as widespread solutions for global health challenges. Hence, this review discusses technological improvements, practical uses, and prospects while also focusing on the challenges surrounding standardization, clinical validation, and interdisciplinary collaboration for widespread application. Finally, ongoing efforts to address these challenges are key to ensure that aptasensors can be effectively implemented in diverse healthcare systems.},
}

Humans
*Biosensing Techniques/methods/trends
*Aptamers, Nucleotide
Machine Learning
Point-of-Care Systems
Precision Medicine/methods
Artificial Intelligence
CRISPR-Cas Systems

@article {pmid38243923,
year = {2025},
author = {Mehrabadi, S and Izadi, FS and Pasha, S and Pourali, R and Khazaei, M and Hassanian, SM and Ferns, GA and Avan, A},
title = {The Potential Therapeutic Applications of CRISPR/Cas9 in the Treatment of Gastrointestinal Cancers.},
journal = {Current molecular medicine},
volume = {25},
number = {3},
pages = {278-288},
pmid = {38243923},
issn = {1875-5666},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gastrointestinal Neoplasms/therapy/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; },
abstract = {Gastrointestinal (GI) cancer is one the most prevalent types of cancer. Despite current chemotherapy's success, patients with GI cancer continue to have a dismal outcome. The onset and progression of cancer are caused by alterations and the abnormal expression of several families of genes, like tumor-suppressor genes, oncogenes, and chemotherapy-resistant genes. The final purpose of tumor therapy is to inhibit cellular development by modifying mutations and editing the irregular expression of genes It has been reported that CDH1, TP53, KRAS, ARID1A, PTEN, and HLA-B are the commonly mutated genes in GI cancer. Gene editing has become one potential approach for cases with advanced or recurrent CRC, who are nonresponsive to conventional treatments and a variety of driver mutations along with progression cause GI progression. CRISPR/Cas9 technique is a reliable tool to edit the genome and understand the functions of mutations driving GI cancer development. CRISPR/Cas9 can be applied to genome therapy for GI cancers, particularly with reference to molecular-targeted medicines and suppressors. Moreover, it can be used as a therapeutic approach by knocking in/out multiple genes. The use of CRISPR/ Cas9 gene editing method for GI cancer therapy has therefore resulted in some improvements. There are several research works on the role of CRISPR/Cas9 in cancer treatment that are summarized in the following separate sections. Here, the use of CRISPR/Cas9-based genome editing in GI and the use of CRISPR/Cas9 is discussed in terms of Targeting Chemotherapy Resistance-related Genes like; KRAS, TP53, PTEN, and ARID1A.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Gastrointestinal Neoplasms/therapy/genetics
*Genetic Therapy/methods
Animals
Mutation

@article {pmid40277083,
year = {2025},
author = {Dillard, KE and Zhang, H and Dubbs, LZ and Chou, CW and Terrace, C and Javanmardi, K and Kim, W and Forsberg, KJ and Finkelstein, IJ},
title = {Mechanism of Cas9 inhibition by AcrIIA11.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
pmid = {40277083},
issn = {1362-4962},
support = {F-1808//Welch Foundation/ ; R01GM124141/GF/NIH HHS/United States ; //Searle Scholars Award/ ; //University of Texas Southwestern Medical Center/ ; //Howard Hughes Medical Institute Emerging Pathogens Initiative/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/antagonists & inhibitors/genetics/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; Staphylococcus aureus/genetics/enzymology ; Streptococcus pyogenes/genetics/enzymology ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics/antagonists & inhibitors ; Francisella/genetics/enzymology ; DNA/metabolism ; HEK293 Cells ; },
abstract = {Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but generally coevolve with a narrow subset of Cas effectors that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), and Francisella novicida (Fn) Cas9s in vitro and in human cells. Single-molecule imaging reveals that AcrIIA11 hinders SaCas9 target search by reducing its diffusion on nonspecific DNA. DNA cleavage is inhibited because the AcrIIA11:SaCas9 complex binds to protospacer adjacent motif (PAM)-rich off-target sites, preventing SaCas9 from reaching its target. AcrIIA11 also greatly slows down DNA cleavage after SaCas9 reaches its target site. A negative-stain electron microscopy reconstruction of an AcrIIA11:SaCas9 RNP complex reveals that the heterodimer assembles with a 1:1 stoichiometry. Physical AcrIIA11-Cas9 interactions across type IIA and IIB Cas9s correlate with nuclease inhibition and support its broad-spectrum activity. These results add a kinetic inhibition mechanism to the phage-CRISPR arms race.},
}

*CRISPR-Associated Protein 9/metabolism/antagonists & inhibitors/genetics/chemistry
Humans
*CRISPR-Cas Systems/genetics
Staphylococcus aureus/genetics/enzymology
Streptococcus pyogenes/genetics/enzymology
DNA Cleavage
*Bacterial Proteins/metabolism/genetics/antagonists & inhibitors
Francisella/genetics/enzymology
DNA/metabolism
HEK293 Cells

@article {pmid40235216,
year = {2025},
author = {Zuo, T and Chen, X and Yu, Y and Qin, L and Xu, G and Wei, F and Yang, J and Zhou, C and Fan, L and Hu, Q and Zhao, Z and Tang, BZ and Cen, Y},
title = {BE-CATCH: Bioamplifier-Equipped CRISPR-Cas12a Transduction System Coupled with Commercial Pregnancy Test Strips to Harness Signal-on Point-of-Care Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {16},
pages = {8947-8956},
doi = {10.1021/acs.analchem.5c00342},
pmid = {40235216},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Humans ; Female ; Pregnancy ; *Point-of-Care Testing ; *Pregnancy Tests/methods ; *Reagent Strips ; Animals ; *Endodeoxyribonucleases/genetics/metabolism ; *Point-of-Care Systems ; Chorionic Gonadotropin ; *CRISPR-Associated Proteins/metabolism/genetics ; Colorimetry ; Bacterial Proteins ; },
abstract = {Repurposing existing commercial diagnostic equipment to enable portable analysis of diverse targets is driving the development of affordable point-of-care testing (POCT). Interestingly, we found that goat antimouse IgG could replace human chorionic gonadotropin (hCG) to make the T line of pregnancy test strips (PTS) appear red color and accordingly synthesized a novel signal output probe, which eliminated the intricate hCG covalent coupling steps, and could meet the multiple needs of expanded POCT. Given this, we introduced a novel separation-free universal POCT strategy termed bioamplifier-equipped CRISPR-Cas12a transduction system coupled with PTS to harness signal-on detection (BE-CATCH). Specifically, target inputs were converted and amplified by the multiplied strand displacement amplification-based bioamplifier, thereby activating Cas12a's trans-cleavage activity. Then, the activated Cas12a would cleave the connector indiscriminately, which ultimately kept the signal output probe in a free state; thus, the inputs could be translated into a colorimetric signal on the PTS. This strategy not only provided boosted sensitivity and specificity but also enhanced user-friendliness by maintaining the signal-on detection mode. We also demonstrated the versatility of the BE-CATCH strategy through selectively detecting miR-155 and flap endonuclease 1. Given its broad adaptability, the BE-CATCH strategy could provide an appealing option to broaden the application of PTS in biomedical diagnostics.},
}

*CRISPR-Cas Systems
Humans
Female
Pregnancy
*Point-of-Care Testing
*Pregnancy Tests/methods
*Reagent Strips
Animals
*Endodeoxyribonucleases/genetics/metabolism
*Point-of-Care Systems
Chorionic Gonadotropin
*CRISPR-Associated Proteins/metabolism/genetics
Colorimetry
Bacterial Proteins

@article {pmid40176603,
year = {2025},
author = {Ham, S and Lee, M and Jeong, D and Son, J and Kim, Y and Lee, T and Ko, K and Moh, SH and Ko, K},
title = {Potential use of human pluripotency-related gene expression reporter cell line for screening small molecules to enhance induction of pluripotency.},
journal = {BMB reports},
volume = {58},
number = {4},
pages = {183-189},
pmid = {40176603},
issn = {1976-670X},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Genes, Reporter/genetics ; Cellular Reprogramming/drug effects/genetics ; Cell Line ; *Small Molecule Libraries/pharmacology ; Octamer Transcription Factor-3/genetics/metabolism ; High-Throughput Screening Assays/methods ; CRISPR-Cas Systems/genetics ; Nanog Homeobox Protein/genetics/metabolism ; Green Fluorescent Proteins/metabolism/genetics ; Fibroblasts/metabolism/cytology ; },
abstract = {The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is a crucial development in regenerative medicine, providing patient-specific cells for therapeutic uses. Traditional methods often utilize viral vectors and transcription factors that pose tumorigenic risks, rendering them unsuitable for clinical applications. This study explored the use of chemicals as a non-tumorigenic alternative for cell reprogramming. Utilizing CRISPR/Cas9 technology, we previously created iPSCs expressing OCT4-EGFP and NANOG-tdTomato, and derived OCT4-EGFP and NANOG-tdTomato fibroblastic cells (ON-FCs). These cells were reprogrammed using episomal vectors, and their pluripotency was validated by fluorescence and FACS analyses. High-content screening was employed to assess small molecules that improve reprogramming efficiency, confirming the usefulness of ON-FCs as a dual reporter cell line for identifying small molecules effective in generating human iPSCs. This study underscores the utility of a dual reporter system and high-content screening in identifying effective reprogramming chemicals, establishing a scalable platform for high-throughput screening. Discovering new chemicals that can reprogram iPSCs would provide a non-tumorigenic method to advance the field of regenerative medicine. [BMB Reports 2025; 58(4): 183-189].},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects
Genes, Reporter/genetics
Cellular Reprogramming/drug effects/genetics
Cell Line
*Small Molecule Libraries/pharmacology
Octamer Transcription Factor-3/genetics/metabolism
High-Throughput Screening Assays/methods
CRISPR-Cas Systems/genetics
Nanog Homeobox Protein/genetics/metabolism
Green Fluorescent Proteins/metabolism/genetics
Fibroblasts/metabolism/cytology

@article {pmid40172222,
year = {2025},
author = {Rimple, PA and Olafsson, EB and Markus, BM and Wang, F and Augusto, L and Lourido, S and Carruthers, VB},
title = {Metabolic adaptability and nutrient scavenging in Toxoplasma gondii: insights from ingestion pathway-deficient mutants.},
journal = {mSphere},
volume = {10},
number = {4},
pages = {e0101124},
doi = {10.1128/msphere.01011-24},
pmid = {40172222},
issn = {2379-5042},
support = {R01AI120607//National Institute of Allergy and Infectious Diseases/ ; R01AI144369//National Institute of Allergy and Infectious Diseases/ ; P20GM130447/GM/NIGMS NIH HHS/United States ; //Boehringer Ingelheim Fonds/ ; },
mesh = {*Toxoplasma/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Mutation ; Vacuoles/parasitology ; *Nutrients/metabolism ; Endocytosis ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host endosomal sorting complex required for transport (ESCRT) machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the plant vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo, and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen in wild-type (WT) parasites and Δgra14, Δcpl, and Δcrt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that potentially become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that Δcpl and Δcrt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, tricarboxylic acid (TCA) cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway's potential role in host resource scavenging. Interestingly, Δcpl and Δcrt showed an exacerbated growth defect when cultured in amino acid-depleted media, suggesting that disrupting proteolysis or the export of proteolytic products from the PLVAC affects parasite survival during nutrient scarcity.

IMPORTANCE: Toxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases, but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study suggests that the ingestion pathway contributes to sustaining parasite metabolites and parasite replication under amino acid-limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma.},
}

*Toxoplasma/genetics/metabolism
Protozoan Proteins/genetics/metabolism
Mutation
Vacuoles/parasitology
*Nutrients/metabolism
Endocytosis
CRISPR-Cas Systems

@article {pmid40172186,
year = {2025},
author = {Watson, RG and Hole, CR},
title = {Simple growth conditions improve targeted gene deletion in Cryptococcus neoformans.},
journal = {mSphere},
volume = {10},
number = {4},
pages = {e0107024},
doi = {10.1128/msphere.01070-24},
pmid = {40172186},
issn = {2379-5042},
support = {1K22 AI148724//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Cryptococcus neoformans/genetics/growth & development ; *Gene Deletion ; CRISPR-Cas Systems ; *Culture Media/chemistry ; Fungal Proteins/genetics ; Transformation, Genetic ; Electroporation ; },
abstract = {Cryptococcus neoformans infections are a significant cause of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. The cryptococcal cell wall is very dynamic and can be modulated depending on growth conditions. It was reported that when C. neoformans is grown in unbuffered yeast nitrogen base (YNB) for 48 hours, the pH of the media drastically drops, and the cells start to shed their cell walls. With this observation, we sought to determine if YNB-grown cells could be used directly for genetic transformation. To test this, we targeted ADE2 using TRACE (transient CRISPR-Cas9 coupled with electroporation) in YNB-grown or competent cells. Deletion of the ADE2 gene results in red-pigmented colonies, allowing visual confirmation of disruption. We were able to successfully delete ADE2 in YNB-grown cells with better efficiency compared to competent cells. Recent studies have shown that gene deletion can be accomplished using short (50 bp) homology arms in place of the normal long arms (~1 kb). However, it was inefficient, leading to more insertions and gene disruption than gene deletions. We tested short homology with YNB-grown cells vs. competent cells and found that gene deletion was significantly improved in YNB-grown cells, at around 60% compared to 6% in competent cells. This was also observed when we deleted LAC1 with the short arms. Altogether, using simple growth conditions, we have greatly improved the speed and efficiency of cryptococcal genetic transformations.IMPORTANCEThe World Health Organization recently ranked C. neoformans as the highest-priority fungal pathogen based on unmet research and development needs and its public health importance. Understanding cryptococcal pathogenicity is key for developing treatments. We found that using simple growth conditions can greatly improve the speed and efficiency of cryptococcal genetic transformations. This finding will advance the field by expanding the ease of cryptococcal genetic manipulations.},
}

*Cryptococcus neoformans/genetics/growth & development
*Gene Deletion
CRISPR-Cas Systems
*Culture Media/chemistry
Fungal Proteins/genetics
Transformation, Genetic
Electroporation

@article {pmid39952507,
year = {2025},
author = {Chang, J and Li, Q and Zhang, T and Sun, H and Jia, Z and Li, Y and Zhang, S and Sun, T and Ma, S and Cao, J},
title = {Genome-wide CRISPR screening of genes and pathways for insect cell responding to abnormal environmental pH.},
journal = {International journal of biological macromolecules},
volume = {305},
number = {Pt 1},
pages = {141000},
doi = {10.1016/j.ijbiomac.2025.141000},
pmid = {39952507},
issn = {1879-0003},
mesh = {Animals ; Hydrogen-Ion Concentration ; Signal Transduction/genetics ; *Bombyx/genetics/cytology ; Cell Line ; *CRISPR-Cas Systems ; *Genome, Insect ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Cells are bathed in the extracellular fluids in which the extracellular pH (pHe) is maintained to a narrow range, and abnormal pHe is related to multiple diseases. However, the genes and signaling pathways underlying cell response to abnormal pHe remain unclear. Identification of genes responsive to extreme pHe challenge has great value in both basic research and medicine. Here, we performed genome-wide CRISPR screening to reveal genes and pathways related to insect cell response to abnormal pHe. Cells of the Bombyx mori embryonic cell line (BmE) genome-scale CRISPR screening cell library (BmEGCKLib) were cultured in different pHe (the physiological pH 6.3 as control; pH 5.0, 5.5, 6.6 and 6.95 as abnormal pHe). In the four extreme pH groups, we identified 44 overlapped fitness genes and 24 overlapped positive selected genes respectively. We also performed Kyoto Encyclopedia of Genes and Genomes pathways enrichment analysis for the selected genes. The "phosphatidylinositol signaling system", "mRNA surveillance pathway" and "spliceosome pathway" were significantly enriched in the negative selection, suggesting that cellular signal transduction and mRNA quality play essential roles for cells to resist to abnormal pHe. This is the first time to provide insight into insect cell response to abnormal pHe on a genome-scale.},
}

