Tf0215

Actin was purified from rabbit skeletal muscle as previously described14 (link). Actin (0.1 mg/ml) was incubated for 30 min at room temperature in a solution containing 50 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4), 1 mM MgCl₂ and 1 mM DTT. To increase its affinity for the actin filament, a glass slide (TF0215; Matsunami Glass Ind., Ltd., Osaka, Japan) was coated with polylysine as described below. The glass slide was washed twice with ethanol, a polylysine solution (Mw 30,000–50,000; Sigma-Aldrich, St. Louis, MO, USA; 0.4 mg/ml dissolved in water) was applied, and then the slide was washed three times with the buffer. Polymerized actin was diluted 10 times with buffer containing 1 μM phalloidin and applied to the polylysine-coated glass slide. The AFM measurement was performed as described above.

The plant tissues were transferred into 150 μl of Cas9 reaction buffer placed on microscope slides containing hydrophilic circular windows with a diameter of 15 mm surrounded by a framework of printed hydrophobic black ink (TF0215; Matsunami, Osaka, Japan) and maintained at room temperature for 5 min for blocking. Then, the same volume of Cas9 reaction buffer containing RNP was added to the slide and mixed by pipetting several times. The slides were placed in a moisture chamber and incubated at 26, 37, or 42 °C for 3–5 h or at 4 °C for 16 h. After the incubation, the reaction was stopped by removing the buffer, and the tissues were washed twice in PBS for 10 min at room temperature. To prevent dissociation of the RNP complex, the tissues were post-fixed with 3.0% PFA for 5–10 min at room temperature, and then washed twice in PBS for 5 min at room temperature. The tissues were mounted with an anti-fade mountant, SlowFade Diamond containing 1 μg/ml DAPI (Thermo Fisher Scientific, Waltham, MA, USA). The slides were placed at 4 °C for at least 8 h until the anti-fading solution had penetrated completely into the tissues.

Glass slides containing hydrophilic circular windows with a diameter of 15 mm surrounded by a framework of printed hydrophobic black ink (TF0215; Matsunami Glass Industry, Ltd., Osaka, Japan) were used for both cell culture and the AFM measurements. When culture medium (D-MEM: Sigma-Aldrich Co. St. Louise, MO, USA) was added to this hydrophilic circular window, the medium produced droplet because of the hydrophobicity of the surrounding framework, as depicted in Fig. 1A. Normal rat kidney (NRK) cells were cultured in this swelled medium for 2 days in a CO₂ incubator and then used in the subsequent experiments. To move the cantilever exactly onto the target area in the cells, a thin sapphire disc (3 mm in diameter, 0.05 mm thick; Rudolf Brügger, Swiss Micro Technology, Lugano, Switzerland) with coordinates was glued in the centre of each hydrophilic circle prior to cell culture. The coordinates on the sapphire discs were printed by carbon shadowing after an EM finder grid was placed on the disc.