Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

Fuguo Jiang; Jun-Jie Liu; Beatriz A Osuna; Michael Xu; Joel D Berry; Benjamin J Rauch; Eva Nogales; Joseph Bondy-Denomy; Jennifer A Doudna

doi:10.1016/j.molcel.2018.11.016

Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

Mol Cell. 2019 Feb 7;73(3):601-610.e5. doi: 10.1016/j.molcel.2018.11.016. Epub 2018 Dec 27.

Authors

Fuguo Jiang¹, Jun-Jie Liu², Beatriz A Osuna³, Michael Xu⁴, Joel D Berry⁵, Benjamin J Rauch⁵, Eva Nogales⁶, Joseph Bondy-Denomy⁷, Jennifer A Doudna⁸

Affiliations

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: [email protected].
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: [email protected].

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.

Publication types

Comparative Study
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Binding, Competitive
CRISPR-Associated Protein 9 / antagonists & inhibitors
CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism*
CRISPR-Associated Protein 9 / ultrastructure
CRISPR-Cas Systems*
Cryoelectron Microscopy
DNA / genetics
DNA / metabolism*
DNA / ultrastructure
Escherichia coli / enzymology
Escherichia coli / genetics
Gene Editing / methods*
Gene Expression Regulation, Bacterial
Models, Molecular
Nucleic Acid Conformation
Protein Binding
Protein Conformation
Pseudomonas Phages / genetics
Pseudomonas Phages / metabolism*
Pseudomonas aeruginosa / enzymology*
Pseudomonas aeruginosa / genetics
Pseudomonas aeruginosa / virology
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism*
RNA, Guide, CRISPR-Cas Systems / ultrastructure
Structure-Activity Relationship
Temperature*
Viral Proteins / genetics
Viral Proteins / metabolism*
Viral Proteins / ultrastructure

Substances

RNA, Guide, CRISPR-Cas Systems
Viral Proteins
DNA
CRISPR-Associated Protein 9
Cas9 endonuclease Streptococcus pyogenes

Abstract

Publication types

MeSH terms

Substances

Grants and funding