Cas13d-mediated viral RNA degradation leads to potent antiviral control against both positive and negative sense RNA viruses

Respiratory viral infections continue to pose a substantial global health challenge, with a pressing need for effective treatment options. The rapid mutation rate of RNA viruses presents a significant risk of escape from existing treatments. Here, we harness the rapid adaptability and high sequence specificity of the RNA-targeting CRISPR-Cas13 nuclease technology to target and cleave the genome of different RNA viruses, disrupting replication and impairing propagation. We identified potent CRISPR-RfxCas13d/crRNA (Cas13d/crRNA) combinations that can efficiently restrict viral replication in both pre- and post-viral exposure treatment settings. In vitro treatment with lipid nanoparticles (LNPs) co-encapsulating Cas13d mRNA and crRNAs targeting coding and non-coding regions of positive-sense (+) RNA coronaviruses including SARS-CoV-2 effectively inhibited viral replication. In addition, both pre- and post-viral exposure treatments with the LNP-Cas13d/crRNA against the negative-sense (−) RNA Nipah virus resulted in 99% restriction of this highly pathogenic virus. Hence, the Cas13d/crRNA antiviral method holds promise as a potential therapeutic approach against severe viral infections caused by both (+) and (−) RNA viruses. Furthermore, the multi-locus targeting of viruses by Cas13d could offer a possibility to prevent virus breakthrough and increase the therapeutic arsenal against many emerging RNA viruses in the future.