NgAgo DNA endonuclease activity enhances homologous recombination in E. coli

Abstract

15Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not 16 require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One 17 promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), however, has 18 been subject to intense debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme 19 and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with the 20 majority of protein being insoluble and inactive even after refolding. However, we report that the soluble 21 fraction does indeed act as a DNA endonuclease. Structural homology modelling revealed that NgAgo 22shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized 23 single stranded DNA binding domain (repA). Both repA and the canonical PIWI domain participate in DNA 24 cleavage activities. We also found that these endonuclease activities are essential for enhanced NgAgo-25 guided homologous recombination, or gene-editing, in E. coli. Collectively, our results provide insight into 26 the poorly characterized NgAgo for subsequent gene-editing tool development and sheds new light on 27 seemingly contradictory reports.28 29 Long prokaryotic Argonaute proteins (pAgos) are programmable endonucleases that have recently been 31 proposed as flexible tools for genome editing 1 . Like Cas9-based gene editing strategies, single-stranded 32 nucleic acids bind to pAgos and enhance pAgo cleavage of complementary target nucleic sequences, 33 enabling DNA repair and editing. However, pAgos have the distinct advantage of not requiring a 34 protospacer adjacent motif (PAM) for function 2-5 , which means that pAgos are not limited to targets flanked 35 by PAM sites and can potentially cut any DNA target regardless of composition. Despite this potential, no 36 pAgo has been developed that rivals the simplicity and function of Cas9-based strategies. 37 Long pAgos are predicted to serve as a form of adaptive defense against invading nucleic acids such as 38 phage/viral DNA and RNA 6,7 . With a single-stranded DNA and/or RNA as a guide, long pAgos cleave 39 complementary target DNA, RNA, or both via the conserved catalytic tetrad, DEDX 1 . To create a double-40 stranded DNA break, long pAgos require two guides. Target recognition and cleavage is enabled by four 41 canonical domains 3 : N (N-terminal), PAZ (PIWI-Argonaute-Zwille), MID (middle), and PIWI (P element-42 induced wimpy testis). The N-terminal domain is essential in target cleavage 8,9 and dissociation of cleaved 43 strands 9,10 , though the detailed mechanism remains poorly understood. The MID domain interacts with the 44 5'-end of the guide 11 and promotes binding of the guide to its target nucleic acids 12 . The PAZ domain 45 interacts with the 3' end of a guide 13-16 , protecting it from degradation 17 . The PIWI domain plays a pivotal 46 role in nucleic acid cleavage via the conserved catalyti...

Genome manipulation by guide-directed Argonaute cleavage

“…This method may extend to RecA-active strains, if RecA activity can be efficiently inhibited by, for example, expressing RecA inhibitor (36). A recent study also reported NgAgo-assisted recombineering (NgAgo, pAgo from Natronobacterium gregoryi), but the fold change was smaller than 2 and the enhancement of editing was not dependent on NgAgo endonuclease activity (37).…”