論文 - 詳細
| RRC ID | 57727 |
|---|---|
| 著者 | Iriki H, Kawata T, Muramoto T. |
| タイトル | Generation of deletions and precise point mutations in Dictyostelium discoideum using the CRISPR nickase. |
| ジャーナル | PLoS One |
| Abstract |
The CRISPR/Cas9 system enables targeted genome modifications across a range of eukaryotes. Although we have reported that transient introduction of all-in-one vectors that express both Cas9 and sgRNAs can efficiently induce multiple gene knockouts in Dictyostelium discoideum, concerns remain about off-target effects and false-positive amplification during mutation detection via PCR. To minimise these effects, we modified the system to permit gene deletions of greater than 1 kb via use of paired sgRNAs and Cas9 nickase. An all-in-one vector expressing the Cas9 nickase and sgRNAs was transiently introduced into D. discoideum, and the resulting mutants showed long deletions with a relatively high efficiency of 10-30%. By further improving the vector, a new dual sgRNA expression vector was also constructed to allow simultaneous insertion of two sgRNAs via one-step cloning. By applying this system, precise point mutations and genomic deletions were generated in the target locus via simultaneous introduction of the vector and a single-stranded oligonucleotide template without integrating a drug resistance cassette. These systems enable simple and straightforward genome editing that requires high specificity, and they can serve as an alternative to the conventional homologous recombination-based gene disruption method in D. discoideum. |
| 巻・号 | 14(10) |
| ページ | e0224128 |
| 公開日 | 2019-10-17 |
| DOI | 10.1371/journal.pone.0224128 |
| PII | PONE-D-19-24103 |
| PMID | 31622451 |
| PMC | PMC6797129 |
| MeSH | Base Sequence CRISPR-Cas Systems / genetics* Deoxyribonuclease I / genetics Deoxyribonuclease I / metabolism Dictyostelium / genetics* Gene Editing / methods Point Mutation RNA, Guide, Kinetoplastida / genetics RNA, Guide, Kinetoplastida / metabolism |
| IF | 2.74 |
| 引用数 | 1 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 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 | |
| 細胞性粘菌 | G90484 G90485 G90486 G90487 |