Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
Methods Mol Biol. 2021;2196:27-37. doi: 10.1007/978-1-0716-0868-5_3.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5 days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.
成簇规律间隔短回文重复序列（CRISPR）/Cas9 系统已能够在多种生物体中实现高效、无标记的基因组编辑。然而，该系统存在脱靶效应，并且精确编辑的区域有限。可以精确编辑的碱基仅限于 20 个碱基对 gRNA 靶向位点和间隔区相邻基序（PAM）序列。我们开发了一种 CRISPR 核酸酶系统，该系统可使用单个 Cas9 核酸酶在酿酒酵母中进行精确的全基因组碱基编辑。通过避免双链断裂（DSB）和随后的非同源末端连接（NHEJ），该系统可以精确编辑更广泛的基因组区域。此外，在该系统中未在靶外位点发现意外突变。与酵母缺口修复克隆相结合，该系统可在 5 天内对酵母细胞进行精确的基因组编辑。在这里，我们描述了使用这种新型 CRISPR 核酸酶系统进行精确和方便的基因组编辑的方法。
