Inhibition of Bacterial Immunity with Anti-CRISPR Proteins

Bacteria use CRISPR-Cas immune systems to defend against viral attack. In response, bacterial viruses have evolved a diverse suite of anti-CRISPR proteins that have previously been shown to inhibit (Type I) CRISPR-Cas3 systems in the bacterium Pseudomonas aeruginosa. To find anti-CRISPR proteins that inhibit (Type II) CRISPR-Cas9 systems, we developed a bioinformatics approach: Because anti-CRISPRs allow a CRIPSR targeted virus to integrate into the bacterial chromosome, we searched for genomes in which a Cas9 associated CRISPR array targeted a mobile element in the same genome, a circumstance we call “self-targeting”. Using this approach, we discovered four unique CRISPR-Cas9-inhibitors encoded in Listeria monocytogenes integrated viruses. Given the success of this approach, we returned to examine CRISPR-Cas3 self-targeting in P. aeruginosa. We were surprised to see that greater than 10% (74 /713) of self-targeting events in P. aeruginosa could not be explained by the presence of known inhibitors, implying the existence of novel anti-CRISPRs. To find these novel inhibitors, we employ bioinformatics coupled with functional tests to uncover and characterize new strategies and mechanisms for CRISPR-Cas inhibition. These discoveries shed light on the interactions between CRISPR and bacterial viruses in the natural world.