@article {pmid40615444,
year = {2025},
author = {Novotná, M and Tinti, M and Faria, JRC and Horn, D},
title = {Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6194},
pmid = {40615444},
issn = {2041-1723},
support = {217105/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {*Histones/genetics/metabolism ; *Trypanosoma brucei brucei/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; RNA Polymerase II/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems ; *Gene Expression Regulation ; Gene Editing ; Mutation ; Acetylation ; Lysine/metabolism ; Protein Processing, Post-Translational ; Methylation ; Gene Expression Profiling ; Chromatin/metabolism ; },
abstract = {Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting "hist[one]H4" strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4[lys10] mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4[lys4] or H4[lys14] mutants. Proteomic and transcriptomic analysis of H4[lys4] glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.},
}

*Histones/genetics/metabolism
*Trypanosoma brucei brucei/genetics/metabolism
Protozoan Proteins/genetics/metabolism
RNA Polymerase II/metabolism/genetics
Promoter Regions, Genetic/genetics
CRISPR-Cas Systems
*Gene Expression Regulation
Gene Editing
Mutation
Acetylation
Lysine/metabolism
Protein Processing, Post-Translational
Methylation
Gene Expression Profiling
Chromatin/metabolism

@article {pmid40615387,
year = {2025},
author = {Lou, S and DJiake Tihagam, R and Wasko, UN and Equbal, Z and Venkatesan, S and Braczyk, K and Przanowski, P and Il Koo, B and Saltani, I and Singh, AT and Likhite, S and Powers, S and Souza, GMPR and Maxwell, RA and Yu, J and Zhu, LJ and Beenhakker, M and Abbott, SBG and Lu, Z and Green, MR and Meyer, KC and Tushir-Singh, J and Bhatnagar, S},
title = {Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6169},
pmid = {40615387},
issn = {2041-1723},
mesh = {*Rett Syndrome/genetics/therapy ; *MicroRNAs/genetics/metabolism ; *X Chromosome Inactivation/genetics ; Animals ; Female ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Humans ; Mice ; RNA, Long Noncoding/genetics/metabolism ; Phenotype ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; Male ; },
abstract = {X chromosome inactivation (XCI) is induced by Xist long non-coding RNA and protein-coding genes. However, the role of small non-coding RNA function in XCI remains unidentified. Our genome-wide, loss-of-function CRISPR/Cas9 screen in female fibroblasts identified microRNAs (miRNAs) as regulators of XCI. A striking finding is the identification of miR106a among the top candidates from the screen. Loss of miR106a is accompanied by altered Xist interactome, leading to dissociation and destabilization of Xist. XCI interference via miR106a inhibition has therapeutic implications for Rett syndrome (RTT) girls with a defective X-linked MECP2 gene. Here, we discovered that the inhibition of miR106a significantly improves several facets of RTT pathology: it increases the life span, enhances locomotor activity and exploratory behavior, and diminishes breathing variabilities. Our results suggest that miR106a targeting offers a feasible therapeutic strategy for RTT and other monogenic X-linked neurodevelopmental disorders.},
}

*Rett Syndrome/genetics/therapy
*MicroRNAs/genetics/metabolism
*X Chromosome Inactivation/genetics
Animals
Female
Methyl-CpG-Binding Protein 2/genetics/metabolism
Humans
Mice
RNA, Long Noncoding/genetics/metabolism
Phenotype
Fibroblasts/metabolism
CRISPR-Cas Systems
Disease Models, Animal
Male

@article {pmid40613815,
year = {2025},
author = {Pinto, D and Mendes, I and Cunha, MV},
title = {Genomic Survey Reveals no Detectable Bacteriophage Activity in Mycobacterium bovis Across a Large Population.},
journal = {FEMS microbiology ecology},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsec/fiaf072},
pmid = {40613815},
issn = {1574-6941},
abstract = {Phages are major drivers of bacterial evolution, yet their ecological and evolutionary interactions with Mycobacterium bovis, a key member of the Mycobacterium tuberculosis complex (MTBC), remain understudied. In this work, we investigate the elusive phage-bacterium interface in M. bovis by integrating comparative genomics of 200 isolates from infected animals with molecular analyses of M. bovis-positive environmental samples. Despite employing diverse and complementary approaches, we found no evidence of active or recent phage infections: no novel prophages beyond the conserved phiRv1, no expansion of CRISPR arrays, and no co-occurrence of M. bovis and mycobacteriophages in host tissues or environmental matrices. Intriguingly, we identified multiple independent excision events of phiRv1 across closely related lineages, suggesting recent prophage mobilization driven by unidentified ecological or genomic triggers. These findings echo previous observations in M. tuberculosis and point toward a stable, phage-scarce landscape across MTBC members. Our results raise compelling questions about the barriers to phage predation in M. bovis, the functionality of its CRISPR-Cas system, and the selective pressures underlying prophage retention and loss. By shedding light on these underexplored dynamics, our study reveals critical gaps in the ecological understanding of M. bovis and highlights opportunities for phage-based innovation in TB control.},
}

@article {pmid40613714,
year = {2025},
author = {Wu, D and Snead, S and Ganguly, C and Babu, K and Li, Y and Kathiresan, V and Shen, R and Zhang, X and Rajan, R and Qin, PZ},
title = {Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf557},
pmid = {40613714},
issn = {1362-4962},
support = {MCB-1716744//National Science Foundation/ ; MCB-1818107//National Science Foundation/ ; MCB-1716423//National Science Foundation/ ; MCB-2424888//National Science Foundation/ ; R35-GM145341/NH/NIH HHS/United States ; //Arnold and Mabel Beckman Foundation/ ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Substrate Specificity ; CRISPR-Cas Systems ; Models, Molecular ; Protein Binding ; DNA Cleavage ; Protein Conformation ; },
abstract = {CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix-loop-helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH's conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9-sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure-dynamic-function relationship governing DNA target discrimination by Cas9.},
}

*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
DNA/metabolism/chemistry/genetics
Substrate Specificity
CRISPR-Cas Systems
Models, Molecular
Protein Binding
DNA Cleavage
Protein Conformation

@article {pmid40613710,
year = {2025},
author = {Hibshman, GN and Taylor, DW},
title = {Structural basis of a dual-function type II-B CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf585},
pmid = {40613710},
issn = {1362-4962},
support = {R35GM138348/NH/NIH HHS/United States ; F-1938//Welch Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *Francisella/genetics/enzymology ; Gene Editing ; *Bacterial Proteins/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; DNA/genetics/chemistry/metabolism ; DNA Cleavage ; },
abstract = {Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
*Francisella/genetics/enzymology
Gene Editing
*Bacterial Proteins/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Streptococcus pyogenes/genetics/enzymology
DNA/genetics/chemistry/metabolism
DNA Cleavage

@article {pmid40613705,
year = {2025},
author = {Schwartz, CI and Abell, NS and Li, A and Aradhana, and Tycko, J and Truong, A and Montgomery, SB and Hess, GT},
title = {Toward optimizing diversifying base editors for high-throughput mutational scanning studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf620},
pmid = {40613705},
issn = {1362-4962},
support = {R35GM150462/NH/NIH HHS/United States ; NIH-1F99DK126120-01/NH/NIH HHS/United States ; NIH-4K00DK126120-03/NH/NIH HHS/United States ; S10RR025518-01/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; DNA Mutational Analysis/methods ; AICDA (Activation-Induced Cytidine Deaminase) ; },
abstract = {Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems
Cytidine Deaminase/genetics/metabolism
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
Point Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
Mutation
DNA Mutational Analysis/methods
AICDA (Activation-Induced Cytidine Deaminase)

