Nature:基于CRISPR/Cas系统通过直接注射单细胞的胚胎来介导大鼠基因编辑
邦耀生物
CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos
基于CRISPR/Cas系统通过直接注射单细胞的胚胎来介导大鼠基因编辑
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.
传统的基于胚胎干细胞(ESC)的基因打靶技术,以及锌指核酸酶(ZFN)和转录激活因子样效应物核酸酶(TALEN)介导的基因编辑技术都是构建遗传修饰动物模型的有效方法。近来,高效、便捷的CRISPR/Cas 系统的出现,替代了上述技术。
我们利用CRISPR/Cas系统,通过直接向单细胞胚胎注射多基因的靶点RNAs,成功构建了多基因突变的大鼠模型,从而证明Cas9介导的基因编辑技术在构建多基因多靶点的突变大鼠模型过程中的高效性。
在此我们系统性地描述了构建基因敲除和敲入大鼠的流程。
这为筛选基因靶标、合成向导RNAs、设计和构建同源重组模板载体、胚胎显微注射、鉴定F0代大鼠及其后代的基因突变与插入提供了指导方法。使用这一方法,从靶标设计到F0代大鼠鉴定只需要不到6周的时间,而其中实际需要动手操作的时间不超过10天。