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Foreign DNA capture during CRISPR–Cas adaptive immunity

Nature volume 527, pages 535–538 (2015)Cite this article

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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.

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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.

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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).

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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

Author notes

  1. James K. Nuñez and Lucas B. Harrington: These authors contributed equally to this work.

Authors and Affiliations

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

    James K. Nuñez, Lucas B. Harrington, Philip J. Kranzusch & Jennifer A. Doudna

  2. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, 94720, California, USA

    Philip J. Kranzusch & Jennifer A. Doudna

  3. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    Alan N. Engelman

  4. Department of Medicine, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Alan N. Engelman

  5. Department of Chemistry, University of California, Berkeley, Berkeley, 94720, California, USA

    Jennifer A. Doudna

  6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Jennifer A. Doudna

  7. Innovative Genomics Initiative, University of California, Berkeley, Berkeley, 94720, California, USA

    Jennifer A. Doudna

  8. Center for RNA Systems Biology, University of California, Berkeley, Berkeley, 94720, California, USA

    Jennifer A. Doudna

Authors
  1. James K. Nuñez
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  4. Alan N. Engelman
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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.

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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
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Supplementary information

Supplementary Information

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

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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

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