Characterization of Argonaute Nuclease from Mesophilic Bacterium Chroococcidiopsis

Mesophilic microbial sources of prokaryotic Argonaute (pAgo) programmable nucleases have garnered considerable attention for their potential applications in genome editing and molecular diagnostics. In this study, we characterized a novel pAgo from the mesophilic bacterium <i>Chroococcidiopsis</i> sp. (ChAgo), which can cleave single-stranded DNA (ssDNA) using both 5'-phosphorylated guide DNA (5'P-gDNA) and 5'-hydroxylated guide DNA (5'OH-gDNA). Efficient cleavage occurs using 14-25 nt 5'P-gDNA and 13-20 nt 5'OH-gDNA in the presence of Mn²⁺ ions at temperatures ranging from 25 to 75 °C, with optimal activity at 55 °C. ChAgo demonstrates low tolerance for single-base mismatches, similar to other pAgo proteins. The cleavage efficiency varies based on the guide/target pair, with mismatches at specific positions significantly reducing activity. For instance, mismatches at positions 4, 5, or 12 in T-gDNA/target pairs and at positions 5 or 8-10 in g38NT-gDNA/target pairs notably decrease efficiency. ChAgo's sensitivity to mismatches makes it a promising tool for nucleic acid manipulation and detection, requiring initial screening for high cleavage efficiency sites and subsequent identification of mismatch positions.