1. Volume 163, Issue 4, Pages 854-865 (November 2015)
Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System 
Sy Redding, Samuel H. Sternberg, Myles Marshall, Bryan Gibb, Prashant Bhat, Chantal K. Guegler, Blake Wiedenheft, Jennifer A. Doudna, Eric C. Greene 
Cell 
Volume 163, Issue 4, Pages (November 2015)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Programmed Target Binding by E. coli Cascade
(A) Overview of DNA curtains.
(B) Schematic of E. coli Cascade programmed with a crRNA targeting one of three different binding sites (designated λ1, λ2, and λ3) on λWT.
(C) Wide-field TIRF microscopy image showing QD-tagged Cascade (magenta) bound to DNA (green) at λ1.
(D) Wide-field image showing Cascade bound at λ3.
(E) Binding distribution for Cascade targeted to each of the three protospacers; error bars here and all subsequent binding distributions represent 95% confidence intervals obtained through bootstrap analysis.
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4. Figure 2 Cascade Searches for PAMs while Interrogating Foreign DNA
(A) Kymographs highlighting examples of Cascade binding events over two different time regimes (see scale bars). Examples of transient sampling and stable recognition are highlighted.
(B–D) Distribution of PAMs (blue line) and transient binding events for Cascade programmed with (B) the λ1-crRNA, (C) the λ3-crRNA, or (D) a P7-crRNA. Count refers to number of occurrences within 1 kbp of DNA. The locations of the λ1 and λ3 target sites are indicated, and the heat map color-coding reflects the binding dwell time (ti) relative to the mean dwell time (t¯).
(E–G) Correlation of PAMs with the transient binding events for Cascade programmed with (E) the λ1-crRNA, (F) the λ3-crRNA, or (G) P7-crRNA, as indicated. Outlying data points (colored green and boxed) reflect underrepresented binding events at PAM sites near the ends of the DNA; detection of binding at these sites is hindered by the chromium barriers.
See also Figure S1.
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5. Figure 3 Recognition of Escape PAM Mutants
(A) Schematic of λePAM bearing two identical protospacers, one with a cognate PAM (λ3) and the other with an escape PAM (mutλ3).
(B) Kymograph highlighting example of Cascade binding to the mutλ3 through 3D diffusion.
(C) Wide-field images showing binding to each of the two targets at different Cascade concentrations following a 10-min incubation. Arrowheads indicate the locations of the λ3 (green) and mutλ3 (magenta) targets.
(D) Binding distributions showing relative occupancy at each Cascade concentration.
(E) Quantification of percent occupancy; Ø indicates no detectable binding.
(F) Survival probability plots for Cascade bound to the two targets; error bars here and all subsequent survival probability plots represent 70% confidence intervals obtained through bootstrap analysis.
See also Table S1.
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6. Figure 4 Cas3 Generates an ssDNA Gap at the λ3 Protospacer
(A) Image showing RPA-eGFP foci at λ3 for reactions with unlabeled Cascade and unlabeled Cas3.
(B) Control images showing that RPA-eGFP foci are not present when Cas3 is omitted from the reactions; the upper and lower panels show the same field of view.
(C) Requirements for RPA-eGFP foci formation at λ3.
(D) Distribution of RPA-eGFP foci in reactions containing both Cascade and Cas3; Count refers to the number of occurrences within 1 kbp of DNA.
(E) Signal intensities for RPA-eGFP foci. The intensity of a focus comprised of three molecules of RPA-eGFP is indicated, and each successive bin corresponds to ∼1 additional molecule of RPA-eGFP. The heat map color-coding in (D) and (E) are the same.
(F) Representative stepwise photobleaching curve used to estimate the number of RPA-eGFP molecules in each focus.
See also Figure S2.
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7. Figure 5 Cascade-Mediated Recruitment of Cas3
(A) Image showing that QD-tagged Cas3 is recruited to unlabeled Cascade at λ3.
(B) Binding of Cas3 to λ3. The distribution is segregated into the translocation (orange) and stationary (green) Cas3 populations.
