@article {pmid40603034,
year = {2025},
author = {Sugo, T and Shirasago, Y and Yoshimoto, S and Kitajima, M},
title = {[GenAhead Bio: your partner for extensive support of genome editing and co-‍development of nucleic acid delivery].},
journal = {Nihon yakurigaku zasshi. Folia pharmacologica Japonica},
volume = {160},
number = {4},
pages = {274-278},
doi = {10.1254/fpj.25004},
pmid = {40603034},
issn = {0015-5691},
mesh = {*Gene Editing ; Humans ; *Nucleic Acids/administration & dosage ; CRISPR-Cas Systems ; Animals ; RNA, Small Interfering ; },
abstract = {Inspired by my experiences working in research at an overseas biotech venture, I founded GenAhead Bio Inc. in 2018. GenAhead Bio adopts a unique dual-business structure, providing contract services for generating genetically modified cells using highly efficient CRISPR/Cas9 genome editing technology for researchers, while simultaneously pursuing a nucleic acid drug business aiming to develop nucleic acid drugs such as antisense oligonucleotides and siRNAs. Based on the emerging delivery system called Antibody-Nucleic acid Conjugate, where an antibody is covalently linked to a nucleic acid as a targeting ligand, we are conducting drug developmental research by delivering nucleic acids to the organs where antibodies accumulate. Our ultimate goal is to apply this technology to genome editing for gene modification in specific cell types. In this review, we will introduce some case studies of genome editing, including single nucleotide substitutions, as well as the delivery of siRNA to the skeletal muscle using anti-transferrin receptor (CD71) antibody and its therapeutic effects on muscular diseases.},
}

*Gene Editing
Humans
*Nucleic Acids/administration & dosage
CRISPR-Cas Systems
Animals
RNA, Small Interfering

@article {pmid40601773,
year = {2025},
author = {Chafe, SC and Zhai, K and Aghaei, N and Miletic, P and Huang, Z and Brown, KR and Mobilio, D and Young, D and Suk, Y and Grewal, S and McKenna, D and Alizada, Z and Kieliszek, AM and Lam, FC and Escudero, L and Huang, Q and Huebner, A and Lu, J and Ang, P and Anand, A and Custers, S and Apel, E and Slassi, S and Brakel, B and Kim, J and Liu, JKC and Bassey-Archibong, BI and Abdo, R and Shargall, Y and Lu, JQ and Cutz, JC and Zhang, Q and Li, SS and Venugopal, C and Hynds, RE and Dufour, A and Moffat, J and Swanton, C and Bao, S and Singh, SK},
title = {A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis.},
journal = {Science translational medicine},
volume = {17},
number = {805},
pages = {eadu2459},
doi = {10.1126/scitranslmed.adu2459},
pmid = {40601773},
issn = {1946-6242},
mesh = {Humans ; *Brain Neoplasms/secondary/genetics ; *Lung Neoplasms/pathology/genetics ; *Amyloid Precursor Protein Secretases/metabolism/genetics ; *Aspartic Acid Endopeptidases/metabolism/genetics ; Animals ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice ; ErbB Receptors/metabolism ; *CRISPR-Cas Systems/genetics ; Xenograft Model Antitumor Assays ; Gene Expression Regulation, Neoplastic ; },
abstract = {Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation screen to identify molecular drivers of brain metastasis from an orthotopic xenograft model derived from a patient with NSCLC. We found that activating expression of the Alzheimer's disease-associated beta-secretase 1 (BACE1) led to a substantial increase in brain metastases. Furthermore, genetic and pharmacological inhibition of BACE1 blocked NSCLC brain metastasis. Mechanistically, we identified that BACE1 acts through epidermal growth factor receptor to drive this metastatic phenotype. Together, our data highlight the power of in vivo CRISPR activation screening to unveil molecular drivers and potential therapeutic targets of NSCLC brain metastasis.},
}

Humans
*Brain Neoplasms/secondary/genetics
*Lung Neoplasms/pathology/genetics
*Amyloid Precursor Protein Secretases/metabolism/genetics
*Aspartic Acid Endopeptidases/metabolism/genetics
Animals
*Carcinoma, Non-Small-Cell Lung/genetics/pathology
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice
ErbB Receptors/metabolism
*CRISPR-Cas Systems/genetics
Xenograft Model Antitumor Assays
Gene Expression Regulation, Neoplastic

@article {pmid40599263,
year = {2025},
author = {Yang, R and Tang, T and Wulae, and Liu, P and Wang, X and Jiang, Y and Zhang, S and Wang, M and Yang, L and Wang, C},
title = {[One-Step Detection of Human Influenza B Virus Through Recombinase Polymerase Amplification and CRISPR/Cas12a Protein].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {2},
pages = {549-555},
pmid = {40599263},
issn = {1672-173X},
mesh = {*Influenza B virus/isolation & purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Influenza, Human/diagnosis/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: To establish a one-step detection method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a protein for the rapid and sensitive detection of human influenza B virus.

METHODS: RPA amplification primers were designed according to the conserved gene (NS1 gene) of human influenza B virus (Victoria lineage). The reaction system was established using the standard plasmid as the template. First of all, the reaction system was incubated at 37 ℃ for 15 minutes for RPA amplification. Then, the CRISPR/Cas12a system on the tube cap was thoroughly mixed with the RPA amplification product at the bottom of the tube through fast centrifugation, and real-time fluorescence detection was carried out at 37 ℃. The reaction conditions were optimized to establish a one-step RPA-CRISPR/Cas12a detection method for human influenza B virus. The sensitivity of the testing method was evaluated using standard plasmids and pseudoviruses, and the specificity was evaluated using other viruses that may cause febrile respiratory syndrome. The consistency between the results of the one-step detection method and those of RT-qPCR detection was evaluated by testing real samples.

RESULTS: A one-step detection method based on RPA-CRISPR/Cas12a was successfully established. The optimal reaction conditions included a reaction temperature of 37 ℃, a Cas12a/crRNA concentation ratio of 1∶1, a Cas12a concentration of 120 nmol/L, a single-stranded DNA (ssDNA) probe concentration of 300 nmol/L, and a primer concentration of 480 nmol/L. The method could detect standard plasmid DNA as low as 2.8 copies/μL within 25 minutes and pseudoviruses as low as 2.77 copies/μL within 30 minutes. The testing method showed high specificity, and no cross-reaction was observed with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A (H1N1) virus, or respiratory syncytial virus subgroup A. When testing clinical samples, the sensitivity and the specificity for examining clinical samples were 93.33% and 100%, respectively, and consistency with RT-qPCR results was 97.14%.

CONCLUSION: With the one-step detection method based on RPA-CRISPR/Cas12a established in this study, the whole sample detection process, including nucleic acid release, reverse transcription, isothermal amplification, CRISPR/Cas12a system cleavage, and fluorescence signal output, can be completed within 30 minutes. Its high sensitivity, specificity, and successful application in clinical samples highlight its potential for rapid point-of-care testing in clinical settings.},
}

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

@article {pmid40598996,
year = {2025},
author = {Han, HG and Nandre, R and Eom, H and Choi, YJ and Ro, HS},
title = {Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {6},
pages = {e2502011},
doi = {10.71150/jm.2502011},
pmid = {40598996},
issn = {1976-3794},
support = {2023R1A2C1007213//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; RS2024-00322425//Rural Development Administration/ ; },
mesh = {*CRISPR-Cas Systems ; *Paecilomyces/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; Green Fluorescent Proteins/genetics/metabolism ; Fungal Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperatures above 40°C, was isolated from a paper mill and investigated as a host for heterologous protein production. To prevent environmental dissemination of spores, UV mutagenesis was employed to create a conidia-deficient strain, UM7. This strain underwent gene editing using Cas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promoter sequences amplified from the genomic DNA of P. variotii (PH4, PP2, PS8, Ptub, Ptef1, and PgpdA), along with a signal sequence-tagged eGFP, flanked by 5'-upstream (336 bp) and 3'-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP into protoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of 5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutants carrying complete HR donor DNAs with the six different promoter sequences, indicating successful homology-directed repair (HDR). Evaluation of promoter strength revealed that PgpdA was the most effective for intracellular GFP production; however, it resulted in negligible extracellular GFP signal under all promoter conditions. A newly edited strain with an HDR integration module connecting PgpdA directly to eGFP, without the signal sequence, exhibited enhanced GFP expression in both mycelial cells and culture broth, suggesting that the signal peptide negatively affect protein expression and secretion. This work represents the first successful RNP-mediated gene editing in P. variotii, contributing to the application of this thermophilic fungus in protein production.},
}

*CRISPR-Cas Systems
*Paecilomyces/genetics/metabolism
Promoter Regions, Genetic
*Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
*Genetic Engineering/methods
Green Fluorescent Proteins/genetics/metabolism
Fungal Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40598890,
year = {2025},
author = {Wei, W and Gao, CH and Jiang, X and Qiao, J and Zhang, L and Yan, Y and Zhao, G and Yang, K and Yan, J and Yang, M},
title = {CARF-dependent preferential RNA cleavage by Csm6 increases drug susceptibility of mycobacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf622},
pmid = {40598890},
issn = {1362-4962},
support = {32070079//National Natural Science Foundation of China/ ; 32370127//National Natural Science Foundation of China/ ; 2662025ZHPY001//Central Universities/ ; 2020kfyXJJS119//Central Universities/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mycobacterium smegmatis/genetics/drug effects/metabolism ; *RNA Cleavage ; *Mycobacterium tuberculosis/genetics/drug effects ; *Bacterial Proteins/genetics/metabolism/chemistry ; Drug Resistance, Bacterial/genetics ; Mycolic Acids/metabolism ; Gene Expression Regulation, Bacterial ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that defend against invading mobile genetic elements. The type III-A CRISPR-Cas system has been studied in the evolutionary and epidemiological context of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, its biological function remains poorly understood. Here, we demonstrate that heterologous expression of csm6, a single-stranded RNA ribonuclease of the CRISPR-Cas system, exhibits preferential RNA cleavage activity targeting host transcripts. This activity significantly downregulates ribosomal and mycolic acid biosynthesis pathway genes, leading to a global reduction in translation levels and an increased drug susceptibility of Mycobacterium smegmatis. Furthermore, mutagenesis analysis revealed that Csm6's biological function critically depends on its CARF domain rather than its HEPN domain. In conclusion, our study elucidates the biological role of the Csm6 protein in the CRISPR-Cas system, both in vitro and in vivo, highlighting how preferential RNA cleavage impacts multiple mycobacterial processes. These findings provide novel insights into the functional diversity of CRISPR-Cas systems in mycobacteria.},
}

*CRISPR-Cas Systems/genetics
*Mycobacterium smegmatis/genetics/drug effects/metabolism
*RNA Cleavage
*Mycobacterium tuberculosis/genetics/drug effects
*Bacterial Proteins/genetics/metabolism/chemistry
Drug Resistance, Bacterial/genetics
Mycolic Acids/metabolism
Gene Expression Regulation, Bacterial

@article {pmid40598087,
year = {2025},
author = {Garimella, SS and Minami, SA and Khanchandani, AN and Abad Santos, JC and Schaffer, SR and Shah, PS},
title = {A simplified two-plasmid system for orthogonal control of mammalian gene expression using light-activated CRISPR effector.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {58},
pmid = {40598087},
issn = {1472-6750},
support = {n/a//AMPAC Fine Chemicals Summer Research Internship/ ; n/a//Hellman Family Foundation/ ; n/a//Good Food Institute/ ; },
mesh = {*Plasmids/genetics ; HEK293 Cells ; Humans ; *Optogenetics/methods ; Green Fluorescent Proteins/genetics/metabolism ; Light ; *CRISPR-Cas Systems/genetics ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression Regulation ; Gene Expression ; },
abstract = {BACKGROUND: Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.

RESULTS: The two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. Using human HEK293T cells, we optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. We also demonstrate the optimal LACE system also depends on cell type. In mouse myoblast C2C12 cells, 2pLACE displayed less variability compared to 4pLACE, similar to HEK293T cells. However, 2pLACE also had a smaller dynamic range in C2C12 cells compared to 4pLACE.

CONCLUSIONS: This simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications. However, the optimal system may depend on cell type and application.},
}

*Plasmids/genetics
HEK293 Cells
Humans
*Optogenetics/methods
Green Fluorescent Proteins/genetics/metabolism
Light
*CRISPR-Cas Systems/genetics
Animals
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Expression Regulation
Gene Expression

@article {pmid40598062,
year = {2025},
author = {Ajdanian, L and Torkamaneh, D},
title = {Mother transformer: A High-Throughput, Cost-Effective in Planta Hairy Root Transformation Method for Cannabis.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {60},
pmid = {40598062},
issn = {1472-6750},
mesh = {*Plant Roots/genetics/microbiology ; *Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; *Transformation, Genetic ; *Cannabis/genetics ; Cost-Benefit Analysis ; *High-Throughput Screening Assays/methods ; },
abstract = {BACKGROUND: Hairy root (HR) transformation assays mediated by Agrobacterium rhizogenes, both in vitro and ex vitro, are essential tools in plant biotechnology and functional genomics. These assays can be significantly influenced by various factors, which ultimately can enhance the efficiency. In this study, we optimized a two-step ex vitro HR transformation method using the actual mother plant combined with the RUBY system and compared with existing methods.

RESULTS: The two-step ex vitro method proved more efficient than both the one-step ex vitro and in vitro methods, with the highest transformation efficiency of 90% observed in the actual plant. This technique also demonstrated a faster and less complicated approach, reducing time to achieve massive transgenic HR formation by 9-29 days compared to other methods.

CONCLUSIONS: A novel, quicker, less complicated, and more efficient two-step transformation method for cannabis has been established, presenting a significantly lower risk of contamination. This protocol is particularly interesting to produce secondary metabolites using the CRISPR/Cas system in cannabis. We anticipate that this method will facilitate substantial time savings by rapidly producing hundreds of transformed samples.},
}

*Plant Roots/genetics/microbiology
*Plants, Genetically Modified/genetics
*Agrobacterium/genetics
*Transformation, Genetic
*Cannabis/genetics
Cost-Benefit Analysis
*High-Throughput Screening Assays/methods

@article {pmid40597657,
year = {2025},
author = {Malekos, E and Montano, C and Carpenter, S},
title = {CRISPRware: a software package for contextual gRNA library design.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {607},
pmid = {40597657},
issn = {1471-2164},
support = {F31AI179201//National Institute of Allergy and Infectious Diseases/ ; R35GM137801/GM/NIGMS NIH HHS/United States ; },
mesh = {*Software ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; Animals ; },
abstract = {We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and can account for genetic variation, which allows allele-specific guide design on a genome-wide scale. As a demonstration of use and to create a publicly available resource, we use CRISPRware to identify and score gRNAs against coding sequences in six model organisms for Cas9 and Cas12A and host these in a publicly available session on the UCSC Genome Browser.},
}

*Software
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Library
*CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing
Humans
Animals

@article {pmid40596971,
year = {2025},
author = {Martí-Díaz, R and Sánchez-Del-Campo, L and Montenegro, MF and Hernández-Caselles, T and Piñero-Madrona, A and Cabezas-Herrera, J and Rodríguez-López, JN},
title = {Ex vivo engineering of phagocytic signals in breast cancer cells for a whole tumor cell-based vaccine.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1029},
pmid = {40596971},
issn = {1471-2407},
support = {21407/FPI/20//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; },
mesh = {Animals ; Female ; Mice ; *Phagocytosis/immunology ; *Cancer Vaccines/immunology ; *Breast Neoplasms/immunology/therapy/pathology ; Humans ; CD47 Antigen/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; Macrophages/immunology ; Mice, Inbred BALB C ; },
abstract = {BACKGROUND: Today, cell therapies are constantly evolving and providing new options for cancer patients. These therapies are mostly based on the inoculation of immune cells extracted from a person's own tumor; however, some studies using whole tumor cell-based vaccines are approaching the level of maturity required for clinical use. Although these latest therapies will have to be developed further and adapted to overcome many ethical barriers, there is no doubt that therapeutic cancer vaccines are the next frontier of immunotherapy.

METHODS: Ionizing radiation and CD47 knockout via CRISPR-Cas9 genome editing were used to optimize the macrophage-mediated phagocytosis of breast cancer cells. These cells were subsequently used in several mouse models to determine their potential as novel whole-cell-based vaccines to drive antitumor immunity. To improve the recognition of tumor cells by activated immune cells, this cellular therapy was combined with anti-PD-1 antibody treatments.

RESULTS: Here, we showed that irradiation of 4T1 breast cancer cells increases their immunogenicity and, when injected into the blood of immunocompetent mice, elicits a complete antitumor immune response mediated, in part, by the adaptive immune system. Next, to improve the macrophage-mediated phagocytosis of breast cancer cells, we knocked out CD47 in 4T1 cells. When injected in the bloodstream, irradiated CD47 knockout cells activated both the adaptive and the innate immune systems. Therefore, we used these ex vivo engineered cells as a whole tumor cell-based vaccine to treat breast tumors in immunocompetent mice. A better response was obtained when these cells were combined with an anti-PD-1 antibody.

CONCLUSION: These results suggest that tumor cells obtained from surgical samples of a breast cancer patient could be engineered ex vivo and used as a novel cell therapy to drive antitumor immunity.},
}

Animals
Female
Mice
*Phagocytosis/immunology
*Cancer Vaccines/immunology
*Breast Neoplasms/immunology/therapy/pathology
Humans
CD47 Antigen/genetics
Cell Line, Tumor
CRISPR-Cas Systems
Macrophages/immunology
Mice, Inbred BALB C

@article {pmid40596718,
year = {2025},
author = {Schuster, B and Dobiášovská, I and Ćurčić, J and Pajer, P and Borna, Š and Bartůněk, P},
title = {SWITCHER, a CRISPR-inducible floxed wild-type Cre regulating CRISPR activity.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {982},
pmid = {40596718},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; *Integrases/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; },
abstract = {Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.},
}

*CRISPR-Cas Systems/genetics
*Integrases/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing/methods
Humans
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics
Bacterial Proteins

@article {pmid40595661,
year = {2025},
author = {Boswell, CW and Hoppe, C and Sherrard, A and Miao, L and Kojima, ML and Martino, P and Zhao, N and Stasevich, TJ and Nicoli, S and Giraldez, AJ},
title = {Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5503},
pmid = {40595661},
issn = {2041-1723},
support = {R01 HD100035/HD/NICHD NIH HHS/United States ; R35 GM122580/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems/genetics ; Zebrafish Proteins/metabolism/genetics ; Single-Domain Antibodies/genetics/metabolism ; Epitopes/genetics/metabolism ; Gene Knock-In Techniques/methods ; Humans ; Single-Chain Antibodies/genetics/metabolism ; },
abstract = {Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.},
}

Animals
Zebrafish/genetics/embryology/metabolism
CRISPR-Cas Systems/genetics
Zebrafish Proteins/metabolism/genetics
Single-Domain Antibodies/genetics/metabolism
Epitopes/genetics/metabolism
Gene Knock-In Techniques/methods
Humans
Single-Chain Antibodies/genetics/metabolism

@article {pmid40595633,
year = {2025},
author = {Fu, W and Ma, J and Wang, Z and Tang, N and Pan, D and Su, M and Wu, Z and Gan, J and Ji, Q},
title = {Mechanisms and engineering of a miniature type V-N CRISPR-Cas12 effector enzyme.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5667},
pmid = {40595633},
issn = {2041-1723},
support = {22277078//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry/ultrastructure ; Humans ; DNA/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry/ultrastructure ; Protein Engineering ; },
abstract = {Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Cryoelectron Microscopy
Gene Editing/methods
*CRISPR-Associated Proteins/metabolism/genetics/chemistry/ultrastructure
Humans
DNA/metabolism/genetics
*Bacterial Proteins/metabolism/genetics/chemistry/ultrastructure
Protein Engineering

@article {pmid40595632,
year = {2025},
author = {Zhang, Y and Zhang, T and Xiao, X and Wang, Y and Kawalek, A and Ou, J and Ren, A and Sun, W and de Bakker, V and Liu, Y and Li, Y and Yang, L and Ye, L and Jia, N and Veening, JW and Liu, X},
title = {CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5870},
pmid = {40595632},
issn = {2041-1723},
support = {82270012//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200047//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Pseudomonas aeruginosa/drug effects/genetics ; *Gallium/pharmacology/therapeutic use ; Animals ; *Pseudomonas Infections/drug therapy/microbiology ; Mice ; Biofilms/drug effects/growth & development ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; Reactive Oxygen Species/metabolism ; *Bacterial Proteins/genetics/metabolism ; Oxidative Stress/drug effects ; CRISPR-Cas Systems ; Female ; Iron/metabolism ; },
abstract = {With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium gain increasing attention. Intravenous gallium nitrate is under Phase II clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we apply a genome-wide CRISPR interference approach (CRISPRi-seq) to identify potential synergistic targets with gallium. We classify the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identify a highly conserved gene, fprB, encoding a ferredoxin-NADP[+] reductase, whose deletion sensitizes P. aeruginosa to gallium, lowering its MIC by 32-fold and shifting mode of action from bacteriostatic to bactericidal. Further investigation reveals that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of iron homeostasis and reactive oxygen species accumulation. Deleting fprB enhances gallium's efficacy against biofilm formation and improves outcomes in a murine lung infection model of P. aeruginosa, suggesting FprB is a promising drug target in combination with gallium. Overall, our data show CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa, advancing the identification of novel therapeutic targets.},
}

