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.

我们开发了一款用于蛋白质工程的新型体内诱变工具，该工具依托经工程改造的酿酒酵母（S. cerevisiae），可实现靶向目标基因的诱变活性。该诱变策略通过核苷脱氨作用产生DNA损伤，随后借助易出错的DNA跨损伤合成引入额外突变。我们采用两种酵母突变株构建了有丝分裂纺锤体蛋白She1的突变文库，并开展了唯一分子标识符（Unique Molecular Identifier，UMI）关联纳米孔测序，以鉴定并表征所产生的突变。