END nucleases are antiphage defence systems targeting multiple phages with modified genomes.

Prokaryotes carry phage defence systems in 'defence islands' as immunity mechanisms; however, which systems are endogenously active against specific phages is often not known. Here we identify a single gene in a defence island that is necessary for a Pseudomonas aeruginosa cystic fibrosis isolate to block phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defence system, here named END nucleases, has a Type IIS restriction endonuclease-like domain fused to a catalytically inactive endonuclease III (iEndoIII), which normally recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. END nucleases exclusively antagonize the replication of modified phages, but they are agnostic to the modification, targeting up to eight distinct families of phages with modified DNA, with at least ten different known modifications beyond methylation. A small region of the iEndoIII domain is identified that dictates targeting specificity. Modified phages from the Pbunavirus and Wroclawvirus family (Pa5Oct-like) of jumbo phages encode direct-binding END nuclease inhibitors. This work identifies a broad modification-dependent restriction endonuclease and cognate inhibitors that can fortify phage outcomes.