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.