Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

Kaylee E. Dillard, Maxwell W. Brown, Nicole V. Johnson, Yibei Xiao, Adam Dolan, Erik Hernandez, Samuel D. Dahlhauser, Yoori Kim, Logan R. Myler, Eric V. Anslyn, Ailong Ke, Ilya J. Finkelstein

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

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. Single-molecule studies illuminate how the type I-E CRISPR-Cas interference and adaptation complexes interact and function to achieve primed spacer acquisition.

Original languageEnglish
Pages (from-to)934-946.e15
JournalCell
Volume175
Issue number4
DOIs
StatePublished - 1 Nov 2018

Bibliographical note

Publisher Copyright:
© 2018 Elsevier Inc.

Keywords

  • CRISPR
  • Cascade
  • DNA curtains
  • fluorescence microscopy
  • primed acquisition

Fingerprint

Dive into the research topics of 'Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex'. Together they form a unique fingerprint.

Cite this