Construction of Designer Selectable Marker Deletions with CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and Optimized Design of Common Entry Vectors

Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4⁺ and ScLEU2 from Saccharomyces cerevisiae, which complement ura4 and leu1 mutations, respectively. These two auxotrophic markers share no homology with the Sch. pombe genome, however most others can recombine with the genomic due to sequence homology shared between the genomic and plasmid-born copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce into host strains "designer" auxotrophic marker deletions, including leu1-0, his3-0 and lys9-0. Together with ura4-D18, this brings the total number of available designer deletion auxotrophic markers to four. The toolbox consists of a Cas9-gRNA expression vector and a donor DNA plasmid pair for each designer deletion. Using this toolbox, a set of auxotrophic Sch. pombe strains was constructed. Further, we reorganized essential components in the commonly used pREP series of plasmids and assembled the corresponding auxotrophic marker gene onto these plasmids. This toolbox for producing designer deletions, together with the newly developed strains and plasmids will benefit the whole yeast community.

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