A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

The HNH domain visits the checkpoint intermediate before accessing its active conformation

To test the checkpoint hypothesis, we determined the real-time kinetics of HNH activation immediately upon binding to surface-immobilized DNA (Fig. 2A). The complexes were in the R state when initially bound to DNA (Fig. 2B). Individual trajectories recorded at 100 Hz revealed that Cas9 visits the I state (τ = 34 ms) between initial landing and transitioning to the D state (Fig. 2B), consistent with the I state serving as the checkpoint between DNA binding and cleavage. The first transition to the D state occurs rapidly (39 min⁻¹) after initial binding to an on-target DNA (Fig. 2, C, D, and G). Ninety-five percent of the complexes stably remained in the D state (Fig. 2C and fig. S7), and reversible transitions to the I and R states were rarely observed. With a 1–3 bp mm DNA, the first transition to the D state occurs at a ~10-fold slower rate relative to an on-target DNA (Fig. 2, E to G) and most of the complexes underwent reversible transitions after reaching the D state (Fig. 2D and fig. S7).

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Fig. 2

Real-time kinetics of HNH activation immediately after DNA binding.

(A) Schematic for observation of Cas9 conformational dynamics upon landing onto surface-immobilized DNA. (B) Left: A representative smFRET trajectory recorded at 100 Hz shows a brief visit to the I state between initial on-target binding and transitioning into the D state. Right: Single exponential fit (red curve) to the I state dwell time histogram reveals its lifetime (τ, ±95% confidence interval). (C and E) A representative smFRET trajectory at 10 Hz of Cas9 after landing to an on-target and 1–3 bp mm DNA. t = 0 s and dashed vertical lines represent time of landing and acceptor photobleaching, respectively. (D and F) Time-dependent changes in the conformational distribution of Cas9 after DNA landing. (G) Cumulative distribution of first transition to the D state after DNA landing. Red curves show fit to a single exponential function (±95% confidence interval).

HNH activation requires divalent cation but is independent of nuclease activity

Next, we tested the roles of divalent cations and nuclease activity on conformational activation of the HNH domain. DNA cleavage activity of Cas9 requires Mg²⁺ at the catalytic site (6), yet it remains stably bound to the DNA target without Mg²⁺ (fig. S8). It was unclear whether Mg²⁺ enables transition to the D state or stabilizes the D state via interactions with the active site and DNA phosphate backbone (19). We observed that Cas9 is completely unable to transition to the D state without divalent cations (Fig. 3A). At low (10 μM) Mg²⁺, 29% of molecules were in the D conformation (Fig. 3A), and 50% of trajectories revealed I-to-D transitions (fig. S9). At 1 to 5 mM Mg²⁺, Ca²⁺, or Co²⁺, >85% of complexes populated the D state, demonstrating that divalent cations lower the threshold energy for the HNH domain to move to its active state. We observed the same divalent cation-dependent docking for catalytically dead dCas9 (fig. S10). Furthermore, Co²⁺ is unable to support DNA cleavage (fig. S8), but enabled transitions to the D state (Fig. 3A) (6), demonstrating that docking of the HNH domain is independent of nuclease activity.

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Fig. 3

HNH activation requires divalent cation but is independent of nuclease activity.

(A) smFRET histograms of Cas9 bound to on-target dsDNA in the absence and presence of a divalent cation. (B) Top: The on-target DNA was truncated at the 5′ end of the NTS one base after the target sequence (pdDNA1) and four bases after PAM (pdDNA2). Bottom: smFRET histograms of Cas9 bound to pdDNAs in the absence and presence of a divalent cation. (C) Cleavage of pdDNA1 was initiated by replacing EDTA with 5 mM Mg²⁺ and monitored by dissociation of the Cy5-labeled NTS 5′ end from Cas9. (D) Still images of Cy5-pdDNA1 bound to surface-immobilized Cas9 after Mg²⁺ addition (t = 0 s). (E) Percentage of Cy5 spots remain at the surface after Mg²⁺ flow. Red curves represent fit to single exponential decay (mean ± 95% confidence interval).

To explore the stability of the D conformation after NTS release, we truncated to the 5′ end of the spacer in the NTS (pdDNA1) to prevent base pairing beyond the target region and enable dissociation of the NTS 5′ end from the complex after cleavage (Fig. 3B) (28). The complexes bound to pdDNA1 transitioned to the D state upon Co²⁺ addition. Unlike an on-target dsDNA, in which the NTS remains bound to the complex after cleavage, initiating the cleavage of pdDNA1 by Mg²⁺ addition reduced the stability of the D conformation. Furthermore, when Cas9 was bound to a distinct substrate that lacks the entire 5′ end of NTS upstream of the cleavage site (pdDNA2), most of the complexes remained in the I state in Mg²⁺. Because the HNH domain reverts back to the I state upon NTS release and cannot dock in the absence of the NTS, the 5′ end of the NTS is necessary for stabilizing the D state (29).

