Unlabelled: The CRISPR/Cas or Cas9/guide RNA system is a newly developed, easily engineered and highly effective tool for gene targeting; it has considerable off-target effects in cultured human cells and in several organisms. However, the Cas9/guide RNA target site is too short for existing alignment tools to exhaustively and effectively identify potential off-target sites. CasOT is a local tool designed to find potential off-target sites in any given genome or user-provided sequence, with user-specified types of protospacer adjacent motif, and number of mismatches allowed in the seed and non-seed regions.
Availability: http://eendb.zfgenetics.org/casot/ CONTACT: zfgenetics@gmail.com or bzhang@pku.edu.cn Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btt764 | DOI Listing |
mSystems
January 2025
Department of Infectious Diseases, School of Translational Medicine, Monash University, Melbourne, Victoria, Australia.
Unlabelled: Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve the yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich the human pathogen , by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene .
View Article and Find Full Text PDFAppl Microbiol Biotechnol
December 2024
Graduate School of Agriculture, Kyoto University, Sakyo-Ku, Kitashirakawaoiwakecho, Kyoto, 606-8502, Japan.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.
Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient.
View Article and Find Full Text PDFInt J Mol Sci
November 2024
Department of Health Pharmacy, Yokohama University of Pharmacy, 601 Matano, Totsuka, Yokohama 245-0066, Japan.
Mol Ther Methods Clin Dev
December 2024
Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Adenoviruses (Ads) are potent gene delivery vectors for and applications. However, current methods for their construction are time-consuming and inefficient, limiting their rapid production and utility in generating complex genetic libraries. Here, we introduce FastAd, a rapid and easy-to-use technology for inserting recombinant "donor" DNA directly into infectious "receiver" Ads in mammalian cells by the concerted action of two efficient recombinases: Cre and Bxb1.
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