Rationale
Since our previous plasmid for in vivo expression of CRISPR-Cas9 in C. glabrata was genetically unstable because of the presence of sequence repeats (see Materials and Methods), we decided to construct a new plasmid, pCFYF, as described in Materials and Methods, and shown on Figure 1. The MET3 promoter for inducible Cas9 expression, theSNR52 promoter for constitutive gRNA expression, and theURA3 marker are the same as in the previous version.
We decided to test our new expression system on the three pathogenicCandida species of the Nakaseomyces clade, targeting theADE2 gene, since it provides a phenotypic screen of red/white colony color, allowing the detection of inactivating mutations in the gene. All species contain a homolog of the ADE2 gene (table 3). We chose the cut site within the gene so as that it is always situated in the same region of the gene, at a distance of 464 nucleotides from the first nucleotide of the ATG. Even though the gene sequence is diverged and individual gRNAs had to be designed, the environment of the cut sequence is rather well conserved, as observed in the gRNAs sequences (table 3).
Once the DSB has been created, since the ADE2 gene is in single copy in the haploid genome of the strains considered, and since no homologous template for repair is transformed into cells, the DSB cannot be repaired by Homologous Recombination, but only by NHEJ. If NHEJ is faithful and does not introduce a sequence modification, the site can be recut, as long as Cas9 and the gRNA are expressed, i.e. during continuous growth of cells in induction medium and in absence of genetic modification of the plasmid. Cells may then undergo multiple rounds of cut-ligate-recut events, in a futile cycle (Maroc and Fairhead, 2019). If repair occurs by unfaithful NHEJ, the sequence will be modified and the gRNA may not recognize it anymore as a cut-site. Furthermore, most unfaithful NHEJ should result in a non-functional gene, so that cells will become Ade- and the red pigment should accumulate.