1. Biochemistry and Chemical Biology

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
https://doi.org/10.7554/eLife.49110
Share this article
Cite this article
  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
(2019)
Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a
eLife 8:e49110.
https://doi.org/10.7554/eLife.49110
  2. Download BibTeX
  3. Download .RIS

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.

Data availability

The cryo-EM map of the LbCas12a-crRNA-AcrVA4 complex at 2.99 Å resolution (State I) and the refined coordinates model have been deposited to the EMDB and PDB with accession codes EMD-20266 and PDB-6P7M, respectively. The cryo-EM map of the 2[LbCas12a-crRNA-AcrVA4] complex at 4.91 Å resolution (State II) and the refined coordinates have been deposited to the EMDB and PDB with accession codes EMDB-20267 and PDB-6P7N, respectively.All other data generated or analyzed during this study are included in the manuscript or supporting files. PDB maps are available on the eLife dropbox:https://www.dropbox.com/sh/zu4l3cd696uzbq7/AAAyo9sVAp3jBfZsFURA2Hpfa?dl=0

The following data sets were generated
    1. Knott
    2. GJ
    3. Liu
    4. JJ
    5. Doudna
    6. JA
    (2019) LbCas12a-crRNA-AcrVA4 State I
    RCSB Protein Data Bank, 6P7M.
    https://www.rcsb.org

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

Version 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.

Metrics

  • 3,885
    views
  • 735
    downloads
  • 42
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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
(2019)
Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a
eLife 8:e49110.
https://doi.org/10.7554/eLife.49110

Share this article

https://doi.org/10.7554/eLife.49110

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Plant Biology

    Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling

    Henning Mühlenbeck, Yuko Tsutsui ... Cyril Zipfel
    Research Article

    Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

    1. Biochemistry and Chemical Biology
    2. Neuroscience

    Alzheimer’s disease linked Aβ42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling

    Katarzyna Marta Zoltowska, Utpal Das ... Lucía Chávez-Gutiérrez
    Research Article

    Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer’s disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17–42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

    Ya-Juan Wang, Xiao-Jing Di ... Ting-Wei Mu
    Research Article Updated

    Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.