論文 - 詳細
| 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, CRISPR-Cas Systems / genetics* T-Box Domain Proteins / genetics* Transgenes / genetics |
| IF | 3.998 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 2 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 遺伝子材料 | pCriMGET_v1-Tbx3-3xFlag-2A-EGFP (RDB19321) pCriMGET_v1-Clec4f-IRES-hDTR-2A-EGFP (RDB19322) pX330-AAVS1-sgRNA (RDB19323) pCriMGET_v1 (RDB19318) pX330-syn-crRNA-TS-sgRNA (RDB19319) pCriMGET_v1-AAVS1-SA-Neo-pA (RDB19320) |
| ヒト・動物細胞 | 293T(RCB2202) HeLa |