Reversible Control of RNA Guided Nucleic Acid Cleavage Based on the Inverse Electron Demand Diels-Alder Reaction†

RNA plays a pivotal role in genetic information transmission, gene expression regulation, and key biological processes, making its functional regulation critical. In this study, we introduce an iedDA-based strategy for post-synthetic RNA modification, enabling controlled nucleic acid cleavage via the CRISPR-Cas system. By modifying RNA with trans-cyclooctene (TCO), its function is paused, and reactivation is achieved using the repair agent dimethyl-tetrazine (Me₂Tz), triggering the iedDA reaction. We demonstrate reversible on/off switching of CRISPR-Cas activity in vitro and further validate the strategy's applicability to other RNA systems, highlighting its potential for gene editing in cells.