@article {pmid40745002,
year = {2025},
author = {Park, EJ and Kim, H},
title = {Live genome imaging by CRISPR engineering: progress and problems.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745002},
issn = {2092-6413},
abstract = {CRISPR-Cas-based genome imaging opened a new era of genome visualization in living cells. While genomic loci with repetitive sequences, such as centromeres and telomeres, can be reliably imaged, applying the technique to nonrepetitive genomic loci has remained challenging. Recent advancements in the design of CRISPR RNAs and Cas proteins, the development of novel fluorophores and the combination of CRISPR-Cas with other molecular machinery amplified target-specific signals and suppressed background signals, revolutionizing this unique genome imaging technique and enabling the tracking of genomic loci with a small number of CRISPR-Cas complexes, down to a single complex. Here we review the latest advancements in CRISPR-Cas-based genome imaging techniques and their application to imaging nonrepetitive genomic loci. The challenges that these techniques are currently facing are the cellular toxicity and genomic instability induced by the expression of CRISPR-Cas and its interference with DNA metabolism, which impacts DNA replication and genome maintenance. Recently reported adverse effects of CRISPR-Cas-based genome labeling are discussed here, along with perspectives on how to overcome the problem.},
}

@article {pmid40745001,
year = {2025},
author = {Varshney, GK and Burgess, SM},
title = {CRISPR-based functional genomics tools in vertebrate models.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745001},
issn = {2092-6413},
support = {ZIAHG000183-24//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R24OD034438//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {The advent of CRISPR-Cas technologies has revolutionized functional genomics by enabling precise genetic manipulations in various model organisms. In popular vertebrate models, including mice and zebrafish, CRISPR has been adapted to high-throughput mutagenesis workflows, knock-in alleles and large-scale screens, bringing us closer to understanding gene functions in development, physiology and pathology. The development of innovative technologies, such as base editors, capable of single-nucleotide modifications, and prime editors, offering precision edits without double-strand breaks, exemplifies the expanding toolkit. In addition to gene editing, transcriptional modulation, that is, CRISPR interference and CRISPR activation systems, can elucidate the mechanisms of gene regulation. Newer methods, such as MIC-Drop and Perturb-seq, which increase screening throughput in vivo, hold significant promise to improve our ability to dissect complex biological processes and mechanisms. Furthermore, CRISPR-based gene therapies for treating sickle cell disease and other monogenic diseases have already demonstrated their potential for clinical translation. Here this Review covers the transformative impact of CRISPR-based tools in vertebrate models, highlighting their utility in functional genomics research and disease modeling.},
}

@article {pmid40745000,
year = {2025},
author = {Kim, MG and Go, MJ and Kang, SH and Jeong, SH and Lim, K},
title = {Revolutionizing CRISPR technology with artificial intelligence.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745000},
issn = {2092-6413},
support = {RS-2023-00210965 and RS-2024-00339116//National Research Foundation of Korea (NRF)/ ; 2V10573 and 2E33791//Korea Institute of Science and Technology (KIST)/ ; },
abstract = {Genome engineering has made remarkable strides, evolving from DNA-binding proteins such as zinc fingers and transcription activator-like effectors to CRISPR-Cas systems. CRISPR technology has revolutionized the field through its simplicity and ability to target specific genome regions via guide RNA and Cas proteins. Progress in CRISPR tools-CRISPR nucleases, base editors and prime editors-has expanded the toolkit to induce targeted insertions or deletions, nucleotide conversions and a wider array of genetic alterations. Nevertheless, variations in editing outcomes across cell types and unintended off-target effects still present substantial hurdles. Artificial intelligence (AI), which has seen rapid advances, provides high-level solutions to these problems. By leveraging large datasets from diverse experiments, AI enhances guide RNA design, predicts off-target activities and improves editing efficiency. In addition, AI aids in discovering and designing novel CRISPR systems beyond natural limitations. These developments provide new modalities essential for the innovation of personalized therapies and help to ensure efficiency, precision and safety. Here we discuss the transformative role of AI in advancing CRISPR technology. We highlight how AI contributes to refining nuclease-based editing, base editing and prime editing. Integrating AI with CRISPR technology enhances existing tools and opens doors to next-generation medicine for gene therapy.},
}

@article {pmid40744946,
year = {2025},
author = {Lysne, DP and Stewart, MH and Susumu, K and Leski, TA and Stenger, DA and Medintz, IL and Díaz, SA and Green, CM},
title = {Quantum dot molecular beacons achieve sub-10 pM CRISPR-Cas detection in field-ready assays.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27950},
pmid = {40744946},
issn = {2045-2322},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems/genetics ; Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Humans ; },
abstract = {CRISPR-Cas systems have revolutionized molecular diagnostics through their specificity and programmability, yet their broad adoption is hindered by the reliance on expensive and complex instrumentation. Here, we present an optimized quantum dot (QD) molecular beacon (QD-MB) platform that integrates Förster resonance energy transfer (FRET)-based detection with CRISPR-Cas functionality, achieving sub-picomolar sensitivity without the need for target amplification. By systematically tuning components, including His-tag modifications for improved QD conjugation, nucleic acid hairpin structures for enhanced enzyme interaction, and QD surface passivation strategies, we demonstrate a two-order-of-magnitude improvement in detection sensitivity. Using LwaCas13a and RNA targets, the limit of detection (LOD) decreased to under 1 pM with plate-reader-based fluorescence measurements and below 10 pM with a lamp-and-smartphone setup, establishing the feasibility of portable, field-ready applications. This work highlights the transformative potential of QD-MBs in biosensing and sets a foundation for further advances in CRISPR-based diagnostics and nanotechnology-enabled sensing platforms.},
}

*Quantum Dots/chemistry
*CRISPR-Cas Systems/genetics
Fluorescence Resonance Energy Transfer/methods
*Biosensing Techniques/methods
Limit of Detection
Humans

@article {pmid40744920,
year = {2025},
author = {Dib, C and Queenan, JA and Swartzrock, L and Willner, H and Denis, M and Ahmed, N and Moulana Zada, F and Borges, B and Charlesworth, CT and Lum, T and Yates, BP and Kwon, CY and Scorzo, AV and Davis, SC and Davis, JR and He, R and Xie, J and Gao, G and MacKenzie, TC and Liu, DR and Newby, GA and Czechowicz, AD},
title = {GFP-on mouse model for interrogation of in vivo gene editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7017},
doi = {10.1038/s41467-025-61449-y},
pmid = {40744920},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *Green Fluorescent Proteins/genetics/metabolism ; Dependovirus/genetics ; Mice ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; Genes, Reporter ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Codon, Nonsense ; },
abstract = {Gene editing technologies have revolutionized therapies for numerous genetic diseases. However, in vivo gene editing hinges on identifying efficient delivery vehicles for editing in targeted cell types, a significant hurdle in fully realizing its therapeutic potential. A model system to rapidly evaluate systemic gene editing would advance the field. Here, we develop the GFP-on reporter mouse, which harbors a nonsense mutation in a genomic EGFP sequence correctable by adenine base editor (ABE) among other genome editors. The GFP-on system was validated using single and dual adeno-associated virus (AAV9) encoding ABE8e and sgRNA. Intravenous administration of AAV9-ABE8e-sgRNA into adult GFP-on mice results in EGFP expression consistent with the tropism of AAV9. Intrahepatic delivery of AAV9-ABE8e-sgRNA into GFP-on fetal mice restores EGFP expression in AAV9-targeted organs lasting at least six months post-treatment. The GFP-on model provides an ideal platform for high-throughput evaluation of emerging gene editing tools and delivery modalities.},
}

Animals
*Gene Editing/methods
*Green Fluorescent Proteins/genetics/metabolism
Dependovirus/genetics
Mice
CRISPR-Cas Systems
Genetic Vectors/genetics
Genes, Reporter
Female
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Codon, Nonsense

@article {pmid40744728,
year = {2025},
author = {Sabin, CE and Lauderdale, JD and Menke, DB},
title = {Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108535},
pmid = {40744728},
issn = {1559-6095},
abstract = {Anolis lizards are an ecologically diverse group that includes more than 400 described species. These reptiles have been the subject of wide-ranging studies, from speciation and convergent evolution to climate adaptation and tail regeneration. While CRISPR-based gene editing has tremendous potential to reveal new insights into these and other aspects of Anolis biology, the reproductive biology of these reptiles has presented significant barriers to gene editing. Here, we briefly summarize gene editing approaches in vertebrates and discuss some of the major challenges associated with the performance of gene editing in anoles. We then introduce a recently established surgical procedure that enables the injection of CRISPR-Cas into the developing oocytes of female lizards. This approach circumvents the need to manipulate early-stage embryos and permits the production of gene-edited anoles. This method has recently been successfully adapted for use in other reptiles, suggesting that it may be effective in a wide range of species and will broadly enable studies of gene function in reptiles.},
}

@article {pmid40744574,
year = {2025},
author = {Zhang, Z and Wu, S and Long, Y and Huang, W and Bramlett, M and Yang, Y and Wu, Y},
title = {Identification of SfABCC2 as the critical receptor for Cry1Fa and Cry1Ab in Spodoptera frugiperda via CRISPR-mediated gene knockouts.},
journal = {Pesticide biochemistry and physiology},
volume = {213},
number = {},
pages = {106526},
doi = {10.1016/j.pestbp.2025.106526},
pmid = {40744574},
issn = {1095-9939},
mesh = {Animals ; *Spodoptera/genetics/metabolism/drug effects ; *Bacillus thuringiensis Toxins ; *Endotoxins/metabolism/pharmacology ; *Hemolysin Proteins/metabolism/pharmacology ; *Bacterial Proteins/metabolism/pharmacology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance/genetics ; Insecticides/pharmacology ; Bacillus thuringiensis ; },
abstract = {Insecticidal proteins from Bacillus thuringiensis (Bt) have been widely used to control major agricultural pests through genetically modified (GM) Bt crops. However, the development of resistance in target pests could undermine the effectiveness of Bt crops. Understanding the mechanisms of action of Bt insecticidal proteins and the resistance mechanisms in pests is crucial for developing effective resistance management strategies to sustain the use of Bt crops. In this study, CRISPR/Cas9 gene editing was used to investigate the functional roles of four genes (SfABCC2, SfABCC3, SfCad1, and SfAPN1) that encode putative receptors for Cry1 proteins in the fall armyworm, Spodoptera frugiperda, a globally significant pest. We created five homozygous knockouts, each with a substantial fragment deletion: SfCad1-KO, SfAPN1-KO, SfABCC2-KO, SfABCC3-KO, and SfC2/C3-KO (a double knockout of SfABCC2 and SfABCC3). Bioassay results revealed that SfCad1-KO, SfAPN1-KO, and SfABCC3-KO strains exhibited no resistance to Cry1Fa or Cry1Ab. In contrast, SfABCC2-KO and SfC2/C3-KO strains demonstrated high levels of resistance to Cry1Fa (>3300-fold) and Cry1Ab (>450-fold), demonstrating that SfABCC2 is pivotal to the insecticidal action of these two Bt proteins. As anticipated, all five knockouts generated in this study did not significantly impact susceptibility to Vip3Aa compared with the control strain. Our findings underscore the critical role of SfABCC2 in mediating Cry1Ab and Cry1Fa toxicity in S. frugiperda. Therefore, resistance monitoring program and resistance management tactics should focus on SfABCC2 mutations in field populations of S. frugiperda.},
}

Animals
*Spodoptera/genetics/metabolism/drug effects
*Bacillus thuringiensis Toxins
*Endotoxins/metabolism/pharmacology
*Hemolysin Proteins/metabolism/pharmacology
*Bacterial Proteins/metabolism/pharmacology
Gene Knockout Techniques
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
Insecticide Resistance/genetics
Insecticides/pharmacology
Bacillus thuringiensis

@article {pmid40744495,
year = {2025},
author = {Sokirniy, I and Inam, H and Tomaszkiewicz, M and Reynolds, J and McCandlish, D and Pritchard, J},
title = {A side-by-side comparison of variant function measurements using deep mutational scanning and base editing.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf738},
pmid = {40744495},
issn = {1362-4962},
support = {2141650//NSF/ ; T32GM108563/GF/NIH HHS/United States ; R35 GM133613/GF/NIH HHS/United States ; U01CA265709/BC/NCI NIH HHS/United States ; 2033673//NSF RECODE/ ; //Simons Center for Quantitative Biology at Cold Spring Harbor Laboratory/ ; },
mesh = {Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; *Mutation ; DNA, Complementary/genetics ; CRISPR-Cas Systems ; Molecular Sequence Annotation/methods ; Genetic Variation ; Genomics/methods ; Gene Library ; },
abstract = {Variant annotation is a crucial objective in mammalian functional genomics. Deep mutational scanning (DMS) using saturation libraries of complementary DNAs (cDNAs) is a well-established method for annotating human gene variants, but CRISPR base editing (BE) is emerging as an alternative. However, questions remain about how well high-throughput BE measurements can annotate variant function and the extent of downstream experimental validation required. This study is the first direct comparison of cDNA DMS and BE in the same lab and cell line. We focus on how well short guide RNA (sgRNA) depletion or enrichment is explained by the predicted edits within the editing "window" defined by the sgRNA. The most likely predicted edits enhance the agreement between a "gold standard" DMS dataset and a BE screen. A simple filter for sgRNAs making single edits in their window could sufficiently annotate a large proportion of variants directly from sgRNA sequencing of large pools. When multi-edit guides are unavoidable, directly measuring edits in medium-sized validation pools can recover high-quality variant annotation data. Our data show a surprisingly high degree of correlation between base editor data and gold standard DMS. We suggest that the main variable measured in base editor screens is the desired base edits.},
}

Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
*Mutation
DNA, Complementary/genetics
CRISPR-Cas Systems
Molecular Sequence Annotation/methods
Genetic Variation
Genomics/methods
Gene Library

@article {pmid40744490,
year = {2025},
author = {Cattle, MA and Aguado, LC and Sze, S and Venkittu, S and Wang, Y and Papagiannakopoulos, T and Smith, S and Rice, CM and Schneider, WM and Poirier, JT},
title = {An enhanced Eco1 retron editor enables precision genome engineering in human cells without double-strand breaks.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf716},
pmid = {40744490},
issn = {1362-4962},
support = {P30CA016087//NYU Laura & Isaac Perlmutter Cancer Center/ ; P30CA016087//NYU Laura & Isaac Perlmutter Cancer Center/ ; //Robertson Foundation/ ; R35GM149355/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA Breaks, Double-Stranded ; Zika Virus/genetics ; *Retroelements/genetics ; 3' Untranslated Regions/genetics ; HEK293 Cells ; RNA, Untranslated/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Genome, Human ; },
abstract = {Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5' sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5' sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
DNA Breaks, Double-Stranded
Zika Virus/genetics
*Retroelements/genetics
3' Untranslated Regions/genetics
HEK293 Cells
RNA, Untranslated/genetics/metabolism
CRISPR-Associated Protein 9/metabolism/genetics
Genome, Human

@article {pmid40743876,
year = {2025},
author = {Gardoul, M and Rached, B and Mbarki, A and Ajdig, M and Belouad, EM and Chouati, T and Chauiyakh, O and Lahcen Ouchari, and Harlé, O and Al-Nakeeb, K and Melloul, M and El Fahime, E},
title = {Comprehensive whole-genome analysis of Streptococcus infantarius strains from Moroccan farmhouse dairy products: Genomic insights into dairy adaptation, safety, and biotechnological potential.},
journal = {International journal of food microbiology},
volume = {442},
number = {},
pages = {111358},
doi = {10.1016/j.ijfoodmicro.2025.111358},
pmid = {40743876},
issn = {1879-3460},
abstract = {Streptococcus infantarius, a lactic acid bacterium prevalent in Moroccan dairy products, holds significant probiotic and biotechnological potential, yet its genomic features remain poorly characterized. This study aims to provide a comprehensive genomic characterization of six S. infantarius isolates (B50-6, B50-7, B51-6, O53-2, T41-8, K85-8) from Moroccan farmhouse dairy products. Whole-genome sequencing and comparative analysis were conducted using advanced bioinformatics tools to assess their genetic diversity, functional attributes, adaptive mechanisms, probiotic properties, safety profile, and potential applications in food fermentation. All strains were confirmed as S. infantarius with average nucleotide identity (ANI) >98.2 % and digital DNA-DNA hybridization (dDDH) >85.9 %. Genomes sizes ranging from 1.81 (K85-8) to 1.91 (T41-8) Mb, with GC content ∼37.4 %. Functional profiling identified 1343, 1343, 1345, 1342, 1359 and 1345 proteins with functional assignments; 519, 521, 536, 520, 542 and 502 hypothetical proteins; and 1752, 1754, 1778, 1752, 1781 and 1740 proteins with COG (Clusters of Orthologous Groups) assignments, in B50-6, B50-7, B51-6, O53-2, T41-8 and K85-8, respectively. Gene enrichment analysis highlighted key metabolic pathways and functional categories relevant to dairy adaptation and biotechnological potential. The total number of genes encoding carbohydrate-active enzymes was 45 (B50-6), 45 (B50-7), 48 (B51-6), 45 (O53-2), 48 (T41-8) and 48 (K85-8). Safety assessments identified 13 (K85-8) to 16 (B51-6) virulence-associated genes per strain. However, no antimicrobial resistance genes or plasmids were detected, while the presence of CRISPR-Cas systems was detected in most strains. A total of 88 mobile genetic elements were identified ranging from 9 (B51-6) to 18 (O53-2) per strain. In addition, prophages were detected in all strains with the exception of T41-8 and K85-8. All Strains exhibited diverse biosynthetic gene clusters (BGCs), including RiPP-like, T3PKS and terpene precursor pathways. These findings position S. infantarius as a relatively stable core genome, metabolically versatile candidate for dairy fermentation, probiotic and biotechnological applications.},
}

@article {pmid40742466,
year = {2025},
author = {Zhang, K and Wang, D and Hu, S and Li, X and Hong, J},
title = {Gene disruption via a transient hypercompact CRISPR-AsCas12f1 system in Kluyveromyces marxianus.},
journal = {Biotechnology letters},
volume = {47},
number = {4},
pages = {84},
pmid = {40742466},
issn = {1573-6776},
support = {202423m10050005//Anhui Provincial Science and Technology Innovation Program/ ; 231100110300//Major Special Science and Technology Project of Henan Province/ ; 32071469//National Natural Science Foundation of China/ ; },
mesh = {*Kluyveromyces/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Transfer/genetics ; },
abstract = {Kluyveromyces marxianus is an attractive chassis for microbial cell factories due to its rapid growth, thermotolerance, and wide substrate spectrum. However, gene disruption in this organism is challenging primarily due the prevalence of dominant nonhomologous recombination. AsCas12f1, a hypercompact CRISPR-associated protein consisting of 422 amino acids-approximately one-third the size of Cas9 or Cas12a-enables more efficient packaging into delivery vehicles than its larger counterparts. In this study, a gene disruption method using AsCas12f1 was established in K. marxianus through a transient targeting strategy. The integration of tRNA-gRNA into the gRNA construct increased gene disruption efficiency. Additionally, disrupting KmKU70 or KmLIG4 further increased this efficiency, achieving nearly 100%. By combining the disruption of KmKU70 with the AsCas12f1 system, the length of the homologous arm was shortened to 200 bp while maintaining a disruption efficiency of 87.5%. The implementation of the gRNA-tRNA-array system resulted in the successful generation of three single-gene knockout strains from a single transformation, resulting an overall efficiency of 86.4%. This approach leverages the transient transformation of fragments, eliminates the need for extensive time investment in constructing gRNA expression vectors and negates the requirement for the removal of the CRISPR-AsCas12f1 system after gene disruption. This study presents a novel strategy for gene disruption in K. marxianus and demonstrates the applicability of Cas12f in yeast systems.},
}

*Kluyveromyces/genetics
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
RNA, Transfer/genetics

@article {pmid40741393,
year = {2025},
author = {Wei, J and Motawaa, M and Li, Y},
title = {Thermus thermophilus CRISPR Cas6 Heterologous Expression and Purification.},
journal = {Bio-protocol},
volume = {15},
number = {14},
pages = {e5382},
pmid = {40741393},
issn = {2331-8325},
abstract = {The CRISPR-Cas system of Thermus thermophilus has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools. Key features • Robust method for expressing and purifying Thermus thermophilus Cas6 proteins in E. coli. • Seamless assembly cloning and dual affinity tagging system: Offers options for both His-tag and GST-tag purification strategies for increased versatility. • Applicable for diverse heterologous expression and purification of well-folding thermostable proteins in mesophilic host chassis cells [E. coli BL21(DE3)].},
}

@article {pmid40739342,
year = {2025},
author = {Ruffolo, JA and Nayfach, S and Gallagher, J and Bhatnagar, A and Beazer, J and Hussain, R and Russ, J and Yip, J and Hill, E and Pacesa, M and Meeske, AJ and Cameron, P and Madani, A},
title = {Design of highly functional genome editors by modelling CRISPR-Cas sequences.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40739342},
issn = {1476-4687},
abstract = {Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology and human health. CRISPR-based gene editors derived from microorganisms, although powerful, often show notable functional tradeoffs when ported into non-native environments, such as human cells[1]. Artificial-intelligence-enabled design provides a powerful alternative with the potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models[2] trained on biological diversity at scale, we demonstrate successful precision editing of the human genome with a programmable gene editor designed with artificial intelligence. To achieve this goal, we curated a dataset of more than 1 million CRISPR operons through systematic mining of 26 terabases of assembled genomes and metagenomes. We demonstrate the capacity of our models by generating 4.8× the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate that an artificial-intelligence-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 to facilitate broad, ethical use across research and commercial applications.},
}

@article {pmid40739316,
year = {2025},
author = {Callahan, A and Mojumdar, A and Hu, M and Wang, A and Griffith, AA and Huang, N and Chua, XY and Mroz, N and Puterbaugh, RZ and Reilly, SP and Salomon, A},
title = {The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27747},
doi = {10.1038/s41598-025-12951-2},
pmid = {40739316},
issn = {2045-2322},
support = {R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Antigen, T-Cell/metabolism ; *Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism/genetics ; Jurkat Cells ; *T-Lymphocytes/metabolism/immunology ; *Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism/genetics ; *Phosphotyrosine/metabolism ; Feedback, Physiological ; Signal Transduction ; CRISPR-Cas Systems ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; },
abstract = {The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, Lck[Y394]. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.},
}

Humans
*Receptors, Antigen, T-Cell/metabolism
*Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism/genetics
Jurkat Cells
*T-Lymphocytes/metabolism/immunology
*Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism/genetics
*Phosphotyrosine/metabolism
Feedback, Physiological
Signal Transduction
CRISPR-Cas Systems
Protein Tyrosine Phosphatase, Non-Receptor Type 1

