Article | Published:

Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity

Nature volume 519, pages 193198 (12 March 2015) | Download Citation

Abstract

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3′-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

Sequencing data are deposited in Gene Expression Omnibus under accession number GSE64552.

References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

    , & 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)

  6. 6.

    , , , & Self-targeting by CRISPR: gene regulation or autoimmunity? Trends in Genet. 26, 335–340 (2010)

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

    , , & Crystal structure of Cas1 from Archaeoglobus fulgidus and characterization of its nucleolytic activity. Biochem. Biophys. Res. Commun. 441, 720–725 (2013)

  20. 20.

    et al. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem. 283, 20361–20371 (2008)

  21. 21.

    , & Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 1552–1556 (2010)

  22. 22.

    et al. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein. J. Biol. Chem. 287, 35943–35952 (2012)

  23. 23.

    & Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration. J. Biol. Chem. 280, 29334–29339 (2005)

  24. 24.

    LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro. Nucleic Acids Res. 35, 113–124 (2007)

  25. 25.

    et al. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75. PLoS Pathog. 5, e1000259 (2009)

  26. 26.

    , & Positional information within the Mu transposase tetramer: catalytic contributions of individual monomers. Cell 85, 447–455 (1996)

  27. 27.

    , , , & Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell 31, 43–51 (1982)

  28. 28.

    , & Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell 31, 35–42 (1982)

  29. 29.

    , , & Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science 255, 723–726 (1992)

  30. 30.

    , & True reversal of Mu integration. EMBO J. 23, 3408–3420 (2004)

  31. 31.

    , & HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell 67, 1211–1221 (1991)

  32. 32.

    & Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism. Cell 66, 129–140 (1991)

  33. 33.

    & The outs and ins of transposition: from mu to kangaroo. Nature Rev. Mol. Cell Biol. 4, 865–877 (2003)

  34. 34.

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

  35. 35.

    & Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ. Microbiol. 10, 200–207 (2008)

  36. 36.

    & Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA. Nucleic Acids Res. 13, 6137–6154 (1985)

  37. 37.

    , , & Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli. J. Mol. Biol. 423, 14–16 (2012)

  38. 38.

    , , , & High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli. RNA Biol. 10, 716–725 (2013)

  39. 39.

    et al. Pervasive generation of oppositely oriented spacers during CRISPR adaptation. Nucleic Acids Res. 42, 5907–5916 (2014)

  40. 40.

    et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008)

  41. 41.

    et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011)

  42. 42.

    et al. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet. 9, e1003742 (2013)

  43. 43.

    & HIV DNA integration. Cold Spring Harbor Perspect. Med. 2, a006890 (2012)

  44. 44.

    Retroviral integrase superfamily: the structural perspective. EMBO Rep. 10, 144–151 (2009)

  45. 45.

    & Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem. Sci. 40, 58–66 (2015)

  46. 46.

    Local supercoil-stabilized DNA structures. Crit. Rev. Biochem. Mol. Biol. 26, 151–226 (1991)

  47. 47.

    & Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity. J. Virol. 69, 5908–5911 (1995)

  48. 48.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  49. 49.

    , , & WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)

Download references

Acknowledgements

We are grateful to M. Chung, P. J. Kranzusch and A.V. Wright for technical assistance and members of the Doudna laboratory and J. Cate for discussions. This project was funded by US National Science Foundation grant no. 1244557 to J.A.D. and by NIH grant AI070042 to A.E. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 Instrumentation Grants S10RR029668 and S10RR027303. J.K.N. is supported by a US National Science Foundation Graduate Research Fellowship and a UC Berkeley Chancellor’s Graduate Fellowship. A.S.Y.L. is supported as an American Cancer Society Postdoctoral Fellow (PF-14-108-01-RMC). J.A.D. is an Investigator of the Howard Hughes Medical Institute and a member of the Center for RNA Systems Biology.

Author information

Affiliations

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

    • James K. Nuñez
    • , Amy S. Y. Lee
    •  & Jennifer A. Doudna
  2. Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA

    • Amy S. Y. Lee
    •  & Jennifer A. Doudna
  3. Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Alan Engelman
  4. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  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

Authors

  1. Search for James K. Nuñez in:

  2. Search for Amy S. Y. Lee in:

  3. Search for Alan Engelman in:

  4. Search for Jennifer A. Doudna in:

Contributions

J.K.N. performed the biochemical experiments. A.S.Y.L. processed and analysed the high-throughput sequencing data. J.K.N., A.S.Y.L., A.E. and J.A.D. designed the study, analysed the data and wrote the manuscript.

Competing interests

J.A.D. and J.K.N. have filed a related patent application.

Corresponding author

Correspondence to Jennifer A. Doudna.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14237

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.