UC San Diego

CRISPR-Cas13 has been developed as a tool for genome editing by enabling precise RNA manipulation without permanently altering the genome. While its potential in therapeutics and diagnostics is promising, Cas13d’s off-target activities have limited its further use. To enhance our understanding of Cas13d’s binding specificity, we performed a massively parallel filter-binding assay probing the impact of mismatches in the target RNA on binding. Our results revealed that Cas13d binding is greatly affected by the specific position of the mutations in the target sequence, with less dependence on the type of mutation. Mismatches in the 5’ region of the target sequence were well tolerated, while those in the central region abrogated binding. These results emphasize the requirement for a complementary sequence in the central region for specific Cas13d binding. Additionally, this study expands the filter-binding method as a valuable tool for analyzing the binding specificity and off-target effects of Cas13d. Thus, our work lays a solid foundation for future high-throughput profiling of Cas13d binding and applying Cas13d for safe and effective transcriptional perturbation.