Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

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.

Valproic Acid Thermally Destabilizes and Inhibits SpyCas9 Activity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system plays an important role in prokaryotic adaptive immunity. Due to its capacity for sequence-specific gene editing, CRISPR-Cas9 has become one of the most important tools widely used for genome editing in molecular biotechnology. However, its clinical application is currently limited by unwanted mutations at off-target sites. Various strategies have been developed for precise control of Cas9 in order to reduce these off-target effects, including chemical-based approaches. From a chemical screening, I observed that valproic acid (VPA) binds to and destabilizes Streptococcus pyogenes Cas9 (SpyCas9) protein in vitro, as well as in cells, while within its therapeutical concentration range under conditions of hyperthermia as demonstrated. Conditions were generated either by an external heat bag or in combination with the photothermal therapeutic agent indocyanine green activated by a near-infrared laser. Use of other histone deacetylase inhibitors failed, suggesting a histone deacetylase inhibition-independent function of VPA. Thus, this finding provides an uncomplicated thermotherapeutical approach for timely regulation of the activity of the CRISPR-Cas9 system at desired locations.

Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.

CRISPR-Cas systems in bacteria and archaea provide immunity against bacteriophages and plasmids. To overcome CRISPR immunity, phages have acquired anti-CRISPR genes that reduce CRISPR-Cas activity. Using a synthetic genetic circuit, we developed a high-throughput approach to discover anti-CRISPR genes from metagenomic libraries based on their functional activity rather than sequence homology or genetic context. We identified 11 DNA fragments from soil, animal, and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain. Further in vivo and in vitro characterization of a subset of these hits validated the activity of four anti-CRISPRs. Notably, homologs of some of these anti-CRISPRs were detected in seven different phyla, namely Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Spirochaetes, and Balneolaeota, and have high sequence identity suggesting recent horizontal gene transfer. Thus, anti-CRISPRs against type II-A CRISPR-Cas systems are widely distributed across bacterial phyla, suggesting a more complex ecological role than previously appreciated.