CRISPR/Cas9系统诱导的多种基因组修饰的检测

Detection of Multiple Genome Modifications Induced by the CRISPR/Cas9 System.

作者信息

Ota Satoshi, Kawahara Atsuo

机构信息

Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.

出版信息

Methods Mol Biol. 2016;1451:53-63. doi: 10.1007/978-1-4939-3771-4_4.

DOI:10.1007/978-1-4939-3771-4_4

PMID:27464800

Abstract

The recent remarkable innovation of an RNA-guided nuclease system, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, enables us the modification of specific genomic loci in various model animals including zebrafish. With this system, multiple guide RNAs simultaneously injected with the Cas9 nuclease into zebrafish embryos cause multiple genome modifications at different genomic loci with high efficiency; therefore, a simple method to detect individual mutations at distinct loci is desired. In this chapter, we describe a procedure for inducing multiple CRISPR/Cas9-mediated genome modifications in zebrafish and a convenient method to detect CRISPR/Cas9-induced insertion and/or deletion (indel) mutations using a heteroduplex mobility assay (HMA).

摘要

近期，一种RNA引导核酸酶系统——成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统有了显著创新，这使我们能够在包括斑马鱼在内的各种模式动物中对特定基因组位点进行修饰。利用该系统，将多个引导RNA与Cas9核酸酶同时注射到斑马鱼胚胎中，可高效地在不同基因组位点引发多个基因组修饰；因此，需要一种简单的方法来检测不同位点的单个突变。在本章中，我们描述了一种在斑马鱼中诱导多个CRISPR/Cas9介导的基因组修饰的程序，以及一种使用异源双链迁移率分析（HMA）检测CRISPR/Cas9诱导的插入和/或缺失（indel）突变的简便方法。