(C) Survival probabilities of the stationary Cas3 population.
(D) Kymograph illustrating the translocation of Cas3 away from λ3 in a reaction with unlabeled Cascade. The delay period prior to the initiation of Cas3 translocation is indicated.
(E) Two-color experiment showing that Cas3 (green) translocates away from Cascade (magenta).
(F) Survival probability (delay time) of the translocating population of Cas3 prior to moving away from λ3.
(G) Cas3 velocity distribution.
(H) Cas3 processivity distribution.
(I) Kymograph showing an example of Cas3 repeatedly looping the DNA.
(J) Intensity profile showing the increase in Cas3 fluorescence signal coinciding with DNA loop formation.
See also Figure S3 and Movie S1.
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8. Figure 6 Cas1-Cas2-Mediated Recruitment of Cas3 to Escape Targets
(A) Binding distribution of Cas3 on λePAM in the absence of Cas1-Cas2.
(B) Cas3 binding distribution histogram on λePAM in the presence of Cas1-Cas2.
(C) Overlaid trajectories showing examples of Cas3 translocation events originating from either the λ3 protospacer (green) or the mutλ3 protospacer (magenta). Of the trajectories originating from mutλ3, 59% of the Cas3 molecules move toward the downstream anchor points, and the remaining 41% travel in the opposite direction.
See also Figures S4 and S5 and Movies S2, S3, and S4.
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9. Figure 7
Model for Foreign DNA Recognition and Processing by Cascade, Cas1, Cas2, and Cas3
(A) The predominant mechanism for protospacer recognition is through the PAM-dependent pathway.
(B) PAM-dependent processing involves the recruitment of Cas3 to the protospacer by Cascade. Cas3 nicks the R-loop and generates an ssDNA gap; Cas3 can dissociate at either of these two steps. Cas3 then breaks free from Cascade and travels unidirectionally along the DNA.
(C) PAM-independent processing requires Cas1-Cas2 to recruit Cas3. Cas3 is loaded onto the DNA in one of two possible orientations through a mechanism that attenuates Cas3 nuclease activity. Cas3 then travels in either direction along the DNA as part of a spacer acquisition complex.
See also Figure S6.
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10. Figure S1 Cascade Search Intermediate Lifetimes, Related to Figure 2
(A) Lifetime analysis for Cascade loaded with λ3 crRNA. The left plot shows lifetimes across the phage genome (reproduced from Figure 2C), and the right plot shows position specific lifetimes; binding at the λ3 target (green) at a distal site of equal extent (orange) or across the phage genome (magenta). Values correspond to a double exponential fit (solid line), where A is the population weight between lifetimes, and the data represent experimental results collected at 25 mM KCl and 25°C.
(B) Survival probabilities for transient search intermediates (mean ± 70% confidence intervals) that sample the phage DNA but do not stably engage the target site; these data reflect experiments with the λ3 crRNA. These transient intermediates exhibited complex dissociation kinetics, and the simplest model that describes the data is a double-exponential decay (solid line), indicating that at least two, and possibly more, intermediates exist on the path to target recognition. Consistent with our previous work on S. pyogenes Cas9, we ascribe the shorter lifetime (τa) to PAM binding by Cascade, and the longer-lived intermediates (τb) as attempts by Cascade to probe the flanking DNA at non-target sites for sequence complementarity to the crRNA (Sternberg et al., 2014).
(C) Survival probabilities and lifetimes for each of the different crRNAs used in this study.
(D) Survival probabilities and lifetimes for the P7 crRNA for transient sampling data collected under different reaction conditions, as indicated.
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11. Figure S2
Reaction Requirements for ssDNA Formation, Related to Figure 4
(A) RPA-eGFP foci distributions and intensities under different conditions, as indicated. Color-coding corresponds to the calibration data presented in Figures 4E and 4F. Threshold values reflect RPA-eGFP aggregation, and were excluded from further analysis. The lower panel is the data that is shown in Figure 4D, and is presented here for direct comparison to the other datasets.
