RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. 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...

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Bibliographic Details
Published in:Cell reports (Cambridge) 2018-07, Vol.24 (4), p.1025-1036
Main Authors: Tambe, Akshay, East-Seletsky, Alexandra, Knott, Gavin J., Doudna, Jennifer A., O’Connell, Mitchell R.
Format: Article
Language:English
Subjects:
C2c2
Carrier Proteins
Cas13
Cas13a
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems
Endonucleases - metabolism
Humans
RNA biology
RNA specificity
RNA, Guide, CRISPR-Cas Systems - metabolism
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Summary:CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. 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 poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not 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 RNA binding and cleavage behavior for RNA-targeting tool development. [Display omitted] •Cas13a RNA binding and RNase activity are differentially affected by mismatches•Perfect base pairing in the guide seed region is required for stable RNA binding•RNA mismatches in a different guide region result in binding, but not RNase activity•The RNA binding and RNase activities of Cas13a can be decoupled CRISPR-Cas13a is a RNA-guided RNase that has emerged as a powerful tool for programmable RNA recognition. Tambe et al. find that Lbu-Cas13a target RNA binding affinity and RNase activity are differentially affected by mismatches between the guide and the target RNA and that these activities can be decoupled from each other.
ISSN:2211-1247
2211-1247
DOI:10.1016/j.celrep.2018.06.105