用于在小鼠胚胎干细胞中引入大片段基因缺失的Cas12a/Cpf1导向RNA复合物的制备与电穿孔

Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells.

作者信息

Kissling Lucas, Monfort Asun, Swarts Daan C, Wutz Anton, Jinek Martin

机构信息

Department of Biochemistry, University of Zurich, Zurich, Switzerland; Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland.

Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland.

出版信息

Methods Enzymol. 2019;616:241-263. doi: 10.1016/bs.mie.2018.10.028. Epub 2019 Jan 17.

DOI:10.1016/bs.mie.2018.10.028

PMID:30691645

Abstract

CRISPR-Cas12a is a bacterial RNA-guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.

摘要

CRISPR-Cas12a是一种细菌RNA引导的脱氧核糖核酸酶，已被广泛应用于多种生物体的基因工程。在此，我们描述了用于制备和应用AsCas12a引导RNA核糖核蛋白（RNP）复合物以在小鼠胚胎干细胞（ES细胞）中进行基因缺失工程的方案。我们提供了详细的方案，用于纯化含核定位信号的AsCas12a-eGFP融合蛋白、设计引导RNA、组装RNP复合物以及通过电穿孔转染小鼠ES细胞。此外，我们展示了使用成对的Cas12a核酸酶进行大型基因缺失工程的数据，并概述了在ES细胞中应用Cas12a核酸酶的实验注意事项。