Letter | Published:

How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration

Nature volume 550, pages 137141 (05 October 2017) | Download Citation

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1,2,3,4,5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6,7,8,9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR–Cas systems, the spacer integration mechanism is essentially identical7,8,9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer6,10. The prespacer is bound by Cas1–Cas2 as a dual-forked DNA, and the terminal 3′-OH of each 3′ overhang serves as an attacking nucleophile during integration11,12,13,14. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat7,15,16,17,18,19,20. Spacer integration in the well-studied Escherichia coli type I–E CRISPR system also relies on the bacterial integration host factor21,22. In type II–A CRISPR, however, Cas1–Cas2 alone integrates spacers efficiently in vitro18; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers17,23. Here we present four structural snapshots from the type II–A system24 of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1–Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3′ overhangs. Three molecular events take place upon encountering a target: first, the Cas1–Cas2–prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′ overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.

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

References

  1. 1.

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

  2. 2.

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

  3. 3.

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

    & CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008)

  6. 6.

    et al. CRISPR–Cas: adapting to change. Science 356, eaal5056 (2017)

  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. Discovery and functional characterization of diverse class 2 CRISPR–Cas systems. Mol. Cell 60, 385–397 (2015)

  9. 9.

    et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015)

  10. 10.

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

  11. 11.

    et al. Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR–Cas systems. Cell 163, 840–853 (2015)

  12. 12.

    , , , & Foreign DNA capture during CRISPR–Cas adaptive immunity. Nature 527, 535–538 (2015)

  13. 13.

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

  14. 14.

    , , , & Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition. eLife 4, e08716 (2015)

  15. 15.

    , , , & CRISPR–spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR–Cas I-E variants of Escherichia coli. RNA Biol. 10, 792–802 (2013)

  16. 16.

    & CRISPR–Cas systems optimize their immune response by specifying the site of spacer integration. Mol. Cell 64, 616–623 (2016)

  17. 17.

    , , & Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus. Nucleic Acids Res. 43, 1749–1758 (2015)

  18. 18.

    & Protecting genome integrity during CRISPR immune adaptation. Nat. Struct. Mol. Biol. 23, 876–883 (2016)

  19. 19.

    et al. Repeat size determination by two molecular rulers in the type IE CRISPR array. Cell Rep. 16, 2811–2818 (2016)

  20. 20.

    , , , & DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica. Nucleic Acids Res. 44, 4266–4277 (2016)

  21. 21.

    , , , & CRISPR immunological memory requires a host factor for specificity. Mol. Cell 62, 824–833 (2016)

  22. 22.

    , , & Asymmetric positioning of Cas1–2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system. Nucleic Acids Res. 45, 367–381 (2017)

  23. 23.

    et al. Cas9 specifies functional viral targets during CRISPR–Cas adaptation. Nature 519, 199–202 (2015)

  24. 24.

    , & Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity. J. Biol. Chem. 286, 30759–30768 (2011)

  25. 25.

    , , & Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices. Science 266, 763–770 (1994)

  26. 26.

    & Recognition of specific DNA sequences. Mol. Cell 8, 937–946 (2001)

  27. 27.

    Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 44, 1–93 (2011)

  28. 28.

    , , & CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349 (2017)

  29. 29.

    , , & Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009)

  30. 30.

    & Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010)

  31. 31.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  32. 32.

    Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

  33. 33.

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

  34. 34.

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

  35. 35.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  36. 36.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  37. 37.

    et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)

  38. 38.

    & Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014)

  39. 39.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

Download references

Acknowledgements

This work is supported by NIH/NIGMS awards GM118174 and GM102543 to A.K. We thank D. Neau for assistance with data collection, and K. Rajashankar and I. Kriksunov for beamtime allocation. We thank P. Nguyen, R. Battaglia and A. Dolan for technical assistance and discussions. This work is based upon research conducted at NECAT, supported by NIH/NIGMS awards P41-GM103403 and S10-RR029205; and at CHESS and MACCHESS, supported by NSF award DMR-1332208 and NIH/NIGMS award GM-103485. This research used resources of the Advanced Photon Source, a US Department of Energy facility under contract no. DE-AC02-06CH11357.

Author information

Author notes

    • Sherwin Ng
    •  & Ki Hyun Nam

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, New York 14853, USA

    • Yibei Xiao
    • , Sherwin Ng
    •  & Ailong Ke
  2. Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, South Korea

    • Ki Hyun Nam

Authors

  1. Search for Yibei Xiao in:

  2. Search for Sherwin Ng in:

  3. Search for Ki Hyun Nam in:

  4. Search for Ailong Ke in:

Contributions

Y.X. and A.K. designed the research. Y.X. and S.N. are the main contributors to the biochemical and crystallography experiments. Y.X., K.H.N. and A.K. collected diffraction data, determined the structure, and performed structure–function analyses. A.K. and the rest of the authors wrote the manuscript.

Competing interests

Cornell University is in the process of applying for a patent application covering the utilization of the Cas1–Cas2-mediated DNA integration mechanism, as revealed in this study, for potential genome manipulation applications that lists A.K. and Y.X. as inventors.

Corresponding author

Correspondence to Ailong Ke.

Reviewer Information Nature thanks F. Dyda, M. White and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains the uncropped gels.

  2. 2.

    Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature24020

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.