Research ArticleGENOME SEQUENCING

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

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Science Advances  04 Aug 2017:
Vol. 3, no. 8, eaao0027
DOI: 10.1126/sciadv.aao0027
  • Fig. 1 HNH conformational dynamics reveal a distinct I state as a function of PAM-distal mismatches.

    (A) Model shows HNH labeling sites under different conformations of Cas9, using sgRNA-bound (4ZT0) and dsDNA-bound (5F9R) structures. The cysteine-light Cas9 construct is labeled with Cy3 and Cy5 at S867C and S355C positions. (B) Top: Cas9 was incubated with 55-bp-long dsDNA substrates that include PAM and target sequences. Mismatches were introduced at the PAM-distal site. Bottom: DNA binding to Cas9 results in HNH interconversion, determined by a transition from a low to high FRET state. Scissors show the DNA cleavage sites. (C) Steady-state smFRET histograms for Cas9 in the absence and presence of 200 nM DNA targets. A multi-Gaussian fitting (black curve) reveals D, I, and R states of HNH. (D) Representative time traces (top), transition density plots (TDPs; middle), and rates of the major transitions in TDPs (bottom) for various DNA substrates. a.u., arbitrary units.

  • 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).

  • 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).

  • Fig. 4 Truncation of the gRNA traps the HNH domain in the checkpoint intermediate with fewer mismatches on the DNA.

    (A) On-target DNA binding assay with 20-nt and truncated gRNAs. (B) Bulk cleavage rates of the DNA substrates by Cas9 assembled with 20- and 17-nt gRNAs. (C) Steady-state smFRET histograms of Cas9 guided with a 17-nt gRNA. (D) D population of Cas9 guided with 20- and 17-nt gRNAs. (E) Top: A single mismatch was introduced at the 4th bp after the PAM-distal end (blue arrowhead). Bottom: Real-time cleavage of 4th bp mm by Cas9 guided with 20- and 17-nt gRNAs. Black curves represent fit to a single exponential decay (mean ± 95% confidence interval). (F) Steady-state smFRET histograms of Cas9 with 20- and 17-nt gRNAs bound to 4th bp mm. (G) Model for substrate-dependent HNH activation. DNA binding triggers transition from R (blue) to I (green) conformation, which serves as a conformational checkpoint between DNA binding and cleavage. In Mg2+, recognition of an on-target locks HNH in the catalytically active D conformation (red), which is destabilized after NTS release. HNH activation is prohibited when the RNA-DNA complementarity drops below a threshold (red cross).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/eaao0027/DC1

    fig. S1. Steady-state smFRET measurements and bulk cleavage assays of Cas9 labeled with Cy3/Cy5 FRET pairs.

    fig. S2. Representative smFRET trajectories reveal three different stable conformations of Cas9 under saturating DNA concentrations.

    fig. S3. Steady-state smFRET histograms of a reciprocal Cas9 variant.

    fig. S4. DNA immobilization of Cas9-sgRNA to the PEG surface eliminates the complexes that are unable to bind the DNA.

    fig. S5. Dwell time analysis of dynamic transitions of Cas9 in the absence and presence of the DNA substrates.

    fig. S6. Specific binding of Cas9 to the on-target DNA substrate immobilized to the PEG surface.

    fig. S7. Conformational dynamics of the HNH domain observed at 2 Hz.

    fig. S8. DNA cleavage activity of Cas9 in the presence of various divalent cations.

    fig. S9. Conformational dynamics of the HNH domain in the presence of 10 μM Mg2+.

    fig. S10. smFRET histograms of dCas9 bound to on-target dsDNA in the absence and presence of a divalent cation.

    table S1. List of RNA and DNA substrates used in this study.

    table S2. Expected and measured EFRET values for Cas9HNH-1 and Cas9HNH-2.

    movie S1. Real-time cleavage of an on-target pdDNA1 by Cas9.

    movie S2. Real-time cleavage of a 1–3 bp mm pdDNA1 by Cas9.

    Reference (32)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Steady-state smFRET measurements and bulk cleavage assays of Cas9 labeled with Cy3/Cy5 FRET pairs.
    • fig. S2. Representative smFRET trajectories reveal three different stable conformations of Cas9 under saturating DNA concentrations.
    • fig. S3. Steady-state smFRET histograms of a reciprocal Cas9 variant.
    • fig. S4. DNA immobilization of Cas9-sgRNA to the PEG surface eliminates the complexes that are unable to bind the DNA.
    • fig. S5. Dwell time analysis of dynamic transitions of Cas9 in the absence and presence of the DNA substrates.
    • fig. S6. Specific binding of Cas9 to the on-target DNA substrate immobilized to the PEG surface.
    • fig. S7. Conformational dynamics of the HNH domain observed at 2 Hz.
    • fig. S8. DNA cleavage activity of Cas9 in the presence of various divalent cations.
    • fig. S9. Conformational dynamics of the HNH domain in the presence of 10 μM Mg2+.
    • fig. S10. smFRET histograms of dCas9 bound to on-target dsDNA in the absence and presence of a divalent cation.
    • table S1. List of RNA and DNA substrates used in this study.
    • table S2. Expected and measured EFRET values for Cas9HNH-1 and Cas9HNH-2.
    • Legends for movies S1 and S2
    • Reference (32)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Real-time cleavage of an on-target pdDNA1 by Cas9.
    • movie S2 (.avi format). Real-time cleavage of a 1–3 bp mm pdDNA1 by Cas9.

    Files in this Data Supplement: