Programmable Inhibition and Detection of RNA Viruses Using Cas13

Two-thirds of known viruses have RNA genomes, and numerous of these cause human disease yet are lacking virus-specific antivirals or vaccines. Antiviral development is limited by our knowledge of viral and host protein biology and is compounded by viruses’ ability to evolve resistance to existing antivirals. The RNA-guided, RNA-targeting CRISPR-Cas effector Cas13 could enable a new class of CRISPR-based antivirals. Moreover, Cas13 is highly programmable as it can cleave any RNA complementary to its CRISPR RNA (crRNA). Here, we demonstrate that Cas13 can potently reduce viral RNA >15-fold and infectivity >300-fold for two distinct RNA viruses: lymphocytic choriomeningitis virus (LCMV), a model arenavirus for studying the deadly Lassa fever virus, and influenza A virus. We perform a tiling screen along the LCMV genome and identify over 100 cRNAs that reduce LCMV levels by ≥2-fold and tens of crRNAs that reduce levels by ≥10-fold. Analysis of the target sites of these crRNAs reveals targeting “hotspots” along the LCMV genome and helps define a set of crRNA design principles for targeting viral RNA. We demonstrate that targeting efficiency can be enhanced through the use of alternative Cas13 orthologs and multiplexing. Additionally, we develop CARVER (Cas13-Assisted Restriction of Viral Expression and Readout), an end-to-end platform for Cas13-based detection of viral RNA in Cas13-treated samples using SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing). We showcase the remarkable promise of CRISPR-Cas13 to enable both diagnostic and antiviral technologies, which could have widespread implications for future diagnosis and treatment of human infectious disease.