Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

Cell. 2018 Nov 1;175(4):934-946.e15. doi: 10.1016/j.cell.2018.09.039. Epub 2018 Oct 18.


CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Keywords: CRISPR; Cascade; DNA curtains; fluorescence microscopy; primed acquisition.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinomycetales / enzymology*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems*
  • DNA, Viral / metabolism
  • Protein Multimerization
  • Single Molecule Imaging


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • DNA, Viral