@article {pmid40738762,
year = {2025},
author = {Gupta, PK and Kumar, S},
title = {Third-generation novel technologies for gene editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.012},
pmid = {40738762},
issn = {1879-3096},
abstract = {Gene editing technologies have revolutionized the field of biotechnology. CRISPR-Cas methods using RNA-guided enzymes are the most used gene editing tools and have produced gene-edited crops (rice, wheat, corn, etc.) and human therapeutics (Casgevy, approved for commercial use; Vertex Pharmaceuticals). However, these systems have some limitations, including the requirement of a protospacer adjacent motif sequence, generation of undesirable double-strand breaks (DSBs), and the inability to edit long genomic segments. Some of these limitations were partially addressed by the development of second-generation editors, including base editors (BEs) and prime editors (PEs). Third-generation gene editing technologies such as seekRNA and bridgeRNA can overcome most of these limitations and are the subject of this review.},
}

@article {pmid40690516,
year = {2025},
author = {Shigenobu, S and Yoda, S and Ohsawa, S and Suzuki, M},
title = {Refined CRISPR/Cas9 genome editing in the pea aphid uncovers the essential roles of Laccase2 in overwintering egg adaptation.},
journal = {PLoS genetics},
volume = {21},
number = {7},
pages = {e1011557},
doi = {10.1371/journal.pgen.1011557},
pmid = {40690516},
issn = {1553-7404},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Aphids/genetics/physiology ; *Laccase/genetics/metabolism ; Pigmentation/genetics ; *Insect Proteins/genetics ; *Adaptation, Physiological/genetics ; Ovum ; Female ; Seasons ; Diapause/genetics ; Gene Knockout Techniques ; },
abstract = {The production of overwintering eggs is a critical adaptation for winter survival among many insects. Melanization contributes to eggshell pigmentation and hardening, consequently enhancing resistance to environmental stress. The complex life cycle of the pea aphid (Acyrthosiphon pisum), a model hemipteran insect with remarkable reproductive capacity, involves cyclical parthenogenesis. It enables the production of black overwintering eggs that undergo obligate diapause to survive under unfavorable conditions. Laccase2 (Lac2) is essential for cuticle sclerotization and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid eggshell pigmentation and survival during diapause. To test the hypothesis, we used CRISPR/Cas9 ribonucleoprotein microinjections and a novel Direct Parental CRISPR (DIPA-CRISPR) method to knockout Lac2. In Lac2 knockout (KO) crispants (G0), pigment-less eggs correlated with induced indel rates. Additionally, eggshell pigmentation was completely lost in homozygous Lac2 knockouts, leading to embryonic lethality. Observation of late-stage embryos in KO diapause eggs suggested that lethality occurred during late embryogenesis or hatching. Furthermore, eggshell stiffness was significantly reduced in Lac2 KOs, highlighting the role of this gene in eggshell hardening. Moreover, fungal growth was observed in KO eggs. These findings reveal the essential roles of Lac2 in eggshell pigmentation, hardening, late embryonic development, hatching, and fungal protection, which are critical for pea aphid survival during overwintering diapause. This study also advances CRISPR/Cas9-mediated genome editing in pea aphids by addressing the challenges associated with their unique biology, including complex life cycles, obligatory diapause, bacterial endosymbiosis, inbreeding depression, and high nuclease activity. Our optimized protocol achieved efficient targeted mutagenesis and germline transmission, thereby generating stable KO lines. Additionally, we successfully applied DIPA-CRISPR to aphids by inducing mutations via adult oviparous female injections in fertilized eggs. These robust genome-editing protocols will facilitate functional studies in aphids, a key model for research on evolution, ecology, development, and agriculture.},
}

Animals
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Aphids/genetics/physiology
*Laccase/genetics/metabolism
Pigmentation/genetics
*Insect Proteins/genetics
*Adaptation, Physiological/genetics
Ovum
Female
Seasons
Diapause/genetics
Gene Knockout Techniques

@article {pmid40666904,
year = {2025},
author = {Pindi, C and Ahsan, M and Sinha, S and Palermo, G},
title = {Graph Attention Neural Networks Reveal TnsC Filament Assembly in a CRISPR-Associated Transposon.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40666904},
issn = {2692-8205},
abstract = {CRISPR-associated transposons (CAST) enable programmable, RNA-guided DNA integration, marking a transformative advancement in genome engineering. A central player in the type V-K CAST system is the AAA+ ATPase TnsC, which assembles into helical filaments on double-stranded DNA (dsDNA) to orchestrate target site recognition and transposition. Despite its essential role, the molecular mechanisms underlying TnsC filament nucleation and elongation remain poorly understood. Here, multiple-microsecond and free energy simulations are combined with deep learning-based Graph Attention Network (GAT) models to elucidate the mechanistic principles of TnsC filament formation and growth. Our findings reveal that ATP binding promotes TnsC nucleation by inducing DNA remodelling and stabilizing key protein-DNA interactions, particularly through conserved residues in the initiator-specific motif (ISM). Furthermore, GNN-based attention analyses identify a directional bias in filament elongation in the 5'→3' direction and uncover a dynamic compensation mechanism between incoming and bound monomers that facilitate directional growth along dsDNA. By leveraging deep learning-based graph representations, our GAT model provides interpretable mechanistic insights from complex molecular simulations and is readily adaptable to a wide range of biological systems. Altogether, these findings establish a mechanistic framework for TnsC filament dynamics and directional elongation, advancing the rational design of CAST systems with enhanced precision and efficiency.},
}

@article {pmid40570715,
year = {2026},
author = {Fang, T and Deng, Y and Chen, M and Luo, T and Ning, T and Chen, G},
title = {Nanoparticles-mediated intratumoral gene editing targeting PD-L1 and Galectin-9 for improved cancer immunotherapy.},
journal = {Biomaterials},
volume = {324},
number = {},
pages = {123511},
doi = {10.1016/j.biomaterials.2025.123511},
pmid = {40570715},
issn = {1878-5905},
mesh = {*Galectins/genetics ; Animals ; *Nanoparticles/chemistry ; *B7-H1 Antigen/genetics ; *Immunotherapy/methods ; Mice ; *Gene Editing/methods ; Humans ; Calcium Phosphates/chemistry ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems/genetics ; Female ; Mice, Inbred C57BL ; },
abstract = {PD-L1, a typical immune checkpoint expressed on tumor cells, reduces the effectiveness of T cell-mediated killing, which is further aggravated by Galectin-9 (Gal-9) co-expression through the TIM3/Gal-9 pathway. Although immune checkpoint inhibitors have shown promise in cancer therapy, limitations remain including low response rate, systemic toxicities, and the need of frequent treatments. Here, we described a dual knockout approach targeting PD-L1 and Gal-9 in tumor cells, achieved by nanoparticle-assisted CRISPR-Cas9 delivery, aimed at improved cancer immunotherapy. A calcium phosphate nanoparticle (CaP NP) was engineered for co-delivery of CRISPR-Cas9/sgRNA ribonucleoprotein (RNP) and initiation of anti-tumor immunity. Intratumoral administration of RNP-loaded CaP NPs effectively knocked out PD-L1 and Gal-9 in tumor cells, evoking robust anti-tumor immunity. Additionally, Ca[2+] overload due to the degradation of CaP NPs led to release of damage-associated molecular patterns (DAMPs) signals, further enhancing T-cell-mediated antitumor immune responses. Our results demonstrated that this treatment effectively evoked both local and systemic anti-tumor immune responses, significantly inhibiting the growth of primary and distant tumors in mouse models. Importantly, local treatment also altered the phenotypes of circulating tumor cells, as a substantial of circulating tumor cells originated from RNP-CaP-treated primary tumors and exhibited dual knockouts, which led to reduced lung metastasis.},
}

*Galectins/genetics
Animals
*Nanoparticles/chemistry
*B7-H1 Antigen/genetics
*Immunotherapy/methods
Mice
*Gene Editing/methods
Humans
Calcium Phosphates/chemistry
Cell Line, Tumor
*Neoplasms/therapy/immunology/genetics
CRISPR-Cas Systems/genetics
Female
Mice, Inbred C57BL

@article {pmid40550345,
year = {2025},
author = {Bedir, I and Ozturk, K and Telci, D},
title = {Impact of PLA2G2A rs11573156 C > G Variant on Phospholipase Expression and Metastatic Behavior in Prostate Cancer.},
journal = {Gene},
volume = {964},
number = {},
pages = {149641},
doi = {10.1016/j.gene.2025.149641},
pmid = {40550345},
issn = {1879-0038},
mesh = {Male ; Humans ; *Prostatic Neoplasms/genetics/pathology ; *Group II Phospholipases A2/genetics/metabolism ; Neoplasm Metastasis ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic ; PC-3 Cells ; *Polymorphism, Single Nucleotide ; Cell Line, Tumor ; Cell Movement/genetics ; CRISPR-Cas Systems ; Alleles ; },
abstract = {Prostate cancer (PCa) is a major global health concern and a leading cause of cancer-related deaths in men. Despite available treatments, PCa frequently recurs and exhibits high metastatic potential. One key factor in its malignancy is PLA2G2A, a secretory phospholipase A2 enzyme with strong inflammatory properties. Elevated PLA2G2A levels in the bloodstream have been linked to tumor grade, potentially due to the rs11573156 C > G polymorphism in the gene's 5' untranslated region (5'UTR). Previously, we demonstrated that individuals carrying the G allele have a 2.5-fold lower risk of developing metastatic PCa compared to those with the C allele. To further investigate the functional impact of this polymorphism, we employed CRISPR-Cas9 base editing to convert the GC genotype of metastatic PC-3 prostate cancer cells into the homozygous GG genotype. Our findings indicate that the G allele enhances PLA2G2A expression while downregulating genes associated with epithelial-to-mesenchymal transition (EMT), an effect reversed upon siRNA-mediated PLA2G2A silencing. Furthermore, this genetic alteration impaired the migratory capacity of PC-3 cells following interferon-γ (IFN-γ) priming, simulating an inflammatory tumor microenvironment. Notably, siRNA-induced downregulation of PLA2G2A reversed the anti-migratory effects associated with the GG genotype. Our results suggest that increased PLA2G2A expression, driven by the G allele, negatively affects cell proliferation, EMT, and metastatic properties, underscoring its potential anti-tumor role in PCa.},
}

Male
Humans
*Prostatic Neoplasms/genetics/pathology
*Group II Phospholipases A2/genetics/metabolism
Neoplasm Metastasis
Epithelial-Mesenchymal Transition/genetics
Gene Expression Regulation, Neoplastic
PC-3 Cells
*Polymorphism, Single Nucleotide
Cell Line, Tumor
Cell Movement/genetics
CRISPR-Cas Systems
Alleles

@article {pmid38489095,
year = {2025},
author = {Yu, S and Lei, X and Qu, C},
title = {MicroRNA Sensors Based on CRISPR/Cas12a Technologies: Evolution From Indirect to Direct Detection.},
journal = {Critical reviews in analytical chemistry},
volume = {55},
number = {5},
pages = {968-984},
doi = {10.1080/10408347.2024.2329229},
pmid = {38489095},
issn = {1547-6510},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; },
abstract = {MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and a potential therapeutic targets for drug development. The detection of miRNA can serve as a noninvasive tool in diseases diagnosis and predicting diseases prognosis. CRISPR/Cas12a system has great potential in nucleic acid detection due to its high sensitivity and specificity, which has been developed to be a versatile tool for nucleic acid-based detection of targets in various fields. However, conversion from RNA to DNA with or without amplification operation is necessary for miRNA detection based on CRISPR/Cas12a system, because dsDNA containing PAM sequence or ssDNA is traditionally considered as the activator of Cas12a. Until recently, direct detection of miRNA by CRISPR/Cas12a system has been reported. In this review, we provide an overview of the evolution of biosensors based on CRISPR/Cas12a for miRNA detection from indirect to direct, which would be beneficial to the development of CRISPR/Cas12a-based sensors with better performance for direct detection of miRNA.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans

@article {pmid40738541,
year = {2025},
author = {Choudhury, PR and Chakravarti, M and Banerjee, S and Baral, R and Bose, A},
title = {B-cell editing: An emerging alternative of precision oncotherapy.},
journal = {Advances in immunology},
volume = {166},
number = {},
pages = {103-135},
doi = {10.1016/bs.ai.2024.10.002},
pmid = {40738541},
issn = {1557-8445},
mesh = {Humans ; *Precision Medicine/methods ; *B-Lymphocytes/immunology/metabolism ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; Animals ; *Immunotherapy/methods ; *Cancer Vaccines/immunology/therapeutic use ; CRISPR-Cas Systems ; Antibodies, Monoclonal/therapeutic use/immunology ; },
abstract = {Lately, the urgency of precision medicine in cancer care through immunotherapy has reformed the arena of oncology. Although immunomodulatory therapeutics in cancer have been preliminarily concentrated on T-cells, emerging evidences have suggested that intra-tumoral B-cells and plasma cells have significant contributions in cancer prognosis primarily through the production of antibodies. B-cell oriented cancer vaccines have been used in early clinical trials of breast and other cancers after multiple preclinical studies. Passive immunotherapy via administration of monoclonal antibodies (mAbs) and emergence of anti-idiotypic antibodies have led to considerable advancement in oncotherapy. Endogenous production of mAbs would be of significant benefit in recurrent or residual malignancies and permanent infusion would help in the overcoming of issues related to pharmacodynamic variations observed in case of intravenous inoculations of bi or tri specific mAbs. This has directed towards the development of genome reprogrammed B-cells with the capability of yielding therapeutic mAbs independently. Genetic alteration through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) nucleases have enabled the introduction of transgenes into B-cell genome thereby stimulating the plasma cells to produce exogenous remedial antibodies. It also facilitates ex vivo B-cell editing to elevate specificities of antigen receptors and generate target specific antibody responses which cannot normally be evoked in patient's immune system. Hence, genome-altered B-cells possess the potential of engineered therapeutics against certain malignancies. Co-operation of B-cells in T-cell based vaccines are ultimate need for vaccine success. In this chapter, the mechanisms, challenges and potential advantages of B-cell editing in cancer immune therapy shall be explored. The prospects of B-cell editing in onco-therapy will be clearly elucidated with all its strength and weaknesses.},
}

Humans
*Precision Medicine/methods
*B-Lymphocytes/immunology/metabolism
*Neoplasms/therapy/immunology/genetics
*Gene Editing/methods
Animals
*Immunotherapy/methods
*Cancer Vaccines/immunology/therapeutic use
CRISPR-Cas Systems
Antibodies, Monoclonal/therapeutic use/immunology

@article {pmid40737091,
year = {2025},
author = {Jain, S and Planells, J and Regadas, I and Barrett, D and von Euler, A and Sinha, I and Lindberg, BG and Hesson, JC and Swacha, P and Gekara, NO and Pelechano, V and Engström, Y and Mannervik, M and Visa, N},
title = {A U3 snoRNA is required for the regulation of chromatin dynamics and antiviral response in Drosophila melanogaster.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf715},
pmid = {40737091},
issn = {1362-4962},
support = {2019-02335//Swedish Research Council/ ; 2020-01480//Swedish Research Council/ ; 2021-06112//Swedish Research Council/ ; KAW 2021.0167//Wallenberg Academy Fellowship/ ; //Karolinska Institute/ ; 2024-04173//Swedish Research Council/ ; 20 1044 Pj//The Swedish Cancer Society/ ; 23 2963 Pj//The Swedish Cancer Society/ ; 2022-01308_3//Swedish Research Council/ ; CAN 23 3096 Pj//The Swedish Cancer Society/ ; //Stockholm University/ ; //Stockholm University/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/virology/immunology ; *RNA, Small Nucleolar/genetics/metabolism ; *Chromatin/genetics/metabolism ; Drosophila Proteins/genetics/metabolism ; Sindbis Virus/immunology/genetics ; Chromatin Assembly and Disassembly/genetics ; Larva/genetics/virology ; CRISPR-Cas Systems ; Trans-Activators ; Cell Cycle Proteins ; },
abstract = {Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. Here we show that specific snoRNAs are required for the activation of immune response genes and for survival during viral infections in Drosophila melanogaster. We have studied snoRNA:U3:9B, a chromatin-associated snoRNA that binds to a large number of protein coding genes, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B and study its function in vivo. SnoRNA:U3:9B-deficient larvae are viable but failed to develop into pupae when challenged by expression of a Sindbis virus replicon. SnoRNA:U3:9B is localized to immune genes in vivo and the chromatin decompaction and gene activation typically observed at immune genes following infection are abolished in snoRNA:U3:9B-deficient larvae, which suggests that this snoRNA acts locally to regulate chromatin accessibility. Mechanistically, snoRNA:U3:9B is required for the recruitment of the chromatin remodeler Brahma to a set of target immune genes. In summary, these results uncover an antiviral defense mechanism that relies on a snoRNA for the recruitment of a chromatin remodeling factor to immune genes to facilitate immune gene activation.},
}

Animals
*Drosophila melanogaster/genetics/virology/immunology
*RNA, Small Nucleolar/genetics/metabolism
*Chromatin/genetics/metabolism
Drosophila Proteins/genetics/metabolism
Sindbis Virus/immunology/genetics
Chromatin Assembly and Disassembly/genetics
Larva/genetics/virology
CRISPR-Cas Systems
Trans-Activators
Cell Cycle Proteins

@article {pmid40736862,
year = {2025},
author = {Mercer, GD and Ballios, BG and Kertes, PJ},
title = {Mutation Specific Treatments for Inherited Retinal Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1467},
number = {},
pages = {337-341},
pmid = {40736862},
issn = {0065-2598},
mesh = {Humans ; *Genetic Therapy/methods ; *Retinal Diseases/genetics/therapy ; *Gene Editing/methods ; *Mutation ; CRISPR-Cas Systems ; Animals ; Oligonucleotides, Antisense/therapeutic use/genetics ; RNA Interference ; RNA Editing ; },
abstract = {The next frontier in genetic therapy for IRDs is the correction or silencing of specific mutations. This is of relevance to conditions with dominant negative and gain-of-function disease mechanisms, or with causative genes that are too large for gene replacement using AAV vectors. We discuss two approaches that have reached the stage of human clinical trials: CRISPR-Cas9 based gene editing and post-transcriptional gene silencing using antisense oligonucleotides. Other mutation-specific treatment approaches in pre-clinical development include adenosine deaminases acting on RNA (ADAR)-based RNA editing, RNA interference, and translational read through inducing drugs (TRADs). These have been reviewed elsewhere (e.g., Martinez Velazquez and Ballios 2021).},
}

Humans
*Genetic Therapy/methods
*Retinal Diseases/genetics/therapy
*Gene Editing/methods
*Mutation
CRISPR-Cas Systems
Animals
Oligonucleotides, Antisense/therapeutic use/genetics
RNA Interference
RNA Editing

@article {pmid40736578,
year = {2025},
author = {Allard-Chamard, X and Rodríguez, EC and Brais, B and Armstrong, GAB},
title = {Loss of dcst2 expression in male zebrafish is not associated with muscle hypertrophy.},
journal = {Molecular genetics and genomics : MGG},
volume = {300},
number = {1},
pages = {74},
doi = {10.1007/s00438-025-02279-x},
pmid = {40736578},
issn = {1617-4623},
support = {Natural Sciences and Engineering Research Council of Canada//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Male ; Hypertrophy/genetics ; CRISPR-Cas Systems ; *Membrane Proteins/genetics/metabolism ; *Muscle, Skeletal/metabolism/pathology ; Frameshift Mutation ; },
abstract = {Recently, a large family of French-Canadians was found to possess above-average strength and muscle hypertrophy that segregated with a single variant in the gene encoding Dendritic Cell-specific Six Transmembrane domain containing protein 2 (DCST2). To investigate the potential role DCST2 has in muscle cell biology we used the CRISPR/Cas9 mutagenic system and generated a 2-nucleotide deletion in exon 3 of zebrafish dcst2 resulting in a frameshift mutation. Homozygous carriers of the mutation displayed reduced transcriptional expression of dcst2 suggesting that our mutation disrupted gene expression. Homozygous mutant dcst2 zebrafish developed normally to adulthood and displayed no differences in motor function using a free-swim and swim tunnel assays. Furthermore, histological examination of muscle cells revealed no differences in slow-twitch or fast-twitch muscle cell cross-sectional area in our mutants. We did observe that male dcst2[-/-] zebrafish were infertile. The data collected here, suggest that dcst2 does not play a role in zebrafish muscle cell biology.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
Male
Hypertrophy/genetics
CRISPR-Cas Systems
*Membrane Proteins/genetics/metabolism
*Muscle, Skeletal/metabolism/pathology
Frameshift Mutation

@article {pmid40735630,
year = {2025},
author = {Wu, L and Yu, Z and Li, P and Wang, Z and Li, Q and Bao, H and García, P and Letarov, A and Zhou, Y and Zhang, H},
title = {Genome MLST scheme for tracing genetic diversity and multidrug resistance of food animal-derived Clostridium perfringens.},
journal = {Current research in food science},
volume = {11},
number = {},
pages = {101149},
pmid = {40735630},
issn = {2665-9271},
abstract = {Clostridium perfringens is an important food-borne anaerobic bacterium that can cause chronic disease in humans and animals. The complex toxins that it produces can cause diarrhea, necrotizing enteritis, and even death. This study aimed to evaluate the presence of antibiotic resistance genes and determinants of toxin production in various strains of C. perfringens, derived from different geographical locations and animal sources, through whole-genome sequencing (WGS) and bioinformatics analysis. Antimicrobial resistance testing revealed a notable prevalence of resistance among isolates, with 39.5 % being resistant to clindamycin and 32.6 % resistant to tetracycline. Additionally, 29.1 % of the isolates were found to be resistant to at least two classes of commonly used antibiotics. TetA(P) and tetB(P), associated with tetracycline resistance, were the most frequently identified resistance genes, present in 93.0 % and 79.0 % of the isolates, respectively. Virulence genes encoding extracellular collagenases (colA and colSI), alpha-toxin (plc), and sialidase (nanH) were detected in 85 isolates, representing 99 % of all sequenced strains. Notably, the gene encoding C. perfringens enterotoxin (cpe) was identified in only one isolate, which originated from chicken. Multi-locus sequence typing revealed that the 86 representative isolates belonged to 49 sequence types (STs), including 33 unique, previously uncharacterized STs. Furthermore, 30.23 % of these STs were grouped into six clonal complexes (CCs). CgSNP analysis of globally circulating isolates demonstrated that the prevalence of the virulence gene cpe was higher in these isolates than in clinical strains. The identification of multi-drug resistance and toxin-encoding genes among the isolates underscores the concerning spread of antimicrobial resistance among C. perfringens affecting both animals and humans. The diversity of CRISPR/Cas system provided addition insight into complex genetic correlations and evolutionary dynamics of pathogenic C. perfringens. Notably, the phage SD72 exhibited a broad inhibitory effect against C. perfringens isolates, irrespective of STs types and antimicrobial resistance (74/86, 80.1 %). These findings highlight the urgent need for enhanced epidemic surveillance of C. perfringens in livestock settings to mitigate the risks of human transmission via environmental or food sources. Additionally, bacteriophages present a promising avenue as bio-antimicrobial agents for controlling the transmission of C. perfringens from animal sources to humans.},
}

