Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9

  1. Lukasz Nierzwicki
  2. Kyle W East
  3. Uriel N Morzan
  4. Pablo R Arantes
  5. Victor S Batista
  6. George P Lisi  Is a corresponding author
  7. Giulia Palermo  Is a corresponding author
  1. University of California, Riverside, United States
  2. Brown University, United States
  3. International Centre for Theoretical Physics, Italy
  4. Yale University, United States

Abstract

CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

Data availability

Analysis codes and script files can be downloaded from Github: https://github.com/palermolabResonance assignments for the HNH structure are available at bmrb.io under BMRB entry 27949.

Article and author information

Author details

  1. Lukasz Nierzwicki

    Department of Bioengineering, University of California, Riverside, Riverside, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Kyle W East

    Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Uriel N Morzan

    International Centre for Theoretical Physics, Trieste, Italy
    Competing interests
    The authors declare that no competing interests exist.
  4. Pablo R Arantes

    Department of Bioengineering, University of California, Riverside, Riverside, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Victor S Batista

    Department of Chemistry, Yale University, New Heaven, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. George P Lisi

    Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, United States
    For correspondence
    george_lisi@brown.edu
    Competing interests
    The authors declare that no competing interests exist.
  7. Giulia Palermo

    Department of Bioengineering, University of California, Riverside, Riverside, United States
    For correspondence
    giulia.palermo@ucr.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1404-8737

Funding

National Institutes of Health (R01GM141329)

  • Giulia Palermo

National Science Foundation (CHE-1905374)

  • Giulia Palermo

National Institutes of Health (R01GM136815)

  • Victor S Batista
  • George P Lisi
  • Giulia Palermo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Rina Rosenzweig, Weizmann Institute of Science, Israel

Publication history

  1. Received: September 3, 2021
  2. Accepted: December 14, 2021
  3. Accepted Manuscript published: December 15, 2021 (version 1)
  4. Version of Record published: January 7, 2022 (version 2)

Copyright

© 2021, Nierzwicki et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Lukasz Nierzwicki
  2. Kyle W East
  3. Uriel N Morzan
  4. Pablo R Arantes
  5. Victor S Batista
  6. George P Lisi
  7. Giulia Palermo
(2021)
Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9
eLife 10:e73601.
https://doi.org/10.7554/eLife.73601
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