Animals
Hydrogen-Ion Concentration
Signal Transduction/genetics
*Bombyx/genetics/cytology
Cell Line
*CRISPR-Cas Systems
*Genome, Insect
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39756219,
year = {2025},
author = {Mao, Y and Shisler, JL and Nguyen, TH},
title = {Enhanced detection for antibiotic resistance genes in wastewater samples using a CRISPR-enriched metagenomic method.},
journal = {Water research},
volume = {274},
number = {},
pages = {123056},
doi = {10.1016/j.watres.2024.123056},
pmid = {39756219},
issn = {1879-2448},
mesh = {*Wastewater/microbiology ; *Metagenomics/methods ; *Drug Resistance, Microbial/genetics ; High-Throughput Nucleotide Sequencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {The spread of antibiotic resistance genes (ARGs) in the environment is a global public health concern. To date, over 5000 genes have been identified to express resistance to antibiotics. ARGs are usually low in abundance for wastewater samples, making them difficult to detect. Metagenomic sequencing and quantitative polymerase chain reaction (qPCR), two conventional ARG detection methods, have low sensitivity and low throughput limitations, respectively. We developed a CRISPR-Cas9-modified next-generation sequencing (NGS) method to enrich the targeted ARGs during library preparation. The false negative and false positive of this method were determined based on a mixture of bacterial isolates with known whole-genome sequences. Low values of both false negative (2/1208) and false positive (1/1208) proved the method's reliability. We compared the results obtained by this CRISPR-NGS and the conventional NGS method for six untreated wastewater samples. As compared to the ARGs detected in the same samples using the regular NGS method, the CRISPR-NGS method found up to 1189 more ARGs and up to 61 more ARG families in low abundances, including the clinically important KPC beta-lactamase genes in the six wastewater samples collected from different sources. Compared to the regular NGS method, the CRISPR-NGS method lowered the detection limit of ARGs from the magnitude of 10[-4] to 10[-5] as quantified by qPCR relative abundance. The CRISPR-NGS method is promising for ARG detection in wastewater. A similar workflow can also be applied to detect other targets that are in low abundance but of high diversity.},
}

*Wastewater/microbiology
*Metagenomics/methods
*Drug Resistance, Microbial/genetics
High-Throughput Nucleotide Sequencing
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems

@article {pmid39747596,
year = {2025},
author = {Glaus, AN and Brechet, M and Swinnen, G and Lebeigle, L and Iwaszkiewicz, J and Ambrosini, G and Julca, I and Zhang, J and Roberts, R and Iseli, C and Guex, N and Jiménez-Gómez, J and Glover, N and Martin, GB and Strickler, S and Soyk, S},
title = {Repairing a deleterious domestication variant in a floral regulator gene of tomato by base editing.},
journal = {Nature genetics},
volume = {57},
number = {1},
pages = {231-241},
pmid = {39747596},
issn = {1546-1718},
support = {802008//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 310030_212218//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; PCEFP3_181238//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; IOS-1546625//National Science Foundation (NSF)/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development ; *Gene Editing/methods ; *Domestication ; *Plant Proteins/genetics ; Mutation ; *Flowers/genetics ; Transcription Factors/genetics ; Gene Expression Regulation, Plant ; Genome, Plant ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Crop genomes accumulate deleterious mutations-a phenomenon known as the cost of domestication. Precision genome editing has been proposed to eliminate such potentially harmful mutations; however, experimental demonstration is lacking. Here we identified a deleterious mutation in the tomato transcription factor SUPPRESSOR OF SP2 (SSP2), which became prevalent in the domesticated germplasm and diminished DNA binding to genome-wide targets. We found that the action of SSP2 is partially redundant with that of its paralog SSP in regulating shoot and inflorescence architecture. However, redundancy was compromised during tomato domestication and lost completely in the closely related species Physalis grisea, in which a single ortholog regulates shoot branching. We applied base editing to directly repair the deleterious mutation in cultivated tomato and obtained plants with compact growth that provide an early fruit yield. Our work shows how deleterious variants have sensitized modern genotypes for phenotypic tuning and illustrates how repairing deleterious mutations with genome editing may allow predictable crop improvement.},
}

*Solanum lycopersicum/genetics/growth & development
*Gene Editing/methods
*Domestication
*Plant Proteins/genetics
Mutation
*Flowers/genetics
Transcription Factors/genetics
Gene Expression Regulation, Plant
Genome, Plant
CRISPR-Cas Systems
Phenotype

@article {pmid40222687,
year = {2025},
author = {Silver, AJ and Brown, DJ and Olmstead, SD and Watke, JM and Gorska, AE and Tanner, L and Ramsey, HE and Savona, MR},
title = {Interallelic gene conversion of leukemia-associated single nucleotide variants.},
journal = {Gene},
volume = {958},
number = {},
pages = {149493},
doi = {10.1016/j.gene.2025.149493},
pmid = {40222687},
issn = {1879-0038},
mesh = {Humans ; *Leukemia/genetics ; CRISPR-Cas Systems ; Animals ; *Polymorphism, Single Nucleotide ; Mice ; *Gene Conversion ; Alleles ; Cell Line, Tumor ; *Repressor Proteins/genetics ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9 is a useful tool for inserting precise genetic alterations through homology-directed repair (HDR), although current methods largely rely on provision of an exogenous repair template. Here, we tested the possibility of interchanging heterozygous single nucleotide variants (SNVs) using mutation-specific guide RNA, and the cell's own wild-type allele rather than an exogenous template. Using high-fidelity Cas9 to perform allele-specific CRISPR across multiple human leukemia cell lines as well as in primary hematopoietic cells from patients with leukemia, we find high levels of reversion to wild-type in the absence of exogenous template. Moreover, we demonstrate that bulk treatment to revert a truncating mutation in ASXL1 using CRISPR-mediated interallelic gene conversion (IGC) is sufficient to prolong survival in a human cell line-derived xenograft model (median survival 33 days vs 27.5 days; p = 0.0040). These results indicate that IGC is a useful laboratory tool which can be applied to numerous types of leukemia and can meaningfully alter cellular phenotypes at scale. Because our method targets single-base mutations, rather than larger variants targeted by IGC in prior studies, it greatly expands the pool of genetic lesions which could potentially be targeted by IGC. This technique may reduce cost and complexity for experiments modeling phenotypic consequences of SNVs. The principles of SNV-specific IGC demonstrated in this proof-of-concept study could be applied to investigate the phenotypic effects of targeted clonal reduction of leukemogenic SNV mutations.},
}

Humans
*Leukemia/genetics
CRISPR-Cas Systems
Animals
*Polymorphism, Single Nucleotide
Mice
*Gene Conversion
Alleles
Cell Line, Tumor
*Repressor Proteins/genetics
Gene Editing/methods

@article {pmid40193385,
year = {2025},
author = {Breunig, M and Hohwieler, M and Haderspeck, J and von Zweydorf, F and Hauff, N and Pasquini, LP and Wiegreffe, C and Zimmer, E and Mulaw, MA and Julier, C and Simon, E and Gloeckner, CJ and Liebau, S and Kleger, A},
title = {PPDPF is not a key regulator of human pancreas development.},
journal = {PLoS genetics},
volume = {21},
number = {4},
pages = {e1011657},
doi = {10.1371/journal.pgen.1011657},
pmid = {40193385},
issn = {1553-7404},
mesh = {Humans ; *Pancreas/metabolism/growth & development/cytology ; Animals ; Cell Differentiation/genetics ; Zebrafish/genetics ; CRISPR-Cas Systems ; Human Embryonic Stem Cells/metabolism/cytology ; Gene Expression Regulation, Developmental ; Pluripotent Stem Cells/metabolism/cytology ; Gene Knockout Techniques ; Cell Lineage/genetics ; },
abstract = {Given their capability to differentiate into each cell type of the human body, human pluripotent stem cells (hPSCs) provide a unique platform for developmental studies. In the current study, we employed this cell system to understand the role of pancreatic progenitor differentiation and proliferation factor (PPDPF), a protein that has been little explored so far. While the zebrafish orthologue exdpf is essential for exocrine pancreas specification, its importance for mammalian and human development has not been studied yet. We implemented a four times CRISPR/Cas9 nicking approach to knockout PPDPF in human embryonic stem cells (hESCs) and differentiated PPDPFKO/KO and PPDPFWT/WT cells towards the pancreatic lineage. In contrast to data obtained from zebrafish, a very modest effect of the knockout was observed in the development of pancreatic progenitors in vitro, not affecting lineage specification upon orthotopic transplantation in vivo. The modest effect is in line with the finding that genetic variants near PPDPF are associated with random glucose levels in humans, but not with type 2 diabetes risk, supporting that dysregulation of this gene may only result in minor alterations of glycaemic balance in humans. In addition, PPDPF is less organ- and cell type specifically expressed in higher vertebrates and its so far reported functions appear highly context-dependent.},
}

Humans
*Pancreas/metabolism/growth & development/cytology
Animals
Cell Differentiation/genetics
Zebrafish/genetics
CRISPR-Cas Systems
Human Embryonic Stem Cells/metabolism/cytology
Gene Expression Regulation, Developmental
Pluripotent Stem Cells/metabolism/cytology
Gene Knockout Techniques
Cell Lineage/genetics

@article {pmid40113021,
year = {2025},
author = {Xu, J and Xu, J and Sun, C and He, X and Shu, Y and Huangfu, Q and Meng, L and Liang, Z and Wei, J and Cai, M and Wen, J and Wang, B},
title = {Effective delivery of CRISPR/dCas9-SAM for multiplex gene activation based on mesoporous silica nanoparticles for bladder cancer therapy.},
journal = {Acta biomaterialia},
volume = {197},
number = {},
pages = {460-475},
doi = {10.1016/j.actbio.2025.03.032},
pmid = {40113021},
issn = {1878-7568},
mesh = {*Urinary Bladder Neoplasms/therapy/genetics/pathology/metabolism ; *Silicon Dioxide/chemistry ; *Nanoparticles/chemistry/therapeutic use ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; *Genetic Therapy/methods ; Porosity ; Mice ; *Gene Transfer Techniques ; Mice, Nude ; },
abstract = {The molecular complexity of bladder cancer restricts reliance on single-feature or single-gene targeted therapies, necessitating integrated individualized treatments and multi-gene interventions. In this study, we introduced the CRISPR/dCas9-SAM system to BCa treatment, known for its high specificity, low off-target effects, and reduced genetic toxicity, making it ideal for multiplexed gene activation at minimal cost-just 20 nucleotides per target. However, despite its potential in complex gene therapy and cellular engineering, challenges persist due to safety concerns associated with viral vectors and the risk of off-target effects during in vivo delivery, necessitating the development of new vectors. Herein, we reported pH-sensitive hollow mesoporous silica nanoparticles modified with PLZ4 ligands (PLZ4-Lip@AMSN/CRISPR/dCas9-SAM, PLACS NPs) for precise targeting of bladder tumors and co-delivery of CRISPR/dCas9-SAM system. With good stability and high plasmid loading capacity, they efficiently co-delivered dCas9-VP64, MS2-P65-HSF1, and sgRNA. Compared to Lipofectamine 3000, these nanoparticles exhibited superior lysosomal escape capability, significantly enhancing transfection efficiency in bladder cancer cells. Moreover, PLACS NPs simultaneously activated the expression of four target genes, inhibiting proliferation and migration, and promoting apoptosis in bladder cancer cells. In vivo, they achieved efficient gene editing at tumor sites, significantly inhibiting bladder tumor growth. Real-time imaging revealed their substantial accumulation and prolonged retention at bladder tumor sites without significant liver targeting and major organ damage, showcasing good specificity and biosafety. This study overcomes in vivo delivery challenges of multi-component CRISPR/dCas9 systems, enabling precise gene editing and anti-tumor effects, presenting an innovative strategy for targeted therapy in bladder cancer treatment. STATEMENT OF SIGNIFICANCE: This study introduces a newly-developed approach to address key challenges in bladder cancer gene therapy, namely low gene upregulation efficiency, limited targeting specificity, and inefficient nucleic acid delivery. By integrating the CRISPR/dCas9-SAM system, we achieve highly specific gene activation with minimal off-target effects, enabling the addition of treatment targets with just 20 nucleotides per target. To improve bladder cancer targeting, we developed PLACS NPs, a mesoporous silica nanoparticle system that enhances plasmid delivery, transfection efficiency, and endosomal escape. This system shows good tumor targeting and significant anti-tumor effects in bladder cancer, without significant liver targeting and major organ toxicity, offering promising therapeutic potential and broad clinical applications.},
}

*Urinary Bladder Neoplasms/therapy/genetics/pathology/metabolism
*Silicon Dioxide/chemistry
*Nanoparticles/chemistry/therapeutic use
Humans
*CRISPR-Cas Systems/genetics
Animals
Cell Line, Tumor
*Genetic Therapy/methods
Porosity
Mice
*Gene Transfer Techniques
Mice, Nude

@article {pmid39715634,
year = {2025},
author = {Kalanithy, JC and Mingardo, E and Stegmann, JD and Dhakar, R and Dakal, TC and Rosenfeld, JA and Tan, WH and Coury, SA and Woerner, AC and Sebastian, J and Levy, PA and Fleming, LR and Waffenschmidt, L and Lindenberg, TT and Yilmaz, Ö and Channab, K and Babra, BK and Christ, A and Eiberger, B and Hölzel, S and Vidic, C and Häberlein, F and Ishorst, N and Rodriguez-Gatica, JE and Pezeshkpoor, B and Kupczyk, PA and Vanakker, OM and Loddo, S and Novelli, A and Dentici, ML and Becker, A and Thiele, H and Posey, JE and Lupski, JR and Hilger, AC and Reutter, HM and Merz, WM and Dworschak, GC and Odermatt, B},
title = {TFAP2E is implicated in central nervous system, orofacial and maxillofacial anomalies.},
journal = {Journal of medical genetics},
volume = {62},
number = {2},
pages = {126-137},
pmid = {39715634},
issn = {1468-6244},
support = {UM1 HG006542/HG/NHGRI NIH HHS/United States ; R35 NS105078/NS/NINDS NIH HHS/United States ; K08 HG008986/HG/NHGRI NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; U01 HG011758/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Transcription Factor AP-2/genetics/chemistry/metabolism ; Zebrafish/genetics ; Female ; Male ; *Central Nervous System/metabolism/abnormalities ; *Maxillofacial Abnormalities/genetics/pathology ; Pedigree ; CRISPR-Cas Systems ; Mutation, Missense ; },
abstract = {BACKGROUND: Previous studies in mouse, Xenopus and zebrafish embryos show strong tfap2e expression in progenitor cells of neuronal and neural crest tissues suggesting its involvement in neural crest specification. However, the role of human transcription factor activator protein 2 (TFAP2E) in human embryonic central nervous system (CNS), orofacial and maxillofacial development is unknown.

METHODS: Through a collaborative work, exome survey was performed in families with congenital CNS, orofacial and maxillofacial anomalies. Exome variant prioritisation prompted TFAP2E gene for functional analysis in zebrafish embryos. Embryonic morphology and development were assessed after antisense morpholino (MO) knockdown (KD), CRISPR/Cas9 knockout and overexpression of tfap2e in fluorescent zebrafish reporter lines using in vivo microscopy. Computational structural protein modelling of the identified human variants was performed.

RESULTS: In total, exome survey identified novel or ultra-rare heterozygous missense variants in TFAP2E in seven individuals from five independent families with predominantly CNS, orofacial and maxillofacial anomalies. One variant was found de novo and another variant segregated in an affected multiplex family. Protein modelling of the identified variants indicated potential distortion of TFAP2E in the transactivation or dimerisation domain. MO KD and CRISPR/Cas9 knockout of tfap2e in zebrafish revealed hydrocephalus and a significant reduction of brain volume, consistent with a microencephaly phenotype. Furthermore, mRNA overexpression of TFAP2E indicates dosage-sensitive phenotype expression. In addition, zebrafish showed orofacial and maxillofacial anomalies following tfap2e KD, recapitulating the human phenotype.

