CRISPR/Cas9介导的斑马鱼中外源报告基因的靶向敲入

CRISPR/Cas9-Mediated Targeted Knockin of Exogenous Reporter Genes in Zebrafish.

作者信息

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. 2017;1630:165-173. doi: 10.1007/978-1-4939-7128-2_14.

DOI:10.1007/978-1-4939-7128-2_14

PMID:28643258

Abstract

Genome editing technologies such as ZFN, TALEN, and CRISPR/Cas9 efficiently induce DNA double-stranded breaks (DSBs) at a targeted genomic locus, often resulting in a frameshift-mediated target gene disruption. It remains difficult to perform targeted integration of exogenous genes by genome editing technologies. DSBs can be restored through DNA repair mechanisms, such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). It is well known that HR facilitates homology-dependent integration of donor DNA template into a targeted locus. Recently, both NHEJ-mediated and MMEJ-mediated targeted integrations of exogenous genes have been developed in zebrafish. This chapter summarizes the application of CRISPR/Cas9-mediated knock-in technology in zebrafish.

摘要

诸如锌指核酸酶（ZFN）、转录激活样效应因子核酸酶（TALEN）和规律成簇间隔短回文重复序列/ Cas9（CRISPR/Cas9）等基因组编辑技术能够在目标基因组位点高效诱导DNA双链断裂（DSB），常常导致移码介导的靶基因破坏。通过基因组编辑技术进行外源基因的靶向整合仍然具有挑战性。DSB可通过DNA修复机制来修复，如非同源末端连接（NHEJ）、微同源性介导的末端连接（MMEJ）和同源重组（HR）。众所周知，HR有助于将供体DNA模板以同源依赖性方式整合到目标位点。最近，在斑马鱼中已经开发出了NHEJ介导和MMEJ介导的外源基因靶向整合方法。本章总结了CRISPR/Cas9介导的敲入技术在斑马鱼中的应用。