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Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons

Michael Schmitz, Irma Querques, Seraina Oberli, Christelle Chanez, View ORCID ProfileMartin Jinek
doi: https://doi.org/10.1101/2022.06.17.496590
Michael Schmitz
1Department of Biochemistry, University of Zurich, Zurich, 8057, Switzerland
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Irma Querques
1Department of Biochemistry, University of Zurich, Zurich, 8057, Switzerland
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Seraina Oberli
1Department of Biochemistry, University of Zurich, Zurich, 8057, Switzerland
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Christelle Chanez
1Department of Biochemistry, University of Zurich, Zurich, 8057, Switzerland
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Martin Jinek
1Department of Biochemistry, University of Zurich, Zurich, 8057, Switzerland
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  • ORCID record for Martin Jinek
  • For correspondence: jinek@bioc.uzh.ch
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Summary

CRISPR systems have been co-opted by Tn7-like elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprising RNA-guided Cas12k, TniQ, TnsC and, unexpectedly, the ribosomal protein S15. Complex assembly on target DNA results in complete R-loop formation mediated by critical interactions between TniQ and the trans-activating crRNA, and is coupled with TniQ-dependent nucleation of a TnsC filament. In vivo transposition assays corroborate our structural findings, and biochemical and functional analyses of S15 supports its role as a bona fide component of the type V crRNA-guided transposition machinery. Altogether, our work uncovers key aspects of the mechanisms underpinning RNA-mediated assembly of CRISPR-associated transposons that will guide their development as programmable site-specific gene insertion tools.

Competing Interest Statement

The authors have filed a related patent application.

Copyright 
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-ND 4.0 International license.
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Posted June 17, 2022.
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Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons
Michael Schmitz, Irma Querques, Seraina Oberli, Christelle Chanez, Martin Jinek
bioRxiv 2022.06.17.496590; doi: https://doi.org/10.1101/2022.06.17.496590
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Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons
Michael Schmitz, Irma Querques, Seraina Oberli, Christelle Chanez, Martin Jinek
bioRxiv 2022.06.17.496590; doi: https://doi.org/10.1101/2022.06.17.496590

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