Ultraufoil grid

Cryo-EM samples were thawed and diluted to 3 μM (Cas9:sgRNA:DNA) or 1.5 μM (Cas9:sgRNA) in cryo-EM buffer. An UltrAuFoil grid (1.2/1.3-μm, 300 mesh, Electron Microscopy Sciences, catalog no. Q350AR13A) was glow-discharged in a PELCO easiGlow for 15 s at 25 mA, then loaded into an FEI Vitrobot Mark IV equilibrated to 8°C with 100% humidity. From the sample, kept on ice up until use, 3.6 μL was applied to the grid, which was immediately blotted (Cas9:sgRNA:DNA{0 RNA:DNA matches} and Cas9:sgRNA: blot time 4.5 s, blot force 8; Cas9:sgRNA:DNA{3 RNA:DNA matches}: blot time 3 s, blot force 6) and plunged into liquid-nitrogen-cooled ethane. Micrographs for Cas9:sgRNA were collected on a Talos Arctica TEM operated at 200 kV and x36,000 magnification (1.115 Å/pixel), at −0.8 to −2 μm defocus, using the super-resolution camera setting (0.5575 Å/pixel) on a Gatan K3 Direct Electron Detector. Micrographs for Cas9:sgRNA:DNA complexes were collected on a Titan Krios G3i TEM operated at 300 kV with energy filter, x81,000 nominal magnification (1.05 Å/pixel), −0.8 μm to −2 μm defocus, using the super-resolution camera setting (0.525 Å/pixel) in CDS mode on a Gatan K3 Direct Electron Detector. All images were collected using beam shift in SerialEM v.3.8.7 software.