Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

Publication date: 26 December 2017
Source:Cell Reports, Volume 21, Issue 13
Author(s): Chaoyou Xue, Yicheng Zhu, Xiangmei Zhang, Yeon-Kyun Shin, Dipali G. Sashital
CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the type I-E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of genetic material en route to target recognition.

Graphical abstract

Teaser

Xue et al. show that the Cascade surveillance complex rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at PAM sites that may be adjacent to the target. They identify three Cascade motifs that are essential for this process and together enable efficient target recognition and immune response.


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