Discovery of multiple anti-CRISPRs uncovers anti-defense gene clustering in mobile genetic elements

Many prokaryotes employ CRISPR-Cas systems to combat invading mobile genetic elements (MGEs). In response, MGEs have evolved Anti-CRISPR (Acr) proteins to bypass this immunity. Here, we uncover 11 new type I anti-CRISPR genes encoded on numerous mobile genetic elements mainly within Enterobacteriaceae and Pseudomonas. Candidate genes were identified adjacent to anti-CRISPR associated gene 5 (aca5) and assayed against a panel of six type I systems: I-F (Pseudomonas, Pectobacterium, and Serratia), I-E (Pseudomonas and Serratia), and I-C (Pseudomonas), revealing the type I-F and/or I-E acr genes and a new aca (aca9). We find that acr genes not only associate with other acr genes, but also with inhibitors of distinct bacterial systems. These genomic regions appear to be islands, reminiscent of the clustered arrangement of defense islands in prokaryotic genomes. Our findings expand on the diversity of CRISPR-Cas inhibitors and reveal the potential exploitation of acr loci neighborhoods for identifying new anti-defense systems.