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Structural basis for CRISPR RNA-guided DNA recognition by Cascade

Nature Structural & Molecular Biology volume 18, pages 529–536 (2011)Cite this article

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Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

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Figure 1: Core complexes of Cascade retain crRNA.
Figure 2: Architecture of crRNA.
Figure 3: Target recognition by Cascade.
Figure 4: R-loop formation by Cascade.
Figure 5: Subunit composition of Cascade.
Figure 6: EM structure of Cascade.

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Acknowledgements

We thank M.H. Lai for experimental contributions, and E. Schaible, P. Zwart and M. Bokhove for technical support and for assistance with post processing of SAXS data. This work was financially supported by The Netherlands Organisation for Scientific Research (NWO) Vici grant to J.v.d.O (865.05.001), Veni grants to S.J.J.B. (863.08.014) and E.v.D. (700.58.402), NWO TOP grant to E.J.B., and UK Engineering and Physical Sciences Research Council and Biotechnology and Biological Sciences Research Council grants to M.J.D. M.L. was financially supported by the Wenner-Gren Foundation, E.R.W. by Spinoza resources awarded to W.M. de Vos, A.P.L.S. by the Research Councils UK, B.W. by the Life Sciences Research Foundation of HHMI and Ü.P. by the Deutsche Forschungsgemeinschaft PU 435/1-1. We thank The Netherlands Proteomics Center for financial support.

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Author notes

  1. Magnus Lundgren & Ambrosius P L Snijders

    Present address: Present addresses: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden (M.L.); MRC Clinical Sciences Centre, Imperial College London, London, UK (A.P.L.S.).,

  2. Matthijs M Jore, Magnus Lundgren, Esther van Duijn and Jelle B Bultema: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Agrotechnology and Food Sciences, Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands

    Matthijs M Jore, Magnus Lundgren, Edze R Westra, Marieke R Beijer, John van der Oost & Stan J J Brouns

  2. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and the Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Esther van Duijn, Arjan Barendregt & Albert J R Heck

  3. The Netherlands Proteomics Center, Utrecht, The Netherlands

    Esther van Duijn, Arjan Barendregt & Albert J R Heck

  4. Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands

    Jelle B Bultema & Egbert J Boekema

  5. Department of Chemical and Process Engineering, ChELSI Institute, University of Sheffield, Sheffield, UK

    Sakharam P Waghmare, Ambrosius P L Snijders & Mark J Dickman

  6. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.,

    Blake Wiedenheft, Kaihong Zhou & Jennifer A Doudna

  7. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California.,

    Blake Wiedenheft, Kaihong Zhou & Jennifer A Doudna

  8. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Blake Wiedenheft, Kaihong Zhou & Jennifer A Doudna

  9. Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

    Ümit Pul, Reinhild Wurm & Rolf Wagner

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Contributions

M.M.J., M.L., E.R.W., M.R.B., J.J.B. and J.v.d.O. purified Cascade and performed binding assays and plaque assays. E.v.D., A.B. and A.J.R.H. conducted protein MS experiments. J.B.B. and E.J.B. performed EM. S.P.W., A.P.L.S. and M.J.D. conducted RNA MS experiments. B.W., K.Z. and J.A.D. performed SAXS. Ü.P., R. Wurm and R. Wagner did the footprint analyses. All the authors analyzed the data, and S.J.J.B., M.M.J. and J.v.d.O. wrote the manuscript with input from all authors.

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Correspondence to John van der Oost.

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Jore, M., Lundgren, M., van Duijn, E. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18, 529–536 (2011). https://doi.org/10.1038/nsmb.2019

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