@article {pmid40613203,
year = {2025},
author = {Criollo Delgado, L and Zamalutdinov, A and Potokina, E},
title = {Identification of Soybean E1-E4 Gene Orthologs in the Guar Genome Using Comprehensive Transcriptome Assembly and Annotation.},
journal = {Frontiers in bioscience (Scholar edition)},
volume = {17},
number = {2},
pages = {26548},
doi = {10.31083/FBS26548},
pmid = {40613203},
issn = {1945-0524},
support = {24-26-00073//Russian Science Foundation/ ; },
mesh = {*Glycine max/genetics ; *Cyamopsis/genetics ; *Transcriptome/genetics ; *Genome, Plant ; Molecular Sequence Annotation ; Photoperiod ; Gene Expression Regulation, Plant ; Plant Proteins/genetics ; *Genes, Plant ; Gene Expression Profiling ; Galactans ; Mannans ; Plant Gums ; },
abstract = {BACKGROUND: We publish the first available transcriptome assembly of guar (Cyamopsis tetragonoloba (L.) Taub.), a well-known source of guar gum (food additive E 412). At high latitudes, e.g., in Russia, the main challenge for guar cultivation is the long photoperiod during summer, which delays flowering and maturation of guar plants. Meanwhile, identifying of genes affecting the photoperiod sensitivity of guar would have a major impact on the development of marker-assisted breeding of this valuable food crop.

METHODS: RNA isolated from leaves of early and late flowering guar plants grown under long-day conditions were used to generate de novo transcriptome assembly. A similarity search was conducted using BLASTN 2.2.31+ with default settings to identify homologous sequences of soybean maturity genes E1-E4 in guar transcriptome and genome assembly. Gene prediction tools such as AUGUSTUS and FGENESH+ were used to predict the exon-intron structure of the candidate genes. Functional annotation of the amino acid sequence was performed using InterProScan v. 5.68-100.

RESULTS: The transcriptome assembly contained sequences of 96,447 clustered transcript isoforms in the leaves of guar plants grown under long-day conditions. The transcriptome assembly was annotated using BLAST against the Glycine max genome, and 42,615 guar transcripts (44.2%) were found to be similar to soybean genes. We used the developed transcriptome assembly to discover orthologs of the E1-E4 soybean loci in the guar genome that have the greatest impact on the flowering and maturation of this closely related, short-day legume crop. A high level of identity was detected between peptide sequences encoding by orthologous genes E1 and CtE1 (80%), E2 and CtE2 (93%), E3 and CtE3 (83%), and E4 and CtE4 (91%). The sequences and the intron-exon structure of the genes in soybean and guar were similar, suggesting that the genetic pathways underlying basic flowering mechanisms are conserved between these two legume crops.

CONCLUSIONS: The revealed intron-exon structure of the guar genes CtE1-CtE4 creates possibilities for their targeted mutagenesis, e.g., using CRISPR-Cas and developing new guar germplasm with low sensitivity to photoperiod.},
}

*Glycine max/genetics
*Cyamopsis/genetics
*Transcriptome/genetics
*Genome, Plant
Molecular Sequence Annotation
Photoperiod
Gene Expression Regulation, Plant
Plant Proteins/genetics
*Genes, Plant
Gene Expression Profiling
Galactans
Mannans
Plant Gums

@article {pmid40611477,
year = {2025},
author = {Bulle, M and Abbagani, S and Raza, A},
title = {Genome blaze: engineering chilli pepper chloroplasts for sustainable production of capsaicinoids through organellar genome editing.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {},
number = {},
pages = {},
doi = {10.1111/plb.70067},
pmid = {40611477},
issn = {1438-8677},
support = {PDF/2018/001226//Science and Engineering Research Board/ ; },
abstract = {The quest for superior parental lines of chilli pepper, enriched with heightened levels of phytochemicals, such as capsinoids (CATs) and capsaicinoids (CAPs), has recently gained momentum. Plant scientists now pay more attention to generating pepper cultivars that offer both increased yield and richness in these crucial ingredients. Indeed, the average pepper yield per hectare, as well as richness of these ingredients, are affected by multiple environmental stimuli. However, expression of specific genes is crucial to stimulate the CAT and CAP levels in response to environmental conditions. Recently, new technologies, like genome editing using CRISPR/Cas and engineering of chloroplasts, have been tested in chilli pepper. The advances in genome editing tools, such as the prime editor and base editor methods, have tremendous potential for plant organelle genome engineering. In this article, we highlight recent advancements in CRISPR/Cas and plastid engineering within Capsicum, coupled with application of base editing approaches for editing plant organelle DNA. We also assess the challenges and opportunities in the pursuit of commercial and sustainable production of bioactive compounds specific to Capsicum species.},
}

@article {pmid40611420,
year = {2025},
author = {Rezaei, M and Jalali, A and Sadah Al-Azzawi, DH},
title = {Engineered Bacteriophages: Advances in Phage Genome Redesign Strategies for Therapeutic and Environmental Applications.},
journal = {Protein and peptide letters},
volume = {},
number = {},
pages = {},
doi = {10.2174/0109298665372719250616085616},
pmid = {40611420},
issn = {1875-5305},
abstract = {Bacteriophages, or phages, have emerged as powerful platforms in synthetic biology, offering innovative solutions for therapeutic and environmental challenges through advanced genome redesign strategies. This review explores a wide range of phage engineering techniques, including CRISPR (clustered regularly-interspaced short palindromic repeats)-Cas systems, phage display, random and site-directed mutagenesis, retrons, and rebooting approaches, highlighting their potential to create phages with tailored functionalities. CRISPR-Cas systems enable precise genome editing, allowing the development of phages with expanded host ranges, biofilm degradation capabilities, and targeted antimicrobial activity. Phage display facilitates the presentation of peptides on phage surfaces, enabling applications in targeted drug delivery, tumor imaging, and bioremediation. Beyond these, techniques like retron-mediated recombination and homologous recombination offer additional avenues for precise phage genome modification. In the therapeutic realm, engineered phages show promise in combating drug-resistant infections, modulating the microbiome, and delivering targeted therapies for cancer and other diseases. Environmentally, phage-based strategies, such as the use of phage-displayed metal-binding peptides, provide innovative solutions for bioremediation and reducing exposure to toxic heavy metals. This review also addresses challenges, such as phage resistance, immune responses, and the limitations of current engineering methods, while exploring future directions, including the development of improved CRISPR systems, phage-based biosensors, and high-throughput screening platforms. By integrating cutting-edge genome redesign strategies with diverse applications, this review underscores the transformative potential of engineered bacteriophages in addressing global healthcare and environmental sustainability challenges.},
}

@article {pmid40611351,
year = {2025},
author = {Smidler, AL and Akbari, OS},
title = {CRISPR technologies for the control and study of malaria-transmitting anopheline mosquitoes.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {252},
pmid = {40611351},
issn = {1756-3305},
support = {R01AI151004/GF/NIH HHS/United States ; R01AI151004/GF/NIH HHS/United States ; },
mesh = {*Anopheles/genetics/parasitology/physiology ; Animals ; *Mosquito Control/methods ; *Mosquito Vectors/genetics/parasitology/physiology ; *CRISPR-Cas Systems ; *Malaria/transmission/prevention & control ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Female ; Animals, Genetically Modified ; },
abstract = {Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.},
}

*Anopheles/genetics/parasitology/physiology
Animals
*Mosquito Control/methods
*Mosquito Vectors/genetics/parasitology/physiology
*CRISPR-Cas Systems
*Malaria/transmission/prevention & control
Gene Editing
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Female
Animals, Genetically Modified

