title: Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.
abstract: Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Rauch
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Melanie R
authors.initials: MR
authors.lastname: Silvis
authors.affiliation: Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA.
authors.forename: Judd F
authors.initials: JF
authors.lastname: Hultquist
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Waters
authors.affiliation: Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McGregor
authors.affiliation: Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA.
authors.forename: Nevan J
authors.initials: NJ
authors.lastname: Krogan
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, QBI, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: joseph.bondy-denomy@ucsf.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bondy-Denomy
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors; D001435:Bacteriophages / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D004720:Endonucleases / Q000037:antagonists & inhibitors*; D004926:Escherichia coli; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000276:immunology / Q000821:virology; D039002:Prophages
chemical_list: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR-Cas; Cas9; Cas9 inhibitor; Listeria monocytogenes; anti-CRISPR; bacteriophage; dCas9; gene editing; prophage