Laughery Marian F, Wyrick John J
School of Molecular Biosciences, Washington State University, Pullman, Washington.
Curr Protoc Mol Biol. 2019 Dec;129(1):e110. doi: 10.1002/cpmb.110.
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.
CRISPR-Cas9已成为在多种物种中编辑基因组的一种强大方法，因为当与Cas9结合的引导RNA靶向时，它可以产生特定的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，以便快速产生模板DNA中编码的基因组编辑（例如，DNA替换、插入或缺失）。由于重复的Cas9诱导的DNA断裂会筛选未编辑的细胞，因此Cas9可用于产生无标记的基因组编辑。在这里，我们描述了一种构建包含用户设计的引导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高效基因组编辑的方案。©2019约翰威立父子公司。基本方案1：构建引导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：筛选编辑细胞 基本方案5：去除sgRNA/CAS9表达载体 替代方案2：去除pML107衍生的sgRNA/CAS9表达载体。