@article {pmid40610423,
year = {2025},
author = {Fuhrmeister, ER and Kim, S and Mairal, SA and McCormack, C and Chieng, B and Swarthout, JM and Paulos, AH and Njenga, SM and Pickering, AJ},
title = {Context-Seq: CRISPR-Cas9 targeted nanopore sequencing for transmission dynamics of antimicrobial resistance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5898},
pmid = {40610423},
issn = {2041-1723},
support = {OPP1129535//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; UL1TR002544//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1906957//National Science Foundation (NSF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Nanopore Sequencing/methods ; *Drug Resistance, Bacterial/genetics ; Kenya ; Anti-Bacterial Agents/pharmacology ; Dogs ; *Bacteria/genetics/drug effects ; Klebsiella pneumoniae/genetics/drug effects ; Child ; Adult ; Escherichia coli/genetics/drug effects ; Poultry/microbiology ; beta-Lactamases/genetics ; Genome, Bacterial ; Haemophilus influenzae/genetics/drug effects ; },
abstract = {Precisely understanding how and to what extent antimicrobial resistance (AMR) is exchanged between animals and humans is needed to inform control strategies. Metagenomic sequencing has low detection for rare targets such as antibiotic resistance genes, while whole genome sequencing of isolates misses exchange between uncultured bacterial species. We introduce Context-Seq, CRISPR-Cas9 targeted sequencing of ARGs and their genomic context with long-reads. Using Context-Seq, we investigate genetically similar AMR elements containing the ARGs blaCTX-M and blaTEM between adults, children, poultry, and dogs in Nairobi, Kenya. We identify genetically distinct clusters containing blaTEM and blaCTX-M that are shared between animals and humans within and between households. We also uncover potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumoniae, and Haemophilus influenzae in this study context. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.},
}

*CRISPR-Cas Systems/genetics
Animals
Humans
*Nanopore Sequencing/methods
*Drug Resistance, Bacterial/genetics
Kenya
Anti-Bacterial Agents/pharmacology
Dogs
*Bacteria/genetics/drug effects
Klebsiella pneumoniae/genetics/drug effects
Child
Adult
Escherichia coli/genetics/drug effects
Poultry/microbiology
beta-Lactamases/genetics
Genome, Bacterial
Haemophilus influenzae/genetics/drug effects

@article {pmid40610421,
year = {2025},
author = {Sek, K and Chen, AXY and Cole, T and Armitage, JD and Tong, J and Yap, KM and Munoz, I and Dunbar, PA and Wu, S and van Elsas, MJ and Hidajat, O and Scheffler, C and Giuffrida, L and Henderson, MA and Meyran, D and Souza-Fonseca-Guimaraes, F and Nguyen, D and Huang, YK and de Menezes, MN and Derrick, EB and Chan, CW and Todd, KL and Chan, JD and Li, J and Lai, J and Petley, EV and Mardiana, S and Bosco, A and Waithman, J and Parish, IA and Mølck, C and Stewart, GD and Kats, L and House, IG and Darcy, PK and Beavis, PA},
title = {Tumor site-directed A1R expression enhances CAR T cell function and improves efficacy against solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6123},
pmid = {40610421},
issn = {2041-1723},
support = {836379//Cancer Council Victoria/ ; },
mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment/immunology ; Cell Line, Tumor ; Female ; Receptor, Adenosine A2A/genetics/metabolism ; Mice, Inbred C57BL ; },
abstract = {The efficacy of Chimeric Antigen Receptor T cells against solid tumors is limited by immunosuppressive factors in the tumor microenvironment including adenosine, which suppresses Chimeric Antigen Receptor T cells through activation of the A2A receptor. To overcome this, Chimeric Antigen Receptor T cells are engineered to express A1 receptor, a receptor that signals inversely to A2A receptor. Using murine and human Chimeric Antigen Receptor T cells, constitutive A1 receptor overexpression significantly enhances Chimeric Antigen Receptor T cell effector function albeit at the expense of Chimeric Antigen Receptor T cell persistence. Through a CRISPR/Cas9 homology directed repair "knock-in" approach we demonstrate that Chimeric Antigen Receptor T cells engineered to express A1 receptor in a tumor-localized manner, enhances anti-tumor therapeutic efficacy. This is dependent on the transcription factor IRF8 and is transcriptionally unique when compared to A2A receptor deletion. This data provides a novel approach for enhancing Chimeric Antigen Receptor T cell efficacy in solid tumors and provides proof of principle for site-directed expression of factors that promote effector T cell differentiation.},
}

Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
*Neoplasms/therapy/immunology/genetics
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems
Tumor Microenvironment/immunology
Cell Line, Tumor
Female
Receptor, Adenosine A2A/genetics/metabolism
Mice, Inbred C57BL

@article {pmid40610153,
year = {2025},
author = {Ren, W and Li, M and Liu, X and You, W and You, Q and Li, B and Ye, H and Zhang, R},
title = {Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA.},
journal = {Analytica chimica acta},
volume = {1367},
number = {},
pages = {344204},
doi = {10.1016/j.aca.2025.344204},
pmid = {40610153},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *RNA/analysis/genetics ; MicroRNAs/analysis/genetics ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.

RESULTS: We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an "AND" logic gate detection platform that processes multiple inputs within 40 min. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.

SIGNIFICANCE: SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA/analysis/genetics
*RNA/analysis/genetics
MicroRNAs/analysis/genetics
*CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40609997,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {804-817},
doi = {10.1134/S0006297925600139},
pmid = {40609997},
issn = {1608-3040},
mesh = {*RNA Interference ; *CRISPR-Cas Systems ; *Plant Diseases/virology/genetics/prevention & control ; Gene Editing/methods ; *Plants/virology/genetics ; *Disease Resistance/genetics ; *Biotechnology/methods ; Plant Viruses/genetics ; },
abstract = {Established genome editing technologies, such as CRISPR-Cas and RNA interference (RNAi), have significantly advanced research studies in nearly all fields of life sciences, including biotechnology and medicine, and have become increasingly in demand in plant biology. In the review, we present the main principles of the CRISPR-Cas and RNAi technologies and their application in model plants and crops for the control of viral diseases. The review explores the antiviral effects they provide, including direct suppression of genomes of DNA- and RNA-containing viruses and inhibition of activity of host genes that increase plant susceptibility to viruses. We also provide a detailed comparison of the effectiveness of CRISPR-Cas and RNAi methods in plant protection, as well as discuss their advantages and disadvantages, factors limiting their application in practice, and possible approaches to overcome such limitations.},
}

*RNA Interference
*CRISPR-Cas Systems
*Plant Diseases/virology/genetics/prevention & control
Gene Editing/methods
*Plants/virology/genetics
*Disease Resistance/genetics
*Biotechnology/methods
Plant Viruses/genetics

@article {pmid40609996,
year = {2025},
author = {Kazakova, AA and Leonova, EI and Sopova, JV and Chirinskaite, AV and Minskaya, ES and Kukushkin, IS and Ivanov, RA and Reshetnikov, VV},
title = {Progress in CRISPR/CAS13-Mediated Suppression of Influenza A and SARS-CoV-2 Virus Infection in in vitro and in vivo Models.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {786-803},
doi = {10.1134/S0006297925601212},
pmid = {40609996},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; Animals ; *SARS-CoV-2/genetics ; Humans ; *COVID-19/therapy/virology ; *Influenza A virus/genetics ; *Influenza, Human/therapy/virology/genetics ; Disease Models, Animal ; RNA, Viral/genetics/metabolism ; Antiviral Agents ; },
abstract = {The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.},
}

*CRISPR-Cas Systems
Animals
*SARS-CoV-2/genetics
Humans
*COVID-19/therapy/virology
*Influenza A virus/genetics
*Influenza, Human/therapy/virology/genetics
Disease Models, Animal
RNA, Viral/genetics/metabolism
Antiviral Agents

