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Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance

MPG-Autoren
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Turkowyd,  Bartosz
Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Müller-Esparza,  Hanna
Max Planck Research Group Prokaryotic small RNA Biology, Alumni, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Climenti,  Vanessa
Max Planck Research Group Prokaryotic small RNA Biology, Alumni, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

Steube,  Niklas
Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Endesfelder,  Ulrike
Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Randau,  Lennart
Max Planck Research Group Prokaryotic small RNA Biology, Alumni, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Zitation

Turkowyd, B., Müller-Esparza, H., Climenti, V., Steube, N., Endesfelder, U., & Randau, L. (2019). Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance. In CRISPR-CAS ENZYMES (pp. 133-171).


Zitierlink: https://hdl.handle.net/21.11116/0000-0008-BF60-5
Zusammenfassung
Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to
scan DNA strands for target regions. Different crRNAs are bound by
several CRISPR-associated (Cas) protein subunits that form the stable
ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance
process requires a sufficient degree of base-complementarity between
crRNA and target sequences and relies on the recognition of small DNA
motifs, termed protospacer adjacent motifs. Recently, superresolution
microscopy and single-particle tracking methods have been developed to
follow individual protein complexes in live cells. Here, we described
how this technology can be adapted to visualize the DNA scanning process
of Cascade assemblies in Escherichia coli cells. The activity of
recombinant Type I-Fv Cascade complexes of Shewanella putrefaciens CN-32
serves as a model system that facilitates comparative studies for many
of the diverse CRISPR-Cas systems.