1. Biochemistry and Chemical Biology
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Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a

  1. Gavin J Knott
  2. Brady F Cress
  3. Jun-Jie Liu
  4. Brittney W Thornton
  5. Rachel J Lew
  6. Basem Al-Shayeb
  7. Daniel J Rosenberg
  8. Michal Hammel
  9. Benjamin A Adler
  10. Marco J Lobba
  11. Michael Xu
  12. Adam P Arkin
  13. Christof Fellmann
  14. Jennifer A Doudna  Is a corresponding author
  1. University of California, Berkeley, United States
  2. University of California, San Francisco, United States
  3. Lawrence Berkeley National Laboratory, United States
  4. Howard Hughes Medical Institute, University of California, Berkeley, United States
Research Article
  • Cited 17
  • Views 2,653
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Cite this article as: eLife 2019;8:e49110 doi: 10.7554/eLife.49110

Abstract

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The A. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

Article and author information

Author details

  1. Gavin J Knott

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Gavin J Knott, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9007-6273
  2. Brady F Cress

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Jun-Jie Liu

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Jun-Jie Liu, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors..
  4. Brittney W Thornton

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Brittney W Thornton, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors..
  5. Rachel J Lew

    Gladstone Institutes, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  6. Basem Al-Shayeb

    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  7. Daniel J Rosenberg

    Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8017-8156
  8. Michal Hammel

    Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States
    Competing interests
    No competing interests declared.
  9. Benjamin A Adler

    Department of Bioengineering, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  10. Marco J Lobba

    Department of Chemistry, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  11. Michael Xu

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  12. Adam P Arkin

    Department of Bioengineering, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4999-2931
  13. Christof Fellmann

    Gladstone Institutes, University of California, San Francisco, San Francisco, United States
    Competing interests
    Christof Fellmann, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9545-5723
  14. Jennifer A Doudna

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    For correspondence
    doudna@berkeley.edu
    Competing interests
    Jennifer A Doudna, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences, and Inari. J.A.D. is a Director at Johnson & Johnson and has sponsored research projects by Pfizer, Roche Biopharma, and Biogen. C.F. is a co-founder of Mirimus, Inc..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9161-999X

Funding

Defense Advanced Research Projects Agency (HR0011-17-2-0043)

  • Jennifer A Doudna

Paul G Allen Frontiers Group

  • Jennifer A Doudna

National Science Foundation (MCB-1244557)

  • Jennifer A Doudna

Howard Hughes Medical Institute

  • Jennifer A Doudna

National Institutes of Health (P50GM082250)

  • Jun-Jie Liu

National Science Foundation (Graduate Research Fellowship,DGE 1752814)

  • Basem Al-Shayeb

National Institutes of Health (RM1HG009490)

  • Marco J Lobba

National Institute of General Medical Sciences (R00GM118909)

  • Christof Fellmann

William M Keck Foundation

  • Gavin J Knott

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

Reviewing Editor

  1. Douglas L Black, University of California, Los Angeles, United States

Publication history

  1. Received: June 6, 2019
  2. Accepted: August 9, 2019
  3. Accepted Manuscript published: August 9, 2019 (version 1)
  4. Version of Record published: August 27, 2019 (version 2)

Copyright

© 2019, Knott 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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