Reprogramming RNA Degraders as Intracellular Molecule Sensors for Tumor Cell Sensing and Manipulation.

Abstract

Engineering transgene expression to respond specifically to intracellular cues holds transformative potential for monitoring cell states and modulating cellular functions. Here we introduce TRADER (Target-induced Transgene Activators via Reprogramming of dCas6-barnase Degrader), a versatile and modular platform that enables transgene activation in response to intracellular molecules at the post-transcriptional level. TRADER leverages a proximity-dependent RNA degrader system, in which a catalytically inactive Cas6 (dCas6) is fused to an engineered RNase, barnase, to suppress exogenous gene expression through targeted mRNA degradation. This repression can be relieved through two distinct mechanisms: (1) incorporating microRNA (miRNA) target sites into the dCas6-barnase transcript allows for endogenous miRNA-mediated downregulation of the degrader, and (2) inserting protease-cleavable linkers enables protease-triggered degrader disassembly, and further engineering a positive-feedback loop achieves signal amplification. As a proof of concept, we demonstrate the utility of TRADER in two applications: selective sensing of intracellular miRNA and ATP levels in target cells using fluorescent proteins, and conditional elimination of tumor cells overexpressing cathepsin B protease (CaBp) through induced expression of a cytotoxic protein. Collectively, TRADER offers a flexible and expandable toolkit for cell-state-specific control of transgene expression, with broad implications for synthetic biology, cell-based diagnostics, and precision therapeutics.