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 the literature by means of SpCas9 or FnCas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III 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 Saccharomyces cerevisiae. Using FnCas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex 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：基于 CRISPR 的多重基因组编辑的无克隆和聚合酶链反应的方法

即使对于基因可及的酵母酿酒酵母，CRISPR-Cas DNA 编辑技术也极大地加速和促进了菌株的构建。几种方法已被验证用于快速和高效的单一编辑事件，并且文献中已经描述了通过Sp Cas9 或Fn进行多重基因组编辑的多种方法Cas12a 核酸内切酶及其相关的向导 RNA (gRNA)。用于将 Cas 核酸内切酶引导至编辑位点的 gRNA 通常由使用天然 Pol II 或 Pol III RNA 聚合酶的质粒表达。这些 gRNA 表达质粒需要费力、耗时的克隆步骤，这阻碍了它们在学术和应用方面的实施。在这项研究中，我们探索了使用 T7 RNA 聚合酶 (T7RNAP) 从线性 DNA 片段中表达 gRNA 的潜力，用于在酿酒酵母中进行单一和多重基因组编辑。使用FnCas12a，这项工作表明，在表达 T7RNAP 的酵母菌株中转化编码 gRNA 的短线性 DNA 片段可促进高效的单链和双链 DNA 编辑。这些 DNA 片段可以定制，这使得这种方法非常适合高通量菌株构建。这项工作扩展了 CRISPR 工具箱，用于在酿酒酵母中进行大规模菌株构建计划，并有望与其他遗传性较差的酵母物种相关。