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Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein

Nature volume 546, pages 436–439 (2017)Cite this article

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Abstract

CRISPR–Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA1,2,3,4,5. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells6. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing ‘off-switch’ tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.

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Figure 1: AcrIIA2 and AcrIIA4 inhibit SpyCas9 activity by directly interacting with the sgRNA-bound SpyCas9.
Figure 2: Overall structure of AcrIIA4–SpyCas9–sgRNA.
Figure 3: Structural mechanism of AcrIIA4 recognition by SpyCas9.
Figure 4: AcrIIA4 and PAM sequence overlap when interacting with SpyCas9

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References

  1. Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015)

    Article  CAS  Google Scholar 

  2. Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012)

    Article  ADS  CAS  Google Scholar 

  3. Marraffini, L. A. CRISPR–Cas immunity in prokaryotes. Nature 526, 55–61 (2015)

    Article  ADS  CAS  Google Scholar 

  4. Westra, E. R. et al. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 (2012)

    Article  CAS  Google Scholar 

  5. Sorek, R., Lawrence, C. M. & Wiedenheft, B. CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 82, 237–266 (2013)

    Article  CAS  Google Scholar 

  6. Rauch, B. J. et al. Inhibition of CRISPR–Cas9 with bacteriophage proteins. Cell 168, 150–158.e10 (2017)

    Article  CAS  Google Scholar 

  7. Barrangou, R. & Marraffini, L. A. CRISPR–Cas systems: prokaryotes upgrade to adaptive immunity. Mol. Cell 54, 234–244 (2014)

    Article  CAS  Google Scholar 

  8. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010)

    Article  ADS  CAS  Google Scholar 

  9. van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M. & Brouns, S. J. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem. Sci. 34, 401–407 (2009)

    Article  CAS  Google Scholar 

  10. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

    Article  ADS  CAS  Google Scholar 

  11. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012)

    Article  ADS  CAS  Google Scholar 

  12. Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014)

    Article  ADS  CAS  Google Scholar 

  13. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014)

    Article  CAS  Google Scholar 

  14. Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014)

    Article  Google Scholar 

  15. Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J. A. Structural biology. A Cas9–guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015)

    Article  ADS  CAS  Google Scholar 

  16. Jiang, W. & Marraffini, L. A. CRISPR–Cas: new tools for genetic manipulations from bacterial immunity systems. Annu. Rev. Microbiol. 69, 209–228 (2015)

    Article  CAS  Google Scholar 

  17. Sternberg, S. H. & Doudna, J. A. Expanding the biologist’s toolkit with CRISPR–Cas9. Mol. Cell 58, 568–574 (2015)

    Article  CAS  Google Scholar 

  18. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014)

    Article  CAS  Google Scholar 

  19. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014)

    Article  CAS  Google Scholar 

  20. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013)

    Article  CAS  Google Scholar 

  21. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013)

    Article  CAS  Google Scholar 

  22. Yen, S. T. et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 393, 3–9 (2014)

    Article  CAS  Google Scholar 

  23. Orthwein, A. et al. A mechanism for the suppression of homologous recombination in G1 cells. Nature 528, 422–426 (2015)

    Article  ADS  CAS  Google Scholar 

  24. Wright, A. V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015)

    Article  ADS  CAS  Google Scholar 

  25. Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR–Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015)

    Article  CAS  Google Scholar 

  26. Nuñez, J. K., Harrington, L. B. & Doudna, J. A. Chemical and biophysical modulation of Cas9 for tunable genome engineering. ACS Chem. Biol. 11, 681–688 (2016)

    Article  Google Scholar 

  27. Pawluk, A. et al. Naturally occurring off-switches for CRISPR–Cas9. Cell 167, 1829–1838.e9 (2016)

    Article  CAS  Google Scholar 

  28. Bondy-Denomy, J. et al. Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins. Nature 526, 136–139 (2015)

    Article  ADS  CAS  Google Scholar 

  29. Chowdhury, S. et al. Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-guided surveillance complex. Cell 169, 47–57.e11 (2017)

    Article  CAS  Google Scholar 

  30. Jorgensen, I., Rayamajhi, M. & Miao, E. A. Programmed cell death as a defence against infection. Nat. Rev. Immunol. 17, 151–164 (2017)

    Article  CAS  Google Scholar 

  31. Otwinowski, Z. M. W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  32. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  33. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  34. Adams, P. D . et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  35. DeLano, W. L. PyMOL Molecular Viewer (http://www.pymol.org) (2002)

Download references

Acknowledgements

We thank J. He at Shanghai Synchrotron Radiation Facility (SSRF) for help with data collection. We thank J. Chai for critical reading of the manuscript. This research was funded by the National Natural Science Foundation of China grant no. 31422014, 31450001 and 31300605 to Z.H.