CONCLUSION: Our human genetic data and analysis of Tfap2e manipulation in zebrafish indicate a potential role of TFAP2E in human CNS, orofacial and maxillofacial anomalies.},
}

Animals
Humans
*Transcription Factor AP-2/genetics/chemistry/metabolism
Zebrafish/genetics
Female
Male
*Central Nervous System/metabolism/abnormalities
*Maxillofacial Abnormalities/genetics/pathology
Pedigree
CRISPR-Cas Systems
Mutation, Missense

@article {pmid39705796,
year = {2025},
author = {Bian, S and Cai, Q and Wang, S and Xie, Y and Chen, N and Song, Q and Li, H and Zhao, N and Zhang, X},
title = {Evaluation of the toxoplasma Urm1 gene deletion mutant (PruΔUrm1) as a promising vaccine candidate against toxoplasmosis in mice.},
journal = {Vaccine},
volume = {45},
number = {},
pages = {126632},
doi = {10.1016/j.vaccine.2024.126632},
pmid = {39705796},
issn = {1873-2518},
mesh = {Animals ; *Toxoplasma/genetics/immunology/pathogenicity ; *Protozoan Vaccines/immunology/genetics/administration & dosage ; Mice ; Gene Deletion ; *Protozoan Proteins/genetics/immunology ; Female ; *Toxoplasmosis, Animal/prevention & control/immunology ; Antibodies, Protozoan/blood ; Mice, Inbred BALB C ; Cytokines/metabolism ; *Toxoplasmosis/prevention & control ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Toxoplasmosis is a significant zoonotic disease that poses a serious threat to both human and animal health. Despite ongoing research, developing an effective vaccine for toxoplasmosis remains a challenge. In this study, we evaluated the vaccine potential of the Toxoplasma Urm1 gene deletion mutant (PruΔUrm1) by assessing its pathogenicity and protective efficacy in mice. Using CRISPR/Cas9 technology, we successfully created a type II Toxoplasma gondii Pru mutant strain with a deleted Urm1 gene. Compared to the wild-type parasite, the PruΔUrm1 strain exhibited significantly reduced invasive and proliferative abilities in vitro. In in vivo studies, mice intraperitoneally infected with the parental Pru strain showed severe symptoms including emaciation, hunching, and high mortality rates. In contrast, mice infected with PruΔUrm1 tachyzoites demonstrated a 100 % survival rate, no overt symptoms, and a markedly reduced parasite burden in the liver, spleen, and lungs, indicating reduced pathogenicity. Notably, PruΔUrm1 vaccination triggered a strong immune response, characterized by significantly elevated cytokine levels, including TNF-α, IFN-γ and IL-10. Furthermore, we assessed the immunoprotective efficacy of PruΔUrm1 vaccination in mice against type I strains. Mice immunized with PruΔUrm1 were able to resist the tachyzoites of type I RH wild-type parasites, achieving a 100 % survival rate and significantly reduced parasite loads in the liver, spleen and lungs. These data demonstrate that PruΔUrm1 immunization provides effective protection against acute Toxoplasma infections and holds promise as a potential vaccine candidate for toxoplasmosis.},
}

Animals
*Toxoplasma/genetics/immunology/pathogenicity
*Protozoan Vaccines/immunology/genetics/administration & dosage
Mice
Gene Deletion
*Protozoan Proteins/genetics/immunology
Female
*Toxoplasmosis, Animal/prevention & control/immunology
Antibodies, Protozoan/blood
Mice, Inbred BALB C
Cytokines/metabolism
*Toxoplasmosis/prevention & control
Disease Models, Animal
CRISPR-Cas Systems

@article {pmid38659323,
year = {2025},
author = {Xue, P and Peng, Y and Wang, R and Wu, Q and Chen, Q and Yan, C and Chen, W and Xu, J},
title = {Advances, challenges, and opportunities for food safety analysis in the isothermal nucleic acid amplification/CRISPR-Cas12a era.},
journal = {Critical reviews in food science and nutrition},
volume = {65},
number = {13},
pages = {2473-2488},
doi = {10.1080/10408398.2024.2343413},
pmid = {38659323},
issn = {1549-7852},
mesh = {*Food Safety/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Food Contamination/analysis ; Food Microbiology/methods ; Humans ; Mycotoxins/analysis ; Food, Genetically Modified ; Biosensing Techniques/methods ; },
abstract = {Global food safety stands out as a prominent public concern, affecting populations worldwide. The recurrent challenge of food safety incidents reveals the need for a robust inspection framework. In recent years, the integration of isothermal nucleic acid amplification with CRISPR-Cas12a techniques has emerged as a promising tool for molecular detection of food hazards, presenting next generation of biosensing for food safety detection. This paper provides a comprehensive review of the current state of research on the synergistic application of isothermal nucleic acid amplification and CRISPR-Cas12a technology in the field of food safety. This innovative combination not only enriches the analytical tools, but also improving assay performance such as sensitivity and specificity, addressing the limitations of traditional methods. The review summarized various detection methodologies by the integration of isothermal nucleic acid amplification and CRISPR-Cas12a technology for diverse food safety concerns, including pathogenic bacterium, viruses, mycotoxins, food adulteration, and genetically modified foods. Each section elucidates the specific strategies employed and highlights the advantages conferred. Furthermore, the paper discussed the challenges faced by this technology in the context of food safety, offering insightful discussions on potential solutions and future prospects.},
}

*Food Safety/methods
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Food Contamination/analysis
Food Microbiology/methods
Humans
Mycotoxins/analysis
Food, Genetically Modified
Biosensing Techniques/methods

@article {pmid39674517,
year = {2025},
author = {Chen, X and Yao, S and Xie, L and Li, J and Xiong, L and Yang, X and Chen, Y and Cao, F and Hou, Q and You, M and Liu, Y and Gurr, GM and You, S},
title = {Disruption of the odorant receptor co-receptor (Orco) reveals its critical role in multiple olfactory behaviors of a cosmopolitan pest.},
journal = {Insect biochemistry and molecular biology},
volume = {177},
number = {},
pages = {104248},
doi = {10.1016/j.ibmb.2024.104248},
pmid = {39674517},
issn = {1879-0240},
mesh = {Animals ; *Receptors, Odorant/genetics/metabolism ; *Moths/genetics/physiology ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Sexual Behavior, Animal ; Oviposition ; Sex Attractants/metabolism ; Smell ; CRISPR-Cas Systems ; },
abstract = {The olfactory system of insects plays a pivotal role in multiple, essential activities including feeding, mating, egg laying, and host localization. The capacity of odorant receptors to recognize odor molecules relies on odorant receptor co-receptors forming heterodimers. Here we report the successful engineering a homozygous mutant strain of diamondback moth (Plutella xylostella) in which the odorant receptor co-receptor PxOrco was silenced using CRISPR/Cas9. This insect is a globally important crop pest for which novel control methods are urgently required. Behavioral assays demonstrated that PxOrco knockout males exhibited abolished courtship behaviors, inability to mate, and loss of selective preference for P. xylostella's key sex pheromone components. Whilst female mating behavior and fecundity remained unaffected by PxOrco knockout, oviposition response to leaf alcohol, a key cue for normal oviposition behavior, was lost. Electroantennography revealed drastically reduced responses to sex pheromones and plant volatiles in PxOrco-deficient adults but food location by larvae was unaffected. Moreover, expression analysis of PxOrco-deficient pheromone receptors (PRs) indicated varied regulation patterns, with down-regulation observed in several PRs in both sexes. These findings underscore the critical role of PxOrco in regulating multiple olfactory aspects in P. xylostella, including feeding, mating, and host location. Our study identifies the potential of disrupting the Orco gene in this and other pest species to provide novel avenues for future pest control.},
}

Animals
*Receptors, Odorant/genetics/metabolism
*Moths/genetics/physiology
Female
Male
*Insect Proteins/genetics/metabolism
Sexual Behavior, Animal
Oviposition
Sex Attractants/metabolism
Smell
CRISPR-Cas Systems

@article {pmid39674177,
year = {2025},
author = {Lou, H and Li, S and Shi, Z and Zou, Y and Zhang, Y and Huang, X and Yang, D and Yang, Y and Li, Z and Xu, C},
title = {Engineering source-sink relations by prime editing confers heat-stress resilience in tomato and rice.},
journal = {Cell},
volume = {188},
number = {2},
pages = {530-549.e20},
doi = {10.1016/j.cell.2024.11.005},
pmid = {39674177},
issn = {1097-4172},
mesh = {*Oryza/genetics/physiology/metabolism ; *Solanum lycopersicum/genetics/physiology/metabolism ; *Gene Editing/methods ; *Heat-Shock Response/genetics ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; },
abstract = {A 2°C climate-warming scenario is expected to further exacerbate average crop losses by 3%-13%, yet few heat-tolerant staple-crop varieties are available toward meeting future food demands. Here, we develop high-efficiency prime-editing tools to precisely knockin a 10-bp heat-shock element (HSE) into promoters of cell-wall-invertase genes (CWINs) in elite rice and tomato cultivars. HSE insertion endows CWINs with heat-responsive upregulation in both controlled and field environments to enhance carbon partitioning to grain and fruits, resulting in per-plot yield increases of 25% in rice cultivar Zhonghua11 and 33% in tomato cultivar Ailsa Craig over heat-stressed controls, without fruit quality penalties. Up to 41% of heat-induced grain losses were rescued in rice. Beyond a prime-editing system for tweaking gene expression by efficiently delivering bespoke changes into crop genomes, we demonstrate broad and robust utility for targeted knockin of cis-regulatory elements to optimize source-sink relations and boost crop climate resilience.},
}

*Oryza/genetics/physiology/metabolism
*Solanum lycopersicum/genetics/physiology/metabolism
*Gene Editing/methods
*Heat-Shock Response/genetics
Plants, Genetically Modified
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
Promoter Regions, Genetic
CRISPR-Cas Systems
Crops, Agricultural/genetics

@article {pmid39656538,
year = {2024},
author = {Zhang, Y and David, NL and Pesaresi, T and Andrews, RE and Kumar, GVN and Chen, H and Qiao, W and Yang, J and Patel, K and Amorim, T and Sharma, AX and Liu, S and Steinhauser, ML},
title = {Noncoding variation near UBE2E2 orchestrates cardiometabolic pathophenotypes through polygenic effectors.},
journal = {JCI insight},
volume = {10},
number = {2},
pages = {},
pmid = {39656538},
issn = {2379-3708},
support = {R01 DK120659/DK/NIDDK NIH HHS/United States ; T32 DK007052/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Mice ; *Diabetes Mellitus, Type 2/genetics/metabolism ; *Obesity/genetics ; Genome-Wide Association Study ; Humans ; Polymorphism, Single Nucleotide ; Adipogenesis/genetics ; Male ; Multifactorial Inheritance ; Mice, Inbred C57BL ; Female ; CRISPR-Cas Systems ; },
abstract = {Mechanisms underpinning signals from genome-wide association studies remain poorly understood, particularly for noncoding variation and for complex diseases such as type 2 diabetes mellitus (T2D) where pathogenic mechanisms in multiple different tissues may be disease driving. One approach is to study relevant endophenotypes, a strategy we applied to the UBE2E2 locus where noncoding single nucleotide variants (SNVs) are associated with both T2D and visceral adiposity (a pathologic endophenotype). We integrated CRISPR targeting of SNV-containing regions and unbiased CRISPR interference (CRISPRi) screening to establish candidate cis-regulatory regions, complemented by genetic loss of function in murine diet-induced obesity or ex vivo adipogenesis assays. Nomination of a single causal gene was complicated, however, because targeting of multiple genes near UBE2E2 attenuated adipogenesis in vitro; CRISPR excision of SNV-containing noncoding regions and a CRISPRi regulatory screen across the locus suggested concomitant regulation of UBE2E2, the more distant UBE2E1, and other neighborhood genes; and compound heterozygous loss of function of both Ube2e2 and Ube2e1 better replicated pathological adiposity and metabolic phenotypes compared with homozygous loss of either gene in isolation. This study advances a model whereby regulatory effects of noncoding variation not only extend beyond the nearest gene but may also drive complex diseases through polygenic regulatory effects.},
}

Animals
*Ubiquitin-Conjugating Enzymes/genetics/metabolism
Mice
*Diabetes Mellitus, Type 2/genetics/metabolism
*Obesity/genetics
Genome-Wide Association Study
Humans
Polymorphism, Single Nucleotide
Adipogenesis/genetics
Male
Multifactorial Inheritance
Mice, Inbred C57BL
Female
CRISPR-Cas Systems

@article {pmid39645650,
year = {2025},
author = {Moffa, L and Mannino, G and Bevilacqua, I and Gambino, G and Perrone, I and Pagliarani, C and Bertea, CM and Spada, A and Narduzzo, A and Zizzamia, E and Velasco, R and Chitarra, W and Nerva, L},
title = {CRISPR/Cas9-driven double modification of grapevine MLO6-7 imparts powdery mildew resistance, while editing of NPR3 augments powdery and downy mildew tolerance.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {2},
pages = {e17204},
doi = {10.1111/tpj.17204},
pmid = {39645650},
issn = {1365-313X},
support = {101135088//HORIZON EUROPE European Innovation Council/ ; BIOTECH-VITECH//Ministero delle Politiche Agricole Alimentari e Forestali/ ; },
mesh = {*Vitis/genetics/microbiology/immunology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/immunology/genetics ; Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; *Ascomycota/physiology ; Plants, Genetically Modified ; Erysiphe/physiology ; },
abstract = {The implementation of genome editing strategies in grapevine is the easiest way to improve sustainability and resilience while preserving the original genotype. Among others, the Mildew Locus-O (MLO) genes have already been reported as good candidates to develop powdery mildew-immune plants. A never-explored grapevine target is NPR3, a negative regulator of the systemic acquired resistance. We report the exploitation of a cisgenic approach with the Cre-lox recombinase technology to generate grapevine-edited plants with the potential to be transgene-free while preserving their original genetic background. The characterization of three edited lines for each target demonstrated immunity development against Erysiphe necator in MLO6-7-edited plants. Concomitantly, a significant improvement of resilience, associated with increased leaf thickness and specific biochemical responses, was observed in defective NPR3 lines against E. necator and Plasmopara viticola. Transcriptomic analysis revealed that both MLO6-7 and NPR3 defective lines modulated their gene expression profiles, pointing to distinct though partially overlapping responses. Furthermore, targeted metabolite analysis highlighted an overaccumulation of stilbenes coupled with an improved oxidative scavenging potential in both editing targets, likely protecting the MLO6-7 mutants from detrimental pleiotropic effects. Finally, the Cre-loxP approach allowed the recovery of one MLO6-7 edited plant with the complete removal of transgene. Taken together, our achievements provide a comprehensive understanding of the molecular and biochemical adjustments occurring in double MLO-defective grape plants. In parallel, the potential of NPR3 mutants for multiple purposes has been demonstrated, raising new questions on its wide role in orchestrating biotic stress responses.},
}

*Vitis/genetics/microbiology/immunology
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology/immunology/genetics
Gene Editing/methods
*Disease Resistance/genetics
*Plant Proteins/genetics/metabolism
*Ascomycota/physiology
Plants, Genetically Modified
Erysiphe/physiology

@article {pmid40275527,
year = {2025},
author = {Chen, Y and Li, M and Liu, X and Duan, Q and Xiao, L and Wang, L and Huang, C and Song, H and Cao, Y},
title = {Establishment of CRISPR-STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica.},
journal = {Microbial biotechnology},
volume = {18},
number = {4},
pages = {e70151},
pmid = {40275527},
issn = {1751-7915},
support = {2021YFC2104400//National Key Research and Development Program of China/ ; NSFC22478294//the National Natural Science Foundation of China/ ; NSFC22078240//the National Natural Science Foundation of China/ ; },
mesh = {*Yarrowia/genetics/metabolism ; *Transcriptional Activation ; *Gene Expression Regulation, Fungal ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The ability to regulate gene expression in multiple directions is crucial to maximise the production of microbial cell factories. However, the lack of a regulatory tool that can simultaneously activate and repress multiple genes restricts the manipulation diversity of Yarrowia lipolytica, which is an industrial workhorse for bioproduction. To address this issue, we developed a CRISPR scaffold RNAs (scRNAs)-mediated transcriptional activation and repression (CRISPR-STAR) platform. Firstly, we evaluated different methods for bidirectional regulation using CRISPR on both endogenous and synthetic promoters in Y. lipolytica, and chose the utilisation of orthogonal scRNAs to recruit activation and inhibition domains. Secondly, CRISPR-STAR was optimised by the introduction of alternative dCas proteins, scRNA structures and activators. 2.6-fold and 54.9-fold activation were achieved for synthetic and endogenous promoters, respectively, when the VPR transcriptional activator was recruited via MS2 hairpin. The repression of several genes was successfully achieved, with repression levels ranging from 3% to 32%, when the MXI1 transcriptional repressor was recruited via PP7 hairpin. Finally, CRISPR-STAR was applied to enhance fatty alcohol production by activating the FAR gene (encodes fatty acyl-CoA reductase) and repression of the PEX10 gene (encodes an integral membrane protein required for peroxisome biogenesis and matrix protein import). Compared to the non-targeting control, the bidirectionally regulated strain showed a 55.7% increase in yield to 778.8 mg/L. Our findings demonstrate that the CRISPR-STAR platform enables multi-mode regulation of genes, offering engineering opportunities to improve the productive performance of Y. lipolytica.},
}

*Yarrowia/genetics/metabolism
*Transcriptional Activation
*Gene Expression Regulation, Fungal
*CRISPR-Cas Systems
Promoter Regions, Genetic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40275470,
year = {2025},
author = {Kelly, G and Plesser, E and Bdolach, E and Arroyave, M and Belausov, E and Doron-Faigenboim, A and Rozen, A and Zemach, H and Zach, YY and Goldenberg, L and Arad, T and Yaniv, Y and Sade, N and Sherman, A and Eyal, Y and Carmi, N},
title = {In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {2},
pages = {e70155},
pmid = {40275470},
issn = {1365-313X},
mesh = {*Citrus/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; Plant Shoots/genetics/growth & development ; },
abstract = {Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.},
}

