Diverse viral cas genes antagonize CRISPR immunity

Prokaryotic CRISPR-Cas immunity is subverted by anti-CRISPRs (Acrs), which inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids¹. Acrs often bind to specific cognate Cas proteins, and hence inhibition is typically limited to a single CRISPR-Cas subtype². Furthermore, although acr genes are frequently organized together in phage-associated gene clusters³, how such inhibitors initially evolve has remained unclear. Here we investigated the Acr content and inhibition specificity of diverse Listeria isolates, which naturally harbour four CRISPR-Cas systems (types I-B, II-A, II-C and VI-A). We observed widespread antagonism of CRISPR, which we traced to 11 previously unknown and 4 known acr gene families encoded by endogenous mobile elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins, one of which assembles into a defective interference complex. Surprisingly, an additional type I-B Cas homologue did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system by means of CRISPR RNA (crRNA) degradation. By probing viral sequence databases, we detected abundant orphan cas genes located within putative anti-defence gene clusters. Among them, we verified the activity of a particularly broad-spectrum cas3 homologue that inhibits type I-B, II-A and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution by cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.