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Protecting genome integrity during CRISPR immune adaptation

Abstract

Bacterial CRISPR–Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1–Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal. S. pyogenes Cas1–Cas2 is highly specific for the leader-proximal repeat and recognizes the repeat's palindromic ends, thus fitting a model of independent recognition by distal Cas1 active sites. These findings suggest that DNA-insertion sites are less common than suggested by previous work, thereby preventing toxicity during CRISPR immune adaptation and maintaining host genome integrity.

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Figure 1: S. pyogenes Cas1–Cas2 integrates only into plasmids with the target locus.
Figure 2: SpyCas1–Cas2 integration is highly specific for the leader-proximal repeat.
Figure 3: SpyCas1–Cas2 recognizes sequences at the repeat ends for integration.
Figure 4: Improper substrates are arrested as half-site intermediates.
Figure 5: Off-target half sites are resolved only by disintegration.
Figure 6: Model for maintenance of genome stability by SpyCas1–Cas2.

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Acknowledgements

We are grateful to J.K. Nuñez for technical assistance and members of the Doudna laboratory for discussion and input on the manuscript. This project was funded by US National Science Foundation grant no. 1244557 (J.A.D.) and National Institute of General Medicine Sciences grant no. 1P50GM102706-01 (J.H. Cate). A.V.W. is supported by a US National Science Foundation Graduate Fellowship. J.A.D. is supported as an Investigator of the Howard Hughes Medical Institute and as a Paul Allen Distinguished Investigator. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 Instrumentation Grants S10RR029668 and S10RR027303.

Author information

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Authors

Contributions

A.V.W. performed experiments. A.V.W. and J.A.D. designed experiments, analyzed data, and wrote the manuscript.

Corresponding author

Correspondence to Jennifer A Doudna.

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Competing interests

J.A.D. has filed a related patent with the United States Patent and Trademark Office. J.A.D. is a cofounder and Scientific Advisory Board member of Caribou Biosciences and Intellia Therapeutics and a cofounder of Editas Medicine, all of which develop CRISPR-based technologies.

Integrated supplementary information

Supplementary Figure 1 High-throughput sequencing of SpyCas1–Cas2 integration shows preference for leader-proximal repeats.

(a-f) Mapped reads from the integration reaction into pUC19 (a,b), pEcoCR (c,d), and pSpyCR (e,f). Plasmid features are shown below pile-up.

Supplementary Figure 2 SpyCas1–Cas2 requires divalent cations and 3′-OH for integration.

(a) Integration with radiolabeled protospacer and divalent cations. (b) Integration with protospacers lacking a 3′ nucleophile. 3′ deoxy strands are noted as “H”, and the labeled strand is indicated with an asterisk. Uncropped gels are shown in Supplementary Data Set 1.

Supplementary Figure 3 4-nt overhangs allow for more rapid integration.

(a) Integration assay with labeled target and protospacers with 3′ overhangs. Uncropped gel is shown in Supplementary Data Set 1. (b,c) Quantification of leader-side and spacer-side integration in (a). Source data is available online.

Source data

Supplementary Figure 4 Single-nucleotide mutations have minor effects on integration.

(a-c) Integration assays using targets with single-nucleotide transversions (C→G, A→T) at the indicated position in the leader (a), leader-proximal repeat (b), and spacer-proximal repeat (c). The WT target sequence is shown below for reference. Uncropped gels are shown in Supplementary Data Set 1.

Supplementary Figure 5 Full-site integration requires proper-length substrates.

(a) Integration assay with hairpin target and radiolabeled variable-length protospacer. Product bands are indicated. (b) Integration assay with variable-length hairpin target and radiolabeled protospacer. Product bands are indicated. Uncropped gels are shown in Supplementary Data Set 1.

Supplementary Figure 6 Wild-type leader-side and spacer-side half sites both support full-site integration.

(a) Quantification of disintegration reaction shown in Figure 5b,c. Reaction was performed in triplicate. Means are plotted, with error bars representing standard deviation. (b) Disintegration and full-site integration assay with 3′ deoxy leader fragment and labeled protospacer strands on leader-side WT half-site. (c,d) Disintegration assays with wild-type (WT) and off-target (OT) spacer-side half-site substrates with labeled spacer fragment (c) and labeled integrated protospacer strand (d). (e,f) Full-site integration assays with spacer-side half site with labeled plus strand (e) and labeled unintegrated protospacer (f). (g) Disintegration and full-site integration assay with 3′ deoxy spacer fragment and labeled protospacer strands on spacer-side half-site.

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 697 kb)

Supplementary Data Set 1

Uncropped gels (PDF 14718 kb)

Source data

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Wright, A., Doudna, J. Protecting genome integrity during CRISPR immune adaptation. Nat Struct Mol Biol 23, 876–883 (2016). https://doi.org/10.1038/nsmb.3289

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