@article {pmid40733833,
year = {2025},
author = {Cheng, Y and Wang, Z and Cao, M and Liu, S and Zhang, X and Cheng, C and Cai, C and Zhang, X and Zheng, J},
title = {Structure-activity relationships study on inhibition of CRISPR-Cas9 by glycosaminoglycans.},
journal = {Carbohydrate polymers},
volume = {366},
number = {},
pages = {123912},
doi = {10.1016/j.carbpol.2025.123912},
pmid = {40733833},
issn = {1879-1344},
mesh = {*CRISPR-Cas Systems/drug effects ; Structure-Activity Relationship ; *Heparin/chemistry/pharmacology ; *Chondroitin Sulfates/chemistry/pharmacology ; *Glycosaminoglycans/chemistry/pharmacology ; Molecular Dynamics Simulation ; Gene Editing ; *CRISPR-Associated Protein 9/antagonists & inhibitors/metabolism ; Humans ; },
abstract = {The CRISPR-Cas9 system is a revolutionary genome editing system known for its precision, simplicity and efficiency, playing a crucial role in gene-editing. It has enabled applications ranging from biomedicine to agriculture. However, the uncontrollable activity of Cas9 has raised significant safety concerns in clinical settings, limiting its broader application. Consequently, regulating CRISPR-Cas9 activity holds substantial promise for enhancing the safety and efficacy of gene-editing technologies. In this study, we demonstrated that sulfated glycosaminoglycans (GAGs) exhibit inhibitory effects of Cas9. Specifically, both chondroitin sulfate (CS) and heparin (HP) can inhibit CRISPR/Cas9 activity, with heparin showing a stronger inhibitory effect that correlates positively with its concentration. Molecular dynamics simulations indicate that sulfated heparin residues might inhibit Cas9 function by binding to essential DNA-binding sites, which are crucial for functional interactions, potentially impairing activity. Additionally, higher molecular weight GAGs exhibit enhanced inhibitory effects under the same sulphation. Notably, the sulfation site also influenced activity. C6-sulfation of chondroitin sulfate is more favorable for Cas9 inhibition, and N-sulfation of heparin enhances its inhibitory effect on Cas9 activity. These findings provide valuable insights into the development of carbohydrate-based inhibitors for CRISPR-Cas9, offering a foundation for further exploration in this field.},
}

*CRISPR-Cas Systems/drug effects
Structure-Activity Relationship
*Heparin/chemistry/pharmacology
*Chondroitin Sulfates/chemistry/pharmacology
*Glycosaminoglycans/chemistry/pharmacology
Molecular Dynamics Simulation
Gene Editing
*CRISPR-Associated Protein 9/antagonists & inhibitors/metabolism
Humans

@article {pmid40733529,
year = {2025},
author = {Anastassopoulou, C and Tsakri, D and Panagiotopoulos, AP and Saldari, C and Sagona, AP and Tsakris, A},
title = {Armed Phages: A New Weapon in the Battle Against Antimicrobial Resistance.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070911},
pmid = {40733529},
issn = {1999-4915},
mesh = {*Bacteriophages/genetics/physiology ; Humans ; *Phage Therapy/methods ; *Bacterial Infections/therapy/microbiology ; *Bacteria/virology/drug effects ; Drug Resistance, Multiple, Bacterial ; CRISPR-Cas Systems ; Animals ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The increasing prevalence of multidrug-resistant (MDR) bacterial infections necessitates the exploration of alternative antimicrobial strategies, with phage therapy emerging as a viable option. However, the effectiveness of naturally occurring phages can be significantly limited by bacterial defense systems that include adsorption blocking, restriction-modification, CRISPR-Cas immunity, abortive infection, and NAD+ depletion defense systems. This review examines these bacterial defenses and their implications for phage therapy, while highlighting the potential of phages' bioengineering to overcome these barriers. By leveraging synthetic biology, genetically engineered phages can be tailored to evade bacterial immunity through such modifications as receptor-binding protein engineering, anti-CRISPR gene incorporation, methylation pattern alterations, and enzymatic degradation of bacterial protective barriers. "Armed phages", enhanced with antimicrobial peptides, CRISPR-based genome-editing tools, or immune-modulating factors, offer a novel therapeutic avenue. Clinical trials of bioengineered phages, currently SNIPR001 and LBP-EC01, showcase their potential to safely and effectively combat MDR infections. SNIPR001 has completed a Phase I clinical trial evaluating safety in healthy volunteers, while LBP-EC01 is in Phase II trials assessing its performance in the treatment of Escherichia coli-induced urinary tract infections in patients with a history of drug-resistant infections. As "armed phages" progress toward clinical application, they hold great promise for precision-targeted antimicrobial therapies and represent a critical innovation in addressing the global antibiotic resistance crisis.},
}

*Bacteriophages/genetics/physiology
Humans
*Phage Therapy/methods
*Bacterial Infections/therapy/microbiology
*Bacteria/virology/drug effects
Drug Resistance, Multiple, Bacterial
CRISPR-Cas Systems
Animals
Anti-Bacterial Agents/pharmacology

@article {pmid40733516,
year = {2025},
author = {Lin, L and Lui, WY and Ong, CP and Yau, MY and Jin, DY and Yuen, KS},
title = {CRISPR/Cas13-Mediated Inhibition of EBNA1 for Suppression of Epstein-Barr Virus Transcripts and DNA Load in Nasopharyngeal Carcinoma Cells.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070899},
pmid = {40733516},
issn = {1999-4915},
support = {UGC/FDS17/M07/22//Research Grants Council of the Hong Kong Special Administrative Region/ ; },
mesh = {Humans ; *Herpesvirus 4, Human/genetics/drug effects ; *Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/virology ; Cell Line, Tumor ; *Nasopharyngeal Neoplasms/virology ; *Viral Load ; *Epstein-Barr Virus Infections/virology/therapy ; DNA, Viral/genetics ; },
abstract = {Epstein-Barr virus (EBV), a double-stranded DNA virus, is implicated in nasopharyngeal carcinoma (NPC), with particularly high incidence in regions such as southern China and Hong Kong. Although NPC is typically treated with radio- and chemotherapy, outcomes remain poor for advanced-stage diagnoses, highlighting the need for targeted therapies. This study explores the potential of CRISPR/CRISPR-associated protein 13 (Cas13) technology to target essential EBV RNA in NPC cells. Previous research demonstrated that CRISPR/Cas9 could partially reduce EBV load, but suppression was incomplete. Here, the combination of CRISPR/Cas13 with CRISPR/Cas9 shows enhanced viral clearance. Long-term EBNA1 suppression via CRISPR/Cas13 reduced the EBV genome, improved CRISPR/Cas9 effectiveness, and identified suitable AAV serotypes for delivery. Furthermore, cotreatment increased NPC cell sensitivity to 5-fluorouracil and cisplatin. These findings underscore the potential of CRISPR/Cas13 as an anti-EBV therapeutic approach, effectively targeting latent EBV transcripts and complementing existing treatments. The study suggests a promising new direction for developing anti-EBV strategies, potentially benefiting therapies for NPC and other EBV-associated malignancies.},
}

Humans
*Herpesvirus 4, Human/genetics/drug effects
*Epstein-Barr Virus Nuclear Antigens/genetics/metabolism
*CRISPR-Cas Systems
*Nasopharyngeal Carcinoma/virology
Cell Line, Tumor
*Nasopharyngeal Neoplasms/virology
*Viral Load
*Epstein-Barr Virus Infections/virology/therapy
DNA, Viral/genetics

@article {pmid40733494,
year = {2025},
author = {Wo, L and Qi, S and Guo, Y and Sun, C and Yin, X},
title = {TRIM5α/Cyclophilin A-Modified MDBK Cells for Lentiviral-Based Gene Editing.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070876},
pmid = {40733494},
issn = {1999-4915},
support = {32370162//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Cyclophilin A/genetics/metabolism ; Cattle ; *Lentivirus/genetics ; Cell Line ; CRISPR-Cas Systems ; *Tripartite Motif Proteins/genetics/metabolism ; HIV-1/genetics ; Ubiquitin-Protein Ligases/genetics ; Humans ; },
abstract = {The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin-Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of TRIM5α, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a TRIM5α knock-out MDBK-iCas9 cell line MDBK-iCas9[TRIM5α-/-] without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9[TRIM5α-/-] displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9[TRIM5α-/-] and MDBK-WT cell lines, suggesting that CypA functions independently with TRIM5α as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine TRIM5α and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells.},
}

Animals
*Gene Editing/methods
*Cyclophilin A/genetics/metabolism
Cattle
*Lentivirus/genetics
Cell Line
CRISPR-Cas Systems
*Tripartite Motif Proteins/genetics/metabolism
HIV-1/genetics
Ubiquitin-Protein Ligases/genetics
Humans

@article {pmid40733490,
year = {2025},
author = {Jiang, X and Huang, Y and Jiang, Y and Yang, G and Zheng, X and Gao, S},
title = {CRISPR-Cas12a/RPA Dual-Readout Assay for Rapid Field Detection of Porcine Rotavirus with Visualization.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070872},
pmid = {40733490},
issn = {1999-4915},
support = {Grant No: SCKJ-JYRC-2023-29//the Project of Sanya Yazhou Bay Science and Technology City/ ; Grant No: 324QN343.//the Youth Fund Project of Hainan Provincial Natural Science Foundation/ ; },
mesh = {Animals ; Swine ; *Swine Diseases/virology/diagnosis ; *Rotavirus/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Rotavirus Infections/diagnosis/veterinary/virology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/metabolism/genetics ; Capsid Proteins/genetics ; Endodeoxyribonucleases/genetics ; Antigens, Viral/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a's collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10[2] copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management.},
}

Animals
Swine
*Swine Diseases/virology/diagnosis
*Rotavirus/genetics/isolation & purification
*CRISPR-Cas Systems
*Rotavirus Infections/diagnosis/veterinary/virology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/metabolism/genetics
Capsid Proteins/genetics
Endodeoxyribonucleases/genetics
Antigens, Viral/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins

@article {pmid40732725,
year = {2025},
author = {Naveed, S and Brown, JK and Mubin, M and Javed, N and Nawaz-Ul-Rehman, MS},
title = {Potential for Duplexed, In-Tandem gRNA-Mediated Suppression of Two Essential Genes of Tomato Leaf Curl New Delhi Virus in Crop Plants.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/pathogens14070679},
pmid = {40732725},
issn = {2076-0817},
support = {6427//Higher Education Commission of Pakistan/ ; },
mesh = {*Begomovirus/genetics ; *Plant Diseases/virology ; *Solanum lycopersicum/virology ; Capsicum/virology ; Nicotiana/virology ; CRISPR-Cas Systems ; Genome, Viral ; Capsid Proteins/genetics ; },
abstract = {Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1-4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293-993 nt) and rep (1561-2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species.},
}

*Begomovirus/genetics
*Plant Diseases/virology
*Solanum lycopersicum/virology
Capsicum/virology
Nicotiana/virology
CRISPR-Cas Systems
Genome, Viral
Capsid Proteins/genetics

@article {pmid40732701,
year = {2025},
author = {Hanssens, C and Van Cleemput, J},
title = {Applying CRISPR Technologies for the Treatment of Human Herpesvirus Infections: A Scoping Review.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/pathogens14070654},
pmid = {40732701},
issn = {2076-0817},
support = {202409/004//Ghent University/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Herpesviridae Infections/therapy/virology/genetics ; Gene Editing/methods ; *Herpesviridae/genetics ; Antiviral Agents/therapeutic use ; *Genetic Therapy/methods ; Virus Latency/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Human herpesviruses are double-stranded DNA viruses of which eight types have been identified at present. Herpesvirus infection comprises an active lytic phase and a lifelong latency phase with the possibility of reactivation. These infections are highly prevalent worldwide and can lead to a broad spectrum of clinical manifestations, ranging from mild symptoms to severe disease, particularly in immunocompromised individuals. Clustered regularly interspaced palindromic repeats (CRISPR)-based therapy is an interesting alternative to current antiviral drugs, which fail to cure latent infections and are increasingly challenged by viral resistance.

OBJECTIVE: This scoping review aimed to summarize the current state of CRISPR-based antiviral strategies against herpesvirus infections, highlighting the underlying mechanisms, study design and outcomes, and challenges for clinical implementation.

DESIGN: A literature search was conducted in the databases PubMed and Web of Science, using both a general and an individual approach for each herpesvirus.

RESULTS: This scoping review identified five main mechanisms of CRISPR-based antiviral therapy against herpesvirus infections in vitro and/or in vivo. First, CRISPR systems can inhibit the active lytic replication cycle upon targeting viral lytic genes or host genes. Second, CRISPR technologies can remove latent viral genomes from infected cells by targeting viral genes essential for latency maintenance or destabilizing the viral genome. Third, reactivation of multiple latent herpesvirus infections can be inhibited by CRISPR-Cas-mediated editing of lytic viral genes, preventing a flare-up of clinical symptoms and reducing the risk of viral transmission. Fourth, CRISPR systems can purposefully induce viral reactivation to enhance recognition by the host immune system or improve the efficacy of existing antiviral therapies. Fifth, CRISPR technology can be applied to develop or enhance the efficiency of cellular immunotherapy.

CONCLUSIONS: Multiple studies demonstrate the potential of CRISPR-based antiviral strategies to target herpesvirus infections through various mechanisms in vitro and in vivo. However, aspects regarding the delivery and biosafety of CRISPR systems, along with the time window for treatment, require further investigation before broad clinical implementation can be realized.},
}

Humans
*CRISPR-Cas Systems
*Herpesviridae Infections/therapy/virology/genetics
Gene Editing/methods
*Herpesviridae/genetics
Antiviral Agents/therapeutic use
*Genetic Therapy/methods
Virus Latency/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40732192,
year = {2025},
author = {Yang, K and Wu, X and Ding, H and Ma, B and Li, Z and Wang, Y and Yang, Z and Yao, X and Luo, Y},
title = {Isolation, Identification, and Antibiotic Resistance, CRISPR System Analysis of Escherichia coli from Forest Musk Deer in Western China.},
journal = {Microorganisms},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/microorganisms13071683},
pmid = {40732192},
issn = {2076-2607},
abstract = {Escherichia coli (E. coli) is an opportunistic pathogen widely distributed in nature, and multi-drug resistance (MDR) E. coli has been widely recognized as a critical reservoir of resistance genes, posing severe health threats to humans and animals. A total of 288 E. coli strains were isolated and purified from fresh fecal samples of forest musk deer collected from farms in Sichuan, Shaanxi, and Yunnan Provinces of China between 2013 and 2023. This study aimed to conduct antibiotic susceptibility testing and resistance gene detection on the isolated forest musk deer-derived E. coli, analyze the correlations between them, investigate the presence of CRISPR systems within the strains, and perform bioinformatics analysis on the CRISPR systems carried by the strains. Results showed that 138 out of 288 E. coli strains were MDR, with the highest resistance to tetracycline (48.3%), cefalexin (45.1%), and doxycycline (41.7%). Prevalent genes were tetA (41.0%), sul2 (30.2%), blaTEM (27.1%), with 29 gene-phenotype pairs correlated. CRISPR system-negative strains had higher resistance rates to 16 antibiotics and lower detection rates only for aac (6')-Ib-cr, qnrA, and qnrB compared to CRISPR system-positive strains. Regional analysis showed that the problem of drug resistance in Sichuan and Shaanxi was more serious, and that the detection rate of antibiotic resistance genes was relatively high. This study guides E. coli infection control in forest musk deer and enriches resistance research data.},
}

@article {pmid40732175,
year = {2025},
author = {Di, T and Zhang, H and Zhang, C and Tian, L and Chang, M and Han, W and Qiao, R and Li, M and Zhang, S and Yang, G},
title = {Complete Genome and Characterization Analysis of a Bifidobacterium animalis Strain Isolated from Wild Pigs (Sus scrofa ussuricus).},
journal = {Microorganisms},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/microorganisms13071666},
pmid = {40732175},
issn = {2076-2607},
support = {092102110088, 212102110001, 22210320010//This study was supported by the Key R&D and Promotion Program in Henan Province of China/ ; },
abstract = {Bifidobacterium is a predominant probiotic in animals that is associated with host intestinal health. The protective mechanisms of the Bifidobacterium animalis (B. animalis) strain, specifically those related to functional gene-host interactions in intestinal homeostasis, remain poorly elucidated. This study reports the complete genome sequence and characterization of a B. animalis strain isolated from wild pig feces, which comprised a single circular chromosome (1,944,022 bp; GC content 60.49%) with 1567 protein-coding genes, and the B. animalis strain had certain acid resistance, bile salt resistance, gastrointestinal fluid tolerance, and antibacterial characteristics. Genomic annotation revealed three putative genomic islands and two CRISPR-Cas systems. Functional characterization identified genes encoding carbohydrate-active enzymes (CAZymes) and associated metabolic pathways, indicating that this strain can degrade complex dietary carbohydrates and synthesize bioactive metabolites for gut homeostasis. Although the antibiotic resistance genes were predicted, phenotypic assays demonstrated discordant resistance patterns, indicating complex regulatory networks. This study indicated the genomic basis of Bifidobacterium-host crosstalk in intestinal protection, providing a framework for developing novel probiotic interventions.},
}

@article {pmid40731998,
year = {2025},
author = {Gagaletsios, LA and Kikidou, E and Galbenis, C and Bitar, I and Papagiannitsis, CC},
title = {Exploring Virulence Characteristics of Clinical Escherichia coli Isolates from Greece.},
journal = {Microorganisms},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/microorganisms13071488},
pmid = {40731998},
issn = {2076-2607},
abstract = {The aim of this study was to examine the genetic characteristics that could be associated with the virulence characteristics of Escherichia coli collected from clinical samples. A collection of 100 non-repetitive E. coli isolates was analyzed. All isolates were typed by MLST. String production, biofilm formation and serum resistance were examined for all isolates. Twenty E. coli isolates were completely sequenced Illumina platform. The results showed that the majority of E. coli isolates (87%) produced significant levels of biofilm, while none of the isolates were positive for string test and resistance to serum. Additionally, the presence of CRISPR/Cas systems (type I-E or I-F) was found in 18% of the isolates. Analysis of WGS data found that all sequenced isolates harbored a variety of virulence genes that could be implicated in adherence, invasion, iron uptake. Also, WGS data confirmed the presence of a wide variety of resistance genes, including ESBL- and carbapenemase-encoding genes. In conclusion, an important percentage (87%) of the E. coli isolates had a significant ability to form biofilm. Biofilms, due to their heterogeneous nature and ability to make microorganisms tolerant to multiple antimicrobials, complicate treatment strategies. Thus, in combination with the presence of multidrug resistance, expression of virulence factors could challenge antimicrobial therapy of infections caused by such bacteria.},
}

@article {pmid40731176,
year = {2025},
author = {Wang, Y and Zhu, K and Wang, Y and Wang, Y and Xu, J and Li, Y and Liu, F and Long, J and Yang, H},
title = {The application of the CRISPR-Cas system in Klebsiella pneumoniae infections.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {766},
pmid = {40731176},
issn = {1573-4978},
support = {2023YFC2605603//National Key Research and Development Project/ ; 82273696//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Klebsiella pneumoniae/genetics/drug effects/pathogenicity ; Humans ; Gene Editing/methods ; *Klebsiella Infections/genetics/microbiology/therapy/drug therapy ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Klebsiella pneumoniae (Kp) is an important conditional pathogen that causes bacteremia, pneumonia, sepsis, urinary tract infections, and liver abscesses. The emergence of multidrug-resistant Kp, especially carbapenem-resistant Kp (CRKP), has become a major challenge to global public health. There is a need for early identification and diagnosis of Kp pathogens as well as precise treatment of Kp infections. The CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and associated Cas proteins) system is an adaptive immune mechanism for bacteria. Studies have shown that the presence of the CRISPR-Cas system is negatively correlated with drug resistance in Kp strains, and CRISPR-Cas system-based technologies have been developed for the detection, gene editing, the development of therapeutic drugs and vaccines in Kp infections. However, no review has been published on this area; therefore, this review describes the role of the CRISPR-Cas system in the development of antibiotic resistance in Kp, the application of CRISPR-Cas system-based technology in detection and gene editing, and the therapeutic potential of CRISPR-Cas system in Kp infections.},
}

*CRISPR-Cas Systems/genetics
*Klebsiella pneumoniae/genetics/drug effects/pathogenicity
Humans
Gene Editing/methods
*Klebsiella Infections/genetics/microbiology/therapy/drug therapy
Drug Resistance, Multiple, Bacterial/genetics
Anti-Bacterial Agents/pharmacology

@article {pmid40731099,
year = {2025},
author = {Santinha, AJ and Strano, A and Platt, RJ},
title = {Methods and applications of in vivo CRISPR screening.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41576-025-00873-8},
pmid = {40731099},
issn = {1471-0064},
abstract = {A fundamental goal in genetics is to understand the connection between genotype and phenotype in health and disease. Genetic screens in which dozens to thousands of genetic elements are perturbed in a pooled fashion offer the opportunity to generate large-scale, information-rich and unbiased genotype-phenotype maps. Although typically applied in reductionist in vitro settings, methods enabling pooled CRISPR-Cas perturbation screening in vivo are gaining attention as they have the potential to accelerate the discovery and annotation of gene function across cells, tissues, developmental stages, disease states and species. In this Review, we discuss essential criteria for understanding, designing and implementing in vivo screening experiments, with a focus on pooled CRISPR-based screens in mice. We also highlight how the resulting datasets, combined with advances in multi-omics and artificial intelligence, will accelerate progress and enable fundamental discoveries across basic and translational sciences.},
}

@article {pmid40731026,
year = {2025},
author = {Geng, XL and Li, JY and Xu, HY and Wu, JP and Tao, DL and Chen, JM and Fan, YY and Yang, X and Song, JK and Zhao, GH},
title = {Proline dehydrogenase, a rate-limiting catabolic enzyme, affecting the growth and pathogenicity of Toxoplasma gondii tachyzoites by regulating the proline metabolism and mitochondrial function of the parasite.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {309},
doi = {10.1186/s13071-025-06966-x},
pmid = {40731026},
issn = {1756-3305},
mesh = {*Toxoplasma/pathogenicity/growth & development/enzymology/genetics/metabolism ; *Proline Oxidase/genetics/metabolism ; *Proline/metabolism ; *Mitochondria/metabolism ; Animals ; Virulence ; Mice ; Reactive Oxygen Species/metabolism ; Membrane Potential, Mitochondrial ; Mice, Inbred BALB C ; Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Female ; },
abstract = {BACKGROUND: The pathogenicity of Toxoplasma gondii is closely associated with its intracellular lytic cycle in host cells. Currently, the mechanisms by which T. gondii completes the lytic cycle remain unclear. The proline metabolism has been reported to be crucial for intracellular growth of pathogens by providing energy and nutrients. However, it remains unclear whether the intracellular growth and pathogenicity of T. gondii are related to proline metabolism.

METHODS: The gene-edited strains of proline dehydrogenase (Tgprodh) were constructed by using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. The effects of the Tgprodh gene on the growth in vitro and pathogenicity in vivo of the tachyzoites for T. gondii were studied through proliferation, plaque, invasion, egress and virulence assays. The effects of the Tgprodh gene on mitochondrial function were studied by using reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψm), adenosine triphosphate (ATP) assay kits, mitochondrial DNA (mtDNA) copy numbers, transmission electron microscopy (TEM) analysis, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The effects of the Tgprodh gene on proline metabolism were studied by using L-proline (L-Pro), L-glutamic acid (L-Glu), L-glutamine (L-Gln) assay kits, and RT-qPCR.

