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Structure and function of a bacterial type III-E CRISPR–Cas7-11 complex

Nature Microbiology volume 7, pages 2078–2088 (2022)Cite this article

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Abstract

The type III-E CRISPR–Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR–Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA–target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.

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Fig. 1: Overall structure of SbCas7-11–crRNA complex.
Fig. 2: Identification of the active site for pre-crRNA processing and target RNA cleavage.
Fig. 3: Overall structure of SbCas7-11–crRNA–Csx29 ternary complex.
Fig. 4: Overall structure of the SbCas7-11–crRNA–target RNA–Csx29 quaternary complex.
Fig. 5: Proposed mechanism for the type III-E CRISPR–Cas system.

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Data availability

The atomic coordinates and EM maps have been deposited in the Protein Data Bank under accession codes 7X7A (SbCas7-11–crRNA), 7X7R (SbCas7-11–crRNA–target RNA), 7X8A (SbCas7-11–crRNA–Csx29) and 7XC7 (SbCas7-11–crRNA–target RNA–Csx29), and in the Electron Microscopy Data Bank under corresponding accession codes EMD-33040, EMD-33046, EMD-33056 and EMD-33114. All data supporting the conclusions of this paper have been included. Source data are provided with this paper.

References

  1. Shmakov, S. et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).

    Article  CAS  Google Scholar 

  2. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).

    Article  CAS  Google Scholar 

  3. Özcan, A. et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7-11. Nature 597, 720–725 (2021).

    Article  Google Scholar 

  4. van Beljouw, S. P. et al. The gRAMP CRISPR–Cas effector is an RNA endonuclease complexed with a caspase-like peptidase. Science 373, 1349–1353 (2021).

    Article  Google Scholar 

  5. Samai, P. et al. Co-transcriptional DNA and RNA cleavage during type III CRISPR–Cas immunity. Cell 161, 1164–1174 (2015).

    Article  CAS  Google Scholar 

  6. Staals, R. H. et al. RNA targeting by the type III-A CRISPR–Cas Csm complex of Thermus thermophilus. Mol. Cell 56, 518–530 (2014).

    Article  CAS  Google Scholar 

  7. Tamulaitis, G. et al. Programmable RNA shredding by the type III-A CRISPR–Cas system of Streptococcus thermophilus. Mol. Cell 56, 506–517 (2014).

    Article  CAS  Google Scholar 

  8. Osawa, T., Inanaga, H., Sato, C. & Numata, T. Crystal structure of the CRISPR–Cas RNA silencing Cmr complex bound to a target analog. Mol. Cell 58, 418–430 (2015).

    Article  CAS  Google Scholar 

  9. Guo, M. et al. Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR–Csm revealed by cryo-EM and biochemistry. Cell Res. 29, 305–312 (2019).

    Article  CAS  Google Scholar 

  10. You, L. et al. Structure studies of the CRISPR–Csm complex reveal mechanism of co-transcriptional interference. Cell 176, 239–253. e216 (2019).

    Article  CAS  Google Scholar 

  11. Jia, N. et al. Type III-A CRISPR–Cas Csm complexes: assembly, periodic RNA cleavage, DNase activity regulation, and autoimmunity. Mol. Cell 73, 264–277. e265 (2019).

    Article  CAS  Google Scholar 

  12. Sofos, N. et al. Structures of the Cmr-β complex reveal the regulation of the immunity mechanism of type III-B CRISPR–Cas. Mol. Cell 79, 741–757. e747 (2020).

    Article  CAS  Google Scholar 

  13. Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, Č. & Siksnys, V. Spatiotemporal control of type III-A CRISPR–Cas immunity: coupling DNA degradation with the target RNA recognition. Mol. Cell 62, 295–306 (2016).

    Article  CAS  Google Scholar 

  14. Elmore, J. R. et al. Bipartite recognition of target RNAs activates DNA cleavage by the type III-B CRISPR–Cas system. Genes Dev. 30, 447–459 (2016).

    Article  CAS  Google Scholar 

  15. Estrella, M. A., Kuo, F.-T. & Bailey, S. RNA-activated DNA cleavage by the type III-B CRISPR–Cas effector complex. Genes Dev. 30, 460–470 (2016).

