Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
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DOI：
10.21769/bioprotoc.2765
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发表时间：
2018-03-20
期刊：
影响因子：
0.8
通讯作者：
Marcotte, Edward M.
中科院分区：
文献类型：
--
作者：
Akhmetov, Azat;Laurent, Jon M.;Marcotte, Edward M.
芽殖酵母的基因组修饰非常成功，很大程度上是由于其高效的同源定向DNA修复机制。以前已经描述了几种修饰酵母基因组的方法，其中许多方法至少涉及两个步骤：插入一个可选择的标记，并将该标记替换为预期的修饰。在这里，我们描述了一种CRISPR-Cas9介导的基因组编辑方案，用于在没有任何可选择标记的单步转化中修改任何感兴趣的酵母基因（必要或非必要）。在该系统中，Cas9核酸酶在选择位点处产生双链断裂，这在酵母细胞中通常是致命的，而不管目标位点的重要性，由于低效的非同源末端连接修复。这种致死性导致通过同源重组使用来自PCR的修复模板进行有效修复。在涉及基本基因的情况下，用功能性等位基因编辑基因组病变的必要性作为额外的选择层。作为一个激励的例子，我们描述了使用这种策略来替换HEM2，一个重要的酵母基因，用它对应的人类同源基因ALAD。
Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.