ABSTRACT
The main (Mpro) and papain-like (PLpro) proteases encoded by SARS-CoV-2 are essential to process viral polyproteins into functional units, thus representing key targets for anti-viral drug development. There is a need for an efficient inhibitor screening system that can identify drug candidates in a cellular context. Here we describe modular, tunable autoproteolytic gene switches (TAGS) relying on synthetic transcription factors that self-inactivate, unless in the presence of coronavirus protease inhibitors, consequently activating transgene expression. TAGS rapidly report the impact of drug candidates on Mpro and PLpro activities with a high signal-to-noise response and a sensitivity matching concentration ranges inhibiting viral replication. The modularity of the TAGS enabled the study of other Coronaviridae proteases, characterization of mutations and multiplexing of gene switches in human cells. Mice implanted with Mpro or PLpro TAGS-engineered cells enabled analysis of the activity and bioavailability of protease inhibitors in vivo in a virus-free setting.