RESULTS: TgPRODH, the first rate-limiting enzyme in proline metabolism, was identified to be encoded by T. gondii and localized in the cytoplasm of T. gondii. Deletion of the Tgprodh gene resulted in significant growth inhibition in vitro and reduced pathogenicity in vivo of T. gondii. Further study found that deletion of the Tgprodh gene caused damage to the mitochondrial morphology, decreased membrane potential, mtDNA copy numbers, and the production of ATP and ROS. The expression of genes for maintaining mitochondrial integrity was downregulated in the Tgprodh-knockout strain of T. gondii, while complementation of the Tgprodh gene restored these defects in this parasite. Meantime, the deletion of the Tgprodh gene resulted in the accumulation of proline, reduced the contents of glutamate and glutamine, and affected the expression of genes related to proline catabolism in T. gondii.

CONCLUSIONS: The present study found the significance of the Tgprodh gene for the intracellular growth and pathogenicity of T. gondii through regulating mitochondrial function and the proline metabolism and provided a novel insight to reveal intracellular survival strategies of T. gondii.},
}

*Toxoplasma/pathogenicity/growth & development/enzymology/genetics/metabolism
*Proline Oxidase/genetics/metabolism
*Proline/metabolism
*Mitochondria/metabolism
Animals
Virulence
Mice
Reactive Oxygen Species/metabolism
Membrane Potential, Mitochondrial
Mice, Inbred BALB C
Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
Female

@article {pmid40730819,
year = {2025},
author = {Schertzer, MD and Stirn, A and Isaev, K and Pereira, L and Park, SH and Das, A and Réal, A and Jeffery, ED and Harbison, C and Wessels, HH and Sheynkman, GM and Sanjana, NE and Knowles, DA},
title = {Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6948},
doi = {10.1038/s41467-025-62066-5},
pmid = {40730819},
issn = {2041-1723},
support = {F32GM142213//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R21CA272345//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01AI176601//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Exons/genetics ; Protein Isoforms/genetics/metabolism ; *Computational Biology/methods ; Alternative Splicing ; HEK293 Cells ; *RNA/genetics/metabolism ; },
abstract = {Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we develop and methodically test an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions. In parallel, we provide computational tools for experimental design and analysis. In this study, we demonstrate that junction-targeting achieves robust and isoform-specific RNA knockdown across diverse alternative isoform events, genes, and cell types.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Knockdown Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Exons/genetics
Protein Isoforms/genetics/metabolism
*Computational Biology/methods
Alternative Splicing
HEK293 Cells
*RNA/genetics/metabolism

@article {pmid40730529,
year = {2025},
author = {Wang, P and Wu, M and Du, J},
title = {[Generation of a dense granule protein 3 gene-deficient strain of Toxoplasma gondii and its virulence testing].},
journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control},
volume = {37},
number = {3},
pages = {304-309},
doi = {10.16250/j.32.1915.2024293},
pmid = {40730529},
issn = {1005-6661},
support = {82472313//National Natural Science Foundation of China/ ; AHWJ2023A30013//Scientific Research Project of Anhui Provincial Health Commission/ ; },
mesh = {*Toxoplasma/genetics/pathogenicity ; *Protozoan Proteins/genetics/metabolism ; Virulence ; Animals ; Mice ; Humans ; CRISPR-Cas Systems ; *Toxoplasmosis/parasitology ; *Antigens, Protozoan/genetics ; },
abstract = {OBJECTIVE: To generate a dense granule protein 3 (GRA3) gene-deficient mutant of the Toxoplasma gondii ME49 strain and to test the virulence of the mutant.

METHODS: Gene-deficient parasites were generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Guide RNA (gRNA) was designed using the E-CRISPR software, and mutated on the pSAG1::Cas9-U6::sgUPRT plasmid using the Q5 site-directed mutagenesis kit to generate the pSAG1::Cas9-U6::sgGRA3 plasmid. A GRA3 donor plasmid containing GRA3 gene upstream sequences, pyrimethamine resistant gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) and GRA3 gene downstream sequence was generated, and GRA3 donor DNA was amplified using PCR assay. The pSAG1::Cas9-U6::sgGRA3 plasmid and GRA3 donor DNA were electroporated into tachyzoites of the wild-type T. gondii ME49 strain. Then, parasite suspensions were inoculated into human foreskin fibroblast (HFF) cells and screened with pyrimethamine to yield pyrimethamine-resistant parasites for monoclonal screening. The GRA3 gene deficient monoclonal strain (ME49Δgra3) of T. gondii was identified using PCR and Western blotting assays, and the expression of GRA3 protein was determined in the T. gondii ME49Δgra3 strain using Western blotting. Subsequently, 1 000 freshly lysed tachyzoites of T. gondii ME49 and ME49Δgra3 strains were transferred to 12-well plates seeded with HFF cells, and incubated at 37 °C containing 5% CO2 for 7 days, and the number of plaques was counted by staining with crystal violet solutions. HFF cells infected with tachyzoites of T. gondii ME49 and ME49Δgra3 strains were stained using Giemsa solutions, and the numbers of cells containing 1, 2, 4, and > 4 T. gondii parasitophorous vacuoles were counted. In addition, the survival rates of C57BL/6 mice infected with T. gondii ME49 and ME49Δgra3 strains were compared 35 days post-infection.

RESULTS: PCR assay revealed successful amplification of both the upstream and downstream homologous arm bands of the DHFR-TS gene in the T. gondii ME49Δgra3 strain, and no corresponding bands were amplified in the ME49 strain. The GRA3 band was amplified in the ME49 strain, and the DHFR-TS band, rather than GRA3 band, was amplified in the ME49Δgra3 strain. Western blotting determined absence of GRA3 protein expression in the ME49Δgra3 strain. Crystal violet staining showed that the T. gondii ME49 strain produced more plaques than the ME49Δgra3 strain [(352.67 ± 26.39) plaques vs. (235.00 ± 26.29) plaques; t = 5.472, P < 0.01], and Giemsa staining revealed that the proportion of T. gondii parasitophorous vacuoles containing at least four T. gondii tachyzoites was higher in HFF cells infected with the ME49 strain than in those infected with the T. gondii ME49Δgra3 strain [(75.67 ± 2.52)% vs. (59.67 ± 2.31)%; t = 8.113, P < 0.01], and the proportion of T. gondii parasitophorous vacuoles containing at least 1 or 2 T. gondii tachyzoites was higher in HFF cells infected with the T. gondii ME49 strain than in those infected with the T. gondii ME49Δgra3 strain [(24.33 ± 2.52)% vs. (40.33 ± 2.31)%; t = -8.113, P < 0.01]. In addition, mice infected with the T. gondii ME49 and ME49Δgra3 strains started to die 8 and 9 days post-infection, and the 35-day mortality rates of mice infected with T. gondii ME49 and ME49Δgra3 strains were 10.00% and 70.00% post-infection (χ[2] = 6.762, P < 0.01).

CONCLUSIONS: The T. gondii ME49Δgra3 strain has been successfully generated, and GRA3 protein may increase the virulence of the T. gondii ME49 strain.},
}

*Toxoplasma/genetics/pathogenicity
*Protozoan Proteins/genetics/metabolism
Virulence
Animals
Mice
Humans
CRISPR-Cas Systems
*Toxoplasmosis/parasitology
*Antigens, Protozoan/genetics

@article {pmid40728780,
year = {2025},
author = {Irum, S and Biswas, S and Cilkiz, M and Tsakirpaloglou, N and Thomson, MJ and Septiningsih, EM},
title = {Multiplex CRISPR-Cas9 editing of chlorophyll biosynthesis genes in chickpea via protoplast and Agrobacterium-mediated transformation.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {163},
pmid = {40728780},
issn = {1438-7948},
mesh = {*Cicer/genetics/metabolism ; *CRISPR-Cas Systems ; *Chlorophyll/biosynthesis/genetics ; *Gene Editing/methods ; Protoplasts/metabolism ; Agrobacterium/genetics ; Transformation, Genetic ; Plant Proteins/genetics/metabolism ; },
abstract = {Chickpea is an important legume consumed worldwide and a rich source of protein. Chickpea is less amenable to recent gene editing techniques despite its economic significance. Accelerating the improvement process and enabling novel trait development in chickpea will require new approaches for genetic intervention. The CRISPR system has been used in different plant species to generate genetic variation and manipulate gene functions, facilitating studies on gene function and crop improvement. To implement genome editing in chickpea, genes involved in the chlorophyll biosynthesis pathway were selected as targets for gene editing. A construct (pTrans_100-Chbio) carrying gRNAs for chlorophyllide a oxygenase (CAO) and chlorophyll synthase (CHLG), along with the Cas9 protein, was introduced into chickpea protoplasts via PEG-mediated transformation. Multiple edits containing deletions and base insertions were identified after protoplast transformation, as confirmed by Sanger sequencing. Afterward, Agrobacterium transformation of explants was performed, resulting in the successful regeneration of pale and chimeric yellow tissues, subsequently confirmed as containing largely substitutions, as detected through deep amplicon sequencing. Edited plants showed yellowish leaves and lower chlorophyll content. Our results indicated that chlorophyll biosynthesis pathway genes played an essential role in chlorophyll degradation and ROS scavenging to regulate both natural and induced chickpea senescence. We established an efficient and feasible CRISPR/Cas9-based editing system in chickpea that successfully generates allelic mutations and phenotypic variation. The established platform can be a foundation for future functional studies and precise genome editing of additional agronomic traits, ultimately contributing to chickpea crop improvement and sustainable agriculture.},
}

*Cicer/genetics/metabolism
*CRISPR-Cas Systems
*Chlorophyll/biosynthesis/genetics
*Gene Editing/methods
Protoplasts/metabolism
Agrobacterium/genetics
Transformation, Genetic
Plant Proteins/genetics/metabolism

@article {pmid40728527,
year = {2025},
author = {Pelea, O and Mayes, S and Ferry, QRV and Fulga, TA and Sauka-Spengler, T},
title = {Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {40728527},
issn = {2050-084X},
support = {grant EP/L016494/1//UKERC/ ; 10.35802/215615/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Zebrafish/embryology/genetics ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; Humans ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/metabolism ; Streptococcus pyogenes/genetics/enzymology ; Gene Editing/methods ; *Transcriptional Activation ; },
abstract = {Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Engineered sgRNAs become activated upon recognising complementary RNAs, thus enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimisations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAs in vivo. Our sensors open up novel opportunities for developing new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers.},
}

*Zebrafish/embryology/genetics
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
Humans
*CRISPR-Cas Systems
Embryo, Nonmammalian/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*RNA/metabolism
Streptococcus pyogenes/genetics/enzymology
Gene Editing/methods
*Transcriptional Activation

@article {pmid40725091,
year = {2025},
author = {Bi, M and Wang, Z and Li, K and Ren, Y and Ma, D and Mo, X},
title = {CRISPR/Cas12a-Based One-Tube RT-RAA Assay for PoRV Genotyping.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
pmid = {40725091},
issn = {1422-0067},
support = {32071476//National Natural Science Foundation in China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Swine ; *Rotavirus/genetics ; Genotype ; *Genotyping Techniques/methods ; *Swine Diseases/virology/diagnosis ; *Rotavirus Infections/virology/veterinary/diagnosis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Porcine rotavirus (PoRV), a primary etiological agent of viral diarrhea in piglets, frequently co-infects with other enteric pathogens, exacerbating disease severity and causing substantial economic losses. Its genetic recombination capability enables cross-species transmission potential, posing public health risks. Globally, twelve G genotypes and thirteen P genotypes have been identified, with G9, G5, G3, and G4 emerging as predominant circulating strains. The limited cross-protective immunity between genotypes compromises vaccine efficacy, necessitating genotype surveillance to guide vaccine development. While conventional molecular assays demonstrate sensitivity, they lack rapid genotyping capacity and face technical limitations. To address this, we developed a novel diagnostic platform integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-Cas12a. This system employs universal primers for the simultaneous amplification of G4/G5/G9 genotypes in a single reaction, coupled with sequence-specific CRISPR recognition, achieving genotyping within 50 min at 37 °C with 10[0] copies/μL sensitivity. Clinical validation showed a high concordance with reverse transcription quantitative polymerase chain reaction (RT-qPCR). This advancement provides an efficient tool for rapid viral genotyping, vaccine compatibility evaluation, and optimized epidemic control strategies.},
}

*CRISPR-Cas Systems/genetics
Animals
Swine
*Rotavirus/genetics
Genotype
*Genotyping Techniques/methods
*Swine Diseases/virology/diagnosis
*Rotavirus Infections/virology/veterinary/diagnosis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40685572,
year = {2025},
author = {Liang, Z and Yu, Y and Chen, X and Qin, L and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {Breakthrough in LbCas12a Chemical Inhibitor Screening: The Firefly Probe and Its Application in Dual-Mode Biosensors.},
journal = {Analytical chemistry},
volume = {97},
number = {29},
pages = {16050-16057},
doi = {10.1021/acs.analchem.5c03437},
pmid = {40685572},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Fluorescent Dyes/chemistry ; Hydrogen Peroxide/pharmacology/chemistry ; *CRISPR-Associated Proteins/antagonists & inhibitors/metabolism ; Glucose/analysis ; Molecular Docking Simulation ; Catechols/pharmacology/chemistry ; Catalase/analysis/metabolism ; Hydroquinones/pharmacology/chemistry ; CRISPR-Cas Systems/drug effects ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Biosensors employing anti-CRISPR (Acr) proteins have been designed, and prohibitive manufacturing expenses and unfavorable storage conditions still restrict Acr protein applications. Since chemical inhibitors have lower production costs and are relatively insensitive to storage conditions, they are more accessible and easier to store and transport than protein inhibitors. Furthermore, they could be efficiently manufactured industrially for future applications. To screen the chemical inhibitors of CRISPR/Cas12a, we constructed a label-free fluorescent probe, dubbed the firefly probe. Then, three highly effective chemical inhibitors (H2O2, catechol, and hydroquinone) against LbCas12a were discovered based on this probe. These small molecule inhibitors could preferentially bind with LbCas12a to interrupt the assembly of the LbCas12a/crRNA binary complex. Thus, the cis- and trans-cleavage activities of LbCas12a were simultaneously inhibited. The inhibitory mechanisms were also explored through electrophoresis and molecular docking experiments. Subsequently, a universal sensing platform based on the inhibitor H2O2 was built, which enabled fluorescence analysis of catalase and glucose. The limits of detection for catalase and glucose in the fluorescence mode were as low as 0.080 U/mL and 0.0059 mM, respectively. Furthermore, the platform supported visual analysis by integrating smartphones with the RGB measurement software. In summary, small molecule inhibitors of LbCas12a were found, and their inhibition mechanism was identified in this research. These inhibitors can serve as efficient tools for blocking the CRISPR/Cas12a system and facilitate the application of LbCas12a inhibitors in biosensors.},
}

*Biosensing Techniques/methods
*Fluorescent Dyes/chemistry
Hydrogen Peroxide/pharmacology/chemistry
*CRISPR-Associated Proteins/antagonists & inhibitors/metabolism
Glucose/analysis
Molecular Docking Simulation
Catechols/pharmacology/chemistry
Catalase/analysis/metabolism
Hydroquinones/pharmacology/chemistry
CRISPR-Cas Systems/drug effects
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40682549,
year = {2025},
author = {Yang, L and Liu, R and Meng, Y and Deng, Z and Bu, S and Liu, J and Huang, A and Wu, S and Kan, X},
title = {Fruit Phenotype Analysis of SlSAHH2-CRISPR Tomato and Methylation Mechanism of SlSAHH2 Promoting Fruit Ripening.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {30},
pages = {18691-18705},
doi = {10.1021/acs.jafc.5c05207},
pmid = {40682549},
issn = {1520-5118},
mesh = {*Solanum lycopersicum/genetics/growth & development/metabolism/enzymology ; *Fruit/genetics/growth & development/metabolism/enzymology ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Ethylenes/biosynthesis/metabolism ; *Adenosylhomocysteinase/genetics/metabolism ; *Plants, Genetically Modified/genetics/metabolism/growth & development/enzymology ; DNA Methylation ; Methylation ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {S-adenosyl-l-homocysteine hydrolase (SAHH EC 3.3.1.1) is a key enzyme that maintains methylation homeostasis. In this study, the knockout of SlSAHH2 delayed tomato fruit ripening. Compared with the wild type (WT), most ripening-related biochemical characteristics were significantly reduced in SlSAHH2-CRISPR fruits. Additionally, the expression of genes related to ethylene synthesis and ripening was generally down-regulated. In transcriptome data, a total of 3701 up-regulated and 3134 down-regulated differentially expressed genes (DEGs) were identified and analyzed. Bisulfite sequencing PCR (BSP) results showed that the methylation levels of the promoters of ethylene-related genes in SlSAHH2-OE fruits were higher. Furthermore, in the presence of a methylation inhibitor, the expression of E4, E8, ACO1, ACO3, and ACS2 was generally up- or down-regulated in SlSAHH2-OE or SlSAHH2-CRISPR fruits, respectively. In summary, it can be inferred that in the balance between methylation and ethylene synthesis, SlSAHH2 was more inclined to promote the latter process during tomato fruit ripening.},
}

*Solanum lycopersicum/genetics/growth & development/metabolism/enzymology
*Fruit/genetics/growth & development/metabolism/enzymology
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Ethylenes/biosynthesis/metabolism
*Adenosylhomocysteinase/genetics/metabolism
*Plants, Genetically Modified/genetics/metabolism/growth & development/enzymology
DNA Methylation
Methylation
Phenotype
CRISPR-Cas Systems

@article {pmid40680452,
year = {2025},
author = {Yang, T and Zhang, S and Nie, K and Cheng, C and Peng, X and Huo, J and Zhang, Y},
title = {ZNF207-driven PRDX1 lactylation and NRF2 activation in regorafenib resistance and ferroptosis evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {82},
number = {},
pages = {101274},
doi = {10.1016/j.drup.2025.101274},
pmid = {40680452},
issn = {1532-2084},
mesh = {Humans ; *NF-E2-Related Factor 2/metabolism/genetics ; *Ferroptosis/drug effects ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Peroxiredoxins/metabolism/genetics ; *Liver Neoplasms/drug therapy/pathology/genetics ; *Carcinoma, Hepatocellular/drug therapy/pathology/genetics ; *Phenylurea Compounds/pharmacology ; *Pyridines/pharmacology ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Regorafenib (RGF) is a critical second-line therapy for advanced hepatocellular carcinoma (HCC) following disease progression on sorafenib; however, the rapid onset of RGF resistance poses a significant barrier to enhancing patient outcomes. In this study, CRISPR/Cas9 screening in RGF-treated HCC cells identified Zinc Finger Protein 207 (ZNF207) as a primary driver of resistance. Further analysis revealed that ZNF207 promotes resistance by inducing antioxidant responses that inhibit ferroptosis, a form of iron-dependent cell death. Mechanistically, ZNF207 facilitates the lactylation of peroxiredoxin 1 (PRDX1) at lysine 67, enhancing nuclear translocation and activation of nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant pathways. This ZNF207-PRDX1-NRF2 pathway creates a ferroptosis-resistant, pro-survival environment under RGF treatment, enabling HCC cells to evade cell death. Functional assays demonstrated that ZNF207 knockdown significantly enhances RGF sensitivity by restoring ferroptosis, with additional findings showing that disrupting PRDX1 lactylation or NRF2 activity similarly reverses resistance. Together, these findings establish a critical link between protein lactylation and RGF resistance, positioning the ZNF207-PRDX1-NRF2 axis as a promising therapeutic target to enhance treatment efficacy in HCC. The implications of this research extend beyond HCC, indicating that targeting ferroptosis-suppressive pathways may offer a broader approach to overcoming resistance in various cancers.},
}

Humans
*NF-E2-Related Factor 2/metabolism/genetics
*Ferroptosis/drug effects
*Drug Resistance, Neoplasm/genetics/drug effects
*Peroxiredoxins/metabolism/genetics
*Liver Neoplasms/drug therapy/pathology/genetics
*Carcinoma, Hepatocellular/drug therapy/pathology/genetics
*Phenylurea Compounds/pharmacology
*Pyridines/pharmacology
Cell Line, Tumor
Antineoplastic Agents/pharmacology
CRISPR-Cas Systems

@article {pmid40588867,
year = {2025},
author = {Rahimi, A and Karimipoor, M and Mahdian, R and Alipour, A and Hosseini, S and Mohammadi, M and Kaghazian, H and Shahsavarani, H and Shokrgozar, MA},
title = {Engineering of the Caspase-3 Gene in Recombinant CHO Cells Caused More Apoptosis Resistance and enhanced Recombinant Protein Production Than the BAX Gene.},
journal = {Iranian biomedical journal},
volume = {29},
number = {3},
pages = {1-10},
doi = {10.61186/ibj.4934},
pmid = {40588867},
issn = {2008-823X},
mesh = {Animals ; *Apoptosis/genetics ; *Caspase 3/genetics/metabolism ; *bcl-2-Associated X Protein/genetics/metabolism ; CHO Cells ; Cricetulus ; *Recombinant Proteins/biosynthesis/genetics ; Erythropoietin/biosynthesis/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Cell Survival/genetics ; },
abstract = {BACKGROUND: BAX and caspase-3 are essential genes in the apoptotic pathway of cells, promoting the apoptotic cascade through different mechanisms. Inhibition of these genes can increase the longevity of cells in cell culture. This study aimed to compare the effects of CRISPR-Cas9-mediated knockdown of BAX and caspase-3 genes on apoptosis inhibition, cell lifespan, and EPO production in CHO cell lines.

METHODS: The BAX and caspase-3 gene expression was evaluated in the rCHO cell lines producing EPO using the CRISPR-Cas9 method. Their anti-apoptotic effects and the level of EPO expression were also compared. In addition, OP as an apoptosis inducer, was introduced to the manipulated cell line to assess the stability and viability of the manipulated cell lines.

RESULTS: The rCHO cells with the manipulated BAX gene exhibited a higher cell density than those with the manipulated caspase-3 gene (152% vs. 142%). Despite the increased cell density in the cells with the BAX gene manipulation, EPO production was higher in the cells with the manipulated caspase-3 gene (1.58-fold increase in the BAX-manipulated cells compared to a 1.70-fold increase in the caspase-3-manipulated cells).