@article {pmid40609995,
year = {2025},
author = {Volodina, OV and Demchenko, AG and Anuchina, AA and Ryzhkova, OP and Kovalskaya, VA and Kondrateva, EV and Tabakov, VY and Lavrov, AV and Smirnikhina, SA},
title = {Selection of Optimal pegRNAs to Enhance Efficiency of Prime Editing in AT-Rich Genome Regions.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {773-785},
doi = {10.1134/S0006297924604672},
pmid = {40609995},
issn = {1608-3040},
mesh = {*Gene Editing/methods ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Cystic Fibrosis/genetics/therapy ; CRISPR-Cas Systems ; },
abstract = {Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.},
}

*Gene Editing/methods
Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*Cystic Fibrosis/genetics/therapy
CRISPR-Cas Systems

@article {pmid40609268,
year = {2025},
author = {Allan, AC and Scott, B and Tate, W and Jameson, PE and MacRae, E and Conner, AJ and Drummond, R and Foster, A and Martin, C and Caradus, J},
title = {Human-mediated outdoor genome editing is not possible so therefore poses no risk to the environment.},
journal = {Ecotoxicology and environmental safety},
volume = {302},
number = {},
pages = {118609},
doi = {10.1016/j.ecoenv.2025.118609},
pmid = {40609268},
issn = {1090-2414},
abstract = {There is a world-wide re-examination of the regulations that surround genetic technologies, including gene edited organisms. In many countries, crop plants with small gene edits and where no foreign DNA is introduced are exempt from detailed regulatory assessment. This will allow these types of plants to be released, after assessment for benefit and risk, by plant breeders or plant scientists. The full regulatory risk assessment and risk management of novel (including transgenic) plants is well established and focusses on five key criteria (weediness, gene flow, plant pests, non-target impact, biodiversity). However, plants produced by "traditional" plant breeding technologies, many of which have been subject to random mutagenesis or wide crosses that may introduce enormous numbers of DNA changes, are almost never considered novel so are not assessed for risk through a regulatory system. The most targeted, versatile and widely used gene editing technique involves the enzyme-RNA complex, CRISPR-Cas. This method can produce far more precise and targeted changes than "traditional" mutagenesis techniques. Getting the CRISPR-Cas machinery into plant or animal cells requires highly sterile tissue culture and sophisticated delivery tools. Therefore, gene editing, in the open environment by "field spraying", is not currently possible. While other uses of nucleotide chemistry - such as double stranded RNA - have been applied to plants to knock down gene expression, this is not gene editing and produces no DNA change. Suggestions that gene editing using CRISPR-Cas can occur through spraying directly on to plants in the outside environment is fanciful, incorrect and misleading.},
}

@article {pmid40608406,
year = {2025},
author = {Ito, K and Ito, Y},
title = {Comparative genomic analysis of Latilactobacillus sakei strains provides new insights into their association with different niche adaptations.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {7},
pages = {},
doi = {10.1099/mic.0.001578},
pmid = {40608406},
issn = {1465-2080},
mesh = {*Genome, Bacterial ; Phylogeny ; *Adaptation, Physiological/genetics ; Plasmids/genetics ; *Latilactobacillus sakei/genetics/classification/physiology/growth & development/metabolism ; Genomics ; Carbohydrate Metabolism/genetics ; Fermentation ; Genetic Variation ; },
abstract = {Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 29 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominate in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakei's ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family amongst sugar metabolism-related genes present on chromosomes and plasmids varies between strains and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. blast analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.},
}

*Genome, Bacterial
Phylogeny
*Adaptation, Physiological/genetics
Plasmids/genetics
*Latilactobacillus sakei/genetics/classification/physiology/growth & development/metabolism
Genomics
Carbohydrate Metabolism/genetics
Fermentation
Genetic Variation

@article {pmid40608358,
year = {2025},
author = {Li, YG and Haeusser, D and Margolin, W and Christie, PJ},
title = {Conjugative delivery of toxin genes ccdB and kil confers synergistic killing of bacterial recipients.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0016825},
doi = {10.1128/jb.00168-25},
pmid = {40608358},
issn = {1098-5530},
abstract = {The bacterial type IV secretion systems (T4SS) are medically problematic for their roles in the dissemination of mobile genetic elements or effector proteins, but they also have great potential for new antimicrobial therapies. Recent studies have deployed the T4SS subfamily of conjugation systems to deliver gene editing CRISPR/Cas systems to disrupt drug resistance genes or kill targeted bacterial recipients. However, the therapeutic potential of conjugative CRISPR/Cas delivery is compromised by mutations or host repair systems that diminish the efficiency with which CRISPR/Cas induces double-strand breaks in new transconjugants. Here, we compared the efficiencies of conjugation-based killing systems based on the delivery of CRISPR-Cas9 elements or toxin genes encoding the bacteriophage lambda Kil peptide or the F plasmid-encoded CcdB. Escherichia coli equipped with one of two efficient conjugation systems, pKM101 (IncN) or F (IncF), served as donors to mobilize plasmids carrying the cognate oriT sequence and one or more toxic elements. Overall, toxin gene delivery proved significantly more effective than CRISPR-Cas9 in killing of transconjugant population, but the highest levels of growth suppression of both E. coli and Klebsiella pneumoniae recipients were achieved by a combination of CRISPR-Cas9 plus one or two toxin genes. By contrast, capsule production conferred no or very slight protective effects on plasmid acquisition and killing of either species. We propose that the conjugative co-transfer of two or more toxic elements with distinct mechanisms of action has strong potential for growth suppression of targeted species in environmental or clinical settings.IMPORTANCEThe prevalence of antibiotic resistance emphasizes the need for alternative antimicrobial intervention strategies. We engineered Escherichia coli for conjugative transmission of plasmids encoding CRISPR-Cas9 elements or genes encoding the cell division inhibitor Kil or gyrase poisoner CcdB. Delivery of toxin genes more effectively suppressed the growth of E. coli recipients than CRISPR-Cas9, but the combinatorial delivery of CRISPR-Cas9 and a toxin gene or two toxin genes elicited the strongest killing effects. Capsule production by E. coli or Klebsiella pneumoniae recipient cells had no or little protective effect on plasmid acquisition or growth suppression. Our findings suggest that probiotic donor strains equipped for conjugative delivery of two or more toxic elements may prove effective as an alternative or adjunct to traditional antimicrobials.},
}

@article {pmid40607431,
year = {2025},
author = {Wang, JW and Liu, JH and Xun, JJ},
title = {CCR5 gene editing and HIV immunotherapy: current understandings, challenges, and future directions.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1590690},
pmid = {40607431},
issn = {1664-3224},
mesh = {*Receptors, CCR5/genetics/immunology ; Humans ; *Gene Editing/methods ; *HIV Infections/therapy/immunology/genetics ; *Immunotherapy/methods ; *HIV-1/immunology ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Human immunodeficiency virus (HIV) infection remains a major global public health challenge. Although highly active antiretroviral therapy (HAART or ART) can effectively control viral replication, it fails to eradicate latent viral reservoirs and poses limitations such as lifelong medication and cumulative drug toxicity. This study focuses on the pivotal role of C-C chemokine receptor 5 (CCR5) gene editing in HIV immunotherapy, particularly highlighting the natural resistance to R5-tropic HIV strains observed in the "Berlin" and "London" patients carrying the homozygous CCR5-Δ32 mutation. We further explore the synergistic potential of multiplex gene editing strategies-including CCR5, CXCR4, and HIV LTR loci-and the combinatorial mechanisms between gene editing technologies and immunotherapy. A personalized treatment framework is proposed to address the clinical heterogeneity among people living with HIV. In addition, we assess the balance between long-term safety and global accessibility of gene-editing approaches such as CRISPR/Cas9, emphasizing strategies to enhance therapeutic efficacy while reducing cost and off-target effects. Our findings suggest that the integration of CCR5-targeted gene editing with immune-based interventions holds great promise for overcoming current therapeutic limitations and achieving functional HIV cure. However, key challenges-such as immune rejection, viral tropism switching, and economic feasibility-must be resolved. This integrative approach provides a robust theoretical and technical foundation for the next generation of HIV treatment paradigms.},
}

