Chen Ping, Chen Minjie, Chen Yujie, Jing Xinyun, Zhang Niubing, Zhou Xiaojuan, Li Xuan, Long Gang, Hao Pei
Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China; Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China.
Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
Virus Res. 2022 Apr 15;312:198707. doi: 10.1016/j.virusres.2022.198707. Epub 2022 Feb 9.
Zika virus (ZIKV) outbreaks occurred in recent years on an unprecedented scale, which caused fever and severe complications like Guillain-Barré syndrome in adults and fetal abnormalities. No vaccines or other effective treatments against ZIKV are available to date. The CRISPR-Cas13 family has the unique ability to target single-strand RNA molecules and mediate RNA cleavage. In the present study, we sought to exploit CRISPR-Cas13b for developing an anti-ZIKV system in mammalian cells. We first generated a ZIKV infection and reporting system by: (1) fusing mCherry to the ZIKV capsid protein for reporting infection by fluorescence; and (2) deriving a 293T cell line (293T-DC-SIGN) stably expressing DC-SIGN (Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) that became highly susceptible to ZIKV infection. The CRISPR Cas13b expression was reported to be in the cytoplasm of 293T-DC-SIGN cells using a Cas13b-GFP fusion expression vector. Fourteen CRISPR RNAs (crRNAs) were designed to target the most conserved regions of the ZIKV genome through bioinformatics analysis of 1138 ZIKV genome sequences. Five crRNAs were found to have significant effects (p < 0.001; two-sided t test) for Cas13b-targeted inhibition on ZIKV infection in 293T-DC-SIGN cells. Our study demonstrated an exciting example of using the CRISPR-Cas13b system for the treatment and prevention of ZIKV infection, highlighting CRISPR-Cas13 as a promising therapeutic anti-RNA virus strategy.
寨卡病毒（ZIKV）近年来爆发的规模空前，导致发热以及严重并发症，如成人的吉兰-巴雷综合征和胎儿异常。迄今为止，尚无针对寨卡病毒的疫苗或其他有效治疗方法。CRISPR-Cas13家族具有靶向单链RNA分子并介导RNA切割的独特能力。在本研究中，我们试图利用CRISPR-Cas13b在哺乳动物细胞中开发一种抗寨卡病毒系统。我们首先通过以下方式建立了寨卡病毒感染和报告系统：（1）将mCherry与寨卡病毒衣壳蛋白融合，通过荧光报告感染情况；（2）获得稳定表达DC-SIGN（树突状细胞特异性细胞间粘附分子-3抓取非整合素）的293T细胞系（293T-DC-SIGN），该细胞系对寨卡病毒感染高度敏感。使用Cas13b-GFP融合表达载体，报告CRISPR Cas13b在293T-DC-SIGN细胞中的细胞质中表达。通过对1138个寨卡病毒基因组序列进行生物信息学分析，设计了14条CRISPR RNA（crRNA）靶向寨卡病毒基因组中最保守的区域。发现5条crRNA对Cas13b靶向抑制293T-DC-SIGN细胞中的寨卡病毒感染有显著效果（p < 0.001；双侧t检验）。我们的研究展示了一个使用CRISPR-Cas13b系统治疗和预防寨卡病毒感染的令人兴奋的例子，突出了CRISPR-Cas13作为一种有前途的治疗抗RNA病毒策略。
