No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Protein purification and dye labelling

S. pyogenes Cas9 and truncation derivatives were cloned into a custom pET-based expression vector containing an N-terminal His 6 -tag, maltose-binding protein (MBP) and TEV protease cleavage site. Point mutations were introduced by Gibson assembly or around-the-horn PCR and verified by DNA sequencing. Proteins were purified as described23, with the following modifications: after Ni-NTA affinity purification and overnight TEV cleavage at 4 °C, proteins were purified over an MBPTrap HP column connected to a HiTrap Heparin HP column for cation exchange chromatography. The final gel filtration step (Superdex 200) was carried out in elution buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5% (v/v) glycerol and 1 mM TCEP. For FRET experiments, Cy3/Cy5-dye positions were selected within a cysteine-free Cas9 protein on the basis of a structural alignment of the sgRNA-bound (4ZT0) to dsDNA-bound (5F9R) structures. Each FRET pair consisted of one cysteine substitution within the ‘mobile’ domain (HNH, REC2 or REC3) and another within the relatively ‘stationary’ domain (REC1, arginine-rich helix or RuvC), such that the inter-residue distance change from the sgRNA-bound to dsDNA-bound states was between 10 and 90 Å (Extended Data Fig. 10). Dye-labelled Cas9 samples were subsequently prepared as described12. A list of all protein variants and truncations is in Supplementary Table 2.

Nucleic acid preparation

sgRNA templates were PCR amplified from a pUC19 vector containing a T7 promoter, 20 nucleotide target sequence and optimized sgRNA scaffold. The amplified PCR product was extracted with phenol:chloroform:isoamyl alcohol and served as the DNA template for sgRNA transcription reactions, which were performed as described24. DNA oligonucleotides and 5′ end biotinylated DNAs (Supplementary Table 3) were synthesized commercially (Integrated DNA Technologies), and DNA duplexes were prepared and purified by native PAGE as described23.

DNA cleavage and binding assays

DNA duplex substrates were 5′-32P-radiolabelled on both strands. For cleavage experiments, Cas9 and sgRNA were pre-incubated at room temperature for at least 10 min in 1× binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 5% glycerol, 50 μg ml−1 heparin) before initiating the cleavage reaction by addition of DNA duplexes. For REC3 in vitro complementation experiments, 100 nM SpCas9∆REC3–sgRNA complexes were pre-incubated with tenfold molar excess of REC3 for at least 10 min at room temperature before addition of 1 nM radiolabelled substrate. For REC3 titration experiments, 100 nM SpCas9∆REC3-sgRNA complexes were separately pre-incubated with 0, 10, 20, 50, 100, 200, 500, and 1,000 nM REC3 for at least 10 min at room temperature before addition of 1 nM radiolabelled substrate; reactions were quenched after 10 min with an equal volume of buffer containing 50 mM EDTA, 0.02% bromophenol blue, and 90% (vol/vol) formamide. DNA cleavage experiments were performed and analysed as previously described12.

DNA binding assays were conducted using increasing concentrations of SpCas9 variant–sgRNA complexes (0.1, 1, 3, 10, 30, 100, 300, and 1,000 nM) in 1× binding buffer without MgCl 2 + 1 mM EDTA, and reactions were incubated with <0.1 nM radiolabelled duplex substrate at room temperature for 2 h. DNA-bound complexes were resolved on 8% native PAGE (0.5× TBE + 1 mM EDTA, without MgCl 2 ) at 4 °C, as previously described10. Experiments were replicated at least three times, and presented gels are representative results.

Bulk FRET experiments

All bulk FRET assays were performed at room temperature in 1× binding buffer, containing 50 nM SpCas9 HNH (C80S/S355C/C574S/S867C labelled with Cy3/Cy5), SpCas9∆REC3 HNH (M1–N497,GGS,V713–D1368 + C80S/S355C/C574S/S867C) or SpCas9 REC2 (E60C/C80S/D273C/C574S labelled with Cy3/Cy5) with 200 nM sgRNA and DNA substrate where indicated. Fluorescence measurements were collected and analysed as described12. For REC3 in vitro complementation FRET experiments, SpCas9∆REC3 HNH and sgRNA were pre-incubated with tenfold molar excess of REC3 for at least 10 min at room temperature before measuring bulk fluorescence.

Sample preparation for smFRET assay

MicroSurfaces supplied 99% PEG and 1% biotinylated-PEG coated quartz slides. Sample preparation was performed as previously described10. Briefly, the glass surface was pre-blocked with casein (10 mg ml−1) for 10 min. The sample chamber was washed with 1× binding buffer, then incubated with 20 μl streptavidin (1 mg ml−1) for 10 min. Unbound streptavidin was washed away with 40 μl of 1× binding buffer.

To immobilize SpCas9 on its DNA substrate, 2.5 nM biotinylated DNA substrate was introduced and incubated in the sample chamber for 5 min. Excess DNA was washed with 1× binding buffer. SpCas9–sgRNA complexes were prepared by mixing 50 nM Cas9 and 50 nM sgRNA in 1× binding buffer and incubated for 10 min at room temperature. SpCas9–sgRNA was diluted to 100 pM, introduced to sample chamber and incubated for 10 min. Before data acquisition, 20 μl imaging buffer (1 mg ml−1 glucose oxidase, 0.04 mg ml−1 catalase, 0.8% dextrose (w/v) and 2 mM Trolox in 1× binding buffer) was flown into chamber. The REC3 in vitro complementation assay was performed similarly to steady-state FRET experiments: 2.5 nM biotinylated DNA substrate (on-target) was immobilized on the surface, and excess DNA was washed with 1× binding buffer. SpCas9–sgRNA complexes were prepared by mixing 50 nM SpCas9ΔREC3 and 50 nM sgRNA in 1× binding buffer and incubated for 10 min at room temperature. SpCas9–sgRNA was diluted to 100 pM, introduced to the sample chamber and incubated for 10 min. Before data acquisition, 20 μl imaging buffer was flowed into the chamber. After data acquisition, the sample chamber was washed with 1× binding buffer. Imaging buffer (20 μl) supplemented with 1 μM REC3 was flowed into the sample chamber and incubated for 10 min. After incubation, data for REC3 complementation were collected.

