Conformational control of DNA target cleavage by CRISPR–Cas9

Journal name:
Nature
Volume:
527,
Pages:
110–113
Date published:
DOI:
doi:10.1038/nature15544
Received
Accepted
Published online

Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR)1, 2. Together with single-guide RNAs3, Cas9 also functions as a powerful genome engineering tool in plants and animals4, 5, 6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7, 8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9, 10, 11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition12 and RNA–DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation.

At a glance

Figures

  1. Full-length sgRNA drives inward lobe closure of Cas9.
    Figure 1: Full-length sgRNA drives inward lobe closure of Cas9.

    a, Domain organization of S. pyogenes Cas9 (top) and X-ray crystal structure of sgRNA/DNA-bound Cas9 (Protein Data Bank (PDB) accession number 4UN3, ref. 16) (bottom), with HNH domain omitted for clarity. BH, bridge helix; REC, recognition; PI, PAM-interacting. b, Design of Cas9hinge FRET construct. Measured distances between D435 and E945 in apo (PDB 4CMP, ref. 13) and sgRNA/DNA-bound Cas9 structures are indicated. Inward lobe closure is exemplified by movement of the BH (arrow). Regions of the PI domain, sgRNA, and DNA are omitted for clarity. c, Fluorescence emission spectra for Cas9hinge in the presence of the indicated substrates. d, (Ratio)A data for Cas9hinge. Inset: schematic of full-length sgRNA coloured by motif 28. Error bars, s.d.; n = 3.

  2. FRET experiments reveal an activated conformation of the HNH nuclease domain.
    Figure 2: FRET experiments reveal an activated conformation of the HNH nuclease domain.

    a, Design of Cas9HNH-1 FRET construct. Measured distances between S355 and S867 in the sgRNA/DNA-bound Cas9 structure16 and a model of the HNH domain docked at the cleavage site are indicated, as are putative conformational changes of the HNH domain (arrow). The model was generated using an HNH homologue structure (PDB 2QNC, ref. 21). b, Fluorescence emission spectra for dCas9HNH-1 in the presence of the indicated substrates. Inset: (ratio)A values. c, (Ratio)A data for dCas9HNH-1. Mismatches (mm) were introduced sequentially from the PAM-distal end of the target. d, Cleavage rate constants using wild-type Cas9. ND, cleavage not detected. e, (Ratio)A data for catalytically active Cas9HNH-1 and Cas9HNH-2. Error bars in b–e, s.d.; n = 3.

  3. RuvC nuclease activity is allosterically controlled by HNH conformational changes.
    Figure 3: RuvC nuclease activity is allosterically controlled by HNH conformational changes.

    a, Tested DNA substrates, with on-target (1) at top. Matched and mismatched positions of DNA target strand sequences relative to the sgRNA are coloured red and black, respectively, with the PAM in yellow. Some substrates contain internal mismatches between the two DNA strands; dashed lines indicate additional flanking sequences. Schematic at bottom right depicts identical non-target strand substrates presented to the RuvC nuclease domain in substrates 5 and 7. b, Non-target (black) and target (red) strand cleavage time courses for the indicated DNA substrates using wild-type Cas9. Exponential fits are shown as solid lines. c, (Ratio)A data for Cas9HNH-1 (pink bars, left y axis) and non-target strand cleavage kinetics of the RuvC domain (blue bars, right y axis) for the indicated DNA substrates. d, Non-target and target strand cleavage time courses for the indicated DNA substrates using wild-type Cas9. Exponential fits are shown as solid lines. e, (Ratio)A data for Cas9HNH-1. Error bars in be, s.d.; n = 3.

  4. Mechanism of communication between the HNH and RuvC nuclease domains to achieve concerted DNA cleavage.
    Figure 4: Mechanism of communication between the HNH and RuvC nuclease domains to achieve concerted DNA cleavage.

    a, Target DNA binding assay with dCas9 and ΔHNH–Cas9, resolved by native polyacrylamide gel electrophoresis (PAGE) (top); for gel source data, see Supplementary Fig. 1. Quantified data are below; binding fits are shown as solid lines. b, Target DNA cleavage assay with dCas9, wild-type (WT) Cas9, and ΔHNH–Cas9, resolved by denaturing PAGE. S, substrate; NT, cleaved non-target strand; T, cleaved target strand. c, Magnified view of the sgRNA/DNA-bound Cas9 structure16 (top) highlights two α-helices connecting the HNH domain carboxy (C) terminus and RuvC III amino (N) terminus. Bottom shows sequence alignment29 of this region, and residues mutated to proline or alanine are indicated (arrows). d, Target DNA cleavage assay with the indicated Cas9 variants, resolved by denaturing PAGE. e, Target (red) and non-target (black) strand cleavage time courses with the indicated Cas9 variants (for WT-Cas9 data, see Fig. 3b). Exponential fits are shown as solid lines. Error bars in a and e, s.d.; n = 5 and 3, respectively. f, Model for conformational control of target cleavage by CRISPR–Cas9.