*Pseudomonas aeruginosa/drug effects/genetics
*Gallium/pharmacology/therapeutic use
Animals
*Pseudomonas Infections/drug therapy/microbiology
Mice
Biofilms/drug effects/growth & development
*Anti-Bacterial Agents/pharmacology
Humans
Microbial Sensitivity Tests
Reactive Oxygen Species/metabolism
*Bacterial Proteins/genetics/metabolism
Oxidative Stress/drug effects
CRISPR-Cas Systems
Female
Iron/metabolism

@article {pmid40595611,
year = {2025},
author = {Irie, N and Takeda, N and Satou, Y and Araki, K and Ono, M},
title = {Machine learning-assisted decoding of temporal transcriptional dynamics via fluorescent timer.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5720},
pmid = {40595611},
issn = {2041-1723},
support = {DCRPGF\100007//Cancer Research UK (CRUK)/ ; JP21K07082, JP21H00433, JP24K10259, JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16H0627601, JP22H04922//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24gm1810001s0103//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; *Machine Learning ; *Forkhead Transcription Factors/genetics/metabolism ; Mice ; *Transcription, Genetic ; Flow Cytometry/methods ; Gene Expression Regulation ; Mice, Inbred C57BL ; Single-Cell Analysis/methods ; Enhancer Elements, Genetic ; T-Lymphocytes, Regulatory/metabolism ; Genes, Reporter ; Neural Networks, Computer ; Spleen/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein, the Timer-of-cell-kinetics-and-activity system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer fluorescence data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a convolutional neural network-based method that incorporates image conversion and class-specific feature visualisation for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3 fluorescent Timer reporter mice lacking the enhancer Conserved Non-coding Sequence 2, which revealed new roles of this enhancer in regulating Foxp3 transcription frequency under specific conditions. Furthermore, analysis of wild-type Foxp3 fluorescent Timer reporter mice at different ages uncovered distinct patterns of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3[+] T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, single-cell dynamics analysis, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.},
}

Animals
*Machine Learning
*Forkhead Transcription Factors/genetics/metabolism
Mice
*Transcription, Genetic
Flow Cytometry/methods
Gene Expression Regulation
Mice, Inbred C57BL
Single-Cell Analysis/methods
Enhancer Elements, Genetic
T-Lymphocytes, Regulatory/metabolism
Genes, Reporter
Neural Networks, Computer
Spleen/metabolism/cytology
CRISPR-Cas Systems

@article {pmid40595537,
year = {2025},
author = {Hofmann, R and Herman, C and Mo, CY and Mathai, J and Marraffini, LA},
title = {Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5730},
pmid = {40595537},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Bacterial Proteins/genetics/metabolism ; Bacteriophages/genetics ; *Endonucleases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.},
}

*CRISPR-Cas Systems/genetics
*Streptococcus pyogenes/genetics/virology
*CRISPR-Associated Proteins/genetics/metabolism
Mutation
*Bacterial Proteins/genetics/metabolism
Bacteriophages/genetics
*Endonucleases/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40595093,
year = {2025},
author = {Wei, T and Yan, Y and Niu, M and Dong, X and Li, H and Sun, Y and Fa, Y},
title = {A rapid LASV detection method based on CRISPR-Cas13a and recombinase aided amplification with special lateral-flow test strips.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {20640},
pmid = {40595093},
issn = {2045-2322},
support = {2023YFC2605100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Lassa virus/isolation & purification/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *Lassa Fever/diagnosis/virology ; Reagent Strips ; Animals ; },
abstract = {Lassa virus (LASV) is a high-risk pathogen associated with severe viral hemorrhagic fever in both humans and animals. Owing to its significant treatment challenges and high infectivity, LASV is classified as a biosafety level 4 (BSL-4) pathogen. It is essential to establish a rapid LASV detection method to prevent and control the disease. To address the biosecurity threats caused by LASV, in this study, we developed a new test method for LASV detection by combining the recombinase-mediated isothermal amplification (RAA) and CRISPR-Cas13a detection technology. The detection efficiency of this method was evaluated and compared with existing methods. The results demonstrate that this new detection maintains relatively high sensitivity and specificity, while having excellent simplicity and rapidity. The sensitivity of the method for detecting the LASV can achieve a threshold of 10[1] copies/µL using fluorescence detection in 90 min and 10[2] copies/µL with lateral flow strip detection in just an hour, which only needs a simple constant temperature equipment to achieve. The application of this detection method holds substantial biosecurity significance for underdeveloped regions (e.g., West Africa), as well as for countries like China, which have a vast territory and uneven development of medical testing levels in various regions.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
*Lassa virus/isolation & purification/genetics
Humans
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
*Lassa Fever/diagnosis/virology
Reagent Strips
Animals

@article {pmid40594904,
year = {2025},
author = {Sayem, M and Rafi, MA and Mishu, ID and Mahmud, Z},
title = {Comprehensive genomic analysis reveals virulence and antibiotic resistance genes in a multidrug-resistant Bacillus cereus isolated from hospital wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {22915},
pmid = {40594904},
issn = {2045-2322},
mesh = {*Bacillus cereus/genetics/pathogenicity/isolation & purification/drug effects ; *Wastewater/microbiology ; Bangladesh ; *Drug Resistance, Multiple, Bacterial/genetics ; Phylogeny ; Hospitals ; Virulence/genetics ; Genome, Bacterial ; Whole Genome Sequencing ; Genomics/methods ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Humans ; },
abstract = {Hospital wastewater represents a significant reservoir for antimicrobial-resistant bacteria, including multidrug-resistant (MDR) Bacillus cereus, a pathogen of growing concern due to its potential impact on public health and environmental safety. This study characterizes the genomic features, antimicrobial resistance (AMR) mechanisms, and virulence potential of Bacillus cereus MBC, isolated from hospital wastewater in Dhaka, Bangladesh. Using whole-genome sequencing (WGS) and advanced bioinformatics, we analyzed the isolate's taxonomy, phylogenetics, functional annotation, and biosynthetic potential. The genome, spanning 5.6 Mb with a GC content of 34.84%, contained 5,881 protein-coding sequences, including 1,424 hypothetical proteins, and 28 genes associated with AMR. Phylogenetic analysis revealed a close genetic relationship with Bacillus cereus ATCC 14579, sharing virulence factors such as hemolysin BL (HBL), non-hemolytic enterotoxin (NHE), and cytotoxin K (CytK), all contributing to its pathogenicity. The ability to form biofilms further enhances the strain's persistence and resistance in hospital environments. AMR profiling identified genes conferring resistance to beta-lactams (e.g., BcI, BcII, BcIII), tetracyclines (tetB(P)), glycopeptides (vanY), and fosfomycin, highlighting the bacterium's capacity to resist a wide array of antibiotics. Functional annotation revealed metabolic pathways involved in iron acquisition and the biosynthesis of siderophores such as petrobactin and bacillibactin, reinforcing the bacterium's adaptability in nutrient-limited environments. Mobile genetic elements, including prophages, CRISPR-Cas systems, and transposable elements, suggest significant horizontal gene transfer (HGT), enhancing genetic plasticity and resistance spread. Pangenomic analysis, involving 125 B. cereus strains, revealed a high degree of genetic diversity and close relationships with strains from clinical, food, and agricultural environments, emphasizing the overlap between clinical and environmental reservoirs of resistance. The strain's isolation from hospital wastewater underscores the complex interplay between environmental contaminants and bacterial evolution, which fosters MDR traits. Our findings underscore the urgent need for enhanced genomic surveillance and wastewater management strategies to mitigate the spread of MDR B. cereus and AMR genes in hospital environments.},
}

*Bacillus cereus/genetics/pathogenicity/isolation & purification/drug effects
*Wastewater/microbiology
Bangladesh
*Drug Resistance, Multiple, Bacterial/genetics
Phylogeny
Hospitals
Virulence/genetics
Genome, Bacterial
Whole Genome Sequencing
Genomics/methods
Anti-Bacterial Agents/pharmacology
Virulence Factors/genetics
Humans

@article {pmid40594722,
year = {2025},
author = {Ren, X and Yao, XR and Chen, K and Xiao, WT and He, JY},
title = {CRISPR-Cas9 screening identifies a gene signature predictive of prognosis in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {21077},
pmid = {40594722},
issn = {2045-2322},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; *Glioblastoma/genetics/pathology/mortality ; Prognosis ; *CRISPR-Cas Systems/genetics ; *Brain Neoplasms/genetics/pathology/mortality ; Gene Expression Regulation, Neoplastic ; Biomarkers, Tumor/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; Gene Regulatory Networks ; *Transcriptome ; },
abstract = {Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by a poor prognosis and limited therapeutic options. Identifying essential genes and pathways involved in GBM proliferation is important for developing prognostic biomarkers and potential therapeutic targets. In this study, genome-wide CRISPR-Cas9 screening data from the dependency map (DepMap) database were analyzed to explore proliferation-related essential genes and pathways in GBM. A five-gene prognostic signature-CLSPN, HSP90B1, MED10, SAMM50, and TOMM20-was constructed using univariate, LASSO, and multivariate Cox regression analyses, and its prognostic value was evaluated in independent cohorts. Weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) suggested that the E2F targets pathway may be involved in GBM proliferation, consistent with the CRISPR screening results. Among the identified genes, MED10 was preliminarily implicated in regulating GBM cell proliferation and migration, as supported by functional assays. These findings propose a proliferation-related gene signature with potential prognostic relevance in GBM and indicate the E2F targets pathway as a biological process potentially associated with tumor progression. MED10 warrants further investigation as a candidate gene in the context of GBM biology and therapy.},
}

Humans
*Glioblastoma/genetics/pathology/mortality
Prognosis
*CRISPR-Cas Systems/genetics
*Brain Neoplasms/genetics/pathology/mortality
Gene Expression Regulation, Neoplastic
Biomarkers, Tumor/genetics
Cell Proliferation/genetics
Gene Expression Profiling
Gene Regulatory Networks
*Transcriptome

@article {pmid40593724,
year = {2025},
author = {Hu, M and Zhang, B and Shan, Y and Cao, F and Wang, Y and Qi, W and Wang, X and Shen, Y and Guo, X and Zhang, M and Tian, T and Xie, W and Zhang, M and Liang, F and Pei, D and Zhou, X},
title = {Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5939},
pmid = {40593724},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32300692//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92068201//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M760985//China Postdoctoral Science Foundation/ ; GZB20240235//China Postdoctoral Science Foundation/ ; 2024T170292//China Postdoctoral Science Foundation/ ; 2023M741235//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Phosphorothioate Oligonucleotides/metabolism/chemistry/genetics ; *DNA/metabolism/chemistry/genetics ; Light ; Humans ; },
abstract = {The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&PS DNA and restored through light-triggered chemical bond breakage of PC&PS DNA. The universality of DNACas is demonstrated by adopting the PC&PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Phosphorothioate Oligonucleotides/metabolism/chemistry/genetics
*DNA/metabolism/chemistry/genetics
Light
Humans

@article {pmid40593661,
year = {2025},
author = {Hwang, HY and Lee, M and Yi, H and Seok, C and Lim, K and Na, YR and Kang, JS and Park, JH and Kim, D},
title = {Engineered Sdd7 cytosine base editors with enhanced specificity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5881},
pmid = {40593661},
issn = {2041-1723},
support = {2020R1A2C2101714//National Research Foundation of Korea (NRF)/ ; RS-2025-00521074//National Research Foundation of Korea (NRF)/ ; HI21C1314, HR22C1363, RS-2024-02507183,21A0202L1-12//Korea Health Industry Development Institute (KHIDI)/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Cytosine Deaminase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7, a recently developed cytosine deaminase, exhibits high activity across a broad protospacer range but induces unintended off-target effects, including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here, we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this, we engineer two Sdd7 variants, Sdd7e1 and Sdd7e2, enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing, narrowed editing windows, and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity, nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2, especially when delivered via eVLP, as high-fidelity CBEs poised for safe, precise therapeutic genome editing.},
}

*Gene Editing/methods
*Cytosine/metabolism
Humans
CRISPR-Cas Systems/genetics
HEK293 Cells
*Cytosine Deaminase/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40593653,
year = {2025},
author = {Pastuszka, A and Mazzuoli, MV and Crestani, C and Deborde, L and Sismeiro, O and Lemaire, C and Rong, V and Gominet, M and Jacquemet, E and Legendre, R and Lanotte, P and Firon, A},
title = {The virulence regulator CovR boosts CRISPR-Cas9 immunity in Group B Streptococcus.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5678},
pmid = {40593653},
issn = {2041-1723},
support = {ANR-22-CE15-0024//Agence Nationale de la Recherche (French National Research Agency)/ ; LabEx IBEID, ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*Streptococcus agalactiae/genetics/immunology/pathogenicity ; *CRISPR-Cas Systems/genetics/immunology ; *Bacterial Proteins/genetics/metabolism/immunology ; Virulence/genetics ; Gene Expression Regulation, Bacterial ; Streptococcal Infections/immunology/microbiology ; Promoter Regions, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; Operon ; Host-Pathogen Interactions/immunology/genetics ; Mice ; Animals ; Humans ; },
abstract = {CRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements in Streptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of the cas operon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.},
}

*Streptococcus agalactiae/genetics/immunology/pathogenicity
*CRISPR-Cas Systems/genetics/immunology
*Bacterial Proteins/genetics/metabolism/immunology
Virulence/genetics
Gene Expression Regulation, Bacterial
Streptococcal Infections/immunology/microbiology
Promoter Regions, Genetic
*CRISPR-Associated Protein 9/genetics/metabolism
Operon
Host-Pathogen Interactions/immunology/genetics
Mice
Animals
Humans

@article {pmid40593649,
year = {2025},
author = {Thorpe, C and Luo, W and Ji, Q and Eggenberger, AL and Chicowski, AS and Xu, W and Sandhu, R and Lee, K and Whitham, SA and Qi, Y and Wang, K and Jiang, S},
title = {Enhancing biolistic plant transformation and genome editing with a flow guiding barrel.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5624},
pmid = {40593649},
issn = {2041-1723},
support = {2019-67013-29016//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04714//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04308//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOS-2132693//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
mesh = {*Gene Editing/methods ; *Biolistics/methods/instrumentation ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Triticum/genetics ; *Transformation, Genetic ; Onions/genetics ; Genome, Plant ; Seedlings/genetics ; Genetic Engineering/methods ; },
abstract = {The biolistic delivery system is an essential tool in plant genetic engineering, capable of delivering DNAs, RNAs, and proteins independent of tissue type, genotype, or species. However, its efficiency and consistency remain longstanding challenges despite decades of widespread use. Here, through advanced simulations, we identify gas and particle flow barriers as the root cause of these limitations. We show that a flow guiding barrel (FGB) achieves a 22-fold enhancement in transient transfection efficiency, a 4.5-fold increase in CRISPR-Cas9 ribonucleoprotein editing efficiency in onion epidermis, and a 17-fold improvement in viral infection efficiency in maize seedlings. Furthermore, stable transformation frequency in maize using B104 immature embryos increases over 10-fold, while in planta CRISPR-Cas12a-mediated genome editing efficiency in wheat meristems doubles in both T0 and T1 generations. This study provides insights into the fundamental mechanisms underlying biolistic inefficiency and demonstrates a practical solution that enables broader and more reliable applications in plant genetic engineering.},
}

*Gene Editing/methods
*Biolistics/methods/instrumentation
Zea mays/genetics
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Triticum/genetics
*Transformation, Genetic
Onions/genetics
Genome, Plant
Seedlings/genetics
Genetic Engineering/methods

@article {pmid40593618,
year = {2025},
author = {Lin, M and Qiu, Z and Hao, M and Qi, W and Zhang, T and Shen, Y and Xiao, H and Liang, C and Xie, L and Jiang, Y and Cheng, M and Tian, T and Zhou, X},
title = {Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5597},
pmid = {40593618},
issn = {2041-1723},
support = {2023M741238//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; Sensitivity and Specificity ; *Bacterial Proteins/metabolism/genetics ; *Nucleic Acids/genetics/analysis ; Nucleic Acid Hybridization ; },
abstract = {CRISPR technology holds significant promise for advancing nucleic acid assays. However, current CRISPR diagnostic techniques, reliant on indiscriminate trans-cleavage mechanisms, face challenges in developing multiplex detection formats. Moreover, chaotic trans-cleavage activity often results from mismatched targets, leading to specificity issues. To address these limitations, here we exploit a double-key recognition mechanism based on CRISPR-Cas12a cis-cleavage and invasive hybridization identification of released sticky-end DNA products. By integrating multiplexed nucleic acid amplification, the double-key Cas12a detection mechanism, and a lateral flow detection platform, we develop a method termed Cas12a cis-cleavage mediated lateral flow assay (cc-LFA). We demonstrate that the cc-LFA exhibited superior specificity compared to three mainstream trans-cleavage-based CRISPR diagnostic techniques, achieving single-base resolution detection free from high-concentration wild-type DNA background interference. cc-LFA is also applied for highly specific detection of multiple respiratory pathogen samples and precise multiplexed detection of nine high-risk human papillomavirus (HPV) subtypes, achieving over 90% sensitivity and 100% specificity, respectively. Additionally, we present a portable device to automate nucleic acid amplification and strip detection procedures, showcasing the potential of cc-LFA for future applications in decentralized laboratory scenarios.},
}

*CRISPR-Cas Systems/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics
Sensitivity and Specificity
*Bacterial Proteins/metabolism/genetics
*Nucleic Acids/genetics/analysis
Nucleic Acid Hybridization

@article {pmid40593576,
year = {2025},
author = {Kiernan, KA and Taylor, DW},
title = {Visualization of a multi-turnover Cas9 after product release.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5681},
pmid = {40593576},
issn = {2041-1723},
support = {R35GM138348//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry/ultrastructure ; Gene Editing/methods ; Streptococcus pyogenes/enzymology/genetics ; Cryoelectron Microscopy ; Kinetics ; DNA/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/genetics ; },
abstract = {While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.},
}

RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry/ultrastructure
Gene Editing/methods
Streptococcus pyogenes/enzymology/genetics
Cryoelectron Microscopy
Kinetics
DNA/metabolism/chemistry/genetics
Bacterial Proteins/metabolism/genetics

@article {pmid40593519,
year = {2025},
author = {Abe, I and Ohno, H and Mochizuki, M and Hayashi, K and Saito, H},
title = {Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5362},
pmid = {40593519},
issn = {2041-1723},
support = {JP20H05626//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25H00970//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20H05701//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20K12644//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25K03464//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22bm0104001//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1223002h0002//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123040//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Humans ; MicroRNAs/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation ; Gene Editing/methods ; HEK293 Cells ; *Protein Processing, Post-Translational ; RNA, Messenger/genetics/metabolism ; },
abstract = {RNA switch is a synthetic RNA-based technology that controls gene expression in response to cellular RNAs and proteins, thus enabling cell type-specific gene regulation and holding promise for gene therapy, regenerative medicine, and cell therapy. However, individual RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we present "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing. We show that split RNA switches significantly improve the ON/OFF ratio of microRNA-responsive ON switch system by canceling leaky OFF level in human cells. Using this approach, we achieve efficient cell purification using drug-resistance genes based on endogenous microRNA profiles and CRISPR-mediated genome editing with minimal off-target-cell effects. Additionally, we demonstrate RNA-based synthetic circuits using split RNA switches to enable the detection of multiple microRNAs and proteins with logical operations. Split RNA switches highlight the potential of post-translational processing as a versatile and comprehensive strategy for advancing mRNA-based therapeutic technologies.},
}

Humans
MicroRNAs/genetics/metabolism
CRISPR-Cas Systems/genetics
*Gene Expression Regulation
Gene Editing/methods
HEK293 Cells
*Protein Processing, Post-Translational
RNA, Messenger/genetics/metabolism

@article {pmid40570069,
year = {2025},
author = {Speth, ZJ and Rehard, DG and Norton, PJ and Franz, AWE},
title = {Performance of two low-threshold population replacement gene drives in cage populations of the yellow fever mosquito, Aedes aegypti.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011757},
pmid = {40570069},
issn = {1553-7404},
mesh = {Animals ; *Aedes/genetics/virology ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/virology ; Male ; Female ; *Yellow Fever/virology/transmission/genetics ; Promoter Regions, Genetic ; Mosquito Control/methods ; Animals, Genetically Modified ; },
abstract = {Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement in which arbovirus-susceptible mosquitoes in the field are replaced by laboratory-engineered refractory mosquitoes represents a novel genetic control measure to interrupt arboviral disease cycles. For this approach, the engineered mosquitoes need to harbor two genetic components: an antiviral effector construct which is linked to a gene drive (GD). We tested the performance of two single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement in small cage populations for up to 16 generations. Starting from a low release threshold of 1:9 GD bearing males to wild-type males, we observed two GD constructs in which Cas9 was expressed from two different germline promoters, nanos and zpg, to increase in frequency in all cage populations. By G16, an average of 72% and 82% of individuals from the zpg-GD and nanos-GD populations, respectively, harbored at least one GD copy with corresponding increases in allele frequencies. This indicated that the two single-locus, CRISPR/Cas9-based homing GD exhibited continuous super-Mendelian inheritance in populations of Ae. aegypti. Gene drive blocking indel (GDBI, a.k.a. "resistant alleles") frequency was measured for each discrete generation in pooled samples from the six populations harboring GD. We found that populations with Cas9 expression under control of the nanos-promoter accumulated GDBI at more than twice the rate of those populations harboring the zpg-promoter driven GD. Based on preexisting data sets for homing and GDBI frequencies in addition to the cage trial observations, the relative contributions of sex-specific homing rates, maternal Cas9 deposition and potential fitness effects were modeled in MGDrivE for both GD, further explaining their divergent performance. Our study demonstrates the feasibility of low-threshold, single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement.},
}