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

@article {pmid40275435,
year = {2025},
author = {Yang, Z and Bai, W and Guo, G and Huang, S and Wang, Y and Zhou, Y and Zhang, Y and Sun, J},
title = {The Q-interacted protein QIP3 recruits TaTPL to regulate spike architecture in wheat.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {2},
pages = {e70149},
doi = {10.1111/tpj.70149},
pmid = {40275435},
issn = {1365-313X},
support = {32472096//National Natural Science Foundation of China Grant/ ; 2023YFF1000402//National Key Research and Development Program of China/ ; //Innovation Program of the Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Triticum/genetics/metabolism/anatomy & histology/growth & development ; *Plant Proteins/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Spike architecture is a critical determinant of grain yield in wheat; yet the regulatory mechanisms remain poorly understood. Here, we demonstrate that the AP2 transcription factor Q directly represses the expression of TaMYB30-6A, a gene associated with spike length in wheat. We further identify QIP3 as a Q-interacting protein harboring an N-terminal EAR motif. Simultaneously, we reveal that QIP3 exhibits transcriptional repression activity, dependent on the EAR motif, and physically interacts with the transcriptional corepressor TaTPL. Importantly, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-generated qip3-aabbdd mutants exhibit reduced plant height and increased spike length phenotypes. Furthermore, RNA-seq and RT-qPCR assays show that QIP3 negatively regulates the expression of the Q target gene TaMYB30-6A in wheat. Collectively, we propose that the EAR motif-containing QIP3 interacts with Q to regulate spike architecture by recruiting the transcriptional corepressor TaTPL in wheat.},
}

*Triticum/genetics/metabolism/anatomy & histology/growth & development
*Plant Proteins/metabolism/genetics
*Transcription Factors/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid40274952,
year = {2025},
author = {Zhang, Z and Abreu, B and Brothwood, JL and Alexander, J and Sims, MJ and Lyons, JF and Munck, JM and Hindley, CJ},
title = {The identification of functional regions of MEK1 using CRISPR tiling screens.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {656},
pmid = {40274952},
issn = {2399-3642},
mesh = {*MAP Kinase Kinase 1/genetics/antagonists & inhibitors/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Discovery/methods ; Cell Survival/drug effects ; },
abstract = {CRISPR tiling screen is a powerful tool to identify protein regions relevant to its biological function. Understanding the functional relevance of the regions of target protein is of great help for structure-based drug discovery. Studying the drug resistance mechanisms of small-molecule inhibitors is important for the development and clinical application of the compounds. Using MEK1 and MEK inhibitors as example here, we demonstrate the utility of CRISPR tiling to identify regions essential for cancer cell viability and regions where mutations are resistant to MEK inhibitors. We study the drug resistance mechanisms of the regions and discussed the potential, as well as limitations, of applying the technology to drug development. Our findings demonstrate the value and prompt the utilization of CRISPR tiling technology in structure-based drug discovery.},
}

*MAP Kinase Kinase 1/genetics/antagonists & inhibitors/metabolism/chemistry
Humans
*CRISPR-Cas Systems
Protein Kinase Inhibitors/pharmacology
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
Drug Discovery/methods
Cell Survival/drug effects

@article {pmid40274871,
year = {2025},
author = {Chai, R and Guo, J and Yang, C and Zhu, D and Li, T and Yang, W and Liu, X and Chen, X and Huang, S and Wang, H and Yao, X and Gao, Y and Qiu, L},
title = {Enhanced chemotaxis and degradation of nonylphenol in Pseudoxanthomonas mexicana via CRISPR-mediated receptor modification.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {14296},
pmid = {40274871},
issn = {2045-2322},
support = {25B610014//the Key scientific research projects of colleges and universities in Henan Province/ ; GTTXM202412//the Consortium Construction Research Project of China Biotechnology Vocational Education Teaching Steering Committee/ ; 2403107//the Kaifeng Science and Technology Development Project/ ; 252102320066//the Key Research and Development Special Project of Henan Provincial Science and Technology/ ; },
mesh = {*Chemotaxis/genetics ; *Phenols/metabolism ; Biodegradation, Environmental ; Phylogeny ; Bacterial Proteins/genetics/metabolism ; Wastewater/microbiology ; Sewage/microbiology ; *CRISPR-Cas Systems ; },
abstract = {In this study, a novel nonylphenol (NP)-degrading bacterium, Pseudoxanthomonas mexicana CH, was isolated from wastewater treatment plant effluent. Phylogenetic analysis showed its close relationship to P. mexicana AMX 26BT. The strain displayed chemotaxis toward NP, with Mcp24 as the key chemoreceptor. The Mcp24 deletion mutant (CH- 1) had weaker chemotaxis and NP degradation (over 30% lower in solution and 8% lower in sludge than the wild type). In vitro, Mcp15's C-terminal pentapeptide DWQEF was methylated by CheR. Using CRISPR, this pentapeptide was added to Mcp24 to create CH- 2. CH- 2 showed better NP chemotaxis (17% higher in plate assays and 39% higher in capillary assays) and higher NP degradation rates (23.5% and 24.2% higher in solution and sludge, respectively). These findings demonstrate that NP acts as a bacterial chemoattractant, with Mcp24 as the receptor. Enhancing Mcp24's C-terminal pentapeptide improves chemotaxis and degradation efficiency, representing a significant advancement in bioremediation by strengthening bacterial responses to pollutants.},
}

*Chemotaxis/genetics
*Phenols/metabolism
Biodegradation, Environmental
Phylogeny
Bacterial Proteins/genetics/metabolism
Wastewater/microbiology
Sewage/microbiology
*CRISPR-Cas Systems

@article {pmid40274854,
year = {2025},
author = {Balakrishnan, A and Hunziker, M and Tiwary, P and Pandey, V and Drew, D and Billker, O},
title = {A CRISPR homing screen finds a chloroquine resistance transporter-like protein of the Plasmodium oocyst essential for mosquito transmission of malaria.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3895},
pmid = {40274854},
issn = {2041-1723},
support = {LT000131/2020-L//Human Frontier Science Program (HFSP)/ ; LT000131/2020-L//Human Frontier Science Program (HFSP)/ ; 895744//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; 788516//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 895744//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; P2SKP3_187635//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Plasmodium berghei/genetics/drug effects/metabolism ; Animals ; *Malaria/transmission/parasitology ; *Oocysts/metabolism/genetics/drug effects/growth & development ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; Mice ; Mosquito Vectors/parasitology ; Drug Resistance/genetics ; Chloroquine/pharmacology ; Female ; Anopheles/parasitology ; },
abstract = {Genetic screens with barcoded PlasmoGEM vectors have identified thousands of Plasmodium berghei gene functions in haploid blood stages, gametocytes and liver stages. However, the formation of diploid cells by fertilisation has hindered similar research on the parasites' mosquito stages. In this study, we develop a scalable genetic system that uses barcoded gene targeting vectors equipped with a CRISPR-mediated homing mechanism to generate homozygous loss-of-function mutants after one parent introduces a modified allele into the zygote. To achieve this, we use vectors additionally expressing a target gene specific gRNA. When integrated into one of the parental alleles it directs Cas9 to the intact allele after fertilisation, leading to its disruption. This homing strategy is 90% effective at generating homozygous gene editing of a fluorescence-tagged reporter locus in the oocyst. A pilot screen identifies PBANKA_0916000 as a chloroquine resistance transporter-like protein (CRTL) essential for oocyst growth and sporogony, pointing to an unexpected importance for malaria transmission of the poorly understood digestive vacuole of the oocyst that contains hemozoin granules. Homing screens provide a method for the systematic discovery of malaria transmission genes whose first essential functions are after fertilisation in the bloodmeal, enabling their potential as targets for transmission-blocking interventions to be assessed.},
}

*Plasmodium berghei/genetics/drug effects/metabolism
Animals
*Malaria/transmission/parasitology
*Oocysts/metabolism/genetics/drug effects/growth & development
*Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Editing/methods
Mice
Mosquito Vectors/parasitology
Drug Resistance/genetics
Chloroquine/pharmacology
Female
Anopheles/parasitology

@article {pmid39809840,
year = {2025},
author = {Li, XG and Zhu, GS and Cao, PJ and Huang, H and Chen, YH and Chen, C and Chen, PJ and Wu, D and Ding, C and Zhang, ZH and Zhang, RH and Hu, ZX and Zhao, WH and Liu, MH and Li, YW and Liu, HY and Chen, J},
title = {Genome-wide CRISPR-Cas9 screening identifies ITGA8 responsible for abivertinib sensitivity in lung adenocarcinoma.},
journal = {Acta pharmacologica Sinica},
volume = {46},
number = {5},
pages = {1419-1432},
pmid = {39809840},
issn = {1745-7254},
mesh = {Humans ; *CRISPR-Cas Systems ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; *Adenocarcinoma of Lung/drug therapy/genetics/pathology/metabolism ; Animals ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Cell Movement/drug effects ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mice ; *Antineoplastic Agents/pharmacology/therapeutic use ; Mice, Nude ; ErbB Receptors/antagonists & inhibitors ; Mice, Inbred BALB C ; },
abstract = {The emergence of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has improved the prognosis for lung cancer patients with EGFR-driven mutations. However, acquired resistance to EGFR-TKIs poses a significant challenge to the treatment. Overcoming the resistance has primarily focused on developing next-generation targeted therapies based on the molecular mechanisms of resistance or inhibiting the activation of bypass pathways to suppress or reverse the resistance. In this study we developed a novel approach by using CRISPR-Cas9 whole-genome library screening to identify the genes that enhance the sensitivity of lung adenocarcinoma cells to EGFR-TKIs. Through this screening, we revealed integrin subunit alpha 8 (ITGA8) as the key gene that enhanced sensitivity to abivertinib in lung adenocarcinoma. Notably, ITGA8 expression was significantly downregulated in lung adenocarcinoma tissues compared to adjacent normal tissues. Bioinformatics analyses revealed that ITGA8 was positively correlated with the sensitivity of lung adenocarcinoma to abivertinib. We showed that knockdown of ITGA8 significantly enhanced the proliferation, migration and invasion of H1975 cells. Conversely, overexpression of ITGA8 reduced the proliferation migration and invasion of H1975/ABIR cells. Furthermore, we demonstrated that ITGA8 sensitized lung adenocarcinoma cells to EGFR-TKIs by attenuating the downstream FAK/SRC/AKT/MAPK signaling pathway. In H1975 cell xenograft mouse models, knockdown of ITGA8 significantly increased tumor growth and reduced the sensitivity to abivertinib, whereas overexpression of ITGA8 markedly suppressed tumor proliferation and enhanced sensitivity to the drug. This study demonstrates that ITGA8 inhibits the proliferation, invasion and migration of lung adenocarcinoma cells, enhances the sensitivity to EGFR-TKIs, improves treatment efficacy, and delays the progression of acquired resistance. Thus, ITGA8 presents a potential therapeutic candidate for addressing acquired resistance to EGFR-TKIs from a novel perspective.},
}

Humans
*CRISPR-Cas Systems
*Lung Neoplasms/drug therapy/genetics/pathology/metabolism
*Adenocarcinoma of Lung/drug therapy/genetics/pathology/metabolism
Animals
Cell Proliferation/drug effects
Drug Resistance, Neoplasm/genetics/drug effects
Cell Line, Tumor
Cell Movement/drug effects
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Mice
*Antineoplastic Agents/pharmacology/therapeutic use
Mice, Nude
ErbB Receptors/antagonists & inhibitors
Mice, Inbred BALB C

@article {pmid39609561,
year = {2025},
author = {Hołubowicz, R and Du, SW and Felgner, J and Smidak, R and Choi, EH and Palczewska, G and Menezes, CR and Dong, Z and Gao, F and Medani, O and Yan, AL and Hołubowicz, MW and Chen, PZ and Bassetto, M and Risaliti, E and Salom, D and Workman, JN and Kiser, PD and Foik, AT and Lyon, DC and Newby, GA and Liu, DR and Felgner, PL and Palczewski, K},
title = {Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {57-78},
pmid = {39609561},
issn = {2157-846X},
support = {F30 EY033642/EY/NEI NIH HHS/United States ; FENG.02.01-IP.05-T005/23//Fundacja na rzecz Nauki Polskiej (Foundation for Polish Science)/ ; R01 EY032948/EY/NEI NIH HHS/United States ; R01EY032948, R21NS113264//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R21 NS113264/NS/NINDS NIH HHS/United States ; R01 EY030873/EY/NEI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; UG3AI150551, U01AI142756, R35GM118062, RM1HG009490//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 EY034501/EY/NEI NIH HHS/United States ; N66001-21-C-4013//United States Department of Defense | Defense Threat Reduction Agency (DTRA)/ ; T32GM008620, F30EY033642//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32GM148383//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P30EY034070//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; I01BX004939//U.S. Department of Veterans Affairs (Department of Veterans Affairs)/ ; UG3 AI150551/AI/NIAID NIH HHS/United States ; 75N93022C00054/AI/NIAID NIH HHS/United States ; R01EY009339, R01EY030873, P30EY034070, P30CA062203//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 EY009339/EY/NEI NIH HHS/United States ; P30 EY034070/EY/NEI NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; R00HL163805//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; I01 BX004939/BX/BLRD VA/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; T32 GM148383/GM/NIGMS NIH HHS/United States ; P30 CA062203/CA/NCI NIH HHS/United States ; 2022/47/B/NZ5/03023, 2020/39/D/NZ4/01881, 2019/34/E/NZ5/00434//Narodowe Centrum Nauki (National Science Centre)/ ; },
mesh = {*Gene Editing/methods ; *Ribonucleoproteins/genetics/chemistry/metabolism ; *Nanoparticles/chemistry ; *Lipids/chemistry ; Humans ; Animals ; Mice ; Cell-Penetrating Peptides/chemistry ; HEK293 Cells ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.},
}

*Gene Editing/methods
*Ribonucleoproteins/genetics/chemistry/metabolism
*Nanoparticles/chemistry
*Lipids/chemistry
Humans
Animals
Mice
Cell-Penetrating Peptides/chemistry
HEK293 Cells
CRISPR-Cas Systems
Liposomes

@article {pmid39578174,
year = {2025},
author = {Rosenberg-Mogilevsky, A and Siegfried, Z and Karni, R},
title = {Generation of tumor neoantigens by RNA splicing perturbation.},
journal = {Trends in cancer},
volume = {11},
number = {1},
pages = {12-24},
doi = {10.1016/j.trecan.2024.10.008},
pmid = {39578174},
issn = {2405-8025},
mesh = {Humans ; *Neoplasms/immunology/genetics/therapy ; *Antigens, Neoplasm/genetics/immunology ; *RNA Splicing/genetics/drug effects ; *Immunotherapy/methods ; Oligonucleotides, Antisense ; Nonsense Mediated mRNA Decay/drug effects ; RNA, Small Interfering/genetics ; RNA Splicing Factors/genetics ; Animals ; Alternative Splicing ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Immunotherapy has revolutionized cancer treatment, but the limited availability of tumor-specific neoantigens still remains a challenge. The potential of alternative mRNA splicing-derived neoantigens as a source of new immunotherapy targets has gained significant attention. Tumors exhibit unique splicing changes and splicing factor mutations which are prevalent in various cancers and play a crucial role in neoantigen production. We present advances in splicing modulation approaches, including small-molecule drugs, decoy and splice-switching antisense oligonucleotides (SSOs), CRISPR, small interfering RNAs (siRNAs), and nonsense-mediated RNA decay (NMD) inhibition, that can be adapted to enhance antitumor immune responses. Finally, we explore the clinical implications of these approaches, highlighting their potential to transform cancer immunotherapy and broaden its efficacy.},
}

Humans
*Neoplasms/immunology/genetics/therapy
*Antigens, Neoplasm/genetics/immunology
*RNA Splicing/genetics/drug effects
*Immunotherapy/methods
Oligonucleotides, Antisense
Nonsense Mediated mRNA Decay/drug effects
RNA, Small Interfering/genetics
RNA Splicing Factors/genetics
Animals
Alternative Splicing
Mutation
CRISPR-Cas Systems

@article {pmid39532588,
year = {2025},
author = {Michicich, M and Traylor, Z and McCoy, C and Valerio, DM and Wilson, A and Schneider, M and Davis, S and Barabas, A and Mann, RJ and LePage, DF and Jiang, W and Drumm, ML and Kelley, TJ and Conlon, RA and Hodges, CA},
title = {A W1282X cystic fibrosis mouse allows the study of pharmacological and gene-editing therapeutics to restore CFTR function.},
journal = {Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society},
volume = {24},
number = {1},
pages = {164-174},
pmid = {39532588},
issn = {1873-5010},
support = {F31 HL166193/HL/NHLBI NIH HHS/United States ; T32 GM135081/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Cystic Fibrosis/genetics/therapy/drug therapy ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; *Genetic Therapy/methods ; Codon, Nonsense ; Humans ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: People with cystic fibrosis carrying two nonsense alleles lack CFTR-specific treatment. Growing evidence supports the hypothesis that nonsense mutation identity affects therapeutic response, calling for mutation-specific CF models. We describe a novel W1282X mouse model and compare it to an existing G542X mouse.

METHODS: The W1282X mouse was created using CRISPR/Cas9 to edit mouse Cftr. In this model, Cftr transcription was assessed using qRT-PCR and CFTR function was measured in the airway by nasal potential difference and in the intestine by short circuit current. Growth, survival, and intestinal motility were examined as well. Correction of W1282X CFTR was assessed pharmacologically and by gene-editing using a forskolin-induced swelling (FIS) assay in small intestine-derived organoids.

