A programmable pAgo nuclease with high activity and specificity for efficient DNA and RNA manipulation

Prokaryotic Argonaute proteins (pAgos) are programmable nucleases with potential for the development of novel nucleic acid manipulation tools. Previous studies have shown that several pAgos exhibit strong preferences for either DNA or RNA targets, and effective cleavage of plasmid or linear double-stranded DNA (dsDNA) is typically limited to regions with low GC content. Here, we characterize a pAgo, GgeAgo from the thermophilic organism Geobacillus genomosp. 3. GgeAgo utilizes 5' phosphorylated DNA guides to cleave both DNA and RNA with high efficiency and specificity under a wide range of conditions. Notably, GgeAgo efficiently cleaves both plasmid and linear dsDNA with GC content up to 53% at elevated temperatures, and retains detectable activity even at 64% GC. Furthermore, GgeAgo demonstrates robust activity in practical applications, including DNA cloning, nucleic acid detection, and enrichment of rare variants, with high specificity and sensitivity. These results suggest that GgeAgo holds promise as an effective DNA and RNA manipulation tool for biotechnology.