Structural basis for CRISPR RNA-guided DNA recognition by Cascade

Journal name:
Nature Structural & Molecular Biology
Volume:
18,
Pages:
529–536
Year published:
DOI:
doi:10.1038/nsmb.2019
Received
Accepted
Published online

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.

At a glance

Figures

  1. Core complexes of Cascade retain crRNA.
    Figure 1: Core complexes of Cascade retain crRNA.

    (a) Schematic diagram of the CRISPR-Cas locus in E. coli K12 containing cas3 (ygcB), casA (cse1, ygcL), casB (cse2, ygcK), casC (cse4, ygcJ), casD (cas5e, ygcI), casE (cse3, ygcH), cas1 (ygbT) and cas2 (ygbF)9, 10. (b) Coomassie blue–stained SDS-polyacrylamide gel showing StrepTactin-purified Cascade, CasBCDE and CasCDE. Protein marker sizes in kDa. Asterisk, Strep-tagged subunits. (c) Ethidium bromide–stained denaturing PAA-gel showing nucleic acids isolated from purified Cascade (sub)complexes. RNA marker sizes in nucleotides. (d) RNase A or DNase I treatment of Cascade-bound nucleic acids. (e) Size exclusion elution profiles of CasCDE, CasBCDE and Cascade before and after DNase I treatment.

  2. Architecture of crRNA.
    Figure 2: Architecture of crRNA.

    (a) Ion-pair reversed-phase HPLC purification of mature R44 crRNA at 75 °C. (b) Multiple-charged ESI-MS spectrum of the purified mature crRNA. (c) Enhanced view of the −18 charged species before (top) and after (bottom) acid treatment indicating hydrolysis of the 2′,3′-cyclic phosphate. (d) Diagram of mature crRNA derived from the R44 CRISPR.

  3. Target recognition by Cascade.
    Figure 3: Target recognition by Cascade.

    (a,b) Effect of the type of crRNA bound. Cascade was loaded with either targeting crRNA (R44 CRISPR, Supplementary Fig. 2) or nontargeting crRNA (K12 CRISPR). The binding of these two types of Cascade complex to one type of probe is shown. DNA probes are 86-nucleotide ssDNA or dsDNA sequences containing the R44 protospacer (32 nucleotides) flanked by 27 nucleotides on either end. (ch) Effect of uniform crRNA-loaded (sub)complexes (R44 CRISPR) on the binding of single- or double-stranded target and nontarget DNA. Nontarget DNA probes contain a scrambled R44 protospacer sequence. (i,j) Effect of uniform crRNA-loaded Cascade (R44 CRISPR) on the binding of target and nontarget ssRNA and dsRNA. (ah) Labeled probe concentration 1 nM; DNA competitor concentration 2,500, 500, 50, 5 and 0.5 ng μl−1 (highest concentration not used for CasCDE); protein concentration 200–300 nM except in i,j where the Cascade concentrations were 200, 100, 50, 25 and 12.5 nM.

  4. R-loop formation by Cascade.
    Figure 4: R-loop formation by Cascade.

    (a) Competition assay between R44-crRNA–loaded Cascade and CasBCDE for R44 ssDNA target. Total protein concentration was 500 nM in each reaction, and the Cascade:CasBCDE ratio was 1:0, 100:1, 10:1, 1:1, 1:10, 1:100 and 0:1. DNA competitor concentration was 1 μg μl−1. (b,c) Effect of labeling the complementary or noncomplementary strand of a target dsDNA with 27-bp flanks (b) or without protospacer-flanking sequences (c). Cascade concentrations were 1,500, 300, 60 and 12.5 nM. (d,e) Mapping of ssDNA regions in the Cascade–target DNA complex using nuclease P1 and KMnO4. Sensitive regions are indicated by dashed lines and the protospacer by a solid line according to the G+A sequencing lanes of each strand. Cascade loaded with K12-derived crRNA was used as a control. (f) Exonuclease III mapping of accessible dsDNA regions upstream and downstream of the Cascade–DNA complex. The borders of the Cascade-protected regions are indicated by arrows. (g) Detection of the R-loop in a target plasmid. Agarose gel indicating the mobility of the different plasmid forms (SC, supercoiled; L, linear; OC, open circular) and the mobility shifts caused by R44-Cascade and R44-crRNA binding. (h) Schematic diagram of the R-loop formed in crRNA-guided dsDNA recognition by Cascade. Regions sensitive to nuclease P1 and KMnO4 are indicated by hash and asterisk signs, respectively.

