RNA structure modulates Cas13 activity and enables mismatch detection

The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging^1-6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA^1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured^2,8. Understanding Cas13's mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown⁹. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3' to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.