Fig. 6: Plasmid DNA cleavage and site-specific insertion of DNA fragments using GgeAgo.

a Schematic overview of site-specific insertion of DNA fragments into target plasmid using GgeAgo. First, the plasmid was digested by GgeAgo with a pair designed gDNAs at the desired location. Second, two homologous regions of 20 bp compatible with the ends of the digested plasmid were introduced into the destination DNA (the insert) via PCR. The PCR products were then mixed with the digested plasmid and treated with T5 exonuclease, following transformation into E. coli as our previously established TLCL method⁴⁴. b The representative transformation plates showing cloning results with 10-fold dilution. GFP expression cassette as the insert fragment cloned into the different site of the pUC19 plasmid, digested by the GgeAgo-gDNA complex. The positive clones are green. c The cloning efficiency of the transformation. Colonies are defined as the total colony-forming unit (CFU). Cloning efficiency is the percentage of the number of positive clones (green) to the total number of clones. Data are the means ± SD from two experimental replicates.