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Mol Cell. 2018 Dec 19. pii: S1097-2765(18)30986-9. doi: 10.1016/j.molcel.2018.11.016. [Epub ahead of print]

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
2
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
3
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
4
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
5
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA.
6
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; Howard Hughes Medical Institute, Berkeley; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
7
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: joseph.bondy-denomy@ucsf.edu.
8
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, Berkeley, CA 94704, USA; Howard Hughes Medical Institute, Berkeley; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA. Electronic address: doudna@berkeley.edu.

Abstract

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.

KEYWORDS:

AcrIIA2; CRISPR-Cas; CRISPR-Cas9; Cas9; Cas9 inhibitors; PAM blockage; anti-CRISPR; bacteriophage; genome editing; temperature-dependent inhibition

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