@article {pmid40208959,
year = {2025},
author = {Baca, CF and Majumder, P and Hickling, JH and Patel, DJ and Marraffini, LA},
title = {Cat1 forms filament networks to degrade NAD[+] during the type III CRISPR-Cas antiviral response.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {eadv9045},
doi = {10.1126/science.adv9045},
pmid = {40208959},
issn = {1095-9203},
abstract = {Type III CRISPR-Cas systems defend against viral infection in prokaryotes using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligo-adenylate (cOA) messengers to activate CARF immune effectors. Here we investigated a protein containing a CARF domain fused Toll/interleukin-1 receptor (TIR) domain, Cat1. We found that Cat1 provides immunity by cleaving and depleting NAD[+] molecules from the infected host, inducing a growth arrest that prevents viral propagation. Cat1 forms dimers that stack upon each other to generate long filaments that are maintained by bound cOA ligands, with stacked TIR domains forming the NAD[+] cleavage catalytic sites. Further, Cat1 filaments assemble into unique trigonal and pentagonal networks that enhance NAD[+] degradation. Cat1 presents an unprecedented chemistry and higher-order protein assembly for the CRISPR-Cas response.},
}

@article {pmid40207628,
year = {2025},
author = {Yuan, K and Xi, X and Han, S and Han, J and Zhao, B and Wei, Q and Zhou, X},
title = {Selict-seq profiles genome-wide off-target effects in adenosine base editing.},
journal = {Nucleic acids research},
volume = {53},
number = {7},
pages = {},
pmid = {40207628},
issn = {1362-4962},
support = {2023YFC3402200//National Key R&D Program of China/ ; 92153303//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Adenosine/genetics/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Inosine/genetics ; Genome, Human ; Anemia, Sickle Cell/genetics ; Mutation ; },
abstract = {Adenosine base editors (ABEs) facilitate A·T to G·C base pair conversion with significant therapeutic potential for correcting pathogenic point mutations in human genetic diseases, such as sickle cell anemia and β-thalassemia. Unlike CRISPR-Cas9 systems that induce double-strand breaks, ABEs operate through precise deamination, avoiding chromosomal instability. However, the off-target editing effects of ABEs remain inadequately characterized. In this study, we present a biochemical method Selict-seq, designed to evaluate genome-wide off-target editing by ABEs. Selict-seq specifically captures deoxyinosine-containing single-stranded DNA and precisely identifies deoxyadenosine-to-deoxyinosine (dA-to-dI) mutation sites, elucidating the off-target effects induced by ABEs. Through investigations involving three single-guide RNAs, we identified numerous unexpected off-target edits both within and outside the protospacer regions. Notably, ABE8e(V106W) exhibited distinct off-target characteristics, including high editing rates (>10%) at previously unreported sites (e.g. RNF2 and EMX1) and out-of-protospacer mutations. These findings significantly advance our understanding of the off-target landscape associated with ABEs. In summary, our approach enables an unbiased analysis of the ABE editome and provides a widely applicable tool for specificity evaluation of various emerging genome editing technologies that produce intermediate products as deoxyinosine.},
}

*Gene Editing/methods
Humans
*Adenosine/genetics/metabolism
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Inosine/genetics
Genome, Human
Anemia, Sickle Cell/genetics
Mutation

@article {pmid40207041,
year = {2025},
author = {Hoffmann, S and Seeger, T},
title = {Advances in human induced pluripotent stem cell (hiPSC)-based disease modelling in cardiogenetics.},
journal = {Medizinische Genetik : Mitteilungsblatt des Berufsverbandes Medizinische Genetik e.V},
volume = {37},
number = {2},
pages = {137-146},
pmid = {40207041},
issn = {1863-5490},
abstract = {Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level. hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms. In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles. Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.},
}

@article {pmid40205022,
year = {2025},
author = {Gracia-Rodriguez, C and Martínez-Medina, AE and Torres-Cosio, L and Lopez-Ortiz, C and Nimmakayala, P and Luévanos-Escareño, MP and Hernández-Almanza, AY and Castro-Alonso, MJ and Sosa-Martínez, JD and Reddy, UK and Balagurusamy, N},
title = {Can the molecular and transgenic breeding of crops be an alternative and sustainable technology to meet food demand?.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {83},
pmid = {40205022},
issn = {1438-7948},
mesh = {*Crops, Agricultural/genetics/growth & development ; *Plant Breeding/methods ; *Plants, Genetically Modified/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {The gradual increase in the worldwide population represents various challenges, and one of the most alarming being the food demand. Historically technological advances led to the development of crops that meets the requirements and demands. Currently, molecular breeding unlocks the genetic potential of crops for their improvement, positioning it as a key technology for the development of new crops. The implementation of OMICs sciences, such spatial and single cell transcriptomics is providing a large and precise information, which can be exploited for crop improvement related to increasing yield, improving the nutritional value; designing new strategies for diseases resistance and management and for conserving biodiversity. Furthermore, the use of new technologies such CRISPR/CAS9 brought us the ability to modify the selected regions of the genome to select the superior's genotypes at a short time and the use of artificial intelligence aid in the analysis of big data generated by OMICS sciences. On the other hand, the application of molecular improvement technologies open up discussion on global regulatory measures, the socio-economic and socio-ethics, as the frameworks on its global regulation and its impact on the society create the public perception on its acceptance. In this review, the use and impact of OMICs sciences and genetic engineering in crops development, the regulatory measures, the socio-economic impact and as well as the mediatic information on genetically modified crops worldwide is discussed along with comprehensive insights on the potential of molecular plant breeding as an alternative and sustainable technology to meet global food demand.},
}

*Crops, Agricultural/genetics/growth & development
*Plant Breeding/methods
*Plants, Genetically Modified/genetics
Gene Editing
CRISPR-Cas Systems

@article {pmid40205014,
year = {2025},
author = {Tan, JYP and Thevendran, R and Quek, YS and Maheswaran, S},
title = {Studying the cellular efficacy and tolerability of using CRISPR-gRNA ribonucleoprotein (RNP) complex for in-vitro knockdown of TRIB2 in acute myeloid leukaemia (AML) cells as preliminary clinical evaluations.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {379},
pmid = {40205014},
issn = {1573-4978},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques/methods ; Cell Line, Tumor ; *Calcium-Calmodulin-Dependent Protein Kinases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Intracellular Signaling Peptides and Proteins/genetics ; },
abstract = {BACKGROUND: Acute Myeloid Leukemia (AML) is a complex, genetically driven cancer disease with several mutations that complicate therapy regimes. The Tribbles gene family, specifically the TRIB2 gene, has garnered substantial interest as a crucial oncogenic factor of AML progression. Studying cancer through gene expression studies and focusing on the primary oncogenes provide accurate information for future therapies. Here, CRISPR stands as the most exceptional tool used to explore both oncogene functionalities and therapeutic utilities. The ribonucleoprotein (RNP) mode of CRISPR formation and usage are preferred compared to plasmid-mediated CRISPR systems, however, it can lead to complications post-transfection to sensitive cellular entities such as human cancer cells in-vitro, transcribing to similar outcomes in-vivo as well. Therefore, this study describes the use of in-house designed CRISPR-RNP systems targeting the TRIB2 oncogene and evaluates their post-transfection cellular safety and efficacy aspects for future clinical applications.

METHODS: This study uses a designed guide RNA targeting the TRIB2 gene assembled via In-vitro synthesis. The gRNA with Cas9 protein leads to the formation of CRISPR-RNP structures, which target and cleave the TRIB2 gene. The assembled CRISPR-RNP system is transfected into target AML cell and control cell lines (i.e. HEK cells), and the subsequent gene cleavage and resulting changes to the AML cells in terms of cellular safety/tolerability and gene knockdown efficacy were studied via RT-qPCR, flow cytometry, and cell viability analysis.

RESULTS: The outcome demonstrates the well-tolerated transfection of the in-vitro assembled CRISPR RNP system with no signs of cellular toxicity and disruptions towards the AML cell's metabolic activities, promoting the safety aspects of CRISPR RNP post-transfection to human cells. The study further highlights the in-vitro efficacy of the CRISPR RNP in targeting the TRIB2 oncogene, where a statistically significant gene knockdown of more than 80% was detected via qPCR analysis of TRIB2 gene expression with minimal to no background effects from individual RNP components, equating to their targeted gene cleavage effects. In addition, the CRISPR TRIB2 gene knockdown also indicated the possibilities of induced AML cell death measured via flow cytometry markers, translating to favourable outcomes in eliminating cancerous growths.

CONCLUSIONS: This study research contributes to the refinement of the CRISPR-RNP strategy and preliminary evaluation for future clinical uses.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/metabolism
*Ribonucleoproteins/genetics/metabolism
CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockdown Techniques/methods
Cell Line, Tumor
*Calcium-Calmodulin-Dependent Protein Kinases/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Intracellular Signaling Peptides and Proteins/genetics

@article {pmid40204947,
year = {2025},
author = {Andel, D and Nouwens, AJ and Klaassen, S and Laoukili, J and Viergever, B and Verheem, A and Intven, MPW and Zandvliet, M and Hagendoorn, J and Borel Rinkes, IHM and Kranenburg, O},
title = {Rational design of alternative treatment options for radioresistant rectal cancer using patient-derived organoids.},
journal = {British journal of cancer},
volume = {},
number = {},
pages = {},
pmid = {40204947},
issn = {1532-1827},
abstract = {BACKGROUND: Resistance to radiation therapy is a common challenge in the field of oncology. Cancer cells with an increased ability to effectively repair DNA or cells with higher levels of antioxidants are more resistant to radiation. As cancer cells rely on these traits for survival, they may offer vulnerabilities that could be exploited.

METHODS: In the current study, rectal cancer organoids that showed different responses to radiation treatment were identified. RNA sequencing was used to compare radioresistant and radiosensitive organoids. In vitro combination drug screens were performed. The selection of drugs was guided by the RNA sequencing results.

RESULTS: Radioresistant organoids exhibited superior transcriptional adaptability and activated more DNA repair pathways when irradiated. Additionally, radioresistant organoids displayed enhanced antioxidant metabolism, including pathways related to the detoxification of reactive oxygen species and the synthesis of glutathione. Combinatorial drug screens identified the combination of RRx-001 (an inducer of oxidative stress) with GCLC inhibitor BSO as a highly effective and synergistic drug combination in killing radioresistant organoids. CRISPR-CAS-mediated knockout of GCLC sensitised organoids to RRx-001.

CONCLUSION: Combining RRx-001 with the inhibition of GCLC may be a promising alternative treatment strategy in radioresistant rectal cancer.},
}

@article {pmid40204795,
year = {2025},
author = {Wang, Y and Cheng, Y and Sun, H and Wang, Z and Chen, N and Shi, C and Liu, H and Yang, J and Xu, Y},
title = {Genome editing in spinocerebellar ataxia type 3 cells improves Golgi apparatus structure.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {12106},
pmid = {40204795},
issn = {2045-2322},
support = {82001973//the Youth Fund of National Natural Science Foundation of China grant/ ; SBGJ2020003017//the Youth Project Co-established by Henan Province and the Ministry grant/ ; YXKC2020057//the Health Science and Technology Innovation Excellent Youth Training Project in Henan province grant/ ; 2017YFA0105000//National Key R&D Program of China grant/ ; U1904207//the National Natural Science Foundation of China grants/ ; },
mesh = {*Machado-Joseph Disease/genetics/therapy/pathology ; *Gene Editing/methods ; *Golgi Apparatus/metabolism/genetics/ultrastructure ; Humans ; *Ataxin-3/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; Mutation ; Repressor Proteins ; },
abstract = {Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract, referred to as mutant ataxin-3. In this study, we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First, we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently, the pluripotency of GE-iPSCs was confirmed, and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons, restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells), and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion, our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.},
}

*Machado-Joseph Disease/genetics/therapy/pathology
*Gene Editing/methods
*Golgi Apparatus/metabolism/genetics/ultrastructure
Humans
*Ataxin-3/genetics/metabolism
Induced Pluripotent Stem Cells/metabolism
CRISPR-Cas Systems
Mutation
Repressor Proteins

@article {pmid40204736,
year = {2025},
author = {Antoniou, P and Dacquay, L and Mårtensson, H and Madeyski-Bengtson, K and Loyd, AL and Shiriaeva, A and Gordon, E and Mustfa, S and Thom, G and Hsieh, PP and Šviković, S and Firth, M and Akrap, N and Maresca, M and Peterka, M},
title = {Modified pegRNAs mitigate scaffold-derived prime editing by-products.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3374},
pmid = {40204736},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA-Directed DNA Polymerase/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Prime editors (PEs) employ reverse transcriptase (RT) to install genomic edits using a template within the prime editing guide RNA (pegRNA). RT creates a 3' genomic flap containing the intended edit. However, reverse transcription can continue beyond the template, incorporating the pegRNA scaffold sequence into the 3' flap. These scaffold-derived by-products can be installed alongside the intended edit, reducing prime editing precision. Here, we develop a method that prevents RT from accessing the scaffold, thereby mitigating such by-products. We demonstrate that an internal abasic spacer or 2'-O-methylation within the pegRNAs terminates RT at the end of the template. This prevents scaffold-derived sequences from being incorporated into the target locus. We benchmark these pegRNAs in different cell types and demonstrate that they can be used with processive PEs such as PE6d or PE**. Our findings provide a simple approach to mitigate a common prime editing by-product and improve prime editing precision.},
}

*Gene Editing/methods
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*RNA-Directed DNA Polymerase/metabolism/genetics
CRISPR-Cas Systems
HEK293 Cells

@article {pmid40203649,
year = {2025},
author = {Moyo, B and Brown, LBC and Khondaker, II and Bao, G},
title = {Engineering adeno-associated viral vectors for CRISPR/Cas based in vivo therapeutic genome editing.},
journal = {Biomaterials},
volume = {321},
number = {},
pages = {123314},
doi = {10.1016/j.biomaterials.2025.123314},
pmid = {40203649},
issn = {1878-5905},
abstract = {The recent approval of the first gene editing therapy for sickle cell disease and transfusion-dependent beta-thalassemia by the U.S. Food and Drug Administration (FDA) demonstrates the immense potential of CRISPR (clustered regularly interspaced short palindromic repeats) technologies to treat patients with genetic disorders that were previously considered incurable. While significant advancements have been made with ex vivo gene editing approaches, the development of in vivo CRISPR/Cas gene editing therapies has not progressed as rapidly due to significant challenges in achieving highly efficient and specific in vivo delivery. Adeno-associated viral (AAV) vectors have shown great promise in clinical trials as vehicles for delivering therapeutic transgenes and other cargos but currently face multiple limitations for effective delivery of gene editing machineries. This review elucidates these challenges and highlights the latest engineering strategies aimed at improving the efficiency, specificity, and safety profiles of AAV-packaged CRISPR/Cas systems (AAV-CRISPR) to enhance their clinical utility.},
}

@article {pmid40114442,
year = {2025},
author = {Gregor, A and Distel, L and Ekici, AB and Kirchner, P and Uebe, S and Krumbiegel, M and Turan, S and Winner, B and Zweier, C},
title = {Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11-deficiency.},
journal = {HGG advances},
volume = {6},
number = {2},
pages = {100425},
doi = {10.1016/j.xhgg.2025.100425},
pmid = {40114442},
issn = {2666-2477},
mesh = {Humans ; Animals ; *Neurons/metabolism/cytology/drug effects ; *F-Box Proteins/genetics/metabolism ; Phenotype ; *Proteasome Endopeptidase Complex/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Drosophila ; CRISPR-Cas Systems ; Protein-Arginine N-Methyltransferases ; },
abstract = {Haploinsufficiency of FBXO11, encoding a ubiquitin ligase complex subunit, is associated with a variable neurodevelopmental disorder. So far, the underlying nervous system-related pathomechanisms are poorly understood, and specific therapies are lacking. Using a combined approach, we established an FBXO11-deficient human stem cell-based neuronal model using CRISPR-Cas9 and a Drosophila model using tissue-specific knockdown techniques. We performed transcriptomic analyses on iPSC-derived neurons and molecular phenotyping in both models. RNA sequencing revealed disrupted transcriptional networks related to processes important for neuronal development, such as differentiation, migration, and cell signaling. Consistently, we found that loss of FBXO11 leads to neuronal phenotypes such as impaired neuronal migration and abnormal proliferation/differentiation balance in human cultured neurons and impaired dendritic development and behavior in Drosophila. Interestingly, application of three different proteasome-activating substances could alleviate FBXO11-deficiency-associated phenotypes in both human neurons and flies. One of these substances is the long-approved drug Verapamil, opening the possibility of drug repurposing in the future. Our study shows the importance of FBXO11 for neurodevelopment and highlights the reversibility of related phenotypes, opening an avenue for potential development of therapeutic approaches through drug repurposing.},
}

Humans
Animals
*Neurons/metabolism/cytology/drug effects
*F-Box Proteins/genetics/metabolism
Phenotype
*Proteasome Endopeptidase Complex/metabolism
Induced Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
Drosophila
CRISPR-Cas Systems
Protein-Arginine N-Methyltransferases

@article {pmid40202350,
year = {2025},
author = {Dudley, EG},
title = {The E. coli CRISPR-Cas conundrum: are they functional immune systems or genomic singularities?.},
journal = {EcoSal Plus},
volume = {},
number = {},
pages = {eesp00402020},
doi = {10.1128/ecosalplus.esp-0040-2020},
pmid = {40202350},
issn = {2324-6200},
abstract = {The discovery and subsequent characterization and applications of CRISPR-Cas is one of the most fascinating scientific stories from the past two decades. While first identified in Escherichia coli, this microbial workhorse often took a back seat to other bacteria during the early race to detail CRISPR-Cas function as an adaptive immune system. This was not a deliberate slight, but the result of early observations that the CRISPR-Cas systems found in E. coli were not robust phage defense systems as first described in Streptococcus thermophilus. This apparent lack of activity was discovered to result from transcriptional repression by the nucleoid protein H-NS. Despite extensive evidence arguing against such roles, some studies still present E. coli CRISPR-Cas systems in the context of anti-phage and/or anti-plasmid activities. Here, the studies that led to our understanding of its cryptic nature are highlighted, along with ongoing research to uncover potential alternative functions in E. coli.},
}

@article {pmid40202271,
year = {2025},
author = {Sun, T and He, W and Chen, X and Shu, X and Liu, W and Ouyang, G},
title = {Nanomaterials-Integrated CRISPR/Cas Systems: Expanding the Toolbox for Optical Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00020},
pmid = {40202271},
issn = {2379-3694},
abstract = {Nanomaterials-integrated CRISPR/Cas systems have rapidly emerged as powerful next-generation platforms for optical biosensing. These integrated platforms harness the precision of CRISPR/Cas-mediated nucleic acid detection while leveraging the unique properties of nanomaterials to achieve enhanced sensitivity and expanded analytical capabilities, thereby broadening their diagnostic potential. By incorporating a diverse range of nanomaterials, these systems effectively expand the analytical toolbox for optical detection, offering adaptable solutions tailored to various diagnostic challenges. This review provides a comprehensive overview of the nanomaterials successfully integrated into CRISPR/Cas-based optical sensing systems. It examines multiple optical detection modalities, including fluorescence, electrochemiluminescence, colorimetry, and surface-enhanced Raman spectroscopy, highlighting how nanomaterials facilitate signal amplification, enable multiplexing, and support the development of point-of-care applications. Additionally, practical applications of these integrated systems in critical fields such as healthcare diagnostics and environmental monitoring are showcased. While these platforms offer considerable advantages, several real-world challenges such as the complexity of assay workflows, environmental impact of nanomaterials, cost, and regulatory hurdles must be addressed before widespread implementation can be achieved. By identifying these critical obstacles and proposing strategic solutions, we aim to pave the way for the continued advancement and adoption of nanomaterial-integrated CRISPR/Cas optical biosensing technologies.},
}

@article {pmid40199340,
year = {2025},
author = {Miao, X and Cao, F and Yu, XF and Li, TP and Su, HY and Guo, J and Hu, GL and Chen, BW and You, MS and Liu, YY and Lei, GK and You, S},
title = {A zinc finger protein shapes the temperature adaptability of a cosmopolitan pest.},
journal = {Open biology},
volume = {15},
number = {4},
pages = {240346},
pmid = {40199340},
issn = {2046-2441},
support = {//The Youth Teachers Visit and Study Program at the Haixia Institute of Science and Technology/ ; //Fujian Provincial Science and Technology Department/ ; //The Natural Science Foundation for Distinguished Young Scholars of Fujian Province/ ; },
mesh = {Animals ; *Zinc Fingers ; *Insect Proteins/genetics/metabolism/chemistry ; *Moths/genetics/physiology ; *Thermotolerance/genetics ; Female ; Temperature ; Male ; *Adaptation, Physiological ; CRISPR-Cas Systems ; },
abstract = {Global climate change is characterized by increased extreme temperatures affecting insects at all trophic levels. Zinc finger proteins (ZFPs) are key regulators of gene expression and cell differentiation in eukaryotes, essential for stress resistance in both animals and plants. Using CRISPR/Cas9 for gene deletion, this study predicted and examined the structure of ZFP320 in the diamondback moth (Plutella xylostella) and investigated its function in temperature stress response through a comprehensive age-stage, two-sex life table analysis. We found ZFP320 encodes a 387 amino acid protein (43 kDa) with no transmembrane domains, featuring a ZnF-C2H2 domain. Quantitative fluorescence analysis showed that ZFP320 expression increased under high temperatures. ZFP320 knockout altered antioxidant gene expression, resulting in higher levels of superoxide dismutase and catalase in mutant strains compared with wild-type strain. Life table analysis revealed that the mutant strains had shorter fecundity and oviposition periods under both normal and high temperatures. Additionally, mutant strains exhibited lower parameters (r, λ, R0), as well as reduced survival rates and critical thermal maxima. Notably, PxZFP320 plays a crucial role in temperature adaptation, paving the way for future investigations on the significance of ZFPs in P. xylostella's temperature tolerance.},
}

Animals
*Zinc Fingers
*Insect Proteins/genetics/metabolism/chemistry
*Moths/genetics/physiology
*Thermotolerance/genetics
Female
Temperature
Male
*Adaptation, Physiological
CRISPR-Cas Systems

@article {pmid40198566,
year = {2025},
author = {Kaur, K and Biswal, AK and Kaur, R and Singh, M and Dhugga, KS and Singh, J},
title = {A Protocol for High-efficiency Transformation and Genome Editing in Elite Wheat Cultivars.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2898},
number = {},
pages = {307-320},
pmid = {40198566},
issn = {1940-6029},
mesh = {*Triticum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Genetic Vectors/genetics ; },
abstract = {CRISPR-Cas9-based editing offers a simple yet efficient method for introducing targeted mutations in the genes of interest. Low efficiency of transformation and a lack of reproducibility in editing genes in elite wheat cultivars are the bottlenecks in improving simply inherited traits. We describe a high-efficiency transformation protocol for editing elite wheat cultivars using JD633-GRF4-GIF1 CRISPR vector. This vector reduces the regeneration time of transformants to less than 90 days and enables transformation of elite cultivars.},
}

*Triticum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Transformation, Genetic
Plants, Genetically Modified/genetics
*Genome, Plant
Genetic Vectors/genetics

@article {pmid40197046,
year = {2025},
author = {Irfan, M and Solbiati, J and Duran-Pinedo, A and Rocha, FG and Gibson, FC and Frias-Lopez, J},
title = {Erratum for Irfan et al., "A Porphyromonas gingivalis hypothetical protein controlled by the type I-B CRISPR-Cas system is a novel adhesin important in virulence".},
journal = {mSystems},
volume = {},
number = {},
pages = {e0029525},
doi = {10.1128/msystems.00295-25},
pmid = {40197046},
issn = {2379-5077},
}