*Receptors, CCR5/genetics/immunology
Humans
*Gene Editing/methods
*HIV Infections/therapy/immunology/genetics
*Immunotherapy/methods
*HIV-1/immunology
CRISPR-Cas Systems
Genetic Therapy/methods

@article {pmid40605043,
year = {2025},
author = {Salazar-García, LM and Damas-Ramos, LC and Trejo-Alarcón, LM and Rago, D and Ahonen, L and Cruz-Morales, P and Ponce-Noyola, P and Licona-Cassani, C},
title = {CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {151},
pmid = {40605043},
issn = {1475-2859},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; },
mesh = {*Pseudomonas aeruginosa/metabolism/genetics/isolation & purification ; Glycolipids/metabolism/biosynthesis ; *CRISPR-Cas Systems ; *Hydrocarbons/metabolism/chemistry ; Bacterial Proteins/genetics/metabolism ; Pyocyanine/metabolism/biosynthesis ; Emulsions ; Gene Editing ; Sigma Factor/genetics ; Petroleum Pollution ; },
abstract = {BACKGROUND: Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains of Pseudomonas aeruginosa have demonstrated significant potential for producing rhamnolipids (RMLs) and pyocyanin (PYO), secondary metabolites associated to hydrocarbon emulsification. In this study, we isolated and characterized an environmental strain from an oil-contaminated site in the Gulf of Mexico. Upon genome sequencing and taxonomic classification, we developed genetic engineering tools and assessed their capacity to produce PYO and RMLs, molecules relevant for hydrocarbon emulsification.

RESULTS: Using the CRISPR/Cas9-APOBEC1-UGI system, we generated a targeted cytosine to thymine transition in the rpoS gene to generate a premature STOP codon. The resulting mutant exhibited increased production of PYO and RMLs, along with enhanced gasoline emulsification in cell-free supernatants, demonstrating successful modulation of a key regulatory gene. While the strain IGLPR01 retains certain virulence-associated features, this study contributes to the exploration of environmental isolates as future candidate chassis for biosurfactant production, emphasizing the need for further safety evaluation and rational attenuation strategies.

CONCLUSION: This study provides a successful example of implementing CRISPR-based editing in an environmental P. aeruginosa strain. Despite the technical challenges, a genetic editing system was established and validated through a proof of concept to increase production of relevant metabolites. Our work demonstrates the applicability of genetic engineering tools in non-model environmental isolates, facilitating further developments. Importantly, the presence of virulence-associated features highlights the need for in-depth evaluation of pathogenicity and containment strategies before considering any future biotechnological applications.},
}

*Pseudomonas aeruginosa/metabolism/genetics/isolation & purification
Glycolipids/metabolism/biosynthesis
*CRISPR-Cas Systems
*Hydrocarbons/metabolism/chemistry
Bacterial Proteins/genetics/metabolism
Pyocyanine/metabolism/biosynthesis
Emulsions
Gene Editing
Sigma Factor/genetics
Petroleum Pollution

@article {pmid40605002,
year = {2025},
author = {Chou, J and Esmaeili Anvar, N and Elghaish, R and Chen, J and Hart, T},
title = {Z-scores outperform similar methods for analyzing CRISPR paralog synthetic lethality screens.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {188},
pmid = {40605002},
issn = {1474-760X},
support = {R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; R35CA274234/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Synthetic Lethal Mutations ; Cell Line, Tumor ; },
abstract = {Genetic screens offer a promising strategy for identifying tumor-specific therapeutic targets, but single-gene knockout screens often miss functionally redundant paralogs. Multiplex Cas9 and Cas12a CRISPR systems have been deployed to assay genetic interactions, but analysis pipelines vary considerably. Here we evaluate data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using delta log fold change, Z-transformed dLFC, and rescaled dLFC approaches to identify synthetic lethal interactions. Both ZdLFC and RdLFC provide more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method, while ZdLFC benefits from not requiring a training set of known interactors.},
}

Humans
*CRISPR-Cas Systems
*Synthetic Lethal Mutations
Cell Line, Tumor

@article {pmid40577485,
year = {2025},
author = {Rissone, A and La Spina, M and Bresciani, E and Syed, ZA and Combs, CA and Kirby, M and Elkahloun, A and Chen, V and Sood, R and Burgess, SM and Puertollano, R},
title = {The transcription factors Tfeb and Tfe3 are required for survival and embryonic development of pancreas and liver in zebrafish.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011754},
pmid = {40577485},
issn = {1553-7404},
mesh = {Animals ; *Zebrafish/genetics/embryology/growth & development ; *Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Pancreas/embryology/metabolism/growth & development ; *Liver/embryology/metabolism/growth & development ; *Embryonic Development/genetics ; Autophagy/genetics ; Gene Expression Regulation, Developmental ; Apoptosis/genetics ; Oxidative Stress/genetics ; CRISPR-Cas Systems ; },
abstract = {The transcription factors TFEB and TFE3 modulate expression of lysosomal, autophagic, and metabolic genes to restore energy and cellular homeostasis in response to a variety of stress conditions. Since their role during vertebrate development is less characterized, we used CRISPR/Cas9 to deplete tfeb, tfe3a, and tfe3b in zebrafish. The simultaneous lack of these genes compromised embryo survival during early development, with an almost complete lethality of the larvae by 8-10 dpf. The knockout animals showed apoptosis in brain and retina and alterations in pancreas, liver, and gut. Exocrine pancreas presented the most severe defects, with accumulation of abnormal zymogen granules leading to acinar atrophy in embryos and pancreatitis-like phenotypes in adults; likely due to a block of the autophagy machinery implicated in removal of damaged granules. Knockout animals displayed increased susceptibility to oxidative and heat-shock stress. Our work reveals an essential role of Tfeb and Tfe3 in maintaining cellular and tissue homeostasis during development.},
}

Animals
*Zebrafish/genetics/embryology/growth & development
*Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism
*Zebrafish Proteins/genetics/metabolism
*Pancreas/embryology/metabolism/growth & development
*Liver/embryology/metabolism/growth & development
*Embryonic Development/genetics
Autophagy/genetics
Gene Expression Regulation, Developmental
Apoptosis/genetics
Oxidative Stress/genetics
CRISPR-Cas Systems

@article {pmid40516528,
year = {2025},
author = {Matthews, RE and Danac, JMC and Naden, EL and Farleigh Smith, LE and Lestari, S and Gungi, A and Appert, A and Buttress, T and Verma, A and Sinclair, O and Chong, F and Suberu, J and Antrobus, R and Bonev, B and Dawson, MA and Reid, AJ and Timms, RT and Ahringer, J and Tchasovnikarova, IA},
title = {CRAMP1 drives linker histone expression to enable Polycomb repression.},
journal = {Molecular cell},
volume = {85},
number = {13},
pages = {2503-2516.e8},
doi = {10.1016/j.molcel.2025.05.031},
pmid = {40516528},
issn = {1097-4164},
mesh = {*Histones/genetics/metabolism ; *Polycomb Repressive Complex 2/genetics/metabolism ; Animals ; Humans ; Mice ; Promoter Regions, Genetic ; Heterochromatin/genetics/metabolism ; CRISPR-Cas Systems ; *Epigenetic Repression ; },
abstract = {In contrast to the well-understood role of core histones in DNA packaging, the function of the linker histone (H1) remains enigmatic. Challenging the prevailing view that linker histones are a general feature of heterochromatin, here we show a critical requirement for H1 in Polycomb repressive complex 2 (PRC2) function. A CRISPR-Cas9 genetic screen using a fluorescent PRC2 reporter identified an essential role for the poorly characterized gene CRAMP1 in PRC2-mediated repression. CRAMP1 localizes to the promoters of expressed H1 genes and positively regulates their transcription. CRAMP1 ablation simultaneously depletes all linker histones, which results in selective decompaction of H3K27me3-marked loci and derepression of PRC2 target genes without concomitant loss of PRC2 occupancy or enzymatic activity. Strikingly, we find that linker histones preferentially localize to genomic loci marked by H3K27me3 across diverse cell types and organisms. Altogether, these data demonstrate a prominent role for linker histones in epigenetic repression by PRC2.},
}

