Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Publication date: Available online 29 December 2016
Source:Cell
Author(s): Benjamin J. Rauch, Melanie R. Silvis, Judd F. Hultquist, Christopher S. Waters, Michael J. McGregor, Nevan J. Krogan, Joseph Bondy-Denomy
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.

Graphical abstract

Teaser

Four CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages prevent Cas9 binding and gene editing in bacteria and human cells, including currently the most widely used Cas9 from Streptococcus pyogenes.


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