RESULTS: Homozygous W1282X mice demonstrate decreased Cftr mRNA, little to no CFTR function, and reduced survival, growth, and intestinal motility. W1282X organoids treated with various combinations of pharmacologic correctors display a significantly different amount of CFTR function than that of organoids from G542X mice. Successful gene editing of W1282X to wildtype sequence in intestinal organoids was achieved leading to restoration of CFTR function.

CONCLUSIONS: The W1282X mouse model recapitulates common human manifestations of CF similar to other CFTR null mice. Despite the similarities between the congenic W1282X and G542X models, they differ meaningfully in their response to identical pharmacological treatments. This heterogeneity highlights the importance of studying therapeutics across genotypes.},
}

Animals
*Cystic Fibrosis/genetics/therapy/drug therapy
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
Mice
*Gene Editing/methods
Disease Models, Animal
*Genetic Therapy/methods
Codon, Nonsense
Humans
CRISPR-Cas Systems

@article {pmid40274667,
year = {2025},
author = {Mukherjee, A and Samanta, S and Das, S and Haque, MZ and Jana, PS and Samanta, I and Kar, I and Das, S and Nanda, PK and Thomas, P and Dandapat, P},
title = {Leveraging CRISPR-Cas-Enhanced Isothermal Amplification Tools for Quick Identification of Pathogens Causing Livestock Diseases.},
journal = {Current microbiology},
volume = {82},
number = {6},
pages = {260},
pmid = {40274667},
issn = {1432-0991},
support = {STBT-11012(27)/5/2024-ST SEC//Department of Science and Technology, Government of West Bengal/ ; },
mesh = {Animals ; *Nucleic Acid Amplification Techniques/methods ; *Livestock/microbiology/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Animal Diseases/diagnosis/microbiology/virology ; *Bacteria/genetics/isolation & purification ; },
abstract = {Prompt and accurate diagnosis of infectious pathogens of livestock origin is of utmost importance for epidemiological surveillance and effective therapeutic strategy formulation. Among various methods, nucleic acid-based detection of pathogens is the most sensitive and specific; but the majority of these assays need expensive equipment and skilled workers. Due to the rapid advancement of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based nucleic acid detection methods, these are now being widely used for pathogen detection. CRISPR-Cas is a bacterial counterpart of "adaptive immunity", generally used for editing genome. Many CRISPR systems have been modified for nucleic acid detection due to their excellent selectivity in detecting DNA and RNA sequences. The combination of CRISPR with suitable isothermal amplification technologies has made it more sensitive, specific, versatile, and reproducible for the detection of pathogen nucleic acids at the point of care. Amplification of pathogen nucleic acid by isothermal amplification followed by CRISPR-Cas-based detection has several advantages, including short sample-to-answer times and no requirement for laboratory set-up. They are also significantly less expensive than the existing nucleic acid detection methods. This review focuses on the recent trends in the use of this precision diagnostic method for diagnosis of a wide range of animal pathogens with or without zoonotic potential, particularly various isothermal amplification strategies, and visualization methods for sensing bacteria, viruses, and parasites of veterinary and public health importance.},
}

Animals
*Nucleic Acid Amplification Techniques/methods
*Livestock/microbiology/virology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
*Animal Diseases/diagnosis/microbiology/virology
*Bacteria/genetics/isolation & purification

@article {pmid40273916,
year = {2025},
author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA},
title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2025.03.024},
pmid = {40273916},
issn = {1097-4164},
abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.},
}

@article {pmid40273800,
year = {2025},
author = {Zheng, M and Bao, N and Wang, Z and Song, C and Jin, Y},
title = {Alternative splicing in autism spectrum disorder: Recent insights from mechanisms to therapy.},
journal = {Asian journal of psychiatry},
volume = {108},
number = {},
pages = {104501},
doi = {10.1016/j.ajp.2025.104501},
pmid = {40273800},
issn = {1876-2026},
abstract = {Alternative splicing (AS) is a vital and highly dynamic RNA regulatory mechanism that allows a single gene to generate multiple mRNA and protein isoforms. Dysregulation of AS has been identified as a key contributor to the pathogenesis of autism spectrum disorders (ASD). A comprehensive understanding of aberrant splicing mechanisms and their functional consequences in ASD can help uncover the molecular basis of the disorder and facilitate the development of therapeutic strategies. This review focuses on the major aberrant splicing events and key splicing regulators associated with ASD, highlighting their roles in linking defective splicing to ASD pathogenesis. In addition, a discussion of how emerging technologies, such as long-read sequencing, single-cell sequencing, spatial transcriptomics and CRISPR-Cas systems are offering novel insights into the role and mechanisms of AS in ASD is presented. Finally, the RNA splicing-based therapeutic strategies are evaluated, emphasizing their potential to address unmet clinical needs in ASD treatment.},
}

@article {pmid40273258,
year = {2025},
author = {Hayes, VM and Zhang, JT and Katz, MA and Li, Y and Kocsis, B and Brinkley, DM and Jia, N and Meeske, AJ},
title = {RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6745},
pages = {387-391},
doi = {10.1126/science.adr3656},
pmid = {40273258},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Molecular Mimicry ; *Listeria/virology/genetics ; *CRISPR-Associated Proteins/chemistry/antagonists & inhibitors ; Nucleic Acid Conformation ; *RNA, Viral/chemistry/genetics ; Bacteriophages/genetics ; Plasmids ; },
abstract = {To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.},
}

*CRISPR-Cas Systems
Cryoelectron Microscopy
*Molecular Mimicry
*Listeria/virology/genetics
*CRISPR-Associated Proteins/chemistry/antagonists & inhibitors
Nucleic Acid Conformation
*RNA, Viral/chemistry/genetics
Bacteriophages/genetics
Plasmids

@article {pmid40273189,
year = {2025},
author = {Akiyama, C and Sakata, S and Ono, F},
title = {Normal locomotion in zebrafish lacking the sodium channel NaV1.4 suggests that the need for muscle action potentials is not universal.},
journal = {PLoS biology},
volume = {23},
number = {4},
pages = {e3003137},
pmid = {40273189},
issn = {1545-7885},
mesh = {Animals ; *Zebrafish/physiology/genetics ; *NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology ; *Action Potentials/physiology ; *Locomotion/physiology/genetics ; Muscle Contraction/physiology ; *Zebrafish Proteins/genetics/metabolism ; Muscle, Skeletal/physiology/metabolism ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Tetrodotoxin/pharmacology ; Calcium/metabolism ; },
abstract = {Extensive studies over decades have firmly established the concept that action potentials (APs) in muscles are indispensable for muscle contraction. To re-examine the significance of APs, we generated zebrafish lacking APs by editing the scn4aa and scn4ab genes, which together encode NaV1.4 (NaVDKO), using the CRISPR-Cas9 system. Surprisingly, the escape response of NaVDKOs to tactile stimuli, both in the embryonic and adult stages, was indistinguishable from that of wild-type (WT) fish. Ca2+ imaging using the calcium indicator protein GCaMP revealed that myofibers isolated from WT fish could be excited by the application of acetylcholine (ACh), even in the presence of tetrodotoxin (TTX) indicating that NaVs are dispensable for skeletal muscle contraction in zebrafish. Mathematical simulations showed that the end-plate potential was able to elicit a change in membrane potential large enough to activate the dihydropyridine receptors of the entire muscle fiber owing to the small fiber size and the disseminated distribution of neuromuscular synapses in both adults and embryos. Our data demonstrate that NaVs are not essential for muscle contraction in zebrafish and that the physiological significance of NaV1.4 in muscle is not uniform across vertebrates.},
}

Animals
*Zebrafish/physiology/genetics
*NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology
*Action Potentials/physiology
*Locomotion/physiology/genetics
Muscle Contraction/physiology
*Zebrafish Proteins/genetics/metabolism
Muscle, Skeletal/physiology/metabolism
Animals, Genetically Modified
CRISPR-Cas Systems
Tetrodotoxin/pharmacology
Calcium/metabolism

@article {pmid40270959,
year = {2025},
author = {Caras, I and Ionescu, IE and Pantazica, AM and van Eerde, A and Steen, H and Heldal, I and Haugslien, S and Tucureanu, C and Chelmus, RE and Tofan, VC and Costache, A and Onu, A and Su, H and Branza-Nichita, N and Liu-Clarke, J and Stavaru, C},
title = {Humanized mouse model reveals the immunogenicity of Hepatitis B Virus vaccine candidates produced in CRISPR/Cas9-edited Nicotiana benthamiana.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1479689},
pmid = {40270959},
issn = {1664-3224},
mesh = {Animals ; *Nicotiana/genetics ; *Hepatitis B Vaccines/immunology/genetics ; Humans ; Mice ; *Hepatitis B virus/immunology ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Hepatitis B/immunology/prevention & control ; Mice, Inbred NOD ; *Immunogenicity, Vaccine ; Hepatitis B Antibodies/immunology ; Antibodies, Neutralizing/immunology ; Hepatitis B Surface Antigens/immunology/genetics ; Female ; Gene Editing ; Leukocytes, Mononuclear/immunology ; Mice, Inbred BALB C ; },
abstract = {INTRODUCTION: Hepatitis B Virus (HBV) infection is still an ongoing public health issue worldwide. The most efficient tool in preventing HBV infection remains vaccination and significant efforts have been made in the last decade to improve current HBV vaccines. Owing to the strict HBV tropism for the human liver, developing animal models for preclinical screening of vaccine candidates is extremely challenging. To date, there are only a few reports regarding the use of humanized mouse models for the evaluation of the immunogenic properties of viral antigens.

METHODS: Previously we showed that a Nicotiana benthamiana-produced HBV-S/preS1[16-42] antigen elicited strong HBV-specific immune responses in BALB/c mice. In the current study, we used immunodeficient NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl]/SzJ (NSG) mice as recipients of human peripheral blood mononuclear cells (hPBMCs), to evaluate the immunogenicity of the recently developed chimeric HBV immunogen produced in CRISPR/Cas9-edited N. benthamiana, under more "humanized" conditions.

RESULTS: Analysis of the immune response in NSG mice immunized with the chimeric antigen demonstrated induction of virus infection-neutralizing antibodies, indicating activation of antigen-specific B cells.

DISCUSSION: The ability of hPBMCs-engrafted NSG mice to mount specific humoral immune responses after immunization with viral antigens supports this animal model as a promising tool for pre-clinical evaluation of human vaccine antigens.},
}

Animals
*Nicotiana/genetics
*Hepatitis B Vaccines/immunology/genetics
Humans
Mice
*Hepatitis B virus/immunology
*CRISPR-Cas Systems
Disease Models, Animal
*Hepatitis B/immunology/prevention & control
Mice, Inbred NOD
*Immunogenicity, Vaccine
Hepatitis B Antibodies/immunology
Antibodies, Neutralizing/immunology
Hepatitis B Surface Antigens/immunology/genetics
Female
Gene Editing
Leukocytes, Mononuclear/immunology
Mice, Inbred BALB C

@article {pmid40270035,
year = {2025},
author = {Lin, XL and Zhou, YM and Meng, K and Yang, JY and Zhang, H and Lin, JH and Wu, HY and Wang, XY and Zhao, H and Feng, SS and Park, KS and Cai, DQ and Zheng, L and Qi, XF},
title = {CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.},
journal = {Cell & bioscience},
volume = {15},
number = {1},
pages = {52},
pmid = {40270035},
issn = {2045-3701},
support = {21623110//Fundamental Research Funds for Central Universities of the Jinan University/ ; ZSYXM202303//Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University/ ; ZSYXM202402//Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University/ ; 82370247, 82070257, 81770240//National Natural Science Foundation of China/ ; 2023A1515012147//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
abstract = {The Xenopus tropicalis (Western clawed frog) is an important amphibian model for genetics, developmental and regenerative biology, due to its diploid genetic background and short generation time. CRISPR-Cas13 and CRISPR interference (CRISPRi) systems have recently been employed to suppress mRNA expression in many organisms such as yeast, plants, and mammalian cells. However, no systematic study of these two systems has been carried out in Xenopus tropicalis. Here, we show that CRISPRi rather than CRISPR-Cas13 is an effective and suitable approach to suppress specific mRNA transcription in Xenopus tropicalis embryos. We demonstrated that CRISPRi composed of dCas9 and KRAB-MeCP2 (dCas9-KM) can efficiently target exogenous and endogenous transcripts in Xenopus tropicalis embryos. Moreover, our data suggest that the new KRAB domain from ZIM3 protein (ZIM3-KRAB, ZIM3K) alone has a comparable transcript targeting capacity in Xenopus tropicalis embryos to the traditional fusion repressor KRAB-MeCP2 in which the KRAB domain from KOX1 protein. In conclusion, our results demonstrate that CRISPRi rather than CRISPR-Cas13 is an efficient knockdown platform to explore specific gene function in Xenopus tropicalis embryos.},
}

@article {pmid40269480,
year = {2025},
author = {Chen, Z and Pilehvar, E and Sadeghi, H and Pilehvar, Y},
title = {Precision Reimagined: CRISPR and Multiomics Transform Systemic Lupus Erythematosus Diagnosis and Therapy.},
journal = {International journal of rheumatic diseases},
volume = {28},
number = {4},
pages = {e70189},
doi = {10.1111/1756-185X.70189},
pmid = {40269480},
issn = {1756-185X},
mesh = {Humans ; *Lupus Erythematosus, Systemic/genetics/diagnosis/therapy/immunology ; *Precision Medicine/methods ; *Gene Editing/methods ; *Genomics/methods ; Artificial Intelligence ; *CRISPR-Cas Systems ; Predictive Value of Tests ; *Proteomics/methods ; Phenotype ; Genetic Predisposition to Disease ; *Genetic Therapy/methods ; Multiomics ; },
abstract = {Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with diverse clinical manifestations and unpredictable progression, posing significant challenges to accurate diagnosis and effective treatment. Traditional biomarkers and treatments often fail to address the disease's molecular and clinical heterogeneity. Recent advancements in CRISPR gene-editing technology and multiomics approaches offer transformative opportunities for personalized SLE care by unraveling its underlying molecular complexity and enabling precise therapeutic interventions. CRISPR technology allows targeted editing of SLE-associated genetic mutations, addressing disease drivers directly, while multiomics-including genomics, transcriptomics, and proteomics-provides insights into dysregulated immune networks, identifying biomarkers and therapeutic targets. Integrating these approaches can refine patient stratification and enhance the precision of treatments. Artificial intelligence (AI) complements these technologies by synthesizing high-dimensional data, enabling personalized treatment plans, predicting disease trajectories, and optimizing therapeutic strategies. However, the integration of CRISPR and multiomics in clinical settings raises challenges, including technical limitations, ethical concerns, and economic barriers. Emerging clinical trials and case studies demonstrate the potential of these innovations to personalize care and improve outcomes. Nonetheless, the transition from experimental research to routine clinical application requires robust regulatory frameworks and strategies to address these challenges. This review aims to explore the potential of CRISPR and multiomics technologies to revolutionize SLE diagnosis and therapy, emphasizing their integration with AI to advance personalized care. By addressing existing barriers, the review envisions a future where precision medicine transforms SLE management, paving the way for individualized, patient-centered autoimmune therapy.},
}

Humans
*Lupus Erythematosus, Systemic/genetics/diagnosis/therapy/immunology
*Precision Medicine/methods
*Gene Editing/methods
*Genomics/methods
Artificial Intelligence
*CRISPR-Cas Systems
Predictive Value of Tests
*Proteomics/methods
Phenotype
Genetic Predisposition to Disease
*Genetic Therapy/methods
Multiomics

@article {pmid40268754,
year = {2025},
author = {Liu, Y and Yao, F and Zou, J and Guo, D and Jiang, W and Fan, J and Li, R and Yang, Z and Ma, Y and Deng, H and Huang, J and Tan, L},
title = {RPAD locus controls prostrate growth habit in Oryza nivara.},
journal = {The plant genome},
volume = {18},
number = {2},
pages = {e70032},
pmid = {40268754},
issn = {1940-3372},
support = {2023YFF1000401//National Key Research and Development Program of China/ ; 32125029//National Natural Science Foundation of China/ ; PC2023A01003//Pinduoduo-China Agricultural University Research Fund/ ; },
mesh = {*Oryza/genetics/growth & development ; *Quantitative Trait Loci ; Domestication ; CRISPR-Cas Systems ; Chromosomes, Plant/genetics ; Transcription Factors/genetics ; Chromosome Mapping ; Plant Breeding ; Plant Proteins/genetics ; },
abstract = {The development of ideal plant architecture is crucial for optimizing grain yield in crop breeding. The transition from prostrate growth habit in wild rice to erect growth habit in cultivated rice is one of the important events during rice domestication. Here, we identified a yield-related quantitative trait locus (QTL) cluster on the short arm of chromosome 7 using Teqing/W2014 (Oryza nivara) derived BC3F6 population. The introgression line TIL81 containing this QTL cluster exhibited significantly larger tiller angle, increased tiller numbers, and prostrate growth habit compared to the recipient parent Teqing. Using a segregating F2 population derived from a cross between TIL81 and Teqing, this yield-related QTL cluster was mapped to a similar position as the known rice plant architecture domestication (RPAD) locus controlling rice plant architecture domestication. CRISPR/Cas9-mediated genome (where CRISPR is clustered regularly interspaced short palindromic repeats) editing of four zinc finger transcription factors (OnZnF1, OnZnF6, OnZnF8, and OnZnF9) within the RPAD locus demonstrated their collective involvement in regulating plant architecture and yield-related traits. Notably, the knockout lines harboring all four zinc finger gene mutations exhibited plant architecture traits and grain yield per plant comparable to the control Teqing. These findings demonstrated that RPAD locus in O. nivara functions in prostrate growth habit and provided new insights into the molecular mechanism of plant architecture during rice domestication.},
}

