{Reference Type}: Journal Article {Title}: Regulatory sequence-based discovery of anti-defense genes in archaeal viruses. {Author}: Bhoobalan-Chitty Y;Xu S;Martinez-Alvarez L;Karamycheva S;Makarova KS;Koonin EV;Peng X; {Journal}: Nat Commun {Volume}: 15 {Issue}: 1 {Year}: 2024 May 2 {Factor}: 17.694 {DOI}: 10.1038/s41467-024-48074-x {Abstract}: In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs 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 identify 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.