Identification of Closantel as a small-molecule inhibitor of the compact CRISPR-Cas RNA editor Cas13bt3

The type VI CRISPR-Cas systems are widely employed for programmable RNA editing, and the ultra-compact Cas13bt3 ribonuclease offers particular advantages for cellular delivery due to its minimal molecular size. However, its therapeutic potential is hindered by nonspecific collateral RNA cleavage activity and the lack of small-molecule inhibitors to enable spatiotemporal regulation of its function. Here, we performed a high-throughput screen for Cas13bt3 inhibitors using a fluorescence resonance energy transfer (FRET)-based RNA cleavage assay. From a library of 17,760 compounds, we identified Closantel as a specific Cas13bt3 inhibitor, with an IC₅₀ of 7.48 µM. Biochemical assays confirmed that Closantel abrogates both on-target and collateral RNA cleavage by Cas13bt3, while exerting negligible inhibitory activity against Cas13a, a closely related Cas13 ortholog. Combined molecular docking and electrophoretic mobility shift assay (EMSA) analyses further revealed that Closantel binds to the cavity of Cas13bt3 that accommodates the direct repeat region of crRNA, thereby competitively interfering with crRNA-Cas13bt3 binding. Finally, to minimize nonspecific RNA cleavage of Cas13bt3, we engineered a K748A mutant that retains robust on-target RNA cleavage activity with reduced collateral activity in vitro. Our findings provide a selective small-molecule chemical probe for Cas13bt3 and an optimized variant with improved targeting precision, collectively advancing the utility of Cas13bt3 for precise RNA editing applications.