  5. Subunit composition of Cascade.
    Figure 5: Subunit composition of Cascade.

    (a) Native nano-ESI mass spectrum of Cascade. Two charge state distributions are present at high m/z values, corresponding to complexes of 405 kDa (purple) and 349 kDa (pink). The charge state distribution in red corresponds to the CasB dimer. (b) Cascade (sub)complexes analyzed by native mass spectrometry. The subcomplexes were formed in solution after adding 5% 2-propanol to the buffer solution containing Cascade.

  6. EM structure of Cascade.
    Figure 6: EM structure of Cascade.

    (ac,s) Three Cascade projections showing an elongated, seahorse-shaped particle with dimensions 20 × 10 nm. (di) Six Cascade projections bound to target ssDNA. The arrow indicates an anticipated rotation along the vertical axis. The six regularly arranged CasC subunits are indicated by asterisks. (jl) Difference map (l) of Cascade (j) and Cascade with target ssDNA bound (k) indicating morphological changes upon DNA binding in mainly the head and back areas. (mo) Difference map (o) of Cascade (m) and CasBCDE (n) with target ssDNAs bound, showing the location of the CasA subunit. (pr) Difference map (r) of CasBCDE (p) and CasCDE (q), showing the location of the CasB subunits. (s) Enlargement of a defining the seahorse-like morphological features of Cascade. A typical indentation that contributes to the head feature of the seahorse is indicated with a hash sign. The number of particle projections to create the average shown is depicted on the bottom-right corner of each image. The § sign indicates that the depicted particle is a mirrored view of the original to match the predominant particle orientation in a. Raw electron micrographs and an overview of the particle analysis method are given in Supplementary Figures 7 and 8, respectively. Scale bar, 10 nm. (t) Structural model of Cascade.

