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Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons

View ORCID ProfileIrma Querques, View ORCID ProfileMichael Schmitz, Seraina Oberli, View ORCID ProfileChristelle Chanez, View ORCID ProfileMartin Jinek
doi: https://doi.org/10.1101/2021.07.06.451292
Irma Querques
1Department of Biochemistry, University of Zurich, Zurich, 8057, Switzerland
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Michael Schmitz
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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  • For correspondence: jinek@bioc.uzh.ch
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SUMMARY

Although the canonical function of CRISPR-Cas systems is to provide adaptive immunity against mobile genetic elements1, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion2–7. Type V-K CRISPR-associated transposons rely on the activities of the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ7. However, the molecular and structural details of RNA-directed DNA transposition have remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. The assembly of the TnsC helical filament is ATP-dependent and accompanied by structural remodeling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments further show that TniQ restricts TnsC polymerization, while the TnsB transposase interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest a mechanistic model whereby RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodeling, generating a recruitment platform for TnsB to catalyze site-specific transposon insertion. The present work advances our mechanistic understanding of the cross-talk between CRISPR effectors and the transposition machinery and will inform design efforts to harness CRISPR-associated transposons as programmable site-specific gene insertion tools for genome engineering applications.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 06, 2021.
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Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons
Irma Querques, Michael Schmitz, Seraina Oberli, Christelle Chanez, Martin Jinek
bioRxiv 2021.07.06.451292; doi: https://doi.org/10.1101/2021.07.06.451292
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Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons
Irma Querques, Michael Schmitz, Seraina Oberli, Christelle Chanez, Martin Jinek
bioRxiv 2021.07.06.451292; doi: https://doi.org/10.1101/2021.07.06.451292

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