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bioRxiv · 10.1101/190603

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

Tambe, A.·East-Seletsky, A.·Knott, G. J.·Doudna, J. A.·O'Connell, M. R.
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Abstract

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.

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Tambe, A., East-Seletsky, A., Knott, G. J., Doudna, J. A., O'Connell, M. R.. 2017-09-19. RNA binding and HEPN nuclease activation are decoupled in CRISPR-Cas13a. https://doi.org/10.1101/190603

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