An M-trap optical tweezer microscope from LUMICKS (Amsterdam, Netherlands) was used to perform the single-molecule optical tweezer assays. The sample chamber preparation was similar to that previously described (35). Briefly, glass coverslips were cleaned and functionalized with partially biotinylated polyethylene glycol (Laysan Bio) as described previously (37), followed by coating with streptavidin (Thermo Fisher Scientific). DNA tethers were then formed by incubation with biotin-tagged DNA. Antidigoxigenin-coated 0.48-mm polystyrene microspheres (Polysciences) were then added to the chamber. Cas9 and sgRNA were preassembled by mixing them at a ratio of 1:2.5 in unzipping buffer [50 mM tris-HCl (pH 7.9), 100 mM NaCl, and 10 mM MgCl2]. This solution was then diluted to obtain the final experimental concentration of 30 nM. The resulting solution was added to the chamber just prior to data acquisition.

The experiments were conducted in a climate-controlled room at a temperature of 23.3°C; however, owing to the local laser trap heating, the temperature increased slightly to 25° ± 1°C. Each experiment was conducted by mechanically unzipping the dsDNA at a slow velocity of 50 nm/s to probe the potential interactions at the fork.

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