References

  1. 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, 401407 (2009).
  2. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167170 (2010).
  3. Karginov, F.V. & Hannon, G.J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol. Cell 37, 719 (2010).
  4. Marraffini, L.A. & Sontheimer, E.J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11, 181190 (2010).
  5. Deveau, H., Garneau, J.E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475493 (2010).
  6. Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174182 (2005).
  7. Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653663 (2005).
  8. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 25512561 (2005).
  9. Jansen, R., Embden, J.D., Gaastra, W. & Schouls, L.M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 15651575 (2002).
  10. Haft, D.H., Selengut, J., Mongodin, E.F. & Nelson, K.E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLOS Comput. Biol. 1, e60 (2005).
  11. Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I. & Koonin, E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7 (2006).
  12. Kunin, V., Sorek, R. & Hugenholtz, P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 8, R61 (2007).
  13. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 17091712 (2007).
  14. Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 14011412 (2008).
  15. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 6771 (2010).
  16. Wiedenheft, B. et al. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17, 904912 (2009).
  17. Tang, T.H. et al. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. USA 99, 75367541 (2002).
  18. Hale, C., Kleppe, K., Terns, R.M. & Terns, M.P. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14, 25722579 (2008).
  19. Lillestøl, R.K. et al. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72, 259272 (2009).
  20. Brouns, S.J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960964 (2008).
  21. 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, 34893496 (2008).
  22. Haurwitz, R.E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 13551358 (2010).
  23. Hale, C.R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945956 (2009).
  24. Marraffini, L.A. & Sontheimer, E.J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 18431845 (2008).
  25. Dickman, M.J. & Hornby, D.P. Enrichment and analysis of RNA centered on ion pair reverse phase methodology. RNA 12, 691696 (2006).
  26. Waghmare, S.P., Pousinis, P., Hornby, D.P. & Dickman, M.J. Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions. J. Chromatogr. A 1216, 13771382 (2009).
  27. Raghavan, S.C., Tsai, A., Hsieh, C.L. & Lieber, M.R. Analysis of non-B DNA structure at chromosomal sites in the mammalian genome. Methods Enzymol. 409, 301316 (2006).
  28. Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733740 (2009).
  29. Wilson-Sali, T. & Hsieh, T.S. Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIbeta. Proc. Natl. Acad. Sci. USA 99, 79747979 (2002).
  30. Thomas, M., White, R.L. & Davis, R.W. Hybridization of RNA to double-stranded DNA: formation of R-loops. Proc. Natl. Acad. Sci. USA 73, 22942298 (1976).
  31. Heck, A.J.R. Native mass spectrometry: a bridge between interactomics and structural biology. Nat. Methods 5, 927933 (2008).
  32. Lorenzen, K., Vannini, A., Cramer, P. & Heck, A.J. Structural biology of RNA polymerase III: mass spectrometry elucidates subcomplex architecture. Structure 15, 12371245 (2007).
  33. Zhou, M. et al. Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3. Proc. Natl. Acad. Sci. USA 105, 1813918144 (2008).
  34. Agari, Y., Yokoyama, S., Kuramitsu, S. & Shinkai, A. X-ray crystal structure of a CRISPR-associated protein, Cse2, from Thermus thermophilus HB8. Proteins 73, 10631067 (2008).
  35. Ma, J.B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666670 (2005).
  36. Li, J., Yang, Z., Yu, B., Liu, J. & Chen, X. Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr. Biol. 15, 15011507 (2005).
  37. Houwing, S. et al. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129, 6982 (2007).
  38. Marraffini, L.A. & Sontheimer, E.J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568571 (2010).
  39. Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 13901400 (2008).
  40. Singleton, M.R., Dillingham, M.S. & Wigley, D.B. Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 2350 (2007).
  41. Itoh, T. & Tomizawa, J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc. Natl. Acad. Sci. USA 77, 24502454 (1980).
  42. Baker, T.A. & Kornberg, A. Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell 55, 113123 (1988).
  43. Lee, D.Y. & Clayton, D.A. Initiation of mitochondrial DNA replication by transcription and R-loop processing. J. Biol. Chem. 273, 3061430621 (1998).
  44. Yu, K., Chedin, F., Hsieh, C.L., Wilson, T.E. & Lieber, M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442451 (2003).
  45. Gnatt, A.L., Cramer, P., Fu, J., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution. Science 292, 18761882 (2001).
  46. Noy, A., Perez, A., Marquez, M., Luque, F.J. & Orozco, M. Structure, recognition properties, and flexibility of the DNA.RNA hybrid. J. Am. Chem. Soc. 127, 49104920 (2005).
  47. Han, D. & Krauss, G. Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2. FEBS Lett. 583, 771776 (2009).
  48. Tahallah, N., Pinkse, M., Maier, C.S. & Heck, A.J. The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. Rapid Commun. Mass Spectrom. 15, 596601 (2001).
  49. van den Heuvel, R.H. et al. Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. Anal. Chem. 78, 74737483 (2006).
  50. Maxam, A.M. & Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499560 (1980).
  51. Oostergetel, G.T., Keegstra, W. & Brisson, A. Automation of specimen selection and data acquisition for protein electron crystallography. Ultramicroscopy 74, 4759 (1998).
  52. van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307369 (2000).

Download references

Author information

  1. These authors contributed equally to this work.

    • Matthijs M Jore,
    • Magnus Lundgren,
    • Esther van Duijn &
    • Jelle B Bultema

Affiliations

  1. Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, 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. ChELSI Institute, Department of Chemical and Process Engineering, 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
  10. 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.).

    • Magnus Lundgren &
    • Ambrosius P L Snijders

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (5M)

    Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary Methods

Additional data