  5. Biochemical preparation and DNA cleavage activity of dye-labelled Cas9.
    Extended Data Fig. 1: Biochemical preparation and DNA cleavage activity of dye-labelled Cas9.

    a, Size-exclusion chromatograms of Cy3/Cy5-labelling reactions with cysteine-free Cas9 (C80S/C574S) or the two double-cysteine Cas9 variants used to generate Cas9hinge and Cas9HNH-1. Reactions contained 10 μM Cas9 and 200 μM Cy3- and Cy5-maleimide, and were separated on a Superdex 200 10/300 column (GE Healthcare). Cysteine-free Cas9 was unreactive. b, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of unlabelled and dye-labelled Cas9 variants. The gel was scanned for Cy3 and Cy5 fluorescence (right) before being stained with Coomassie blue (left). For gel source data, see Supplementary Fig. 1. c, Representative radiolabelled DNA cleavage assay with wild-type (WT) Cas9 and doubly labelled Cas9 variants used in this study, resolved by denaturing PAGE (left); quantified data and exponential fits are shown on the right. S, substrate; NT, cleaved non-target strand; T, cleaved target strand. Error bars, s.d.; n = 3.

  6. Fluorescence control experiments with Cas9hinge and dCas9hinge, and representative analysis of fluorescence emission spectra to calculate (ratio)A.
    Extended Data Fig. 2: Fluorescence control experiments with Cas9hinge and dCas9hinge, and representative analysis of fluorescence emission spectra to calculate (ratio)A.

    a, Fluorescence emission spectra of 50 nM Cas9hinge in the presence of increasing concentrations of full-length sgRNA. Protein and sgRNA concentrations were calculated under non-denaturing conditions using theoretical extinction coefficients. b, Fluorescence emission spectra of (1) Cy3-labelled Cas9hinge, (2) Cy5-labelled Cas9hinge, and (3) an equal mixture of Cy3-Cas9hinge and Cy5-Cas9hinge upon excitation at 530 nm. The minor fluorescence peak for Cy5 in the mixed sample results from residual absorbance of Cy5-Cas9hinge at 530 nm and not from intermolecular FRET (compare spectra 3 with 4, which is a sum of spectra 1 and 2). c, Fluorescence emission spectra of Cas9hinge in the presence of sgRNA substrates specific to S. pyogenes (Spy) or N. meningitidis (Nme) Cas9. d, Determination of the (ratio)A parameter, which is proportional to FRET efficiency. Shown for apo-Cas9hinge are (1) an emission spectrum of Cy3/Cy5-Cas9hinge upon excitation of the donor at 530 nm; (2) an emission spectrum of donor only Cy3-Cas9hinge upon excitation of the donor at 530 nm, normalized to 1; (3) the extracted fluorescence of the acceptor via energy transfer, obtained by subtracting 2 from 1; and (4) an emission spectrum of Cy3/Cy5-Cas9hinge upon direct excitation of the acceptor at 630 nm. (Ratio)A is calculated by dividing the integrated intensity (650–800 nm) of 3 by the integrated intensity of 4. e, (Ratio)A data for dCas9hinge in the presence of the same sgRNA substrates tested with nuclease-active Cas9hinge in Fig. 1e. Error bars, s.d.; n = 3.

  7. Modelling of the HNH domain docked at the cleavage site, and design of the Cas9HNH-2 FRET construct.
    Extended Data Fig. 3: Modelling of the HNH domain docked at the cleavage site, and design of the Cas9HNH-2 FRET construct.

