Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

Abstract

Summary CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the Type I–E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of genetic material en route to target recognition.

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

“…Tfu Cse1 encodes positive charges at five of these eight sites (Figure 1A). Notably, this positive patch is disrupted in Ec Cse1, likely explaining the conflicting reports regarding facilitated diffusion of Ec Cascade proposed by prior studies (Figure S1A)(Redding et al, 2015; Xue et al, 2017). To test the importance of the Cse1 positive patch on facilitated diffusion, we purified Cascade harboring Cse1(5A), a variant with all five positive residues mutated to alanine (Figure S1A).…”

“…The interference efficiency of Cas7(3A), containing the K144A, K145A, K148A substitutions, was also reduced and the Cse1(5A)/Cas7(3A) double mutant had the most severe interference defect (8-fold lower than WT Cascade). These results, along with complementary smFRET studies in the Ec Cascade system, suggest that both Cse1 and Cas7 stabilize Cascade on non-specific DNA during target search via facilitated 1D diffusion (Xue et al, 2017). …”

“…Positive residues in the Cas7 subunit (K144, K145, K148) are positioned to interact with DNA and may contribute additional stabilization during target search (van Erp et al, 2015; Xiao et al, 2017a; Xue et al, 2017). We adapted an in vivo interference assay using Tfu Cascade and Tfu Cas3 to determine the functional significance of the Cse1 and Cas7 positive patches (Figure 1I,J and S1G,H).…”

“…Neutralizing mutations in these positive patches reduce the lifetimes of diffusing Cascade complexes on non-specific DNA and decrease the in vivo interference efficiency. Facilitated diffusion is likely a conserved search mechanism among all CRISPR systems (Globyte et al, 2018; Xue et al, 2017). Cascade target recognition and stable R-loop locking proceeds via at least two temporally distinct intermediates.…”

Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

“…Compared to the unbound conformation, the length of the backbone as measured from Cas7f.1 to Cas7f.6 is extended ∼18Å in the target-bound structure, which opens the tight helical spiral, exposing an average of ∼145Å 2 of formerly buried surface area between adjacent Cas7 subunits. The elongated conformation also creates a gap between the head and the tail of the complex that 32 P-labeled dsDNA substrates show that charge-swap mutations in Cas8f residues R282/R293/R299/R302 result in reduced dsDNA binding. However, high-affinity binding is rescued by DNA targets with 10-nucleotide protospacer "bubbles".…”