Materials and methods

Protein expression and purification

Cas9 was expressed and purified as described previously25, with slight modifications. TEV protease digestion was performed overnight in Ni-NTA elution buffer at 4°C without dialysis. Before loading onto the heparin ion exchange column, the digested protein solution was diluted with one volume of low-salt ion exchange buffer. Protein-purification size exclusion buffer was 20 mM HEPES (pH 7.5), 150 mM KCl, 10% glycerol, 1 mM dithiothreitol (DTT).

Nucleic acid preparation

All DNA oligonucleotides were synthesized by Integrated DNA Technologies except the cystamine-functionalized target strand, which was synthesized by TriLink Biotechnologies (with HPLC purification). DNA oligonucleotides that were not HPLC-purified by the manufacturer were PAGE-purified in house (unless a downstream preparative step involved another PAGE purification), and all DNA oligonucleotides were stored in water. Duplex DNA substrates were annealed by heating to 95°C and cooling to 25°C over the course of 40 min on a thermocycler. Guide RNAs were transcribed and purified as described previously25, except no ribozyme was included in the transcript. All sgRNA molecules were annealed (80°C for 2 min, then moved directly to ice) in RNA storage buffer (0.1 mM EDTA, 2 mM sodium citrate, pH 6.4) prior to use. For both DNA and RNA, A260 was measured on a NanoDrop (Thermo Scientific), and concentration was estimated according to extinction coefficients reported previously38.

Cryo-EM construct preparation

DNA duplexes were pre-annealed in water at 10X concentration (60 µM target strand, 75 µM non-target strand). Cross-linking reactions were assembled with 300 µL water, 100 µL 5X disulfide reaction buffer (250 mM Tris-Cl, pH 7.4 at 25°C, 750 mM NaCl, 25 mM MgCl2, 25% glycerol, 500 µM DTT), 50 µL 10X DNA duplex, 25 µL 100 µM sgRNA, and 25 µL 80 µM Cas9. Cross-linking was allowed to proceed at 25°C for 24 hours (0 RNA:DNA matches) or 8 hours (3 RNA:DNA matches). Sample was then purified by size exclusion (Superdex 200 Increase 10/300 GL, Cytiva) in cryo-EM buffer (20 mM Tris-Cl, pH 7.5 at 25°C, 200 mM KCl, 100 µM DTT, 5 mM MgCl2, 0.25% glycerol). Peak fractions were pooled, concentrated to an estimated 6 µM, snap-frozen in 10-µL aliquots in liquid nitrogen, and stored at −80°C until grid preparation. For the Cas9:sgRNA structural construct, which lacked a cross-link, the reaction was assembled with 350 µL water, 100 µL 5X disulfide reaction buffer, 0.45 µL 1 M DTT, 25 µL 100 µM sgRNA, and 25 µL 80 µM Cas9. The complex was allowed to form at 25°C for 30 minutes. The Cas9:sgRNA sample was then size-exclusion-purified and processed as described for the DNA-containing constructs. For Cas9:sgRNA, cryo-EM buffer contained 1 mM DTT instead of 100 µM DTT.

SDS-PAGE analysis

For non-reducing SDS-PAGE, thiol exchange was first quenched by the addition of 20 mM S-methyl methanethiosulfonate (S-MMTS). Then, 0.25 volumes of 5X non-reducing SDS-PAGE loading solution (0.0625% w/v bromophenol blue, 75 mM EDTA, 30% glycerol, 10% SDS, 250 mM Tris-Cl, pH 6.8) were added, and the sample was heated to 90°C for 5 minutes before loading of 3 pmol onto a 4-15% Mini-PROTEAN TGX Stain-Free Precast Gel (Bio-Rad), alongside PageRuler Prestained Protein Ladder (Thermo Scientific). Gels were imaged using the Stain-Free imaging protocol on a Bio-Rad ChemiDoc (5-min activation, 3-s exposure). For reducing SDS-PAGE, no S-MMTS was added, and 5% β-mercaptoethanol (βME) was added along with the non-reducing SDS-PAGE loading solution. For radioactive SDS-PAGE analysis, a 4-20% Mini-PROTEAN TGX Precast Gel (Bio-Rad) was pre-run for 20 min at 200 V (to allow free ATP to migrate ahead of free DNA), run with radioactive sample for 15 min at 200 V, dried (80°C, 3 hours) on a gel dryer (Bio-Rad), and exposed to a phosphor screen, subsequently imaged on an Amersham Typhoon (Cytiva).

