A one-step platform for screening high-efficient and minimal off-target CRISPR/Cas13 crRNAs to eradicate SARS-CoV-2 virus for treatment of COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is a new respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and now spreads globally. Currently, therapeutics and effective treatment options remain scarce and there is no proven drug to treat COVID-19. Targeting the positive-sense RNA genome and viral mRNAs of SARS-CoV-2 to simultaneously degrade viral genome templates for replication and viral mRNAs for essential gene expression would be a strategy to completely realize virus elimination. Type VI CRISPR enzymes Cas13 have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allows for RNA cleavage and degradation. The precise viral RNA detection and antiviral application of the CRISPR/Cas13 system depend on high-efficient and minimal off-target crRNAs. Although a computer-based algorithm has been applied for the design of crRNAs targeting SRAS-CoV-2, the experimental screening system to identify optimal crRNA is not available. We develop a one-step experimental screening system to identify high-efficient crRNAs with minimal off-target effects for CRISPR/Cas13-based SARS-CoV-2 elimination. This platform provides the foundation for CRISPR/Cas13-based diagnostics and therapeutics for COVID-19. This platform is versatile and could also be applied for crRNAs screening for other RNA viruses.