Wang Yinghua, Ding Yifu, Li Jinsong
State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China.
School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai, 201210, China.
Methods Mol Biol. 2017;1622:293-305. doi: 10.1007/978-1-4939-7108-4_20.
Precise genome editing is a powerful tool for analysis of gene function. However, in spermatogonial stem cells (SSCs), this still remains a big challenge mainly due to low efficiency and complexity of currently available gene editing techniques. The CRISPR-Cas9 system from bacteria has been applied to modifying genome in different species at a very high efficiency and specificity. Here we describe CRISPR-Cas9-mediated gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in SSCs. This protocol provides guidelines for derivation of SSCs, nucleofection of SSCs with the CRISPR-Cas9 system, transplantation of the gene-modified SSCs into the recipient testes, and production of mice using transplanted SSC-derived round spermatids.
精确的基因组编辑是分析基因功能的强大工具。然而，在精原干细胞（SSCs）中，这仍然是一个巨大的挑战，主要原因是目前可用的基因编辑技术效率低且操作复杂。来自细菌的CRISPR-Cas9系统已被应用于以非常高的效率和特异性修饰不同物种的基因组。在这里，我们描述了通过非同源末端连接（NHEJ）或同源定向修复（HDR）在精原干细胞中进行CRISPR-Cas9介导的基因编辑。本方案提供了精原干细胞的分离、用CRISPR-Cas9系统对精原干细胞进行核转染、将基因修饰的精原干细胞移植到受体睾丸以及使用移植的精原干细胞衍生的圆形精子细胞生产小鼠的指导方针。