Animals
*Aedes/genetics/virology
CRISPR-Cas Systems/genetics
*Gene Drive Technology/methods
*Mosquito Vectors/genetics/virology
Male
Female
*Yellow Fever/virology/transmission/genetics
Promoter Regions, Genetic
Mosquito Control/methods
Animals, Genetically Modified

@article {pmid40488470,
year = {2025},
author = {Deng, Z and Weng, X and Tang, H and Zou, T and Zhou, X and Liu, H and Wen, P and Luo, G and Gan, T and He, J},
title = {Rapid and sensitive detection of Mycobacterium tuberculosis using the RPA/Cas12f1_ge4.1 system with fluorescence and lateral flow readouts.},
journal = {Microbiology spectrum},
volume = {13},
number = {7},
pages = {e0265224},
pmid = {40488470},
issn = {2165-0497},
mesh = {*Mycobacterium tuberculosis/isolation & purification/genetics ; Humans ; *Tuberculosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Recombinases/genetics/metabolism ; DNA, Bacterial/genetics ; Limit of Detection ; },
abstract = {UNLABELLED: Tuberculosis remains a major global health threat, with existing detection methods often limited by efficiency and resource demands. Our previous PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR) fluorescence sensing platform, while effective for PAM-dependent pathogen detection, was constrained by its reliance on specialized fluorescence equipment and lack of visual output, limiting its use in resource-limited settings. To overcome these limitations, we introduce an innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system for rapid Mycobacterium tuberculosis detection. This system combines engineered Cas12f_ge4.1 with recombinase polymerase amplification (RPA), offering both fluorescent and lateral flow detection. It achieves high sensitivity with detection limits of 10 copies/µL (fluorescence) and 100 copies/µL (lateral flow), alongside 100% specificity. In clinical validation, compared with a commercial qPCR kit, the fluorescent and lateral flow approaches demonstrate sensitivities of 94.52% (69/73, 95% confidence interval [CI]: 85.84%-98.23%) and 90.41% (66/73, 95% CI: 80.67%-95.73%), respectively, while maintaining 100% (40/40, 95% CI: 89.09%-100%) specificity and high concordance (kappa values: 0.924 and 0.878). Detection is completed within 1 h, providing a rapid, sensitive, and specific solution for M. tuberculosis identification. This dual-mode capability represents a significant advancement in current tuberculosis diagnostics, enabling both sensitive laboratory confirmation and rapid point-of-care screening. Our versatile and efficient method promises to transform tuberculosis diagnostics, particularly in resource-constrained environments.

IMPORTANCE: Tuberculosis (TB) remains a significant global health challenge, demanding rapid and accurate detection for effective management. The innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system represents a major advancement in TB diagnostics, offering highly sensitive and specific detection of Mycobacterium tuberculosis DNA. This adaptable system, incorporating both fluorescent and lateral flow detection modes, is designed for use in both advanced laboratories and resource-limited settings. Its high performance, rigorously validated through clinical trials, holds the potential to revolutionize TB diagnosis, particularly in high-burden, low-resource areas. By facilitating earlier treatment and enhancing control of TB transmission, this system could significantly contribute to global efforts in combating this persistent public health threat.},
}

*Mycobacterium tuberculosis/isolation & purification/genetics
Humans
*Tuberculosis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Fluorescence
CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Recombinases/genetics/metabolism
DNA, Bacterial/genetics
Limit of Detection

@article {pmid40422178,
year = {2025},
author = {Siddiqui, FA and Chim-Ong, A and Wang, C and Miao, J and Cui, L},
title = {The PfK13 G533S mutation confers artemisinin partial resistance in multiple genetic backgrounds of Plasmodium falciparum.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {7},
pages = {e0016225},
pmid = {40422178},
issn = {1098-6596},
support = {U19AI181593/NH/NIH HHS/United States ; },
mesh = {*Plasmodium falciparum/genetics/drug effects ; *Artemisinins/pharmacology ; *Antimalarials/pharmacology ; *Drug Resistance/genetics ; *Protozoan Proteins/genetics ; Mutation/genetics ; Malaria, Falciparum/drug therapy/parasitology ; Humans ; CRISPR-Cas Systems ; },
abstract = {Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are the key determinant of artemisinin partial resistance. While more than 200 PfK13 mutations have been identified in global parasite populations, only 13 have been validated to confer in vivo or in vitro artemisinin partial resistance. In the western Greater Mekong Subregion, the prevalence of the PfK13 G533S mutation has significantly increased in recent years. Field isolates carrying the PfK13 G533S mutation showed slower parasite clearance and higher day-3 positivity rates after artemisinin treatment, while culture-adapted isolates displayed significantly elevated ring-stage survival rates. Here, the PfK13 G533S mutation was introduced using CRISPR/Cas9 into four parasite strains: Dd2, 3D7, GB4, and F09N25 (a recent culture-adapted field isolate from the China-Myanmar border area). Across all four genetic backgrounds, the PfK13 G533S mutation conferred ring-stage survival rates of 12%-23% with a minimal fitness cost, explaining its rising prevalence in the region. In contrast, the PfK13 G533A mutation, sporadically detected in world P. falciparum populations, did not increase ring-stage survival rates when engineered into the 3D7 and Dd2 strains. These findings validate the PfK13 G533S mutation as a critical marker for artemisinin resistance surveillance and underscore the importance of monitoring its spread.},
}

*Plasmodium falciparum/genetics/drug effects
*Artemisinins/pharmacology
*Antimalarials/pharmacology
*Drug Resistance/genetics
*Protozoan Proteins/genetics
Mutation/genetics
Malaria, Falciparum/drug therapy/parasitology
Humans
CRISPR-Cas Systems

@article {pmid40592986,
year = {2025},
author = {Elhamouly, NA and Atta, N and Liu, S and Tu, Y and Peng, D},
title = {A novel assay incorporating CRISPR with RPA in a single pot for visual and accurate detection of Aphelenchoides besseyi in soybean.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {21217},
pmid = {40592986},
issn = {2045-2322},
mesh = {*Glycine max/parasitology ; *Nucleic Acid Amplification Techniques/methods ; Animals ; *Plant Diseases/parasitology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Tylenchida/genetics/isolation & purification ; },
abstract = {Aphelenchoides besseyi is a highly prevalent plant parasitic nematode which has a substantial impact and poses an economic risk to soybean cultivation, with a reported 2017 outbreak resulting in significant yield losses of up to 60%. Therefore, more effective control of this nematode depends on early and accurate nucleic acid detection. One of the promising detection approaches is to combine the CRISPR technology with the isothermal RPA. However, incorporating the RPA amplicon with the CRISPR ingredients in a single pot remains a significant challenge due to their incompatibility. In the current research, we propose a visual nucleic acid detection technique that takes less than thirty minutes and is highly sensitive for detecting A. besseyi. First, we conduct the RPA amplification, then we perform the CRISPR reaction using either a portable thermal cup or our body heat temperature. We tested this new assay on forty-four soybean samples exhibiting GSFR syndrome symptoms, and it effectively detected samples containing the A. besseyi. We designed three different ways for data collection and visualization to suit the requirements of various environments. Our findings confirm that the suggested new low-instrumentation portable single-pot RPA-CRISPR assay is durable, specific, and has strong nucleic acid sensitivity in the open field.},
}

*Glycine max/parasitology
*Nucleic Acid Amplification Techniques/methods
Animals
*Plant Diseases/parasitology
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Tylenchida/genetics/isolation & purification

@article {pmid40591069,
year = {2025},
author = {Anari, E and Anari, R and Pazoki-Toroudi, H},
title = {A decade of scientific advancements and collaborations on CRISPR-Cas9 application in cancer research: a bibliometric review.},
journal = {Discover oncology},
volume = {16},
number = {1},
pages = {1232},
pmid = {40591069},
issn = {2730-6011},
support = {25550//Iran University of Medical Sciences/ ; },
abstract = {The CRISPR-Cas system, a novel gene-editing technique, was a focus of research on cancer diagnosis and treatment during the last decade. A systematic review of the research trends and collaborations of the literature published in PubMed database between 2014 and 2024 resulted in 3241 papers. Bibliometric analysis and data visualization were performed using Excel, RStudio, and VOSviewer software. An increasing trend in the number of publications and citations has been observed since 2014, with a moderate fall after 2023. China and the USA have contributed the most publications and international collaborations, and high-income nations involved the highest contributions in the literature. The most productive institutes were Harvard Medical School and Dana-Farber Cancer Institute. Wang and Li contributed the maximum number of publications in this field. The prominent sources were Cancer Research, and Cancers. The application of CRISPR technique in breast and colorectal cancers has been frequently studied during the last decade. Future trials should explore the specificity and safety of CRISPR in various cancers.},
}

@article {pmid40590986,
year = {2025},
author = {K, MMP and Pagariya, MC and Jadhav, PR and Gawade, NS and Sarode, DK and Karkute, SG and Kardile, HB and Deshmukh, R and Penna, S and Kawar, PG},
title = {Advancing ornamental plant breeding through genomic technologies: opportunities, challenges, and future directions.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {140},
pmid = {40590986},
issn = {1438-7948},
mesh = {*Plant Breeding/methods ; *Genome, Plant ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; *Plants/genetics ; },
abstract = {The ornamental plants constitute an important sector of horticulture industry, which are worth billions of dollars worldwide. There is a growing demand for new and improved cultivars and hence, breeders employ new tools and methods to address the problem of plant improvement. Recent advancements in Ornamental plant genomics have seen a great revolution due to new technologies of whole genome sequencing which have created previously unheard-of breeding program prospects. Research into gene regulation, genomic variations, genome evolution, and other biological processes are now aided by the use of complete genome sequencing data. The assembly of high-quality genomes for various ornamental species has facilitated the identification of genes controlling desirable traits such as flower color, shape, fragrance, biotic and abiotic stress resistance. The CRISPR/Cas9 based genome editing technology has offered immense scope for ornamental plant improvement through the enhancement of floral characteristics. Herein, we discuss how these genomic resources can be leveraged to improve breeding efficiency, accelerate the development of novel cultivars to augment the sustainability of the ornamental plant industry. This review aims to provide a viewpoint for the application of whole genome sequencing in ornamental plant breeding, highlighting the opportunities, challenges, and future prospects.},
}

*Plant Breeding/methods
*Genome, Plant
Gene Editing
*Genomics/methods
CRISPR-Cas Systems
*Plants/genetics

@article {pmid40589866,
year = {2025},
author = {Chen, X and Zhang, S and Lin, S and Wang, S and Huang, M and Chen, S and Chen, S},
title = {A combination of recombinase polymerase amplification with CRISPR technology rapidly detects goose parvovirus with high accuracy and sensitivity.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1566603},
pmid = {40589866},
issn = {2235-2988},
mesh = {Animals ; Sensitivity and Specificity ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Poultry Diseases/diagnosis/virology ; Geese/virology ; Ducks/virology ; *Parvovirinae/genetics/isolation & purification ; *Parvovirus/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Goose parvovirus (GPV) poses a significant threat to the waterfowl industry, necessitating reliable detection methods. However, conventional techniques are often time-consuming, equipment-dependent, or lack sufficient sensitivity for detecting early-stage infection. In contrast, emerging CRISPR/Cas12a-based systems offer a promising alternative for rapid, sensitive, and on-site diagnostics.

METHODS: We developed and optimized a recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting the conserved VP3 gene of GPV. The analytical and diagnostic performance of this assay was rigorously validated using plasmid standards and clinical specimens from both experimentally infected and field-collected ducklings.

RESULTS: Our developed assay combines RPA with CRISPR/Cas12a technology for rapid GPV nucleic acids detection. This method achieves a detection limit of 10 copies/μL of the VP3 gene within one hour, demonstrating high sensitivity and rapid turnaround. The assay exhibited exceptional specificity, with no cross-reactivity against other waterfowl viruses, and showed robust reproducibility, with intra- and inter-assay coefficients of variation consistently below 5.0%. Clinical validation using 42 field samples confirmed a diagnostic sensitivity of 100% and 95.5% specificity, showing superior performance to real-time quantitative PCR (qPCR) in both metrics. Furthermore, the assay supports flexible visual readouts using portable blue light transilluminators, facilitating on-site interpretation.

CONCLUSIONS: This study established a highly field-deployable RPA-CRISPR/Cas12a assay for rapid, visual detection of GPV with outstanding sensitivity and specificity. Its capability for instrument-free on-site diagnosis via blue light transillumination makes this approach particularly promising for resource-limited settings.},
}

Animals
Sensitivity and Specificity
*Parvoviridae Infections/diagnosis/veterinary/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism/genetics
*Poultry Diseases/diagnosis/virology
Geese/virology
Ducks/virology
*Parvovirinae/genetics/isolation & purification
*Parvovirus/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods

@article {pmid40588792,
year = {2025},
author = {Ma, Q and Zhang, Y and Chen, L and Bao, Y and Guo, W and Feng, K and Huang, T and Cai, YD},
title = {Machine Learning-Driven Discovery of Essential Binding Preference in Anti-CRISPR Proteins.},
journal = {Proteomics. Clinical applications},
volume = {},
number = {},
pages = {e70013},
doi = {10.1002/prca.70013},
pmid = {40588792},
issn = {1862-8354},
support = {XDB38050200//Strategic Priority Research Program of Chinese Academy of Sciences/ ; XDA26040304//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 2022YFF1203202//National Key R&D Program of China/ ; GZNL2024A01003//Major Project of Guangzhou National Laboratory/ ; SRPG22-007//Self-Supporting Program of Guangzhou Laboratory/ ; 202002//Key Laboratory of Tissue Microenvironment and Tumor of Chinese Academy of Sciences/ ; ZR2022MC072//Shandong Provincial Natural Science Foundation/ ; 2024KCXTD081//Key Scientific Research Project of General Universities in Guangdong Province/ ; },
abstract = {PURPOSE: Anti-CRISPR (Acr) proteins can evade CRISPR-Cas immunity, yet their molecular determinants remain poorly understood. This study aimed to uncover key features driving Acr activity, thereby advancing both fundamental knowledge and the rational design of robust CRISPR-based tools.

EXPERIMENTAL DESIGN: We compiled a binary-encoded matrix of 761 InterPro-annotated domains and binding-site features for known Acr proteins. Seven feature ranking algorithms were applied to prioritize determinant features, and an incremental feature selection strategy, coupled with four distinct classifiers, was used to identify optimal subsets. Consensus key features were defined by intersecting the top subsets across all methods.

RESULTS: Key identified features include the DUF2829 domain, the Lambda repressor-like domain and Sulfolobus islandicus virus proteins, the Cro/C1-type helix-turn-helix domain, phage protein, and replication initiator A. These findings illuminate novel structural modules and regulatory motifs that underpin Acr inhibition.

CONCLUSIONS: This study provides critical theoretical support for deciphering Acr mechanisms and offers actionable insights for engineering next-generation CRISPR-Cas applications in clinical and biotechnological settings.

SUMMARY: The CRISPR system is a part of the antiviral immune defense initially discovered in bacteria and archaea. At present, the CRISPR system has become the cornerstone of genome editing technologies such as CRISPR-Cas9, widely used in clinical, agricultural, and biological research. Anti-CRISPR proteins are a group of proteins that inhibit the normal activity of CRISPR-Cas system in certain bacteria or archaea and avoid having the phages' genomes destroyed by the prokaryotic cells. The anti-CRISPR protein family has various components, but with similar functions to help exogenous DNA escape from the immune system. This study tried to uncover molecular mechanisms for anti-CRISPR proteins.},
}

@article {pmid40588529,
year = {2025},
author = {Park, BS and Lee, M and Kim, J and Kim, T},
title = {Perturbomics: CRISPR-Cas screening-based functional genomics approach for drug target discovery.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40588529},
issn = {2092-6413},
support = {CAP23011-000//National Research Council of Science and Technology (National Research Council of Science & Technology)/ ; 2E33131//Korea Institute of Science and Technology (KIST)/ ; RS-2024-00452719//National Research Foundation of Korea (NRF)/ ; },
abstract = {Despite more than two decades since the completion of the first draft of the Human Genome Project, a substantial proportion of human genes remain poorly characterized in terms of their functions. Functional genomics aims to elucidate the roles and interactions of genes and genetic elements, providing insights into their involvement in various biological processes. In this context, the perturbomics approach-a systematic analysis of phenotypic changes resulting from gene function modulation-offers valuable insights into the function of unannotated genes. With the advent of CRISPR-Cas-based genome and epigenome editing, CRISPR screens have become the method of choice for perturbomics studies, enabling the identification of target genes whose modulation may hold therapeutic potential for diseases such as cancer, cardiovascular disorders and neurodegeneration. These findings contribute to the development of targeted drug therapies and the design of gene and cell therapies for regenerative medicine. Here we highlight recent technical advances in CRISPR-based perturbomics, focusing on more physiologically relevant, single-cell-level analyses and their successful applications in discovering novel therapeutic strategies.},
}

@article {pmid40588516,
year = {2025},
author = {Malong, L and Roskosch, J and Hager, C and Fortin, JP and Schmucki, R and Callow, MG and Weile, C and Romeo, V and Patsch, C and Martin, S and Costa, M and Modrusan, Z and Villaseñor, R and Koller, E and Haley, B and Spang, A and Roudnicky, F},
title = {A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5378},
pmid = {40588516},
issn = {2041-1723},
mesh = {*Endosomes/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; *Golgi Apparatus/metabolism ; Animals ; Lysosomes/metabolism ; *Oligonucleotides, Antisense/metabolism/genetics ; Mice ; GTPase-Activating Proteins/metabolism/genetics ; HeLa Cells ; HEK293 Cells ; Adaptor Protein Complex 1/metabolism/genetics ; },
abstract = {Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.},
}

*Endosomes/metabolism
*CRISPR-Cas Systems/genetics
Humans
*Golgi Apparatus/metabolism
Animals
Lysosomes/metabolism
*Oligonucleotides, Antisense/metabolism/genetics
Mice
GTPase-Activating Proteins/metabolism/genetics
HeLa Cells
HEK293 Cells
Adaptor Protein Complex 1/metabolism/genetics

@article {pmid40588042,
year = {2025},
author = {Long, R and Tang, D and Yang, T and Li, M and Lu, Y and Liu, W and Jiang, L},
title = {Advanced research and exploration of CRISPR technology in the field of directed evolution.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108633},
doi = {10.1016/j.biotechadv.2025.108633},
pmid = {40588042},
issn = {1873-1899},
abstract = {Directed molecular evolution is the key technology for obtaining enzymes, proteins, metabolic pathways, and other components of living organisms that have specific functions or desirable properties, which are indispensable in a variety of industrial and medical applications. Despite the success of traditional methods, they are often limited by low efficiency and the high cost of obtaining desired mutants. The advent of CRISPR technology has significantly advanced the field by enabling precise and efficient gene targeting, offering new prospects for directed evolution. This review provides a comprehensive overview of CRISPR tools and their applications in directed evolution, highlighting the principles, technological advancements, and specific applications of CRISPR-based mutation and screening platforms. We discuss the key findings from the use of CRISPR in enzyme and genome evolution, showcasing its ability to generate genetic diversity and select for improved phenotypes. The study underscores the unique value of CRISPR in directed evolution, particularly in its flexibility to target and edit various species' genomes, and its potential to accelerate the discovery of novel biomolecules with enhanced properties.},
}

