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

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

Author information

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

PMID:
28041849
PMCID:
PMC5235966
DOI:
10.1016/j.cell.2016.12.009
[Indexed for MEDLINE]
Free PMC Article

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