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

Jochem N.A. Vink, View ORCID ProfileKoen J.A. Martens, View ORCID ProfileMarnix Vlot, View ORCID ProfileRebecca E. McKenzie, Cristóbal Almendros, Boris Estrada Bonilla, Daan J.W. Brocken, View ORCID ProfileJohannes Hohlbein, View ORCID ProfileStan J.J. Brouns
doi: https://doi.org/10.1101/589119
Jochem N.A. Vink
1Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, Netherlands
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Koen J.A. Martens
2Laboratory of Biophysics, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, Netherlands
3Laboratory of Bionanotechnology, Wageningen University & Research, Bornse Weilanden 9, 6708 WG Wageningen, Netherlands
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Marnix Vlot
4Laboratory of Microbiology, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, Netherlands
5Tropic Health Sciences, Transistorweg 5-C02, 6534 AT Nijmegen, Netherlands
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Rebecca E. McKenzie
1Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, Netherlands
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Cristóbal Almendros
1Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, Netherlands
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Boris Estrada Bonilla
1Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, Netherlands
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Daan J.W. Brocken
6Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2333 CC Leiden, Netherlands
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Johannes Hohlbein
2Laboratory of Biophysics, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, Netherlands
7Microspectroscopy Research Facility, Wageningen University & Research, Stippeneng 4, 6708 WE, Wageningen, Netherlands
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  • For correspondence: stanbrouns@gmail.com johannes.hohlbein@wur.nl
Stan J.J. Brouns
1Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, Netherlands
4Laboratory of Microbiology, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, Netherlands
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  • For correspondence: stanbrouns@gmail.com johannes.hohlbein@wur.nl
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Abstract

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 summary The results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted March 25, 2019.
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Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism
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
bioRxiv 589119; doi: https://doi.org/10.1101/589119
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Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism
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
bioRxiv 589119; doi: https://doi.org/10.1101/589119

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