    Article  CAS  Google Scholar 

  16. Niewoehner, O. et al. Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017).

    Article  CAS  Google Scholar 

  17. Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR–Cas systems. Science 357, 605–609 (2017).

    Article  CAS  Google Scholar 

  18. Schmid, M. et al. Candidatus ‘Scalindua brodae’, sp. nov., Candidatus ‘Scalindua wagneri’, sp. nov., two new species of anaerobic ammonium oxidizing bacteria. Syst. Appl. Microbiol. 26, 529–538 (2003).

    Article  CAS  Google Scholar 

  19. Li, H. Structural principles of CRISPR RNA processing. Structure 23, 13–20 (2015).

    Article  CAS  Google Scholar 

  20. Carte, J., Wang, R., Li, H., Terns, R. M. & Terns, M. P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489–3496 (2008).

    Article  CAS  Google Scholar 

  21. Kato, K. et al. Structure and engineering of the type III-E CRISPR–Cas7-11 effector complex. Cell 185, 2324–2337 (2022).

    Article  CAS  Google Scholar 

  22. Hennig, J. et al. Structural basis for the assembly of the Sxl–Unr translation regulatory complex. Nature 515, 287–290 (2014).

    Article  CAS  Google Scholar 

  23. McLuskey, K. & Mottram, J. C. Comparative structural analysis of the caspase family with other clan CD cysteine peptidases. Biochem. J. 466, 219–232 (2015).

    Article  CAS  Google Scholar 

  24. Johnson, A. G. et al. Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375, 221–225 (2022).

    Article  CAS  Google Scholar 

  25. Hochstrasser, M. L. & Nuñez, J. K. CRISPR meets caspase. Nat. Microbiol. 6, 1481–1482 (2021).

    Article  CAS  Google Scholar 

  26. Rouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S. & White, M. F. Control of cyclic oligoadenylate synthesis in a type III CRISPR system. eLife 7, e36734 (2018).

    Article  Google Scholar 

  27. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  28. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  Google Scholar 

  29. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  Google Scholar 

  30. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  Google Scholar 

  31. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).

    Article  CAS  Google Scholar 

  32. Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D 74, 814–840 (2018).

    Article  CAS  Google Scholar 

  33. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  34. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  Google Scholar 

  35. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  Google Scholar 

  36. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  37. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  38. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

  39. Williams, C. J. et al. MolProbity: More and better reference data for improved all‐atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32071218 to H.Z. and 32200496 to G.Y.) and the CAS Pioneer Hundred Talents Program to Z.D. We thank C. Xie and J. Jin for critical suggestions. We thank G. Rao for his help in cryo-EM data collection. We thank the Center for Instrumental Analysis and Metrology of Wuhan Institute of Virology for providing technical assistance.

Author information

Author notes

  1. These authors contributed equally: Guimei Yu, Xiaoshen Wang, Yi Zhang, Qiyin An.

Authors and Affiliations

  1. Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Haihe Laboratory of Cell Ecosystem, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Institute of Immunology, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China

    Guimei Yu, Xiaoshen Wang, Yi Zhang, Yanan Wen, Xuzichao Li & Heng Zhang

  2. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China

    Qiyin An & Zengqin Deng

  3. University of Chinese Academy of Sciences, Beijing, China

    Qiyin An

  4. Department of Pharmacology, Tianjin Medical University, Tianjin, China

    Hang Yin

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Contributions

H.Z., Z.D. and G.Y. conceived the study. X.W. and Y.Z. performed molecular cloning, protein purification and biochemical assays with the help of Y.W., X.L. and H.Y. Z.D. and Q.A. collected and analysed cryo-EM data. G.Y. built and refined the structure model with input from Z.D. and H.Z. H.Z. wrote the manuscript with contributions from G.Y. and Z.D.

Corresponding authors

Correspondence to Zengqin Deng or Heng Zhang.

