CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication
CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication
复制标题
CRISPR-Cas13a 切割登革热病毒 NS3 基因可有效抑制病毒复制
DOI：
10.1016/j.omtn.2020.01.028
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发表时间：
2020-03-06
影响因子：
8.8
通讯作者：
Kou, Zhihua
中科院分区：
文献类型：
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作者：
Li, Hao;Wang, Shan;Kou, Zhihua
CRISPR-Cas9系统已在真核和原核生物系统中精确地应用于DNA编辑，但它不能直接编辑RNA。最近开发的CRISPR-Cas13a系统已被证明能够有效地降低哺乳动物和植物细胞中的RNA表达。在本研究中，我们利用CRISPR-Cas13a系统在哺乳动物细胞中实现登革热病毒的可编程失活。定量反转录PCR （qRT-PCR）、荧光活化细胞分选（FACS）和斑块分析表明，在登革热病毒基因组RNA的10个不同区域中，靶向NS3区的CRISPR RNA （crRNA）对病毒的抑制作用最大。NS3- crrna /Cas13a复合物转染后，在NS3区域附近也发现了缺失和插入。我们的研究结果表明，CRISPR-Cas13a系统是抑制登革热病毒复制的一种新颖有效的技术，这表明这种可编程方法可能进一步发展成为登革热和其他RNA病毒的一种新的治疗策略。
The CRISPR-Cas9 system has been applied to DNA editing with precision in eukaryotic and prokaryotic systems, but it is unable to edit RNA directly. A recently developed CRISPR-Cas13a system has been shown to be capable of effectively knocking down RNA expression in mammalian and plant cells. In this study, we employ the CRISPR-Cas13a system to achieve reprogrammable inactivation of dengue virus in mammalian cells. Quantitative reverse transcription PCR (qRT-PCR), fluorescence-activated cell sorting (FACS), and plaque assays showed that CRISPR RNA (crRNA) targeting the NS3 region led to the greatest viral inhibition among 10 crRNAs targeting different regions along the dengue viral genomic RNA. Deletions and insertions had also been found adjacent to the NS3 region after NS3-crRNA/Cas13a complex transfection. Our results demonstrate that the CRISPR-Cas13a system is a novel and effective technology to inhibit dengue viral replication, suggesting that such a programmable method may be further developed into a novel therapeutic strategy for dengue and other RNA viruses.