Regulatory sequence-based discovery of anti-defense genes in archaeal viruses

<title>Abstract</title> In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method with known Acrs or anti-CRISPR associated proteins (Acas) used as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Acas hinders our ability to identify Acrs using guilt-by-association. Here, based on an observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesized that these genes, and many other unidentified anti-defense genes, are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes (ADG) in archaeal viruses. Using this consensus sequence based method, we identified 354 potential anti-defense related genes in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identified regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.