使用CRISPR-Cas9系统的基因组工程。

Genome engineering using CRISPR-Cas9 system.

作者信息

Cong Le, Zhang Feng

机构信息

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA, 02142, USA,

出版信息

Methods Mol Biol. 2015;1239:197-217. doi: 10.1007/978-1-4939-1862-1_10.

DOI:10.1007/978-1-4939-1862-1_10

PMID:25408407

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

摘要

成簇规律间隔短回文重复序列（CRISPR）-Cas9系统是一种存在于多种微生物中的适应性免疫系统。它可以被改造后在真核细胞中发挥作用，成为一种用于操纵基因组序列的快速、低成本、高效且可扩展的工具。在本章中，我们描述了利用化脓性链球菌的CRISPR-Cas9系统在哺乳动物细胞中实现RNA引导的基因组工程应用的详细方案。我们介绍了所有相关方法，包括初始位点选择、分子克隆、将向导RNA（gRNA）和Cas9导入哺乳动物细胞、靶标切割验证以及检测包括插入缺失和同源重组在内的基因组修饰的测定方法。这些工具为研究人员提供了新的手段，加速了正向和反向遗传学研究工作。