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Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

Kaylee E. Dillard, Cynthia Terrace, Kamyab Javanmardi, Wantae Kim, Kevin J. Forsberg, View ORCID ProfileIlya J. Finkelstein
doi: https://doi.org/10.1101/2021.09.15.460536
Kaylee E. Dillard
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
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  • For correspondence: kaylee.dillard@utexas.edu ilya@finkelsteinlab.org
Cynthia Terrace
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
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Kamyab Javanmardi
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
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Wantae Kim
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
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Kevin J. Forsberg
3Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390, USA
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Ilya J. Finkelstein
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
2Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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  • ORCID record for Ilya J. Finkelstein
  • For correspondence: kaylee.dillard@utexas.edu ilya@finkelsteinlab.org
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Abstract

Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but are generally specific for a narrow subset of Cas nucleases that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits diverse Cas9 sub-types in vitro and human cells. Single-molecule fluorescence imaging reveals that AcrIIA11 interferes with the first steps of target search by reducing S. aureus Cas9’s diffusion on non-specific DNA. DNA cleavage is inhibited because the AcrIIA11:Cas9 complex is kinetically trapped at PAM-rich decoy sites, preventing Cas9 from reaching its target. This work establishes that DNA trapping can be used to inhibit a broad spectrum of Cas9 orthologs in vitro and during mammalian genome editing.

Competing Interest Statement

The authors have declared no competing interest.

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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. All rights reserved. No reuse allowed without permission.
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Posted September 15, 2021.
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Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11
Kaylee E. Dillard, Cynthia Terrace, Kamyab Javanmardi, Wantae Kim, Kevin J. Forsberg, Ilya J. Finkelstein
bioRxiv 2021.09.15.460536; doi: https://doi.org/10.1101/2021.09.15.460536
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Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11
Kaylee E. Dillard, Cynthia Terrace, Kamyab Javanmardi, Wantae Kim, Kevin J. Forsberg, Ilya J. Finkelstein
bioRxiv 2021.09.15.460536; doi: https://doi.org/10.1101/2021.09.15.460536

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