GoKawiilCloning, protein expression and purification
The DNA encoding ThermoCas9 with a C-terminal His 6 tag was integrated into the pML-1B vector and expressed in the Escherichia coli NiCo21(DE3) strain. Cells were grown in Luria–Bertani (LB) medium with 0.2% d-(+)-glucose at 37 °C until optical density at 600 nm reached 0.8, at which point addition, isopropyl-β-d-thiogalactopyranoside was added to 0.5 mM concentration. Cells were grown for an additional 16–18 h at 20 °C and harvested by centrifugation and stored in −80 °C. Previously frozen cells were lysed via sonication in a lysis buffer (500 mM NaCl, 50 mM phosphate buffer pH 8.0 (sodium phosphate dibasic and sodium phosphate monobasic), 5 mM imidazole and 1 mM β-mercaptoethanol) containing 1 tablet of cOmplete Mini Protease Inhibitor Cocktail (Sigma-Aldrich) per 100 ml. The lysate was centrifuged at a speed of 16,000 rpm for 60 min at 4 °C, after which the supernatant was loaded on a pre-equilibrated 5-ml HisTrap HP His tag protein purification column (Cytiva Life Sciences). The resin was subsequently washed with 200 ml wash buffer (500 mM NaCl, 50 mM phosphate buffer pH 8.0, 30 mM imidazole and 1 mM β-mercaptoethanol), before being eluted with elution buffer (500 mM NaCl, 50 mM phosphate buffer pH 8.0, 250 mM imidazole and 1 mM β-mercaptoethanol). The resultant eluate was transferred onto a pre-equilibrated HiTrap Heparin HP affinity column (Cytiva Life Sciences) and eluted with a 100 mM to 2 M NaCl gradient. The purified protein was then concentrated and stored at −80 °C until further use.
For purification of ThermoCas9 used in human gene-editing experiments, the DNA encoding 3×-nuclear localization sequence (2× SV40 NLS and 1× nucleoplasm NLS) fused with ThermoCas9 with a C-terminal His 6 tag was integrated into the pML-1B vector and expressed in E. coli Rosetta (DE3) cells. The same purification method was used with the exception that the gel-filtration buffer was made with cytotoxin-free water.
In vitro RNA transcription
We used the T7 in vitro transcription method to produce the sgRNA for both ThermoCas9 and AceCas9. The sgRNA templates containing a T7 promotor were purchased from Eurofins Genomics. A 149 nt sgRNA for ThermoCas9 and a 106 nt sgRNA for AceCas9 (Supplementary Table 1), respectively, were transcribed by T7 RNA polymerase in a transcription buffer (5 mM NTPs, 50 mM Tris-HCl pH 7.5, 15 mM MgCl 2 , 5 mM dithiothreitol and 2 mM spermidine) and purified via the Monarch RNA Cleanup Kits (New England Biolabs). The DNA used in cryo-EM and biochemical assays was purchased from Eurofins Genomics.
Cryo-EM sample preparation, data collection and 3D reconstruction
The heparin-purified protein was incubated with sgRNA at a 1:1.5 molar ratio at 37 °C for 30 min, and the resulting RNP was further purified via size-exclusion chromatography with a Superdex 200 10/300 GL column (Cytiva Life Sciences) in gel-filtration buffer (300 mM NaCl, 30 mM HEPES pH 7.5 and 1 mM dithiothreitol). The Cas9–RNA–DNA ternary complex was assembled by adding pre-annealed substrate dsDNA into the RNP at a 2:1 molar ratio with the presence of 10 mM magnesium chloride. The reactive ternary complex was incubated at 37–50 °C for 15–30 min. Of the sample, 4 µl was added to glow-discharged Gold 300 mesh R1.2/1.3 grids, which was then allowed to adsorb for 30 s before blotting for 2.5 s under conditions of 20 °C and 100% humidity. These grids were rapidly frozen in liquid nitrogen cooled ethane within Vitrobot Mark IV.
Raw micrographs of ThermoCas9 bound with DNA containing 5′-NNNNCCA-3′ PAM and AceCas9 bound with DNA containing 5′-NNN5mCC-3′ PAM were collected at the Laboratory for Biomolecular Structure of the Brookhaven National Laboratory using a Titan Krios G3i cryo transmission electron microscope equipped with a Gatan K3 direct electron detector. Raw micrographs of ThermoCas9 bound with DNA containing 5′-NNNNCGA-3′ PAM were collected at the Pacific Northwester Center for Cryo-EM using a Titan Krios Electron Microscope equipped with a Gatan K3 direct electron detector (Thermo Fisher Scientific). Movies were recorded at a nominal magnification of 105,000 in a super-resolution mode with an energy filter of 15 eV, corresponding to a corrected physical pixel size of 0.82 Å per pixel. A total dose of 50–60 e− Å−2 was spread over 60 frames with random defocus set to −0.8 to −2.5 µm. Motion correction was executed in bin 2 via MotionCorr2 and contrast transfer function (CTF) estimation was carried out with Gctf61. A total of 6,080 micrographs were collected and 2,516,939 particles were picked using Topaz62, followed by multiple rounds of 2D classification using cryoSPARC63, resulting in 2,015,088 good particles for 3D classification. After heterogenous refinement in cryoSPARC, the dataset was classified into five classes. Several rounds of 3D refinement and 3D classification were then performed using Relion 4.0 (ref. 64) to obtain high-quality particles. Finally, several rounds of non-uniform refinement65 were performed using cryoSPARC to reach the final 3D structures. Structural models were built in COOT66 and refined in PHENIX67 to satisfactory stereochemistry and real-space map correlation parameters. Note that water molecules were only modelled based on both density and interaction chemistry in the two high-resolution structures.
Bacterial survival assay
The survival assay in bacterial cells followed a previously outlined procedure44 with minor modifications. In brief, electrocompetent E. coli BW25141 cells, harbouring the modified p11-LacY-wtx1 plasmid encoding toxic ccdB protein, were transformed with 60 ng of WT or mutant ThermoCas9 plasmids. Afterwards, the cells were recovered in LB for 30 min with shaking at 37 °C. Subsequently, 0.05 mM isopropyl-β-d-thiogalactopyranoside was introduced, and the recovery process continued for an additional 60 min. The recovered cells were then plated on LB agar plates containing either chloramphenicol (15 mg ml−1) or a combination of chloramphenicol and 10 mM arabinose. The plates were incubated at 37 °C for 16–20 h. Manual counting of colonies was performed on both plates, and survival rates were determined by dividing the CFUs on arabinose-containing plates by those on chloramphenicol-only plates. For directed evolution of ThermoCas9, a library of ThermoCas9 linker II variants were transformed into BW25141 cells harbouring a modified p11-LacY-wtx1 plasmid containing a PAM-distal truncated protospacer of 17 nucleotides (17-mer) in the same manner as stated above. CFUs that grew on arabinose in the 17-mer cells were selected for Sanger sequencing.
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