In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo

In spite of the development of genome-editing tools using CRISPR–Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens ( Ttd Ago) was characterized in vitro . Our results showed that Ttd Ago has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. Ttd Ago can utilize 5′-phosphorylated (5′-P) or 5′- hydroxylated (5′-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn 2+ or Mg 2+ and displayed no obvious preference for the 5′-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, Ttd Ago performed programmable cleavage of double-stranded DNA at 75°C. We further introduced Ttd Ago into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo . Our preliminary results indicated that Ttd Ago showed cell toxicity toward Z. mobilis , resulting in a reduced growth rate and final biomass. In conclusion, we characterized Ttd Ago in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.