PMC

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.

Cascade tolerates 4 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 to either 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.

BiFC analysis reveals that Cascade and Cas3 interact upon target recognition. A) Venus fluorescence of cells expressing CascadeΔCse1 and CRISPR 7Tm, which targets 7 protospacers on the phageλ genome, and Cse1-N155Venus and Cas3-C85Venus fusion proteins. B) Brightfield 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) Brightfield image of the cells in (D). F) Overlay of (D) and (E). G) Venus fluorescence of phage λ infected cells expressing CascadeΔCse1 and non-targeting CRISPR R44, and N155Venus and C85Venus proteins. H) Brightfield image of the cells in (G). I) Overlay of (G) and (H). J) Average of the fluorescence intensity of 4–7 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.

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.

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 lane 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 non-targeting (R44) crRNA. D) Gel-shift as in (A) with Nt.BspQI nicked pUC-λE) Gel-shift as in (A) with PdmI linearized pUC-λF) Specific binding of Cascade to the protospacer monitored by BsmI footprinting at a pUC-λ : Cascade molar ratio of 10:1. Lane 1 and 5 contain only pUC-λ. Lane 2 and 6 contain pUC-λ mixed with Cascade. Lane 3 and 7 contain pUC-λ mixed with Cascade and subsequent BsmI addition. Lane 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. Lane 1 and 6 contain only pUC-λ. Lane 2 and 7 contain pUC-λ mixed with Cascade. Lane 3 and 8 contain pUC-λ mixed with Cascade and a subsequent BsmI footprint. Lane 4 and 9 contain pUC-λ mixed with Cascade, followed by nicking with Nt.BspQI and a BsmI footprint. Lane 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. Lane 1 and 6 contain only pUC-λ. Lane 2 and 7 contain pUC-λ mixed with Cascade. Lane 3 and 8 contain pUC-λ mixed with Cascade and a subsequent BsmI footprint. Lane 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 a ~200 nt fragment; the latter is not visible on this gel). Lane 5 and 10 contain pUC-λ cleaved with EcoRI.

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 (; ). (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″ ().

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 non-targeting (R44) CRISPR. Cells expressing non-fused Cascade and Cas3 with a targeting (J3) CRISPR is given 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 non-target 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 non-target plasmid (pUC-P7, right) with J3-Cascade-Cas3 in the presence of 10 mM MgCl₂. Lane 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 panel (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′ labelled 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 .