Continuous induction on plates
For these experiments, cells are plated in parallel on induction medium
(SC-Ind, without methionine and cysteine) and repression medium (SC-Rep,
with methionine and cysteine), both of which select for the presence of
the URA3 plasmid. Counting colonies on both types of plates
allows estimation of the lethality caused by the CRISPR-Cas9-induced
DSB.
As shown on figure 2, controls without the plasmid and with the plasmid
that does not express the gRNA, show that there is no major deleterious
effect of the induction medium, nor of the expression of Cas9, since
survival ranges between 84 and 100%. for all species When the gRNA is
expressed in addition to Cas9, survival drops to approximately 17% forC. nivariensis and 34% for C. bracarensis , showing that
the continuous induction of a DSB is lethal for most cells of these two
species. Survival is much higher, almost 90%, in C. glabrata .
This could be due to inefficient cutting, or to a high level of
unfaithful NHEJ (see below and discussion).
One way of testing the efficiency of the cut is to observe how many are
repaired by unfaithful NHEJ, which we expect to result in a
non-functional ADE2 gene, and therefore in a red colony
phenotype. We observe that in C. glabrata , 100% of colonies are
red, and in the two other Candida species, more than 96% of
colonies are red (not shown). This shows that the large majority of
surviving cells in all species has repaired the cut by unfaithful NHEJ,
thus inactivating the ADE2 gene. This is much higher than what we
reported with our previous system in C. glabrata (Maroc and
Fairhead, 2019), and probably reflects a more efficient cutting because
of a different choice of gRNA, or because the plasmid is more stable in
this yeast.