@article {pmid40180235,
year = {2025},
author = {Zheng, HH and Wang, LQ and Hou, CY and Song, YP and Liu, S and Zheng, LL and Ma, SJ and Chen, HY},
title = {Construction and characterization of a gE/gI/TK-gene-deleted recombinant pseudorabies virus variant expressing the GP5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and NADC30-like PRRSV.},
journal = {Microbial pathogenesis},
volume = {203},
number = {},
pages = {107522},
doi = {10.1016/j.micpath.2025.107522},
pmid = {40180235},
issn = {1096-1208},
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics/immunology/pathogenicity ; Animals ; Swine ; *Viral Envelope Proteins/genetics/immunology ; *Herpesvirus 1, Suid/genetics/immunology ; Antibodies, Viral/blood ; Porcine Reproductive and Respiratory Syndrome/prevention & control/virology/immunology ; *Viral Vaccines/immunology/genetics/administration & dosage ; Antibodies, Neutralizing/blood ; Gene Deletion ; China ; Vaccines, Synthetic/immunology/genetics ; Pseudorabies/prevention & control/virology ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) and pseudorabies (PR) are still the major problems of the worldwide pork industry. Previous studies showed that PR virus (PRV) and PRRS virus (PRRSV) commercial vaccines available in China could not provide complete protection against PRV variants and currently prevalent PRRSV strains. In the present study, a recombinant pseudorabies virus rPRV-GP5/HP-GP5/NA expressing the GP5 of the highly pathogenic PRRSV (HP-PRRSV) and NADC30-like PRRSV was constructed by transfecting the transfer plasmid pG-GP5/HP-GP5/NA-EGFP into ST cells inoculated with gE/gI/TK-gene-deleted rPRV NY-gE[-]/gI[-]/TK[-] using homologous recombination and CRISPR/Cas9 gene editing technique. The recombinant virus rPRV-GP5/HP was also constructed. The expression of the GP5 protein was confirmed by Western blot and indirect immunofluorescence assay. These two viruses were similar to the parental virus rPRV-gE[-]/gI[-]/TK[-] in terms of growth curve, morphogenesis and virus plaque sizes, and proliferated in different cell types. The animal test results showed that ELISA antibodies against PRRSV could be detected in piglets immunized with these two recombinant viruses, and the antibody levels were slightly lower than those of commercial vaccines, but these two recombinant viruses elicited high levels of PRV ELISA antibody and neutralizing antibody, as is the case with commercial vaccine. These two recombinant viruses could provide some protection against virulent PRRSV and PRV, and could effectively inhibit virus proliferation in tissues. These findings provide insights that these two viruses need to be optimally engineered as promising bivalent vaccine candidates against PRV and PRRSV for the control and eradication of the variant PRV and currently prevalent PRRSV. IMPORTANT: Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine pseudorabies virus (PRV) can infect pigs of all ages with high mortality. Due to the appearance of the PRRSV variant (HP-PRRSV and NADC30-Like-PRRSV) and the outbreak of PRV variant in China, the current commercial vaccines available cannot provide complete protection against PRV variants and prevalent PRRSV strains, which causes a major economic loss in pig industry worldwide. Therefore, safe and effective new vaccines are urgently developed to simultaneously control and even eradicate the two viruses. This study intends to use the modified attenuated PRV strain as the carrier and the main antigen gene ORF5 of PRRSV as the exogenous gene to construct the recombinant virus strain, and further validate the protective effect of recombinant strains in vitro and in vivo against the challenge of PRRSV and PRV, which is expected to become a candidate vaccine strain.},
}

*Porcine respiratory and reproductive syndrome virus/genetics/immunology/pathogenicity
Animals
Swine
*Viral Envelope Proteins/genetics/immunology
*Herpesvirus 1, Suid/genetics/immunology
Antibodies, Viral/blood
Porcine Reproductive and Respiratory Syndrome/prevention & control/virology/immunology
*Viral Vaccines/immunology/genetics/administration & dosage
Antibodies, Neutralizing/blood
Gene Deletion
China
Vaccines, Synthetic/immunology/genetics
Pseudorabies/prevention & control/virology
Cell Line
CRISPR-Cas Systems
Gene Editing

@article {pmid40151930,
year = {2025},
author = {Gao, M and Yang, C and Si, W and Xi, X and Chen, L and Zeng, Z and Rong, Y and Yang, Y and Wang, F and Yuan, C},
title = {Combining CRISPR-Cas12a with Microsphere Array-Enhanced Fluorescence for Portable Pathogen Nucleic Acid Detection.},
journal = {ACS applied materials & interfaces},
volume = {17},
number = {14},
pages = {20932-20942},
doi = {10.1021/acsami.5c00655},
pmid = {40151930},
issn = {1944-8252},
mesh = {*Microspheres ; *CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation & purification ; Fluorescence ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques ; *CRISPR-Associated Proteins/genetics ; Lab-On-A-Chip Devices ; Food Contamination/analysis ; Limit of Detection ; RNA, Viral/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The detection of food contamination in a swift and sensitive manner is essential for safeguarding public health. Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays for nucleic acid detection are renowned for their high specificity and convenient, related studies have focused on refining the Cas protein and optimizing the CRISPR (cr)RNAs design within CRISPR-based assays for enhancing the sensitivity of nucleic acid detection. Our research offers innovative insights into enhancing the fluorescence signal output intensity from a physical standpoint, thereby presenting a practical and cost-effective strategy to lower the detection thresholds in CRISPR-based assays. By a layer of microsphere arrays was spread onto the bottom of the microfluidic chip to enhance the fluorescence signal of the sample via self-assembly of the microspheres. Recombinase polymerase amplification (RPA) was used to amplify target sequences, followed by crRNA binding to activate Cas enzyme, cleaving fluorescein amidite (FAM)-labeled reporters and emitting a fluorescent signal. The method successfully identified SARS-CoV-2 positive samples (10 clinical samples and 8 environmental contamination samples) and distinguished them from negative samples. Meanwhile, it successfully detected 4 food contamination Shigella samples and 5 clinical Shigella samples. In this study, the developed method exhibited a detection limit (LoD) of 75 fM for SARS-CoV-2 (POCT with USB camera: 50 fM) and 100 fM for Shigella (POCT with USB camera: 75 fM). It also demonstrated promising sensitivity (100%) and specificity (100%) in a small-sample validation. Combined portable and automated detection was achieved using a smartphone to receive and process the fluorescent signals obtained from the samples. The detection platform developed in this study is not only applicable for the detection of pathogens in cold-chain food products, but also extends to pathogen detection in community hospitals and resource-limited areas, providing an efficient solution for rapid pathogen screening in different settings. Moreover, different nucleic acid samples can be detected by changing the RPA primer and CRISPR crRNA. This method provides a paradigm for studying enhanced fluorescence signaling and holds significant potential to advance the commercialization and practical use of CRISPR fluorescence sensors.},
}

*Microspheres
*CRISPR-Cas Systems/genetics
Humans
*SARS-CoV-2/genetics/isolation & purification
Fluorescence
*COVID-19/diagnosis/virology
Nucleic Acid Amplification Techniques
*CRISPR-Associated Proteins/genetics
Lab-On-A-Chip Devices
Food Contamination/analysis
Limit of Detection
RNA, Viral/genetics/analysis
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40120586,
year = {2025},
author = {Herger, M and Kajba, CM and Buckley, M and Cunha, A and Strom, M and Findlay, GM},
title = {High-throughput screening of human genetic variants by pooled prime editing.},
journal = {Cell genomics},
volume = {5},
number = {4},
pages = {100814},
doi = {10.1016/j.xgen.2025.100814},
pmid = {40120586},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *High-Throughput Screening Assays/methods ; *Genetic Variation/genetics ; MutL Protein Homolog 1/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; },
abstract = {Multiplexed assays of variant effect (MAVEs) enable scalable functional assessment of human genetic variants. However, established MAVEs are limited by exogenous expression of variants or constraints of genome editing. Here, we introduce a pooled prime editing (PE) platform to scalably assay variants in their endogenous context. We first improve efficiency of PE in HAP1 cells, defining optimal prime editing guide RNA (pegRNA) designs and establishing enrichment of edited cells via co-selection. We next demonstrate negative selection screening by testing over 7,500 pegRNAs targeting SMARCB1 and observing depletion of efficiently installed loss-of-function (LoF) variants. We then screen for LoF variants in MLH1 via 6-thioguanine selection, testing 65.3% of all possible SNVs in a 200-bp region including exon 10 and 362 non-coding variants from ClinVar spanning a 60-kb region. The platform's overall accuracy for discriminating pathogenic variants indicates that it will be highly valuable for identifying new variants underlying diverse human phenotypes across large genomic regions.},
}

Humans
*Gene Editing/methods
*High-Throughput Screening Assays/methods
*Genetic Variation/genetics
MutL Protein Homolog 1/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
Loss of Function Mutation

@article {pmid40118067,
year = {2025},
author = {Takesue, H and Okada, S and Doi, G and Sugiyama, Y and Kusumoto, E and Ito, T},
title = {Strategic targeting of Cas9 nickase expands tandem gene arrays.},
journal = {Cell genomics},
volume = {5},
number = {4},
pages = {100811},
doi = {10.1016/j.xgen.2025.100811},
pmid = {40118067},
issn = {2666-979X},
mesh = {Humans ; *Tandem Repeat Sequences/genetics ; *CRISPR-Cas Systems/genetics ; *Deoxyribonuclease I/metabolism/genetics ; DNA Replication/genetics ; *CRISPR-Associated Protein 9/metabolism ; Saccharomyces cerevisiae/genetics ; Animals ; },
abstract = {Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here, we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has just replicated the array, forming a single-ended double-strand break. This break is subsequently end-resected to become single stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching ∼1 Mb. Furthermore, appropriate splint DNAs allow BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.},
}

Humans
*Tandem Repeat Sequences/genetics
*CRISPR-Cas Systems/genetics
*Deoxyribonuclease I/metabolism/genetics
DNA Replication/genetics
*CRISPR-Associated Protein 9/metabolism
Saccharomyces cerevisiae/genetics
Animals

@article {pmid40112818,
year = {2025},
author = {Wala, J and Dalin, S and Webster, S and Shapira, O and Busanovich, J and Sarmashghi, S and Beroukhim, R and Bandopadhayay, P and Rendo, V},
title = {Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification.},
journal = {Cell genomics},
volume = {5},
number = {4},
pages = {100815},
doi = {10.1016/j.xgen.2025.100815},
pmid = {40112818},
issn = {2666-979X},
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; Cell Cycle Proteins/genetics ; Female ; Cell Line, Tumor ; *Gene Amplification/genetics ; *Nuclear Proteins/genetics/metabolism ; Ovarian Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Animals ; Cyclin E/genetics ; Protein Isoforms/genetics/metabolism ; Mice ; Bromodomain Containing Proteins ; },
abstract = {Recent work by the ICGC-PCAWG consortium identified recurrent focal deletions in the BRD4 gene, decreasing expression despite increased copy number. We show that these focal deletions occur in the context of cyclin E1 amplification in breast, ovarian, and endometrial cancers, and serve to disrupt BRD4 regulatory regions and gene expression across isoforms. We analyze open reading frame screen data and find that overexpression of BRD4 long (BRD4-L) and short isoform BRD4-S(a) impairs cell growth across cell lines. We confirm these results in OVSAHO ovarian cancer cells, where the overexpression of BRD4 isoforms significantly reduces tumor growth. Next, we mimic BRD4 focal deletions using CRISPR-Cas9 technology and show that these focal deletions rescue ovarian cancer cells from toxicity associated with BRD4 overexpression, suggesting that BRD4 levels must be fine-tuned for cancer cell proliferation. Our study provides experimental evidence for the first recurrent deletion reducing toxicity in cancer, expanding the landscape of cancer progression mechanisms.},
}

Humans
*Transcription Factors/genetics/metabolism
Cell Cycle Proteins/genetics
Female
Cell Line, Tumor
*Gene Amplification/genetics
*Nuclear Proteins/genetics/metabolism
Ovarian Neoplasms/genetics/pathology
Cell Proliferation/genetics
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Animals
Cyclin E/genetics
Protein Isoforms/genetics/metabolism
Mice
Bromodomain Containing Proteins

@article {pmid40097027,
year = {2025},
author = {Ahmed, S and Azli, B and Abdul Razak, M and Hair-Bejo, M and Omar, AR and Ideris, A and Mat Isa, N},
title = {Delayed nuclear localization of CRISPR/Cas9-modified fiber of fowl adenovirus serotype 8b reduces pathogenicity in Specific pathogen-free chicken embryonic liver cells.},
journal = {Microbial pathogenesis},
volume = {203},
number = {},
pages = {107482},
doi = {10.1016/j.micpath.2025.107482},
pmid = {40097027},
issn = {1096-1208},
mesh = {Animals ; *Adenoviridae Infections/veterinary/prevention & control/virology ; *Poultry Diseases/virology/prevention & control ; Chick Embryo ; *CRISPR-Cas Systems ; *Aviadenovirus/genetics/pathogenicity/immunology ; Chickens ; Specific Pathogen-Free Organisms ; Serogroup ; *Liver/virology/cytology ; *Capsid Proteins/genetics/immunology/metabolism ; Virulence ; Vaccines, Attenuated/immunology/genetics ; Viral Vaccines/immunology/genetics ; *Cell Nucleus/virology ; Gene Editing ; Malaysia ; },
abstract = {Fowl adenovirus (FAdV) poses incessant outbreaks to poultry production worldwide, and Inclusion body hepatitis (IBH) is a predominant FAdV infectious disease. Currently, limited vaccines are available in Malaysia to fight against the local predominant FAdV strain 8b isolate (FAdV-8b), posing a desperate demand for efficient vaccine development. The fiber protein of FAdV is one of the major constituents of the adenoviral capsid involved in the virulence of pathogens. Hence, the aim was to modify the fiber gene of FAdV-8b UPMT27 to develop a live attenuated FAdV vaccine via the gene-editing CRISPR/Cas9 technology. Primary specific pathogen-free (SPF) chicken embryonic liver cells (CELs) infected with the modified isolated (cfUPMT27) were reported with significantly reduced cytopathic effects, delayed viral localization into the nucleus, and low apoptotic rates. cfUPMT27 isolate also exhibited constant amino acid substitution of Y179D in subsequent passages. Meanwhile, the liver of cfUPMT27 inoculated-SPF chicken embryonic eggs (CEE) was observed with mild hydropericardium and reported with a delayed mortality at 6-days post-infection (dpi). This holistic, integrative study incorporating genetic, pathology, and immunology analysis proposed cfUPMT27 isolate as a candidate vaccine for FAdV infections, providing efficient future protection in chickens.},
}

Animals
*Adenoviridae Infections/veterinary/prevention & control/virology
*Poultry Diseases/virology/prevention & control
Chick Embryo
*CRISPR-Cas Systems
*Aviadenovirus/genetics/pathogenicity/immunology
Chickens
Specific Pathogen-Free Organisms
Serogroup
*Liver/virology/cytology
*Capsid Proteins/genetics/immunology/metabolism
Virulence
Vaccines, Attenuated/immunology/genetics
Viral Vaccines/immunology/genetics
*Cell Nucleus/virology
Gene Editing
Malaysia

@article {pmid40049338,
year = {2025},
author = {Liu, K and Hu, S and Wufuer, R and Zhang, Q and Qiu, L and Zhang, Z and Wang, M and Zhang, Y},
title = {Deficiency of DDI2 suppresses liver cancer progression by worsening cell survival conditions.},
journal = {Free radical biology & medicine},
volume = {232},
number = {},
pages = {200-213},
doi = {10.1016/j.freeradbiomed.2025.03.003},
pmid = {40049338},
issn = {1873-4596},
mesh = {*Liver Neoplasms/pathology/genetics/metabolism ; Humans ; Reactive Oxygen Species/metabolism ; Animals ; DNA Damage ; NF-E2-Related Factor 2/genetics/metabolism ; Mice ; Cell Survival/genetics ; Apoptosis/genetics ; Cell Proliferation ; CRISPR-Cas Systems ; Disease Progression ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Nuclear Respiratory Factor 1/genetics/metabolism ; *Carcinoma, Hepatocellular/pathology/genetics/metabolism ; NF-E2-Related Factor 1/genetics/metabolism ; Oxidative Stress ; },
abstract = {The levels of reactive oxygen species (ROS) and the extent of ensuing DNA damage significantly influence cancer initiation and progression. Of crucial importance, the aspartate protease DDI2 has been proposed to play a pivotal role in monitoring intracellular ROS levels (to trigger oxidative eustress or distress), as well as in the oxidative DNA damage repair, through redox homeostasis-determining factor Nrf1 (encoded by NFE2L1). However, the specific role of DDI2 in the multi-step process resulting in the development and progression of liver cancer remains elusive to date. In the present study, we employed the CRISPR/Cas9 gene editing system to create two nuanced lines of DDI2 knockout (i.e., DDI2[-/-] and DDI2[insG/-]) from liver cancer cells. Subsequent experiments indicate that the knockout of DDI2 leads to increased ROS levels in hepatoma cells by downregulating two major antioxidant transcription factors Nrf1 and Nrf2 (encoded by NFE2L2), exacerbating endogenous DNA damages caused by ROS and not-yet-identified factors, thereby inhibiting cell proliferation and promoting apoptosis, and ultimately hindering in vivo malignant growth of xenograft tumor cells. Conversely, the restoration of DDI2 expression reverses the accumulation of ROS and associated DNA damage caused by DDI2 knockout, eliminating the subsequent inhibitory effects of DDI2 deficiency on both in vitro and in vivo growth of liver cancer cells. Collectively, these findings demonstrate that DDI2 deficiency impedes liver tumor growth by disrupting its survival environment, suggesting that DDI2 may serve as a novel therapeutic target for anti-cancer strategies aimed at modulating ROS or DNA damage processes.},
}

*Liver Neoplasms/pathology/genetics/metabolism
Humans
Reactive Oxygen Species/metabolism
Animals
DNA Damage
NF-E2-Related Factor 2/genetics/metabolism
Mice
Cell Survival/genetics
Apoptosis/genetics
Cell Proliferation
CRISPR-Cas Systems
Disease Progression
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Nuclear Respiratory Factor 1/genetics/metabolism
*Carcinoma, Hepatocellular/pathology/genetics/metabolism
NF-E2-Related Factor 1/genetics/metabolism
Oxidative Stress

@article {pmid40032004,
year = {2025},
author = {Kang, H and Yang, X and Jiang, R and Gao, P and Zhang, Y and Zhou, L and Ge, X and Han, J and Guo, X and Yang, H},
title = {Ultrasensitive and visual detection of pseudorabies virus based on CRISPR-Cas12b system.},
journal = {Microbial pathogenesis},
volume = {203},
number = {},
pages = {107447},
doi = {10.1016/j.micpath.2025.107447},
pmid = {40032004},
issn = {1096-1208},
mesh = {*Herpesvirus 1, Suid/isolation & purification/genetics ; *Pseudorabies/diagnosis/virology ; Swine ; Animals ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Swine Diseases/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; *Molecular Diagnostic Techniques/methods ; Real-Time Polymerase Chain Reaction ; },
abstract = {Aujeszky's disease (AD) is an acute infectious disease that infects pigs and other animals, resulting in significant economic losses and posing a threat to human health. Reliable and rapid detection methods are essential for the prevention of AD. In this study, a RAA-Cas12b assay based on recombinase-aided amplification (RAA) and CRISPR-Cas12b system was established, optimized and evaluated for the rapid detection of wild-type Pseudorabies Virus (PRV). The results can not only be detected by real-time fluorescence readout, but also can be visualized by a portable blue light instrument. There was no cross-reaction with PRV Bartha-K61 strain or other swine infectious viruses. The analytical sensitivities of the real-time PRV RAA-Cas12b assay and visual PRV RAA-Cas12b assay were determined to be 15 copies/μL with 95 % confidence interval and 140 copies/μL with 95 % confidence interval, respectively. A total of 31 clinical samples were detected and compared with PRV qPCR assay to evaluate the diagnostic performance of the PRV RAA-Cas12b assay. The diagnostic coincidence rate of the two assays was 100 %. In summary, this convenient and reliable assay has great potential for rapid detection of wild type PRV in point-of-care testing (POCT).},
}

*Herpesvirus 1, Suid/isolation & purification/genetics
*Pseudorabies/diagnosis/virology
Swine
Animals
*CRISPR-Cas Systems
Sensitivity and Specificity
*Swine Diseases/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
Recombinases/metabolism
*Molecular Diagnostic Techniques/methods
Real-Time Polymerase Chain Reaction

@article {pmid40025867,
year = {2025},
author = {Koodamvetty, A and Thangavel, S},
title = {Advancing Precision Medicine: Recent Innovations in Gene Editing Technologies.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {14},
pages = {e2410237},
doi = {10.1002/advs.202410237},
pmid = {40025867},
issn = {2198-3844},
support = {IA/TSG/22/1/600431//DBT/Wellcome Trust India Alliance/ ; BT/PR38267/GET/119/348/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Gene Editing/methods/trends ; Humans ; *Precision Medicine/methods/trends ; *Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {The advent of gene editing has significantly advanced the field of medicine, opening new frontiers in the treatment of genetic disorders, cancer, and infectious diseases. Gene editing technology remains a dynamic and promising area of research and development. Recent advancements in protein and RNA engineering within this field have addressed critical issues such as imprecise edits, poor editing efficiency, and off-target effects. Advancements in delivery methods have allowed the achievement of therapeutic or even selection-free gene editing efficiency with reduced toxicity in primary cells, thereby enhancing the safety and efficacy of gene manipulation. This progress paves the way for transformative changes in molecular biology, medicine, and other fields. This review provides a comprehensive overview of the advancements in gene editing techniques, focusing on prime editor proteins and their engineered variants. It also explores alternative systems that expand the toolkit for precise genomic modifications and highlights the potential of these innovations in treating hematological disorders, while also discussing the limitations and challenges that remain.},
}

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

@article {pmid39965128,
year = {2025},
author = {Shen, J and Wang, Y and Liu, Y and Lan, J and Long, S and Li, Y and Chen, D and Yu, P and Zhao, J and Wang, Y and Wang, S and Yang, F},
title = {Behavioral Abnormalities, Cognitive Impairments, Synaptic Deficits, and Gene Replacement Therapy in a CRISPR Engineered Rat Model of 5p15.2 Deletion Associated With Cri du Chat Syndrome.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {14},
pages = {e2415224},
doi = {10.1002/advs.202415224},
pmid = {39965128},
issn = {2198-3844},
support = {L222077//Beijing Natural Science Foundation/ ; IS23097//Beijing Natural Science Foundation/ ; },
mesh = {Animals ; Disease Models, Animal ; Rats ; *Cognitive Dysfunction/genetics/therapy ; *Genetic Therapy/methods ; *Cri-du-Chat Syndrome/genetics/therapy ; CRISPR-Cas Systems/genetics ; Behavior, Animal/physiology ; Male ; Chromosome Deletion ; Social Behavior ; },
abstract = {The Cri du Chat Syndrome (CdCS), a devastating genetic disorder caused by a deletion on chromosome 5p, faces challenges in finding effective treatments and accurate animal models. Using CRISPR-Cas9, a novel CdCS rat model with a 2q22 deletion is developed, mirroring a common genetic alteration in CdCS patients. This model exhibits pronounced deficits in social behavior, cognition, and anxiety, accompanied by neuronal abnormalities and immune dysregulation in key brain regions such as the hippocampus and medial prefrontal cortex (mPFC). The immunostaining and RNA-seq analyses provide new insights into CdCS pathogenesis, revealing inflammatory and immune processes. Importantly, it is demonstrated that early gene replacement therapy with AAV-Ctnnd2 alleviates cognitive impairments in CdCS rats, highlighting the potential for early intervention. However, the effectiveness of this therapy is confined to the early developmental stages and does not fully restore all CdCS symptoms. The findings deepen the understanding of CdCS pathogenesis and suggest promising therapeutic directions.},
}

Animals
Disease Models, Animal
Rats
*Cognitive Dysfunction/genetics/therapy
*Genetic Therapy/methods
*Cri-du-Chat Syndrome/genetics/therapy
CRISPR-Cas Systems/genetics
Behavior, Animal/physiology
Male
Chromosome Deletion
Social Behavior

@article {pmid39960330,
year = {2025},
author = {Zheng, S and Liu, Y and Xia, X and Xiao, J and Ma, H and Yuan, X and Zhang, Y and Chen, Z and Peng, G and Li, W and Fei, JF and Liu, Y},
title = {Sequence Context-Agnostic TadA-Derived Cytosine Base Editors for Genome-Wide Editing in Zebrafish.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {14},
pages = {e2411478},
doi = {10.1002/advs.202411478},
pmid = {39960330},
issn = {2198-3844},
support = {32070819//National Natural Science Foundation of China/ ; 92268114//National Natural Science Foundation of China/ ; 31970782//National Natural Science Foundation of China/ ; 2021YFA0805000//National Key R&D Program of China/ ; 2023YFA1800600//National Key R&D Program of China/ ; DFJHBF202103//High-level Hospital Construction Project of Guangdong Provincial People's Hospital/ ; KJ012021012//High-level Hospital Construction Project of Guangdong Provincial People's Hospital/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Cytosine/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; },
abstract = {Single-nucleotide variants (SNVs) represent a significant form of genetic variation linked to various diseases. CRISPR-mediated base editing has emerged as a powerful method for modeling diseases caused by SNVs, particularly in zebrafish, which serve as an excellent platform for investigating disease mechanisms and conducting drug screenings. However, existing cytosine base editors (CBEs) for zebrafish often have broad editing windows and strong sequence preferences, limiting their effectiveness. In this study, zebrafish (z) TadA-derived cytosine base editors, termed zTadA-CBEs, are developed by introducing key mutations into the TadA8e enzyme. These novel editors demonstrate improved efficiency and precision in cytosine base editing. Specifically, zTadA-BE4max and zTadA-BEmv offer complementary editing windows, while zTadA-SpRY-BE4max allows for PAM-flexible editing. Using zTadA-CBEs, a precise disease model for Axenfeld-Rieger syndrome is established, and created two new models for Hermansky-Pudlak syndrome. Additionally, a novel albinism model carrying two pathogenic SNVs in the F0 generation is developed. By employing specifically designed sgRNA, the fms[ts±] missense mutation is corrected back to the wild-type nucleotide (C > T), successfully restoring macrophage levels to normal. These findings underscore the potential of zTadA-CBEs to enhance genome editing techniques and their applications in developing therapies for SNV-related disorders.},
}

