<div> <div> <div> <p>Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene- editing as they do not require sequence motifs adjacent to their targets for function. One promising pAgo candidate from the halophilic archaeon Natronobacterium gregoryi (NgAgo) has been the subject of intense debate regarding its potential in eukaryotic systems. NgAgo was initially claimed to edit genes in mammalian cells, but the report was retracted due to replication failure. Due to low solubility, subsequent studies refolded NgAgo and suggested that it cuts RNA but not DNA; however, mutation of the conserved active site does not abolish cleavage activity, raising the possibility of nuclease contamination. Another independent study demonstrated gene-editing via NgAgo in bacteria. These inconsistent results underscore the knowledge gap and roadblock for NgAgo-based gene-editing tool development. <br><br> </p><div> <div> <div> <p>In this work, I revisit this enzyme and characterize its function in vitro and in a bacterial system. The halophilic features of NgAgo have been neglected in the literature, leading to inconclusive results. Like other halophilic proteins, NgAgo has modified amino acid composition, leading to failure of domain identification/function prediction via sequence alignment. Indeed, using more sensitive structural alignments, I identified a new single-stranded DNA binding domain, repA, in NgAgo and other halophilic pAgos. Due to its halophilic nature, NgAgo expresses poorly in low-salt environments, with the majority of protein being insoluble and inactive even after refolding. However, soluble NgAgo indeed cuts DNA. NgAgo DNA-cleaving activity can only be abolished via mutation in the canonical PIWI domain and repA deletion, revealing a new catalytic behavior in pAgos. Moreover, NgAgo requires both repA and PIWI domains to create double- stranded DNA breaks, leading to cell death or enhancing homologous recombination, or gene- editing, at a modest level in bacteria. Rational protein engineering of NgAgo was also pursued to increase solubility. Although three out of seven mutants showed significant increases in solubility, they lost the ability to cleave DNA in E.coli. Structural modeling revealed some subtle but important differences in the protein structures, explaining why the mutants lose their function. Besides, a selection system for improving endonuclease activity was optimized for future pAgo optimization. Collectively, this work revealed that NgAgo possesses unique catalytic behavior in the pAgo family and has some gene-editing application potential. More importantly, this work expands knowledge of the pAgo family, providing a foundation for future pAgo-based gene- editing tool development. </p> </div> </div> </div> </div> </div> </div>