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

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex.

Author information

1
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
2
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
3
Department of Chemistry, University of Texas at Austin, Austin, TX 78712, USA.
4
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA. Electronic address: ilya@finkelsteinlab.org.

Abstract

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

PMID:
30343903
DOI:
10.1016/j.cell.2018.09.039

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