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Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

By Jochem N.A. Vink, Koen J.A. Martens, Marnix Vlot, Rebecca E. McKenzie, Cristóbal Almendros, Boris Estrada Bonilla, Daan J.W. Brocken, Johannes Hohlbein, Stan J.J. Brouns

Posted 25 Mar 2019
bioRxiv DOI: 10.1101/589119 (published DOI: 10.1016/j.molcel.2019.10.021)

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (~30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

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