Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR/Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events and diverse approaches for multiplex genome editing have been described in literature by means of Cas9 or Cas12a endonucleases and their associated gRNAs. The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native PolII or PolIII RNA polymerases. These gRNA-expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in S. cerevisiae. Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single DNA editing. These DNA fragments can be custom-ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR-toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other, less genetically accessible yeast species.

中文翻译：

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gEL DNA，一种无需克隆和PCR的方法，用于基于CRISPR的多重基因组编辑

甚至对于可遗传获得的酵母酿酒酵母，CRISPR / Cas DNA编辑技术极大地加速和促进了菌株的构建。已经通过快速和高效的单次编辑事件验证了几种方法，文献中已经通过Cas9或Cas12a核酸内切酶及其相关的gRNA描述了用于多重基因组编辑的多种方法。通常使用天然PolII或PolIII RNA聚合酶从质粒表达用于将Cas核酸内切酶引导至编辑位点的gRNA。这些表达gRNA的质粒需要费力，耗时的克隆步骤，这阻碍了它们在学术和应用方面的实现。在这项研究中，我们探索了使用T7 RNA聚合酶（T7RNAP）从线性DNA片段表达gRNA的潜力，用于酿酒酵母中的单基因组和多重基因组编辑。使用Cas12a，这项工作证明了在表达T7RNAP的酵母菌株中转化编码gRNA的短而线性的DNA片段可促进高效的单DNA编辑。这些DNA片段可以定制，这使得该方法非常适合高通量菌株构建。这项工作扩展了用于酿酒酵母大规模菌株构建程序的CRISPR-toolbox，并有望与其他遗传性较差的酵母物种相关。