*Oryza/genetics/growth & development
*Quantitative Trait Loci
Domestication
CRISPR-Cas Systems
Chromosomes, Plant/genetics
Transcription Factors/genetics
Chromosome Mapping
Plant Breeding
Plant Proteins/genetics

@article {pmid40268745,
year = {2025},
author = {Zhang, X and Ma, D and Liu, F},
title = {CRISPR Technology and Its Emerging Applications.},
journal = {Genomics, proteomics & bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1093/gpbjnl/qzaf034},
pmid = {40268745},
issn = {2210-3244},
abstract = {The discovery and iteration of clustered regularly interspaced short palindromic repeats (CRISPR) systems have revolutionized genome editing due to their remarkable efficiency and easy programmability, enabling precise manipulation of genomic elements. Owing to these unique advantages, CRISPR technology has the transformative potential to elucidate biological mechanisms and clinical treatments. This review provides a comprehensive overview of the development and applications of CRISPR technology. After describing the three primary CRISPR-Cas systems-CRISPR-associated protein 9 (Cas9) and Cas12a targeting DNA, and Cas13 targeting RNA-which serve as the cornerstone for technological advancements, we describe a series of novel CRISPR-Cas systems that offer new avenues for research, and then explore the applications of CRISPR technology in large-scale genetic screening, lineage tracing, genetic diagnosis, and gene therapy. As this technology evolves, it holds significant promise for studying gene functions and treating human diseases in the near future.},
}

@article {pmid40268248,
year = {2025},
author = {Biber, J and Gandor, C and Becirovic, E and Michalakis, S},
title = {Retina-directed gene therapy: Achievements and remaining challenges.},
journal = {Pharmacology & therapeutics},
volume = {},
number = {},
pages = {108862},
doi = {10.1016/j.pharmthera.2025.108862},
pmid = {40268248},
issn = {1879-016X},
abstract = {Gene therapy is an innovative medical approach that offers new treatment options for congenital and acquired diseases by transferring, correcting, inactivating or regulating genes to supplement, replace or modify a gene function. The approval of voretigene neparvovec (Luxturna), a gene therapy for RPE65-associated retinopathy, has marked a milestone for the field of retinal gene therapy, but has also helped to accelerate the development of gene therapies for genetic diseases affecting other organs. Voretigene neparvovec is a vector based on adeno-associated virus (AAV) that delivers a functional copy of RPE65 to supplement the missing function of this gene. The AAV-based gene delivery has proven to be versatile and safe for the transfer of genetic material to retinal cells. However, challenges remain in treating additional inherited as well as acquired retinopathies with this technology. Despite the high level of activity in this field, no other AAV gene therapy for retinal diseases has been approved since voretigene neparvovec. Ongoing research focuses on overcoming the current restraints through innovative strategies like AAV capsid engineering, dual-AAV vector systems, or CRISPR/Cas-mediated genome editing. Additionally, AAV gene therapy is being explored for the treatment of complex acquired diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) by targeting molecules involved in the pathobiology of the degenerative processes. This review outlines the current state of retinal gene therapy, highlighting ongoing challenges and future directions.},
}

@article {pmid40146774,
year = {2025},
author = {Fidelito, G and Todorovski, I and Cluse, L and Vervoort, SJ and Taylor, RA and Watt, MJ},
title = {Lipid-metabolism-focused CRISPR screens identify enzymes of the mevalonate pathway as essential for prostate cancer growth.},
journal = {Cell reports},
volume = {44},
number = {4},
pages = {115470},
doi = {10.1016/j.celrep.2025.115470},
pmid = {40146774},
issn = {2211-1247},
mesh = {Male ; *Mevalonic Acid/metabolism ; *Prostatic Neoplasms/pathology/genetics/metabolism/enzymology ; Humans ; Cell Line, Tumor ; *Lipid Metabolism/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Cell Proliferation ; Mice ; *Dimethylallyltranstransferase/metabolism/genetics ; Endoplasmic Reticulum Stress ; },
abstract = {Dysregulated lipid metabolism plays an important role in prostate cancer, although the understanding of the essential regulatory processes in tumorigenesis is incomplete. We employ a CRISPR-Cas9 screen using a custom human lipid metabolism knockout library to identify essential genes for prostate cancer survival. Screening in three prostate cancer cell lines reveals 63 shared dependencies, with enrichment in terpenoid backbone synthesis and N-glycan biosynthesis. Independent knockout of key genes of the mevalonate pathway reduces cell proliferation. Further investigation focuses on NUS1, a subunit of cis-prenyltransferase required for dolichol synthesis. NUS1 knockout decreases tumor growth in vivo and viability in patient-derived xenograft (PDX)-derived organoids. Mechanistic studies reveal that loss of NUS1 promotes oxidative stress, lipid peroxidation and ferroptosis sensitivity, endoplasmic reticulum (ER) stress, and G1 cell-cycle arrest, and it dampens androgen receptor (AR) signaling, collectively leading to growth arrest. This study highlights the critical role of the mevalonate-dolichol-N-glycan biosynthesis pathway, particularly NUS1, in prostate cancer survival and growth.},
}

Male
*Mevalonic Acid/metabolism
*Prostatic Neoplasms/pathology/genetics/metabolism/enzymology
Humans
Cell Line, Tumor
*Lipid Metabolism/genetics
Animals
*CRISPR-Cas Systems/genetics
Cell Proliferation
Mice
*Dimethylallyltranstransferase/metabolism/genetics
Endoplasmic Reticulum Stress

@article {pmid40267200,
year = {2025},
author = {Brumage, L and Best, S and Hippe, DS and Grunblatt, E and Chanana, P and Wu, F and Lee, MC and Ying, Z and Ibrahim, A and Chung, JH and Vigil, A and Fatherree, J and Beronja, S and Paddison, P and Sullivan, L and Nabet, B and MacPherson, D},
title = {In vivo functional screens reveal KEAP1 loss as a driver of chemoresistance in small cell lung cancer.},
journal = {Science advances},
volume = {11},
number = {17},
pages = {eadq7084},
doi = {10.1126/sciadv.adq7084},
pmid = {40267200},
issn = {2375-2548},
mesh = {*Small Cell Lung Carcinoma/drug therapy/genetics ; Humans ; *Drug Resistance, Neoplasm ; *Kelch-Like ECH-Associated Protein 1/genetics ; DNA, Complementary/analysis ; Proto-Oncogene Proteins c-myc/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Signal Transduction ; *Lung Neoplasms/drug therapy/genetics ; Male ; Female ; Immunologic Deficiency Syndromes ; HEK293 Cells ; },
abstract = {Exquisitely chemosensitive initially, small cell lung cancer (SCLC) exhibits dismal outcomes owing to rapid transition to chemoresistance. Elucidating the genetic underpinnings has been challenging owing to limitations with cellular models. As SCLC patient-derived xenograft (PDX) models mimic therapeutic responses, we perform genetic screens in chemosensitive PDX models to identify drivers of chemoresistance. cDNA overexpression screens identify MYC, MYCN, and MYCL, while CRISPR deletion screens identify KEAP1 loss as driving chemoresistance. Deletion of KEAP1 switched a chemosensitive SCLC PDX model to become chemoresistant and resulted in sensitivity to inhibition of glutamine metabolism. Data from the IMpower133 clinical trial revealed ~6% of patients with extensive-stage SCLC exhibit KEAP1 genetic alterations, with activation of a KEAP1/NRF2 transcriptional signature associated with reduced survival upon chemotherapy treatment. While roles for KEAP1/NRF2 have been unappreciated in SCLC, our genetic screens revealed KEAP1 loss as a driver of chemoresistance, while patient genomic analyses demonstrate clinical importance.},
}

*Small Cell Lung Carcinoma/drug therapy/genetics
Humans
*Drug Resistance, Neoplasm
*Kelch-Like ECH-Associated Protein 1/genetics
DNA, Complementary/analysis
Proto-Oncogene Proteins c-myc/metabolism
Animals
Mice
CRISPR-Cas Systems
Signal Transduction
*Lung Neoplasms/drug therapy/genetics
Male
Female
Immunologic Deficiency Syndromes
HEK293 Cells

@article {pmid40266686,
year = {2025},
author = {Zeng, Y and Tan, X and Xiao, P and Gao, P and Wang, L and Zhang, A},
title = {Natronobacterium gregoryi Argonaute inhibits class 1 integron integrase-mediated excision and integration.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
doi = {10.1093/nar/gkaf248},
pmid = {40266686},
issn = {1362-4962},
support = {2021YFD1800402//National Key Research and Development Program of China/ ; 32172848//National Natural Science Foundation of China/ ; },
mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; *Integrases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; Protein Binding ; *Integrons/genetics ; },
abstract = {Argonaute (Ago) proteins, ubiquitous in all domains of life, serve as key components in defense against foreign nucleic acids. While eukaryotic Agos (eAgos) are well characterized for guide RNA-mediated RNA targeting, prokaryotic Agos (pAgos) exhibit diverse functions, particularly in protecting bacteria from invasive DNA. The previous study identified Class 1 integron integrase (IntI-1), a tyrosine site-specific recombinase involved in horizontal transfer of antibiotic resistance genes, as a potential interaction partner of Natronobacterium gregoryi Argonaute (NgAgo), a member of pAgos. Here, we demonstrated that this interaction was direct, depended on the PIWI domain, and was independent of the catalytic activity of NgAgo. Notably, no interaction occurred between NgAgo and Cre (another tyrosine site-specific recombinase), highlighting the specificity of NgAgo-IntI-1 interaction. Furthermore, NgAgo could inhibit binding of IntI-1 to its target DNA, and then impede IntI-1-mediated integration and excision. Consistent with the above finding, few pAgos could be found in prokaryotic genomes containing IntI, whereas IntI showed significant co-occurrence with another bacterial defense system, CRISPR-Cas. In summary, our study elucidated a novel defense mechanism of pAgos through interaction with IntI-1 for inhibiting IntI-1-mediated gene excision/integration process.},
}

*Argonaute Proteins/metabolism/genetics/chemistry
*Integrases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics/chemistry
Protein Binding
*Integrons/genetics

@article {pmid40265805,
year = {2025},
author = {Han, X and Deng, Z and Liu, H and Ji, X},
title = {Current Advancement and Future Prospects in Simplified Transformation-Based Plant Genome Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {6},
pages = {},
doi = {10.3390/plants14060889},
pmid = {40265805},
issn = {2223-7747},
support = {32370432//National Natural Science Foundation of China/ ; },
abstract = {Recent years have witnessed remarkable progress in plant biology, driven largely by the rapid evolution of CRISPR/Cas-based genome editing (GE) technologies. These tools, including versatile CRISPR/Cas systems and their derivatives, such as base editors and prime editors, have significantly enhanced the universality, efficiency, and convenience of plant functional genomics, genetics, and molecular breeding. However, traditional genetic transformation methods are essential for obtaining GE plants. These methods depend on tissue culture procedures, which are time-consuming, labor-intensive, genotype-dependent, and challenging to regenerate. Here, we systematically outline current advancements in simplifying plant GE, focusing on the optimization of tissue culture process through developmental regulators, the development of in planta transformation methods, and the establishment of nanomaterial- and viral vector-based delivery platforms. We also discuss critical challenges and future directions for achieving genotype-independent, tissue culture-free plant GE.},
}

@article {pmid40264801,
year = {2025},
author = {Zueva, AS and Shevchenko, AI and Medvedev, SP and Elisaphenko, EA and Sleptcov, AA and Nazarenko, MS and Tmoyan, NA and Zakian, SM and Zakharova, IS},
title = {Isogenic induced pluripotent stem cell line ICGi036-A-1 from a patient with familial hypercholesterolaemia, derived by correcting a pathogenic variant of the gene LDLR c.530C>T.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {2},
pages = {189-199},
doi = {10.18699/vjgb-25-22},
pmid = {40264801},
issn = {2500-0462},
abstract = {Familial hypercholesterolaemia is a common monogenic disorder characterized by high plasma cholesterol levels leading to chronic cardiovascular disease with high risk and often early manifestation due to atherosclerotic lesions of the blood vessels. The atherosclerotic lesions in familial hypercholesterolaemia are mainly caused by pathogenic variants of the low-density lipoprotein receptor (LDLR) gene, which plays an important role in cholesterol metabolism. Normally, cholesterol-laden low-density lipoproteins bind to the LDLR receptor on the surface of liver cells to be removed from the bloodstream by internalisation with hepatocytes. In familial hypercholesterolaemia, the function of the receptor is impaired and the uptake of low-density lipoproteins is significantly reduced. As a result, cholesterol accumulates in the subendothelial space on the inner wall of blood vessels, triggering atherogenesis, the formation of atherosclerotic plaques. At present, there are no effective and universal approaches to the diagnosis and treatment of familial hypercholesterolaemia. A relevant approach to study the molecular genetic mechanisms of the disease and to obtain systems for screening chemical compounds as potential drugs is the generation of cellular models based on patient-specific induced pluripotent stem cells. The aim of our work was to derive an isogenic genetically modified induced pluripotent stem cell line by correcting the pathogenic allelic variant c.530C of the LDLR gene in the original iPSC previously obtained from a compound heterozygote patient with familial hypercholesterolaemia. The resulting isogenic iPSC line differs from the original by only one corrected nucleotide substitution, allowing us to study the direct effect of this pathogenic genetic variant on physiological changes in relevant differentiated cells. CRISPR/Cas-mediated base editing was used to correct the single nucleotide substitution. The resulting genetically modified iPSC line has pluripotency traits, a normal karyotype, a set of short tandem repeats identical to that in the original line and can be used to obtain differentiated derivatives necessary for the elaboration of relevant cell models.},
}

@article {pmid40264105,
year = {2025},
author = {Yahata, T and Toujima, S and Sasaki, I and Iwahashi, N and Fujino, M and Nishioka, K and Noguchi, T and Tanizaki-Horiuchi, Y and Kaisho, T and Ino, K},
title = {Adeno-associated virus-clustered regularly interspaced short palindromic repeats/cas9‑mediated ovarian cancer treatment targeting PD-L1.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {749},
pmid = {40264105},
issn = {1471-2407},
mesh = {Female ; Animals ; *B7-H1 Antigen/genetics/antagonists & inhibitors/metabolism ; *Ovarian Neoplasms/therapy/genetics/immunology/pathology ; *Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Humans ; Cell Line, Tumor ; Genetic Vectors/genetics ; Immunotherapy/methods ; },
abstract = {The response rate of antibody therapy targeting immune checkpoint molecules in ovarian cancer is insufficient. This study aimed to develop a novel gene immunotherapy model targeting programmed death ligand 1 (PD-L1) in vivo in ovarian cancer using adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and investigate its efficacy. In vitro, we produced PD-L1-AAV particles to knock out PD-L1. PD-L1-AAV particles were transduced into the murine ovarian cancer cell line ID8. PD-L1 expression at the cellular level was significantly decreased following treatment with PD-L1-AAV particles compared with control-AAV particles. In the peritoneal dissemination model, the survival time was significantly longer in the PD-L1-AAV particles intraperitoneally injected group than that in the control group. Furthermore, intratumoral lymphocyte recruitment was analyzed by immunohistochemistry, and the number of intratumoral CD4[+] and CD8[+] T cells was significantly higher, whereas that of Foxp3[+] Treg cells was significantly lower in the PD-L1-AAV particles injected group than in the control group. No severe adverse events in normal organs, such as the lungs, spleen, liver, and kidney, were observed. These results suggest that PD-L1-targeted therapy by genome editing using AAV-CRISPR/Cas9 is a novel gene-immune therapeutic strategy for ovarian cancer.},
}

Female
Animals
*B7-H1 Antigen/genetics/antagonists & inhibitors/metabolism
*Ovarian Neoplasms/therapy/genetics/immunology/pathology
*Dependovirus/genetics
Mice
*CRISPR-Cas Systems
*Genetic Therapy/methods
Humans
Cell Line, Tumor
Genetic Vectors/genetics
Immunotherapy/methods

@article {pmid40264050,
year = {2025},
author = {Pang, KL and Li, P and Yao, XR and Xiao, WT and Ren, X and He, JY},
title = {Deciphering a proliferation-essential gene signature based on CRISPR-Cas9 screening to predict prognosis and characterize the immune microenvironment in HNSCC.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {756},
pmid = {40264050},
issn = {1471-2407},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; Prognosis ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/pathology/mortality ; *Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Head and Neck Neoplasms/genetics/immunology/pathology/mortality ; Cell Proliferation/genetics ; *Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Female ; *Genes, Essential ; Gene Expression Profiling ; Male ; Gene Regulatory Networks ; },
abstract = {BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive malignancy with a poor prognosis. Identifying reliable prognostic biomarkers and therapeutic targets is crucial for improving patient outcomes. This study aimed to systematically identify proliferation-essential genes (PEGs) associated with HNSCC prognosis using CRISPR-Cas9 screening data.