@article {pmid40532220,
year = {2025},
author = {Lu, Q and Ye, C and Mao, W and Zeng, F and Liu, Z and Chen, R and Cheng, X and Gao, Y and Wang, M and Liu, M and Tang, S and Song, Y},
title = {Targeting Senescent Alveolar Type 2 Cells with a Gene-Editable FePt Dual-Atom Catalyst for Mitigating Idiopathic Pulmonary Fibrosis.},
journal = {ACS nano},
volume = {19},
number = {25},
pages = {23162-23176},
doi = {10.1021/acsnano.5c04686},
pmid = {40532220},
issn = {1936-086X},
mesh = {*Idiopathic Pulmonary Fibrosis/drug therapy/pathology/metabolism/genetics ; *Cellular Senescence/drug effects ; Animals ; Mice ; Humans ; Hyaluronic Acid/chemistry ; Gene Editing ; *Alveolar Epithelial Cells/drug effects/metabolism/pathology ; *Platinum/chemistry/pharmacology ; CRISPR-Cas Systems ; Catalysis ; *Iron/chemistry ; Mice, Inbred C57BL ; },
abstract = {Idiopathic pulmonary fibrosis (IPF) remains an age-related, fatal, incurable, epithelial-driven fibrotic lung disease despite the availability of approved antifibrotic drugs. The medical need for effective antipulmonary fibrotic therapies is thus very high. A promising therapeutic intervention for IPF is to target key cellular senescence processes in alveolar type 2 (AT2) cells. Herein, we introduce an inhalable gene-editable nanoplatform, comprising a CRISPR-Cas9 gene-editing system linked to a core FePt diatomic catalyst, encapsulated within a biocompatible hyaluronic acid (HA) surface layer (FePtR@HA). The FePt diatomic site facilitates H2O2 bridge adsorption, enabling efficient O-O bond cleavage and rapid catalytic conversion. The strong Fe-Pt interaction modulates the metal's d-band center, optimizing the adsorption of oxygen-containing intermediates. This precise regulation efficiently clears ROS, delivering robust antioxidant and antisenescence effects to AT2 cells. Simultaneously, the CRISPR-Cas9 gene editing system knocks down the pro-aging gene KAT7, reducing senescence-associated secretory phenotype (SASP) factors and further reversing AT2 cell senescence. Additionally, we demonstrated the antifibrotic efficacy of FePtR@HA in a lung-on-a-chip model, where it reprogrammed the fibrotic microenvironment, prevented myofibroblast recruitment to AT2 cells. Moreover, FePtR@HA showed satisfactory results in IPF mouse models, alleviating fibrosis and presenting a highly promising approach to combat the progression of IPF.},
}

*Idiopathic Pulmonary Fibrosis/drug therapy/pathology/metabolism/genetics
*Cellular Senescence/drug effects
Animals
Mice
Humans
Hyaluronic Acid/chemistry
Gene Editing
*Alveolar Epithelial Cells/drug effects/metabolism/pathology
*Platinum/chemistry/pharmacology
CRISPR-Cas Systems
Catalysis
*Iron/chemistry
Mice, Inbred C57BL

@article {pmid40517335,
year = {2025},
author = {Wang, Q and Yang, S and Chen, X and Song, S and Wu, Y and Li, Y and Meng, X and Dai, L and Yang, Z and Wu, H and Xia, J and Xu, J},
title = {Mutually Activated Dual-Exponential Amplification DNA Machine Enhances Robust CRISPR/Cas12a Feedback Propagation for Ultrasensitive miRNA Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {25},
pages = {13504-13513},
doi = {10.1021/acs.analchem.5c01953},
pmid = {40517335},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *DNA/genetics/chemistry ; *Breast Neoplasms/diagnosis/genetics ; Limit of Detection ; Female ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Breast cancer (BC) remains a critical global health challenge, necessitating ultrasensitive methods for detecting biomarkers such as miR-155, a key regulator in BC progression. Here, we present a mutually activated dual-exponential amplification DNA machine (MADEA-DNA machine) for ultrasensitive miR-155 detection. This system integrates exponential rolling circle amplification (E-RCA) and autocatalytic incremental strand displacement amplification (AI-SDA), driven by a bidirectional activation mechanism. Target miR-155 initiates E-RCA via a functional primer probe (FPP) or AI-SDA through a functional hairpin probe (FHP), with amplification products cross-activating the counterpart system to establish a self-reinforcing loop. The resultant amplicons further activate CRISPR/Cas12a, enabling the trans-cleavage of fluorescent reporters for signal amplification. The MADEA-DNA machine achieves a detection limit of 1.26 fM, with a dynamic range spanning 5 fM-10 nM, and demonstrates exceptional specificity against mismatched and nontarget miRNAs. Validation in human serum revealed significantly elevated miR-155 levels in BC patients versus healthy donors, corroborated by qRT-PCR. This system combines machine-like operational efficiency, dual-amplification synergy, and CRISPR-enhanced sensitivity, offering a robust platform for liquid biopsy applications in early BC diagnostics.},
}

*MicroRNAs/analysis/genetics/blood
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*DNA/genetics/chemistry
*Breast Neoplasms/diagnosis/genetics
Limit of Detection
Female
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40511702,
year = {2025},
author = {Sun, X and Li, X and Jiang, H and Shan, Y and Zhou, S and Wang, Z},
title = {Accurate Diagnosis of Colorectal Cancer Using a Combination of Lectin-Induced Recombinase Polymerase Amplification and CRISPR/Cas12a Assay on a Point-of-Care Testing Platform with Deep Learning Assistant.},
journal = {Analytical chemistry},
volume = {97},
number = {25},
pages = {13086-13094},
doi = {10.1021/acs.analchem.5c00468},
pmid = {40511702},
issn = {1520-6882},
mesh = {*Colorectal Neoplasms/diagnosis ; Humans ; *CRISPR-Cas Systems ; Animals ; *Point-of-Care Testing ; Mice ; *Deep Learning ; *Nucleic Acid Amplification Techniques/methods ; *Lectins/metabolism/chemistry ; *Recombinases/metabolism ; Exosomes/metabolism ; Cell Line, Tumor ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Specific glycosylation patterns on exosome surfaces represent novel diagnostic biomarkers for cancer liquid biopsy. Lectins can induce exosome aggregation through multiple bindings with exosomal glycoproteins. In this work, we developed a one-pot lectin-induced recombinase polymerase amplification (RPA) and CRISPR/Cas12a-mediated cleavage assay (LI-RPA-CRISPR/Cas12a) for diagnosing colorectal cancer (CRC) through the interactions of abundant α-fucose residues on CRC cell-derived exosome surfaces with Ulex Europaeus Agglutinin I (UEA-I). The combination of a homemade portable isothermal amplification device, the as-proposed LI-RPA-CRISPR/Cas12a exhibits a wide detection range from 2 × 10[6] to 1 × 10[2] extracellular vehicles (EVs) μL[-1] with a visual limit of detection (LOD) as low as 1.0 × 10[2] EVs μL[-1], and has been successfully utilized to dynamically monitor the progression of tumors in mice-bearing SW480 CRC subtype at an early stage. After integration with a long short-term memory (LSTM) deep learning model, the LI-RPA-CRISPR/Cas12a achieves accurate diagnosis of primary colorectal cancer with a drop of blood through a smartphone-based data analysis application, reaching an accuracy of 95% in 100 clinical samples. This rapid, sensitive, and user-friendly approach provides a promising platform for point-of-care testing (POCT) diagnosis of CRC, enabling early detection and monitoring of disease progression through a minimally invasive liquid biopsy.},
}

*Colorectal Neoplasms/diagnosis
Humans
*CRISPR-Cas Systems
Animals
*Point-of-Care Testing
Mice
*Deep Learning
*Nucleic Acid Amplification Techniques/methods
*Lectins/metabolism/chemistry
*Recombinases/metabolism
Exosomes/metabolism
Cell Line, Tumor
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40434897,
year = {2025},
author = {Chen, L and Chen, X and Kashina, A},
title = {Amino acid-level differences in alpha-tubulin sequences are uniquely required for meiosis.},
journal = {Molecular biology of the cell},
volume = {36},
number = {7},
pages = {ar89},
doi = {10.1091/mbc.E24-11-0529},
pmid = {40434897},
issn = {1939-4586},
mesh = {Schizosaccharomyces/metabolism/genetics ; *Meiosis/physiology/genetics ; *Tubulin/metabolism/genetics ; Schizosaccharomyces pombe Proteins/metabolism/genetics ; Amino Acid Sequence ; CRISPR-Cas Systems ; Protein Isoforms/metabolism/genetics ; Amino Acids/metabolism/genetics ; Spores, Fungal/metabolism ; Spindle Apparatus/metabolism ; },
abstract = {Members of the tubulin gene family members are essential components of the cytoskeleton; however, functional diversity of tubulin isoforms is poorly understood. Here, we addressed this question using Schizosaccharomyces pombe as a model system. These yeast encode two α-tubulins, nda2 and atb2, that are very similar at the amino acid level but differ in their roles in organism's survival: nda2 deletion is lethal, while lack of atb2 does not interfere with cell viability. Using CRISPR-Cas9 gene editing, we generated a yeast strain expressing atb2 amino acid sequence utilizing nda2 codon usage in the native nda2 locus. Such nda2-coded atb2 (NCA) yeast, unlike nda2 knockout, were viable and displayed no visible abnormalities in vegetative life cycle. Instead, they displayed strong impairments in sporulation and meiosis, linked to altered balance of several spindle proteins. Our data indicate that nda2 protein is uniquely required for normal meiosis, and identify novel protein- and nucleotide-level determinants driving functional distinction between closely related tubulin isoforms.},
}

Schizosaccharomyces/metabolism/genetics
*Meiosis/physiology/genetics
*Tubulin/metabolism/genetics
Schizosaccharomyces pombe Proteins/metabolism/genetics
Amino Acid Sequence
CRISPR-Cas Systems
Protein Isoforms/metabolism/genetics
Amino Acids/metabolism/genetics
Spores, Fungal/metabolism
Spindle Apparatus/metabolism

@article {pmid40378737,
year = {2025},
author = {Zheng, H and Guo, J and Wang, H and Wang, Y and Zhou, W and Liu, K and Ma, Q and Qi, Y},
title = {A zeolitic imidazolate framework-90 enhanced ultrasensitive ATP sensing platform with HCR and CRISPR-Cas12a dual signal amplification for live bacteria detection.},
journal = {Journal of hazardous materials},
volume = {494},
number = {},
pages = {138612},
doi = {10.1016/j.jhazmat.2025.138612},
pmid = {40378737},
issn = {1873-3336},
mesh = {*Adenosine Triphosphate/analysis ; *Zeolites/chemistry ; *Imidazoles/chemistry ; CRISPR-Cas Systems ; *Staphylococcus aureus/isolation & purification ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; *Metal-Organic Frameworks/chemistry ; },
abstract = {Bacteria are prevalent environmental pollutants. Live bacteria can proliferate and spread under appropriate conditions, presenting higher risks compared to non-viable counterparts. However, detecting live bacteria remains a challenge. Adenosine triphosphate (ATP), an energy currency in organisms, offers a reliable biomarker for the live bacteria sensing. Herein, we developed a zeolitic imidazolate framework-90 (ZIF-90) enhanced ATP sensing platform to detect live bacteria. The nanosystem which based on ZIF-90, encapsulating the DNA decorated magnetic beads (MB@S1) through self-assembly. When the S. aureus aptamers on ZIF-90 bonded to bacteria, the skeleton structure of ZIF-90 was disrupted by ATP leakage from live bacteria, leading to the release of MB@S1. Then, the MB@S1 initiated the hybridization chain reaction (HCR) and they were transduced by CRISPR-Cas12a to amplify the signal twice. The proposed ZIF-90 blocking sensing strategy (ZIF-90 strategy) exhibited remarkable sensitivity with the limit of detection (LOD) down to 0.223 pM for ATP detection, about 500 folds lower than the traditional ATP aptamer blocking sensing strategy (aptamer strategy). Furthermore, we used a smartphone for on-site analysis, realizing the quantification of live S. aureus with the LOD of 2.0 CFU/mL. Therefore, the approach possessed great application potential for public health, environment monitoring, bioanalysis and food safety.},
}

*Adenosine Triphosphate/analysis
*Zeolites/chemistry
*Imidazoles/chemistry
CRISPR-Cas Systems
*Staphylococcus aureus/isolation & purification
*Biosensing Techniques/methods
Aptamers, Nucleotide/chemistry
Limit of Detection
Nucleic Acid Hybridization
*Metal-Organic Frameworks/chemistry

@article {pmid40586312,
year = {2025},
author = {Soderholm, A and Vunjak, M and de Almeida, M and Popitsch, N and Podvalnaya, N and Araguas-Rodriguez, P and Scinicariello, S and Nischwitz, E and Butter, F and Ketting, RF and Ameres, SL and Müller-McNicoll, M and Zuber, J and Versteeg, GA},
title = {ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf545},
pmid = {40586312},
issn = {1362-4962},
support = {10.55776/P36572//Austrian Science Fund/ ; 10.55776/P30415//Austrian Science Fund/ ; 10.55776/P30231//Austrian Science Fund/ ; 10.55776/P36945//Austrian Science Fund/ ; 10.55776/F79//Austrian Science Fund/ ; 10.55776/W1261//Austrian Science Fund/ ; //Austrian Academy of Sciences/ ; FFG-852936//Austrian Research Promotion Agency/ ; FFG-852936//Österreichische Forschungsförderungsgesellschaft/ ; },
mesh = {*Janus Kinase 2/genetics/metabolism ; Humans ; *Interferon-gamma/metabolism/immunology/genetics ; Signal Transduction/genetics ; *RNA, Messenger/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; Introns ; Immunity, Innate/genetics ; CRISPR-Cas Systems ; Serine-Arginine Splicing Factors/genetics ; HEK293 Cells ; RNA Splicing ; Animals ; },
abstract = {Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way, we identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response.},
}

*Janus Kinase 2/genetics/metabolism
Humans
*Interferon-gamma/metabolism/immunology/genetics
Signal Transduction/genetics
*RNA, Messenger/genetics/metabolism
*RNA Processing, Post-Transcriptional
Introns
Immunity, Innate/genetics
CRISPR-Cas Systems
Serine-Arginine Splicing Factors/genetics
HEK293 Cells
RNA Splicing
Animals

@article {pmid40585259,
year = {2025},
author = {Zheng, Z and Yu, M and Ai, L and Wang, B and Lian, S and Liu, J and Li, L and Tsai, S and Kleinstiver, B and Ip, L},
title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-4552906/v1},
pmid = {40585259},
issn = {2693-5015},
abstract = {Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. To overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.},
}

@article {pmid40585234,
year = {2025},
author = {Jain, P and Orosco, C and Rananaware, S and Huang, B and Hanna, M and Ahmadimashhadi, MR and Lewis, J and Baugh, M and Bodin, A and Flannery, S and Langue, I and Fang, Z and Karalkar, V and Meister, K},
title = {DNA-guided CRISPR/Cas12 for RNA targeting.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-5507198/v1},
pmid = {40585234},
issn = {2693-5015},
abstract = {CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12's potential for diagnostic applications and for targeted RNA modulation in cellular environments.},
}

@article {pmid40587425,
year = {2025},
author = {Li, X and Peng, Y and Xue, W and Liu, X and Luo, S and Wei, P and Wang, J},
title = {CRISPR-based Shuttle Cloning: A High-throughput Cloning Method.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {220},
pages = {},
doi = {10.3791/68503},
pmid = {40587425},
issn = {1940-087X},
mesh = {*Cloning, Molecular/methods ; Plasmids/genetics ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; DNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.},
}

*Cloning, Molecular/methods
Plasmids/genetics
*Genetic Vectors/genetics
*CRISPR-Cas Systems
DNA/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40587016,
year = {2025},
author = {Choudhary, A and Kumar, A and Munshi, A},
title = {Genetic variants in oncogenic miRNA and 3' untranslated region of tumor suppressor genes: emerging insight into cancer genetics.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {8},
pages = {303},
pmid = {40587016},
issn = {1559-131X},
support = {211610155703//University Grants Commission/ ; BT/PR45460/MED/12/952/2022//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Humans ; *MicroRNAs/genetics ; *Genes, Tumor Suppressor ; *3' Untranslated Regions/genetics ; *Neoplasms/genetics ; *Breast Neoplasms/genetics ; *Genetic Variation ; Female ; Gene Expression Regulation, Neoplastic ; Oncogenes ; },
abstract = {The miRNAs are key regulators of post-transcriptional gene expression. These are associated with the different molecular mechanisms which are engaged in the pathogenesis of various cancers, including breast cancer. The tumor suppressor and oncogenic miRNAs have a significant impact on cell proliferation, metastasis, angiogenesis, and apoptotic pathways. Variation in oncogenic miRNA encoding and the 3' UTR of the tumor suppressor genes associated with the development and prognosis of the cancer is being explored. These genetic variants alter oncogenic miRNAs' stability, target recognition, and binding ability, thereby resulting in the dysregulation of their target tumor suppressor gene, leading to uncontrolled cell division, a significant hallmark of cancer. The current review has been compiled to explore the genetic variation reported in the oncogenic miRNA encoding and 3' UTR of their target tumor suppressor genes associated with the development of breast cancer and its progression, focusing on the associated molecular mechanisms. Further, an effort has been made to discuss the possible therapeutic strategies, especially anti-miR, RNA interference, CRISPR/Cas, and ASOs, that have the potential to restore the function of the dysregulated tumor suppressor as well as oncogenic miRNA encoding genes.},
}

Humans
*MicroRNAs/genetics
*Genes, Tumor Suppressor
*3' Untranslated Regions/genetics
*Neoplasms/genetics
*Breast Neoplasms/genetics
*Genetic Variation
Female
Gene Expression Regulation, Neoplastic
Oncogenes

@article {pmid40586829,
year = {2025},
author = {Hussain, MS and Maqbool, M and Arab, MM and Rana, AJ and Ashique, S and Khan, Y and Jakhmola, V and Gupta, G},
title = {Transforming Hemophilia Management: Lessons from Gene Therapy Clinical Trials.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {40586829},
issn = {1559-0305},
abstract = {Gene therapy signifies a transformative revolution in hemophilia care, providing the possibility for sustained endogenous synthesis of coagulation factors and limiting the need for external factor supplementation. Preliminary experiments in hemophilia B via adeno-associated viral (AAV) vectors encountered constraints owing to immunological reactions and temporary translation. Progress in vector technology, particularly via self-complementary AAV innovation and codon-optimized mini-factor IX (FIX) concepts, has markedly improved transduction performance and prolonged FIX activity. Initial investigations have shown encouraging outcomes, with certain individuals sustaining consistent FIX expressions for more than 8 years; hence, decreasing yearly bleeding incidents and requiring preventive therapy. The development of gene therapy for hemophilia A has encountered substantial obstacles owing to the enormous size of the factor VIII (FVIII) gene. The recent experiments using AAV serotypes 5 (AAV5) vectors that encode B-domain-deleted FVIII constructs have shown sustained levels along with substantial decreases in hemorrhage incidents. Research has shown prolonged FVIII expression, with some individuals attaining almost normal coagulation efficiency. Phase III studies have validated long-term effectiveness and safety, with transient transaminase elevations being the most common adverse event. Notwithstanding these advancements, difficulties persist, including immunological reactions to vector capsids, hepatotoxicity, and unpredictability in translation levels. Innovative approaches including lentiviral vectors, gene-editing technologies, and novel customized connection strategies demonstrate possibilities for enhancing the effectiveness of gene therapy. Continuous clinical research and improvement in delivery systems will be crucial in substantiating gene therapy as a definitive approach for hemophilia.},
}

@article {pmid40581000,
year = {2025},
author = {Fraikin, N and Samuel, B and Burstein, D and Lesterlin, C},
title = {Strategies for zygotic gene expression during plasmid establishment.},
journal = {Plasmid},
volume = {},
number = {},
pages = {102754},
doi = {10.1016/j.plasmid.2025.102754},
pmid = {40581000},
issn = {1095-9890},
abstract = {Conjugative plasmids are key drivers of horizontal gene transfer and the spread of antimicrobial resistance. Their successful establishment in new hosts requires overcoming diverse bacterial defence mechanisms, such as restriction-modification systems, CRISPR-Cas systems, and the SOS response. Plasmids achieve this through a leading region-encoded zygotic program of anti-defence genes expressed early in conjugation. This program employs diverse strategies, including single-stranded promoters, repressed double-stranded promoters, and protein translocation. This review explores the diversity of these zygotic programs, the mechanisms underlying their timely regulation, and the array of anti-defence functions they encode. Further investigation of leading region genes is crucial for discovering novel counter-defence strategies and understanding their tailored regulation across diverse plasmid and bacterial species, ultimately enabling us to better understand and potentially manipulate plasmid transfer.},
}

@article {pmid40580284,
year = {2025},
author = {Manjunatha, C and Aditya, K and Prasannakumar, MK and Pramesh, D and Buella, P and Patil, SS and Venkateshbabu, G and Harish, J and Karan, R and Ramegowda, HV},
title = {Isothermal Amplification Techniques: An Emerging Tool for On-Site Detection of Phytopathogens in Field Conditions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2943},
number = {},
pages = {47-64},
pmid = {40580284},
issn = {1940-6029},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology/virology ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Systems ; *Plants/microbiology ; CRISPR-Cas Systems ; },
abstract = {This chapter reviews various isothermal amplification techniques, which are alternative to PCR for detecting plant pathogens. These methods, including NASBA, SDA, LAMP, HDA, and RPA, amplify nucleic acids at a constant temperature, making them potentially more suitable for point-of-care (POC) applications. The review compares these techniques regarding their mechanisms, advantages, and limitations, highlighting their potential for rapid, cost-effective, and on-site plant disease diagnosis, including the integration of these techniques with CRISPR-Cas systems and lab-on-a-chip technologies.},
}

*Nucleic Acid Amplification Techniques/methods
*Plant Diseases/microbiology/virology
*Molecular Diagnostic Techniques/methods
Point-of-Care Systems
*Plants/microbiology
CRISPR-Cas Systems

@article {pmid40580153,
year = {2025},
author = {Sharan, P and Kumar, BK and Kumar, A and Rai, P},
title = {Isothermal amplification for rapid and sensitive detection of hepatitis B virus: what we know so far? and way forward.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2025.2527634},
pmid = {40580153},
issn = {1744-8352},
abstract = {INTRODUCTION: Despite vaccine availability, Hepatitis B Virus (HBV) remains a major global health threat, especially in areas with low vaccination coverage and poor healthcare. Around 250 million people are chronically infected. Achieving the World Health Organisation's 2030 eradication goal is difficult, particularly due to diagnostic challenges in low-resource settings. While HBsAg detection is standard, low antigen levels and mutations hinder its reliability. Though molecular methods for HBV DNA offer high specificity, their cost and complexity limit use in under-resourced areas. Isothermal amplification emerges as a promising alternative, offering a more affordable, effective, and simplified approach to HBV detection, potentially improving access to timely diagnosis and care.

