Efficient RNA virus targeting via CRISPR-CasRx in fish.
Efficient RNA virus targeting via CRISPR-CasRx in fish.
复制标题
通过 CRISPR-CasRx 在鱼类中高效靶向 RNA 病毒。
DOI：
10.1128/jvi.00461-21
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发表时间：
2021
影响因子：
5.4
通讯作者：
Qin Qiwei
中科院分区：
文献类型：
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作者：
Wang Qing;Liu Yun;Han Chong;Yang Min;Huang Fengqi;Duan Xuzhuo;Wang Shaowen;Yu Yepin;Liu Jiaxin;Yang Huirong;Lu Danqi;Zhao Huihong;Zhang Yong;Qin Qiwei
CRISPR/Cas系统作为一种技术的出现改变了我们修饰核酸的能力，CRISPR/Cas 13系统已用于靶向RNA。CasRx是一种小的VI-D型效应子（Cas 13 d），具有RNA敲低效率，可能对RNA病毒具有干扰作用。然而，CasRx的RNA病毒靶向活性仍需要在脊椎动物体内验证。在这项研究中，我们成功地设计了一个高效的CasRx系统用于鱼类病毒干扰。我们设计了编码CasRx的合成mRNA，并使用CRISPR RNA指导它靶向红斑石斑鱼神经坏死病毒（RGNNV）。该技术在体外和体内均对病毒感染产生了显著的干扰。这些结果表明，CRISPR/CasRx可用于设计针对鱼类RNA病毒的干扰，为脊椎动物针对其他RNA病毒的RNA导向免疫提供了潜在的新机制。IMPORTANCERNA病毒是感染脊椎动物和哺乳动物的重要病毒病原体。RNA病毒群体是高度动态的，由于短的世代时间，大的群体大小和高突变频率。因此，目前还难以找到广泛有效的抑制RNA病毒的方法，迫切需要开发有效的抗病毒方法。CasRx是一种具有RNA敲低效率的小型VI-D效应子（Cas 13 d），可对RNA病毒产生干扰作用。神经坏死病毒（Nervous Necrosis Virus，NNV）是一种无囊膜的正链RNA病毒，是世界上最严重的病毒性病原之一，目前已感染40多种养殖鱼类，给人类造成了巨大的经济损失。在这里，我们建立了一个新的有效的CasRx系统的RNA病毒干扰使用NNV和石斑鱼（Epineopyrus coioides）作为模型。我们的数据表明，CasRx是最强大的RNA病毒干扰应用在鱼类，我们证明了其适用于研究与病毒生物学相关的关键问题。
The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verifiedin vivoin vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections bothin vitroandin vivo. These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates.IMPORTANCERNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.