METHODS: CRISPR-Cas9 screening data from the DepMap database were used to identify PEGs in HNSCC cells. A prognostic PEGs signature was constructed using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) Cox regression, and multivariate Cox regression analyses. The predictive accuracy of the signature was validated in internal and external datasets. Weighted gene co-expression network analysis (WGCNA), gene set enrichment analysis (GSEA), and immune infiltration analysis were used to investigate the underlying mechanism between high and low-risk patients. Random forest analysis and functional experiments were conducted to investigate the role of key proliferation essential genes in HNSCC progression.

RESULTS: A total of 1511 PEGs were identified. A seven-gene prognostic PEGs signature (MRPL33, NAT10, PSMC1, PSMD11, RPN2, TAF7, and ZNF335) was developed and validated, demonstrating robust prognostic performance in stratifying HNSCC patients by survival risk. WGCNA and GSEA analyses revealed a marked downregulation of immune-related pathways in high-risk patients. Immune infiltration analysis validated those high-risk patients had reduced immune scores, stromal scores, and ESTIMATE scores, as well as decreased infiltration of multiple immune cell types. Among the identified genes, PSMC1 was highlighted as a pivotal regulator of HNSCC proliferation and migration, as confirmed by functional experiments.

CONCLUSIONS: This study identifies a novel PEGs signature that effectively predicts HNSCC prognosis and stratifies patients by survival risk. PSMC1 was identified as a key gene promoting malignant progression, offering potential as a therapeutic target for HNSCC.},
}

Humans
Prognosis
*Squamous Cell Carcinoma of Head and Neck/genetics/immunology/pathology/mortality
*Tumor Microenvironment/immunology/genetics
*CRISPR-Cas Systems
*Head and Neck Neoplasms/genetics/immunology/pathology/mortality
Cell Proliferation/genetics
*Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic
Female
*Genes, Essential
Gene Expression Profiling
Male
Gene Regulatory Networks

@article {pmid40263874,
year = {2025},
author = {Huang, X and Li, R and Xu, J and Kang, J and Chen, X and Han, B and Xue, Y},
title = {Integrated multi-omics uncover viruses, active fermenting microbes and their metabolic profiles in the Daqu microbiome.},
journal = {Food research international (Ottawa, Ont.)},
volume = {208},
number = {},
pages = {116061},
doi = {10.1016/j.foodres.2025.116061},
pmid = {40263874},
issn = {1873-7145},
mesh = {*Fermentation ; *Microbiota ; Fungi/metabolism/genetics ; *Bacteria/metabolism/genetics ; *Viruses/genetics/classification ; Metagenomics ; Food Microbiology ; *Fermented Foods/microbiology/virology ; Bacteriophages/genetics ; *Metabolome ; Multiomics ; },
abstract = {The coexistence and coevolution of viruses and fermenting microbes have a significant impact on the structure and function of microbial communities. Although the presence of viruses in Daqu, the fermentation starter for Chinese Baijiu, has been documented, their specific effects on the community composition and metabolic functions of low, medium, and high-temperature Daqu remain unclear. In this study, we employed multi-omics technology to explore the distribution of viruses and active bacteria and fungi in various Daqu and their potential metabolic roles. Viral metagenomic sequencing showed a predominance of Parvoviridae in High-Temperature Daqu (HTQ), while Genomoviridae were dominant in Medium-Temperature Daqu (MTQ) and Low- Temperature Daqu (LTQ). Phages belonging to the Siphoviridae, Podoviridae, Herelleviridae, and Myoviridae families showed significantly different abundances across three Daqu groups. Metatranscriptomic analysis showed that fungal communities were most active in LTQ, whereas bacterial communities were dominant in MTQ and HTQ. By employing the CRISPR-Cas spacer, a higher predicted number of phage-host linkages was identified in LTQ, particularly with hosts including Lactobacillus, Staphylococcus, Acinetobacter, Enterobacter, and Bacillus. Correlation analysis showed that bacteria like Acinetobacter, Lactobacillus, and Streptococcus exhibited the strongest associations with metabolites, particularly amino acids and organic acids. The potential phage-induced metabolic differences in the three Daqu groups were mainly linked to pathways involved in the metabolism of amino acids, sugars, and organic acids. Overall, our study elucidates the impact of viruses on shaping microbial composition and influencing metabolic functions in Daqu. These results improve our comprehension of viruses and microbes in Daqu microbial communities and provide valuable insights for enhancing quality control in Daqu production.},
}

*Fermentation
*Microbiota
Fungi/metabolism/genetics
*Bacteria/metabolism/genetics
*Viruses/genetics/classification
Metagenomics
Food Microbiology
*Fermented Foods/microbiology/virology
Bacteriophages/genetics
*Metabolome
Multiomics

@article {pmid40263707,
year = {2025},
author = {Zhu, Z and Li, X and Ding, L and Wu, T},
title = {Exploring the effect of activator topology on CRISPR-Cas12a trans-cleavage activity.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
doi = {10.1093/nar/gkaf311},
pmid = {40263707},
issn = {1362-4962},
support = {22474045//National Natural Science Foundation of China/ ; //Fundamental Research Funds for the Central Universities/ ; 2024JYCXJJ010//Huazhong University of Science and Technology/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {The CRISPR-Cas12a system is widely used in nucleic acid detection and biosensing due to its high sensitivity, selectivity, and simple design. However, traditional CRISPR-Cas12a sensors, which rely on linear activators, face challenges such as limited operability and low stability. This study explored the impact of three different activator topologies-linear, planar, and steric-on the trans-cleavage activity of Cas12a. We developed a Cas12a-based switch using a planar activator, which demonstrated superior operability and maintained higher activity compared to linear activators. Using this planar activator, we achieved highly sensitive detection of hypochlorous acid, with a detection limit as low as 88 nM, outperforming chemical probe-based methods. The introduction of topological activators will open new avenues for the development of CRISPR-Cas12a-based biosensors, offering broad potential for diverse applications.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Bacterial Proteins/metabolism/chemistry/genetics

@article {pmid40263616,
year = {2025},
author = {Wood, TWP and Henriques, WS and Cullen, HB and Romero, M and Blengini, CS and Sarathy, S and Sorkin, J and Bekele, H and Jin, C and Kim, S and Wang, X and Laureau, R and Chemiakine, A and Khondker, RC and Isola, JVV and Stout, MB and Gennarino, VA and Mogessie, B and Jain, D and Schindler, K and Suh, Y and Wiedenheft, B and Berchowitz, LE},
title = {The retrotransposon-derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.},
journal = {Nature aging},
volume = {},
number = {},
pages = {},
pmid = {40263616},
issn = {2662-8465},
support = {R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; R35 GM146725/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM146725/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; NA//Irma T. Hirschl Trust (Irma T. Hirschl Charitable Trust)/ ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 AG069742/AG/NIA NIH HHS/United States ; R01 AG069742/AG/NIA NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 NS109858/NS/NINDS NIH HHS/United States ; R01 NS109858/NS/NINDS NIH HHS/United States ; 213470/A/18/Z//Wellcome Trust (Wellcome)/ ; NA//M.J. Murdock Charitable Trust (Murdock Charitable Trust)/ ; },
abstract = {Almost half of the human genome consists of retrotransposons-'parasitic' sequences that insert themselves into the host genome via an RNA intermediate. Although most of these sequences are silenced or mutationally deactivated, they can present opportunities for evolutionary innovation: mutation of a deteriorating retrotransposon can result in a gene that provides a selective advantage to the host in a process termed 'domestication'[1-3]. The PNMA family of gag-like capsid genes was domesticated from an ancient vertebrate retrotransposon of the Metaviridae clade at least 100 million years ago[4,5]. PNMA1 and PNMA4 are positively regulated by the master germ cell transcription factors MYBL1 and STRA8, and their transcripts are bound by the translational regulator DAZL during gametogenesis[6]. This developmental regulation of PNMA1 and PNMA4 expression in gonadal tissue suggested to us that they might serve a reproductive function. Through the analysis of donated human ovaries, genome-wide association studies (GWASs) and mouse models, we found that PNMA1 and PNMA4 are necessary for the maintenance of a normal reproductive lifespan. These proteins self-assemble into capsid-like structures that exit human cells, and we observed large PNMA4 particles in mouse male gonadal tissue that contain RNA and are consistent with capsid formation.},
}

@article {pmid40263581,
year = {2025},
author = {Weiss, T and Kamalu, M and Shi, H and Li, Z and Amerasekera, J and Zhong, Z and Adler, BA and Song, MM and Vohra, K and Wirnowski, G and Chitkara, S and Ambrose, C and Steinmetz, N and Sridharan, A and Sahagun, D and Banfield, JF and Doudna, JA and Jacobsen, SE},
title = {Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {40263581},
issn = {2055-0278},
support = {2334027//National Science Foundation (NSF)/ ; },
abstract = {Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation[1]. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.},
}

@article {pmid40233430,
year = {2025},
author = {Li, Z and Wang, N and Wang, H and Yang, L and Li, Y and Gu, L and Fu, C and Yue, P and Yu, H},
title = {Single-cell transcriptomics reveals the mechanisms of lung injury induced by galt gene editing in mouse.},
journal = {Biochemical and biophysical research communications},
volume = {763},
number = {},
pages = {151780},
doi = {10.1016/j.bbrc.2025.151780},
pmid = {40233430},
issn = {1090-2104},
mesh = {Animals ; Mice ; *Gene Editing ; *Single-Cell Analysis ; *Lung Injury/genetics/pathology/metabolism ; *Transcriptome ; Lung/pathology/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Galactosemia, caused by mutations in the GALT gene, leads to multi-organ damage. This study investigates the impact of Galt c.847 + 1G > T mutation on lung tissue using single-cell transcriptomics. We employed CRISPR/Cas9 to generate a Galt gene-edited mouse model with the Galt c. 847 + 1G > T mutation and assessed Galt expression through PCR and Western blotting. Histopathological analysis revealed significant structural lung changes, including alveolar congestion and inflammation. Single-cell RNA sequencing demonstrated a marked reduction in immune cells (NK, T, macrophages, B cells) and an increase in alveolar type II cells, vascular endothelial cells, and myofibroblasts in the GAL mouse. The increased abundance of alveolar type II cells indicated impaired differentiation and repair. Metabolic analysis revealed significant abnormalities linked to Galt c.847 + 1G > T mutation, with disruptions in TGF-β1, FGF, and Mif pathways contributing to cellular dysfunction and exacerbated lung injury. This model provides insights into the molecular mechanisms of lung injury in galactosemia, highlighting significant alterations in lung cell populations and key signaling pathways.},
}

Animals
Mice
*Gene Editing
*Single-Cell Analysis
*Lung Injury/genetics/pathology/metabolism
*Transcriptome
Lung/pathology/metabolism
Mice, Inbred C57BL
Disease Models, Animal
CRISPR-Cas Systems

@article {pmid40209323,
year = {2025},
author = {Zhang, Y and Lu, Z and Yang, H and Cheng, L and Zaklyazminskaya, E and Sokolova, O and Tan, H and Zhang, JZ},
title = {Generation of stable Cas9-EGFP expressing human induced pluripotent stem cell lines based on SeLection by Essential-gene Exon Knock-in technology.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103710},
doi = {10.1016/j.scr.2025.103710},
pmid = {40209323},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Exons/genetics ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Gene Editing ; *Genes, Essential ; Cell Differentiation ; *CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {Here, we used SeLection by Essential-gene Exon Knock-in technology to generate the iPSC line with constitutive expression of Cas9-EGFP, while retaining all functions of the essential gene. Cas9-EGFP was inserted into the GAPDH exon9 via the homologous recombination, avoiding Cas9 silencing that often occurs during iPSC differentiation. The edited cell line shows precise knock-in locus with the typical characteristics and pluripotency of iPSCs. Therefore, this iPSC line is valuable for CRISPR screening or related experiments and could be widely used in the CRISPR/Cas9-based gene editing.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Exons/genetics
*Gene Knock-In Techniques/methods
*CRISPR-Cas Systems/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Gene Editing
*Genes, Essential
Cell Differentiation
*CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid40205037,
year = {2025},
author = {Fielden, J and Siegner, SM and Gallagher, DN and Schröder, MS and Dello Stritto, MR and Lam, S and Kobel, L and Schlapansky, MF and Jackson, SP and Cejka, P and Jost, M and Corn, JE},
title = {Comprehensive interrogation of synthetic lethality in the DNA damage response.},
journal = {Nature},
volume = {640},
number = {8060},
pages = {1093-1102},
pmid = {40205037},
issn = {1476-4687},
mesh = {Humans ; *Synthetic Lethal Mutations/genetics ; *DNA Damage/genetics ; DNA-Binding Proteins/metabolism ; Proliferating Cell Nuclear Antigen/metabolism ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Chromosome Breakage ; Flap Endonucleases/metabolism/deficiency/genetics ; Proteolysis ; Genomic Instability/genetics ; Homeostasis/genetics ; *DNA Repair/genetics ; },
abstract = {The DNA damage response (DDR) is a multifaceted network of pathways that preserves genome stability[1,2]. Unravelling the complementary interplay between these pathways remains a challenge[3,4]. Here we used CRISPR interference (CRISPRi) screening to comprehensively map the genetic interactions required for survival during normal human cell homeostasis across all core DDR genes. We captured known interactions and discovered myriad new connections that are available online. We defined the molecular mechanism of two of the strongest interactions. First, we found that WDR48 works with USP1 to restrain PCNA degradation in FEN1/LIG1-deficient cells. Second, we found that SMARCAL1 and FANCM directly unwind TA-rich DNA cruciforms, preventing catastrophic chromosome breakage by the ERCC1-ERCC4 complex. Our data yield fundamental insights into genome maintenance, provide a springboard for mechanistic investigations into new connections between DDR factors and pinpoint synthetic vulnerabilities that could be exploited in cancer therapy.},
}

Humans
*Synthetic Lethal Mutations/genetics
*DNA Damage/genetics
DNA-Binding Proteins/metabolism
Proliferating Cell Nuclear Antigen/metabolism
CRISPR-Cas Systems/genetics
Endonucleases/metabolism
Chromosome Breakage
Flap Endonucleases/metabolism/deficiency/genetics
Proteolysis
Genomic Instability/genetics
Homeostasis/genetics
*DNA Repair/genetics

@article {pmid40199100,
year = {2025},
author = {Su, M and Zhang, HS and Liu, H and Yang, K and Ying, ZM},
title = {Allosteric ribozyme-driven crRNA switch for the amplification-free detection of biomolecules.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117450},
doi = {10.1016/j.bios.2025.117450},
pmid = {40199100},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *RNA, Catalytic/chemistry/genetics ; Humans ; *MicroRNAs/blood/genetics/isolation & purification/analysis ; *Adenosine Triphosphate/isolation & purification/blood/analysis ; Allosteric Regulation ; CRISPR-Cas Systems/genetics ; Limit of Detection ; CRISPR-Associated Proteins/genetics/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Currently, CRISPR-mediated biosensors are concentrating on the design of the crRNA or the activator strand to regulate the trans-cleavage activity of Cas12a. Herein, we report an allosteric ribozyme-driven crRNA switch-regulated CRISPR/Cas12a sensor for amplification-free detection of biomolecules. An allosteric ribozyme is meticulously engineered to connect the target recognition sequence with the 5' binding arm of the hammerhead ribozyme, resulting in the formation of a hairpin structure through complementary hybridization. The presence of target induces the conformational change in the allosteric module and disrupts the hairpin structure, restoring multiple-turnover cleavage RNA activity of ribozyme. Then, the activated ribozyme specifically cuts the cleavage site of the substrate-locked crRNA and releases the native crRNA to initiate CRISPR/Cas12a functions for signal reporting. The reported biosensor exhibited high sensitivity and excellent specificity for miR-155 and adenosine triphosphate (ATP) detection, giving the detection limits of 256 fM and 160 nM, respectively. For clinical validation, our proposed strategy can quantify miR-155 expression levels in cells and serum of cancer patients. Furthermore, we also demonstrate that the allosteric ribozyme-driven crRNA switch can be easily compatible with lateral flow assays, realizing visualization and the portable monitoring of target. Hence, the biosensor not only has outstanding potential in point-of-care testing, but also enables the detection of various biomolecules by flexibly substituting target recognition sequences for molecular diagnosis in the clinic.},
}

