PT - JOURNAL ARTICLE AU - Akshay Tambe AU - Alexandra East-Seletsky AU - Gavin J. Knott AU - Jennifer A. Doudna AU - Mitchell R. O’Connell TI - RNA-binding and HEPN-nuclease activation are decoupled in CRISPRCas13a AID - 10.1101/190603 DP - 2018 Jan 01 TA - bioRxiv PG - 190603 4099 - http://biorxiv.org/content/early/2018/04/16/190603.short 4100 - http://biorxiv.org/content/early/2018/04/16/190603.full AB - 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 Cas13a’s 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 Cas13a’s RNA binding and cleavage behavior for RNA-targeting tool development.