Animals
*Zebrafish/genetics
*Gene Editing/methods
*Cytosine/metabolism
CRISPR-Cas Systems/genetics
Disease Models, Animal

@article {pmid39882846,
year = {2025},
author = {Kanie, T and Liu, B and Love, JF and Fisher, SD and Gustavsson, AK and Jackson, PK},
title = {A hierarchical pathway for assembly of the distal appendages that organize primary cilia.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
doi = {10.7554/eLife.85999},
pmid = {39882846},
issn = {2050-084X},
support = {P20GM103447/GM/NIGMS NIH HHS/United States ; 1R35GM151013/GM/NIGMS NIH HHS/United States ; R00GM134187/GM/NIGMS NIH HHS/United States ; C-2064-20210327//Welch Foundation/ ; RR200025//Cancer Prevention and Research Institute of Texas/ ; R01GM114276/GM/NIGMS NIH HHS/United States ; R01GM121565/GM/NIGMS NIH HHS/United States ; P20GM103447/GM/NIGMS NIH HHS/United States ; 1R35GM151013/GM/NIGMS NIH HHS/United States ; R00GM134187/GM/NIGMS NIH HHS/United States ; R01GM114276/GM/NIGMS NIH HHS/United States ; R01GM121565/GM/NIGMS NIH HHS/United States ; R35GM155365/GM/NIGMS NIH HHS/United States ; },
mesh = {*Cilia/metabolism ; *Centrioles/metabolism ; Humans ; CRISPR-Cas Systems ; Animals ; },
abstract = {Distal appendages are ninefold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for the formation of the primary cilium, by regulating at least four critical steps: preciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here, we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in the RAB34[+] vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.},
}

*Cilia/metabolism
*Centrioles/metabolism
Humans
CRISPR-Cas Systems
Animals

@article {pmid40196388,
year = {2025},
author = {Luo, L and Miao, L and Ma, X and Hu, J and Li, S and Yang, W and Ma, S and Chen, R and Liu, X},
title = {Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing.},
journal = {Plant direct},
volume = {9},
number = {4},
pages = {e70070},
pmid = {40196388},
issn = {2475-4455},
abstract = {Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in Nicotiana benthamiana using the CRISPR-Cas12a multiplex gene editing system. We employed two effector proteins LbCas12a and FnCas12a coupled with six guide RNAs targeting virus genome and a novel mRNA-gRNA nucleic acid complex to transport gRNA efficiently. Compared with the BSCTV accumulation in the wild-type N. benthamiana, it was reduced by more than 90% by most transgenic events derived at 7 days post-inoculation. Additionally, the shoot-tip leaves were normal in the transgenic plants, whereas they appeared severely curled and stunted in wild-type N. benthamiana at 15 days post-infection. Target sites evaluation revealed that the editing system can directly destroy the structure of BSCTV viral genomes via large fragment deletions. We quantified TMV virus accumulation in the transgenic N. benthamiana lines by monitoring dynamic changes in GFP fluorescence and quantitative analysis by qPCR showed that the CRISPR-Cas12a system can introduce TMV virus resistance to N. benthamiana by preventing its systemic spread. Our study provides an innovative strategy-an mRNA-gRNA nucleic acid complex-which has proven to be highly effective in the gene-editing system and offers an efficient antiviral approach for generating virus-resistant plants.},
}

@article {pmid40195838,
year = {2025},
author = {Tran, QG and Le, TT and Choi, DY and Cho, DH and Yun, JH and Choi, HI and Kim, HS and Lee, YJ},
title = {Progress and challenges in CRISPR/Cas applications in microalgae.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {3},
pages = {e2501028},
doi = {10.71150/jm.2501028},
pmid = {40195838},
issn = {1976-3794},
support = {RS-2024-00459155//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; },
mesh = {*Microalgae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Biotechnology/methods ; Metabolic Engineering/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have emerged as powerful tools for precise genome editing, leading to a revolution in genetic research and biotechnology across diverse organisms including microalgae. Since the 1950s, microalgal production has evolved from initial cultivation under controlled conditions to advanced metabolic engineering to meet industrial demands. However, effective genetic modification in microalgae has faced significant challenges, including issues with transformation efficiency, limited target selection, and genetic differences between species, as interspecies genetic variation limits the use of genetic tools from one species to another. This review summarized recent advancements in CRISPR systems applied to microalgae, with a focus on improving gene editing precision and efficiency, while addressing organism-specific challenges. We also discuss notable successes in utilizing the class 2 CRISPR-associated (Cas) proteins, including Cas9 and Cas12a, as well as emerging CRISPR-based approaches tailored to overcome microalgal cellular barriers. Additionally, we propose future perspectives for utilizing CRISPR/Cas strategies in microalgal biotechnology.},
}

*Microalgae/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Biotechnology/methods
Metabolic Engineering/methods

@article {pmid40195420,
year = {2025},
author = {Gabaev, I and Rowland, A and Jovanovic, E and Gawden-Bone, CM and Crozier, TWM and Teixeira-Silva, A and Greenwood, EJD and Gerber, PP and Wit, N and Nathan, JA and Matheson, NJ and Lehner, PJ},
title = {CRISPR-Cas9 genetic screens reveal regulation of TMPRSS2 by the Elongin BC-VHL complex.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {11907},
pmid = {40195420},
issn = {2045-2322},
support = {MR/V011561/1/MRC_/Medical Research Council/United Kingdom ; MR/V011561/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Serine Endopeptidases/genetics/metabolism ; SARS-CoV-2 ; COVID-19/virology/genetics/metabolism ; },
abstract = {The TMPRSS2 cell surface protease is used by a broad range of respiratory viruses to facilitate entry into target cells. Together with ACE2, TMPRSS2 represents a key factor for SARS-CoV-2 infection, as TMPRSS2 mediates cleavage of viral spike protein, enabling direct fusion of the viral envelope with the host cell membrane. Since the start of the COVID-19 pandemic, TMPRSS2 has gained attention as a therapeutic target for protease inhibitors which would inhibit SARS-CoV-2 infection, but little is known about TMPRSS2 regulation, particularly in cell types physiologically relevant for SARS-CoV-2 infection. Here, we performed an unbiased genome-wide CRISPR-Cas9 library screen, together with a library targeted at epigenetic modifiers and transcriptional regulators, to identify cellular factors that modulate cell surface expression of TMPRSS2 in human colon epithelial cells. We find that endogenous TMPRSS2 is regulated by the Elongin BC-VHL complex and HIF transcription factors. Depletion of Elongin B or treatment of cells with PHD inhibitors resulted in downregulation of TMPRSS2 and inhibition of SARS-CoV-2 infection. We show that TMPRSS2 is still utilised by SARS-CoV-2 Omicron variants for entry into colonic epithelial cells. Our study enhances our understanding of the regulation of endogenous surface TMPRSS2 in cells physiologically relevant to SARS-CoV-2 infection.},
}

Humans
*CRISPR-Cas Systems
*Serine Endopeptidases/genetics/metabolism
SARS-CoV-2
COVID-19/virology/genetics/metabolism

@article {pmid40195317,
year = {2025},
author = {Allen, TP and Roennfeldt, AE and Reckdharajkumar, M and Sullivan, AE and Liu, M and Quinn, RJ and Russell, DL and Peet, DJ and Whitelaw, ML and Bersten, DC},
title = {dFLASH; dual FLuorescent transcription factor activity sensor for histone integrated live-cell reporting and high-content screening.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3298},
pmid = {40195317},
issn = {2041-1723},
support = {OPP1171844, INV-001616//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; },
mesh = {Humans ; *Histones/metabolism ; *Transcription Factors/metabolism/genetics ; Receptors, Progesterone/metabolism/genetics ; HEK293 Cells ; High-Throughput Screening Assays/methods ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Drug Discovery/methods ; },
abstract = {Live-cell transcription factor (TF) activity reporting is crucial for synthetic biology, drug discovery and functional genomics. Here we present dFLASH (dual FLuorescent transcription factor Activity Sensor for Histone-integrated live-cell reporting), a modular, genome-integrated TF sensor. dFLASH homogeneously and specifically detects endogenous Hypoxia Inducible Factor (HIF) and Progesterone Receptor (PGR) activities, as well as coactivator recruitment to synthetic TFs. The dFLASH system produces dual-color nuclear fluorescence, enabling normalized, dynamic, live-cell TF activity sensing with strong signal-to-noise ratios and robust screening performance (Z' = 0.61-0.74). We validate dFLASH for functional genomics and drug screening, demonstrating HIF regulation via CRISPRoff and application to whole-genome CRISPR KO screening. Additionally, we apply dFLASH for drug discovery, identifying HIF pathway modulators from a 1600-compound natural product library using high-content imaging. Together, this versatile platform provides a powerful tool for studying TF activity across diverse applications.},
}

Humans
*Histones/metabolism
*Transcription Factors/metabolism/genetics
Receptors, Progesterone/metabolism/genetics
HEK293 Cells
High-Throughput Screening Assays/methods
*Biosensing Techniques/methods
CRISPR-Cas Systems
Drug Discovery/methods

@article {pmid40195165,
year = {2025},
author = {Rad, MB and Mohebbi, SR and Yadegar, A and Ghourchian, H},
title = {Porous GNPs assisted LAMP-CRISPR/Cas12a amperometric biosensor as a potential point of care testing system for SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {192},
number = {5},
pages = {280},
pmid = {40195165},
issn = {1436-5073},
mesh = {*Biosensing Techniques/methods ; *SARS-CoV-2/isolation & purification/genetics ; Gold/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *Point-of-Care Testing ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; Electrochemical Techniques/methods ; Porosity ; Limit of Detection ; CRISPR-Associated Proteins/metabolism ; Molecular Diagnostic Techniques/methods ; Electrodes ; Endodeoxyribonucleases ; Bacterial Proteins ; },
abstract = {A simple and ultrasensitive amperometric biosensor is introduced which has the potential to be applied as a point of care test for SARS-CoV-2 monitoring. It was prepared by integrating the reverse transcription loop-mediated isothermal amplification (RT‑LAMP) and CRISPR/Cas12a nuclease activity on a modified gold screen-printed electrode (GSPE). The GSPE is modified with double-end thiolated oligonucleotide reporters conjugated to porous gold nanoparticles (PGNPs) and inserted into a homemade poly-methyl methacrylate cartridge. This biosensor was integrated with a low-cost electronic kit to make a platform with the potential to be applied as a point-of-care testing system. The PGNPs on the reporters create a dense, negatively charged barrier that repels the redox couple of [Fe(CN)6][3-/4-] from the GSPE surface. Upon the addition of a real sample, followed by LAMP amplification and Cas12a nuclease activity on disposable GSPE, in the presence of SARS-CoV-2, the single-guide RNA binds to the target sequence and activates Cas12a. The activated Cas12a then cleaves the reporters, releasing the PGNPs. This removal of electrostatic hindrance allows the redox couple of [Fe(CN)6][3-/4-] to approach the positively charged GSPE, enhancing the amperometric signal. This biosensor offers an outstanding detection limit of 143 zM (~ 86 copies/mL) and a linear response from 4.7 to 7062 aM for SARS-CoV-2 real samples. By using double-end thiolated reporters and porous GNPs, this novel testing system makes it possible to minimize the required sample volume and reagent costs.},
}

*Biosensing Techniques/methods
*SARS-CoV-2/isolation & purification/genetics
Gold/chemistry
*Nucleic Acid Amplification Techniques/methods
Humans
*Metal Nanoparticles/chemistry
*Point-of-Care Testing
*COVID-19/diagnosis
CRISPR-Cas Systems
Electrochemical Techniques/methods
Porosity
Limit of Detection
CRISPR-Associated Proteins/metabolism
Molecular Diagnostic Techniques/methods
Electrodes
Endodeoxyribonucleases
Bacterial Proteins

@article {pmid40193707,
year = {2025},
author = {Cueny, RR and Voter, AF and McKenzie, AM and Morgenstern, M and Myers, KS and Place, MM and Peters, JM and Coon, JJ and Keck, JL},
title = {Altering translation allows E. coli to overcome G-quadruplex stabilizers.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
pmid = {40193707},
issn = {1362-4962},
support = {R01GM098885/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *DNA Transposable Elements ; *DNA, Bacterial/chemistry/genetics ; *Escherichia coli/chemistry/genetics ; Escherichia coli Proteins/genetics/metabolism ; *G-Quadruplexes ; Gene Deletion ; Peptide Elongation Factor Tu/genetics/metabolism ; Protein Biosynthesis ; Proteomics ; Ribosomes/metabolism ; Gene Expression Regulation, Bacterial ; },
abstract = {G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes, including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of translation elongation factor Tu or slowing translation initiation or elongation with kasugamycin, chloramphenicol, or spectinomycin suppress the effects of G4-stabilizing compounds. In contrast, reducing the expression of specific translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses reveal decreased protein and transcript levels, respectively, for ribosome assembly factors and proteins associated with translation in the presence of G4 stabilizer. Our results support a model in which reducing the rate of translation by altering translation initiation, translation elongation, or ribosome assembly can compensate for G4-related stress in E. coli.},
}

CRISPR-Cas Systems
*DNA Transposable Elements
*DNA, Bacterial/chemistry/genetics
*Escherichia coli/chemistry/genetics
Escherichia coli Proteins/genetics/metabolism
*G-Quadruplexes
Gene Deletion
Peptide Elongation Factor Tu/genetics/metabolism
Protein Biosynthesis
Proteomics
Ribosomes/metabolism
Gene Expression Regulation, Bacterial

@article {pmid40193303,
year = {2025},
author = {Coutinho-Abreu, IV and Chen, F and Li, HH and Rose, NH and Akbari, OS},
title = {Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {217},
pages = {},
doi = {10.3791/67732},
pmid = {40193303},
issn = {1940-087X},
mesh = {Animals ; *Aedes/genetics/embryology ; *CRISPR-Cas Systems ; Microinjections/methods ; *Gene Editing/methods ; Yellow Fever/transmission ; Female ; Genome, Insect ; },
abstract = {The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.},
}

Animals
*Aedes/genetics/embryology
*CRISPR-Cas Systems
Microinjections/methods
*Gene Editing/methods
Yellow Fever/transmission
Female
Genome, Insect

@article {pmid40191992,
year = {2025},
author = {Qu, X and Yue, Y and Tang, Y and Jiao, J and Miao, P},
title = {Polydopamine Powered Droplet Electricity Generator for Protein Assay with CRISPR/Cas Enabled Amplification.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.5c00877},
pmid = {40191992},
issn = {1530-6992},
abstract = {Protein function investigation and clinical assay are fundamental to modern biology and medical diagnostics. Flap endonuclease 1 (FEN1), a key enzyme in DNA replication and repair, plays a critical role in the progression of many diseases. Taking FEN1 as an example, we present a novel protein detection platform combining triboelectric nanogenerator (TENG) and CRISPR/Cas technologies. As a specific form of TENG, the transistor-droplet electricity generator (TDEG) is explored, which offers a low-cost, simple fabrication approach with real-time detection capability. Meanwhile, the FEN1 activated CRISPR/Cas system catalyzes the reactions on the three-dimensional DNA tetrahedron interface, promising the high sensitivity. This work not only demonstrates a powerful method for rapid protein detection but also pioneers the integration of CRISPR/Cas with TENG. It has a great prospect for future development of TENG sensors.},
}

@article {pmid40191453,
year = {2025},
author = {Patnaik, A and Rai, SK and Dhaked, RK},
title = {Analytical techniques and molecular platforms for detection and surveillance of antimicrobial resistance: advancements of the past decade.},
journal = {3 Biotech},
volume = {15},
number = {5},
pages = {108},
pmid = {40191453},
issn = {2190-572X},
abstract = {Developing countries have been able to control and minimise the mortality rates caused by pathogenic infections by ensuring affordable access to antibiotics. However, a large number of bacterial ailments are treated with wrong antibiotic prescription due to improper disease diagnosis. Apart from healthcare, antibiotics are also imprudently utilised in crop processing and animal husbandry. This unsupervised usage of antibiotics has propelled the generation of multidrug-resistant species of bacteria. Presently, several traditional antimicrobial susceptibility/resistance tests (AST/ART) are available; however, the accuracy and reproducibility of these tests are often debatable. Rigorous efforts are essential to develop techniques and methods which substantially decrease turnaround time for resistance screening. The present review has comprehensively incorporated the improvements in instrumentation and molecular methods for antimicrobial resistance studies. We have enlisted some innovative takes on conventional techniques such as isothermal calorimetry, Raman spectroscopy, mass spectrometry and microscopy. The contributions of modern molecular tools such as CRISPR-Cas, aptamers and Oxford-MinION sequencers have also been discussed. Persistent evolution has been observed towards adding innovation in diagnostic platforms for drug resistome screening, with the major attraction being the involvement of non-conventional analytical methods and technological improvements in existing setups. This review highlights these updates and provides a detailed account of principal developments in molecular methods for the testing of drug resistance in bacteria.},
}

@article {pmid40191137,
year = {2025},
author = {Liu, H and Xu, L and Xiu, Y and Ta, N and Xu, Q and Fan, Y and Li, K and Zhao, H and Piao, D and Ren, F and Jiang, H},
title = {A CRISPR/cas13a-assisted precise and portable test for Brucella nucleic acid detection.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1545953},
pmid = {40191137},
issn = {2235-2988},
mesh = {*Brucellosis/diagnosis/microbiology/veterinary ; Animals ; *Brucella/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; Sheep ; Sensitivity and Specificity ; Milk/microbiology ; Female ; *Molecular Diagnostic Techniques/methods ; DNA, Bacterial/genetics ; Nucleic Acid Amplification Techniques/methods ; Vagina/microbiology ; Bacterial Proteins/genetics ; },
abstract = {INTRODUCTION: Brucella infection in humans or animals can lead to brucellosis, which has the potential to significantly impact both the economy and public health. Currently, molecular biological methods for diagnosing brucellosis are either complex or have low sensitivity, and it is difficult to apply them in real-life settings in the field. Therefore, this study aims to establish a rapid and convenient nucleic acid-based molecular biology method for on-site rapid detection of Brucella and early clinical screening of brucellosis.

METHODS: Based on the conserved sequence of the Brucella Bcsp31 gene, we designed CRISPR RNA (crRNA) and RAA primers. We developed a fluorescence detection method and a paper strip detection method by integrating RAA amplification with CRISPR/Cas13a detection. We applied these methods to analyze 100 samples of suspected brucellosis-infected milk, 123 samples of human whole blood, and 100 samples of sheep vaginal swabs in order to validate their practical utility.

RESULTS: The RAA-CRISPR/Cas13a Brucella fluorescence detection method and the strip test method had detection limits of 100 copies/μL and 101 copies/μL, respectively, and both methods had a specificity of 100%. The positivity rate of the RAA-CRISPR/Cas13a fluorescence detection method for the milk, human whole blood, and sheep vaginal swab samples was 93% (93/100), 82.12% (101/123), and 91% (91/100), respectively; the strip test method, 87% (87/100), 64.23% (79/123), and 76% (76/100), respectively.

CONCLUSION: In this study, we have developed a RAA-CRISPR detection method based on the Brucella BCSP31 gene, with potential applications in the identification of Brucella nucleic acid and implications for clinical diagnosis of brucellosis.},
}

*Brucellosis/diagnosis/microbiology/veterinary
Animals
*Brucella/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
Sheep
Sensitivity and Specificity
Milk/microbiology
Female
*Molecular Diagnostic Techniques/methods
DNA, Bacterial/genetics
Nucleic Acid Amplification Techniques/methods
Vagina/microbiology
Bacterial Proteins/genetics

@article {pmid40191042,
year = {2025},
author = {Kim, HK and Cheong, H and Kim, MY and Jin, HE},
title = {Therapeutic Targeting in Ovarian Cancer: Nano-Enhanced CRISPR/Cas9 Gene Editing and Drug Combination Therapy.},
journal = {International journal of nanomedicine},
volume = {20},
number = {},
pages = {3907-3931},
pmid = {40191042},
issn = {1178-2013},
mesh = {Humans ; *Ovarian Neoplasms/genetics/therapy/drug therapy ; Female ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Nanoparticles/chemistry ; *Genetic Therapy/methods ; Animals ; Drug Delivery Systems/methods ; Drug Resistance, Neoplasm/genetics ; Molecular Targeted Therapy/methods ; Antineoplastic Agents/administration & dosage/therapeutic use ; },
abstract = {Ovarian cancer is the third most common gynecological cancer worldwide. Due to the high recurrence rate of advanced-stage ovarian cancer, often resulting from drug-resistant and refractory disease, various treatment strategies are under investigation. Genome editing of therapeutic target genes holds promise in enhancing cancer treatment efficacy by elucidating gene functions and mechanisms involved in cancer progression. The CRISPR/Cas9 system, in particular, shows great potential in ovarian cancer gene therapy and drug development. Targeting therapeutic genes such as BRCA1/2, P53, Snai1 etc, could improve the therapeutic strategy in ovarian cancer. CRISPR/Cas9 is a powerful gene-editing tool that there are many on-going clinical trials to treat various diseases including cancer. Nano-based delivery systems for CRISPR/Cas9 offer further therapeutic benefits, leveraging the unique properties of nanoparticles to improve delivery efficiency. Nano-based delivery systems could enhance the stability of CRISPR/Cas9 delivery formats (such as plasmid, mRNA, etc) and improve the delivery precision of delivery to target tumors. Additionally, combining CRISPR/Cas9 with targeted drug treatments, especially those aimed at genes associated with drug resistance, may significantly improve therapeutic outcomes in ovarian cancer. In this review, we discuss therapeutic target genes and their mechanisms in ovarian cancer, advances in nano-based CRISPR/Cas9 delivery, and the therapeutic potential of combining CRISPR/Cas9 with drug treatments for ovarian cancer.},
}

Humans
*Ovarian Neoplasms/genetics/therapy/drug therapy
Female
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Nanoparticles/chemistry
*Genetic Therapy/methods
Animals
Drug Delivery Systems/methods
Drug Resistance, Neoplasm/genetics
Molecular Targeted Therapy/methods
Antineoplastic Agents/administration & dosage/therapeutic use

@article {pmid40162522,
year = {2025},
author = {Pan, Y and Wang, D and Wei, R and Wang, S and Li, Y and Pan, W and Zhou, P and Li, N and Tang, B},
title = {Lateral Flow Platform for Lung Cancer Diagnosis through Simultaneous Detection of ctDNA and MicroRNA.},
journal = {Analytical chemistry},
volume = {97},
number = {13},
pages = {7063-7070},
doi = {10.1021/acs.analchem.4c05502},
pmid = {40162522},
issn = {1520-6882},
mesh = {*Lung Neoplasms/diagnosis/genetics ; Humans ; *MicroRNAs/analysis/genetics ; *Circulating Tumor DNA/analysis/genetics ; Gold/chemistry ; Biomarkers, Tumor/genetics ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; Proto-Oncogene Proteins p21(ras)/genetics ; CRISPR-Cas Systems ; },
abstract = {Early cancer screening is essential for reducing cancer-related mortality and improving survival rates. Simultaneous detection of multiple tumor markers can enhance the accuracy and specificity of cancer diagnosis, helping us to mitigate false-positive results associated with single-marker analysis. Here, we have developed a lateral flow detection platform that combines recombinase polymerase amplification (RPA), CRISPR Cas9, and catalyzed hairpin assembly (CHA) for the simultaneous detection of KRAS ctDNA and miRNA-223 in lung cancer. The CRISPR Cas9 system acts as a linking element, enabling specific recognition and binding to RPA amplicons of KRAS ctDNA while facilitating the capture of Au-DNA-Bio nanoparticles (NPs), thereby producing a stronger detection signal through Au NPs aggregation. The CHA system enhances this platform by providing sensitive detection of miRNA-223. Our platform was tested on a limited number of clinical saliva samples, demonstrating feasibility but requiring further validation with larger cohorts.},
}

*Lung Neoplasms/diagnosis/genetics
Humans
*MicroRNAs/analysis/genetics
*Circulating Tumor DNA/analysis/genetics
Gold/chemistry
Biomarkers, Tumor/genetics
Metal Nanoparticles/chemistry
Nucleic Acid Amplification Techniques
Proto-Oncogene Proteins p21(ras)/genetics
CRISPR-Cas Systems

