RNA binding and HEPN nuclease activation are decoupled in CRISPR-Cas13a

CRISPR-Cas13a enzymes are RNA-guided, RNA-activated ribonucleases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging and RNA regulation. However, the relationship between target RNA binding and HEPN (higher-eukaryotes-and-prokaryotes nucleotide-binding)- domain nuclease activation is not well understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13as target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number of and position of mismatches between the guide and target. We identify a central binding seed where perfect base pairing is absolutely required for target binding, and a separate nuclease switch where imperfect base-pairing results in tight binding but no HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13as RNA binding and cleavage behavior for RNA-targeting tool development.