1. Volume 24, Issue 4, Pages 1025-1036 (July 2018)
RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a 
Akshay Tambe, Alexandra East-Seletsky, Gavin J. Knott, Jennifer A. Doudna, Mitchell R. O’Connell 
Cell Reports 
Volume 24, Issue 4, Pages (July 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 1025-1036DOI: (10.1016/j.celrep.2018.06.105)
Copyright © 2018 The Authors Terms and Conditions

3. Figure 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 pull-down workflow.
(C) Empirical mismatch distribution profile for the input X and Y activator-RNA libraries (before Cas13a binding).
(D) Scatterplot displaying fold change in abundance of each RNA-X and RNA-Y library member between samples with 10 nM crRNA-bound Cas13a programmed to target RNA-X or RNA-Y sequences and those with crRNA-unbound apo-Cas13a.
(E) Scatterplot displaying fold change in abundance of each RNA-X and RNA-Y library member between samples with 100 nM crRNA-bound Cas13a programmed to target RNA-X or RNA-Y sequences and those with crRNA-unbound apo-Cas13a.
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4. Figure 2
dCas13a High-Throughput RNA Mismatch Profiling Reveals Binding Mismatch Sensitivity Hotspots for Single- and Double-Mismatched Activator-RNAs
(A and B) Regularized fold-change enrichment (relative to apo-Lbu-dCas13a) for activator-RNA members of both libraries that contain only a single mismatch across the shown spacer-targeting and spacer-flanking regions when (A) targeting the activator-RNA-X library with 100 nM dCas13a:crRNA-X and (B) targeting the activator-RNA-Y library with 100 nM dCas13a:crRNA-Y. In each plot, each 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 mismatch combinations) in that position. The fold-change enrichment of the perfectly complementary activator-RNA (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 statistically 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 the regularization and permutation test procedure. See Figure S2 for 100 nM Cas13a:crRNA data plotted as individual data points from three experiments, as well as individual and averaged 10 nM Cas13a:crRNA data.
(C and D) Regularized fold-change enrichment analysis (relative to apo-Lbu-dCas13a) for activator-RNA members of both libraries for all pairs of mismatches across the shown spacer-targeting and spacer-flanking regions when (C) targeting the activator-RNA-X library with 100 nM dCas13a:crRNA-X, and (D) targeting the Y activator-RNA library with 100 nM dCas13a:crRNA-Y. For comparison, single mismatch data are plotted along the diagonal axis, and the fold-change value for a perfectly complementary target is indicated on the heatmap scale bar (noMM∗).
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5. Figure 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, covalently attached 6-carboxyfluorescein fluorophore.
(B) Fluorescence anisotropy binding curves for the interaction between Lbu-dCas13a:crRNA-X and activator-RNA-Xs depicted in (A). Binding data fits (see Experimental Procedures) are shown as solid lines. Error bars represent the SD of the anisotropy from three independent experiments. Dissociation constants (KD) and their associated SEs from three independent experiments are shown.
(C) Data from (B) normalized as a percent binding affinity relative to the affinity to noMM.
(D) Filter binding curves for the interaction between Lbu-dCas13a:crRNA-X (or Y) and X (or Y) 5′-32P-radiolabeled and 3′-labeled 6-FAM fluorophore-labeled activator-RNAs. 32P phosphor-imaging was used to detect the activator-RNAs in this experiment. Binding data fits (see Experimental Procedures) are shown as solid lines. A binding curve could not be fit for RNA-Y g9-12MM. Error bars represent the SD of the fraction bound from three independent experiments.
(E) Dissociation constants (KD) and their associated SEs from three independent experiments.
Data from (D) normalized as a percent binding affinity relative to the affinity to a noMM, with error bars representing the normalized SD from three independent experiments. In all panels, g5-8MM binding data are marked with an asterisk.
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6. Figure 4
Position of crRNA:Activator-RNA Mismatches Differentially Affects 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 activator-RNA-Xs 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 cleavage rate constants (kobs) (mean ± SD, n = 3) as follows: noMM, ± 0.003 min−1; g1-4MM, ± 0.002 min−1; g13-16MM, ± 0.003 min−1; g17-20MM, ± 0.007 min−1; g5-8MM and g9-12 were not fit.
(B) Data from (A) normalized as a percent cleavage rate relative to a noMM, with error bars representing the normalized SD from three independent experiments.
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7. Figure 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 percent cleavage rate relative to a noMM, with error bars representing the normalized SD from three independent experiments. Representative time course data can be found in Figure 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 ≥ in the permutation test are not shown.
(C) Revised model for Lbu-Cas13a HEPN-nuclease activation based on the key findings in this study.
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8. Figure 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 nucleotide 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: 5XWP.
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