@article {pmid40152782,
year = {2025},
author = {Guo, C and Yang, L and Zhang, T and Xie, L and Long, X and Ma, L and Zhao, Q and Cui, Y and Zhang, Y and He, Y},
title = {Target-Induced Enzymatic Cascade Reaction Method: Integrating Strand Displacement Amplification and CRISPR/Cas12a for Homogeneous Assays of Biotin.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {14},
pages = {8651-8659},
doi = {10.1021/acs.jafc.5c02441},
pmid = {40152782},
issn = {1520-5118},
mesh = {*Biotin/analysis/metabolism ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Streptavidin/chemistry ; Food Contamination/analysis ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Bacterial Proteins/genetics/metabolism/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; },
abstract = {The important biological role of biotin emphasizes the need for a sensitive method to detect it in foodstuffs. This article introduces a homogeneous and sensitive biotin analysis method that leverages a target-induced enzymatic cascade reaction, incorporating strand-displacement amplification (SDA) and the CRISPR/Cas12a system. Without target biotin, streptavidin (SA) specifically binds to the biotinylated probe DNA, hindering Klenow polymerase from extending the primer single-stranded DNA (ssDNA) due to the steric hindrance created by the SA-biotin complex, resulting in low fluorescence. Conversely, competition between the target biotin and the biotin label for binding to SA reduces the amount of SA captured on the primer ssDNA. The SDA process, which involves Klenow polymerase and the Nb.BbvCI enzyme, proceeds smoothly, thereby activating the CRISPR/Cas12a system and producing an intense fluorescence signal. Utilizing this principle, precise and sensitive biotin detection in food matrices was achieved, with a limit of detection of 0.01 nM.},
}

*Biotin/analysis/metabolism
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Streptavidin/chemistry
Food Contamination/analysis
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
*Endodeoxyribonucleases/genetics/metabolism/chemistry
Bacterial Proteins/genetics/metabolism/chemistry
DNA, Single-Stranded/chemistry/genetics
Limit of Detection

@article {pmid40145799,
year = {2025},
author = {Thanh, LM and Lan, VTH and Cuong, CQ and Lam, HT and Han, LK and Trang, NTH and Nghia, NH},
title = {Development of CRISPR/Cas9-Mediated Saccharomyces cerevisiae Strains for the Cell-Surface Display of a Novel Fusion Acid-Alkaline Phytase.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {14},
pages = {8458-8468},
doi = {10.1021/acs.jafc.5c00550},
pmid = {40145799},
issn = {1520-5118},
mesh = {*6-Phytase/genetics/metabolism/chemistry ; *Saccharomyces cerevisiae/genetics/enzymology/metabolism/chemistry ; CRISPR-Cas Systems ; Phytic Acid/metabolism ; Animal Feed/analysis ; *Saccharomyces cerevisiae Proteins/genetics/metabolism/chemistry ; Kinetics ; Hydrogen-Ion Concentration ; Glycine max/metabolism/chemistry ; Zea mays/metabolism ; Oryza/metabolism ; },
abstract = {Phytases enhance phosphorus bioavailability in animal feed, but their limited reusability hinders their application. To overcome this, Saccharomyces cerevisiae was engineered to display a fusion phytase combining acid and alkaline phytases on its cell surface by using CRISPR/Cas9. The enzyme was anchored via the α-agglutinin-GPI system in two marker-free strains, BY4743::GAP-mGEB and BY4743::GAP-aGEB, employing MFα and Aga2p signal peptides, respectively. Both strains exhibited robust surface activity across a broad pH range, retaining >50% relative activity between pH 1.0 and 7.0, with dual optima at pH 2.0 and 5.0-6.0. Kinetic analysis revealed a Km of 0.377-0.989 mM and a kcat of 0.014-0.019 μmol/min/mg wet-cell weight, with the Aga2p strain showing the highest efficiency. The fusion phytase exhibited ∼ 3.5-4 times higher activity than the single acid phytase. These strains effectively degraded phytate in soybean, corn flour, and rice bran, demonstrating a sustainable approach for improving phosphorus utilization in animal feed.},
}

*6-Phytase/genetics/metabolism/chemistry
*Saccharomyces cerevisiae/genetics/enzymology/metabolism/chemistry
CRISPR-Cas Systems
Phytic Acid/metabolism
Animal Feed/analysis
*Saccharomyces cerevisiae Proteins/genetics/metabolism/chemistry
Kinetics
Hydrogen-Ion Concentration
Glycine max/metabolism/chemistry
Zea mays/metabolism
Oryza/metabolism

@article {pmid40116487,
year = {2025},
author = {Firestone, K and Gopalakrishna, KP and Rogers, LM and Peters, A and Gaddy, JA and Nichols, CM and Hall, MH and Varela, HN and Carlin, SM and Hillebrand, GH and Giacobe, EJ and Aronoff, DM and Hooven, TA},
title = {A CRISPRi library screen in group B Streptococcus identifies surface immunogenic protein (Sip) as a mediator of multiple host interactions.},
journal = {Infection and immunity},
volume = {93},
number = {4},
pages = {e0057324},
pmid = {40116487},
issn = {1098-5522},
support = {R21AI178067/NH/NIH HHS/United States ; R01AI182835,R01AI177991/NH/NIH HHS/United States ; R01HD090061,R01HD113675/NH/NIH HHS/United States ; 6-FY24-0009//March of Dimes Foundation/ ; 1275387//Burroughs Wellcome Fund/ ; I01BX005352,I01BX007005//U.S. Department of Veterans Affairs/ ; R01AI134036/NH/NIH HHS/United States ; T32GM007347,T32GM152284/NH/NIH HHS/United States ; },
mesh = {*Streptococcus agalactiae/genetics/immunology ; Animals ; Humans ; *Streptococcal Infections/microbiology/immunology ; Mice ; *Bacterial Proteins/genetics/metabolism/immunology ; Female ; *Host-Pathogen Interactions/immunology ; Macrophages/immunology/microbiology ; Biofilms/growth & development ; Interleukin-1beta/metabolism ; *Membrane Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cytokines/metabolism ; },
abstract = {Group B Streptococcus (GBS; Streptococcus agalactiae) is an important pathobiont capable of colonizing various host environments, contributing to severe perinatal infections. Surface proteins play critical roles in GBS-host interactions; however, comprehensive studies of these proteins' functions have been limited by genetic manipulation challenges. This study leveraged a CRISPR interference (CRISPRi) library to target genes encoding surface-trafficked proteins in GBS, identifying their roles in modulating macrophage cytokine responses. Bioinformatic analysis of 654 GBS genomes revealed 66 conserved surface protein genes. Using a GBS strain expressing chromosomally integrated dCas9, we generated and validated CRISPRi strains targeting these genes. THP-1 macrophage-like cells were exposed to ethanol-killed GBS variants, and pro-inflammatory cytokines TNF-⍺ and IL-1β were measured. Notably, knockdown of the sip gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Furthermore, Δsip mutants demonstrated impaired biofilm formation, reduced adherence to human fetal membranes, and diminished uterine persistence in a mouse colonization model. These findings suggest that Sip modulates GBS-host interactions critical for pathogenesis, underscoring its potential as a therapeutic target or vaccine component.},
}

*Streptococcus agalactiae/genetics/immunology
Animals
Humans
*Streptococcal Infections/microbiology/immunology
Mice
*Bacterial Proteins/genetics/metabolism/immunology
Female
*Host-Pathogen Interactions/immunology
Macrophages/immunology/microbiology
Biofilms/growth & development
Interleukin-1beta/metabolism
*Membrane Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Cytokines/metabolism

@article {pmid40099627,
year = {2025},
author = {Li, Y and Ren, X and Wu, D and Ma, H and Wei, Q and Ju, H and Gao, Z},
title = {CRISPR/Cas12a-powered nanoconfined biosensing platform with hybrid chain reaction cascading guanine nanowire amplification for ultrasensitive dual-mode detection of lipopolysaccharide.},
journal = {The Analyst},
volume = {150},
number = {8},
pages = {1571-1577},
doi = {10.1039/d5an00184f},
pmid = {40099627},
issn = {1364-5528},
mesh = {*Nanowires/chemistry ; *Biosensing Techniques/methods ; *Lipopolysaccharides/analysis ; *CRISPR-Cas Systems ; Limit of Detection ; *Guanine/chemistry ; Electrochemical Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Silicon Dioxide/chemistry ; Luminescent Measurements/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Traditional endotoxin detection methods face challenges in sensitivity, interference resistance, and reliability. This study develops a CRISPR/Cas12a-powered nanoconfined biosensing system that integrates mesoporous nanoengineering with a hybrid chain reaction (HCR) cascading guanine nanowire (G-wire) dual amplification strategy for ultrasensitive dual-mode detection of lipopolysaccharide (LPS). By leveraging a vertically ordered mesoporous silica membrane (VMSM) as a molecular sieve and CRISPR trans-cleavage activity modulator, the system achieves precise regulation of Ru(bpy)3[2+] adsorption via LPS-suppressed HCR assembly. This architecture enables physical confinement-mediated electrochemiluminescence (ECL) and fluorescence (FL) signal transduction, with dual-mode outputs providing mutual validation for enhanced reliability. The biosensor exhibits superior sensitivity with detection limits of 3.4 pg mL[-1] for ECL and 1.4 pg mL[-1] for FL, while also offering a broad dynamic range (0.005-100 ng mL[-1]), significantly outperforming conventional LPS assays. The CRISPR-triggered HCR cascading G-wire dual amplification synergizes with nanoconfinement of VMSM to ensure robust anti-interference performance in complex matrices, validated by recovery rates of 97.8-102.5% in real samples. By integrating CRISPR programmability with nanoengineered signal amplification, this work establishes a transformative paradigm for portable, high-precision endotoxin detection in clinical diagnostics, industrial monitoring, and environmental safety applications.},
}

*Nanowires/chemistry
*Biosensing Techniques/methods
*Lipopolysaccharides/analysis
*CRISPR-Cas Systems
Limit of Detection
*Guanine/chemistry
Electrochemical Techniques/methods
Nucleic Acid Amplification Techniques/methods
Silicon Dioxide/chemistry
Luminescent Measurements/methods
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40078160,
year = {2025},
author = {Wu, Z and Li, J and Zhang, T and Zhang, K and Liu, X and Yang, Z and Xu, L and Han, K},
title = {One-pot synthesized three-way junction based multiple strand displacement amplification for sensitive assay of H5N1 DNA.},
journal = {The Analyst},
volume = {150},
number = {8},
pages = {1541-1552},
doi = {10.1039/d4an01586j},
pmid = {40078160},
issn = {1364-5528},
mesh = {*Influenza A Virus, H5N1 Subtype/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; *DNA, Viral/analysis/genetics ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The rapid and sensitive detection of H5N1, a highly pathogenic avian influenza virus, is crucial for controlling its spread and minimizing its impact on public health. In this study, we developed a novel biosensor based on strand displacement amplification (SDA) coupled with CRISPR/Cas12a for highly sensitive detection of H5N1 DNA. The biosensor utilizes a combination of a three-way junction structure, composed of three hairpins (H1, H2, H3), to initiate amplification through SDA, resulting in the production of numerous activators. These activators then trigger CRISPR/Cas12a's collateral cleavage activity, which generates a detectable fluorescence signal. The biosensor demonstrated a linear detection range from 100 fM to 800 pM, with a detection limit as low as 72.87 fM. The optimized biosensor exhibited excellent sensitivity, high specificity, and a broad dynamic range, making it a promising tool for the early detection of H5N1 DNA in complex biological samples. Additionally, the use of CRISPR/Cas12a's trans-cleavage activity significantly improved signal amplification and specificity, allowing for more reliable detection compared to traditional methods. The results highlight the advantages of the integrated SDA and CRISPR/Cas12a approach, which addresses the limitations of conventional detection methods, such as low sensitivity, lengthy analysis times, and high costs. The biosensor's ability to perform well in complex sample matrices demonstrates its potential for point-of-care diagnostics, especially in resource-limited settings. Future applications of this technology could extend to the detection of other pathogens, offering a versatile and adaptable platform for disease surveillance and management.},
}

*Influenza A Virus, H5N1 Subtype/genetics/isolation & purification
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
Limit of Detection
*DNA, Viral/analysis/genetics
Humans
CRISPR-Associated Proteins/genetics/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39994482,
year = {2025},
author = {Huang, L and Simonian, R and Lopez, MA and Karuppasamy, M and Sanders, VM and English, KG and Fabian, L and Alexander, MS and Dowling, JJ},
title = {X-linked myopathy with excessive autophagy: characterization and therapy testing in a zebrafish model.},
journal = {EMBO molecular medicine},
volume = {17},
number = {4},
pages = {823-840},
pmid = {39994482},
issn = {1757-4684},
support = {T32 HD071866/HD/NICHD NIH HHS/United States ; U54 OD030167/OD/NIH HHS/United States ; R01AG075059//HHS | NIH | National Institute on Aging (NIA)/ ; K08NS120812//HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; K08 NS120812/NS/NINDS NIH HHS/United States ; R01AR076227//HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; U54OD030167//HHS | NIH | NIH Office of the Director (OD)/ ; R01HD095897//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R21AR074006//HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Animals ; Zebrafish/genetics ; *Autophagy ; Disease Models, Animal ; *Muscular Diseases/pathology/genetics/drug therapy ; *Genetic Diseases, X-Linked/pathology/drug therapy/genetics ; Morpholines ; CRISPR-Cas Systems ; Humans ; Zebrafish Proteins/genetics ; Lysosomal Storage Diseases ; },
abstract = {X-linked myopathy with excessive autophagy (XMEA), a rare childhood-onset autophagic vacuolar myopathy caused by mutations in VMA21, is characterized by proximal muscle weakness and progressive vacuolation. VMA21 encodes a protein chaperone of the vacuolar hydrogen ion ATPase, the loss of which leads to lysosomal neutralization and impaired function. At present, there is an incomplete understanding of XMEA, its mechanisms, consequences on other systems, and therapeutic strategies. A significant barrier to advancing knowledge and treatments is the lack of XMEA animal models. Therefore, we used CRISPR-Cas9 editing to engineer a loss-of-function mutation in zebrafish vma21. The vma21 mutant zebrafish phenocopy the human disease with impaired motor function and survival, liver dysfunction, and dysregulated autophagy indicated by lysosomal de-acidification, the presence of characteristic autophagic vacuoles in muscle fibers, altered autophagic flux, and reduced lysosomal marker staining. As proof-of-concept, we found that two drugs, edaravone and LY294002, improve swim behavior and survival. In total, we generated and characterized a novel preclinical zebrafish XMEA model and demonstrated its suitability for studying disease pathomechanisms and identifying potential therapeutic targets.},
}

Animals
Zebrafish/genetics
*Autophagy
Disease Models, Animal
*Muscular Diseases/pathology/genetics/drug therapy
*Genetic Diseases, X-Linked/pathology/drug therapy/genetics
Morpholines
CRISPR-Cas Systems
Humans
Zebrafish Proteins/genetics
Lysosomal Storage Diseases

@article {pmid39906987,
year = {2025},
author = {Hou, Z and Ding, Z and Zhang, Y and Pang, B and Gui, L and Liu, Y and Lu, L and Roengjitd, P and Wang, H},
title = {Establishment and Application of a Rapid Detection Method of Cyprinid Herpesvirus 2 (CyHV-2) in Aquacultural Waters by Using a Novel One-Pot RAA-CRISPR/Cas12a Combined With Fe-Iron Flocculation Technology.},
journal = {Journal of fish diseases},
volume = {48},
number = {5},
pages = {e14089},
doi = {10.1111/jfd.14089},
pmid = {39906987},
issn = {1365-2761},
support = {BE2021369//Key Research and Development Plan of Jiangsu Province/ ; CARS-45-16//Earmarked Fund for China Agriculture Research System/ ; 2022-02-08-00-12-F01112//Shanghai Agriculture Applied Technology Development Program, China/ ; },
mesh = {Animals ; *Herpesviridae/isolation & purification/genetics ; *Fish Diseases/virology/diagnosis ; *Herpesviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; Aquaculture ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Carps ; *Goldfish ; Iron/chemistry ; *Molecular Diagnostic Techniques/veterinary/methods ; },
abstract = {Cyprinid herpesvirus 2 (CyHV-2) poses a substantial global threat to goldfish (Carassius auratus) and crucian carp (Carassius carassius). Despite the development of several sensitive molecular diagnostic techniques, there is an ongoing demand for alternative visualisation platforms to streamline the workflow, enhance safety profiles, and improve accessibility for end-users. In this study, we have integrated recombinase-aided amplification (RAA) technology with the CRISPR/Cas12a system to establish a rapid diagnostic system for CyHV-2, termed one-pot RAA-CRISPR/Cas12a. This method enables the results of detection within 60 min. The RAA-CRISPR/Cas12a platform is capable of detecting as few as 10 copies of CyHV-2 per reaction cycle without exhibiting cross-reactivity with other pathogens. The positive detection rate in clinical samples exceeds that of conventional PCR approaches, underscoring its high precision. Furthermore, the method could be used in conjunction with iron flocculation for the concentration and detection of viruses within aquaculture settings. This approach minimises the dissection of aquatic organisms, thereby maximising animal welfare and bolstering detection efficiency. Collectively, our findings validate the RAA-CRISPR/Cas12a method as a robust, specific, confirmatory, user-friendly and promising approach for on-site diagnosis of CyHV-2.},
}

Animals
*Herpesviridae/isolation & purification/genetics
*Fish Diseases/virology/diagnosis
*Herpesviridae Infections/veterinary/diagnosis/virology
*CRISPR-Cas Systems
Aquaculture
*Nucleic Acid Amplification Techniques/veterinary/methods
*Carps
*Goldfish
Iron/chemistry
*Molecular Diagnostic Techniques/veterinary/methods

@article {pmid39776254,
year = {2025},
author = {Amarilla-Quintana, S and Navarro, P and Hernández, I and Ramos, A and Montero-Calle, A and Cabezas-Sainz, P and Barrero, MJ and Megías, D and Vilaplana-Martí, B and Epifano, C and Gómez-Dominguez, D and Monzón, S and Cuesta, I and Sánchez, L and Barderas, R and García-Donas, J and Martín, A and Pérez de Castro, I},
title = {CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds.},
journal = {Molecular oncology},
volume = {19},
number = {4},
pages = {1092-1116},
pmid = {39776254},
issn = {1878-0261},
support = {BecaGethi-RamóndelasPeñas//Grupo Español de Tumores Huerfanos e Infrecuentes/ ; ProyectosSemillaAECC//Fundación Científica Asociación Española Contra el Cáncer/ ; },
mesh = {*Forkhead Box Protein L2/genetics ; *Granulosa Cell Tumor/genetics/pathology/drug therapy ; Female ; Humans ; Cell Line, Tumor ; *Mutation/genetics ; Phenotype ; *Antineoplastic Agents/pharmacology ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; *CRISPR-Cas Systems ; Cell Proliferation/drug effects/genetics ; Apoptosis/drug effects/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {Forkhead box L2 (FOXL2) encodes a transcription factor essential for sex determination, and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation and invasion. Furthermore, these modified cells are more susceptible to dasatinib and ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type-like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing GCTs, highlighting its therapeutic promise for treating this rare ovarian cancer.},
}

*Forkhead Box Protein L2/genetics
*Granulosa Cell Tumor/genetics/pathology/drug therapy
Female
Humans
Cell Line, Tumor
*Mutation/genetics
Phenotype
*Antineoplastic Agents/pharmacology
*Ovarian Neoplasms/genetics/pathology/drug therapy
*CRISPR-Cas Systems
Cell Proliferation/drug effects/genetics
Apoptosis/drug effects/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation, Neoplastic/drug effects

@article {pmid39560750,
year = {2025},
author = {Noruzi, S and Mohammadi, R and Jamialahmadi, K},
title = {CRISPR/Cas9 system: a novel approach to overcome chemotherapy and radiotherapy resistance in cancer.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {398},
number = {4},
pages = {3373-3408},
pmid = {39560750},
issn = {1432-1912},
mesh = {*CRISPR-Cas Systems ; Humans ; *Neoplasms/genetics/therapy/radiotherapy/drug therapy ; *Drug Resistance, Neoplasm/genetics ; Animals ; Gene Editing/methods ; *Radiation Tolerance/genetics ; Antineoplastic Agents/therapeutic use/pharmacology ; },
abstract = {Cancer presents a global health challenge with rising incidence and mortality. Despite treatment advances in cancer therapy, radiotherapy and chemotherapy remained the most common treatments for all types of cancers. However, resistance phenotype in cancer cells leads to unsatisfactory results in the efficiency of therapeutic strategies. Therefore, researchers strive to propose effective solutions to overcome treatment failure, which requires a deep knowledge of treatment-resistant mechanisms. The progression and occurrence of tumors can be attributed to gene mutation. Over the past decade, the emergence of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) genome editing has revolutionized cancer research. This versatile technology enables cancer modeling, manipulation of specific DNA sequences, and genome-wide screening. CRISPR/Cas9 is an effective tool for identifying radio- and chemoresistance genes and offering potential adjunctive treatments to overcome tumor recurrence after chemo- and radiotherapy. This article aims to explain the potential of the CRISPR/Cas9 system in improving the effectiveness of chemo- and radiotherapy and ultimately overcoming treatment failure.},
}

*CRISPR-Cas Systems
Humans
*Neoplasms/genetics/therapy/radiotherapy/drug therapy
*Drug Resistance, Neoplasm/genetics
Animals
Gene Editing/methods
*Radiation Tolerance/genetics
Antineoplastic Agents/therapeutic use/pharmacology

@article {pmid40188791,
year = {2025},
author = {Huang, X and Tan, Z and Wei, J and Bai, X},
title = {Super-robust synthetic microorganism can get chlorine resistance in advance and transfer their inserted DNA sequence in genome to indigenous bacteria in water.},
journal = {Water research},
volume = {281},
number = {},
pages = {123594},
doi = {10.1016/j.watres.2025.123594},
pmid = {40188791},
issn = {1879-2448},
abstract = {CRISPR-Cas gene editing tools have brought us to an era of synthetic biology that will change the world. Synthetic microorganisms (SMs) have brought enormous economic benefits and will contribute more in the future. Among them, super-robust SMs can overcome the stresses in bioproduction and further increase yield. However, when they are released into the environments, little is known about their fates and risks to human health. In this study, it was found that the gene editing super-robust SM could transfer its inserted DNA sequence in genome to the indigenous bacteria in surface water and showed stronger resistance to chlorine compared with wild-type bacteria. Chlorine disinfection did slight damage on cell membrane of super-robust SM, which decreased ATP leakage and DNA damage, and thereby promoted bacterial survival. Chlorine-injured super-robust SM retained high respiratory activity, and could resuscitate and regenerate. Less damage on super-robust SM cell membrane could prevent chlorine from entering the cells and resulted in lower ROS generation. Its DNA repair system and antioxidant system could still function under high concentrations of chlorine exposure. These findings provided new insights into the fates and environmental risks of SMs as an emerging biological pollutant in water supply system.},
}

@article {pmid40187879,
year = {2025},
author = {Ding, F and Hang, X and Tian, S and Cao, W and Wu, J and Wang, L},
title = {Nicking endonuclease-mediated primer exchange reaction for rapid and sensitive miRNA detection.},
journal = {Analytica chimica acta},
volume = {1351},
number = {},
pages = {343902},
doi = {10.1016/j.aca.2025.343902},
pmid = {40187879},
issn = {1873-4324},
mesh = {*MicroRNAs/blood/analysis/genetics ; Humans ; Limit of Detection ; CRISPR-Cas Systems ; *Endonucleases/metabolism ; DNA/chemistry/metabolism ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acid Hybridization ; *DNA Primers/chemistry/metabolism ; },
abstract = {Primer exchange reaction (PER) is a novel and simple nucleic acid-templated extension technique that has recently attracted much attention in the field of biosensing. However, current PER reactions have shown relatively slow rates and low amplification performances, resulting in long assay times and limited detection sensitivities. Here we report a nicking endonuclease-mediated PER reaction (named NEPER) that rapidly releases amplified DNA products by adding a nicking endonuclease to hydrolyze the hybridized double-stranded DNA (dsDNA), and consequently has a maximum speed that is thirty orders of magnitude greater than the maximum for conventional PER. We further combined a CRISPR/Cas12a signal readout technique and developed a cascade NEPER-CRISPR/Cas12a method that can detect miRNA-155 with a limit of detection (LOD) down to 3.1 fM. We also show that the NEPER-CRISPR/Cas12a can be used to detect targets in serum samples.},
}

*MicroRNAs/blood/analysis/genetics
Humans
Limit of Detection
CRISPR-Cas Systems
*Endonucleases/metabolism
DNA/chemistry/metabolism
*Biosensing Techniques/methods
Nucleic Acid Amplification Techniques/methods
Nucleic Acid Hybridization
*DNA Primers/chemistry/metabolism

@article {pmid40187291,
year = {2025},
author = {Meng, T and Kang, Q and Xu, J and Zhao, S and Liu, T and Zhou, D and Gong, X and Zhang, J},
title = {A hairpin reporter-driven feedback CRISPR/Cas signal amplification loop for terminal deoxynucleotidyl transferase activity detection.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128061},
doi = {10.1016/j.talanta.2025.128061},
pmid = {40187291},
issn = {1873-3573},
abstract = {The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans-cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10[-6] U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.},
}

