Full details of experimental procedures are provided in the supplementary materials . SpyCas9 and its point mutants were expressed in Escherichia coli Rosetta 2 strain and purified essentially as described (8 (link)). SpyCas9 crystals were grown using the hanging drop vapor diffusion method from 0.1 M tris-Cl (pH 8.5), 0.2 to 0.3 M Li2SO4, and 14 to 15% (w/v) PEG 3350 (polyethylene glycol, molecular weight 3350) at 20°C. Diffraction data were measured at beamlines 8.2.1 and 8.2.2 of the Advanced Light Source (Lawrence Berkeley National Laboratory), and at beamlines PXI and PXIII of the Swiss Light Source (Paul Scherrer Institut) and processed using XDS (50 (link)). Phasing was performed with crystals of selenomethionine (SeMet)–substituted SpyCas9 and native Cas9 crystals soaked individually with 10 mM Na2WO4, 10 mM CoCl2, 1 mM thimerosal, and 1 mM Er(III) acetate. Phases were calculated using autoSHARP (51 (link)) and improved by density modification using Resolve (52 (link)). The atomic model was built in Coot (53 (link)) and refined using phenix.refine (54 (link)).
A. naeslundii Cas9 (AnaCas9) was expressed in E. coli Rosetta 2 (DE3) as a fusion protein containing an N-terminal His10 tag followed by MBP and a TEV (tobacco etch virus) protease cleavage site. The protein was purified by Ni-NTA (nickel–nitrilotriacetic acid) and heparin affinity chromatography, followed by a gel filtration step. Crystals of native and SeMet-substituted AnaCas9 were grown from 10% (w/v) PEG 8000, 0.25 M calcium acetate, 50 mM magnesium acetate, and 5 mM spermidine. Native and SeMet single-wavelength anomalous diffraction (SAD) data sets were collected at beamline 8.3.1 of the Advanced Light Source, processed using Mosflm (55 (link)), and scaled in Scala (56 (link)). Phases were calculated in Solve/Resolve (52 (link)), and the atomic model was built in Coot and refined in Refmac (57 (link)) and phenix.refine (54 (link)).
For biochemical assays, crRNAs were synthesized by Integrated DNA Technologies, and tracrRNA was prepared by in vitro transcription as described (8 (link)). The sequences of RNA and DNA reagents used in this study are listed intable S2 . Cleavage reactions were performed at room temperature in reaction buffer [20 mM tris-Cl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM dithiothreitol] using 1 nM radio-labeled dsDNA substrates and 1 nM or 10 nM Cas9:crRNA:tracrRNA. Cleavage products were resolved by 10% denaturing (7 M urea) PAGE and visualized by phosphorimaging. Cross-linked peptide-DNA heteroconjugates were obtained by incubating 200 pmol of catalytically inactive (D10A/H840A) Cas9 with crRNA:tracrRNA guide and 10-fold molar excess of BrdU containing dsDNA substrate for 30 min at room temperature, followed by irradiation with UV light (308 nm) for 30 min. S1 nuclease and phosphatase–treated tryptic digests were analyzed using a Dionex UltiMate3000 RSLCnano liquid chromatograph connected in-line with an LTQ Orbitrap XL mass spectrometer equipped with a nanoelectrospray ionization source (Thermo Fisher Scientific).
For negative-stain EM, apo-SpyCas9, SpyCas9: RNA, and SpyCas9:RNA:DNA complexes were reconstituted in reaction buffer, diluted to a concentration of ~25 to 60 nM, applied to glow-discharged 400-mesh continuous carbon grids, and stained with 2% (w/v) uranyl acetate solution. Data were acquired using a Tecnai F20 Twin transmission electron microscope operated at 120 keV at a nominal magnification of either ×80,000 (1.45 Å at the specimen level) or ×100,000 (1.08 Å at the specimen level) using low-dose exposures (~20 e− Å−2) with a randomly set defocus ranging from −0.5 to −1.3 μm. A total of 300 to 400 images of each Cas9 sample were automatically recorded on a Gatan 4k × 4k CCD (charge-coupled device) camera using the MSI-Raster application within the automated macromolecular microscopy software Leginon (58 (link)). Particles were preprocessed in Appion (45 (link)) before projection matching refinement with libraries from EMAN2 and SPARX (59 (link), 60 (link)) using RCT reconstructions (34 (link)) as initial models.
A. naeslundii Cas9 (AnaCas9) was expressed in E. coli Rosetta 2 (DE3) as a fusion protein containing an N-terminal His10 tag followed by MBP and a TEV (tobacco etch virus) protease cleavage site. The protein was purified by Ni-NTA (nickel–nitrilotriacetic acid) and heparin affinity chromatography, followed by a gel filtration step. Crystals of native and SeMet-substituted AnaCas9 were grown from 10% (w/v) PEG 8000, 0.25 M calcium acetate, 50 mM magnesium acetate, and 5 mM spermidine. Native and SeMet single-wavelength anomalous diffraction (SAD) data sets were collected at beamline 8.3.1 of the Advanced Light Source, processed using Mosflm (55 (link)), and scaled in Scala (56 (link)). Phases were calculated in Solve/Resolve (52 (link)), and the atomic model was built in Coot and refined in Refmac (57 (link)) and phenix.refine (54 (link)).
For biochemical assays, crRNAs were synthesized by Integrated DNA Technologies, and tracrRNA was prepared by in vitro transcription as described (8 (link)). The sequences of RNA and DNA reagents used in this study are listed in
For negative-stain EM, apo-SpyCas9, SpyCas9: RNA, and SpyCas9:RNA:DNA complexes were reconstituted in reaction buffer, diluted to a concentration of ~25 to 60 nM, applied to glow-discharged 400-mesh continuous carbon grids, and stained with 2% (w/v) uranyl acetate solution. Data were acquired using a Tecnai F20 Twin transmission electron microscope operated at 120 keV at a nominal magnification of either ×80,000 (1.45 Å at the specimen level) or ×100,000 (1.08 Å at the specimen level) using low-dose exposures (~20 e− Å−2) with a randomly set defocus ranging from −0.5 to −1.3 μm. A total of 300 to 400 images of each Cas9 sample were automatically recorded on a Gatan 4k × 4k CCD (charge-coupled device) camera using the MSI-Raster application within the automated macromolecular microscopy software Leginon (58 (link)). Particles were preprocessed in Appion (45 (link)) before projection matching refinement with libraries from EMAN2 and SPARX (59 (link), 60 (link)) using RCT reconstructions (34 (link)) as initial models.
