Functional Metagenomic Identification of Anti-CRISPR Proteins

CRISPR-technologies represent effective biotechnological tools that are broadly impacting life sciences. Yet, the control over CRISPR-technologies remains poorly understood. Inhibitors of CRISPR-Cas systems offer opportunities to modulate its activity, yet only few such systems are available. 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 DNA fragments from soil, animal and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain. Further characterization of a subset of these hits validate the activity of novel anti-CRISPRs. Notably, homologues of these anti-CRISPRs found in diverse hosts from seven different phyla have high sequence identity indicating recent horizontal gene transfer. Collectively our data demonstrates that anti-CRISPRs against type II-A CRISPR-Cas systems are widely distributed across bacterial phyla suggestive of a more complex ecological role than previously appreciated.