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Cell. 2017 Sep 7;170(6):1224-1233.e15. doi: 10.1016/j.cell.2017.07.037. Epub 2017 Aug 24.

A Broad-Spectrum Inhibitor of CRISPR-Cas9.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
2
Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720, USA.
3
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
4
RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
5
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
6
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
7
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
8
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.

Abstract

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

KEYWORDS:

CRISPR; CRISPR-Cas9; Cas9; anti-CRISPR; gene editing

PMID:
28844692
PMCID:
PMC5875921
DOI:
10.1016/j.cell.2017.07.037
[Indexed for MEDLINE]
Free PMC Article

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