High-speed AFM Imaging of CRISPR-Cas9 Complexes

The laboratory-built high-speed AFM was used in the tapping mode^{31 (link)}. The cantilever deflection was detected with an optical beam deflection detector, on which a 0.7 mW, 780 nm infrared laser was mounted. The infrared laser beam was focused onto the back side of the cantilever (Olympus: BL-AC7DS-KU4) through a ×60 objective lens (Nikon: CFI S Plan Fluor ELWD 60×). The reflected laser from the cantilever was detected with a two-segmented PIN photodiode. The spring constant of the cantilever was ~100 pN nm⁻¹. The resonant frequency and the quality factor of the cantilever in liquid were ~800 kHz and ~2, respectively. An amorphous carbon tip was fabricated on the original AFM tip by electron beam deposition (EBD). The length of the additional AFM tip was ~500 nm, and the radius of the apex of the tip was ~4 nm. The free oscillation amplitude of the cantilever was ~1 nm and the set-point amplitude was set to 90% of the free amplitude. For HS-AFM observations of Cas9, a mica surface was treated for 3 min with 0.011% (3-aminopropyl)triethoxysilane (APTES) (Sigma-Aldrich). The complex of Cas9, RNA and DNA was pre-assembled (Cas9:RNA:DNA = 1:1:1 mole ratio) in AFM-imaging buffer. HS-AFM observations of apo-Cas9 and Cas9–RNA were performed in buffer, consisting of 20 mM Tris-HCl, pH 8.0, 100 mM KCl and 0.01 mM EDTA. HS-AFM observations of the Cas9–RNA–DNA and GFP-dCas9–RNA–DNA complexes were performed in buffer, consisting of 20 mM Tris-HCl, pH 8.0, 30 mM KCl and 0.01 mM EDTA. All HS-AFM experiments were performed at room temperature.