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. 2018 Jul 24;24(4):1025-1036.
doi: 10.1016/j.celrep.2018.06.105.

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

Affiliations
Free PMC article

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

Akshay Tambe et al. Cell Rep. .
Free PMC article

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.

Keywords: C2c2; CRISPR-Cas systems; Cas13; Cas13a; RNA biology; RNA specificity.

Figures

Fig. 1.
Fig. 1.. Development of High-throughput RNA mismatch profiling to determine activator-RNA binding preferences for Lbu-Cas13a.
(A) Schematic of the high-throughput RNA mismatch profiling library design, see Experimental Procedures for details. (B) Schematic of the high-throughput RNA mismatch profiling pulldown workflow. (C) Empirical mismatch distribution profile for the input X and Y activator-RNA libraries (prior to Cas13a binding). (D) A scatter plot displaying fold-change in abundance of each RNA-X and -Y library members between samples with 10 nM crRNA-bound Cas13a (vs. crRNA-unbound apo-Cas13a) programmed to target RNA-X or -Y sequences. (E) A scatter plot displaying fold-change in abundance of each RNA-X and -Y library members between samples with 100 nM crRNA-bound Cas13a (vs. crRNA-unbound apo-Cas13a) programmed to target RNA-X or -Y sequences.
Fig.2.
Fig.2.. dCas13a high-throughput RNA mismatch profiling reveals binding mismatch sensitivity hotspots for single- and double- mismatched activator-RNAs.
dCas13a Regularized fold-change enrichment (relative to Apo-dCas13a) for activator-RNA members of both libraries that contain only a single mismatch across the shown spacer-targeting and flanking regions when (A) targeting activator-RNA-X library with 100 nM dCas13a: crRNA-X, (B) targeting activator-RNA-Y library with 100 nM dCas13a: crRNA-Y. In each plot, each individual point in a position represents the average regularized fold-change value for each unique mismatch combination possible in that position (A: hexagon, C: square, G: triangle, T: circle; from n=3 experiments). The solid line represents average fold-enrichment (of all mismatches combinations) in that position. The fold-change enrichment of the perfectly complementary (noMM) targets are shown on the right of each plot, and as a dotted gray line throughout the plot. The crRNA-complementary on-target library sequence is shown above each plot. Permutation tests (100,000 permutations) were carried out to determine whether statistical significant differences between mean fold changes of each library exist at each mismatch position. Positions shaded in yellow indicate where statistically significant (p < 0.05) lack of enrichment between guide-complementary and guide-noncomplementary sequences was observed. See Experimental Procedures for regularization and permutation test procedure. See Fig. S2 for 100 nM Cas13a: crRNA data plotted as individual data points from three experiments, as well as individual and averaged 10nM Cas13a: crRNA data. Regularized fold-change enrichment analysis (relative to Apo-dCas13a) for activator-RNA members of both libraries for all pairs of mismatches across the shown spacer-targeting and flanking regions when (C) targeting X activator-RNA library with 100 nM dCas13a: crRNA-X, and (D) targeting Y activator-RNA library with 100 nM dCas13a: crRNA-Y. For comparison, single mismatch data is plotted along the diagonal axis and the fold-change value for a perfectly complementary target is indicated on the heat-map scale bar (noMM*).
Fig. 3.
Fig. 3.. Biochemical validation of Lbu-dCas13 binding profiles
(A) Schematic of the Lbu-Cas13a crRNA: ssRNA activator interaction highlighting the different activator-RNAs tested in this study. 6-FAM: a covalently attached 6-carboxyfluorescein fluorophore. (B) Fluorescence anisotropy binding curves for the interaction between Lbu-dCas13a: crRNA-X and X activator-RNAs depicted in (A). Binding data fits (see Experimental Procedures) shown as solid lines. Error-bars represent the s.d of the anisotropy from three independent experiments. Dissociation constants (KD) and their associated standard errors (SE) from three independent experiments are shown. (C) Data from (B) normalized as a percentage binding affinity relative to the affinity to a perfectly complementary activator-RNAs (no MM). (D) filter binding curves for the interaction between Lbu-dCas13a: crRNA-X (or Y) and X (or Y) 5’ 32P-labelled and 3’−6FAM fluorophore- labelled activator-RNAs. 32P phosphor-imaging was used to detect the activator-RNAs in this experiment. Binding data fits (see Experimental Procedures) shown as solid lines. A binding curve could not be fit RNA-Y g9–12MM. Error-bars represent the s.d of the fraction bound from three independent experiments. Dissociation constants (KD) and their associated standard errors (SE) from three independent experiments are shown in the Fig. (E) Data from (D) normalized as a percentage binding affinity relative to the affinity to a perfectly complementary activator-RNA X (no MM) with error bars representing the normalized s.d from three independent experiments. In all panels, g5–8MM binding data is marked with an (*).
Fig. 4.
Fig. 4.. Position of crRNA: activator-RNA mismatches differentially impact Lbu-dCas13a HEPN-nuclease activation
(A) representative time course of background corrected fluorescence measurements generated by Lbu- Cas13a: crRNA-X activation by the addition of 100 pM X activator-RNAs with either zero (noMM) or four consecutive mismatches across the crRNA-spacer targeting region. Quantified data were fitted with single-exponential decays (solid line) with calculated apparent rate constants (kobs) (mean ± s.d., n =3) as follows: noMM 0.023 ± 0.003 min−1, g1–4MM 0.028 ± 0.002 min−1, g13–16MM 0.050 ± 0.003 min−1, g17–20MM 0.004 ± 0.007 min−1, while g5–8MM and g9–12 were not fit. (B) Data from (A) normalized as a percent cleavage rate relative to a perfectly complementary activator-RNA X (no MM) with error bars representing the normalized s.d from three independent experiments.
Fig. 5.
Fig. 5.. Lbu-Cas13a HEPN-nuclease activation is gated by ‘unfavorable’ base-pairing within the context of the Cas13a crRNA: activator-RNA interaction.
(A) Background corrected apparent cleavage rates generated by Lbu- Cas13a: crRNA-X activation by the addition of 100 pM activator-RNA X with either zero (noMM) or single mismatches in positions 1–20 across the crRNA-spacer. Apparent cleavage rates are plotted as a normalized percentage cleavage rate relative to a perfectly complementary activator-RNA X (noMM) with error bars representing the normalized s.d from three independent experiments. Representative time-course data can be found in Fig. S6. (B) Change in nucleotide frequency in each position shown between the top 500 enriched members and the bottom 500 enriched members (as determined by fold-change relative to Apo Lbu-dCas13a) for 100 nM dCas13a: crRNA-X targeting the activator-RNA X library. The activator-RNA X crRNA-target sequence is shown above. Values with p ≥ 0.001 in the permutation test are not shown. (C) A revised model for Lbu-Cas13a HEPN-nuclease activation based on the key findings in this study.
Fig. 6.
Fig. 6.. Putative structural basis of Cas13a detection of crRNA: target-RNA base-pairing in the g5–8 region.
(A) Overall view of the ternary Cas13a: crRNA:target-RNA crystal structure. Features implicated in the recognition of the crRNA-spacer nucleotides 5–8: target nucleotide 5*−8* (complementary to g5–8) RNA duplex region are labeled. (B) Close-up view of the crRNA g5–8: target 5*−8* (complementary to g5–8) RNA duplex region with important features labeled. (C) same as B, except the ternary complex has been rotated 90° clockwise. Figure generated from PDB ID:5XWP.

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