@article {pmid40186758,
year = {2025},
author = {Guo, X and Sun, K and Wu, Z and Xiao, D and Song, Y and Li, S and Wei, G and Li, W and Hao, Y and Xu, B and Zhang, K and Liao, N and Hu, D and Liu, YG and Zong, W and Guo, J},
title = {Improving yield-related traits by editing the promoter and distal regulatory region of heading date genes Ghd7 and PRR37 in elite rice variety Mei Xiang Zhan 2.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {4},
pages = {92},
pmid = {40186758},
issn = {1432-2242},
support = {2023ZD04073//National Key R&D Program of China/ ; 2019B030302006//Major Program of Guangdong Basic and Applied Research/ ; 2022SDZG05//open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province/ ; },
mesh = {*Oryza/genetics/growth & development ; *Promoter Regions, Genetic ; *Gene Editing ; Plant Breeding ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Phenotype ; *Genes, Plant ; },
abstract = {We revealed that editing the promoter and distal regulatory region of the pleiotropic genes Ghd7 and PRR37 reduces their ability to delay heading date while improving their capacity to boost crop yield, offering valuable resources for rice breeding. Heading date is a crucial agronomic characteristic in rice that governs the adaptability to different latitudes and the yield of various varieties. Optimizing the heading date of superior cultivars in breeding practice can significantly broaden their potential planting areas. Ghd7 and PRR37 are pivotal genes that control heading date and enhance agronomic traits. In the elite indica rice variety Mei Xiang Zhan 2 (MXZ2), we used CRISPR/Cas9 technology to effectively generate homozygous mutant lines with a gradient change in heading date by multi-target editing the promoter and distal regulatory region of Ghd7 and PRR37. Various degrees of down-regulation of Ghd7 or PRR37 expression, impaired gene functions, and advancement of the heading date were observed in the mutant lines. Certain mutant lines exhibited an early heading date and increased yield while preserving the exceptional quality of MXZ2. Our study revealed that editing the promoter and distal regulatory region of the pleiotropic genes Ghd7 and PRR37 reduces their ability to delay heading date while improving their capacity to boost crop yield, offering valuable resources for rice breeding.},
}

*Oryza/genetics/growth & development
*Promoter Regions, Genetic
*Gene Editing
Plant Breeding
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Phenotype
*Genes, Plant

@article {pmid40186543,
year = {2025},
author = {Nievergelt, AP},
title = {Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {1},
pages = {e70140},
pmid = {40186543},
issn = {1365-313X},
support = {ALTF 891-2018//European Molecular Biology Organization/ ; LT000515/2019//Human Frontier Science Program/ ; },
mesh = {*Gene Editing/methods/trends ; *Chlamydomonas/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Genetic Engineering/methods ; },
abstract = {The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.},
}

*Gene Editing/methods/trends
*Chlamydomonas/genetics
CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
Genetic Engineering/methods

@article {pmid40186294,
year = {2025},
author = {Jia, J and Hao, Y and Zhang, L and Cao, X and An, L and Wang, H and Ma, Q and Jin, X and Ma, X},
title = {Development and validation of optimized lentivirus-like particles for gene editing tool delivery with Gag-Only strategy.},
journal = {European journal of medical research},
volume = {30},
number = {1},
pages = {242},
pmid = {40186294},
issn = {2047-783X},
support = {2016YFC1000307//This work was funded by grants from the National Key Research and Development Program of China/ ; },
mesh = {Humans ; *Lentivirus/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; HEK293 Cells ; *Gene Transfer Techniques ; *gag Gene Products, Human Immunodeficiency Virus/genetics ; },
abstract = {BACKGROUND: The development of gene editing tools such as CRISPR-Cas9 and base editors (BE) is critical for genetic diseases and cancer. Lentivirus-like particles (LVLPs) grows into an auspicious platform for delivering mRNA or ribonucleic proteins (RNPs) due to it integrates the advantage of viral and non-viral vectors. Current LVLP systems predominantly utilize HIV-Gag and Pol proteins. However, the reverse transcriptase and integrase of Pol, pose risks of genomic integration and potential tumorigenesis. Enhancing the safety of VLP system is essential. This study focuses on improving the LVLP to minimize these risks.

METHODS: We implemented a Gag-Only strategy, constructing LVLPs with HIV-Gag protein, thereby eliminating the integration risks linked to Pol. By leveraging the interactions between MS2-MCP (MS2 coat protein), PP7 and PP7 BP (PP7 binding protein), and the psi (HIV packaging signal) with HIV-Gag, we encapsulated PAMless andesine base editor (CE-8e-SpRY) mRNA and sgRNA targeting the PD1 start codon (ATG) into the LVLP. Using recombinant lentiviral vector technology, we developed a stable PD1-expressing 293T cell line (PD1-293T) to assess the editing efficiency of LVLP.

RESULTS: The psi-LVLP demonstrated effective packaging capabilities, achieving 15% base editing efficiency in 293T cells. By optimizing plasmid ratios, we observed increased CE-8e-SpRY mRNA copy numbers, with 30% base editing efficiency. Additionally, the integration of HDVrz (hepatitis delta virus ribozyme) and psi into sgRNA (HDVrz-psi-LVLP) substantially enhanced sgRNA copy numbers, resulting in approximately 50% base editing efficiency in 293T cells and 20% base editing efficiency in Jurkat cells. Mendelian randomization analyses revealed significant genetic correlations between PD1, B2M, CIITA, and TIGIT genes with various cancer risks. Furthermore, HDVrz-psi-LVLPs targeting the start codons of B2M, CIITA, and TIGIT exhibited high base editing activity in both Jurkat and 293T cells.

CONCLUSION: In conclusion, this optimized platform effectively encapsulates CE-8e-SpRY mRNA and sgRNA, achieving high editing efficiencies across multiple genes and cell types. We introduce a safer and more efficient gene editing tool delivery system by constructing LVLPs based on the Gag-Only strategy. Our study presents a promising implication for cancer immunotherapy.},
}

Humans
*Lentivirus/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Genetic Vectors/genetics
HEK293 Cells
*Gene Transfer Techniques
*gag Gene Products, Human Immunodeficiency Virus/genetics

@article {pmid40089636,
year = {2025},
author = {Cervera, ST and Martínez, S and Iranzo-Martínez, M and Notario, L and Melero-Fernández de Mera, RM and Alonso, J},
title = {Targeted inactivation of EWSR1 : : FLI1 gene in Ewing sarcoma via CRISPR/Cas9 driven by an Ewing-specific GGAA promoter.},
journal = {Cancer gene therapy},
volume = {32},
number = {4},
pages = {437-449},
pmid = {40089636},
issn = {1476-5500},
mesh = {*Sarcoma, Ewing/genetics/therapy/pathology ; Humans ; *CRISPR-Cas Systems ; *RNA-Binding Protein EWS/genetics ; Animals ; *Proto-Oncogene Protein c-fli-1/genetics ; *Promoter Regions, Genetic ; Cell Line, Tumor ; Mice ; Gene Editing ; *Oncogene Proteins, Fusion/genetics ; *Bone Neoplasms/genetics/therapy ; Cell Proliferation ; },
abstract = {We have recently demonstrated that genetic inactivation of EWSR1 : : FLI1 by CRISPR/Cas9, successfully blocks cell proliferation in a cell model of Ewing sarcoma. However, CRISPR/Cas9-mediated gene editing can exhibit off-target effects, and thus, precise regulation of Cas9 expression in target cells is essential to develop gene-editing strategies to inactivate EWSR1 : : FLI1 in Ewing sarcoma cells. In this study, we demonstrate that Cas9 can be specifically expressed in Ewing sarcoma cells when located downstream a promoter consisting of GGAA repeats and a consensus TATA box (GGAAprom). Under these conditions, Cas9 is selectively expressed in Ewing sarcoma cells that express EWSR1 : : FLI1 oncoproteins, but not in cells expressing wild-type FLI1. Consequently, Ewing sarcoma cells infected with GGAAprom>Cas9 and a specific gRNA designed to inactivate EWSR1 : : FLI1, showed successful EWSR1 : : FLI1 inactivation and the subsequent blockade of cell proliferation. Notably, GGAAprom>Cas9 can be efficiently delivered to Ewing sarcoma cells via adenoviral vectors both in vitro and in vivo, highlighting the potential of this approach for Ewing sarcoma treatment. Our results demonstrate that the CRISPR/Cas9 machinery is safe and specific for Ewing sarcoma cells when driven under a GGAAprom, paving the way for the development of cancer gene therapies based on the selective expression of genes with therapeutic potential.},
}

*Sarcoma, Ewing/genetics/therapy/pathology
Humans
*CRISPR-Cas Systems
*RNA-Binding Protein EWS/genetics
Animals
*Proto-Oncogene Protein c-fli-1/genetics
*Promoter Regions, Genetic
Cell Line, Tumor
Mice
Gene Editing
*Oncogene Proteins, Fusion/genetics
*Bone Neoplasms/genetics/therapy
Cell Proliferation

@article {pmid39979422,
year = {2025},
author = {Xu, S and Neupane, S and Wang, H and Pham, TP and Snyman, M and Huynh, TV and Wang, L},
title = {The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.},
journal = {Heredity},
volume = {134},
number = {3-4},
pages = {221-233},
pmid = {39979422},
issn = {1365-2540},
support = {R35GM133730//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MCB-2042490/2348390//National Science Foundation (NSF)/ ; 2220695/2324639//National Science Foundation (NSF)/ ; },
mesh = {Animals ; *Mosaicism ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Daphnia/genetics ; *Mutation ; *Genetic Pleiotropy ; Microinjections ; Transcriptome ; *Decorin/genetics ; },
abstract = {The effective use of CRISPR technologies in emerging model organisms faces significant challenges in efficiently generating heritable mutations and in understanding the genomic consequences of induced DNA damages and the inheritance patterns of induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for gene editing in the microcrustacean Daphnia pulex; 2) assessing the editing dynamics of Cas9 and Cas12a nucleases in the scarlet knock-out mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet gene. Our reengineered microinjection method results in efficient biallelic editing with both nucleases. Our data suggest site-specific DNA cleavage mostly occurs in a stepwise fashion. Indels dominate the induced mutations. A few, unexpected on-site large deletions (>1 kb) are also observed. Notably, genome-wide analyses reveal no off-target mutations. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progeny. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes involved in human neurodegenerative diseases.},
}

Animals
*Mosaicism
*CRISPR-Cas Systems
Gene Editing/methods
*Daphnia/genetics
*Mutation
*Genetic Pleiotropy
Microinjections
Transcriptome
*Decorin/genetics

@article {pmid39955067,
year = {2025},
author = {Severino, MB and Morelli, AP and Pavan, ICB and Mancini, MCS and Góis, MM and Borges, RJ and Braga, RR and da Silva, LGS and Quintero-Ruiz, N and Costa, MM and Oliveira, WL and Bezerra, RMN and Ropelle, ER and Simabuco, FM},
title = {A CRISPR-edited isoform of the AMPK kinase LKB1 improves the response to cisplatin in A549 lung cancer cells.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {3},
pages = {108308},
pmid = {39955067},
issn = {1083-351X},
mesh = {Humans ; *Cisplatin/pharmacology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; AMP-Activated Protein Kinase Kinases ; A549 Cells ; *Lung Neoplasms/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; AMP-Activated Protein Kinases/metabolism/genetics ; *Antineoplastic Agents/pharmacology ; Autophagy/drug effects ; Drug Resistance, Neoplasm/genetics ; TOR Serine-Threonine Kinases/metabolism ; NF-E2-Related Factor 2 ; },
abstract = {Lung cancer presents the highest mortality rate in the world when compared to other cancer types and often presents chemotherapy resistance to cisplatin. The A549 nonsmall cell lung cancer line is widely used as a model for lung adenocarcinoma studies since it presents a high proliferative rate and a nonsense mutation in the STK11 gene. The LKB1 protein, encoded by the STK11 gene, is one of the major regulators of cellular metabolism through AMPK activation under nutrient deprivation. Mutation in the STK11 gene in A549 cells potentiates cancer hallmarks, such as deregulation of cellular metabolism, aside from the Warburg effect, mTOR activation, autophagy inhibition, and NRF2 and redox activation. In this study, we investigated the integration of these pathways associated with the metabolism regulation by LKB1/AMPK to improve cisplatin response in the A549 cell line. We first used the CRISPR/Cas9 system to generate cell lines with a CRISPR-edited LKB1 isoform (called Super LKB1), achieved through the introduction of a +1 adenine insertion in the first exon of the STK11 gene after NHEJ-mediated repair. This insertion led to the expression of a higher molecular weight protein containing an alternative exon described in the Peutz-Jeghers Syndrome. Through metabolic regulation by Super LKB1 expression and AMPK activation, we found an increase in autophagy flux (LC3 GFP/RFP p < 0.05), as well as a reduction in the phosphorylation of mTORC1 downstream targets (S6K2 phospho-serine 423; p < 0.05; and S6 ribosomal protein phospho-serine 240/244; p < 0.03). The NRF2 protein exhibited increased levels and more nuclear localization in A549 WT cells compared to the edited cells (p < 0.01). We also observed lower levels of H2O2 in the WT A549 cells, as a possible result of NRF2 activation, and a higher requirement of cisplatin to achieve the IC50 (WT: 10 μM; c2SL+: 5.5 μM; c3SL+: 6 μM). The data presented here suggests that the regulation of molecular pathways by the novel Super LKB1 in A549 cells related to metabolism, mTORC1, and autophagy promotes a better response of lung cancer cells to cisplatin. This NHEJ-CRISPR-based approach may be potentially used for lung cancer gene therapy.},
}

Humans
*Cisplatin/pharmacology
*Protein Serine-Threonine Kinases/genetics/metabolism
AMP-Activated Protein Kinase Kinases
A549 Cells
*Lung Neoplasms/genetics/drug therapy/pathology/metabolism
*CRISPR-Cas Systems
*Gene Editing
AMP-Activated Protein Kinases/metabolism/genetics
*Antineoplastic Agents/pharmacology
Autophagy/drug effects
Drug Resistance, Neoplasm/genetics
TOR Serine-Threonine Kinases/metabolism
NF-E2-Related Factor 2

@article {pmid39947471,
year = {2025},
author = {Elsharkasy, OM and de Voogt, WS and Tognoli, ML and van der Werff, L and Gitz-Francois, JJ and Seinen, CW and Schiffelers, RM and de Jong, OG and Vader, P},
title = {Integrin beta 1 and fibronectin mediate extracellular vesicle uptake and functional RNA delivery.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {3},
pages = {108305},
doi = {10.1016/j.jbc.2025.108305},
pmid = {39947471},
issn = {1083-351X},
mesh = {*Integrin beta1/metabolism/genetics ; *Extracellular Vesicles/metabolism/genetics ; Humans ; *Fibronectins/metabolism/genetics ; *RNA/metabolism/genetics ; CRISPR-Cas Systems ; Animals ; },
abstract = {Extracellular vesicles (EVs) are cell-derived vesicles secreted by all cell types into the extracellular spaces. EVs comprise a heterogenous population of vesicles that carry bioactive molecules, such as proteins, lipids, and RNAs, which they can deliver to recipient cells. Over the past few years, EVs have been recognized for their vital role in intercellular communication, and thereby in various physiological and pathological processes. In addition, EVs are increasingly being studied as potential drug delivery vehicles. It is therefore crucial to understand the mechanisms and molecular players underlying EV uptake and functional cargo delivery. Several studies have investigated various EV uptake pathways; nonetheless, molecular mechanisms governing uptake and cargo transfer remain largely lacking. Here, we show, using a CRISPR/Cas9-mediated reporter system, that integrin β1 on recipient cells plays an important role in EV uptake and EV-mediated RNA delivery. Additionally, using both RNA interference and blocking antibodies, we show that association of integrin β1 with integrin α4 is essential for this process. We demonstrate that α4β1 on recipient cells interacts with EVs through surface localized fibronectin via binding to its leucine-aspartic acid-valine motif, and that blocking of this interaction reduces both EV uptake and RNA delivery. Thus, we identify a key mechanism in EV uptake and cargo delivery which could potentially facilitate research into EV biology and pave the way for the development of novel therapeutic approaches by targeting pathways that lead to functional cargo delivery.},
}

*Integrin beta1/metabolism/genetics
*Extracellular Vesicles/metabolism/genetics
Humans
*Fibronectins/metabolism/genetics
*RNA/metabolism/genetics
CRISPR-Cas Systems
Animals

@article {pmid40186064,
year = {2025},
author = {Zainab, R and Mukhtar, A and Saleem, Z and Kaul, H and Ahmad, A and Majeed, M},
title = {Editing gliA, gliP and gliZ of Aspergillus fumigatus Using CRISPR/Cas System Renders Fungus Incapable to Produce Gliotoxin.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {40186064},
issn = {1559-0305},
support = {13224//Higher Education Commision, Pakistan/ ; },
abstract = {Aspergillus fumigatus is a saprophytic fungus that causes respiratory infections in human, animals, and birds. This fungus produces gliotoxin which is a secondary metabolite that triggers pathogenicity. Gliotoxin is encoded by a 13-gene cluster including gliA, gliP and gliZ. The purpose of this study was to determine whether the fungus produces gliotoxin after these genes are edited using CRISPR/Cas system. For this, crRNAs for gliA, gliP and gliZ were designed using EuPaGDT, while tracrRNA and Cas9 protein were purchased ready-made. These crRNAs were individually annealed with the tracrRNA to make three gRNAs which were then individually combined with the Cas9 to make three ribonucleoprotein (RNP) complexes. A. fumigatus protoplasts were enzymatically generated and transfected with each of the RNP complexes (group 1) in PEGylated conditions. Non-treated protoplasts were simultaneously run as control (group 2). Transfected protoplasts showed reduced growth on SDA plates as compared to their control. Gliotoxin extraction through thin-layer chromatography was carried out for both the groups which showed the absence of gliotoxin in group 1. Sequencing results confirmed the indels in target genes which shows that the CRISPR/Cas9 system effectively targeted A. fumigatus' gliotoxin-related genes that rendered fungus incapable to produce gliotoxin. This work may pave the way to develop effective strategies to control the infections caused by A. fumigatus.},
}

@article {pmid40185749,
year = {2025},
author = {Manjunath, L and Santiago, G and Ortega, P and Sanchez, A and Oh, S and Garcia, A and Li, J and Duong, D and Bournique, E and Bouin, A and Semler, BL and Setiaputra, D and Buisson, R},
title = {Cooperative role of PACT and ADAR1 in preventing aberrant PKR activation by self-derived double-stranded RNA.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3246},
pmid = {40185749},
issn = {2041-1723},
support = {RSG-24-1249960-01-DMC//American Cancer Society (American Cancer Society, Inc.)/ ; },
mesh = {*Adenosine Deaminase/metabolism/genetics ; *eIF-2 Kinase/metabolism/genetics ; *RNA, Double-Stranded/metabolism/genetics ; *RNA-Binding Proteins/metabolism/genetics ; Humans ; Animals ; CRISPR-Cas Systems ; Enzyme Activation ; HEK293 Cells ; Mice ; },
abstract = {Double-stranded RNAs (dsRNAs) produced during viral infections are recognized by the innate immune sensor protein kinase R (PKR), triggering a host translation shutoff that inhibits viral replication and propagation. Given the harmful effects of uncontrolled PKR activation, cells must tightly regulate PKR to ensure that its activation occurs only in response to viral infections, not endogenous dsRNAs. Here, we use CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that exploits translation levels as a readout and identifies PACT as a key inhibitor of PKR during viral infection. We find that PACT-deficient cells hyperactivate PKR in response to different RNA viruses, raising the question of why cells need to limit PKR activity. Our results demonstrate that PACT cooperates with ADAR1 to suppress PKR activation from self-dsRNAs in uninfected cells. The simultaneous deletion of PACT and ADAR1 results in synthetic lethality, which can be fully rescued in PKR-deficient cells. We propose that both PACT and ADAR1 act as essential barriers against PKR, creating a threshold of tolerable levels to endogenous dsRNA in cells without activating PKR-mediated translation shutdown and cell death.},
}

*Adenosine Deaminase/metabolism/genetics
*eIF-2 Kinase/metabolism/genetics
*RNA, Double-Stranded/metabolism/genetics
*RNA-Binding Proteins/metabolism/genetics
Humans
Animals
CRISPR-Cas Systems
Enzyme Activation
HEK293 Cells
Mice

@article {pmid40185080,
year = {2025},
author = {Knight, AL and Lisi, GP},
title = {Spy-ing on nucleic acids: Atomic resolution of the S. pyogenes CRISPR-Cas9 surveillance state.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {4},
pages = {636-638},
doi = {10.1016/j.str.2025.03.001},
pmid = {40185080},
issn = {1878-4186},
mesh = {*CRISPR-Cas Systems ; *Streptococcus pyogenes/enzymology/genetics ; *DNA/metabolism/chemistry ; *Bacterial Proteins/chemistry/metabolism ; *Endonucleases/chemistry/metabolism ; },
abstract = {In a recent issue of Cell Chemical Biology, De Paula et al.[1] report an extensive methyl-TROSY solution NMR study of the CRISPR-Cas9 holoenzyme. Studying millisecond-to-second protein dynamics using individual domain constructs of Cas9 coupled to structural interrogations of the full-length enzyme, the authors describe the Cas9 "surveillance state," a molecular mechanism driving the discrimination between on- and off-target DNA.},
}

*CRISPR-Cas Systems
*Streptococcus pyogenes/enzymology/genetics
*DNA/metabolism/chemistry
*Bacterial Proteins/chemistry/metabolism
*Endonucleases/chemistry/metabolism

@article {pmid40183586,
year = {2025},
author = {Arshad, F and Abdillah, AN and Shivanand, P and Ahmed, MU},
title = {Dual-Mode RPA/CRISPR-Cas12a Biosensor Based on Silica and Magnetic Hybrid Nanobeads for Rapid Detection of Campylobacter jejuni.},
journal = {ACS applied bio materials},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsabm.4c01810},
pmid = {40183586},
issn = {2576-6422},
abstract = {In this study, we developed a biosensor that makes use of recombinase polymerase amplification (RPA) along with a CRISPR/Cas12a system integrated with silica nanobeads and a magnetic nanoparticle nanohybrid complex that displayed peroxidase-mimicking properties. This nanohybrid nanozyme (NZ) integration with the CRISPR/Cas system allowed dual-mode fluorometric and colorimetric responses . The nanohybrid NZ was a conjugated ssDNA quencher probe sequence with inherent fluorometric properties. In the presence of target RPA amplicons, the CRISPR/Cas12a system gets activated, cleaving the probe sequence attached to the NZ complex and leading to fluorescence signal generation. Post-CRISPR/Cas12a assay, the presence of the NZ in the reaction mixture, after being cleaved away from the probe sequence, gave a colourimetric response directly proportional to the target DNA concentration, as the ssDNA probe sequence no longer hindered its catalytic activity. Therefore, the dual-mode detection using the CRISPR/Cas12a-based fluorometric response and NZ-based colorimetric detection conferred high sensitivity and selectivity toward Campylobacter detection. The developed sensor could detect the pathogenic DNA at concentrations as low as 0.98 pg/μL and 0.96 pg/μL via fluorescence and absorbance spectroscopy, respectively. In addition, our method was also tested in raw food analysis and showed good recovery.},
}

@article {pmid40180891,
year = {2025},
author = {Cakiroglu, E and Eris, S and Oz, O and Karakülah, G and Senturk, S},
title = {Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma.},
journal = {Cell death & disease},
volume = {16},
number = {1},
pages = {241},
pmid = {40180891},
issn = {2041-4889},
support = {117Z227//Türkiye Bilimsel ve Teknolojik Araştirma Kurumu (Scientific and Technological Research Council of Turkey)/ ; 117Z227//Türkiye Bilimsel ve Teknolojik Araştirma Kurumu (Scientific and Technological Research Council of Turkey)/ ; TSA-2024-3533//Dokuz Eylül Üniversitesi (Dokuz Eylul University)/ ; },
mesh = {Humans ; *Protein Serine-Threonine Kinases/genetics/metabolism/antagonists & inhibitors ; Cell Line, Tumor ; *Mesothelioma, Malignant/genetics/pathology/drug therapy ; Apoptosis/genetics/drug effects ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems/genetics ; *Pleural Neoplasms/genetics/pathology/drug therapy ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Malignant pleural mesothelioma (MPM) is a rare yet highly aggressive malignancy with a severe prognosis. Compounded by the lack of effective treatment modalities, MPM remains a formidable health challenge. Therefore, the identification of actionable liabilities is critical for advancing precision medicine to combat this lethal disease. Here, we exploit an unbiased genome-wide CRISPR screen, integrating and cross-comparing three MPM cell lines with nonmalignant mesothelial cells, to selectively map the gene targets whose depletion indicates a common dependency in MPM cells. This systematic approach unveils a cohort of verifiable genes, among which BUB1, a mitotic checkpoint serine/threonine kinase, emerges as a high-confidence hit in cancer cells. Cellular and molecular studies demonstrate that genetic depletion or pharmacological inhibition of BUB1 profoundly impairs MPM cell survival and growth while inducing G2/M cell cycle arrest, cellular senescence, and apoptosis, and attenuating functional hallmarks of aggressive cancer cells. Transcriptomic profiling of BUB1-depleted cells discloses differential gene expression signatures congruent with cell fate phenotypes, including the reprogramming of mitotic network genes. Mechanistically, BUB1 is indispensable for the proper localization of essential mitotic regulators MAD1, MAD2, and Shugoshin (SGO1), thereby ensuring the functionality of the spindle assembly checkpoint (SAC). Furthermore, BUB1 ablation leads to cytokinesis failure and multinucleation, a phenotype characterized by the downregulation of CDC20, Cyclin A, and Cyclin B, and a reciprocal upregulation of the cyclin-dependent kinase inhibitor p21. Clinically, MPM tumors exhibit elevated levels of BUB1, and high BUB1 expression is associated with shorter patient survival. Our novel findings accentuate comparative CRISPR screens as a powerful platform to explore tumor cell-selective gene essentiality and propose BUB1 kinase as a potential marker and druggable vulnerability with therapeutic implications for MPM.},
}