CONCLUSION: Our observations suggest that the downregulation of the BAX and caspase-3 genes using the CRISPR method, inhibits apoptosis and enhances the yield of recombinant EPO, even in the presence of an apoptosis inducer. Additionally, reduction of caspase-3 expression was proved to be more effective than BAX in extending the lifespan of cells and producing heterologous recombinant proteins.},
}

Animals
*Apoptosis/genetics
*Caspase 3/genetics/metabolism
*bcl-2-Associated X Protein/genetics/metabolism
CHO Cells
Cricetulus
*Recombinant Proteins/biosynthesis/genetics
Erythropoietin/biosynthesis/genetics
CRISPR-Cas Systems/genetics
*Genetic Engineering
Cell Survival/genetics

@article {pmid40460025,
year = {2025},
author = {Solayappan, M and Azlan, A and Khor, KZ and Yik, MY and Jaishanker, A and Ramesh, T and Saleem, M and Yusoff, NM and Moses, EJ},
title = {Multifaceted roles of CRISPR technology in blood cancer research.},
journal = {Current opinion in hematology},
volume = {32},
number = {5},
pages = {287-299},
doi = {10.1097/MOH.0000000000000855},
pmid = {40460025},
issn = {1531-7048},
mesh = {Humans ; *Hematologic Neoplasms/genetics/therapy ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; Genomics/methods ; },
abstract = {PURPOSE OF REVIEW: Blood cancers are one of the most common cancers worldwide. These diseases stem from defects in blood components having cytogenetic aberrations and genetic mutations. There have been vast improvements in terms of treatment options and survival outcomes. Nevertheless, due to the clonal nature and heterogeneity of the diseases, the number of cases reported exhibit a rising pattern due to chemoresistance and disease relapse thus posing a healthcare burden. Therefore, the need for more specific forms of targeted therapies is ever-present.

RECENT FINDINGS: CRISPR has emerged as a key player and is the epitome of gene editing technology in this post genomic era. In line with the current trend, numerous studies in blood cancer research have extensively utilized CRISPR-based applications to understand the functional genomics of hematologic malignancies and identify potential therapeutic targets for development of novel therapeutic applications.

SUMMARY: The importance of comprehending the utilities of state-of-the-art technologies such as CRISPR for studying hematologic malignancies has never been more apparent and timelier. Therefore, this review attempts to scrutinize the versatility of CRISPR applications which range from functional genomics to immunotherapeutic applications.},
}

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

@article {pmid40449371,
year = {2025},
author = {Wang, M and Zhang, X and Fan, J and Zhang, C and Xian, Y},
title = {AND logic gate-based alternating PER-Cas12a signal amplification system for ultrasensitive detection of sEVs.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128411},
doi = {10.1016/j.talanta.2025.128411},
pmid = {40449371},
issn = {1873-3573},
mesh = {Humans ; *Breast Neoplasms/diagnosis/pathology ; *Extracellular Vesicles/metabolism/chemistry ; Female ; *Biomarkers, Tumor ; Mucin-1/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Epithelial Cell Adhesion Molecule/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Logic ; Biosensing Techniques/methods ; CRISPR-Cas Systems ; Bacterial Proteins ; },
abstract = {Protein biomarkers on breast cancer-derived small extracellular vesicles (BC-sEVs) hold great promise in liquid biopsy. However, it remains challenging due to their inherent heterogeneity and low abundance. Herein, we developed an AND logic gate-based DNA cascade signal amplification strategy, termed Alternating Primer Exchange Reaction-activated Cas12a (Alt-PER-Cas12a), for the ultrasensitive detection of BC-sEVs in clinic samples. This dual-protein recognition system employs EpCAM/MUC1-specific capture probes to release two DNA hairpins (Hep and Hmu) as AND gate inputs in Alt-PER. The corresponding Hep and Hmu hairpins can initiate the Alt-PER with a large amount of primers to generate long single-stranded DNA products with alternating repeat units. Each repeating unit serves as a CRISPR activator, inducing the trans-cleavage activity of Cas12a and enabling cascade signal amplification. The as-constructed strategy exhibits excellent sensitivity with LOD of 2.6 × 10[3] particles/mL. It has been successfully used to discriminate breast cancer patients from healthy donors (AUC = 0.992) in clinical validation, and shows great potential for liquid biopsy.},
}

Humans
*Breast Neoplasms/diagnosis/pathology
*Extracellular Vesicles/metabolism/chemistry
Female
*Biomarkers, Tumor
Mucin-1/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
Epithelial Cell Adhesion Molecule/metabolism
*Nucleic Acid Amplification Techniques/methods
*Logic
Biosensing Techniques/methods
CRISPR-Cas Systems
Bacterial Proteins

@article {pmid40393239,
year = {2025},
author = {Lin, G and Li, J and Zhang, K},
title = {Multiple CRISPR zones-driven ultrasensitive detection of DNA via CRISPR-Cas12a and ligation-rolling circle amplification.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128336},
doi = {10.1016/j.talanta.2025.128336},
pmid = {40393239},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; Polymorphism, Single Nucleotide ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {The ability to detect specific DNA, including single nucleotide variants (SNVs), with high sensitivity is essential for advancing genetic research, diagnostics, and personalized medicine. This study presents a novel method for ultrasensitive DNA detection, combining ligation-rolling circle amplification (L-RCA) with CRISPR-Cas12a. While L-RCA systems have been widely used for nucleic acid detection, the sensitivity of conventional L-RCA generally reaches approximately 100 pM. Here, we demonstrate that the sensitivity of RCA-Cas12a systems can be markedly enhanced by incorporating multiple CRISPR target regions into the padlock probe. This method achieves remarkable sensitivity, detecting DNA at concentrations as low as 1 aM (6 copies per reaction), and is capable of identifying single nucleotide variants (SNVs) with allele fractions as low as 1 %. Unlike many current complex RCA-Cas12a strategies, this approach is simple and does not require advanced labeling or instrumentation, making it a promising tool for ultrasensitive DNA detection in various applications.},
}

*CRISPR-Cas Systems/genetics
*DNA/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
*CRISPR-Associated Proteins/metabolism/genetics
Humans
Polymorphism, Single Nucleotide
*Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@article {pmid39998382,
year = {2025},
author = {Lin, K and Zou, C and Hubbard, A and Sengelmann, S and Goudy, L and Wang, IC and Sharma, R and Pak, J and Foster, K and Ozawa, T and de Groot, JF and Phillips, J and Vasudevan, HN and Raleigh, DR and Marson, A and Murthy, N and Gilbert, LA and Berger, MS and Liu, SJ},
title = {Multiplexed epigenetic memory editing using CRISPRoff sensitizes glioblastoma to chemotherapy.},
journal = {Neuro-oncology},
volume = {27},
number = {6},
pages = {1443-1457},
doi = {10.1093/neuonc/noaf055},
pmid = {39998382},
issn = {1523-5866},
support = {P50 CA097257/CA/NCI NIH HHS/United States ; //CRISPR/ ; P50 CA097257/GF/NIH HHS/United States ; },
mesh = {*Glioblastoma/genetics/drug therapy/pathology ; Humans ; Animals ; *Brain Neoplasms/genetics/drug therapy/pathology ; Mice ; *Epigenesis, Genetic ; Temozolomide/pharmacology ; Xenograft Model Antitumor Assays ; *Gene Editing/methods ; Lomustine/pharmacology ; Tumor Cells, Cultured ; *Drug Resistance, Neoplasm/genetics ; DNA Repair Enzymes/genetics ; *CRISPR-Cas Systems ; Tumor Suppressor Proteins/genetics ; DNA Modification Methylases/genetics ; Mice, Nude ; Epigenetic Memory ; },
abstract = {BACKGROUND: Glioblastoma (GBM) carries a poor prognosis, and new therapeutic strategies are necessary to improve outcomes for patients with this disease. Alkylating chemotherapies including temozolomide (TMZ) and lomustine (CCNU) are critical for treating GBM, but resistance mechanisms, including hypomethylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter, undermine treatment. CRISPRoff is a programmable epigenetic memory editor that can induce stable and heritable gene silencing after transient delivery, and we hypothesize that CRISPRoff could potentiate the activity of TMZ and CCNU through long-term suppression of target genes.

METHODS: We transiently delivered CRISPRoff mRNA along with sgRNAs against target genes using both electroporation and lipid nanoparticles (LNPs) into established GBM cell lines, patient-derived primary GBM cultures, and orthotopic GBM xenografts. Gene repression, specificity, and stability were measured by RT-qPCR, Western blot, bisulfite sequencing, and RNA sequencing. Sensitivity to chemotherapies was measured by cell viability dose-response, microscopy, and bioluminescence imaging. Genome-wide mapping of CCNU sensitizers was performed using CRISPRi screens.

RESULTS: CRISPRoff induced complete suppression of MGMT and sensitization to TMZ that was stable for over 8 months of continuous cell propagation. GBM orthotopic tumors treated with CRISPRoff against MGMT demonstrated sensitivity to TMZ in vivo, and CRISPRoff delivery resulted in chemosensitivity in patient-derived primary GBM. Genome-wide CRISPRi screens identified combinatorial genetic vulnerabilities (BRIP1, FANCE) that were targetable by multiplexed CRISPRoff to achieve sensitization to CCNU.

CONCLUSION: Transient delivery of a site-specific epigenetic memory can induce stable, complete, and multiplexed suppression of target genes for therapeutic application in GBM.},
}

*Glioblastoma/genetics/drug therapy/pathology
Humans
Animals
*Brain Neoplasms/genetics/drug therapy/pathology
Mice
*Epigenesis, Genetic
Temozolomide/pharmacology
Xenograft Model Antitumor Assays
*Gene Editing/methods
Lomustine/pharmacology
Tumor Cells, Cultured
*Drug Resistance, Neoplasm/genetics
DNA Repair Enzymes/genetics
*CRISPR-Cas Systems
Tumor Suppressor Proteins/genetics
DNA Modification Methylases/genetics
Mice, Nude
Epigenetic Memory

@article {pmid40727907,
year = {2025},
author = {Arifah, AQ and Vento, JM and Kurrer, I and Achmedov, T and Beisel, CL},
title = {Cas9-independent tracrRNA cytotoxicity in Lacticaseibacillus paracasei.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf013},
pmid = {40727907},
issn = {2633-6693},
abstract = {CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from Streptococcus pyogenes (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the S. pyogenes CRISPR-Cas locus is cytotoxic in Lacticaseibacillus paracasei, even in the absence of SpyCas9. Deleting a putative transcription regulator in L. paracasei alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.},
}

@article {pmid40726316,
year = {2025},
author = {Alary, B and Mortada, M and Mas, P},
title = {Gene editing of clock components in Solanum lycopersicum: Effects on gene expression, development, and productivity.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {2},
pages = {e70383},
doi = {10.1111/tpj.70383},
pmid = {40726316},
issn = {1365-313X},
support = {2021-SGR-01131//Agència de Gestió d'Ajuts Universitaris i de Recerca/ ; PID2022-137770NB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; V6423//Fundación Ramón Areces/ ; CEX2019-000902-S//Ministerio de Economía y Competitividad/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development/physiology ; *Gene Editing ; *Circadian Clocks/genetics ; Gene Expression Regulation, Plant ; Plant Leaves/genetics/physiology/growth & development ; CRISPR-Cas Systems ; Circadian Rhythm/genetics ; Arabidopsis/genetics ; Plant Proteins/genetics/metabolism ; Fruit/genetics/growth & development/physiology ; },
abstract = {The circadian clock plays a crucial role in regulating key biological processes, including growth and development. While studies in the model plant Arabidopsis thaliana have significantly advanced our understanding of circadian function, recent research has also focused on crop species for improved yield and quality. In this study, we examined the rhythmic behavior and regulatory function of circadian clock components in tomato (Solanum lycopersicum). Time course analyses of gene expression over the circadian cycle revealed robust rhythmic oscillations in tomato leaves under free-running conditions. Comparative analyses showed similar peak phases for several clock genes in Arabidopsis and tomato, suggesting functional conservation. Rhythms in tomato fruits, however, showed reduced amplitude, slight phase changes, or arrhythmia, indicating organ-specific circadian variations. By using CRISPR-Cas9 gene editing strategies (clock[crispr]), we also showed that proper clock gene expression is essential for setting the phase in tomato plants. Leaf movement analyses also showed a phase change in the clock[crispr] lines, correlating with shorter or longer periods. The clock[crispr] lines also displayed distinct growth and developmental phenotypes that differ from those reported in the Arabidopsis clock mutant counterparts. Our transcriptomic analyses identified species-specific regulation of key target genes. The results offer mechanistic insights into the conserved and divergent molecular pathways governing circadian phenotypic variations between Arabidopsis and tomato plants.},
}

*Solanum lycopersicum/genetics/growth & development/physiology
*Gene Editing
*Circadian Clocks/genetics
Gene Expression Regulation, Plant
Plant Leaves/genetics/physiology/growth & development
CRISPR-Cas Systems
Circadian Rhythm/genetics
Arabidopsis/genetics
Plant Proteins/genetics/metabolism
Fruit/genetics/growth & development/physiology

@article {pmid40725481,
year = {2025},
author = {Cosiquien, RJS and Whalen, IJ and Wong, P and Sorensen, RJ and Shetty, AV and Liang, SQ and Steer, CJ},
title = {Detecting Methylation Changes Induced by Prime Editing.},
journal = {Genes},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/genes16070825},
pmid = {40725481},
issn = {2073-4425},
mesh = {Humans ; *DNA Methylation/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CpG Islands/genetics ; *Epigenesis, Genetic ; },
abstract = {While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Molecular function analyses including Gene Ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.},
}

Humans
*DNA Methylation/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
CpG Islands/genetics
*Epigenesis, Genetic

@article {pmid40725400,
year = {2025},
author = {Kim, JY and Jung, YJ and Kim, DH and Kang, KK},
title = {Transcriptomic Insights into GABA Accumulation in Tomato via CRISPR/Cas9-Based Editing of SlGAD2 and SlGAD3.},
journal = {Genes},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/genes16070744},
pmid = {40725400},
issn = {2073-4425},
support = {2021R1I1A4A01057295//the National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *gamma-Aminobutyric Acid/metabolism/genetics ; *CRISPR-Cas Systems ; *Glutamate Decarboxylase/genetics/metabolism ; Gene Editing/methods ; *Transcriptome/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Fruit/genetics/metabolism ; Plant Leaves/genetics/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with key roles in plant metabolism, stress responses, and fruit nutritional quality. In tomato (Solanum lycopersicum), GABA levels are dynamically regulated during fruit development but decline in the late ripening stages.

METHODS: To enhance GABA accumulation, we used CRISPR/Cas9 to edit the calmodulin-binding domain (CaMBD) of SlGAD2 and SlGAD3, which encode glutamate decarboxylases (GADs). The resulting truncated enzymes were expected to be constitutively active. We quantified GABA content in leaves and fruits and performed transcriptomic analysis on edited lines at the BR+7 fruit stage.

RESULTS: CaMBD truncation significantly increased GABA levels in both leaves and fruits. In gad2 sg1 lines, GABA levels increased by 3.5-fold in leaves and 3.2-fold in BR+10 fruits; in gad3 sg3 lines, increases of 2.8- and 2.5-fold were observed, respectively. RNA-seq analysis identified 1383 DEGs in gad2 #1-5 and 808 DEGs in gad3 #3-8, with 434 DEGs shared across both lines. These shared DEGs showed upregulation of GAD, GABA-T, and SSADH, and downregulation of stress-responsive transcription factors including WRKY46, ERF, and NAC. Notably, total free amino acid content and fruit morphology remained unchanged despite elevated GABA.

CONCLUSIONS: CRISPR/Cas9-mediated editing of the CaMBD in SlGAD genes selectively enhances GABA biosynthesis in tomato without adverse effects on development or fruit quality. These lines offer a useful platform for GABA-centered metabolic engineering and provide insights into GABA's role in transcriptional regulation during ripening.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*gamma-Aminobutyric Acid/metabolism/genetics
*CRISPR-Cas Systems
*Glutamate Decarboxylase/genetics/metabolism
Gene Editing/methods
*Transcriptome/genetics
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Fruit/genetics/metabolism
Plant Leaves/genetics/metabolism
Plants, Genetically Modified/genetics

@article {pmid40725376,
year = {2025},
author = {Kumar, H and Gal'chinsky, N and Sweta, V and Negi, N and Filatov, R and Chandel, A and Ali, J and Oberemok, V and Laikova, K},
title = {Perspectives of RNAi, CUADb and CRISPR/Cas as Innovative Antisense Technologies for Insect Pest Control: From Discovery to Practice.},
journal = {Insects},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/insects16070746},
pmid = {40725376},
issn = {2075-4450},
support = {FZEG-2024-0001//This research results obtained within the framework of a state assignment V.I. Vernadsky Cri-mean Federal University for 2024 and the planning period of 2024-2026/ ; },
abstract = {Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, offering exceptional precision for targeted pest control. While RNA-guided mechanisms such as RNAi and CRISPR/Cas were initially characterized in non-insect systems, primarily as innate defenses against viral infections, the DNA-guided CUADb pathway was first identified in insect pests as a functional pest control strategy. Its broader role in ribosomal RNA (rRNA) biogenesis was recognized later. Together, these discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite sharing a common principle of sequence-specific targeting RNAi, CUADb, and CRISPR/Cas differ in several key aspects, including their mechanisms of action, target specificity, and applicability. Rather than serving as universal solutions, each technology is likely to be optimally effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By integrating foundational biological discoveries with applied innovations, this work provides a new perspectives on incorporating antisense-based strategies into next-generation integrated pest management systems.},
}

@article {pmid40725259,
year = {2025},
author = {Wolny, E and Mur, LAJ and Ohmido, N and Yin, Z and Wang, K and Hasterok, R},
title = {Thriving or Withering? Plant Molecular Cytogenetics in the First Quarter of the 21st Century.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26147013},
pmid = {40725259},
issn = {1422-0067},
mesh = {*Cytogenetic Analysis/methods ; Genome, Plant ; Chromosomes, Plant/genetics ; *Plants/genetics ; In Situ Hybridization, Fluorescence/methods ; *Cytogenetics/methods/trends ; Polyploidy ; Genomics/methods ; },
abstract = {Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping.},
}

*Cytogenetic Analysis/methods
Genome, Plant
Chromosomes, Plant/genetics
*Plants/genetics
In Situ Hybridization, Fluorescence/methods
*Cytogenetics/methods/trends
Polyploidy
Genomics/methods

@article {pmid40725213,
year = {2025},
author = {Covache-Busuioc, RA and Toader, C and Rădoi, MP and Șerban, M},
title = {Precision Recovery After Spinal Cord Injury: Integrating CRISPR Technologies, AI-Driven Therapeutics, Single-Cell Omics, and System Neuroregeneration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146966},
pmid = {40725213},
issn = {1422-0067},
mesh = {*Spinal Cord Injuries/therapy/genetics/physiopathology ; Humans ; *Artificial Intelligence ; Animals ; Gene Editing/methods ; *Nerve Regeneration ; Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Recovery of Function ; Regenerative Medicine/methods ; Precision Medicine/methods ; },
abstract = {Spinal cord injury (SCI) remains one of the toughest obstacles in neuroscience and regenerative medicine due to both severe functional loss and limited healing ability. This article aims to provide a key integrative, mechanism-focused review of the molecular landscape of SCI and the new disruptive therapy technologies that are now evolving in the SCI arena. Our goal is to unify a fundamental pathophysiology of neuroinflammation, ferroptosis, glial scarring, and oxidative stress with the translation of precision treatment approaches driven by artificial intelligence (AI), CRISPR-mediated gene editing, and regenerative bioengineering. Drawing upon advances in single-cell omics, systems biology, and smart biomaterials, we will discuss the potential for reprogramming the spinal cord at multiple levels, from transcriptional programming to biomechanical scaffolds, to change the course from an irreversible degeneration toward a directed regenerative pathway. We will place special emphasis on using AI to improve diagnostic/prognostic and inferred responses, gene and cell therapies enabled by genomic editing, and bioelectronics capable of rehabilitating functional connectivity. Although many of the technologies described below are still in development, they are becoming increasingly disruptive capabilities of what it may mean to recover from an SCI. Instead of prescribing a particular therapeutic fix, we provide a future-looking synthesis of interrelated biological, computational, and bioengineering approaches that conjointly chart a course toward adaptive, personalized neuroregeneration. Our intent is to inspire a paradigm shift to resolve paralysis through precision recovery and to be grounded in a spirit of humility, rigor, and an interdisciplinary approach.},
}

*Spinal Cord Injuries/therapy/genetics/physiopathology
Humans
*Artificial Intelligence
Animals
Gene Editing/methods
*Nerve Regeneration
Single-Cell Analysis/methods
*CRISPR-Cas Systems
Recovery of Function
Regenerative Medicine/methods
Precision Medicine/methods

@article {pmid40725041,
year = {2025},
author = {Fizikova, A and Prokhorova, A and Churikova, D and Konstantinov, Z and Ivanov, R and Karabelsky, A and Rybtsov, S},
title = {Hepatocytes as Model for Investigating Natural Senotherapeutic Compounds and Their Effects on Cell Cycle Dynamics and Genome Stability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146794},
pmid = {40725041},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//the state program of the "Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {Humans ; *Genomic Instability/drug effects ; Animals ; *Senotherapeutics/pharmacology ; *Cell Cycle/drug effects ; *Hepatocytes/drug effects/metabolism/cytology ; Cellular Senescence/drug effects ; DNA Damage ; },
abstract = {DNA is inherently unstable and is susceptible to damage from both endogenous sources (such as reactive oxygen species) and exogenous factors (including UV, ionizing radiation, and chemicals). The accumulation of DNA damage manifests as genetic mutations, chromosomal instability, and the stalling of DNA replication and transcription processes. Accumulated DNA damage influences apoptosis and cell cycle checkpoints, serving as one of the key triggers for the manifestation of the senescent phenotype. Both aging and cancer are associated with the accumulation of mutations in somatic cells. Disruption of cell cycle control and uncontrolled proliferation are fundamental characteristics of any cancer cell, with the majority of anticancer drugs acting as inhibitors of cyclin-dependent kinases, thereby inducing a transition of cells into a senescent state. Consequently, disturbances in the dynamics and regulation of inflammatory responses, oxidative stress, cell proliferation, DNA damage repair, and epigenetic anomalies, along with the influence of retroviruses and transposons, lead to the accumulation of senescent cells within the human body, characterized by blocked replication and cell cycle, as well as a distinct secretory phenotype. The age-related or disease-associated accumulation of these senescent cells significantly alters the physiology of tissues and the organism as a whole. Many secondary metabolites of higher plants exhibit senolytic and senomorphic activities, although most of them are not fully characterized. In this review, we will explore the principal signaling pathways in mammalian cells that govern the cell cycle and cellular senescence, with a particular emphasis on how their dynamics, expression, and regulation have been modified through the application of senotherapeutic compounds. The second section of the review will identify key target genes for the metabolic engineering, primarily aimed at enhancing the accumulation of plant secondary metabolites with potential therapeutic benefits. Lastly, we will discuss the rationale for utilizing liver cells as a model system to investigate the effects of senolytic compounds on human physiology and health, as well as how senotherapeutic substances can be leveraged to improve gene therapy approaches based on CRISPR/Cas9 and prime-editing technologies.},
}

Humans
*Genomic Instability/drug effects
Animals
*Senotherapeutics/pharmacology
*Cell Cycle/drug effects
*Hepatocytes/drug effects/metabolism/cytology
Cellular Senescence/drug effects
DNA Damage

