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.

中文翻译：

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CRISPR-Cas13a 切割登革热病毒 NS3 基因可有效抑制病毒复制


CRISPR-Cas9系统已应用于真核和原核系统中的DNA精确编辑，但无法直接编辑RNA。最近开发的 CRISPR-Cas13a 系统已被证明能够有效地抑制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重编程灭活。定量逆转录 PCR (qRT-PCR)、荧光激活细胞分选 (FACS) 和噬菌斑测定表明，针对登革热病毒基因组不同区域的 10 种 crRNA 中，针对 NS3 区域的 CRISPR RNA (crRNA) 产生了最大的病毒抑制作用核糖核酸。 NS3-crRNA/Cas13a 复合物转染后，在 NS3 区域附近也发现了缺失和插入。我们的结果表明，CRISPR-Cas13a系统是一种抑制登革热病毒复制的新颖而有效的技术，这表明这种可编程方法可以进一步开发为登革热和其他RNA病毒的新型治疗策略。