Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Foreign DNA capture during CRISPR–Cas adaptive immunity

Abstract

Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments1,2,3,4,5,6. The universally conserved Cas1–Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7,8,9,10,11,12,13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2,3,4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overall architecture and active site positioning of 3′-OH nucleophile.
Figure 2: Coordination of protospacer DNA within the complex.
Figure 3: Mechanism of protospacer DNA end separation.
Figure 4: Model of protospacer DNA integration.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited at the Protein Data Bank under accession codes 5DS4 (no Mg2+), 5DS5 (with Mg2+) and 5DS6 (splayed DNA).

References

  1. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007)

    CAS  ADS  Article  Google Scholar 

  2. 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, 174–182 (2005)

    CAS  ADS  Article  Google Scholar 

  3. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005)

    CAS  Article  Google Scholar 

  4. 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, 653–663 (2005)

    CAS  Article  Google Scholar 

  5. Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010)

    CAS  ADS  Article  Google Scholar 

  6. van der Oost, J., Westra, E. R., Jackson, R. N. & Wiedenheft, B. Unravelling the structural and mechanistic basis of CRISPR–Cas systems. Nature Rev. Microbiol. 12, 479–492 (2014)

    CAS  Google Scholar 

  7. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli . Nucleic Acids Res. 40, 5569–5576 (2012)

    CAS  Article  Google Scholar 

  8. Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nature Comm. 3, 945 (2012)

    ADS  Article  Google Scholar 

  9. Swarts, D. C., Mosterd, C., van Passel, M. W. & Brouns, S. J. CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7, e35888 (2012)

    CAS  ADS  Article  Google Scholar 

  10. Nuñez, J. K. et al. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nature Struct. Mol. Biol. 21, 528–534 (2014)

    Article  Google Scholar 

  11. Arslan, Z., Hermanns, V., Wurm, R., Wagner, R. & Pul, U. Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system. Nucleic Acids Res. 42, 7884–7893 (2014)

    CAS  Article  Google Scholar 

  12. Nuñez, J. K., Lee, A. S., Engelman, A. & Doudna, J. A. Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity. Nature 519, 193–198 (2015)

    ADS  Article  Google Scholar 

  13. Rollie, C., Schneider, S., Brinkmann, A. S., Bolt, E. L. & White, M. F. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition. eLife 4, 10.7554/eLife.08716 (2015)

  14. Heler, R., Marraffini, L. A. & Bikard, D. Adapting to new threats: the generation of memory by CRISPR–Cas immune systems. Mol. Microbiol. 93, 1–9 (2014)

    CAS  Article  Google Scholar 

  15. Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008)

    CAS  ADS  Article  Google Scholar 

  16. 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)

    CAS  Article  Google Scholar 

  17. Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010)

    CAS  ADS  Article  Google Scholar 

  18. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011)

    CAS  ADS  Article  Google Scholar 

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

    CAS  ADS  Article  Google Scholar 

  20. Levy, A. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015)

    CAS  ADS  Article  Google Scholar 

  21. Wiedenheft, B. et al. Structural basis for DNase activity of a conserved protein implicted in CRISPR-mediated genome defense. Structure 17, 904–912 (2009)

    CAS  Article  Google Scholar 

  22. Savilahti, H., Rice, P. A. & Mizuuchi, K. The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J. 14, 4893–4903 (1995)

    CAS  Article  Google Scholar 

  23. Scottoline, B. P., Chow, S., Ellison, V. & Brown, P. O. Disruption of the terminal base pairs of retroviral DNA during integration. Genes Dev. 11, 371–382 (1997)

    CAS  Article  Google Scholar 

  24. Katz, R. A., Merkel, G., Andrake, M. D., Roder, H. & Skalka, A. M. Retroviral integrases promote fraying of viral DNA ends. J. Biol. Chem. 286, 25710–25718 (2011)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  27. Babu, M. et al. A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair. Mol. Microbiol. 79, 484–502 (2011)

    CAS  Article  Google Scholar 

  28. Makarova, K. S. et al. Evolution and classification of the CRISPR–Cas systems. Nature Rev. Microbiol. 9, 467–477 (2011)

    CAS  Google Scholar 

  29. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank G. Meigs and the 8.3.1 beamline staff at the Advanced Light Source for assistance with data collection, J. Chen for input on experimental design and members of the Doudna laboratory for comments and discussions. The 8.3.1 beamline is supported by UC Office of the President, Multicampus Research Programs and Initiatives grant MR-15-328599 and Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation. This project was funded by US National Science Foundation grant No. 1244557 to J.A.D. and by NIH grant AI070042 to A.N.E. J.K.N. and L.B.H. are supported by US National Science Foundation Graduate Research Fellowships and J.K.N. by a UC Berkeley Chancellor’s Graduate Fellowship. P.J.K. is supported as a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. J.A.D. is an Investigator of the Howard Hughes Medical Institute and a member of the Center for RNA Systems Biology.