AREAS COVERED: This review evaluates the efficacy of various isothermal techniques to give insights into their benefits and limits, guiding researchers and clinicians in selecting the most effective assays for HBV molecular diagnostics.

EXPERT OPINION: RPA and PSR are the most promising isothermal assays for HBV detection in field settings. RPA is faster (∼20 min), works at low temperatures (37-42 °C), and uses stable lyophilized reagents, while PSR is simple, can be clubbed with visual detection, making both ideal for a low-resource setup.},
}

@article {pmid40580034,
year = {2025},
author = {Lee, H and Rashid, F and Hwang, J and London, JA and Fishel, R and Berger, JM and Myong, S and Ha, T},
title = {A high-throughput single-molecule platform to study DNA supercoiling effect on protein-DNA interactions.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf581},
pmid = {40580034},
issn = {1362-4962},
support = {R35 GM122569/NH/NIH HHS/United States ; R35 CA263778/NH/NIH HHS/United States ; R37 GM071747/NH/NIH HHS/United States ; R35 CA263778/NH/NIH HHS/United States ; R01 GM149729/NH/NIH HHS/United States ; R01 CA067007/NH/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*DNA, Superhelical/metabolism/chemistry/genetics ; DNA Gyrase/metabolism ; Plasmids/genetics/chemistry/metabolism ; Protein Binding ; *MutS DNA Mismatch-Binding Protein/metabolism/genetics ; *Single Molecule Imaging/methods ; Escherichia coli Proteins/metabolism ; Adenosine Triphosphate/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; DNA/metabolism/chemistry ; },
abstract = {DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced physiological levels of negative or positive supercoiling in these plasmids using gyrase or reverse gyrase, respectively. By comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and the mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed that both negative and positive DNA supercoiling enhance the binding interaction between MutS and a mismatched base pair but do not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also open new avenues for exploring the intricate dynamics of protein-DNA interactions under the influence of supercoiling.},
}

*DNA, Superhelical/metabolism/chemistry/genetics
DNA Gyrase/metabolism
Plasmids/genetics/chemistry/metabolism
Protein Binding
*MutS DNA Mismatch-Binding Protein/metabolism/genetics
*Single Molecule Imaging/methods
Escherichia coli Proteins/metabolism
Adenosine Triphosphate/metabolism
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems
DNA/metabolism/chemistry

@article {pmid40491322,
year = {2025},
author = {Cao, J and Guo, Z and Xu, X and Li, P and Fang, Y and Deng, S},
title = {Advances in CRISPR-Cas9 in lineage tracing of model animals.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {6},
pages = {1004-1022},
pmid = {40491322},
issn = {2576-2095},
support = {22SH19//Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Collaborative Innovation Program of the Chinese Academy of Sciences/ ; 2023-PT180-01//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cell Lineage/genetics ; *Models, Animal ; Mice ; Swine ; Zebrafish ; *Cell Tracking/methods ; },
abstract = {Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Humans
*Cell Lineage/genetics
*Models, Animal
Mice
Swine
Zebrafish
*Cell Tracking/methods

@article {pmid40481182,
year = {2025},
author = {Yu, L and Liu, Y and Lin, X},
title = {Transitioning from native to synthetic receptors: broadening T-cell engineering and beyond.},
journal = {Cellular & molecular immunology},
volume = {22},
number = {7},
pages = {712-729},
pmid = {40481182},
issn = {2042-0226},
support = {82341212//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82293664//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *T-Lymphocytes/immunology ; Animals ; *Receptors, Antigen, T-Cell/genetics/immunology ; Gene Editing ; *Immunotherapy, Adoptive/methods ; *Cell Engineering/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; CRISPR-Cas Systems ; *Receptors, Artificial/genetics/immunology ; },
abstract = {T-cell immunotherapy has progressed rapidly, evolving from native T-cell receptor biology to the development of innovative synthetic receptors that extend therapeutic applications beyond cancer. This review explores engineering strategies, ranging from natural TCRs to synthetic receptors, that increase T-cell activation and therapeutic potential. We begin by highlighting the foundational role of native receptors in the T-cell response, emphasizing how these structural and functional insights inform the design of next-generation synthetic receptors. Comparisons between CAR and TCR-like synthetic receptors underscore their respective advantages in specificity, efficacy, and safety, as well as potential areas for further improvement. In addition, gene editing technologies such as CRISPR-Cas9 enable precise modifications to the T-cell genome, enhancing receptor performance and minimizing immunogenic risks. In addition to tumors, these engineered T cells can be directed against viral infections, autoimmune disorders, and other diseases. We also explore advanced strategies that engage multiple immune cell types to achieve synergistic, durable responses. By demonstrating how native and synthetic receptors collectively drive innovation, this review aims to inspire new research directions and ultimately expand the scope of T-cell engineering for universal therapeutic applications.},
}

Humans
*T-Lymphocytes/immunology
Animals
*Receptors, Antigen, T-Cell/genetics/immunology
Gene Editing
*Immunotherapy, Adoptive/methods
*Cell Engineering/methods
*Receptors, Chimeric Antigen/genetics/immunology
CRISPR-Cas Systems
*Receptors, Artificial/genetics/immunology

@article {pmid40299756,
year = {2025},
author = {An, Y and Wang, SQ and Jia, XY and Jiao, X and Qu, MQ and Dong, Y and Wang, ZY and Ma, ZY and Yang, S and Han, X and Huang, LC and Chen, NN and Jiang, C and Lu, MZ and Dai, JF and Zhang, J},
title = {Bioengineered poplar fibres via PagGLR2.8 editing: A synergistic design for high-performance biocomposites.},
journal = {Plant biotechnology journal},
volume = {23},
number = {7},
pages = {2824-2838},
pmid = {40299756},
issn = {1467-7652},
support = {32371902//National Natural Science Foundation of China/ ; 51903222//National Natural Science Foundation of China/ ; 2021YFD2200205//National Key Research and Development Program of China/ ; 2021C02070-1//the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties/ ; 2018FR013//the research foundation of Zhejiang A&F University/ ; //Zhejiang students' technology and innovation program/ ; },
mesh = {*Populus/genetics/metabolism ; Plants, Genetically Modified/genetics ; Polyesters ; Gene Editing ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bioengineering ; },
abstract = {The urgent need to replace petroleum-derived materials with sustainable alternatives drives innovation at the nexus of plant biotechnology and materials science. Here, we engineered Populus alba × P. glandulosa '84 K' through CRISPR-Cas9-mediated knockout of PagGLR2.8, a glutamate receptor gene regulating vascular development, to investigate its role in fibre biosynthesis and composite performance. Knockout of PagGLR2.8 improved the quality of poplar fibre by altering the structure and development mode of poplar vascular tissue. Our study established the relationship between fibre quantity and structure and the performance of polylactic acid (PLA) composites. The mechanical and fire-resistance properties of these transgenic plant fibres/PLA composites significantly outperformed those of pure PLA, demonstrating the potential of phloem fibres to reinforce toughened composites. Notably, we also evaluated flammability and dripping behaviours, with findings indicating that our optimised fibre/PLA composites exhibit superior strengths, modulus, fire resistance, and anti-dripping, surpassing those of PLA. This research unveils a groundbreaking approach to regulating composite properties through genetic manipulation and highlights the promising potential of plant-derived materials in enriching forest resources and advancing the sustainable utilisation of poplar fibres and polymers.},
}

*Populus/genetics/metabolism
Plants, Genetically Modified/genetics
Polyesters
Gene Editing
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Bioengineering

@article {pmid39397560,
year = {2025},
author = {Gou, S and Liu, Y and Li, Q and Yang, J and Qiu, L and Zhao, Y},
title = {CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.},
journal = {Foodborne pathogens and disease},
volume = {22},
number = {7},
pages = {459-466},
doi = {10.1089/fpd.2024.0078},
pmid = {39397560},
issn = {1556-7125},
mesh = {*Rotavirus/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; *Food Contamination/analysis ; RNA, Viral/genetics ; },
abstract = {Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.},
}

*Rotavirus/isolation & purification/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Food Microbiology/methods
*Molecular Diagnostic Techniques/methods
Humans
*Food Contamination/analysis
RNA, Viral/genetics

@article {pmid40579649,
year = {2025},
author = {Liu, ZG and Wang, N and Wang, WJ and Huang, L and Chi, SS and Nie, YX and Yuan, MS and Sun, YR and Mu, YL and Li, K},
title = {Transcriptome analysis reveals no obvious unintended effects in the spleen of CRISPR/Cas9-mediated CD163 and pAPN double-knockout pigs.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {139},
pmid = {40579649},
issn = {1438-7948},
support = {2022ZD04020//Biological Breeding-National Science and Technology Major Project/ ; CAAS-CSAB-202401//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 202102//Outstanding Talents Training Fund in Shenzhen/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; CD163 Antigen ; *Antigens, Differentiation, Myelomonocytic/genetics/metabolism ; *Receptors, Cell Surface/genetics/metabolism ; *Spleen/metabolism ; Swine/genetics ; *Antigens, CD/genetics/metabolism ; *Transcriptome ; Gene Editing ; Gene Knockout Techniques ; Gene Expression Profiling ; Membrane Proteins ; },
abstract = {Gene editing provides powerful tools for farm animal breeding. Our group previously obtained CD163/pAPN double-knockout (DKO) pigs via CRISPR/Cas9 and somatic cell nuclear transfer. These pigs are not only resistant to three infectious viruses but also maintain normal production performance. However, unintended effects of CRISPR/Cas9 tools may pose potential risks to animal well-being or safety. This study aimed to characterize the differences in splenic gene expression between wild-type (WT) and DKO pigs, providing a basis for safety evaluation of gene-edited animals. A comprehensive transcriptional panorama reflected considerable congruence in the aggregate gene expression profiles of the DKO and WT pigs. Comparisons between 35-day-old and 11-month-old DKO pigs and their WT equivalents revealed 225 and 242 differentially expressed genes (DEGs), respectively, a count significantly lower than that of the DEGs in the disparate developmental stage comparison groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the majority of DEGs between DKO and WT pigs correlated with the biological functions of CD163 and pAPN, without any alterations in the expression of tumor suppressor genes in DKO pigs. This revealed a less pronounced effect of dual gene editing on the gene expression profile of porcine spleens than the effect of animal maturation, with no evident unanticipated consequences observed in DKO pigs.},
}

Animals
*CRISPR-Cas Systems
CD163 Antigen
*Antigens, Differentiation, Myelomonocytic/genetics/metabolism
*Receptors, Cell Surface/genetics/metabolism
*Spleen/metabolism
Swine/genetics
*Antigens, CD/genetics/metabolism
*Transcriptome
Gene Editing
Gene Knockout Techniques
Gene Expression Profiling
Membrane Proteins

@article {pmid40579133,
year = {2025},
author = {Mo, Y and Shu, Y and Mo, Y and Liu, J and Xu, O and Deng, H and Wang, Q},
title = {[CRISPR-Cas9-mediated CDC20 gene knockout inhibits cervical cancer cell proliferation, invasion and metastasis].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {45},
number = {6},
pages = {1200-1211},
doi = {10.12122/j.issn.1673-4254.2025.06.09},
pmid = {40579133},
issn = {1673-4254},
mesh = {Humans ; *Uterine Cervical Neoplasms/pathology/genetics/metabolism ; Female ; *Cdc20 Proteins/genetics ; *Cell Proliferation ; Animals ; Cell Movement ; Neoplasm Invasiveness ; Apoptosis ; Mice, Nude ; beta Catenin/metabolism ; *CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; },
abstract = {OBJECTIVES: To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism.

METHODS: CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and β-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, naïve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and β-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays.

RESULTS: Cervical cancer tissues expressed significantly higher CDC20 and β‑Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and β-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and β‑Catenin.

CONCLUSIONS: CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/β-Catenin signaling pathway.},
}

Humans
*Uterine Cervical Neoplasms/pathology/genetics/metabolism
Female
*Cdc20 Proteins/genetics
*Cell Proliferation
Animals
Cell Movement
Neoplasm Invasiveness
Apoptosis
Mice, Nude
beta Catenin/metabolism
*CRISPR-Cas Systems
Mice
Cell Line, Tumor
Gene Knockout Techniques
Neoplasm Metastasis

@article {pmid40578341,
year = {2025},
author = {Mahler, M and Cui, L and Smith, LM and Wandera, KG and Dietrich, O and Mayo-Muñoz, D and Balamkundu, S and Lee, ME and Ye, H and Liu, CF and Wu, J and Mathew, J and Dubrulle, J and Malone, LM and Jackson, SA and Fairbanks, AJ and Dedon, PC and Brouns, SJJ and Fineran, PC},
title = {Phage arabinosyl-hydroxy-cytosine DNA modifications result in distinct evasion and sensitivity responses to phage defense systems.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.06.005},
pmid = {40578341},
issn = {1934-6069},
abstract = {Bacteria encode diverse anti-phage systems, such as CRISPR-Cas and restriction modification (RM), which limit infection by targeting phage DNA. We identified a DNA modification in phages, i.e., 5-arabinosyl-hydroxy-cytosine (5ara-hC), which adds arabinose to cytosines via a hydroxy linkage and protects phage from DNA targeting. The hydroxy linkage was common among arabinoslyated phages, with some arabinosylated phages encoding arabinose-5ara-hC transferases (Aat) that add a second or third arabinose to DNA. DNA arabinosylation enables evasion from DNA-targeting type I CRISPR-Cas and type II RM systems. However, arabinosylated phages remain sensitive to RNA-targeting CRISPR-Cas (type III and VI) and promiscuous type IV restriction endonucleases. 5ara-hC enables evasion of glycosylase defenses that target phages with glucosylated hydroxymethyl cytosines, and 5ara-ara-hC protects against some defenses capable of targeting 5ara-hC-modified phages. Collectively, this work identifies DNA modifications that enable phages to evade multiple defenses yet remain vulnerable to some systems that target RNA or modified nucleobases.},
}

@article {pmid40575705,
year = {2025},
author = {Chen, S and Liu, Z and Lo, CH and Wang, Q and Ning, K and Zhang, Q and Zhao, J and Shen, Y and Sun, Y},
title = {Gene therapy for ocular hypertension using hfCas13d-mediated mRNA targeting.},
journal = {PNAS nexus},
volume = {4},
number = {6},
pages = {pgaf168},
pmid = {40575705},
issn = {2752-6542},
abstract = {Glaucoma is a major global cause of irreversible vision loss. It is marked by elevated intraocular pressure (IOP) and the loss of retinal ganglion cells (RGC). While there are medical and surgical therapies for glaucoma aiming to reduce aqueous humor production or enhance its drainage, these treatments are often inadequate for effectively managing the disease. In this study, we developed a targeted therapy for glaucoma by knocking down two genes associated with aqueous humor production (aquaporin 1 [AQP1] and carbonic anhydrase type 2 [CA2]) using Cas13 RNA editing systems. We demonstrate that hfCas13d-mediated knockdown of AQP1 and CA2 significantly lowers IOP in wild-type mice and in a corticosteroid-induced glaucoma mouse model. We show that the lowered IOP results from decreasing aqueous production without affecting the outflow facility; this treatment also significantly promotes RGC survival as compared with untreated control groups. Therefore, CRISPR-Cas-based gene editing may be an effective treatment to lower IOP for glaucomatous optic neuropathy.},
}

@article {pmid40575544,
year = {2025},
author = {Choi, JH and Yoon, J and Chen, M and Shin, M and Goldston, LL and Lee, KB and Choi, JW},
title = {CRISPR/Cas-Based Nanobiosensor Using Plasmonic Nanomaterials to Detect Disease Biomarkers.},
journal = {Biochip journal},
volume = {19},
number = {2},
pages = {167-181},
pmid = {40575544},
issn = {1976-0280},
abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) technology (CRISPR/Cas) as a gene-editing tool has the potential to revolutionize nucleic acid analysis. Recently, CRISPR/Cas systems have demonstrated considerable promise in the development of biosensors for the detection of essential disease biomarkers because they exhibit nonspecific collateral cleavage properties upon target sequence recognition. However, the CRISPR/Cas-based biosensors developed thus far have limitations, such as complicated steps, low sensitivity, low selectivity, and low signal-to-noise ratios. These limitations can be overcome by incorporating the unique characteristics of plasmonic nanomaterials into CRISPR/Cas systems to enhance the signal and improve the sensitivity of these biosensors. From this perspective, current interdisciplinary studies on CRISPR/Cas-based nanobiosensors comprising plasmonic nanomaterials can contribute to the development of highly sensitive CRISPR/Cas-based nanobiosensors. These nanobiosensors can detect attractive disease biomarkers, such as viral nucleic acids, small molecules, and proteins. This review article provides a thorough overview of nanobiosensors that incorporate CRISPR/Cas systems combined with plasmonic nanomaterials to enhance biosensing performance. We believe this review will inspire novel approaches and further innovation in the fields of molecular diagnostics and biomedicine aimed at using CRISPR/Cas systems and plasmonic nanomaterials for more personalized and effective medical treatments.},
}

@article {pmid40573878,
year = {2025},
author = {Ullah, F and Ali, S and Siraj, M and Akhtar, MS and Zaman, W},
title = {Plant Microbiomes Alleviate Abiotic Stress-Associated Damage in Crops and Enhance Climate-Resilient Agriculture.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/plants14121890},
pmid = {40573878},
issn = {2223-7747},
abstract = {Plant microbiomes, composed of a diverse array of microorganisms such as bacteria, fungi, archaea, and microalgae, are critical to plant health and resilience, playing key roles in nutrient cycling, stress mitigation, and disease resistance. Climate change is expected to intensify various abiotic stressors, such as drought, salinity, temperature extremes, nutrient deficiencies, and heavy metal toxicity. Plant-associated microbiomes have emerged as a promising natural solution to help mitigate these stresses and enhance agricultural resilience. However, translating laboratory findings into real-world agricultural benefits remains a significant challenge due to the complexity of plant-microbe interactions under field conditions. We explore the roles of plant microbiomes in combating abiotic stress and discuss advances in microbiome engineering strategies, including synthetic biology, microbial consortia design, metagenomics, and CRISPR-Cas, with a focus on enhancing their practical application in agriculture. Integrating microbiome-based solutions into climate-smart agricultural practices may contribute to long-term sustainability. Finally, we underscore the importance of interdisciplinary collaboration in overcoming existing challenges. Microbiome-based solutions hold promise for improving global food security and promoting sustainable agricultural practices in the face of climate change.},
}

@article {pmid40573432,
year = {2025},
author = {Hu, G and Wei, Z and Guo, J and Zhao, K and Qiao, Q and Zhu, X and Wu, T and Rong, H and Ning, S and Hao, Z and Chi, Y and Cui, L and Ge, Y},
title = {A Single-Tube Two-Step MIRA-CRISPR/Cas12b Assay for the Rapid Detection of Mpox Virus.},
journal = {Viruses},
volume = {17},
number = {6},
pages = {},
doi = {10.3390/v17060841},
pmid = {40573432},
issn = {1999-4915},
support = {2023YFC2605100, 2023YFC2605104//the National Key R&D Program of China/ ; BK20231374, BK20221413//the natural science foundation of Jiangsu Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Animals ; Humans ; Limit of Detection ; *Capripoxvirus/isolation & purification/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics ; },
abstract = {Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein-Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination.},
}

*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Animals
Humans
Limit of Detection
*Capripoxvirus/isolation & purification/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins/genetics

@article {pmid40573367,
year = {2025},
author = {Workman, AM and Heaton, MP and Vander Ley, BL},
title = {CD46 Gene Editing Confers Ex Vivo BVDV Resistance in Fibroblasts from Cloned Angus Calves.},
journal = {Viruses},
volume = {17},
number = {6},
pages = {},
doi = {10.3390/v17060775},
pmid = {40573367},
issn = {1999-4915},
support = {3040-32000-036-00D//USDA-ARS appropriated funds/ ; },
mesh = {Animals ; Cattle ; *Gene Editing ; *Fibroblasts/virology/immunology ; *Bovine Virus Diarrhea-Mucosal Disease/genetics/virology/immunology/prevention & control ; *Membrane Cofactor Protein/genetics ; Cloning, Organism ; *Diarrhea Viruses, Bovine Viral ; CRISPR-Cas Systems ; *Disease Resistance/genetics ; Female ; },
abstract = {A previous study demonstrated that a 19-nucleotide edit, encoding a six amino acid substitution in the bovine CD46 gene, dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned Gir (Bos indicus) heifer. The present study aimed to replicate this result in American Angus (Bos taurus) using genetically matched controls and larger sample sizes. CRISPR/Cas9-mediated homology-directed repair introduced the identical CD46 edit, encoding the A82LPTFS amino acid sequence, into exon 2 of CD46 in primary Angus fibroblasts. Thirty-three cloned embryos (22 CD46-edited and 11 unedited) were transferred to recipient cows. However, all pregnancies resulted in pre- and perinatal losses due to cloning-related abnormalities, preventing in vivo BVDV challenge. Consequently, ex vivo BVDV susceptibility assays were performed on primary fibroblast cell lines rescued from deceased cloned Angus calves. Infection studies revealed significantly reduced susceptibility in the edited lines, comparable to the resistance previously observed from the edited Gir heifer. These studies extend the applicability of this finding from Gir to the most common US beef breed, Angus, suggesting the potential for broad application of CD46 editing in BVDV control. Continued advancements in cloning technology will enhance the potential of gene-editing for producing disease-resistant livestock.},
}

