In vitro characterization of a pAgo nuclease Ttd Ago 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 (Ago) was characterized . Our results showed that 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. 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 or Mg 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, Ago performed programmable cleavage of double-stranded DNA at 75°C. We further introduced Ago into an industrial ethanologenic bacterium to evaluate its effect . Our preliminary results indicated that Ago showed cell toxicity toward , resulting in a reduced growth rate and final biomass. In conclusion, we characterized Ago and investigated its effect on in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.