Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae

Tara N. Stuecker, Stephanie E. Hood, Julio Molina Pineda, Sonali Lenaduwe, Joshua Winter, Meru J. Sadhu, Jeffrey A. Lewis
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Abstract

In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas ‘cutting’ combined with homologous recombination of a ‘repair’ template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.
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用于在酿酒酵母中进行 retron 介导的 CRISPR-Cas9 基因组编辑的改良载体
体内定点突变是一种强大的遗传工具,可用于测试特定等位基因在其正常基因组环境中的影响。虽然芽殖酵母拥有经典的定点诱变工具,但最近基于 CRISPR 的方法使用 Cas "切割 "结合 "修复 "模板的同源重组来引入所需的编辑,效率更高。然而,目前的方法仅限于完全原养的酵母菌株,而且依赖于效率相对较低的短 gRNA 克隆。因此,我们希望将 gRNA 及其同源修复模板顺式结合到单个寡核苷酸上,从而简化这一过程。此外,我们还希望利用一种新方法,即使用大肠杆菌重构子(EcRT)在体内扩增修复模板为多拷贝单链(ms)DNA,这是一种更有效的同源重组模板。为此,我们创建了一套表达 Cas9-EcRT 的质粒,只需一个步骤就能与 gRNA 修复模板质粒共同转化。我们的这套质粒包含不同的抗生素(Nat、Hyg、Kan)或辅助营养(HIS3、URA3)可选择性标记,可以编辑完全原营养的野生酵母菌株。除了传统的半乳糖诱导外,我们还生成了每种质粒的β-雌二醇诱导版本,以便于编辑在半乳糖上生长不良的酵母菌株。这种基于质粒的系统能以最少的步骤和时间实现 95% 的点突变编辑效率和 50% 的无标记缺失编辑效率。我们提供了如何使用该系统的详细分步指南。
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