Humans
*Protein Serine-Threonine Kinases/genetics/metabolism/antagonists & inhibitors
Cell Line, Tumor
*Mesothelioma, Malignant/genetics/pathology/drug therapy
Apoptosis/genetics/drug effects
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems/genetics
*Pleural Neoplasms/genetics/pathology/drug therapy
Cell Proliferation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40110701,
year = {2025},
author = {Liang, H and Li, T and Chen, Y and Wang, J and Aslam, M and Qin, H and Fan, W and Du, H and Kao, SJ and Lin, S},
title = {Urea Amidolyase as an Enzyme for Urea Utilisation in Phytoplankton: Functional Display in Chlamydomonas reinhardtii.},
journal = {Molecular ecology},
volume = {34},
number = {8},
pages = {e17734},
doi = {10.1111/mec.17734},
pmid = {40110701},
issn = {1365-294X},
support = {42206116//Natural Science Foundation of China/ ; 2024A1515011467；2025A1515010095//Natural Science Foundation of Guangdong Province, China/ ; 2021B1212050025//Science and Technology Plan Projects of Guangdong Province/ ; 2022KCXTD008//Program for University Innovation Team of Guangdong Province/ ; 2024-MRB-00-001//Research on Breeding Technology of Candidate Species for Guangdong Modern Marine Ranching/ ; },
mesh = {*Chlamydomonas reinhardtii/genetics/enzymology/metabolism ; *Urea/metabolism ; *Phytoplankton/genetics/enzymology/metabolism ; *Amidohydrolases/genetics/metabolism ; Nitrogen/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Urease/genetics/metabolism ; Metagenome ; },
abstract = {Urea is an important source of nitrogen for many phytoplankton with the potential to stimulate harmful algal blooms, but the molecular machinery underpinning urea uptake and assimilation by algae is not fully understood. Urease (URE) is commonly regarded as the responsible enzyme, but urea amidolyase (UAL), albeit known to exist, has hardly been studied. Here, the species distribution, expression patterns and functional roles of UAL are examined. We found a widespread occurrence of UAL across six major phytoplankton lineages, along with evidence of a potential URE-independent evolutionary trajectory and lineage-specific losses. Quantitative analyses based on marine planktonic metagenomes and metatranscriptomes revealed that UAL is as prevalent as URE, but exhibits higher expression levels in phytoplankton than in bacteria, suggesting that UAL plays a crucial role in nitrogen nutrition in marine phytoplankton. Furthermore, using the CRISPR/Cas9 genome editing method and Chlamydomonas reinhardtii as the algal model, we showed that DUR2 in UAL is essential for urea utilisation, as its knockout completely abolishes the ability of algae to grow under urea as the sole nitrogen source. This study unveils an unappreciated mechanism in algae for utilising urea as a nutrient, underscores the need to consider both URE and UAL enzyme systems to model urea utilisation by algae and provides a crucial gene (DUR2) as a potential genetic marker for detecting the contribution of UAL to urea utilisation in phytoplankton.},
}

*Chlamydomonas reinhardtii/genetics/enzymology/metabolism
*Urea/metabolism
*Phytoplankton/genetics/enzymology/metabolism
*Amidohydrolases/genetics/metabolism
Nitrogen/metabolism
Phylogeny
CRISPR-Cas Systems
Urease/genetics/metabolism
Metagenome

@article {pmid39910976,
year = {2025},
author = {Gou, L and Liu, D and Fan, TP and Deng, H and Cai, Y},
title = {Construction of a CRISPR-Cas9-Based Genetic Editing Tool for Serratia marcescens Using a Stationary Phase Promoter and Its Application in Putrescine Production.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {5},
pages = {1233-1244},
doi = {10.1002/bit.28949},
pmid = {39910976},
issn = {1097-0290},
support = {//The authors received no specific funding for this work./ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Serratia marcescens/genetics/metabolism ; *Promoter Regions, Genetic/genetics ; *Putrescine/biosynthesis/metabolism ; },
abstract = {Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in Serratia marcescens. S. marcescens has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in S. marcescens. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from Streptococcus pyogenes protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various S. marcescens strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in S. marcescens HBQA7, optimizing the expression of ornithine decarboxylase from Clostridium aceticum DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances S. marcescens gene editing and metabolic engineering.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Serratia marcescens/genetics/metabolism
*Promoter Regions, Genetic/genetics
*Putrescine/biosynthesis/metabolism

@article {pmid40179700,
year = {2025},
author = {Shen, D and Guo, H and Zhang, F and Chen, X and Tong, X and Li, H and Wu, W and Mei, S},
title = {Highly-sensitive and logic platform based on spatially-constrained T7 transcription enhanced Cas13a for DNA repair enzyme detection and intracellular imaging.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117406},
doi = {10.1016/j.bios.2025.117406},
pmid = {40179700},
issn = {1873-4235},
abstract = {The activity of DNA repair enzymes, particularly Flap endonuclease 1 (FEN1) and apurinic/apyrimidinic endonuclease 1 (APE1), plays a critical role in disease prevention, diagnosis, and prognosis. Accurate detection of these enzymes is therefore essential. Recent advancements in CRISPR-Cas technology, particularly its programmable and trans-cleavage activity, have paved the way for the development of innovative detection methods. However, there is a need for a simple, low-background, highly sensitive detection platform with logical capabilities for FEN1 and APE1. In this study, we present a novel detection platform that integrates spatially constrained T7 transcription with the CRISPR-Cas13a system. This biosensor minimizes background interference and achieves high sensitivity, with limits of detection as low as 5 × 10[-7] U/μL for FEN1 and 2 × 10[-8] U/μL for APE1, making it one of the most sensitive methods available for detecting these enzymes. The platform supports both OR and logic detection, offering enhanced versatility. It demonstrates robustness by detecting FEN1 activity at concentrations as low as 1 cell/μL and screening enzyme inhibitors. Additionally, the system was successfully used for intracellular imaging of FEN1 activity in cells and reliably measured APE1 activity in ovarian tissue samples, confirming its clinical applicability. This biosensor represents a promising tool for detecting FEN1 and APE1, further expanding the potential of CRISPR-Cas13a in diagnostic applications.},
}

@article {pmid40163950,
year = {2025},
author = {Fang, L and Yang, X and Li, Y and Xue, C and Li, Z and Jiang, H and Li, X and Lu, S and Wang, D and He, H and Huang, Z and Guo, X and Luo, G},
title = {SPECIAL: Phosphorothioate dNTP assisted RPA equipped with CRISPR/Cas12a amplifier enables high-specific nucleic acid testing.},
journal = {Biosensors & bioelectronics},
volume = {279},
number = {},
pages = {117421},
doi = {10.1016/j.bios.2025.117421},
pmid = {40163950},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; *Recombinases/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Recombinase polymerase amplification (RPA) is one of the most widely used isothermal amplification methods and considered to be a promising tool for point-of-care testing (POCT) molecular diagnosis. However, RPA is prone to have nonspecific amplification occur, due to the poor recognition accuracy of polymerase and recombinase, which severely hindered its clinical application. It is important to improve the specificity of RPA further. Herein, we developed a novel nucleic acid testing method termed phosphorothioate dNTP (dNTPαS) assisted RPA (S-RPA) that employs dNTPαS as substrates to suppress nonspecific amplification effectively. We found that dNTPαS could improve the recognition accuracy of Bsu polymerase and recombinase, thereby enhancing their amplification specificity. Our S-RPA provided much higher specificity (approximately 40 % improvement compared to classical RPA), realizing detection target with single nucleotide mutation. Based on its outstanding performance, we further combined the S-RPA with CRISPR/Cas12a to achieve highly specific and sensitive fluorescence detection, namely S-RPA equipped with CRISPR/Cas12a amplifier (SPECIAL). Our SPECIAL was more sensitive (10-fold higher) than the classical RPA-CRISPR/Cas12a assay, offering 100 % agreement with the qPCR during clinical validation. In summary, a strategy based on dNTPαS was established to enhance the specificity of RPA, thereby improving its practicability and providing a potential POCT tool for molecular diagnosis.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Humans
*Recombinases/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40179192,
year = {2025},
author = {Martin-Rufino, JD and Caulier, A and Lee, S and Castano, N and King, E and Joubran, S and Jones, M and Goldman, SR and Arora, UP and Wahlster, L and Lander, ES and Sankaran, VG},
title = {Transcription factor networks disproportionately enrich for heritability of blood cell phenotypes.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6742},
pages = {52-59},
doi = {10.1126/science.ads7951},
pmid = {40179192},
issn = {1095-9203},
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; *Gene Regulatory Networks ; *Chromatin/metabolism/genetics ; Phenotype ; Single-Cell Analysis ; CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; *Blood Cells ; Cell Differentiation/genetics ; *Erythropoiesis/genetics ; Erythroid Cells/cytology ; },
abstract = {Most phenotype-associated genetic variants map to noncoding regulatory regions of the human genome, but their mechanisms remain elusive in most cases. We developed a highly efficient strategy, Perturb-multiome, to simultaneously profile chromatin accessibility and gene expression in single cells with CRISPR-mediated perturbation of master transcription factors (TFs). We examined the connection between TFs, accessible regions, and gene expression across the genome throughout hematopoietic differentiation. We discovered that variants within TF-sensitive accessible chromatin regions in erythroid differentiation, although representing <0.3% of the genome, show a ~100-fold enrichment for blood cell phenotype heritability, which is substantially higher than that for other accessible chromatin regions. Our approach facilitates large-scale mechanistic understanding of phenotype-associated genetic variants by connecting key cis-regulatory elements and their target genes within gene regulatory networks.},
}

Humans
*Transcription Factors/genetics/metabolism
*Gene Regulatory Networks
*Chromatin/metabolism/genetics
Phenotype
Single-Cell Analysis
CRISPR-Cas Systems
Genome, Human
Genetic Variation
*Blood Cells
Cell Differentiation/genetics
*Erythropoiesis/genetics
Erythroid Cells/cytology

@article {pmid40178896,
year = {2025},
author = {Kim, DY and Lee, SY and Ha, HJ and Park, HH},
title = {AcrIE7 inhibits the CRISPR-Cas system by directly binding to the R-loop single-stranded DNA.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {14},
pages = {e2423205122},
doi = {10.1073/pnas.2423205122},
pmid = {40178896},
issn = {1091-6490},
support = {2021R1A2C3003331//National Research Foundation of Korea (NRF)/ ; },
mesh = {*DNA, Single-Stranded/metabolism/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Protein Binding ; *Bacterial Proteins/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; },
abstract = {The CRISPR-Cas system is a well-known adaptive immune system in bacteria, and a prominent mechanism for evading this immunity involves anti-CRISPR (Acr) proteins, which employ various methods to neutralize the CRISPR-Cas system. In this study, using structural and biochemical analyses, we revealed that AcrIE7 binds to the single-stranded DNA in the R-loop formed when Cascade encounters the target DNA, thereby preventing Cas3 from cleaving the DNA. This represents a different inhibition strategy distinct from previously reported Acr mechanisms and offers insights into CRISPR-Cas inhibition.},
}

*DNA, Single-Stranded/metabolism/chemistry/genetics
*CRISPR-Cas Systems/genetics
Protein Binding
*Bacterial Proteins/metabolism/chemistry/genetics
*CRISPR-Associated Proteins/metabolism/chemistry/genetics

@article {pmid40176181,
year = {2025},
author = {Yamamoto, A and Tanaka, Y and Ishibashi, S and Ikeda, M and Sugita, K and Ono, M and Nishi, H and Kurata, M},
title = {Modified screening of MYC promotor region elements using the CRISPR library in ovarian cancer.},
journal = {Journal of ovarian research},
volume = {18},
number = {1},
pages = {68},
pmid = {40176181},
issn = {1757-2215},
support = {21K16800//KAKENHI/ ; },
mesh = {Humans ; Female ; *Ovarian Neoplasms/genetics/pathology ; *Promoter Regions, Genetic ; Proto-Oncogene Mas ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-myc/genetics ; Gene Expression Regulation, Neoplastic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Gene Library ; },
abstract = {Ovarian cancer remains one of the most lethal gynecological malignancies owing to its high recurrence rate and chemotherapeutic resistance. MYC is a well-known proto-oncogene that is frequently amplified in ovarian cancer and has been implicated in drug resistance. Previously, we established a new promoter-reporter system combined with a CRISPR activation library to identify unknown MYC regulators, and M1AP was identified as a novel MYC regulator. However, considering the insufficient explanation for the absence of guide RNA (gRNA) of MYC, this present study explored methods to prevent the gRNA of MYC itself from binding. This study first modified the promoter-reporter vector to improve its quality, then conducted CRISPR screening and analyzed candidate genes as MYC promoter regulators using next-generation sequencing in OVSAHO ovarian cancer cells. Eighty-six genes had ≥ 1000 reads, and Pearson's correlation coefficient analysis was performed on the cBioPortal of the Cancer Genomics database. Fourteen genes were identified as candidate MYC regulators with positive and significant correlations with MYC. Seven genes, including CYP4v2, ASPH, ANP32D, PCED1A, ABI1, FUZ, and HOOK2, demonstrated significantly higher luciferase activity than the control genes. Four genes, including ABI1, PCED1A, HOOK2, and CYP4v2, activated the MYC promoter, which showed over twofold higher activity than the control when overexpressed using a vector. In conclusion, four genes that activate MYC promoters were identified in an ovarian cancer cell line using the CRISPR library system with a modified promoter-reporter tool. These results will prove helpful in the development of novel treatment strategies for ovarian cancer.},
}

Humans
Female
*Ovarian Neoplasms/genetics/pathology
*Promoter Regions, Genetic
Proto-Oncogene Mas
Cell Line, Tumor
*Proto-Oncogene Proteins c-myc/genetics
Gene Expression Regulation, Neoplastic
*Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Gene Library

@article {pmid40176174,
year = {2025},
author = {Okesanya, OJ and Ahmed, MM and Ogaya, JB and Amisu, BO and Ukoaka, BM and Adigun, OA and Manirambona, E and Adebusuyi, O and Othman, ZK and Oluwakemi, OG and Ayando, OD and Tan, MIRS and Idris, NB and Kayode, HH and Oso, TA and Ahmed, M and Kouwenhoven, MBN and Ibrahim, AM and Lucero-Prisno, DE},
title = {Reinvigorating AMR resilience: leveraging CRISPR-Cas technology potentials to combat the 2024 WHO bacterial priority pathogens for enhanced global health security-a systematic review.},
journal = {Tropical medicine and health},
volume = {53},
number = {1},
pages = {43},
pmid = {40176174},
issn = {1348-8945},
abstract = {BACKGROUND: Antimicrobial resistance (AMR) poses a global health threat, particularly in low- and middle-income countries (LMICs). Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system technology offers a promising tool to combat AMR by targeting and disabling resistance genes in WHO bacterial priority pathogens. Thus, we systematically reviewed the potential of CRISPR-Cas technology to address AMR.

METHODS: This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive literature search was conducted using the Scopus and PubMed databases, focusing on publications from 2014 to June 2024. Keywords included "CRISPR/Cas," "antimicrobial resistance," and "pathogen." The eligibility criteria required original studies involving CRISPR/Cas systems that targeted AMR. Data were extracted from eligible studies, qualitatively synthesized, and assessed for bias using the Joanna Briggs Institute (JBI)-standardized tool.

RESULTS: Data from 48 eligible studies revealed diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas3, targeting various AMR genes, such as blaOXA-232, blaNDM, blaCTX-M, ermB, vanA, mecA, fosA3, blaKPC, and mcr-1, which are responsible for carbapenem, cephalosporin, methicillin, macrolide, vancomycin, colistin, and fosfomycin resistance. Some studies have explored the role of CRISPR in virulence gene suppression, including enterotoxin genes, tsst1, and iutA in Staphylococcus aureus and Klebsiella pneumoniae. Delivery mechanisms include bacteriophages, nanoparticles, electro-transformation, and conjugative plasmids, which demonstrate high efficiency in vitro and in vivo. CRISPR-based diagnostic applications have demonstrated high sensitivity and specificity, with detection limits as low as 2.7 × 10[2] CFU/mL, significantly outperforming conventional methods. Experimental studies have reported significant reductions in resistant bacterial populations and complete suppression of the targeted strains. Engineered phagemid particles and plasmid-curing systems have been shown to eliminate IncF plasmids, cured plasmids carrying vanA, mcr-1, and blaNDM with 94% efficiency, and restore antibiotic susceptibility. Gene re-sensitization strategies have been used to restore fosfomycin susceptibility in E. coli and eliminate blaKPC-2-mediated carbapenem resistance in MDR bacteria. Whole-genome sequencing and bioinformatics tools have provided deeper insights into CRISPR-mediated defense mechanisms. Optimization strategies have significantly enhanced gene-editing efficiencies, offering a promising approach for tackling AMR in high-priority WHO pathogens.

CONCLUSIONS: CRISPR-Cas technology has the potential to address AMR across priority WHO pathogens. While promising, challenges in optimizing in vivo delivery, mitigating potential resistance, and navigating ethical-regulatory barriers must be addressed to facilitate clinical translation.},
}

@article {pmid40175930,
year = {2025},
author = {Xiao, G and Liu, H and Xu, H and Shi, H and Liu, D and Ou, M and Liu, P and Zhang, G},
title = {Direct detection from sputum for drug-resistant Mycobacterium tuberculosis using a CRISPR-Cas14a-based approach.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {188},
pmid = {40175930},
issn = {1471-2180},
support = {82300128//National Natural Science Foundation of China/ ; 82170009//National Natural Science Foundation of China/ ; 2020YFA0907200//National Key Research and Development Program of China/ ; KCXFZ20211020163545004//Shenzhen Scientific and Technological Foundation/ ; JCYJ20210324130009024//Shenzhen Scientific and Technological Foundation/ ; JCYJ20220530163216036//Shenzhen Scientific and Technological Foundation/ ; RCJC20221008092726022//Shenzhen Scientific and Technological Foundation/ ; 0620220214//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; 2020B1111170014//Science and Technology Planning Project of Guangdong Province/ ; SZZYSM202311009//Sanming Project of Medicine in Shenzen Municipality/ ; },
mesh = {*Sputum/microbiology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; Rifampin/pharmacology ; Microbial Sensitivity Tests ; Sensitivity and Specificity ; Antitubercular Agents/pharmacology ; Isoniazid/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics ; Mutation ; },
abstract = {The increasing prevalence of multidrug-resistant tuberculosis (MDR-TB) highlights the urgent need for an efficient approach to identify Mycobacterium tuberculosis complex (MTBC) strains resistant to rifampicin (RIF) and isoniazid (INH). In response, we developed a CRISPR-Cas14a MTB RIF/INH platform that can detect the most common mutations associated with RIF and INH resistance. To evaluate the sensitivity and specificity of our CRISPR-Cas14a MTB RIF/INH platform, we carried out a comprehensive assessment using clinical isolates of M. tuberculosis and sputum samples from TB patients, making direct comparisons with phenotypic drug susceptibility testing (pDST). A total of 60 clinical isolates from TB patients were utilized, consisting of 18 RIF mono-resistant, 15 INH mono-resistant, 24 MDR isolates, and 3 fully susceptible isolates. Among the 42 RIF-resistant isolates, our platform accurately identified 39, achieving a sensitivity of 93.3% (95% CI, 80.0-98.5) and a specificity of 100% (95% CI, 81.6-100). Similarly, out of the 39 INH-resistant isolates, the platform successfully identified 38, demonstrating a sensitivity of 97.5% (95% CI, 86.5-99.9) and a specificity of 100% (95% CI, 83.8-100) when compared with pDST. Moreover, in the analysis of 55 sputum samples, our platform accurately identified RIF resistance in 10 out of 12 samples (85.7%) and INH resistance in all 11 samples (100%). Notably, excluding the nucleic acid extraction step, the entire testing procedure can be completed in approximately 1.5 h. These results suggest that the CRISPR-Cas14a MTB RIF/INH platform is a reliable and promising novel tool for detecting RIF and INH resistance in isolates or directly from sputum samples.},
}

*Sputum/microbiology
*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
Humans
*CRISPR-Cas Systems
*Tuberculosis, Multidrug-Resistant/microbiology/diagnosis
Rifampin/pharmacology
Microbial Sensitivity Tests
Sensitivity and Specificity
Antitubercular Agents/pharmacology
Isoniazid/pharmacology
Drug Resistance, Multiple, Bacterial/genetics
Mutation

@article {pmid40175837,
year = {2025},
author = {Moreno, DS and Cunha, J and de Melo, LDR and Tanaka, K and Bamba, T and Hasunuma, T and Azeredo, J and Domingues, L},
title = {CRISPR-Cas9 engineered Saccharomyces cerevisiae for endolysin delivery to combat Listeria monocytogenes.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {81},
pmid = {40175837},
issn = {1432-0614},
support = {UIDB/04469/2020//Fundação para a Ciência e a Tecnologia/ ; UI/BD/151411/2021//Fundação para a Ciência e a Tecnologia/ ; },
mesh = {*Listeria monocytogenes/drug effects ; *Saccharomyces cerevisiae/genetics/metabolism ; *Endopeptidases/genetics/metabolism/pharmacology ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology/metabolism ; Bacteriophages/enzymology/genetics ; Listeriosis/prevention & control/microbiology ; },
abstract = {Listeriosis is an infection caused by the consumption of food contaminated with Listeria monocytogenes. It leads to febrile gastroenteritis, central nervous system infections, and even death in risk populations. Bacteriophage endolysins selectively kill bacteria hydrolyzing their cell walls and have emerged as a potential tool for listeriosis control. Ply511 is an anti-Listeria endolysin that has activity against all serovars of L. monocytogenes. The yeast Saccharomyces cerevisiae has been used to produce endolysins for biocontrol, but prior efforts relied on plasmids, which can lead to gene loss and include selection markers unsuitable for therapeutic use. Integration of endolysins in its genome has also been previously demonstrated, relying however, on selection markers for selection and maintenance of the modifications. This study explores S. cerevisiae as a generally regarded as safe (GRAS) platform for producing and displaying Ply511 through CRISPR-Cas9 integration, offering a marker-free and stable solution for Listeria biocontrol. Our results demonstrate that the surface display of Ply511 does not lead to bacterial reduction. In contrast, we show that yeast secreting endolysin significantly reduces L. monocytogenes in cells, supernatants, and cell extracts. The strongest effect was observed with concentrated spent supernatant and cell extract, which reduced L. monocytogenes below the lower limit of quantification. Additionally, the spent supernatant exhibited active anti-Listeria activity in milk. This study highlights yeast-secreted endolysins as a promising platform for listeriosis control and demonstrates the yeast secretion of endolysins can be used for the biocontrol of pathogenic bacteria. KEY POINTS: • S. cerevisiae was edited using CRISPR-Cas9 to display or secrete endolysin Ply511. • Cells, supernatants, and extracts of yeast secreting Ply511 act against L. monocytogenes. • Demonstrates the yeast-based delivery of endolysins to control L. monocytogenes.},
}

*Listeria monocytogenes/drug effects
*Saccharomyces cerevisiae/genetics/metabolism
*Endopeptidases/genetics/metabolism/pharmacology
*CRISPR-Cas Systems
*Anti-Bacterial Agents/pharmacology/metabolism
Bacteriophages/enzymology/genetics
Listeriosis/prevention & control/microbiology

@article {pmid40175136,
year = {2025},
author = {Adachi, Y},
title = {[Identification of genes regulating human CAR-T cell proliferation by genome-wide CRISPR screening].},
journal = {[Rinsho ketsueki] The Japanese journal of clinical hematology},
volume = {66},
number = {3},
pages = {145-152},
doi = {10.11406/rinketsu.66.145},
pmid = {40175136},
issn = {0485-1439},
mesh = {Humans ; Cell Proliferation ; *T-Lymphocytes/immunology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Receptors, Chimeric Antigen/immunology ; Immunotherapy, Adoptive ; },
abstract = {In vivo expansion and long-term maintenance of CAR-T cells are considered to be the hallmark of treatment success after CD19 CAR-T cell therapy. Genome-wide CRISPR screening has emerged as a powerful tool for large-scale gene screens. Genome-wide CRISPR screening revealed that CUL5 gene knockout (KO) improved the proliferation of CD19 CAR-T cells. CUL5KO improved not only the proliferation but also the effector function of CAR-T cells. The JAK-STAT pathway was upregulated in CUL5KO CAR-T cells, and CUL5 was associated with the degradation of JAK3 upon activation through IL-2 signaling. CUL5KO CD19 CAR-T cells efficiently suppressed in vivo tumor progression as compared to control CD19 CAR-T cells.},
}

