Mutation spectrum and frequencies of gene libraries generated with in vivo directed evolution in S. cerevisiae
We developed a novel in vivo mutagenesis tool for protein engineering that leverages S. cerevisiae engineered to exhibit mutagenic activity directed to the gene of interest. Mutagenesis is achieved by generating DNA damage through nucleoside deamination followed by introduction of further mutations by harnessing error-prone DNA translesion synthesis. We generated libraries of mitotic spindle protein she1 using two yeast mutator strains and performed UMI-linked nanopore sequencing to identify and characterize the generated mutations.