Reversible Control of <scp>RNA</scp> Guided Nucleic Acid Cleavage Based on the Inverse Electron Demand Diels‐Alder Reaction^†

Comprehensive Summary 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 (Me 2 Tz), 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.