A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.
A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.
复制标题
用于丙型肝炎病毒实时监测和高通量 CRISPR/Cas9 文库筛选的双报告系统。
DOI：
10.1038/srep08865
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发表时间：
2015-03-09
影响因子：
4.6
通讯作者：
Wei W
中科院分区：
文献类型：
--
作者：
Ren Q;Li C;Yuan P;Cai C;Zhang L;Luo GG;Wei W
丙型肝炎病毒是慢性肝炎、肝硬变和肝细胞癌的主要病因之一，全世界约有1.7亿人感染丙型肝炎病毒。尽管已经开发了几种报告系统，但许多缺点限制了它们在评估丙型肝炎病毒感染方面的使用。在这里，我们报告了一个实时活细胞报告系统，称为NRID(NS3-4A可诱导RTTA介导的双报告系统)，它提供了一个开关，专门针对丙型肝炎病毒感染。利用NIRD系统和聚焦CRISPR/Cas9文库，我们确定CLDN1、OCLN和CD81是丙型肝炎病毒无细胞进入和细胞间传播的必需基因。这种超灵敏的报告系统和CRISPR基因敲除筛查相结合，为识别丙型肝炎病毒感染的关键宿主成分提供了一种强大的高通量策略。
The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.