論文 - 詳細
| RRC ID | 50484 |
|---|---|
| 著者 | Zhang XR, He JB, Wang YZ, Du LL. |
| タイトル | A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast. |
| ジャーナル | G3 (Bethesda) |
| Abstract |
The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast. |
| 巻・号 | 8(6) |
| ページ | 2067-2077 |
| 公開日 | 2018-5-31 |
| DOI | 10.1534/g3.118.200164 |
| PII | g3.118.200164 |
| PMID | 29703785 |
| PMC | PMC5982833 |
| MeSH | Base Sequence CRISPR-Cas Systems / genetics* Cloning, Molecular DNA Repair / genetics Gene Editing / methods* Gene Knock-In Techniques Point Mutation / genetics Schizosaccharomyces / genetics* Sequence Deletion Temperature |
| IF | 2.781 |
| 引用数 | 3 |
| 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 | X(Twitter) |
| 各媒体での言及数の合計 | 6 |
| 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 | |
| 酵母 | FY29043(JB938) |