The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. colifabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.

CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34-base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing. Here, we report a novel two-step precise gene-editing method by leveraging the SSA-mediated repair mechanism into the CRISPR/Cas9-mediated gene-editing system. An integrating cassette was developed with positive and negative selection markers, which was flanked by direct repeat sequences with desired mutations as SSA arms. After the targeted integration of the cassette mediated by CRISPR/Cas9-induced homologous-directed repair, cell clones were first selected through the positive selection. In the second round targeting, the selection cassette was removed by the SSA-mediated DNA double-strand break (DSB) repair without any scar left behind. The novel seamless genome editing technique was tested on CCR5 and APP loci, and finally demonstrated, respectively, up to 45.83% and 68% of precise genome editing efficiency. This study provides a new efficient approach for precise genome editing and gene correction.

We revealed the mechanism that explains how the NgAgo eliminates the invading foreign DNA and bacteriophage in bacterial cells at 37°C, and by leveraging this discovery, NgAgo can be programmed to target a plasmid or a chromosome locus.