A CRISPR-based SARS-CoV-2 diagnostic assay that is robust against viral evolution and RNA editing

Abstract Extensive testing is essential to break the transmission of the new coronavirus SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Recently, CRISPR-based diagnostics have emerged as attractive alternatives to quantitative real-time PCR due to their faster turnaround time and their potential to be used in point-of-care testing scenarios. However, existing CRISPR-based assays for COVID-19 have not considered viral genome mutations and RNA editing in human cells. Here, we present the VaNGuard (Variant Nucleotide Guard) test that is not only specific and sensitive for SARS-CoV-2, but can also detect the virus when its genome or transcriptome has evolved or has been edited by deaminases in infected human cells. We show that an engineered AsCas12a enzyme is more tolerant of mismatches than wildtype LbCas12a and that multiplexed Cas12a targeting can overcome the presence of single nucleotide variations. Our assay can be completed in 30 minutes with a dipstick for a rapid point-of-care test.