Multina microchip electrophoresis device

The pUC119 plasmid, containing the 24-nt target sequence and the PAMs, was used as the substrate for in vitro cleavage assays (Supplementary Table 6). The EcoRI-linearized pUC119 plasmid (100 ng, 4.7 nM) was incubated at 37 °C for 0.25–30 min with the CdCas9-sgRNA (50 nM) in 10 μL of reaction buffer, containing 20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl₂, 1 mM dithiothreitol, and 5% glycerol. The reaction was stopped by the addition of quench buffer, containing EDTA (40 mM final concentration) and proteinase K (4 µg). Reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (Shimadzu). For the measurement of the cleavage activity of the CdCas9 D10A mutant, the circular pUC119 target plasmid (500 ng, 4.7 nM) was incubated at 37 °C for 0.5–5 min with the CdCas9-sgRNA (50 nM), in 50 μL of the reaction buffer, and the reaction was then stopped by the addition of the quench buffer. The reaction products were resolved on an ethidium bromide-stained 1% agarose gel, and then visualized using an Amersham Imager 600 (GE Healthcare).

For the in vitro cleavage assay, the TnpB–ωRNA complexes (wild-type or mutants) were purified in a similar manner to that for the complex prepared for the cryo-EM analysis. Protein concentrations were measured using a Bradford Protein Assay Kit (TAKARA). The DNA cleavage activity of TnpB was measured by in vitro DNA cleavage assays. The TnpB–ωRNA complex (2 μl, final concentration 250 nM) was mixed with the 3-kb linearized plasmid target containing the 16-nt target sequence and the TTGAT TAM (8 μl, 100 ng) (Supplementary Table 1), and incubated at 37 or 50 °C for 30 min in 10 μl reaction buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 5% glycerol). The reaction was stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (Shimadzu). In vitro cleavage experiments were performed at least three times.

First, in vitro cleavage experiments were performed, using the purified CjCas9, four sgRNAs with different guide lengths (20–23-nt), and a linearized pUC plasmid containing the target sequence (complementary to the 20–23-nt guides) with the T₃AACAC PAM (Supplementary Table 1). Next, the cleavage activities CjCas9 were measured, using the optimal 22-nt guide sgRNA and linearized pUC plasmids with 16 different PAMs. The linearized plasmid DNA (100 ng, 4.7 nM) was incubated at 37 °C for 0.5–5 min with the CjCas9–sgRNA complex (50 nM) in 10 μL of reaction buffer, containing 20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 5% glycerol. The reaction was stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (SHIMADZU).