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
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
- Douglas L Black, University of California, Los Angeles, United States
Publication history
- Received: June 6, 2019
- Accepted: August 9, 2019
- Accepted Manuscript published: August 9, 2019 (version 1)
- 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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