Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Benjamin J Rauch; Melanie R Silvis; Judd F Hultquist; Christopher S Waters; Michael J McGregor; Nevan J Krogan; Joseph Bondy-Denomy

doi:10.1016/j.cell.2016.12.009

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Cell. 2017 Jan 12;168(1-2):150-158.e10. doi: 10.1016/j.cell.2016.12.009. Epub 2016 Dec 29.

Authors

Benjamin J Rauch¹, Melanie R Silvis², Judd F Hultquist³, Christopher S Waters⁴, Michael J McGregor³, Nevan J Krogan³, Joseph Bondy-Denomy⁵

Affiliations

¹ 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.
² 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.
³ 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.
⁴ 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.
⁵ 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.

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.

Keywords: CRISPR-Cas; Cas9; Cas9 inhibitor; Listeria monocytogenes; anti-CRISPR; bacteriophage; dCas9; gene editing; prophage.

MeSH terms

Bacterial Proteins / antagonists & inhibitors
Bacteriophages / metabolism*
CRISPR-Associated Protein 9
CRISPR-Cas Systems*
Endonucleases / antagonists & inhibitors*
Escherichia coli
Genetic Engineering*
HEK293 Cells
Humans
Listeria monocytogenes / enzymology*
Listeria monocytogenes / immunology
Listeria monocytogenes / virology
Prophages

Substances

Bacterial Proteins
CRISPR-Associated Protein 9
Cas9 endonuclease Streptococcus pyogenes
Endonucleases

Abstract

MeSH terms

Substances

Grants and funding