Ttago

For TtAgo reactions, 5’-phosphorylated DNA guides were either purchased from Integrated DNA technologies or generated by incubating an unmodified oligonucleotide with T4 Polynucleotide Kinase (NEB) at 37 °C for 30 minutes, followed by heat-activation at 65°C for 20 min. Complexes of TtAgo programmed with ssDNA guides were prepared by combining final concentrations of 1 pmol TtAgo (NEB) and 2 pmol 5’-phosphorylated ssDNA guides and incubating at 70 °C for 20 minutes. Cleavage reactions were performed by combining TtAgo complexes with either 79.85 fmol of linearized KAC833 plasmid substrate (digested with PvuI, NEB) or 79.85 fmol supercoiled plasmid DNA (KAC1151 or MNW95) in ThermoPol buffer (NEB) with a final concentration of 10mM MgSO₄. Reactions were performed at 80 °C for 60 minutes and terminated by the addition of 1 μL of Proteinase K (NEB). Cleavage fragments from pre-linearized substrates were purified using paramagnetic beads and quantified and analyzed as described above. Cleavage fragments from scaled-up plasmid DNA digests were resolved by 0.8% agarose gel electrophoresis and visualized by ethidium bromide staining.