WMD Threats and Aerosol Science, Sandia National Laboratories, Albuquerque, New Mexico, USA; National Strategic Research Institute, Omaha, Nebraska, USA.
University of Nebraska Medical Center, Omaha, Nebraska, USA; and National Strategic Research Institute, Omaha, Nebraska, USA.
CRISPR J. 2023 Aug;6(4):359-368. doi: 10.1089/crispr.2022.0095. Epub 2023 Mar 13.
CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.
基于 CRISPR 的技术已被广泛用作抗病毒策略，包括作为广谱人类冠状病毒（HCoV）治疗方法。在这项工作中，我们设计了一种带有可交叉反应于几种 HCoV 物种的向导 RNA（gRNA）的 CRISPR-CasRx 效应子系统。我们通过评估不同 CRISPR 靶点在 HCoV-OC43、HCoV-229E 和严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）中与病毒活力降低相关的效果来测试这种泛冠状病毒效应子系统的功效。我们确定了几种 CRISPR 靶点，尽管 gRNA 中存在单核苷酸多态性，但与非靶向、阴性对照 gRNA 相比，显著降低了病毒滴度。与未处理的病毒对照相比，CRISPR 靶点使 HCoV-OC43 中的病毒滴度降低了 85%至>99%，HCoV-229E 中的病毒滴度降低了 78%至>99%，SARS-CoV-2 中的病毒滴度降低了 70%至 94%。这些数据为能够降低 2 级和 3 级风险组 HCoV 病原体中存活病毒的泛冠状病毒 CRISPR 效应子系统建立了一个概念验证。
