Mutational impact of PolE exonuclease domain mutants

Several PolE exonuclease-domain mutants were introduce in RPE-1 cells by CRISPR-Cas9 gene editing in a homozygous manner. As a control, the wildtype sequence was edited similarly, but in a way to keep the wildtype sequence. Single cell clones were established and cultivated for around 60 cell cycles. Then, single cell clones from these cultures were expanded to purify enough gDNA for whole genome sequencing. This approach let us quantify the amount of mutations acquired in PolE mutant clones compared with the amount of PolE wildtype clones and thus the mutational impact of the knock-in PolE mutation.