VanDusen Nathan J, Guo Yuxuan, Gu Weiliang, Pu William T
Department of Cardiology, Boston Children's Hospital, Boston, Massachusetts.
Department of Pharmacology, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Curr Protoc Mol Biol. 2017 Oct 2;120:31.11.1-31.11.14. doi: 10.1002/cpmb.46.
In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV-based somatic mutagenesis) platform, in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre-dependent Cas9-P2A-GFP mice. Because Cre is under the control of a tissue-specific promoter, this system allows temporally controlled, cell type-selective knockout of virtually any gene to be obtained within a month using only one mouse line. Here, we focus on gene disruption in cardiomyocytes, but the system could easily be adapted to inactivate genes in other cell types transduced by AAV. © 2017 by John Wiley & Sons, Inc.
目前，体内功能丧失研究受到对合适的条件性敲除等位基因需求的限制。CRISPR/Cas9是一种常用于在体外诱导功能丧失突变的强大工具。然而，CRISPR组件在体内难以应用。为了解决这个问题，我们开发了CASAAV（基于CRISPR/Cas9/AAV的体细胞诱变）平台，其中重组腺相关病毒（AAV）用于将串联引导RNA和Cre重组酶递送至依赖Cre的Cas9-P2A-GFP小鼠。由于Cre受组织特异性启动子的控制，该系统允许在仅使用一个小鼠品系的情况下，在一个月内获得几乎任何基因的时间可控、细胞类型选择性敲除。在这里，我们专注于心肌细胞中的基因破坏，但该系统可以很容易地适用于使由AAV转导的其他细胞类型中的基因失活。© 2017年约翰·威利父子公司版权所有