Humans
Cell Proliferation
*T-Lymphocytes/immunology/cytology
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Receptors, Chimeric Antigen/immunology
Immunotherapy, Adoptive

@article {pmid40172964,
year = {2025},
author = {Patel, AC and Sinha, S and Arantes, PR and Palermo, G},
title = {Unveiling Cas8 dynamics and regulation within a transposon-encoded Cascade-TniQ complex.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {14},
pages = {e2422895122},
doi = {10.1073/pnas.2422895122},
pmid = {40172964},
issn = {1091-6490},
support = {R01GM141329//HHS | National Institutes of Health (NIH)/ ; CHE-2144823//National Science Foundation (NSF)/ ; FG-2023-20431//Alfred P. Sloan Foundation (APSF)/ ; TC-24-063//Camille and Henry Dreyfus Foundation (Dreyfus Foundation)/ ; },
mesh = {*Vibrio cholerae/genetics/metabolism ; *DNA Transposable Elements/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; CRISPR-Associated Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Protein Conformation ; Models, Molecular ; },
abstract = {The Vibrio cholerae Cascade-TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of "knocking-in" genes at desired sites, the mechanisms underlying DNA binding and transposition remain elusive. In this system, a conformational change of the Cas8 protein is essential for DNA binding, yet how it occurs is unclear. Here, structural modeling and free energy simulations reconstruct the Cas8 helical bundle and reveal an open-closed conformational change that is key for the complex's function. We show that when Cascade-TniQ binds RNA, the Cas8 bundle changes conformation mediated by the interaction with the Cas7.1 protein. This interaction promotes the bundle's transition toward the open state, priming the complex for DNA binding. As the target DNA binds the guide RNA, the opening of the Cas8 bundle becomes more favorable, exposing positively charged residues and facilitating their interaction with DNA, which ultimately leads the DNA-binding process to completion. These outcomes provide a dynamic representation of a critical conformational change in one of the largest CRISPR systems and illustrate its role at critical steps of the Cascade-TniQ biophysical function, advancing our understanding of nucleic acid binding and transposition mechanisms.},
}

*Vibrio cholerae/genetics/metabolism
*DNA Transposable Elements/genetics
*Bacterial Proteins/metabolism/genetics/chemistry
CRISPR-Associated Proteins/metabolism/chemistry/genetics
CRISPR-Cas Systems
Protein Binding
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Protein Conformation
Models, Molecular

@article {pmid39970912,
year = {2025},
author = {Zilberzwige-Tal, S and Altae-Tran, H and Kannan, S and Wilkinson, ME and Vo, SC and Strebinger, D and Edmonds, KK and Yao, CJ and Mears, KS and Shmakov, SA and Makarova, KS and Macrae, RK and Koonin, EV and Zhang, F},
title = {Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins.},
journal = {Cell},
volume = {188},
number = {7},
pages = {1925-1940.e20},
doi = {10.1016/j.cell.2025.01.034},
pmid = {39970912},
issn = {1097-4172},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Toxin-Antitoxin Systems ; Cryoelectron Microscopy ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Evolution, Molecular ; Bacterial Proteins/metabolism/genetics/chemistry ; *Antitoxins/metabolism/chemistry/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Untranslated/metabolism/genetics ; },
abstract = {Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*Toxin-Antitoxin Systems
Cryoelectron Microscopy
CRISPR-Associated Proteins/metabolism/genetics/chemistry
Evolution, Molecular
Bacterial Proteins/metabolism/genetics/chemistry
*Antitoxins/metabolism/chemistry/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Untranslated/metabolism/genetics

@article {pmid40171918,
year = {2025},
author = {Wu, W and Miao, R and Li, Z and Fang, Z},
title = {CRISPR/Cas9-mediated editing of BADH2 and Wx genes for the development of novel aromatic and soft-textured black and red rice.},
journal = {Physiologia plantarum},
volume = {177},
number = {2},
pages = {e70194},
doi = {10.1111/ppl.70194},
pmid = {40171918},
issn = {1399-3054},
support = {Qiankehezhongyindi (2023) 008//the Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province/ ; qiankehechengguo (2024) General 116//the Guizhou Provincial Science and Technology Projects/ ; qiankehejichu-ZK (2021) General 128//the Guizhou Provincial Science and Technology Projects/ ; qiankehejichu-ZK (2022) Key 008//the Guizhou Provincial Science and Technology Projects/ ; qiankehepingtai-YWZ (2024) 004//the Guizhou Provincial Science and Technology Projects/ ; qiankehepingtairencai-BQW (2024) 001//the Guizhou Provincial Science and Technology Projects/ ; Qiandongnan Kehe Support (2023)06//the Qiandongnan Science and Technology Support Project/ ; Qianjiaoji (2023)007//the Key Laboratory of Functional Agriculture of Guizhou Provincial Department of Education/ ; qiankehepingtairencai-YQK (2023) 002//the Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; 32260498/32060064//the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Amylose/metabolism/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Odorants/analysis ; Gas Chromatography-Mass Spectrometry ; Pyrroles ; },
abstract = {Black and red rice are known for their rich nutritional content, yet most varieties suffer from a firm texture and insufficient fragrance. In this study, we aimed to develop a fragrant and soft-textured black and red rice variety using the CRISPR/Cas9 technology to knock out the OsWx gene, which is associated with amylose content (AC), and the OsBADH2 gene, responsible for rice aroma. Our results showed that, compared to wild-type, CRISPR lines of XHZ, HM, NWZ, and PGZ targeting OsWx and OsBADH2 exhibited a reduction in AC content, altered gel consistency, and a more than 50% increase in gel consistency. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed that the 2-acetyl-1-pyrroline (2-AP) content in the grains of xhz-c[BADH2 Wx] and hm-c[BADH2 Wx] reached 189.04 μg kg[-1] and 309.03 μg kg[-1], respectively. Furthermore, we observed a slight increase in anthocyanins and proanthocyanidins in these co-edited lines, without significant effects on their agronomic traits. Furthermore, to investigate the genes involved in the quality formation of black and red rice for the knockout of OsBADH2 and OsWx, we conducted RNA-seq analysis. The results indicated that knockout of OsBADH2 and OsWx affected the expression of genes involved in carotenoid biosynthesis, multiple amino acid metabolism genes, and endosperm starch and sucrose metabolic pathways. These findings suggest that the CRISPR/Cas9 technology can effectively target OsBADH2 and OsWx to develop high-quality black and red rice varieties with enhanced aroma and softer texture, providing a new strategy for the improvement of colored rice.},
}

*Oryza/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Amylose/metabolism/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified/genetics
Odorants/analysis
Gas Chromatography-Mass Spectrometry
Pyrroles

@article {pmid40127118,
year = {2025},
author = {Dhinoja, S and Qaryoute, AA and Deebani, A and De Maria, A and Jagadeeswaran, P},
title = {CRISPR/Cas9 mediated generation of zebrafish f9a mutant as a model for hemophilia B.},
journal = {Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis},
volume = {36},
number = {3},
pages = {90-98},
doi = {10.1097/MBC.0000000000001355},
pmid = {40127118},
issn = {1473-5733},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Hemophilia B/genetics ; *Factor IX/genetics/metabolism ; Disease Models, Animal ; Mutation ; Gene Knockout Techniques ; Humans ; },
abstract = {AIM: This study aimed to develop a zebrafish model for hemophilia B by creating a f9a knockout, as f9a has previously demonstrated functional similarity to human Factor IX.

METHODS: Using CRISPR/Cas9 technology, two gRNAs targeting exon 8 of the f9a gene, were injected along with Cas9 protein into single-cell zebrafish wild-type embryos. DNA was harvested from the tail tips of the resulting adult zebrafish and screened for mutations using PCR. The founder mutant was crossed with wild-type fish to confirm heritability and subsequently reared to homozygosity. Homozygous mutants were analyzed through quantitative RT-PCR and Western blot to assess f9a RNA and F9a protein levels, respectively. Functional assays like kinetic partial thromboplastin time (kPTT), bleeding assay in adult mutants, and venous laser injury on mutant larvae were performed to assess the hemostatic role.

RESULTS: Around 61 adults from the CRISPR/Cas9 knockouts were screened, which resulted in a mutant line with a 72 bp deletion in the exon 8 encoding catalytic domain. Quantitative RT-PCR and Western Blot analysis showed reduced levels of f9a RNA and F9a protein in the homozygous mutants compared to wild-type siblings. At five dpf, f9a homozygous mutant larvae demonstrated prolonged venous occlusion times in a laser injury assay. Additionally, plasma from the mutants displayed delayed fibrin formation in kPTT assays and exhibited increased bleeding after mechanical injury.

CONCLUSION: This study created a zebrafish f9a knockout model that mimics the bleeding phenotype observed in hemophilia B patients, which will be valuable for evaluating novel therapeutic approaches for hemophilia B.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Hemophilia B/genetics
*Factor IX/genetics/metabolism
Disease Models, Animal
Mutation
Gene Knockout Techniques
Humans

@article {pmid40110772,
year = {2025},
author = {Yang, W and Peng, M and Wang, Y and Zhang, X and Li, W and Zhai, X and Wu, Z and Hu, P and Chen, L},
title = {Deletion of hepcidin disrupts iron homeostasis and hematopoiesis in zebrafish embryogenesis.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {7},
pages = {},
doi = {10.1242/dev.204307},
pmid = {40110772},
issn = {1477-9129},
support = {32200414//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Hepcidins/genetics/metabolism ; *Hematopoiesis/genetics ; *Iron/metabolism ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Developmental ; Ferroptosis/genetics ; Gene Deletion ; Embryo, Nonmammalian/metabolism ; Gene Knockout Techniques ; GATA1 Transcription Factor/metabolism/genetics ; },
abstract = {Iron is essential for cell growth and hematopoiesis, which is regulated by hepcidin (hamp). However, the role of hamp in zebrafish hematopoiesis remains unclear. Here, we have created a stable hamp knockout zebrafish model using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 system (CRISPR/Cas9 system). Our study revealed that hamp deletion led to maternal iron overload in embryos, significantly downregulating hemoglobin genes and reducing hemoglobin content. Single-cell RNA sequencing identified abnormal expression patterns in blood progenitor cells, with a specific progenitor subtype showing increased ferroptosis and delayed development. By crossing hamp knockout zebrafish with a gata1+ line (blood cells labeled fish line), we confirmed ferroptosis in blood progenitor cells. These findings underscore the crucial role of hamp in iron regulation and hematopoiesis, offering novel insights into developmental biology and potential therapeutic targets for blood disorders.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Hepcidins/genetics/metabolism
*Hematopoiesis/genetics
*Iron/metabolism
*Homeostasis/genetics
*Embryonic Development/genetics
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Expression Regulation, Developmental
Ferroptosis/genetics
Gene Deletion
Embryo, Nonmammalian/metabolism
Gene Knockout Techniques
GATA1 Transcription Factor/metabolism/genetics

@article {pmid40054857,
year = {2025},
author = {Kougnassoukou Tchara, PE and Loehr, J and Lambert, JP},
title = {Coupling Proximity Biotinylation with Genomic Targeting to Characterize Locus-Specific Changes in Chromatin Environments.},
journal = {Journal of proteome research},
volume = {24},
number = {4},
pages = {1845-1860},
doi = {10.1021/acs.jproteome.4c00931},
pmid = {40054857},
issn = {1535-3907},
mesh = {Humans ; *Chromatin/genetics/metabolism/chemistry ; Biotinylation/methods ; Cell Line, Tumor ; Azepines/pharmacology ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Triazoles/pharmacology ; *Proteomics/methods ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Melanoma/genetics ; Genetic Loci ; Bromodomain Containing Proteins ; Proteins ; },
abstract = {Regulating gene expression involves significant changes in the chromatin environment at the locus level, especially at regulatory sequences. However, their modulation following pharmacological treatments or pathological conditions remain mostly undetermined. Here, we report versatile locus-specific proteomics tools to address this knowledge gap, which combine the targeting ability of the CRISPR/Cas9 system and the protein-labeling capability of the highly reactive biotin ligases TurboID (in CasTurbo) and UltraID (in CasUltra). CasTurbo and CasUltra enabled rapid chromatin protein labeling at repetitive sequences like centromeres and telomeres, as well as nonamplified genes. We applied CasUltra to A375 melanoma cell lines to decipher the protein environment of the MYC promoter and characterize the molecular effects of the bromodomain inhibitor JQ1, which targets bromodomain and extra-terminal (BET) proteins that regulate MYC expression. We quantified the consequences of BET protein displacement from the MYC promoter and found that it was associated with a considerable reorganization of the chromatin composition. Additionally, BET protein retention at the MYC promoter was consistent with a model of increased JQ1 resistance. Thus, through the combination of proximity biotinylation and CRISPR/Cas9 genomic targeting, CasTurbo and CasUltra have successfully demonstrated their utility in profiling the proteome associated with a genomic locus in living cells.},
}

Humans
*Chromatin/genetics/metabolism/chemistry
Biotinylation/methods
Cell Line, Tumor
Azepines/pharmacology
Promoter Regions, Genetic
CRISPR-Cas Systems
Triazoles/pharmacology
*Proteomics/methods
Proto-Oncogene Proteins c-myc/genetics/metabolism
Melanoma/genetics
Genetic Loci
Bromodomain Containing Proteins
Proteins

@article {pmid40014809,
year = {2025},
author = {Li, M and Lin, Y and Cheng, Q and Wei, T},
title = {Prime Editing: A Revolutionary Technology for Precise Treatment of Genetic Disorders.},
journal = {Cell proliferation},
volume = {58},
number = {4},
pages = {e13808},
doi = {10.1111/cpr.13808},
pmid = {40014809},
issn = {1365-2184},
support = {2023ZD0500601//Noncommunicable Chronic Diseases-National Science and Technology Major Project/ ; 2023IOZ0204//Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences/ ; 22JCZXJC00030//Beijing-Tianjin-Hebei Basic Research Cooperation Project/ ; NCTIB2023XB02001//the "Open Competition to Select the Best Candidates" Key Technology Program for Nucleic Acid Drugs of NCTIB/ ; //the Fundamental Research Funds for the Central Universities, Peking University/ ; 2023000CC0040//the Beijing Life Science Academy/ ; 2023M740101//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems/genetics ; },
abstract = {Genetic diseases have long posed significant challenges, with limited breakthroughs in treatment. Recent advances in gene editing technologies offer new possibilities in gene therapy for the treatment of inherited disorders. However, traditional gene editing methods have limitations that hinder their potential for clinical use, such as limited editing capabilities and the production of unintended byproducts. To overcome these limitations, prime editing (PE) has been developed as a powerful tool for precise and efficient genome modification. In this review, we provide an overview of the latest advancements in PE and its potential applications in the treatment of inherited disorders. Furthermore, we examine the current delivery vehicles employed for delivering PE systems in vitro and in vivo, and analyze their respective benefits and limitations. Ultimately, we discuss the challenges that need to be addressed to fully unlock the potential of PE for the remission or cure of genetic diseases.},
}

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

@article {pmid39731215,
year = {2025},
author = {Zhou, Z and Chen, Y and Ba, Y and Xu, H and Zuo, A and Liu, S and Zhang, Y and Weng, S and Ren, Y and Luo, P and Cheng, Q and Zuo, L and Zhu, S and Zhou, X and Zhang, C and Chen, Y and Han, X and Pan, T and Liu, Z},
title = {Revolutionising Cancer Immunotherapy: Advancements and Prospects in Non-Viral CAR-NK Cell Engineering.},
journal = {Cell proliferation},
volume = {58},
number = {4},
pages = {e13791},
doi = {10.1111/cpr.13791},
pmid = {39731215},
issn = {1365-2184},
support = {221100310100//Henan Provincial Science and Technology Research Project/ ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *Cell Engineering/methods ; *Immunotherapy, Adoptive/methods ; *Immunotherapy/methods ; Gene Editing ; Animals ; CRISPR-Cas Systems ; },
abstract = {The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.},
}

Humans
*Killer Cells, Natural/immunology/transplantation
*Neoplasms/therapy/immunology
*Receptors, Chimeric Antigen/immunology/genetics/metabolism
*Cell Engineering/methods
*Immunotherapy, Adoptive/methods
*Immunotherapy/methods
Gene Editing
Animals
CRISPR-Cas Systems

@article {pmid40171792,
year = {2025},
author = {Diril, MK and Esmen, K and Sehitogullari, T and Őztürk, G},
title = {Generation of albino C57BL/6J mice by CRISPR embryo editing of the mouse tyrosinase locus.},
journal = {Journal of genetics},
volume = {104},
number = {},
pages = {},
pmid = {40171792},
issn = {0973-7731},
mesh = {Animals ; *Gene Editing/methods ; *Monophenol Monooxygenase/genetics ; *CRISPR-Cas Systems ; Mice ; *Mice, Inbred C57BL ; Embryo, Mammalian/metabolism ; Zygote/metabolism ; Electroporation/methods ; Female ; Gene Knock-In Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Loci ; INDEL Mutation ; },
abstract = {After the arrival of the CRISPR/Cas9 genome editing technology, genetic engineering of model organisms has become much faster and more efficient. The development of genetically modified mouse models is also facilitated by the application of various CRISPR methodologies. Although the very first studies utilized pronuclear injection (PNI) of Cas9 mRNA and sgRNAs into the zygote stage embryos to create knockout and knockin mutations, the repertoire of techniques and collection of reagents for CRISPR editing has rapidly expanded. This presents researchers in the field with a versatility of choices for genetic engineering. However, there are not many comparative studies that analysed the efficacy of gene editing when Cas9 and sgRNA/ssDNA oligos were transferred to the embryos by different methodologies. Here, we aimed to compare two different methods, electroporation and PNI. One of the recent developments gaining wide use in mouse model research is the application of electroporation for the introduction of Cas9/sgRNA ribonucleoprotein complexes into zygote stage embryos. Here, we have used this technique to generate albino coat-coloured C57BL/6J mice by targeted inactivation of the mouse tyrosinase gene through indel or knockin mutations. We have also applied the PNI protocol with the same set of reagents, to compare the efficiency of the two techniques in generation of indel and knockin mutations. Although PNI results in signifi- cantly higher efficiency for knockin mutations, it requires specialized equipment setup and advanced training in embryo micromanipulation and microinjection. Therefore, for the generation of simple gene knockouts by indel mutations, electroporation can be used.},
}

Animals
*Gene Editing/methods
*Monophenol Monooxygenase/genetics
*CRISPR-Cas Systems
Mice
*Mice, Inbred C57BL
Embryo, Mammalian/metabolism
Zygote/metabolism
Electroporation/methods
Female
Gene Knock-In Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Loci
INDEL Mutation

@article {pmid40171546,
year = {2025},
author = {Kaur, N and Qadir, M and Francis, DV and Alok, A and Tiwari, S and Ahmed, ZFR},
title = {CRISPR/Cas9: a sustainable technology to enhance climate resilience in major Staple Crops.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1533197},
pmid = {40171546},
issn = {2673-3439},
abstract = {Climate change is a global concern for agriculture, food security, and human health. It affects several crops and causes drastic losses in yield, leading to severe disturbances in the global economy, environment, and community. The consequences on important staple crops, such as rice, maize, and wheat, will worsen and create food insecurity across the globe. Although various methods of trait improvements in crops are available and are being used, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) mediated genome manipulation have opened a new avenue for functional genomics and crop improvement. This review will discuss the progression in crop improvement from conventional breeding methods to advanced genome editing techniques and how the CRISPR/Cas9 technology can be applied to enhance the tolerance of the main cereal crops (wheat, rice, and maize) against any harsh climates. CRISPR/Cas endonucleases and their derived genetic engineering tools possess high accuracy, versatile, more specific, and easy to design, leading to climate-smart or resilient crops to combat food insecurity and survive harsh environments. The CRISPR/Cas9-mediated genome editing approach has been applied to various crops to make them climate resilient. This review, supported by a bibliometric analysis of recent literature, highlights the potential target genes/traits and addresses the significance of gene editing technologies in tackling the vulnerable effects of climate change on major staple crops staple such as wheat, rice, and maize.},
}

@article {pmid40170307,
year = {2025},
author = {Mao, Y and Chu, G and Liang, Q and Liu, Y and Yang, Y and Liao, X and Wang, M},
title = {[Artificial intelligence-assisted design, mining, and modification of CRISPR-Cas systems].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {3},
pages = {949-967},
doi = {10.13345/j.cjb.240865},
pmid = {40170307},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Engineering/methods ; Data Mining ; Synthetic Biology/methods ; },
abstract = {With the rapid advancement of synthetic biology, CRISPR-Cas systems have emerged as a powerful tool for gene editing, demonstrating significant potential in various fields, including medicine, agriculture, and industrial biotechnology. This review comprehensively summarizes the significant progress in applying artificial intelligence (AI) technologies to the design, mining, and modification of CRISPR-Cas systems. AI technologies, especially machine learning, have revolutionized sgRNA design by analyzing high-throughput sequencing data, thereby improving the editing efficiency and predicting off-target effects with high accuracy. Furthermore, this paper explores the role of AI in sgRNA design and evaluation, highlighting its contributions to the annotation and mining of CRISPR arrays and Cas proteins, as well as its potential for modifying key proteins involved in gene editing. These advancements have not only improved the efficiency and precision of gene editing but also expanded the horizons of genome engineering, paving the way for intelligent and precise genome editing.},
}

*CRISPR-Cas Systems
*Artificial Intelligence
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Engineering/methods
Data Mining
Synthetic Biology/methods

@article {pmid40170086,
year = {2025},
author = {Wang, H and Qi, Z and Wang, J and He, Z and Lu, L and Chen, Z and Shao, Y and Wang, G and Wang, Z and Tu, J and Song, X},
title = {Rapid and visual detection of transmissible gastroenteritis virus using a CRISPR/Cas12a system combined with loop-mediated isothermal amplification.},
journal = {BMC veterinary research},
volume = {21},
number = {1},
pages = {234},
pmid = {40170086},
issn = {1746-6148},
support = {GXXT-2022-057//University Synergy Innovation Program of Anhui Province/ ; YQYB2023001//Anhui Provincial University Excellent Young Talents Support Program/ ; 2023SJY01//Research Funds of Joint Research Center for Food Nutrition and Health of IHM/ ; },
mesh = {*Transmissible gastroenteritis virus/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/veterinary ; Animals ; *Gastroenteritis, Transmissible, of Swine/diagnosis/virology ; Swine ; Sensitivity and Specificity ; Molecular Diagnostic Techniques/methods/veterinary ; Endodeoxyribonucleases/genetics ; },
abstract = {BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV). The primary techniques for identifying TGEV involve enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescent quantitative PCR (qPCR). However, these approaches are complex, demanding specialized tools and significant time. Therefore, a precise, swift, and effective differential diagnosis method is crucial for TGEV prevention. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas-associated proteins have become popular for their high specificity, unique cleavage activity, and ease of detection. CRISPR-Cas12a, a novel RNA-guided nucleic acid endonuclease, is emerging as a powerful molecular scissor.

RESULTS: In this study, we designed three pairs of crRNA targeting the N gene of TGEV. Following the selection of the most appropriate crRNA, we established the loop-mediated isothermal (LAMP) amplification method with a sensitivity of 10[2] copies/µL. And based on this, we established the CRISPR-Cas12a fluorescence assay with a sensitivity of 10[0] copies/µL. Furthermore, we established a CRISPR/Cas12a lateral-flow dipstick assay with a sensitivity of 10[2] copies/µL. Importantly, none of these methods exhibited cross-reactivity with other related viruses, enabling quicker and more straightforward observation of experimental results.