Author information

Authors and Affiliations

Authors

Contributions

J.K.N. and L.B.H. conducted the crystallography, biochemistry and in vivo spacer acquisition assays. J.K.N., L.B.H. and P.J.K. collected the X-ray diffraction data and determined the crystal structures. J.K.N., L.B.H., P.J.K., A.N.E. and J.A.D. designed the study, analysed all data and wrote the manuscript.

Corresponding author

Correspondence to Jennifer A. Doudna.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Effect of overhang length on integration efficiency.

a, A plot of the per cent integration of protospacers ± standard deviation with varying 3′ single-stranded DNA extensions. A representative gel is shown in Fig. 1a. b, Protospacer sequences used for the assays described in a and Fig. 1a, with the red nucleotides indicating the 3′ overhang regions.

Extended Data Figure 2 Assembly of Cas1–Cas2 complex bound to protospacer DNA.

a, Gel filtration chromatogram of pre-assembled Cas1–Cas2 complex with protospacer DNA containing five-nucleotide 3′ overhangs. The dotted lines indicate the peak fractions of the Cas1–Cas2 complex without DNA, as shown in d. The dotted lines indicate the peak fractions of the Cas1–Cas2 complex bound to DNA (first peak) and excess, unbound DNA (second peak). b, c, The fractions from peak 1 (~12 ml) and peak 2 (~15 ml) were analysed by Coomassie-stained SDS–PAGE (b) and 12% urea-PAGE (c) to confirm the presence of Cas1, Cas2 and protospacer DNA. d, Gel-filtration chromatogram of assembled Cas1–Cas2 without protospacer DNA. e, Coomassie-stained SDS–PAGE of the peak fractions from d. Supplementary Information contains the full images for b, c and e.

Extended Data Figure 3 Conformational dynamics upon protospacer DNA binding.

a, An overlay of the DNA-bound Cas1–Cas2 structure with the apo Cas1–Cas2 (grey, PDB 4P6I). b, Vector lines depicting the conformational changes the Cas1–Cas2 complex undergoes upon protospacer DNA binding compared to the apo complex (PDB 4P6I). The Cas1 subunits rotate towards the direction of the arrows.

Extended Data Figure 4 Omit maps of the protospacer DNA.

a, Simulated annealing Fo − Fc omit electron density map of the entire protospacer DNA using the ‘no Mg2+’ map and model. b, c, Simulated annealing Fo − Fc omit electron density maps of the terminal five nucleotides in the active sites of the structures (a) with Mg2+ or (b) without Mg2+ in the crystallization condition. The maps are contoured at 2.0σ.

Extended Data Figure 5 Sequence alignment of Cas1 proteins in type I CRISPR systems.

Sequence alignments of Cas1 from representative organisms with type I CRISPR systems. The E. coli sequence is displayed at the top. The dots indicate the residues described in this study, with the red dots indicating the metal-binding residues. The box highlights the non-universal conservation of the E. coli Y22 residue in the β1 region of type I CRISPR systems. The secondary structure representations shown are for the E. coli Cas1.

Extended Data Figure 6 Integration of protospacer substrates with splayed ends.

a, Representative agarose gel of in vitro integration reactions using increasing lengths of splayed ends. The average per cent integration of three independent experiments is plotted in Fig. 3d. b, Sequences of protospacers used in the integration assays in a. c, A 12% denaturing polyacrylamide gel of protospacers after incubation with Cas1–Cas2 for 1 h at 37 °C in integration assay buffer conditions. The indicated DNA substrates are radiolabelled at the 5′ end. Supplementary Information contains the full images for a and c. nt, nucleotide.

Extended Data Figure 7 Crystallographic packing of the complex bound to Mg2+.

a, View of the symmetry mates (grey) contacting the non-catalytic Cas1 subunits (green). Catalytic Cas1 subunits are shown in blue, Cas2 in yellow and DNA is shown in salmon and red. b, Superposition of our two crystal structures, with or without Mg2+, shows a slight DNA kink in the structure bound to Mg2+ (dotted box). This region contacts α-helix 7 of a symmetry mate, as described in the text.

Extended Data Table 1 Summary of X-ray crystallography data collection and refinement

Supplementary information

Supplementary Information

This file contains uncropped gel images with size marker indications. (PDF 1129 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nuñez, J., Harrington, L., Kranzusch, P. et al. Foreign DNA capture during CRISPR–Cas adaptive immunity. Nature 527, 535–538 (2015). https://doi.org/10.1038/nature15760

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature15760

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing