SRA

It is well known that ssDNA is more susceptible to mutagenesis than dsDNA due to the exposure of nucleobases. Previous studies have shown that DSBs induced in the presence of UV, alkylating agents, bisulfite, or APOBEC can lead to mutagenesis in their flanking regions, resembling kataegis or clustered mutations observed in cancer genomes. Our lab has demonstrated that dSpCas9 can stall the progression of the DSB resection machinery. Based on this, we hypothesized that dSpCas9 could be applied to control or restrict the region susceptible to experimentally induced kataegis: the DSB distal side of the dSpCas9 binding site exhibits a lower mutation rate than the DSB proximal side and is thus protected from kataegis. We termed this approach of region restricted random mutagenesis controlled kataegis. We performed a proof of concept experiment for controlled kataegis using the CAN1 gene in a reporter Saccharomyces cerevisiae strain. In the presence of bisulfite, binding dSpCas9 to the region between an upstream DSB and the CAN1 gene resulted in a reduced frequency of Canavanine resistant mutants compared to the control strain, which contained the DSBs but no the dSpCas9 binding. To confirm that the Canavanine resistant mutations arose from ssDNA generated by resection rather than ssDNA from transcription of CAN1, and to assess the distribution of mutations in the mutants, we performed targeted sequencing of the CAN1 region.