遗传 ›› 2023, Vol. 45 ›› Issue (1): 78-87.doi: 10.16288/j.yczz.22-354

• 实验操作指南 • 上一篇    下一篇

利用CRISPR/Cas9技术构建基因编辑大鼠模型

刘梅珍(), 王立人(), 李咏梅, 马雪云, 韩红辉, 李大力()   

  1. 华东师范大学生命科学学院,上海 200241
  • 收稿日期:2022-11-10 修回日期:2022-12-23 出版日期:2023-01-20 发布日期:2023-01-01
  • 通讯作者: 李大力 E-mail:mzliu@bio.ecnu.edu.cn;lrwang@bio.ecnu.edu.cn;dlli@bio.ecnu.edu.cn
  • 作者简介:刘梅珍,硕士,工程师,研究方向:动物模型构建。E-mail: mzliu@bio.ecnu.edu.cn;
  • 基金资助:
    国家自然科学基金杰出青年基金项目编号(32025023);国家自然科学基金面上项目编号(81873685);国家自然科学基金面上项目编号(31971366);国家重点研发计划项目编号(2019YFA0110802);国家重点研发计划项目(2019YFA0802802);国家重点研发计划项目(2019YFA0802802);上海市科技创新行动计划实验项目(20140900200);华东师范大学闵行实验动物中心平台(011)

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

Meizhen Liu(), Liren Wang(), Yongmei Li, Xueyun Ma, Honghui Han, Dali Li()   

  1. School of Life Sciences, East China Normal University, Shanghai 200241, China
  • Received:2022-11-10 Revised:2022-12-23 Online:2023-01-20 Published:2023-01-01
  • Contact: Li Dali E-mail:mzliu@bio.ecnu.edu.cn;lrwang@bio.ecnu.edu.cn;dlli@bio.ecnu.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(32025023);Supported by the National Natural Science Foundation of China(81873685);Supported by the National Natural Science Foundation of China(31971366);the National Key Research and Development Program of China(2019YFA0110802);the National Key Research and Development Program of China(2019YFA0802802);the National Key Research and Development Program of China(2019YFA0802802);the Shanghai Municipal Commission for Science and Technology(20140900200);the Animal Center of East China Normal University(011)

摘要:

RNA介导的CRISPR/Cas9基因编辑系统由单链引导RNA(sgRNA)与核酸酶Cas9构成。在细胞内,sgRNA能够按照碱基互补配对的原则引导Cas9与靶点结合,由Cas9切割目标DNA,造成双链DNA断裂(double stranded break, DSB)。在随后的DNA修复过程中,细胞主要进行非同源末端连接(non-homologous end joining, NHEJ)或在有修复模板存在的情况下进行重组修复(homology directed repair, HDR)。如果将CRISPR/Cas9系统以及修复模板通过显微注射的方式导入大鼠的胚胎内,就能借助细胞的修复机制实现大鼠胚胎的基因编辑,由此构建各种基因修饰大鼠模型。本文详细介绍了利用CRISPR/Cas9基因编辑技术构建大鼠模型的具体操作步骤,以期为相关领域的科研人员提供一种大鼠基因修饰模型的构建方法。

关键词: 大鼠, CRISPR/Cas9, 基因编辑, 基因敲除

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.

Key words: rat, CRISPR/Cas9, genome editing, gene knockout