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 language | English |
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Pages (from-to) | 934-946.e15 |
Journal | Cell |
Volume | 175 |
Issue number | 4 |
DOIs | |
State | Published - 1 Nov 2018 |
Bibliographical note
Publisher Copyright:© 2018 Elsevier Inc.
Keywords
- CRISPR
- Cascade
- DNA curtains
- fluorescence microscopy
- primed acquisition