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

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

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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 ultrastar dc hc550 mating-types, diploids and polyploids.We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, 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 polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy.

The CRISPR/Cas9 mating-type switching method will be useful in click here 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/α/α isogenic tetraploids.