DISCUSSION
Our results show that the CRISPR-Cas9 system can be used to induce DSBs
in all three species studied here, when desired, after transformation of
cells with the same plasmid in which the appropriate gRNA sequence is
cloned. All species here are haploid, and since we did not provide an
intact template for homologous repair of the cut-site in the ADE2 gene, repair must occur through NHEJ. Our red/white screening strategy,
in parallel with monitoring of cell death on plates and in time-course
experiments, allows us to estimate the efficiency of cut and repair in
each species. Repair by faithful NHEJ results in a regenerated, intact,
cut-site, and thus cutting by CRISPR-Cas9 can occur once more. If cells
do not die from this, through cell-cycle checkpoint arrest, or through
degradation of chromosomal DNA at some point, they can undergo a futile
cut-ligate-recut cycle (Maroc and Fairhead, 2019). Escaping this
potential futile cycle can occur through plasmid mutation/rearrangement,
or through plasmid loss, accompanied by integration of the URA3 gene (whose presence is selected in the medium), and finally through
events of repair by unfaithful NHEJ that modify the cut-site recognition
sequence. In our experiments targeting the ADE2 gene, we observe
a very high percentage of red colonies in survivors, indicating a very
efficient chromosomal cutting followed by unfaithful NHEJ.
In S. cerevisiae , a CRISPR-Cas9-created chromosomal cut, in a
haploid strain in a unique region is lethal for most cells (Yarrington
et al., 2018). In C. bracarensis and C. nivariensis , even
though survival is higher than in the model yeast, only 17%-34% of
cells survive continuous induction on plates. In temporary liquid medium
induction, we observe that survival drops with increasing exposure and
that long exposure results in survival rates similar to induction on
plates. The percentage of cells repaired by unfaithful NHEJ (red
colonies) increases with exposure time, reflecting either the time
course of cutting by CRISPR-Cas9, or the prevalence of faithful NHEJ at
the beginning of exposure, gradually replaced by unfaithful NHEJ events
which are resistant to further cutting.
It is remarkable that C. glabrata has a much higher survival rate
than the other two Candida . We have previously determined that
cells of C. glabrata undergo futile cycles of cut-religation and
that the cell-cycle chekpoint must be “leaky” (Maroc and Fairhead,
2019). The situation may well be different in the other two otherCandida studied here. Another, non exclusive, reason for lower
survival may be that unfaithful NHEJ is much less efficient in the other
two species than in C. glabrata , preventing the creation of
non-cuttable ade2 - alleles in the former. On the other hand,
perhaps the cell cycle checkpoints are less ”leaky” in C.
bracarensis and C. nivariensis , and cells with unrepaired DSBs
die more frequently.
Sequencing of the new junctions created by this type of NHEJ, i.e. from
red colonies, shows that cells have repaired the DSB with varying
amounts of rearrangements, that differ between species. There is more
variation in the types of unfaithful NHEJ repair events in C.
bracarensis and C. nivariensis than in C. glabrata , where
survival is also much higher. Perhaps an efficient and reproducible
unfaithful NHEJ mechanism in C. glabrata allows this survival
rate, accompanied by leakiness of checkpoints. This may signify
different adaptation mechanisms, and remains to be investigated
thoroughly.