    a, The scissile phosphate and flanking nucleotides of a DNA substrate co-crystallized with the phage T4 endonuclease VII (endo VII; PDB 2QNC; left) were aligned with the scissile phosphate and flanking nucleotides of the DNA target strand in the sgRNA/DNA-bound Cas9 crystal structure (PDB 4UN3; middle). Structural alignment of the Cas9 HNH domain with endonuclease VII (middle) results in a model of how the Cas9 HNH domain docks at the cleavage site (right). Catalytic residues are labelled, target strands are shown in red and pink, and a magnesium ion is depicted as a blue sphere. b, Conservation rendering of the sgRNA/DNA-bound Cas9 crystal structure, generated using ConSurf, shows that the most highly conserved patches of the HNH domain, including the active site, are solvent-exposed in the observed conformation. The HNH domain is omitted from the view on the left for clarity. c, Magnified view of the HNH domain in its observed conformation (left) and the model for the docked state (right), coloured as in b. The DNA target strand fits snugly in a groove on the HNH domain in the model, with the most highly conserved patches located in the immediate vicinity of the scissile phosphate. DNA and sgRNA are coloured red and orange, respectively. d, The conformational flexibility of the HNH domain in available Cas9 crystal structures is revealed by structural alignment of the nuclease lobe (RuvC and PI domains) from two sgRNA/DNA-bound structures (PDB accession numbers 4UN3 and 4OO8) and the sgRNA-bound structure (PDB 4ZT0). The modelled docked state from a is shown. e, Design of Cas9HNH-2 FRET construct. Measured distances between ~N1054 and S867 in the sgRNA/DNA-bound Cas9 structure and a model of the HNH domain docked at the cleavage site are indicated. Putative conformational changes of the HNH domain are shown with a black arrow.

  8. Evidence that variable (ratio)A values for dCas9HNH-1 reflect distinct conformational states/dynamics, and FRET data for Cas9HNH-2.
    Extended Data Fig. 4: Evidence that variable (ratio)A values for dCas9HNH-1 reflect distinct conformational states/dynamics, and FRET data for Cas9HNH-2.

    a, DNA binding assay with dCas9 and either on-target DNA or off-target DNAs containing 2, 4, or 8-bp mismatches at the PAM-distal end. Binding fits are shown as solid lines and yield equilibrium dissociation constants (Kd) of 0.80, 6.7, 19, and 20 nM, respectively. Given these values, 99%, 96%, 89%, and 89% of dCas9 should be bound to DNA under the conditions used for FRET experiments in Fig. 2c (50 nM dCas9HNH-1, 200 nM DNA). b, (Ratio)A data for 50 nM dCas9HNH-1 in the presence of 1 μM sgRNA and either 200 nM, 400 nM, or 1 μM off-target DNAs containing 2- or 4-bp mismatches. Data for sgRNA only and on-target DNA are shown for comparison. c, DNA cleavage time courses for the indicated DNA substrates using wild-type Cas9. Exponential fits are shown as solid lines, and extracted rate constants are shown in Fig. 2d. d, Fluorescence emission spectra of Cas9HNH-2 in the presence of the indicated substrates. The inset shows (ratio)A values; mut, mutation. Error bars in a and bd, s.d.; n = 3–5 and 3, respectively.

  9. Additional experimental support for dependence of RuvC nuclease activity on HNH conformational changes.
    Extended Data Fig. 5: Additional experimental support for dependence of RuvC nuclease activity on HNH conformational changes.

    a, Panel of DNA substrates tested in b, with on-target (1) at top. Matched and mismatched positions of DNA target strand sequences relative to the sgRNA are coloured red and black, respectively, with the PAM in yellow. Some substrates contain internal mismatches between the two DNA strands; dashed lines indicate additional flanking sequence. b, Kinetics of non-target (black) and target (red) strand cleavage for the indicated DNA substrates. c, Panel of DNA substrates tested in d and e, depicted as in a. d, (Ratio)A data for Cas9HNH-1 in the presence of the indicated DNA substrates. e, Non-target strand cleavage kinetics of the RuvC domain for the indicated DNA substrates. Error bars in b, d, e, s.d.; n = 3.

  10. Design, purification, and DNA cleavage activity of ΔHNH-Cas9.
    Extended Data Fig. 6: Design, purification, and DNA cleavage activity of ΔHNH-Cas9.

    a, Domain organization of WT- and ΔHNH-Cas9 (top), showing the residues that were replaced with a GGS2 linker to generate ΔHNH-Cas9. Magnified view of connections between the HNH domain and RuvC II and III motifs in the apo (left) and sgRNA/DNA-bound (right) Cas9 crystal structures, as well as in the ΔHNH-Cas9 construct. Disordered linkers and the introduced GGS2 linker are shown as dashed lines. b, Size-exclusion chromatograms of WT- and ΔHNH-Cas9 using a Superdex 200 16/60 column (GE Healthcare). c, SDS–PAGE analysis of dCas9 (D10A/H840A), WT-Cas9, ΔHNH-Cas9, and the purified HNH domain (residues 776–907). Expected molecular masses are 159 kDa, 159 kDa, 142 kDa, and 16 kDa, respectively. For gel source data, see Supplementary Fig. 1. d, Representative radiolabelled DNA cleavage assay with WT-Cas9, ΔHNH-Cas9, ΔHNH-Cas9 in the presence of excess HNH domain, and HNH domain alone, resolved by denaturing PAGE.

