| RRC ID |
63013
|
| 著者 |
Ishibashi R, Abe K, Ido N, Kitano S, Miyachi H, Toyoshima F.
|
| タイトル |
Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence.
|
| ジャーナル |
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.
|
| 巻・号 |
10(1)
|
| ページ |
14120
|
| 公開日 |
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, Kinetoplastida / genetics*
T-Box Domain Proteins / genetics*
Transgenes / genetics
|
| IF |
3.998
|
| リソース情報 |
| ヒト・動物細胞 |
293T(RCB2202) |
| 遺伝子材料 |
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) |