Animals
Cattle
*Gene Editing
*Fibroblasts/virology/immunology
*Bovine Virus Diarrhea-Mucosal Disease/genetics/virology/immunology/prevention & control
*Membrane Cofactor Protein/genetics
Cloning, Organism
*Diarrhea Viruses, Bovine Viral
CRISPR-Cas Systems
*Disease Resistance/genetics
Female

@article {pmid40572275,
year = {2025},
author = {Kalu, MC and Acharya, A and Jorth, P and Wong-Beringer, A},
title = {ESBL-Producing Escherichia coli and Klebsiella pneumoniae Exhibit Divergent Paths During In-Human Evolution Towards Carbapenem Resistance.},
journal = {Microorganisms},
volume = {13},
number = {6},
pages = {},
pmid = {40572275},
issn = {2076-2607},
support = {not applicable//Merck & Co., Inc., Rahway, NJ, USA (United States)/ ; },
abstract = {Treatment of infections caused by ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) with carbapenem antibiotics can lead to the development of carbapenem resistance over time through the acquisition of porin mutations and plasmids bearing blaKPC. However, the impact of genetic background and the presence of CRISPR-Cas systems on the evolutionary path towards carbapenem resistance in EC and KP has yet to be investigated. The in-human evolution following repeated carbapenem treatment among ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) clinical pairs (n = 45 pairs) was examined to determine the relationship between strain genetic background (MLST, CRISPR-Cas) and the evolved genetic mutations related to resistance, virulence, and metabolism by whole genome sequencing. ST131 and ST258 were predominant among seven distinct STs in EC (70%, 19/27) and 11 STs in KP (33%, 6/18), respectively. Complete CRISPR-Cas systems were present in 22% EC (6/27) and 27.8% (5/18) KP pairs, but none in strains belonging to ST131 or ST258; partial loss of CRISPR-Cas was associated with increased carbapenem resistance. Porin, virulence, and metabolism-related genetic mutations were present on the chromosome in both the EC and KP evolved strains, but their presence was differentially associated with the CRISPR-Cas system. Future research on the role of antibiotic exposure in the species-specific resistance evolution of the Enterobacterales could guide antimicrobial stewardship efforts.},
}

@article {pmid40572209,
year = {2025},
author = {Antequera-Zambrano, L and Parra-Sánchez, Á and González-Paz, L and Fernandez, E and Martinez-Navarrete, G},
title = {Distribution of Genetic Determinants Associated with CRISPR-Cas Systems and Resistance to Antibiotics in the Genomes of Archaea and Bacteria.},
journal = {Microorganisms},
volume = {13},
number = {6},
pages = {},
pmid = {40572209},
issn = {2076-2607},
abstract = {The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material-reflected in the higher complexity and diversity of CRISPR loci in Archaea-and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures.},
}

@article {pmid40493452,
year = {2025},
author = {Xue, H and Mishra, MK and Liu, Y and Liu, P and Grzybowski, M and Pandey, R and Usa, K and Vanden Avond, MA and Bala, N and Alli, AA and Cowley, AW and Qiu, Q and Greene, AS and Rao, S and O'Meara, CC and Geurts, AM and Liang, M},
title = {Physiological role and mechanisms of action for a long noncoding haplotype region.},
journal = {Cell reports},
volume = {44},
number = {6},
pages = {115805},
doi = {10.1016/j.celrep.2025.115805},
pmid = {40493452},
issn = {2211-1247},
mesh = {Humans ; *Haplotypes/genetics ; Polymorphism, Single Nucleotide/genetics ; Animals ; Induced Pluripotent Stem Cells/metabolism ; Blood Pressure/genetics ; Gene Editing/methods ; Mice ; CRISPR-Cas Systems ; },
abstract = {Direct targeting of noncoding genomic regions harboring common sequence variants associated with human traits through in vivo animal model studies and precise genome editing in human cells is essential for closing the critical gap between genetic discoveries and physiological understanding. However, such investigation has been impractical for many of these variants as they are in haplotypes containing multiple single-nucleotide polymorphisms (SNPs) spanning thousands of base pairs and have small effect sizes. We developed an integrated approach to address this challenge, combining an efficient two-step technique to precisely edit large haplotypes in human induced pluripotent stem cells and orthologous region deletion in phenotypically permissive animal models. As proof of principle, we applied this approach to examine a blood pressure-associated locus with a noncoding haplotype containing 11 SNPs spanning 17.4 kbp. We found a robust blood pressure effect of nearly 10 mmHg and identified the physiological and molecular mechanisms involved.},
}

Humans
*Haplotypes/genetics
Polymorphism, Single Nucleotide/genetics
Animals
Induced Pluripotent Stem Cells/metabolism
Blood Pressure/genetics
Gene Editing/methods
Mice
CRISPR-Cas Systems

@article {pmid40434888,
year = {2025},
author = {Guo, J and Zhou, Z and Li, R and Xing, Z and Zhang, L and Zhao, S and Wei, W and Wang, J and Deng, T},
title = {A genome-wide base-editing screen uncovers a pivotal role of paxillin δ ubiquitination in influenza virus infection.},
journal = {Cell reports},
volume = {44},
number = {6},
pages = {115748},
doi = {10.1016/j.celrep.2025.115748},
pmid = {40434888},
issn = {2211-1247},
mesh = {Animals ; *Ubiquitination ; *Paxillin/metabolism/genetics ; Humans ; Mice ; Virus Replication ; *Influenza A virus/physiology ; *Gene Editing ; *Orthomyxoviridae Infections/virology/genetics/metabolism ; Madin Darby Canine Kidney Cells ; CRISPR-Cas Systems ; Virus Internalization ; Dogs ; Protein Isoforms/metabolism ; },
abstract = {Dissecting host factors critical for viral infection and understanding their mechanisms of action is critical for identifying drug targets. Here, we leverage a genome-wide CRISPR base-editing screen to identify functional lysine residues in host factors required for influenza A virus (IAV) replication. Multiple host genes, including GSTM4, FLNC, HMGB1, ZNF236, GRIP1, and PXN, along with regulatory lysine codons, are identified. Among these, paxillin (encoded by PXN) is identified as an important host entry factor. Depletion of paxillin significantly reduces IAV infection in both cell cultures and mice. Further analysis suggests that the δ isoform of paxillin, rather than the canonical β isoform, plays the key role. Additionally, our data indicate that lysine 68 of paxillin δ undergoes K6-linked ubiquitination and regulates influenza virus replication via modulating endosome-dependent viral entry. These observations contribute to understanding how influenza viruses interact with host factors and may inform therapeutic development.},
}

Animals
*Ubiquitination
*Paxillin/metabolism/genetics
Humans
Mice
Virus Replication
*Influenza A virus/physiology
*Gene Editing
*Orthomyxoviridae Infections/virology/genetics/metabolism
Madin Darby Canine Kidney Cells
CRISPR-Cas Systems
Virus Internalization
Dogs
Protein Isoforms/metabolism

@article {pmid40384639,
year = {2025},
author = {Ye, J and Shen, Y and Lin, Z and Xu, L and Wang, L and Lin, X and Huang, B and Ma, Z and Yu, Z and Lin, D and Chen, W and Feng, S},
title = {A CRISPR/Cas12a-Assisted SERS Nanosensor for Highly Sensitive Detection of HPV DNA.},
journal = {ACS sensors},
volume = {10},
number = {6},
pages = {4286-4296},
doi = {10.1021/acssensors.5c00547},
pmid = {40384639},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *Human papillomavirus 16/genetics/isolation & purification ; *Biosensing Techniques/methods ; Humans ; *Human papillomavirus 18/genetics/isolation & purification ; Biotin/chemistry ; Limit of Detection ; },
abstract = {The lack of timely and effective screening and diagnosis is a major contributing factor to the high mortality rate of cervical cancer in low-income countries and resource-limited regions. Therefore, the development of a rapid, sensitive, and easily deployable diagnostic tool for HPV DNA is of critical importance. In this study, we present a novel high-sensitivity and high-specificity detection method for HPV16 and HPV18 by integrating the CRISPR/Cas12a system with surface-enhanced Raman scattering (SERS) technology. This method leverages the trans-cleavage activity of the CRISPR/Cas12a system, which cleaves biotin-modified spherical nucleic acids (Biotin-SNA) in the presence of target DNA, releasing free Biotin-DNA. The released Biotin-DNA preferentially binds to streptavidin-modified magnetic beads (SAV-MB), reducing the capture of Biotin-SNA by SAV-MB and thereby significantly enhancing detection sensitivity. This method offers the potential for point-of-care diagnostics as it operates efficiently at 37 °C without the need for thermal cycling. Using standard DNA samples, we demonstrated that this biosensor achieved detection limits as low as 209 copies/μL and 444 copies/μL within 95 min. When combined with recombinase polymerase amplification (RPA), the sensor demonstrated enhanced sensitivity, enabling detection of target DNA at concentrations as low as 1 copy/μL within approximately 50 min. Furthermore, validation with clinical samples confirmed the feasibility and practical applicability of this method. This novel SERS-based sensor offers a new and effective tool in the prevention and detection of cervical cancer.},
}

*Spectrum Analysis, Raman/methods
*CRISPR-Cas Systems
*DNA, Viral/analysis/genetics
*Human papillomavirus 16/genetics/isolation & purification
*Biosensing Techniques/methods
Humans
*Human papillomavirus 18/genetics/isolation & purification
Biotin/chemistry
Limit of Detection

@article {pmid40300601,
year = {2025},
author = {Kang, X and Li, X and Zhou, J and Zhang, Y and Qiu, L and Tian, C and Deng, Z and Liang, X and Zhang, Z and Du, S and Hu, S and Wang, N and Yue, Z and Xu, Y and Gao, Y and Dai, J and Wang, Z and Yu, C and Chen, J and Wu, Y and Chen, L and Yao, Y and Yao, S and Yang, X and Yan, L and Wen, Q and Depies, OM and Chan, K and Liang, X and Li, G and Zi, Z and Liu, X and Gan, H},
title = {Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors.},
journal = {Cell},
volume = {188},
number = {13},
pages = {3405-3421.e27},
doi = {10.1016/j.cell.2025.04.012},
pmid = {40300601},
issn = {1097-4172},
mesh = {Humans ; *DNA Replication ; Ataxia Telangiectasia Mutated Proteins/metabolism ; DNA End-Joining Repair ; DNA Breaks, Double-Stranded ; *Neoplasms/genetics/metabolism/pathology ; *DNA Damage ; Cell Line, Tumor ; DNA Ligase ATP/metabolism ; DNA-Directed DNA Polymerase/metabolism ; DNA, Circular/metabolism/genetics ; DNA Polymerase theta ; CRISPR-Cas Systems ; },
abstract = {Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells. However, the functional significance of ecDNA and the molecular components involved in its replication and maintenance remain largely unknown. Here, using CRISPR-C technology, we generated ecDNA-carrying (ecDNA+) cell models. By leveraging these models alongside other well-established systems, we demonstrated that ecDNA can replicate and be maintained in ecDNA+ cells. The replication of ecDNA activates the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) pathway. Topoisomerases, such as TOP1 and TOP2B, play a role in ecDNA replication-induced DNA double-strand breaks (DSBs). A subset of these elevated DSBs persists into the mitotic phase and is primarily repaired by the alternative non-homologous end joining (alt-NHEJ) pathway, which involves POLθ and LIG3. Correspondingly, ecDNA maintenance requires DDR, and inhibiting DDR impairs the circularization of ecDNA. In summary, we demonstrate reciprocal interactions between ecDNA maintenance and DDR, providing new insights into the detection and treatment of ecDNA+ tumors.},
}

Humans
*DNA Replication
Ataxia Telangiectasia Mutated Proteins/metabolism
DNA End-Joining Repair
DNA Breaks, Double-Stranded
*Neoplasms/genetics/metabolism/pathology
*DNA Damage
Cell Line, Tumor
DNA Ligase ATP/metabolism
DNA-Directed DNA Polymerase/metabolism
DNA, Circular/metabolism/genetics
DNA Polymerase theta
CRISPR-Cas Systems

@article {pmid40571317,
year = {2025},
author = {Karinen, S and Forero-Rodríguez, J and Järvinen, A and Eklund, KK and Barreto, G and Salem, A},
title = {CRISPR/Cas9-mediated Knockout of LYVE1 In Human Tongue Cancer Cells Reveals Transcriptomic Changes in Metastasis-associated Pathways.},
journal = {Cancer genomics & proteomics},
volume = {22},
number = {4},
pages = {525-537},
doi = {10.21873/cgp.20519},
pmid = {40571317},
issn = {1790-6245},
mesh = {Humans ; *Tongue Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; *Transcriptome ; *Vesicular Transport Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; *Squamous Cell Carcinoma of Head and Neck/genetics ; },
abstract = {BACKGROUND/AIM: Tongue squamous cell carcinoma (TSCC), a highly aggressive subtype of head and neck cancers, is characterized by frequent lymphatic metastasis and poor prognosis. Recently, we showed that lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is involved in TSCC progression, yet the underlying molecular mechanisms remain unclear.

MATERIALS AND METHODS: CRISPR/Cas9 gene editing was employed to generate LYVE1 knockout (KO) TSCC cell lines. Single-cell clones were isolated, screened, and validated through sequencing and Inference of CRISPR Edits (ICE) analysis and qRT-PCR. RNA sequencing was performed on LYVE1 KO and wild-type (WT) cells to identify differentially expressed genes (DEGs). Bioinformatic analyses, including Gene Ontology (GO) enrichment and protein-protein interaction (PPI) network mapping, were conducted to explore affected pathways. Finally, network topology was examined using NetworkAnalyzer and cytoHubba plugins.

RESULTS: Transcriptomic analysis revealed significant down-regulation of pro-metastatic pathways, including epithelial-mesenchymal transition (EMT), extracellular matrix remodeling, and immune modulation. DEG analysis identified 263 genes, with key down-regulated targets such as WNT5A, TGFB2, and MMP2, and up-regulation of tumor-suppressive genes including PTGS2. GO and PPI analyses highlighted LYVE1's pivotal role in regulating cell adhesion, migration, and immune response.

CONCLUSION: LYVE1 KO reduces TSCC invasive potential by disrupting EMT and tumor-stroma interactions, aligning with previous experimental findings. These results suggest LYVE1 as a critical driver of metastasis, highlighting its potential as a therapeutic target.},
}

Humans
*Tongue Neoplasms/genetics/pathology/metabolism
*CRISPR-Cas Systems
*Transcriptome
*Vesicular Transport Proteins/genetics/metabolism
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition
Cell Line, Tumor
Gene Knockout Techniques
Neoplasm Metastasis
*Squamous Cell Carcinoma of Head and Neck/genetics

@article {pmid40571168,
year = {2025},
author = {Jeong, JH and Kim, SH and Kim, JY},
title = {Empowering Agrobacterium: Ternary vector systems as a new arsenal for plant transformation and genome editing.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108631},
doi = {10.1016/j.biotechadv.2025.108631},
pmid = {40571168},
issn = {1873-1899},
abstract = {The continuous evolution of plant transformation technologies is pivotal for accelerating genetic advancements in agriculture. Among these, ternary vector systems have emerged as a transformative innovation, significantly enhancing Agrobacterium-mediated plant transformation by overcoming critical biological barriers. Unlike traditional binary vectors, ternary vector systems incorporate accessory virulence genes and immune suppressors that overcome the intrinsic transformation barriers of recalcitrant crops. This has enabled 1.5- to 21.5-fold increases in stable transformation efficiency in species previously resistant to Agrobacterium-mediated transformation, such as maize, sorghum, and soybean, thereby expanding the effective host range of plant genetic engineering. Furthermore, the fusion of ternary vectors with advanced genome editing technologies like CRISPR/Cas is revolutionizing precision breeding, facilitating unprecedented control over genetic modifications. Future innovations are likely to focus on expanding the capabilities of ternary vectors, including transient delivery of morphogenic factors to enhance regeneration and organelle-targeted transformation for broader genetic modifications. Additionally, refining Agrobacterium engineering, such as developing auxotrophic strains for improved biosafety and optimizing secretion systems for enhanced protein delivery, presents exciting opportunities for further advancements. This review highlights the recent breakthroughs in ternary vector engineering, including its potential to optimize regeneration pathways via morphogenic factors and extend genetic transformation to previously unamenable plant species. By bridging the gap between transformation efficiency and targeted genome modifications, these advancements are reshaping the landscape of plant biotechnology, driving more resilient and high-performing crops in an era of global agricultural challenges.},
}

@article {pmid40569097,
year = {2025},
author = {Jenkins, K and Layton, D and Gough, T and O'Neil, T and Malaver Otega, L and Mishra, K and Bruce, K and Morris, K and Wise, T and Challagulla, A and Doran, T and Bean, A},
title = {Production of immune receptor knockout chickens via direct in vivo transfection of primordial germ cells.},
journal = {Animal biotechnology},
volume = {36},
number = {1},
pages = {2523027},
doi = {10.1080/10495398.2025.2523027},
pmid = {40569097},
issn = {1532-2378},
mesh = {Animals ; *Chickens/genetics ; *Receptor, Interferon alpha-beta/genetics ; *Gene Knockout Techniques/veterinary/methods ; *Transfection/veterinary/methods ; *Animals, Genetically Modified/genetics ; *Germ Cells ; *Receptors, Interleukin-1 Type I/genetics ; CRISPR-Cas Systems ; Chick Embryo ; Male ; Female ; },
abstract = {The advancement of genetic engineering in chickens has enabled significant advancement in developmental biology, bioreactors, and disease resilience. The development of CRISPR/Cas9 genome engineering technology has further expanded the potential applications of genetic engineering in poultry. In this study we aimed to evaluate the efficacy of a direct in vivo transfection method, previously demonstrated to produce transgenic chickens, in generating gene knockout (KO) chickens. Specifically, we targeted the Interferon-α/β Receptor 1 (IFNAR1) and Interleukin 1 receptor, type I (IL1R1), both critical pathways in the inflammatory and antiviral responses. We designed guide RNAs targeting the genes and validated their efficiency in vivo via microinjection into the developing embryos. PCR analysis confirmed the presence of gene deletions in chimeric roosters, which were subsequently bred to produce G1 germline heterozygote KO offspring. Homozygous KO chickens were generated and subjected to phenotypic and functional analyses. Our results demonstrated successful generation of functional knockouts of both IFNAR1 and IL1R1 using a direct in vivo transfection. Overall, this study demonstrates that direct in vivo transfection provides a robust and predictable method for generating KO chickens, facilitating further research into avian immune responses and the development of antiviral strategies.},
}

Animals
*Chickens/genetics
*Receptor, Interferon alpha-beta/genetics
*Gene Knockout Techniques/veterinary/methods
*Transfection/veterinary/methods
*Animals, Genetically Modified/genetics
*Germ Cells
*Receptors, Interleukin-1 Type I/genetics
CRISPR-Cas Systems
Chick Embryo
Male
Female

@article {pmid40568938,
year = {2025},
author = {Liu, X and Tan, H and Wang, J and Cao, Y and Li, P and Fan, X and Wang, Q and Zhang, H and Zhang, J and Yang, T and Zhao, G and Zhang, X and Duan, X and Zi, L and Liu, L and Ma, L and Chen, Z and Liang, L and Liu, R},
title = {SELECT: high-precision genome editing strategy via integration of CRISPR-Cas and DNA damage response for cross-species applications.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf595},
pmid = {40568938},
issn = {1362-4962},
support = {2023YFC3402300//National Key R&D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT23YG219//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Saccharomyces cerevisiae/genetics ; Escherichia coli/genetics ; *DNA Damage ; Promoter Regions, Genetic ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR-based methods enable genome modifications for diverse applications but often face challenges, such as inconsistent efficiencies, reduced performance in iterative modifications, and difficulties generating high-quality datasets for high-throughput genome engineering. Here, we present SELECT (SOS Enhanced programmabLE CRISPR-Cas ediTing), a novel strategy integrating the CRISPR-Cas system with the DNA damage response. By employing designed and optimized double-strand break induced promoters that are activated upon genome editing, SELECT enables a counter-selection process to eliminate unedited cells, ensuring high-fidelity editing. This approach achieves up to 100% efficiency for point mutations, iterative knockouts, and insertions. In high-throughput library editing, SELECT achieved up to 94.2% efficiency and preserved higher library diversity compared with conventional methods. Application of SELECT in flaviolin biosynthesis resulted in a 3.97-fold increase in production. Furthermore, integration with machine learning tools allowed rapid mapping of genotype-phenotype relationships. SELECT provides a versatile platform for precision genome engineering in Escherichia coli and Saccharomyces cerevisiae.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Saccharomyces cerevisiae/genetics
Escherichia coli/genetics
*DNA Damage
Promoter Regions, Genetic
DNA Breaks, Double-Stranded

