利用CRISPR-Cas13a技术靶向抑制登革病毒复制的实验研究

近年来，新一代的RNA靶向技术CRISPR-Cas13a已被证实可在植物细胞及哺乳动物细胞内靶向剪切单链RNA，并且该技术已被用于在植物细胞内的抗病毒研究。为探索CRISPR-Cas13a技术用于人类病毒性疾病中抗病毒研究的可行性以及更好地优化该技术的抗病毒方案，本研究以登革病毒为研究对象，通过筛选高效crRNA靶点、探索最佳使用剂量及给药时间，建立并优化基于CRISPR-Cas13a的抗登革病毒技术方案，通过深度测序和单细胞转录组测序探索该技术剪切病毒RNA的作用原理并评估其在抗病毒时的脱靶效应。本研究将为抗登革病毒的研究提供一种新的策略，同时也为今后利用该技术开展抑制其他RNA病毒的研究提供理论及技术支持。

A recently developed RNA targeting tool “CRISPR-Cas13a” has shown to effectively binding and disrupt single-strand RNA expression in mammalian and plant cells, and this tool has been used to inhibit viral replication in plant cells. To explore its potential for developing into a novel reprogrammable anti-viral therapeutic strategy in human viral diseases and optimize the technology schemes in the anti-viral application, we use dengue virus (DENV) as a research object and establish an optimized anti-viral method based on CRISPR-Cas13a through screening efficiency crRNAs and finding suitable dose and time. Besides, using deep sequencing and single-cell transcriptome sequencing, we explore the mechanism of its anti-viral effect and find a more accurate way to evaluate the off-target effect of CRISPR-Cas13a. This study will provide a novel and effective way to inhibit DENV infection, and lay a theoretical foundation for inhibition of other RNA virus using CRISPR-Cas13a technology.