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Author notes

  1. De Dong, Minghui Guo and Sihan Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150080, China

    De Dong, Minghui Guo, Sihan Wang, Yuwei Zhu, Shuo Wang, Zhi Xiong, Jianzheng Yang, Zengliang Xu & Zhiwei Huang

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Contributions

D.D., M.G. and S.W. expressed, purified, characterized and crystallized the AcrIIA4–SpyCas9–sgRNA complex with the help of S.W., Z.X., J.Y. and Z.X. D.D., M.G., Y.Z. and Z.H. carried out crystallographic studies. D.D., M.G. and Z.H. prepared the figures. D.D. and M.G. performed in vitro transcription of sgRNA, in vitro dsDNA cleavage, GST pull-down, gel filtration, microscale thermophoresis and electrophoretic mobility shift assay experiments with the help of S.W., Z.X., J.Y. and Z.X. Z.H., D.D., M.G. and S.W. wrote the paper. All authors contributed to the manuscript preparation. Z.H. designed the experiments.

Corresponding author

Correspondence to Zhiwei Huang.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks S. Bailey, J. van der Oost and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 AcrIIA2 and AcrIIA4 specifically interact with sgRNA-bound SpyCas9.

Binding assays were carried out between the anti-CRISPR protein of AcrIIA2 or AcrIIA4 and the GST-tagged CRISPR protein of SpyCas9 or NmeCas9 in the presence or absence of cognate sgRNA. The purified GST–SpyCas9 or GST–NmeCas9 protein was first bound to glutathione sepharose beads in the presence or absence of sgRNA, and then the beads were incubated them with AcrIIA2 or AcrIIA4 protein. After extensive washing, the bound proteins were visualized by Coomassie staining following SDS–PAGE. Data shown are the representative of three replicates.

Extended Data Figure 2 Alignment of Cas9 protein sequences.

Multiple sequence alignment of the amino acid sequences of type II-A Cas9 proteins from Streptococcus pyogenes (GI 15675041), Listeria monocytogenes J0161 (GI 345535315), Listeria innocua Clip11262 (GI 16414891), and type II-C Cas9 proteins of Neisseria meningitidis (GI 518572566), Pasteurella multocida subsp. multocida str. Pm70 (GI 218767588), aligned using MUSCLE. Residues with more than 70% similarity are shown in red and boxed in blue. Residues involved in interaction with AcrIIA4 are indicated.

Extended Data Figure 3 Structural comparison of SpyCas9–sgRNA–DNA and SpyCas9–sgRNA–AcrIIA4.

a, Structural superimposition of SpyCas9–sgRNA–DNA (PDB code, 4UN3) and SpyCas9–sgRNA–AcrIIA4. b, GST pull-down assays to verify the structural determinants for preferential binding of AcrIIA4 to SpyCas9. Wild-type or mutant GST-fused AcrIIA4 proteins were first bound to glutathione sepharose beads and incubated with sgRNA-preloaded SpyCas9 (or mutant) protein as indicated. After extensive washing, the bound proteins were visualized by Coomassie staining following SDS–PAGE. c, Enzymatic activity assays to verify structural determinants for specific AcrIIA4–SpyCas9 interaction. The assays were performed as described in Fig. 1b. Data shown are representative of three independent experiments. d, AcrIIA2 and AcrIIA4 compete with PAM-containing dsDNA for binding to the SpyCas9–sgRNA. sgRNA-preloaded GST–SpyCas9 protein was first mixed with AcrIIA2 or AcrIIA4 at 4 °C and incubated for 15 min, followed by addition of PAM-containing dsDNA into the reaction mixtures. After 15 min incubation, the reactions were stopped by adding loading buffer for denaturing gel and the reaction mixtures were loaded onto glutathione sepharose beads and incubated for 15 min. After extensive washing, the bound proteins were visualized by Coomassie staining following SDS–PAGE. Data shown are representative of three independent experiments. e, Electrophoretic mobility shift assay results showing that AcrIIA2 and AcrIIA4 compete with PAM-containing dsDNA for binding to the SpyCas9–sgRNA. The fluorophore-labelled dsDNA and AcrIIA2 (upper panel) or AcrIIA4 (lower panel) were added to sgRNA-preloaded inactive SpyCas9(D10A/H840A) simultaneously. Molar ratios of SpyCas9–anti-CRISPR protein are shown at the top of each lane. The reaction mixtures were run on 6% native polyacrylamide gels and visualized by fluorescence imaging (800 nm). Data shown are representative of three independent experiments.

Extended Data Figure 4 Structural comparison of SpyCas9–sgRNA and SpyCas9.

Structural superimposition of SpyCas9–sgRNA (PDB code, 4ZT0) and SpyCas9 (PDB code, 4CMP). The interface of AcrIIA4 and SpyCas9 is circled in black dashed line.

Extended Data Table 1 Data collection, phasing and refinement statistics
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Extended Data Table 2 Nucleic acid sequences used in the study
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Dong, D., Guo, M., Wang, S. et al. Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein. Nature 546, 436–439 (2017). https://doi.org/10.1038/nature22377

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