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Nature Microbiology thanks Andreas Boland and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 SbCas7-11 domain structures.

a-d, Atomic models and the cryo-EM densities of Cas7.1-4 domains, displaying the thumb-palm-finger architecture. e, Overall structure, and density of Cas11 domain. f, Cryo-EM density map of resolved crRNA. The repeat and spacer regions are colored in grey and orange, respectively. g, A continuous density protruding from Cas7.1 connected to the 5’ side of the 18-nt repeat at a lower threshold.

Extended Data Fig. 2 Sequence alignment of the Cas7-11 orthologs.

The residues crucial for the pre-crRNA maturation are indicated by green dots. The active-site residues in Cas7.2 and Cas7.3 are indicated by pink and cyan triangles, respectively. The residues critical for crRNA recognition and target RNA cleavage are indicated by orange stars. CjcCas7-11, Cas7-11 from Candidatus Jettenia caeni; CbfCas7-11, Cas7-11 from Candidatus Brocadia fulgida; SstCas7-11, Cas7-11 from Sorted cell/s from Southern Trench hydrothermal vent microbial mat; HsmCas7-11, Cas7-11 from Hydrothermal sediment microbial communities from Guaymas Basin; DsbaCas7-11, Cas7-11 from Desulfobactera ceae bacterium.

Extended Data Fig. 3 Structural and biochemical studies of Cas7.1 domain in pre-crRNA processing.

a, The polar residues in SbCas7-11 equivalent to those selected for mutagenesis in DiCas7-11 are shown in stick representations. b, In vitro pre-crRNA cleavage assay with WT and mutated DiCas7-11 proteins. The corresponding residues in SbCas7-11 are shown in parentheses (green). These gels are representative of three replicate experiments. c, In vitro target RNA cleavage assay of WT and mutant DiCas7-11 proteins. These gels are representative of three replicate experiments.

Source data

Extended Data Fig. 4 Structural comparison between SbCas7-11-crRNA-target RNA ternary complex and DiCas7-11-crRNA-target RNA ternary complex.

a, Superposition of the target RNA-bound SbCas7-11 ternary complex (white) and the target RNA-bound DiCas7-11 ternary complex (salmon, PDB:7WAH). b, Detailed comparison of the Cas7.1 (left), Cas7.2 (middle) and Cas7.3 (right) domains responsible for pre-crRNA maturation and target RNA cleavage at site 1 and 2, respectively. The residues in the active sites are shown in stick representation.

Extended Data Fig. 5 Recognition of crRNA by SbCas7-11.

a, The recognition of crRNA by SbCas7-11. The finger and thumb regions are shown in ribbon representations. The other areas have been omitted for clarity. The 5-nt bracket sequence is indicated by arrows. b, Close-up view of the interactions between the crRNA repeat region and SbCas7-11. The crRNA-interacting residues of SbCas7-11 are shown as sticks. c, Close-up view of the interactions between the crRNA spacer region and SbCas7-11. d, In vitro target RNA cleavage assay of WT and mutant SbCas7-11 proteins. These gels are representative of three replicate experiments.

Source data

Extended Data Fig. 6 IPD may associate with crRNA and regulate target RNA cleavage.

a, Cryo-EM density of SbCas7-11-crRNA complex with the focused refined IPD region. An atomic model predicted with AlphaFold2 was docked and refined with the density. An extra density (circled in magenta) was observed in IPD, potentially attributed to crRNA. b, Superimposition of the predicted IPD domain and CSD1 domain in the Upstream-of-N-ras (Unr) protein (PDB: 4QQB). c, In vitro target RNA cleavage assay shows that the IPD domain is dispensable for the target RNA cleavage activity of SbCas7-11. This gel is a representative of three replicate experiments. Del-IPD-1: The IPD region (aa 1032-1387) was replaced with a GGGS linker in SbCas7-11. Del-IPD-2: The IPD region (aa 1038-1383) was replaced with a GSAS linker in SbCas7-11.