*Histones/genetics/metabolism
*Polycomb Repressive Complex 2/genetics/metabolism
Animals
Humans
Mice
Promoter Regions, Genetic
Heterochromatin/genetics/metabolism
CRISPR-Cas Systems
*Epigenetic Repression

@article {pmid40399675,
year = {2025},
author = {Han, M and Fu, ML and Zhu, Y and Choi, AA and Li, E and Bezney, J and Cai, S and Miles, L and Ma, Y and Qi, LS},
title = {Programmable control of spatial transcriptome in live cells and neurons.},
journal = {Nature},
volume = {643},
number = {8070},
pages = {241-251},
pmid = {40399675},
issn = {1476-4687},
mesh = {Animals ; *Neurons/metabolism/cytology ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; Mice ; Neurites/metabolism ; Humans ; RNA Transport/genetics ; Ribosomes/metabolism ; Neuronal Outgrowth/genetics ; Actins/genetics/metabolism ; Rats ; Protein Biosynthesis ; Cells, Cultured ; Microtubules/metabolism ; Female ; Axons/metabolism ; },
abstract = {Spatial RNA organization has a pivotal role in diverse cellular processes and diseases[1-4]. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions[1,5]. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells. CRISPR-TO enables targeted localization of endogenous RNAs to diverse subcellular compartments, including the outer mitochondrial membrane, p-bodies, stress granules, telomeres and nuclear stress bodies, across various cell types. It allows for inducible and reversible bidirectional RNA transport along microtubules via motor proteins, facilitating real-time manipulation and monitoring of RNA localization dynamics in living cells. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips, and co-transport with ribosomes, with β-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. CRISPR-TO-enabled screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.},
}

Animals
*Neurons/metabolism/cytology
*Transcriptome/genetics
*CRISPR-Cas Systems/genetics
RNA, Messenger/metabolism/genetics
Mice
Neurites/metabolism
Humans
RNA Transport/genetics
Ribosomes/metabolism
Neuronal Outgrowth/genetics
Actins/genetics/metabolism
Rats
Protein Biosynthesis
Cells, Cultured
Microtubules/metabolism
Female
Axons/metabolism

@article {pmid40359943,
year = {2025},
author = {Wang, Y and Wu, J and Yang, S and Li, X and Wang, J and Lv, Q and Zhu, X and Lu, G and Zhang, J and Shen, WH and Liu, B and Lin, J and Dong, A},
title = {Structural and functional interrelationships of histone H2A with its variants H2A.Z and H2A.W in Arabidopsis.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {7},
pages = {1240-1249.e5},
doi = {10.1016/j.str.2025.04.015},
pmid = {40359943},
issn = {1878-4186},
mesh = {*Histones/chemistry/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/chemistry/metabolism/genetics ; Nucleosomes/metabolism/chemistry ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; Mutation ; Protein Isoforms/chemistry/metabolism/genetics ; },
abstract = {Multiple histone H2A variants are known in eukaryotes. However, the functional relationship between H2A and its variants in plants remains largely obscure. Using CRISPR-Cas9 editing, we generated a mutant lacking four H2A isoforms in Arabidopsis and analyzed the functional and structural relationships between H2A, H2A.Z, and H2A.W. RNA sequencing and phenotype analyses revealed mild changes in gene transcription and plant development in mutants lacking H2A, H2A.Z, or H2A.W compared with the wild-type plants. Chromatin immunoprecipitation sequencing analysis showed that H2A can substitute for both H2A.Z and H2A.W across the genome, including in euchromatin and heterochromatin regions. However, H2A.Z replaced both H2A and H2A.W primarily within the euchromatin regions. By using DNA and histones from Arabidopsis, we constructed nucleosomes containing H2A, H2A.Z, or H2A.W and resolved their cryogenic electron microscopy (cryo-EM) structures at near-atomic resolution. Collectively, the results reveal the structural similarity and functional redundancy of H2A and its variants in Arabidopsis.},
}

*Histones/chemistry/genetics/metabolism
*Arabidopsis/genetics/metabolism
*Arabidopsis Proteins/chemistry/metabolism/genetics
Nucleosomes/metabolism/chemistry
Cryoelectron Microscopy
CRISPR-Cas Systems
Mutation
Protein Isoforms/chemistry/metabolism/genetics

@article {pmid40344384,
year = {2025},
author = {Yao, Z and Li, W and He, K and Wang, H and Xu, Y and Wu, Q and Wang, L and Nie, Y},
title = {Facilitating crRNA Design by Integrating DNA Interaction Features of CRISPR-Cas12a System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {25},
pages = {e2501269},
pmid = {40344384},
issn = {2198-3844},
support = {32172175//National Natural Science Foundation of China/ ; 32172307//National Natural Science Foundation of China/ ; 2023C02006//Key R&D Program of Zhejiang/ ; KYCX24_2592//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; NO.111-2-06//Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics/metabolism ; Molecular Dynamics Simulation ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR-Cas12a system has gained significant attention as a rapid nucleic acid diagnostic tool due to its crRNA-guided trans-cleavage activity. Accurately predicting the activity of different targets is significant to facilitate the crRNA availability but remains challenging. In this study, a novel approach is presented that combines molecular dynamics simulations and neural network modeling to predict the trans-cleavage activity. Unlike conventional tools that rely solely on the base sequences, our method integrated sequence features and molecular interaction features of DNA in the CRISPR-Cas12a system, significantly improving prediction accuracy. Through feature importance analysis, key sequence features that influence Cas12a trans-cleavage activity are identified. Additionally, a crRNA-DNA library with over 23 456 feature sequences from representative viruses and bacteria is established, and validated the high predictive accuracy of the model (Pearson's r = 0.9328) by screening crRNAs from reference targets. This study offers new insights into the molecular interactions of Cas12a/crRNA-DNA and provides a reliable framework for optimizing crRNA design, facilitating the application of the CRISPR-Cas12a in rapid nucleic acid diagnostics.},
}

