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NgAgo-enhanced homologous recombination in E. coli is mediated by DNA endonuclease activity

By Kok Zhi Lee, Michael A Mechikoff, Archana Kikla, Arren Liu, Paula Pandolfi, Frederick S Gimble, Kevin Solomon

Posted 04 Apr 2019
bioRxiv DOI: 10.1101/597237

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, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), however, has been subject to intense debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with the majority of protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a DNA endonuclease. Structural homology modelling revealed that NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single stranded DNA binding domain (repA). Both repA and the canonical PIWI domain participate in DNA cleavage activities. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results provide insight into the poorly characterized NgAgo for subsequent gene-editing tool development and sheds new light on seemingly contradictory reports.

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