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Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9

Nature Structural & Molecular Biology volume 26, pages 679–685 (2019)Cite this article

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Abstract

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9–sgRNA–DNA complex in the presence of Mg2+. In the precatalytic state, Cas9 adopts the ‘checkpoint’ conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.

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Fig. 1: Cryo-EM structures of three states of Cas9–sgRNA–dsDNA complex.
Fig. 2: The central channel of Cas9 accommodates the R-loop structure in state I.
Fig. 3: HNH domain adopts catalytic conformation in state II.
Fig. 4: The HNH active site conformation in state II.
Fig. 5: Proposed mechanism for the concerted series of domain movements involved in Cas9-mediated DNA cleavage.

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Data availability

All data needed to assess and evaluate the conclusions in the paper are available in the main text and supplementary information. The coordinates and electron density maps are deposited in the Protein Data Bank and EMDB with the following accession numbers (respectively): 6O0Z and 0585 for precatalytic complex (state I), 6O0Y and 0584 for postcatalytic complex (state II) and 6O0X and 0583 for product complex (state III). Uncropped gel images for Supplementary Fig. 3 are shown in Supplementary Data Set 1. All other data are available upon request.

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Acknowledgements

This work was supported by the intramural research program of the National Cancer Institute (X.Z., S.C. and S.S.), NIH grants (no. GM097042 to M.S. and no. HD081534 to B.J.M.), UIC Center for Clinical and Translational Sciences (R.C. and A.K.P.) and a Canada Excellence Research Chair Award (to S.S.).

Author information

Author notes

  1. Xing Zhu & Sagar Chittori

    Present address: University of British Columbia, Vancouver, British Columbia, Canada

  2. Alan Merk

    Present address: Frederick National Laboratory for Cancer Research, Frederick, MD, USA

  3. These authors contributed equally: Xing Zhu, Ryan Clarke, Anupama K. Puppala.

Authors and Affiliations

  1. Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Xing Zhu, Sagar Chittori & Alan Merk

  2. Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA

    Ryan Clarke, Anupama K. Puppala, Bradley J. Merrill & Miljan Simonović

  3. Genome Editing Core, University of Illinois at Chicago, Chicago, IL, USA

    Bradley J. Merrill

  4. University of British Columbia, Vancouver, British Columbia, Canada

    Sriram Subramaniam

  5. Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Sriram Subramaniam

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Contributions

R.C., B.J.M., M.S. and S.S. conceived the project. R.C. and A.K.P. purified the complex. S.C. prepared cryo-EM grids. A.M. collected cryo-EM data. X.Z. carried out cryo-EM image processing. X.Z., A.K.P. and M.S. built and refined atomic models. M.S. and S.S. provided overall supervision and guidance at all stages of the project. All authors contributed to the experimental design and wrote the manuscript.

Corresponding authors

Correspondence to Bradley J. Merrill, Miljan Simonović or Sriram Subramaniam.

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The authors declare no competing interests.

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Peer review information: Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Representative cryo-EM density maps and models corresponding to states I-III.

(a) Atomic-resolution models of states I-III of the Cas9-sgRNA-dsDNA ternary complex. Arginine-rich bridge of Cas9 and PAM-distal DNA duplex are shown in the upper and lower panels, respectively. (b) Unsharpened (top) and sharpened (bottom) EM maps for each structure are presented. Coloring scheme is the same as in Fig. 1.

Supplementary Figure 2 Steric occlusion of NTS from the tunnel between HNH and RuvC in state I.

(Left) Close-up view of the HNH-RuvC interface where the two domains create a tunnel for the NTS in the previously reported “Mg2+-free” ternary complex crystal structure (5F9R). (Right) Close-up view of the same tunnel in state I, showing closure of the gap, making it inaccessible for the NTS.

Supplementary Figure 3 In vitro analyses of the Cas9-sgRNA-DNA ternary complex.

