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Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity

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.

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Figure 1: The Cas1–Cas2 complex integrates protospacers in vitro.
Figure 2: Half-site, full-site integration and pCRISPR topoisomer products.
Figure 3: Integration requires 3′-OH protospacer ends and supercoiled target DNA.
Figure 4: Protospacers are specifically integrated into the CRISPR locus.
Figure 5: Model of protospacer integration during CRISPR–Cas adaptive immunity.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

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

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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.

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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.

Corresponding author

Correspondence to Jennifer A. Doudna.

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

Extended data figures and tables

Extended Data Figure 1 The integration reaction is dependent on the presence of protospacers, low salt and divalent metal ions.

a, In vitro integration assay alongside EcoRI- and Nb.BbvCI nickase-treated pCRISPR. b, Salt-dependence assay using Cas1 or Cas2 only and Cas1+Cas2. The titration corresponds to 0, 25, 50, 100 and 200 nM KCl, in addition to the salt carried in from the reaction reagents. c, Integration assays in the presence of 10 mM EDTA, Mg2+, Mn2+ or no additive. d, Integration assays with increasing protospacer concentrations. e, A comparison of post-reaction treatments as indicated. The data presented in ae are representative of at least three replicates.

Extended Data Figure 2 Cas1 requires Cas2 for robust protospacer integration.

a, Schematic of the integration assays using 32P-labelled protospacers (PDB code 4P6I for Cas1–Cas2). b, Integration assays in the presence of increasing protein and 10 mM MnCl2. The titration corresponds to 0, 50, 100 and 200 nM protein. c, Same as b except in the presence of 10 mM MgCl2. The data presented in b and c are representative of at least three replicates.

Extended Data Figure 3 The catalytic activity of Cas1 is required for integration.

a, Close-up view of the Cas1 active site with the conserved residues shown in stick configurations (PDB 4P6I). b, Integration assays of purified Cas1 active site mutants complexed with wild-type Cas2. c, The same as b except using radiolabelled protospacers. The data presented in b and c are representative of at least three replicates.

Extended Data Figure 4 Band X corresponds to topoisomers of pCRISPR.

a, Agarose gel of purified relaxed and band X integration products. b, Analysis of the total reaction products, after phenol chloroform extraction and ethanol precipitation, on a pre-stained agarose gel. c, Same as b except ethidium bromide staining was performed after electrophoresis. d, PCR amplification products of various segments of pCRISPR using the relaxed, band X or pCRISPR template shown in a. The laddering effect of minor products using CRISPR locus primers likely reflects the propensity of CRISPR repeats to form DNA hairpins. The data presented in ad are representative of at least three replicates.

Extended Data Figure 5 Cas1 catalyses the disintegration of half-site integrated protospacers.

a, Schematic of the four strands constituting the Y DNA substrate used in the disintegration assays. b, Native polyacrylamide gel analysis of the annealing products with either strand A or strand C radiolabelled. c, Native polyacrylamide gel analysis of disintegration assay products using Y DNA substrates with strand A labelled. d, Denaturing gel analysis of the disintegration assay products with strand A labelled.

Extended Data Figure 6 Cas1–Cas2 can integrate various lengths of double-stranded DNA with blunt- or 3′-overhang ends into a supercoiled target plasmid.

a, Integration assays using the indicated lengths of protospacer DNA. b, Integration assays using varying 5′ or 3′ overhang lengths. c, d, A comparison of integration assays using pCRISPR or Nb.BbvCI-nicked pCRISPR target. e, Integration assay using different target plasmids with or without a CRISPR locus. The green arrows correspond to the relaxed product of each target and the cyan arrows correspond to the band X product. The data presented in ae are representative of at least three replicates.

Extended Data Figure 7 Cas1 tyrosine mutants support integration activity in vitro.

a, A close-up of the Cas1 active site with the tyrosine residues labelled in blue. b, Structure-based sequence alignment of Cas1 proteins, highlighting the tyrosine residues mutated to alanine in this study. c, Radiolabelled protospacer integration assay of Cas1 tyrosine mutants complexed with wild-type Cas2. The gel presented in c is representative of at least three replicates.

Extended Data Figure 8 High-throughput sequencing of integration products reveals sequence-specific integration.

a, Schematic of the workflow for high-throughput sequencing analysis of the integration sites. b, Raw map of the total reads along pCRISPR before collapsing into single peaks of protospacer–pCRISPR junctions depicted in Fig. 4. c, Same as b, except for the pUC19 target. d, Sequence of the leader-end of the CRISPR locus in E. coli. e, f, WebLogo analysis from the −5 to +5 positions surrounding the protospacer integration sites on the plus (e) and minus (f) of pCRISPR. The arrow points to the nucleotide that is covalently joined to the protospacer. g, h, Same as e, f, except for the pUC19 target.

Extended Data Figure 9 Cas1–Cas2 correctly orients the protospacer DNA during integration.

af, Mapped integration sites along the CRISPR locus of pCRISPR when using protospacer DNA with nucleotide ends ‘wild-type’ 3′ C and 3′ T (a), 3′ A and 3′ T (c), and 3′ C and 3′ C (e). The red arrow in c and e points to the nucleotide change in the protospacer DNA compared to the ‘wild-type’ sequence in a. The protospacer DNA 3′ nucleotide and the CRISPR locus strand biases in a, c, e are plotted in b, d and f, respectively, as percentages of integration events within the CRISPR locus. The black and clear bars represent the (−) and (+) strands of the CRISPR locus, respectively. NS corresponds to not significant and P < 0.0001 by chi-square test. The n values for b, d and f are 5,623, 5,685 and 12,453 reads along the CRISPR locus, respectively.

Extended Data Figure 10 Model of the CRISPR–Cas adaptive immunity pathway in E. coli.

Mature double-stranded protospacers bearing a 3′ C-OH are site-specifically integrated into the leader-end of the CRISPR locus. Correct protospacer integration (left) results in the 5′G/3′C as the first nucleotide of the spacer, proximal to the leader. After transcription of the CRISPR locus and subsequent crRNA processing, foreign DNA destruction is initiated by strand-specific recognition of the 3′-TTC-5′ PAM sequence in the target strand by the crRNA-guided Cascade complex. Incorrect protospacer integration (right) cannot initiate foreign DNA destruction due to the inability for the crRNA to recognize the strand with the 3′-TTC-5′ PAM. Thus, foreign DNA interference during CRISPR–Cas adaptive immunity relies on the Cas1–Cas2 complex for correctly orienting the protospacer during integration.

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Nuñez, J., Lee, A., Engelman, A. et al. Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity. Nature 519, 193–198 (2015). https://doi.org/10.1038/nature14237

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