Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism
| Autor: | Rebecca E. McKenzie, Cristóbal Almendros, Johannes Hohlbein, Marnix Vlot, Jochem N.A. Vink, Koen Martens, Daan J.W. Brocken, Stan J. J. Brouns, Boris Estrada Bonilla |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
DNA Replication
Protein subunit CRISPR-Associated Proteins Biophysics Gene Dosage Computational biology single molecule Biology ENCODE PALM Microbiology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Microbiologie Cleave CRISPR Nucleoid Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Cas Molecular Biology 030304 developmental biology VLAG 0303 health sciences CRISPR interference Bacteria Mechanism (biology) Cell Biology DNA Biofysica chemistry Cascade arms race target search PAM single-particle tracking EPS CRISPR-Cas Systems photo-activation localization microscopy 030217 neurology & neurosurgery RNA Guide Kinetoplastida |
| Zdroj: | Molecular Cell 77 (2020) 1 Molecular Cell, 77(1), 39-50.e10 Molecular Cell |
| ISSN: | 1097-2765 |
| Popis: | 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.One sentence summaryThe results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences. |
| Databáze: | OpenAIRE |
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