(a) Assessment of Cas9 stability during DNA cleavage. The ternary complex sample (22.5 μL) corresponding to each timepoint was loaded onto a 4–20% precast SDS-PA gel. The gel shows lack of proteolytic degradation of Cas9 even after 960 min at +37 °C. (b) Probing the ternary complex stability and DNA cleavage levels using an EMSA. Untreated and RNaseA/proteinase K-treated samples (18 μL) corresponding to each time point were analyzed on an 8% 0.5x TBE-PA gel. The same gel was stained for nucleic acids (left) and then for protein (right). The ternary complex is stable after addition of Mg2+ and exhibits single-turnover kinetics until the 240 min timepoint, thus explaining how different structural states of the complex may have been captured at 30 min. (c) Analysis of the Cas9-catalyzed DNA cleavage. Untreated and RNaseA/proteinase K-treated samples (20 μL) corresponding to each time point were analyzed on a denaturing 15% TBE-urea PA gel. The gel demonstrates that Cas9 is catalytically active. Control samples contained 400 nM Cas9, 600 nM sgRNA, or 600 nM DNA only. For more detail, see on-line Methods. Uncropped gel images are shown in Supplementary Data Set 1.

Supplementary Figure 4 Interactions between RuvC and extended DNA duplex, and a likely path for the single stranded NTS that closes the R-loop.

(a) In states II and III, the RuvC domain (light blue) interacts with the 10 bp-long extension of the DNA duplex (TS is blue, NTS is purple) via the solvent-exposed loop that carries three positively charged residues. A segment of RuvC encompassing residues 1000–1076 is disordered (dashed line). Structural comparison shows that the longer duplex used in our study would clash with the 1000–1076 segment of the “Mg2+-free” ternary complex (PDB ID 5F9R; grey), thus causing the disorder in this segment in states II and III. (b) Low-resolution cryo-EM density (purple transparent surface) extends from the cleaved NTS (purple cartoon) and threads between RuvC and HNH (pink) domains. The density points towards the distal TS-NTS duplex, which is sandwiched between RuvC and REC3, suggesting that this is the path for the single-stranded NTS that closes the R-loop. sgRNA is orange cartoon, TS is blue cartoon, NTS is purple cartoon, HNH domain is pink transparent surface, and the rest of the Cas9 protein is white transparent surface.

Supplementary Figure 5 Structural changes after Cas9-catalyzed DNA cleavage.

Structural comparison of states II and III of the Cas9-sgRNA-dsDNA ternary complex. HNH and REC2 domains are excluded from analysis because of the alternate structural disorder in two states. Analysis and representation were completed as in Fig. 2a. Cas9 domains and nucleic acids are colored as in Fig. 1a.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Supplementary Notes 1 and 2, and Supplementary Data Set 1

Reporting Summary

Supplementary Video 1

Conformational changes in the Cas9 ternary complex during transition from state I (‘conformation’ checkpoint) to state II (‘postcatalytic’). HNH domain (pink) swings about the axis perpendicular to the plane of view and it docks at the cleavage site in the TS DNA (blue). Segments of the complex that become disordered (for example, REC2 domain and parts of RuvC) and ordered (for example, loops in REC3 and distal duplex) upon transition, flicker at the start and the end of the video, respectively. Coloring scheme is the same as in Fig. 1.

Supplementary Video 2

Structural rearrangements in the Cas9 ternary complex during transition from state II (‘postcatalytic’) to state III (‘product’). HNH domain (pink) dissociates from cleaved TS DNA (blue) and moves towards solvent, leading to its disorder. REC2 domain (green) now returns to its location as in state I. The REC lobe and distal duplex slightly rotate around the horizontal axis causing the complex to open up a bit more when compared to state II. Segments of the complex that become disordered (for example, HNH and HNH-REC2 linker) and ordered (for example, REC2) upon transition, flicker at the start and the end of the video, respectively. Coloring scheme is the same as in Fig. 1.

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Zhu, X., Clarke, R., Puppala, A.K. et al. Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Nat Struct Mol Biol 26, 679–685 (2019). https://doi.org/10.1038/s41594-019-0258-2

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