Fig. 1: GgeAgo is a DNA-guided DNA and RNA endonuclease.

a Schematic diagram of the workflow during in vitro cleavage assay. GgeAgo cleaves a 45 nt substrate with 18 nt guides to generate 34 nt product. b Scheme of the guide and target nucleic acids. 5’-phosphorylated DNA guides were used in most experiments; the black triangle indicates the cleavage site. M1 and M2 correspond to the cleavage products of FAM-labeled DNA and RNA targets. c GgeAgo exhibits DNA-guided DNA endonuclease activity. d GgeAgo exhibits DNA-guided RNA endonuclease activity. e Catalytic dead mutant GgeAgo_DM with substitutions of two out of four catalytic tetrad residues (D502A/D571A) causes the loss of activity. Positions of the cleavage products and target are indicated on the left of the gels. GgeAgo or GgeAgo_DM, guide and target were mixed in a 4:2:1 molar ratio (800 nM GgeAgo preloaded with 400 nM guide in 5 mM Mn²⁺ for 10 min at 50 °C, plus 200 nM target) and incubated for 30 min at 50 °C. Lanes M1 and M2 contain chemically synthesized 34 nt DNA and RNA corresponding to the cleavage products of the DNA and the RNA target, respectively.