Mutation spectrum and frequencies of gene libraries generated with in vivo directed evolution in S. cerevisiae. YeastIT

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.

我们开发了一种用于蛋白质工程的新型体内诱变工具，该工具借助经过工程改造的酿酒酵母（Saccharomyces cerevisiae，简称S. cerevisiae），使其能够针对目标基因展现定向诱变活性。该诱变过程通过核苷脱氨作用产生DNA损伤，再利用易错DNA跨损伤合成（error-prone DNA translesion synthesis）引入额外突变来完成。我们使用两株酵母诱变菌株构建了有丝分裂纺锤体蛋白SHE1的突变文库，并通过连接唯一分子标识符（Unique Molecular Identifier，UMI）的纳米孔测序技术对所产生的突变进行鉴定与表征。