  11. Structural analysis and perturbation of the HNH–RuvC III linker.
    Extended Data Fig. 7: Structural analysis and perturbation of the HNH–RuvC III linker.

    a, Molecules A (left) and B (right) of the sgRNA/DNA-bound Cas9 crystal structure (PDB 4OO8). Molecule A has an ordered HNH domain and HNH–RuvC III linker, whereas these are both disordered in molecule B; the missing density for the HNH domain is replaced with the modelled docked state (right). Another prominent difference is the N-terminal region of the RuvC III motif (blue helices), which rearranges from a helix–loop–helix in molecule A into an extended helix in molecule B. Proline pairs were inserted to prevent formation of this extended helix. b, Target (red) and non-target (black) strand cleavage time courses with the indicated Cas9 variant. Exponential fits are shown as solid lines. c, Kinetics of target (red) and non-target (black) strand cleavage for the indicated Cas9 mutants. ND, cleavage not detected. Error bars in b and c, s.d.; n = 3.

Tables

  1. Measured distances between residues labelled with FRET pairs
    Extended Data Table 1: Measured distances between residues labelled with FRET pairs
  2. RNA and DNA substrates used in this study
    Extended Data Table 2: RNA and DNA substrates used in this study

Videos

  1. Morph between different Cas9 conformational states
    Video 1: Morph between different Cas9 conformational states
    Cas9 molecules from available crystal structures (apo, PDB ID 4CMP; sgRNA-bound, 4ZT0; sgRNA/DNA-bound, 4UN3) were aligned in the RuvC and PI domains to highlight conformational changes induced by guide RNA and target DNA binding. A model for the docked state of the HNH domain was generated using a homologous structure of endonuclease VII bound to DNA (PDB ID 2QNC).

References

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Author information

  1. Present address: Department of Bioengineering, Stanford University, Stanford, California 94305, USA.

    • Matias Kaplan

Affiliations

  1. Department of Chemistry, University of California, Berkeley, California 94720, USA

    • Samuel H. Sternberg &
    • Jennifer A. Doudna
  2. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

    • Benjamin LaFrance &
    • Jennifer A. Doudna
  3. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

    • Matias Kaplan &
    • Jennifer A. Doudna
  4. Innovative Genomics Initiative, University of California, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Jennifer A. Doudna

Contributions

S.H.S. designed and conducted all experiments. B.L. and M.K. assisted with protein purification, dye labelling, cleavage assays, and fluorescence experiments. All authors discussed the data; S.H.S. and J.A.D. wrote the manuscript.

Competing financial interests

S.H.S. and J.A.D. are inventors on a related patent application.

Corresponding author

Correspondence to:

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Biochemical preparation and DNA cleavage activity of dye-labelled Cas9. (201 KB)

    a, Size-exclusion chromatograms of Cy3/Cy5-labelling reactions with cysteine-free Cas9 (C80S/C574S) or the two double-cysteine Cas9 variants used to generate Cas9hinge and Cas9HNH-1. Reactions contained 10 μM Cas9 and 200 μM Cy3- and Cy5-maleimide, and were separated on a Superdex 200 10/300 column (GE Healthcare). Cysteine-free Cas9 was unreactive. b, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of unlabelled and dye-labelled Cas9 variants. The gel was scanned for Cy3 and Cy5 fluorescence (right) before being stained with Coomassie blue (left). For gel source data, see Supplementary Fig. 1. c, Representative radiolabelled DNA cleavage assay with wild-type (WT) Cas9 and doubly labelled Cas9 variants used in this study, resolved by denaturing PAGE (left); quantified data and exponential fits are shown on the right. S, substrate; NT, cleaved non-target strand; T, cleaved target strand. Error bars, s.d.; n = 3.