CONCLUSIONS: We have successfully developed a CRISPR-Cas12a fluorescence assay and a CRISPR/Cas12a lateral-flow dipstick assay for clinical TGEV detection. Overall, we created a portable, quick, and sensitive TGEV assay with strong specificity utilizing the CRISPR-Cas12a system.},
}

*Transmissible gastroenteritis virus/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/veterinary
Animals
*Gastroenteritis, Transmissible, of Swine/diagnosis/virology
Swine
Sensitivity and Specificity
Molecular Diagnostic Techniques/methods/veterinary
Endodeoxyribonucleases/genetics

@article {pmid40169710,
year = {2025},
author = {Palomino, SM and Gabriel, KA and Mwirigi, JM and Cervantes, A and Horton, P and Funk, G and Moutal, A and Martin, LF and Khanna, R and Price, TJ and Patwardhan, A},
title = {Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {11116},
pmid = {40169710},
issn = {2045-2322},
support = {NS116694/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Ganglia, Spinal/metabolism/cytology ; *Neurons/metabolism ; *TRPV Cation Channels/genetics/metabolism ; Cells, Cultured ; Transfection/methods ; },
abstract = {CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca[2+] accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Ganglia, Spinal/metabolism/cytology
*Neurons/metabolism
*TRPV Cation Channels/genetics/metabolism
Cells, Cultured
Transfection/methods

@article {pmid40166066,
year = {2025},
author = {Saeed, U and Piracha, ZZ and Khan, M and Tariq, MN and Gilani, SS and Raza, M and Munusamy, R and Bose, N and Ozsahin, DU and Özşahin, İ and Nauli, SM},
title = {Cracking the code of HBV persistence: cutting-edge approaches to targeting cccDNA in chronic hepatitis B with or without pyogenic liver Abscesses.},
journal = {Frontiers in medicine},
volume = {12},
number = {},
pages = {1504736},
pmid = {40166066},
issn = {2296-858X},
abstract = {Chronic Hepatitis B Virus (HBV) infection remains a formidable global health challenge, driving severe liver complications such as hepatocellular carcinoma (HCC) and pyogenic liver abscesses (PLA). At the core of HBV persistence lies covalently closed circular DNA (cccDNA), a viral reservoir that fuels ongoing infection despite antiviral treatments. This review highlights molecular mechanisms governing cccDNA formation, maintenance, and clearance, spotlighting innovative therapeutic strategies to disrupt this key viral element. We explore cutting-edge approaches, including epigenetic modulation to silence cccDNA, RNA interference (RNAi) for viral RNA degradation, and CRISPR/Cas genome editing to excise cccDNA directly. Additionally, emerging antiviral therapies and immunotherapies, such as therapeutic vaccines and immune checkpoint inhibitors, offer new avenues for enhanced treatment efficacy. Special attention is given to the clinical complexities of managing HBV in patients with co-morbid conditions like HCC and PLA, emphasizing the necessity of a multidisciplinary approach. The interplay between antibacterial and antiviral therapies in PLA-associated HBV cases is critically examined to prevent treatment antagonism, ensuring optimal patient outcomes. Advanced therapeutic strategies, including nucleos(t)ide analogs, interferon therapy, and novel genomic interventions, are explored in both isolated HBV infection and PLA co-infections. Personalized regimens remain pivotal in enhancing therapeutic efficacy and long-term disease control. Current review advocates for a shift toward precision medicine, highlighting the critical need for interdisciplinary collaboration to bridge molecular discoveries with clinical innovations. Ultimately, these advancements promise to revolutionize the management of chronic HBV, paving the way for potential cures and improved patient outcomes.},
}

@article {pmid40165637,
year = {2025},
author = {Guo, Q and Shen, Q and Hao, Q and Jiang, XL and Zou, LP and Xue, YP and Zheng, YG},
title = {Resolving the Trade-Off Between Toxicity and Efficiency of CRISPR-Cas9 System for Genome Editing Within Escherichia coli.},
journal = {Biotechnology journal},
volume = {20},
number = {4},
pages = {e70010},
doi = {10.1002/biot.70010},
pmid = {40165637},
issn = {1860-7314},
support = {2021YFC2102100//National Key Research and Development Program/ ; 2024C03011//Zhejiang Provincial Science and Technology Plan Project/ ; },
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plasmids/genetics ; Genome, Bacterial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Efficient gene editing of Escherichia coli BL21 (DE3) holds significant practical value as a host for heterologous protein expression. Recently reported CRISPR-Cas9 editing systems for this strain exhibit a trade-off between efficiency and toxicity. In this study, we addressed this trade-off by employing the strategy to transiently induce Cas9 expression in the high-copy plasmid during the editing stage. Furthermore, we demonstrated that eliminating the sgRNA-expressing plasmid using a temperature-sensitive replicon, combined with SacB for removing the Cas9-expressing plasmid, exhibited higher efficiency compared to previously reported strategies for editing system removal. We assigned this optimized CRISPR-Cas9 genome editing system as the pEBcas9/pEBsgRNA system, which has successfully achieved efficient five rounds of genome editing and simultaneous editing of multiple loci in E. coli BL21 (DE3). Using this system, we identified several loci suitable for multi-copy integrated expression of exogenous genes. Overall, the pEBcas9/pEBsgRNA system may facilitate the application of E. coli in both industrial and academic fields.},
}

*Escherichia coli/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Plasmids/genetics
Genome, Bacterial/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40165508,
year = {2025},
author = {Zhang, T and Zhao, Y and Zhu, C and Zhu, X and Zhu, X and Qiu, Y and Nie, Z and Lei, C},
title = {CRISPR/Cas12a Protein Switch Powered Label-Free Electrochemical Biosensor for Sensitive Viral Protease Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c00547},
pmid = {40165508},
issn = {1520-6882},
abstract = {Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor's mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.},
}

@article {pmid40126138,
year = {2025},
author = {Pirali, A and Jafarpour, F and Hajian, M and Hosseini Moghaddam, SH and Moradi, R and Tanhaie-Vash, N and Rahimi Andani, M and Izadi, T and Shiralian-Esfahani, H and Safaeinejad, Z and Kues, W and Nasr-Esfahani, MH and Eghbalsaied, S},
title = {Editing the CYP19 Gene in Goat Embryos Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer Techniques.},
journal = {Cellular reprogramming},
volume = {27},
number = {2},
pages = {86-93},
doi = {10.1089/cell.2024.0109},
pmid = {40126138},
issn = {2152-4998},
mesh = {Animals ; *Nuclear Transfer Techniques/veterinary ; *CRISPR-Cas Systems ; *Aromatase/genetics ; *Gene Editing/methods ; *Goats/genetics/embryology ; Female ; Blastocyst/metabolism/cytology ; *Embryo, Mammalian/metabolism ; Embryonic Development/genetics ; Gene Knockout Techniques ; Male ; Cloning, Organism ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system is revolutionizing genome engineering and is expected to bring significant advancements in livestock traits, including the treatment of genetic diseases. This study focuses on CRISPR/Cas9-mediated modifications of the CYP19 gene, which encodes aromatase, an enzyme crucial for converting testosterone to estrogen and essential for steroid metabolism. Guide RNAs (gRNAs) were designed to target the CYP19 gene and cloned into the pX459 vector. The recombinant plasmid was then electrotransfected into fibroblast cells from a Lori-Bakhtiari buck, and these transfected cells were used for embryo production via somatic cell nuclear transfer (SCNT). The cloned embryos were evaluated for their progression through embryonic stages, showing no significant difference in blastocyst development between knock-out and unedited groups. The knockout efficiency was 78.4% in cells and 68.9% in goat blastocysts, demonstrating the successful depletion of CYP19. We successfully achieved a high rate of CYP19 gene-edited embryos through the combined application of cell electrotransfection and SCNT technologies, while maintaining the normal developmental rate of the embryos. These embryos can be used for transfer to generate knock-out goats, providing a foundation for further studies on CYP19's role in male fertility and production traits.},
}

Animals
*Nuclear Transfer Techniques/veterinary
*CRISPR-Cas Systems
*Aromatase/genetics
*Gene Editing/methods
*Goats/genetics/embryology
Female
Blastocyst/metabolism/cytology
*Embryo, Mammalian/metabolism
Embryonic Development/genetics
Gene Knockout Techniques
Male
Cloning, Organism

@article {pmid40022449,
year = {2025},
author = {Wallace, KA and Gerstenberg, TL and Ennis, CL and Perez-Bermejo, JA and Partridge, JR and Bandoro, C and Matern, WM and Andreoletti, G and Krassovsky, K and Kabir, S and Lalisan, CD and Churi, AR and Chew, GM and Corbo, L and Vincelette, JE and Klasson, TD and Silva, BJ and Strukov, YG and Quejarro, BJ and Hill, KA and Treusch, S and Grogan, JL and Dever, DP and Porteus, MH and Wienert, B},
title = {A differentiated β-globin gene replacement strategy uses heterologous introns to restore physiological expression.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1407-1419},
doi = {10.1016/j.ymthe.2025.02.036},
pmid = {40022449},
issn = {1525-0024},
mesh = {*beta-Globins/genetics/metabolism ; *Introns ; Humans ; CRISPR-Cas Systems ; *beta-Thalassemia/genetics/therapy ; Animals ; *Anemia, Sickle Cell/genetics/therapy ; Mice ; Genetic Therapy/methods ; Gene Editing ; Recombinational DNA Repair ; },
abstract = {β-Hemoglobinopathies are common monogenic disorders. In sickle cell disease (SCD), a single mutation in the β-globin (HBB) gene results in dysfunctional hemoglobin protein, while in β-thalassemia, over 300 mutations distributed across the gene reduce β-globin levels and cause severe anemia. Genetic engineering replacing the whole HBB gene through homology-directed repair (HDR) is an ideal strategy to restore a benign genotype and rescue HBB expression for most genotypes. However, this is technically challenging because (1) the insert must not be homologous to the endogenous gene and (2) synonymous codon-optimized, intron-less sequences may not reconstitute adequate β-globin levels. Here, we developed an HBB gene replacement strategy using CRISPR-Cas9 that successfully addresses these challenges. We determined that a DNA donor containing a diverged HBB coding sequence and heterologous introns to avoid sequence homology provides proper physiological expression. We identified a DNA donor that uses truncated γ-globin introns, results in 34% HDR, and rescues β-globin expression in in vitro models of SCD and β-thalassemia in hematopoietic stem and progenitor cells (HSPCs). Furthermore, while HDR allele frequency dropped in vivo, it was maintained at ∼15%, demonstrating editing of long-term repopulating HSPCs. In summary, our HBB gene replacement strategy offers a differentiated approach by restoring naturally regulated adult hemoglobin expression.},
}

*beta-Globins/genetics/metabolism
*Introns
Humans
CRISPR-Cas Systems
*beta-Thalassemia/genetics/therapy
Animals
*Anemia, Sickle Cell/genetics/therapy
Mice
Genetic Therapy/methods
Gene Editing
Recombinational DNA Repair

@article {pmid39955618,
year = {2025},
author = {Baatz, F and Ghosh, A and Herbst, J and Polten, S and Meyer, J and Rhiel, M and Maetzig, T and Geffers, R and Rothe, M and Bastone, AL and John-Neek, P and Frühauf, J and Eiz-Vesper, B and Bonifacius, A and Falk, CS and Kaisenberg, CV and Cathomen, T and Schambach, A and van den Brink, MRM and Hust, M and Sauer, MG},
title = {Targeting BCL11B in CAR-engineered lymphoid progenitors drives NK-like cell development with prolonged anti-leukemic activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1584-1607},
doi = {10.1016/j.ymthe.2025.02.024},
pmid = {39955618},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; CRISPR-Cas Systems ; *Killer Cells, Natural/immunology/metabolism/cytology ; *Lymphoid Progenitor Cells/metabolism/immunology/cytology ; *Repressor Proteins/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *Leukemia/therapy/immunology/genetics ; Immunotherapy, Adoptive ; Cell Differentiation ; },
abstract = {Chimeric antigen receptor (CAR)-induced suppression of the transcription factor B cell CLL/lymphoma 11B (BCL11B) propagates CAR-induced killer (CARiK) cell development from lymphoid progenitors. Here, we show that CRISPR-Cas9-mediated Bcl11b knockout in human and murine early lymphoid progenitors distinctively modulates this process either alone or in combination with a CAR. Upon adoptive transfer into hematopoietic stem cell recipients, Bcl11b-edited progenitors mediated innate-like antigen-independent anti-leukemic immune responses. With CAR expression allowing for additional antigen-specific responses, the progeny of double-edited lymphoid progenitors acquired prolonged anti-leukemic activity in vivo. These findings give important insights into how Bcl11b targeting can be used to tailor anti-leukemia functionality of CAR-engineered lymphoid progenitor cells.},
}

Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
CRISPR-Cas Systems
*Killer Cells, Natural/immunology/metabolism/cytology
*Lymphoid Progenitor Cells/metabolism/immunology/cytology
*Repressor Proteins/genetics/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
*Leukemia/therapy/immunology/genetics
Immunotherapy, Adoptive
Cell Differentiation

@article {pmid39935177,
year = {2025},
author = {Cheng, Y and Zhang, J and Mu, W and Ye, S and Cheng, J and Zhu, L and Wang, G and Cao, Y and Li, D and Hu, G and Huang, L and Wang, J and Zhou, J},
title = {Dasatinib-resistant universal CAR-T cells proliferate in the presence of host immune cells and exhibit antitumor activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1535-1551},
doi = {10.1016/j.ymthe.2025.02.012},
pmid = {39935177},
issn = {1525-0024},
mesh = {*Dasatinib/pharmacology ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Cell Proliferation/drug effects ; *T-Lymphocytes/immunology/metabolism/drug effects ; *Drug Resistance, Neoplasm ; Protein Kinase Inhibitors/pharmacology ; Receptors, Antigen, T-Cell/genetics ; Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics ; CRISPR-Cas Systems ; Mutation ; },
abstract = {The universal chimeric antigen receptor T cell (UCAR-T) immunotherapy derived from healthy donors holds great promise in pan-cancer treatment. However, UCAR-T cell therapy faces a challenge in the rapid elimination of allogeneic cells by the host immune system. To address this, we introduced a T316I mutation in the leukocyte-specific protein tyrosine kinase (LCK) locus in CAR-T cells using the cytosine base editor (CBE) system. Concurrently, we disrupted endogenous T cell receptor alpha chain (TRAC) and beta-2 microglobulin (B2M) with the CRISPR-Cas9 system, along with dasatinib to overcome host immune rejection, an Src family kinase (SFK) inhibitor. The resulting LCK mutated UCAR-T (KM UCAR-T) cells exhibited normal phenotypes in activation, proliferation, differentiation, and tumor cytotoxicity in vitro. Moreover, KM UCAR-T cells demonstrated sustained expansion in mixed lymphocyte reactions (MLR) when incubated with T cells or peripheral blood mononuclear cells (PBMCs) from HLA-mismatched donors upon dasatinib treatment. Additionally, we illustrated that KM UCAR-T cells displayed antitumor activity in a xenograft murine model and verified the expansion and cytotoxicity of KM UCAR-T over traditional UCAR-T in the presence of allogeneic PBMCs when treated with dasatinib in vivo. These findings offer a novel strategy for UCAR-T cells to resist host immune rejection and achieve sustained expansion.},
}

*Dasatinib/pharmacology
Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Immunotherapy, Adoptive/methods
Xenograft Model Antitumor Assays
Cell Line, Tumor
Cell Proliferation/drug effects
*T-Lymphocytes/immunology/metabolism/drug effects
*Drug Resistance, Neoplasm
Protein Kinase Inhibitors/pharmacology
Receptors, Antigen, T-Cell/genetics
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics
CRISPR-Cas Systems
Mutation

@article {pmid39921871,
year = {2025},
author = {Lv, J and Fan, Q and Zhang, Y and Zhou, X and Yu, P and Yu, X and Xin, C and Hong, J and Cheng, Y},
title = {A Serum Resistant Polymer with Exceptional Endosomal Escape and mRNA Delivery Efficacy for CRISPR Gene Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {13},
pages = {e2413006},
doi = {10.1002/advs.202413006},
pmid = {39921871},
issn = {2198-3844},
support = {2019YFA0904500//National Key R&D Program of China/ ; 2023YFA0915000//National Key R&D Program of China/ ; 52373134//National Natural Science Foundation of China/ ; 82171102//National Natural Science Foundation of China/ ; 82271044//National Natural Science Foundation of China/ ; 2019YFA0904500//National Key Research and Development Program of China/ ; 2023YFA0915000//National Key Research and Development Program of China/ ; 22Y21900900//Shanghai Medical Innovation Research Program/ ; },
mesh = {Animals ; *RNA, Messenger/genetics/metabolism/administration & dosage ; *Genetic Therapy/methods ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Endosomes/metabolism ; *Polymers/chemistry ; Gene Editing/methods ; Nanoparticles/chemistry ; *Gene Transfer Techniques ; Choroidal Neovascularization/therapy/genetics ; },
abstract = {Nanoparticle-based mRNA delivery offers a versatile platform for innovative therapies. However, most of the current delivery systems are limited by poor serum tolerance, suboptimal endosomal escape and mRNA delivery efficacy. Herein, a highly efficient mRNA-delivering material is identified from a library of fluoropolymers. The lead material FD17 shows exceptional serum stability and endosomal escape, enabling efficient mRNA delivery into various cell types, surpassing commercial mRNA delivery reagents such as Lipofectamine 3000. The formed mRNA nanoparticles adsorb abundant serum albumin on the surface, which facilitates cellular uptake via scavenger receptor-mediated endocytosis. FD17 enables the delivery of mRNAs encoding CRE, Cas9, and base editor hyCBE for efficient genome editing. The material mediates CRISPR/Cas9 gene therapy via intraocular injection effectively down-regulates vascular endothelial growth factor A in retinal pigment epithelial cells of mice, yielding promising therapeutic responses against laser-induced choroidal neovascularization. The discovered material in this study shows great promise for the development of mRNA therapeutics to combat a wide range of diseases.},
}

Animals
*RNA, Messenger/genetics/metabolism/administration & dosage
*Genetic Therapy/methods
Mice
*CRISPR-Cas Systems/genetics
Humans
*Endosomes/metabolism
*Polymers/chemistry
Gene Editing/methods
Nanoparticles/chemistry
*Gene Transfer Techniques
Choroidal Neovascularization/therapy/genetics

@article {pmid39881543,
year = {2025},
author = {Tachida, Y and Manian, KV and Butcher, R and Levy, JM and Pendse, N and Hennessey, E and Liu, DR and Pierce, EA and Liu, Q and Comander, J},
title = {Systematic empirical evaluation of individual base editing targets: Validating therapeutic targets in USH2A and comparison of methods.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1466-1484},
doi = {10.1016/j.ymthe.2025.01.042},
pmid = {39881543},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *Extracellular Matrix Proteins/genetics ; Mice ; Mutation ; Genetic Therapy/methods ; *Usher Syndromes/genetics/therapy ; High-Throughput Nucleotide Sequencing ; Disease Models, Animal ; Dependovirus/genetics ; CRISPR-Cas Systems ; },
abstract = {Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids vs. transgenes) and assays (next-generation sequencing vs. Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knockin mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955∗). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65% ± 3% at the mutant base pair and of 52% ± 3% excluding bystander amino acid changes. This efficiency is higher than that seen in a retinal gene editing program testing in a clinical trial. These results demonstrate the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.},
}

Humans
*Gene Editing/methods
Animals
*Extracellular Matrix Proteins/genetics
Mice
Mutation
Genetic Therapy/methods
*Usher Syndromes/genetics/therapy
High-Throughput Nucleotide Sequencing
Disease Models, Animal
Dependovirus/genetics
CRISPR-Cas Systems

@article {pmid39825209,
year = {2025},
author = {Jin, Q and Feng, X and Hong, M and Wang, K and Chen, X and Cheng, J and Kuang, Y and Si, X and Xu, M and Huang, X and Guang, S and Zhu, C},
title = {Peri-centrosomal localization of small interfering RNAs in C. elegans.},
journal = {Science China. Life sciences},
volume = {68},
number = {4},
pages = {895-911},
pmid = {39825209},
issn = {1869-1889},
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; Caenorhabditis elegans Proteins/metabolism/genetics ; *Centrosome/metabolism ; *RNA, Small Interfering/metabolism/genetics ; RNA Interference ; RNA-Dependent RNA Polymerase/metabolism/genetics ; Argonaute Proteins/metabolism/genetics ; CRISPR-Cas Systems ; RNA-Binding Proteins ; },
abstract = {The centrosome is the microtubule-organizing center and a crucial part of cell division. Centrosomal RNAs (cnRNAs) have been reported to enable precise spatiotemporal control of gene expression during cell division in many species. Whether and how cnRNAs exist in C. elegans are unclear. Here, using the nuclear RNAi Argonaute protein NRDE-3 as a reporter, we observed potential peri-centrosome localized small interfering (si)RNAs in C. elegans. NRDE-3 was previously shown to associate with pre-mRNAs and pre-rRNAs via a process involving the presence of complementary siRNAs. We generated a GFP-NRDE-3 knock-in transgene through CRISPR/Cas9 technology and observed that NRDE-3 formed peri-centrosomal foci neighboring the tubulin protein TBB-2, other centriole proteins and pericentriolar material (PCM) components in C. elegans embryos. The peri-centrosomal accumulation of NRDE-3 depends on RNA-dependent RNA polymerase (RdRP)-synthesized 22G siRNAs and the PAZ domain of NRDE-3, which is essential for siRNA binding. Mutation of eri-1, ergo-1, or drh-3 significantly increased the percentage of pericentrosome-enriched NRDE-3. At the metaphase of the cell cycle, NRDE-3 was enriched in both the peri-centrosomal region and the spindle. Moreover, the integrity of centriole proteins and pericentriolar material (PCM) components is also required for the peri-centrosomal accumulation of NRDE-3. Therefore, we concluded that siRNAs could accumulate in the pericentrosomal region in C. elegans and suggested that the peri-centrosomal region may also be a platform for RNAi-mediated gene regulation.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
Caenorhabditis elegans Proteins/metabolism/genetics
*Centrosome/metabolism
*RNA, Small Interfering/metabolism/genetics
RNA Interference
RNA-Dependent RNA Polymerase/metabolism/genetics
Argonaute Proteins/metabolism/genetics
CRISPR-Cas Systems
RNA-Binding Proteins

@article {pmid39462750,
year = {2025},
author = {Liang, L and Tan, H and Liu, R},
title = {Emerging gene editing in industrial microbiology beyond CRISPR-Cas9.},
journal = {Trends in biotechnology},
volume = {43},
number = {4},
pages = {742-744},
doi = {10.1016/j.tibtech.2024.09.012},
pmid = {39462750},
issn = {1879-3096},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Industrial Microbiology/methods/trends ; },
abstract = {The CRISPR-Cas9 system has been widely applied for industrial microbiology but is not effective in certain microorganisms. This forum explores the strategies aimed at overcoming these challenges, including the use of the Cas12a system, Cas9 variants, and non-CRISPR techniques, to provide more effective strategies for expanding applications in microbial engineering.},
}

*Gene Editing/methods/trends
*CRISPR-Cas Systems/genetics
*Industrial Microbiology/methods/trends

@article {pmid39327106,
year = {2025},
author = {Yan, Y and Ahmed, HMM and Wimmer, EA and Schetelig, MF},
title = {Biotechnology-enhanced genetic controls of the global pest Drosophila suzukii.},
journal = {Trends in biotechnology},
volume = {43},
number = {4},
pages = {826-837},
doi = {10.1016/j.tibtech.2024.09.005},
pmid = {39327106},
issn = {1879-3096},
mesh = {Animals ; *Drosophila/genetics ; *Biotechnology/methods ; *Pest Control, Biological/methods ; Male ; CRISPR-Cas Systems ; Female ; Animals, Genetically Modified ; },
abstract = {Spotted wing Drosophila (Drosophila suzukii Matsumura, or SWD), an insect pest of soft-skinned fruits native to East Asia, has rapidly spread worldwide in the past 15 years. Genetic controls such as sterile insect technique (SIT) have been considered for the environmentally friendly and cost-effective management of this pest. In this review, we provide the latest developments for the genetic control strategies of SWD, including sperm-marking strains, CRISPR-based sex-ratio distortion, neoclassical genetic sexing strains, transgenic sexing strains, a sex-sorting incompatible male system, precision-guided SIT, and gene drives based on synthetic Maternal effect dominant embryonic arrest (Medea) or homing CRISPR systems. These strategies could either enhance the efficacy of traditional SIT or serve as standalone methods for the sustainable control of SWD.},
}

Animals
*Drosophila/genetics
*Biotechnology/methods
*Pest Control, Biological/methods
Male
CRISPR-Cas Systems
Female
Animals, Genetically Modified

@article {pmid40165374,
year = {2025},
author = {Butt, H and Sathish, S and London, E and Lee Johnson, T and Essawi, K and Leonard, A and Tisdale, JF and Demirci, S},
title = {Genome Editing Strategies for Targeted Correction of β-globin Mutation in Sickle Cell Disease: From Bench to Bedside.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.03.047},
pmid = {40165374},
issn = {1525-0024},
abstract = {Sickle cell disease (SCD) includes a range of genotypes that result in a clinical syndrome, where abnormal red blood cell (RBC) physiology leads to widespread complications affecting nearly every organ system. Treatment strategies for SCD can be broadly categorized into disease-modifying therapies and those aimed toward a cure. Although several disease-modifying drugs have been approved, they do not fully address the complexity and severity of SCD. Recent advances in allogeneic transplantation and autologous gene therapy show promising outcomes in terms of efficacy and safety. While these approaches have improved the lives of many patients, achieving a durable and comprehensive cure for all remains challenging. To address this, gene-editing technologies, including zinc finger nucleases, TALENs, CRISPR-Cas, base editing, and prime editing, have been explored both ex vivo and in vivo for targeted correction of the β-globin gene (HBB) in SCD. However, direct correction of HBB and its translation from the laboratory to the clinic presents ongoing limitations, with challenges involved in achieving robust mutation correction efficiency, off-target effects, and high costs of therapies. The optimal strategy for curing SCD remains uncertain, but several promising approaches are emerging. This review will touch on past, present and future developments in HBB correction.},
}