Gene editing tools harness prokaryotic defense mechanisms, such as CRISPR and Argonaute, which protect the host from invading nucleic acids. Unlike CRISPR, prokaryotic Argonautes (pAgos) have recently been demonstrated to cleave single and double-stranded nucleic acids without regard for an adjacent sequence motif at gene loci via both targeted guide-dependent and non-specific guide-independent mechanisms. However, little is known about the protein domains that mediate the transition between guide-dependent and guide-independent mechanisms, or how they may be altered to control cleavage specificity. Thus, we heterologously expressed a mesophilic pAgo and engineered mutants in E. coli as a model to derive mechanistic insight. To better understand the mechanism of our model pAgo, we created an in vivo survival assay that targets either essential or non-essential genes to determine whether the pAgo interacts with DNA or RNA. Results from the survival assay reveal that targeted pAgo guide-dependent activity reduces survival via a DNA interaction. Further experiments suggest this may involve DNA cleavage as in vitro incubation of purified pAgo with plasmid DNA cleaves the DNA in the absence of a targeting guide. We also observe similar guide-independent cleavage activity in vivo; strains expressing pAgo without guide display reduced plasmid yield that is highly mutated. Based on our protein structure modeling, the model pAgo has a well-conserved catalytic tetrad, which is required for nucleic acid cleavage. Interestingly, we found that the pAgo also possesses an additional uncharacterized N-terminal domain, which is absent in all well-characterized pAgos. Deletion of this domain abolishes all cleavage activity in vivo suggesting a critical role in pAgo function. Similar point mutations of conserved residues also reduced pAgo activity. Collectively, our work provides insight into the structure and function of our model pAgo, while providing a roadmap for its future exploitation as a novel tool for gene editing and genome engineering.

DNA cleavage via prokaryote argonaute mediated by an uncharacterized N-terminal domain

255th National Meeting (2018)

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