1. Volume 46, Issue 5, Pages 595-605 (June 2012)
CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3 
Edze R. Westra, Paul B.G. van Erp, Tim Künne, Shi Pey Wong, Raymond H.J. Staals, Christel L.C. Seegers, Sander Bollen, Matthijs M. Jore, Ekaterina Semenova, Konstantin Severinov, Willem M. de Vos, Remus T. Dame, Renko de Vries, Stan J.J. Brouns, John van der Oost 
Molecular Cell 
Volume 46, Issue 5, Pages (June 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Cascade Only Binds nSC Plasmid DNA with High Affinity
(A) Gel shift of nSC plasmid DNA with J3-Cascade, containing a targeting (J3) crRNA. pUC-λ was mixed with 2-fold increasing amounts of J3-Cascade, from a pUC-λ:Cascade molar ratio of 1:0.5 up to a 1:256 molar ratio. The first and last lanes contain only pUC-λ.
(B) Gel shift as in (A) with an escape mutant of pUC-λ containing a single point mutation in the PAM (CAT to CGT).
(C) Gel shift as in (A) with R44-Cascade containing a nontargeting (R44) crRNA.
(D) Gel shift as in (A) with Nt.BspQI-nicked pUC-λ.
(E) Gel shift as in (A) with PdmI-linearized pUC-λ.
(F) Specific binding of Cascade to the protospacer monitored by BsmI footprinting at a pUC-λ:Cascade molar ratio of 10:1. Lanes 1 and 5 contain only pUC-λ. Lanes 2 and 6 contain pUC-λ mixed with Cascade. Lanes 3 and 7 contain pUC-λ mixed with Cascade and subsequent BsmI addition. Lanes 4 and 8 contain pUC-λ mixed with BsmI.
(G) BsmI footprint as in (F) with Nt.BspQI cleavage of one strand of the plasmid subsequent to Cascade binding. Lanes 1 and 6 contain only pUC-λ. Lanes 2 and 7 contain pUC-λ mixed with Cascade. Lanes 3 and 8 contain pUC-λ mixed with Cascade and a subsequent BsmI footprint. Lanes 4 and 9 contain pUC-λ mixed with Cascade, followed by nicking with Nt.BspQI and a BsmI footprint. Lanes 5 and 10 contain pUC-λ nicked with Nt.BspQI.
(H) BsmI footprint as in (F) with EcoRI cleavage of both strands of the plasmid subsequent to Cascade binding. Lanes 1 and 6 contain only pUC-λ. Lanes 2 and 7 contain pUC-λ mixed with Cascade. Lanes 3 and 8 contain pUC-λ mixed with Cascade and a subsequent BsmI footprint. Lanes 4 and 9 contain pUC-λ mixed with Cascade, followed by cleavage with EcoRI and a BsmI footprint (combined cleavage of BsmI and EcoRI produces a 2.8 kb fragment and an ∼200 nt fragment; the latter is not visible on this gel). Lanes 5 and 10 contain pUC-λ cleaved with EcoRI.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Cascade Induces Bending of Target DNA upon Protospacer Binding
(A–P) Scanning force microscopy images of nSC plasmid DNA with J3-Cascade containing a targeting (J3) crRNA. pUC-λ was mixed with J3-Cascade at a pUC-λ:Cascade ratio of 1:7. Each image shows a 500 × 500 nm surface area. White dots correspond to Cascade.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Cascade Tolerates Four Distinct PAMs that Are Recognized on the Target Strand
(A) Mutagenesis of the PAM flanking the previously described M13 protospacer on the phage M13 genome gives rise either to mutants that escape CRISPR interference (sequences shown in red) or to mutants that are still subject to CRISPR interference (sequences shown in black).
(B–F) Gel shift assays to monitor M13-Cascade binding to 65 nt dsDNA probes containing the M13 protospacer flanked by the PAM sequences indicated by an asterisk in (A), corresponding to four PAMs (B–E) that are tolerated and a single escape PAM mutant (F).
(G) Gel shift assays using 65 nt dsDNA probes containing the M13 protospacer flanked by an escape PAM sequence (GGG/CCC) on both the target and the displaced strand.
(H) Gel shift as in (G) with the M13 protospacer being flanked by a tolerated PAM sequence (CTT/AAG) on both the target and the displaced strand.
(I) Gel shift assays as in (G) with the M13 protospacer being flanked by an escape PAM sequence (GGG) on the target strand and a tolerated PAM (AAG) on the displaced strand.
(J) Gel shift assays as in (G) with the M13 protospacer being flanked by a tolerated PAM sequence (CTT) on the target strand and an escape PAM (CCC) on the displaced strand.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
BiFC Analysis Reveals that Cascade and Cas3 Interact upon Target Recognition
(A) Venus fluorescence of cells expressing CascadeΔCse1 and CRISPR 7Tm, which targets seven protospacers on the phage λ genome, and Cse1-N155Venus and Cas3-C85Venus fusion proteins.
(B) Bright-field image of the cells in (A).
(C) Overlay of (A) and (B).
(D) Venus fluorescence of phage λ-infected cells expressing CascadeΔCse1 and CRISPR 7Tm, and Cse1-N155Venus and Cas3-C85Venus fusion proteins.
