RdRp activity test using CRISPR/Cas13a enzyme (RACE) for screening of SARS-CoV-2 inhibitors

The coronavirus disease 2019 pandemic has highlighted the need for efficient antiviral drug screening tech-nologies, particularly for targeting RNA-dependent RNA polymerase (RdRp). Here, we present a novel RdRp activity assay using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) enzyme (RACE) for screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibitors. This is the first application of the CRISPR/Cas complex for RdRp activity-based drug screening. The RACE system combines RdRp, RNA template, target inhibitor, CRISPR/Cas13a, and reporter probe, enabling accurate detection of the inhibitory effect on RdRp activity. This is demonstrated by the system's ability to provide half-maximal inhibitory concentration values of 7.5 +/- 0.5 and 8.9 +/- 0.6 mu M for remdesivir triphosphate and C646, respectively. In addition, the RACE system requires a detection time of 5 min, making it more efficient than traditional endpoint assays. The real-time fluorometric output of the RACE system allows monitoring of reactions, providing insight into the kinetics of RdRp activity. Furthermore, the versatility of the RACE system extends to lateral flow assay formats, improving convenience. We anticipate that the RACE system has the potential to identify antiviral compounds that target RdRp, thereby accelerating the development of effective antiviral therapies.