Characterization and rational engineering of Butyrivibrio crossotus argonaute for improved cleavage activity

Prokaryotic Argonautes (pAgos) are becoming more popular in biotechnology due to their versatility in molecular cloning, nucleic acid detection, and gene editing. However, the increasing demand for pAgos necessitates mining or engineering more robust and efficient Agos with high cleavage activity. In this study, we focused on characterization and engineering a prokaryotic Argonaute nuclease from bacteria Butyrivibrio crossotus (BcAgo) and investigated its features, particularly its ability to process 5 '-phosphorylated guides. Our findings reveal that BcAgo can efficiently employ single-stranded DNA (ssDNA) guides to slice target ssDNA, with an optimal guide interval of 15-20 nucleotides. We also discovered that mismatches within the central and 3 ' supplementary regions of the guide considerably reduce BcAgo's cleavage activity. To further enhance BcAgo activity, we produced a mutant Leu678Glu using in silico docking. The mutant showed 30% and 56% improved cleavage activity at 65 degrees C and 37 degrees C, respectively, compared to the wild type. Molecular modeling studies revealed the Leu678Glu protrusion toward the catalytic pocket, and the orientation of the catalytic residue offers a structural basis for the mutant enhanced activity. These findings provide a foundation for designing pAgos with effective activity and specificity, essential for their use in biotechnological application.