Identification of New Anti-CRISPERS Uncovers Anti-Defense Gene Clustering

Abstract

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 describe the analysis of gene neighbors of the anti-CRISPR associated gene 5 (aca5) in search of new Acrs on chromosomal and extrachromosomal MGEs. Functional testing of candidates was performed against a panel of six type I systems, revealing 11 type I-F and/or I-E Acrs 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.