Wettstein Rahel, Bodak Maxime, Ciaudo Constance
Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology Zürich, HPL G28, Otto-Stern-Weg 7, CH-8093, Zürich, Switzerland.
Methods Mol Biol. 2016;1341:321-43. doi: 10.1007/7651_2015_213.
CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.
CRISPR/Cas9最初作为一种细菌免疫系统被发现，最近已被改造成一种最新工具，用于在多种不同生物体中成功引入位点特异性突变。该系统仅由Cas9蛋白及其一个用于发挥功能的工程化向导RNA组成，与其他引导核酸酶（如锌指核酸酶或转录激活因子样效应物核酸酶）相比，其设置要简单得多，操作也更容易。在此，我们描述了在小鼠胚胎干细胞（mESCs）中同时转染两个配对的CRISPR sgRNAs-Cas9质粒，从而导致所选靶基因的敲除。结合四引物评估系统，这是一种生成新的独立基因敲除小鼠胚胎干细胞系的有效方法。