Source data

Extended Data Fig. 7 Catalytic sites of Cas7.2 and Cas7.3 for target RNA cleavage.

a, Structural comparisons of Cas7.2 and Cas7.3 domains in SbCas7-11 with Csm3 protein (white, PDB: 6NUE). b, Close-up view of the catalytic sites. The catalytic D33 residues in Csm3 are indicated by arrows. Corresponding residues and those in close proximity of SbCas7-11 are marked. c, In vitro target RNA cleavage with SbCas7-11 variants bearing mutations in Cas11 and Cas7.2 domains. These gels are representative of three replicate experiments. d, Representative target RNA cleavage assay for SbCas7-11 WT and mutants. Time courses represent 5, 15, and 30 min. This gel is a representative of three replicate experiments. e, In vitro target RNA cleavage with DiCas7-11 variants bearing mutations in Cas7.2 domain. The gel is a representative of three replicate experiments. f, Superimposition of the SbCas7-11-crRNA binary complex (white) and the SbCas7-11-crRNA-target RNA ternary complex. The Cas11 domain rotates outward upon binding of target RNA.

Source data

Extended Data Fig. 8 Structural analysis of Csx29 and SbCas7-11 in different complexes.

a, Superimposition of the PS1 and PS2 domains in Csx29. The PS1 subdomain is roughly similar to PS2 despite the lack of the catalytic dyad. b, In vitro pull-down of Csx29 WT and mutants by MBP-tagged SbCas7-11 proteins. The Csx29 bands are indicated by red stars. This gel is a representative of three replicate experiments. c, Superimposition of the SbCas7-11-crRNA complex (white) and the SbCas7-11-crRNA-Csx29 complex. d, Superimposition of the SbCas7-11-crRNA-taget RNA complex (ruby) and SbCas7-11-crRNA-Csx29 complex.

Source data

Extended Data Fig. 9 Biochemical characterizations of the effect of Csx29 on SbCas7-11 target RNA cleavage and the potential protease activity of Csx29.

a, In vitro target RNA cleavage assay in the presence or absence of Csx29. This gel is a representative of three replicate experiments. b, Representative target RNA cleavage assay. The assay was carried out at room temperature. Time courses represent 1, 5, 15 and 30 min. This gel is representative of three replicate experiments. c, The EMSA assay with catalytic dead SbCas7-11 proteins. The EMSA was performed using the Cy3-labeled target RNA as a probe. The triangle indicates the increasing concentrations of SbCas7-11-crRNA binary complex or SbCas7-11-crRNA-Csx29 ternary complex proteins. This gel is a representative of three replicate experiments. d, Csx29 failed to cleave the tested candidate substrate proteins. Human GSDMD bearing the XXXD (FLTD) motif, one of the caspase substrates, was used for the assay. We also mutated the XXXD motif to EXXR (ELTR) motif in human GSDMD to mimic the recognition motif of caspase-like protease Separase. This gel is a representative of three replicate experiments.

Source data

Extended Data Fig. 10 Csx29 undergoes substantial conformational changes in the presence of target RNA.

a, Structural alignment of Csx29-bound complexes. The Csx29 model in SbCas7-11-crRNA-Csx29 complex is shown in ribbon representations and colored in white. The SbCas7-11-crRNA model in SbCas7-11-crRNA-Csx29 complex has been omitted for clarity. The SbCas7-11-crRNA-target RNA model is shown in surface representations. b, Target RNA binding triggers the movements of Csx29. c, The protease domain is projected outward, and the catalytic dyad is upper shifted upon binding of target RNA. d-f, Close-up view of the β6-α19 loop positions of Csx29 in the absence (d) or presence (e) of target RNA. The β6-α19 loop, especially Asp543, has the potential to block the access to the catalytic pocket (d), while the β6-α19 loop is shifted away from SbCas7-11 upon binding of target RNA (e). Additionally, although the aromatic residues in the β6-α19 loop occupy the P1 position of the substrate, these residues move away and empty the P1-binding pocket in the presence of target RNA. Binding of target RNA induce the conformational dynamic of the loop harboring H585, the disordered region is indicated by red circle (f).

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Supplementary text, references, Figs. 1–6, Table 1 and uncropped gel images.

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Yu, G., Wang, X., Zhang, Y. et al. Structure and function of a bacterial type III-E CRISPR–Cas7-11 complex. Nat Microbiol 7, 2078–2088 (2022). https://doi.org/10.1038/s41564-022-01256-z

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