Methenamine

Cas9-sgRNA-DNA crystals were grown at 20 °C using the hanging drop vapor diffusion method. The drops were composed of equal volumes (1 μl + 1 μl) of purified complex (diluted to 1.25–2 mg ml⁻¹ in 20 mM HEPES pH 7.5, 250 mM KCl, 5 mM MgCl₂) and reservoir solution (0.1 M Tris-acetate, pH 8.5, 200-400 mM KSCN, 14-18 % PEG 3350). Iterative microseeding was used to improve crystal nucleation, growth and morphology. For cryoprotection, crystals were transferred into 0.1 M Tris-acetate, pH 8.5, 200 mM KSCN, 30 % PEG 3350, 10 % ethylene glycol, and flash-cooled in liquid nitrogen. Diffraction data were measured at beamlines PXI and PXIII of the Swiss Light Source (Paul Scherer Institute, Villigen, Switzerland) and processed using XDS^{30 (link)}. The crystals belonged to space group C2 and contained one complex in the asymmetric unit. Native data extended to a resolution of 2.59 Å. Data collection statistics are summarized in Extended Data Table 1. Phases were obtained from a single-wavelength anomalous diffraction (SAD) experiment using complex crystals containing SeMet-substituted Cas9 and measured at Se K-edge wavelength (0.97965 Å). Three data sets were measured by exposing different parts of the same crystal, rotating the crystal through 360° in each dataset. Selenium sites were located using the Hybrid Substructure Search (HySS) module of the Phenix package^{31 (link)}. Additional phasing information came from a SAD experiment carried out with complex crystals soaked with 5 mM iridium hexamine chloride for 2 h and measured at the Ir L-III edge (1.10501 Å). The native and SAD data sets were combined for substructure refinement and phase calculations using the MIRAS procedure in AutoSHARP^{32 (link)}, yielding readily interpretable electron density maps. Fragments of apo-Cas9 structure^{20 (link)} were docked using MOLREP^{33 (link)}. Model building was completed in COOT^{34 (link)}. The atomic model was refined using Phenix.refine^{35 (link)}. The final model includes Cas9 residues 4–710, 719–764, 776–1012, 1030–1050, 1059–1241, 1253–1364; nucleotides 1–81 of the sgRNA; nucleotides (−8)–20 of the target DNA strand and nucleotides (−3)–8 of the non-target DNA strand. The structures of dCas9–sgRNA–mismatch DNA complexes were solved by molecular replacement using the Phaser module of the Phenix package^{31 (link)} using the atomic structures of Cas9 and sgRNA as separate search models and omitting DNA from the initial search.