Assembly and translocation of a CRISPR-Cas primed acquisition complex

Abstract

Bacteria and archaea destroy foreign nucleic acids by mounting an RNA-based CRISPR-Cas adaptive immune response [1][2][3] . In type I CRISPR-Cas systems, the most frequently found type of CRISPR in bacteria and archaea 3,4 , foreign DNAs that trigger efficient immunity can also provoke primed acquisition of protospacers into the CRISPR locus [5][6][7][8][9][10][11][12] . Both interference and primed acquisition require Cascade (CRISPR-associated complex for antiviral defense) and the Cas3 helicase/nuclease. Primed acquisition also requires the Cas1-Cas2 integrase; however, the biophysical mechanisms of how interference and primed acquisition are coordinated have remained elusive. Here, we present single-molecule characterization of the type I-E Thermobifida fusca (Tfu) primed acquisition complex (PAC). TfuCascade rapidly samples non-specific DNA for its target via facilitated one-dimensional (1D) diffusion. An evolutionary-conserved positive patch on the Cse1 subunit increases the target recognition efficiency by promoting this 1D diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex initiates processive translocation via a looped DNA intermediate. Moving Cascade/Cas3 complexes stall and release the DNA loop at protein roadblocks. Cas1-Cas2 samples DNA transiently via 3D collisions, but stably associates with target-bound Cascade. Cas1-Cas2 also remains associated with translocating Cascade/Cas3, forming the PAC. By directly imaging all key subcomplexes involved in target recognition, interference, and primed acquisition, this work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

“…Our real-time analysis of DNA reeling by Cas3 revealed, that Cas3 could go through multiple cycles of reeling on a single substrate, by slipping back to its initial location ( Fig. 2b & 2c) 25 . Analysis of this repetitive behavior showed that Cas3 undergoes an average of 1.8 ± 0.1 cycles per substrate ( Fig.…”

“…1b & Extended Data Fig. 1a-c) 25 . Notably, the immobilization of Cas3 did not appreciably affect its capability to degrade dsDNA substrates (Extended Data Fig.…”

“…Thereby Cas3 translocates away from the Cascade binding site and degrades single-stranded DNA fragments along the way (Fig 1a) [13][14][15][16]21,24 . In the reeling model, Cas3 and Cascade remain in tight contact while Cas3 unwinds the DNA, which may result in loops in the target strand (Fig 1a) 21,25 . To distinguish between these two models, we sought to visualize the DNA unwinding activity of Cas3 with a high spatiotemporal resolution.…”

“…2a) 15,25 . This finding is consistent with DNA curtain experiments where no stable interaction between Cascade and Cas3 was observed when cobalt was omitted from the assay 21,25 . To focus on the Cas3 helicase activity, experiments were performed in the absence of cobalt, unless stated otherwise.…”

CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

“…Due to diffraction limit (~100 nm) of the DNA curtain technique, it remains unknown whether the model of exclusive 3-dimensional target search is valid for the length scale of nucleotides. Other single-molecule studies have shown that RNA-guided proteins such as Argonaute or CRISPR type I Cascade protein complex use facilitated 1-dimensional diffusion during their target search 21,22 Using single-molecule FRET (Foerster Resonance Energy Transfer), we investigate the target search mechanism of SpCas9 and demonstrate that it uses 1-D diffusion along the DNA strand during its target search.…”