Simple CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae.
Simple CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae.
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DOI：
10.1002/cpmb.110
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发表时间：
2019-12-01
影响因子：
--
通讯作者：
Wyrick, John J
中科院分区：
文献类型：
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作者：
Laughery, Marian F;Wyrick, John J
CRISPR-Cas9已经成为编辑各种物种基因组的强大方法，因为它可以在被Cas9结合的指导RNA靶向时产生特异性DNA断裂。在酵母中，Cas9靶向的DNA断裂用于促进与诱变模板DNA的同源重组，以便快速产生基因组编辑（例如，DNA取代、插入或缺失）。由于重复的Cas9诱导的DNA断裂针对未编辑的细胞进行选择，因此Cas9可用于产生无标记的基因组编辑。在这里，我们描述了一种用于构建含有用户设计的指导RNA的Cas9表达质粒的简单方案，以及使用这些质粒在酵母中进行有效基因组编辑的方案。John Wiley & Sons，Inc._关于我们基本方案1：构建指导RNA表达载体基本方案2：制备双链寡核苷酸修复模板替代方案1：制备单链寡核苷酸修复模板基本方案3：通过共转化酵母诱导基因组编辑基本方案4：筛选编辑的细胞基本方案5：去除sgRNA/CAS 9表达载体替代方案2：去除pML 107衍生的sgRNA/CAS9表达载体。
CRISPR-Cas9 has emerged as a powerful method for editing the genome in a wide variety of species, since it can generate a specific DNA break when targeted by the Cas9-bound guide RNA. In yeast, Cas9-targeted DNA breaks are used to promote homologous recombination with a mutagenic template DNA, in order to rapidly generate genome edits (e.g., DNA substitutions, insertions, or deletions) encoded in the template DNA. Since repeated Cas9-induced DNA breaks select against unedited cells, Cas9 can be used to generate marker-free genome edits. Here, we describe a simple protocol for constructing Cas9-expressing plasmids containing a user-designed guide RNA, as well as protocols for using these plasmids for efficient genome editing in yeast. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Constructing the guide RNA expression vector Basic Protocol 2: Preparing double-stranded oligonucleotide repair template Alternate Protocol 1: Preparing a single-stranded oligonucleotide repair template Basic Protocol 3: Induce genome editing by co-transformation of yeast Basic Protocol 4: Screening for edited cells Basic Protocol 5: Removing sgRNA/CAS9 expression vector Alternate Protocol 2: Removing pML107-derived sgRNA/CAS9 expression vector.