@article {pmid40724990,
year = {2025},
author = {Montagna, C and Maiani, E and Pieroni, L and Consalvi, S},
title = {Duchenne Muscular Dystrophy: Integrating Current Clinical Practice with Future Therapeutic and Diagnostic Horizons.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146742},
pmid = {40724990},
issn = {1422-0067},
abstract = {Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by progressive muscle degeneration due to mutations in the dystrophin gene. Despite major advancements in understanding its pathophysiology, there is still no curative treatment. This review provides an up-to-date overview of current and emerging therapeutic approaches-including antisense oligonucleotides, gene therapy, gene editing, corticosteroids, and histone deacetylases(HDAC) inhibitors-aimed at restoring dystrophin expression or mitigating disease progression. Special emphasis is placed on the importance of early diagnosis, the utility of genetic screening, and the innovations in pre-and post-natal testing. As the field advances toward personalized medicine, the integration of precision therapies with cutting-edge diagnostic technologies promises to improve both prognosis and quality of life for individuals with DMD.},
}

@article {pmid40724964,
year = {2025},
author = {Ruiz-Moreno, HA and Valderrama-Rincon, JD and Cala, MP and Fernández-Niño, M and Valderruten Cajiao, M and Villegas-Torres, MF and González Barrios, AF},
title = {Enhanced Outer Membrane Vesicle Production in Escherichia coli: From Metabolic Network Model to Designed Strain Lipidomic Profile.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146714},
pmid = {40724964},
issn = {1422-0067},
support = {822-2017//Colciencias/ ; },
mesh = {*Escherichia coli/metabolism/genetics ; *Lipidomics/methods ; *Metabolic Networks and Pathways ; *Bacterial Outer Membrane/metabolism ; Mutation ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Extracellular Vesicles/metabolism ; Bacterial Outer Membrane Proteins/metabolism/genetics ; Gene Editing ; },
abstract = {Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings.},
}

*Escherichia coli/metabolism/genetics
*Lipidomics/methods
*Metabolic Networks and Pathways
*Bacterial Outer Membrane/metabolism
Mutation
Escherichia coli Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
*Extracellular Vesicles/metabolism
Bacterial Outer Membrane Proteins/metabolism/genetics
Gene Editing

@article {pmid40724866,
year = {2025},
author = {Nidhi, F and Tomatsu, S},
title = {Integrase-Deficient Lentiviral Vector as a Platform for Efficient CRISPR/Cas9-Mediated Gene Editing for Mucopolysaccharidosis IVA.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146616},
pmid = {40724866},
issn = {1422-0067},
support = {1R01HD102545-01A1/GF/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; *Mucopolysaccharidosis IV/therapy/genetics ; *Genetic Vectors/genetics ; Humans ; NIH 3T3 Cells ; Genetic Therapy/methods ; *Chondroitinsulfatases/genetics/metabolism ; *Integrases/genetics/deficiency ; Fibroblasts/metabolism ; Disease Models, Animal ; },
abstract = {Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder causing systemic skeletal dysplasia due to a deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) enzyme activity, leading to the impaired degradation and accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate. While treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available, they have significant limitations regarding efficacy in skeletal tissues and long-term safety, highlighting the need for more effective therapies. We evaluated a novel gene therapy approach using a dual Integrase-deficient lentiviral vector (IDLV) to deliver an expression cassette that includes human GALNS cDNA and Cas9 sgRNA, targeting the upstream region of the mouse Galns initial codon. This approach leverages the endogenous promoter to drive transgene expression. We assessed in vitro transduction, editing, and functional correction in NIH3T3 and MPS IVA mouse fibroblasts. In vivo efficacy was successfully evaluated via the facial vein injection in MPS IVA newborn mice. In vitro, this IDLV platform demonstrated supraphysiological GALNS activity in cell lysate, resulting in the normalization of KS levels. In vivo direct IDLV platform in newborn MPS IVA mice led to sustained plasma GALNS activity, reduced plasma KS, and favorable biodistribution. Partial correction of heart and bone pathology was observed, with no vector toxicity and minimal antibody responses. This dual IDLV-CRISPR/Cas9 approach effectively mediated targeted GALNS knock-in, yielding sustained enzyme activity, reduced KS storage, and partial pathological amelioration in MPS IVA mice. In conclusion, IDLVs represent an efficient, safe platform for delivering the CRISPR/Cas9 gene editing system for MPS IVA.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Lentivirus/genetics
*Mucopolysaccharidosis IV/therapy/genetics
*Genetic Vectors/genetics
Humans
NIH 3T3 Cells
Genetic Therapy/methods
*Chondroitinsulfatases/genetics/metabolism
*Integrases/genetics/deficiency
Fibroblasts/metabolism
Disease Models, Animal

@article {pmid40722665,
year = {2025},
author = {Zhang, A and Zhang, I and Liu, F},
title = {Applications of CRISPR-Cas-Based Genome Editing Approaches Against Human Cytomegalovirus Infection.},
journal = {Biomedicines},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/biomedicines13071590},
pmid = {40722665},
issn = {2227-9059},
support = {Start-Up Fund 101//University of California, Berkeley/ ; },
abstract = {Human cytomegalovirus (HCMV), a globally ubiquitous herpesvirus with the ability to carry out both lytic productive and lifelong latent infections, is a major cause of congenital infections, often leading to intellectual disabilities and neurological disorders. Moreover, HCMV is an opportunistic pathogen commonly found in immunocompromised individuals such as organ transplant recipients, HIV-positive individuals, and cancer patients, causing severe and life-threatening complications. While effective in inhibiting viral lytic infection, current FDA-approved compounds cannot eliminate the latent viral genome and have little effect on viral latent infection. Developing novel antiviral therapeutic approaches to eliminate HCMV lytic and latent infections is a major public health priority for controlling HCMV infection and preventing viral-associated diseases. The genome-editing technology based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) RNA-guided nuclease system represents a novel and promising antiviral approach through modifying or destroying the genetic material of human viruses. This review summarizes the recently published progress in using the CRISPR-Cas approach to study and inhibit HCMV infections and discusses prospects for developing the CRISPR-based genome-editing technology for therapeutic applications against HCMV infection and associated diseases.},
}

@article {pmid40721002,
year = {2025},
author = {Ahi, EP and Khorshid, M},
title = {Potentials of RNA biosensors in developmental biology.},
journal = {Developmental biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ydbio.2025.07.011},
pmid = {40721002},
issn = {1095-564X},
abstract = {RNA-based/associated biosensors represent a rapidly expanding area of research, providing highly sensitive tools for detecting and monitoring RNA in diverse biological contexts. These sensors offer the ability to track RNA localization, modifications, and interactions in real-time, making them particularly well-suited for developmental biology research. Despite their demonstrated utility in fields such as diagnostics, synthetic biology and environmental science, the application of RNA biosensors in developmental biology has only begun to emerge within the past decade. This gap is notable given the potential of these tools to address key questions about spatiotemporal RNA regulation and cellular signaling during development. This perspective review presents a selection of RNA biosensors, including fluorescent RNA aptamers, CRISPR-Cas-based systems, riboswitches, and catalytic RNA sensors, which have gained attraction in other scientific disciplines. These tools can be used not only to study intrinsic RNA biology, such as RNA expression, splicing, and localization, but also to detect the effects of extrinsic physical and chemical factors, including pH, temperature, redox state, and mechanical stress, on RNA behavior during developmental processes. These examples illustrate how RNA biosensors could be adapted to study developmental mechanisms in model organisms, enabling investigations into RNA dynamics and their role in shaping developmental processes. By revisiting these underutilized tools, this review highlights their relevance for advancing the understanding of molecular mechanisms in developmental biology studies.},
}

@article {pmid40716775,
year = {2025},
author = {Nalefski, EA and Hedley, S and Rajaraman, K and Kooistra, RM and Parikh, I and Sinan, S and Finklestein, IJ and Madan, D},
title = {Unleashing high trans-substrate cleavage kinetics of Cas12a for nucleic acid diagnostics.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf712},
pmid = {40716775},
issn = {1362-4962},
support = {//College of Natural Sciences Catalyst/ ; //Global Health Labs/ ; F-1808//Welch Foundation/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Kinetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Substrate Specificity ; *Nucleic Acids/genetics/analysis ; Ribonucleoproteins/metabolism ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid diagnostics enable rapid, sensitive pathogen detection. Cas12a is frequently used in these assays because target-activated trans cleavage of a reporter molecule generates an easily detectable signal. However, variable activity across assays suggests that the catalytic potential of Cas12a has been limited via unknown mechanisms. Here, we show that Cas12a trans-nuclease activity is auto-inhibited by long PAM-proximal DNA (>120 bp) following cis-cleavage of targets. Short targets (<100 bp), optimized trans cleavage substrates, and low salt buffers unleash high catalytic efficiency (≈108 M-1 s-1) and turnover (≈1 s-1) across Cas12a orthologs. Pooling multiple Cas12a ribonucleoproteins (RNPs) targeting clustered protospacers overcomes cis-cleavage auto-inhibition, further boosting sensitivity. Optimized CRISPR RNA pools enable sub-femtomolar sensitivity for target detection without any pre-amplification. This mechanistic insight and mitigation strategy broaden the application of CRISPR-Cas enzymes for nucleic acid diagnostics.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*CRISPR-Cas Systems
Kinetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
DNA/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems/genetics
Substrate Specificity
*Nucleic Acids/genetics/analysis
Ribonucleoproteins/metabolism

@article {pmid40716264,
year = {2025},
author = {Souza-Neves, M and Pórfido, JL and Crispo, M and Menchaca, A},
title = {Electroporation of sheep zygotes as an alternative to microinjection for the generation of CRISPR/Cas genome edited models.},
journal = {Theriogenology},
volume = {248},
number = {},
pages = {117603},
doi = {10.1016/j.theriogenology.2025.117603},
pmid = {40716264},
issn = {1879-3231},
abstract = {Zygote microinjection is considered the most suitable technique to introduce CRISPR/Cas9 reagents for efficient genome editing in livestock. In this study, zygote electroporation was evaluated as an alternative to microinjection for CRISPR/Cas9-mediated genome editing in sheep. Four experiments were conducted on 3548 cumulus-oocyte complexes. Acid Tyrode's solution (AT) was used to partially degrade the zona pellucida (ZP) to improve reagent entry, resulting in ZP thinning with longer AT exposure (P < 0.05). Although early embryo development was impaired by AT exposure (P < 0.05), blastocyst rates were similar across all groups by day 8. Electroporation conditions were optimized by testing pulse length (1 or 3 ms), with the best results from 6 pulses of 20 V for 3 ms with AT during 60 s. Electroporation with 500 ng/μL Cas9 and 300 ng/μL sgRNA with AT during 60 s achieved a 38.5 % mutation rate. When compared with conventional microinjection, electroporation had higher developmental rates but a lower mutation rate (21.4 % vs. 60.0 %; P < 0.05). These findings suggest that electroporation is a viable, cost-effective technique for genome editing in sheep. Nevertheless, further research will be required to fine-tune electroporation conditions and enhance efficiency in terms of mutation rate.},
}

@article {pmid40715925,
year = {2025},
author = {Westarp, P and Keller, T and Brand, J and Horvat, S and Albrecht, K and Beisel, C and Groll, J},
title = {Efficient encapsulation of CRISPR-Cas9 RNP in bioreducible nanogels and release in a cytosol-mimicking environment.},
journal = {Discover nano},
volume = {20},
number = {1},
pages = {119},
pmid = {40715925},
issn = {2731-9229},
abstract = {CRISPR/Cas9-mediated programmable gene editing has disrupted the biotechnology industry since it was first described in 2012. Safe in vivo delivery is a key bottleneck for its therapeutic use. Viral vector-mediated delivery raises concerns due to immunogenicity, long-term expression, and genomic disruption. Delivery of pre-complexed ribonucleoprotein (RNP) reduces off-target effects, and recombinant Cas9 production is more cost-effective than viral vector synthesis. CRISPR-Cas RNPs do not possess intrinsic cell entry mechanisms, and physical delivery methods are confined to ex vivo editing, necessitating non-viral delivery approaches. Nanogels (NG) are biocompatible polymeric nanoparticles capable of entrapping proteins. Here, we report the first proof of principle that NGs from thiol-functionalized polyglycidol can entrap active RNPs with high efficiency (60 ± 2%). We call these particles CRISPR-Gels. A commercially available E. coli lysate for cell-free transcription and translation (TXTL) was used to mimic the intracellular reductive degradation of NGs while providing a real-time fluorescence readout of RNP activity. Degradation and RNP activity were observed within 30-90 min. The described TXTL assay can be utilized to evaluate the release of RNP in a cytosol-mimicking environment from redox-sensitive nanoparticles in a high-throughput and cost-effective way. Further studies are needed to assess the in vitro and in vivo performance of CRISPR-Gels.},
}

@article {pmid40715906,
year = {2025},
author = {Garg, A and Chauhan, P and Kaur, C and Arora, PK and Garg, SK and Singh, VP and Singh, KP and Srivastava, A},
title = {Comprehensive heavy metal remediation mechanisms with insights into CRISPR-Cas9 and biochar innovations.},
journal = {Biodegradation},
volume = {36},
number = {4},
pages = {69},
pmid = {40715906},
issn = {1572-9729},
support = {DST/INSPIRE/03/2021/002510//DST-INSPIRE INDIA/ ; },
mesh = {*Metals, Heavy/metabolism ; *Charcoal/chemistry ; *CRISPR-Cas Systems ; Biodegradation, Environmental ; *Soil Pollutants/metabolism ; Plants/metabolism/genetics ; Gene Editing ; Bacteria/metabolism/genetics ; },
abstract = {Heavy metal contamination of the environment is a serious issue, and more efficient and effective bioremediation techniques are needed. This review introduces current heavy metal bioremediation techniques, with focus on phytoremediation and microbial remediation, and recent developments in biochar and CRISPR-Cas9 technology. Phytoremediation employs the natural process of plants to accumulate and detoxify metals as an eco-friendly and sustainable technique. Microbial remediation by fungi and bacteria provides an additional approach through reduction, sequestration, and transformation of metals. Biochar as a high-carbon value-added pyrolytic biomass product improves soil quality, increases microbial activity, and adsorbs heavy metals, making bioremediation more effective. The discovery of CRISPR-Cas9 revolutionized gene engineering by allowing gene editing of plants and microbes to improve their metal tolerance and degradation. This review outlines recent developments, synergistic uses of biochar and CRISPR-Cas9, and how they might enhance phytoremediation and microbial remediation. By combining such novel technologies, strong, sustainable, and scalable solutions could be built for curbing heavy metal pollution and safeguarding environmental health.},
}

*Metals, Heavy/metabolism
*Charcoal/chemistry
*CRISPR-Cas Systems
Biodegradation, Environmental
*Soil Pollutants/metabolism
Plants/metabolism/genetics
Gene Editing
Bacteria/metabolism/genetics

@article {pmid40720830,
year = {2025},
author = {Šimečková, H and Bárdy, P and Kuntová, L and Macháčová, E and Botka, T and Bíňovský, J and Houser, J and Farka, Z and Plevka, P and Pantůček, R and Mašlaňová, I},
title = {CRISPR-Cas10-Assisted Structural Modification of Staphylococcal Kayvirus for Imaging and Biosensing Applications.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00387},
pmid = {40720830},
issn = {2161-5063},
abstract = {Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.},
}

@article {pmid40720017,
year = {2025},
author = {Maruyama, R and Yokota, T},
title = {Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2964},
number = {},
pages = {157-162},
pmid = {40720017},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Gene Editing/methods ; *Exons/genetics ; *Dystrophin/genetics ; *Oligonucleotides, Antisense/genetics ; Cell Line ; Mutation ; },
abstract = {Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.},
}

Humans
*CRISPR-Cas Systems
*Muscular Dystrophy, Duchenne/genetics/therapy/pathology
*Gene Editing/methods
*Exons/genetics
*Dystrophin/genetics
*Oligonucleotides, Antisense/genetics
Cell Line
Mutation

@article {pmid40719726,
year = {2025},
author = {Deivarajan, HR and Senthilkumar, K and Sekar, HV and Elamurugan, V and Pandian, J and Venugopal, A and Kuppamuthu, D and Prajna, L and Prajna, VN and Narendran, S},
title = {Rapid one-tube RPA-coupled CRISPR/Cas12a-based RID-MyC assay for the diagnosis of fungal keratitis.},
journal = {Indian journal of ophthalmology},
volume = {73},
number = {8},
pages = {1208-1212},
doi = {10.4103/IJO.IJO_1613_24},
pmid = {40719726},
issn = {1998-3689},
mesh = {Humans ; *Eye Infections, Fungal/diagnosis/microbiology ; Prospective Studies ; Cross-Sectional Studies ; Female ; Male ; *CRISPR-Cas Systems ; Middle Aged ; *Nucleic Acid Amplification Techniques/methods ; *Keratitis/diagnosis/microbiology ; Adult ; *Cornea/microbiology/pathology ; *DNA, Fungal/analysis/genetics ; *Fungi/genetics/isolation & purification ; Aged ; *Recombinases/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {PURPOSE: This study introduces and evaluates the single-tube rapid identification of mycoses using CRISPR (ST-RID-MyC) assay. This novel diagnostic tool combines recombinase polymerase amplification (RPA) with CRISPR/Cas12a for the rapid and precise diagnosis of fungal keratitis (FK).

DESIGN: Prospective cross-sectional study.

METHODS: Corneal scrapings from 61 patients with suspected microbial keratitis were collected at the Cornea Department of a Tertiary Eye Care Center. The study assessed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ST-RID-MyC assay. Additional measures included concordance rates with traditional diagnostic methods and the time to diagnosis.

RESULTS: The ST-RID-MyC assay exhibited a sensitivity of 90% and a specificity of 90.48%, with a PPV of 94.74% and an NPV of 82.61%. The ST-RID-MyC showed substantial agreement with culture and microscopy and perfect concordance with conventional RID-MyC. The mean time to diagnosis was significantly reduced (P < 0.001) using the ST-RID-MyC assay, compared to the traditional RID-MyC assay (6 vs. 32 minutes). Visual assessments demonstrated a high level of inter-observer agreement (kappa = 0.832).

CONCLUSIONS: The ST-RID-MyC assay, combining RPA and CRISPR/Cas12a in a single-tube system, offers a rapid, accurate, and resource-efficient diagnostic method for FK, potentially transforming clinical management of this condition by enabling faster therapeutic decisions.},
}

Humans
*Eye Infections, Fungal/diagnosis/microbiology
Prospective Studies
Cross-Sectional Studies
Female
Male
*CRISPR-Cas Systems
Middle Aged
*Nucleic Acid Amplification Techniques/methods
*Keratitis/diagnosis/microbiology
Adult
*Cornea/microbiology/pathology
*DNA, Fungal/analysis/genetics
*Fungi/genetics/isolation & purification
Aged
*Recombinases/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40715281,
year = {2025},
author = {Lee, S and Kyung, M and Park, M and Park, S and Lee, J and Kim, S and Lee, S and Jo, M and Jung, ST and Lee, HW},
title = {Advanced human FcRn knock-in mice for pharmacokinetic profiling of therapeutic antibodies.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27186},
pmid = {40715281},
issn = {2045-2322},
support = {2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Receptors, Fc/genetics/metabolism ; *Histocompatibility Antigens Class I/genetics/metabolism ; Humans ; Mice ; *Gene Knock-In Techniques ; *Immunoglobulin G ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {IgG-based therapeutic antibodies are increasingly adopted for diverse human diseases, such as cancer and autoimmune disorders displaying remarkable therapeutic performance. A key factor in their success lies in the extended half-life of IgG molecules, which is regulated by the pH-dependent interaction between IgG and neonatal Fc receptor (FcRn). This interaction prevents lysosomal degradation of IgG. Despite the frequent use of humanized rodent models expressing human FcRn (hFcRn) in preclinical studies, these models often fail to accurately replicate human antibody pharmacokinetics (PK) due to the use of non-native promoters that influence FcRn expression. To overcome this limitation, we developed an innovative humanized FcRn knock-in (hiFcRn) mouse model using CRISPR/Cas9 technology. This model integrates hFcRn cDNA into the endogenous locus of the mouse Fcgrt gene, completely replacing native mouse FcRn (mFcRn) expression. The hiFcRn mouse model offers a more human-relevant platform for the preclinical evaluation of therapeutic antibodies and Fc-fusion proteins.},
}

Animals
*Receptors, Fc/genetics/metabolism
*Histocompatibility Antigens Class I/genetics/metabolism
Humans
Mice
*Gene Knock-In Techniques
*Immunoglobulin G
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid40715076,
year = {2025},
author = {Bell, HW and Feng, R and Shah, M and Yao, Y and Douglas, J and Doerfler, PA and Mayuranathan, T and O'Dea, MF and Li, Y and Wang, YD and Zhang, J and Mackay, JP and Cheng, Y and Quinlan, KGR and Weiss, MJ and Crossley, M},
title = {Removal of promoter CpG methylation by epigenome editing reverses HBG silencing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6919},
pmid = {40715076},
issn = {2041-1723},
support = {2020861//Department of Health | National Health and Medical Research Council (NHMRC)/ ; K01DK132453//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 156647//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; *Promoter Regions, Genetic/genetics ; *DNA Methylation/genetics ; *CpG Islands/genetics ; *Gene Silencing ; *Gene Editing/methods ; CRISPR-Cas Systems ; Ubiquitin-Protein Ligases/genetics/metabolism ; CCAAT-Enhancer-Binding Proteins/genetics/metabolism ; *gamma-Globins/genetics/metabolism ; *Epigenome/genetics ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism/genetics ; DNA-Binding Proteins/genetics/metabolism ; Epigenome Editing ; },
abstract = {β-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood, and toxicity limits their use. We identify the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression through a CRISPR/Cas9 screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 causes global demethylation and HBG activation that is reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activates their genes in HUDEP2 or primary CD34[+] cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD co-repressor complex, recapitulates the effects of promoter demethylation. Our findings demonstrate that localized CpGmethylation at the HBG promoters facilitates gene silencing and identify a potential therapeutic approach for β-hemoglobinopathies via epigenomic editing.},
}

Humans
*Promoter Regions, Genetic/genetics
*DNA Methylation/genetics
*CpG Islands/genetics
*Gene Silencing
*Gene Editing/methods
CRISPR-Cas Systems
Ubiquitin-Protein Ligases/genetics/metabolism
CCAAT-Enhancer-Binding Proteins/genetics/metabolism
*gamma-Globins/genetics/metabolism
*Epigenome/genetics
Cell Line
DNA (Cytosine-5-)-Methyltransferase 1/metabolism/genetics
DNA-Binding Proteins/genetics/metabolism
Epigenome Editing

@article {pmid40613420,
year = {2025},
author = {Yuan, T and Du, J and Hu, J and Luo, J and Pu, X and Zhu, X and Chen, B and Teng, Y and Li, H and Li, S and Jiang, L and Xiong, E},
title = {CRISPR-Cas13a-based dual-channel AND-logic gated biosensor for the simultaneous assay of APE1 and miRNA-224.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {62},
pages = {11673-11676},
doi = {10.1039/d5cc03222a},
pmid = {40613420},
issn = {1364-548X},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics ; Humans ; },
abstract = {Simultaneous detection of multiple targets is of great significance for the precise diagnosis of diseases. Herein, we report a dual-channel AND-logic gated biosensing platform based on the CRISPR-Cas13a system for the simultaneous detection of APE1 and miRNA-224, which showed superior specificity, sensitivity and potential for practical applications. Our study not only expands the CRISPR toolbox beyond nucleic acid assay but also establishes a new paradigm for multi-analyte diagnostic systems.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics
Humans

