RRC ID 73354
Author Ishibashi R, Maki R, Kitano S, Miyachi H, Toyoshima F.
Title Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a.
Journal Sci Rep
Abstract The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.
Volume 12(1)
Pages 17775
Published 2022-10-22
DOI 10.1038/s41598-022-22639-6
PII 10.1038/s41598-022-22639-6
PMID 36272994
PMC PMC9588054
MeSH Animals CRISPR-Cas Systems* DNA Endonucleases / genetics Gene Editing* / methods Mice Plasmids / genetics RNA / genetics Transgenes
IF 3.998
DNA material CSII-EF-mRFP1-RfA (RDB012128)