Prokaryotic Argonautes (pAgos) have recently been shown to cleave single and double-stranded nucleic acids, and have been proposed as alternative gene-editing tools that do not require a protospacer adjacent motif sequence. However, all currently characterized pAgos are thermophilic, making them infeasible for use in common mesophilic model organisms. Here, we characterize a mesophilic pAgo and demonstrate its potential for gene editing. Computational structure modeling revealed that our pAgo candidate consists of canonical N-terminal, PAZ, MID, and PIWI domains found in other catalytically active pAgos, such as the programmable restriction enzyme PfAgo. However, our mesophilic protein also contains a putative single-stranded DNA binding or repA domain. Phylogenetic analysis of pAgos showed that this repA domain is characteristic of a new class of mesophilic pAgos currently unrecognized in the literature. Our pAgo candidate nicks and cuts plasmid DNA non-specifically in the absence of guide in in vitro assays, presumably due to the presence of single-stranded DNA that co-purifies with the protein. Truncation and substitution mutants suggest that this cleavage may be independently attributed to the repA and PIWI domains. In vivo, we are able to program our pAgo candidate with 5’ phosphorylated DNA guides to cleave both plasmid and chromosomal targets to reduce survival under selective conditions in E.coli. This effect is observed regardless of target gene function suggesting that lethality is due to unrepaired double-stranded DNA breaks. In the presence of homologous donor DNA and pAgo, homologous recombination is increased by up to 50% at targeted gene loci, suggesting that gene-editing applications are feasible. Collectively, this study demonstrates that mesophilic pAgos may have potential applications for gene editing, and suggests the role of a hitherto unrecognized domain within this class of enzymes.

Characterization of a mesophilic prokaryotic Argonaute for gene-editing

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.