@article {pmid40495486,
year = {2025},
author = {Jeong, YY and Han, JH and Yu, J and Bae, S and Seo, PJ},
title = {Identification of optimal adenine and cytosine base editors for genome editing in Arabidopsis and soybean.},
journal = {BMB reports},
volume = {58},
number = {7},
pages = {288-292},
pmid = {40495486},
issn = {1976-670X},
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; *Glycine max/genetics ; *Cytosine/metabolism ; *Adenine/metabolism ; Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are widely used in numerous organisms to introduce site-specific sequence modifications in genomic DNA without causing double-strand breaks (DSBs). However, these editors exhibit low editing efficiencies, particularly in dicot plants, thereby limiting their application in dicot plant genome engineering. In this study, we assessed the editing efficiencies of various base editors to identify those optimal for base editing in dicot plants. We discovered that ABE8e, an ABE variant, demonstrated superior A-to-G base editing efficiency within A5-A8 windows, and A3A/Y130F-V04, a CBE variant, exhibited the highest C-to-T base editing efficiency within C4-C15 windows in both Arabidopsis and soybean protoplasts. Overall, we recommend these two base editors as prime choices for efficient genome engineering in a range of crop plants. [BMB Reports 2025; 58(7): 288-292].},
}

*Gene Editing/methods
*Arabidopsis/genetics
*Glycine max/genetics
*Cytosine/metabolism
*Adenine/metabolism
Genome, Plant/genetics
CRISPR-Cas Systems/genetics

@article {pmid40441113,
year = {2025},
author = {Shen, C and Chen, X and Yu, Y and Qin, L and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {A novel fluorescent sensing platform for miRNA-210 detection based on signal amplification via SDA and self-assembly Cas12a system via RCT.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128382},
doi = {10.1016/j.talanta.2025.128382},
pmid = {40441113},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Spectrometry, Fluorescence ; *Fluorescent Dyes/chemistry ; Fluorescence ; Bacterial Proteins ; },
abstract = {In this work, a biosensing platform for miR-210 which is a potent biomarker for the early diagnosis of breast cancer was innovatively designed based on CRISPR/Cas12a by integrating strand displacement amplification and rolling circle transcription. The target opened the hairpin through toe-hold, allowing polymerization, incision and extension to occur which involved SDA. This process produced two chains: cycle chain and trigger. The cycle chain then complemented to the loop of the hairpin to open it, repeating the above process to generate additional trigger circularly. The trigger was bound to the notched dumbbell through base complementary pairing and then the dumbbell became intact by T4 DNA ligase. The closed dumbbell strand served as the initiator of transcription and the template for crRNA under the influence of T7 enzyme, and was responsible for the transcription of numerous crRNA sequences in a process called RCT. These sequences subsequently bound to Cas12a proteins, forming a binary complex. In the transcriptional state, the dumbbell was in an unwinding configuration, with the stem portion served as activator binding to the binary complex to facilitate trans-cleavage activity of Cas12a, which resulted in cleavage of the F-Q, generating fluorescent signals. The above platform could sensitively detect miR-210 with a detection limit of 6.67 fM. The platform has the advantages of being easy to use and flexible to sequence according to different target, making it feasible to detect different biomarkers in clinic settings.},
}

*MicroRNAs/analysis/genetics
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
Spectrometry, Fluorescence
*Fluorescent Dyes/chemistry
Fluorescence
Bacterial Proteins

@article {pmid40435758,
year = {2025},
author = {Ren, K and Ding, S and Shi, J and Dong, J and Du, F and Tang, Z},
title = {Detection of lead contamination using DNAzyme and split activator-triggered CRISPR/Cas12a.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128385},
doi = {10.1016/j.talanta.2025.128385},
pmid = {40435758},
issn = {1873-3573},
mesh = {*Lead/analysis ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis ; Humans ; Drinking Water/analysis ; },
abstract = {Widespread Pb[2+] contamination represents a significant global health threat, particularly to children, highlighting the critical need for accurate monitoring and quantification to mitigate its adverse effects. The integration of DNAzymes with the programmable nuclease Cas12a has emerged as a promising approach for achieving specific and ultrasensitive detection of Pb[2+]. However, conventional DNAzyme-Cas12a systems suffer from inevitable background signals caused by dynamic instability between DNAzymes and Cas12a activators, which compromises analytical reliability. Herein, we present a novel DNAzyme-Cas12a assay featuring a split activator-based Cas12a switch. We demonstrate that the split activator containing a flap region effectively prevents Cas12a activation, thereby suppressing background noise induced by "DNA breathing" phenomena. Upon Pb[2+]-dependent activation of the GR-5 DNAzyme, the flap is cleaved, enabling reconstitution of the Cas12a activator and triggering trans-cleavage activity for signal amplification. This strategy achieves a detection limit of 615 pM for Pb[2+] while maintaining high specificity against interfering metal ions. Notably, the assay eliminates requirements for DNA amplification or nanoparticle modification, enabling rapid Pb[2+] detection at ambient temperature. The method demonstrated high accuracy in detecting contaminated tap and drinking water, suggesting its potential as a reliable analytical tool for monitoring Pb2+ contamination in practical samples.},
}

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

@article {pmid40398044,
year = {2025},
author = {Xu, J and Zhang, Y and Yuan, B and Wang, Y and Wang, J and Yuan, Y},
title = {A novel one-tube RPA/CRISPR melting curve detection sensing system based on unique 3'-toehold nucleic acid aptamer for Bacillus anthracis detection.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128306},
doi = {10.1016/j.talanta.2025.128306},
pmid = {40398044},
issn = {1873-3573},
mesh = {*Bacillus anthracis/isolation & purification/genetics ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Spores, Bacterial/isolation & purification ; },
abstract = {Developing effective detection methods for Bacillus anthracis is essential for our public health system to accurately detect hidden anthrax outbreaks. Herein, we introduce a unique 3'-toehold nucleic acid aptamer (probes) into Cas12a biosensor, combined with RPA, to establish a rapid (1h), specific, and sensitive (1copy/μL) detection method for B. anthracis. The design behind this approach is that the target sequence is amplified via RPA, and the amplification product triggers the crRNA/Cas12a complex to degrade the 3' toehold probes, which are analyzed using melting curve analysis on a specific instrument, naming a one-tube RPA/CRISPR melting curve detection (ORCMD) sensing system. Furthermore, ORCMD is used to detect the B. anthracis spores-positive or negative soil samples from the location of world War-II site (Harbin, China), B. anthracis was precisely identified as other methods, suggesting its significant practical application potential. This system enriches the CRISPR detection technology toolbox, compared to other CRISPR-based sensing strategies, the concept of the 3' toehold probes offers distinct advantages in the development of CRISPR-based multi-target detection methods.},
}

*Bacillus anthracis/isolation & purification/genetics
*Aptamers, Nucleotide/chemistry/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Spores, Bacterial/isolation & purification

@article {pmid40185317,
year = {2025},
author = {Lee, EG and Kim, KH},
title = {Transposition of transposable element IS1 in Edwardsiella piscicida mutant generated by CRISPR/Cas9 along with λ-Red recombineering system.},
journal = {Research in microbiology},
volume = {176},
number = {5-6},
pages = {104297},
doi = {10.1016/j.resmic.2025.104297},
pmid = {40185317},
issn = {1769-7123},
mesh = {*CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; *Edwardsiella/genetics ; Mutation ; Recombination, Genetic ; Genome, Bacterial ; },
abstract = {This study aimed to investigate unintended mutations introduced by the CRISPR/Cas9 genome editing system in Edwardsiella piscicida. Whole-genome sequencing was conducted on the wild-type E. piscicida NH1 and its alanine racemase knockout mutants (E. piscicida Δalr325 NH1 and E. piscicida Δalr50 NH1) generated using CRISPR/Cas9 with a λ-Red recombineering system. Comparative genomic analyses revealed that the insertion sequence 1 (IS1) transpositions occurred in the CRISPR/Cas9-edited mutants, disrupting the type I restriction-modification system subunit M gene, in addition to the targeted gene deletion. Interestingly, no IS1 transpositions were detected in mutants produced via conventional plasmid-based allelic exchange, indicating the potential link between CRISPR/Cas9-mediated editing and transposition events. These results suggest that genome editing via CRISPR/Cas9 could trigger IS1 transposition, potentially due to double-stranded DNA breaks. The lack of sequence similarity between the single guide RNA (sgRNA) and the transposed regions suggests that transpositions are not CRISPR/Cas9 off-target effects. This study provides evidence of interactions between mobile genetic elements and genome editing systems, requiring further investigation into their underlying mechanisms.},
}

*CRISPR-Cas Systems
*DNA Transposable Elements/genetics
*Gene Editing/methods
*Edwardsiella/genetics
Mutation
Recombination, Genetic
Genome, Bacterial

@article {pmid40719891,
year = {2025},
author = {Rather, GA and Ayzenshtat, D and Kumar, M and Aisemberg, E and Bocobza, S},
title = {Direct haploid formation in Arabidopsis using transgenic CENH3-based inducers.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {182},
pmid = {40719891},
issn = {1432-203X},
support = {20-01-0245//Chief Scientist - Ministry of Agriculture and Rural Development/ ; },
abstract = {This study introduces a streamlined transgenic method for generating haploid inducers using a single T-DNA construct, combining CENH3 disruption, functional complementation, and a visual marker for efficient haploid screening. The development of doubled haploid lines is crucial for plant breeding programs, but conventional inbreeding methods are laborious and costly. Centromere-mediated genome elimination using modified CENH3 histones offers an efficient single-generation approach to induce haploidy. However, this approach necessitates the generation of haploid inducer lines, which typically involves cumbersome random mutagenesis screens. In this study, we implemented a transgenic strategy to circumvent this and directly create haploid inducers in Arabidopsis. This was achieved by knocking out endogenous AtCENH3 using CRISPR/Cas while complementing it with mutated AtCENH3 variants on the same T-DNA. Four constructs with truncated or full-length AtCENH3 harboring the G83E mutation alone or with the L130F mutation, and one negative control without mutations, were transformed into Arabidopsis. Stable homozygous transgenic lines were obtained and pollinated with a glabra mutant (Atgl1). Progenies lacking RFP fluorescence and exhibiting a glabrate phenotype were recovered, and flow cytometry analyses showed their haploidy, suggesting genome elimination. Comparatively, the G83E variants showed the highest haploid induction rate. This transgenic approach directly generated haploid inducer lines in Arabidopsis while avoiding random mutagenesis. This novel transgenic strategy provides a powerful tool to rapidly establish haploid inducer lines in additional transformable crops.},
}

@article {pmid40719158,
year = {2025},
author = {Torres-Barceló, C and Boyer, C and Garneau, JR and Poussier, S and Robène, I and Moury, B},
title = {A Phylogenetic Host-Range Index Reveals Ecological Constraints in Phage Specialisation and Virulence.},
journal = {Molecular ecology},
volume = {},
number = {},
pages = {e70052},
doi = {10.1111/mec.70052},
pmid = {40719158},
issn = {1365-294X},
abstract = {Phages are typically known for having a limited host range, targeting particular strains within a bacterial species, but accurately measuring their specificity remains challenging. Factors like the genetic diversity or population dynamics of host bacteria are often disregarded despite their potential influence on phage specialisation and virulence. This study focuses on the Ralstonia solanacearum species complex (RSSC), which comprises genetically diverse bacteria responsible for a major plant disease. It uses a diversified collection of RSSC phages to develop new host-range analysis methods and to test ecological and evolutionary hypotheses on phage host range. We introduce a new 'phylogenetic host-range index' that employs an ecological diversity index to account for the genetic diversity of bacterial hosts, allowing systematic classification of phages along a continuum between specialists and generalists. We propose and provide evidence that generalist phages are more likely to be represented in CRISPR-Cas immune system of bacteria than specialist phages. We explore the hypothesis that generalist phages might exhibit lower virulence than specialist ones due to potential evolutionary trade-offs between host-range breadth and virulence. Importantly, contrasted correlations between phage virulence and host range depend on the epidemiological context. A trade-off was confirmed in a context of low bacterial diversity, but not in a context of higher bacterial diversity, where no apparent costs were detected for phages adapted to a wide range of hosts. This study highlights the need for genetic analyses in phage host range and of investigating ecological trade-offs that could improve both fundamental phage knowledge and applications in biocontrol or therapy.},
}

@article {pmid40717612,
year = {2025},
author = {Bagi, M and Jamalzadegan, S and Steksova, A and Wei, Q},
title = {CRISPR-Cas based platforms for RNA detection: fundamentals and applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03257a},
pmid = {40717612},
issn = {1364-548X},
abstract = {The detection of RNA biomarkers is crucial for diagnosing many urgent diseases such as infections and cancer. Conventional RNA detection techniques such as RT-PCR, LAMP, and microarrays are effective, but often face limitations in terms of speed, sensitivity, and equipment demands. In recent years, CRISPR/Cas systems have emerged as versatile platforms for RNA detection, which offer high specificity, programmability, and adaptability across a wide range of diagnostic applications. This review first categorizes different CRISPR-based RNA detection systems according to the CRISPR effectors employed, including Cas13, Cas12, Cas14, Cas9, and newly characterized enzymes such as Cas7-11 and Cas10, detailing their mechanisms of target recognition, cleavage activity, and signal generation. The CRISPR detection platforms are coupled with or without pre-amplification steps to meet the different sensitivity needs. Preamplification-based systems integrate CRISPR with methods like RT-PCR and isothermal amplification to enhance sensitivity. In parallel, preamplification-free strategies, such as split-crRNA or split-activator systems, are gaining attention for their balanced assay performance and simplicity, which are especially attractive for point-of-care (POC) settings. Then, the diagnostic applications of these technologies are explored across two major domains: infectious disease detection and cancer biomarker identification via miRNAs, demonstrating the clinical potential of CRISPR-based RNA detection platforms. In addition, we explore ongoing challenges such as improving sensitivity in amplification-free formats, and developing field-deployable, cost-effective systems. The review concludes by outlining emerging trends and future directions in CRISPR-based RNA diagnostics, emphasizing their transformative potential in clinical settings.},
}

@article {pmid40713745,
year = {2025},
author = {Jiang, M and Chen, X and Song, Y and Wei, M and Cao, J and Lu, W and Lan, F and Bai, Y and Cui, M},
title = {NEXN deficiency leads to dilated cardiomyopathy in human pluripotent stem cell-derived cardiomyocytes.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {402},
pmid = {40713745},
issn = {1757-6512},
support = {7232088//Beijing Natural Science Foundation/ ; BRWEP2024W014090201//Beijing Research Ward Excellence Clinical Study Program/ ; 2024003//Key Clinical Projects of Peking University Third Hospital/ ; 82070272//National Natural Science Foundation of China/ ; },
mesh = {*Myocytes, Cardiac/metabolism/pathology/cytology ; Humans ; *Cardiomyopathy, Dilated/pathology/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {BACKGROUND: Dilated cardiomyopathy (DCM) constitutes a major cause of heart failure, characterized by high mortality rates and a limited availability of effective therapeutic options. A substantial body of evidence indicates that mutations in the Nexilin (NEXN) gene are significant pathogenic contributors to DCM, but the pathogenic mechanism for dilated cardiomyopathy is unclear.

METHODS: A human NEXN homozygous knockout cardiomyocyte model was established by combining CRISPR/Cas9 gene editing technology and human induced pluripotent stem cells (hiPSCs)-directed differentiation technology. Cell model phenotypic assays were done to characterize the pathological features of the resulting NEXN-deficient cardiomyocytes.

RESULTS: NEXN gene knockout did not affect the pluripotency and differentiation efficiency of hiPSCs. NEXN-deficient cardiomyocytes showed disordered junctional membrane complexes, abnormal excitation-contraction coupling, increased oxidative stress and decreased energy metabolism level. Moreover, levo-carnitine and sarcoplasmic reticulum calcium ATPase (SERCA2a) Activator 1 were identified as promising therapeutic agents for the treatment of DCM.

CONCLUSION: We demonstrated that NEXN was one of the important components in maintaining the structure and function of cardiomyocyte junctional membrane complexes (JMCs), excitation-contraction coupling and energy metabolism of cardiomyocytes, while the loss of its function would lead to DCM. This model represents an important tool to gain insight into the mechanism of DCM, elucidate the gene-phenotype relationship of NEXN deficiency and facilitate drug screening.},
}

*Myocytes, Cardiac/metabolism/pathology/cytology
Humans
*Cardiomyopathy, Dilated/pathology/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
CRISPR-Cas Systems
Oxidative Stress

@article {pmid40713739,
year = {2025},
author = {Amina, S and Wei, M and Zhang, S and Jiaqi, F and Ma, S and Abbas, MK and Lan, F and Jiang, H},
title = {Metabolism-related gene PDK1 regulates myocardial cell remodeling and its mechanism.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {404},
doi = {10.1186/s13287-025-04518-9},
pmid = {40713739},
issn = {1757-6512},
support = {NSFC 82370440//Beijing Natural Science Foundation/ ; },
mesh = {*Myocytes, Cardiac/metabolism/cytology ; Humans ; *Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; Apoptosis ; Cell Differentiation ; Oxidative Stress ; Gene Knockout Techniques ; Cell Hypoxia ; *Myocardium/metabolism ; },
abstract = {BACKGROUND: The progression of ischemic heart disease results from various forms of cardiomyopathies, which begin with cardiac remodelling. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one of the basic kinase family components responsible for oxidative phosphorylation. However, due to the lack of a suitable research model, there is no evidence that remodelling pathogenesis in humans causes death by PDK1 knockout (KO). In the current study, we established a PDK1-deficient human cardiomyocyte (CM) model under conditions imitating the human PDK1-KO model. We determined the role of PDK1 in myocardial apoptosis induced by hypoxia and its implicit mechanism.

METHODS: A human PDK1-KO CM's model was established by combining CRISPR/Cas-9 gene-editing and human induced pluripotent stem cells (hiPSC) directed differentiation technology. The pathological features of PDK1-KO cardiomyocytes were assessed using a phenotypic cell model under basal and hypoxic conditions.

RESULTS: We found that pluripotency and differentiation efficiency of hiPSCs after PDK1 knockout remain intact. Cardiomyocytes with a PDK1 gene knockout showed hypoxia-induced myocardial apoptosis by disturbing mitochondrial metabolism, increased oxidative stress levels, and decreased cell energy and viability. In addition, lentivirus transfection significantly improved the metabolism and cell viability in PDK1-deficient cardiomyocytes.

CONCLUSIONS: Our study established a PDK1 knockout model under hypoxia that exhibits mitochondrial metabolism dysregulation, elevated oxidative stress, and decreased cell viability. This model is an important tool for understanding the mechanism of hypoxia-induced myocardial apoptosis, elucidating the gene-phenotype relationship of PDK1 deficiency, and providing evidence to mitigate the damage against hypoxia.},
}

*Myocytes, Cardiac/metabolism/cytology
Humans
*Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics/metabolism
Induced Pluripotent Stem Cells/metabolism/cytology
Apoptosis
Cell Differentiation
Oxidative Stress
Gene Knockout Techniques
Cell Hypoxia
*Myocardium/metabolism

@article {pmid40711918,
year = {2025},
author = {Roberts, A and Adler, BA and Cress, BF and Doudna, JA and Barrangou, R},
title = {Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {30},
pages = {e2504853122},
doi = {10.1073/pnas.2504853122},
pmid = {40711918},
issn = {1091-6490},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/virology ; *Bacteriophage lambda/genetics ; Genome, Bacterial ; },
abstract = {Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infect Escherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted E. coli cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.},
}

*Gene Editing/methods
*Transposases/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/virology
*Bacteriophage lambda/genetics
Genome, Bacterial

@article {pmid40709174,
year = {2025},
author = {Han, B and Liu, W and Wang, W and Li, Z and You, B and Liu, D and Nan, Y and Ding, T and Dai, Z and Zhang, Y and Zhang, W and Liu, Q and Li, X},
title = {CRISPR/Cas9-based discovery of ccRCC therapeutic opportunities through molecular mechanism and immune microenvironment analysis.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1619361},
pmid = {40709174},
issn = {1664-3224},
mesh = {Humans ; *Tumor Microenvironment/immunology/genetics ; *Carcinoma, Renal Cell/genetics/immunology/drug therapy/pathology ; *Kidney Neoplasms/genetics/immunology/drug therapy/pathology ; *CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Prognosis ; Mutation ; },
abstract = {INTRODUCTION: Clear cell renal cell carcinoma is a common and aggressive form of renal cell carcinoma. Its incidence continues to rise, and metastatic recurrence leads to poor clinical outcomes. Current prognostic biomarkers lack reliability. We integrated multi-omics data to discover key ccRCC genes and build a prognostic model to improve risk prediction and guide treatment decisions.

METHODS: Our study integrated genome-wide CRISPR screening data from DepMap and transcriptomic profiles from TCGA to identify key genes associated with ccRCC pathogenesis. Initial screening identified 11 candidate genes through differential expression analysis and CRISPR functional validation. Using LASSO and Cox regression, we selected five key genes (GGT6, HAO2, SLPI, MELK, and EIF4A1) for model construction. The functional role of MELK was tested by knockdown experiments. Additional analyses included tumor mutation burden, immune microenvironment assessment, and drug response prediction.

RESULTS: The model stratified patients into high-risk and low-risk groups with distinct survival outcomes. High-risk cases showed higher mutation loads, immunosuppressive features, and activated cytokine pathways, whereas low-risk cases displayed metabolic pathway activity. MELK knockdown reduced cancer cell proliferation and migration. High-risk patients exhibited better responses to targeted drugs such as pazopanib and sunitinib.

