Yeast Genome Mutagenesis With Multi‐Mismatch PCR: A Rapid and Efficient Strategy for Site‐Directed Mutagenesis in Saccharomyces cerevisiae

Saccharomyces cerevisiae (S. cerevisiae) provides an array of cost-effective and time-efficient methods for diverse genome modifications. Among these techniques, site-directed mutagenesis of target genes is a powerful strategy to elucidate intricate structure-function relationships and create specific mutations. While various PCR-based and CRISPR/Cas9-based methods have been developed for introducing point mutations into the S. cerevisiae genome, they often involve multiple steps. In this study, we presented a rapid and effective site-directed mutagenesis strategy using one-step multi-mismatch PCR, termed Yeast Genome Mutagenesis with Multi-mismatch PCR (YGMMP). YGMMP incorporated multiple synonymous mutations proximal to the target point mutations, along with a selection marker cassette and flanking homologous sequences, into the gene segment spanning from the desired mutation to the gene's terminus through overlap PCR. The resulting PCR product was introduced into yeast cells to facilitate the selection of target variants. As a proof of concept, we applied YGMMP to generate an ADE2 mutant. The results demonstrated that the introduction of five and nine synonymous mutations, in addition to the desired single-point mutation, yielded mutagenesis efficiencies of approximately 20% and 30%, respectively. This rapid, straightforward, and efficient method has the potential to greatly simplify site-specific modifications within the S. cerevisiae genome.