To determine whether stable docking of HNH is rate-limiting for the DNA cleavage activity, we designed a single-molecule stopped flow assay to monitor DNA cleavage activity of individual Cas9 complexes in real time. In this assay, pdDNA1 was labeled with Cy5 at the 5′ end of NTS and preassembled with Cas9 in 10 mM EDTA, and then complexes were immobilized via the sgRNA (Fig. 3C). Replacement of EDTA with 5 mM Mg²⁺ resulted in disappearance of 80% of the Cy5 spots at a rate of 0.16 s⁻¹ (Fig. 3D and movie S1), which represents DNA cleavage and NTS release. Because docking of the HNH domain occurs about fourfold faster than DNA cleavage, it is not rate-limiting for cleavage of the on-target DNA. However, when Cas9 was preassembled with a 1–3 bp mm DNA, HNH docking and DNA cleavage (Figs. 2G and ​and3E3E and movie S2) occurred at comparable rates, suggesting that HNH docking is a rate-limiting step for the cleavage of this substrate.

Nucleic acid preparation

sgRNA templates were polymerase chain reaction–amplified and cloned into Eco RI and Bam HI sites in pUC19 (table S1), transcribed, and purified in vitro, as previously described (12). DNA oligonucleotides and 5′ biotinylated NTSs (table S1) were ordered synthetically (Integrated DNA Technologies), and DNA duplexes were prepared and purified by native polyacrylamide gel electrophoresis (PAGE), as described.

DNA bulk cleavage and binding assays

DNA duplex substrates were 5′-[³²P]–radiolabeled on both strands. For cleavage experiments, Cas9 and sgRNA were preincubated at room temperature for 10 min in 1× binding buffer [20 mM tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 5% glycerol, and heparin (50 μg/ml)] before initiating the reaction by addition of DNA duplexes. DNA cleavage experiments were performed and analyzed as previously described (12).

Binding assays were performed as previously described (12), with the following modifications: For the 0 mM MgCl₂ condition, binding reactions were conducted in 1× binding buffer + 1 mM EDTA in the absence of MgCl₂ [20 mM tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, and heparin (50 μg/ml)] and resolved on 8% native PAGE [0.5× tris-borate EDTA (TBE) + 1 mM EDTA, without MgCl₂] at 4°C. For the 5 mM MgCl₂ condition, binding reactions were conducted in 1× binding buffer and resolved on 8% native PAGE (0.5× TBE + 5 mM MgCl₂) at 4°C. Experiments were replicated at least three times, and presented gels are representative results.

Sample preparation for smFRET assay

Quartz slides coated with 99% polyethylene glycol (PEG) and 1% biotinylated PEG were acquired from MicroSurfaces Inc. Air-tight sample chamber was prepared by sandwiching double-sided tape between quartz slides and coverslips. To prepare the slides for molecule deposition, the PEG surface was preblocked with casein (10 mg/ml) incubated for 10 min. Flow chamber was washed with 1× binding buffer and then incubated with 20 μl of streptavidin (1 mg/ml) for 10 min. Excess streptavidin was washed away with 40 μl of 1× binding buffer. All the smFRET experiments were performed using Cas9_HNH-1, with the exception that data shown in figs. S3 and S10 were collected using Cas9_HNH-2 and dCas9_HNH-1 constructs, respectively.

Two separate experimental geometries were used for smFRET experiments. To surface immobilize Cas9 from its sgRNA, 50 nM sgRNA was hybridized to a 5′ biotin-DNA tether. Cas9-sgRNA complexes were preassembled by mixing 50 nM Cas9, 50 nM sgRNA-biotin, and 200 nM DNA substrate in 1× binding buffer and incubating for 10 min. The sample was spun at 16,000g at 4°C for 5 min. The supernatant was diluted to 100 pM, flown into the sample chamber, and incubated for 10 min. To surface immobilize Cas9 from its DNA substrate, 1 nM biotinylated DNA substrate [NTS (5′ biotin) hybridized to TS] was flown into the sample chamber and incubated for 10 min. The chamber was washed with 1× binding buffer. Cas9 (50 nM) and sgRNA (50 nM) were mixed in 1× binding buffer and incubated for 10 min. The sample was spun at 16,000g at 4°C for 5 min. The supernatant was diluted to 100 pM, flown into the sample chamber, and incubated for 10 min. Before data acquisition, the sample chamber was washed with 1× binding buffer and 20 μl of imaging buffer [glucose oxidase (1 mg/ml), catalase (0.04 mg/ml), 0.8% dextrose, and 2 mM Trolox in 1× binding buffer].

