Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

• Kaylee E. Dillard ^[1] ; Maxwell W. Brown ^[1] ; Nicole V. Johnson ^[1]
  1. [1] University of Texas at Austin

     University of Texas at Austin

     Estados Unidos

     
• Localización: Cell, ISSN 0092-8674, Vol. 175, Nº. 4, 2018, págs. 934-946
• Idioma: inglés
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• Resumen

  • 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.