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DNA interference states of the hypercompact CRISPR–CasΦ effector

Nature Structural & Molecular Biology volume 28, pages 652–661 (2021)Cite this article

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Abstract

CRISPR–CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.

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Fig. 1: Structure of the crRNA-bound CasΦ poised for DNA recognition.
Fig. 2: Minimal domains mediate DNA recognition by CasΦ.
Fig. 3: DNA unwinding and target recognition activate CasΦ for DNA cutting.
Fig. 4: Structure of CasΦ with a trapped substrate in the active site.
Fig. 5: Helix α7 of the RecI domain regulates substrate accessibility of the RuvC.
Fig. 6: Helix α7 adjusts fidelity and can be engineered for sensitive nucleic acid detection.

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Data availability

The cryo-EM maps and model coordinates have been deposited to the EMDB (codes EMD-23600, EMD-23601 and EMD-23678) and PDB (codes 7LYS, 7LYT and 7M5O), respectively. Source data are provided with this paper.

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Acknowledgements

We thank E. Charles, C. Huang, G. Knott, D. Smock and members of the Doudna and Nogales laboratories for discussion. We thank J. Cofsky and G. Knott for critical reading and comments on the manuscript. We thank J. Remis, D. Toso and G. Knott for electron microscopy assistance and A. Chintangal for computational support. EM data were collected at the Cal-Cryo facility located at UC Berkeley. P.P. is supported by the NIH Somatic Cell Genome Editing consortium (NIH U01AI142817-02). C.A.T. is supported by Campus Executive Grants 2101705 and 1655264 through Sandia National Laboratories. B.A.-S. was supported by an NSF Graduate Research Fellowship (DGE 1752814). J.A.D. receives funding from the Centers for Excellence in Genomic Science of the National Institutes of Health under award number RM1HG009490, from the Somatic Cell Genome Editing Program of the Common Fund of the National Institutes of Health under award number U01AI142817-02, and from the National Science Foundation under award number 1817593. D.A.H. was supported by the EMBO (ALTF 1002-2018) and SNSF (P2BSP3_181878). J.A.D. and E.N. are Howard Hughes Medical Institute Investigators.

Author information

Author notes

  1. Patrick Pausch

    Present address: VU LSC-EMBL Partnership for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania

  2. These authors contributed equally: Patrick Pausch, Katarzyna M. Soczek.

Authors and Affiliations

  1. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA

    Patrick Pausch, Katarzyna M. Soczek, Connor A. Tsuchida, Basem Al-Shayeb, Jillian F. Banfield & Jennifer A. Doudna

  2. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Patrick Pausch, Katarzyna M. Soczek, Eva Nogales & Jennifer A. Doudna

  3. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA

    Patrick Pausch, Katarzyna M. Soczek, Dominik A. Herbst, Eva Nogales & Jennifer A. Doudna

  4. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Dominik A. Herbst, Eva Nogales & Jennifer A. Doudna

  5. University of California, Berkeley–University of California, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA

    Connor A. Tsuchida

  6. Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA

    Basem Al-Shayeb

  7. Department of Earth and Planetary Sciences, University of California, Berkeley, Berkeley, CA, USA

    Jillian F. Banfield

  8. Howard Hughes Medical Institute, Baltimore, MD, USA

    Eva Nogales & Jennifer A. Doudna

  9. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Jennifer A. Doudna

  10. Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA

    Jennifer A. Doudna

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Contributions

P.P. conceived the study with input from J.A.D. P.P. designed experiments and analyzed data. P.P. cloned constructs, purified proteins and performed biochemical experiments. K.M.S. and P.P. prepared cryo-EM grids. K.M.S. collected and processed cryo-EM data for 3D image reconstruction and 3DVA with input from E.N. and D.A.H. P.P. built and refined structure models. C.A.T. provided materials. B.A.-S. and J.F.B. provided the sequence information and bioinformatics analysis for CRISPR–CasΦ homologs, before publication of refs. 7,8. P.P. wrote the manuscript and prepared figures with input from K.M.S. and J.A.D.. The manuscript was reviewed and approved by all co-authors.

