Ligand-Induced Trigger RNA Cleavage Enables Programmable Gene Expression Regulation via Strand Displacement in Prokaryotic and Eukaryotic Cells

Background: Synthetic RNA circuits offer powerful tools for reprogramming cellular behavior, but constructing ligand-responsive RNA switches that function reliably inside living cells remains challenging. Existing cis-acting designs often lack modularity and programmability due to tight coupling between sensing and output domains. Methods: We developed a generalizable strategy termed Ligand-Induced Trigger RNA Cleavage (LITC). This approach integrates an aptamer-embedded hammerhead ribozyme (aptazyme) as a trans-acting trigger. The aptazyme sequence is inserted into the spacer region of an RNA trigger strand, separating its toehold and displacement domains. Ligand-induced aptazyme self-cleavage inactivates the trigger, thereby controlling downstream toehold-mediated strand displacement reactions. We validated this system in both prokaryotic (E. coli) and eukaryotic (HEK-293T) cells using translation-controlling toehold switches and gRNA switches within the CRISPR/Cas9 system. Results: The LITC strategy successfully enabled programmable, dose-dependent regulation of gene expression. A spacer inserted between toehold and displacement domains did not impair trigger function. Embedding self-cleaving ribozymes (HHR, sTRSV) constitutively silenced trigger activity. Using a theophylline-responsive aptazyme, we achieved ligand-controlled regulation of a toehold switch, with different communication modules (CMs) yielding varied regulatory performance and theophylline concentrations up to 4 mM providing graded control. Furthermore, this approach was extended to control CRISPR interference (CRISPRi) in E. coli and CRISPR activation (CRISPRa) of the endogenous ASCL1 gene in HEK-293T cells, demonstrating cross-system portability. Conclusions: The LITC platform provides a general, modular, and transferable strategy for small-molecule control of toehold-mediated strand displacement reactions. It enables precise conditional regulation of RNA-based devices, including translation switches and CRISPR-Cas9 systems, across both prokaryotic and eukaryotic cells, thereby offering a versatile framework for constructing intelligent genetic circuits.