  2. Extended Data Figure 2: Fluorescence control experiments with Cas9hinge and dCas9hinge, and representative analysis of fluorescence emission spectra to calculate (ratio)A. (182 KB)

    a, Fluorescence emission spectra of 50 nM Cas9hinge in the presence of increasing concentrations of full-length sgRNA. Protein and sgRNA concentrations were calculated under non-denaturing conditions using theoretical extinction coefficients. b, Fluorescence emission spectra of (1) Cy3-labelled Cas9hinge, (2) Cy5-labelled Cas9hinge, and (3) an equal mixture of Cy3-Cas9hinge and Cy5-Cas9hinge upon excitation at 530 nm. The minor fluorescence peak for Cy5 in the mixed sample results from residual absorbance of Cy5-Cas9hinge at 530 nm and not from intermolecular FRET (compare spectra 3 with 4, which is a sum of spectra 1 and 2). c, Fluorescence emission spectra of Cas9hinge in the presence of sgRNA substrates specific to S. pyogenes (Spy) or N. meningitidis (Nme) Cas9. d, Determination of the (ratio)A parameter, which is proportional to FRET efficiency. Shown for apo-Cas9hinge are (1) an emission spectrum of Cy3/Cy5-Cas9hinge upon excitation of the donor at 530 nm; (2) an emission spectrum of donor only Cy3-Cas9hinge upon excitation of the donor at 530 nm, normalized to 1; (3) the extracted fluorescence of the acceptor via energy transfer, obtained by subtracting 2 from 1; and (4) an emission spectrum of Cy3/Cy5-Cas9hinge upon direct excitation of the acceptor at 630 nm. (Ratio)A is calculated by dividing the integrated intensity (650–800 nm) of 3 by the integrated intensity of 4. e, (Ratio)A data for dCas9hinge in the presence of the same sgRNA substrates tested with nuclease-active Cas9hinge in Fig. 1e. Error bars, s.d.; n = 3.

  3. Extended Data Figure 3: Modelling of the HNH domain docked at the cleavage site, and design of the Cas9HNH-2 FRET construct. (597 KB)

    a, The scissile phosphate and flanking nucleotides of a DNA substrate co-crystallized with the phage T4 endonuclease VII (endo VII; PDB 2QNC; left) were aligned with the scissile phosphate and flanking nucleotides of the DNA target strand in the sgRNA/DNA-bound Cas9 crystal structure (PDB 4UN3; middle). Structural alignment of the Cas9 HNH domain with endonuclease VII (middle) results in a model of how the Cas9 HNH domain docks at the cleavage site (right). Catalytic residues are labelled, target strands are shown in red and pink, and a magnesium ion is depicted as a blue sphere. b, Conservation rendering of the sgRNA/DNA-bound Cas9 crystal structure, generated using ConSurf, shows that the most highly conserved patches of the HNH domain, including the active site, are solvent-exposed in the observed conformation. The HNH domain is omitted from the view on the left for clarity. c, Magnified view of the HNH domain in its observed conformation (left) and the model for the docked state (right), coloured as in b. The DNA target strand fits snugly in a groove on the HNH domain in the model, with the most highly conserved patches located in the immediate vicinity of the scissile phosphate. DNA and sgRNA are coloured red and orange, respectively. d, The conformational flexibility of the HNH domain in available Cas9 crystal structures is revealed by structural alignment of the nuclease lobe (RuvC and PI domains) from two sgRNA/DNA-bound structures (PDB accession numbers 4UN3 and 4OO8) and the sgRNA-bound structure (PDB 4ZT0). The modelled docked state from a is shown. e, Design of Cas9HNH-2 FRET construct. Measured distances between ~N1054 and S867 in the sgRNA/DNA-bound Cas9 structure and a model of the HNH domain docked at the cleavage site are indicated. Putative conformational changes of the HNH domain are shown with a black arrow.

  4. Extended Data Figure 4: Evidence that variable (ratio)A values for dCas9HNH-1 reflect distinct conformational states/dynamics, and FRET data for Cas9HNH-2. (206 KB)

    a, DNA binding assay with dCas9 and either on-target DNA or off-target DNAs containing 2, 4, or 8-bp mismatches at the PAM-distal end. Binding fits are shown as solid lines and yield equilibrium dissociation constants (Kd) of 0.80, 6.7, 19, and 20 nM, respectively. Given these values, 99%, 96%, 89%, and 89% of dCas9 should be bound to DNA under the conditions used for FRET experiments in Fig. 2c (50 nM dCas9HNH-1, 200 nM DNA). b, (Ratio)A data for 50 nM dCas9HNH-1 in the presence of 1 μM sgRNA and either 200 nM, 400 nM, or 1 μM off-target DNAs containing 2- or 4-bp mismatches. Data for sgRNA only and on-target DNA are shown for comparison. c, DNA cleavage time courses for the indicated DNA substrates using wild-type Cas9. Exponential fits are shown as solid lines, and extracted rate constants are shown in Fig. 2d. d, Fluorescence emission spectra of Cas9HNH-2 in the presence of the indicated substrates. The inset shows (ratio)A values; mut, mutation. Error bars in a and bd, s.d.; n = 3–5 and 3, respectively.

