RRC ID 63013
Author Ishibashi R, Abe K, Ido N, Kitano S, Miyachi H, Toyoshima F.
Title Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence.
Journal Sci Rep
Abstract CRISPR/Cas-mediated genome editing is a powerful tool for generating genetically mutated cells and organisms. Linearisation of donor cassettes with this system has been shown to facilitate both transgene donor insertion and targeted knock-in. Here, we developed a donor plasmid that we name pCriMGET (plasmid of synthetic CRISPR coded RNA target sequence-equipped donor plasmid-mediated gene targeting), in which an off-target free synthetic CRISPR coded RNA-target sequence (syn-crRNA-TS) is incorporated with a multi-cloning site, where a donor cassette can be inserted. With co-expression of Cas9 and the syn-crRNA-TS guide RNA (gRNA), pCriMGET provides a linearised donor cassette in vivo, thereby promoting the transgene donor insertion and targeted knock-in. When co-injected with Cas9 protein and gRNA into murine zygotes, pCriMGET yielded around 20% transgene insertion in embryos. This method also achieved more than 25% in-frame knock-in at the mouse Tbx3 gene locus without predicted insertion-deletion mutations using a transgene donor with 400-bp homology arms. pCriMGET is therefore useful as a versatile CRISPR/Cas9-cleavable donor plasmid for efficient integration and targeted knock-in of exogenous DNA in mice.
Volume 10(1)
Pages 14120
Published 2020-8-24
DOI 10.1038/s41598-020-70804-6
PII 10.1038/s41598-020-70804-6
PMID 32839482
PMC PMC7445171
MeSH Animals CRISPR-Cas Systems / genetics* Female Gene Editing / methods* Gene Knock-In Techniques / methods* Gene Targeting Genetic Engineering / methods Genome / genetics HEK293 Cells HeLa Cells Humans INDEL Mutation / genetics Mice Mice, Inbred C57BL Mice, Inbred ICR Mice, Transgenic Plasmids / genetics* RNA, Guide / genetics* T-Box Domain Proteins / genetics* Transgenes / genetics
IF 3.998
Resource
Human and Animal Cells 293T(RCB2202)
DNA material pCriMGET_v1 (RDB19318) pX330-syn-crRNA-TS-sgRNA (RDB19319) pCriMGET_v1-AAVS1-SA-Neo-pA (RDB19320) pCriMGET_v1-Tbx3-3xFlag-2A-EGFP (RDB19321) pCriMGET_v1-Clec4f-IRES-hDTR-2A-EGFP (RDB19322) pX330-AAVS1-sgRNA (RDB19323)