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.

这项工作的目的是快速有效地将目标 DNA 序列插入酿酒酵母的预定基因组位点。在本研究中，我们基于CRISPR/Cas9系统设计了两条将基因插入酿酒酵母基因组的技术路线，将CRISPR阵列插入AmppCRCT质粒的位点和crRNA位点，分别。CRISPR 阵列由一个 100 bp 的供体序列、靶基因和指导序列组成。一个 100 bp 的供体序列被设计为在 Cas9 切割位点两侧具有两个 50 bp 的同源臂，并包含 8 bp 或 1000 bp 的缺失，包括 PAM 序列，其中还插入了靶基因。结果表明，仅使用一个pCRCTG质粒和一个100 bp dsDNA诱变同源重组供体，我们就可以在酿酒酵母基因组的靶位点成功插入一个2.9 kb的基因片段。然而，将CRISPR阵列插入crRNA位点比插入Amp具有更高的重组效率地点。这种重组策略代表了一种强大的工具，用于创建带有目标基因插入的酵母菌株。