Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a
- University of California, Berkeley, United States
- University of California, San Francisco, United States
- Lawrence Berkeley National Laboratory, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
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
Article and author information
Author details
Daniel J Rosenberg
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.
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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Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a
eLife 8:e49110.
https://doi.org/10.7554/eLife.49110