(E) Bright-field image of the cells in (D).
(F) Overlay of (D) and (E).
(G) Venus fluorescence of phage λ-infected cells expressing CascadeΔCse1 and nontargeting CRISPR R44, and Cse1-N155Venus and Cas3-C85Venus fusion proteins.
(H) Bright-field image of the cells in (G).
(I) Overlay of (G) and (H).
(J) Average of the fluorescence intensity of four to seven individual cells of each strain, as determined using the profile tool of LSM viewer (Carl Zeiss). Error bars represent the standard deviation of the mean.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Role of Cas3 Nuclease and Helicase Activities during CRISPR Interference
(A) Competent BL21-AI cells expressing Cascade, a Cas3 mutant, and CRISPR J3 were transformed with pUC-λ. Colony-forming units per microgram pUC-λ (cfu/μg DNA) are depicted for each of the strains expressing a Cas3 mutant. Cells expressing WT Cas3 and CRISPR J3 or CRISPR R44 serve as positive and negative controls, respectively. Experiments were performed in triplicate. Error bars represent the standard deviation of the mean.
(B) BL21-AI cells carrying Cascade-, Cas3 mutant-, and CRISPR-encoding plasmids as well as pUC-λ are grown under conditions that suppress expression of the cas genes and CRISPR. At t = 0, expression of the CRISPR and cas genes is induced. The fraction of cells that retain pUC-λ over time is shown, as determined by the ratio of ampicillin-resistant and total cell counts.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Cascade-Cas3 Fusion Complex Provides In Vivo Resistance and Has In Vitro Nuclease Activity
(A) Coomassie blue-stained SDS-PAGE of purified Cascade and Cascade-Cas3 fusion complex.
(B) Efficiency of plaquing of phage λ on cells expressing Cascade-Cas3 fusion complex and a targeting (J3) or nontargeting (R44) CRISPR. Cells expressing nonfused Cascade and Cas3 with a targeting (J3) CRISPR serve as a control.
(C) Gel shift (in the absence of divalent metal ions) of nSC target plasmid with J3-Cascade-Cas3 fusion complex. pUC-λ was mixed with 2-fold increasing amounts of J3-Cascade-Cas3, from a pUC-λ:J3-Cascade-Cas3 molar ratio of 1:0.5 up to 1:128. The first and last lanes contain only pUC-λ.
(D) Gel shift (in the absence of divalent metal ions) of nSC nontarget plasmid with J3-Cascade-Cas3 fusion complex. pUC-P7 was mixed with 2-fold increasing amounts of J3-Cascade-Cas3, from a pUC-P7:J3-Cascade-Cas3 molar ratio of 1:0.5 up to 1:128. The first and last lanes contain only pUC-P7.
(E) Incubation of nSC target plasmid (pUC-λ, left) or nSC nontarget plasmid (pUC-P7, right) with J3-Cascade-Cas3 in the presence of 10 mM MgCl2. Lanes 1 and 7 contain only plasmid.
(F) Assay as in (E) in the presence of 2 mM ATP.
(G) Assay as in (E) with the mutant J3-Cascade-Cas3K320N complex.
(H) Assay as in (G) in the presence of 2 mM ATP.
(I) Schematic overview of the three DNA substrates used in the in vitro nuclease assay shown in (J). The substrates are 89 nt and contain a 39 nt double-stranded region at a variable position. Asterisks at the 5′ end indicate the presence of the 32P label.
(J) Incubation of Cascade-Cas3 with the 5′-labeled substrates shown in (I) in the presence of 10 mM MgCl2. The endonuclease products that run low in the gel are better visible in the overexposed version shown in Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Model of the CRISPR-Interference Type I Pathway in E. coli
Steps that are not well-understood are depicted with dashed arrows. (1) Cascade (blue) carrying a crRNA (orange). (2) Cascade associates nonspecifically with the nSC plasmid DNA and scans for a protospacer (red), with protospacer adjacent motif (PAM) (yellow). (3) Sequence-specific binding to a protospacer is achieved through base pairing between the crRNA and the complementary strand of the DNA, forming an R loop. Upon binding, Cascade induces bending of the DNA, and Cascade itself undergoes conformational changes (Jore et al., 2011; Wiedenheft et al., 2011a). (4) The Cse1 subunit of Cascade recruits the nuclease/helicase Cas3 (brown). This may be triggered by the conformational changes of Cascade and the target DNA. (5) The HD domain (dark brown) of Cas3 catalyzes Mg2+-dependent nicking of the target DNA at an unknown position, possibly within or near to the R loop. (6) Plasmid nicking alters the topology of the target plasmid from nSC to relaxed OC, causing a reduced affinity of Cascade for the target. Dissociation of Cascade from the target may involve Cas3 helicase activity. Cascade may then remain associated with Cas3 or may be remobilized to locate new targets. (7) Cas3 degrades the entire plasmid in an ATP-dependent manner as it progressively moves (in the 3′ to 5′ direction) along, unwinds, and cleaves the target dsDNA. Exonucleolytic degradation takes place in the 3′-5′ direction, as was also reported for the combined activities of the helicase MjaCas3′ and the nuclease MjaCas3″ (Beloglazova et al., 2011).
Molecular Cell  , DOI: ( /j.molcel )
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