1] Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. [2].
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
Nat Protoc. 2014 Oct;9(10):2493-512. doi: 10.1038/nprot.2014.171. Epub 2014 Sep 25.
Conventional embryonic stem cell (ESC)-based gene targeting, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
传统的胚胎干细胞（ESC）基因靶向、锌指核酸酶（ZFN）和转录激活因子样效应核酸酶（TALEN）技术是用于生成遗传修饰动物的强大策略。最近，CRISPR/Cas 系统已成为这些方法的有效替代方法。我们使用 CRISPR/Cas 系统通过直接将 RNA 注射到单细胞胚胎中，生成携带多个基因突变的大鼠品系，证明了 Cas9 介导的基因编辑在大鼠中具有高效性，可同时生成复合基因突变模型。在此，我们描述了一种通过直接注射 RNA 生成敲除和敲入大鼠的逐步程序。该方案提供了用于选择基因组靶标、合成向导 RNA、设计和构建同源重组 (HR) 模板载体、胚胎微注射以及在创始人或其后代中检测突变和插入的指南。从目标设计到鉴定创始人的过程可能只需 6 周，其中实际动手时间不到 10 天。