Corresponding author

Correspondence to Jennifer A. Doudna.

Ethics declarations

Competing interests

The Regents of the University of California, Berkeley have patents pending for CRISPR technologies on which the authors are inventors. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Vertex, Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences, The Column Group and Inari. J.A.D. is a director at Johnson & Johnson and Tempus and has research projects sponsored by Biogen, Pfizer, AppleTree Partners and Roche. J.F.B. is a founder of Metagenomi. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Structural and Molecular Biology thanks Ryan Jackson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Cryo-EM data processing for CasΦ in the binary state.

a, Cryo-EM data processing schematic. b, Local resolution map for the final cryoSPARC map calculated in cryoSPARC v3.1 with FSC threshold 0.5. Figure was generated in Chimera v.1.14 using the Surface color function and Chimera map sigma level 3.67 with dust removal size 5. c, Particle orientation distribution plot. d, Left: Gold standard FSC curves for the binary complex from the final round of the refinement in cryoSPARC v.3.1. Right: Map vs model FSC plots of the final binary model refined to the LocSpiral map and plotted with the final cryoSPARC sharp experimental map.

Source data

Extended Data Fig. 2 The architecture of CasΦ is similar to, but distinct from the architecture of large type V effectors.

For comparison to CasΦ (above), ternary structures of a representative set of type V effectors in the crRNA (Cas12a and Cas12i), or crRNA/tracrRNA (Cas12b, CasX and Cas14), and DNA bound states are shown. Ternary states were selected for comparison, since binary structures were not available for all effectors. Structures are shown as colored cartoons. Domains are color coded according to the legend on the right. Following models were used to prepare the figure: CasΦ binary structure (this study); Cas12a (PDB-ID: 6I1K23); Cas12b (PDB-ID: 5WTI28); CasX (PDB-ID: 6NY217); Cas14 (PDB-ID: 7C7L18) and Cas12i (PDB-ID: 6W5C26).

Extended Data Fig. 3 A Cas12-typical OBD domain recruits the crRNA to CasΦ.

For comparison to CasΦ (above, left), the OBD domains from representative type V effectors in the crRNA (Cas12a and Cas12i), or crRNA/tracrRNA (Cas12b, CasX and Cas14), and DNA bound states are shown. Following models were used to prepare the figure: CasΦ binary structure (this study); Cas12a (PDB-ID: 6I1K23); Cas12b (PDB-ID: 5WTI28); CasX (PDB-ID: 6NY217); Cas14 (PDB-ID: 7C7L18) and Cas12i (PDB-ID: 6W5C26).

Extended Data Fig. 4 Cryo-EM data processing for CasΦ in the ternary state.

a, Cryo-EM data processing schematic. b, Local resolution map for the final cryoSPARC map calculated in cryoSPARC v3.1 with FSC threshold 0.5. Figure was generated in Chimera v.1.14 using the Surface color function and Chimera map sigma level 3.71 with dust removal size 5. c, Particle orientation distribution plot. d, Left: Gold standard FSC curves for the binary complex from the final round of the refinement in cryoSPARC v.3.1. Right: Map vs model FSC plots of the final binary model refined to the LocSpiral map and plotted with the final cryoSPARC sharp experimental map.

Source data

Extended Data Fig. 5 Superhelical DNA is efficiently cut in the presence of alternative PAMs.

a, dsDNA cleavage assay in probing the ability of CasΦ to cleave linear PCR fragments (left) and supercoiled plasmid targets (right) in dependence of different PAM motifs. b, Quantified cleavage efficiencies for linear PCR fragments (left) and supercoiled plasmid targets (right) in dependence of different PAM motifs. (n = 3 independent reaction replicates; means ± SD). c, Analytical agarose gel electrophoresis images of three subsequently run independent technical replicates corresponding to the plot shown in b. Samples were processed in parallel.

Source data

Extended Data Fig. 6 Helix α7 repositions close to the NTS upon transition from the binary to the ternary state.

