清脆的
基因组编辑
Cas9
质粒
波姆裂殖酵母
生物
引导RNA
裂殖酵母
克隆(编程)
遗传学
DNA
计算生物学
酵母
酿酒酵母
基因
计算机科学
程序设计语言
作者
Xiao-Ran Zhang,Jia-Bei He,Wang Y,Li-Lin Du
标识
DOI:10.1534/g3.118.200164
摘要
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.
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