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, some MGEs have evolved Anti-CRISPR (Acr) proteins to bypass this immunity, yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we uncover 11 new type I anti-CRISPR genes encoded on numerous chromosomal and extrachromosomal mobile genetic elements 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 defense systems. These genomic regions appear to be “anti-defense 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.