DISCUSSION: Our study demonstrates the pivotal role of MELK in ccRCC progression. This multi-omics-driven model elucidates MELK-mediated mechanisms and their interactions with the tumor microenvironment, providing novel strategies for risk stratification and targeted therapy. Future studies will validate these findings in independent cohorts and investigate the regulatory networks of MELK to identify potential therapeutic targets.},
}

Humans
*Tumor Microenvironment/immunology/genetics
*Carcinoma, Renal Cell/genetics/immunology/drug therapy/pathology
*Kidney Neoplasms/genetics/immunology/drug therapy/pathology
*CRISPR-Cas Systems
Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic
Prognosis
Mutation

@article {pmid40707831,
year = {2025},
author = {Sereika, M and Mussig, AJ and Jiang, C and Knudsen, KS and Jensen, TBN and Petriglieri, F and Yang, Y and Jørgensen, VR and Delogu, F and Sørensen, EA and Nielsen, PH and Singleton, CM and Hugenholtz, P and Albertsen, M},
title = {Genome-resolved long-read sequencing expands known microbial diversity across terrestrial habitats.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {40707831},
issn = {2058-5276},
support = {130690//Villum Fonden (Villum Foundation)/ ; 50093//Villum Fonden (Villum Foundation)/ ; },
abstract = {The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the terrestrial microbial genome catalogue has been challenging due to the enormous complexity of these environments. Here we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected during the Microflora Danica project, yielding genomes of 15,314 previously undescribed microbial species, recovered using our custom mmlong2 workflow. The recovered microbial genomes span 1,086 previously uncharacterized genera and expand the phylogenetic diversity of the prokaryotic tree of life by 8%. The long-read assemblies also enabled the recovery of thousands of complete ribosomal RNA operons, biosynthetic gene clusters and CRISPR-Cas systems. Furthermore, the incorporation of the recovered genomes into public genomic databases substantially improved species-level classification rates for soil and sediment metagenomic datasets. These findings demonstrate that long-read sequencing allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain an untapped source of biodiversity.},
}

@article {pmid40661409,
year = {2025},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, H and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologs drive RNA-guided transcription.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40661409},
issn = {2692-8205},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP) [1] . Although the seven σ factors in E. coli have been extensively characterized [2] , Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ [E]) whose precise roles are unknown, pointing to additional layers of regulatory potential [3] . Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ [E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ [E] homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif (TAM). Recruitment of σ [E] was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ [E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ [E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans [4,5] .},
}

@article {pmid40661357,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40661357},
issn = {2692-8205},
abstract = {UNLABELLED: Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify Cas1 and the Cas2/3 fusion protein from Pseudomonas aeruginosa . We determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Taken together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.

HIGHLIGHTS: - A positively charged channel on the Cas1-2/3 complex captures fragments of DNA- A loop in the RecA1 domain controls access to the Cas3 nuclease active site- Foreign DNA binding allosterically regulates access to additional DNA binding sites- Distortion of the CRISPR repeat sequence licenses complete foreign DNA integration.},
}

@article {pmid40652511,
year = {2025},
author = {Wei, T and Li, J and Lei, X and Lin, R and Wu, Q and Zhang, Z and Shuai, S and Tian, R},
title = {Multimodal CRISPR screens uncover DDX39B as a global repressor of A-to-I RNA editing.},
journal = {Cell reports},
volume = {44},
number = {7},
pages = {116009},
doi = {10.1016/j.celrep.2025.116009},
pmid = {40652511},
issn = {2211-1247},
mesh = {*RNA Editing/genetics ; *DEAD-box RNA Helicases/metabolism/genetics ; Humans ; *Adenosine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; *Inosine/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.},
}

*RNA Editing/genetics
*DEAD-box RNA Helicases/metabolism/genetics
Humans
*Adenosine/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells
*Inosine/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics

@article {pmid40600880,
year = {2025},
author = {Kristof, A and Karunakaran, K and Ferry, Y and Briggs, S and Allen, C and Mizote, P and Jian, Z and Arvanitis, C and Blazeck, J},
title = {Dissection of the MeCP2 repressor protein enables CRISPR platform optimization via localization engineering.},
journal = {Protein engineering, design & selection : PEDS},
volume = {38},
number = {},
pages = {},
doi = {10.1093/protein/gzaf008},
pmid = {40600880},
issn = {1741-0134},
mesh = {*Methyl-CpG-Binding Protein 2/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Protein Engineering/methods ; Nuclear Localization Signals/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; Animals ; Protein Domains ; },
abstract = {Clustered regularly interspaced short palindromic repeat interference (CRISPRi), the fusion of nuclease-inactive Cas9 with transcriptional repressor domains, is a powerful platform enabling site-specific gene knockdown across diverse biological contexts. Previously described CRISPRi systems typically utilize two distinct domain classes: (1) Krüppel-associated box domains and (2) truncations of the multifunctional protein, MeCP2. Despite widespread adoption of MeCP2 truncations for developing CRISPRi platforms, individual contributions of subdomains within MeCP2's transcriptional repression domain (TRD) toward enhancing gene knockdown remain unclear. Here, we dissect MeCP2's TRD and observe that two subdomains, the expected NcoR/SMRT interaction domain (NID) and an embedded nuclear localization signal (NLS), can separately enhance gold-standard CRISPRi platform performance beyond levels attained with the canonical MeCP2 protein truncation. Incorporating side-by-side analyses of nuclear localization and gene knockdown for over 30 constructs featuring MeCP2 subdomains or virus-derived NLS sequences, we demonstrate that appending C-terminal NLS motifs to dCas9-based transcriptional regulators, both repressors and activators, can significantly improve their effector function across several cell lines. We also observe that NLS placement greatly impacts CRISPRi repressor performance, and that modifying the subdomain configuration natively found within MeCP2 can also enhance gene suppression capabilities in certain contexts. Overall, this work demonstrates the interplay of two complimentary chimeric protein design considerations, transcriptional domain 'dissection' and NLS motif placement, for optimizing CRISPR-mediated transcriptional regulation in mammalian systems.},
}

*Methyl-CpG-Binding Protein 2/genetics/metabolism/chemistry
Humans
*CRISPR-Cas Systems
HEK293 Cells
*Protein Engineering/methods
Nuclear Localization Signals/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockdown Techniques
Animals
Protein Domains

@article {pmid40466269,
year = {2025},
author = {Jiang, C and Lei, D and Xu, B and Wang, Z and Fang, R and Tang, Y and Wang, H},
title = {High-efficient molecular detection system termed RAA-based CRISPR-Cas13a for novel duck orthoreovirus.},
journal = {Poultry science},
volume = {104},
number = {8},
pages = {105327},
pmid = {40466269},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Ducks ; *Reoviridae Infections/veterinary/diagnosis/virology ; *Orthoreovirus, Avian/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {The novel duck orthoreovirus (NDRV) is an immunosuppressive pathogen that significantly impacts the health of waterfowl breeding. Accurate, efficient, and convenient detection techniques are crucial for the prevention and control of NDRV, particularly in terms of field detection. By employing recombinase aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we have developed a highly sensitive enzymatic molecular system that combines Cas13a with T7 in vitro transcription and RAA, enabling efficient and accurate detection of NDRV at a sensitivity level of 10° copies/μL. Furthermore, the integration of portable lateral flow dipstick can effectively reduce the point-of-care testing time to 40 min, while exhibiting no cross-reactivity with duck hepatitis a virus, Tembusu virus and novel duck-origin goose parvovirus. Both this system and the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR) method demonstrated a consistent 100% accuracy in clinical samples. This study facilitated the development of an optimized assay, which enables specific detection of NDRV through a simplified procedure and significantly reduces the risk of contamination. This highlights the potential applicability of this assay for point-of-care testing.},
}

Animals
*Poultry Diseases/diagnosis/virology
*Ducks
*Reoviridae Infections/veterinary/diagnosis/virology
*Orthoreovirus, Avian/isolation & purification/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
Recombinases/metabolism

@article {pmid40449106,
year = {2025},
author = {Sun, D and Zhu, Y and Wang, M and Wang, J and Cheng, W and Li, Z and Deng, Y and Ou, X and Jia, R and Chen, S and Zhu, D and Liu, M and Zhao, X and Yang, Q and Wu, Y and Zhang, S and Huang, J and He, Y and Wu, Z and Cheng, A},
title = {A RT-ERA-CRISPR/Cas12a assay for rapid point-of-care duck hepatitis A virus detection.},
journal = {Poultry science},
volume = {104},
number = {8},
pages = {105316},
pmid = {40449106},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Poultry Diseases/diagnosis/virology ; *Hepatitis Virus, Duck/isolation & purification ; Point-of-Care Systems ; *Hepatitis, Viral, Animal/diagnosis/virology ; *Picornaviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Duck hepatitis A virus (DHAV) is a severe pathogen that threatens the duck industry. DHAV is transmitted primarily through the respiratory and gastrointestinal tracts. Therefore, developing accurate and rapid diagnostic technologies is crucial to prevent the spread of this infectious disease. Currently, the most widely used DHAV detection methods in clinical diagnosis include PCR, real-time PCR (RT-PCR) and ELISA, which require specialized equipment or trained professionals. Isothermal amplification technologies are often combined with lateral flow assays or visual readout methods for detecting pathogen nucleic acids in non-laboratory settings. CRISPR nucleases have accelerated the development of nucleic acid detection, increasing the sensitivity to a higher degree. Here, we applied reverse transcription-enzymatic recombinase amplification (RT-ERA) assisted by a Cas12a-fluorescence assay and a Cas12a-lateral flow assay for the detection of DHAV-1. Based on the sequence of DHAV-1, RT-ERA primers and crRNAs were designed, and different concentrations of ssDNA/Cas12a/crRNA were established to optimize the CRISPR reaction. The LoD for the Cas12a-fluorescence assay was 10 copies/μL, and this assay effectively differentiated DHAV-1 from other avian pathogens, exhibiting high sensitivity and specificity. Additionally, the Cas12a-lateral flow assay is user-friendly and can achieve point-of-care detection. Sixty-four clinical samples were tested and compared with quantitative real-time PCR (RT-PCR). This accurate and rapid point-of-care assay has significant potential for detecting DHAV-1 in clinical applications, especially for duck farms in rural areas.},
}

Animals
*Ducks
*Poultry Diseases/diagnosis/virology
*Hepatitis Virus, Duck/isolation & purification
Point-of-Care Systems
*Hepatitis, Viral, Animal/diagnosis/virology
*Picornaviridae Infections/veterinary/diagnosis/virology
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
CRISPR-Cas Systems

@article {pmid40707593,
year = {2025},
author = {Mahajan, D and Chia, HM and Lu, L},
title = {Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26980},
pmid = {40707593},
issn = {2045-2322},
support = {Tier 1 RG 25/22//Ministry of Education - Singapore/ ; },
mesh = {*Cilia/metabolism/genetics ; *ADP-Ribosylation Factors/genetics/metabolism ; CRISPR-Cas Systems ; Hedgehog Proteins/metabolism ; Signal Transduction ; Cell Line ; Animals ; Humans ; Gene Knockout Techniques ; Mice ; Smoothened Receptor/metabolism ; },
abstract = {Small G protein ARL13B localizes to the cilium and plays essential roles in cilium biogenesis, organization, trafficking, and signaling. Here, we established multiple ARL13B knockout cell lines using the CRISPR/Cas9 system. Surprisingly, all our cell lines lost their cilia completely, in contrast to the reported short cilium and reduced ciliogenesis phenotype. We found that multiple regions of ARL13B are necessary for a complete rescue. Additionally, we found that ARL13B knockout cells also lost their response to SMO-mediated hedgehog stimulation. Our work demonstrates the critical requirement of ARL13B for ciliogenesis and hedgehog signaling, at least in cultured cells, and suggests that ARL13B plays a more crucial role in ciliary function than previously understood.},
}

*Cilia/metabolism/genetics
*ADP-Ribosylation Factors/genetics/metabolism
CRISPR-Cas Systems
Hedgehog Proteins/metabolism
Signal Transduction
Cell Line
Animals
Humans
Gene Knockout Techniques
Mice
Smoothened Receptor/metabolism

@article {pmid40707336,
year = {2025},
author = {Li, J and Lau, CH and Zhu, H},
title = {CRISPR/Cas-based personal glucose meters for nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.006},
pmid = {40707336},
issn = {1879-3096},
abstract = {Through integration with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems, personal glucose meters (PGMs) have been repurposed to detect non-glucose targets. PGMs enable the intuitive readout of electrochemical signals, while CRISPR/Cas offers rapid detection and signal amplification capability. Their integration can realize point-of-care (POC) diagnostics. Herein, we critically discuss their advances, pitfalls, and future perspectives.},
}

@article {pmid40704512,
year = {2025},
author = {Baig, MMFA and Chien, WT and Chair, SY},
title = {Nanotechnological approaches for the targeted delivery of CRISPR-Cas systems for genomic modifications, biomolecular sensing, and precision medicine.},
journal = {Biomaterials science},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5bm00711a},
pmid = {40704512},
issn = {2047-4849},
abstract = {The integration strategies of CRISPR-Cas gene editing systems with nanotechnological approaches have achieved synergistic effects in targeting genes; correcting genetic disorders; and treating, sensing, and diagnosing a variety of cancers and metabolic, immunological, and complex infectious diseases-all having connectivity with distinct genetic cues and mutations. Numerous recent studies have demonstrated the use of the nano-scale properties of nanomaterials to tremendously improve the genomic-editing efficiencies of CRISPR/Cas systems for achieving 50% enhanced bioavailability, improved cell targetability, and gene-level specificity while minimizing immunogenicity, compared with conventional/ordinary delivery techniques. Thus, nano-delivery methods utilizing the unique properties of nanomaterials, molecular interactions, biocompatibility, targeted cellular uptake, and nuclear delivery capability effectively overcame the challenges of inefficient biomolecular delivery, and off-target effects were effectively overcome. Nano -carriers made up of materials such as DNA lattices, lipids, dendrimers, polymers, peptides, and metals (gold, silver, etc.) that were explored for facilitating the precise delivery of CRISPR/Cas components, sensing biomolecules, and diagnostic purposes are discussed in this review report. The ability of DNA scaffold materials to incorporate nano-CRISPR systems, to sense biomolecules, and for targeted cellular delivery of payloads (e.g., Cas9, Cas12, Cas13, and Cas14 proteins and single-guide RNAs (sgRNAs)) maximized gene targeting and improved therapeutic outcomes while achieving up to 90% efficiency compared with common/trivial delivery methods.},
}

@article {pmid40704023,
year = {2025},
author = {Niu, M and Dong, Z and Yu, L and Dong, X and An, J and Han, Y and Yan, Y and Yi, W and Sun, Y and Li, H},
title = {Anti-RNA virus crRNA targets efficient screening platform based on bioinformatics and CRISPR detection.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102619},
pmid = {40704023},
issn = {2162-2531},
abstract = {The mutation and evolution of RNA viruses pose significant challenges in treatment efforts. The CRISPR-Cas system is a promising antiviral tool because of its powerful programmability. However, traditional cell screening methods for CRISPR targets are time-consuming, limiting their application. Here, we developed a rapid and efficient screening platform for crRNA targets by combining the CaSilico-based bioinformatics method with CRISPR in vitro detection technology. Using a bioinformational approach to design and screen crRNAs, the characteristics of crRNAs and the corresponding target sequences can be rapidly determined. CRISPR is used for secondary screening in vitro, enabling swift identification of the target site with optimal cleavage efficiency. This method significantly reduces the screening time for antiviral targets compared with traditional cell screening. We successfully designed and screened effective crRNA targeting SARS-CoV-2 conserved N gene regions and demonstrated its inhibition function in HEK 293T cells. We also designed and screened crRNAs targeting DENV to validate the feasibility of the platform. E-2330 crRNA reduced more than 90% of the DENV RNA load in multiple mammalian cell lines and effectively inhibited the replication of all four DENV serotypes. This study provides a new approach for screening antiviral crRNAs for antivirus research.},
}

@article {pmid40702552,
year = {2025},
author = {Vahabi, M and Xu, G and Sarkisjan, D and Hassouni, BE and Mantini, G and Donati, V and Wang, B and Lencioni, G and Honeywell, RJ and Deng, D and Strano, S and Peters, GJ and Blandino, G and Giovannetti, E},
title = {Unraveling resistance mechanisms to the novel nucleoside analog RX-3117 in lung cancer: insights into DNA repair, cell cycle dysregulation and targeting PKMYT1 for improved therapy.},
journal = {Journal of experimental & clinical cancer research : CR},
volume = {44},
number = {1},
pages = {217},
pmid = {40702552},
issn = {1756-9966},
support = {IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {Humans ; *Lung Neoplasms/drug therapy/genetics/pathology ; *Drug Resistance, Neoplasm ; *DNA Repair ; Cell Line, Tumor ; Cell Cycle/drug effects ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology ; *Cytidine/analogs & derivatives/pharmacology ; *Nucleoside-Phosphate Kinase/antagonists & inhibitors ; CRISPR-Cas Systems ; *Nucleosides/pharmacology ; },
abstract = {BACKGROUND: Nucleoside analogues are crucial in treating non-small cell lung cancer (NSCLC), but resistance hampers patient outcomes. The cytidine analogue RX-3117 shows promise in gemcitabine-resistant cancers, yet mechanisms underlying acquired resistance to this drug remain unexplored. This study includes a comprehensive investigation into RX-3117 resistance mechanisms by leveraging new preclinical models and cutting-edge genomic tools, including a CRISPR-Cas9 knockout screen and transcriptomics.

METHODS: NSCLC cell lines A549 and SW1573 were exposed to stepwise increasing concentrations of RX-3117 to establish stable resistant subclones, confirmed by SRB and clonogenic assays. Intracellular RX-3117 nucleotide levels were measured via LC/MS-MS, prompting the evaluation and modulation of the expression of key metabolic enzymes by Western blot and siRNA. A CRISPR-Cas9 screen identified genes whose loss increased RX-3117 sensitivity, while RNA-sequencing with differential expression analyses revealed resistance-related pathways, further investigated through cell cycle distribution, knock-out, and ELISA assays.

RESULTS: Resistant clones exhibited decreased accumulation of RX-3117 nucleotides, which however, was not associated to reduced expression of activation enzymes (UCK2, UMPK, CMPK, NME1/NDPK, RR1 and RR2). Instead, increased expression was observed in certain DNA repair and deactivation enzymes (NT5C3) but pharmacological inhibition and silencing of the latter did not circumvent resistance. Remarkably, a comprehensive approach with CRISPR-Cas9 screen highlighted DNA-repair and cell cycle determinants as key sensitizing genes. XL-PCR and RNA-sequencing confirmed aberrations in DNA-repair and pathways involved in cell cycle regulation. Knock-out and pharmacological inhibition validated the role of PKMYT1, a protein kinase involved in G2/M transition and genomic stability. RX-3117-resistant A549 cells showed enhanced sensitivity to the PKMYT1 inhibitor lunresertib and its synergism with RX-3117, suggesting further studies, especially in patients with high PKMYT1 expression who have significantly shorter survival rates, as observed in public databases and validated in an internal cohort of NSCLC patients.

CONCLUSION: By integrating CRISPR-Cas9 with functional assays and transcriptomics, our study established a framework for decoding resistance mechanisms and highlights potential therapeutic strategies to enhance RX-3117 efficacy in NSCLC. We demonstrated for the first time that aberrant DNA repair and cell cycle dysregulation led resistance, identifying PKMYT1 as a promising target.},
}

Humans
*Lung Neoplasms/drug therapy/genetics/pathology
*Drug Resistance, Neoplasm
*DNA Repair
Cell Line, Tumor
Cell Cycle/drug effects
*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology
*Cytidine/analogs & derivatives/pharmacology
*Nucleoside-Phosphate Kinase/antagonists & inhibitors
CRISPR-Cas Systems
*Nucleosides/pharmacology

@article {pmid40702188,
year = {2025},
author = {Baglaenko, Y and Mu, Z and Curtis, M and Mire, HM and Jayanthi, V and Al Suqri, M and Liu, C and Agnew, R and Nathan, A and Mah-Som, AY and Liu, DR and Newby, GA and Raychaudhuri, S},
title = {Precisely defining disease variant effects in CRISPR-edited single cells.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40702188},
issn = {1476-4687},
abstract = {Genetic studies have identified thousands of individual disease-associated non-coding alleles, but the identification of the causal alleles and their functions remains a critical bottleneck[1]. CRISPR-Cas editing has enabled targeted modification of DNA to introduce and test disease alleles. However, the combination of inefficient editing, heterogeneous editing outcomes in individual cells and nonspecific transcriptional changes caused by editing and culturing conditions limits the ability to detect the functional consequences of disease alleles[2,3]. To overcome these challenges, we present a multi-omic single-cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome and measures cell-surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, non-coding single-nucleotide polymorphism alleles and multiplexed editing. We identify the effects of individual single-nucleotide polymorphisms, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single-cell assays, including DNA sequencing, enable the identification of causal variation in primary human cells and bridge a crucial gap in our understanding of complex human diseases.},
}

@article {pmid40617066,
year = {2025},
author = {Wang, L and He, X and Tian, T and Cheng, J and Cao, R and Hou, J and Lin, H and Li, Y},
title = {Functional characterization of CEL3C reveals its critical role in regulating cellulase gene expression in Trichoderma reesei Rut C30.},
journal = {Enzyme and microbial technology},
volume = {190},
number = {},
pages = {110706},
doi = {10.1016/j.enzmictec.2025.110706},
pmid = {40617066},
issn = {1879-0909},
mesh = {*Cellulase/genetics/metabolism ; *Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics/metabolism ; *Hypocreales/genetics/enzymology ; *beta-Glucosidase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The nuclear-localized β-glucosidase CEL3C in Trichoderma reesei plays a pivotal role in cellulase regulation, though its mechanism remains poorly understood. To address this, we disrupted CEL3C in the hypercellulolytic strain T. reesei Rut C30 via CRISPR-Cas9 and evaluated cellulase production under sophorose-rich MGD induction. Deletion of CEL3C significantly enhanced total cellulase activity by 31.28 % (p < 0.05), with β-glucosidase, endoglucanase, and cellobiohydrolase activities increasing by 94.97 %, 19.40 %, and 28.99 %, respectively. These improvements were driven by transcriptional upregulation of core cellulase genes (CEL7A: 2.01-fold; CEL6A: 1.5-fold; CEL12A: 2.0-fold; CEL5A: 1.32-fold) and β-glucosidases (CEL3A: 6.41-fold; CEL3B: 5.02-fold), confirming transcriptional-level control as the dominant regulatory mechanism. Transcriptomic profiling identified 688 differentially expressed genes (399 upregulated, 299 downregulated), with key changes including activation of transcriptional activators XYR1 (59.6 % increase), ACE3 (75.49 % increase), and RXE1 (161.56 % increase), suppression of repressors RCE1 (65.86 % decrease) and RCE2 (65.23 % decrease), and induction of sugar transporters (TrireC30_133589: 13.41-fold) and ER chaperones (BIP1: 1.26-fold; PDI1: 1.55-fold). These alterations collectively enhanced inducer uptake, enzyme maturation, and secretion while alleviating MAPK-mediated repression (TMK2: 110.54 % decrease). Intracellular sugar profiling revealed that gentiobiose and cellobiose were undetectable in the T. reesei ΔCEL3C, whereas glucose and sophorose levels increased by 31.71 % and 13.45 % (p < 0.05), respectively. These results suggest that CEL3C deletion enhances β-glucosidase-mediated hydrolysis of disaccharides into glucose and possibly promotes sophorose formation via transglycosylation. In parallel, the upregulation of disaccharide transporters may facilitate sophorose uptake. Together, these two mechanisms contributed to the intracellular enrichment of sophorose, thereby amplifying cellulase gene induction and enzyme production. Our findings establish CEL3C as a dual-function nuclear regulator that balances cellulase synthesis through transcriptional and enzymatic pathways, providing actionable targets for engineering T. reesei with optimized industrial cellulase yields.},
}

*Cellulase/genetics/metabolism
*Gene Expression Regulation, Fungal
*Fungal Proteins/genetics/metabolism
*Hypocreales/genetics/enzymology
*beta-Glucosidase/genetics/metabolism
CRISPR-Cas Systems