Building Ligand‐Responsive Artificial Signaling Pathways Through Programmable Trans ‐Acting RNA Circuits in Mammalian Cells

Abstract Synthetic RNA circuits provide powerful tools to reprogram genetic networks for customized cellular functions. However, the construction of ligand‐induced complex synthetic signaling pathways in mammalian cells remains challenging due to the lack of modular and scalable RNA‐based components. Here, we report a generalizable strategy to engineer ligand‐responsive artificial signaling pathways (ASPs) using programmable RNA circuits. By designing aptamer‐embedded circular RNAs as trans ‐acting triggers coupled with controllable CRISPR functions as outputs, we demonstrate that various small molecules and proteins can be sensed and transduced into manipulation of originally unrelated endogenous genes through amplifiable, logical and multiplexed RNA circuits. Integration of this RNA system into the cellular genetic network endows cells with state/type‐specific phenotype responses regulated by endogenous metabolites and proteins. This study establishes a universal RNA platform for engineering ASPs induced by ligands in mammalian cells, with broad potential of cellular signaling and response engineering for diagnostic and therapeutic applications.