Metagenomic mining reveals novel Cas12 subtypes and their evolutionary diversification.

We identified 4112 Cas12 Proteins and 6 new Cas12 subtypes, revealed their significant diversity in N-terminal regions, repeat sequences, and sequences motifs. We developed an AI-driven algorithm, Cas12fam, which allows precise annotation of 18 distinct domains in Cas12 proteins. Through detailed structural analysis, we identified two novel glycine residues in the RuvC-like nuclease domains (RuvC-I, RuvC-II), which were experimentally validated to play a critical role in the DNA-cleavage activity of Cas12. Our analyses revealed that Cas12 proteins are enriched in specific environments, with significant variation in their abundance across ecological niches.