Nucleic acid radiolabeling

Standard 5′ radiolabeling of DNA oligonucleotides was performed as described previously25. For 5′ radiolabeling of sgRNAs, the 5′ triphosphate was first removed by treatment with Quick CIP (New England BioLabs, manufacturer’s instructions). The reaction was then supplemented with 5 mM DTT and the same concentrations of T4 polynucleotide kinase (New England BioLabs) and γ-32P-ATP (PerkinElmer) used for DNA radiolabeling, and the remainder of the protocol was completed as for DNA.

Radiolabeled target-strand cleavage rate measurements

DNA duplexes at 10X concentration (20 nM radiolabeled target strand, 75 µM unlabeled non-target strand) were annealed in water with 60 µM cystamine dihydrochloride (pH 7). A 75-µL reaction was assembled from 15 µL 5X Mg-free disulfide reaction buffer (250 mM Tris-Cl, pH 7.4 at 25°C, 750 mM NaCl, 5 mM EDTA, 25% glycerol, 500 µM DTT), 7.5 µL 600 µM cystamine dihydrochloride (pH 7), 37.5 µL water, 3.75 µL 80 µM Cas9, 3.75 µL 100 µM sgRNA, 7.5 µL 10X DNA duplex. The reaction was incubated at 25°C for 2 hours, at which point the cross-linked fraction had fully equilibrated. To non-reducing or reducing reactions, 5 µL of 320 mM S-MMTS or 80 mM DTT (respectively) in 1X Mg-free disulfide reaction buffer was added. Samples were incubated at 25°C for an additional 5 min, then cooled to 16°C and allowed to equilibrate for 15 min. One aliquot was quenched into 0.25 volumes 5X non-reducing SDS-PAGE solution and subject to SDS-PAGE analysis to assess the extent of cross-linking (for the reduced sample, no βME was added, as the DTT had already effectively reduced the sample). Another aliquot was quenched for reducing urea-PAGE analysis as timepoint 0. DNA cleavage was initiated by combining the remaining reaction volume with 0.11 volumes 60 mM MgCl2. Aliquots were taken at the indicated timepoints for reducing urea-PAGE analysis.

Urea-PAGE analysis

To each sample was added 1 volume of 2X urea-PAGE loading solution (92% formamide, 30 mM EDTA, 0.025% bromophenol blue, 400 µg/mL heparin). For reducing urea-PAGE analysis, 5% βME was subsequently added. Samples were heated to 90°C for 5 minutes, then resolved on a denaturing polyacrylamide gel (10% or 15% acrylamide:bis-acrylamide 29:1, 7 M urea, 0.5X TBE). For radioactive samples, gels were dried (80°C, 3 hr) on a gel dryer (Bio-Rad), exposed to a phosphor screen, and imaged on an Amersham Typhoon (Cytiva). For samples containing fluorophore-conjugated DNA, gels were directly imaged on the Typhoon without further treatment. For unlabeled samples, gels were stained with 1X SYBR Gold (Invitrogen) in 0.5X TBE prior to Typhoon imaging.

Fluorescence and autoradiograph data analysis

Band volumes in fluorescence images and autoradiographs were quantified in Image Lab 6.1 (Bio-Rad). Data were fit by the least-squares method in Prism 7 (GraphPad Software).

Cryo-EM grid preparation and data collection

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 a 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.

Cryo-EM data processing and model building

Details of cryo-EM data processing and model building can be found in the Supplementary Information.

