Wiegand, TannerWilkinson, RoyceSantiago-Frangos, AndrewLynes, MackenzieHatzenpichler, RolandWiedenheft, Blake2023-07-312023-07-312023-04Wiegand, T., Wilkinson, R., Santiago-Frangos, A., Lynes, M., Hatzenpichler, R., & Wiedenheft, B. (2023). Functional and Phylogenetic Diversity of Cas10 Proteins. The CRISPR Journal, 6(2), 152-162.2573-1599https://scholarworks.montana.edu/handle/1/18016Final publication is available from Mary Ann Liebert, Inc., publishers https://dx.doi.org/10.1089/crispr.2022.0085Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin–antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.en-UScopyright Mary Ann Liebert Inc 2023https://web.archive.org/web/20200107105118/https://home.liebertpub.com/authors/policies/152Cas10 ProteinsProteinsPhylogenetic DiversityFunctional DiversityArticle