CasΦ in the ternary state is shown as a colored cartoon. To highlight the rearrangement of Helix α7 (arrow), the structure of CasΦ in the binary state (purple) was superimposed to the ternary state structure. For clarity, only the RecI domain of the CasΦ binary structure is shown.

Extended Data Fig. 7 The lid-loop associates with the crRNA:TS duplex in the ternary state.

a, CasΦ in the ternary state is shown as a colored cartoon. The lid-loop element is highlighted in purple and the corresponding LocSpiral cryo-EM map around residues 610-638 is shown as a translucent surface, contoured at 12 σ. b, dsDNA cleavage assay probing the ability of WT and mutant CasΦ to cleave linear PCR fragments. Shown is the analytical agarose gel electrophoresis image of three independent reaction replicates that were processed in parallel. Analyzed time point, t = 1h. c, analytical size-exclusion chromatogram showing that the analyzed variants elute as single peaks. d, FQ-assay testing the ability of wild type and variant CasΦ to indiscriminately cut the FQ-reporter in dependence of a crRNA complementary ssDNA activator at a concentration of 2 nM. (n = 3 independent reaction replicates; means).

Source data

Extended Data Fig. 8 Cryo-EM data processing for CasΦ in the ternary state with phosphorothioate DNA and Mg2+.

a, Cryo-EM data processing schematic. b, Local resolution map for the final cryoSPARC map calculated in cryoSPARC v3.1 with FSC threshold 0.5. Figure was generated in Chimera v.1.14 using the Surface color function and Chimera map sigma level 4.75 with dust removal size 5. c, Particle orientation distribution plot. d, Left: Gold standard FSC curves for the binary complex from the final round of the refinement in cryoSPARC v.3.1. Right: Map vs model FSC plots of the final binary model refined to the LocSpiral map and plotted with the final cryoSPARC sharp experimental map.

Source data

Extended Data Fig. 9 The PAM-distal TS is single-stranded.

Above: Overview of the LocSpiral map (left panel, colored volume, contoured at 7.6 σ) and model of CasΦ (right panel) in the ternary state in presence of the phosphorothioate NTS-DNA and the magnesium cofactors (purple spheres). Below: Close up onto the DNA arrangement observed in the ternary structure. The hexagons (magenta) highlight the active site (AS).

Extended Data Fig. 10 3D variability analysis of heterogeneous DNA states around the active site.

Shown are two 90°-rotated views of the states observed in the 3DVA for the CasΦ ternary complexes in absence (above) and presence (below) of the magnesium cofactor. Two distinct states (frame 1 and frame 20) for each mode are shown to highlight the structural heterogeneity. Purple density indicates density corresponding to dynamic DNA, not accounted for by our model.

Supplementary information

Supplementary Information

Supplementary Notes, Supplementary Figs. 1–12, Supplementary Tables 1–4 and Source Data for Supplementary Figs. 1, 2, 7, 9 and 10.

Reporting Summary

Peer Review File

Source data

Source Data Fig. 2

Source data for binding curves (numerical values).

Source Data Fig. 3

Source data for binding curves (numerical values).

Source Data Fig. 5

Source data for cleavage kinetics (numerical values).

Source Data Fig. 6

Source data for DNA cleavage assay and FQ reporter kinetics (numerical values).

Source Data Extended Data Fig. 1

Source data for FSC curves (numerical values).

Source Data Extended Data Fig. 4

Source data for FSC curves (numerical values).

Source Data Extended Data Fig. 5

Source data for DNA cleavage assay (numerical values).

Source Data Extended Data Fig. 5

Source data for DNA cleavage assay (images).

Source Data Extended Data Fig. 7

Source data for FQ reporter kinetics (numerical values).

Source Data Extended Data Fig. 7

Source data for DNA cleavage assay (images).

Source Data Extended Data Fig. 8

Source data for FSC curves (numerical values).

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Pausch, P., Soczek, K.M., Herbst, D.A. et al. DNA interference states of the hypercompact CRISPR–CasΦ effector. Nat Struct Mol Biol 28, 652–661 (2021). https://doi.org/10.1038/s41594-021-00632-3

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