@article {pmid40567005,
year = {2025},
author = {Zeng, Z and Wang, H and Luo, Y and Chen, W and Xu, M and Wei, H and Chen, Z and Xiang, T and Wang, L and Han, N and Huang, X and Bian, H},
title = {CRISPR/Cas9-mediated editing of barley lipoxygenase genes promotes grain fatty acid accumulation and storability.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {482-497},
doi = {10.1080/21645698.2025.2523069},
pmid = {40567005},
issn = {2164-5701},
mesh = {*Hordeum/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; Gene Editing ; *Lipoxygenase/genetics/metabolism ; *Fatty Acids/metabolism ; *Edible Grain/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Plant lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids, which can adversely affect grain storability. Although the genetic engineering of LOXs holds great potential for improving grain storage quality, this approach remains largely unexplored in barley. In this study, we identified five LOX genes in the barley genome: HvLOXA, HvLOXB, and HvLOXC1-3. HvLOXC1 exhibited the highest expression in early developing grains, roots, and shoots; HvLOXA was predominantly expressed in embryos, whereas HvLOXB and HvLOXC3 were weakly expressed across tissues. Transgene-free homozygous barley mutants of loxB, loxC1, and loxAloxC1 were generated using CRISPR/Cas9-mediated genome editing. Compared to the wild-type, all mutants displayed normal plant height, tiller number, and grain size, although the loxC1 and loxAloxC1 mutants exhibited significantly lower thousand grain weights. Notably, the total LOX activity in mature grains decreased by 36-42% in loxC1 mutants and by 94% in loxAloxC1 mutants, with no significant change observed in loxB mutants. Additionally, the loxAloxC1 double mutants had a significantly lower malondialdehyde content and accumulated 10-21% more fatty acids than the wild-type. Artificial aging treatment experiments revealed that loxAloxC1 mutants had enhanced grain storability, demonstrated by significantly higher germination rates, reduced lipid peroxidation, and improved seedling growth. Our findings highlight that the targeted knockout of LOX genes, particularly the double mutation of HvLOXA and HvLOXC1, represents a promising genetic strategy for improving grain storability and nutritional value in barley.},
}

*Hordeum/genetics/metabolism/enzymology
*CRISPR-Cas Systems
Gene Editing
*Lipoxygenase/genetics/metabolism
*Fatty Acids/metabolism
*Edible Grain/genetics/metabolism
Plants, Genetically Modified/genetics/metabolism
*Plant Proteins/genetics/metabolism
Seeds/genetics/metabolism
Gene Expression Regulation, Plant

@article {pmid40566742,
year = {2025},
author = {Ni, H and Kelley, K and Xie, N and Zou, H and Friedel, RH},
title = {Generation of Plexin-B1 Conditional Knockout Mouse With CRISPR/Cas9 Technology.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {3},
pages = {e70019},
doi = {10.1002/dvg.70019},
pmid = {40566742},
issn = {1526-968X},
support = {NS092735/NS/NINDS NIH HHS/United States ; NS134159/NS/NINDS NIH HHS/United States ; AG077828/AG/NIA NIH HHS/United States ; CA196521/CA/NCI NIH HHS/United States ; DOH01-C39068GG//New York State Department of Health/ ; DOH01-C38330GG//New York State Department of Health/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Nerve Tissue Proteins/genetics ; Alleles ; Gene Targeting ; *Receptors, Cell Surface/genetics ; Gene Editing/methods ; Female ; },
abstract = {Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles that enable conditional deletion are needed to facilitate cell type-specific functional analysis. We report here the generation of a conditional floxed allele of Plexin-B1 (gene symbol Plxnb1) in mouse using CRISPR/Cas9 technology to insert two loxP sites flanking critical exons. Targeting reagents (Cas9 protein, sgRNAs, ssODNs) were delivered into single-cell embryos by electroporation. After screening a total of 128 mouse pups by PCR and Sanger sequencing, two mice were identified carrying both loxP sites in the targeted Plxnb1 locus (success rate ~ 1.6%). The usage of Alt-R modified ssODNs increased targeting frequencies at one loxP site, but not the other. We also tested homology directed repair (HDR) enhancer V2 reagent, but addition of the enhancer reduced the viability of mouse embryos. The Plxnb1[flox] allele was successfully transmitted through the germline in Mendelian ratios, and effective excision of the floxed region was confirmed by breeding with Cre recombinase strains.},
}

Animals
Mice
*CRISPR-Cas Systems
Mice, Knockout
*Nerve Tissue Proteins/genetics
Alleles
Gene Targeting
*Receptors, Cell Surface/genetics
Gene Editing/methods
Female

@article {pmid40566685,
year = {2025},
author = {Liu, J and Zhang, R and Chai, N and Su, L and Zheng, Z and Liu, T and Guo, Z and Ma, Y and Xie, Y and Xie, X and Lin, Q and Chen, L and Liu, YG and Zhu, Q},
title = {Programmable genome engineering and gene modifications for plant biodesign.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101427},
doi = {10.1016/j.xplc.2025.101427},
pmid = {40566685},
issn = {2590-3462},
abstract = {Plant science has entered a transformative era with genome editing by enabling precise DNA alterations to address global challenges such as climate adaptability and food safety. These alterations are primarily driven by the integration of three modular components that can be activated or suppressed: DNA-targeting modules, effector modules, and control modules. The field has evolved from protein-centric systems (zinc finger nucleases and transcription activator-like effector nucleases) to RNA-focused platforms (CRISPR-Cas and other nucleases), which facilitate diverse control over genetic and epigenetic contexts. The modular design of DNA-targeting modules paired with effector domains, with or without inducible systems, provides scientists with superior precision in regulating transcription and altering chromatin states. The present review article examines these three modules and highlights various optimization methods. Additionally, it outlines innovative tools such as optogenetic systems and receptor-integrated systems that enable spatiotemporal control of genome editor expression. These modular instruments overcome traditional boundaries and allow scientists to create plants with favorable characteristics, decipher complex gene networks, and adopt sustainable farming practices.},
}

@article {pmid40565548,
year = {2025},
author = {Xavier, KVM and Silva, AMA and Luz, ACO and da Silva, FSC and de Melo, BST and Pitta, JLLP and Leal-Balbino, TC},
title = {Diversity and Role of Prophages in Pseudomonas aeruginosa: Resistance Genes and Bacterial Interactions.},
journal = {Genes},
volume = {16},
number = {6},
pages = {},
doi = {10.3390/genes16060656},
pmid = {40565548},
issn = {2073-4425},
mesh = {*Pseudomonas aeruginosa/genetics/virology/drug effects/pathogenicity ; *Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; Humans ; Pseudomonas Infections/microbiology ; Genome, Bacterial ; Brazil ; },
abstract = {Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, understanding phage-host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of prophages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hospitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, the presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Results: A total of 841 viral sequences were identified by the VIBRANT tool, of which 498 were confirmed by CheckV, with a predominance of the class Caudoviricetes and high overall phage diversity. No statistically significant difference was observed in the number of prophages between isolates with and without CRISPR/Cas systems. Prophages carrying resistance genes such as rsmA, OXA-56, SPM-1, and others were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage-host relationship. The dual role of prophages-as vectors of antimicrobial resistance and as potential therapeutic agents-reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance.},
}

*Pseudomonas aeruginosa/genetics/virology/drug effects/pathogenicity
*Prophages/genetics
CRISPR-Cas Systems/genetics
*Drug Resistance, Bacterial/genetics
Humans
Pseudomonas Infections/microbiology
Genome, Bacterial
Brazil

@article {pmid40565267,
year = {2025},
author = {Chakraborty, M and Nielsen, L and Nash, D and Bruder, MR and Nissimov, JI and Charles, TC and Aucoin, MG},
title = {Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations.},
journal = {International journal of molecular sciences},
volume = {26},
number = {12},
pages = {},
doi = {10.3390/ijms26125805},
pmid = {40565267},
issn = {1422-0067},
support = {RGPIN 355513-2017/RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Baculoviridae/genetics ; *Viral Envelope Proteins/genetics ; Genetic Vectors/genetics ; Genome, Viral ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Sf9 Cells ; Mutation ; },
abstract = {The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS.},
}

*CRISPR-Cas Systems
Animals
*Baculoviridae/genetics
*Viral Envelope Proteins/genetics
Genetic Vectors/genetics
Genome, Viral
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
Sf9 Cells
Mutation

@article {pmid40565111,
year = {2025},
author = {Bouchard, C and Rousseau, J and Lamothe, G and Dubost, M and Bastrenta, L and Ramezani, S and Tremblay, JP},
title = {In Vitro Correction of Point Mutations in the DYSF Gene Using Prime Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {12},
pages = {},
doi = {10.3390/ijms26125647},
pmid = {40565111},
issn = {1422-0067},
support = {TREM-01//Jain Foundation/ ; },
mesh = {Humans ; *Point Mutation ; *Gene Editing/methods ; HEK293 Cells ; *Dysferlin/genetics ; Genetic Therapy/methods ; Myoblasts/metabolism ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; },
abstract = {Dysferlinopathy is caused by over 500 mutations in the gene encoding dysferlin, including close to 300 point mutations. One option to cure the disease is to use a gene therapy to correct these mutations at the root. Prime editing is a technique which can replace the mutated nucleotide with the wild-type nucleotide. In this article, prime editing is used to correct several point mutations in the DYSF gene responsible for dysferlinopathy. In vitro editing of HEK293T cells reaches up to 31%. Notably, editing was more efficient in myoblasts than in patient-derived fibroblasts. The prime editing technique was also used to create a new myoblast clone containing a patient mutation from a healthy myoblast cell line.},
}

Humans
*Point Mutation
*Gene Editing/methods
HEK293 Cells
*Dysferlin/genetics
Genetic Therapy/methods
Myoblasts/metabolism
Fibroblasts/metabolism
CRISPR-Cas Systems

@article {pmid40521923,
year = {2025},
author = {Cao, Y and Huang, C and Li, K and Zhang, X and Tong, M and Tu, J and Yang, K and Yuan, S and Zhang, H},
title = {CRISPR/Cas12a-assisted electrochemiluminescent detection of ochratoxin A based on COF@Ru coupled with a DNA tetrahedral scaffold.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {25},
pages = {5298-5307},
doi = {10.1039/d5ay00427f},
pmid = {40521923},
issn = {1759-9679},
mesh = {*Ochratoxins/analysis ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; Gold/chemistry ; *DNA/chemistry ; Metal Nanoparticles/chemistry ; Aptamers, Nucleotide/chemistry ; DNA Probes/chemistry ; },
abstract = {To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2'-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)3[2+]/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL[-1] in a linear detection range of 10[-5]-100 ng mL[-1]. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.},
}

*Ochratoxins/analysis
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
*Biosensing Techniques/methods
*Luminescent Measurements/methods
*Metal-Organic Frameworks/chemistry
Limit of Detection
Gold/chemistry
*DNA/chemistry
Metal Nanoparticles/chemistry
Aptamers, Nucleotide/chemistry
DNA Probes/chemistry

@article {pmid40518166,
year = {2025},
author = {Ge, L and Xiong, H},
title = {[Advances in CRISPR-Cas9 genome editing for the treatment of muscular dystrophies].},
journal = {Zhonghua er ke za zhi = Chinese journal of pediatrics},
volume = {63},
number = {7},
pages = {808-811},
doi = {10.3760/cma.j.cn112140-20250421-00356},
pmid = {40518166},
issn = {0578-1310},
mesh = {Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; *Muscular Dystrophies/therapy/genetics ; Animals ; *Gene Editing/methods ; Muscular Dystrophy, Duchenne/therapy/genetics ; Disease Models, Animal ; },
}

Humans
*Genetic Therapy/methods
*CRISPR-Cas Systems
*Muscular Dystrophies/therapy/genetics
Animals
*Gene Editing/methods
Muscular Dystrophy, Duchenne/therapy/genetics
Disease Models, Animal

@article {pmid40501421,
year = {2025},
author = {Zheng, Y and Liu, B and Zuo, Q and Deng, F and Lan, C},
title = {Detection of Streptococcus anginosus in fecal samples using PCR-CRISPR /Cas12a system.},
journal = {Bioanalysis},
volume = {17},
number = {11},
pages = {737-745},
doi = {10.1080/17576180.2025.2518042},
pmid = {40501421},
issn = {1757-6199},
mesh = {*Feces/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *Streptococcus anginosus/genetics/isolation & purification ; DNA, Bacterial/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {OBJECTIVE: To develop a highly sensitive and specific detection method based on PCR-CRISPR/Cas12a for the detection of Streptococcus anginosus (S. anginosus) in feces and to evaluate its detection rate in the general population as well as its potential as a gastrointestinal tumor marker.

MATERIALS AND METHODS: Specific primers and crRNA targeting the 16S rDNA of S. anginosus were designed to construct a PCR-CRISPR/Cas12a detection system. A total of 230 fecal samples were collected from the general population, and bacterial DNA was extracted. The target gene was detected using this system to verify its sensitivity, specificity, and stability.

RESULTS: The established detection system demonstrated strong specificity, with stable recognition of S. anginosus, and a minimum detection limit of 10[-7] ng/μL. The detection rate of S. anginosus in fecal samples from the general population was 51.7%.

CONCLUSION: The PCR-CRISPR/Cas12a system can efficiently detect S. anginosus in feces, providing a reliable technical tool for exploring its association with gastrointestinal tumors.},
}

*Feces/microbiology
Humans
*CRISPR-Cas Systems/genetics
*Polymerase Chain Reaction/methods
*Streptococcus anginosus/genetics/isolation & purification
DNA, Bacterial/genetics
RNA, Ribosomal, 16S/genetics

@article {pmid40490360,
year = {2025},
author = {Kagawa, N and Umesono, Y and Suzuki, KT and Mochii, M},
title = {Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression.},
journal = {Development, growth & differentiation},
volume = {67},
number = {5},
pages = {293-302},
doi = {10.1111/dgd.70011},
pmid = {40490360},
issn = {1440-169X},
support = {//University of Hyogo/ ; JP18K06266//Japan Society for the Promotion of Science/ ; JP21H03829//Japan Society for the Promotion of Science/ ; 22NIBB331//NIBB Collaborative Research Program/ ; 23NIBB340//NIBB Collaborative Research Program/ ; JPMJCR 2025//JST, CREST/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {We established a novel knock-in technique, New and Easy Xenopus Targeted integration (NEXTi), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene (egfp) into the target 5' untranslated region (UTR) of the Xenopus laevis genome. It enables us to track eGFP expression under the regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L, myod1.S, sox2.L, and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5' UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection, and genotyping of F1 animals for the NEXTi procedure.},
}

Animals
*Xenopus laevis/genetics
*Gene Knock-In Techniques/methods
CRISPR-Cas Systems/genetics
Green Fluorescent Proteins/genetics/metabolism

@article {pmid39883121,
year = {2025},
author = {Paryani, M and Gupta, N and Jain, SK and Butani, S},
title = {Lowering LDL cholesterol by PCSK9 inhibition: a new era of gene silencing, RNA, and alternative therapies.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {398},
number = {6},
pages = {6597-6615},
pmid = {39883121},
issn = {1432-1912},
mesh = {Humans ; *Antibodies, Monoclonal/pharmacology/therapeutic use ; Cholesterol, LDL/blood ; CRISPR-Cas Systems ; Gene Silencing ; Hypercholesterolemia/drug therapy/economics ; *Oligonucleotides, Antisense/pharmacology/therapeutic use ; *Plant Extracts/pharmacology/therapeutic use ; Proprotein Convertase 9/genetics/metabolism ; *Proprotein Convertases/antagonists & inhibitors ; Protein Subunit Vaccines/pharmacology/therapeutic use ; *RNA, Small Interfering/pharmacology/therapeutic use ; *Anticholesteremic Agents/pharmacology/therapeutic use ; Fibronectin Type III Domain ; Recombinant Proteins ; },
abstract = {Proprotein convertase subtilisin/kexin type 9 (PCSK9) discovery has added a new paradigm to our understanding of cholesterol homeostasis and lipid metabolism. Since its discovery, PCSK9 inhibitors have become a widely investigated therapeutic class for lipid management in cardiovascular diseases and hypercholesterolemia. Scientists have explored different approaches for PCSK9 inhibition, such as monoclonal antibodies (mAbs), gene silencing and gene editing techniques, vaccines, mimetic peptides, and small molecules. European Medicines Agency (EMA) and United States Food and Drug Administration (US FDA) have approved only three PCSK9 inhibitors, including two monoclonal antibodies and one small interfering ribonucleic acid (siRNA). Despite the efficacy of approved large molecules, high costs and the need for regular injection have limited their adherence to the patient. This review aims to provide an understanding of PCSK9's function in Low-Density Lipoprotein Cholesterol (LDL-C) management, its current treatment, recent advancements, and potential future development of small molecules in the class of PCSK9 inhibitors.},
}

Humans
*Antibodies, Monoclonal/pharmacology/therapeutic use
Cholesterol, LDL/blood
CRISPR-Cas Systems
Gene Silencing
Hypercholesterolemia/drug therapy/economics
*Oligonucleotides, Antisense/pharmacology/therapeutic use
*Plant Extracts/pharmacology/therapeutic use
Proprotein Convertase 9/genetics/metabolism
*Proprotein Convertases/antagonists & inhibitors
Protein Subunit Vaccines/pharmacology/therapeutic use
*RNA, Small Interfering/pharmacology/therapeutic use
*Anticholesteremic Agents/pharmacology/therapeutic use
Fibronectin Type III Domain
Recombinant Proteins

@article {pmid30965672,
year = {2019},
author = {Song, HY and Chien, CS and Yarmishyn, AA and Chou, SJ and Yang, YP and Wang, ML and Wang, CY and Leu, HB and Yu, WC and Chang, YL and Chiou, SH},
title = {Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy.},
journal = {Cells},
volume = {8},
number = {4},
pages = {},
pmid = {30965672},
issn = {2073-4409},
mesh = {Apoptosis ; *Autophagy ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology ; Cell Line ; Exosomes ; Fabry Disease/*complications ; Gene Knockout Techniques ; Human Embryonic Stem Cells/metabolism ; Humans ; *Models, Biological ; Myocytes, Cardiac/metabolism ; Reactive Oxygen Species/metabolism ; Trihexosylceramides/metabolism ; alpha-Galactosidase/*genetics ; },
abstract = {Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.},
}

Apoptosis
*Autophagy
CRISPR-Cas Systems
Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology
Cell Line
Exosomes
Fabry Disease/*complications
Gene Knockout Techniques
Human Embryonic Stem Cells/metabolism
Humans
*Models, Biological
Myocytes, Cardiac/metabolism
Reactive Oxygen Species/metabolism
Trihexosylceramides/metabolism
alpha-Galactosidase/*genetics

@article {pmid28565744,
year = {2017},
author = {Cheng, LH and Liu, Y and Niu, T},
title = {[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {38},
number = {5},
pages = {427-431},
pmid = {28565744},
issn = {0253-2727},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosome Deletion ; DNA ; Gene Deletion ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; Multigene Family ; Mutagenesis, Insertional ; NIH 3T3 Cells ; Plasmids ; },
abstract = {Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.},
}

Animals
*CRISPR-Cas Systems
Chromosome Deletion
DNA
Gene Deletion
*Gene Editing
HEK293 Cells
Humans
Mice
Multigene Family
Mutagenesis, Insertional
NIH 3T3 Cells
Plasmids

@article {pmid28549865,
year = {2017},
author = {Powell, SK and Gregory, J and Akbarian, S and Brennand, KJ},
title = {Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease.},
journal = {Molecular and cellular neurosciences},
volume = {82},
number = {},
pages = {157-166},
pmid = {28549865},
issn = {1095-9327},
support = {P50 MH096890/MH/NIMH NIH HHS/United States ; R01 MH101454/MH/NIMH NIH HHS/United States ; R01 MH106056/MH/NIMH NIH HHS/United States ; },
mesh = {Brain/*growth & development ; Brain Diseases/therapy ; CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Gene Expression/*genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology ; },
abstract = {CRISPR/Cas9 technology has transformed our ability to manipulate the genome and epigenome, from efficient genomic editing to targeted localization of effectors to specific loci. Through the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators, the role of gene-regulatory and epigenetic pathways in basic biology and disease processes can be directly queried. Here, we discuss emerging CRISPR-based methodologies, with specific consideration of neurobiological applications of human induced pluripotent stem cell (hiPSC)-based models.},
}

Brain/*growth & development
Brain Diseases/therapy
CRISPR-Cas Systems/*genetics
*Gene Editing/methods
Gene Expression/*genetics
Humans
Induced Pluripotent Stem Cells/*cytology

