Prokaryotic Argonautes (pAgos) have been proposed as more flexible gene-editing tools as they do not require a sequence motif adjacent to their targets for activity, unlike popular CRISPR/Cas systems. One promising candidate from the halophilic archaeon Natronobacterium gregoryi (NgAgo), however, has been subject to intense debate regarding its endonuclease activity and gene-editing potential in eukaryotes. Here, we develop methods and characterize NgAgo in vitro and in vivo, which revealed several unrecognized features that may explain the inconsistent results in the literature. First, NgAgo expresses poorly in non-halophilic hosts with the majority of protein being insoluble and inactive even after refolding, similar to other halophilic proteins. While our data suggest that soluble NgAgo does indeed act as a DNA endonuclease, it does not display significant levels of targeted activity in vitro. Second, structure homology modeling revealed that NgAgo not only shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Phylogenetic analysis showed that this repA domain is a hallmark of halophilic pAgos, which form a unique clade that is distinct from other characterized pAgos. We showed that both repA and conserved PIWI domains are required for DNA cleavage in vitro and in vivo. In vivo, these domains can be programmed with guides to induce targeted double-stranded DNA breaks that reduce survival ~51% when targeted to loci. In the absence of such loci, survival remains unaffected relative to unguided controls. Similarly, this targeted cleavage enhances homologous recombination, or gene-editing, in E. coli by 107%. In summary, our results provide new insight into poorly characterized NgAgo for subsequent gene-editing tool development and shed new light on seemingly contradictory reports. Meanwhile, the methods we develop can be applied to other endonucleases, providing new tools for screening potential programmable endonucleases.

Characterizing the DNA-cleaving and gene-editing abilities of NgAgo and other programmable endonucleases

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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