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. Author manuscript; available in PMC: 2021 Mar 13.
Published in final edited form as: Mol Cell. 2018 Aug 16;71(5):791–801.e3. doi: 10.1016/j.molcel.2018.07.013

Figure 6: Extended complementarity inhibits Cas13 cis- and trans-cleavage in vitro.

Figure 6:

A. A schematic showing protospacer-containing target RNA (left) with noncomplementary 3’ extension, and anti-target RNAs bearing 8nt anti-tag that extends the crRNA:target duplex. B. In vitro cis-RNA cleavage time course with 10 nM purified LbuCas13 and 10 nM labeled target, non-target or anti-target RNA substrates. Reaction products were analyzed at 0, 5, 20, and 60 minutes incubation. C. Cas13 trans-RNA cleavage assay. Labeled nontarget RNA substrates (10 nM) were incubated with 10 nM Cas13 RNP and the indicated unlabeled activating RNA target (10 nM). D. Trans-cleavage of 10 nM labeled anti-target by preincubation of 10 nM Cas13 with increasing amounts (0.01, 0.1, 1, 10, 100, 1000 nM) of activating target RNA. E. Inhibition of target cleavage by addition of competing anti-target RNA. 10 nM target RNA was cleaved by Cas13 RNP in reactions pre-incubated with 10, 25, 50, or 100 nM nontarget or anti-target RNA. F. EMSA testing interaction between 1 nM crRNA and 0, 10, 100 nM Cas13 in the presence of 100 nM competitor RNA. See also Figure S5.