To perform smFRET measurements of Cas9-sgRNA complexes that landed onto a DNA-immobilized surface, 400 pM Cas9-sgRNA mixture was flown into the sample chamber in 100 μl of imaging buffer during data acquisition. After recording each movie, the imaged area was photobleached with an intense laser beam, and fresh Cas9-sgRNA mixture in imaging buffer was flown into the chamber while imaging the bleached region. This approach prevented crowding of the imaged area with fluorescent spots and enabled more reliable detection of the molecules that landed onto the surface during data acquisition.

Microscopy and data acquisition

A prism-type total internal reflection fluorescence microscope was set up using a Nikon Ti-E Eclipse inverted fluorescence microscope equipped with a 60× 1.20 N.A. 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 signals were 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 × 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. For DNA landing assays, smFRET data were acquired at 100, 10, and 2 Hz under 1.0, 0.3, and 0.05 kW cm⁻² 532-nm excitation, respectively.

Observation of real-time DNA cleavage

The on-target (pdDNA1-Cy5) and 1–3 bp mm [pdDNA1 (1- to 3-bp mm)–Cy5] DNA substrates were truncated at the 5′ end of NTS, missing the flanking sequence at the PAM-distal site, and labeled with Cy5. Cy3-Cas9 (50 nM), 50 nM sgRNA-biotin, and 200 nM DNA were mixed in precleavage buffer [20 mM tris-HCl (pH 7.5), 100 mM KCl, 10 mM EDTA, 1 mM DTT, 5% glycerol, and heparin (50 μg/ml)] and incubated for 10 min. The sample was spun at 16,000g at 4°C for 5 min. The supernatant was diluted to 100 pM, flown into the sample chamber, and incubated for 10 min. Before data acquisition, the sample chamber was washed with precleavage buffer, and 20 μl of imaging buffer was prepared in precleavage buffer. Movies were acquired at 10 Hz under 0.05 kW cm⁻² excitation intensity of the 633-nm beam. To initiate cleavage, flow chamber was washed with imaging buffer prepared in 1× binding buffer (containing 5 mM Mg²⁺) 5 s after the start of image acquisition. The number of molecules on the surface at the beginning of a movie was normalized to 100%. The percent of Cy5 spots that remained on the surface after Mg²⁺ addition was fitted to a single exponential decay to calculate the cleavage rate. Cy5 photobleaching was corrected by performing the assay under the same imaging conditions without initiating the DNA cleavage.

We would like to thank S. Can and B. LaFrance for helpful discussions and revision of our manuscript and T. Ha for the data acquisition software. Funding: J.S.C. is supported by the NSF Graduate Research Fellowship. This work was supported by NIH [GM094522 and GM118773 (to A.Y.)], NSF [MCB-1617028 (to A.Y.) and MCB-1244557 (to J.A.D.)], and the Howard Hughes Medical Institute (to J.A.D.). Author contributions: Y.S.D., J.S.C., S.H.S., and A.Y. designed experiments; J.S.C. performed protein expression, labeling, and biochemical characterization; Y.S.D. performed single-molecule fluorescence assays and analyzed the data; and Y.S.D., J.S.C., S.H.S., J.A.D., and A.Y. wrote the manuscript. Competing interests: J.A.D. is employed by the Howard Hughes Medical Institute and works at the University at California (UC), Berkeley, which have patents pending for CRISPR technologies on which she is an inventor. J.A.D. is the executive director of the Innovative Genomics Institute at UC Berkeley and UC San Francisco. J.A.D. is a cofounder of Editas Medicine, Intellia Therapeutics, and Caribou Biosciences Inc. and a scientific advisor to Caribou Biosciences Inc., Intellia Therapeutics, eFFECTOR Therapeutics, and Driver. S.H.S. is a current employee of Caribou Biosciences Inc. S.H.S., J.S.C., and J.A.D. are inventors on a patent application related to this work filed by the UC (application no. PCT/US2016/036754, filed 9 June 2016). Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. All relevant plasmids and experimental data may be requested from the authors.