(B) Example illustrating how the relative RPA-eGFP signals reported in Figure 4C was calculated from the different RPA-eGFP foci distribution histograms. The Gaussian profile was first determined for reactions containing the full complement of cofactors (upper panel), and the bins under this curve contained the total number of RPA foci at the protospacer (colored magenta; also see Figure 4C). This same profile was then overlaid with the RPA-eGFP binding distributions collected under different reaction conditions (e.g., bottom panel), and the bins under the curve contained the total number of RPA foci at the protospacer under a given set of conditions (colored magenta; also see Figure 4C).
(C) Plasmid digest assay under different reaction conditions. Reactions were performed as described in the Supplemental Experimental Procedures, and the products were resolved by alkaline gel electrophoresis. The positions of the circular (C) input DNA, the nicked circle (N) product, and the smaller degradation products (P) are all indicated.
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12. Figure S3 Characterization of Fluorescent Cas3, Related to Figure 5
(A) Plasmid digest assays with Cas3 and Cascade in the absence or presence of QDs, as indicated. There is reduced digestion efficiency when both Cascade and Cas3 are labeled with QDs, therefore DNA curtain experiments used only one labeled component (unless otherwise indicated).
(B) Lifetimes of stationary Cas3 molecules bound to Cascade (green), superimposed on the dwell times for the translocating population of Cas3 prior to moving away from Cascade (orange).
(C) Summary of lifetime values from double exponential fits to the data shown in (B).
(D) Cartoon illustrating how a translocating molecule of Cas3 bound to a stationary molecule of Cascade will lead to a looped DNA intermediate, and the increased tension on the DNA will pull the complexes closer to the slide surface. Pi and Pt correspond to the initial and translocated positions, respectively.
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13. Figure S4
Cas3 Is Not Recruited to the Escape Target, Related to Figure 6
(A) Wide-field TIRF microscopy images showing QD-tagged Cascade (upper panel), QD-tagged Cas3 (middle panel), and an overlay (lower panel). The locations of the λ3 and mutλ3 protospacers are indicated.
(B) RPA-eGFP foci distribution for λWT phage DNA containing only the λ3 protospacer; note that this panel is reproduced from the lower panel of Figure S2A to allow side-by-side comparison with data for λePAM phage DNA containing both targets.
(C) RPA-eGFP foci distribution for phage DNA containing both the λ3 and mutλ3.
(D) RPA-eGFP foci quantitation for λwt phage DNA containing only λ3. (E) RPA-eGFP foci distribution for λePAM phage DNA containing both protospacers.
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14. Figure S5
Properties of Cas3 in the Presence of Cas1-Cas2, Related to Figure 6
(A) Cas3 translocation velocities on the λePAM DNA in the absence of Cas1-Cas2.
(B) Cas3 translocation velocities on the λePAM DNA in the presence of Cas1-Cas2.
(C) Survival probability (delay time) of Cas3 prior to moving away from mutλ3 in the presence of Cascade, and Cas1-Cas2.
(D) RPA-eGFP foci measurements on the λePAM DNA after incubation with unlabeled Cascade, Cas1-Cas2, and Cas3. The locations of the mutλ3 and λ3 protospacers are indicated.
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15. Figure S6 Targets Bearing an ePAM Are Not Cleaved, Related to Figure 7
(A and B) Plasmid digest assays in the (A) absence of Cas1-Cas2, and in the (B) presence of Cas1-Cas2, resolved by alkaline gel electrophoresis. In (A) and (B), plasmid substrates with the indicated protospacers (Table S1) were incubated with Cascade at a 6:1 molar ratio. Digestion reactions were then initiated with the addition of Cas3 in the presence or absence of Cas1-Cas2, as indicated. Reactions were terminated, and products were resolved by alkaline agarose gel electrophoresis.
(C) Control gel shift assays using ∼250-bp Atto488 dye labeled DNA substrates demonstrating that Cascade binds to the λ3 and mutλ3 targets in bulk. Reactions were performed in the presence or absence of a 10-fold molar excess of unlabeled competitor DNA containing a control P7 protospacer to verify the specificity of binding.
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