Abstract：
: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.
journal_name
Cell Repjournal_title
Cell reportsauthors
Tambe A,East-Seletsky A,Knott GJ,Doudna JA,O'Connell MRdoi
10.1016/j.celrep.2018.06.105subject
Has Abstractpub_date
2018-07-24 00:00:00pages
1025-1036issue
4issn
2211-1247pii
S2211-1247(18)31047-7journal_volume
24pub_type
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