*Biosensing Techniques/methods
*RNA, Catalytic/chemistry/genetics
Humans
*MicroRNAs/blood/genetics/isolation & purification/analysis
*Adenosine Triphosphate/isolation & purification/blood/analysis
Allosteric Regulation
CRISPR-Cas Systems/genetics
Limit of Detection
CRISPR-Associated Proteins/genetics/chemistry
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40194617,
year = {2025},
author = {Wu, Z and Xu, Y and Zhou, W and Shi, L and Shi, W and Pu, L and Jiang, J},
title = {Rapid detection of Klebsiella pneumoniae based on one-tube RPA-CRISPR/Cas12a system.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {573},
number = {},
pages = {120281},
doi = {10.1016/j.cca.2025.120281},
pmid = {40194617},
issn = {1873-3492},
mesh = {*Klebsiella pneumoniae/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Time Factors ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Klebsiella pneumoniae (KP) is a prevalent pathogen implicated in both community-acquired and nosocomial infections, often leading to severe clinical outcomes. The conventional methods for KP identification are characterized by intricacy and suboptimal efficiency. In this research, we have engineered a novel One-Tube RPA- CRISPR/Cas12a system, integrating recombinase polymerase amplification (RPA) method with the CRISPR/Cas12a diagnostic platform, to facilitate the detection of K. pneumoniae. To minimize the likelihood of aerosol-based contamination, the RPA components are positioned at the base of the tube, while the CRISPR/Cas12a components are placed at the tube's cap. The systems are combined post-RPA amplification through a brief centrifugation step, ensuring that RPA reactions are conducted independently to produce an adequate amount of target DNA before interaction with the CRISPR/Cas12a system. This method was validated using both fluorescent and lateral flow strip assays, achieving a limit of detection (LOD) of 10[0] copies/μL and 10[1] copies/μL respectively. The specificity for KP detection was found to be 100 %. Furthermore, the system demonstrated a positivity rate of 78 % (18/23) when directly extracting DNA from sputum samples, corroborated by culture and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The simplicity and rapidity of the assay are augmented by a straightforward sample processing without extraction. The complete assay duration from specimen receipt to result is approximately 40 min, significantly reducing the turnaround time (TAT). Collectively, this system presents a streamlined, expeditious, and highly specific diagnostic approach for the detection of Klebsiella pneumoniae strains.},
}

*Klebsiella pneumoniae/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Humans
*Recombinases/metabolism
Limit of Detection
Time Factors
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40194350,
year = {2025},
author = {Hu, H and Xue, H and Dong, K and Li, Y and Liu, P and Wang, H and Li, L and Xiao, X and Chen, H},
title = {Strand displacement-enhanced CRISPR-Cas13a system for ultra-specific detection of RNA single nucleotide variation.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117445},
doi = {10.1016/j.bios.2025.117445},
pmid = {40194350},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Polymorphism, Single Nucleotide/genetics ; *COVID-19/virology/diagnosis ; *MicroRNAs/genetics/analysis ; *RNA, Viral/genetics/analysis ; Limit of Detection ; },
abstract = {RNA plays a critical role in biological systems, mediating genetic information transfer and regulating gene expression. However, RNA is susceptible to variations from endogenous and exogenous sources, with potentially profound biological consequences. The CRISPR-Cas13a system has emerged as a promising tool for RNA variation detection due to its cost-effectiveness, sensitivity, and user-friendly nature. Despite this, designing a simple, universal system with high discrimination factor (DF) for single-nucleotide variations remains a challenge. Here, we present the strand displacement-enhanced Cas13a single-nucleotide variation detection assay (SECND), a sensitive, universal, and easy-to-implement method with a high DF for RNA variations. Using SECND, we detected 5 types of single-nucleotide variations, achieving a maximum DF of 1083.2. We validated the assay's effectiveness on miRNA and SARS-CoV-2 genomic RNA simulants, incorporating a 4-way strand displacement mechanism to enhance detection limits to 10 pmol/L and 50 pmol/L, and to identify variations at frequencies as low as 0.01 % and 0.1 %. Additionally, we demonstrated SECND's utility in quantifying single-nucleotide variants of miR-200b and miR-200c in ovarian cancer and retinal glioma cells. This versatile tool not only advances RNA variation detection but also has significant implications for disease research, diagnostics, and viral classification, enhancing our understanding of the CRISPR-Cas13a system and its potential applications.},
}

*CRISPR-Cas Systems/genetics
Humans
*SARS-CoV-2/genetics/isolation & purification
*Biosensing Techniques/methods
*Polymorphism, Single Nucleotide/genetics
*COVID-19/virology/diagnosis
*MicroRNAs/genetics/analysis
*RNA, Viral/genetics/analysis
Limit of Detection

@article {pmid40179699,
year = {2025},
author = {Hu, F and Zhang, Y and Yang, Y and Peng, L and Cui, S and Ma, Q and Wang, F and Wang, X},
title = {A rapid and ultrasensitive RPA-assisted CRISPR-Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117428},
doi = {10.1016/j.bios.2025.117428},
pmid = {40179699},
issn = {1873-4235},
mesh = {Humans ; *Smartphone/instrumentation ; *SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation ; *Biosensing Techniques/instrumentation ; *COVID-19 Nucleic Acid Testing/instrumentation ; Equipment Design ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The spread of infectious diseases can be controlled by early identification of the source of infection and timely diagnosis to stop transmission. Real-time fluorescence quantitative polymerase chain reaction (PCR) is the current gold standard for pathogen diagnosis, with high detection sensitivity and accuracy. However, due to the need for specialized equipment, laboratories, and personnel, it is difficult to achieve rapid and immediate diagnosis during large-scale infectious disease outbreaks. Herein, an optimized CRISPR-based nucleic acid detection method was developed that reduces the CRISPR detection time to 15 min while maintaining high sensitivity. By using nucleic acid extraction-free and lyophilization techniques, the 'sample-in-result-out' detection of the two target genes of SARS-CoV-2, the human internal reference gene, and the negative quality control sample can be completed in 20 min, with a sensitivity of 0.5 copies/μL. Additionally, to facilitate the application, a smartphone-based reverse transcription-recombinase polymerase amplification (RT-RPA)-assisted CRISPR-rapid, portable nucleic acid detection device was developed, integrating functions such as heating, centrifugation, mixing, optical detection and result output. Process control, output, and uploading of detection results were conducted through smartphones. The device is not dependent on a power supply and can perform on-site rapid virus detection in resource-limited settings. Real-time uploading of results helps to rapidly implement epidemic prevention and control measures, providing an innovative means of detection, control, and prevention of virus-based infectious diseases. This important work provides a new and effective tool to manage potential future outbreaks of infectious diseases.},
}

Humans
*Smartphone/instrumentation
*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*COVID-19/diagnosis/virology
*Nucleic Acid Amplification Techniques/instrumentation
*Biosensing Techniques/instrumentation
*COVID-19 Nucleic Acid Testing/instrumentation
Equipment Design
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40157320,
year = {2025},
author = {Li, J and Yin, L and Wang, C and Xu, Y},
title = {Generation of a homozygous ABCA7 knockout cell line (AHMUCNi002-A) in human iPSCs using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103700},
doi = {10.1016/j.scr.2025.103700},
pmid = {40157320},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *ATP-Binding Cassette Transporters/genetics/metabolism/deficiency ; Homozygote ; Cell Line ; *Gene Knockout Techniques ; Gene Editing ; },
abstract = {ABCA7, located on chromosome 19, encodes an ATP-binding cassette transporter. Loss-of-function variants of ABCA7 are associated with an increased risk of Alzheimer's disease. To explore the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, CRISPR/Cas9 genome-editing technology was utilized to generate a homozygous ABCA7 knockout in human induced pluripotent stem cells (hiPSCs). The resulting ABCA7 knockout cell line exhibited normal pluripotency, a stable karyotype, and the ability to differentiate into all three germ layers.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*ATP-Binding Cassette Transporters/genetics/metabolism/deficiency
Homozygote
Cell Line
*Gene Knockout Techniques
Gene Editing

@article {pmid40147061,
year = {2025},
author = {Zhou, H and Liu, Y and Chen, Q and Huang, K and Ren, PG},
title = {Generation of TP53 knock out induced pluripotent stem cell using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103699},
doi = {10.1016/j.scr.2025.103699},
pmid = {40147061},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Tumor Suppressor Protein p53/genetics/deficiency/metabolism ; Animals ; Mice ; Humans ; *Gene Knockout Techniques ; Cell Line ; Teratoma/pathology ; },
abstract = {The TP53 gene is an important tumor suppressor gene. Through CRISPR/Cas9 technology, we have established a TP53 gene knockout cell line in iPSCs (SIIBRi001-A). This cell line maintains normal stem cell-like morphology, karyotype, expresses markers of pluripotency, and is capable of generating teratomas in immunodeficient mice. Quantitative analysis of pluripotency gene expression remains normal. This cell line can be utilized for studying the mechanisms underlying tumorigenesis.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Tumor Suppressor Protein p53/genetics/deficiency/metabolism
Animals
Mice
Humans
*Gene Knockout Techniques
Cell Line
Teratoma/pathology

@article {pmid40120557,
year = {2025},
author = {Ludwik, KA and Opitz, R and Jyrch, S and Megges, M and Kühnen, P and Stachelscheid, H},
title = {Correction of the Allan-Herndon-Dudley syndrome-causing SLC16A2 mutation G401R in a patient derived hiPSC line.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103698},
doi = {10.1016/j.scr.2025.103698},
pmid = {40120557},
issn = {1876-7753},
mesh = {Humans ; *X-Linked Intellectual Disability/genetics/pathology/therapy ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monocarboxylic Acid Transporters/genetics/metabolism ; *Muscle Hypotonia/genetics/pathology/therapy ; *Muscular Atrophy/genetics/pathology/therapy ; Cell Line ; *Mutation ; Symporters/genetics ; Male ; CRISPR-Cas Systems ; Gene Editing ; Mutation, Missense ; },
abstract = {The X-linked Allan-Herndon-Dudley syndrome (AHDS) is a genetic disorder characterized by severe psychomotor impairment, resulting from mutations in the SLC16A2 gene, which encodes the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). Previously, we established a hiPSC line from a patient carrying the SLC16A2:R401G mutation (BIHi045-A). Using CRISPR/Cas9-mediated gene editing, we targeted exon 3 of SLC16A2 and used single-stranded oligodeoxynucleotides as homology-directed repair templates to correct the R401G missense mutation, generating an isogenic control cell line.},
}

Humans
*X-Linked Intellectual Disability/genetics/pathology/therapy
*Induced Pluripotent Stem Cells/metabolism/cytology
*Monocarboxylic Acid Transporters/genetics/metabolism
*Muscle Hypotonia/genetics/pathology/therapy
*Muscular Atrophy/genetics/pathology/therapy
Cell Line
*Mutation
Symporters/genetics
Male
CRISPR-Cas Systems
Gene Editing
Mutation, Missense

@article {pmid40108474,
year = {2025},
author = {Yuan, S and Sun, R and Shi, H and Chapman, NM and Hu, H and Guy, C and Rankin, S and Kc, A and Palacios, G and Meng, X and Sun, X and Zhou, P and Yang, X and Gottschalk, S and Chi, H},
title = {VDAC2 loss elicits tumour destruction and inflammation for cancer therapy.},
journal = {Nature},
volume = {640},
number = {8060},
pages = {1062-1071},
pmid = {40108474},
issn = {1476-4687},
mesh = {Animals ; *Voltage-Dependent Anion Channel 2/deficiency/genetics/metabolism ; Mice ; Interferon-gamma/immunology/pharmacology ; *Neoplasms/immunology/therapy/pathology/genetics ; Membrane Proteins/metabolism ; *Inflammation/immunology/pathology/genetics ; Humans ; Mitochondria/metabolism/pathology ; Signal Transduction ; Female ; *Immunotherapy ; Tumor Microenvironment/immunology ; bcl-2 Homologous Antagonist-Killer Protein/metabolism ; Nucleotidyltransferases/metabolism ; Cell Line, Tumor ; Male ; CRISPR-Cas Systems/genetics ; CD8-Positive T-Lymphocytes/immunology ; BH3 Interacting Domain Death Agonist Protein/metabolism ; Mice, Inbred C57BL ; },
abstract = {Tumour cells often evade immune pressure exerted by CD8[+] T cells or immunotherapies through mechanisms that are largely unclear[1,2]. Here, using complementary in vivo and in vitro CRISPR-Cas9 genetic screens to target metabolic factors, we established voltage-dependent anion channel 2 (VDAC2) as an immune signal-dependent checkpoint that curtails interferon-γ (IFNγ)-mediated tumour destruction and inflammatory reprogramming of the tumour microenvironment. Targeting VDAC2 in tumour cells enabled IFNγ-induced cell death and cGAS-STING activation, and markedly improved anti-tumour effects and immunotherapeutic responses. Using a genome-scale genetic interaction screen, we identified BAK as the mediator of VDAC2-deficiency-induced effects. Mechanistically, IFNγ stimulation increased BIM, BID and BAK expression, with VDAC2 deficiency eliciting uncontrolled IFNγ-induced BAK activation and mitochondrial damage. Consequently, mitochondrial DNA was aberrantly released into the cytosol and triggered robust activation of cGAS-STING signalling and type I IFN response. Importantly, co-deletion of STING signalling components dampened the therapeutic effects of VDAC2 depletion in tumour cells, suggesting that targeting VDAC2 integrates CD8[+] T cell- and IFNγ-mediated adaptive immunity with a tumour-intrinsic innate immune-like response. Together, our findings reveal VDAC2 as a dual-action target to overcome tumour immune evasion and establish the importance of coordinately destructing and inflaming tumours to enable efficacious cancer immunotherapy.},
}

Animals
*Voltage-Dependent Anion Channel 2/deficiency/genetics/metabolism
Mice
Interferon-gamma/immunology/pharmacology
*Neoplasms/immunology/therapy/pathology/genetics
Membrane Proteins/metabolism
*Inflammation/immunology/pathology/genetics
Humans
Mitochondria/metabolism/pathology
Signal Transduction
Female
*Immunotherapy
Tumor Microenvironment/immunology
bcl-2 Homologous Antagonist-Killer Protein/metabolism
Nucleotidyltransferases/metabolism
Cell Line, Tumor
Male
CRISPR-Cas Systems/genetics
CD8-Positive T-Lymphocytes/immunology
BH3 Interacting Domain Death Agonist Protein/metabolism
Mice, Inbred C57BL

@article {pmid40088038,
year = {2025},
author = {Blomme, J and Arraiza Ribera, J and De Clerck, O and Jacobs, TB},
title = {Consolidating Ulva functional genomics: gene editing and new selection systems.},
journal = {The New phytologist},
volume = {246},
number = {4},
pages = {1710-1723},
doi = {10.1111/nph.70068},
pmid = {40088038},
issn = {1469-8137},
support = {BOF/STA/202209/016//Bijzonder Onderzoeksfonds UGent/ ; BOF20/PDO/016//Bijzonder Onderzoeksfonds UGent/ ; 9329//Gordon and Betty Moore Foundation/ ; 12T3418N//Fonds Wetenschappelijk Onderzoek/ ; G015623N//Fonds Wetenschappelijk Onderzoek/ ; G0AHR24N//Fonds Wetenschappelijk Onderzoek/ ; GOH3817N//Fonds Wetenschappelijk Onderzoek/ ; I001621N//Fonds Wetenschappelijk Onderzoek/ ; },
mesh = {*Ulva/genetics/microbiology ; *Gene Editing/methods ; *Genomics/methods ; *Selection, Genetic ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; },
abstract = {The green seaweed Ulva compressa is a promising model for functional biology. In addition to historical research on growth and development, -omics data and molecular tools for stable transformation are available. However, more efficient tools are needed to study gene function. Here, we expand the molecular toolkit for Ulva. We screened the survival of Ulva and its mutualistic bacteria on 14 selective agents and established that Blasticidin deaminases (BSD or bsr) can be used as selectable markers to generate stable transgenic lines. We show that Cas9 and Cas12a RNPs are suitable for targeted mutagenesis and can generate genomic deletions of up to 20 kb using the marker gene ADENINE PHOSPHORIBOSYLTRANSFERASE (APT). We demonstrate that the targeted insertion of a selectable marker via homology-directed repair or co-editing with APT is possible for nonmarker genes. We evaluated 31 vector configurations and found that the bicistronic fusion of Cas9 to a resistance marker or the incorporation of introns in Cas9 led to the most mutants. We used this to generate mutants in three nonmarker genes using a co-editing strategy. This expanded molecular toolkit now enables us to reliably make gain- and loss-of-function mutants; additional optimizations will be necessary to allow for vector-based multiplex genome editing in Ulva.},
}

*Ulva/genetics/microbiology
*Gene Editing/methods
*Genomics/methods
*Selection, Genetic
CRISPR-Cas Systems/genetics
Mutation/genetics