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Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity

Nature Structural & Molecular Biology volume 21, pages 528534 (2014) | Download Citation

Abstract

The initial stage of CRISPR–Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1–Cas2 complex. Mutations that perturb Cas1–Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1–Cas2 complexes specify sites of CRISPR spacer integration.

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Acknowledgements

We are grateful for the input on this work provided by members of the Doudna laboratory. We thank S. Floor, A.S. Lee, H.Y. Lee, R. Wilson, R. Wu and K. Zhou for technical assistance, the 8.3.1 beamline staff at the Advanced Light Source and A. Iavarone (University of California, Berkeley) for MS. We thank D. King (Howard Hughes Medical Institute, University of California, Berkeley) for Flag and HA peptides. This project was funded by a US National Science Foundation grant to J.A.D. (no. 1244557). J.K.N. and A.V.W. are supported by US National Science Foundation Graduate Research Fellowships and J.K.N. by a University of California, Berkeley Chancellor's Fellowship. P.J.K. is supported as a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. J.N. is supported by a Long-Term Postdoctoral Fellowship from the Human Frontier Science Program Organization. J.A.D. is supported as an Investigator of the Howard Hughes Medical Institute.

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Affiliations

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

    • James K Nuñez
    • , Philip J Kranzusch
    • , Jonas Noeske
    • , Addison V Wright
    • , Christopher W Davies
    •  & Jennifer A Doudna
  2. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.

    • Philip J Kranzusch
    •  & Jennifer A Doudna
  3. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.

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

    • Jennifer A Doudna

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Contributions

J.K.N. performed the protein purification, biochemical and crystallography experiments. X-ray diffraction data were collected by J.K.N., P.J.K. and J.N., and structure determination was performed by J.K.N. and P.J.K. A.V.W. assisted J.K.N. with in vivo acquisition and immunoprecipitation assays. C.W.D. performed and analyzed analytical ultracentrifugation experiments. J.K.N. and J.A.D. designed the study, analyzed all data and wrote the manuscript.

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Jennifer A Doudna.

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https://doi.org/10.1038/nsmb.2820

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