@article {pmid28549739,
year = {2017},
author = {Deng, H and Gao, R and Liao, X and Cai, Y},
title = {Characterization of a major facilitator superfamily transporter in Shiraia bambusicola.},
journal = {Research in microbiology},
volume = {168},
number = {7},
pages = {664-672},
doi = {10.1016/j.resmic.2017.05.002},
pmid = {28549739},
issn = {1769-7123},
mesh = {Ascomycota/chemistry/drug effects/*genetics/pathogenicity ; CRISPR-Cas Systems ; Membrane Transport Proteins/*genetics/*metabolism ; Mutation ; Perylene/*analogs & derivatives/metabolism/pharmacology ; Phenol ; Quinones/*metabolism/pharmacology ; Reactive Oxygen Species/metabolism ; Sasa/microbiology ; },
abstract = {Reactive oxygen species (ROS) generated by photo-activated hypocrellin from Shiraia bambusicola are detrimental to cellular macromolecules. However, S. bambusicola can still maintain excellent morphology during continuous hypocrellin production, indicating an extraordinary autoresistance system that protects against the harmful ROS. In this study, a major facilitator superfamily transporter (MFS) was isolated from S. bambusicola and deleted using the clustered regularly interspaced short palindromic repeat sequences (CRISPR)/Cas9 system. The ΔMFS mutant abolished hypocrellin production and was slightly sensitive to 40-μM hypocrellin, while the ΔMFS compliment strain restored hypocrellin production and resistance. Hypocrellin treatment also enhanced the relative expression of MFS in wild-type S. bambusicola. Subsequent pathogenicity assays showed that MFS deletion reduced damage to bamboo leaves. By contrast, restoration of hypocrellin production in the MFS compliment strain generated similar necrotic lesions on bamboo leaves to those observed with the wild-type strain. These results revealed that the identified MFS is involved in efflux of hypocrellin from cells, which reduces the hypocrellin toxicity. Furthermore, hypocrellin contributed to the virulence of S. bambusicola on bamboo leaves. These findings could help to reduce plant loss by disrupting hypocrellin biosynthesis in S. bambusicola, or overexpressing the associated resistance gene in transgenic plants.},
}

Ascomycota/chemistry/drug effects/*genetics/pathogenicity
CRISPR-Cas Systems
Membrane Transport Proteins/*genetics/*metabolism
Mutation
Perylene/*analogs & derivatives/metabolism/pharmacology
Phenol
Quinones/*metabolism/pharmacology
Reactive Oxygen Species/metabolism
Sasa/microbiology

@article {pmid40562814,
year = {2025},
author = {Qiao, JH and Zang, Y and Gao, Q and Liu, S and Zhang, XW and Hu, W and Wang, Y and Han, C and Li, D and Wang, XB},
title = {Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {40562814},
issn = {2055-0278},
support = {32425046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas genome editing technology is a cutting-edge strategy for crop breeding. However, the delivery of genome-editing reagents remains to be a technological bottleneck in monocot plants[1]. Here we engineered barley yellow striate mosaic virus (BYSMV) into a negative-strand RNA virus-based vector system[2] for delivery of both Cas9 and single guide RNA to achieve heritable gene editing in different wheat cultivars. We found that fusion of a mobile transfer RNA sequence[3] to the Cas9 messenger RNA and single guide RNAs could deliver them into the growth points of axillary meristems to achieve gene editing before tiller generation. The resulting nascent tillers contained simultaneous mutations in the three homoeoalleles. Moreover, the progeny seedlings are virus-free and harbour bi-allelic or homozygous mutations. Given BYSMV infects 26 monocot species[4], the BYSMV delivery system could have wide applicability for achieving highly efficient, non-transgenic and less genotype-dependent heritable genome editing, thereby facilitating genomic studies and crops breeding.},
}

@article {pmid40560955,
year = {2025},
author = {Leprince, A and Moineau, S},
title = {CRISPR-Cas in the Cheese Industry.},
journal = {Annual review of virology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-virology-092623-111016},
pmid = {40560955},
issn = {2327-0578},
abstract = {Bacteria have evolved a wide range of defense systems to combat phage infections. In the cheese industry, lactic acid bacteria (LAB) used for milk fermentation continuously face threats from phages. Therefore, selecting or developing industrial strains with enhanced phage resistance requires a focus on robust defense systems. Among these systems, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are notably prevalent in LAB. The early characterization of this adaptive immune system was closely tied to the cheese industry, particularly with Streptococcus thermophilus in which CRISPR-Cas systems are ubiquitous and highly active. This review underscores the contributions of S. thermophilus and its virulent phages to our understanding of the function and mechanisms of CRISPR-Cas systems. Additionally, we review the diversity of CRISPR-Cas systems in LAB used in the cheese industry, the counter-defense strategies employed by dairy phages, and the applications of CRISPR-Cas systems within this sector.},
}

@article {pmid40560862,
year = {2025},
author = {Nakamura, K and Ito, S and Ohguchi, Y and Jimbo, T and Wada, Y and Nakajima, R and Kanou, M and Yamana, K and Ueda, H},
title = {Establishment of Cre/LoxP-mediated multifunctional reporter knock-in rats with the CRISPR system.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325444},
doi = {10.1371/journal.pone.0325444},
pmid = {40560862},
issn = {1932-6203},
mesh = {Animals ; Rats ; *Integrases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Genes, Reporter ; Rats, Transgenic ; Male ; },
abstract = {Rats and mice are essential experimental animals in preclinical research, serving as models for various human diseases and contributing significantly to drug development. Rats offer distinct advantages over mice due to their larger size, which allows for more complex surgical procedures, repeated blood sampling, or sophisticated behavioral analysis. However, unlike the case with mice, genetically modified rat lines for achieving complex experimental objectives-such as tissue-specific gene knockout or visualization of specific cell populations-are still limited. We here established LoxP-mediated multifunctional reporter KI rats, enabling us to evaluate fluorescence, bioluminescence, and cell-killing assays simultaneously with only one gene-modified rat line. CRISPR/Cas12a, also known as CRISPR/Cpf1, was successfully used to insert the Cre sequence into a target locus to generate Cre driver rats. These results will contribute to the application of gene-modified rats for a more comprehensive understanding of physiology, and for extrapolation of their capabilities in preclinical research.},
}

Animals
Rats
*Integrases/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Genes, Reporter
Rats, Transgenic
Male

@article {pmid40560492,
year = {2025},
author = {Lv, D and Xu, Y and Wang, ZX and Zhang, QL and Yan, JP and Xu, JW},
title = {CRISPR/Cas9-mediated genome editing in Ganoderma lucidum: recent advances and biotechnological opportunities.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {7},
pages = {223},
pmid = {40560492},
issn = {1573-0972},
support = {32360014//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Reishi/genetics/metabolism ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal ; },
abstract = {Ganoderma lucidum is a well-known traditional medicinal mushroom that has attracted considerable attention due to its potential as a promising cell factory for producing high-value bioactive compounds. However, conventional methods for the genetic manipulation of G. lucidum are often time-consuming and labor-intensive, hindering research into the biosynthesis and regulatory mechanisms of its valuable natural products. In recent years, the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system has emerged as a powerful genome editing tool, extensively utilized in life sciences research due to its high efficiency and user-friendliness. This review provides a structured overview of advancements in CRISPR/Cas9-mediated genome editing technology in G. lucidum. We first discuss the development and optimization of the CRISPR system, focusing on the various expression strategies for Cas9 and guide RNA established in G. lucidum. Furthermore, we highlight the application of this system for targeted gene deletion, insertion, and replacement in genome editing, as well as its use in the functional analysis of genes in G. lucidum. In addition, we discuss the limitations and challenges associated with employing CRISPR/Cas9 tools in G. lucidum and provide an outlook on the future development of the CRISPR/Cas9 system and its applications in this organism.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Reishi/genetics/metabolism
*Biotechnology/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Fungal

@article {pmid40560319,
year = {2025},
author = {Peer, LA and Mir, BA},
title = {Molecular mechanisms and genetic regulation of self-incompatibility in flowering plants: implications for crop improvement and evolutionary biology.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {76},
pmid = {40560319},
issn = {1573-5028},
mesh = {*Self-Incompatibility in Flowering Plants/genetics ; *Crops, Agricultural/genetics ; Biological Evolution ; *Gene Expression Regulation, Plant ; Evolution, Molecular ; Pollination/genetics ; Self-Fertilization/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Self-incompatibility is a fundamental biological mechanism in flowering plants that prevents self-fertilization, thereby promoting outcrossing and enhancing genetic diversity. This complex system has independently evolved across multiple angiosperm lineages and is crucial in maintaining plant reproductive success. Recent research has expanded our understanding of self-incompatibility's molecular basis and uncovered key genes and signaling pathways involved in self-incompatibility responses, such as S-RNase in Solanaceae and PrsS-PrpS in Papaveraceae, as well as the SRK-SCR interaction in Brassicaceae. However, despite significant advances, many aspects of self-incompatibility, such as the interplay between gene duplications, polyploidization, and the evolution of novel self-incompatibility mechanisms, remain underexplored. This review integrates findings from various plant families, including Solanaceae, Rosaceae, Papaveraceae, and Brassicaceae, and discusses the evolutionary dynamics of self-incompatibility systems, highlighting the role of gene duplication, recombination, and translocation events in shaping self-incompatibility diversity. Special emphasis is placed on understanding how modern molecular techniques, such as CRISPR/Cas9 and marker-assisted selection, can be employed to transition self-incompatibility to self-compatibility in economically significant crops. Additionally, the role of epigenetic changes and modifier genes in mediating transitions from self-incompatibility to self-compatibility is addressed, offering insights into how these mechanisms can be leveraged for crop breeding and hybrid seed production. Future research should focus on elucidating the molecular mechanisms underlying self-incompatibility responses, exploring the potential of targeted gene editing to overcome reproductive barriers, and understanding the evolutionary resilience of self-incompatibility systems to environmental changes.},
}

*Self-Incompatibility in Flowering Plants/genetics
*Crops, Agricultural/genetics
Biological Evolution
*Gene Expression Regulation, Plant
Evolution, Molecular
Pollination/genetics
Self-Fertilization/genetics
Plant Proteins/genetics/metabolism

@article {pmid40559612,
year = {2025},
author = {Jurić, I and Jelić, M and Markanović, M and Kanižaj, L and Bošnjak, Z and Budimir, A and Kuliš, T and Tambić-Andrašević, A and Ivančić-Baće, I and Mareković, I},
title = {CRISPR-Cas Dynamics in Carbapenem-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Clinical Isolates from a Croatian Tertiary Hospital.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {6},
pages = {},
doi = {10.3390/pathogens14060604},
pmid = {40559612},
issn = {2076-0817},
support = {10106-24-1295 to I.M. and 20286539 to I.I.B.//School od Medicine, University of Zagreb and Faculty of Science, University of Zagreb/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification/classification ; Humans ; *CRISPR-Cas Systems/genetics ; *Carbapenems/pharmacology ; *Klebsiella Infections/microbiology ; beta-Lactamases/genetics ; Croatia ; Tertiary Care Centers ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Bacterial Proteins/genetics ; Multilocus Sequence Typing ; Drug Resistance, Multiple, Bacterial/genetics ; *Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects/isolation & purification ; },
abstract = {(1) Background: CRISPR-Cas systems provide adaptive immunity against mobile genetic elements (MGEs) carrying antimicrobial resistance (AMR) genes. Carbapenem-resistant (CR) Klebsiella pneumoniae is a major public health concern, and the role of CRISPR-Cas in its resistance is understudied. This study explored CRISPR-Cas associations with multidrug resistance in clinical K. pneumoniae. (2) Methods: 400 K. pneumoniae isolates (200 CR and 200 carbapenem susceptible (CS)) were analyzed. Carbapenemase genes (blaOXA-48, blaNDM-1, blaKPC-2), cas1, rpoB, and CRISPR1-3 loci were identified by PCR, while only CRISPR loci were sequenced. Genetic relatedness was assessed via PFGE, MLST, and spacer analysis. Statistical analysis utilized chi-squared and Fisher's exact tests. (3) Results: CRISPR-Cas was present in 15.8% of isolates, mainly subtypes I-E and I-E* (93.3%), with CRISPR3 loci showing the greatest spacer diversity. Clonal complexes ST14/15/101 (CR) and ST35 (CS) were identified. blaOXA-48 was linked to CRISPR-Cas-negative strains, while blaNDM-1 and blaKPC-2 were more frequent in CRISPR-Cas-positive strains (p < 0.0001). Imipenem/relebactam resistance was higher in CRISPR-Cas-negative isolates. (4) Conclusions: K. pneumoniae CRISPR-Cas systems correlate with specific carbapenemase profiles, suggesting pressure against blaOXA-48 acquisition. The coexistence of I-E and I-E* subtypes highlight synergies in targeting MGEs. CRISPR loci could be tools for subtyping organisms following MLST.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification/classification
Humans
*CRISPR-Cas Systems/genetics
*Carbapenems/pharmacology
*Klebsiella Infections/microbiology
beta-Lactamases/genetics
Croatia
Tertiary Care Centers
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Bacterial Proteins/genetics
Multilocus Sequence Typing
Drug Resistance, Multiple, Bacterial/genetics
*Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects/isolation & purification

@article {pmid40558493,
year = {2025},
author = {Parikh, SJ and Terron, HM and Burgard, LA and Butler, DD and LaFerla, FM and Lane, S and Leissring, MA},
title = {5' DREDGE: Direct Repeat-Enabled Downregulation of Gene Expression via the 5' UTR of Target Genes.},
journal = {Cells},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/cells14120866},
pmid = {40558493},
issn = {2073-4409},
support = {1R01AG066928-04A1/NH/NIH HHS/United States ; },
mesh = {*5' Untranslated Regions/genetics ; *Down-Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; },
abstract = {Despite the availability of numerous methods for controlling gene expression, there remains a strong need for technologies that maximize two key properties: selectivity and reversibility. To this end, we developed a novel approach that exploits the highly sequence-specific nature of CRISPR-associated endoribonucleases (Cas RNases), which recognize and cleave short RNA sequences known as direct repeats (DRs). In this approach, referred to as DREDGE (direct repeat-enabled downregulation of gene expression), selective control of gene expression is enabled by introducing one or more DRs into the untranslated regions (UTRs) of target mRNAs, which can then be cleaved upon expression of the cognate Cas RNase. We previously demonstrated that the expression of target genes with DRs in their 3' UTRs are efficiently controlled by the DNase-dead version of Cas12a (dCas12a) with a high degree of selectivity and complete reversibility. Here, we assess the feasibility of using DREDGE to regulate the expression of genes with DRs inserted in their 5' UTRs. Among the five different Cas RNases tested, Csy4 was found to be the most efficient in this context, yielding robust downregulation with rapid onset in doxycycline-regulatable systems targeting either a stably expressed fluorescent protein or an endogenous gene, both in a fully reversible manner. Unexpectedly, dCas12a was also found to be modestly effective despite binding essentially irreversibly to the cut mRNA on its 5' end and thereby boosting mRNA levels. Our results expand the utility of DREDGE as an attractive method for regulating gene expression in a targeted, highly selective, and fully reversible manner.},
}

*5' Untranslated Regions/genetics
*Down-Regulation/genetics
Humans
CRISPR-Cas Systems/genetics
RNA, Messenger/genetics/metabolism

@article {pmid40558461,
year = {2025},
author = {Jeddoub, O and Touil, N and Nyabi, O and El Fahime, E and Ennibi, K and Gala, JL and Benjouad, A and Belayachi, L},
title = {The Use of CRISPR-Cas Systems for Viral Detection: A Bibliometric Analysis and Systematic Review.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060379},
pmid = {40558461},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Bibliometrics ; Humans ; *Viruses/isolation & purification/genetics ; *Virus Diseases/diagnosis ; },
abstract = {Viral infections impose a significant burden on global public health and the economy. This study examines the current state of CRISPR-Cas system research, focusing on their applications in viral detection and their evolution over recent years. A bibliometric analysis and systematic review were conducted using articles published between 2019 and 2024, retrieved from Web of Science, Scopus, and PubMed databases. Out of 2713 identified articles, 194 were included in the analysis. The findings reveal substantial growth in scientific output related to CRISPR-Cas systems, with the United States leading in research and development in this field. The rapid increase in CRISPR-Cas research during this period underscores its immense potential to transform viral diagnostics. With advantages such as speed, precision, and suitability for deployment in resource-limited settings, CRISPR-Cas systems outperform many traditional diagnostic methods. The concerted efforts of scientists worldwide further highlight the promising future of this technology. CRISPR-Cas systems are emerging as a powerful alternative, offering the possibility of expedited and accessible point-of-care testing and paving the way for more equitable and effective diagnostics on a global scale.},
}

*CRISPR-Cas Systems
Bibliometrics
Humans
*Viruses/isolation & purification/genetics
*Virus Diseases/diagnosis

@article {pmid40558442,
year = {2025},
author = {Du, K and Zeng, Q and Jiang, M and Hu, Z and Zhou, M and Xia, K},
title = {CRISPR/Cas12a-Based Biosensing: Advances in Mechanisms and Applications for Nucleic Acid Detection.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060360},
pmid = {40558442},
issn = {2079-6374},
support = {No. 82330035//National Natural Science Foundation of China/ ; No. 82130043//National Natural Science Foundation of China/ ; No. 82361138573//National Natural Science Foundation of China/ ; No. 2021SK1010//National Natural Science Foundation of Hunan Province/ ; No. 2024JJ5340//Natural Science Foundation of Hunan Province/ ; No. 2024RC3207//Science and Technology innovation Program of Hunan Province/ ; No. 23B0437//Scientific Research Fund of Hunan Provincial Education Department/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acids/analysis ; DNA/analysis ; },
abstract = {Nucleic acid detection technology is crucial for molecular diagnosis. The advent of CRISPR/Cas12a-based nucleic acid detection has considerably broadened its scope, from the identification of infectious disease-causing microorganisms to the detection of disease-associated biomarkers. This innovative system capitalizes on the non-specific single-strand cleavage activity of Cas12a upon target DNA recognition. By employing a fluorescent probe in the form of a single-stranded DNA/RNA, this technology enables the observation of fluorescence changes resulting from nonspecific cleavage, thereby facilitating detection. CRISPR/Cas12a-based detection systems can be regarded as a new type of biosensor, offering a practical and efficient approach for nucleic acid analysis in various diagnostic settings. CRISPR/Cas12a-based biosensors outperform conventional nucleic acid detection methods in terms of portability, simplicity, speed, and efficiency. In this review, we elucidate the detection principle of CRISPR/Cas12a-based biosensors and their application in disease diagnostics and discuss recent innovations and technological challenges, aiming to provide insights for the research and further development of CRISPR/Cas12a-based biosensors in personalized medicine. Our findings show that although CRISPR/Cas12a-based biosensors have considerable potential for various applications and theoretical research, certain challenges remain. These include simplifying the reaction process, enhancing precision, broadening the scope of disease detection, and facilitating the translation of research findings into clinical practice. We anticipate that ongoing advancements in CRISPR/Cas12a-based biosensors will address these challenges.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
*Nucleic Acids/analysis
DNA/analysis

@article {pmid40558428,
year = {2025},
author = {De Silva, PIT and Hiniduma, K and Canete, R and Bhalerao, KS and Shawky, SM and Gunathilaka, H and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Diniz, BS and Rusling, JF},
title = {Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060346},
pmid = {40558428},
issn = {2079-6374},
support = {2231490//U.S. National Science Foundation/ ; },
mesh = {*MicroRNAs/blood/analysis ; Humans ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer's disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls.},
}

*MicroRNAs/blood/analysis
Humans
*Biosensing Techniques
*CRISPR-Cas Systems
Limit of Detection

@article {pmid40558037,
year = {2025},
author = {Taran, JA and Mintaev, RR and Glazkova, DV and Belugin, BV and Bogoslovskaya, EV and Shipulin, GA},
title = {[Influence of Homology Arm Length and Structure on the Efficiency of Long Transgene Integration into a Cleavage Site Induced by SpCas9 or AsCpf1].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {2},
pages = {255-265},
pmid = {40558037},
issn = {0026-8984},
mesh = {Humans ; *Receptors, CCR5/genetics/metabolism ; Dependovirus/genetics ; *CRISPR-Cas Systems ; *Transgenes ; Green Fluorescent Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genetic Vectors/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {One of the promising new approaches to the treatment of HIV infection is CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and differed significantly from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite the lower efficiency of CCR5 knockout. The maximum percentage of cells with the integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.},
}

Humans
*Receptors, CCR5/genetics/metabolism
Dependovirus/genetics
*CRISPR-Cas Systems
*Transgenes
Green Fluorescent Proteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
Genetic Vectors/genetics
*Bacterial Proteins/genetics/metabolism

@article {pmid40558035,
year = {2025},
author = {Mintaev, RR and Glazkova, DV and Taran, JA and Bogoslovskaya, EV and Shipulin, GA},
title = {[Improving the Efficiency and Safety of Human CCR5 Gene Editing by Selection of Optimal Guide RNAs for SpCAS9 and CAS12A].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {2},
pages = {234-243},
pmid = {40558035},
issn = {0026-8984},
mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *HIV Infections/genetics/therapy ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9- HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60-72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.},
}

*Receptors, CCR5/genetics/metabolism
Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
*HIV Infections/genetics/therapy
*CRISPR-Associated Proteins/genetics/metabolism
HEK293 Cells
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism