Using Saccharomyces cerevisiae to study the role of the X family DNA polymerase POL4 in CRISPR/Cas9-induced mutation

The yeast repair DNA polymerase POL4 is part of the X family of DNA polymerases that function in DNA repair, specifically in gap filling for non-homologous end joining (NHEJ). POL4 is the only Pol X family polymerase in Saccharomyces cerevisiae and has been shown to have a role in creating deletions or insertions after double-strand break (DSB) repair by NHEJ, an error-prone mechanism. I investigated the role of the yeast POL4 genotype on mutations induced by a previously described yeast CRISPR/Cas9 system. In this system a galactose-inducible Cas9 is used in combination with a gRNA targeted to the yeast CAN1 gene. CAN1 mutations are easily selected by resistance to canavanine. Our initial analysis suggested that the pol4 mutant genotype resulted in larger deletions compared to wild-type POL4. However, we found that the Cas9 plasmid induced mutations in the absence of galactose indicating that basal Cas9 expression was sufficient for inducing mutations. This suggested that the mutations I identified were in many cases clonal and not independent from each other. This hypothesis was supported by similar mutations being enriched for within an experiment performed on the same day. After controlling for independence, we did not detect a POL4 genotype-dependent effect on mutation type. We also asked whether the POL4 genotype affected the overall mutation frequency in both haploid and diploid yeast. We found pol4 haploids had less overall CRISPR/Cas9 induced mutations than POL4 haploids while in diploids the overall mutation frequency was lower than in haploids but did not vary by genotype. Our results suggest that a requirement for POL4 in repair of DSBs caused by CRISPR/Cas9 is minor at best