Permanganate reactivity measurements

DNA duplexes were annealed at 50X concentration (100 nM radiolabeled target strand, 200 nM unlabeled non-target strand) in 1X annealing buffer (10 mM Tris-Cl, pH 7.9 at 25°C, 50 mM KCl, 1 mM EDTA), then diluted to 10X concentration in water. A Cas9 titration at 5X was prepared by diluting an 80 µM Cas9 stock solution with protein-purification size exclusion buffer. An sgRNA titration at 5X was prepared by diluting a 100 µM sgRNA stock solution with RNA storage buffer. For all reactions, the sgRNA concentration was 1.25 times the Cas9 concentration, and the reported ribonucleoprotein concentration is that of Cas9. Reactions were assembled with 11 µL 5X permanganate reaction buffer (100 mM Tris-Cl, pH 7.9 at 25°C, 120 mM KCl, 25 mM MgCl2, 5 mM TCEP, 500 µg/mL UltraPure BSA, 0.05% Tween-20), 11 µL water, 11 µL 5X Cas9, 11 µL 5X sgRNA, 5.5 µL 10X DNA. A stock solution of KMnO4 was prepared fresh in water, and its concentration was corrected to 100 mM (10X reaction concentration) based on 8 averaged NanoDrop readings (ε526 = 2.4 x 103 M-1 cm-1). Reaction tubes and KMnO4 (or water, for reactions lacking permanganate) were equilibrated to 30°C for 15 minutes. To initiate the reaction, 22.5 µL of Cas9:sgRNA:DNA was added to 2.5 µL 100 mM KMnO4 or water. After 2 min, 25 µL 2X stop solution (2 M βME, 30 mM EDTA) was added to stop the reaction, and 50 µL of water was added to each quenched reaction. The remainder of the protocol was conducted as described previously25, except an additional wash with 500 µL 70% ethanol was added to decrease the salt concentration in the final samples. Data analysis was similar to that described previously25. Let vi denote the volume of band i in a lane with n total bands (band 1 is the shortest cleavage fragment, band n is the topmost band corresponding to the starting/uncleaved DNA oligonucleotide). The probability of cleavage at thymine i is defined as: . Oxidation probability of thymine i is defined as: , where +pm indicates the experiment that contained 10 mM KMnO4 and −pm indicates the no-permanganate experiment.

Preparation of DNA cyclization substrates

Each variant DNA cyclization substrate precursor was assembled by PCR from two amplification primers (one of which contained a fluorescein-dT) and two assembly primers. Each reaction was 400 µL total (split into 4 x 100-µL aliquots) and contained 1X Q5 reaction buffer (New England BioLabs), 200 µM dNTPs, 200 nM forward amplification primer, 200 nM reverse amplification primer, 1 nM forward assembly primer, 1 nM reverse assembly primer, 0.02 U/µL Q5 polymerase. Thermocycle parameters were as follows: 98°C, 30 s; {98°C, 10 s; 55°C, 20 s; 72°C, 15 s}; {98°C, 10 s; 62°C, 20 s; 72°C, 15 s}; {98°C, 10 s; 72°C, 35 s}x25; 72°C, 2 min; 10°C, ∞. PCR products were phenol-chloroform-extracted, ethanol-precipitated, and resuspended in 80 µL water. To this was added 15 µL 10X CutSmart buffer, 47.5 µL water, and 7.5 µL ClaI restriction enzyme (10,000 units/mL, New England BioLabs), and digestion was allowed to proceed overnight at 37°C. Samples were then combined with 0.25 volumes 5X native quench solution (25% glycerol, 250 µg/mL heparin, 125 mM EDTA, 1.2 mg/mL proteinase K, 0.0625% w/v bromophenol blue), incubated at 55°C for 15 minutes, and resolved on a preparative native PAGE gel (8% acrylamide:bis-acrylamide 37.5:1, 0.5X TBE) at 4°C. Fluorescent bands, made visible on a blue LED transilluminator, were cut out, and DNA was extracted, ethanol-precipitated, and resuspended in water.

Cyclization efficiency measurements

Each cyclization reaction contained the following components: 1 µL 10X T4 DNA ligase reaction buffer (New England BioLabs), 2 µL water, 1 µL 10X ligation buffer additives (400 µg/mL UltraPure BSA, 100 mM KCl, 0.1% NP-40), 2 µL 80 µM Cas9 (or protein-purification size exclusion buffer), 2 µL 100 µM sgRNA (or RNA storage buffer), 1 µL 25 nM cyclization substrate, 1 µL T4 DNA ligase (400,000 units/mL, New England BioLabs) (or ligase storage buffer). All reaction components were incubated together at 20°C for 15 minutes prior to reaction initiation except for the ligase, which was incubated separately. Reactions were initiated by combining the ligase with the remainder of the components, allowed to proceed at 20°C for 30 minutes, then quenched with 2.5 µL 5X native quench solution. Samples were then incubated at 55°C for 15 minutes, resolved on an analytical native PAGE gel (8% acrylamide:bis-acrylamide 37.5:1, 0.5X TBE) at 4°C, and imaged for fluorescein on an Amersham Typhoon (Cytiva). Monomolecular cyclization efficiency (MCE) for a given lane is defined as (band volume of circular monomers)/(sum of all band volumes). Bimolecular ligation efficiency (BLE) is defined as (sum of band volumes of all linear/circular n-mers, for n≥2)/(sum of all band volumes). The non-specific degradation products indicated in Extended Data Fig. 6a were not included in the analysis.