通过 CRISPR/Cas9 技术生成基因修饰大鼠模型。

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

机构信息

School of Life Sciences, East China Normal University, Shanghai 200241, China.

出版信息

Yi Chuan. 2023 Jan 20;45(1):78-87. doi: 10.16288/j.yczz.22-354.

DOI:10.16288/j.yczz.22-354

Abstract

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

摘要

RNA 引导的 CRISPR/Cas9 基因组编辑系统由单指导 RNA（sgRNA）和 Cas9 核酸酶组成。这两个组件在细胞中形成复合物，靶向与 sgRNA 互补的基因组位点。Cas9 核酸酶切割靶位点，产生双链 DNA 断裂（DSB）。在哺乳动物细胞中，DSB 通常通过易错的非同源末端连接（NHEJ）或在存在供体 DNA 模板的情况下通过同源定向修复（HDR）进行修复。将 CRISPR/Cas9 系统微注射到大鼠胚胎中，可产生基因修饰的大鼠模型。在这里，我们描述了一种通过 CRISPR/Cas9 技术创建基因敲除或敲入大鼠模型的详细方案。