Programmable eukaryotic protein expression with RNA sensors

The diversity of cell types and states can be scalably measured and defined by expressed RNA transcripts. However, approaches to programmably sense and respond to the presence of specific RNAs within living biological systems with high sensitivity are lacking. RNA sensors that gate expression of reporter or cargo genes would have diverse applications for basic biology, diagnostics and therapeutics by enabling cell-state specific control of transgene expression. Here, we engineer a novel programmable RNA-sensing technology, Reprogrammable ADAR Sensors (RADARS), which leverages RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a protein payload on the presence of endogenous RNA transcripts. In mammalian cells, we engineer RADARS with diverse payloads, including luciferase and fluorescent proteins, with up to 164-fold activation and quantitative detection in the presence of target RNAs. We show RADARS are functional either expressed from DNA or as synthetic mRNA. Importantly, RADARS can function with endogenous cellular ADAR. We apply RADARS to multiple contexts, including RNA-sensing induced cell death via caspases, cell type identification, and in vivo control of synthetic mRNA translation, demonstrating RADARS as a tool with significant potential for gene and cell therapy, synthetic biology, and biomedical research. One Sentence Summary A new technology utilizing ADAR mediated RNA-editing enables robust reprogrammable protein expression based on target RNA transcripts in mammalian cells, leading to broad applications in basic science research, cell engineering, and gene therapy. ### Competing Interest Statement A patent application has been filed related to this work. J.S.G. and O.O.A. are co-founders of Sherlock Biosciences, Proof Diagnostics, Moment Biosciences, and Tome Biosciences. J.S.G. and O.O.A. were advisors for Beam Therapeutics during the course of this project. F.C. is a paid consultant of Atlas bio.