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A Simple PCR-based Strategy for the Introduction of Point Mutations in the Yeast Saccharomyces cerevisiae via CRISPR/Cas9

Guohui Hu, Shiwen Luo, Hai Rao, Haili Cheng and Xin Gan

The methods currently employed for in vivo site-directed mutagenesis in yeast are laborious and/or inefficient. Recent developments of the CRISPR-based approaches hold great promise for genome editing, but its application in the yeast S. cerevisiae remains a time-consuming affair. The rate-limiting step in CRISPR-mediated genetic engineering in yeast is the incorporation of the guide sequences, which target Cas9 to relevant chromosomal locus, into the relevant yeast vectors. Here we present a PCR-based strategy to introduce specific point mutation into the yeast CDC48 gene via CRISPR. Our method eliminates the need for special dam- strain and markedly shortens the elaborate multi-step cloning process, leading to significant savings in time, labor and cost.