Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae

Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations such as the construction of strains, for instance isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break (DSB) at the MAT locus, and in a single co-transformation, both haploid and diploid cells were switched to the specified mating-type at ~80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLα and HMRa cryptic mating loci.  Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid poly-synthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example in the construction of fully a/a/a/a isogenic tetraploids.

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