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

Vollständiger Abstract

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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.