Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in  Schizosaccharomyces pombe  and New 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  S. pombe  genome; however, most others can recombine with the genome due to sequence homology shared between the genomic and plasmid-borne copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce "designer" auxotrophic marker deletions into host strains, including  leu1 -Δ 0 ,  his3 -Δ 0 , and  lys9 -Δ 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  S. 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.
Zhao Y, Boeke JD