Microscopy and data analysis

A prism-type TIRF microscope was set up using a Nikon Ti-E Eclipse inverted fluorescent microscope equipped with a 60×, 1.20 numerical aperture Plan Apo water objective and the Perfect Focus System (Nikon). A 532-nm solid state laser (Coherent Compass) and a 633-nm HeNe laser (JDSU) were used for Cy3 and Cy5 excitation, respectively. Cy3 and Cy5 fluorescence was split into two channels using an Optosplit II image splitter (Cairn Instruments) and imaged separately on the same electron-multiplied charged-coupled device camera (512 pixels × 512 pixels, Andor Ixon EM+). Effective pixel size of the camera was set to 267 nm after magnification. Movies for steady-state FRET measurements were acquired at 10 Hz under 0.3 kW cm−2 532-nm excitation. Steady-state and dynamic FRET data analysis was performed as described previously10. Briefly, for steady-state FRET analysis, two fluorescent channels were registered with each other using fiducial markers (20 nm diameter Nile Red Beads, Life Technologies) to determine the Cy3/Cy5 FRET pairs. Cy3/Cy5 pairs that photobleached in one step and showed anti-correlated signal changes were used to build histograms. FRET values were corrected for donor leakage and the histograms were normalized to determine the percentage of distinct FRET populations. Only samples showing greater than 3% of molecules with active transitions were subjected to dynamic FRET analysis.

Human cell culture and transfection

Descriptions of nuclease and guide RNA plasmids used for human cell culture are available in Supplementary Tables 2 and 3. Nuclease variants were generated by isothermal assembly into JDS246 (Addgene 43861)5, and guide RNAs were cloned into BsmBI-digested BPK1520 (Addgene 65777)25. Both U2OS cells (a gift from T. Cathomen, Freiburg) and U2OS–eGFP cells (encoding a single integrated copy of a pCMV–eGFP–PEST cassette)26 were cultured at 37 °C with 5% CO 2 in advanced DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax, penicillin–streptomycin, and 400 μg ml−1 Geneticin (for U2OS–eGFP cells only). Cell culture reagents were purchased from Thermo Fisher Scientific, cell line identities were validated by STR profiling (American Type Culture Collection, ATCC) and deep-sequencing, and cell culture supernatant was tested twice a month for mycoplasma. Transfections were performed using a Lonza 4-D Nucleofector with the SE Kit and the DN-100 program on ~200,000 cells with 750 ng of nuclease and 250 ng of guide RNA plasmids.

Human cell eGFP disruption assay

eGFP disruption experiments were performed as previously described5,26. Briefly, transfected cells were analysed ~52 h after transfection for loss of eGFP fluorescence using a Fortessa flow cytometer (BD Biosciences). Background loss was determined by gating a negative control transfection (containing nuclease and empty guide RNA plasmid) at ~2.5% for all experiments.

T7 endonuclease I assay

Roughly 72 h after transfection, genomic DNA was extracted from U2OS cells using an Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics), and T7 endonuclease I (T7E1) assays were performed as previously described26. Briefly, 600- to 800-nucleotide amplicons surrounding on-target sites were amplified from ~100 ng of genomic DNA using Phusion Hot-Start Flex DNA Polymerase (New England Biolabs, NEB) using the primers listed in Supplementary Table 3. PCR products were visualized (using a QIAxcel capillary electrophoresis instrument, Qiagen) and purified (Agencourt Ampure XP cleanup, Beckman Coulter Genomics). Denaturation and annealing of ~200 ng of the PCR product was followed by digestion with T7EI (NEB). Digestion products were purified (Ampure) and quantified (QIAxcel) to approximate the mutagenesis frequencies induced by Cas9–sgRNA complexes.

GUIDE-seq

GUIDE-seq experiments were performed with WT SpCas9, SpCas9-HF1, eSpCas9(1.1) and HypaCas9 for six different sgRNAs, essentially as previously described6. Briefly, U2OS cells were transfected as described above with the addition of 100 pmol of an end-protected double-stranded oligonucleotide (dsODN) GUIDE-seq tag. Approximately 72 h after nucleofection, genomic DNA was extracted and gene disruption was quantified by T7E1 assay (as described above). GUIDE-seq tag-integration efficiencies were assessed using restriction fragment length polymorphism (RFLP) assays as previously described9. Briefly, PCR reactions amplified from ~100 ng of genomic DNA from GUIDE-seq treated samples, using Phusion Hot-Start Flex DNA Polymerase (NEB), were treated with 20 U NdeI (NEB) for 3 h. Digested products were purified (Ampure) and quantified (QIAxcel) to approximate GUIDE-seq tag-integration efficiencies. To perform GUIDE-seq, sample libraries were assembled as previously described6 and sequenced on an Illumina MiSeq machine. Data were analysed using open-source guideseq software (version 1.1)27. GUIDE-seq data can be found in Supplementary Table 1, and are deposited with the NCBI Sequence Read Archive. Potential alternative alignments shown in Supplementary Table 1, resulting from RNA or DNA bulges28, depict one of many possible alternative alignments.

Data availability

Plasmids encoding the high-fidelity SpCas9 variants described in this manuscript have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/). All sequencing data from this study are available through the NCBI Sequence Read Archive under accession number SRP116962. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files.