Accéder au contenu
Merck

Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism.

Molecular cell (2019-11-19)
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
RÉSUMÉ

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 visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation 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 affect 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.

MATÉRIAUX
Référence du produit
Marque
Description du produit

Sigma-Aldrich
Acide sulfurique, ACS reagent, 95.0-98.0%
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
Chlorure de calcium dihydrate, ACS reagent, ≥99%
Sigma-Aldrich
Sulfate de magnésium heptahydraté, ACS reagent, ≥98%
Sigma-Aldrich
Chloramphénicol, ≥98% (HPLC)
Sigma-Aldrich
L-(+)-Arabinose, ≥99% (GC)
Sigma-Aldrich
Uracile, ≥99.0%
Sigma-Aldrich
M9, Minimal Salts, 5X, powder, minimal microbial growth medium
Sigma-Aldrich
Céfalexine hydrate, first-generation cephalosporin antibiotic
Sigma-Aldrich
Streptomycin sulfate salt, powder
Sigma-Aldrich
Streptomycin Ready Made Solution, 100 mg/mL in water