A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast. Issue 6 (1st June 2018)

• Record Type: Journal Article
• Authors: Zhang, Xiao-Ran; He, Jia-Bei; Wang, Yi-Zheng; Du, Li-Lin
• Is Part Of: G3. Volume 8:Issue 6(2018)
• Page Start: 2067
• Page End: 2077
• Publication Date: 2018-06-01
• Subjects: CRISPR/Cas9 -- genome editing -- gap repair -- fission yeast -- Schizosaccharomyces pombe; Genetics -- Research -- Periodicals; Genomics -- Periodicals; Genetics; Genomics; Genes; Genetics -- Research; Genomics; Electronic journals; Periodical; Periodicals; Fulltext; Internet Resources; Periodicals; 572.8
• DOI: 10.1534/g3.118.200164
• Languages: English
• ISSNs: 2160-1836

Abstract

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.