*CRISPR-Cas Systems/genetics
*DNA/genetics/metabolism
Molecular Dynamics Simulation
*CRISPR-Associated Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40302201,
year = {2025},
author = {Hao, JH and Kang, X and Zhang, L and Chen, J and Wang, D and Dong, S and Li, X and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-Mediated SiEPF2 Mutagenesis Attenuates Drought Tolerance and Yield in Foxtail Millet (Setaria italica).},
journal = {Plant, cell & environment},
volume = {48},
number = {8},
pages = {6043-6046},
doi = {10.1111/pce.15597},
pmid = {40302201},
issn = {1365-3040},
support = {//This study was supported by the National Key Research and Development Programme of China (2023YFD1202702), National Natural Science Foundation (32400217 and 32200222) and Start-up Fund of Shanxi Agricultural University (2021BQ84)./ ; },
mesh = {*Setaria Plant/genetics/physiology/growth & development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Mutagenesis ; Plant Stomata/physiology ; Gene Expression Regulation, Plant ; Drought Resistance ; },
abstract = {Plants employ peptide ligands to coordinate development and integrate environmental signals via dedicated cascades (#ref-0013). Epidermal patterning factor (EPF), plays a significant role in regulating stomatal density, seed germination and panicle development (#ref-0008). EPF/EPFL enhance drought tolerance by reducing stomatal density have been reported in multiple species, including Hordeum vulgare, Arabidopsis thaliana, Populus spp., Vitis vinifera, Sorghum bicolor and Brassica napus (#ref-0001). Our previous study has shown that EPF can mediate drought resistance in foxtail millet by regulating stomatal density (#ref-0004). Furthermore, evidence suggests that plants can regulate photosynthesis through stomatal modification, ultimately enhancing yield (#ref-0005). Beyond stomatal density regulation, the EPF/EPFL gene family modulates seed germination through phytohormone signalling and regulates inflorescence development via ligand-receptor interactions (#ref-0007). OsEPFLs act as upstream ligands for the OsER1 receptor, activating the MAPK signalling cascade to regulate panicle morphogenesis (#ref-0002). In this study, we sought to elucidate how SiEPF2 balances drought resistance and yield in foxtail millet by modulating stomatal density and panicle morphology. Our findings not only provide novel insights into SiEPF2's role in abiotic stress responses but also contribute valuable genetic resources for high-yield breeding programmes in millet crops.},
}

*Setaria Plant/genetics/physiology/growth & development
Droughts
*Plant Proteins/genetics/metabolism/physiology
*CRISPR-Cas Systems/genetics
Mutagenesis
Plant Stomata/physiology
Gene Expression Regulation, Plant
Drought Resistance

@article {pmid40280813,
year = {2025},
author = {Hu, N and Tian, H and Li, Y and Li, X and Li, D and Li, L and Wang, S and Zhang, Y and Shi, X and Huang, B and Lu, Q and Wang, T and Pan, X and Tu, L and Dai, D and Zhang, B and Peng, R and Yan, F},
title = {pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1788-1808},
doi = {10.1016/j.tibtech.2025.03.016},
pmid = {40280813},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Solanum lycopersicum/genetics ; Arabidopsis/genetics ; DNA, Bacterial/genetics ; Promoter Regions, Genetic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant-specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P35SIC47, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly(A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genome, Plant/genetics
Nicotiana/genetics
Plants, Genetically Modified/genetics
Solanum lycopersicum/genetics
Arabidopsis/genetics
DNA, Bacterial/genetics
Promoter Regions, Genetic

@article {pmid40263591,
year = {2025},
author = {Rohde, T and Demirtas, TY and Süsser, S and Shaw, AH and Kaulich, M and Billmann, M},
title = {BaCoN (Balanced Correlation Network) improves prediction of gene buffering.},
journal = {Molecular systems biology},
volume = {21},
number = {7},
pages = {807-824},
pmid = {40263591},
issn = {1744-4292},
support = {397484323 - TRR259//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Gene Regulatory Networks ; Cell Line, Tumor ; Algorithms ; Transcriptome ; *Computational Biology/methods ; Neoplasms/genetics ; },
abstract = {Buffering between genes, where one gene can compensate for the loss of another gene, is fundamental for robust cellular functions. While experimentally testing all possible gene pairs is infeasible, gene buffering can be predicted genome-wide under the assumption that a gene's buffering capacity depends on its expression level and its absence primes a severe fitness phenotype of the buffered gene. We developed BaCoN (Balanced Correlation Network), a post hoc unsupervised correction method that amplifies specific signals in expression-vs-fitness correlation networks. We quantified 147 million potential buffering relationships by associating CRISPR-Cas9-screening fitness effects with transcriptomic data across 1019 Cancer Dependency Map (DepMap) cell lines. BaCoN outperformed state-of-the-art methods, including multiple linear regression based on our compiled gene buffering prediction metrics. Combining BaCoN with batch correction or Cholesky data whitening further boosts predictive performance. We characterized 808 high-confidence buffering predictions and found that in contrast to buffering gene pairs overall, buffering paralogs were on different chromosomes. BaCoN performance increases with more screens and genes considered, making it a valuable tool for gene buffering predictions from the growing DepMap.},
}

Humans
CRISPR-Cas Systems
*Gene Regulatory Networks
Cell Line, Tumor
Algorithms
Transcriptome
*Computational Biology/methods
Neoplasms/genetics

@article {pmid40205048,
year = {2025},
author = {Fell, CW and Villiger, L and Lim, J and Hiraizumi, M and Tagliaferri, D and Yarnall, MTN and Lee, A and Jiang, K and Kayabolen, A and Krajeski, RN and Schmitt-Ulms, C and Ramani, H and Yousef, SM and Roberts, N and Vakulskas, CA and Nishimasu, H and Abudayyeh, OO and Gootenberg, JS},
title = {Reprogramming site-specific retrotransposon activity to new DNA sites.},
journal = {Nature},
volume = {642},
number = {8069},
pages = {1080-1089},
pmid = {40205048},
issn = {1476-4687},
mesh = {*Retroelements/genetics ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Finches/genetics ; *Gene Editing/methods ; *DNA/genetics ; Genome/genetics ; *Mutagenesis, Insertional/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Mice ; Reverse Transcription ; },
abstract = {Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes[1-6]. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR-Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologues, we select an orthologue, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9[H840A]-R2Tocc, a system we name site-specific target-primed insertion through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7 kilobases, gene replacement and use of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.},
}

*Retroelements/genetics
Animals
Humans
CRISPR-Cas Systems/genetics
Finches/genetics
*Gene Editing/methods
*DNA/genetics
Genome/genetics
*Mutagenesis, Insertional/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Mice
Reverse Transcription

@article {pmid40199626,
year = {2025},
author = {Lu, Y and Wang, J and Xu, Y and Xu, M and Li, B and Fan, Z and Liu, J and Li, X and Cai, Z and Zheng, Y and Wang, W and Yang, J and Zhang, Z and Liu, Z},
title = {Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1743-1764},
doi = {10.1016/j.tibtech.2025.02.020},
pmid = {40199626},
issn = {1879-3096},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; Genetic Therapy/methods ; Gene Knock-In Techniques/methods ; *Gene Targeting/methods ; DNA Repair ; Hepatocytes/metabolism ; Deoxyribonuclease I/metabolism/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based targeted insertion of DNA fragments holds great promise for gene therapy. However, designing highly efficient and precise integration of large DNA segments in somatic cells while avoiding unpredictable products remains challenging. Here, we devised a novel long-offset paired nicking target integration (LOTI) strategy, which enhances the capacity of Cas9 nickase (Cas9n) in targeted gene integration in somatic cells, yielding higher knock-in (KI) efficiency compared with classical nickase-based approaches. The underlying repair mechanism involves the DNA repair proteins Rad51 and Rad52, and Ligase I/III. Moreover, we achieved efficient KI of at least 1.5-kb gene fragments in hepatocytes and recovery 55% FIX activity in a hemophilia B mouse model using only one-dose plasmid DNA delivery. Compared with the Cas9-based strategy, LOTI reduces off-target activity and restricts the formulation of unwanted insertions and deletions (indels) at the target site. Thus, LOTI provides a precise and efficient strategy for gene integration in somatic cells in vivo.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
Genetic Therapy/methods
Gene Knock-In Techniques/methods
*Gene Targeting/methods
DNA Repair
Hepatocytes/metabolism
Deoxyribonuclease I/metabolism/genetics

