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A kinetic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity

Behrouz Eslami-Mossallam, Misha Klein, Constantijn v.d. Smagt, Koen v.d. Sanden, Stephen K. Jones Jr., John A. Hawkins, View ORCID ProfileIlya J. Finkelstein, Martin Depken
doi: https://doi.org/10.1101/2020.05.21.108613
Behrouz Eslami-Mossallam
1Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, the Netherlands
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Misha Klein
1Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, the Netherlands
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Constantijn v.d. Smagt
1Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, the Netherlands
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Koen v.d. Sanden
1Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, the Netherlands
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Stephen K. Jones Jr.
2Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA
3Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
4Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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John A. Hawkins
2Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA
3Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
4Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
5Oden Institute for Computational Engineering and Science, University of Texas at Austin, Austin, Texas 78712, USA
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Ilya J. Finkelstein
2Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA
3Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
4Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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  • ORCID record for Ilya J. Finkelstein
Martin Depken
1Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft 2629HZ, the Netherlands
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  • For correspondence: S.M.Depken@tudelft.nl
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Abstract

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites via a single guide RNA (sgRNA). However, SpCas9 also binds and cleaves genomic off-target sites that are partially matched to the sgRNA. Here, we report a microscopic kinetic model that simultaneously captures binding and cleavage dynamics for SpCas9 and Sp-dCas9 in free-energy terms. This model not only outperforms state-of-the-art off-target prediction tools, but also details how Sp-Cas9’s structure-function relation manifests itself in binding and cleavage dynamics. Based on the biophysical parameters we extract, our model predicts SpCas9’s open, intermediate, and closed complex configurations and indicates that R-loop progression is tightly coupled with structural changes in the targeting complex. We show that SpCas9 targeting kinetics are tuned for extended sequence specificity while maintaining on-target efficiency. Our extensible approach can characterize any CRISPR-Cas nuclease – benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

Competing Interest Statement

The authors have declared no competing interest.

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Posted July 21, 2020.
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A kinetic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity
Behrouz Eslami-Mossallam, Misha Klein, Constantijn v.d. Smagt, Koen v.d. Sanden, Stephen K. Jones Jr., John A. Hawkins, Ilya J. Finkelstein, Martin Depken
bioRxiv 2020.05.21.108613; doi: https://doi.org/10.1101/2020.05.21.108613
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A kinetic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity
Behrouz Eslami-Mossallam, Misha Klein, Constantijn v.d. Smagt, Koen v.d. Sanden, Stephen K. Jones Jr., John A. Hawkins, Ilya J. Finkelstein, Martin Depken
bioRxiv 2020.05.21.108613; doi: https://doi.org/10.1101/2020.05.21.108613

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