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Elife. 2019 Aug 9;8. pii: e49110. doi: 10.7554/eLife.49110. [Epub ahead of print]

Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
2
Gladstone Institutes, University of California, San Francisco, San Francisco, United States.
3
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, United States.
4
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
5
Department of Bioengineering, University of California, Berkeley, Berkeley, United States.
6
Department of Chemistry, University of California, Berkeley, Berkeley, United States.
7
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, 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.

KEYWORDS:

E. coli; biochemistry; chemical biology

PMID:
31397669
DOI:
10.7554/eLife.49110
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Conflict of interest statement

GK Gavin J Knott, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors.. BC, RL, BA, DR, MH, BA, ML, MX, AA The other authors declare that no competing interests exist. JL Jun-Jie Liu, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors.. BT Brittney W Thornton, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors.. CF Christof Fellmann, The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors.. JD 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..

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