@article {pmid40192038,
year = {2025},
author = {Shi, K and Huang, W and Zhu, M and Teng, S and Zhang, J and Duan, Z and Zhu, C and Hu, T and Wang, K and Wang, Z},
title = {Efficient genetic transformation and genome editing via an Agrobacterium-mediated in commercial oat (Avena sativa L.) cultivars.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {7},
pages = {1697-1699},
doi = {10.1111/jipb.13915},
pmid = {40192038},
issn = {1744-7909},
support = {2005DKA21003//the National Center for Forestry and Grassland Genetic Resources/ ; },
mesh = {*Avena/genetics/microbiology ; *Gene Editing/methods ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
abstract = {An optimized Agrobacterium-mediated transformation protocol for immature and mature oat embryos increased transformation efficiencies and the number of transformable cultivars and enabled highly efficient CRISPR/Cas9 and CRISPR/Cas12i genome editing to accelerate oat biotechnology breeding.},
}

*Avena/genetics/microbiology
*Gene Editing/methods
*Transformation, Genetic
*Agrobacterium/genetics
Plants, Genetically Modified
CRISPR-Cas Systems/genetics
Genome, Plant/genetics

@article {pmid40187931,
year = {2025},
author = {Jiang, Y and Xiao, Z and Luo, Z and Zhou, S and Tong, C and Jin, S and Liu, X and Qin, R and Xu, R and Pan, L and Li, J and Wei, P},
title = {Improving plant C-to-G base editors with a cold-adapted glycosylase and TadA-8e variants.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1765-1787},
doi = {10.1016/j.tibtech.2025.03.001},
pmid = {40187931},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; Oryza/genetics ; Plants, Genetically Modified/genetics ; *Cytidine Deaminase/genetics/metabolism ; *Uracil-DNA Glycosidase/genetics/metabolism ; Cytosine/metabolism ; Guanine/metabolism ; Humans ; CRISPR-Cas Systems ; Animals ; Cold Temperature ; },
abstract = {Plant cytosine (C)-to-guanine (G) base editors (CGBEs) have been established but suffer from limited editing efficiencies and low outcome purities. This study engineered a cod uracil DNA glycosylase (cod UNG, coUNG) from the cold-adapted fish Gadus morhua for plant CGBE, demonstrating 1.71- to 2.54-fold increases in C-to-G editing efficiency compared with the CGBE using human UNG (hUNG). Further engineering took advantage of TadA-8e-derived cytidine deaminases (TadA-CDs). These variants induced C substitutions with efficiencies ranging from 26.28% to 30.82% in rice cells, whereas adenine (A) conversion was negligible. By integrating coUNG and TadA-CDc elements with SpCas9 nickase, the resulting CDc-CGBEco achieved pure C-to-G editing without byproducts in up to 52.08% of transgenic lines. Whole-genome sequencing (WGS) analysis revealed no significant off-target effects of the CDc-BEs in rice. In addition, CDc-CGBEco enabled precise C-to-G editing in soybean and tobacco. These engineered CGBEs enhanced editing efficiency, purity, and specificity, suggesting their broad potential for applications in scientific research and crop breeding.},
}

*Gene Editing/methods
Oryza/genetics
Plants, Genetically Modified/genetics
*Cytidine Deaminase/genetics/metabolism
*Uracil-DNA Glycosidase/genetics/metabolism
Cytosine/metabolism
Guanine/metabolism
Humans
CRISPR-Cas Systems
Animals
Cold Temperature

@article {pmid40119518,
year = {2025},
author = {He, X and Yan, T and Song, Z and Xiang, L and Xiang, J and Yang, Y and Ren, K and Bu, J and Xu, X and Li, Z and Guo, X and Lin, B and Zhou, Q and Lin, G and Gu, F},
title = {Correcting a patient-specific Rhodopsin mutation with adenine base editor in a mouse model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {7},
pages = {3101-3113},
doi = {10.1016/j.ymthe.2025.03.021},
pmid = {40119518},
issn = {1525-0024},
mesh = {Animals ; Disease Models, Animal ; Mice ; *Gene Editing/methods ; *Adenine/metabolism ; Humans ; *Retinitis Pigmentosa/genetics/therapy ; *Mutation ; *Rhodopsin/genetics ; Gene Knock-In Techniques ; Dependovirus/genetics ; Genetic Vectors/genetics ; Electroretinography ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Retina/metabolism/pathology ; Genetic Therapy/methods ; },
abstract = {Genome editing offers a great promise to treating human genetic diseases. To assess genome-editing-mediated therapeutic effects in vivo, an animal model is indispensable. The genomic disparities between mice and humans often impede the direct clinical application of genome-editing-mediated treatments using conventional mouse models. Thus, the generation of a mouse model with a humanized genomic segment containing a patient-specific mutation is highly sought after for translational research. In this study, we successfully developed a knockin mouse model for autosomal-dominant retinitis pigmentosa (adRP), designated as hT17M knockin, which incorporates a 75-nucleotide DNA segment with the T17M mutation (Rhodopsin-c.C50T; p.T17M). This model demonstrated significant reductions in electroretinogram amplitudes and exhibited disruptions in retinal structure. Subsequently, we administered an adeno-associated virus vectors carrying an adenine base editor (ABE) and a single-guide RNA specifically targeting the T17M mutation, achieving a peak correction rate of 39.7% at the RNA level and significantly improving retinal function in ABE-injected mice. These findings underscore that the hT17M knockin mouse model recapitulates the clinical features of adRP patients and exhibits therapeutic effects with ABE-mediated treatments. It offers a promising avenue for the development of gene-editing therapies for RP.},
}

Animals
Disease Models, Animal
Mice
*Gene Editing/methods
*Adenine/metabolism
Humans
*Retinitis Pigmentosa/genetics/therapy
*Mutation
*Rhodopsin/genetics
Gene Knock-In Techniques
Dependovirus/genetics
Genetic Vectors/genetics
Electroretinography
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Retina/metabolism/pathology
Genetic Therapy/methods

@article {pmid38944889,
year = {2025},
author = {Kang, ES and Kim, NH and Lim, HK and Jeon, H and Han, K and No, YH and Kim, K and Khaleel, ZH and Shin, D and Eom, K and Nam, J and Lee, BS and Kim, HJ and Suh, M and Lee, J and Thach, TT and Hyun, J and Kim, YH},
title = {Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {26},
pages = {e2404680},
doi = {10.1002/adma.202404680},
pmid = {38944889},
issn = {1521-4095},
support = {//National Research Foundation of Korea/ ; NRF-2023R1A2C3005731//Korean government/ ; RS-2023-00302458)//Korean government/ ; IBS-R015-D1//Institute for Basic Science/ ; },
mesh = {Animals ; Mice ; *Protein Engineering/methods ; Thermodynamics ; Humans ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; DNA/metabolism ; },
abstract = {Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.},
}

Animals
Mice
*Protein Engineering/methods
Thermodynamics
Humans
Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
CRISPR-Cas Systems
HEK293 Cells
DNA/metabolism

@article {pmid40612691,
year = {2024},
author = {Lin, J and Yang, J},
title = {CRISPR-Cas systems: A revolution in genome editing and its diverse applications.},
journal = {Journal of biomed research},
volume = {5},
number = {1},
pages = {108-114},
pmid = {40612691},
issn = {2693-5910},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) Cas (CRISPR6 associated protein) system is an advanced adaptive immune system found in prokaryotes. First discovered in1987, CRISPR Cas has revolutionized genetic research in the past two decades. CRISPR-Cas9 the most widespread system enables precise gene editing by creating double strand breaks. Its ease of use and cost-effectiveness has lowered the barrier to entry for genetic research. CRISPR holds immense potential in many fields from agriculture to medicine. In agriculture, CRISPR has accelerated crop improvement by enabling precise gene edits for desirable traits. In medicine, CRISPR holds promise in xenotransplant, cancers and infectious diseases (HIV) treatment. This review traces the historical development of CRISPR-Cas systems, explores their unique applications, and discusses future advancements aimed at enhancing CRISPR's precision and expanding its applications through technologies like prime and base editing.},
}

@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