The aim of this work was to rapidly and efficiently insert target DNA sequences into predetermined genomic sites in Saccharomyces cerevisiae. In this study, we designed two technical routes for gene insertion in the S. cerevisiae genome based on the CRISPR/Cas9 system, and a CRISPR array was inserted into the Amp site and the crRNA site of the pCRCT plasmid, respectively. The CRISPR array consists of a 100 bp donor sequence, the target gene and guide sequence. A 100 bp donor sequence was designed to have two 50 bp homology arms flanking the Cas9 cutting site and incorporate 8 bp or 1000 bp deletions including the PAM sequence, where the target gene was also inserted. The results showed that using only one pCRCTG plasmid and a 100 bp dsDNA mutagenizing homologous recombination donor, we can successfully insert a 2.9 kb gene fragment at the target site of the S. cerevisiae genome. However, inserting the CRISPR array into the crRNA site has a higher recombination efficiency than inserting into the Amp site. This recombination strategy represents a powerful tool for creating yeast strains with target gene inserts.
Keywords: CRISPR/Cas9; Gene insertion; Homologous recombination; Saccharomyces cerevisiae; pCRCTG.
© King Abdulaziz City for Science and Technology 2021.