Letter | Published:

Foreign DNA capture during CRISPR–Cas adaptive immunity

Nature volume 527, pages 535538 (26 November 2015) | Download Citation


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

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).


  1. 1.

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

  2. 2.

    , , & Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005)

  3. 3.

    , , & Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005)

  4. 4.

    , & 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)

  5. 5.

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

  6. 6.

    , , & Unravelling the structural and mechanistic basis of CRISPR–Cas systems. Nature Rev. Microbiol. 12, 479–492 (2014)

  7. 7.

    , & Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012)

  8. 8.

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

  9. 9.

    , , & CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7, e35888 (2012)

  10. 10.

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

  11. 11.

    , , , & Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system. Nucleic Acids Res. 42, 7884–7893 (2014)

  12. 12.

    , , & Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity. Nature 519, 193–198 (2015)

  13. 13.

    , , , & Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition. eLife 4, 10.7554/eLife.08716 (2015)

  14. 14.

    , & Adapting to new threats: the generation of memory by CRISPR–Cas immune systems. Mol. Microbiol. 93, 1–9 (2014)

  15. 15.

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

  16. 16.

    , , , & Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489–3496 (2008)

  17. 17.

    , , , & Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010)

  18. 18.

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

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

    , & The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J. 14, 4893–4903 (1995)

  23. 23.

    , , & Disruption of the terminal base pairs of retroviral DNA during integration. Genes Dev. 11, 371–382 (1997)

  24. 24.

    , , , & Retroviral integrases promote fraying of viral DNA ends. J. Biol. Chem. 286, 25710–25718 (2011)

  25. 25.

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

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

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

Download references


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

Author notes

    • James K. Nuñez
    •  & Lucas B. Harrington

    These authors contributed equally to this work.


  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA

    • James K. Nuñez
    • , Lucas B. Harrington
    • , Philip J. Kranzusch
    •  & Jennifer A. Doudna
  2. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA

    • Philip J. Kranzusch
    •  & Jennifer A. Doudna
  3. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA

    • Alan N. Engelman
  4. Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Alan N. Engelman
  5. Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  7. Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California 94720, USA.

    • Jennifer A. Doudna
  8. Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA

    • Jennifer A. Doudna


  1. Search for James K. Nuñez in:

  2. Search for Lucas B. Harrington in:

  3. Search for Philip J. Kranzusch in:

  4. Search for Alan N. Engelman in:

  5. Search for Jennifer A. Doudna in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jennifer A. Doudna.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains uncropped gel images with size marker indications.

About this article

Publication history






Further reading


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.