New technologies to target nucleotide diversification in vivo are promising enabling strategies to perform directed evolution for engineering applications and forward genetics for addressing biological questions. Recently, we reported EvolvR—a system that employs CRISPR-guided Cas9 nickases fused to nick-translating, error-prone DNA polymerases to diversify targeted genomic loci—in E. coli. As CRISPR-Cas9 has shown activity across diverse cell types, EvolvR has the potential to be ported into other organisms, including eukaryotes, if nick-translating polymerases can be active across species. Here, we implement and characterize EvolvR’s function in Saccharomyces cerevisiae, representing a key first step to enable EvolvR-mediated mutagenesis in eukaryotes. This advance will be useful for mutagenesis of user-defined loci in the yeast chromosomes for both engineering and basic research applications, and it furthermore provides a platform to develop the EvolvR technology for performance in higher eukaryotes.

中文翻译：

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利用CRISPR指导的DNA聚合酶I在酿酒酵母基因组中进行靶向多样化。

靶向体内核苷酸多样化的新技术有望使战略得以进行工程应用的定向进化，并提出遗传学解决生物学问题。最近，我们报道了EvolvR-一种在大肠杆菌中使用CRISPR引导的Cas9尼克酶的系统，该酶与尼克翻译易错的DNA聚合酶融合，从而使目标基因组位点多样化。由于CRISPR-Cas9已显示出对多种细胞类型的活性，因此如果切口平移聚合酶可以跨物种活跃，EvolvR有可能被移植到包括真核生物在内的其他生物体中。在这里，我们实现和表征酿酒酵母中EvolvR的功能，这是在真核生物中启用EvolvR介导的诱变的关键第一步。这项进展对于工程和基础研究应用中的酵母染色体中用户定义基因座的诱变将是有用的，并且它还提供了开发EvolvR技术以在高等真核生物中发挥作用的平台。