  5. Extended Data Figure 5: Additional experimental support for dependence of RuvC nuclease activity on HNH conformational changes. (258 KB)

    a, Panel of DNA substrates tested in b, with on-target (1) at top. Matched and mismatched positions of DNA target strand sequences relative to the sgRNA are coloured red and black, respectively, with the PAM in yellow. Some substrates contain internal mismatches between the two DNA strands; dashed lines indicate additional flanking sequence. b, Kinetics of non-target (black) and target (red) strand cleavage for the indicated DNA substrates. c, Panel of DNA substrates tested in d and e, depicted as in a. d, (Ratio)A data for Cas9HNH-1 in the presence of the indicated DNA substrates. e, Non-target strand cleavage kinetics of the RuvC domain for the indicated DNA substrates. Error bars in b, d, e, s.d.; n = 3.

  6. Extended Data Figure 6: Design, purification, and DNA cleavage activity of ΔHNH-Cas9. (284 KB)

    a, Domain organization of WT- and ΔHNH-Cas9 (top), showing the residues that were replaced with a GGS2 linker to generate ΔHNH-Cas9. Magnified view of connections between the HNH domain and RuvC II and III motifs in the apo (left) and sgRNA/DNA-bound (right) Cas9 crystal structures, as well as in the ΔHNH-Cas9 construct. Disordered linkers and the introduced GGS2 linker are shown as dashed lines. b, Size-exclusion chromatograms of WT- and ΔHNH-Cas9 using a Superdex 200 16/60 column (GE Healthcare). c, SDS–PAGE analysis of dCas9 (D10A/H840A), WT-Cas9, ΔHNH-Cas9, and the purified HNH domain (residues 776–907). Expected molecular masses are 159 kDa, 159 kDa, 142 kDa, and 16 kDa, respectively. For gel source data, see Supplementary Fig. 1. d, Representative radiolabelled DNA cleavage assay with WT-Cas9, ΔHNH-Cas9, ΔHNH-Cas9 in the presence of excess HNH domain, and HNH domain alone, resolved by denaturing PAGE.

  7. Extended Data Figure 7: Structural analysis and perturbation of the HNH–RuvC III linker. (388 KB)

    a, Molecules A (left) and B (right) of the sgRNA/DNA-bound Cas9 crystal structure (PDB 4OO8). Molecule A has an ordered HNH domain and HNH–RuvC III linker, whereas these are both disordered in molecule B; the missing density for the HNH domain is replaced with the modelled docked state (right). Another prominent difference is the N-terminal region of the RuvC III motif (blue helices), which rearranges from a helix–loop–helix in molecule A into an extended helix in molecule B. Proline pairs were inserted to prevent formation of this extended helix. b, Target (red) and non-target (black) strand cleavage time courses with the indicated Cas9 variant. Exponential fits are shown as solid lines. c, Kinetics of target (red) and non-target (black) strand cleavage for the indicated Cas9 mutants. ND, cleavage not detected. Error bars in b and c, s.d.; n = 3.

Extended Data Tables

  1. Extended Data Table 1: Measured distances between residues labelled with FRET pairs (106 KB)
  2. Extended Data Table 2: RNA and DNA substrates used in this study (562 KB)

Supplementary information

Video

  1. Video 1: Morph between different Cas9 conformational states (25.57 MB, Download)
    Cas9 molecules from available crystal structures (apo, PDB ID 4CMP; sgRNA-bound, 4ZT0; sgRNA/DNA-bound, 4UN3) were aligned in the RuvC and PI domains to highlight conformational changes induced by guide RNA and target DNA binding. A model for the docked state of the HNH domain was generated using a homologous structure of endonuclease VII bound to DNA (PDB ID 2QNC).

PDF files

  1. Supplementary Information (694 KB)

    This file contains Supplementary Figure 1, which shows uncropped gel images from polyacrylamide gel electrophoresis experiments presented in the manuscript.

Additional data